CN116322760A - Coronavirus antigen composition and use thereof - Google Patents

Abstract

The present disclosure provides compositions and methods comprising cyclic polyribonucleotides comprising sequences encoding coronavirus antigens, and compositions and methods comprising linear polyribonucleotides comprising sequences encoding coronavirus antigens. Compositions and methods relating to the generation of polyclonal antibodies, for example, using the disclosed cyclic polyribonucleotides or the disclosed linear polyribonucleotides, are provided.

Description

Coronavirus antigen composition and use thereof

Sequence listing

The present application contains a sequence listing that has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy name created at 5.20 of 2021 is 51509-020wo6_sequence_listing_5.20.21_st25 and is 207,385 bytes in size.

Background

There is an urgent need for vaccines and therapeutic agents that are active against coronaviruses.

SUMMARY

The present disclosure relates generally to cyclic polyribonucleotides comprising sequences encoding coronavirus antigens and immunogenic compositions comprising the same. The disclosure further relates to methods of using the cyclic polyribonucleotides and the immunogenic composition comprising sequences encoding coronavirus antigens. In some embodiments, the cyclic polyribonucleotides and immunogenic compositions of the present disclosure are used in methods of generating polyclonal antibodies. The polyclonal antibodies produced may be used in a method of preventing a subject (e.g., a human subject) or in a method of treating a subject having a coronavirus infection (e.g., a human subject). The polyclonal antibodies produced may be administered to a subject at high risk of exposure to coronavirus infection.

The disclosure also relates to linear polyribonucleotides comprising a sequence encoding a SEQ ID NO selected from Table 3 and immunogenic compositions comprising the linear polyribonucleotides. The disclosure further relates to methods of using the linear polyribonucleotides comprising sequences encoding coronavirus antigens and the immunogenic compositions comprising the linear polyribonucleotides. In some embodiments, the linear polyribonucleotides and immunogenic compositions of the present disclosure are used in methods of generating polyclonal antibodies. The polyclonal antibodies produced may be used in a method of preventing a subject (e.g., a human subject to be treated) or in a method of treating a subject having a coronavirus infection (e.g., a human subject to be treated). The polyclonal antibodies produced may be administered to a subject to be treated at high risk of exposure to coronavirus infection.

In one aspect, the invention features a composition (e.g., an immunogenic composition) that includes (a) a cyclic polyribonucleotide that includes a sequence that encodes a coronavirus antigen, such as a sequence selected from the group consisting of SEQ ID nos. in table 1 or table 2, or (b) a linear polyribonucleotide that includes a sequence selected from the group consisting of SEQ ID nos. in table 3.

In one embodiment, the composition further comprises plasma from a non-human animal (e.g., a non-human animal comprising a humanized immune system) or a human subject (e.g., after immunization of a subject to be immunized).

In one embodiment, the composition further comprises plasma from a non-human animal (e.g., a non-human animal comprising a humanized immune system) and coronavirus antigen (e.g., after immunization of a non-human animal subject to be immunized). In one embodiment, the composition further comprises plasma from a human subject (e.g., after immunization of the human subject to be immunized) and a coronavirus antigen.

In some embodiments, the composition further comprises a non-human B cell comprising a humanized immunoglobulin locus and a humanized B cell receptor, wherein the humanized B cell receptor binds to the coronavirus antigen. In some embodiments, the composition or immunogenic composition further comprises a plurality of non-human B cells, wherein the non-human B cells of the plurality of non-human B cells comprise a humanized immunoglobulin locus, wherein the plurality of non-human B cells comprises a first B cell that binds to a first epitope of a coronavirus antigen and a second B cell that binds to a second epitope of the coronavirus antigen.

In some embodiments, the coronavirus antigen is from a β coronavirus or fragment thereof, or from a sabbevac virus (sarbecovirus) or fragment thereof. In some embodiments, the coronavirus antigen is derived from Severe Acute Respiratory Syndrome (SARS) -associated coronavirus or a fragment thereof. In some embodiments, the coronavirus antigen is from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a fragment thereof, severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) or a fragment thereof, or middle east respiratory syndrome coronavirus (MERS-CoV) or a fragment thereof.

In some embodiments, the coronavirus antigen is a membrane protein or variant or fragment thereof, an envelope protein or variant or fragment thereof, a spike protein or variant or fragment thereof, a nucleocapsid protein or variant or fragment thereof, a helper protein or variant or fragment thereof. In some embodiments, the coronavirus antigen is a receptor binding domain of a spike protein or variant or fragment thereof. In some embodiments, the spike protein lacks a cleavage site. In some embodiments, the helper protein of the coronavirus is selected from the group consisting of ORF3a, ORF7b, ORF8, ORF10, or any variant or fragment thereof. In some embodiments, the coronavirus antigen comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from table 1 or a sequence selected from SEQ ID No. of table 2. In some embodiments, the cyclic polyribonucleotide comprises a sequence that has at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID nos. of table 2.

In some embodiments, the polyribonucleotide comprises a plurality of sequences, each sequence encoding an antigen, and at least one sequence of the plurality of sequences encodes a coronavirus antigen. In some embodiments, the circular polyribonucleotide comprises two or more ORFs. In some embodiments, the circular polyribonucleotide comprises at least five sequences, each sequence encoding an antigen and at least one of the antigens is a coronavirus antigen. In some embodiments, the circular polyribonucleotide comprises at least two ORFs, e.g., at least 2, 3, 4, or 5 ORFs. In some embodiments, the cyclic polyribonucleotide comprises 5 to 20 sequences, each sequence encoding an antigen and at least one of the antigens is a coronavirus antigen. In some embodiments, the cyclic polyribonucleotide comprises 5 to 10 sequences, each sequence encoding an antigen and at least one of the antigens is a coronavirus antigen. In some embodiments, the cyclic polyribonucleotide comprises a sequence that encodes an antigen from at least two different microorganisms, and at least one microorganism is a coronavirus. In some embodiments, the linear polyribonucleotide comprises a sequence that encodes two or more antigens and at least one antigen is a coronavirus antigen encoded by the sequence of SEQ ID No. in table 3. In some embodiments, the linear polyribonucleotide comprises a sequence that encodes at least 2, 3, 4, or 5 antigens, and at least one antigen is a coronavirus antigen encoded by the sequence of SEQ ID No. in table 3. In some embodiments, the coronavirus antigen comprises an epitope. In some embodiments, the coronavirus antigen comprises an epitope recognized by B cells. In some embodiments, the coronavirus antigen comprises at least two epitopes.

In some embodiments, the composition or immunogenic composition comprising a cyclic polyribonucleotide further comprises a second cyclic polyribonucleotide that comprises a sequence that encodes a second antigen. In some embodiments, the composition or immunogenic composition further comprises a second circular polyribonucleotide comprising a second ORF. In some embodiments, the composition or immunogenic composition further comprises a third, fourth, or fifth cyclic polyribonucleotide comprising a sequence that encodes a third, fourth, or fifth antigen. In some embodiments, the composition or immunogenic composition further comprises a second linear polyribonucleotide comprising a sequence that encodes a second antigen. In some embodiments, the composition or immunogenic composition comprising the linear polyribonucleotide further comprises a second linear polyribonucleotide comprising a second ORF. In some embodiments, the composition or immunogenic composition further comprises a third, fourth, or fifth linear polyribonucleotide comprising a sequence that encodes a third, fourth, or fifth antigen. In some embodiments, the first antigen, the second antigen, the third antigen, the fourth antigen, and the fifth antigen are different antigens.

In some embodiments, the composition or immunogenic composition further comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the polyribonucleotide is administered in the absence of a carrier ("naked"). In other embodiments, the polyribonucleotide is formulated with a carrier such as LNP, VLP, liposome, and the like.

In some embodiments, the composition further comprises an adjuvant. In some embodiments, the composition or immunogenic composition further comprises a diluent. In some embodiments, the composition or immunogenic composition further comprises protamine.

In another aspect, the invention features a method including the steps of: (a) Administering to a non-human animal or to a human subject a composition described herein (e.g., a composition comprising (i) a cyclic polyribonucleotide comprising a sequence encoding a coronavirus antigen, such as a sequence selected from the group consisting of SEQ ID nos. in tables 1, 2, or 3, or (ii) a linear polyribonucleotide comprising a sequence selected from the group consisting of SEQ ID nos. in table 3) (e.g., to induce an immune response against the antigen or to produce polyclonal antibodies against the antigen in a non-human animal or human subject to be immunized) and (b) optionally collecting antibodies against the antigen from the non-human animal or the human subject (e.g., a non-human animal or human subject to be immunized).

In some embodiments, the method further comprises administering an adjuvant (e.g., adavax) to the non-human animal or to a human subject (e.g., a non-human animal or human subject to be immunized)^TM Adjuvant, MF59, AS03, freund's complete adjuvant). The adjuvant may be co-formulated and co-administered with the polyribonucleotide or it may be formulated and administered separately.

In some embodiments, the method further comprises pre-administering (priming) a non-human animal or human subject (e.g., a non-human animal or human subject to be immunized) with an agent (e.g., an antigen) to improve the immunogenic response. For example, the method comprises administering a protein antigen to a non-human animal or human subject (e.g., a non-human animal or human subject to be immunized) prior to administering a polyribonucleotide comprising a sequence encoding the antigen (e.g., 1-7 days, e.g., 1, 2, 3, 4, 5, 6, 7 days ago). The protein antigen may be administered as a protein formulation, or encoded in a plasmid (pDNA), or present in a virus-like particle (VLP), formulated in the form of a Lipid Nanoparticle (LNP), and the like.

In some embodiments, the method further comprises administering or immunizing a subject (e.g., a subject to be immunized) with protamine.

In some embodiments, the polyribonucleotide is administered in the absence of a carrier ("naked"). In other embodiments, the polyribonucleotide is formulated with a carrier such as LNP, VLP, liposome, and the like.

In some embodiments, the method further comprises administering or immunizing a subject (e.g., a subject to be immunized) with a polyribonucleotide (e.g., a circular or linear polyribonucleotide) at least twice, e.g., 2, 3, 4, 5 times.

In some embodiments, the method further comprises collecting plasma from the subject (e.g., after the subject to be immunized is immunized). In some embodiments, the method further comprises purifying the polyclonal antibody from the subject (e.g., after immunization of the subject to be immunized). In some embodiments, the method further comprises administering or immunizing a subject (e.g., a subject to be immunized). In some embodiments, the vaccine is a pneumococcal polysaccharide vaccine (e.g., PCV13 or PPSV 23). In some embodiments, the vaccine is for bacterial infection. In some embodiments, the subject (e.g., a subject to be immunized) is vaccinated with the loop RNA by injection. In some embodiments, the subject (e.g., a subject to be immunized) is immunized by injection with linear RNA.

In embodiments, the subject is a human subject (e.g., a human subject to be immunized). In some embodiments, the human subject (e.g., a human subject to be immunized) is a subject at risk of a coronavirus-related disease, e.g., a human over 50 years old; immunocompromised persons; a person suffering from a chronic healthy condition such as obesity, diabetes, cancer, etc.; health care workers.

In embodiments, the subject is a non-human animal (e.g., a non-human animal to be vaccinated). In some embodiments, the non-human animal (e.g., a non-human animal to be vaccinated) is an agricultural animal, such as a cow, pig, sheep, horse, goat; pets, such as cats or dogs; or zoo animals, such as felines.

In some embodiments, the non-human animal (e.g., a non-human animal to be immunized) is a mammal, such as a rodent (e.g., a rabbit, rat, or mouse), or an ungulate (e.g., a pig, cow, goat, or sheep). In some embodiments, the non-human animal (e.g., a non-human animal to be immunized) is a transchromosomal non-human animal comprising a humanized immunoglobulin locus. In some embodiments, the non-human animal is a transchromosomal bovine comprising a Human Artificial Chromosome (HAC) vector comprising a humanized immunoglobulin locus. In some embodiments, the humanized immunoglobulin locus encodes an immunoglobulin heavy chain. In some embodiments, the humanized immunoglobulin heavy chain comprises an IgG isotype heavy chain. In some embodiments, the humanized immunoglobulin heavy chain comprises an IgG1, igG2, igG3, or IgG4 isotype heavy chain.

In some embodiments, the non-human animal (e.g., a non-human animal to be immunized) comprises B cells having a B cell receptor that binds to a coronavirus antigen. In some embodiments, the non-human animal comprises a plurality of B cells including a first B cell that binds to a first epitope of a coronavirus antigen and a second B cell that binds to a second epitope of the coronavirus antigen.

In some embodiments, the non-human animal (e.g., a non-human animal to be immunized) comprises T cells, wherein the T cells comprise a T cell receptor that binds to a coronavirus antigen. In some embodiments, the T cells enhance production of antibodies that bind to the antigen after activation. In some embodiments, the T cells enhance antibody production by B cells that bind to the coronavirus antigen after activation. In some embodiments, the T cells, upon activation, enhance survival, proliferation, plasma cell differentiation, somatic hypermutation, immunoglobulin class switching, or a combination thereof, of B cells that bind to the coronavirus antigen.

In some embodiments, the non-human animal or human subject (e.g., a non-human animal or human subject to be immunized) produces antibodies that specifically bind to coronavirus antigens. In some embodiments, the antibody is a humanized antibody or a fully human antibody. In some embodiments, the antibody is an IgG, igA, or IgM isotype antibody. In some embodiments, the antibody is an IgG1, igG2, igG3, or IgG4 isotype antibody. In some embodiments, the non-human animal (e.g., a non-human animal to be vaccinated) comprises a plurality of polyclonal antibodies that specifically bind to at least two epitopes encoded by the cyclic polyribonucleotides. In some embodiments, the non-human animal or human subject (e.g., a non-human animal or human subject to be immunized) comprises a plurality of polyclonal antibodies that specifically bind to at least two epitopes encoded by linear RNAs. In some embodiments, the plurality of antibodies comprises humanized antibodies. In some embodiments, the plurality of polyclonal antibodies comprises fully human antibodies. In some embodiments, the plurality of polyclonal antibodies comprises IgG antibodies, igG1 antibodies, igG2 antibodies, igG3 antibodies, igG4 antibodies, igM antibodies, igA antibodies, or a combination thereof. In some embodiments, the immunoglobulin heavy chain comprises an IgM or IgA isotype heavy chain. In some embodiments, the humanized immunoglobulin locus encodes an immunoglobulin light chain. In some embodiments, the immunoglobulin light chain comprises a kappa light chain or a lambda light chain.

In some embodiments, the method further comprises collecting blood from the non-human animal or human subject (e.g., after immunization of the non-human animal or human subject to be immunized) and purifying antibodies to the antigen from the blood.

In another aspect, the invention features an anti-coronavirus antibody formulation (e.g., a polyclonal antibody formulation) produced by: (a) Administering a composition comprising a polyribonucleotide described herein to a non-human animal (e.g., a bovine having a humanized immune system as described herein) or a human subject (e.g., a non-human animal or human subject to be immunized) described herein, and (b) collecting antibodies to the antigen from the non-human animal or human subject (e.g., after immunization of the non-human animal or human subject to be immunized).

In embodiments, the polyribonucleotide is (a) a cyclic polyribonucleotide comprising a sequence that encodes a coronavirus antigen, such as a sequence selected from the group consisting of SEQ ID nos. in tables 1, 2, or 3, or (b) a linear polyribonucleotide comprising a sequence selected from the group consisting of SEQ ID nos. in table 3.

In embodiments, the antibody formulation is formulated into a pharmaceutical composition.

In another aspect, the invention features a method of delivering an antibody to a coronavirus to a subject (e.g., a subject to be treated) having, at risk of being exposed to, or in need of a coronavirus infection, e.g., a method of preventing or treating a coronavirus infection in a subject (e.g., a subject to be treated). The method comprises administering to a subject having, at risk of exposure to, or in need of, a coronavirus infection (e.g., a subject to be treated) polyclonal antibodies raised from an animal (e.g., mammal) having a human or humanized immune system that has been immunized with a polyribonucleotide described herein, e.g., (a) a cyclic polyribonucleotide comprising a sequence encoding a coronavirus antigen, e.g., a sequence selected from SEQ ID No. in table 1, 2, or 3, or (b) a linear polyribonucleotide comprising a sequence selected from SEQ ID No. in table 3.

In certain embodiments, the method further comprises one or more of the following: immunization of a non-human animal (e.g., a non-human animal to be immunized) that has been genetically modified to produce a human antibody with the polyribonucleotides disclosed herein, collecting blood from the non-human animal, purifying the antibody from the non-human animal, formulating the antibody for pharmaceutical use, and administering the formulated antibody to a human subject (e.g., a human subject to be treated).

In some embodiments, the mammal having a human or humanized immune system is a human (e.g., a human subject to be immunized).

In some embodiments, the mammal having a human or humanized immune system is a non-human animal that has been genetically modified to produce human antibodies, e.g., a non-human animal comprising a humanized immunoglobulin locus, e.g., a transchromosomal bovine comprising a Human Artificial Chromosome (HAC) vector comprising a human immunoglobulin locus.

In embodiments, the subject having a coronavirus infection or in need thereof (e.g., a subject to be treated) is a human subject diagnosed with a coronavirus-related disease, e.g., covid-19, SARS, MERS. In some embodiments, the subject at risk of exposure to or in need of a coronavirus infection (e.g., a subject to be treated) is a subject at risk of a coronavirus-related disease, e.g., a human over 50 years old; immunocompromised persons; a person suffering from a chronic healthy condition such as obesity, diabetes, cancer, etc.; health care workers.

In some embodiments, administration or immunization is performed before, after, or simultaneously with exposure to the risk of coronavirus.

In some embodiments, the method further comprises monitoring the human subject (e.g., a subject to be treated) for the presence of antibodies to the coronavirus, e.g., before and/or after administration.

Exemplary embodiments of the present invention are described in the paragraphs enumerated below.

E1. An immunogenic composition comprising:

a) A circular polyribonucleotide comprising a sequence that encodes a coronavirus antigen; or (b)

b) A linear polyribonucleotide comprising a sequence selected from any one of SEQ ID NOs 13, 15 and 12.

E2. An immunogenic composition comprising a cyclic polyribonucleotide comprising a sequence that encodes a coronavirus antigen, wherein the coronavirus antigen comprises a sequence that has at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to a coronavirus antigen selected from any one of SEQ ID NOs 1-10, 13, 15, 17, 19, 21, 23, 25-30, 48 and 49, or the cyclic polyribonucleotide comprises a sequence that has at least about 80%, 85%, 90%, 95%, 99% or 100% sequence identity to a cyclic polyribonucleotide selected fromSEQ ID NOs 12, 14, 16, 18, 20, 22 and 24.

E3. The immunogenic composition of any one of the preceding embodiments, further comprising plasma from a non-human animal (e.g., a non-human animal comprising a humanized immune system; e.g., a non-human animal to be immunized) or a human subject (e.g., a human subject to be immunized).

E4. The immunogenic composition of any one of the preceding embodiments, further comprising the coronavirus antigen.

E5. The immunogenic composition of any one of the preceding embodiments, wherein the composition further comprises non-human B cells comprising a humanized immunoglobulin locus and a humanized B cell receptor, wherein the humanized B cell receptor binds to the coronavirus antigen.

E6. The immunogenic composition of any one of the preceding embodiments, wherein the composition further comprises a plurality of non-human B cells, wherein a non-human B cell of the plurality of non-human B cells comprises a humanized immunoglobulin locus, wherein the plurality of B cells comprises a first B cell that binds to a first epitope of the coronavirus antigen and a second B cell that binds to a second epitope of the coronavirus antigen.

E7. The immunogenic composition of any one of the preceding embodiments, wherein the coronavirus antigen is from a beta coronavirus or fragment thereof, or from a sabcomev or fragment thereof.

E8. The immunogenic composition of any one of the preceding embodiments, wherein the coronavirus antigen is derived from severe acute respiratory syndrome-associated coronavirus or a fragment thereof.

E9. The immunogenic composition of any one of the preceding embodiments, wherein the coronavirus antigen is derived from Severe Acute Respiratory Syndrome (SARS) -associated coronavirus or a fragment thereof.

E10. The immunogenic composition of any one of the preceding embodiments, wherein the coronavirus antigen is from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a fragment thereof, severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) or a fragment thereof, or middle east respiratory syndrome coronavirus (MERS-CoV) or a fragment thereof.

E11. The immunogenic composition of any one of the preceding embodiments, wherein the coronavirus antigen is a membrane protein or variant or fragment thereof, an envelope protein or variant or fragment thereof, a spike protein or variant or fragment thereof, a nucleocapsid protein or variant or fragment thereof, a helper protein or variant or fragment thereof.

E12. The immunogenic composition of any one of the preceding embodiments, wherein the coronavirus antigen is a receptor binding domain of a spike protein or variant or fragment thereof.

E13. The immunogenic composition of example 8, wherein the spike protein lacks a cleavage site.

E14. The immunogenic composition according to any one of the preceding embodiments, wherein the helper protein of the coronavirus is selected from the group consisting of ORF3a, ORF7b, ORF8, ORF10, or any variant or fragment thereof.

E15. The immunogenic composition of any one of the preceding embodiments, wherein the cyclic polyribonucleotide comprises a plurality of sequences, each sequence encoding an antigen, and at least one sequence encoding a coronavirus antigen.

E16. The immunogenic composition of any one of the preceding embodiments, wherein the cyclic polyribonucleotide comprises two or more ORFs.

E17. The immunogenic composition of any one of the preceding embodiments, wherein the cyclic polyribonucleotide comprises at least five sequences, each sequence encoding an antigen, and at least one antigen is a coronavirus antigen.

E18. The immunogenic composition of any one of the preceding embodiments, wherein the cyclic polyribonucleotide comprises at least two ORFs (e.g., at least 2, 3, 4, or 5).

E19. The immunogenic composition of any one of the preceding embodiments, wherein the cyclic polyribonucleotide comprises a sequence that encodes an antigen from at least two different microorganisms, and at least one microorganism is a coronavirus.

E20. The immunogenic composition of any one of the preceding embodiments, wherein the linear polyribonucleotide comprises a sequence that encodes two or more antigens and at least one antigen is the coronavirus antigen.

E21. The immunogenic composition of any one of the preceding embodiments, wherein the linear polyribonucleotide comprises a sequence that encodes at least 2, 3, 4 or 5 antigens, and at least one antigen is a coronavirus antigen encoded by the sequence of SEQ ID No. in table 3.

E22. The immunogenic composition of any one of the preceding embodiments, wherein the coronavirus antigen comprises an epitope.

E23. The immunogenic composition of any one of the preceding embodiments, wherein the coronavirus antigen comprises an epitope recognized by B cells.

E24. The immunogenic composition of any one of the preceding embodiments, wherein the coronavirus antigen comprises at least two epitopes.

E25. The immunogenic composition of any one of the preceding embodiments, further comprising a second cyclic polyribonucleotide comprising a sequence that encodes a second antigen.

E26. The immunogenic composition of any one of the preceding embodiments, further comprising a second cyclic polyribonucleotide comprising a second ORF.

E27. The immunogenic composition of any one of the preceding embodiments, further comprising a third, fourth, or fifth cyclic polyribonucleotide comprising a sequence that encodes a third, fourth, or fifth antigen.

E28. The immunogenic composition of any one of the preceding embodiments, further comprising a second linear polyribonucleotide comprising a sequence that encodes a second antigen.

E29. The immunogenic composition of any one of the preceding embodiments, further comprising a second linear polyribonucleotide comprising a second ORF.

E30. The immunogenic composition of any one of the preceding embodiments, further comprising a third, fourth, or fifth linear polyribonucleotide comprising a sequence that encodes a third, fourth, or fifth antigen.

E31. The immunogenic composition of any one of the preceding embodiments, wherein the first antigen, second antigen, third antigen, fourth antigen, and fifth antigen are different antigens.

E32. The immunogenic composition of any one of the preceding embodiments, wherein the immunogenic composition further comprises a pharmaceutically acceptable carrier or excipient.

E33. The immunogenic composition of any one of the preceding embodiments, wherein the immunogenic composition further comprises a pharmaceutically acceptable excipient and does not contain any carrier.

E34. The immunogenic composition of any one of the preceding embodiments, wherein the cyclic polyribonucleotide, linear polyribonucleotide, or immunogenic composition is formulated with a carrier (e.g., a lipid nanoparticle, virus-like particle, or liposome).

E35. The immunogenic composition of any one of the preceding embodiments, wherein the immunogenic composition further comprises an adjuvant.

E36. The immunogenic composition ofembodiment 35, wherein the adjuvant is a saponin or an oil emulsion.

E37. The immunogenic composition of embodiment 36, wherein the oil emulsion is a squalene-water emulsion (e.g., adavax^TM Adjuvant, MF59 or AS 03).

E38. The immunogenic composition of any one of the preceding embodiments, wherein the immunogenic composition further comprises a diluent.

E40. A Lipid Nanoparticle (LNP) comprising the immunogenic composition of any of the preceding embodiments.

E41. The LNP ofembodiment 40, comprising an ionizable lipid.

E42. The LNP of example 40, comprising a cationic lipid.

E43. The LNP of example 42, wherein the cationic lipid has a structure according to:

E44. The LNP of any one of embodiments 40-43, further comprising one or more neutral lipids, e.g., DSPC, DPPC, DMPC, DOPC, POPC, DOPE, SM, a steroid, e.g., cholesterol, and/or one or more polymer conjugated lipids, e.g., a pegylated lipid, e.g., PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkoxypropyl carbamate.

E45. A method of delivering an immunogenic composition to a non-human animal or human subject (e.g., a non-human animal or human subject to be immunized), the method comprising: a) Administering the immunogenic composition of any one of the preceding embodiments to the non-human animal or human subject, and b) optionally, collecting antibodies to the coronavirus antigen from the non-human animal or human subject.

E46. A method of inducing an immune response against a coronavirus antigen in a non-human animal or human subject (e.g., a non-human animal or human subject to be immunized), the method comprising: a) Administering the immunogenic composition of any one of the preceding embodiments to the non-human animal or human subject, and b) optionally, collecting antibodies to the coronavirus antigen from the non-human animal or human subject.

E47. The method of any one of the preceding embodiments, further comprising administering an adjuvant to the non-human animal or human subject (e.g., a non-human animal or human subject to be immunized).

E48. The method of example 47, wherein the adjuvant is co-formulated and co-administered with the immunogenic composition or formulated and administered separately from the immunogenic composition.

E49. The method of any one of the preceding embodiments, further comprising administering (e.g., pre-administering or priming) the non-human animal or human subject (e.g., a non-human animal or human subject to be immunized) with the coronavirus antigen prior to administering the immunogenic composition.

E50. The method of any one of the preceding embodiments, further comprising administering the coronavirus antigen to the non-human animal or human subject (e.g., the non-human animal or human subject to be immunized) 1 to 7 days (e.g., 1, 2, 3, 4, 5, 6, or 7 days prior to administration of the immunogenic composition).

E51. The method of any one of the preceding embodiments, wherein the coronavirus antigen is administered as a protein formulation, encoded in a plasmid (pDNA), present in a virus-like particle (VLP), or formulated in the form of a Lipid Nanoparticle (LNP), or the like.

E52. The method of any one of the preceding embodiments, further comprising administering the cyclic or linear polyribonucleotide in the absence of a carrier.

E53. The method of any one of the preceding embodiments, further comprising formulating the immunogenic composition with a carrier (e.g., a lipid nanoparticle, a virus-like particle, or a liposome).

E54. The method of any one of the preceding embodiments, further comprising administering or immunizing the cyclic or linear polyribonucleotide at least twice (e.g., 2, 3, 4, or 5 times) to the non-human animal or human subject (e.g., a non-human animal or human subject to be immunized).

E55. The method of any one of the preceding embodiments, further comprising collecting plasma from the non-human animal or human subject (e.g., a non-human animal or human subject to be immunized).

E56. The method of any one of the preceding embodiments, further comprising purifying the polyclonal antibody from the plasma of a non-human animal or human subject (e.g., a non-human animal or human subject to be immunized).

E57. The method of any one of the preceding embodiments, further comprising administering or immunizing the non-human animal or human subject (e.g., a non-human animal or human subject to be immunized).

E58. The method of embodiment 51, wherein the vaccine is a pneumococcal polysaccharide vaccine (e.g., PCV13 or PPSV 23).

E59. The method of embodiment 57, wherein the vaccine is for bacterial infection.

E60. The method of any one of the preceding embodiments, wherein the non-human animal or human subject (e.g., a non-human animal or human subject to be immunized) is immunized by injection with the cyclic or linear polyribonucleotide.

E61. The method of any one of the preceding embodiments, wherein the human subject (e.g., a human subject to be immunized) is at risk of developing a coronavirus-related disease.

E62. The method of any one of the preceding embodiments, wherein the human subject (e.g., human subject to be immunized) is a human over 50 years old, immunocompromised, a human suffering from a chronic health condition (e.g., obesity, diabetes, cancer), or a health care worker.

E63. The method of any one of the preceding embodiments, wherein the non-human animal (e.g., a non-human animal subject to be immunized) is an agricultural animal (e.g., a pig, cow, goat, chicken, sheep).

E64. The method of any one of the preceding embodiments, wherein the non-human animal (e.g., a non-human animal subject to be immunized) is a pet (e.g., a dog or cat), a zoo animal (e.g., a feline), a mammal (e.g., an ungulate (e.g., a pig, cow, goat, sheep), a rodent (e.g., a rabbit, rat, mouse).

E65. The method of any one of the preceding embodiments, wherein the non-human animal is a transchromosomal non-human animal comprising a humanized immunoglobulin locus.

E66. The method of embodiment 65, wherein the non-human animal is a transchromosomal bovine comprising a Human Artificial Chromosome (HAC) vector comprising a humanized immunoglobulin locus.

E67. The method of any one of embodiments 65 or 66, wherein the humanized immunoglobulin locus encodes an immunoglobulin heavy chain.

E68. The method of embodiment 67, wherein the humanized immunoglobulin heavy chain comprises an IgG isotype heavy chain.

E69. The method of any one of embodiments 67 or 68, wherein the humanized immunoglobulin heavy chain comprises an IgG1, igG2, igG3, or IgG4 isotype heavy chain.

E70. The method of any one of embodiments 65-69, wherein the humanized immunoglobulin locus encodes an immunoglobulin light chain.

E71. The method of embodiment 70, wherein the immunoglobulin light chain comprises a kappa light chain or a lambda light chain.

E72. The method of any one of the preceding embodiments, wherein the non-human animal comprises a B cell having a B cell receptor, and wherein the B cell receptor binds to the antigen.

E73. The method of any one of the preceding embodiments, wherein the non-human animal comprises a plurality of B cells comprising a first B cell that binds to a first epitope of a coronavirus antigen and a second B cell that binds to a second epitope of the coronavirus antigen.

E74. The method of any one of the preceding embodiments, wherein the non-human animal comprises a T cell, and wherein the T cell comprises a T cell receptor that binds to the coronavirus antigen.

E75. The method of any one of the preceding embodiments, wherein the T cells enhance production of antibodies that bind to the coronavirus antigen after activation.

E76. The method of any one of the preceding embodiments, wherein upon activation, the T cell enhances antibody production by a B cell that binds to the coronavirus antigen.

E77. The method of any one of the preceding embodiments, wherein upon activation, the T cells enhance survival, proliferation, plasma cell differentiation, somatic hypermutation, immunoglobulin class switching, or a combination thereof, of B cells that bind to a coronavirus antigen.

E78. The method of any one of the preceding embodiments, further comprising purifying polyclonal antibodies to the coronavirus antigen from the plasma of the non-human animal or human subject (e.g., the non-human animal or human subject to be immunized).

E79. The method of any one of the preceding embodiments, wherein one of the polyclonal antibodies specifically binds to the coronavirus antigen.

E80. The method of any one of the preceding embodiments, wherein one of the polyclonal antibodies is a humanized antibody or a fully human antibody.

E81. The method of any one of the preceding embodiments, wherein one of the polyclonal antibodies is an IgG, igG, igA or IgM isotype antibody.

E82. The method of any one of the preceding embodiments, wherein one of the polyclonal antibodies is an IgG1, igG2, igG3, or IgG4 isotype antibody.

E83. The method of any one of the preceding embodiments, wherein the non-human animal comprises a plurality of polyclonal antibodies that specifically bind to at least two epitopes encoded by the cyclic polyribonucleotide.

E84. The method of any one of the preceding embodiments, wherein the non-human animal comprises a plurality of polyclonal antibodies that specifically bind to at least two epitopes encoded by the linear polyribonucleotide.

E85. The method of any one of embodiments 81 or 82, wherein the plurality of polyclonal antibodies comprises humanized antibodies.

E86. The method of any one of embodiments 83 or 84, wherein the plurality of polyclonal antibodies comprises fully human antibodies.

E87. The method of any one of embodiments 83-86, wherein the plurality of polyclonal antibodies comprises IgG antibodies, igG1 antibodies, igG2 antibodies, igG3 antibodies, igG4 antibodies, igM antibodies, igA antibodies, or a combination thereof.

E88. The method of any one of embodiments 83-86, wherein the plurality of polyclonal antibodies comprises humanized immunoglobulin loci that comprise IgM or IgA isotype heavy chains.

E89. The method of any one of embodiments 83-88, wherein the plurality of polyclonal antibodies comprises humanized immunoglobulin loci encoding immunoglobulin light chains.

E90. The method of example 89, wherein the immunoglobulin light chains comprise kappa light chains or lambda light chains.

E91. The method of any one of the preceding embodiments, further comprising collecting blood from the non-human animal or human subject (e.g., a non-human animal or human subject to be immunized) and purifying antibodies to the coronavirus antigen from the blood.

E92. A method of producing a polyclonal antibody preparation (e.g., an anti-coronavirus antibody preparation) directed against a coronavirus antigen, the method comprising:

a) Administering the immunogenic composition of any one of the preceding embodiments to a non-human animal or human subject (e.g., a non-human animal or human subject to be immunized); and

b) Blood or plasma is collected from the non-human animal or human subject.

E93. The method of embodiment 92, wherein the polyclonal antibody preparation is formulated as a pharmaceutical composition or a veterinary composition.

E94. A method of delivering a polyclonal antibody preparation against a coronavirus to a subject having a coronavirus infection (e.g., a subject to be treated), the method comprising administering the polyclonal antibody preparation of any one of the preceding embodiments to a subject having a coronavirus infection.

E95. A method of delivering a polyclonal antibody preparation to a subject at risk of exposure to a coronavirus infection (e.g., a subject to be treated), the method comprising administering the polyclonal antibody preparation of any one of the preceding embodiments to a subject at risk of exposure to a coronavirus infection.

E96. A method of preventing or treating a coronavirus infection in a subject in need thereof (e.g., a subject to be treated), the method comprising administering to a subject in need thereof the polyclonal antibody preparation of any one of the preceding embodiments.

E97. The method of any of the preceding embodiments, further comprising:

a) Immunizing a non-human animal that has been genetically modified to produce a human antibody with a cyclic polyribonucleotide according to any of the preceding embodiments or a linear polyribonucleotide according to any of the preceding embodiments;

b) Collecting blood from the non-human animal;

c) Purifying antibodies from the non-human animal;

d) Preparing the antibodies for pharmaceutical use; and

e) Administering the formulated antibody to a human subject.

E98. The method of embodiment 97, wherein the non-human animal has a humanized immune system.

E99. The method of embodiment 97, wherein the non-human animal has a humanized immunoglobulin locus.

E100. The method of embodiment 97, wherein the non-human animal is a transchromosomal bovine comprising a Human Artificial Chromosome (HAC) vector comprising a human immunoglobulin locus.

E101. The method of any one of the preceding embodiments, wherein the administering or immunizing is performed before, after, or simultaneously with a subject in need thereof being at risk of exposure to coronavirus.

E102. The method of any one of the preceding embodiments, wherein the subject having a coronavirus infection (e.g., a subject to be treated), the subject at risk of exposure to a coronavirus infection, or the subject in need thereof is a human subject.

E103. The method of embodiment 102, wherein the human subject (e.g., human subject to be treated) is a human over 50 years old, an immunocompromised human, a human suffering from a chronic health condition (e.g., obesity, diabetes, or cancer), or a health care worker.

E104. The method of any one of the preceding embodiments, wherein the subject at risk of exposure to a coronavirus infection (e.g., a subject to be treated) or in need thereof is a human subject at risk of a coronavirus-related disease.

E105. The method of any one of the preceding embodiments, wherein the subject having a coronavirus infection (e.g., a subject to be treated), the subject at risk of exposure to a coronavirus infection, or the subject in need thereof is a human subject diagnosed with a coronavirus-related disease (e.g., covid-19, SARS, MERS).

E106. The method of any one of the preceding embodiments, wherein the subject having a coronavirus infection (e.g., a subject to be treated), the subject at risk of exposure to a coronavirus infection (e.g., a subject to be treated), or the subject in need thereof (e.g., a subject to be treated) is a non-human animal subject.

E107. The method of any one of the preceding embodiments, wherein the subject having a coronavirus infection (e.g., a subject to be treated), the subject at risk of exposure to a coronavirus infection (e.g., a subject to be treated), or the subject in need thereof (e.g., a subject to be treated) is an agricultural animal (e.g., a cow, pig, sheep, horse, goat), a pet (e.g., a cat or dog), or a zoo animal (e.g., a feline).

E108. The method of any one of the preceding embodiments, further comprising monitoring a subject having a coronavirus infection (e.g., a subject to be treated), a subject at risk of exposure to a coronavirus infection (e.g., a subject to be treated), or a subject in need thereof for the presence or absence of the polyclonal antibodies.

E109. The method of any one of the preceding embodiments, wherein the monitoring is prior to and/or after administration of the polyclonal antibodies.

Definition of the definition

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Unless otherwise indicated, the terms set forth below are generally to be understood as being a consensus thereof.

As used herein, the terms "circRNA", "circular polyribonucleotide" and "circular RNA" are used interchangeably and refer to a polyribonucleotide molecule having a structure without a free end (i.e., without a free 3 'end and/or 5' end), e.g., a polyribonucleotide that forms a circular or endless structure by covalent or non-covalent bonds.

As used herein, the terms "circRNA formulation," "circular polyribonucleotide formulation," and "circular RNA formulation" are used interchangeably and refer to a composition comprising a circRNA molecule and a diluent, carrier, first adjuvant, or combination thereof. An "immunogenic" circRNA formulation is a formulation that, when introduced into an animal, causes the animal's immune system to become reactive to antigens expressed by the circRNA.

As used herein, the terms "linear RNA," "linear polyribonucleotide," and "linear polyribonucleotide molecule" are used interchangeably and refer to a single or polyribonucleotide molecule having a 5 'end and a 3' end. One or both of the 5 'and 3' ends may be free ends or may be linked to another moiety. In some embodiments, the linear RNA has a 5 'end or a 3' end that is modified or protected from degradation (e.g., protected by a 5 'end protecting agent or a 3' end protecting agent). In some embodiments, the linear RNA has a non-covalently linked 5 'or 3' end. Linear RNAs can be used as starting materials for circularization by, for example, splint ligation or chemical, enzymatic, ribozyme or splice-catalyzed circularization methods.

As used herein, the terms "linear RNA formulation" and "linear polyribonucleotide formulation" are used interchangeably and refer to a composition comprising a linear RNA molecule and a diluent, carrier, first adjuvant, or combination thereof. An "immunogenic" linear RNA formulation is a formulation that, when introduced into an animal, causes the animal's immune system to become reactive to antigens expressed by the circRNA.

As used herein, the term "total ribonucleotide molecule" means the total amount of any ribonucleotide molecule as measured by the total mass of the ribonucleotide molecule, including linear polyribonucleotide molecules, cyclic polyribonucleotide molecules, monomeric ribonucleotides, other polyribonucleotide molecules, fragments thereof and modified variants thereof.

As used herein, the term "fragment" means any portion of a nucleotide molecule that is at least one nucleotide shorter than the nucleotide molecule. For example, the nucleotide molecule may be a linear polyribonucleotide molecule and the fragment thereof may be a single ribonucleotide or any number of consecutive polyribonucleotides as part of a linear polyribonucleotide molecule. For another example, the nucleotide molecule may be a cyclic polyribonucleotide molecule and the fragment thereof may be a polyribonucleotide or any number of consecutive polyribonucleotides as part of a cyclic polyribonucleotide molecule. Fragments of a nucleotide molecule comprise at least 10 nucleic acid residues, e.g., at least 20 nucleic acid residues, at least 50 nucleic acid residues, and at least 100 nucleic acid residues. Fragment also means any portion of a polypeptide molecule that is at least one peptide shorter than the polypeptide molecule. For example, a fragment of a polypeptide may be a polypeptide or any number of consecutive amino acids that are part of a full-length polypeptide molecule. Fragments of a polypeptide comprise at least 5 amino acid residues, e.g., at least 10 amino acid residues, at least 20 amino acid residues, at least 50 amino acid residues, at least 100 amino acid residues.

As used herein, the term "expression sequence" is a nucleic acid sequence encoding a product, such as a peptide or polypeptide or regulatory nucleic acid. An exemplary expression sequence encoding a peptide or polypeptide may comprise a plurality of nucleotide triplets, each of which may encode an amino acid, and is referred to as a "codon".

As used herein, the term "modified ribonucleotide" is a nucleotide that has at least one modification to a sugar, nucleobase or internucleoside linkage.

As used herein, the phrase "quasi-helical structure" is a higher order structure of a cyclic polyribonucleotide in which at least a portion of the cyclic polyribonucleotide is folded into a helical structure.

As used herein, the phrase "quasi-double stranded secondary structure" is a higher order structure of a cyclic polyribonucleotide, wherein at least a portion of the cyclic polyribonucleotide creates an internal double strand.

As used herein, the term "regulatory element" is a portion, such as a nucleic acid sequence, that modifies the expression of an expression sequence within a circular polyribonucleotide.

As used herein, the term "repetitive nucleotide sequence" is a repetitive nucleic acid sequence within a piece of DNA or RNA or within the entire genome. In some embodiments, the repetitive nucleotide sequence comprises a poly CA sequence or a poly TG (UG) sequence. In some embodiments, the repetitive nucleotide sequence comprises a repetitive sequence in the Alu family of introns.

As used herein, the term "replicating element" is a sequence and/or motif that can be used to replicate or initiate transcription of a circular polyribonucleotide.

As used herein, the term "staggered element" is a moiety, such as a nucleotide sequence, that induces a ribosome pause during translation. In some embodiments, the staggered elements are non-conserved sequences of amino acids with strong alpha-helix propensity, followed by consensus sequence-D (V/I) ExNPG P, where x = any amino acid. In some embodiments, the staggered elements may include chemical moieties, such as glycerol, non-nucleic acid linking moieties, chemical modifications, modified nucleic acids, or any combination thereof.

As used herein, the term "substantially resistant to … …" may refer to a substance that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% resistant to an effector as compared to a reference.

As used herein, the term "stoichiometric translation" is the substantially equal production of an expression product resulting from translation of a circular polyribonucleotide. For example, for a cyclic polyribonucleotide having two expressed sequences, stoichiometric translation of the cyclic polyribonucleotide means that the expression products of the two expressed sequences can have substantially equal amounts, e.g., the difference (e.g., molar difference) between the two expressed sequences can be about 0, or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20%, or any percentage in between.

As used herein, the term "translation initiation sequence" is a nucleic acid sequence that initiates translation of an expressed sequence in a cyclic polyribonucleotide.

As used herein, the term "termination element" is a portion, such as a nucleic acid sequence, that terminates translation of an expressed sequence in a circular polyribonucleotide.

As used herein, the term "translational efficiency" is the rate or amount of production of a protein or peptide from a ribonucleotide transcript. In some embodiments, translation efficiency may be expressed as the amount of protein or peptide produced by a given amount of a transcript encoding a protein or peptide, for example, over a given period of time, for example, in a given translation system, for example, an in vitro translation system (like rabbit reticulocyte lysate) or an in vivo translation system (like eukaryotic or prokaryotic cells).

As used herein, the term "cyclization efficiency" is a measure of the resulting cyclic polyribonucleotides relative to their non-cyclic starting materials.

As used herein, the term "adaptive immune response" refers to a humoral or cell-mediated immune response. Humoral immune responses (also known as antibody immune responses) are mediated by B lymphocytes, which release antibodies that bind specifically to antigens. Cell-mediated immune responses (also known as cellular immune responses) involve the binding of Cytotoxic T Lymphocytes (CTLs) to foreign or infected cells, which are then lysed.

As used herein, the term "adjuvant" refers to a compound that, when used in combination with a circular RNA molecule, increases or otherwise alters or modifies the immune response generated. Modification of the immune response includes boosting or amplifying the specificity of either or both of the antibody and the cellular immune response. Modification of an immune response may also mean reducing or suppressing certain antigen-specific immune responses.

As used herein, the terms "human antibody," "human immunoglobulin," and "human polyclonal antibody" are used interchangeably and refer to one or more antibodies produced in a non-human animal that are otherwise indistinguishable from antibodies produced in a human vaccinated with the same circular RNA formulation. This is in contrast to "humanized antibodies," which are modified to have human characteristics (such as by producing chimeras), but retain the properties of the host animal from which they were produced. Because the human antibodies prepared according to the methods disclosed herein consist of fully human IgG, no enzymatic treatment is required to eliminate the risk of allergic reactions and serological disorders associated with heterologous species IgG.

As used herein, the term "linear counterpart" is a polyribonucleotide molecule (and fragments thereof) that has the same or similar nucleotide sequence as a cyclic polyribonucleotide (e.g., 100%, 95%, 90%, 85%, 80%, 75% or any percentage sequence similarity therebetween) and has two free ends (i.e., the uncyclized form of the cyclic polyribonucleotide (and fragments thereof)). In some embodiments, the linear counterpart (e.g., the pre-circularised form) is a polynucleic acid molecule (and fragments thereof) having the same or similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80%, 75% or any percentage sequence similarity therebetween) as the cyclic polyribonucleotide and the same or similar nucleic acid modification and having two free ends (i.e., the uncyclized form of the cyclic polyribonucleotide (and fragments thereof)). In some embodiments, the linear counterpart is a polynucleic acid molecule (and fragments thereof) having the same or similar nucleotide sequence as the cyclic polynucleic acid (e.g., 100%, 95%, 90%, 85%, 80%, 75% or any percentage sequence similarity therebetween) and a different nucleic acid modification or no nucleic acid modification and having two free ends (i.e., the uncyclized form of the cyclic polynucleic acid (and fragments thereof)). In some embodiments, the fragment of a polynucleic acid molecule that is a linear counterpart is any portion of the linear counterpart polynucleic acid molecule that is shorter than the linear counterpart polynucleic acid molecule. In some embodiments, the linear counterpart further comprises a 5' cap. In some embodiments, the linear counterpart further comprises a poly-a tail. In some embodiments, the linear counterpart further comprises a 3' utr. In some embodiments, the linear counterpart further comprises a 5' utr.

As used herein, the term "carrier" means a compound, composition, agent, or molecule that facilitates the transport or delivery of a composition (e.g., a cyclic polyribonucleotide) into a cell by covalent modification of the cyclic polyribonucleotide via a partial or complete encapsulating agent or a combination thereof. Non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride modified phytoglycogen or glycogen-based materials), nanoparticles (e.g., nanoparticles encapsulated or covalently linked to cyclic polyribonucleotides, such as lipid nanoparticles or LNPs), liposomes, fusions (fusome), ex vivo differentiated reticulocytes, exosomes, protein carriers (e.g., proteins covalently linked to cyclic polyribonucleotides), or cationic carriers (e.g., cationic lipopolymers or transfection reagents).

As used herein, the terms "naked," "naked delivery," and homologs thereof mean that the formulation is delivered to the cell without the aid of a carrier and without covalent modification of the moiety that contributes to delivery to the cell. The naked delivery formulation does not contain any transfection reagent, cationic carrier, carbohydrate carrier, nanoparticle carrier, or protein carrier. For example, a naked delivery formulation of a cyclic polyribonucleotide is a formulation comprising a cyclic polyribonucleotide that is not covalently modified and that is free of a carrier. The naked delivery formulation may comprise a non-carrier pharmaceutical excipient or diluent.

The term "diluent" means a vehicle (vehicle) comprising an inactive solvent in which the compositions described herein (e.g., compositions comprising cyclic polyribonucleotides) may be diluted or dissolved. The diluent may be an RNA solubilising agent, a buffer, an isotonic agent or a mixture thereof. The diluent may be a liquid diluent or a solid diluent. Non-limiting examples of liquid diluents include water or other solvents, solubilizing agents and emulsifiers (such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, peanut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and 1, 3-butylene glycol non-limiting examples of solid diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium lactose phosphate, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch or powdered sugar.

As used herein, a "subject to be immunized" is a subject to whom an immunogenic composition (e.g., a composition comprising cyclic polyribonucleotides comprising a sequence encoding a coronavirus antigen, or a composition comprising linear polyribonucleotides comprising a sequence selected from the group consisting of SEQ ID nos. in table 3) is administered. The subject to be immunized is a non-human animal ("non-human animal subject to be immunized") (e.g., agricultural animal, pet, zoo animal, etc.) or a human subject ("human subject to be immunized").

As used herein, a "subject to be treated" is a subject to whom polyclonal antibodies against coronavirus (e.g., polyclonal antibody preparations against coronavirus) are administered as a prophylactic treatment or treatment of coronavirus infection. Prophylactic treatment includes administering polyclonal antibodies to coronaviruses to a subject at risk of exposure to coronaviruses (e.g., health care workers) or at risk of suffering from coronavirus-related diseases (e.g., people over 50 years old; immunocompromised; people suffering from chronic healthy conditions such as obesity, diabetes, cancer, etc.). The subject to be treated is a non-human animal ("non-human animal subject to be treated") (e.g., agricultural animal, pet, zoo animal, etc.) or a human subject ("human subject to be treated").

As used herein, "variant" refers to a polypeptide that includes at least one alteration, e.g., substitution, insertion, deletion, and/or fusion, at one or more residue positions as compared to the parent or wild-type polypeptide. Variants may include 1 to 10, 10 to 20, 20 to 50, 50 to 100, or more changes.

Incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 shows exemplary cyclic polyribonucleotides comprising sequences encoding coronavirus antigens (e.g., spike proteins, receptor Binding Domain (RBD) proteins of spike proteins).

FIG. 2 shows a schematic representation of the generation of human polyclonal antibodies that bind to coronavirus antigens for administration to a human subject.

FIG. 3 shows that RBD antigen encoded by circular RNA was detected in BJ fibroblasts and HeLa cells, but not in BJ fibroblasts and HeLa cells controlled with vehicle.

Figure 4 shows that in a mouse model, a sustainable anti-RBD antibody response is obtained after administration of a circular RNA encoding SARS-CoV-2RBD antigen formulated with a cationic polymer (e.g., protamine).

Figure 5 shows that in a mouse model, an anti-spike response is obtained following administration of a circular RNA encoding a SARS-CoV-2RBD antigen formulated with a cationic polymer (e.g., protamine).

FIG. 6 shows anti-RBD IgG2a and IgG1 isotype levels obtained after administration of a circular RNA encoding SARS-CoV-2RBD antigen formulated with a cationic polymer (e.g., protamine) in a mouse model.

Figure 7 shows long term expression of proteins from in vivo circular RNAs following intramuscular injection of circular RNA preparations (Trans-IT formulated, protamine formulated, unfused), protamine vehicle alone and in uninjected control mice.

Figure 8 shows simultaneous intramuscular delivery of adavax^TM Adjuvants with (i) an unformulated circular RNA preparation (left panel), (ii) a TransIT-formulated circular RNA (middle panel), and (iii) a protamine-eggAfter the white formulated circular RNA (right panel), the protein was expressed from the circular RNA in vivo for a long period of time. In each case Addavax^TM The adjuvants were delivered as separate injections at 0 and 24 hours.

FIG. 9 shows the results of the injection of Addavax at (i) a loop RNA formulated with protamine, (ii) a loop RNA formulated with protamine (24 hours injection of Addavax^TM Adjuvant), (iii) post intradermal delivery of protamine vehicle alone, and (iv) non-injected control mice, protein was expressed from the in vivo circular RNA for a prolonged period.

FIG. 10 is a schematic representation of an exemplary circular RNA comprising two expression sequences, wherein each expression sequence encodes an antigen and wherein one or both expression sequences encode a coronavirus antigen. The circular RNA comprises two Open Reading Frames (ORFs), each ORF encoding an expression sequence, wherein each ORF is operably linked to an IRES.

FIG. 11 is a schematic representation of an exemplary circular RNA comprising two expression sequences, wherein each expression sequence is an antigen, and wherein one or both expression sequences encode a coronavirus antigen. The circular RNA comprises two expressed sequences separated by a 2A sequence, all operably linked to an IRES.

FIG. 12 shows a schematic of a plurality of polyribonucleotides, wherein each polynucleotide comprises an ORF that encodes an antigen, and wherein one or both ORFs encode a coronavirus antigen.

Fig. 13A shows multi-antigen expression of cyclic polyribonucleotides. RBD antigen expression was detected from circular RNAs encoding SARSs-CoV-2RBD antigen and GLuc polypeptide.

Fig. 13B shows multi-antigen expression of cyclic polyribonucleotides. GLuc activity was detected from circular RNA encoding SARSs-CoV-2RBD antigen and GLuc polypeptide.

Figure 14A demonstrates the immunogenicity of various antigens from circular RNAs in a mouse model. Mice were vaccinated with a first circular RNA encoding SARS-CoV-2RBD antigen and a second circular RNA encoding GLuc polypeptide. anti-RBD antibodies were obtained 17 days after injection.

Figure 14B demonstrates the immunogenicity of various antigens from circular RNAs in a mouse model. Mice were vaccinated with a first circular RNA encoding SARS-CoV-2RBD antigen and a second circular RNA encoding GLuc polypeptide. GLuc activity was detected 2 days after injection.

Figure 15A demonstrates the immunogenicity of various antigens from circular RNAs in a mouse model. Mice were vaccinated with a first circular RNA encoding the SARS-CoV-2RBD antigen and a second circular RNA encoding the influenza virus Hemagglutinin (HA) antigen. anti-RBD antibodies were obtained 17 days after injection.

Figure 15B demonstrates the immunogenicity of various antigens from circular RNAs in a mouse model. Mice were vaccinated with a first circular RNA encoding the SARS-CoV-2RBD antigen and a second circular RNA encoding the influenza virus Hemagglutinin (HA) antigen. anti-HA antibodies were obtained 17 days after injection.

Figure 16A demonstrates the immunogenicity of various antigens from circular RNAs in a mouse model. Mice were vaccinated with a first circular RNA encoding SARS-CoV-2 spike antigen and a second circular RNA encoding influenza virus Hemagglutinin (HA) antigen. anti-RBD (spike domain) antibodies were obtained 17 days after injection.

Figure 16B demonstrates the immunogenicity of various antigens from circular RNAs in a mouse model. Mice were vaccinated with a first circular RNA encoding SARS-CoV-2 spike antigen and a second circular RNA encoding influenza virus Hemagglutinin (HA) antigen. anti-HA antibodies were obtained 17 days after injection.

Figure 17 demonstrates anti-HA antibody responses in mice administered with circular RNAs encoding multiple antigens. Mice were administered a circular RNA encoding: SARS-CoV-2RBD antigen, SARS-CoV-2 spike antigen, influenza HA antigen, SARS-CoV-2RBD antigen and GLuc polypeptide, or SARS-CoV-2RBD antigen and SARS-CoV-2 spike antigen. Anti-influenza HA antibodies were measured using the hemagglutination inhibition assay (HAI). FIG. 24 shows that HAI titers occur in samples that were administered with circular RNA preparations encoding influenza HA antigen, when administered alone or in combination with SARS-CoV-2 antigen, such as RBD or spike.

Detailed Description

The present disclosure relates generally to cyclic polyribonucleotides comprising sequences encoding antigens and/or epitopes from coronaviruses, immunogenic compositions comprising cyclic polyribonucleotides encoding coronavirus antigens and/or epitopes, and methods for producing cyclic polyribonucleotides encoding coronavirus antigens and/or epitopes and compositions comprising cyclic polyribonucleotides encoding coronavirus antigens and/or epitopes. In some embodiments, the cyclic polyribonucleotides and/or immunogenic compositions are used in a method of generating an immune response against an antigen and/or epitope from a coronavirus by administering the cyclic polyribonucleotides and/or immunogenic compositions to a subject or immunizing a subject with a cyclic polyribonucleotide comprising a sequence encoding a coronavirus antigen and/or epitope. The subject (e.g., a subject to be immunized) can be a mammal, such as an ungulate. The subject to be immunized can be a human. In some embodiments, the subject to be immunized is a non-human animal having a humanized immune system.

The present disclosure also relates generally to linear polyribonucleotides comprising sequences encoding antigens and/or epitopes from coronaviruses, immunogenic compositions comprising linear polyribonucleotides encoding coronavirus antigens and/or epitopes, and methods for producing linear polyribonucleotides encoding coronavirus antigens and/or epitopes and compositions comprising linear polyribonucleotides encoding coronavirus antigens and/or epitopes. In some embodiments, the linear polyribonucleotides and/or immunogenic compositions are used in a method of generating an immune response against an antigen and/or epitope from a coronavirus by administering the linear polyribonucleotides and/or immunogenic compositions to a subject or immunizing a subject with a linear polyribonucleotide comprising a sequence encoding a coronavirus antigen and/or epitope. The subject (e.g., a subject to be immunized) can be a mammal, such as an ungulate. The subject to be immunized can be a human. In some embodiments, the subject to be immunized is a non-human animal having a humanized immune system.

The disclosure also generally relates to methods of using the cyclic polyribonucleotides or immunogenic compositions described herein to generate or produce polyclonal antibodies that bind to antigens and/or epitopes from coronaviruses in a subject. In some embodiments, the subject to be vaccinated is a human. In some embodiments, the subject to be immunized is a non-human animal (e.g., ungulate). In some embodiments, the non-human animal has a humanized immune system. In a particular embodiment, cyclic polyribonucleotides encoding antigens and/or epitopes from coronavirus and/or immunogenic compositions comprising cyclic polyribonucleotides encoding antigens and/or epitopes from coronavirus are administered to a non-human animal having a humanized immune system, thereby stimulating the production of human polyclonal antibodies that bind to antigens and/or epitopes from coronavirus.

The disclosure also generally relates to methods of using the linear polyribonucleotides or immunogenic compositions described herein to generate or produce polyclonal antibodies that bind to antigens and/or epitopes from coronaviruses in a subject. In some embodiments, the subject to be vaccinated is a human. In some embodiments, the subject to be immunized is a non-human animal (e.g., ungulate). In one embodiment, linear polyribonucleotides encoding antigens and/or epitopes from coronavirus and/or immunogenic compositions comprising linear polyribonucleotides encoding coronavirus antigens and/or epitopes are administered to a non-human animal having a humanized immune system, thereby stimulating the production of human polyclonal antibodies that bind to antigens and/or epitopes from coronavirus.

In further embodiments, the polyclonal antibodies produced are purified. The purified polyclonal antibodies are suitable for use as a prophylactic against coronaviruses or in the treatment of coronavirus infections. The purified polyclonal antibody may be administered to a subject to be treated. An illustrative example of the method described herein is provided in fig. 2.

Cyclic polyribonucleotides

The cyclic polyribonucleotides as disclosed herein comprise sequences encoding antigens and/or epitopes from coronaviruses. Such cyclic polyribonucleotides express sequences encoding antigens and/or epitopes from coronaviruses in a subject (e.g., a subject to be immunized). In some embodiments, a circular polyribonucleotide comprising a coronavirus antigen and/or epitope is used to generate an immune response in a subject (e.g., a subject to be immunized). In some embodiments, cyclic polyribonucleotides that comprise coronavirus antigens and/or epitopes are used to generate polyclonal antibodies as described herein.

Coronavirus antigens and epitopes

The circular polyribonucleotides comprise a sequence that encodes a coronavirus antigen or epitope. The antigens and/or epitopes disclosed herein are associated with coronaviruses. In some embodiments, these antigens and/or epitopes are expressed by or derived from coronaviruses.

An antigen is a molecule that contains one or more epitopes (linear epitopes, conformational epitopes, or both) that will elicit an adaptive immune response in a subject (e.g., a subject to be immunized). An epitope may be a portion of an antigen that is recognized, targeted, or bound by a given antibody or T cell receptor. The epitope may be a linear epitope, e.g. a contiguous sequence of amino acids. The epitope may be a conformational epitope, e.g., an epitope comprising amino acids that form an epitope in the folded conformation of the protein. Conformational epitopes may contain non-contiguous amino acids from the primary amino acid sequence. Typically, an epitope will comprise about 3-15, typically about 5-15 amino acids. B cell epitopes are typically about 5 amino acids but may be as small as 3-4 amino acids. T cell epitopes (such as CTL epitopes) will comprise at least about 7-9 amino acids, while helper T cell epitopes comprise at least about 12-20 amino acids. Typically, an epitope will comprise about 7 to 15 amino acids, such as 9, 10, 12 or 15 amino acids.

The coronavirus antigen or epitope may be or comprise all or a portion of a protein, peptide, glycoprotein, lipoprotein, phosphoprotein, ribonucleoprotein, carbohydrate (e.g., polysaccharide), lipid (e.g., phospholipid or triglyceride), or nucleic acid (e.g., DNA, RNA).

The coronavirus antigen or epitope may comprise a protein antigen or epitope (e.g., a peptide antigen or peptide epitope from a protein, glycoprotein, lipoprotein, phosphoprotein, or ribonucleoprotein). The antigen or epitope may include amino acids, sugars, lipids, phosphoryl or sulfonyl groups or combinations thereof.

The coronavirus protein antigen or epitope may comprise post-translational modifications such as glycosylation, ubiquitination, phosphorylation, nitrosylation, methylation, acetylation, amidation, hydroxylation, sulfation, or lipidation.

In some embodiments, the coronavirus is a pathogenic coronavirus. In some embodiments, the coronavirus is a respiratory pathogen. In some embodiments, the coronavirus is a blood-borne pathogen. In some embodiments, the coronavirus is an enteropathogen.

Non-limiting examples of coronaviruses of the present disclosure include severe acute respiratory syndrome-associated coronaviruses (SARS-CoV, e.g., SARS-CoV-1, SARS-CoV-2), middle east respiratory syndrome coronaviruses (MERS-CoV), bats coronaviruses, zoonotic coronaviruses that can infect humans or other animals, emerging or newly discovered coronaviruses, and other coronaviruses.

In some embodiments, the cyclic polyribonucleotide comprises a severe acute respiratory syndrome associated coronavirus (SARS-CoV) antigen and/or epitope. In some embodiments, the circular polyribonucleotide comprises a SARS-CoV-1 antigen and/or epitope. In some embodiments, the circular polyribonucleotide comprises a SARS-CoV-2 antigen and/or epitope. In some embodiments, the circular polyribonucleotide comprises a middle east respiratory syndrome coronavirus (MERS-CoV) antigen and/or epitope. In some embodiments, the circular polyribonucleotides comprise human-animal co-coronavirus antigens and/or epitopes that can infect humans or other animals. In some embodiments, the circular polyribonucleotides comprise an antigen and/or epitope from a newly emerged coronavirus.

In some embodiments, the circular polyribonucleotide comprises a Coronaviridae (Coronaviridae) antigen and/or epitope.

In some embodiments, the circular polyribonucleotide comprises an antigen and/or epitope from a genus or subgenera of an alpha coronavirus (alphacoronavir), beta coronavirus (betacoronavir), gamma coronavirus (Gammacoronavirus), delta coronavirus (deltacoronavir), mebecovirus (Mebecovirus), or Sha Beike virus (Sarbecovirus). In some embodiments, the circular polyribonucleotide comprises a β -coronavirus antigen and/or epitope. In some embodiments, the circular polyribonucleotide comprises Sha Beike viral antigen and/or epitope. In some embodiments, the cyclic polyribonucleotides comprise a mebaceae virus antigen and/or epitope.

In some embodiments, the circular polyribonucleotide comprises a sequence from an antigen of a coronavirus that is a biosafety level 2 (BSL-2) pathogen. In some embodiments, the circular polyribonucleotide comprises a sequence from a coronavirus that is a biosafety level 3 (BSL-3) pathogen. In some embodiments, the coronavirus is a biosafety class 4 (BSL-4) pathogen. In some embodiments, no approved drug (e.g., antiviral or antibiotic drug) may be used to treat the coronavirus infection from which the antigen expressed by the cyclic polyribonucleotide is derived. In some embodiments, no approved vaccine can be used to prevent or reduce the risk of coronavirus infection from which the antigen expressed by the cyclic polyribonucleotide is derived.

The antigen and/or epitope may be from a coronavirus surface protein, a coronavirus membrane protein, a coronavirus envelope protein, a coronavirus capsid protein, a coronavirus nucleocapsid protein, a coronavirus spike protein, a spike coronavirus Receptor Binding Domain (RBD) protein, a coronavirus entry protein, a coronavirus membrane fusion protein, a coronavirus structural protein, a coronavirus nonstructural protein, a coronavirus regulatory protein, a coronavirus helper protein, a secreted coronavirus protein, a coronavirus polymerase protein, a coronavirus RNA polymerase, a coronavirus protease, a coronavirus glycoprotein, a coronavirus fusogenic, a coronavirus helical capsid protein, a coronavirus icosahedral capsid protein, a coronavirus matrix protein, a coronavirus replicase, a coronavirus transcription factor, or a coronavirus enzyme.

Antigens and/or epitopes from many coronaviruses are expressed by the cyclic polyribonucleotides. In some cases, the antigen and/or epitope is associated with or expressed by a coronavirus as disclosed herein. In some embodiments, the antigen and/or epitope is associated with or expressed by two or more coronaviruses disclosed herein.

In some cases, two or more coronaviruses are phenotypically related. For example, the compositions and methods of the present disclosure may utilize antigens and/or epitopes from the following coronaviruses: two or more coronaviruses that are respiratory pathogens, two or more coronaviruses that are associated with severe disease, two or more coronaviruses that are associated with adverse consequences in an immunocompromised subject (e.g., a subject to be vaccinated), two or more coronaviruses that are associated with Acute Respiratory Distress Syndrome (ARDS), two or more coronaviruses that are associated with Severe Acute Respiratory Syndrome (SARS), two or more coronaviruses that are associated with Middle East Respiratory Syndrome (MERS), or a combination of these.

The circular polyribonucleotide may comprise or encode, for example, antigens and/or epitopes from at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 or more coronaviruses.

In some embodiments, the circular polyribonucleotide comprises or encodes an antigen and/or epitope from at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 15, at most 20, at most 25, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90, at most 100, or less coronaviruses.

In some embodiments, the circular polyribonucleotide comprises or encodes an antigen and/or epitope from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 coronaviruses.

In some embodiments, the antigen and/or epitope is from a coronavirus, such as a severe acute respiratory syndrome-associated coronavirus (SARS-CoV, e.g., SARS-CoV-1, SARS-CoV-2), middle east respiratory syndrome coronavirus (MERS-CoV), or another coronavirus. In some embodiments, the antigen and/or epitope of the disclosure is from a predicted open reading frame of a coronavirus genome.

The novel SARS isolate can be identified by a percentage of homology of the polynucleotide sequence of the novel viral specific genomic region with 99%, 98%, 97%, 95%, 92%, 90%, 85% or 80% homology to the polynucleotide sequence of a known SARS virus specific genomic region. Alternatively, new SARS isolates can be identified by a percentage of homology of the polypeptide sequence encoded by the polynucleotide of a particular genomic region of the new SARS virus to 99%, 98%, 97%, 95%, 92%, 90%, 85% or 80% of the polypeptide sequence encoded by the polynucleotide of a particular region of a known SARS virus. These genomic regions may include regions commonly shared among many coronaviruses (e.g., gene products or ORFs), as well as group-specific regions (e.g., antigen groups), such as any of the following genomic regions that can be readily identified by virologists skilled in the art: a 5 'untranslated region (UTR), a leader sequence, ORF1a, ORF1b, nonstructural protein 2 (NS 2), hemagglutinin esterase glycoprotein (HE) (also known as E3), spike glycoprotein (S) (also known as E2), ORF3a, ORF3b, nonstructural protein 4 (NS 4), envelope (small membrane) protein (E) (also known as sM), membrane glycoprotein (M) (also known as E1), ORF5a, ORF5b, nucleocapsid phosphoprotein (N), ORF6, ORF7a, ORF7b, ORF8a, ORF8b, ORF9a, ORF9b, ORF10, intergenic sequences, receptor Binding Domains (RBD) of spike proteins, 3' UTRs, or RNA-dependent RNA polymerase (pol). The SARS virus can have an identifiable genomic region and one or more of the above identified genomic regions. SARS virus antigens include proteins encoded by any of these genomic regions. The SARS virus antigen may be a protein or fragment thereof that is highly conserved with coronavirus. The SARS virus antigen may be a protein or fragment thereof that is specific for the SARS virus (as compared to known coronaviruses).

In some embodiments, the antigen and/or epitope of the present disclosure is from a predicted transcript of the SARS-CoV genome. In some embodiments, the antigen and/or epitope of the present disclosure is from a protein encoded by an open reading frame from the SARS-CoV genome. Non-limiting examples of open reading frames in the SARS-CoV genome can include ORF1a, ORF1b, spike (S), ORF3a, ORF3b, envelope (E), membrane (M), ORF6, ORF7a, ORF7b, ORF8a, ORF8b, ORF9a, ORF9b, nucleocapsids (N) and ORF10.

ORF1a and ORF1b encode 16 nonstructural proteins (nsp), for example nsp1, nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsp10, nsp11, nsp12, nsp13, nsp14, nsp15 and nsp16. For example, the nonstructural proteins facilitate viral replication, viral assembly, modulation of immune response, or a combination thereof. In some embodiments, the antigen is a non-structural protein or an antigen sequence encoding a non-structural protein. In some embodiments, the epitope is from a coronavirus nonstructural protein.

The spike (S) encodes a spike protein, which in some embodiments aids in binding to a host cell receptor, fusion of the virus with a host cell membrane, entry of the virus into a host cell, or a combination thereof. The spike protein may be an antigen. In some embodiments, the epitope of the disclosure is from a spike protein. In some embodiments, the epitopes of the disclosure comprise the receptor binding domain of a spike protein. In some embodiments, the epitope of the disclosure comprises the ACE2 binding domain of a spike protein.

Envelope (E) encodes an envelope protein, which in some embodiments aids in viral assembly and morphogenesis. The envelope protein may be an antigen. In some embodiments, the epitope of the disclosure is from a coronavirus envelope protein.

Membrane (M) encodes a membrane protein that, in some embodiments, aids in viral assembly. The membrane protein may be an antigen. In some embodiments, the epitope of the disclosure is from a coronavirus membrane protein.

Nucleocapsid (N) encodes a nucleocapsid protein, which in some embodiments may form a complex with genomic RNA and facilitate viral assembly, and/or interact with M protein. The nucleocapsid protein may be an antigen. In some embodiments, the epitope of the disclosure is from a coronavirus nucleocapsid protein.

ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8a, ORF8b, ORF9a, ORF9b and ORF10 encode an accessory protein. In some embodiments, the helper protein may modulate host cell signaling, modulate host cell immune response, be incorporated into mature virions as a secondary structural protein, or a combination of these. The accessory protein may be an antigen. In some embodiments, the epitope of the disclosure is from a coronavirus helper protein.

The compositions and methods of the present disclosure may utilize antigens and/or epitopes encoded by or derived from one or more open reading frames of the SARS-CoV genome. For example, the antigen and/or epitope may be encoded by or derived from ORF1a, ORF1b, spike (S), ORF3a, ORF3b, envelope (E), membrane (M), ORF6, ORF7a, ORF7b, ORF8a, ORF8b, ORF9a, ORF9b, nucleocapsid (N), ORF10, or any combination thereof.

In some embodiments, the epitope of the disclosure is from a spike protein. In some embodiments, the epitopes of the disclosure comprise the Receptor Binding Domain (RBD) of spike proteins. In some embodiments, the epitope of the disclosure comprises the ACE2 binding domain of a spike protein. In some embodiments, the epitopes of the disclosure comprise the S1 subunit of spike protein, the S2 subunit of spike protein, or a combination thereof. In some embodiments, the epitope of the disclosure comprises the extracellular domain of a spike protein. In some embodiments, the epitope of the disclosure comprises Gln498, thr500, asn501, or a combination thereof from a coronavirus spike protein. In some embodiments, the epitope of the disclosure comprises Lys417, tyr453, or a combination thereof from a coronavirus spike protein. In some embodiments, the epitope of the disclosure comprises Gln474, phe486, or a combination thereof from a coronavirus spike protein. In some embodiments, the epitope of the disclosure comprises Gln498, thr500, asn501, lys417, tyr453, gln474, phe486, one or more equivalent amino acids from a spike protein variant or derivative of a coronavirus spike protein, or a combination thereof. In some embodiments, a spike protein of the disclosure comprises a D614G mutation, i.e., having the amino acid glycine (G) at position 614 instead of aspartic acid (D). In some embodiments, the epitope of the present disclosure comprises Gly614 from a spike protein variant or derivative of a coronavirus spike protein, or a combination thereof. In some cases, the D614G mutation may result in reduced S1 shedding and increased coronavirus infectivity.

In some embodiments, the antigen and/or epitope is encoded by ORF1a or derived from ORF1a. In some embodiments, the antigen and/or epitope is encoded by or derived from SARS-CoV ORF1b. In some embodiments, the antigen and/or epitope is encoded by or derived from SARS-CoV spike. In some embodiments, the antigen and/or epitope is encoded by or derived from SARS-CoV ORF3a. In some embodiments, the antigen and/or epitope is encoded by or derived from SARS-CoV ORF3b. In some embodiments, the antigen and/or epitope is encoded by or derived from SARS-CoV envelope (E). In some embodiments, the antigen and/or epitope is encoded by or derived from a SARS-CoV membrane (M). In some embodiments, the antigen and/or epitope is encoded by or derived from SARS-CoV ORF6. In some embodiments, the antigen and/or epitope is encoded by SARS-CoV ORF7a or derived from SARS-CoV ORF7a. In some embodiments, the antigen and/or epitope is encoded by or derived from SARS-CoV ORF7b. In some embodiments, the antigen and/or epitope is encoded by SARS-CoV ORF8 or derived from SARS-CoV ORF8. In some embodiments, the antigen and/or epitope is encoded by SARS-CoV ORF8a or derived from SARS-CoV ORF8a. In some embodiments, the antigen and/or epitope is encoded by SARS-CoV ORF9a or derived from SARS-CoV ORF9a. In some embodiments, the antigen and/or epitope is encoded by SARS-CoV ORF9b or derived from SARS-CoV ORF9b. In some embodiments, the antigen and/or epitope is encoded by or derived from SARS-CoV nucleocapsid (N). In some embodiments, the antigen and/or epitope is encoded by SARS-CoV ORF10 or derived from SARS-CoV ORF10. In some embodiments, the antigen and/or epitope is encoded by or derived from SARS-CoV spike (S), envelope (E), membrane (M) and nucleocapsid (N).

In some embodiments, the antigen and/or epitope is not encoded by SARS-CoV ORF1a or is not derived from SARS-CoV ORF1a. In some embodiments, the antigen and/or epitope is not encoded by SARS-CoV ORF1b or is not derived from SARS-CoV ORF1b. In some embodiments, the antigen and/or epitope is not encoded by or derived from SARS-CoV spike. In some embodiments, the antigen and/or epitope is not encoded by SARS-CoV ORF3a or is not derived from SARS-CoV ORF3a. In some embodiments, the antigen and/or epitope is not encoded by SARS-CoV ORF3b or is not derived from SARS-CoV ORF3b. In some embodiments, the antigen and/or epitope is not encoded by or derived from SARS-CoV envelope (E). In some embodiments, the antigen and/or epitope is not encoded by or derived from SARS-CoV membrane (M). In some embodiments, the antigen and/or epitope is not encoded by or derived from SARS-CoV ORF6. In some embodiments, the antigen and/or epitope is not encoded by SARS-CoV ORF7a or is not derived from SARS-CoV ORF7a. In some embodiments, the antigen and/or epitope is not encoded by SARS-CoV ORF7b or is not derived from SARS-CoV ORF7b. In some embodiments, the antigen and/or epitope is not encoded by SARS-CoV ORF8 or is not derived from SARS-CoV ORF8. In some embodiments, the antigen and/or epitope is not encoded by SARS-CoV ORF8a or is not derived from SARS-CoV ORF8a. In some embodiments, the antigen and/or epitope is not encoded by SARS-CoV ORF9a or is not derived from SARS-CoV ORF9a. In some embodiments, the antigen and/or epitope is not encoded by SARS-CoV ORF9b or is not derived from SARS-CoV ORF9b. In some embodiments, the antigen and/or epitope is not encoded by or derived from SARS-CoV nucleocapsid (N). In some embodiments, the antigen and/or epitope is not encoded by SARS-CoV ORF10 or is not derived from SARS-CoV ORF10. In some embodiments, the antigen and/or epitope is not encoded by or derived from SARS-CoV spike (S), envelope (E), membrane (M) and nucleocapsid (N).

The antigen and/or epitope may be encoded by SARS-CoV2 or derived from SARS-CoV2.

A non-limiting example of the SARS-CoV-2 genome is provided in DB source accession number MN908947.3, which is the complete genomic sequence of a severe acute respiratory syndrome coronavirus 2 isolate, the contents of which are incorporated herein by reference in their entirety. DB Source accession No. MN908947.3:21563-25384 corresponds to protein S, the contents of which are incorporated herein by reference in their entirety. Non-limiting examples of SARS-CoV-2 spike protein are provided in GenBank sequences: QHD43416.1, the sequence of the spike protein of severe acute respiratory syndrome coronavirus 2 isolate, the contents of which are incorporated herein by reference in their entirety.

A non-limiting example of the SARS-CoV-2 genome is provided in sequence NCBI reference sequence accession No. NC_045512, version NC_045512.2, which is the complete genomic sequence of Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, the contents of which are incorporated herein by reference in their entirety.

A non-limiting example of the SARS-CoV-2 genome is provided in sequence NCBI reference sequence accession number MW450666, which is the complete genomic sequence of a severe acute respiratory syndrome coronavirus 2 isolate, the contents of which are incorporated herein by reference in their entirety.

A non-limiting example of the SARS-CoV-2 genome is provided in sequence NCBI reference sequence accession number MW487270, which is the complete genomic sequence of severe acute respiratory syndrome coronavirus 2 lineage b.1.1.7 virus, the contents of which are incorporated herein by reference in their entirety.

A non-limiting example of the SARS-CoV-2 genome is provided in sequence GISAID reference sequence accession number epi_isl_792683, which is the complete genomic sequence of severe acute respiratory syndrome coronavirus 2 lineage p.1 virus, the contents of which are incorporated herein by reference in their entirety.

A non-limiting example of the SARS-CoV-2 genome is provided in sequence GISAID reference sequence accession number epi_isl_678615, which is the complete genomic sequence of severe acute respiratory syndrome coronavirus 2 lineage b.1.351 virus, the contents of which are incorporated herein by reference in their entirety.

Non-limiting examples of SARS-CoV-2 genome are provided in sequence NCBI reference sequence accession number MW972466-MW974550, which is the complete genomic sequences of Severe acute respiratory syndrome coronavirus 2 lineage B.1.427 and B.1.429 viruses, the contents of which are incorporated herein by reference in their entirety.

A non-limiting example of the SARS-CoV-2 genome is provided in sequence NCBI reference sequence accession No. MZ156756-MZ226428, which is the complete genomic sequence of Severe acute respiratory syndrome coronavirus 2 virus, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the SAR-CoV-2 genome is provided in the GISAID database of www.gisaid.org.. In some embodiments, the SARS-CoV-2 genome is provided in International nucleotide sequence database collaboration (International Nucleotide Sequence Database Collaboration, INSDC) at www.insdc.org.

In some embodiments, the antigen and/or epitope of the present disclosure is derived from a predicted transcript of the SARS-CoV-2 genome. In some embodiments, the antigen and/or epitope of the present disclosure is from a protein encoded by an open reading frame from the SARS-CoV-2 genome, or a derivative thereof. Non-limiting examples of open reading frames in the SARS-CoV-2 genome include ORF1a, ORF1b, spike (S), ORF3a, envelope (E), membrane (M), ORF6, ORF7a, ORF7b, ORF8, nucleocapsid (N) and ORF10. In some embodiments, the SARS-CoV-2 genome encodes ORF3b, ORF9a, ORF9b, or a combination thereof. In some embodiments, the SARS-CoV-2 genome does not encode ORF3b, ORF9a, ORF9b, or any combination thereof.

Non-limiting examples of amino acid sequences are provided in table 1. In some embodiments, the antigen comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to a sequence from table 1.

Table 1: examples of amino acid sequences of proteins encoded by the SARS-CoV-2 genome.

Other non-limiting examples of proteins encoded by the SARS-CoV-2 genome include those having the contents of NCBI accession numbers: the method comprises the steps of selecting a specific material from a plurality of materials, wherein each material comprises a plurality of materials, and each material comprises a plurality of materials, wherein each material comprises a plurality of materials, each material comprises a material selected from the group consisting of a material, a material the method comprises the steps of selecting a specific material from a plurality of materials, wherein each material comprises a plurality of materials, and each material comprises a plurality of materials, wherein each material comprises a plurality of materials, each material, and each material; MT, MT MT, MT326137, MT-L-shaped metal oxide film the method comprises the steps of selecting a specific material from a plurality of materials, wherein each material comprises a metal material selected from the group consisting of metal, metal alloy, metal alloy, metal or metal, metal alloy, metal or metal, metal the system comprises a plurality of groups of components, each group of components comprises a plurality of groups of components, each group comprises a plurality of groups of components, each group of components, each MT, MT MT, MT325627, MT-A-B-C, MT, the method comprises the steps of (1) selecting a first signal to be transmitted from a first source to a second source, selecting a second signal to be transmitted from the first source to the second source, and selecting a third signal to be transmitted from the second source to the second source, wherein the third signal is a signal to be transmitted from the first source to the second source, and the fourth signal is a signal to be transmitted from the second source to the second source, wherein the third signal is a signal to the third source, and the fourth signal is a signal to the fourth source, and the fourth source is a signal to be transmitted from the fourth source to the fourth source, and the fourth source, to the fourth source, and the fourth source is a signal to the fourth source The method comprises the steps of (1) selecting a first signal to be transmitted from a first source to a second source, selecting a second signal to be transmitted from the first source to the second source, and selecting a third signal to be transmitted from the second source to the second source, wherein the third signal is a signal to be transmitted from the first source to the second source, and the fourth signal is a signal to be transmitted from the second source to the second source, and the third source is a signal to the third source, and the fourth source is a signal to the fourth source MT, LC, MT050416, MT042774, MT-A-B-C, MT-C, LC, MT-A-B-C-B MT, MT050414, MT050415, MT, LC522350, MT, 522350, MT MT, LC, MT050416, MT042774, MT050414, MT050415, MT, LC522350, MT 05074, MT, LC, respectively, may be provided with a plurality of functions as a function of a MT, MT019532, MT, LR, MT008022, MT, MN, each of which is incorporated herein by reference in its entirety.

In a particular embodiment, the circular polyribonucleotide comprises the SARS-CoV-2 antigen described in Table 2. In some embodiments, the antigen comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to a sequence from table 2.

TABLE 2 description of constructs and SARS-CoV-2ORF

In Table 2, "proline substitution" means proline substitution at residues 986 and 987, and "GSAS" substitution at furin cleavage sites (residues 682-685). For "clone optimization," single base substitutions were made at coordinates 2541 to disrupt the BsaI site to aid in the golden gate clone construction of the plasmid DNA template (Golden Gate Cloning construction). For "cyclization optimization": four mononucleotides-at positions 2307, 2790, 159 and 315-are substituted to disrupt the site that can potentially bind to the circularization element of the splint nucleic acid sequence, thereby potentially inhibiting effective ligation. For constructs with type II terminator removed (e.g., p33, p35, p36, p39, p41, p44, and p 45): two mononucleotides-at positions 1047, 1049-are substituted to disrupt the type II terminator site. For constructs with GC optimization (e.g., p39 and p 41), GC optimization was performed to make the GC content about 50%. All single base pair substitutions were designed for translational silencing. In Table 2, IRES is EMCV (SEQ ID NO: 31) or CVB3 (SEQ ID NO: 45).

In some embodiments, the antigen or epitope is from a host subject (e.g., a subject to be immunized) cell. For example, antibodies blocking coronavirus entry may be generated by using an antigen or epitope from a host cell component of the virus that serves as an entry factor.

In some embodiments, the coronavirus epitope comprises or contains at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids, or more. In some embodiments, the coronavirus epitope comprises or contains at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, or at most 30 amino acids, or less. In some embodiments, the coronavirus epitope comprises or contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, the coronavirus epitope contains 5 amino acids. In some embodiments, the coronavirus epitope contains 6 amino acids. In some embodiments, the epitope contains 7 amino acids. In some embodiments, the coronavirus epitope contains 8 amino acids. In some embodiments, an epitope may be about 8 to about 11 amino acids. In some embodiments, the epitope may be about 9 to about 22 amino acids.

Coronavirus antigens may include antigens recognized by B cells, antigens recognized by T cells, or a combination thereof. In some embodiments, the antigen comprises an antigen recognized by B cells. In some embodiments, the coronavirus antigen is an antigen recognized by B cells. In some embodiments, the coronavirus antigen comprises an antigen recognized by T cells. In some embodiments, the antigen is an antigen recognized by T cells.

Coronavirus epitopes include antigens recognized by B cells, antigens recognized by T cells, or combinations thereof. In some embodiments, the coronavirus epitope comprises an epitope recognized by B cells. In some embodiments, the epitope is an epitope recognized by B cells. In some embodiments, the coronavirus epitope comprises an epitope recognized by T cells. In some embodiments, the coronavirus epitope is an epitope recognized by T cells.

For example, techniques for identifying antigens and epitopes via computer modeling such as those described in Sanchez-Trincado et al (2017), fundamentals and methods for T-and B-cell epitope prediction [ basic principles and methods of T-cell and B-cell epitope prediction ], journal of immunology research [ journal of immunology; grifoni, alba et al, A Sequence Homology and Bioinformatic Approach Can Predict Candidate Targets for Immune Responses to SARS-CoV-2[ sequence homology and bioinformatics methods can predict candidate targets for SARS-CoV-2 immune response ]. Cell host & microbe [ Cell host and microorganism ] (2020); computer simulation prediction of T-cell and B-cell epitopes in PmpD, russi et al, in silico prediction of T-and B-cell epitopes in PmpD: first step towards to the design of a Chlamydia trachomatis vaccine: first step in designing Chlamydia trachomatis vaccine, biomedical journal [ journal of biomedicine ]41.2 (2018): 109-117; baruah et al, immunoinformation-aided identification of T cell and B cell epitopes in the surface glycoprotein of 2019-nCoV [ Immunoinformatics aid in identifying T-and B-cell epitopes in 2019-nCoV surface glycoproteins ]. Journal of Medical Virology [ journal of medical virology ] (2020); each of which is incorporated herein by reference in its entirety.

The cyclic polyribonucleotides of the present disclosure may comprise sequences of a number of coronavirus antigens and/or epitopes. The circular polyribonucleotides comprise, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, or more coronavirus antigens or epitopes.

In some embodiments, the circular polyribonucleotides comprise, for example, sequences of up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 15, up to 20, up to 25, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 120, up to 140, up to 160, up to 180, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, up to 500, or less coronavirus antigens or epitopes.

In some embodiments, the circular polyribonucleotide comprises, for example, a sequence of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 coronavirus antigens or epitopes.

The cyclic polyribonucleotides may comprise sequences from one or more coronavirus epitopes of a coronavirus antigen. For example, a coronavirus antigen may comprise an amino acid sequence that may contain multiple coronavirus epitopes (e.g., epitopes recognized by B cells and/or T cells), and a cyclic polyribonucleotide may contain or encode one or more of those coronavirus epitopes.

The circular polyribonucleotides comprise a sequence from, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500 or more epitopes of a coronavirus antigen.

In some embodiments, the circular polyribonucleotides comprise a sequence of, for example, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 15, up to 20, up to 25, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 120, up to 140, up to 160, up to 180, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, or up to 500 or less coronavirus epitopes from one coronavirus antigen.

In some embodiments, the circular polyribonucleotide comprises a sequence from, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 coronavirus epitopes of a coronavirus antigen.

The cyclic polyribonucleotide may encode a variant of a coronavirus antigen or epitope. The variants may be naturally occurring variants (e.g., variants identified in sequence data from different coronaviruses, species, isolates, or quasispecies), or may be derived sequences as disclosed herein that have been generated via computer simulation (e.g., antigens or epitopes having one or more amino acid insertions, deletions, substitutions, or combinations thereof as compared to the wild-type antigen or epitope).

The circular polyribonucleotides comprise, for example, the sequence of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, or more variants of a coronavirus antigen or epitope.

In some embodiments, the circular polyribonucleotides comprise the sequence of up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 15, up to 20, up to 25, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 120, up to 140, up to 160, up to 180, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, up to 500, or less variants of a coronavirus antigen or epitope, for example.

In some embodiments, the circular polyribonucleotide comprises, for example, the sequence of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 variants of a coronavirus antigen or epitope.

The coronavirus antigen and/or epitope sequences of the cyclic polyribonucleotides may also be referred to as coronavirus expression sequences. In some embodiments, the circular polyribonucleotide comprises one or more coronavirus expression sequences, each of which may encode a coronavirus polypeptide. Coronavirus polypeptides can be produced in large quantities. The coronavirus polypeptide may be a coronavirus polypeptide secreted from a cell or localized to the cytoplasm, nucleus or membrane compartment of a cell. Some coronavirus polypeptides include, but are not limited to, antigens as disclosed herein, epitopes as disclosed herein, at least a portion of coronavirus proteins (e.g., viral envelope proteins, viral matrix proteins, viral spike proteins, viral Receptor Binding Domains (RBDs) of viral spike proteins, viral membrane proteins, viral nucleocapsid proteins, viral helper proteins, fragments thereof, or combinations thereof). In some embodiments, a coronavirus polypeptide encoded by a cyclic polyribonucleotide of the present disclosure comprises a fragment of a coronavirus antigen disclosed herein. In some embodiments, a coronavirus polypeptide encoded by a cyclic polyribonucleotide of the present disclosure comprises a fusion protein comprising two or more coronavirus antigens or fragments thereof as disclosed herein. In some embodiments, a coronavirus polypeptide encoded by a cyclic polyribonucleotide of the present disclosure comprises a coronavirus epitope. In some embodiments, the polypeptide encoded by a cyclic polyribonucleotide of the present disclosure comprises a fusion protein comprising two or more coronavirus epitopes of the disclosure, e.g., an artificial peptide sequence comprising a plurality of predicted epitopes from one or more coronaviruses of the disclosure.

In some embodiments, exemplary coronavirus proteins expressed from the cyclic polyribonucleotides disclosed herein include secreted proteins, such as proteins that naturally include a signal peptide (e.g., an antigen and/or epitope), or proteins that do not normally encode a signal peptide, but that are modified to contain a signal peptide.

In some cases, the circular polyribonucleotide expresses a secreted coronavirus protein that has a short half-life in blood, or may be a protein with subcellular localization signals, or a protein with secretion signal peptides. In some cases, the cyclic polyribonucleotide expresses a transmembrane domain that has a short half-life in blood, or can be a protein with a subcellular localization signal, or a protein with a secretory peptide.

In some embodiments, the circular polyribonucleotides comprise one or more coronavirus expression sequences and are configured for sustained expression in cells in a subject (e.g., a subject to be immunized). In some embodiments, the circular polyribonucleotides are configured such that expression of the one or more coronavirus expression sequences in the cell at a later time point is equal to or higher than expression at an earlier time point. In such embodiments, the expression of the one or more coronavirus expression sequences may be maintained at a relatively stable level or may increase over time. In some embodiments, expression of the coronavirus expression sequences is relatively stable over a long period of time.

In some embodiments, the cyclic-polyribonucleotide is, e.g., transiently or chronically expressed in a subject (e.g., a subject to be immunized) as one or more coronavirus antigens and/or epitopes. In certain embodiments, expression of the coronavirus expression sequence is continued for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or any time in between. In certain embodiments, expression of the coronavirus antigen and/or epitope lasts for no more than about 30 minutes to about 7 days, or no more than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 45 days, 60, 75 days, or any time between 90 days.

In some embodiments, the coronavirus expression sequence is less than 5000bp (e.g., less than about 5000bp, 4000bp, 3000bp, 2000bp, 1000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300bp, 200bp, 100bp, 50bp, 40bp, 30bp, 20bp, 10bp, or less) in length. In some embodiments, the coronavirus expression sequences independently or additionally have a length of greater than 10bp (e.g., at least about 10bp, 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000kb, 1.1kb, 1.2kb, 1.3kb, 1.4kb, 1.5kb, 1.6kb, 1.7kb, 1.8kb, 1.9kb, 2kb, 2.1kb, 2.2kb, 2.3kb, 2.4kb, 2.5kb, 2.6kb, 2.7kb, 2.8kb, 2.9kb, 3.1kb, 3.2kb, 3.3kb, 3.5kb, 3.6kb, 3.7kb, 3.8kb, 3.9kb, 4kb, 4.1.1 kb, 4.5kb, 4.7kb, 4.5kb, 4.6kb, 4.5kb, 4.8kb or more).

Derivatives and fragments

An antigen or epitope of the disclosure may comprise a wild-type sequence. When describing an antigen or epitope, the term "wild-type" refers to a sequence (e.g., an amino acid sequence) that occurs naturally and is encoded by a genome (e.g., a coronavirus genome). Coronaviruses may have one wild-type sequence or two or more wild-type sequences (e.g., there is one canonical wild-type sequence in the reference coronavirus genome and there are wild-type sequences of other variants resulting from mutations).

When describing an antigen or epitope, the terms "derivative" and "derived from" refer to a sequence (e.g., an amino acid sequence) that differs from the wild-type sequence in one or more amino acids, e.g., contains one or more amino acid insertions, deletions, and/or substitutions relative to the wild-type sequence.

An antigen or epitope derivative sequence is a sequence that has at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a wild-type sequence (e.g., a wild-type protein, antigen or epitope sequence).

"sequence identity" and "sequence similarity" are determined by aligning two peptides or two nucleotide sequences using global or local alignment algorithms. Sequences may be said to be "substantially identical" or "substantially similar" when they have at least some minimum percentage of sequence identity (e.g., when optimally aligned using default parameters by programs GAP or BESTFIT). GAP uses Needleman and Wunsch global alignment algorithms to align two sequences over their entire length, thereby maximizing the number of matches and minimizing the number of GAPs. Typically, GAP creation penalty = 50 (nucleotides)/8 (proteins), GAP extension penalty = 3 (nucleotides)/2 (proteins) using GAP default parameters. For nucleotides, the default scoring matrix used is nwsgapdna and for proteins, blosum62 (Henikoff and Henikoff,1992, PNAS [ Proc. Natl. Acad. Sci. USA ]89,915-919). The scores for sequence alignment and percent sequence identity may be determined using a computer program, such as GCG Wisconsin software package version 10.3 or EmbossWin version 2.10.0 (using the program "needle") available from aske Le De company (Accelrys inc.,9685Scranton Road,San Diego,CA) of san diego, ca. Alternatively or additionally, the similarity or percent identity is determined by searching the database using an algorithm such as FASTA, BLAST, or the like. Sequence identity refers to sequence identity over the entire length of the sequence.

In some embodiments, the antigen or epitope contains one or more amino acid insertions, deletions, substitutions, or combinations thereof that affect the structure of the encoded protein. In some embodiments, the antigen or epitope contains one or more amino acid insertions, deletions, substitutions, or combinations thereof that affect the function of the encoded protein. In some embodiments, an antigen or epitope contains one or more amino acid insertions, deletions, substitutions, or combinations thereof that affect the expression or processing of the encoded protein by a cell.

Amino acid insertions, deletions, substitutions, or combinations thereof may introduce sites of post-translational modification (e.g., to introduce glycosylation, ubiquitination, phosphorylation, nitrosylation, methylation, acetylation, amidation, hydroxylation, sulfation, or lipidation sites, or to target sequences for cleavage). In some embodiments, the insertion, deletion, substitution, or combination thereof of an amino acid removes a site of post-translational modification (e.g., removes a glycosylation, ubiquitination, phosphorylation, nitrosylation, methylation, acetylation, amidation, hydroxylation, sulfation, or lipidation site, or a sequence targeted for cleavage). In some embodiments, the insertion, deletion, substitution, or combination thereof of an amino acid modifies the site of post-translational modification (e.g., modifies the site to alter the efficiency or characteristics of glycosylation, ubiquitination, phosphorylation, nitrosylation, methylation, acetylation, amidation, hydroxylation, sulfation, or lipidation sites, or cleavage).

Amino acid substitutions may be conservative or non-conservative substitutions. Conservative amino acid substitutions may be one amino acid substituted for another amino acid that has similar biochemical properties (e.g., charge, size, and/or hydrophobicity). A non-conservative amino acid substitution may be a substitution of one amino acid with another amino acid having different biochemical properties (e.g., charge, size, and/or hydrophobicity). Conservative amino acid changes may be, for example, substitutions that have minimal effect on the secondary or tertiary structure of the polypeptide. The conservative amino acid change may be an amino acid change from one hydrophilic amino acid to another hydrophilic amino acid. Hydrophilic amino acids may include Thr (T), ser (S), his (H), glu (E), asn (N), gln (Q), asp (D), lys (K), and Arg (R). The conservative amino acid change may be an amino acid change from one hydrophobic amino acid to another hydrophilic amino acid. Hydrophobic amino acids may include Ile (I), phe (F), val (V), leu (L), trp (W), met (M), ala (A), gly (G), tyr (Y) and Pro (P). The conservative amino acid change may be an amino acid change from one acidic amino acid to another acidic amino acid. The acidic amino acids may include Glu (E) and Asp (D). Conservative amino acid changes may be amino acid changes from one basic amino acid to another. Basic amino acids may include His (H), arg (R) and Lys (K). The conservative amino acid change may be an amino acid change from one polar amino acid to another. Polar amino acids may include Asn (N), gln (Q), ser (S), and Thr (T). Conservative amino acid changes may be amino acid changes from one nonpolar amino acid to another nonpolar amino acid. Nonpolar amino acids can include Leu (L), val (V), ile (I), met (M), gly (G), and Ala (A). The conservative amino acid change may be an amino acid change from one aromatic amino acid to another. Aromatic amino acids may include Phe (F), tyr (Y), and Trp (W). The conservative amino acid change may be an amino acid change from one aliphatic amino acid to another. Aliphatic amino acids may include Ala (A), val (V), leu (L) and Ile (I). In some embodiments, conservative amino acid substitutions are amino acid changes from one amino acid to another amino acid of one of the following classes: class I: ala, pro, gly, gln, asn, ser, thr; class II: cys, ser, tyr, thr; class III: val, ile, leu, met, ala, phe; class IV: lys, arg, his; class V: phe, tyr, trp, his; and class VI: asp, glu.

In some embodiments, an antigen derivative or epitope derivative of the present disclosure comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acid deletions relative to a sequence disclosed herein (e.g., a wild-type sequence).

In some embodiments, an antigen derivative or epitope derivative of the present disclosure comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acid substitutions relative to a sequence disclosed herein (e.g., a wild-type sequence).

In some embodiments, an antigen derivative or epitope derivative of the present disclosure comprises up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, or up to 50 amino acid substitutions relative to a sequence disclosed herein (e.g., a wild-type sequence).

In some embodiments, the antigen derivatives or epitope derivatives of the present disclosure include 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40, 10-15, 15-20, or 20-25 amino acid substitutions relative to the sequences disclosed herein (e.g., wild-type sequences).

In some embodiments, an antigen derivative or epitope derivative of the present disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions relative to a sequence disclosed herein (e.g., a wild-type sequence).

The one or more amino acid substitutions may be within the N-terminal, C-terminal, amino acid sequence, or a combination thereof. These amino acid substitutions may be continuous, discontinuous, or a combination thereof.

In some embodiments, an antigen derivative or epitope derivative of the present disclosure includes at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, at most 50, at most 60, at most 70, at most 80, at most 90, at most 100, at most 120, at most 140, at most 160, at most 180, or at most 200 amino acid deletions relative to a sequence disclosed herein (e.g., a wild-type sequence).

In some embodiments, the antigen derivative or epitope derivative of the present disclosure includes 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40, 10-15, 15-20, 20-25, 20-30, 30-50, or 100 amino acids deleted relative to the sequences disclosed herein (e.g., wild-type sequences).

In some embodiments, an antigen derivative or epitope derivative of the present disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid deletions relative to a sequence disclosed herein (e.g., a wild-type sequence).

The one or more amino acid deletions may be within the N-terminal, C-terminal, amino acid sequence, or a combination thereof. These amino acid deletions may be contiguous, non-contiguous, or a combination thereof.

In some embodiments, an antigen derivative or epitope derivative of the present disclosure comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acid insertions relative to a sequence disclosed herein (e.g., a wild-type sequence).

In some embodiments, an immunogenic derivative or epitope derivative of the present disclosure includes up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, or up to 50 amino acid insertions relative to a sequence disclosed herein (e.g., a wild-type sequence).

In some embodiments, the antigen derivative or epitope derivative of the present disclosure comprises 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40, 10-15, 15-20, or 20-25 amino acid insertions relative to the sequences disclosed herein (e.g., wild-type sequences).

In some embodiments, an immunogenic derivative or epitope derivative of the disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid insertions relative to a sequence disclosed herein (e.g., a wild-type sequence).

The one or more amino acid insertions may be within the N-terminal, C-terminal, amino acid sequence, or a combination thereof. These amino acid insertions may be contiguous, non-contiguous, or a combination thereof.

Cyclic polyribonucleotides

The circular polyribonucleotides comprise elements as described below and coronavirus antigens or epitopes as described herein.

In some embodiments, the cyclic polynucleic acid is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides.

In some embodiments, the circular polyribonucleotides can be of sufficient size to accommodate the binding site of the ribosome. In some embodiments, the maximum size of the cyclic polyribonucleotide can be as large as within the technical limitations of generating and/or using cyclic polyribonucleotides. Without being bound by any particular theory, it is possible that multiple segments of RNA may be produced from DNA and that their 5 'and 3' free ends anneal to produce a "string" of RNA that may eventually be circularized when only one 5 'and one 3' free end remain. In some embodiments, the maximum size of the circular polyribonucleotide may be limited by the ability to package the RNA and deliver it to the target. In some embodiments, the size of the cyclic polyribonucleotide is a length sufficient to encode a useful polypeptide (such as an antigen and/or epitope of the present disclosure), and thus a length of at least 20,000 nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides, at least 7,500 nucleotides, or at least 5,000 nucleotides, at least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000 nucleotides, at least 1,000 nucleotides, at least 500 nucleotides, at least 400 nucleotides, at least 300 nucleotides, at least 200 nucleotides, at least 100 nucleotides, or at least 70 nucleotides may be useful.

Cyclic polyribonucleotide elements

In some embodiments, the cyclic polyribonucleotides comprise one or more of the elements as described herein in addition to sequences encoding coronavirus antigens and/or epitopes. In some embodiments, the circular polyribonucleotide lacks a poly-a sequence, lacks a free 3' terminus, lacks an RNA polymerase recognition motif, or any combination thereof. In some embodiments, the cyclic polyribonucleotides include any feature or any combination of features disclosed in WO 2019/118919, which is hereby incorporated by reference in its entirety. For example, a circular polyribonucleotide comprises a regulatory element, such as a sequence that modifies the expression of an expressed sequence within the circular polyribonucleotide. Regulatory elements may include sequences that are positioned adjacent to an expression sequence encoding an expression product. The regulatory element may be operably linked to the adjacent sequence. The regulatory element may increase the amount of the expressed product compared to the amount of the expressed product in the absence of the regulatory element. In addition, one regulatory element may increase the amount of product expressed by a plurality of expression sequences connected in series. Thus, a regulatory element may enhance expression of one or more expression sequences. A variety of regulatory elements may also be used, for example, to differentially regulate expression of different expression sequences. In some embodiments, regulatory elements provided herein may include a selective translation sequence. As used herein, the term "selectively translated sequence" refers to a nucleic acid sequence, such as certain riboswitch aptamer enzymes, that selectively initiates or activates translation of an expressed sequence in a circular polyribonucleotide. Regulatory elements may also include selective degradation sequences. As used herein, the term "selectively degrading sequence" refers to a nucleic acid sequence that initiates degradation of a cyclic polyribonucleotide or an expression product of a cyclic polyribonucleotide. In some embodiments, the regulatory element is a translational regulator. The translational regulator may regulate translation of the expressed sequence of the cyclic polyribonucleotide. The translational regulator may be a translational enhancer or a translational repressor. In some embodiments, the translation initiation sequence may act as a regulatory element. Further examples of regulatory elements are described in paragraphs [0154] - [0161] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

In some embodiments, the cyclic-polyribonucleotide encodes an antigen that produces a human polyclonal antibody of interest and comprises a translation initiation sequence, such as an initiation codon. In some embodiments, the translation initiation sequence comprises a kozak or a summer-darcino (Shine-Dalgarno) sequence. In some embodiments, the cyclic-polyribonucleotide includes a translation initiation sequence, such as a kozak sequence, adjacent to the expression sequence. In some embodiments, the translation initiation sequence is a non-coding initiation codon. In some embodiments, a translation initiation sequence (e.g., a kozak sequence) is present on one or both sides of each expression sequence, resulting in a separation of the expression products. In some embodiments, the cyclic-polyribonucleotide includes at least one translation initiation sequence adjacent to the expression sequence. In some embodiments, the translation initiation sequence provides conformational flexibility to the circular polyribonucleotide. In some embodiments, the translation initiation sequence is substantially within a single stranded region of the circular polyribonucleotide. Further examples of translation initiation sequences are described in paragraphs [0163] - [0165] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

In some embodiments, the circular polyribonucleotides described herein comprise an Internal Ribosome Entry Site (IRES) element. A suitable IRES element included in a cyclic polyribonucleotide may be an RNA sequence capable of engaging eukaryotic ribosomes. Other examples of IRES are described in paragraphs [0166] - [0168] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

The cyclic polyribonucleotides may include one or more expression sequences (e.g., encoding an antigen), and each expression sequence may or may not have a termination element. Further examples of termination elements are described in paragraphs [0169] - [0170] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

The cyclic polyribonucleotides of the present disclosure may comprise staggered elements. The term "staggered element" refers to a portion, such as a nucleotide sequence, that induces a ribosome pause during translation. In some embodiments, the staggered elements are non-conserved sequences of amino acids with strong alpha-helix propensity, followed by consensus sequence-D (V/I) ExNPGP, where x = any amino acid (SEQ ID NO: 52). In some embodiments, the staggered elements may include chemical moieties, such as glycerol, non-nucleic acid linking moieties, chemical modifications, modified nucleic acids, or any combination thereof.

In some embodiments, the cyclic polyribonucleotide comprises at least one staggered element adjacent to the expression sequence. In some embodiments, the cyclic polyribonucleotides include staggered elements adjacent to each expressed sequence. In some embodiments, staggered elements are present on one or both sides of each expressed sequence, resulting in, for example, separation of expression products of one or more peptides and/or one or more polypeptides. In some embodiments, the interleaving element is part of one or more expression sequences. In some embodiments, the circular polyribonucleotide comprises one or more expression sequences, and each of the one or more expression sequences is separated from a subsequent expression sequence by a staggered element on the circular polyribonucleotide. In some embodiments, the staggering element prevents (a) two-round translation of a single expressed sequence or (b) one or more rounds of translation of two or more expressed sequences from generating a single polypeptide. In some embodiments, the staggered elements are sequences that are spaced apart from the one or more expressed sequences. In some embodiments, the interleaving element comprises a portion of an expression sequence of the one or more expression sequences.

Examples of interlaced elements are described in paragraphs [0172] - [0175] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

In some embodiments, the circular polyribonucleotides include one or more regulatory nucleic acid sequences or include one or more expression sequences encoding a regulatory nucleic acid (e.g., a nucleic acid that modifies expression of an endogenous gene and/or an exogenous gene). In some embodiments, the expression sequences of the circular polyribonucleotides provided herein can comprise sequences antisense to regulatory nucleic acids like non-coding RNAs such as, but not limited to tRNA, lncRNA, miRNA, rRNA, snRNA, microRNA, siRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, Y RNA and hnRNA.

Exemplary regulatory nucleic acids are described in paragraphs [0177] - [0194] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

In some embodiments, the cyclic polyribonucleotides provided herein have a greater translational efficiency than a reference, such as a linear counterpart, a linear expression sequence, or a linear cyclic polyribonucleotide. In some embodiments, a circular polyribonucleotide provided herein has a translational efficiency that is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 70%, 800%, 900%, 1000%, 2000%, 5000%, 10000%, 100000% or more greater than the translational efficiency of the reference. In some embodiments, the translational efficiency of the circular polyribonucleotide is 10% higher than the translational efficiency of the linear counterpart. In some embodiments, the translational efficiency of the circular polyribonucleotide is 300% greater than the translational efficiency of the linear counterpart.

In some embodiments, the cyclic polyribonucleotides produce stoichiometric expression products. Rolling circle translation continuously produces expression products at substantially equal rates. In some embodiments, the cyclic polyribonucleotides have stoichiometric translational efficiencies such that the expression products are produced at substantially equal rates. In some embodiments, the cyclic polyribonucleotides have stoichiometric translational efficiencies of multiple expression products (e.g., products from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more expression sequences).

In some embodiments, once translation of the cyclic polyribonucleotide is initiated, the ribosome bound to the cyclic polyribonucleotide will not detach from the cyclic polyribonucleotide before at least one round of translation of the cyclic polyribonucleotide is completed. In some embodiments, a circular polyribonucleotide as described herein is capable of rolling circle translation. In some embodiments, once translation of a cyclic polyribonucleotide is initiated during rolling circle translation, the ribosome that is bound to the cyclic polyribonucleotide will not be detached from the cyclic polyribonucleotide before translation of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, at least 1000, at least 1500, at least 2000, at least 5000, at least 10000, at least 105, or at least 106 of the cyclic polyribonucleotide is completed.

In some embodiments, rolling circle translation of the cyclic polyribonucleotides results in the production of a polypeptide product that results from more than one round of translation of the cyclic polyribonucleotides ("sequential" expression products). In some embodiments, the cyclic polyribonucleotides comprise staggered elements, and rolling circle translation of the cyclic polyribonucleotides results in the production of a polypeptide product that is produced by a single round of translation or less than a single round of translation of the cyclic polyribonucleotides ("discrete" expression product). In some embodiments, the cyclic polyribonucleotides are configured such that at least 10%, 20%, 30%, 40%, 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the total polypeptide (moles/mole) generated during rolling circle translation of the cyclic polyribonucleotides is a discrete polypeptide. In some embodiments, the amount ratio of discrete products to total polypeptide is tested in an in vitro translation system. In some embodiments, the in vitro translation system for the test dose ratio comprises rabbit reticulocyte lysate. In some embodiments, the quantitative ratio is tested in cells in an in vivo translation system, such as eukaryotic or prokaryotic cells, cultured cells, or organisms.

In some embodiments, the cyclic polyribonucleotide comprises an untranslated region (UTR). The UTR comprising the genomic region of the gene may be transcribed but not translated. In some embodiments, the UTR may be included upstream of the translation initiation sequences of the expression sequences described herein. In some embodiments, UTRs may be included downstream of the expression sequences described herein. In some cases, one UTR of a first expressed sequence is identical to or contiguous with or overlaps with another UTR of a second expressed sequence. In some embodiments, the intron is a human intron. In some embodiments, the intron is a full-length human intron, e.g., ZKSCAN1.

Exemplary untranslated regions are described in paragraphs [0197] to [201] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

In some embodiments, the cyclic-polyribonucleotide comprises a poly-a sequence. Exemplary poly-A sequences are described in paragraphs [0202] - [0205] of WO 2019/118919, which is hereby incorporated by reference in its entirety. In some embodiments, the cyclic polyribonucleotide lacks a poly a sequence.

In some embodiments, the circular polyribonucleotide comprises one or more riboswitches. Exemplary riboswitches are described in paragraphs [0232] to [0252] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

In some embodiments, the circular polyribonucleotide comprises an aptamer enzyme. Exemplary aptamer enzymes are described in paragraphs [0253] to [0259] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

In some embodiments, the circular polyribonucleotide comprises one or more RNA binding sites. Microrna (or miRNA) antigens are short non-coding RNAs that bind to the 3' utr of a nucleic acid molecule and down-regulate gene expression by reducing the stability of the nucleic acid molecule or by inhibiting translation. The cyclic polyribonucleotide may comprise one or more microrna target sequences, microrna sequences, or microrna seeds. Such sequences may correspond to any known microRNA, such as those taught in U.S. publication No. US 2005/0261218 and U.S. publication No. US 2005/0059005, the contents of which are incorporated herein by reference in their entirety. Other examples of RNA binding sites are described in paragraphs [0206] - [0215] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

In some embodiments, the circular polyribonucleotide comprises one or more protein binding sites, such that a protein, e.g., ribose, is capable of binding to internal sites in the RNA sequence. Other examples of protein binding sites are described in paragraphs [0218] to [0221] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

In some embodiments, the circular polyribonucleotide comprises a spacer sequence. In some embodiments, the elements of the polyribonucleotides may be separated from each other by a spacer sequence or linker. Exemplary spacer sequences are described in paragraphs [0293] - [0302] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

The circular polyribonucleotides described herein may also comprise non-nucleic acid linkers. Exemplary non-nucleic acid linkers are described in paragraphs [0303] - [0307] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

In some embodiments, the cyclic-polyribonucleotide further comprises another nucleic acid sequence. In some embodiments, the circular polyribonucleotides may comprise other sequences, including DNA, RNA, or artificial nucleic acids. Other sequences may include, but are not limited to, genomic DNA, cDNA, or sequences encoding tRNA, mRNA, rRNA, miRNA, gRNA, siRNA or other RNAi molecules. In some embodiments, the cyclic-polyribonucleotide comprises siRNA to target a different locus of the same gene expression product as the cyclic-polyribonucleotide. In some embodiments, the cyclic polyribonucleotide comprises an siRNA to target a gene expression product that is different from a gene expression product present in the cyclic polyribonucleotide.

In some embodiments, the cyclic polyribonucleotide lacks a 5' -UTR. In some embodiments, the cyclic polyribonucleotide lacks a 3' -UTR. In some embodiments, the cyclic polyribonucleotide lacks a poly a sequence. In some embodiments, the cyclic polyribonucleotide lacks a terminating element. In some embodiments, the cyclic polyribonucleotide lacks an internal ribosome entry site. In some embodiments, the cyclic polyribonucleotide lacks susceptibility to degradation by exonuclease. In some embodiments, the fact that the cyclic polyribonucleotide lacks susceptibility to degradation may mean that the cyclic polyribonucleotide is not degraded by exonuclease or is degraded to a limited extent in the presence of exonuclease only, e.g. comparable or similar to in the absence of exonuclease. In some embodiments, the cyclic polyribonucleotide is not degraded by exonuclease. In some embodiments, cyclic polyribonucleotide degradation is reduced when exposed to an exonuclease. In some embodiments, the cyclic polyribonucleotide lacks binding to a cap binding protein. In some embodiments, the cyclic polyribonucleotide lacks a 5' cap.

In some embodiments, the cyclic polyribonucleotide lacks a 5' -UTR and is capable of expressing a protein from one or more of its expression sequences. In some embodiments, the cyclic-polyribonucleotide lacks a 3' -UTR and is capable of expressing a protein from one or more of its expression sequences. In some embodiments, the cyclic-polyribonucleotide lacks a poly-a sequence and is capable of expressing a protein from one or more of its expression sequences. In some embodiments, the cyclic polyribonucleotide lacks a termination element and is capable of expressing a protein from one or more of its expression sequences. In some embodiments, the cyclic polyribonucleotide lacks an internal ribosome entry site and is capable of expressing a protein from one or more of its expression sequences. In some embodiments, the cyclic-polyribonucleotide lacks a cap and is capable of expressing a protein from one or more of its expression sequences. In some embodiments, the cyclic polyribonucleotides lack 5'-UTR, 3' -UTR, and IRES, and are capable of expressing a protein from one or more of its expression sequences. In some embodiments, the circular polyribonucleotide further comprises one or more of the following sequences: a sequence encoding one or more mirnas, a sequence encoding one or more replication proteins, a sequence encoding a foreign gene, a sequence encoding a therapeutic agent, a regulatory element (e.g., a translational regulator such as a translational enhancer or repressor), a translation initiation sequence, one or more regulatory nucleic acids targeting an endogenous gene (e.g., siRNA, lncRNA, shRNA), and a sequence encoding a therapeutic mRNA or protein.

As a result of its circularization, a cyclic polyribonucleotide may contain certain features that distinguish it from linear RNA. For example, cyclic polyribonucleotides are less susceptible to exonuclease degradation than linear RNAs. In this way, cyclic polyribonucleotides can be more stable than linear RNA, especially when incubated in the presence of exonuclease. The increased stability of the cyclic polyribonucleotides as compared to linear RNAs may make the cyclic polyribonucleotides more useful as a cell transforming reagent for producing polypeptides (e.g., antigens and/or epitopes that elicit an antibody response). The improved stability of the cyclic polyribonucleotides compared to linear RNA allows the cyclic polyribonucleotides to be stored more easily for longer than linear RNA. The stability of the exonuclease treated cyclic polyribonucleotides can be tested using methods standard in the art to determine whether RNA degradation has occurred (e.g., by gel electrophoresis).

Furthermore, unlike linear RNAs, cyclic polyribonucleotides may be less prone to dephosphorylation when incubated with phosphatases such as calf intestinal phosphatase.

In some embodiments, the circular polyribonucleotide comprises a particular sequence feature. For example, a cyclic polyribonucleotide may comprise a specific nucleotide composition. In some such embodiments, the cyclic polynucleic acid may include one or more purine (adenine or guanine) enrichment regions. In some such embodiments, the cyclic polyribonucleotides can include one or more purine-rich regions. In some embodiments, the cyclic polyribonucleotides may include one or more AU enrichment regions or elements (ARE). In some embodiments, the cyclic polynucleic acid may include one or more adenine-rich regions.

In some embodiments, a circular polyribonucleotide can include one or more repeat elements described elsewhere herein. In some embodiments, the circular polyribonucleotide comprises one or more modifications described elsewhere herein.

The cyclic polyribonucleotides may include one or more substitutions, insertions and/or additions, deletions and covalent modifications relative to the reference sequence. For example, cyclic polyribonucleotides having one or more insertions, additions, deletions, and/or covalent modifications relative to the parent polyribonucleotide are included within the scope of the present disclosure. Exemplary modifications are described in paragraphs [0310] to [0325] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

In some embodiments, the cyclic polyribonucleotide comprises a higher order structure, such as a secondary or tertiary structure. In some embodiments, the complementary segment of the circular polyribonucleotide folds itself into a double-stranded segment, paired with hydrogen bonding (e.g., A-U and C-G). In some embodiments, a helix, also referred to as a stem, is formed intramolecularly, with a double stranded segment attached to the end loop. In some embodiments, the cyclic polyribonucleotide has at least one segment with a quasi-double stranded secondary structure.

In some embodiments, one or more sequences of the circular polyribonucleotides include a region that is substantially single-stranded and double-stranded. In some embodiments, the ratio of single strand to double strand may affect the function of the cyclic polyribonucleotide.

In some embodiments, one or more sequences of the circular polyribonucleotides are substantially single-stranded. In some embodiments, one or more sequences of the substantially single-stranded circular polyribonucleotides may include a protein or RNA binding site. In some embodiments, the substantially single-stranded circular polyribonucleotide sequence may be conformationally flexible to allow for increased interaction. In some embodiments, the sequence of the circular polyribonucleotide is purposefully engineered to include such secondary structures, thereby binding or increasing protein or nucleic acid binding.

In some embodiments, the circular polyribonucleotide sequence is substantially double-stranded. In some embodiments, one or more sequences of the substantially double-stranded circular polyribonucleotides may include a conformational recognition site, such as a riboswitch or an aptamer enzyme. In some embodiments, the substantially double-stranded circular polyribonucleotide sequence may be conformationally rigid. In some such examples, the conformational rigid sequence may sterically hinder the cyclic polyribonucleotide binding protein or nucleic acid. In some embodiments, the sequence of the circular polyribonucleotide is purposefully engineered to include such secondary structures, thereby avoiding or reducing protein or nucleic acid binding.

There are 16 possible base pairs, but six of them (AU, GU, GC, UA, UG, CG) are likely to form the actual base pairs. The rest, called mismatch, occurs in the spiral at very low frequencies. In some embodiments, the structure of the cyclic polyribonucleotide is not easily disrupted, does not affect its function and has no fatal consequences, which provides the option of maintaining the secondary structure. In some embodiments, the primary structure of the stem (i.e., its nucleotide sequence) may still be varied while still maintaining the helical region. The nature of the bases is the second position of the higher order structure and substitution can be made as long as they retain the second structure. In some embodiments, the circular polyribonucleotide has a quasi-helical structure. In some embodiments, the cyclic polyribonucleotide has at least one segment with a quasi-helical structure. In some embodiments, the cyclic-polyribonucleotide comprises at least one of a U-rich or a-rich sequence or a combination thereof. In some embodiments, the U-rich and/or a-rich sequences are arranged in a manner that will result in a triple quasi-helical structure. In some embodiments, the circular polyribonucleotide has a double helix structure. In some embodiments, the circular polyribonucleotide has one or more segments (e.g., 2, 3, 4, 5, 6, or more) with a duplex structure. In some embodiments, the cyclic-polyribonucleotide comprises at least one of a C-rich and/or G-rich sequence. In some embodiments, the C-rich and/or G-rich sequences are arranged in a manner that will produce a triple quasi-helical structure. In some embodiments, the cyclic polyribonucleotides have an intramolecular triple quasi-helical structure that contributes to stability.

In some embodiments, the cyclic polyribonucleotides have two quasiccrew structures (e.g., separated by a phosphodiester linkage) such that the base pairs at their ends are stacked, and the quasiccrew structures become co-linear, resulting in a "coaxially stacked" substructure.

In some embodiments, the circular polyribonucleotide comprises a tertiary structure with one or more motifs, such as pseudojunctions, g-quadruplexes, helices, and coaxial stacks.

Other examples of the structure of cyclic polyribonucleotides as disclosed herein are described in paragraphs [0326] - [0333] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

Stability and half-life

In some embodiments, the cyclic polyribonucleotides provided herein have an increased half-life than a reference, e.g., a linear polyribonucleotide (linear counterpart) that has the same nucleotide sequence, that is not cyclized. In some embodiments, the cyclic polyribonucleotide is substantially resistant to degradation (e.g., degradation by exonucleases). In some embodiments, the cyclic polyribonucleotide is resistant to self-degradation. In some embodiments, the cyclic polyribonucleotide lacks an enzymatic cleavage site, such as a dicer cleavage site. Other examples of stability and half-life of cyclic polyribonucleotides as disclosed herein are described in paragraphs [0308] - [0309] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

Production method

In some embodiments, the cyclic polyribonucleotides include non-naturally occurring deoxyribonucleic acid sequences, and can be produced using recombinant techniques (e.g., in vitro derivatization using DNA plasmids) or chemical synthesis, or a combination thereof.

Within the scope of the present disclosure, a DNA molecule for producing an RNA loop may include a DNA sequence of a naturally occurring original nucleic acid sequence, a modified version thereof, or a DNA sequence encoding a synthetic polypeptide that is not normally found in nature (e.g., a chimeric molecule or fusion protein, such as a fusion protein comprising multiple immunogens). DNA and RNA molecules can be modified using a variety of techniques including, but not limited to, classical mutagenesis techniques and recombinant techniques, such as site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, cleavage of nucleic acid fragments by restriction enzymes, ligation of nucleic acid fragments, polymerase Chain Reaction (PCR) amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures, and ligation of mixture groups to "build" a mixture of nucleic acid molecules, and combinations thereof.

The cyclic polyribonucleotides can be prepared according to any available technique, including but not limited to chemical synthesis and enzymatic synthesis. In some embodiments, the linear primary construct or linear mRNA can be circularized or concatenated to produce a circular polyribonucleotide as described herein. The mechanism of cyclization or interlinking may occur by methods such as, but not limited to, chemical, enzymatic, splinting or ribozyme catalysis. The newly formed 5'-/3' -bond may be an intramolecular bond or an intermolecular bond.

Methods of preparing the circular polyribonucleotides described herein are described, for example, in Khudyakov & Fields, artifical DNA: methods and Applications [ Artificial DNA: methods and applications ], CRC Press (2002); zhao, synthetic Biology: tools and Applications [ synthetic biology: tools and applications ], (first edition), academic Press (2013); and Egli & Herhewijn, chemistry and Biology of Artificial Nucleic Acids [ chemical and biological of artificial nucleic acids ], (first edition), wiley-VCH (2012).

Various methods of synthesizing circular polyribonucleotides are also described in the art (see, e.g., U.S. Pat. No. 6210931, U.S. Pat. No. 5773244, U.S. Pat. No. 5766903, U.S. Pat. No. 5712128, U.S. Pat. No. 5426180, U.S. publication No. US 20100137407, international publication No. WO 1992001813, and International publication No. WO 2010084371, the contents of each of which are incorporated herein by reference in their entirety).

In some embodiments, the cyclic polyribonucleotides are purified, e.g., free ribonucleic acids, linear or nicked RNAs, DNA, proteins, and the like are removed. In some embodiments, the cyclic polyribonucleotides can be purified by any known method commonly used in the art. Non-limiting examples of purification methods include column chromatography, gel excision, size exclusion, and the like.

Cyclization

In some embodiments, the linear cyclic polyribonucleotides may be circularized or concatemerized. In some embodiments, the linear cyclic polyribonucleotides can be cyclized in vitro prior to formulation and/or delivery. In some embodiments, the linear cyclic polyribonucleotide may be circularized within the cell.

a. Extracellular cyclization

In some embodiments, the linear cyclic polyribonucleotides are chemically cyclized or concatenated to form cyclic polyribonucleotides. In some chemical methods, the 5 '-end and the 3' -end of the nucleic acid (e.g., linear circular polyribonucleotides) include chemically reactive groups that, when brought into proximity to each other, can form new covalent bonds between the 3 '-end and the 5' -end of the molecule. The 5 '-end may contain a NHS ester reactive group and the 3' -end may contain a 3 '-amino terminal nucleotide such that in an organic solvent, the 3' -amino terminal nucleotide on the 3 '-end of the linear RNA molecule will undergo nucleophilic attack on the 5' -NHS-ester moiety, forming a new 5'-/3' -amide bond.

In some embodiments, a 5 '-phosphorylated nucleic acid molecule (e.g., a linear circular polyribonucleotide) is enzymatically attached to the 3' -hydroxy group of a nucleic acid (e.g., a linear nucleic acid) using a DNA or RNA ligase to form a novel phosphodiester bond. In an exemplary reaction, according to the manufacturer's protocol, The linear circular polyribonucleotides were incubated with 1-10 units of T4 RNA ligase (New England Biolabs, ipswich, mass.) for 1 hour at 37 ℃. Ligation reactions can occur in the presence of linear nucleic acids that are capable of base pairing with juxtaposed 5 '-and 3' -regions to aid in the enzymatic ligation reaction. In some embodiments, the connection is a splinting connection. For example, splint ligases, e.g.

Ligase, which can be used for splint ligation. For splint ligation, a single stranded polynucleotide (splint), such as a single stranded RNA, may be designed to hybridize to both ends of a linear polyribonucleotide such that the two ends may be juxtaposed upon hybridization to the single stranded splint. Thus, the splint ligase may catalyze the ligation of the two ends of the linear polyribonucleotides side by side to generate a cyclic polyribonucleotide.

In some embodiments, DNA or RNA ligase is used for the synthesis of the circular polynucleotide. As a non-limiting example, the ligase may be a circ ligase or a circular ligase.

In some embodiments, the 5 '-or 3' -end of the linear circular polyribonucleotide can encode a ligase ribozyme sequence such that during in vitro transcription, the resulting linear circular polyribonucleotide comprises an active ribozyme sequence that is capable of ligating the 5 '-end of the linear circular polyribonucleotide to the 3' -end of the linear circular polyribonucleotide. The ligase ribozyme may be derived from group I introns, hepatitis delta virus, hairpin ribozymes, or may be selected by SELEX (ligand system evolution by exponential enrichment). The ribozyme ligase reaction may take 1 to 24 hours at a temperature between 0 ℃ and 37 ℃.

In some embodiments, the linear circular polyribonucleotides can be circularized or concatemerized by using at least one non-nucleic acid moiety. In one aspect, at least one non-nucleic acid moiety can react with a region or feature near the 5 'end and/or near the 3' end of the linear circular polyribonucleotide to circularize or concatenate the linear circular polyribonucleotide. In another aspect, at least one non-nucleic acid moiety can be located at or linked to or adjacent to the 5 'end and/or the 3' end of the linear circular polyribonucleotide. Contemplated non-nucleic acid portions may be homologous or heterologous. As one non-limiting example, the non-nucleic acid moiety may be a bond, such as a hydrophobic bond, an ionic bond, a biodegradable bond, and/or a cleavable bond. As another non-limiting example, the non-nucleic acid moiety is a linking moiety. As yet another non-limiting example, the non-nucleic acid moiety may be an oligonucleotide or peptide moiety, an aptamer or a non-nucleic acid linker as described herein.

In some embodiments, the linear cyclic polyribonucleotides are circularized or concatemerized due to non-nucleic acid moieties, resulting in attraction between the surfaces of atoms, molecules located at, adjacent to, or attached to the 5 'and 3' ends of the cyclic polyribonucleotides. As one non-limiting example, one or more linear cyclic polyribonucleotides can be cyclized or interlinked by intermolecular forces or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, van der Waals forces, and dispersive forces. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonance bonds, hydrogen-grasping bonds (diagnostic bonds), dipole bonds, conjugation, super-conjugation, and reverse bonds.

In some embodiments, the linear circular polyribonucleotides may comprise a ribozyme RNA sequence near the 5 'end and near the 3' end. The ribozyme RNA sequence may be covalently linked to the peptide when the sequence is exposed to the remainder of the ribozyme. In one aspect, ribozyme RNA sequences in which the peptide is covalently linked to the 5 'end and near the 3' end can be linked to each other, resulting in cyclization or concatemerization of the linear cyclic polyribonucleotide. In another aspect, covalent attachment of the peptide near the 5 'and 3' ends of the ribozyme RNA sequence may cause cyclization or concatemerization of the linear primary construct or linear mRNA following ligation using methods known in the art, such as, but not limited to, protein ligation. A non-limiting example of a ribozyme, or method of incorporating and/or covalently linking a peptide, for use in the linear primary construct or linear RNA of the present invention is described in U.S. patent application No. US 20030082768, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the linear circular polyribonucleotide may include a 5 'triphosphate of the nucleic acid that is converted to a 5' monophosphate, for example by contacting the 5 'triphosphate with an RNA 5' pyrophosphorohydrolase (RppH) or ATP diphosphorohydrolase (dephosphorizing enzyme). Alternatively, the conversion of the 5 'triphosphate of a linear cyclic polyribonucleotide to a 5' monophosphate may be accomplished by a two step reaction comprising: (a) Contacting the 5' nucleotide of the linear cyclic polyribonucleotide with a phosphatase (e.g., a thermosensitive phosphatase, shrimp alkaline phosphatase, or calf intestinal phosphatase) to remove all three phosphates; and (b) after step (a), contacting the 5' nucleotide with a kinase (e.g., a polynucleotide kinase) to which a single phosphate is added.

In some embodiments, the cyclization methods provided herein have a cyclization efficiency of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100%. In some embodiments, the cyclization methods provided herein have a cyclization efficiency of at least about 40%. In some embodiments, the cyclization process is provided with a cyclization efficiency of between about 10% to about 100%; for example, the cyclization efficiency can be about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 99%. In some embodiments, the cyclization efficiency is between about 20% to about 80%. In some embodiments, the cyclization efficiency is between about 30% to about 60%. In some embodiments, the cyclization efficiency is about 40%.

b. Splice element

In some embodiments, the cyclic polyribonucleotide comprises at least one splice element. Exemplary splice elements are described in paragraphs [0270] - [0275] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

In some embodiments, the cyclic polyribonucleotide comprises at least one splice element. In the circular polyribonucleotides provided herein, the splice element may be an intact splice element that can mediate splicing of the circular polyribonucleotide. Alternatively, the splice element may also be the remaining splice element from the completed splice event. For example, in some cases, the splice elements of a linear polyribonucleotide may mediate a splice event that results in cyclization of a linear polyribonucleotide, such that the resulting cyclic polyribonucleotide comprises the remaining splice elements from such splice-mediated cyclization event. In some cases, the remaining splice elements are unable to mediate any splicing. In other cases, the remaining splice elements may still mediate splicing in some cases. In some embodiments, the splice element is adjacent to at least one expression sequence. In some embodiments, the circular polyribonucleotide comprises a splice element adjacent to each expressed sequence. In some embodiments, splice elements are on one or both sides of each expressed sequence, resulting in, for example, separation of expression products of one or more peptides and/or one or more polypeptides.

In some embodiments, the circular polyribonucleotides include internal splice elements that, when replicated, splice ends are joined together. Some examples may include mini-introns (< 100 nt) with splice site sequences and short inverted repeats (30-40 nt), such as AluSq2, aluJr and AluSz, inverted sequences in flanking introns, alu elements in flanking introns, and motifs found in cis-sequence elements (the selectable 4 enrichment motif) near the reverse splicing event, such as sequences in 200bp before (upstream) or after (downstream) the reverse splice site with flanking exons. In some embodiments, the cyclic-polyribonucleotide includes at least one repeated nucleotide sequence as described elsewhere herein as an internal splice element. In such embodiments, the repetitive nucleotide sequence may include a repetitive sequence from an Alu family intron. In some embodiments, splice-related ribosome binding proteins can regulate the biogenesis of cyclic polyribonucleotides (e.g., blind actin and shock protein (QKI) splicing factors).

In some embodiments, the cyclic-polyribonucleotide can include canonical splice sites flanking the head-to-tail junction of the cyclic-polyribonucleotide.

In some embodiments, the cyclic-polyribonucleotide may include a ridge-helix-Long Qiji sequence comprising two 4-base pair stems flanked by 3-nucleotide ridges. Cleavage occurs at one site in the bulge region, producing a characteristic fragment of 5' -hydroxy and 2',3' -cyclic phosphate ester ending. Cyclization is performed by nucleophilic attack of the 5' -OH group onto the 2',3' -cyclic phosphate of the same molecule that forms the 3',5' -phosphodiester bridge.

In some embodiments, the circular polyribonucleotide may comprise a polynucleic RNA sequence having an HPR element. HPR comprises a 2',3' -cyclic phosphate and a 5' -OH terminus. The HPR element self-processes the 5 '-end and the 3' -end of the linear polyribonucleotide for cyclization, thereby ligating these ends together.

In some embodiments, the cyclic-polyribonucleotide may include a self-splicing element. For example, the cyclic polyribonucleotide may include an intron from the cyanobacteria Anabaena (Anabaena).

In some embodiments, the cyclic-polyribonucleotide may include a sequence that mediates self-ligation. In one embodiment, the cyclic-polyribonucleotide may include an HDV sequence (e.g., an HDV replication domain conserved sequence, GGCUCAUCUCGACAAGAGGCGGCAGUCCUCAGUA CUCUUACUCUUUUCUGUAAAGAGGAGACUGCUGGACUCGCCGCCC AAGUUCGAGCAUGAGCC (SEQ ID NO: 61) or GGCUAGAGGCGGCAGU CCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUGCUGGACU CGCCGCCCGAGCC (SEQ ID NO: 62)) for self-ligation. In one embodiment, the cyclic-polyribonucleotide may include a loop E sequence (e.g., in PSTVd) for self-ligation. In another embodiment, the cyclic polyribonucleotide may include self-circularising introns, such as 5 'and 3' splice junctions, or self-circularising catalytic introns, such as type I, type II or type III introns. Non-limiting examples of type I intron self-splicing sequences may include self-splicing replacement intron-exon sequences derived from T4 phage gene td and tetrahymena Insert (IVS) rRNA.

Other cyclization methods

In some embodiments, the linear circular polyribonucleotides can include complementary sequences, including repeated or non-repeated nucleic acid sequences within an individual intron or within a flanking intron. A repetitive nucleic acid sequence is a sequence that occurs within a segment of a cyclic polyribonucleotide. In some embodiments, the circular polyribonucleotide comprises a repeat nucleic acid sequence. In some embodiments, the repetitive nucleotide sequence comprises a poly CA sequence or a poly UG sequence. In some embodiments, a cyclic polynucleic acid comprises at least one repeated nucleic acid sequence hybridized to a complementary repeated nucleic acid sequence in another segment of the cyclic polynucleic acid, the hybridized segment forming an internal double strand. In some embodiments, the repeated nucleic acid sequences of two separate circular polyribonucleotides and the complementary repeated nucleic acid sequence hybridize to generate a single circularized polyribonucleotide, and the hybridized segments form an internal double strand. In some embodiments, the complementary sequences are located at the 5 'and 3' ends of the linear circular polyribonucleotides. In some embodiments, the complementary sequences comprise about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more paired nucleotides.

In some embodiments, cyclizing chemistry methods can be used to generate cyclic polyribonucleotides. Such methods may include, but are not limited to, click chemistry (e.g., alkyne and azide based methods, or clickable bases), alkene metathesis, phosphoramidate ligation, half imine-imine crosslinking, base modification, and any combination thereof.

In some embodiments, a cyclase method can be used to generate cyclic polyribonucleotides. In some embodiments, a ligase, such as a DNA or RNA ligase, may be used to generate the template of the cyclic polyribonucleotide or complement, the complementary strand of the cyclic polyribonucleotide, or the cyclic polyribonucleotide.

Cyclization of the cyclic polyribonucleotides can be accomplished by methods known in the art, for example, petkovic and Muller, "RNAcircularization strategies in vivo and in vitro [ in vivo and in vitro ribonucleic acid cyclization strategy ]" Nucleic Acids Res [ nucleic acid research ],2015,43 (4): 2454-2465, and Muller and Appel, "In vitro circularization ofRNA [ in vitro cyclization of ribonucleic acid ]" RNA Biol [ RNA biology ],2017,14 (8): 1018-1027.

The cyclic polyribonucleotides may encode sequences and/or motifs that are useful in replication. Exemplary replication elements are described in paragraphs [0280] - [0286] of WO 2019/118919, which is hereby incorporated by reference in its entirety.

Linear polyribonucleotides

The linear polyribonucleotides as disclosed herein comprise sequences encoding antigens and/or epitopes from coronaviruses. Such linear polyribonucleotides express sequences encoding antigens and/or epitopes from coronaviruses in a subject (e.g., a subject to be immunized). In some embodiments, linear polyribonucleotides comprising coronavirus antigens and/or epitopes are used to generate an immune response in a subject (e.g., a subject to be immunized). In some embodiments, linear polyribonucleotides that are mRNA and that comprise coronavirus antigens and/or epitopes are used to generate an immune response in a subject (e.g., a subject to be immunized). In some embodiments, linear polyribonucleotides comprising coronavirus antigens and/or epitopes are used to generate polyclonal antibodies as described herein.

Coronavirus antigens and epitopes

The linear polyribonucleotides comprise a sequence that encodes a coronavirus antigen or epitope. The antigens and/or epitopes disclosed herein are associated with coronaviruses. In some embodiments, these antigens and/or epitopes are expressed by or derived from coronaviruses.

An antigen is a molecule containing one or more epitopes (linear epitopes, conformational epitopes, or both) that elicit an adaptive immune response in a subject (e.g., a subject to be immunized). An epitope may be a portion of an antigen that is recognized, targeted, or bound by a given antibody or T cell receptor. The epitope may be a linear epitope, e.g. a contiguous sequence of amino acids. The epitope may be a conformational epitope, e.g., an epitope comprising amino acids that form an epitope in the folded conformation of the protein. Conformational epitopes may contain non-contiguous amino acids from the primary amino acid sequence. Typically, an epitope will comprise about 3-15, typically about 5-15 amino acids. B cell epitopes are typically about 5 amino acids but may be as small as 3-4 amino acids. T cell epitopes (such as CTL epitopes) will comprise at least about 7-9 amino acids, while helper T cell epitopes comprise at least about 12-20 amino acids. Typically, an epitope will comprise about 7 to 15 amino acids, such as 9, 10, 12 or 15 amino acids.

The coronavirus antigen or epitope may be or comprise all or a portion of a protein, peptide, glycoprotein, lipoprotein, phosphoprotein, ribonucleoprotein, carbohydrate (e.g., polysaccharide), lipid (e.g., phospholipid or triglyceride), or nucleic acid (e.g., DNA, RNA).

The coronavirus antigen or epitope may comprise a protein antigen or epitope (e.g., a peptide antigen or peptide epitope from a protein, glycoprotein, lipoprotein, phosphoprotein, or ribonucleoprotein). The antigen or epitope may include amino acids, sugars, lipids, phosphoryl or sulfonyl groups or combinations thereof.

The coronavirus protein antigen or epitope may comprise post-translational modifications such as glycosylation, ubiquitination, phosphorylation, nitrosylation, methylation, acetylation, amidation, hydroxylation, sulfation, or lipidation.

The antigen and/or epitope may be from a coronavirus surface protein, a coronavirus membrane protein, a coronavirus envelope protein, a coronavirus capsid protein, a coronavirus nucleocapsid protein, a coronavirus spike protein, a spike coronavirus receptor binding domain protein, a coronavirus entry protein, a coronavirus membrane fusion protein, a coronavirus structural protein, a coronavirus nonstructural protein, a coronavirus regulatory protein, a coronavirus helper protein, a secreted coronavirus protein, a coronavirus polymerase protein, a coronavirus RNA polymerase, a coronavirus protease, a coronavirus glycoprotein, a coronavirus fusion antigen, a coronavirus spiral capsid protein, a coronavirus icosahedral capsid protein, a coronavirus matrix protein, a coronavirus replicase, a coronavirus transcription factor, or a coronavirus enzyme.

In some embodiments, the antigen and/or epitope of the present disclosure is from a predicted transcript of the SARS-CoV genome. In some embodiments, the antigen and/or epitope of the present disclosure is from a protein encoded by an open reading frame from the SARS-CoV genome. Non-limiting examples of open reading frames in the SARS-CoV genome can include ORF1a, ORF1b, spike (S), ORF3a, ORF3b, envelope (E), membrane (M), ORF6, ORF7a, ORF7b, ORF8a, ORF8b, ORF9a, ORF9b, nucleocapsids (N) and ORF10. In some embodiments, the open reading frame from the SARS-CoV genome comprises SEQ ID NO. 11.

In a particular embodiment, the linear polyribonucleotides comprise the SARS-CoV-2 antigen described in Table 3.

Table 3: description of the designed linear construct.

In Table 3, "proline substitution" means proline substitution at residues 986 and 987, as well as "GSAS" substitution at furin cleavage sites (residues 682-685). For clone optimization, single base substitutions were made at coordinates 2541 to disrupt the BsaI site to aid in the construction of the gold clone of the plasmid DNA template. For circularization optimization, four mononucleotides-at positions 2307, 2709, 159 and 315-are substituted to disrupt the sites that can potentially bind to the splint nucleic acid sequence circularization element, thereby potentially inhibiting effective ligation. All single base pair substitutions were designed for translational silencing. In Table 3, furthermore, the 5' element is globin (SEQ ID NO: 32); and the 3' element is: globin (SEQ ID NO: 33).

In some embodiments, the coronavirus epitope comprises or contains at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids, or more. In some embodiments, the coronavirus epitope comprises or contains at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, or at most 30 amino acids, or less. In some embodiments, the coronavirus epitope comprises or contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, the coronavirus epitope contains 5 amino acids. In some embodiments, the coronavirus epitope contains 6 amino acids. In some embodiments, the epitope contains 7 amino acids. In some embodiments, the coronavirus epitope contains 8 amino acids. In some embodiments, an epitope may be about 8 to about 11 amino acids. In some embodiments, the epitope may be about 9 to about 22 amino acids.

Coronavirus antigens may include antigens recognized by B cells, antigens recognized by T cells, or a combination thereof. In some embodiments, the antigen comprises an antigen recognized by B cells. In some embodiments, the coronavirus antigen is an antigen recognized by B cells. In some embodiments, the coronavirus antigen comprises an antigen recognized by T cells. In some embodiments, the antigen is an antigen recognized by T cells.

Coronavirus epitopes include epitopes recognized by B cells, epitopes recognized by T cells, or a combination thereof. In some embodiments, the coronavirus epitope comprises an epitope recognized by B cells. In some embodiments, the epitope is an epitope recognized by B cells. In some embodiments, the coronavirus epitope comprises an epitope recognized by T cells. In some embodiments, the coronavirus epitope is an epitope recognized by T cells.

For example, techniques for identifying antigens and epitopes via computer modeling such as those described in Sanchez-Trincado et al (2017), fundamentals and methods for T-and B-cell epitope prediction [ basic principles and methods of T-cell and B-cell epitope prediction ], journal of immunology research [ journal of immunology; grifoni, alba et al, A Sequence Homology and Bioinformatic Approach Can Predict Candidate Targets for Immune Responses to SARS-CoV-2[ sequence homology and bioinformatics methods can predict candidate targets for SARS-CoV-2 immune response ]. Cell host & microbe [ Cell host and microorganism ] (2020); computer simulation prediction of T-cell and B-cell epitopes in PmpD, russi et al, in silico prediction of T-and B-cell epitopes in PmpD: first step towards to the design of a Chlamydia trachomatis vaccine: first step in designing Chlamydia trachomatis vaccine, biomedical journal [ journal of biomedicine ]41.2 (2018): 109-117; baruah et al, immunoinformation-aided identification of T cell and B cell epitopes in the surface glycoprotein of 2019-nCoV [ Immunoinformatics aid in identifying T-and B-cell epitopes in 2019-nCoV surface glycoproteins ]. Journal of Medical Virology [ journal of medical virology ] (2020); each of which is incorporated herein by reference in its entirety.

The linear polyribonucleotides of the present disclosure may comprise sequences of a number of coronavirus antigens and/or epitopes. The linear polyribonucleotides comprise, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, or more sequences of coronavirus antigens or epitopes.

In some embodiments, the linear polyribonucleotides comprise, for example, sequences of up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 15, up to 20, up to 25, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 120, up to 140, up to 160, up to 180, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, up to 500, or less coronavirus antigens or epitopes.

In some embodiments, the linear polyribonucleotide comprises, for example, a sequence of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 coronavirus antigens or epitopes.

The linear polyribonucleotides may comprise sequences from one or more coronavirus epitopes of a coronavirus antigen. For example, a coronavirus antigen may comprise an amino acid sequence that may contain multiple coronavirus epitopes (e.g., epitopes recognized by B cells and/or T cells), and a linear polyribonucleotide may comprise or encode one or more of those coronavirus epitopes.

The linear polyribonucleotides comprise, for example, a sequence from at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500 or more epitopes of a coronavirus antigen.

In some embodiments, the linear polyribonucleotides comprise, for example, sequences of up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 15, up to 20, up to 25, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 120, up to 140, up to 160, up to 180, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, up to 500, or less coronavirus epitopes from one coronavirus antigen.

In some embodiments, the linear polyribonucleotide comprises, for example, a sequence of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 coronavirus epitopes from one coronavirus antigen.

The linear polyribonucleotides may encode variants of a coronavirus antigen or epitope. The variants may be naturally occurring variants (e.g., variants identified in sequence data from different coronaviruses, species, isolates, or quasispecies), or may be derived sequences as disclosed herein that have been generated via computer simulation (e.g., antigens or epitopes having one or more amino acid insertions, deletions, substitutions, or combinations thereof as compared to the wild-type antigen or epitope).

The linear polyribonucleotides comprise, for example, the sequence of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, or more variants of a coronavirus antigen or epitope.

In some embodiments, the linear polyribonucleotides comprise the sequence of up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 15, up to 20, up to 25, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 120, up to 140, up to 160, up to 180, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, up to 500, or less variants of a coronavirus antigen or epitope, for example.

In some embodiments, the linear polyribonucleotide comprises, for example, the sequence of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 variants of a coronavirus antigen or epitope.

The coronavirus antigen and/or epitope sequences of the linear polyribonucleotides may also be referred to as coronavirus expression sequences. In some embodiments, the linear polyribonucleotides comprise one or more coronavirus expression sequences, each of which may encode a coronavirus polypeptide. Coronavirus polypeptides can be produced in large quantities. The coronavirus polypeptide may be a coronavirus polypeptide secreted from a cell or localized to the cytoplasm, nucleus or membrane compartment of a cell. Some coronavirus polypeptides include, but are not limited to, antigens as disclosed herein, epitopes as disclosed herein, at least a portion of a coronavirus protein (e.g., a viral envelope protein, a viral matrix protein, a viral spike protein, a viral membrane protein, a viral nucleocapsid protein, a viral helper protein, a fragment thereof, or a combination thereof). In some embodiments, a coronavirus polypeptide encoded by a linear polyribonucleotide of the present disclosure comprises a fragment of a coronavirus antigen disclosed herein. In some embodiments, a coronavirus polypeptide encoded by a linear polyribonucleotide of the present disclosure comprises a fusion protein comprising two or more coronavirus antigens or fragments thereof as disclosed herein. In some embodiments, a coronavirus polypeptide encoded by a linear polyribonucleotide of the present disclosure comprises a coronavirus epitope. In some embodiments, the polypeptide encoded by a linear polyribonucleotide of the present disclosure comprises a fusion protein comprising two or more coronavirus epitopes of the disclosure, e.g., an artificial peptide sequence comprising a plurality of predicted epitopes from one or more coronaviruses of the disclosure.

In some embodiments, exemplary coronavirus proteins expressed from the linear polyribonucleotides disclosed herein include secreted proteins, such as proteins that naturally include a signal peptide (e.g., an antigen and/or epitope), or proteins that do not normally encode a signal peptide, but that are modified to contain a signal peptide.

Linear polyribonucleotides

The linear polyribonucleotides comprise elements as described below and coronavirus antigens or epitopes as described herein.

Linear polyribonucleotides described herein are polyribonucleotide molecules having a 5 'terminus and a 3' terminus. In some embodiments, the linear RNA has a free 5 'end or 3' end. In some embodiments, the linear RNA has a 5 'end or a 3' end that is modified or protected from degradation. In some embodiments, the linear RNA has a non-covalently linked 5 'or 3' end. In some embodiments, the linear RNA is mRNA.

The linear RNA may be modified at its ends to improve stability and/or reduce degradation. For example, the 5 'free end and/or the 3' free end includes caps, poly-a tails, G-quadruplexes, pseudoknots, stable terminal stem loops, U-rich expression, nuclear retention elements (ENE), or binding moieties. For example, the 5 'free end and/or the 3' free end includes an end protecting agent, such as a cap, poly a tail, g-quadruplex, pseudoknot, stable terminal stem loop, U-rich expression, nuclear retention element (ENE), or binding moiety.

In some embodiments, the linear polyribonucleotide is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides.

In some embodiments, the linear polyribonucleotides can be of sufficient size to accommodate the binding site of the ribosome. In some embodiments, the maximum size of the linear polyribonucleotide can be as large as within the technical limitations of generating and/or using linear polyribonucleotides. Without being bound by any particular theory, it is possible that multiple segments of RNA may be produced from DNA and annealed at their 5 'and 3' free ends to produce a "string" of RNA. In some embodiments, the maximum size of a linear polyribonucleotide may be limited by the ability to package RNA and deliver it to a target. In some embodiments, the size of the linear polyribonucleotides is a length sufficient to encode a useful polypeptide (such as an immunogen of the present disclosure or an epitope thereof), and thus a length of at least 20,000 nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides, at least 7,500 nucleotides or at least 5,000 nucleotides, at least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000 nucleotides, at least 1,000 nucleotides, at least 500 nucleotides, at least 400 nucleotides, at least 300 nucleotides, at least 200 nucleotides, at least 100 nucleotides, or at least 70 nucleotides may be useful.

Linear polyribonucleotide element

In some embodiments, the linear polyribonucleotides comprise one or more of the elements as described herein in addition to sequences encoding coronavirus antigens and/or epitopes. For example, a linear polyribonucleotide comprises a regulatory element, such as a sequence that modifies the expression of an expressed sequence within the linear polyribonucleotide. Regulatory elements may include sequences that are positioned adjacent to an expression sequence encoding an expression product. The regulatory element may be operably linked to the adjacent sequence. The regulatory element may increase the amount of the expressed product compared to the amount of the expressed product in the absence of the regulatory element. In addition, one regulatory element may increase the amount of product expressed by a plurality of expression sequences connected in series. Thus, a regulatory element may enhance expression of one or more expression sequences. A variety of regulatory elements may also be used, for example, to differentially regulate expression of different expression sequences. In some embodiments, regulatory elements provided herein may include a selective translation sequence. As used herein, the term "selectively translated sequence" refers to a nucleic acid sequence, such as certain riboswitch aptamer enzymes, that selectively initiates or activates translation of an expressed sequence in a linear polyribonucleotide. Regulatory elements may also include selective degradation sequences. As used herein, the term "selectively degrading sequence" refers to a nucleic acid sequence that initiates degradation of a linear polyribonucleotide or an expression product of a linear polyribonucleotide. In some embodiments, the regulatory element is a translational regulator. Translation regulators may regulate translation of expressed sequences in linear polyribonucleotides. The translational regulator may be a translational enhancer or a translational repressor. In some embodiments, the translation initiation sequence may act as a regulatory element.

In some embodiments, the linear polyribonucleotide encodes an antigen that produces a polyclonal antibody of interest and comprises a translation initiation sequence, such as an initiation codon. In some embodiments, the translation initiation sequence comprises a kozak or a summer-darcino (Shine-Dalgarno) sequence. In some embodiments, the linear polyribonucleotide includes a translation initiation sequence, such as a kozak sequence, adjacent to the expression sequence. In some embodiments, the translation initiation sequence is a non-coding initiation codon. In some embodiments, a translation initiation sequence (e.g., a kozak sequence) is present on one or both sides of each expression sequence, resulting in a separation of the expression products. In some embodiments, the linear polyribonucleotide comprises at least one translation initiation sequence adjacent to the expression sequence. In some embodiments, the translation initiation sequence provides conformational flexibility to the linear polyribonucleotide. In some embodiments, the translation initiation sequence is substantially within a single stranded region of the linear polyribonucleotide.

In some embodiments, the linear polyribonucleotides described herein comprise an Internal Ribosome Entry Site (IRES) element. Suitable IRES elements included in the linear polyribonucleotides may be RNA sequences capable of engaging eukaryotic ribosomes.

The linear polyribonucleotides may include one or more expression sequences (e.g., encoding an antigen), and each expression sequence may or may not have a termination element.

In some embodiments, the linear polynucleotide comprises a 5 'cap, wherein the 5' cap structure of the mRNA increases mRNA stability. The 5' cap binds to the mRNA cap binding protein (MBP), which associates with the poly a binding protein via CBP to form mature RNA species, contributing to mRNA stability and translation ability in the cell.

In some embodiments, the linear polynucleotide is 5 '-terminally capped and comprises a 5' -ppp-5 'triphosphate linkage between the terminal guanosine cap residue of the linear polynucleotide and the 5' -terminal transcribed sense nucleotide. Such 5 'guanosine caps, also known as 5' guanylate caps, may be methylated to produce an N7-methylguanylate cap.

In some embodiments, the linear polyribonucleotide comprises an untranslated region (UTR). The UTR comprising the genomic region of the gene may be transcribed but not translated. In some embodiments, the UTR may be included upstream of the translation initiation sequences of the expression sequences described herein. In some embodiments, UTRs may be included downstream of the expression sequences described herein. In some cases, one UTR of a first expressed sequence is identical to or contiguous with or overlaps with another UTR of a second expressed sequence. In some embodiments, the intron is a human intron. In some embodiments, the intron is a full-length human intron, e.g., ZKSCAN1.

In some embodiments, the linear polyribonucleotide comprises a poly a sequence. In some embodiments, the poly a sequence is greater than 10 nucleotides in length. In some embodiments, the poly a sequence is greater than 15 nucleotides in length (e.g., at least or greater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the poly a sequence is about 10 to about 3,000 nucleotides (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 750, 30 to 1,000, 30 to 1,500, 30 to 2,000, 30 to 2,500, 50 to 100, 50 to 250, 50 to 500, 50 to 750, 50 to 1,000, 50 to 1,500, 50 to 2,000, 50 to 2,500, 50 to 3,000, 100 to 500, 100 to 750, 100 to 1,000, 100 to 1,500, 100 to 2,000, 100 to 2,500, 100 to 3,000, 500 to 750, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 2,500, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,500, 1,000 to 3,000, 1,500 to 2,000, 1,500 to 2,000, 1,000, 500 to 2,000, 500 and 3,000.

In some embodiments, the poly a sequence is designed relative to the length of the entire linear polyribonucleotide. The design may be based on the length of the coding region, the length of a particular feature or region (such as the first or flanking region), or the length of the final product of linear polyribonucleotide expression. In this context, the length of the poly a sequence may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% longer than the linear polyribonucleotide or a characteristic thereof. The poly-a sequence can also be designed as part of a linear polyribonucleotide. In this context, the poly a sequence may be the total length of the construct or 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the total length of the construct minus the poly a sequence. Further, conjugation of engineered binding sites and linear polyribonucleotides to poly-a binding proteins can enhance expression.

In some embodiments, the linear polyribonucleotides are designed to include poly a-G tetrads (quaternaries). G-tetrads are cyclic hydrogen bond arrays of four guanine nucleotides, which can be formed from G-rich sequences in DNA and RNA. In some embodiments, G-tetrads may be incorporated into the ends of the poly A sequence. The stability, protein yield, and/or other parameters of the resulting linear polyribonucleotide construct can be determined, including half-life at different time points. In some embodiments, the poly a-G tetrads can produce a protein yield that is at least 75% of the protein yield obtained using 120 nucleotide poly a sequences alone.

In some embodiments, the linear polyribonucleotide comprises a UTR with one or more segments of adenosine and uridine embedded therein. AU enrichment signatures can increase the conversion of the expression product.

The introduction, removal or modification of UTR AU enrichment elements (ARE) can be used to modulate the stability or immunogenicity of linear polyribonucleotides. When engineering a particular linear polyribonucleotide, one or more copies of an ARE can be introduced to destabilize the linear polyribonucleotide, and these copies of an ARE can reduce translation and/or reduce the yield of expression products. Similarly, ARE can be identified and removed or mutated to increase intracellular stability, thereby increasing translation and the yield of the resulting protein.

UTR from any gene may be incorporated into the corresponding flanking regions (e.g., at the 5 'end or the 3' end) of the linear polyribonucleotide. In addition, multiple wild-type UTRs of any known gene may be utilized. In some embodiments, an artificial UTR that is not a variant of a wild-type gene may be used. These UTRs or portions thereof may be placed in the same orientation as in the transcripts from which they were selected, or may be changed in orientation or position. Thus, a 5 '-or 3' -UTR can be inverted, shortened, lengthened, or chimeric with one or more other 5 '-UTRs or 3' -UTRs. As used herein, the term "altered" when in relation to a UTR sequence means that the UTR has been altered in some way relative to a reference sequence. For example, the 3 '-or 5' -UTR may be altered relative to a wild-type or natural UTR by alteration of orientation or position as taught above, or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, exchange of nucleotides, or transposition. Any such alterations (whether 3 'or 5') that result in an "altered" UTR include variant UTRs.

In some embodiments, dual UTR, triple UTR, or quad UTR may be used, such as 5 '-or 3' -UTR. As used herein, a "dual" UTR is a case in which two copies of the same UTR are encoded in tandem or substantially in tandem. For example, a double beta-globin 3' -UTR may be used in some embodiments of the invention.

In some embodiments, the linear polyribonucleotides include one or more regulatory nucleic acid sequences or include one or more expression sequences encoding a regulatory nucleic acid (e.g., a nucleic acid that modifies expression of an endogenous gene and/or an exogenous gene). In some embodiments, the expression sequences of the linear polyribonucleotides provided herein can comprise sequences antisense to regulatory nucleic acids like non-coding RNAs such as, but not limited to tRNA, lncRNA, miRNA, rRNA, snRNA, microRNA, siRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, Y RNA and hnRNA.

In some embodiments, the linear polyribonucleotides produce stoichiometric expression products. In some embodiments, the linear polyribonucleotides have stoichiometric translational efficiencies such that the expression products are produced at substantially equal rates. In some embodiments, the linear polyribonucleotides have stoichiometric translational efficiencies of multiple expression products (e.g., products from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more expression sequences).

In some embodiments, the linear polyribonucleotide comprises one or more riboswitches.

In some embodiments, the linear polyribonucleotide comprises an aptamer enzyme.

In some embodiments, the linear polyribonucleotide lacks a 5' -UTR. In some embodiments, the linear polyribonucleotide lacks a 3' -UTR. In some embodiments, the linear polyribonucleotide lacks a poly a sequence. In some embodiments, the linear polyribonucleotide lacks a terminating element. In some embodiments, the linear polyribonucleotide lacks an internal ribosome entry site. In some embodiments, the linear polyribonucleotide lacks binding to the cap binding protein. In some embodiments, the linear polyribonucleotide lacks a 5' cap.

Production method

In some embodiments, the linear polyribonucleotides include non-naturally occurring deoxyribonucleic acid sequences, and can be produced using recombinant techniques (e.g., in vitro derivatization using DNA plasmids) or chemical synthesis, or a combination thereof.

Within the scope of the present disclosure, a DNA molecule for producing RNA may include a DNA sequence of a naturally occurring original nucleic acid sequence, a modified version thereof, or a DNA sequence encoding a synthetic polypeptide that is not normally found in nature (e.g., a chimeric molecule or fusion protein, such as a fusion protein comprising multiple immunogens). DNA and RNA molecules can be modified using a variety of techniques including, but not limited to, classical mutagenesis techniques and recombinant techniques, such as site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, cleavage of nucleic acid fragments by restriction enzymes, ligation of nucleic acid fragments, polymerase Chain Reaction (PCR) amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures, and ligation of mixture groups to "build" a mixture of nucleic acid molecules, and combinations thereof.

The linear polyribonucleotides can be prepared according to any available technique, including but not limited to chemical synthesis and enzymatic synthesis. In some embodiments, the linear primary construct or linear mRNA may be concatemerized to produce the linear polyribonucleotides described herein. The mechanism of concatemerization may occur by methods such as, but not limited to, chemical, enzymatic, splinting or ribozyme catalysis. The newly formed 5'-/3' -bond may be an intramolecular bond or an intermolecular bond.

Methods of preparing linear polyribonucleotides described herein are described, for example, in Khudyakov & Fields, artifical DNA: methods and Applications [ Artificial DNA: methods and applications ], CRC Press (2002); zhao, synthetic Biology: tools and Applications [ synthetic biology: tools and applications ], (first edition), academic Press (2013); and Egli & Herhewijn, chemistry and Biology of Artificial Nucleic Acids [ chemical and biological of artificial nucleic acids ], (first edition), wiley-VCH (2012).

Various methods of synthesizing linear polyribonucleotides are also described in the art (see, e.g., U.S. Pat. No. 6210931, U.S. Pat. No. 5773244, U.S. Pat. No. 5766903, U.S. Pat. No. 5712128, U.S. Pat. No. 5426180, U.S. publication No. US 20100137407, international publication No. WO 1992001813, and International publication No. WO 2010084371, the contents of each of which are incorporated herein by reference in their entirety).

Method for generating immune response

The present disclosure provides immunogenic compositions comprising the cyclic polyribonucleotides described above. The present disclosure provides immunogenic compositions comprising the linear polyribonucleotides described above. The immunogenic compositions of the invention may comprise a diluent or carrier, adjuvant, or any combination thereof. The immunogenic compositions of the invention may also comprise one or more immunomodulators, e.g. one or more adjuvants. Adjuvants may include TH1 adjuvants and/or TH2 adjuvants discussed further below. In some embodiments, the immunogenic composition comprises a diluent that does not contain any carrier, and is used to deliver the cyclic polyribonucleotide to a subject (e.g., a subject to be immunized). In some embodiments, the immunogenic composition comprises a diluent that does not contain any carrier, and is used to deliver the linear polyribonucleotide to the subject.

The immunogenic compositions of the invention are useful for generating an immune response in a subject (e.g., a subject to be immunized). The immune response may include an antibody response (typically including IgG) and/or a cell-mediated immune response. In some embodiments, the immunogenic composition is used to produce polyclonal antibodies as described herein. For example, a subject is immunized with an immunogenic composition comprising cyclic polyribonucleotides that comprise a coronavirus antigen and/or epitope to stimulate the production of polyclonal antibodies that bind to the coronavirus antigen and/or epitope. In another example, a subject is immunized with an immunogenic composition comprising linear polyribonucleotides that comprise coronavirus antigens and/or epitopes to stimulate the production of polyclonal antibodies that bind to the coronavirus antigens and/or epitopes. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the non-human animal has a humanized immune system. In some embodiments, the subject is further vaccinated with an adjuvant. In some embodiments, the subject is further vaccinated with the vaccine. Optionally, after immunization with an immunogenic composition comprising cyclic polyribonucleotides, the polyclonal antibodies produced are collected and purified from the subject. Optionally, after immunization with an immunogenic composition comprising linear polyribonucleotides, the polyclonal antibodies produced are collected and purified from the subject. In some embodiments, the composition comprises plasma collected after administration of an immunogenic composition described herein.

Immunization with

In some embodiments, the methods of the disclosure include immunizing a subject (e.g., a subject to be immunized) with an immunogenic composition comprising a cyclic polyribonucleotide as disclosed herein. In some embodiments, the coronavirus antigen and/or epitope is expressed by a cyclic polyribonucleotide. In some embodiments, immunization induces an immune response in a subject against coronavirus antigens and/or epitopes expressed by the cyclic polyribonucleotides. In some embodiments, immunization induces the production of polyclonal antibodies that bind to coronavirus antigens and/or epitopes expressed by the cyclic polyribonucleotides. In some embodiments, the immunogenic composition comprises a cyclic polyribonucleotide and a diluent, carrier, first adjuvant, or combination thereof in a single composition. In some embodiments, the subject is further vaccinated with a second adjuvant. In some embodiments, the subject is further vaccinated with the vaccine.

In some embodiments, the methods of the disclosure include immunizing a subject (e.g., a subject to be immunized) with an immunogenic composition comprising a linear polyribonucleotide as disclosed herein. In some embodiments, the coronavirus antigen and/or epitope is expressed by a linear polyribonucleotide. In some embodiments, immunization induces an immune response in a subject against coronavirus antigens and/or epitopes expressed by linear polyribonucleotides. In some embodiments, immunization induces the production of polyclonal antibodies that bind to coronavirus antigens and/or epitopes expressed by linear polyribonucleotides. In some embodiments, the immunogenic composition comprises linear polyribonucleotides and diluent, carrier, first adjuvant, or combination thereof in a single composition. In some embodiments, the subject is further vaccinated with a second adjuvant. In some embodiments, the subject is further vaccinated with the vaccine.

The cyclic polyribonucleotides as disclosed herein stimulate the production of human polyclonal antibodies by stimulating an adaptive immune response after immunization of a subject (e.g., a subject to be immunized). In some embodiments, the adaptive immune response of the subject includes stimulating B lymphocytes to release polyclonal antibodies that specifically bind to coronavirus antigens expressed by the cyclic polyribonucleotides. Linear polyribonucleotides as disclosed herein stimulate the production of human polyclonal antibodies by stimulating an adaptive immune response after immunization of a subject. In some embodiments, the adaptive immune response of the subject includes stimulating B lymphocytes to release polyclonal antibodies that specifically bind to coronavirus antigens expressed by linear polyribonucleotides. In some embodiments, the adaptive immune response in the subject includes stimulating a cell-mediated immune response.

A subject (e.g., a subject to be immunized) is immunized with one or more immunogenic compositions comprising any number of cyclic polyribonucleotides. The subject is immunized with one or more immunogenic compositions, e.g., comprising at least 1 cyclic polyribonucleotide. A non-human animal having a non-humanized immune system is vaccinated with one or more immunogenic compositions comprising, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20 different cyclic polyribonucleotides or more different cyclic polyribonucleotides. In some embodiments, the subject is vaccinated with one or more immunogenic compositions comprising up to 1 cyclic polyribonucleotide. In some embodiments, a non-human animal having a humanized immune system is vaccinated with one or more immunogenic compositions comprising at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 20 different cyclic polyribonucleotides, or less than 21 different cyclic polyribonucleotides. In some embodiments, the subject is vaccinated with one or more immunogenic compositions comprising about 1 cyclic polyribonucleotide. In some embodiments, a non-human animal having a humanized immune system is vaccinated with one or more immunogenic compositions comprising about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, or about 20 different cyclic polyribonucleotides. In some embodiments, the subject is immunized with one or more immunogenic compositions comprising about 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 4-4, 4-3, 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 5-10, 10-15, or 15-20 different cyclic polyribonucleotides. Different cyclic polyribonucleotides have different sequences from each other. For example, they may include or encode different antigens and/or epitopes, overlapping antigens and/or epitopes, similar antigens and/or epitopes, or the same antigens and/or epitopes (e.g., with the same or different regulatory elements, initiation sequences, promoters, termination elements, or other elements of the disclosure). Where a subject is vaccinated with one or more immunogenic compositions comprising two or more different cyclic polyribonucleotides, the two or more different cyclic polyribonucleotides may be in the same or different immunogenic compositions and vaccinated simultaneously or at different times. An immunogenic composition comprising two or more different cyclic polyribonucleotides can be administered to the same anatomical site or to different anatomical sites.

The two or more different circularized polyribonucleotides may include or encode antigens and/or epitopes from the same coronavirus, different coronaviruses, or different combinations of coronaviruses disclosed herein. The two or more different cyclic polyribonucleotides may include or encode antigens and/or epitopes from the same coronavirus or from different coronaviruses (e.g., different isolates).

A subject (e.g., a subject to be immunized) is immunized with one or more immunogenic compositions comprising any number of linear polyribonucleotides. The subject is vaccinated with one or more immunogenic compositions comprising, for example, at least 1 linear polyribonucleotide. A non-human animal having a non-humanized immune system is vaccinated with one or more immunogenic compositions comprising, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20 different linear polyribonucleotides or more different linear polyribonucleotides. In some embodiments, the subject is vaccinated with one or more immunogenic compositions comprising up to 1 linear polyribonucleotide. In some embodiments, a non-human animal having a humanized immune system is vaccinated with one or more immunogenic compositions comprising at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 20 different linear polyribonucleotides, or less than 21 different linear polyribonucleotides. In some embodiments, the subject is vaccinated with one or more immunogenic compositions comprising about 1 linear polyribonucleotide. In some embodiments, a non-human animal having a humanized immune system is vaccinated with one or more immunogenic compositions comprising about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, or about 20 different linear polyribonucleotides. In some embodiments, the subject is immunized with one or more immunogenic compositions comprising about 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 4-4, 4-3, 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 5-10, 10-15, or 15-20 different linear polyribonucleotides. Different linear polyribonucleotides have sequences that differ from each other. For example, they may include or encode different antigens and/or epitopes, overlapping antigens and/or epitopes, similar antigens and/or epitopes, or the same antigens and/or epitopes (e.g., with the same or different regulatory elements, initiation sequences, promoters, termination elements, or other elements of the disclosure). Where a subject is vaccinated with one or more immunogenic compositions comprising two or more different linear polyribonucleotides, the two or more different linear polyribonucleotides may be in the same or different immunogenic compositions and vaccinated simultaneously or at different times. An immunogenic composition comprising two or more different linear polyribonucleotides can be administered to the same anatomical site or to different anatomical sites.

The two or more different linear polyribonucleotides may include or encode antigens and/or epitopes from the same coronavirus, different coronaviruses, or different combinations of coronaviruses disclosed herein. The two or more different linear polyribonucleotides can include or encode antigens and/or epitopes from the same coronavirus or from different coronaviruses (e.g., different isolates).

In some embodiments, a subject (e.g., a subject to be immunized) is immunized with one or more immunogenic compositions comprising any number of cyclic polyribonucleotides and one or more immunogenic compositions comprising any number of linear polyribonucleotides as disclosed herein. In some embodiments, the immunogenic compositions disclosed herein comprise one or more cyclic polyribonucleotides and one or more linear polyribonucleotides as disclosed herein.

In some embodiments, the immunogenic composition comprises a cyclic polyribonucleotide and a diluent, carrier, first adjuvant, or combination thereof. In a particular embodiment, the immunogenic composition comprises a cyclic polyribonucleotide described herein and a carrier or diluent that does not contain any carrier. In some embodiments, an immunogenic composition comprising a cyclic polyribonucleotide and a diluent that does not contain any carrier is used to deliver the cyclic polyribonucleotide to a subject in naked form. In another particular embodiment, the immunogenic composition comprises a cyclic polyribonucleotide described herein and a first adjuvant.

In certain embodiments, the subject (e.g., the subject to be immunized) is further administered a second adjuvant. The adjuvant enhances an innate immune response, which in turn enhances an adaptive immune response in the subject to produce polyclonal antibodies. The adjuvant may be any adjuvant as discussed below. In certain embodiments, the adjuvant is formulated with the cyclic polyribonucleotides as part of an immunogenic composition. In certain embodiments, the adjuvant is not part of an immunogenic composition comprising cyclic polyribonucleotides. In certain embodiments, the adjuvant is administered separately from the immunogenic composition comprising the cyclic polyribonucleotide. In this regard, the adjuvant is administered to the subject either concurrently (e.g., simultaneously) or at a different time with an immunogenic composition comprising cyclic polyribonucleotides. For example, the adjuvant is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour in between, after the immunogenic composition comprising the cyclic polyribonucleotide. In some embodiments, the adjuvant is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any number of minutes or hours in between, prior to the immunogenic composition comprising the cyclic polyribonucleotide. For example, the adjuvant is administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any number of days in between, after the immunogenic composition comprising the cyclic polyribonucleotide. In some embodiments, the adjuvant is administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any number of days in between, prior to the immunogenic composition comprising the cyclic polyribonucleotide. The adjuvant is administered to the same anatomical location or a different anatomical location than the immunogenic composition comprising the cyclic polyribonucleotide.

In some embodiments, the immunogenic composition comprises a linear polyribonucleotide and a diluent, carrier, first adjuvant, or combination thereof. In a particular embodiment, the immunogenic composition comprises a linear polyribonucleotide described herein and a carrier or diluent that does not contain any carrier. In some embodiments, an immunogenic composition comprising a linear polyribonucleotide and a diluent that does not contain any carrier is used to deliver the linear polyribonucleotide to a subject (e.g., a subject to be immunized). In another particular embodiment, the immunogenic composition comprises a linear polyribonucleotide described herein and a first adjuvant.

In certain embodiments, the subject (e.g., the subject to be immunized) is further administered a second adjuvant. The adjuvant enhances an innate immune response, which in turn enhances an adaptive immune response in the subject to produce polyclonal antibodies. The adjuvant may be any adjuvant as discussed below. In certain embodiments, the adjuvant is formulated with linear polyribonucleotides as part of an immunogenic composition. In certain embodiments, the adjuvant is not part of an immunogenic composition comprising linear polyribonucleotides. In certain embodiments, the adjuvant is administered separately from the immunogenic composition comprising linear polyribonucleotides. In this regard, the adjuvant is administered to the subject either concurrently (e.g., simultaneously) or at different times with an immunogenic composition comprising linear polyribonucleotides. For example, the adjuvant is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour in between, after the immunogenic composition comprising the linear polyribonucleotide. In some embodiments, the adjuvant is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any number of minutes or hours in between, prior to the immunogenic composition comprising the linear polyribonucleotide. For example, the adjuvant is administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any number of days in between, after the immunogenic composition comprising the linear polyribonucleotide. In some embodiments, the adjuvant is administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any number of days in between, prior to the immunogenic composition comprising the linear polyribonucleotide. The adjuvant is applied to the same anatomical location or a different anatomical location than the immunogenic composition comprising linear polyribonucleotides.

In some embodiments, the subject (e.g., the subject to be immunized) is further immunized with a second agent, e.g., a vaccine that is not a cyclic polyribonucleotide (as described below). The vaccine is administered to the subject either concurrently (e.g., simultaneously) or at different times with an immunogenic composition comprising cyclic polyribonucleotides. For example, a vaccine is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour in between, after an immunogenic composition comprising a cyclic polyribonucleotide. In some embodiments, the vaccine is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any number of minutes or hours in between, prior to the immunogenic composition comprising the cyclic polyribonucleotide. For example, the vaccine is administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any number of days in between, after the immunogenic composition comprising the cyclic polyribonucleotide. In some embodiments, the vaccine is administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any number of days in between, prior to the immunogenic composition comprising the cyclic polyribonucleotide.

In some embodiments, the subject (e.g., the subject to be immunized) is further immunized with a second agent, e.g., a vaccine that is not a linear polyribonucleotide (as described below). The vaccine is administered to the subject either concurrently (e.g., simultaneously) or at different times with an immunogenic composition comprising linear polyribonucleotides. For example, the vaccine is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or hour in between, after the immunogenic composition comprising the linear polyribonucleotide. In some embodiments, the vaccine is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any number of minutes or hours in between, prior to the immunogenic composition comprising linear polyribonucleotides. For example, the vaccine is administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any number of days in between, after the immunogenic composition comprising linear polyribonucleotides. In some embodiments, the vaccine is administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any number of days in between, prior to the immunogenic composition comprising linear polyribonucleotides.

The subject (e.g., the subject to be immunized) can be immunized with the immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof any suitable number of times to achieve the desired response. For example, prime-boost immunization strategies may be utilized to generate hyperimmune plasma containing high concentrations of antibodies that bind to the antigens and/or epitopes of the present disclosure. The subject can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine) of the disclosure, or a combination thereof, e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 15 or more times.

In some embodiments, a subject (e.g., a subject to be immunized) can be immunized with an immunogenic composition, adjuvant, vaccine (e.g., a protein subunit vaccine) of the disclosure, or a combination thereof, up to 2 times, up to 3 times, up to 4 times, up to 5 times, up to 6 times, up to 7 times, up to 8 times, up to 9 times, up to 10 times, up to 15 times, or up to 20 times or less.

In some embodiments, a subject (e.g., a subject to be immunized) can be immunized about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times with an immunogenic composition, adjuvant, vaccine (e.g., a protein subunit vaccine) of the disclosure, or a combination thereof.

In some embodiments, a subject (e.g., a subject to be immunized) can be immunized once with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine) of the disclosure, or a combination thereof. In some embodiments, a subject may be vaccinated twice with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine) of the disclosure, or a combination thereof. In some embodiments, a subject may be vaccinated three times with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine) of the disclosure, or a combination thereof. In some embodiments, a subject may be vaccinated four times with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine) of the disclosure, or a combination thereof. In some embodiments, a subject may be vaccinated five times with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine) of the disclosure, or a combination thereof. In some embodiments, a subject may be vaccinated seven times with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine) of the disclosure, or a combination thereof.

The appropriate time interval may be selected to interval two or more immunizations. The time interval may be suitable for multiple immunizations with the same immunogenic composition, adjuvant or vaccine (e.g., protein subunit vaccine), or a combination thereof, e.g., the same immunogenic composition, adjuvant or vaccine (e.g., protein subunit vaccine), or a combination thereof may be administered in the same amount or different amounts via the same immunization route or different immunization routes. The time interval may be suitable for immunization with different agents, e.g., a first immunogenic composition comprising a first cyclic polyribonucleotide and a second immunogenic composition comprising a second cyclic polyribonucleotide. The time interval may be applicable to a first immunogenic composition comprising a first linear polyribonucleotide and a second immunogenic composition comprising a second linear polyribonucleotide. For a regimen comprising three or more immunizations, the time intervals of the immunizations may be the same or different. In some examples, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 40, 48, or 72 hours elapse between two immunizations. In some embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17, 18, 20, 21, 24, 28, or 30 days elapse between immunizations. In some embodiments, about 1, 2, 3, 4, 5, 6, 7, or 8 weeks elapse between two immunizations. In some embodiments, about 1, 2, 3, 4, 5, 6, 7, or 8 months passes between immunizations.

In some embodiments, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 36 hours, or at least 72 hours or more pass between immunizations. In some embodiments, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours, up to 15 hours, up to 20 hours, up to 24 hours, up to 36 hours, or up to 72 hours, or less passes between two immunizations.

In some embodiments, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 15 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, or at least 30 days or more pass between immunizations. In some embodiments, up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 15 days, up to 20 days, up to 21 days, up to 22 days, up to 23 days, up to 24 days, up to 25 days, up to 26 days, up to 27 days, up to 28 days, up to 29 days, up to 30 days, up to 32 days, up to 34 days, or up to 36 days or less pass between immunizations.

In some embodiments, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks or more pass between immunizations. In some embodiments, up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 5 weeks, up to 6 weeks, up to 7 weeks, up to 8 weeks, or less time passes between immunizations.

In some embodiments, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, or at least 8 months or more pass between immunizations. In some embodiments, up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, up to 7 months, up to 8 months, or less time passes between immunizations.

In some embodiments, the non-human animal with a humanized immune system is immunized 3 times at 3-4 week intervals.

In some embodiments, the method further comprises pre-administering an agent to the non-human animal (e.g., a non-human animal having a humanized immune system) or human subject (e.g., a non-human animal or human subject to be immunized) to enhance the immune response. In some embodiments, the agent is an antigen (e.g., a protein antigen) as disclosed herein. For example, the method comprises administering theprotein antigen 1 to 7 days prior to administering the cyclic polyribonucleotide comprising a sequence encoding the protein antigen. In some embodiments, the protein antigen is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of the cyclic polyribonucleotide comprising a sequence encoding the protein antigen. For example, the method comprises administering theprotein antigen 1 to 7 days prior to administering the linear polyribonucleotide comprising a sequence encoding the protein antigen. In some embodiments, the protein antigen is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of the linear polyribonucleotide comprising a sequence encoding the protein antigen. Protein antigens may be administered as protein formulations, encoded in plasmids (pDNA), present in virus-like particles (VLPs), formulated in the form of lipid nanoparticles, and the like.

The subject (e.g., a subject to be immunized) can be immunized with an immunogenic composition, adjuvant, or vaccine (e.g., a protein subunit vaccine), or a combination thereof, at any suitable number of anatomical sites. The same immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof may be administered to multiple anatomical sites, different immunogenic compositions, adjuvants, vaccines (e.g., protein subunit vaccine), or a combination thereof comprising the same or different cyclic polyribonucleotides may be administered to different anatomical sites, different immunogenic compositions, adjuvants, vaccines (e.g., protein subunit vaccine), or a combination thereof comprising the same or different cyclic polyribonucleotides may be administered to the same anatomical site, or any combination thereof. For example, an immunogenic composition comprising cyclic polyribonucleotides can be administered to two different anatomical sites, and/or an immunogenic composition comprising cyclic polyribonucleotides can be administered to one anatomical site, and an adjuvant can be administered to a different anatomical site. The same immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or a combination thereof may be administered to multiple anatomical sites, different immunogenic compositions, adjuvants, vaccines (e.g., protein subunit vaccine), or a combination thereof comprising the same or different linear polyribonucleotides may be administered to different anatomical sites, different immunogenic compositions, adjuvants, vaccines (e.g., protein subunit vaccine), or a combination thereof comprising the same or different linear polyribonucleotides may be administered to the same anatomical site, or any combination thereof. For example, an immunogenic composition comprising linear polyribonucleotides may be administered to two different anatomical sites, and/or an immunogenic composition comprising linear polyribonucleotides may be administered to one anatomical site, and an adjuvant may be administered to a different anatomical site.

Immunization of any two or more anatomical routes may be via the same immunization route (e.g., intramuscularly) or by two or more immunization routes. In some embodiments, an immunogenic composition, adjuvant, or vaccine (e.g., a protein subunit vaccine) of the disclosure comprising cyclic polyribonucleotides, or a combination thereof, is vaccinated against at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 anatomical sites of a subject. In some embodiments, an immunogenic composition, adjuvant, or vaccine (e.g., a protein subunit vaccine) of the disclosure comprising a cyclic polyribonucleotide, or a combination thereof, is vaccinated against at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 anatomical sites or less of the subject. In some embodiments, an immunogenic composition or adjuvant comprising a cyclic polyribonucleotide of the present disclosure is vaccinated against 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 anatomical sites of a subject. In some embodiments, an immunogenic composition, adjuvant, or vaccine (e.g., a protein subunit vaccine) of the disclosure comprising linear polyribonucleotides, or a combination thereof, is vaccinated against at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 anatomical sites of a subject. In some embodiments, an immunogenic composition, adjuvant, or vaccine (e.g., a protein subunit vaccine) of the disclosure comprising linear polyribonucleotides, or a combination thereof, is vaccinated against at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 anatomical sites or less of the subject. In some embodiments, an immunogenic composition or adjuvant comprising linear polyribonucleotides of the present disclosure is vaccinated against 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 anatomical sites of a subject.

Immunization may be via any suitable route. Non-limiting examples of immunization routes include intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, intracerebral, intraocular, intralesional, intracerebroventricular, intracisternal, or intraparenchymal, such as injection and infusion. In some cases, immunization may be via inhalation. Two or more immunizations may be performed by the same or different routes.

Any suitable amount of cyclic polyribonucleotides can be administered to a subject of the present disclosure (e.g., a subject to be immunized). For example, the subject can be immunized with at least about 1ng, at least about 10ng, at least about 100ng, at least about 1 μg, at least about 10 μg, at least about 100 μg, at least about 1mg, at least about 10mg, at least about 100mg, or at least about 1g of cyclic polyribonucleotides. In some embodiments, the subject may be vaccinated with up to about 1ng, up to about 10ng, up to about 100ng, up to about 1 μg, up to about 10 μg, up to about 100 μg, up to about 1mg, up to about 10mg, up to about 100mg, or up to about 1g of cyclic polyribonucleotides. In some embodiments, the subject may be immunized with about 1ng, about 10ng, about 100ng, about 1 μg, about 10 μg, about 100 μg, about 1mg, about 10mg, about 100mg, or about 1g of cyclic polyribonucleotide.

Any suitable amount of linear polyribonucleotides can be administered to a subject of the present disclosure (e.g., a subject to be immunized). For example, the subject can be immunized with at least about 1ng, at least about 10ng, at least about 100ng, at least about 1 μg, at least about 10 μg, at least about 100 μg, at least about 1mg, at least about 10mg, at least about 100mg, or at least about 1g of linear polyribonucleotide. In some embodiments, the subject may be vaccinated with up to about 1ng, up to about 10ng, up to about 100ng, up to about 1 μg, up to about 10 μg, up to about 100 μg, up to about 1mg, up to about 10mg, up to about 100mg, or up to about 1g of linear polyribonucleotides. In some embodiments, the subject may be immunized with about 1ng, about 10ng, about 100ng, about 1 μg, about 10 μg, about 100 μg, about 1mg, about 10mg, about 100mg, or about 1g of linear polyribonucleotide.

In some embodiments, the method further comprises evaluating the antibody response of the non-human animal or human subject (e.g., the subject to be immunized) to the antigen. In some embodiments, the evaluation is before and/or after administration of a circular polyribonucleotide comprising a sequence that encodes a coronavirus antigen. In some embodiments, the evaluation is before and/or after administration of the linear polyribonucleotides comprising a sequence that encodes a coronavirus antigen.

Diluent agent

In some embodiments, the immunogenic compositions of the invention comprise cyclic polyribonucleotides and a diluent. In some embodiments, the immunogenic compositions of the invention comprise linear polyribonucleotides and a diluent.

The diluent may be a non-carrier excipient. Non-carrier excipients are used as vehicles or mediums for compositions such as the cyclic polyribonucleotides as described herein. Non-carrier excipients are used as vehicles or mediums for compositions such as linear polyribonucleotides as described herein. Non-limiting examples of non-carrier excipients include solvents, aqueous solvents, nonaqueous solvents, dispersion media, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickening agents, emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidase, dispersants, granulating agents, disintegrants, binders, buffers (e.g., phosphate Buffered Saline (PBS)), lubricants, oils, and mixtures thereof. The non-carrier vehicle may be any non-active ingredient approved by the U.S. Food and Drug Administration (FDA) and listed in the non-active ingredient database that does not exhibit cell penetration. The non-carrier vehicle may be any non-active ingredient suitable for administration to a non-human animal (e.g., suitable for veterinary use). Modifications to compositions suitable for administration to humans are well understood in order to render the compositions suitable for administration to a variety of animals, and a veterinarian of ordinary skill can design and/or make such modifications by merely ordinary experimentation, if any.

In some embodiments, the cyclic polyribonucleotides may be delivered in the form of a naked delivery formulation, such as comprising a diluent. The naked delivery formulation delivers the cyclic polyribonucleotide to the cell without the aid of a carrier and without the need to modify or partially or completely encapsulate the cyclic polyribonucleotide, the capped polyribonucleotide, or a complex thereof.

The naked delivery formulation is a vehicle-free formulation and wherein the cyclic polyribonucleotides are not covalently modified by binding to a moiety that facilitates delivery to a cell, or are not partially or fully encapsulated. In some embodiments, the covalently modified cyclic polyribonucleotide that is not bound to a moiety that facilitates delivery to a cell is a polyribonucleotide that is not covalently bound to a protein, small molecule, particle, polymer, or biopolymer. Covalently modified cyclic polyribonucleotides that do not incorporate moieties that facilitate delivery to cells do not contain modified phosphate groups. For example, the covalently modified cyclic polyribonucleotide that does not incorporate moieties that facilitate delivery to a cell is free of phosphorothioates, phosphoroselenos, phosphoroborophosphates, phosphoroborodates, phosphorohydrogen phosphates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, or phosphotriesters.

In some embodiments, the linear polyribonucleotides may be delivered in the form of a naked delivery formulation, such as comprising a diluent. The naked delivery formulation delivers the linear polyribonucleotide to the cell without the aid of a carrier and without the need for modification or partial or complete encapsulation of the linear polyribonucleotide, capped polyribonucleotide, or complexes thereof.

The naked delivery formulation is a vehicle-free formulation and wherein the linear polyribonucleotides are not covalently modified by binding to a moiety that facilitates delivery to a cell, or are not partially or fully encapsulated by the linear polyribonucleotides. In some embodiments, the covalently modified linear polyribonucleotide that is not bound to a moiety that facilitates delivery to a cell is a polyribonucleotide that is not covalently bound to a protein, small molecule, particle, polymer, or biopolymer. Covalently modified linear polyribonucleotides that do not incorporate moieties that facilitate delivery to cells do not contain modified phosphate groups. For example, the covalently modified linear polyribonucleotide that does not incorporate moieties that facilitate delivery to a cell is free of phosphorothioates, phosphoroselenos, phosphoroborophosphates, phosphoroborodates, phosphorohydrogen phosphates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, or phosphotriesters.

In some embodiments, the naked delivery formulation does not contain any or all of: transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers or protein carriers. In some embodiments, the naked delivery formulation is free of phytooctenyl succinate, phytoglycogen beta-dextrin, anhydride modified phytoglycogen beta-dextrin, lipofectamine (lipofectamine), polyethylenimine, poly (trimethylimine), poly (tetramethylimine), polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly (2-dimethylamino) ethyl methacrylate, poly (lysine), poly (histidine), poly (arginine), cationic gelatin, dendrimer, chitosan, 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP), N- [1- (2, 3-dioleoyloxy) propyl group]-N, N, N-trimethylammonium chloride (DO^T MA), 1- [2- (oleoyloxy) ethyl]-2-oleyl-3- (2-hydroxyethyl) imidazolium chloride (DOTIM), 2, 3-dioleoyloxy-N- [2 (spermidine carboxamido) ethyl]-N, N-dimethyl-1-trifluoroacetate propyl ammonium (DOSPA), 3B- [ N- (N\N' -dimethylaminoethane) -carbamoyl]Cholesterol hydrochloride (DC-cholesterol hydrochloride), di-seventeen Alkylaminoglycyl spermidine (DOGS), N-distearyl-N, N-dimethyl ammonium bromide (DDAB), N- (1, 2-dimyristoxyprop-3-yl) -N, N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), N-dioleyl-N, N-dimethyl ammonium chloride (DODAC), human Serum Albumin (HSA), low Density Lipoprotein (LDL), high Density Lipoprotein (HDL) or globulin.

In certain embodiments, the naked delivery formulation comprises a non-carrier excipient. In some embodiments, the non-carrier vehicle comprises an inactive ingredient that does not exhibit cell penetration. In some embodiments, the non-carrier vehicle comprises a buffer, such as PBS. In some embodiments, the non-carrier vehicle is a solvent, non-aqueous solvent, diluent, suspending agent, surfactant, isotonic agent, thickening agent, emulsifying agent, preservative, polymer, peptide, protein, cell, hyaluronidase, dispersing agent, granulating agent, disintegrating agent, binding agent, buffering agent, lubricant, or oil.

In some embodiments, the naked delivery formulation comprises a diluent. The diluent may be a liquid diluent or a solid diluent. In some embodiments, the diluent is an RNA solubilizer, buffer, or isotonic agent. Examples of RNA solubilizing agents include water, ethanol, methanol, acetone, formamide and 2-propanol. Examples of buffers include 2- (N-morpholino) ethanesulfonic acid (MES), bis-Tris, 2- [ (2-amino-2-oxoethyl) - (carboxymethyl) amino ] acetic acid (ADA), N- (2-acetamido) -2-aminoethanesulfonic Acid (ACES), piperazine-N, N' -Bis (2-ethanesulfonic acid) (PIPES), 2- [ [1, 3-dihydroxy-2- (hydroxymethyl) propan-2-yl ] amino ] ethanesulfonic acid (TES), 3- (N-morpholino) propanesulfonic acid (MOPS), 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), tris, tricine, gly-Gly, bicine or phosphate. Examples of isotonic agents include glycerol, mannitol, polyethylene glycol, propylene glycol, trehalose or sucrose.

Carrier agent

In some embodiments, the immunogenic compositions of the invention comprise cyclic polyribonucleotides and a carrier. In some embodiments, the immunogenic compositions of the invention comprise linear polyribonucleotides and a carrier.

In certain embodiments, the immunogenic composition comprises a cyclic polyribonucleotide as described herein in a vesicle or other membrane-based carrier. In certain embodiments, the immunogenic composition comprises linear polyribonucleotides as described herein in a vesicle or other membrane-based carrier.

In other embodiments, the immunogenic composition comprises a cyclic polyribonucleotide in or via a cell, vesicle, or other membrane-based carrier. In other embodiments, the immunogenic composition comprises linear polyribonucleotides in or via a cell, vesicle, or other membrane-based carrier. In one embodiment, the immunogenic composition comprises cyclic polyribonucleotides in liposomes or other similar vesicles. In one embodiment, the immunogenic composition comprises linear polyribonucleotides in liposomes or other similar vesicles. Liposomes are spherical vesicle structures composed of a lipid bilayer of one or more layers surrounding an inner aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be anionic, neutral or cationic. Liposomes are biocompatible, non-toxic, can deliver both hydrophilic and lipophilic drug molecules, avoid their cargo degradation by plasmatic enzymes, and transport their load across the biological membrane and the Blood Brain Barrier (BBB) (for reviews see, e.g., spuch and navaro, journal of Drug Delivery [ journal of drug delivery ], volume 2011, article ID 469679, page 12, 2011.doi:10.1155/2011/469679).

Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to form liposomes as drug carriers. Methods of preparing multilamellar vesicle lipids are known in the art (see, e.g., U.S. patent No. 6,693,086, the teachings of which are incorporated herein by reference for the preparation of multilamellar vesicle lipids). Although vesicle formation is spontaneous when lipid membranes are mixed with aqueous solutions, vesicle formation can also be accelerated by applying force in the form of oscillations using a homogenizer, sonicator or squeeze device (for reviews see, e.g., spuch and Navarro, journal of Drug Delivery [ J. Drug delivery ], volume 2011, article ID 469679, page 12, 2011.doi:10.1155/2011/469679). The extruded lipids may be prepared by extrusion through a filter having a reduced size, as described in Templeton et al, nature Biotech [ Nature Biotech ],15:647-652,1997, the teachings of which are incorporated herein by reference for the preparation of extruded lipids.

In certain embodiments, the immunogenic compositions of the invention comprise cyclic polyribonucleotides and lipid nanoparticles, such as the lipid nanoparticle formulations described herein. In certain embodiments, the immunogenic compositions of the invention comprise linear polyribonucleotides and lipid nanoparticles. Lipid nanoparticles are another example of a carrier that provides a biocompatible and biodegradable delivery system for a cyclic polyribonucleotide molecule as described herein. Lipid nanoparticles are another example of a carrier that provides a biocompatible and biodegradable delivery system for linear polyribonucleotide molecules as described herein. Nanostructured Lipid Carriers (NLCs) are modified Solid Lipid Nanoparticles (SLNs) that retain the properties of SLNs, improve drug stability and drug loading, and prevent drug leakage. Polymeric Nanoparticles (PNPs) are an important component of drug delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipopolymer Nanoparticles (PLNs), a novel carrier that combines liposomes and polymers, can also be used. These nanoparticles have the complementary advantage of PNP and liposomes. PLN is composed of a core-shell structure; the polymer core provides a stable structure and the phospholipid shell provides good biocompatibility. Thus, the two components improve the effective drug encapsulation, promote surface modification, and prevent leakage of water-soluble drugs. For reviews, see, for example, li et al 2017, nanomaterials [ nanomaterials ]7,122; doi 10.3390/nano7060122.

Other non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride modified phytoglycogen or glycogen type materials), protein carriers (e.g., proteins covalently linked to cyclic polyribonucleotides or proteins covalently linked to linear polyribonucleotides), or cationic carriers (e.g., cationic lipopolymers or transfection reagents). Non-limiting examples of carbohydrate carriers include phyto-octenyl succinate, phyto-glycogen beta-dextrin and anhydride modified phyto-glycogen beta-dextrin. Non-limiting examples of cationic carriers include lipofectamine (lipofectamine), polyethylenimine, poly (trimethyl imine), poly (tetramethyl imine), polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino-B-cyclodextrin, spermine, spermidine, poly (2-dimethylamino) ethyl methacrylate, poly (lysine), poly (histidine), poly (arginine), cationic gelatin, dendrimers, chitosan, 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP), N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA), 1- [2- (oleoyloxy) ethyl ] -2-oleyl-3- (2-hydroxyethyl) imidazolium chloride (DOTIM), 2, 3-dioleoyloxy-N- [2 (spermimido) ethyl ] -N, N-dimethyl-1-trifluoroammonium acetate (DOSPA), 3B- [ N- (N\N' -dimethylaminoethane) -carbamoyl ] cholesterol hydrochloride (DC-cholesterol hydrochloride), di-heptadecylaminochromatin (DOGS), N-distearyl-N, N-dimethyl ammonium bromide (DDAB), N- (1, 2-dimyristoxyprop-3-yl) -N, N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE) and N, N-dioleyl-N, N-dimethyl ammonium chloride (DODAC). Non-limiting examples of protein carriers include Human Serum Albumin (HSA), low Density Lipoprotein (LDL), high Density Lipoprotein (HDL), or globulin.

Exosomes may also be used as carriers or drug delivery vehicles for the cyclic polyribonucleotide molecules described herein. Exosomes may also be used as carriers or drug delivery vehicles for the linear polyribonucleotide molecules described herein. For review, see Ha et al, 2016, 7, acta Pharmaceutica Sinica B, journal of pharmacy, volume 6, stage 4, pages 287-296; https:// doi.org/10.1016/j.apsb.2016.02.001.

The ex vivo differentiated erythrocytes can also be used as a carrier for the cyclic polyribonucleotide molecules described herein. Ex vivo differentiated erythrocytes can also be used as carriers for the linear polyribonucleotide molecules described herein. See, for example, WO 2015073587; WO 2017123646; WO 2017123644; WO 2018102740; WO 2016183482; WO 2015153102; WO 2018151829; WO 2018009838; shi et al 2014.Proc Natl Acad Sci USA [ Proc. Natl. Acad. Sci. USA ].111 (28): 10131-10136; us patent 9,644,180; huang et al 2017.Nature Communications [ Nature communication ]8:423; shi et al 2014.Proc Natl Acad Sci USA [ Proc. Natl. Acad. Sci. USA ].111 (28): 10131-10136.

Fusion compositions such as described in WO 2018208728 may also be used as vehicles to deliver the cyclic polyribonucleotide molecules described herein. Fusion compositions such as described in WO 2018208728 may also be used as vehicles to deliver linear polyribonucleotide molecules as described herein.

Virosomes and virus-like particles (VLPs) may also be used as carriers to deliver the cyclic polyribonucleotide molecules described herein to targeted cells. Virosomes and virus-like particles (VLPs) may also be used as carriers to deliver the linear polyribonucleotide molecules described herein to targeted cells.

Plant nanovesicles and Plant Messenger Packages (PMPs), such as described in international patent publication nos. WO 2011097480, WO 2013070324, WO 2017004526 or WO 2020041784, can also be used as vehicles to deliver the cyclic polysaccharides described herein. Plant nanovesicles and Plant Messenger Packages (PMPs) can also be used as vehicles to deliver the linear polyribonucleotide molecules described herein

Microbubbles can also be used as carriers to deliver the cyclic polyribonucleotide molecules described herein. Microbubbles can also be used as carriers to deliver linear polyribonucleotides as described herein. See, for example, US 7115583; beeri, r. Et al, circulation [ cycle ] 10/1/2002; 106 1756 to 1759; bez, M. et al, nat Protoc [ Nature laboratory Manual ] month 4 of 2019; 14 (4) 1015-1026; hennit, s. Et al Adv Drug Deliv Rev [ advanced drug delivery review ]2008, 6 months, 30 days; 60 1153-1166; rychak, J.J. et al, adv Drug Deliv Rev [ advanced drug delivery overview ] month 6 2014; 72:82-93. In some embodiments, the microbubbles are albumin coated perfluorocarbon microbubbles.

Lipid nanoparticles

The compositions, methods, and delivery systems provided herein may take any suitable carrier or delivery form, including in certain embodiments Lipid Nanoparticles (LNPs). In some embodiments, the lipid nanoparticle comprises one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic or zwitterionic lipids); one or more conjugated lipids (such as PEG conjugated lipids described in table 5 of WO 2019217941 or lipids conjugated to polymers; which are incorporated herein by reference in their entirety); one or more sterols (e.g., cholesterol).

Lipids that may be used to form the nanoparticles (e.g., lipid nanoparticles) include those described in table 4, e.g., WO 2019217941 (incorporated by reference) -e.g., lipid-containing nanoparticles may comprise one or more of the lipids in table 4 of WO 2019217941. The lipid nanoparticle may comprise additional elements such as polymers, such as the polymers described in table 5 of WO 2019217941 (incorporated by reference).

In some embodiments, conjugated lipids, when present, may include one or more of the following: PEG-Diacylglycerols (DAG) (such as l- (monomethoxy-polyethylene glycol) -2, 3-dimyristoylglycerol (PEG-DMG)), PEG-Dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramides (Cer), pegylated phosphatidylethanolamine (PEG-PE), PEG succinic diacylglycerols (PEGs-DAG) (such as 4-0- (2 ',3' -di (tetradecanoyloxy) propyl-l-0- (w-methoxy (polyethoxy) ethyl) succinate (PEG-S-DMG)), PEG dialkoxypropyl carbamate, N- (carbonyl-methoxypolyethylene glycol 2000) -1, 2-distearoyl-sn-glycerol-3-phosphate ethanolamine sodium salt, as well as those described in table 2 of WO 2019051289 (incorporated by reference) and combinations of the foregoing.

In some embodiments, sterols that may be incorporated into the lipid nanoparticle include one or more of cholesterol or cholesterol derivatives, such as those in W02009/127060 or US 2010/013588 (incorporated by reference). Additional exemplary sterols include plant sterols, including those described in Eygeris et al (2020), dx.doi.org/10.1021/acs.nanolet.0c01386, which are incorporated herein by reference.

In some embodiments, the lipid particles comprise an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits aggregation of the particles, and a sterol. The amounts of these components may be varied independently to achieve the desired characteristics. For example, in some embodiments, the lipid nanoparticle comprises: an ionizable lipid in an amount of about 20mol% to about 90mol% of the total lipid (in other embodiments, it may be 20% -70% (mol), 30% -60% (mol), or 40% -50% (mol); about 50mol% to about 90 mol%) of the total lipid present in the lipid nanoparticle; a non-cationic lipid in an amount of about 5mol% to about 30mol% of the total lipid; conjugated lipids in an amount of about 0.5mol% to about 20mol% of the total lipids, and sterols in an amount of about 20mol% to about 50mol% of the total lipids. The ratio of total lipid to nucleic acid may be varied as desired. For example, the ratio of total lipid to nucleic acid (mass or weight) may be about 10:1 to about 30:1.

In some embodiments, the ratio of lipid to nucleic acid (mass/mass ratio; w/w ratio) may be in the following range: about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of lipid and nucleic acid can be adjusted to provide a desired N/P ratio, such as an N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher. Typically, the total lipid content of the lipid nanoparticle formulation may range from about 5mg/mL to about 30 mg/mL.

Some non-limiting examples of lipid compounds that can be used (e.g., in combination with other lipid components) to form lipid nanoparticles for delivering compositions described herein, such as nucleic acids (e.g., RNAs (e.g., circular polyribonucleotides, linear polyribonucleotides)) described herein include:

in some embodiments, LNP comprising formula (i) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

In some embodiments, LNP comprising formula (ii) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

In some embodiments, an LNP comprising formula (iii) is used to deliver a polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) composition described herein to a cell.

In some embodiments, LNP comprising formula (v) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

In some embodiments, LNP comprising formula (vi) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

In some embodiments, LNP comprising formula (viii) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

In some embodiments, LNP comprising formula (ix) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

Wherein the method comprises the steps of

X¹ Is O, NR¹ Or a direct bond, X² Is C2-5 alkylene, X³ Is C (=O) or a direct bond, R¹ Is H or Me, R³ Is C1-3 alkyl, R² Is C1-3 alkyl, or R² To which nitrogen atom and X are attached² Together 1-3 carbon atoms of (a) form a 4-, 5-or 6-membered ring, or X¹ Is NR¹ ，R¹ And R is² Together with the nitrogen atom to which they are attached form a 5-or 6-membered ring, or R² And R is R³ Together with the nitrogen atom to which they are attached form a 5-, 6-or 7-membered ring, Y¹ Is C2-12 alkylene, Y² Selected from the group consisting of

(in either orientation),

n is 0 to 3, R⁴ Is C1-15 alkyl, Z¹ Is a C1-6 alkylene group or a direct bond,

Z² is that

(in either orientation) or absent, provided that if Z¹ Is a direct bond, then Z² Absence of;

R⁵ is C5-9 alkyl or C6-10 alkoxy, R⁶ Is C5-9 alkyl or C6-10 alkoxy, W is methylene or a direct bond, and R⁷ H or Me, or salts thereof, provided that if R³ And R is² Is C2 alkyl, X¹ Is O, X² Is a linear C3 alkylene group, X³ C (=0), Y¹ Is a linear Ce alkylene group, (Y)² )n-R⁴ Is that

，R⁴ Is a linear C5 alkyl group, Z¹ Is C2 alkylene, Z² Absent, W is methylene, and R⁷ Is H, then R⁵ And R is⁶ Not Cx alkoxy.

In some embodiments, LNP comprising formula (xii) is used to deliver a polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) composition described herein to a cell.

In some embodiments, LNP comprising formula (xi) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

Wherein->

In some embodiments, the LNP comprises a compound of formula (xiii) and a compound of formula (xiv).

In some embodiments, LNP comprising formula (xv) is used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

In some embodiments, LNP comprising a formulation of formula (xvi) is used to deliver a polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) composition described herein to a cell.

Wherein->

In some embodiments, the lipid compound used to form the lipid nanoparticle for delivering a composition described herein, e.g., a nucleic acid described herein (e.g., RNA (e.g., cyclic polyribonucleotide, linear polyribonucleotide)), is made by one of the following reactions:

in some embodiments, the compositions (e.g., nucleic acids or proteins) described herein are provided in an LNP comprising an ionizable lipid. In some embodiments, the ionizable lipid is heptadec-9-yl 8- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) octanoate (SM-102); for example as described in example 1 of US 9,867,888 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is 9z,12 z) -3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyloctadeca-9, 12-dienoate (LP 01), for example, as synthesized in example 13 of WO 2015/095340 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is 9- ((4-dimethylamino) butyryl) oxy) heptadecanedioic acid di ((Z) -non-2-en-1-yl) ester (L319), e.g., as synthesized in example 7, example 8, or example 9 of US 2012/0027803 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is 1,1' - ((2- (4- (2- ((2- (bis (2-hydroxydodecylamino) ethyl) piperazin-1-yl) ethyl) azetidinediyl) bis (dodecane-2-ol) (C12-200), e.g., as synthesized in examples 14 and 16 of WO 2010/053572 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is an Imidazole Cholesterol Ester (ICE) lipid (3 s,10R,13R, 17R) -10, 13-dimethyl-17- ((R) -6-methylhept-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16,17-decatetrahydro-lH-cyclopenta [ a ] phenanthren-3-yl 3- (1H-imidazol-4-yl) propionate, such as structure (I) from WO 2020/106946 (incorporated herein by reference in its entirety).

In some embodiments, the ionizable lipid may be a cationic lipid, an ionizable cationic lipid, such as a cationic lipid that may exist in a positively charged form or a neutral form depending on pH, or an amine-containing lipid that may be readily protonated. In some embodiments, the cationic lipid is a lipid that is capable of being positively charged, for example, under physiological conditions. Exemplary cationic lipids include one or more positively charged amine groups. In some embodiments, the lipid particles comprise a cationic lipid formulated with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, and polymer conjugated lipids. In some embodiments, the cationic lipid may be an ionizable cationic lipid. Exemplary cationic lipids as disclosed herein may have an effective pKa of greater than 6.0. In embodiments, the lipid nanoparticle may comprise a second cationic lipid having an effective pKa different from (e.g., greater than) the first cationic lipid. The lipid nanoparticle may comprise 40 to 60 mole% of a cationic lipid, neutral lipid, steroid, polymer conjugated lipid, and therapeutic agent encapsulated within or associated with the lipid nanoparticle, e.g., a nucleic acid (e.g., RNA (e.g., cyclic polyribonucleotide, linear polyribonucleotide)) as described herein. In some embodiments, the nucleic acid is co-formulated with a cationic lipid. The nucleic acid may be adsorbed to the surface of an LNP (e.g., an LNP comprising a cationic lipid). In some embodiments, the nucleic acid can be encapsulated in an LNP (e.g., an LNP comprising a cationic lipid). In some embodiments, the lipid nanoparticle may comprise a targeting moiety, e.g., a targeting moiety coated with a targeting agent. In an embodiment, the LNP formulation is biodegradable. In some embodiments, lipid nanoparticles comprising one or more lipids described herein (e.g., formulas (i), (ii), (vii), and/or (ix)) encapsulate at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or 100% of the RNA molecules.

Exemplary ionizable lipids that can be used in the lipid nanoparticle formulation include, but are not limited to, those listed in table 1 of WO 2019051289, which is incorporated herein by reference. Additional exemplary lipids include, but are not limited to, one or more of the following formulas: x of US 2016/0311759; i in US 20150376115 or US 2016/0376224; i, II or III of US 20160151284; i, IA, II or IIA of US 20170210967; i-c of US 20150140070; a of US 2013/0178541; US 2013/0303587 or US 2013/01233338; US 2015/0141678I; II, III, IV or V of US 2015/0239218; i of US 2017/019904; i or II of WO 2017/117528; a of US 2012/0149894; a of US 2015/0057373; a of WO 2013/116126; a of US 2013/0090372; a of US 2013/0274523; a of US 2013/0274504; a of US 2013/0053572; a of W02013/016058; a of W02012/162210; i of US 2008/042973; i, II, III or IV of US 2012/01287870; i or II of US 2014/0200257; i, II or III of US 2015/0203446; i or III of US 2015/0005363; i, IA, IB, IC, ID, II, IIA, IIB, IIC, IID or III-XXIV of US 2014/0308304; US 2013/0338210; i, II, III or IV of W02009/132131; a of US 2012/01011478; i or XXXV of US 2012/0027796; XIV or XVII of US 2012/0058144; US 2013/0323369; i of US 2011/017125; i, II or III of US 2011/0256175; i, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US 2012/0202871; i, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV or XVI of US 2011/0076335; i or II of US 2006/008378; US 2013/012338I; i or X-A-Y-Z of US 2015/0064242; XVI, XVII or XVIII of US 2013/0022649; i, II or III of US 2013/016307; i, II or III of US 2013/016307; i or II of US 2010/0062967; I-X of US 2013/0189351; i of US 2014/0039032; v of US 2018/0028664; i of US 2016/0317458; i of US 2013/0195920; 5, 6 or 10 of US 10,221,127; III-3 of WO 2018/081480; i-5 or I-8 of WO 2020/081938; 18 or 25 of US 9,867,888; a of US 2019/0136131; II of WO 2020/219876; 1 of US 2012/0027803; OF-02 OF US 2019/0240049; 23 of US 10,086,013; cKK-E12/A6 by Miao et al (2020); c12-200 of WO 2010/053572; 7C1 of Dahlman et al (2017); 304-O13 or 503-O13 of Whitehead et al; TS-P4C2 of US 9,708,628; i of WO 2020/106946; WO 2020/106946.

In some embodiments, the ionizable lipid is MC3 (6 z,9z,28z,3 lz) -heptadecane-6, 9,28,3 l-tetraen-l 9-yl-4- (dimethylamino) butyrate (DLin-MC 3-DMA or MC 3), e.g., as described in example 9 of WO 2019051289A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is the lipid ATX-002, e.g., as described in example 10 of WO 2019051289A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is (l 3Z, l 6Z) -a, a-dimethyl-3-nonylbehenyl-l 3, l 6-dien-l-amine (compound 32), e.g., as described in example 11 of WO 2019051289A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is compound 6 or compound 22, e.g., as described in example 12 of WO 2019051289A9 (incorporated herein by reference in its entirety).

Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycerophosphate-ethanolamine, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylcholine (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl phosphatidylcholine (POPC), palmitoyl phosphatidylethanolamine (POPE), dioleoyl phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE) dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidyl ethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidyl ethanolamine (such as 16-O-dimethyl PE), l 8-l-trans-PE, l-stearoyl-2-oleoyl-phosphatidyl ethanolamine (SOPE), hydrogenated Soybean Phosphatidyl Choline (HSPC), lecithin (EPC), dioleoyl phosphatidyl serine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidyl choline (DMPC), dimyristoyl phosphatidyl glycerol (DMPG), distearoyl phosphatidyl glycerol (DSPG), bis-erucic phosphatidylcholine (DEPC), palmitoyl Oleoyl Phosphatidylglycerol (POPG), bis-elapsinyl phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lecithin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphoric acid, lysophosphatidylcholine, di-linoleoyl phosphatidylcholine, or mixtures thereof. It should be understood that other diacyl phosphatidyl choline and diacyl phosphatidyl ethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having a C10-C24 carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl or oleoyl. In certain embodiments, additional exemplary lipids include, but are not limited to, those described in Kim et al (2020) dx.doi.org/10.1021/acs.nanolet.0c01386, which are incorporated herein by reference. In some embodiments, such lipids include plant lipids (e.g., DGTS) that were found to improve liver transfection with mRNA.

Other examples of non-cationic lipids suitable for use in the lipid nanoparticle include, but are not limited to, non-phospholipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glyceryl ricinoleate, cetyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, polyethoxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramides, sphingomyelin, and the like. Other non-cationic lipids are described in WO 2017/099823 or U.S. patent publication US 2018/0028664 (the contents of which are incorporated herein by reference in their entirety).

In some embodiments, the non-cationic lipid is oleic acid or a compound of formula I, II or IV of US 2018/0028664, which is incorporated by reference in its entirety. The non-cationic lipids may comprise, for example, 0-30% (mole) of the total lipids present in the lipid nanoparticle. In some embodiments, the non-cationic lipid content is 5% -20% (mole) or 10% -15% (mole) of the total lipid present in the lipid nanoparticle. In embodiments, the molar ratio of ionizable lipid to neutral lipid is about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).

In some embodiments, the lipid nanoparticle does not comprise any phospholipids.

In some aspects, the lipid nanoparticle may further comprise a component such as a sterol to provide membrane integrity. One exemplary sterol that can be used in the lipid nanoparticle is cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogues such as 5 a-cholestanol, 53-cholestanol, cholestanyl- (2, -hydroxy) -ethyl ether, cholestanyl- (4' -hydroxy) -butyl ether and 6-ketocholestanol; nonpolar analogs such as 5 a-cholestane, cholestenone, 5 a-cholestanone, 5 p-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog, e.g., cholesteryl- (4' -hydroxy) -butyl ether. Exemplary cholesterol derivatives are described in PCT publication W02009/127060 and U.S. patent publication US 2010/013058, each of which is incorporated herein by reference in its entirety.

In some embodiments, the component that provides membrane integrity, such as sterols, may comprise 0-50% (mole) (e.g., 0-10%, 10% -20%, 20% -30%, 30% -40%, or 40% -50%) of the total lipids present in the lipid nanoparticle. In some embodiments, such components comprise 20% -50% (mole), 30% -40% (mole) of the total lipid content of the lipid nanoparticle.

In some embodiments, the lipid nanoparticle may comprise polyethylene glycol (PEG) or conjugated lipid molecules. Typically, these are used to inhibit aggregation of lipid nanoparticles and/or to provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ) -lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic Polymer Lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, such as a (methoxypolyethylene glycol) conjugated lipid.

Exemplary PEG-lipid conjugates include, but are not limited to, PEG-Diacylglycerol (DAG) (such as l- (monomethoxy-polyethylene glycol) -2, 3-dimyristoylglycerol (PEG-DMG)), PEG-Dialkoxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), pegylated phosphatidylethanolamine (PEG-PE), PEG succinic diacylglycerol (PEGs-DAG) (such as 4-0- (2 ',3' -bis (tetradecanoyloxy) propyl-l-0- (w-methoxy (polyethoxy) ethyl) succinate (PEG-S-DMG)), PEG dialkoxypropyl carbamate, N- (carbonyl-methoxypolyethylene glycol 2000) -l, 2-distearoyl-sn-glycero-3-phosphate ethanolamine sodium salt, or mixtures thereof, further exemplary PEG-lipid conjugates are for example described in US 5,885,6l3, US 6,287,59l, US 2003/7829, US/0079, US 2005/01700282, US 2008/0050028, US 2012010/01188, US 011010825, US 011012016/2012016, US 2012016/2012016, and their entirety as described herein by way of examples of such and are included herein and are incorporated by reference of examples of such in their formulas and are included herein of examples of such as PEG-20120120120120120120120157 and 2013 A compound of III-a-2, III-b-1, III-b-2 or V. In some embodiments, the PEG-lipid has formula II of US 20150376115 or US 2016/0376224 (the contents of both are incorporated herein by reference in their entirety). In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristoxypropyl, PEG-dipalmitoxypropyl, or PEG-distearyloxy propyl. The PEG-lipid may be one or more of the following: PEG-DMG, PEG-dilauryl glycerol, PEG-dipalmitoyl glycerol, PEG-distearyl glycerol, PEG-dilauryl glycerolipid amide, PEG-dimyristoyl glycerolipid amide, PEG-dipalmitoyl glycerolipid amide, PEG-distearyl glycerolipid amide, PEG-cholesterol (l- [8' - (cholest-5-en-3 [ beta ] -oxy) carboxamide-3 ',6' -dioxaoctyl ] carbamoyl- [ omega ] -methyl-poly (ethylene glycol), PEG-DMB (3, 4-ditetraalkoxybenzyl- [ omega ] -methyl-poly (ethylene glycol) ether), and 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] in some embodiments, PEG-lipid comprises PEG-DMG, 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000]. In some embodiments, PEG-lipid comprises a structure selected from:

In some embodiments, lipids conjugated to molecules other than PEG may also be used in place of PEG-lipids. For example, polyoxazoline (POZ) -lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic polymer lipid (GPL) conjugates may be used in place of or in addition to PEG-lipids.

Exemplary conjugated lipids, namely PEG-lipids, (POZ) -lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids are described in PCT and LIS patent applications listed in table 2 of WO 2019051289 A9, the contents of all of which are incorporated herein by reference in their entirety.

In some embodiments, the PEG or conjugated lipid may comprise 0-20% (mole) of the total lipid present in the lipid nanoparticle. In some embodiments, the PEG or conjugated lipid is present in an amount of 0.5% -10% or 2% -5% (mole) of the total lipid present in the lipid nanoparticle. The molar ratios of ionizable lipids, non-cationic lipids, sterols, and PEG/conjugated lipids can be varied as desired. For example, the lipid particle may comprise from 30% to 70% of the ionizable lipid by mole or total weight of the composition, from 0% to 60% cholesterol by mole or total weight of the composition, from 0% to 30% of the non-cationic lipid by mole or total weight of the composition, and from 1% to 10% conjugated lipid by mole or total weight of the composition. Preferably, the composition comprises 30% to 40% of ionizable lipids based on the moles or total weight of the composition, 40% to 50% of cholesterol based on the moles or total weight of the composition, and 10% to 20% of non-cationic lipids based on the moles or total weight of the composition. In some other embodiments, the composition is 50% -75% ionizable lipid by mole or total weight of the composition, 20% -40% cholesterol by mole or total weight of the composition, and 5% -10% non-cationic lipid by mole or total weight of the composition, and 1% -10% conjugated lipid by mole or total weight of the composition. The composition may contain 60% to 70% of ionizable lipids based on the moles or total weight of the composition, 25% to 35% of cholesterol based on the moles or total weight of the composition, and 5% to 10% of non-cationic lipids based on the moles or total weight of the composition. The composition may also contain up to 90% by mole or total weight of the composition of an ionizable lipid and from 2% to 15% by mole or total weight of the composition of a non-cationic lipid. The formulation may also be a lipid nanoparticle formulation, for example comprising 8% -30% of ionizable lipids, based on the moles or total weight of the composition, 5% -30% of non-cationic lipids, based on the moles or total weight of the composition, and 0-20% cholesterol, based on the moles or total weight of the composition; 4% -25% by mole or total weight of the composition of ionizable lipids, 4% -25% by mole or total weight of the composition of non-cationic lipids, 2% -25% by mole or total weight of the composition of cholesterol, 10% -35% by mole or total weight of the composition of conjugated lipids, and 5% by mole or total weight of the composition of cholesterol; or 2% -30% of ionizable lipids based on moles or total weight of the composition, 2% -30% of non-cationic lipids based on moles or total weight of the composition, 1% -15% of cholesterol based on moles or total weight of the composition, 2% -35% of conjugated lipids based on moles or total weight of the composition, and 1% -20% of cholesterol based on moles or total weight of the composition; or even up to 90% by moles or total weight of the composition of ionizable lipids and from 2% to 10% by moles or total weight of the composition of non-cationic lipids, or even 100% by moles or total weight of the composition of cationic lipids. In some embodiments, the lipid particle formulation comprises ionizable lipids, phospholipids, cholesterol, and pegylated lipids in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation comprises ionizable lipids, cholesterol, and pegylated lipids in a molar ratio of 60:38.5:1.5.

In some embodiments, the lipid particles comprise an ionizable lipid, a non-cationic lipid (e.g., a phospholipid), a sterol (e.g., cholesterol), and a pegylated lipid, wherein the lipid molar ratio of the ionizable lipid is in the range of 20 to 70 mole percent, targeted at 40-60 mole percent, the molar percentage of the non-cationic lipid is in the range of 0 to 30 mole percent, targeted at 0 to 15 mole percent, the molar percentage of the sterol is in the range of 20 to 70 mole percent, targeted at 30 to 50 mole percent, and the molar percentage of the pegylated lipid is in the range of 1 to 6 mole percent, targeted at 2 to 5 mole percent.

In some embodiments, the lipid particle comprises ionizable lipid/non-cationic lipid/sterol/conjugated lipid in a molar ratio of 50:10:38.5:1.5.

In one aspect, the present disclosure provides lipid nanoparticle formulations comprising phospholipids, lecithins, phosphatidylcholines, and phosphatidylethanolamines.

In some embodiments, one or more additional compounds may also be included. Those compounds may be administered alone or additional compounds may be included in the lipid nanoparticles of the present invention. In other words, the lipid nanoparticle may contain other compounds than the first nucleic acid in addition to the nucleic acid or at least the second nucleic acid. Other additional compounds may be selected from the group consisting of, without limitation: small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biological materials, or any combination thereof.

In some embodiments, the LNP comprises biodegradable ionizable lipids. In some embodiments, the LNP comprises (9 z, l2 z) -3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyloctadeca-9, l 2-dienoate, also known as 3- ((4, 4-bis (octyloxy) butanoyl) oxy) -2- (((((3- (diethylamino) propoxy) carbonyl) oxy) methyl) propyl (9 z, l2 z) -octadeca-9, l 2-dienoate) or another ionizable lipid. See, e.g., WO 2019/067992, WO/2017/173054, WO 2015/095340, and WO 2014/136086, and the lipids of the references provided therein. In some embodiments, the terms cationic and ionizable are interchangeable in the context of LNP lipids, e.g., wherein the ionizable lipid is cationic according to pH.

In some embodiments, the mean LNP diameter of the LNP formulation may be between tens and hundreds of nm, as measured by Dynamic Light Scattering (DLS). In some embodiments, the mean LNP diameter of the LNP formulation can be about 40nm to about 150nm, such as about 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, or 150nm. In some embodiments, the mean LNP diameter of the LNP formulation can be about 50nm to about 100nm, about 50nm to about 90nm, about 50nm to about 80nm, about 50nm to about 70nm, about 50nm to about 60nm, about 60nm to about 100nm, about 60nm to about 90nm, about 60nm to about 80nm, about 60nm to about 70nm, about 70nm to about 100nm, about 70nm to about 90nm, about 70nm to about 80nm, about 80nm to about 100nm, about 80nm to about 90nm, or about 90nm to about 100nm. In some embodiments, the mean LNP diameter of the LNP formulation may be about 70nm to about 100nm. In particular embodiments, the mean LNP diameter of the LNP formulation may be about 80nm. In some embodiments, the mean LNP diameter of the LNP formulation may be about 100nm. In some embodiments, the LNP formulation has an average LNP diameter ranging from about l mm to about 500mm, from about 5mm to about 200mm, from about 10mm to about 100mm, from about 20mm to about 80mm, from about 25mm to about 60mm, from about 30mm to about 55mm, from about 35mm to about 50mm, or from about 38mm to about 42mm.

In some cases, the LNP may be relatively homogeneous. The polydispersity index may be used to indicate the homogeneity of the LNP, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The polydispersity index of the LNP may be from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the LNP may be from about 0.10 to about 0.20.

The zeta potential of the LNP can be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential may describe the surface charge of the LNP. Lipid nanoparticles having a relatively low charge (positive or negative) are generally desirable because higher charged species may undesirably interact with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the LNP may be from about-10 to about +20mV, from about-10 to about +15mV, from about-10 to about +10mV, from about-10 to about +5mV, from about-10 to about 0mV, from about-10 to about-5 mV, from about-5 to about +20mV, from about-5 to about +15mV, from about-5 to about +10mV, from about-5 to about +5mV, from about-5 to about 0mV, from about 0 to about +20mV, from about 0 to about +15mV, from about 0 to about +10mV, from about 0 to about +5mV, from about +5 to about +20mV, from about +5 to about +15mV, or from about +5 to about +10mV.

Encapsulation efficiency of proteins and/or nucleic acids describes the amount of protein and/or nucleic acid that is encapsulated or otherwise associated with the LNP after preparation relative to the initial amount provided. Encapsulation efficiency is desirably high (e.g., near 100%). Encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing lipid nanoparticles before and after disruption of the lipid nanoparticles with one or more organic solvents or detergents. Anion exchange resins can be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence can be used to measure the amount of free protein and/or nucleic acid (e.g., RNA) in a solution. For the lipid nanoparticles described herein, the encapsulation efficiency of the protein and/or nucleic acid may be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

The LNP may optionally comprise one or more coatings. In some embodiments, the LNP may be formulated in a capsule, film, or tablet with a coating. Capsules, films or tablets comprising the compositions described herein may have any useful size, tensile strength, hardness or density.

Additional exemplary lipids, formulations, methods, and LNP characterizations are taught by WO 2020061457, which is incorporated herein by reference in its entirety.

In some embodiments, in vitro or ex vivo cell lipofection is performed using Lipofectamine MessengerMax (sameimer Fisher) or a TransIT-mRNA transfection reagent (Mi Lusi biosystems (Mirus Bio)). In certain embodiments, LNP is formulated using a GenVoy ILM ionizable lipid mixture (precision nanosystems (Precision NanoSystems)). In certain embodiments, LNPs are formulated using 2, 2-dioleyleneyl-4-dimethylaminoethyl- [1,3] -dioxolane (DLin-KC 2-DMA) or dioleylenemethyl-4-dimethylaminobutyrate (DLin-MC 3-DMA or MC 3), the formulation and in vivo use of which are taught in Jayaraman et al, angew Chem Int Ed Engl [ German application chemistry ]51 (34): 8529-8533 (2012) (incorporated herein by reference in its entirety).

LNP formulations optimized for delivery of CRISPR-Cas systems (e.g., cas9-gRNARNP, gRNA, cas mRNA) are described in WO 2019067992 and WO 2019067910, both incorporated by reference, and are useful for delivery of cyclic polyribonucleotides and linear polyribonucleotides described herein.

Additional specific LNP formulations useful for delivering nucleic acids (e.g., cyclic polyribonucleotides, linear polyribonucleotides) are described in US 8158601 and US 8168775, both incorporated by reference, including the formulation sold under the name ontatro used in patrician (patsiran).

Exemplary administrations of the LNP of the polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) can include about 0.1, 0.25, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, or 100mg/kg (RNA). Exemplary administration of an AAV comprising a polyribonucleotide described herein can comprise about 10¹¹ 、10¹² 、10¹³ And 10¹⁴ MOI of vg/kg.

Adjuvant

The adjuvant will enhance the immune response (humoral and/or cellular immune response) elicited in a subject (e.g., a subject to be immunized) receiving the adjuvant and/or an immunogenic composition comprising the adjuvant. In some embodiments, an adjuvant is administered to a subject (e.g., a subject to be immunized) to produce polyclonal antibodies from cyclic polyribonucleotides as disclosed herein. In some embodiments, an adjuvant is administered to a subject to produce polyclonal antibodies from linear polyribonucleotides as disclosed herein. In some embodiments, an adjuvant is used in the methods described herein to produce a polyclonal antibody as described herein. In a particular embodiment, an adjuvant is used to facilitate production of polyclonal antibodies in a subject against a coronavirus antigen and/or epitope expressed by a cyclic polyribonucleotide. In some embodiments, the adjuvant and the cyclic polyribonucleotide are co-administered in separate compositions. In some embodiments, the adjuvant is mixed with the cyclic polyribonucleotide or formulated as a single composition to obtain an immunogenic composition, which is administered to a subject. In a particular embodiment, an adjuvant is used to facilitate the production of polyclonal antibodies in a subject against coronavirus antigens and/or epitopes expressed by linear polyribonucleotides. In some embodiments, the adjuvant and the linear polyribonucleotide are co-administered in separate compositions. In some embodiments, the adjuvant is mixed with the linear polyribonucleotide or formulated as a single composition to obtain an immunogenic composition, which is administered to a subject.

The adjuvant may be a TH1 adjuvant and/or a TH2 adjuvant. Preferred adjuvants include, but are not limited to, one or more of the following:

mineral-containing compositions. Mineral-containing compositions suitable for use as adjuvants in the present invention include mineral salts, such as aluminum salts and calcium salts. The invention includes mineral salts such as hydroxides (e.g., oxyhydroxide), phosphates (e.g., hydroxy phosphate, orthophosphate), sulfates, and the like, or mixtures of different mineral compounds, wherein the compounds are in any suitable form (e.g., gel, crystalline, amorphous, and the like). Calcium salts include calcium phosphates (e.g., "CAP"). Aluminum salts include hydroxides, phosphates, sulfates, and the like.

An oil emulsion composition. Oil emulsion compositions suitable for use AS adjuvants in the present invention include squalene-water emulsions such AS MF59 (5% squalene, 0.5% tween 80 and 0.5% Span, formulated AS submicron particles using a microfluidizer), AS03 (alpha-tocopherol, squalene and polysorbate 80 in oil-in-water emulsions), montanide formulations (e.g., montanide ISA51, montanide ISA 720), incomplete Freund's Adjuvant (IFA), complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA).

A small molecule. Suitable small molecules for use as adjuvants in the present invention include imiquimod or 847, remiquimod or R848, or gardimmod.

Polymer nanoparticles. Polymeric nanoparticles suitable for use as adjuvants in the present invention include poly (a-hydroxy acids), polyhydroxybutyric acid, polylactones (including polycaprolactone), polydioxanone, polypentanolactone, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine derived polycarbonates, polyvinylpyrrolidone or polyester-amides, and combinations thereof.

Saponins (i.e., glycosides, polycyclic aglycones attached to one or more sugar side chains). Saponin formulations suitable for use as adjuvants in the present invention include purified formulations such as QS21, and lipid formulations such as ISCOMs and ISCOM matrices. QS21 is marketed as STIMULON (TM). The saponin formulation may also comprise sterols, such as cholesterol. The combination of saponins and cholesterol can be used to form unique particles known as Immune Stimulating Complexes (ISCOMs). ISCOMs also typically contain a phospholipid, such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the ISCOM comprises one or more of quill, QHA and QHC. Optionally, the ISCOMs may be free of additional detergents.

Lipopolysaccharide. Adjuvants suitable for use in the present invention include non-toxic derivatives of enterobacterial Lipopolysaccharide (LPS). Such derivatives include monophosphoryl lipid A (MPLA), glucopyranosyl Lipid A (GLA) and 3-O-deacylated MPL (3 dMPL). 3dMPL is a mixture of 3 deoxy-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. Other non-toxic LPS derivatives include monophosphoryl lipid A mimics such as aminoalkyl glucosamine phosphate derivatives, e.g., RC-529.

And (3) liposome. Liposomes suitable for use as adjuvants in the present invention include virosomes and CAF01.

Lipid nanoparticles. Adjuvants suitable for use in the present invention include Lipid Nanoparticles (LNPs) and components thereof.

Lipopeptides (i.e., compounds that comprise one or more fatty acid residues and two or more amino acid residues). Lipopeptides suitable for use as adjuvants in the present invention include Pam2 (Pam 2CSK 4) and Pam3 (Pam 3CSK 4).

Glycolipids. Glycolipids suitable for use as adjuvants in the present invention include cord factors (trehalose dimycolate).

Peptides and peptidoglycans derived (synthesized or purified) from gram-negative or gram-positive bacteria, such as MDP (N-acetyl-muramyl-L-alanyl-D-isoglutamine), are suitable for use as adjuvants in the present invention.

Suitable carbohydrates (including carbohydrates) or polysaccharides for use as adjuvants include dextran (e.g., branched chain microbial polysaccharides), dextran sulfate, lentinan, zymosan, beta-glucan, dein, mannans, and chitin.

RNA-based adjuvants. RNA-based adjuvants suitable for use in the invention are poly-ICs, poly-ICs: LCs, hairpin RNAs with or without 5' -triphosphates, viral sequences, sequences containing poly-Us, natural or synthetic RNA sequences of dsRNA, and nucleic acid analogs (e.g., cyclic GMP-AMP or other cyclic dinucleotides (e.g., cyclic di-GMP), immunostimulatory base analogs (e.g., C8-substituted and N7, C8-disubstituted guanine ribonucleotides)).

DNA-based adjuvants. DNA-based adjuvants suitable for use in the present invention include CpG, dsDNA, and natural or synthetic immunostimulatory DNA sequences.

A protein or peptide. Proteins and peptides suitable for use as adjuvants in the present invention include flagellin fusion proteins, MBL (mannose binding lectin), cytokines and chemokines.

Viral particles. Suitable viral particles for use as adjuvants include virosomes (phospholipid cell membrane bilayers).

Adjuvants for use in the present invention may be of bacterial origin, such as flagellin, LPS or bacterial toxins (e.g. enterotoxins (proteins), e.g. heat labile toxins or cholera toxins). Adjuvants for use in the present invention may be hybrid molecules such as CpG conjugated to imiquimod. The adjuvant used in the present invention may be a fungal or oomycete MAMP, such as chitin or beta-glucan. In some embodiments, the adjuvant is an inorganic nanoparticle, such as a gold nanorod or a silica-based nanoparticle (e.g., a Mesoporous Silica Nanoparticle (MSN)). In some embodiments, the adjuvant is a multicomponent adjuvant or adjuvant system stabilized with a glycolipid immunomodulator (trehalose 6, 6-dibehenate (TDB), which may be a synthetic variant of a cord factor located on the cell wall of mycobacteria), such AS01, AS03, AS04 (mlp5+alum), CFA (complete freund's adjuvant: ifa+peptidoglycan or trehalose dimycolate), CAF01 (two-component system of cationic liposome vehicle (dimethyl dioctadecyl ammonium (DDA)).

A cytokine. The adjuvant may be a partial or full length DNA encoding a cytokine such as a pro-inflammatory cytokine (e.g., GM-CSF, IL-1α, IL-1β, TGF- β, TNF- α, TNF- β), a Th-1 inducing cytokine (e.g., IFN- γ, IL-2, IL-12, IL-15, IL-18), or a Th-2 inducing cytokine (e.g., IL-4, IL-5, IL-6, IL-10, IL-13).

Chemokines. The adjuvant may be partial or full length DNA encoding a chemokine, such as MCP-1, MIP-1. Alpha., MIP-1. Beta., rantes or TCA-3.

The adjuvant may be a partial or full length DNA encoding a costimulatory molecule, such as CD80, CD86, CD40-L, CD70 or CD27.

The adjuvant may be a partial or full length DNA encoding an innate immune whistle (partial, full length or mutant) or a constitutively active (ca) innate immune whistle such as callr 4, casting, caTLR3, callr 9, callr 7, callr 8, callr 7, calig-I/DDX 58 or calmda-5/IFIH 1.

The adjuvant may be a partial or full length DNA encoding an aptamer or signaling molecule such as STING, TRIF, TRAM, myD, IPS1, ASC, MAVS, MAPK, IKK- α, IKK complex, TBK1, β -catenin andcaspase 1.

The adjuvant may be a partial or full length DNA encoding a transcriptional activator, such as a transcriptional activator that up-regulates an immune response (e.g., AP1, NF- κ B, IRF3, IRF7, IRF1, or IRF 5). The adjuvant may be a partial or full length DNA encoding a cytokine receptor such as IL-2 beta, IFN-gamma or IL-6.

The adjuvant may be a partial or full length DNA encoding a bacterial component such as flagellin or MBL.

The adjuvant may be a partial or full length DNA encoding any component of the innate immune system.

In a particular embodiment, the adjuvant used in the present invention is a proprietary adjuvant formulation of SAB, SAB-adj-1 or SAB-adj-2.

Vaccine

In some embodiments of the methods described herein, a second agent is also administered to a subject (e.g., a subject to be immunized), e.g., a second vaccine is also administered to a subject (e.g., a subject to be immunized). In some embodiments, the composition administered to a subject comprises a cyclic polyribonucleotide described herein and a second vaccine. In some embodiments, the vaccine and the cyclic polyribonucleotide are co-administered in separate compositions. The vaccine is administered concurrently with the cyclic polyribonucleotide immunization, either before the cyclic polyribonucleotide immunization or after the cyclic polyribonucleotide immunization.

For example, in some embodiments, a subject (e.g., a subject to be vaccinated) is vaccinated with a non-circular polyribonucleotide coronavirus vaccine (e.g., a protein subunit vaccine) and an immunogenic composition comprising a circular polyribonucleotide. In some embodiments, the subject is vaccinated with a non-polyribonucleotide vaccine of a first microorganism (e.g., pneumococcus) and an immunogenic composition comprising a cyclic polyribonucleotide as disclosed herein. The vaccine may be any bacterial infection vaccine or viral infection vaccine. In a particular embodiment, the vaccine is a pneumococcal polysaccharide vaccine, such as PCV13 or PPSV23. In some embodiments, the vaccine is an influenza vaccine. In some embodiments, the vaccine is an RSV vaccine (e.g., palivizumab).

In some embodiments, the composition administered to a subject comprises a linear polyribonucleotide and a vaccine as described herein. In some embodiments, the vaccine and linear polyribonucleotide are co-administered in separate compositions. The vaccine is administered simultaneously with linear polyribonucleotide immunization, either before or after linear polyribonucleotide immunization.

For example, in some embodiments, a subject (e.g., a subject to be vaccinated) is vaccinated with a polyribonucleotide (e.g., a nonlinear polyribonucleotide) coronavirus vaccine (e.g., a protein subunit vaccine) and an immunogenic composition comprising a linear polyribonucleotide comprising a sequence encoding a coronavirus antigen as disclosed herein. In some embodiments, the subject is vaccinated with a non-polyribonucleotide vaccine of a first microorganism (e.g., pneumococcus) and an immunogenic composition comprising a linear polyribonucleotide comprising a sequence that encodes a coronavirus antigen as disclosed herein. The vaccine may be any bacterial infection vaccine or viral infection vaccine. In a particular embodiment, the vaccine is a pneumococcal polysaccharide vaccine, such as PCV13 or PPSV23. In some embodiments, the vaccine is an influenza vaccine. In some embodiments, the vaccine is an RSV vaccine (e.g., palivizumab).

Subjects to be immunized

The present disclosure provides for administering or immunizing a subject (e.g., a subject to be immunized) with an immunogenic composition comprising a cyclic polyribonucleotide that comprises a sequence that encodes a coronavirus antigen and/or epitope. The present disclosure provides for administering or immunizing a subject (e.g., a subject to be immunized) with an immunogenic composition comprising linear polyribonucleotides that comprise a sequence that encodes a coronavirus antigen and/or epitope. In some embodiments, the subject (e.g., a subject to be immunized) is an animal. In a particular embodiment, the subject (e.g., the subject to be immunized) is a mammal. In certain embodiments, the subject (e.g., a subject to be immunized) is a human. In some embodiments, the subject (e.g., the subject to be immunized) is a non-human animal. In some embodiments, the non-human animal has a humanized immune system. The subject's plasma or blood is used to generate hyperimmune plasma, e.g., plasma having a high concentration of antibodies that bind to the target coronavirus antigen and/or epitope.

Non-human animals to be immunized

The present disclosure provides for administering or immunizing an immunogenic composition comprising a cyclic polyribonucleotide comprising a sequence that encodes a coronavirus antigen and/or epitope to a non-human animal (e.g., a non-human animal to be immunized). The present disclosure provides for administering or immunizing an immunogenic composition comprising linear polyribonucleotides comprising sequences encoding coronavirus antigens and/or epitopes to a non-human animal (e.g., a non-human animal to be immunized).

In some embodiments, the non-human animal (e.g., a non-human animal to be vaccinated) is a pet. In some embodiments, the non-human animal (e.g., a non-human animal to be vaccinated) is a livestock animal. In some embodiments, the non-human animal (e.g., a non-human animal to be vaccinated) is a farm animal. In some embodiments, the non-human animal (e.g., the non-human animal to be vaccinated) is a zoo animal (e.g., tiger, lion, wolf, etc.).

In some embodiments, the non-human animal (e.g., a non-human animal to be vaccinated) is a mammal. Non-human animals (e.g., non-human animals to be vaccinated) include ungulates such as donkeys, goats, horses, cows, or pigs. Non-human animals (e.g., non-human animals to be vaccinated) also include rabbits, rats, or mice. In some embodiments, the non-human animal (e.g., the non-human animal to be vaccinated) is a bovine (bovine). In other embodiments, the non-human animal is a goat.

In some embodiments, the non-human animal (e.g., a non-human animal to be vaccinated) is a chicken.

In some embodiments, a non-human animal (e.g., a non-human animal to be immunized) has a humanized immune system and is used to produce human polyclonal antibodies.

Humanized immune system

Non-human animals with a humanized immune system (e.g., non-human animals to be immunized with a humanized immune system) include ungulates such as donkeys, goats, horses, cattle or pigs. Non-human animals with humanized immune systems also include rabbits, rats or mice. In some embodiments, the non-human animal having a humanized immune system is a bovine (bovine). In some embodiments, the non-human animal having a humanized immune system is a goat. In some embodiments, the non-human animal having a humanized immune system is a chicken.

A non-human animal having a humanized immune system (e.g., a non-human animal to be immunized having a humanized immune system) is an animal that produces human antibodies or antibody variants, fragments, and derivatives thereof. The humanized immune system comprises a humanized immunoglobulin locus, or multiple humanized immunoglobulin loci.

In some embodiments, the humanized immunoglobulin loci comprise germline sequences of a human immunoglobulin, allowing a non-human animal to produce humanized antibodies (e.g., fully human antibodies).

In some embodiments, the non-human animal of the present disclosure having a humanized immune system comprises a non-human B cell having a humanized immunoglobulin locus. The humanized immunoglobulin loci undergo VDJ recombination during B cell development, allowing the production of B cells with a variety of antigen binding specificities.

The binding specificity of antibodies is generated by the VDJ recombination process. Exons encoding antigen binding portions (variable regions) are assembled by chromosomal breaks and religation in developing B cells. Exons encoding antigen binding domains are assembled from so-called V (variable), D (diversity) and J (junction) gene segments by "cut and paste" DNA rearrangement. This process, known as V (D) J recombination, selects a pair of segments, introduces a double strand break near each segment, deletes (or in the case selected, inverts) the inserted DNA, and ligates the segments together. Rearrangement can occur in an orderly fashion, with the V segment being linked to the rearranged DJ segment after D and J linkage. This combinatorial assembly process, selecting a segment of each type from several (sometimes multiple) possibilities, is the basic engine driving antigen receptor diversity in mammals. The variability of the characteristics (loss or increase of small nucleotides) of the junctions between different segments greatly expands the diversity. This process converts the germline coding capacity into almost unlimited potential antigen binding specificity with relatively little investment in it.

In some embodiments, the non-human animal with a humanized immune system comprises a plurality of B cells with different specificities generated by VDJ recombination, e.g., of humanized immunoglobulin loci. B cells encoding B cell receptors (and antibodies) that specifically bind to antigens and/or epitopes of the present disclosure are activated upon encountering homologous antigens, e.g., upon encountering antigens and/or epitopes expressed from cyclic polyribonucleotides of the present disclosure. B cells encoding B cell receptors (and antibodies) that specifically bind to antigens and/or epitopes of the present disclosure are activated upon encountering homologous antigens, e.g., upon encountering antigens and/or epitopes expressed from linear polyribonucleotides of the present disclosure. Activated B cells produce antibodies that specifically bind to the antigens and/or epitopes of the disclosure. Activated B cells proliferate. In some embodiments, the activated non-human B cells differentiate into memory B cells and/or plasma cells. In some embodiments, activated non-human B cells undergo class switching to generate antibodies of different isotypes as disclosed herein. In some embodiments, the non-human B cells undergo somatic hypermutation to generate antibodies that bind with higher affinity to the antigen and/or epitope.

Upon immunization with one or more immunogenic compositions comprising one or more cyclic polyribonucleotides of the disclosure that express multiple antigens and/or epitopes, the multiple B cell clones react with their respective cognate antigens, resulting in the generation of polyclonal antibodies with multiple binding specificities. In some embodiments, immunization of a non-human animal of the present disclosure with one or more immunogenic compositions comprising one or more cyclic polyribonucleotides of the present disclosure activates at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 non-human B cell clones. In some embodiments, immunization of a non-human animal comprising one or more cyclic polyribonucleotides of the present disclosure with an immunogenic composition comprising at least one cyclic polyribonucleotide of the present disclosure results in the production of a polyclonal antiserum comprising antibodies that specifically bind to at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 antigens and/or epitopes of the present disclosure.

Upon immunization with one or more immunogenic compositions comprising one or more linear polyribonucleotides of the disclosure that express multiple antigens and/or epitopes, the multiple B cell clones react with their respective cognate antigens, resulting in the generation of polyclonal antibodies with multiple binding specificities. In some embodiments, immunization of a non-human animal of the present disclosure with one or more immunogenic compositions comprising one or more linear polyribonucleotides of the present disclosure activates at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 non-human B cell clones. In some embodiments, immunization of a non-human animal comprising one or more linear polyribonucleotides of the present disclosure with one or more immunogenic compositions of the present disclosure results in the production of polyclonal antisera comprising antibodies that specifically bind to at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 antigens and/or epitopes of the present disclosure.

Various techniques for modifying the genome of a non-human animal (e.g., a non-human animal to be vaccinated) can be employed to develop an animal capable of producing humanized antibodies. The non-human animal may be a transgenic animal, e.g., a transgenic animal comprising all or a majority of one or more humanized immunoglobulin loci. The non-human animal may be a transchromosomal animal, for example a non-human animal comprising a human artificial chromosome or a yeast artificial chromosome.

The humanized immunoglobulin loci may be present on a vector, such as a human artificial chromosome or a Yeast Artificial Chromosome (YAC). Human Artificial Chromosomes (HACs) comprising humanized immunoglobulin loci can be introduced into animals. The vector (e.g., HAC) may contain a germline pool of human antibody heavy chain genes (from human chromosome 14) and human antibody light chain genes (e.g., one or both of a kappa light chain gene (from human chromosome 2) and a lambda light chain gene (from human chromosome 22)). HACs can be transferred into cells of non-human animal species and transgenic animals can be produced by somatic cell nuclear transfer. Transgenic animals can also be bred to produce non-human animals comprising humanized immunoglobulin loci.

In some embodiments, the humanized immunoglobulin locus is integrated into the genome of a non-human animal. For example, techniques including homologous recombination or homology directed repair can be employed to modify an animal genome to introduce a human nucleotide sequence. Tools such as CRISPR/Cas, TALENs, and zinc finger nucleases can be used to target integration.

Methods of generating a non-human animal having a humanized immune system (e.g., a non-human animal to be immunized having a humanized immune system) have been disclosed. For example, a human artificial chromosome can be generated and transferred into a cell that contains other genomic modifications of interest (e.g., a deletion of endogenous non-human immune system genes), and the cell can be used as a nuclear donor to generate a transgenic non-human animal.

In some embodiments, the humanized immune system comprises one or more human antibody heavy chains, wherein each gene encoding an antibody heavy chain is operably linked to a class switching regulatory element. Operably linked may mean that a first DNA molecule (e.g., a heavy chain gene) is linked to a second DNA molecule (e.g., a class switching regulatory element), wherein the first and second DNA molecules are arranged such that the first DNA molecule affects the function of the second DNA molecule. The two DNA molecules may or may not be part of a single continuous DNA molecule, and may or may not be adjacent. For example, a promoter is operably linked to a transcribable DNA molecule if the promoter is capable of affecting the transcription or translation of the transcribable DNA molecule.

In some embodiments, the humanized immune system comprises one or more human antibody light chains. In some embodiments, the humanized immune system comprises one or more human antibodies in place of the light chain.

In some embodiments, the humanized immune system comprises an amino acid sequence derived from a non-human animal, e.g., a constant region, such as a heavy chain constant region or portion thereof. In some embodiments, the humanized immune system comprises IgM heavy chain constant regions from a non-human animal (e.g., igM heavy chain constant regions of ungulate origin). In some embodiments, at least one class switching regulatory element of a gene encoding the one or more human antibody heavy chains is replaced with a non-human (e.g., of ungulate origin) class switching regulatory element, e.g., to allow for antibody class switching when antibodies to the antigens and/or epitopes of the disclosure are produced in a non-human animal.

The humanized immunoglobulin loci may comprise non-human elements incorporated for compatibility with non-human animals. In some embodiments, non-human elements may be present in the humanized immunoglobulin loci to reduce recognition by any remaining elements of the non-human animal immune system. In some embodiments, immunoglobulin genes (e.g., igM) may be partially replaced with amino acid sequences from non-human animals. In some embodiments, a non-human regulatory element is present in a humanized immunoglobulin locus to facilitate expression and regulation of the locus in a non-human animal.

The humanized immunoglobulin locus may comprise a human DNA sequence. The humanized immunoglobulin loci may be codon optimized to facilitate expression of the contained genes (e.g., antibody genes) in non-human animals.

A non-human animal having a humanized immune system (e.g., a non-human animal to be immunized having a humanized immune system) may comprise or lack endogenous non-human immune system components. In some embodiments, a non-human animal having a humanized immune system may lack non-human antibodies (e.g., lack the ability to produce non-human antibodies). The non-human animal having a humanized immune system may lack, for example, one or more non-human immunoglobulin heavy chain genes, one or more non-human immunoglobulin light chain genes, or a combination thereof.

A non-human animal having a humanized immune system (e.g., a non-human animal to be immunized having a humanized immune system) may retain, for example, non-human immune cells. A non-human animal with a humanized immune system may retain non-human innate immune system components (e.g., cells, complement, antimicrobial peptides, etc.). In some embodiments, a non-human animal with a humanized immune system may retain non-human T cells. In some embodiments, a non-human animal with a humanized immune system may retain non-human B cells. In some embodiments, a non-human animal having a humanized immune system can retain non-human antigen presenting cells. In some embodiments, a non-human animal with a humanized immune system may retain non-human antibodies.

In some embodiments, the humanized immune system comprises a human innate immune protein, such as a complement protein.

In some embodiments, the humanized immune system comprises humanized T cells and/or antigen presenting cells.

In some embodiments, the compositions and methods of the present disclosure comprise T cells. For example, the cyclic polyribonucleotides of the present disclosure may comprise antigens that are recognized by B cells and T cells, and after immunization of a non-human animal with a humanized immune system, the T cells may provide T cell help, thereby increasing antibody production in the non-human animal. In another example, the linear polyribonucleotides of the present disclosure can comprise antigens that are recognized by B cells and T cells, and the T cells can provide T cell help after immunization of a non-human animal with a humanized immune system, thereby increasing antibody production in the non-human animal.

In some embodiments, a non-human animal having a humanized immune system (e.g., a non-human animal to be vaccinated having a humanized immune system) comprises any feature or any combination of features or any method of preparation as disclosed in US20170233459, which is hereby incorporated by reference in its entirety. In some embodiments, a non-human animal having a humanized immune system (e.g., a non-human animal to be vaccinated having a humanized immune system) comprises any feature or any combination of features or any method of preparation as disclosed below: kuroiwa, Y et al, nat Biotechnol [ natural biotechnology ], month 2 2009; 27 (2) 173-81; matsushita, H. Et al PLos ONE [ public science library: comprehensive ], 3 months and 6 days of 2014; 9 (3) e90383; hooper, j.w. et al Sci trans l Med [ science conversion medical ], 11, 26, 2014; 264ra162; matsushit, H. Et al, PLoS ONE [ library of public science: comprehensive 2015, 6 months and 24 days; 10 (6) e0130699; luke, t. et al Sci trans l Med [ science of scientific transformation ], month 2, 17 of 2016; 8 (326) 326ra21; dye, j. Et al, sci Rep [ science report ] month 4, 25 of 2016; 6:24897; gardner, c.et al J Virol [ journal of virology ]2017, month 6, 26; 91 (14); stein, d. Et al, antiviral Res [ Antiviral study ] 10 months 2017; 146:164-173; silver, j.n., clin select Dis [ clinical infection ] 3 months 19 days of 2018; 66 (7) 1116-1119; beigel, j.h. et al, lancet Infect Dis [ lancet infection ], month 4 of 2018; 18; (4) 410-418; luke, t. Et al, J infdis [ journal of infectious diseases ]2018, 11, 33;218 (journal 5) S636-S648, each of which is hereby incorporated by reference in its entirety.

Plasma collection

Plasma comprising polyclonal antibodies produced from an immunogenic composition comprising cyclic polyribonucleotides encoding coronavirus antigens and/or epitopes expressed by the cyclic polyribonucleotides as disclosed herein can be collected from a subject immunized with the cyclic polyribonucleotides (e.g., after immunization of a subject to be immunized). These polyclonal antibodies may be used to prevent or treat coronaviruses associated with antigens and/or epitopes expressed by the cyclic polyribonucleotides. Plasma comprising polyclonal antibodies produced by an immunogenic composition comprising linear polyribonucleotides encoding coronavirus antigens and/or epitopes expressed from linear polyribonucleotides as disclosed herein can be collected from a subject immunized with linear polyribonucleotides (e.g., after immunization of a subject to be immunized). These polyclonal antibodies may be used to prevent or treat coronaviruses associated with antigens and/or epitopes expressed by linear polyribonucleotides. Plasma may be collected via plasmapheresis. The plasma may be collected one or more times from the same subject (e.g., after immunization of the subject to be immunized), e.g., multiple times each during a given period of time after immunization, multiple times between immunizations, or any combination thereof.

The plasma may be collected from the subject (e.g., after immunization of the subject to be immunized) at any suitable time after immunization (e.g., first immunization, last immunization, or intermediate immunization). Plasma may be collected from the subject at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 15 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, or at least 30 days or more after immunization. In some embodiments, plasma is collected from the subject at up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 15 days, up to 20 days, up to 21 days, up to 22 days, up to 23 days, up to 24 days, up to 25 days, up to 26 days, up to 27 days, up to 28 days, up to 29 days, up to 30 days, up to 35 days, up to 42 days, up to 49 days, or up to 56 after immunization. In some embodiments, plasma is collected from the subject about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 days or more after immunization. In some embodiments, the composition comprises plasma collected after administration of an immunogenic composition described herein.

The plasma may be frozen (e.g., for frozen storage or transport). In some embodiments, the plasma is kept fresh, or antibodies are purified from fresh plasma.

In some embodiments, the composition comprises collected plasma. For example, the composition comprises plasma from a subject and cyclic polyribonucleotides that contain a sequence that encodes an antigen. In some embodiments, the composition comprises plasma from a subject and a cyclic polyribonucleotide comprising a sequence that encodes an antigen, and an antigen. In one example, the composition comprises plasma from a subject and linear polyribonucleotides comprising a sequence that encodes an antigen. In some embodiments, the composition comprises plasma from a subject and linear polyribonucleotides comprising a sequence that encodes an antigen, and an antigen.

Polyclonal antibody purification

The present disclosure provides polyclonal antibodies specific for a coronavirus antigen and/or epitope of the invention, and methods of treating or preventing a coronavirus-related disease or infection by administering an effective amount of the polyclonal antibodies to a subject (e.g., subject treatment). Polyclonal antibodies are produced as disclosed herein and purified after collection of plasma from a subject immunized with an immunogenic composition comprising cyclic polyribonucleotides (e.g., a subject to be immunized). Polyclonal antibodies are produced as disclosed herein and purified after collection of plasma from a subject immunized with an immunogenic composition comprising linear polyribonucleotides (e.g., a subject to be immunized).

Polyclonal antibodies are purified from plasma using techniques well known to those skilled in the art. For example, plasma pH is adjusted to 4.8 (e.g., 20% acetic acid is added dropwise), fractionated with octanoic acid at a octanoic acid/total protein ratio of 1.0, and then clarified by centrifugation (e.g., centrifugation at 10,000g for 20min at room temperature). The supernatant containing polyclonal antibodies (e.g., igG polyclonal antibodies) was neutralized to pH 7.5,0.22 μm with 1M tris, filtered, and affinity purified with an anti-human immunoglobulin specific column (e.g., an anti-human IgG light chain specific column). Polyclonal antibodies are further purified by affinity columns that specifically bind impurities (e.g., non-human antibodies from non-human animals). Polyclonal antibodies are stored in a suitable buffer, for example a sterile filtration buffer consisting of 10mM monosodium glutamate, 262mM D-sorbitol and Tween (Tween) (0.05 mg/ml) (pH 5.5). The amount and concentration of purified polyclonal antibodies were determined. HPLC size exclusion chromatography was performed to determine if aggregates or multimers were present.

In some embodiments, human polyclonal antibodies are purified from non-human animals having a humanized immune system according to Beigel, JH et al (Lancet Effect. Dis. [ Lancet infectious disease ],18:410-418 (2018), including supplementary appendix), which is incorporated herein by reference in its entirety. Briefly, humanized IgG polyclonal antibodies from non-human animals with humanized immune systems were purified using chromatography. Human IgG is separated from non-human animal IgG as a capture step using a human IgG kappa chain specific affinity column (e.g., kappa select from the general electric Healthcare group (GE Healthcare)). The human IgG kappa chain-specific affinity column specifically binds to whole human IgG with minimal cross-reaction to non-human animal IgG Fc and IgG. Additional non-human animal IgG is removed using an IgG Fc specific affinity column that specifically binds to non-human animal IgG (e.g., capto HC15 from the general electric healthcare group for bovine), which is used as a negative affinity step to specifically clean non-human animal IgG. Anion exchange chromatography steps can also be used to further reduce contaminants such as host DNA, endotoxins, igG aggregates and leached affinity ligands.

Polyclonal antibodies

Polyclonal antibodies produced as disclosed bind to coronavirus antigens and/or epitopes (e.g., SARS-CoV-2 antigen and/or epitope). These polyclonal antibodies are useful in methods of treating or preventing a coronavirus-related disease or infection (e.g., a covd-19 or SARS-CoV-2 infection), for example, the antibodies may provide protection against coronaviruses expressing these antigens and/or epitopes or similar antigens and/or epitopes.

The polyclonal antibodies of the present disclosure bind to, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, or more coronavirus antigens or epitopes.

In some embodiments, a polyclonal antibody of the disclosure binds to, for example, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 15, at most 20, at most 25, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90, at most 100, at most 120, at most 140, at most 160, at most 180, at most 200, at most 250, at most 300, at most 350, at most 400, at most 450, at most 500, or less coronavirus antigens or epitopes.

In some embodiments, the polyclonal antibodies of the disclosure bind to, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 coronavirus antigens or epitopes.

The polyclonal antibodies of the present disclosure bind to one or more epitopes from a coronavirus antigen. For example, coronavirus antigens comprise amino acid sequences that contain multiple epitopes (e.g., epitopes recognized by B cells and/or T cells), and antibody clones bind to one or more of those epitopes.

In some embodiments, the polyclonal antibodies of the disclosure bind to, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, or more epitopes from one coronavirus antigen.

In some embodiments, the polyclonal antibodies of the disclosure bind to, for example, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 15, up to 20, up to 25, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 120, up to 140, up to 160, up to 180, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, or up to 500 or less epitopes from one coronavirus antigen.

In some embodiments, the polyclonal antibodies of the disclosure bind to, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 epitopes from one coronavirus antigen.

The polyclonal antibodies of the present disclosure bind to variants of coronavirus antigens or epitopes. The variants may be naturally occurring variants (e.g., variants identified in sequence data from different coronavirus species, isolates, or quasispecies), or may be derived sequences as disclosed herein that have been generated via computer simulation (e.g., antigens or epitopes having one or more amino acid insertions, deletions, substitutions, or combinations thereof as compared to the wild-type antigen or epitope).

In some embodiments, the polyclonal antibodies of the disclosure bind to, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, or more variants of a coronavirus antigen or epitope.

In some embodiments, polyclonal antibodies of the disclosure bind to, for example, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 15, up to 20, up to 25, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 120, up to 140, up to 160, up to 180, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, up to 500, or less variants of a coronavirus antigen or epitope.

In some embodiments, the polyclonal antibodies of the disclosure bind to, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 variants of a coronavirus antigen or epitope.

In a particular embodiment, the antibodies of the present disclosure are neutralizing antibodies, non-neutralizing antibodies, or a combination thereof.

The humanized antibodies or variants, fragments and derivatives thereof may be antibodies that may be formulated for administration to humans. The humanized antibody may be a chimeric humanized antibody or a fully human antibody.

The humanized antibody may be a chimeric humanized antibody, for example, comprising an amino acid sequence derived from or having similarity to a human antibody amino acid sequence and a non-human amino acid sequence. For example, a portion of the heavy and/or light chain of a chimeric humanized antibody may be identical or similar to a corresponding sequence in a human antibody, while the remainder of the one or more chains may be non-human, e.g., identical or similar to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass. The non-human sequences may be humanized to reduce the likelihood of immunogenicity while preserving target specificity, for example, by incorporating human DNA into the gene sequence of the genes that produce antibodies in the non-human animal.

The humanized antibody may be a fully human antibody, for example, containing an amino acid sequence that is the amino acid sequence of a human antibody.

In some embodiments, the non-human animal with a humanized immune system produces only fully human antibodies.

The antibodies of the disclosure may be antibodies comprising a substantially four-chain antibody unit. A basic four-chain antibody unit may comprise two heavy (H) polypeptide sequences and two light (L) polypeptide sequences. Each heavy chain may comprise an N-terminal variable (V_H ) A region and three or four C-terminal constant regions (C_H 1、C_H 2、C_H 3 and C_H 4) A zone. Each light chain may comprise an N-terminal variable (V_L ) The region and one C-terminal constant (C_L ) A zone. The light chain variable region is aligned with the heavy chain variable region, and the light chain constant region is aligned with the first heavy chain constant region C_H1 Alignment. The heavy chain variable region and the light chain variable region pair together form a single antigen binding site. Each light chain is linked to the heavy chain by a covalent disulfide bond. Depending on the heavy chain isotype, the two heavy chains are linked to each other by one or more disulfide bonds. Each heavy and light chain may also contain regularly spaced intrachain disulfide bridges. The C-terminal constant region of the heavy chain comprises the Fc region of an antibody, which may mediate effector functions, for example, through interactions with Fc receptors or complement proteins.

Based on the amino acid sequence of the constant region, the light chain may be designated kappa or lambda. Based on the amino acid sequence of the constant region, the heavy chain can be named α, δ, ε, γ, or μ. Antibodies are classified into five immunoglobulin classes or isotypes based on heavy chain. IgA comprises an alpha heavy chain, igD comprises a delta heavy chain, igE comprises an epsilon heavy chain, igG comprises a gamma heavy chain, and IgM comprises a mu heavy chain. Antibodies of the IgG, igD and IgE classes comprise monomers of the four-chain units described above (two heavy and two light chains), whereas IgM and IgA classes may comprise multimers of four-chain units. The alpha and gamma classes are further divided into subclasses based on differences in heavy chain constant region sequences and functions. The subclasses of IgA and IgG expressed by humans include IgG1, igG2, igG3, igG4, igA1, and IgA2.

Illustrative amino acid sequences for human constant domain sequences are provided in table 4. In some embodiments, antibodies, non-human animals, or non-human B cells of the disclosure comprise a human IgG1 constant domain sequence, e.g., comprising SEQ ID No. 34 or variants, derivatives, or fragments thereof. In some embodiments, antibodies, non-human animals, or non-human B cells of the disclosure comprise a human IgG2 constant domain sequence, e.g., comprising SEQ ID No. 35 or variants, derivatives, or fragments thereof. In some embodiments, antibodies, non-human animals, or non-human B cells of the disclosure comprise a human IgG3 constant domain sequence, e.g., comprising SEQ ID No. 36 or variants, derivatives, or fragments thereof. In some embodiments, antibodies, non-human animals, or non-human B cells of the disclosure comprise a human IgG4 constant domain sequence, e.g., comprising SEQ ID No. 37 or variants, derivatives, or fragments thereof. In some embodiments, antibodies, non-human animals, or non-human B cells of the disclosure comprise a human IgE constant domain sequence, e.g., comprising SEQ ID NO 38 or a variant, derivative, or fragment thereof. In some embodiments, an antibody, non-human animal or non-human B cell of the disclosure comprises a human IgA1 constant domain sequence, e.g., comprising SEQ ID No. 39 or a variant, derivative or fragment thereof. In some embodiments, an antibody, non-human animal or non-human B cell of the disclosure comprises a human IgA2 constant domain sequence, e.g., comprising SEQ ID No. 40 or a variant, derivative or fragment thereof. In some embodiments, antibodies, non-human animals, or non-human B cells of the disclosure comprise a human IgM constant domain sequence, e.g., comprising SEQ ID NO 41 or a variant, derivative, or fragment thereof. In some embodiments, antibodies, non-human animals, or non-human B cells of the disclosure comprise a human IgD constant domain sequence, e.g., comprising SEQ ID NO. 42 or a variant, derivative, or fragment thereof.

TABLE 4 illustrative amino acid sequences of human constant domain sequences.

Antibodies of the disclosure may comprise human light chain constant domain sequences, such as kappa (IgK) or lambda (IgL) chains. In some embodiments, antibodies, non-human animals, or non-human B cells of the disclosure comprise a human IgK constant domain sequence, e.g., comprising SEQ ID NO 43 or a variant, derivative, or fragment thereof. In some embodiments, antibodies, non-human animals, or non-human B cells of the disclosure comprise a human IgL constant domain sequence, e.g., comprising SEQ ID NO 44 or a variant, derivative, or fragment thereof.

Table 5 provides example light chain constant domain sequences.

The signal peptide may result in higher protein expression and/or secretion by the cell. In some embodiments, the antibodies of the disclosure comprise a signal peptide. The signal peptidase can cleave the signal peptide from the protein, e.g., during secretion, producing a mature protein that does not include the signal peptide sequence. In some embodiments, the signal peptide is cleaved from a compound or antibody of the disclosure. In some embodiments, the mature compounds or antibodies of the present disclosure do not comprise a signal peptide.

The constant region may mediate a variety of effector functions and may minimally be involved in antigen binding. Different IgG isotypes or subclasses can be associated with different effector functions or therapeutic characteristics, for example, due to interactions with different Fc receptors and/or complement proteins. Antibodies comprising an Fc region that binds to an activated Fc receptor may be involved in antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), induction of signaling by immune receptor tyrosine-based activation motifs (ITAMs), and induction of cytokine secretion. For example, antibodies comprising an Fc region that binds to an inhibitory Fc receptor may induce signaling through an immunoreceptor tyrosine-based inhibitory motif (ITIM).

Different antibody subclasses contain different capabilities to elicit immune effector functions. For example, igG1 and IgG3 can effectively recruit complement to activate CDC, and IgG2 elicits minimal ADCC. IgG4 has a low ability to trigger immune effector functions. Modifications to the constant region may also affect antibody characteristics, for example, enhancement or decrease of Fc receptor ligation, enhancement or decrease of ADCC, enhancement or decrease of ADCP, enhancement or decrease of CDC, enhancement or decrease of signaling by ITAM, enhancement or decrease of cytokine induction, enhancement or decrease of signaling by ITIM, enhancement or decrease of half-life, or enhancement or decrease of co-engagement of antigen with Fc receptor. Modifications may include, for example, amino acid mutations, altered post-translational modifications (e.g., glycosylation), combinations of domains from different isoforms or subclasses, or combinations thereof.

Antibodies of the disclosure may comprise a constant region or Fc region selected or modified to provide suitable antibody characteristics, e.g., suitable characteristics for treating a disease or condition as disclosed herein. In some embodiments, igG1 can be used, for example, to promote inflammation, immune activation, and immune effector function to treat infection. In some embodiments, for example, igG4 may be used where antagonistic properties of antibodies with reduced immune effector function are desired (e.g., to neutralize coronavirus antigens and inhibit virus entry into cells without promoting inflammation and immune activation).

Non-limiting examples of antibody modifications and their effects are provided in table 6.

The variable (V) region may mediate antigen binding and define the specificity of a particular antibody for an antigen. The variable regions include relatively invariant sequences known as framework regions and hypervariable regions, which vary greatly in sequence between antibodies of different binding specificities. The variable region of each antibody heavy or light chain comprises four framework regions separated by three hypervariable regions. The variable regions of the heavy and light chains are folded in such a way that the hypervariable regions are in close proximity to each other to create an antigen binding site. The four framework regions are predominantly in an f 3-folded configuration, while the three hypervariable regions form loops that connect the f 3-folded structure, and in some cases form part of the f 3-folded structure.

Within the hypervariable region are amino acid residues that primarily determine the binding specificity of the antibody. Sequences comprising these residues are known as Complementarity Determining Regions (CDRs). An antigen binding site of an antibody may comprise six CDRs, three located in the hypervariable region of the light chain and three located in the hypervariable region of the heavy chain. CDRs in the light chain may be referred to as LCDR1, LCDR2, LCDR3, while CDRs in the heavy chain may be referred to as HCDR1, HCDR2, and HCDR3.

In some embodiments, antibodies of the disclosure include variants, derivatives, and antigen-binding fragments thereof. For example, non-human animals may be genetically modified to produce antibody variants, derivatives, and antigen binding fragments thereof. In some embodiments, the antibody can be a single domain antibody (sdAb), e.g., a heavy chain only antibody (HCAb) VHH, or a nanobody. Non-limiting examples of antigen binding fragments include Fab, fab ', F (ab')₂ Dimers and trimers of Fab conjugates, fv, scFv, minibodies, diabodies, triabodies and tetrabodies, and linear antibodies. Fab and Fab' are light chain V which may contain linkages via disulfide bonds_L And C_L Heavy chain V of the Domain_H AndC_H 1 domain. F (ab')₂ Two fabs or fabs' linked by disulfide bonds may be included. Fv's may comprise V held together by non-covalent interactions_H And V_L A domain. scFv (Single chain variable fragment)) Is can comprise V linked by a peptide linker_H And V_L Domain fusion proteins. For V_H And V_L Manipulation of the orientation of the domains and the linker length can be used to generate different forms of molecules, which can be monomers, dimers (diabodies), trimers (triabodies) or tetramers (tetrabodies). Minibodies are scFv-C assembled into bivalent dimers_H 3 fusion protein.

In some embodiments, the antibodies of the disclosure are anti-coronavirus antibodies produced by administering an immunogenic composition as disclosed herein to a non-human animal or human subject (e.g., a non-human animal or human subject to be immunized). In some embodiments, the plurality of antibodies of the disclosure are a plurality of anti-coronavirus polyclonal antibodies produced by immunization of a non-human animal or human subject (e.g., a non-human animal or human subject to be immunized) with an immunogenic composition as disclosed herein. In some embodiments, the anti-coronavirus antibody or the plurality of anti-coronavirus polyclonal antibodies further comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the non-human animal (e.g., a non-human animal subject to be immunized) is a non-human animal having a humanized immune system.

Pharmaceutical composition

In some embodiments, the immunogenic composition administered to a subject (e.g., a subject to be immunized) is a pharmaceutical composition. Pharmaceutical compositions contemplated by the present invention may also include pharmaceutically acceptable excipients.

The present disclosure also provides pharmaceutical compositions comprising a plurality of polyclonal antibodies or polyclonal antibody preparations against a coronavirus disclosed herein and a pharmaceutically acceptable excipient.

The pharmaceutically acceptable excipient may be a non-carrier excipient. Non-carrier excipients are used as vehicles or mediums for compositions such as the cyclic polyribonucleotides as described herein. Non-carrier excipients are used as vehicles or mediums for compositions such as linear polyribonucleotides as described herein. Non-limiting examples of non-carrier excipients include solvents, aqueous solvents, nonaqueous solvents, dispersion media, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickening agents, emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidase, dispersants, granulating agents, disintegrants, binders, buffers (e.g., phosphate Buffered Saline (PBS)), lubricants, oils, and mixtures thereof. The non-carrier vehicle may be any non-active ingredient approved by the U.S. Food and Drug Administration (FDA) and listed in the non-active ingredient database that does not exhibit cell penetration. The pharmaceutical composition may optionally comprise one or more additional active substances, for example therapeutically and/or prophylactically active substances. The pharmaceutical compositions of the present invention may be sterile and/or pyrogen-free. General considerations in the formulation and/or production of pharmaceutical formulations can be found in the following: for example, remington, the Science and Practice of Pharmacy [ Remington: pharmaceutical science and practice 21 st edition, lippincott Williams & Wilkins,2005 (incorporated herein by reference).

The pharmaceutical compositions of the present disclosure may comprise the polyclonal antibodies of the present disclosure, the cyclic polyribonucleotides of the present disclosure, or a combination thereof. The pharmaceutical compositions of the present disclosure may comprise the polyclonal antibodies of the present disclosure, the linear polyribonucleotides of the present disclosure, or a combination thereof. The pharmaceutical compositions of the present disclosure may comprise the polyclonal antibodies of the present disclosure, the cyclic polyribonucleotides of the present disclosure, the linear polyribonucleotides of the present disclosure, or a combination thereof.

In some embodiments, the pharmaceutical compositions provided herein are suitable for administration to humans. In some embodiments, a pharmaceutical composition provided herein (e.g., comprising a cyclic polyribonucleotide, a linear polyribonucleotide, or an immunogenic composition as described herein) is suitable for administration to a subject (e.g., a subject to be immunized), wherein the subject is a human. In some embodiments, the pharmaceutical compositions provided herein (e.g., comprising a plurality of polyclonal antibodies or polyclonal antibody preparations as described herein) are suitable for administration to a subject (e.g., a subject to be immunized), wherein the subject is a human.

In some embodiments, a pharmaceutical composition provided herein (e.g., comprising a cyclic polyribonucleotide, a linear polyribonucleotide, or an immunogenic composition as described herein) is suitable for administration to a subject (e.g., a subject to be immunized), wherein the subject is a non-human animal, e.g., suitable for veterinary use. Modifications to pharmaceutical compositions suitable for administration to humans in order to adapt the composition to a variety of animals are well known and a typical veterinary pharmacist may design and/or make such modifications by mere routine experimentation, if any. Subjects contemplated for administration of the pharmaceutical compositions include, but are not limited to, any animal, such as humans and/or other primates; mammals, including commercially relevant mammals, e.g., companion and livestock animals, such as cows, pigs, horses, sheep, cats, dogs, mice and/or rats; and/or birds, including birds of commercial relevance, such as parrots, poultry, chickens, ducks, geese, hens or roosters and/or turkeys; zoo animals, such as felines; non-mammalian animals such as reptiles, fish, amphibians, and the like.

In some embodiments, the pharmaceutical compositions provided herein (e.g., comprising a plurality of polyclonal antibodies or polyclonal antibody preparations as described herein) are suitable for administration to a subject (e.g., a subject to be immunized), wherein the subject is a non-human animal, e.g., suitable for veterinary use. Modifications to pharmaceutical compositions suitable for administration to humans in order to adapt the composition to a variety of animals are well known and a typical veterinary pharmacist may design and/or make such modifications by mere routine experimentation, if any. Subjects contemplated for administration of the pharmaceutical compositions include, but are not limited to, any animal, such as humans and/or other primates; mammals, including commercially relevant mammals, e.g., companion and livestock animals, such as cows, pigs, horses, sheep, cats, dogs, mice and/or rats; and/or birds, including birds of commercial relevance, such as parrots, poultry, chickens, ducks, geese, hens or roosters and/or turkeys; zoo animals, such as felines; non-mammalian animals such as reptiles, fish, amphibians, and the like.

Subjects to whom the pharmaceutical composition is contemplated to be administered (e.g., subjects to be vaccinated or subjects to be treated) include any ungulates.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known in the pharmacological arts or later developed. Generally, such a preparation method comprises the following steps: the active ingredient is combined with excipients and/or one or more other auxiliary ingredients, and the product is then separated, shaped and/or packaged, if necessary and/or desired.

The pharmaceutical composition may be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. Examples of suitable aqueous and non-aqueous compositions that may be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., an agent such as a cyclic polyribonucleotide, linear polyribonucleotide or antibody) in the required amount in an appropriate solvent such as one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) to yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The agents of the present disclosure (e.g., cyclic polyribonucleotides, linear polyribonucleotides, or antibodies) can be prepared in compositions that will avoid their rapid release, such as controlled release flat day foods, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Methods for preparing such formulations are generally known to those skilled in the art. See, e.g., sustained and Controlled Release Drug Delivery Systems [ sustained and controlled release drug delivery systems ], J.R. Robinson editions, marcel Dekker, inc. [ Marseldel, inc. ], new York [ New York ],1978. The compositions of the present disclosure may be, for example, in immediate release form or in controlled release formulations. Immediate release formulations can be formulated to allow the compound (e.g., an agent such as a cyclic polyribonucleotide, linear polyribonucleotide, or antibody) to act rapidly. Non-limiting examples of immediate release formulations include readily dissolvable formulations. The controlled release formulation may be a pharmaceutical formulation that is tailored such that the release rate and release profile of the active agent can be matched to physiological and temporal therapeutic requirements, or that has been formulated to achieve release of the active agent at a programmed rate. Non-limiting examples of controlled release formulations include granules, delayed release granules, hydrogels (e.g., of synthetic or natural origin), other gelling agents (e.g., gel-forming dietary fibers), matrix-based formulations (e.g., formulations comprising a polymeric material having at least one active ingredient dispersed therein), granules within a matrix, polymer mixtures, and clusters of granules.

Pharmaceutical formulations for administration may include aqueous solutions of the active compound (e.g., a pharmaceutical agent such as a cyclic polyribonucleotide, linear polyribonucleotide, or antibody) in water-soluble form. Suspensions of the active compounds may be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils (such as sesame oil) or synthetic fatty acid esters (such as ethyl oleate or triglycerides) or liposomes. The aqueous injection suspension may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension may also contain suitable stabilizers or agents that increase the solubility of the agent to allow for the preparation of highly concentrated solutions. The active ingredient may be in powder form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to use.

Methods of preparing compositions comprising the agents described herein include formulating the agents with one or more inert, pharmaceutically acceptable excipients or carriers to form solid, semi-solid, or liquid compositions. Solid compositions include, for example, powders, dispersible granules and cachets. Liquid compositions include, for example, solutions in which the agent is dissolved, emulsions comprising the agent, or solutions containing liposomes, micelles, or nanoparticles comprising the agent as disclosed herein. Semi-solid compositions include, for example, gels, suspensions, and creams. The composition may be in the form of a liquid solution or suspension, a solid suitable for dissolution or suspension in a liquid prior to use, or as an emulsion. These compositions may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically acceptable additives.

Non-limiting examples of dosage forms suitable for use in the present disclosure include liquids, powders, gels, nanosuspensions, nanoparticles, microgels, aqueous or oily suspensions, emulsions, and any combination thereof.

In some embodiments, the formulations of the present disclosure contain a heat stabilizer, such as a sugar or sugar alcohol, e.g., sucrose, sorbitol, glycerol, trehalose, or mannitol, or any combination thereof. In some embodiments, the stabilizing agent is a sugar. In some embodiments, the sugar is sucrose, mannitol, or trehalose.

Pharmaceutical compositions as described herein may be formulated to include, for example, a pharmaceutical excipient or carrier. The pharmaceutical carrier may be a membrane, lipid bilayer, and/or polymeric carrier, e.g., a liposome or particle (such as a nanoparticle, e.g., a lipid nanoparticle), and is delivered to a subject in need thereof (e.g., a subject to be immunized or a subject to be treated) (e.g., a human or non-human agricultural animal or livestock, e.g., bovine, canine, feline, equine, poultry) by known methods, such as via partial or complete encapsulation of the cyclic polyribonucleotide.

Delivery method

The cyclic polyribonucleotides as described herein or the pharmaceutical compositions thereof as described herein can be administered to cells in vesicles or other membrane-based vehicles as described herein. The linear polyribonucleotides as described herein or a pharmaceutical composition thereof as described herein can be administered to cells in vesicles or other membrane-based vehicles as described herein.

In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an ungulate cell. In some embodiments, the cell is an animal cell. In some embodiments, the cell is an immune cell. In some embodiments, the tissue is connective tissue, muscle tissue, nerve tissue, or epithelial tissue. In some embodiments, the tissue is an organ (e.g., liver, lung, spleen, kidney, etc.). In some embodiments, the subject (e.g., a subject to be immunized) is a mammal. In some embodiments, the subject (e.g., the subject to be immunized) is an ungulate.

In some embodiments, the pharmaceutical formulations disclosed herein may comprise: (i) Compounds disclosed herein (e.g., cyclic polyribonucleotides or antibodies); (ii) a buffer; (iii) a nonionic detergent; (iv) a tonicity agent; and (v) a stabilizer. In some embodiments, the pharmaceutical formulations disclosed herein may comprise: (i) Compounds disclosed herein (e.g., linear polyribonucleotides or antibodies); (ii) a buffer; (iii) a nonionic detergent; (iv) a tonicity agent; and (v) a stabilizer. In some embodiments, the pharmaceutical formulations disclosed herein are stable liquid pharmaceutical formulations.

Therapeutic method

The present disclosure provides compositions and methods useful as therapeutic or prophylactic agents, e.g., compositions and methods comprising antibodies useful for protecting a subject (e.g., a subject to be vaccinated or a subject to be treated) from a coronavirus infection. For example, a circular polyribonucleotide of the present disclosure can be administered to a subject (e.g., a subject to be immunized) to stimulate the production of antibodies (e.g., human polyclonal antibodies) that bind to a desired coronavirus antigen and/or epitope. The linear polyribonucleotides of the present disclosure can be administered to a subject (e.g., a subject to be immunized) to stimulate the production of antibodies (e.g., human polyclonal antibodies) that bind to a desired coronavirus antigen and/or epitope. Antibodies can be obtained from a subject (e.g., after immunization of a subject to be immunized) and formulated for administration to a subject (e.g., a subject to be treated, such as a human subject to be treated), e.g., as a therapeutic or prophylactic agent. Antibodies may provide protection against, for example, coronaviruses expressing antigens and/or epitopes. In another example, a cyclic polyribonucleotide can be administered to a human subject (e.g., a subject to be immunized) to stimulate the production of antibodies in the human subject that bind to a desired antigen/epitope. In another example, linear polyribonucleotides can be administered to a human subject (e.g., a subject to be immunized) to stimulate the production of antibodies in the human subject that bind to a desired antigen/epitope. In some embodiments, the present disclosure provides compositions for treating or preventing coronavirus infections.

Non-limiting examples of conditions and diseases that can be treated by the compositions and methods of the present disclosure include those caused by or associated with the coronaviruses disclosed herein, such as coronavirus infections. In some embodiments, the pathology is caused by or associated with SARS-CoV. In some embodiments, the pathology is caused by or associated with SARS-CoV-2. In some embodiments, the condition is 2019 coronavirus disease (covd-19). In some embodiments, the pathology is caused by or associated with MERS-CoV.

In some embodiments, polyclonal antibodies are produced by immunization of a non-human animal or human subject (e.g., a non-human animal or human subject to be immunized) with a cyclic polyribonucleotide of the disclosure, collecting plasma from the non-human animal or human subject (e.g., after immunization of the non-human animal or human subject to be immunized), and purifying the polyclonal antibodies from the plasma. In some embodiments, polyclonal antibodies are produced by immunization of a non-human animal or human subject (e.g., a non-human animal or human subject to be immunized) with a linear polyribonucleotide of the present disclosure, collecting plasma from the non-human animal or human subject (e.g., after immunization of the non-human animal or human subject to be immunized), and purifying the polyclonal antibodies from the plasma. Optionally, polyclonal antibodies purified from one or more non-human animals or human subjects (e.g., after immunization of one or more non-human animals or human subjects to be immunized), multiple polyclonal antibody samples purified from the same non-human animal or human subject (e.g., after immunization of a non-human animal or human subject to be immunized), or a combination thereof, are pooled together and administered to a subject in need thereof (e.g., a subject to be treated), e.g., a human subject in need thereof (e.g., a human subject to be treated). In some embodiments, the polyclonal antibody is formulated as a polyclonal antibody preparation, e.g., a polyclonal antibody preparation against a coronavirus. A method of producing a human polyclonal antibody preparation against a coronavirus, the method comprising (a) administering to an animal capable of producing the antibody (e.g., an animal to be immunized) an immunogenic composition comprising a polyribonucleotide (e.g., a cyclic polyribonucleotide or a linear polyribonucleotide) comprising a sequence that encodes a coronavirus antigen, (b) collecting blood or plasma from the mammal, (c) purifying the polyclonal antibody against the coronavirus from the blood or plasma, and (d) formulating the polyclonal antibody into a therapeutic or pharmaceutical preparation for human use (e.g., administration to a human subject to be treated) or a veterinary preparation for use by a non-human animal (e.g., administration to a non-human animal subject to be treated).

In some embodiments, the method further comprises monitoring whether a subject having a coronavirus infection (e.g., a subject to be treated), a subject at risk of exposure to a coronavirus infection (e.g., a subject to be treated), or a subject in need thereof, e.g., a subject to be treated) is present with a polyclonal antibody to a coronavirus antigen. In some embodiments, the monitoring is prior to and/or after administration of the polyclonal antibodies.

In practicing the methods of treatment or use provided herein, a therapeutically effective amount of a compound described herein (e.g., an agent, such as a cyclic polyribonucleotide or antibody) is administered in the form of a pharmaceutical composition to a subject (e.g., a subject to be immunized or a subject to be treated) having a disease or condition to be treated or in need of prophylaxis. In practicing the methods of treatment or use provided herein, a therapeutically effective amount of a compound described herein (e.g., an agent, such as a linear polyribonucleotide or antibody) is administered in the form of a pharmaceutical composition to a subject (e.g., a subject to be immunized or a subject to be treated) having a disease or condition to be treated or in need of prophylaxis. In some embodiments, the subject (e.g., a subject to be immunized or a subject to be treated) is a mammal, such as a human. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject (e.g., the subject to be immunized or the subject to be treated), the potency of the compound used, the characteristics of a given coronavirus, and other factors.

Methods and routes of administration

The compositions (e.g., pharmaceutical compositions) disclosed herein can be administered in therapeutically effective amounts by a variety of forms and routes, including, for example, oral or topical administration. In some embodiments, the composition may be applied by: parenteral, intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral, subarachnoid, intraocular, intrasternal, ocular, endothelial, topical, intranasal, intrapulmonary, rectal, intraarterial, intrathecal, inhalation, intralesional, intradermal, epidural, intracapsular, subcapsular, intracardiac, transtracheal, subcutaneous, subarachnoid or intraspinal administration (e.g., injection or infusion). In some embodiments, the composition may be administered by epithelial or skin mucosa lining absorption (e.g., oral mucosa, rectal and intestinal mucosa administration). In some embodiments, the composition is delivered via a variety of routes of administration.

In some embodiments, the composition is administered by intravenous infusion. In some embodiments, the composition is administered by slow continuous infusion over a long period of time (such as over 24 hours). In some embodiments, the composition is administered as an intravenous injection or a short infusion.

The pharmaceutical composition may be administered in a topical manner, e.g. via injection of the agent directly into the organ, optionally in the form of a depot or sustained release formulation or implant. The pharmaceutical composition may be provided in the form of a quick release formulation, in the form of a slow release formulation or in the form of an intermediate release formulation. The quick release form may provide immediate release. The slow release formulation may provide controlled release or delayed release. In some embodiments, a pump may be used to deliver the pharmaceutical composition. In some embodiments, pen delivery devices may be used, for example, to deliver the compositions of the present disclosure subcutaneously.

The pharmaceutical compositions provided herein can be administered in combination with other therapies, such as antiviral therapies, antibiotics, cell therapies, cytokine therapies, or anti-inflammatory agents. In some embodiments, the cyclic polyribonucleotides or antibodies described herein can be used alone or in combination with one or more therapeutic agents as a component of a mixture. In some embodiments, the linear polyribonucleotides or antibodies described herein can be used alone or in combination with one or more therapeutic agents as a component of a mixture.

Dose and frequency

The therapeutic agents described herein may be administered before, during, or after the occurrence of a disease or condition, and the time for which the therapeutic agent-containing composition is administered may vary. In some cases, the compositions can be used as a prophylactic agent and can be administered continuously to a subject (e.g., a subject to be immunized or a subject to be treated) who has a susceptibility to coronavirus or who is prone to a coronavirus-related condition or disease. Prophylactic administration may reduce the likelihood of an infection, disease, or condition occurring, or may reduce the severity of an infection, disease, or condition.

The composition may be administered to a subject (e.g., a subject to be immunized or a subject to be treated) after onset of symptoms (e.g., as soon as possible after). The composition may be administered to a subject (e.g., a subject to be immunized or a subject to be treated) after (e.g., as soon as possible after) a test result, such as a test result that provides a diagnosis, a test that indicates the presence of coronavirus in a subject (e.g., a subject to be immunized or a subject to be treated), or a test that indicates the progression of a condition (e.g., reduced blood oxygen levels). The therapeutic agent may be administered after the onset of the detected or suspected disease or condition (e.g., as soon as practicable thereafter). The therapeutic agent may be administered after potential exposure to the coronavirus (e.g., as soon as practicable thereafter), such as after the subject (e.g., the subject to be vaccinated or the subject to be treated) has been contacted with the infected subject, or after knowing that they have been contacted with an infected subject that may be infectious.

The cyclic polyribonucleotides, antibodies, or therapeutic agents described herein are administered at any desired interval. The linear polyribonucleotides, antibodies, or therapeutic agents described herein are administered at any desired interval.

The actual dosage level of the agents of the present disclosure (e.g., cyclic polyribonucleotides, linear polyribonucleotides, antibodies, or therapeutic agents) can be varied in order to obtain an amount of the agent to achieve a desired therapeutic response to a particular subject, composition, and mode of administration without toxicity to the subject (e.g., the subject to be immunized or the subject to be treated). The selected dosage level may depend on a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, the compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and past medical history of the patient being treated, and like factors well known in the medical arts.

The dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic and/or prophylactic response). For example, a single bolus may be administered, several partial doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the emergency situation of the treatment scenario. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for subjects (e.g., a subject to be immunized or a subject to be treated); each unit contains a predetermined amount of active agent calculated to produce the desired therapeutic effect, and the desired pharmaceutical carrier. The specification of the dosage unit form of the present disclosure may be determined by and directly dependent on: (a) Unique characteristics of the active agent and the particular therapeutic effect to be achieved, and (b) inherent limitations in the art of formulating such active agents for use in treating sensitivity in individuals. The dosage may be determined by reference to the plasma or local concentration of cyclic polyribonucleotides or antibodies. The dose may be determined by reference to the plasma or local concentration of linear polyribonucleotides or antibodies.

The pharmaceutical compositions described herein may be in unit dosage form suitable for single administration of precise dosages. In unit dosage forms, the formulation may be divided into unit doses containing appropriate amounts of one or more cyclic polyribonucleotides, antibodies and/or therapeutic agents. In unit dosage forms, the formulation may be divided into unit doses containing appropriate amounts of one or more linear polyribonucleotides, antibodies and/or therapeutic agents. The unit dose may be in the form of a package containing discrete amounts of the formulation. Non-limiting examples are packaged injectables, vials and ampoules. The aqueous suspension compositions disclosed herein may be packaged in single dose non-reclosable containers. Multiple doses of the reclosable container may be used, for example, with or without a preservative. The injectable formulations disclosed herein may be presented in unit dosage form, for example, in ampoules or in multi-dose containers with a preservative.

The dose may be based on the amount of agent per kilogram of body weight of the subject (e.g., the subject to be immunized or the subject to be treated). Doses of the agent (e.g., antibody) are in the range of 10-3000mg/kg, e.g., 100-2000mg/kg, e.g., 300-500 mg/kg/day for 1-10 days or 1-5 days; for example 400 mg/kg/day for 3-6 days; for example, 1 g/kg/day for 2-3 days.

A subject

A composition is provided for treating or preventing a condition disclosed herein, such as a coronavirus infection. The composition may be administered to a subject (e.g., a subject to be immunized or a subject to be treated) having a coronavirus infection or related disease or condition. The composition may be administered as a prophylactic agent to a subject having a propensity for coronavirus infection or a related disorder or susceptibility to a disease (e.g., a subject to be vaccinated or a subject to be treated) in order to reduce the likelihood of an infection, disease or condition, or to reduce the severity of an infection, disease or condition.

The subject (e.g., a subject to be immunized or a subject to be treated) may be a subject infected with a coronavirus. The subject (e.g., the subject to be immunized or the subject to be treated) may be a subject positive for the coronavirus test. The subject (e.g., a subject to be immunized or a subject to be treated) may be a subject that has been exposed to coronavirus. The subject (e.g., a subject to be immunized or a subject to be treated) may be a subject that may have been exposed to coronavirus. The subject (e.g., a subject to be immunized or a subject to be treated) may be a subject exhibiting one or more signs and/or symptoms consistent with a coronavirus infection.

In some embodiments, the subject (e.g., a subject to be immunized or a subject to be treated) is a subject at high risk of being contacted with a coronavirus of the disclosure. For example, the subject (e.g., the subject to be vaccinated or the subject to be treated) may be a health care worker, a laboratory staff, or a field first aid member who is more likely to be exposed to the coronavirus of the disclosure (e.g., SARS-CoV 2). A subject (e.g., a subject to be vaccinated or a subject to be treated) may be working at a healthcare facility, such as a hospital, a medical room, a hospitalization facility, an outpatient facility, an emergency care facility, an nursing home, an elderly care facility, or a nursing home.

In some embodiments, the subject (e.g., a subject to be immunized or a subject to be treated) is a subject at high risk of complications if infected with a coronavirus of the disclosure. For example, a subject (e.g., a subject to be vaccinated or a subject to be treated) may have a co-disease, age over 50 years, havetype 1 diabetes, type 2 diabetes, insulin resistance, or a combination thereof. In some embodiments, the subject is an immunocompromised subject. In some embodiments, the subject (e.g., a subject to be immunized or a subject to be treated) is taking an immunosuppressive drug. In some embodiments, the subject (e.g., a subject to be immunized or a subject to be treated) is a transplant recipient who is taking immunosuppressive drugs. In some embodiments, the subject (e.g., a subject to be immunized or a subject to be treated) is receiving a cancer therapy, such as chemotherapy, that may reduce immune system function.

The subject (e.g., a subject to be immunized or a subject to be treated) can be a mammal. The subject (e.g., a subject to be immunized or a subject to be treated) may be a human. The subject (e.g., a subject to be immunized or a subject to be treated) can be a non-human animal. The non-human animal may be an agricultural animal, such as a cow, pig, sheep, horse or goat; pets, such as cats or dogs; or zoo animals, such as felines.

Examples

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: circular RNA constructs

This example describes the design of novel SARS-CoV-2 Open Reading Frame (ORF) and circRNA constructs.

In this example, the design of the SARS-CoV-2ORF and the circular RNA construct encoding the SARS-CoV-2ORF is as described in Table 2.

Example 2: in vitro production of circular RNA encoding SARS-CoV-2 antigen

This example demonstrates the in vitro production of circular RNAs.

The circular RNA was designed to include one IRES, one ORF encoding the modified SARS-CoV-2 spike antigen or RBD antigen (as described in example 1), and two spacer elements flanking the IRES-ORF. Cyclization enables rolling circle translation, multiple ORFs with alternating staggered elements for discrete ORF expression and controlled protein stoichiometry, and IRES targeting RNA for ribosome entry. An exemplary diagram of a circular polyribonucleotide comprising a sequence that encodes a coronavirus antigen is shown in fig. 1.

In this example, circular RNAs are generated as follows. Unmodified linear RNA was synthesized from the DNA segments by in vitro transcription using T7 RNA polymerase. The transcribed RNA was purified using an RNA purification system (New England Biolabs) and treated with RNA5 'phosphate hydrolase (RppH) (New England Biolabs, M0356) according to the manufacturer's instructions and purified again using an RNA purification system. The RppH treated linear RNA was circularized using splint DNA. Alternatively or in addition to treatment with 5' rp ph, RNA is transcribed under conditions where GMP exceeds GTP.

The splint connection is performed as follows: circular RNA was generated by treating transcribed linear RNA and DNA splint (5'-GTTTTTCGGCTATTCCCAATAGCCGTTTTG-3') (SEQ ID NO: 47) with T4 DNA ligase 1 (New England Biolabs, M0437M). To purify the circular RNAs, the ligation mixture was resolved on 4% denaturing PAGE and the RNAs corresponding to each of the circular RNAs were excised. The excised RNA gel fragments were crushed and RNA eluted with gel elution buffer (0.5M sodium acetate, 0.1% SDS, 1mM EDTA) for one hour at 37 ℃. The eluted buffer was harvested and RNA was again eluted by adding gel elution buffer to the crushed gel and incubated for one hour. Gel fragments were removed by a centrifugal filter and RNA was precipitated with ethanol. Agarose gel electrophoresis was used as a quality control measure to verify purity and cyclization. In addition or alternatively, the circular RNA is purified by column chromatography.

Example 3: mRNA constructs

This example describes the design of a novel mRNA construct encoding the SARS-CoV-2 ORF.

In this example, a linear RNA construct encoding the SARS-CoV-2ORF was designed as described in Table 3.

Example 4: in vitro production of mRNA encoding SARS-CoV-2 antigen

This example demonstrates in vitro production of mRNA.

In this example, the designed mRNA has an ORF encoding a modified SARS-CoV-2 spike antigen or RBD as described in example 3.

In this example, the modified mRNA is prepared by in vitro transcription. Completely replacing RNA with pseudo uridine and 5-methyl-C, and using CleanCap^TM AG caps, including 5 'and 3' human α -globin UTRs, and polyadenylation. mRNA was purified by urea-PAGE, eluted in buffer (0.5M sodium acetate, 0.1% SDS, 1mM EDTA), ethanol precipitated and resuspended in RNA storage solution (Sieimer's technology (ThermoFisher Scientific), catalog number AM 7000). Agarose gel electrophoresis was used as a quality control measure to verify purity and cyclization.

Example 5: expression of secreted SARS-CoV-2 antigen from circular RNA in mammalian cells

This example demonstrates the ability to express viral antigens from circular RNAs in mammalian cells.

In this example, circular RNA encoding SARS-CoV-2RBD antigen is designed, produced and purified by the methods described herein.

Expression of circular RNA encoding RBD was tested by immunoprecipitation in combination with Western blotting (IP-Western). Briefly, RBD-encoding circular RNAs (0.1 picomoles) were transfected into BJ fibroblasts and HeLa cells (10,000 cells per well in 96-well plates) using Lipofectamine MessengerMax (sameimers, LMRNA 015). Messenger Max alone was used as a control. Supernatants were collected at 24 hours and immunoprecipitated using rabbit antibodies specific for SARS-CoV-2 RBD-spike glycoprotein (Sino Biologicals, catalog number 40592-T62) coupled to Protein G-Dai Nuoci beads (Protein G-Dynabeads) (England, 10003D) and immunoprecipitated products resolved by PAGE were detected using the same antibodies. Recombinant RBD (42 ng) immunoprecipitation was used as a control and expression of cellular proteins was quantified. Using Image Studio^TM Lite Western blot quantification software (Li-COR biosciences) quantified membrane chemiluminescence.

RBD antigen encoded by the circular RNA was detected in BJ fibroblasts and HeLa cell supernatants, whereas not in the control (fig. 3).

This example shows that SAR-CoV-2RBD antigen (which is a secreted protein) is expressed by circular RNA in mammalian cells.

Example 6: expression of non-secreted SARS-CoV-2 antigen from RNA in mammalian cells

In this example, a circular RNA or mRNA encoding SARS-CoV-2 spike antigen is designed, produced and purified by the methods described herein. Circular RNAs and mrnas were formulated in messenger max and 0.1 picomoles of circular RNA were transfected into HEK293 cells (10 000 cells per well) according to the manufacturer's instructions.

Spike antigen expression was measured at 24, 48 and 72 hours using SARS-CoV-2 spike antigen specific ELISA. To measure expression, cells were lysed in each well at the appropriate time point using lysis buffer and protease inhibitor. Cell lysates were recovered and centrifuged at 12,000rpm for 10 minutes. The supernatant was collected. In this example, a SARS-CoV-2 2019 spike antigen detection sandwich ELISA KIT (SARS-CoV-2 (2019-nCoV) spike detection ELISA KIT, manufactured by Yiqiao China Biotechnology Co., ltd., KIT 40591) was used according to the manufacturer's instructions.

Example 7: formulation of RNA for administration to non-human animals

In this example, circular RNA or mRNA encoding SARS-CoV-2RBD antigen is designed, produced and purified by the methods described herein.

After purification, the circular RNA or mRNA was formulated as follows:

A. the circular RNA or mRNA was diluted in PBS to a final concentration of 2.5 picomoles or 25 picomoles in 50. Mu.L to obtain circular RNA preparations or linear RNA preparations (not formulated).

B. The circular RNA or mRNA was formulated with a lipid vehicle (e.g., transIT (Mi Lusi Bio Inc. (Mirus Bio))) and mRNA boosting reagent (Mi Lusi Bio Inc.) according to the manufacturer's instructions (15% TransIT, 5% Boost) to obtain a final RNA concentration of 2.5 picomoles or 25 picomoles in 50. Mu.L to obtain a circular RNA formulation or a linear RNA formulation.

C. The circular RNA or mRNA is formulated with a cationic polymer (e.g., protamine). Briefly, circular RNAs or mrnas were diluted in purified water. Protamine sulfate was dissolved in ringer's lactic acid solution (4000 ng/. Mu.L). The protamine-ringer's lactic acid solution was added to the semi-circular RNA or mRNA solution while stirring until the weight ratio of RNA to protamine was 2:1. The solution was stirred for an additional 10 minutes to ensure the formation of a stable complex. The remaining circular RNA or mRNA is then added (i.e., the remaining circular RNA is added to the circular RNA solution and the remaining mRNA is added to the mRNA solution) and the solution is stirred briefly. The final concentration of the mixture (i.e., the circular RNA mixture or mRNA mixture) was adjusted using ringer's lactic acid solution to obtain a circular RNA formulation or linear RNA formulation with a final RNA concentration of 2.5 picomoles or 25 picomoles per 50 μl.

D. The lipid nanoparticle is used to formulate a circular RNA or mRNA. Briefly, the circular RNAs or mrnas were diluted in 25mM acetate buffer at ph=4 (filtered through 0.2 μm filter) to a concentration of 0.2 μg/μl. Lipid Nanoparticles (LNP) were first formulated by dissolving ionizable lipids (e.g. ALC 0315), cholesterol, DSPC and DMG-PEG2000 in ethanol at a molar ratio of 50/38.5/10/1.5mol% (filtered through a 0.2 μm sterile filter). The final ionizable lipid/RNA weight ratio was 8/1w/w. The lipid and RNA solutions were mixed in a micromixer chip using a microfluidic system with a flow ratio of 3/1 buffer/ethanol and a total flow of 1ml/min. LNP was then dialyzed in PBS at ph=7.4 for 3 hours to remove ethanol. RNA concentration and encapsulation efficiency inside LNP were measured using the Ribogreen assay. If necessary, LNP can be concentrated to the desired RNA concentration using an Amicon centrifugal filter with a cut-off of 100 kDa. The size, concentration and charge of the particles were measured using a Zetasizer Ultra (malvern panoraceae (Malvern Pananaytical)). The RNA concentration was adjusted to a final concentration of 0.1 or 0.2. Mu.g/. Mu.l with PBS. For formulations containing both RNA sequences, the RNAs were mixed either before formulation in LNP or after each RNA was formulated separately. For in vivo experiments, the final RNA formulated in LNP was filtered through a sterile 0.2 μm regenerated cellulose filter.

Example 8: administration of RNA to non-human animals

In this example, mice received injections of 50 μl each of either a circular or linear RNA formulation via a single intramuscular injection of the hind legs or a single intradermal injection of the back.

Example 9: detection of secreted antigens in blood

Blood samples (-25. Mu.L) were collected from each mouse by sub-zygomatic aspiration for analysis. Blood was collected intoEDTA tubes 0 hours, 6 hours, 24 hours, 48 hours and 7 days after circular RNA administration. Plasma was isolated by centrifugation at 1300g for 30 minutes at 4 ℃. Expression of secreted antigens was assessed using ELISA or western blot, for example, for RBD antigens, using the method described in example 5.

Example 10: detection of antibodies to antigens

This example describes how the presence of antibodies to antigens can be determined.

ELISA was used as described in Chen X et al (medRxiv, doi: doi. Org/10.1101/2020.04.06.20055475 (2020)). Briefly, SARS-CoV-2 protein in 100uL PBS per well was coated on ELISA plates overnight at 4 ℃. The ELISA plates were then blocked with blocking buffer (5% FBS plus 0.05% tween 20) for 1 hour. 10-fold dilutions of plasma were then added to 100 μl of blocking buffer per well over 1 hour. By using a container containing

After washing with 1X Phosphate Buffered Saline (PBST) of the detergent, the bound antibody was incubated with an anti-mouse IgG HRP detection antibody (invitrogen) for 30 minutes, followed by washing with PBST, then PBS, and tetramethyl benzene was added. The ELISA plate was allowed to react for 5 minutes and then quenched with 1M HCl stop buffer. Optical Density (OD) values were measured at 450 nm.

A. For antibodies against SARS-CoV-2RBD antigen, the SARS-CoV-2 protein used is SARS-CoV-2RBD (Yiqiao China Biotechnology Co., ltd., 40592-V08B).

B. For antibodies against SARS-CoV-2 spike antigen, the SARS-CoV-2 protein used is SARS-CoV-2 spike protein (Yinqiao China Biotechnology Co., ltd., 40591-V08H).

Example 11: evaluation of neutralizing antibody against SARS-CoV-2

Antibodies against SARS-CoV-2 infection were tested for neutralizing capacity using a SARS-CoV-2 virus neutralization assay. Okba NMA et al describe examples of such assays (Emerg information Dis. [ New infectious disease ], doi:10.3201/eid2607.200841 (2020)). The assay detects the production of antibodies that functionally inhibit viral infection as evidenced by a reduction in the number of viral plaques. Slight variations of this assay are described in Gauger PC and Vincent AL (at Animal Influenza Virus: methods and Protocols [ animal influenza virus: methods and protocols ], 3 rd edition, E.Spackman, pages 311-320 (2014)) and Wilson HL et AL (J.Clin. Microbiol. [ J.Clin., 55 (10): 3104-3112 (2017)). Briefly, SARS-CoV-2 virus neutralization assay determines the neutralizing capacity of mice to produce plasma containing anti-SARS-CoV-2 antibodies in response to immunization with circular RNA encoding SARS-CoV-2 antigen. Plasma from naive mice injected with vehicle alone (acyclic RNA) served as control.

Example 12: immunogenicity of SARS-CoV-2RBD antigen in mouse model

The immunogenicity of circular RNAs encoding SARS-CoV-2RBD antigens formulated with cationic polymers (e.g., protamine) was evaluated in a mouse model. The production of antibodies against SARS-CoV-2RBD antigen formulated with cationic polymers was also evaluated in a mouse model.

In this example, the circular RNA is designed to have an IRES and an ORF that encodes the SARS-CoV-2RBD antigen by the methods described herein. Unmodified linear RNA was synthesized from the DNA segment by in vitro transcription using T7 RNA polymerase with an excess of guanosine 5' -monophosphate. The transcribed RNA was purified using an RNA purification system (New England Biolabs) according to the manufacturer's instructions. Purified linear RNA was circularized using splint DNA.

Circular RNAs were generated by split ligation as follows: transcribed linear RNA and DNA splint (5'-GTTTTTCGGCTATTCCCAATAGCCGTTTTG-3') (SEQ ID NO: 47) were mixed and annealed and treated with RNA ligase. To purify the circular RNA, the ligation mixture was resolved by reverse phase chromatography. The circular RNA is selectively eluted from the linear RNA by increasing the organic content of the mobile phase. The eluted RNA was collected by fractionation and the purity of the circular RNA was determined. Selected fractions were pooled and buffer exchanged to remove mobile phase salts and solvent. Acrylamide gel electrophoresis was used as a quality control measure to verify purity and cyclization.

Purified circular RNA was diluted in purified water to a concentration of 1100 ng/. Mu.L. Protamine sulfate was dissolved in ringer's lactic acid solution (4000 ng/. Mu.L). The protamine-ringer's lactic acid solution was added to the semi-circular RNA solution while stirring until the weight ratio of RNA to protamine was 2:1. The solution was stirred for an additional 10 minutes to ensure the formation of a stable complex. The remaining circular RNA is then added (i.e., the remaining circular RNA is added to the circular RNA: protamine solution) and the solution is stirred briefly. The final concentration of the mixture (i.e., the circular RNA mixture) was adjusted using ringer's lactic acid solution to obtain a circular RNA preparation having a final RNA concentration of 2 μg or 10 μg RNA in 50 μl.

Three mice per group were vaccinated intramuscularly or intradermally onday 0 and day 21 with a 2 μg or 10 μg dose of either the circular RNA formulation or the protamine vehicle control. Each mouse was administered Addavax intramuscularly or intradermally 24 hours after the circular RNA formulation was administered onday 0 and day 21^TM Adjuvant (Innovogen) once. Addavax according to the manufacturer's instructions^TM The adjuvant was administered at 50 μl at 50% concentration in 1X PBS.

Blood was collected from each mouse by sub-zygomatic aspiration. Ondays 7, 14, 21, 23, 28, 35, 41, 49, 56, 63, 69, 77, 84, 108 and 115 after circular RNA administration, blood was collected into dry anticoagulant-free tubes. Serum was separated from whole blood by centrifugation at 1200g for 30 minutes at 4 ℃. Serum was heat-inactivated by heating at 56 ℃ for 1 hour. The presence or absence of RBD-specific IgG in each heat-inactivated serum sample was determined by enzyme-linked immunosorbent assay (ELISA). ELISA plates (MaxiSorp 442404 96 wells, nelkin (Nunc)) were used at 4deg.C with SARS-CoV-2RBD (40592-V08B; 100n, biotechnology Co., ltd., yiqiao) in 100. Mu.L PBS g) Coating overnight. Plates were then blocked with blocking buffer (TBS with 2% FBS and 0.05% tween 20) for 1 hour. Serum dilutions were then added to 100 μl of blocking buffer per well and incubated for 1 hour at room temperature. By using a container containing

After washing the detergent three times with 1 XTris buffered saline (TBS-T), the plate was incubated with anti-mouse IgG HRP detection antibody (Jackson 115-035-071) for 1 hour, followed by three washes with TBS-T, and then tetramethylbenzene (Pierce 34021) was added. The ELISA plate was allowed to react for 5 minutes and then quenched with 2N sulfuric acid. Optical Density (OD) values were measured at 450 nm.

The optical density of each serum sample was divided by the optical density of the background (RBD coated, plate incubated with secondary antibody only). Fold for each sample against background was plotted.

The results show that anti-RBD antibodies were obtained ondays 14, 21, 23, 28, 35, 41, 49, 56, 63, 69, 77, 84, 108 and 115 after injection of the circular RNA formulation (fig. 4). No anti-RBD antibodies were obtained after injection of protamine vehicle. These results also show that the circular RNA encoding RBD antigen induces antigen-specific immune responses in mice.

Similar ELISA was used to determine the presence or absence of spike-specific IgG in serum samples. ELISA plates (MaxiSorp 442404 96 wells, nelker) were coated overnight at 4℃with SARS-CoV-2 spike (40589-V08B 1;100 ng) in 100. Mu.L PBS. Plates were then blocked with blocking buffer (TBS with 2% FBS and 0.05% tween 20) for 1 hour. Serum dilutions were then added to 100 μl of blocking buffer per well and incubated for 1 hour at room temperature. By using a container containing

After washing the detergent three times with 1 XTris buffered saline (TBS-T), the plate was incubated with anti-mouse IgG HRP detection antibody (Jackson 115-035-071) for 1 hour, followed by three washes with TBS-T, and then tetramethylbenzene (Pierce 34021) was added. The ELISA plate was allowed to react for 5 minutes and then quenched with 2N sulfuric acid. Optical Density (OD) values were measured at 450 nm. />

The results showed that anti-spike antibodies were obtained 35 days after injection of the circular RNA formulation (fig. 5). No anti-spike antibodies were obtained after injection of vehicle.

On day 14 post-dose, serum antibodies were characterized using an assay that measures the relative IgG1 and IgG2a isotypes (fig. 6), and the ability of the serum antibodies to neutralize virus was characterized using a PRNT neutralization assay. The results show that intramuscular administration of 2 μg of RBD edrna with adjuvant has neutralizing capacity. Example 13: modulation of Gaussian (Gaussia) luciferase production from circular RNA in mice using timed adjuvant delivery

This example demonstrates the expression of proteins from circular RNAs in vivo while delivering an adjuvant to stimulate an immune response.

In this example, the circular RNA is designed to have an IRES and an ORF encoding a gaussian luciferase (GLuc) polypeptide. In this example, the circular RNA is produced and purified by the methods described herein. The circular RNAs were formulated as described in example 7 to obtain circular RNA formulations (e.g., transIT formulated, protamine formulated, PBS/unfused). Each circular RNA formulation was administered intramuscularly to mice as described in example 8.

Addavax was used at 0 or 24 hours to stimulate an immune response^TM Adjuvant (Ing Wig Jie) (an oil-in-water nanoemulsion based on squalene, the formulation of which is similar to that of

Adjuvant) was injected into the hind legs of mice (delivered simultaneously with the circular RNA formulation). Addavax according to manufacturer's instructions^TM The adjuvant was administered at 50 μl.

Blood samples (. About.25. Mu.L) were collected from each mouse by sub-zygomatic aspiration. Blood was collected intoEDTA tubes 0, 6, 24 and 48 hours after circular RNA administration. The activity of the gaussian luciferase, a secretase, was tested by centrifugation at 1300g for 30 minutes at 4 ℃ and using a gaussian luciferase activity assay (sammer technology pierce (Thermo Scientific Pierce)). mu.L of 1 Xgluc substrate was added to 5. Mu.L of plasma to conduct the GLuc luciferase activity assay. The plates were read immediately after mixing in a luminometer (Promega).

This example demonstrates that proteins can be successfully expressed from circular RNAs in vivo over a long period of time using the following method: (a) Intramuscular injection of TransIT formulated, protamine formulated and unfoamed circular RNA formulations, no adjuvant delivered (fig. 7) and adjuvant delivered at 0 and 24 hours (fig. 8); and (b) intradermal injection of protamine formulated circular RNA formulation, no adjuvant delivered and adjuvant delivered at 24 hours (fig. 9).

Example 14: administration of RNA encoding SARS-CoV-2 antigen to transchromosome (Tc) cattle

This example describes the generation of fully human neutralizing polyclonal antibodies to a coronavirus antigen from a circular RNA encoding the coronavirus antigen in a non-human mammal having a humanized immune system.

In this example, circular RNA or mRNA encoding SARS-CoV-2 antigen is designed, produced and purified by the methods described herein.

In this example, in one method, RNA is formulated (e.g., with a lipid carrier (e.g., transIT), with a cationic polymer (e.g., protamine), or not) as described in example 7 to obtain a first set of circular RNA preparations or a first set of linear RNA preparations. In the second method, addavax is applied^TM Adjuvants (Yingweijie Co., ltd.),

Adjuvant, complete Freund's adjuvant, AS03 or SAB-specific adjuvant formulation (SAB-adj-1) is formulated with an RNA-lipid carrier mixture or an unfulfilled RNA formulation (e.g., a circular RNA formulation or a linear RNA formulation), AS described by Beigel JH et al (Lancet select. Dis. [ Lancet infectious disease)]18:410-418 (2018)) to obtain a second set of circular or second set of linear RNA preparations having a final RNA concentration of 25 picomoles in 100 uL. For each method, a total volume of 8mL was generated, corresponding to 2 nanomolar circular or linear RNAs. The circular or linear RNA is formulated to obtain a circular or linear RNA preparation shortly before injection into an animal.

In this example, tc cattle are immunized with a circular RNA formulation (i.e., first circular RNA formulation or second circular RNA formulation), a linear RNA formulation (i.e., first circular RNA formulation or second circular RNA formulation), or a vehicle-only control (i.e., no RNA control) via intramuscular or intradermal injection.

A. Intramuscular injection: a total of 4 injections were made at each time point at the following sites: 2mL (each) of the post-aural injections were made once; and injected 2mL (each) once on both sides of the neck.

B. Intradermal injection: a total of 4 injections were made at each time point at the following sites: four 2mL injections were made into each site of the neck-shoulder boundary.

The following 8 time points were used in total: 0. weeks 3, 6, 9, 12, 15, 18 and 21.

Upon administration of the first set of RNA formulations (e.g., circular RNA formulations or linear RNA formulations), addavax will be administered^TM Adjuvants (Yingweijie Co., ltd.),

The adjuvant, complete Freund's adjuvant, SAB-adj-1 of AS03 or SAB, respectively, was administered adjacent (1-2 cm) each injection site (2 mL total) for the first 3 time points. A volume of pre-injection plasma was collected from each study Tc cattle prior to the first injection (V1) and used as a negative control. Blood samples up to 2.1% of the bovine body weight were collected via jugular venipuncture at each time point of 8, 9, 10, 11, 12 and 14 days post injection and at other time points of 60 days post final injection. Plasma was collected using an automated plasma exchange system (Autophesis C model 200, BAITE health Co.). Antigen-specific antibodies in the plasma were then assayed using an antigen-based ELISA. For antigen-specific polyclonal antibodies, human polyclonal antibodies were purified from plasma using Cohn-Oncley purification and octanoate fractionation as described in example 21) below.

Example 15: detection of secreted antigens expressed from circular RNA administered to Tc cattle

To detect expression of the SARS-CoV-2RBD antigen, secreted proteins from the circular RNA were collected from blood samples up to 2.1% of bovine body weight via jugular vein puncture ondays 1, 3, 5, 7, 14 and 21 post-injection. Plasma was collected using an automated plasma exchange system (Autophesis C model 200, BAITE health Co.). The plasma was then assayed for SARS-CoV-2RBD antigen expression. Expression of RBD antigens was assessed as described in example 5. For these assays, an anti-human IgG HRP detection antibody (invitrogen) was used.

Example 16: detection of non-secreted antigens expressed from circular RNA administered to Tc cattle

To detect the expression of SARS-CoV-2 spike antigen, a non-secreted protein from the circular RNA, tissues were harvested for analysis of protein expression. Atdays 0, 2, 5, 7 and 21 post-dose Tc cattle were sacrificed and liver, spleen and muscle (from the injection site) were harvested. Expression of spike antigens was assessed for proteins extracted from each tissue as described in example 6. In these ELISA, an anti-human IgG HRP detection antibody (invitrogen) was used instead of an anti-mouse IgG HRP detection antibody.

Example 17: production of human polyclonal antibodies specific for SARS-CoV-2 antigen from circular RNA administered to Tc cattle

To determine the presence of antibodies to SARS-CoV-2 antigen, blood samples up to 2.1% of the bovine subject's body weight were collected via jugular vein puncture ondays 8, 9, 10, 11, 12, 14, 20, 40 and 60 post-injection. Plasma was collected using an automated plasma exchange system (Autophesis C model 200, BAITE health Co.). The plasma is then assayed for antigen-specific antibodies. The presence of antibodies to SARS-CoV-2 antigen was determined as described in example 10. In these assays, an anti-human IgG HRP detection antibody (invitrogen) was used.

Example 18: generation of human neutralizing polyclonal antibodies against SARS-CoV-2 from circular RNA administered to Tc cattle

Blood samples up to 2.1% of the bovine subject's body weight were collected via jugular venipuncture at each of thetime points 8, 9, 10, 11, 12 and 14 days post injection and atother time points 60 days post final injection. Plasma was collected using an automated plasma exchange system (Autophesis C model 200, BAITE health Co.). The plasma is then assayed for antigen-specific antibodies. A SARS-CoV-2 virus neutralization assay was performed to determine the neutralizing capacity of antibodies in plasma as described in example 11.

Example 19: RNA encoding SARS-CoV-2 antigen is administered to transchromosome (Tc) cattle following adjuvant administration

In this example, circular RNA or mRNA encoding SARS-CoV-2 antigen is designed, produced and purified by the methods described herein.

The circular RNAs and mrnas were formulated with or without adjuvants as follows:

RNA (e.g., circular RNA or mRNA) and adjuvant are administered independently. RNA was formulated as described in example 7 (e.g., with a lipid carrier (e.g., transIT), with a cationic polymer (e.g., protamine), or not) to obtain a circular or linear RNA formulation. The final RNA concentration was 25 picomoles in 100. Mu.L. The total volume generated was 8mL, corresponding to 2 nanomolar circular RNAs or mrnas. The circular RNA or mRNA is formulated shortly before injection into an animal. For a total of 8 injections, a total of 64mL of circular RNA or mRNA was formulated. In this example, tc cattle are immunized via intramuscular injection or intradermal injection with a circular RNA formulation, a linear RNA formulation, or a vehicle-only control (i.e., a no RNA control).

(i) Intramuscular injection: a total of 4 injections were made at each time point at the following sites: 2mL (each) of the post-aural injections were made once; and 2mL (each) was injected once into each hind leg.

(ii) Intradermal injection: a total of 4 injections were made at each time point at the following sites: each site of the neck-shoulder boundary was injected 4 times with 2mL.

The following 8 time points were used in total: 0. weeks 3, 6, 9, 12, 15, 18 and 21. Addavax (R)^TM Adjuvants (Yingweijie Co., ltd.),

Adjuvant, complete Freund's adjuvant, AS03 or SAB in a proprietary adjuvant formulation (SAB-adj-1) was administered (2 mL total) adjacent (1-2 cm) to each vaccination site at the first 3 time points.

RNA (e.g., circular RNA or mRNA) and an adjuvant. RNA was formulated as described in example 7 (e.g., with a lipid carrier (e.g., transIT), with a cationic polymer (e.g., protamine), or not). ThenAddavax is added to^TM Adjuvants (Yingweijie Co., ltd.),

Adjuvant, complete Freund's adjuvant, AS03 or SAB in proprietary adjuvant formulation (SAB-adj-1) with RNA carrier formulation, RNA-polymer formulation or unfused RNA, AS described by Beigel JH et al (Lancet effect. Dis. [ Lancet infectious disease)]18:410-418 (2018)) to obtain a circular or linear RNA preparation with a final RNA concentration of 25 picomoles in 100 uL. The total volume generated was 8mL, corresponding to 2 nanomolar RNA. The circular RNA and mRNA are formulated shortly before injection into an animal. For a total of 8 injections, a total of 64mL of circular RNA and a total of 64mL of mRNA were formulated.

In this example, tc cattle are immunized via intramuscular injection or intradermal injection with a circular RNA formulation, a linear RNA formulation, or a vehicle-only control (i.e., a no RNA control).

A. Intramuscular injection. A total of 4 injections were made at each time point at the following sites: 2mL (each) of the post-aural injections were made once; and 2mL (each) was injected once into each hind leg.

B. Intradermal injection. A total of 4 injections were made at each time point at the following sites: each site of the neck-shoulder boundary was injected 4 times with 2mL.

Example 20: production of neutralizing polyclonal antibodies specific for SARS-CoV-2 from circular RNA in Tc goats

In this example, circular RNA or mRNA encoding SARS-CoV-2 antigen is designed, produced and purified by the methods described herein.

The circular RNAs and mrnas are formulated as described in example 7 (e.g., with a lipid carrier (e.g., transIT), with a cationic polymer (e.g., protamine), or not) to obtain a circular RNA formulation or a linear RNA formulation. The final RNA concentration was 25 picomoles in 100. Mu.L. The total volume generated was 1mL, corresponding to 0.25 nanomolar circular RNA or 0.25 nanomolar mRNA. The circular RNA and mRNA are formulated to obtain a circular RNA formulation or a linear RNA formulation shortly before injection into an animal. For a total of 4 injections, a total of 4mL of circular RNA and a total of 4mL of linear RNA were formulated.

In this example, a transchromosomal goat (Tc goat) is used, wherein a Human Artificial Chromosome (HAC) comprising a whole human immunoglobulin (Ig) gene pool of germline configuration is introduced into the genetic makeup of a domestic goat. Tc goats produced human polyclonal antibodies in their serum (see Wu H et al (Sci Rep [ scientific report ],9 (1): 366, doi: doi. Org/10.1038/s41598-018-36961-5 (2019)).

In this example, tc goats were immunized via intramuscular or intradermal injection with a circular RNA formulation, a linear RNA formulation, or a vehicle-only control (i.e., a no RNA control).

A. Intramuscular injection. A total of 2 injections were made at each time point at the following sites: the neck was injected once with 0.5mL (each).

B. Intradermal injection. A total of 2 injections were made at each time point at the following sites: one injection of 0.5mL (each) was given to the opposite side of the lower neck shoulder.

The following 4 time points were used in total: 0. 3, 6 and 9 weeks.

Addavax^TM Adjuvants (Yingweijie Co., ltd.),

Adjuvant, complete Freund's adjuvant, AS03 or SAB in a proprietary adjuvant formulation (SAB-adj-1) was administered (0.5 mL total) adjacent (1-2 cm) to each injection site at the first 3 time points.

Blood samples (40 mL) were collected via jugular vein puncture at each time point after injection 8 and 14 and atother time points 60 days after final injection. Plasma was collected using an automated plasma exchange system (Autophesis C model 200, BAITE health Co.). The plasma is then assayed for antigen-specific antibodies.

Example 21: polyclonal antibody fractionation and purification

This example describes the purification of human polyclonal antibodies from non-human mammalian plasma with a humanized immune system.

For purification of human anti-SARS-CoV-2 polyclonal antibodies from the collected plasma and subsequent use in human subjects, inactivation and removal of the protein antigen is required. In this example, human polyclonal anti-SARS-CoV-2 antibodies were purified from plasma using the Cohn-Oncley method, such as (Ofosu et al FA (Thromb. Haemost. [ thrombosis and hemostasis ],99 (5): 851-862 (2008)); buchaner A and Iber G (Biotechnol. J. [ J. Biotechnology J. ],1 (2): 148-163 (2006)), buchaner A and Curling JM (Biopharm. Process. [ biopharmaceutical Process. ], chapter 42, pages 857-876, doi: https:// doi. Org/10.1016/B978-0-08-100623-8.00043-8 (2018)). Fractions (I+) II+III obtained by the Cohn-Oncley method are collected and human polyclonal anti-SARS-CoV-2 antibodies are purified from the fractions using the method described by Lebing et al (Vox Sanguinis [ acoustic ],84 (3): 193-201 (2003)), in brief, fraction II+III is suspended in 12 volumes of water for injection (WFI) sodium (20 mM) and sodium hydroxide is adjusted to pH 5.1. Protein and protein is filtered in this step by a continuous precipitation filter aid, the membrane filtration solution is removed by a filter aid filter under a pH of the filter aid of pH 5.PH 1, and a filter membrane anion exchange membrane is carried out in the presence of a filter aid under a filter membrane filtration membrane under a continuous phase filtration buffer ofpH 1 at a depth of 5.PH 1, and a filter-phase filtration membrane is successively removed by a pH filtration membrane filter aid ofpH 1, then ANX Sepharose FF). The eluate was concentrated by ultrafiltration (BioMax 50KDa cassette, millipore) and diafiltered with WFI using the same system. The purified IgG solution was adjusted to pH 4.25, 0.2M glycine and 100mg/mL protein. Bulk IVIG is sterile filtered and used to fill 10, 50, 100 or 200mL vials. The final product was incubated at 23-27℃for 21 days to inactivate the virus and then stored at 2-8 ℃.

To examine the enrichment of IVIG, cellulose acetate electrophoresis was used. For clinical use, a purity of 95% is typical and expected to be the result of such a purification procedure.

Example 22: formulation of fully human polyclonal antibodies for treatment of human subjects

In this example, the purified antibody was formulated at neutral pH (pH 7.2) and diluted in an ionic solution containing sodium chloride. The United States Pharmacopeia (USP) grade infusion solution, 0.9% sodium chloride, was used.

Clinical formulation may be based on solution compositions comprising:

1. trehalose, sodium citrate, citric acid, polysorbate 80.

2. Sodium succinate, sucrose,polysorbate 20.

3. Sodium chloride, tromethamine, polysorbate 80.

4. Sucrose, sodium chloride, sodium phosphate,dextran 40.

Example 23: treatment of human subjects infected with SARS-CoV-2

This example describes the administration of a fully human anti-SARS-CoV-2 polyclonal antibody to a human subject having SARS-CoV-2.

In this example, an adult human subject with covd-19 was administered a single dose (400 mg/kg) of formulated polyclonal antibody intravenously by infusion. Infusion was started at a rate of 1.0mg/kg/min and increased to 1.5-2.5mg/kg/min after 20 minutes. Other suitable infusion rates known in the art may also be used.

The effect of polyclonal antibodies on covd-19 was assessed by evaluating markers for covd-19, such as viral load, serum antibody titer, body temperature change, sequential Organ Failure Assessment (SOFA) score (range 0-24, higher score indicating more severe disease), pao2/Fio2, blood general biochemical indicators, ARDS, and ventilation and in vitro membrano pulmonary oxygenation (ECMO) support, before and after infusion in human subjects.

Example 24: passive immunization of healthy human subjects against SARS-CoV-2 infection

This example describes the passive immunization of a human subject against SARS-CoV-2 infection with fully human polyclonal antibodies raised against SARS-CoV-2 in a non-human mammal having a humanized immune system.

In this example, healthy human subjects were administered single doses (400 mg/kg) of formulated polyclonal antibody or placebo (saline control) intravenously by infusion. Infusion was started at a rate of 1.0mg/kg/min and increased to 1.5-2.5mg/kg/min after 20 minutes. Other suitable infusion rates known in the art may be used. After 3 days, blood was drawn from the treated subjects and the neutralizing capacity of antibodies in plasma was tested using plaque reduction neutralization assay as described in example 11.

In this example, a serological test is performed on a human subject 14 days after administration of the formulated polyclonal antibody. Serological tests for SARS-CoV-2 are known in the art and include, for example, gonzalez JM et al, midRxiv, (doi: doi. Org/10.1101/2020.04.10.20061150 (2020)).

Example 25: prophylactic treatment of healthy human subjects

This example describes the prophylactic treatment of a human subject for SARS-CoV-2 infection with fully human polyclonal antibodies raised against SARS-CoV-2 in a non-human mammal having a humanized immune system.

For this example, purified human polyclonal antibodies against SARS-CoV-2 were obtained as described in example 21. Purified polyclonal antibodies were formulated as described in example 22 and subsequently administered to healthy human subjects as described in example 24.

After 3 days, blood was drawn from healthy human subjects administered formulated polyclonal antibodies or placebo (saline control) and the neutralizing capacity of the antibodies in plasma was tested using plaque reduction neutralization assay as described in example 11.

Example 26: prophylactic treatment of non-human primates

This example describes prophylactic treatment of non-human primate against SARS-CoV-2 infection with fully human polyclonal antibodies against SARS-CoV-2 produced in a non-human mammal having a humanized immune system.

In this example, purified human polyclonal antibodies against SARS-CoV-2 were obtained as described in example 21. Purified polyclonal antibodies were formulated as described in example 22 and subsequently administered to adult rhesus monkeys. Briefly, polyclonal antibody formulations were administered intravenously to rhesus monkeys at a dose of 10 mg/kg. As a control, polyclonal antibodies from transchromosomal cattle injected with vehicle only (acyclic RNA) were used.

Then at1X 10⁶ Dose of 50% Tissue Culture Infection (TCID)₅₀ ) With SARS-CoV-2 pairRhesus monkeys were challenged intratracheally and tested on time for body weight, body temperature, X-ray, serum sampling, nasal/laryngeal swabs and all primary tissue tests as described by Bao L et al (bioRxiv, doi: doi. Org/10.1101/2020.03.13.990226 (2020)). Samples were taken and viral loads were assessed up to 30 days post challenge.

Example 27: administering RNA encoding SARS-CoV-2 antigen to a human subject

This example describes the administration of circular RNA encoding SARS-CoV-2 antigen to a human subject.

In this example, circular RNA or mRNA encoding SARS-CoV-2 antigen is designed, produced and purified by the methods described herein.

In this example, in one method, RNA is formulated (e.g., with a lipid carrier (e.g., transIT), with a cationic polymer (e.g., protamine), with lipid nanoparticles, or not) as described in example 7 to obtain a first set of circular RNA preparations or a first set of linear RNA preparations. In the second method, addavax^TM Adjuvants (Yingweijie Co., ltd.),

The adjuvant or complete Freund's adjuvant is formulated with an RNA-lipid carrier mixture or an unformulated RNA formulation (e.g., a circular RNA formulation or a linear RNA formulation), as described by Beigel JH et al (Lancet infection. Dis. [ Lancet infectious disease)]18:410-418 (2018)) to obtain a second set of circular RNA preparations or a second set of linear RNA preparations. The circular or linear RNA is formulated to obtain a circular or linear RNA formulation shortly before injection into a human subject.

In this example, the human subject is immunized with a circular RNA formulation (i.e., a first circular RNA formulation or a second circular RNA formulation), a linear RNA formulation (i.e., a first circular RNA formulation or a second circular RNA formulation) via intramuscular or intradermal injection. The circular or linear RNA formulation is administered to the human subject at least once, at least twice, at least 3 times or more to elicit an immunogenic response in the human subject.

Example 28: expression of multiple antigens from circular RNAs in mammalian cells

This example demonstrates the expression of multiple antigens from circular RNAs in mammalian cells. An exemplary schematic of these constructs is shown in fig. 12.

Experiment 1

A first circular RNA (nucleic acid SEQ ID NO:56; amino acid SEQ ID NO: 55) encoding a SARS-CoV-2RBD antigen is designed, produced, and purified by the methods described herein. A second circular RNA (nucleic acid SEQ ID NO:54; amino acid SEQ ID NO: 53) encoding a SARS-CoV-2 spike antigen is designed, produced, and purified by the methods described herein. The first circular RNA and the second circular RNA are mixed together to obtain a mixture. The mixture (1 picomole of circular RNA each) was transfected into HeLa cells (100,000 cells per well in 24 well plates) using Lipofectamine MessengerMax (sameimer, LMRNA 015). As a control, the first and second circular RNAs were also transfected into HeLa cells alone using MessengerMax.

Expression of RBD antigen was measured at 24 hours using SARS-CoV-2RBD antigen specific ELISA. Spike antigen expression was measured by flow cytometry at 24 hours.

By transfection with this mixture, SARS-Co-V-2RBD antigen was detected in HeLa cell supernatant and SARS-CoV-2 spike antigen was detected on the cell surface of HeLa cells. By transfection with the first circular RNA, SARS-CoV-2RBD antigen was detected, but no SARS-CoV-2 spike antigen was detected. By transfection with the second circular RNA, SARS-CoV-2 spike antigen was detected, but SARS-CoV-2RBD antigen was not detected. This demonstrates that both SAR-CoV-2RBD and SARS-CoV-2 spike antigen are expressed in mammalian cells from a combined mixture of circular RNAs.

Experiment 2

A first circular RNA (nucleic acid SEQ ID NO:56; amino acid SEQ ID NO: 55) encoding a SARS-CoV-2RBD antigen is designed, produced, and purified by the methods described herein. A second circular RNA (nucleic acid SEQ ID NO:58; amino acid SEQ ID NO: 57) having an IRES and an ORF encoding a Gaussian luciferase (GLuc) polypeptide as a model antigen was designed and produced and purified by the methods described herein. The first circular RNA and the second circular RNA were separately complexed with Lipofectamine MessengerMax (LMRNA 015, sameimers company) and then mixed together to obtain a mixture. The mixture (0.1 picomolar of each circular RNA) was transfected into HeLa cells (20,000 cells per well in 96-well plates). As a control, the first and second circular RNAs were also transfected into HeLa cells alone using MessengerMax.

Expression of RBD antigen was measured at 24 hours using SARS-CoV-2RBD antigen specific ELISA. GLuc activity was measured at 24 hours using a gaussian luciferase activity assay (zemer technology pierce).

By transfection with this mixture, SARS-CoV-2RBD antigen and GLuc activity were detected in HeLa cell supernatant at 24 hours. By transfection with the first circular RNA, SARS-CoV-2RBD antigen was detected, but no GLuc activity was detected. By transfection with the second circular RNA, GLuc activity was detected, but SARS-CoV-2RBD antigen was not detected. This demonstrates that both SAR-CoV-2RBD and GLuc antigens are expressed in mammalian cells from a combined mixture of circular RNAs.

Experiment 3

A first circular RNA (nucleic acid SEQ ID NO:56; amino acid SEQ ID NO: 55) encoding a SARS-CoV-2RBD antigen is designed, produced, and purified by the methods described herein. The second circular RNA was designed to include an IRES followed by an ORF (nucleic acid SEQ ID NO:60; amino acid SEQ ID NO: 59) encoding a Hemagglutinin (HA) antigen from influenza A H1N 1A/California/07/2009, and was produced and purified by the methods described herein. The first circular RNA and the second circular RNA are mixed together to obtain a mixture. The mixture (1 picomole of circular RNA each) was transfected into HeLa cells (100,000 cells per well in 24 well plates) using Lipofectamine MessengerMax (sameimer, LMRNA 015). As a control, the first and second circular RNAs were also transfected into HeLa cells alone using MessengerMax.

Expression of RBD antigen was measured at 24 hours using SARS-CoV-2RBD antigen specific ELISA. HA antigen expression was measured at 24 hours using immunoblotting. Briefly, for immunoblotting, 24 hours after transfection, cells were lysed and western blotting was performed using influenza a H1N1 influenza HA (a/california/07/2009) monoclonal antibody (MA 5-29920 (sameifeier company)) as primary antibody and goat anti-mouse IgG H & L (HRP) as secondary antibody (Ai Bokang company (Abcam), ab 97023) to detect HA antigen. For loading control, alpha tubulin was used with alpha tubulin (DM 1A) mouse antibody (cell signaling technology company (Cell Signaling Technology), CST # 3873) as primary antibody and goat anti-mouse IgG H & L (HRP) (Ai Bokang company, ab 97023) as secondary antibody.

Both SARS-CoV-2RBD and influenza HA antigen were detected by transfection with the mixture. SARS-CoV-2RBD was detected by transfection with the first circular RNA, but no influenza HA antigen was detected. Influenza HA antigen was detected but SARS-CoV-2RBD antigen was not detected by transfection with the second circular RNA. This demonstrates that both SAR-CoV-2RBD and influenza HA antigen are expressed in mammalian cells from a combined mixture of circular RNAs.

Experiment 4

A first circular RNA encoding SARS-CoV-2 spike antigen (nucleic acid SEQ ID NO:45; amino acid SEQ ID NO: 53) is designed, produced, and purified by the methods described herein. The second circular RNA was designed to include an IRES followed by an ORF (nucleic acid SEQ ID NO:60; amino acid SEQ ID NO: 59) encoding HA from influenza A H1N 1A/California/07/2009 and was produced and purified by the methods described herein. The first circular RNA and the second circular RNA are mixed together to obtain a mixture. The mixture (1 picomole of circular RNA each) was transfected into HeLa cells (100,000 cells per well in 24 well plates) using Lipofectamine MessengerMax (sameimer, LMRNA 015). As a control, the first and second circular RNAs were also transfected into HeLa cells alone using MessengerMax.

Spike antigen expression was measured by flow cytometry at 24 hours. HA antigen expression was measured by immunoblotting at 24 hours as described in experiment 3 above.

Both SARS-CoV-2 spike antigen and influenza HA antigen were detected by transfection with the mixture. By transfection with the first circular RNA, SARS-CoV-2 spike antigen was detected, but no influenza HA antigen was detected. Influenza HA antigen was detected but SARS-CoV-2 spike antigen was not detected by transfection with the second circular RNA. This demonstrates that both SAR-CoV-2 spike antigen and influenza HA antigen are expressed in mammalian cells from a combined mixture of circular RNAs.

This example shows that multiple antigens are expressed in mammalian cells from circular RNA preparations comprising different combinations of circular RNAs.

Example 29: expression of multiple antigens from circular RNA

This example demonstrates the expression of multiple antigens from circular RNAs in mammalian cells. Exemplary schematic diagrams of these constructs are shown in fig. 10 and 11.

Experiment 1

In this example, the circular RNA is designed to include an IRES followed by an ORF encoding a GLuc polypeptide, a stop codon, a spacer, an IRES, an ORF encoding a SARS-CoV-2RBD antigen, and a stop codon. The circular RNA is produced and purified by the methods described herein. As a control, the following circular RNAs were generated as described above: (i) A circular RNA having IRES and an ORF encoding SARS-CoV-2RBD antigen; (ii) a circular RNA having an IRES and an ORF encoding a GLuc polypeptide.

Circular RNAs (0.1 picomoles) were transfected into HeLa cells (10,000 cells per well in 96-well plates) using Lipofectamine MessengerMax (sammer femil, LMRNA 015).

Expression of RBD antigen was measured at 24 hours using SARS-CoV-2RBD antigen specific ELISA. GLuc activity was measured at 24 hours using a gaussian luciferase activity assay (zemer technology pierce).

Expression of the RBD antigen was detected from circular RNA encoding SARS-CoV-2RBD antigen and GLuc polypeptide (FIG. 13A). GLuc activity was detected from circular RNAs encoding GLuc polypeptides (fig. 13B). This demonstrates that SAR-CoV-2RBD and GLuc antigens are expressed from circular RNAs encoding SARS-CoV-2RBD and GLuc antigens in mammalian cells.

Experiment 2

In this example, the circular RNA is designed to include an IRES followed by an ORF encoding the SARS-CoV-2RBD antigen, a stop codon, a spacer, an ORF encoding the Middle East Respiratory Syndrome (MERS) RBD antigen, and a stop codon. The circular RNA is produced and purified by the methods described herein.

Circular RNAs were transfected into HeLa cells (10,000 cells per well in 96-well plates) at various concentrations using Lipofectamine MessengerMax (sammer femto, LMRNA 015).

SARS-CoV-2RBD antigen expression was measured at 24 hours using SARS-CoV-2RBD antigen specific ELISA. MERS RBD antigen expression was measured at 24 hours using MERS RBD antigen specific antibodies that were able to be detected.

Example 30: immunogenicity of multiple antigens from circular RNAs in a mouse model

This example describes the expression of multiple antigens in a subject by administration of multiple circular RNA molecules.

Experiment 1

Immunogenicity of a circular RNA preparation comprising (a) circular RNA encoding SARS-CoV-2RBD antigen and (b) circular RNA encoding GLuc polypeptide as model antigen formulated in lipid nanoparticles was evaluated in a mouse model. Antibody production and GLuc activity against SARS-CoV-2RBD antigen were also evaluated in a mouse model.

A first circular RNA (nucleic acid SEQ ID NO:56; amino acid SEQ ID NO: 55) encoding a SARS-CoV-2RBD antigen is designed, produced, and purified by the methods described herein. A second circular RNA (nucleic acid SEQ ID NO:58; amino acid SEQ ID NO: 57) having an IRES and an ORF encoding a GLuc polypeptide was designed and produced and purified by the methods described herein. The first circular RNA and the second circular RNA are mixed together to obtain a mixture. The mixture was then formulated with lipid nanoparticles as described in example 7 to obtain a first circular RNA formulation. The first and second circular RNAs were formulated separately with lipid nanoparticles as also described in example 7 and then mixed together to obtain a second circular RNA formulation.

Three mice were inoculated intramuscularly with the first circular RNA formulation on day 0 (total dose 10. Mu.g RBD+10. Mu.g GLuc) and with the second circular RNA formulation on day 12 (total dose 10. Mu.g RBD+10. Mu.g GLuc). Additional mice (3 or 4 per group) were also inoculated intramuscularly onday 0 and day 12: (i) A dose of 10 μg of the first circular RNA formulated with lipid nanoparticles; (ii) A dose of 10 μg of a second circular RNA formulated with lipid nanoparticles; or (iii) PBS.

Blood was collected from each mouse by submandibular suction. Blood was collected into dry anticoagulant-free tubes 2 days and 17 days after priming with the first circular RNA formulation. Serum was separated from whole blood by centrifugation at 1200g for 30 minutes at 4 ℃. The presence or absence of RBD-specific IgG in each serum sample was determined by enzyme-linked immunosorbent assay (ELISA). ELISA plates (MaxiSorp 442404 96 wells, nelkin) were coated overnight at 4℃with 100. Mu.L of 1 Xcoating buffer (Biolegend, 421701) in SARS-CoV-2RBD (Yiqiao Biotechnology Co., ltd., 40592-V08B;100 ng). The plates were then blocked with blocking buffer (TBS with 2% BSA and 0.05% tween 20) for 1 hour. Serum dilutions (1:500, 1:1500, 1:4500 and 1:13,500) were then added to 100 μl of blocking buffer per well and incubated for 1 hour at room temperature. By using a container containing

After washing the detergent three times with 1 XTris buffered saline (TBS-T), the plate was incubated with anti-mouse IgG HRP detection antibody (Ai Bokang Co., ab 97023) for 1 hour, followed by three washes with TBS-T, and then tetramethylbenzene (BAOCHINE Co., 421101) was added. ELISA plates were allowed to react for 10-20 minutes and then quenched with 0.2N sulfuric acid. The optical density (o.d.) values were determined at 450 nm.

The optical density of each serum sample was divided by the optical density of the background (RBD coated, plate incubated with secondary antibody only). Fold for each sample against background was plotted.

GLuc activity was tested using a gaussian luciferase activity assay (zemer technology pierce). mu.L of 1 Xgluc substrate was added to 10. Mu.L of serum to conduct the GLuc luciferase activity assay. The plates were read immediately after mixing in a luminometer (Promega).

The results showed that anti-RBD antibodies were obtained 17 days after priming (i.e., 17 days after injection of the first circular RNA formulation) (fig. 14A), and GLuc activity was detected 2 days after priming (i.e., 2 days after injection of the first circular RNA formulation) (fig. 14B).

These results show that a circular RNA preparation comprising two circular RNAs encoding different antigens induces antigen-specific responses in mice.

Experiment 2

Immunogenicity of a circular RNA preparation comprising (a) circular RNA encoding SARS-CoV-2RBD antigen and (b) circular RNA encoding influenza Hemagglutinin (HA) antigen formulated in lipid nanoparticles was evaluated in a mouse model. Antibody production against SARS-CoV-2RBD and influenza HA antigen was also evaluated in a mouse model.

A first circular RNA (nucleic acid SEQ ID NO:56; amino acid SEQ ID NO: 55) encoding a SARS-CoV-2RBD antigen is designed, produced, and purified by the methods described herein. The second circular RNA was designed to include an IRES followed by an ORF (nucleic acid SEQ ID NO:60; amino acid SEQ ID NO: 59) encoding Hemagglutinin (HA) from influenza A H1N 1A/California/07/2009 and was produced and purified by the methods described herein. The first circular RNA and the second circular RNA are mixed together to obtain a mixture. The mixture was then formulated with lipid nanoparticles as described in example 7 to obtain a first circular RNA formulation. The first and second circular RNAs were formulated separately with lipid nanoparticles as also described in example 7 and then mixed together to obtain a second circular RNA formulation.

Three mice were inoculated intramuscularly with the first circular RNA formulation on day 0 (total dose 10. Mu.g RBD+10. Mu.g HA) and with the second circular RNA formulation on day 12 (total dose 10. Mu.g RBD+10. Mu.g HA). Additional mice (3 or 4 per group) were also inoculated intramuscularly onday 0 and day 12: (i) A dose of 10 μg of the first circular RNA formulated with lipid nanoparticles; (ii) A dose of 10 μg of a second circular RNA formulated with lipid nanoparticles; or (iii) PBS.

Blood was collected as described inexperiment 1. The presence of RBD-specific IgG was determined by ELISA as described inexperiment 1.

The presence or absence of HA-specific IgG in each serum sample was determined by ELISA. ELISA plates were coated overnight at 4℃with HA recombinant protein (11085-V08B; 100ng, yiqiao Shenzhou Biotechnology Co., ltd.) and the plates were treated as described inexperiment 1. The optical density of each serum sample was divided by the optical density of the background (plates coated with HA incubated with secondary antibody only). Fold for each sample against background was plotted.

The results showed that anti-RBD and anti-HA antibodies were obtained 17 days after priming (i.e., 17 days after injection of the first circular RNA formulation) (fig. 16A and 16B).

The results also show that a circular RNA preparation comprising two circular RNAs encoding different antigens induces antigen-specific immune responses in mice.

Experiment 3

Immunogenicity of a circular RNA preparation comprising (a) circular RNA encoding SARS-CoV-2 spike antigen and (b) circular RNA encoding influenza Hemagglutinin (HA) antigen formulated in lipid nanoparticles was evaluated in a mouse model. Antibodies against SARS-CoV-2 spike and influenza HA antigen were also evaluated in a mouse model.

A first circular RNA encoding SARS-CoV-2 spike antigen (nucleic acid SEQ ID NO:54; amino acid SEQ ID NO: 53) is designed, produced, and purified by the methods described herein. The second circular RNA was designed to include an IRES followed by an ORF (nucleic acid SEQ ID NO:60; amino acid SEQ ID NO: 59) encoding Hemagglutinin (HA) from influenza A H1N 1A/California/07/2009 and was produced and purified by the methods described herein. The first circular RNA and the second circular RNA are mixed together to obtain a mixture. The mixture was then formulated with lipid nanoparticles as described in example 7 to obtain a first circular RNA formulation. The first and second circular RNAs were formulated separately with lipid nanoparticles as also described in example 7 and then mixed together to obtain a second circular RNA formulation.

Three mice were inoculated intramuscularly with the first circular RNA formulation on day 0 (total dose 10 μg spike+10 μg HA) and with the second circular RNA formulation on day 12 (total dose 10 μg spike+10 μg HA). Additional mice (3 or 4 per group) were also inoculated intramuscularly onday 0 and day 12: (i) A dose of 10 μg of the first circular RNA formulated with lipid nanoparticles; (ii) A dose of 10 μg of a second circular RNA formulated with lipid nanoparticles; or (iii) PBS.

Blood was collected as described inexperiment 1 and serum was separated from whole blood by centrifugation at 1200g for 30 minutes at 4 ℃. The presence or absence of RBD (i.e., spike RBD) -specific IgG in each serum sample was determined by ELISA as described inexperiment 1.

The presence or absence of HA-specific IgG in each serum sample was determined by ELISA. ELISA plates were coated overnight at 4℃with HA recombinant protein (11085-V08B; 100ng, yiqiao Shenzhou Biotechnology Co., ltd.) and the plates were treated as described inexperiment 1. The optical density of each serum sample was divided by the optical density of the background (plates coated with HA incubated with secondary antibody only). Fold for each sample against background was plotted.

The results showed that anti-RBD antibodies and anti-HA antibodies were obtained 17 days after priming (i.e., 17 days after injection of the first circular RNA formulation) (fig. 15A and 15B).

The results also show that a circular RNA preparation comprising two circular RNAs encoding different antigens induces antigen-specific immune responses in mice.

Example 31: immunogenicity of circular RNAs comprising multiple antigens in a mouse model

This example describes the immunogenicity of circular RNAs comprising multiple antigens. This example also describes the generation of antibodies against multiple antigens encoded by a single circular RNA in a mouse model.

Experiment 1

In this example, the circular RNA was designed to include an IRES followed by an ORF encoding a GLuc polypeptide (as model antigen), a stop codon, a spacer, an IRES, an ORF encoding a SARS-CoV-2RBD antigen, and a stop codon, generated and purified as described in example 29. As a control, the following circular RNAs were generated as described above: (i) A circular RNA having IRES and an ORF encoding SARS-CoV-2RBD antigen; (ii) a circular RNA having an IRES and an ORF encoding a GLuc polypeptide.

The circular RNAs were formulated with lipid nanoparticles as described in example 7 to obtain circular RNA preparations.

Three mice per group were intramuscular inoculated with a total dose of either 10 μg or 20 μg of the circular RNA formulation onday 0 and day 12.

Blood was collected as described in example 30. The presence of RBD-specific IgG was determined by ELISA as described in example 30. GLuc activity was measured as described in example 30.

Experiment 2

Immunogenicity of a circular RNA preparation comprising circular RNA formulated in lipid nanoparticles designed to include an IRES followed by an ORF encoding SARS-CoV-2RBD antigen, a stop codon, a spacer, an IRES, an ORF encoding MERS RBD antigen and a stop codon was evaluated in a mouse model. Antibody production against SARS-CoV-2RBD and MERS RBD antigens was also evaluated in a mouse model.

The circular RNA was then formulated with lipid nanoparticles as described in example 7 to obtain a circular RNA formulation.

Mice were vaccinated intramuscularly or intradermally with the circular RNA formulation in an amount of 5 μg, 10 μg, 20 μg or 50 μg onday 0 and at least one day after initial administration.

Blood was collected as described inexperiment 1. The presence of SARS-CoV-2RBD specific IgG was determined by ELISA as described inexperiment 1. The presence of MERS RBD-specific IgG was also determined by ELISA.

Determining the presence or absence of anti-SARS-CoV-2 RBD binding antibodies, anti-MERS RBD binding antibodies, neutralizing antibodies to SARS-CoV-2RBD antigen, neutralizing antibodies to MERS RBD antigen, cellular responses to SARS-CoV-2 antigen, and cellular responses to MERS RBD antigen in each serum sample.

Example 32: evaluation of T cell response

The presence of SARS-CoV-2 spike or RBD specific T cells or influenza HA specific T cells was detected using an ELISPot assay. This assay was performed on the following groups of mice from example 30:

1.RBD

2.GLuc

3.HA

4. spike of a needle

5.RBD+HA

6. spike+HA

7.PBS

The spleens of mice were harvested onday 30 post boost (i.e., 30 days after injection of the first circular RNA formulation) and processed into single cell suspensions. Spleen cells were seeded at 0.5M cells per well on IFN-g or IL-4ELISPot plates (ImmunoSpot). Spleen cells were not stimulated or stimulated with SARS CoV-2 and HA peptide library (JPT, PM-WCPV-SRB and PM-IFNA_HACal). The ELISPOT plate was processed according to the manufacturer's protocol.

Example 33: evaluation of antibody response in mice administered with circular RNAs encoding multiple antigens

This example demonstrates that antibody responses are generated by administration of circular RNAs encoding expression of multiple antigens.

Anti-influenza HA antibodies that interfere with hemagglutination in serum from mice were measured using a hemagglutination inhibition assay (HAI). Administering to the mice a preparation of circular RNAs, each circular RNA designed and produced by the methods described herein and encoding expression of: SARS-CoV-2RBD antigen, SARS-CoV-2 spike antigen, influenza HA antigen, SARS-CoV-2RBD antigen and GLuc polypeptide, or SARS-CoV-2RBD antigen and SARS-CoV-2 spike antigen. Blood collection was as described inexperiment 1, example 30, and was performed on day 2 and day 17 post injection.

Two-fold serial dilutions of samples collected from mice on day 2 and day 17 were prepared. A fixed amount of influenza virus with known Hemagglutinin (HA) titres was added to each well of a 96-well plate to a concentration equivalent to 4 hemagglutinin units, except for the serum control wells, where no virus was added. Plates were allowed to stand at room temperature for 60 minutes after which time the red blood cell sample was added and allowed to incubate at 4 ℃ for 30 minutes. The highest serum dilution that prevented clotting was determined as HAI titer of serum. The samples collected on day 17 showed HAI titers in samples administered with circular RNA preparations encoding influenza HA antigen when administered alone or in combination with SARS-CoV-2 antigen such as RBD or spike (fig. 17). In samples not administered HA antigen (e.g., SARS-CoV-2RBD antigen alone or SARS-CoV-2 spike antigen alone), no HAI titer was seen on day 17.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Many modifications, variations and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

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Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

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Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

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Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

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His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

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Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

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Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

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Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

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Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

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Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

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Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

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Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

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Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

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Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

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Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

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Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

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Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

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Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

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Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

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Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

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Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

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Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

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Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

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Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

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Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

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Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

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Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

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Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

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Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

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Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

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Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

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Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

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Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

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Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

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Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

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Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

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Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

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Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

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His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

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Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

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Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

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Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

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Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

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Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

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Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

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Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

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Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

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Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

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Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

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Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

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Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

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Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

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Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

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Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

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Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

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Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

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Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

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Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

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Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

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Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

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Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

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Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn

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Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

1025 1030 1035

Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

1055 1060 1065

Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

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Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu

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Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met

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Ala Ile Ala Met Ala Cys Leu Val Gly Leu Met Trp Leu Ser Tyr Phe

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Ile Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg Ser Met Trp Ser Phe

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Asn Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu His Gly Thr Ile

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Leu Thr Arg Pro Leu Leu Glu Ser Glu Leu Val Ile Gly Ala Val Ile

130 135 140

Leu Arg Gly His Leu Arg Ile Ala Gly His His Leu Gly Arg Cys Asp

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Ile Lys Asp Leu Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu

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Ser Tyr Tyr Lys Leu Gly Ala Ser Gln Arg Val Ala Gly Asp Ser Gly

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Phe Ala Ala Tyr Ser Arg Tyr Arg Ile Gly Asn Tyr Lys Leu Asn Thr

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Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn

35 40 45

Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu

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Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro

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Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly

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Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr

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Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp

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Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp

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His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln

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Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser

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Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn

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Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala

195 200 205

Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu

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Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln

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Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys

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Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln

260 265 270

Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp

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Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile

290 295 300

Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile

305 310 315 320

Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala

325 330 335

Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu

340 345 350

Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro

355 360 365

Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln

370 375 380

Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu

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Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser

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Thr Gln Ala

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Met Asp Leu Phe Met Arg Ile Phe Thr Ile Gly Thr Val Thr Leu Lys

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Gln Gly Glu Ile Lys Asp Ala Thr Pro Ser Asp Phe Val Arg Ala Thr

20 25 30

Ala Thr Ile Pro Ile Gln Ala Ser Leu Pro Phe Gly Trp Leu Ile Val

35 40 45

Gly Val Ala Leu Leu Ala Val Phe Gln Ser Ala Ser Lys Ile Ile Thr

50 55 60

Leu Lys Lys Arg Trp Gln Leu Ala Leu Ser Lys Gly Val His Phe Val

65 70 75 80

Cys Asn Leu Leu Leu Leu Phe Val Thr Val Tyr Ser His Leu Leu Leu

85 90 95

Val Ala Ala Gly Leu Glu Ala Pro Phe Leu Tyr Leu Tyr Ala Leu Val

100 105 110

Tyr Phe Leu Gln Ser Ile Asn Phe Val Arg Ile Ile Met Arg Leu Trp

115 120 125

Leu Cys Trp Lys Cys Arg Ser Lys Asn Pro Leu Leu Tyr Asp Ala Asn

130 135 140

Tyr Phe Leu Cys Trp His Thr Asn Cys Tyr Asp Tyr Cys Ile Pro Tyr

145 150 155 160

Asn Ser Val Thr Ser Ser Ile Val Ile Thr Ser Gly Asp Gly Thr Thr

165 170 175

Ser Pro Ile Ser Glu His Asp Tyr Gln Ile Gly Gly Tyr Thr Glu Lys

180 185 190

Trp Glu Ser Gly Val Lys Asp Cys Val Val Leu His Ser Tyr Phe Thr

195 200 205

Ser Asp Tyr Tyr Gln Leu Tyr Ser Thr Gln Leu Ser Thr Asp Thr Gly

210 215 220

Val Glu His Val Thr Phe Phe Ile Tyr Asn Lys Ile Val Asp Glu Pro

225 230 235 240

Glu Glu His Val Gln Ile His Thr Ile Asp Gly Ser Ser Gly Val Val

245 250 255

Asn Pro Val Met Glu Pro Ile Tyr Asp Glu Pro Thr Thr Thr Thr Ser

260 265 270

Val Pro Leu

275

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Met Phe His Leu Val Asp Phe Gln Val Thr Ile Ala Glu Ile Leu Leu

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Ile Ile Met Arg Thr Phe Lys Val Ser Ile Trp Asn Leu Asp Tyr Ile

20 25 30

Ile Asn Leu Ile Ile Lys Asn Leu Ser Lys Ser Leu Thr Glu Asn Lys

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Tyr Ser Gln Leu Asp Glu Glu Gln Pro Met Glu Ile Asp

50 55 60

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Met Lys Ile Ile Leu Phe Leu Ala Leu Ile Thr Leu Ala Thr Cys Glu

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Leu Tyr His Tyr Gln Glu Cys Val Arg Gly Thr Thr Val Leu Leu Lys

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Glu Pro Cys Ser Ser Gly Thr Tyr Glu Gly Asn Ser Pro Phe His Pro

35 40 45

Leu Ala Asp Asn Lys Phe Ala Leu Thr Cys Phe Ser Thr Gln Phe Ala

50 55 60

Phe Ala Cys Pro Asp Gly Val Lys His Val Tyr Gln Leu Arg Ala Arg

65 70 75 80

Ser Val Ser Pro Lys Leu Phe Ile Arg Gln Glu Glu Val Gln Glu Leu

85 90 95

Tyr Ser Pro Ile Phe Leu Ile Val Ala Ala Ile Val Phe Ile Thr Leu

100 105 110

Cys Phe Thr Leu Lys Arg Lys Thr Glu

115 120

<210> 8

<211> 43

<212> PRT

<213> Severe acute respiratory syndrome coronavirus 2

<400> 8

Met Ile Glu Leu Ser Leu Ile Asp Phe Tyr Leu Cys Phe Leu Ala Phe

1 5 10 15

Leu Leu Phe Leu Val Leu Ile Met Leu Ile Ile Phe Trp Phe Ser Leu

20 25 30

Glu Leu Gln Asp His Asn Glu Thr Cys His Ala

35 40

<210> 9

<211> 121

<212> PRT

<213> Severe acute respiratory syndrome coronavirus 2

<400> 9

Met Lys Phe Leu Val Phe Leu Gly Ile Ile Thr Thr Val Ala Ala Phe

1 5 10 15

His Gln Glu Cys Ser Leu Gln Ser Cys Thr Gln His Gln Pro Tyr Val

20 25 30

Val Asp Asp Pro Cys Pro Ile His Phe Tyr Ser Lys Trp Tyr Ile Arg

35 40 45

Val Gly Ala Arg Lys Ser Ala Pro Leu Ile Glu Leu Cys Val Asp Glu

50 55 60

Ala Gly Ser Lys Ser Pro Ile Gln Tyr Ile Asp Ile Gly Asn Tyr Thr

65 70 75 80

Val Ser Cys Leu Pro Phe Thr Ile Asn Cys Gln Glu Pro Lys Leu Gly

85 90 95

Ser Leu Val Val Arg Cys Ser Phe Tyr Glu Asp Phe Leu Glu Tyr His

100 105 110

Asp Val Arg Val Val Leu Asp Phe Ile

115 120

<210> 10

<211> 38

<212> PRT

<213> Severe acute respiratory syndrome coronavirus 2

<400> 10

Met Gly Tyr Ile Asn Val Phe Ala Phe Pro Phe Thr Ile Tyr Ser Leu

1 5 10 15

Leu Leu Cys Arg Met Asn Ser Arg Asn Tyr Ile Ala Gln Val Asp Val

20 25 30

Val Asn Phe Asn Leu Thr

35

<210> 11

<211> 3822

<212> DNA

<213> Severe acute respiratory syndrome coronavirus 2

<400> 11

atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60

agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120

aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180

aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240

aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300

ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360

aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420

ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480

tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540

ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600

tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660

tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720

ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780

ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840

gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900

tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020

gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320

cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440

aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500

aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620

ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680

cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740

acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800

ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860

cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920

aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980

gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040

cctcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100

gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160

agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220

tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280

acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 2340

gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400

aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460

ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520

cttggtgata ttgctgctag agacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580

ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640

acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700

caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760

aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 2820

acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880

acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940

ctttcacgtc ttgacaaagt tgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000

cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060

tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120

gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180

gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240

atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300

cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360

tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420

ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480

tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540

aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600

caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660

atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc 3720

tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac 3780

tctgagccag tgctcaaagg agtcaaatta cattacacat aa 3822

<210> 12

<211> 7035

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 12

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 60

atctaaagta tatatgagta aacttggtct gacagttatt agaaaaattc atccagcaga 120

cgataaaacg caatacgctg gctatccggt gccgcaatgc catacagcac cagaaaacga 180

tccgcccatt cgccgcccag ttcttccgca atatcacggg tggccagcgc aatatcctga 240

taacgatccg ccacgcccag acggccgcaa tcaataaagc cgctaaaacg gccattttcc 300

accataatgt tcggcaggca cgcatcacca tgggtcacca ccagatcttc gccatccggc 360

atgctcgctt tcagacgcgc aaacagctct gccggtgcca ggccctgatg ttcttcatcc 420

agatcatcct gatccaccag gcccgcttcc atacgggtac gcgcacgttc aatacgatgt 480

ttcgcctgat gatcaaacgg acaggtcgcc gggtccaggg tatgcagacg acgcatggca 540

tccgccataa tgctcacttt ttctgccggc gccagatggc tagacagcag atcctgaccc 600

ggcacttcgc ccagcagcag ccaatcacgg cccgcttcgg tcaccacatc cagcaccgcc 660

gcacacggaa caccggtggt ggccagccag ctcagacgcg ccgcttcatc ctgcagctcg 720

ttcagcgcac cgctcagatc ggttttcaca aacagcaccg gacgaccctg cgcgctcaga 780

cgaaacaccg ccgcatcaga gcagccaatg gtctgctgcg cccaatcata gccaaacaga 840

cgttccaccc acgctgccgg gctacccgca tgcaggccat cctgttcaat catactcttc 900

ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 960

gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 1020

cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct 1080

cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg 1140

agatagggtt gagtggccgc tacagggcgc tcccattcgc cattcaggct gcgcaactgt 1200

tgggaagggc gtttcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg 1260

tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac 1320

gacggccagt gagcgcgacg taatacgact cactataggg cgaattgaag gaaggccgtc 1380

aaggccgcat gggaagccct cgaccgtcga ttgtccactg gtcaacaata gatgacttac 1440

aactaatcgg aaggtgcaga gactcgacgg gagctaccct aacgtcaaga cgagggtaaa 1500

gagagagtcc aattctcaaa gccaataggc agtagcgaaa gctgcaagag aatgaaaatc 1560

cgttgacctt aaacggtcgt gtgggttcaa gtccctccac ccccacgccg gaaacgcaat 1620

agccgaaaaa caaaaaacaa aaaaaacaaa aaaaaaacca aaaaaacaaa acacaacgtt 1680

actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc 1740

atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc 1800

attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag 1860

gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg 1920

cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat 1980

acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga 2040

gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc 2100

cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg 2160

ttaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat 2220

gataatagcc accatgtttg tttttcttgt tttattgcca ctagtctcta gtcagtgtgt 2280

taatcttaca accagaactc aattaccccc tgcatacact aattctttca cacgtggtgt 2340

ttattaccct gacaaagttt tcagatcctc agttttacat tcaactcagg acttgttctt 2400

acctttcttt tccaatgtta cttggttcca tgctatacat gtctctggga ccaatggtac 2460

taagaggttt gataaccctg tcctaccatt taatgatggt gtttattttg cttccactga 2520

gaagtctaac ataataagag gctggatttt tggtactact ttagattcga agacccagtc 2580

cctacttatt gttaataacg ctactaatgt tgttattaaa gtctgtgaat ttcaattttg 2640

taatgatcca tttttgggtg tttattacca caaaaacaac aaaagttgga tggaaagtga 2700

gttcagagtt tattctagtg cgaataattg cacttttgaa tatgtctctc agccttttct 2760

tatggacctt gaaggaaaac agggtaattt caaaaatctt agggaatttg tgtttaagaa 2820

tattgatggt tattttaaaa tatattctaa gcacacgcct attaatttag tgcgtgatct 2880

ccctcagggt ttttcggctt tagaaccatt ggtagatttg ccaataggta ttaacatcac 2940

taggtttcaa actttacttg ctttacatag aagttatttg actcctggtg attcttcttc 3000

aggttggaca gctggtgctg cagcttatta tgtgggttat cttcaaccta ggacttttct 3060

attaaaatat aatgaaaatg gaaccattac agatgctgta gactgtgcac ttgaccctct 3120

ctcagaaaca aagtgtacgt tgaaatcctt cactgtagaa aaaggaatct atcaaacttc 3180

taactttaga gtccaaccaa cagaatctat tgttagattt cctaatatta caaacttgtg 3240

cccttttggt gaagttttta acgccaccag atttgcatct gtttatgctt ggaacaggaa 3300

gagaatcagc aactgtgttg ctgattattc tgtcctatat aattccgcat cattttccac 3360

ttttaagtgt tatggagtgt ctcctactaa attaaatgat ctctgcttta ctaatgtcta 3420

tgcagattca tttgtaatta gaggtgatga agtcagacaa atcgctccag ggcaaactgg 3480

aaagattgct gattataatt ataaattacc agatgatttt acaggctgcg ttatagcttg 3540

gaattctaac aatcttgatt ctaaggttgg tggtaattat aattacctgt atagattgtt 3600

taggaagtct aatctcaaac cttttgagag agatatttca actgaaatct atcaggccgg 3660

tagcacacct tgtaatggtg ttgaaggttt taattgttac tttcctttac aatcatatgg 3720

tttccaaccc actaatggtg ttggttacca accatacaga gtagtagtac tttcttttga 3780

acttctacat gcaccagcaa ctgtttgtgg acctaaaaag tctactaatt tggttaaaaa 3840

caaatgtgtc aatttcaact tcaatggttt aacaggcaca ggtgttctta ctgagtctaa 3900

caaaaagttt ctgcctttcc aacaatttgg cagagacatt gctgacacta ctgatgctgt 3960

ccgtgatcca cagacacttg agattcttga cattacacca tgttcttttg gtggtgtcag 4020

tgttataaca ccaggaacaa atacttctaa ccaggttgct gttctttatc aggatgttaa 4080

ctgcacagaa gtccctgttg ctattcatgc agatcaactt actcctactt ggcgtgttta 4140

ttctacaggt tctaatgttt ttcaaacacg tgcaggctgt ttaatagggg ctgaacatgt 4200

caacaactca tatgagtgtg acatacccat tggtgcaggt atatgcgcta gttatcagac 4260

tcagactaat tctcctggca gcgccagcag tgtagctagt caatccatca ttgcctacac 4320

tatgtcactt ggtgcagaaa attcagttgc ttactctaat aactctattg ccatacccac 4380

aaattttact attagtgtta ccacagaaat tctaccagtg tctatgacca agacatcagt 4440

agattgtaca atgtacattt gtggtgattc aactgaatgc agcaatcttt tgttgcaata 4500

tggcagtttt tgtacacaat taaaccgtgc tttaactgga atagctgttg aacaagacaa 4560

aaacacccaa gaagtttttg cacaagtcaa acaaatttac aaaacaccac caattaaaga 4620

ttttggtggt tttaattttt cacaaatatt accagatcca tcaaaaccaa gcaagaggtc 4680

atttattgaa gatctacttt tcaacaaagt gacacttgca gatgctggct tcatcaaaca 4740

atatggtgat tgccttggtg atattgctgc tagggacctc atttgtgcac aaaagtttaa 4800

cggccttact gttttgccac ctttgctcac agatgaaatg attgctcaat acacttctgc 4860

actgttagcg ggtacaatca cttctggttg gacctttggt gcaggtgctg cattacaaat 4920

accatttgct atgcaaatgg cttataggtt taatggtatt ggagttacac agaatgttct 4980

ctatgagaac caaaaattga ttgccaacca atttaatagt gctattggca aaattcaaga 5040

ctcactttct tccacagcaa gtgcacttgg aaaacttcaa gatgtggtca accaaaatgc 5100

acaagcttta aacacgcttg ttaaacaact tagctccaat tttggtgcaa tttcaagtgt 5160

tttaaatgat atcctttcac gtcttgaccc tcccgaggct gaagtgcaaa ttgataggtt 5220

gatcacaggc agacttcaaa gtttgcagac atatgtgact caacaattaa ttagagctgc 5280

agaaatcaga gcttctgcta atcttgctgc tactaaaatg tcagagtgtg tacttggaca 5340

atcaaaaaga gttgattttt gtggaaaggg ctatcatctt atgtccttcc ctcagtcagc 5400

acctcatggt gtagtcttct tgcatgtgac ttatgtccct gcacaagaaa agaacttcac 5460

aactgctcct gccatttgtc atgatggaaa agcacacttt cctcgtgaag gtgtctttgt 5520

ttcaaatggc acacactggt ttgtaacaca aaggaatttt tatgaaccac aaatcattac 5580

tacagacaac acatttgtgt ctggtaactg tgatgttgta ataggaattg tcaacaacac 5640

agtttatgat cctttgcaac ctgaattaga ctcattcaag gaggagttag ataaatattt 5700

taagaatcat acatcaccag atgttgattt aggtgacatc tctggcatta atgcttcagt 5760

tgtaaacatt caaaaagaaa ttgaccgcct caatgaggtt gccaagaatt taaatgaatc 5820

tctcatcgat ctccaagaac ttggaaagta tgagcagggg tatatccctg aagcccccag 5880

ggacggccag gcttacgtca gaaaggatgg agagtgggtg ctcttgagca ccttcctgta 5940

aaaaaaacaa aaaacaaaac ggctattatg cgttaccggc gagacgctac ggacttaaat 6000

aattgagcct taaagaagaa attctttaag tggatgctct caaactcagg gaaacctaaa 6060

tctagttata gacaaggcaa tcctgagcca agccgaagta gtaattagta agaccagtgg 6120

acaatcgacg gataacagca tatctagctg ggcctcatgg gccttccttt cactgcccgc 6180

tttccagtcg ggaaacctgt cgtgccagct gcattaacat ggtcatagct gtttccttgc 6240

gtattgggcg ctctccgctt cctcgctcac tgactcgctg cgctcggtcg ttcgggtaaa 6300

gcctggggtg cctaatgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 6360

ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 6420

agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 6480

tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 6540

ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 6600

gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 6660

ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 6720

gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 6780

aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 6840

aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 6900

ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 6960

gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 7020

gggattttgg tcatg 7035

<210> 13

<211> 3708

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 13

atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60

agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120

aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180

aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240

aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300

ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360

aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420

ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480

tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540

ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600

tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660

tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720

ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780

ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840

gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900

tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020

gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320

cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440

aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500

aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620

ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680

cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740

acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800

ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860

cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920

aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980

gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040

cctggcagcg ccagcagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100

gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160

agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220

tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280

acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 2340

gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400

aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460

ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520

cttggtgata ttgctgctag ggacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580

ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640

acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700

caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760

aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 2820

acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880

acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940

ctttcacgtc ttgaccctcc cgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000

cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060

tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120

gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180

gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240

atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300

cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360

tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420

ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480

tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540

aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600

caagaacttg gaaagtatga gcaggggtat atccctgaag cccccaggga cggccaggct 3660

tacgtcagaa aggatggaga gtgggtgctc ttgagcacct tcctgtaa 3708

<210> 14

<211> 7149

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 14

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 60

atctaaagta tatatgagta aacttggtct gacagttatt agaaaaattc atccagcaga 120

cgataaaacg caatacgctg gctatccggt gccgcaatgc catacagcac cagaaaacga 180

tccgcccatt cgccgcccag ttcttccgca atatcacggg tggccagcgc aatatcctga 240

taacgatccg ccacgcccag acggccgcaa tcaataaagc cgctaaaacg gccattttcc 300

accataatgt tcggcaggca cgcatcacca tgggtcacca ccagatcttc gccatccggc 360

atgctcgctt tcagacgcgc aaacagctct gccggtgcca ggccctgatg ttcttcatcc 420

agatcatcct gatccaccag gcccgcttcc atacgggtac gcgcacgttc aatacgatgt 480

ttcgcctgat gatcaaacgg acaggtcgcc gggtccaggg tatgcagacg acgcatggca 540

tccgccataa tgctcacttt ttctgccggc gccagatggc tagacagcag atcctgaccc 600

ggcacttcgc ccagcagcag ccaatcacgg cccgcttcgg tcaccacatc cagcaccgcc 660

gcacacggaa caccggtggt ggccagccag ctcagacgcg ccgcttcatc ctgcagctcg 720

ttcagcgcac cgctcagatc ggttttcaca aacagcaccg gacgaccctg cgcgctcaga 780

cgaaacaccg ccgcatcaga gcagccaatg gtctgctgcg cccaatcata gccaaacaga 840

cgttccaccc acgctgccgg gctacccgca tgcaggccat cctgttcaat catactcttc 900

ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 960

gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 1020

cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct 1080

cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg 1140

agatagggtt gagtggccgc tacagggcgc tcccattcgc cattcaggct gcgcaactgt 1200

tgggaagggc gtttcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg 1260

tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac 1320

gacggccagt gagcgcgacg taatacgact cactataggg cgaattgaag gaaggccgtc 1380

aaggccgcat gggaagccct cgaccgtcga ttgtccactg gtcaacaata gatgacttac 1440

aactaatcgg aaggtgcaga gactcgacgg gagctaccct aacgtcaaga cgagggtaaa 1500

gagagagtcc aattctcaaa gccaataggc agtagcgaaa gctgcaagag aatgaaaatc 1560

cgttgacctt aaacggtcgt gtgggttcaa gtccctccac ccccacgccg gaaacgcaat 1620

agccgaaaaa caaaaaacaa aaaaaacaaa aaaaaaacca aaaaaacaaa acacaacgtt 1680

actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc 1740

atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc 1800

attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag 1860

gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg 1920

cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat 1980

acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga 2040

gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc 2100

cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg 2160

ttaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat 2220

gataatagcc accatgtttg tttttcttgt tttattgcca ctagtctcta gtcagtgtgt 2280

taatcttaca accagaactc aattaccccc tgcatacact aattctttca cacgtggtgt 2340

ttattaccct gacaaagttt tcagatcctc agttttacat tcaactcagg acttgttctt 2400

acctttcttt tccaatgtta cttggttcca tgctatacat gtctctggga ccaatggtac 2460

taagaggttt gataaccctg tcctaccatt taatgatggt gtttattttg cttccactga 2520

gaagtctaac ataataagag gctggatttt tggtactact ttagattcga agacccagtc 2580

cctacttatt gttaataacg ctactaatgt tgttattaaa gtctgtgaat ttcaattttg 2640

taatgatcca tttttgggtg tttattacca caaaaacaac aaaagttgga tggaaagtga 2700

gttcagagtt tattctagtg cgaataattg cacttttgaa tatgtctctc agccttttct 2760

tatggacctt gaaggaaaac agggtaattt caaaaatctt agggaatttg tgtttaagaa 2820

tattgatggt tattttaaaa tatattctaa gcacacgcct attaatttag tgcgtgatct 2880

ccctcagggt ttttcggctt tagaaccatt ggtagatttg ccaataggta ttaacatcac 2940

taggtttcaa actttacttg ctttacatag aagttatttg actcctggtg attcttcttc 3000

aggttggaca gctggtgctg cagcttatta tgtgggttat cttcaaccta ggacttttct 3060

attaaaatat aatgaaaatg gaaccattac agatgctgta gactgtgcac ttgaccctct 3120

ctcagaaaca aagtgtacgt tgaaatcctt cactgtagaa aaaggaatct atcaaacttc 3180

taactttaga gtccaaccaa cagaatctat tgttagattt cctaatatta caaacttgtg 3240

cccttttggt gaagttttta acgccaccag atttgcatct gtttatgctt ggaacaggaa 3300

gagaatcagc aactgtgttg ctgattattc tgtcctatat aattccgcat cattttccac 3360

ttttaagtgt tatggagtgt ctcctactaa attaaatgat ctctgcttta ctaatgtcta 3420

tgcagattca tttgtaatta gaggtgatga agtcagacaa atcgctccag ggcaaactgg 3480

aaagattgct gattataatt ataaattacc agatgatttt acaggctgcg ttatagcttg 3540

gaattctaac aatcttgatt ctaaggttgg tggtaattat aattacctgt atagattgtt 3600

taggaagtct aatctcaaac cttttgagag agatatttca actgaaatct atcaggccgg 3660

tagcacacct tgtaatggtg ttgaaggttt taattgttac tttcctttac aatcatatgg 3720

tttccaaccc actaatggtg ttggttacca accatacaga gtagtagtac tttcttttga 3780

acttctacat gcaccagcaa ctgtttgtgg acctaaaaag tctactaatt tggttaaaaa 3840

caaatgtgtc aatttcaact tcaatggttt aacaggcaca ggtgttctta ctgagtctaa 3900

caaaaagttt ctgcctttcc aacaatttgg cagagacatt gctgacacta ctgatgctgt 3960

ccgtgatcca cagacacttg agattcttga cattacacca tgttcttttg gtggtgtcag 4020

tgttataaca ccaggaacaa atacttctaa ccaggttgct gttctttatc aggatgttaa 4080

ctgcacagaa gtccctgttg ctattcatgc agatcaactt actcctactt ggcgtgttta 4140

ttctacaggt tctaatgttt ttcaaacacg tgcaggctgt ttaatagggg ctgaacatgt 4200

caacaactca tatgagtgtg acatacccat tggtgcaggt atatgcgcta gttatcagac 4260

tcagactaat tctcctggca gcgccagcag tgtagctagt caatccatca ttgcctacac 4320

tatgtcactt ggtgcagaaa attcagttgc ttactctaat aactctattg ccatacccac 4380

aaattttact attagtgtta ccacagaaat tctaccagtg tctatgacca agacatcagt 4440

agattgtaca atgtacattt gtggtgattc aactgaatgc agcaatcttt tgttgcaata 4500

tggcagtttt tgtacacaat taaaccgtgc tttaactgga atagctgttg aacaagacaa 4560

aaacacccaa gaagtttttg cacaagtcaa acaaatttac aaaacaccac caattaaaga 4620

ttttggtggt tttaattttt cacaaatatt accagatcca tcaaaaccaa gcaagaggtc 4680

atttattgaa gatctacttt tcaacaaagt gacacttgca gatgctggct tcatcaaaca 4740

atatggtgat tgccttggtg atattgctgc tagggacctc atttgtgcac aaaagtttaa 4800

cggccttact gttttgccac ctttgctcac agatgaaatg attgctcaat acacttctgc 4860

actgttagcg ggtacaatca cttctggttg gacctttggt gcaggtgctg cattacaaat 4920

accatttgct atgcaaatgg cttataggtt taatggtatt ggagttacac agaatgttct 4980

ctatgagaac caaaaattga ttgccaacca atttaatagt gctattggca aaattcaaga 5040

ctcactttct tccacagcaa gtgcacttgg aaaacttcaa gatgtggtca accaaaatgc 5100

acaagcttta aacacgcttg ttaaacaact tagctccaat tttggtgcaa tttcaagtgt 5160

tttaaatgat atcctttcac gtcttgaccc tcccgaggct gaagtgcaaa ttgataggtt 5220

gatcacaggc agacttcaaa gtttgcagac atatgtgact caacaattaa ttagagctgc 5280

agaaatcaga gcttctgcta atcttgctgc tactaaaatg tcagagtgtg tacttggaca 5340

atcaaaaaga gttgattttt gtggaaaggg ctatcatctt atgtccttcc ctcagtcagc 5400

acctcatggt gtagtcttct tgcatgtgac ttatgtccct gcacaagaaa agaacttcac 5460

aactgctcct gccatttgtc atgatggaaa agcacacttt cctcgtgaag gtgtctttgt 5520

ttcaaatggc acacactggt ttgtaacaca aaggaatttt tatgaaccac aaatcattac 5580

tacagacaac acatttgtgt ctggtaactg tgatgttgta ataggaattg tcaacaacac 5640

agtttatgat cctttgcaac ctgaattaga ctcattcaag gaggagttag ataaatattt 5700

taagaatcat acatcaccag atgttgattt aggtgacatc tctggcatta atgcttcagt 5760

tgtaaacatt caaaaagaaa ttgaccgcct caatgaggtt gccaagaatt taaatgaatc 5820

tctcatcgat ctccaagaac ttggaaagta tgagcagtat ataaaatggc catggtacat 5880

ttggctaggt tttatagctg gcttgattgc catagtaatg gtgacaatta tgctttgctg 5940

tatgaccagt tgctgtagtt gtctcaaggg ctgttgttct tgtggatcct gctgcaaatt 6000

tgatgaagac gactctgagc cagtgctcaa aggagtcaaa ttacattaca cataaaaaaa 6060

acaaaaaaca aaacggctat tatgcgttac cggcgagacg ctacggactt aaataattga 6120

gccttaaaga agaaattctt taagtggatg ctctcaaact cagggaaacc taaatctagt 6180

tatagacaag gcaatcctga gccaagccga agtagtaatt agtaagacca gtggacaatc 6240

gacggataac agcatatcta gctgggcctc atgggccttc ctttcactgc ccgctttcca 6300

gtcgggaaac ctgtcgtgcc agctgcatta acatggtcat agctgtttcc ttgcgtattg 6360

ggcgctctcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggg taaagcctgg 6420

ggtgcctaat gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 6480

gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 6540

aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 6600

gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 6660

ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 6720

cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 6780

ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 6840

actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 6900

tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 6960

gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 7020

ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 7080

cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 7140

ttggtcatg 7149

<210> 15

<211> 3822

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 15

atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60

agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120

aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180

aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240

aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300

ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360

aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420

ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480

tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540

ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600

tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660

tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720

ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780

ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840

gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900

tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020

gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320

cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440

aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500

aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620

ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680

cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740

acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800

ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860

cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920

aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980

gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040

cctggcagcg ccagcagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100

gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160

agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220

tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280

acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 2340

gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400

aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460

ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520

cttggtgata ttgctgctag ggacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580

ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640

acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700

caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760

aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 2820

acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880

acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940

ctttcacgtc ttgaccctcc cgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000

cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060

tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120

gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180

gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240

atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300

cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360

tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420

ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480

tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540

aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600

caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660

atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc 3720

tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac 3780

tctgagccag tgctcaaagg agtcaaatta cattacacat aa 3822

<210> 16

<211> 7038

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 16

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 60

atctaaagta tatatgagta aacttggtct gacagttatt agaaaaattc atccagcaga 120

cgataaaacg caatacgctg gctatccggt gccgcaatgc catacagcac cagaaaacga 180

tccgcccatt cgccgcccag ttcttccgca atatcacggg tggccagcgc aatatcctga 240

taacgatccg ccacgcccag acggccgcaa tcaataaagc cgctaaaacg gccattttcc 300

accataatgt tcggcaggca cgcatcacca tgggtcacca ccagatcttc gccatccggc 360

atgctcgctt tcagacgcgc aaacagctct gccggtgcca ggccctgatg ttcttcatcc 420

agatcatcct gatccaccag gcccgcttcc atacgggtac gcgcacgttc aatacgatgt 480

ttcgcctgat gatcaaacgg acaggtcgcc gggtccaggg tatgcagacg acgcatggca 540

tccgccataa tgctcacttt ttctgccggc gccagatggc tagacagcag atcctgaccc 600

ggcacttcgc ccagcagcag ccaatcacgg cccgcttcgg tcaccacatc cagcaccgcc 660

gcacacggaa caccggtggt ggccagccag ctcagacgcg ccgcttcatc ctgcagctcg 720

ttcagcgcac cgctcagatc ggttttcaca aacagcaccg gacgaccctg cgcgctcaga 780

cgaaacaccg ccgcatcaga gcagccaatg gtctgctgcg cccaatcata gccaaacaga 840

cgttccaccc acgctgccgg gctacccgca tgcaggccat cctgttcaat catactcttc 900

ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 960

gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 1020

cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct 1080

cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg 1140

agatagggtt gagtggccgc tacagggcgc tcccattcgc cattcaggct gcgcaactgt 1200

tgggaagggc gtttcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg 1260

tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac 1320

gacggccagt gagcgcgacg taatacgact cactataggg cgaattgaag gaaggccgtc 1380

aaggccgcat gggaagccct cgaccgtcga ttgtccactg gtcaacaata gatgacttac 1440

aactaatcgg aaggtgcaga gactcgacgg gagctaccct aacgtcaaga cgagggtaaa 1500

gagagagtcc aattctcaaa gccaataggc agtagcgaaa gctgcaagag aatgaaaatc 1560

cgttgacctt aaacggtcgt gtgggttcaa gtccctccac ccccacgccg gaaacgcaat 1620

agccgaaaaa caaaaaacaa aaaaaacaaa aaaaaaacca aaaaaacaaa acacaacgtt 1680

actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc 1740

atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc 1800

attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag 1860

gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg 1920

cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat 1980

acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga 2040

gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc 2100

cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg 2160

ttaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat 2220

gataatagcc accatgtttg tttttcttgt tttattgcca ctagtctcta gtcagtgtgt 2280

taatcttaca accagaactc aattaccccc tgcatacact aattctttca cacgtggtgt 2340

ttattaccct gacaaagttt tcagatcctc agttttacat tcaactcagg acttgttctt 2400

acctttcttt tccaatgtta cttggttcca tgctatacat gtctctggga ccaatggtac 2460

taagaggttt gataaccctg tcctaccatt taatgatggt gtttattttg cttccactga 2520

gaagtctaac ataataagag gctggatttt tggtactact ttagattcga agacccagtc 2580

cctacttatt gttaataacg ctactaatgt tgttattaaa gtctgtgaat ttcaattttg 2640

taatgatcca tttttgggtg tttattacca caaaaacaac aaaagttgga tggaaagtga 2700

gttcagagtt tattctagtg cgaataattg cacttttgaa tatgtctctc agccttttct 2760

tatggacctt gaaggaaaac agggtaattt caaaaatctt agggaatttg tgtttaagaa 2820

tattgatggt tattttaaaa tatattctaa gcacacgcct attaatttag tgcgtgatct 2880

ccctcagggt ttttcggctt tagaaccatt ggtagatttg ccaataggta ttaacatcac 2940

taggtttcaa actttacttg ctttacatag aagttatttg actcctggtg attcttcttc 3000

aggttggaca gctggtgctg cagcttatta tgtgggttat cttcaaccta ggacttttct 3060

attaaaatat aatgaaaatg gaaccattac agatgctgta gactgtgcac ttgaccctct 3120

ctcagaaaca aagtgtacgt tgaaatcctt cactgtagaa aaaggaatct atcaaacttc 3180

taactttaga gtccaaccaa cagaatctat tgttagattt cctaatatta caaacttgtg 3240

cccttttggt gaagttttta acgccaccag atttgcatct gtttatgctt ggaacaggaa 3300

gagaatcagc aactgtgttg ctgattattc tgtcctatat aattccgcat cattttccac 3360

ttttaagtgt tatggagtgt ctcctactaa attaaatgat ctctgcttta ctaatgtcta 3420

tgcagattca tttgtaatta gaggtgatga agtcagacaa atcgctccag ggcaaactgg 3480

aaagattgct gattataatt ataaattacc agatgatttt acaggctgcg ttatagcttg 3540

gaattctaac aatcttgatt ctaaggttgg tggtaattat aattacctgt atagattgtt 3600

taggaagtct aatctcaaac cttttgagag agatatttca actgaaatct atcaggccgg 3660

tagcacacct tgtaatggtg ttgaaggttt taattgttac tttcctttac aatcatatgg 3720

tttccaaccc actaatggtg ttggttacca accatacaga gtagtagtac tttcttttga 3780

acttctacat gcaccagcaa ctgtttgtgg acctaaaaag tctactaatt tggttaaaaa 3840

caaatgtgtc aatttcaact tcaatggttt aacaggcaca ggtgttctta ctgagtctaa 3900

caaaaagttt ctgcctttcc aacaatttgg cagagacatt gctgacacta ctgatgctgt 3960

ccgtgatcca cagacacttg agattcttga cattacacca tgttcttttg gtggtgtcag 4020

tgttataaca ccaggaacaa atacttctaa ccaggttgct gttctttatc aggatgttaa 4080

ctgcacagaa gtccctgttg ctattcatgc agatcaactt actcctactt ggcgtgttta 4140

ttctacaggt tctaatgttt ttcaaacacg tgcaggctgt ttaatagggg ctgaacatgt 4200

caacaactca tatgagtgtg acatacccat tggtgcaggt atatgcgcta gttatcagac 4260

tcagactaat tctcctggca gcgccagcag tgtagctagt caatccatca ttgcctacac 4320

tatgtcactt ggtgcagaaa attcagttgc ttactctaat aactctattg ccatacccac 4380

aaattttact attagtgtta ccacagaaat tctaccagtg tctatgacca agacatcagt 4440

agattgtaca atgtacattt gtggtgattc aactgaatgc agcaatcttt tgttgcaata 4500

tggcagtttt tgtacacaat taaaccgtgc tttaactgga atagctgttg aacaagacaa 4560

aaacacccaa gaagtttttg cacaagtcaa acaaatttac aaaacaccac caattaaaga 4620

ttttggtggt tttaattttt cacaaatatt accagatcca tcaaaaccaa gcaagaggtc 4680

atttattgaa gatctacttt tcaacaaagt gacacttgca gatgctggct tcatcaaaca 4740

atatggtgat tgccttggtg atattgctgc tagggacctc atttgtgcac aaaagtttaa 4800

cggccttact gttttgccac ctttgctcac agatgaaatg attgctcaat acacttctgc 4860

actgttagcg ggtacaatca cttctggttg gacctttggt gcaggtgctg cattacaaat 4920

accatttgct atgcaaatgg cttataggtt taatggtatt ggagttacac agaatgttct 4980

ctatgagaac caaaaattga ttgccaacca atttaatagt gctattggca aaattcaaga 5040

ctcactttct tccacagcaa gtgcacttgg aaaacttcaa gatgtggtca accaaaatgc 5100

acaagcttta aacacgcttg ttaaacaact tagctccaat tttggtgcaa tttcaagtgt 5160

tttaaatgat atcctttcac gtcttgaccc tcccgaggct gaagtgcaaa ttgataggtt 5220

gatcacaggc agacttcaaa gtttgcagac atatgtgact caacaattaa ttagagctgc 5280

agaaatcaga gcttctgcta atcttgctgc tactaaaatg tcagagtgtg tacttggaca 5340

atcaaaaaga gttgattttt gtggaaaggg ctatcatctt atgtccttcc ctcagtcagc 5400

acctcatggt gtagtcttct tgcatgtgac ttatgtccct gcacaagaaa agaacttcac 5460

aactgctcct gccatttgtc atgatggaaa agcacacttt cctcgtgaag gtgtctttgt 5520

ttcaaatggc acacactggt ttgtaacaca aaggaatttt tatgaaccac aaatcattac 5580

tacagacaac acatttgtgt ctggtaactg tgatgttgta ataggaattg tcaacaacac 5640

agtttatgat cctttgcaac ctgaattaga ctcattcaag gaggagttag ataaatattt 5700

taagaatcat acatcaccag atgttgattt aggtgacatc tctggcatta atgcttcagt 5760

tgtaaacatt caaaaagaaa ttgaccgcct caatgaggtt gccaagaatt taaatgaatc 5820

tctcatcgat ctccaagaac ttggaaagta tgagcagtat ataaaatggc catggtacat 5880

ttggctaggt tttatagctg gcttgattgc catagtaatg gtgacaatta tgctttgctg 5940

ttaaaaaaaa caaaaaacaa aacggctatt atgcgttacc ggcgagacgc tacggactta 6000

aataattgag ccttaaagaa gaaattcttt aagtggatgc tctcaaactc agggaaacct 6060

aaatctagtt atagacaagg caatcctgag ccaagccgaa gtagtaatta gtaagaccag 6120

tggacaatcg acggataaca gcatatctag ctgggcctca tgggccttcc tttcactgcc 6180

cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa catggtcata gctgtttcct 6240

tgcgtattgg gcgctctccg cttcctcgct cactgactcg ctgcgctcgg tcgttcgggt 6300

aaagcctggg gtgcctaatg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 6360

gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 6420

tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 6480

agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 6540

ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 6600

taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 6660

gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 6720

gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 6780

ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 6840

ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 6900

gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 6960

caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 7020

taagggattt tggtcatg 7038

<210> 17

<211> 3711

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 17

atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60

agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120

aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180

aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240

aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300

ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360

aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420

ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480

tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540

ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600

tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660

tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720

ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780

ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840

gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900

tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020

gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320

cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440

aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500

aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620

ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680

cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740

acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800

ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860

cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920

aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980

gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040

cctggcagcg ccagcagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100

gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160

agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220

tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280

acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 2340

gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400

aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460

ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520

cttggtgata ttgctgctag ggacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580

ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640

acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700

caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760

aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 2820

acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880

acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940

ctttcacgtc ttgaccctcc cgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000

cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060

tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120

gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180

gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240

atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300

cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360

tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420

ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480

tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540

aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600

caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660

atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtta a 3711

<210> 18

<211> 7035

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 18

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 60

atctaaagta tatatgagta aacttggtct gacagttatt agaaaaattc atccagcaga 120

cgataaaacg caatacgctg gctatccggt gccgcaatgc catacagcac cagaaaacga 180

tccgcccatt cgccgcccag ttcttccgca atatcacggg tggccagcgc aatatcctga 240

taacgatccg ccacgcccag acggccgcaa tcaataaagc cgctaaaacg gccattttcc 300

accataatgt tcggcaggca cgcatcacca tgggtcacca ccagatcttc gccatccggc 360

atgctcgctt tcagacgcgc aaacagctct gccggtgcca ggccctgatg ttcttcatcc 420

agatcatcct gatccaccag gcccgcttcc atacgggtac gcgcacgttc aatacgatgt 480

ttcgcctgat gatcaaacgg acaggtcgcc gggtccaggg tatgcagacg acgcatggca 540

tccgccataa tgctcacttt ttctgccggc gccagatggc tagacagcag atcctgaccc 600

ggcacttcgc ccagcagcag ccaatcacgg cccgcttcgg tcaccacatc cagcaccgcc 660

gcacacggaa caccggtggt ggccagccag ctcagacgcg ccgcttcatc ctgcagctcg 720

ttcagcgcac cgctcagatc ggttttcaca aacagcaccg gacgaccctg cgcgctcaga 780

cgaaacaccg ccgcatcaga gcagccaatg gtctgctgcg cccaatcata gccaaacaga 840

cgttccaccc acgctgccgg gctacccgca tgcaggccat cctgttcaat catactcttc 900

ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 960

gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 1020

cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct 1080

cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg 1140

agatagggtt gagtggccgc tacagggcgc tcccattcgc cattcaggct gcgcaactgt 1200

tgggaagggc gtttcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg 1260

tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac 1320

gacggccagt gagcgcgacg taatacgact cactataggg cgaattgaag gaaggccgtc 1380

aaggccgcat gggaagccct cgaccgtcga ttgtccactg gtcaacaata gatgacttac 1440

aactaatcgg aaggtgcaga gactcgacgg gagctaccct aacgtcaaga cgagggtaaa 1500

gagagagtcc aattctcaaa gccaataggc agtagcgaaa gctgcaagag aatgaaaatc 1560

cgttgacctt aaacggtcgt gtgggttcaa gtccctccac ccccacgccg gaaacgcaat 1620

agccgaaaaa caaaaaacaa aaaaaacaaa aaaaaaacca aaaaaacaaa acacaacgtt 1680

actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc 1740

atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc 1800

attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag 1860

gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg 1920

cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat 1980

acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga 2040

gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc 2100

cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg 2160

ttaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat 2220

gataatagcc accatgtttg tttttcttgt tttattgcca ctagtctcta gtcagtgtgt 2280

taatcttaca accagaactc aattaccccc tgcatacact aattctttca cacgtggtgt 2340

ttattaccct gacaaagttt tcagatcctc agttttacat tcaactcagg atttgttctt 2400

acctttcttt tccaatgtta cttggttcca tgctatacat gtctctggga ccaatggtac 2460

taagaggttt gataaccctg tcctaccatt taatgatggt gtttattttg cttccactga 2520

gaagtctaac ataataagag gctggatctt tggtactact ttagattcga agacccagtc 2580

cctacttatt gttaataacg ctactaatgt tgttattaaa gtctgtgaat ttcaattttg 2640

taatgatcca tttttgggtg tttattacca caaaaacaac aaaagttgga tggaaagtga 2700

gttcagagtt tattctagtg cgaataattg cacttttgaa tatgtctctc agccttttct 2760

tatggacctt gaaggaaaac agggtaattt caaaaatctt agggaatttg tgtttaagaa 2820

tattgatggt tattttaaaa tatattctaa gcacacgcct attaatttag tgcgtgatct 2880

ccctcagggt ttttcggctt tagaaccatt ggtagatttg ccaataggta ttaacatcac 2940

taggtttcaa actttacttg ctttacatag aagttatttg actcctggtg attcttcttc 3000

aggttggaca gctggtgctg cagcttatta tgtgggttat cttcaaccta ggacttttct 3060

attaaaatat aatgaaaatg gaaccattac agatgctgta gactgtgcac ttgaccctct 3120

ctcagaaaca aagtgtacgt tgaaatcctt cactgtagaa aaaggaatct atcaaacttc 3180

taactttaga gtccaaccaa cagaatctat tgttagattt cctaatatta caaacttgtg 3240

cccttttggt gaagttttta acgccaccag atttgcatct gtttatgctt ggaacaggaa 3300

gagaatcagc aactgtgttg ctgattattc tgtcctatat aattccgcat cattttccac 3360

ttttaagtgt tatggagtgt ctcctactaa attaaatgat ctctgcttta ctaatgtcta 3420

tgcagattca tttgtaatta gaggtgatga agtcagacaa atcgctccag ggcaaactgg 3480

aaagattgct gattataatt ataaattacc agatgatttt acaggctgcg ttatagcttg 3540

gaattctaac aatcttgatt ctaaggttgg tggtaattat aattacctgt atagattgtt 3600

taggaagtct aatctcaaac cttttgagag agatatttca actgaaatct atcaggccgg 3660

tagcacacct tgtaatggtg ttgaaggttt taattgttac tttcctttac aatcatatgg 3720

tttccaaccc actaatggtg ttggttacca accatacaga gtagtagtac tttcttttga 3780

acttctacat gcaccagcaa ctgtttgtgg acctaaaaag tctactaatt tggttaaaaa 3840

caaatgtgtc aatttcaact tcaatggttt aacaggcaca ggtgttctta ctgagtctaa 3900

caaaaagttt ctgcctttcc aacaatttgg cagagacatt gctgacacta ctgatgctgt 3960

ccgtgatcca cagacacttg agattcttga cattacacca tgttcttttg gtggtgtcag 4020

tgttataaca ccaggaacaa atacttctaa ccaggttgct gttctttatc aggatgttaa 4080

ctgcacagaa gtccctgttg ctattcatgc agatcaactt actcctactt ggcgtgttta 4140

ttctacaggt tctaatgttt ttcaaacacg tgcaggctgt ttaatagggg ctgaacatgt 4200

caacaactca tatgagtgtg acatacccat tggtgcaggt atatgcgcta gttatcagac 4260

tcagactaat tctcctggca gcgccagcag tgtagctagt caatccatca ttgcctacac 4320

tatgtcactt ggtgcagaaa attcagttgc ttactctaat aactctattg ccatacccac 4380

aaattttact attagtgtta ccacagaaat tctaccagtg tctatgacca agacatcagt 4440

agattgtaca atgtacattt gtggtgattc aactgaatgc agcaatcttt tgttgcaata 4500

tggcagtttt tgtacacaat taaaccgtgc tttaactggg atagctgttg aacaagacaa 4560

aaacacccaa gaagtttttg cacaagtcaa acaaatttac aaaacaccac caattaaaga 4620

ttttggtggt tttaattttt cacaaatatt accagatcca tcaaaaccaa gcaagaggtc 4680

atttattgaa gatctacttt tcaacaaagt gacacttgca gatgctggct tcatcaaaca 4740

atatggtgat tgccttggtg atattgctgc tagggacctc atttgtgcac aaaagtttaa 4800

cggccttact gttttgccac ctttgctcac agatgaaatg attgctcaat acacttctgc 4860

actgttagcg ggtacaatca cttctggttg gacctttggt gcaggtgctg cattacaaat 4920

accatttgct atgcaaatgg cttataggtt taatggtatt ggagttacac agaatgttct 4980

ctatgagaac caaaaattga ttgccaacca atttaatagt gccattggca aaattcaaga 5040

ctcactttct tccacagcaa gtgcacttgg aaaacttcaa gatgtggtca accaaaatgc 5100

acaagcttta aacacgcttg ttaaacaact tagctccaat tttggtgcaa tttcaagtgt 5160

tttaaatgat atcctttcac gtcttgaccc tcccgaggct gaagtgcaaa ttgataggtt 5220

gatcacaggc agacttcaaa gtttgcagac atatgtgact caacaattaa ttagagctgc 5280

agaaatcaga gcttctgcta atcttgctgc tactaaaatg tcagagtgtg tacttggaca 5340

atcaaaaaga gttgattttt gtggaaaggg ctatcatctt atgtccttcc ctcagtcagc 5400

acctcatggt gtagtcttct tgcatgtgac ttatgtccct gcacaagaaa agaacttcac 5460

aactgctcct gccatttgtc atgatggaaa agcacacttt cctcgtgaag gtgtctttgt 5520

ttcaaatggc acacactggt ttgtaacaca aaggaatttt tatgaaccac aaatcattac 5580

tacagacaac acatttgtgt ctggtaactg tgatgttgta ataggaattg tcaacaacac 5640

agtttatgat cctttgcaac ctgaattaga ctcattcaag gaggagttag ataaatattt 5700

taagaatcat acatcaccag atgttgattt aggtgacatc tctggcatta atgcttcagt 5760

tgtaaacatt caaaaagaaa ttgaccgcct caatgaggtt gccaagaatt taaatgaatc 5820

tctcatcgat ctccaagaac ttggaaagta tgagcagggg tatatccctg aagcccccag 5880

ggacggccag gcttacgtca gaaaggatgg agagtgggtg ctcttgagca ccttcctgta 5940

aaaaaaacaa aaaacaaaac ggctattatg cgttaccggc gagacgctac ggacttaaat 6000

aattgagcct taaagaagaa attctttaag tggatgctct caaactcagg gaaacctaaa 6060

tctagttata gacaaggcaa tcctgagcca agccgaagta gtaattagta agaccagtgg 6120

acaatcgacg gataacagca tatctagctg ggcctcatgg gccttccttt cactgcccgc 6180

tttccagtcg ggaaacctgt cgtgccagct gcattaacat ggtcatagct gtttccttgc 6240

gtattgggcg ctctccgctt cctcgctcac tgactcgctg cgctcggtcg ttcgggtaaa 6300

gcctggggtg cctaatgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 6360

ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 6420

agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 6480

tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 6540

ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 6600

gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 6660

ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 6720

gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 6780

aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 6840

aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 6900

ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 6960

gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 7020

gggattttgg tcatg 7035

<210> 19

<211> 3708

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 19

atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60

agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120

aaagttttca gatcctcagt tttacattca actcaggatt tgttcttacc tttcttttcc 180

aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240

aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300

ataagaggct ggatctttgg tactacttta gattcgaaga cccagtccct acttattgtt 360

aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420

ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480

tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540

ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600

tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660

tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720

ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780

ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840

gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900

tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020

gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320

cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440

aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500

aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620

ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680

cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740

acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800

ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860

cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920

aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980

gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040

cctggcagcg ccagcagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100

gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160

agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220

tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280

acacaattaa accgtgcttt aactgggata gctgttgaac aagacaaaaa cacccaagaa 2340

gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400

aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460

ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520

cttggtgata ttgctgctag ggacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580

ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640

acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700

caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760

aaattgattg ccaaccaatt taatagtgcc attggcaaaa ttcaagactc actttcttcc 2820

acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880

acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940

ctttcacgtc ttgaccctcc cgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000

cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060

tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120

gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180

gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240

atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300

cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360

tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420

ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480

tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540

aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600

caagaacttg gaaagtatga gcaggggtat atccctgaag cccccaggga cggccaggct 3660

tacgtcagaa aggatggaga gtgggtgctc ttgagcacct tcctgtaa 3708

<210> 20

<211> 7149

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 20

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 60

atctaaagta tatatgagta aacttggtct gacagttatt agaaaaattc atccagcaga 120

cgataaaacg caatacgctg gctatccggt gccgcaatgc catacagcac cagaaaacga 180

tccgcccatt cgccgcccag ttcttccgca atatcacggg tggccagcgc aatatcctga 240

taacgatccg ccacgcccag acggccgcaa tcaataaagc cgctaaaacg gccattttcc 300

accataatgt tcggcaggca cgcatcacca tgggtcacca ccagatcttc gccatccggc 360

atgctcgctt tcagacgcgc aaacagctct gccggtgcca ggccctgatg ttcttcatcc 420

agatcatcct gatccaccag gcccgcttcc atacgggtac gcgcacgttc aatacgatgt 480

ttcgcctgat gatcaaacgg acaggtcgcc gggtccaggg tatgcagacg acgcatggca 540

tccgccataa tgctcacttt ttctgccggc gccagatggc tagacagcag atcctgaccc 600

ggcacttcgc ccagcagcag ccaatcacgg cccgcttcgg tcaccacatc cagcaccgcc 660

gcacacggaa caccggtggt ggccagccag ctcagacgcg ccgcttcatc ctgcagctcg 720

ttcagcgcac cgctcagatc ggttttcaca aacagcaccg gacgaccctg cgcgctcaga 780

cgaaacaccg ccgcatcaga gcagccaatg gtctgctgcg cccaatcata gccaaacaga 840

cgttccaccc acgctgccgg gctacccgca tgcaggccat cctgttcaat catactcttc 900

ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 960

gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 1020

cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct 1080

cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg 1140

agatagggtt gagtggccgc tacagggcgc tcccattcgc cattcaggct gcgcaactgt 1200

tgggaagggc gtttcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg 1260

tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac 1320

gacggccagt gagcgcgacg taatacgact cactataggg cgaattgaag gaaggccgtc 1380

aaggccgcat gggaagccct cgaccgtcga ttgtccactg gtcaacaata gatgacttac 1440

aactaatcgg aaggtgcaga gactcgacgg gagctaccct aacgtcaaga cgagggtaaa 1500

gagagagtcc aattctcaaa gccaataggc agtagcgaaa gctgcaagag aatgaaaatc 1560

cgttgacctt aaacggtcgt gtgggttcaa gtccctccac ccccacgccg gaaacgcaat 1620

agccgaaaaa caaaaaacaa aaaaaacaaa aaaaaaacca aaaaaacaaa acacaacgtt 1680

actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc 1740

atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc 1800

attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag 1860

gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg 1920

cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat 1980

acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga 2040

gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc 2100

cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg 2160

ttaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat 2220

gataatagcc accatgtttg tttttcttgt tttattgcca ctagtctcta gtcagtgtgt 2280

taatcttaca accagaactc aattaccccc tgcatacact aattctttca cacgtggtgt 2340

ttattaccct gacaaagttt tcagatcctc agttttacat tcaactcagg atttgttctt 2400

acctttcttt tccaatgtta cttggttcca tgctatacat gtctctggga ccaatggtac 2460

taagaggttt gataaccctg tcctaccatt taatgatggt gtttattttg cttccactga 2520

gaagtctaac ataataagag gctggatctt tggtactact ttagattcga agacccagtc 2580

cctacttatt gttaataacg ctactaatgt tgttattaaa gtctgtgaat ttcaattttg 2640

taatgatcca tttttgggtg tttattacca caaaaacaac aaaagttgga tggaaagtga 2700

gttcagagtt tattctagtg cgaataattg cacttttgaa tatgtctctc agccttttct 2760

tatggacctt gaaggaaaac agggtaattt caaaaatctt agggaatttg tgtttaagaa 2820

tattgatggt tattttaaaa tatattctaa gcacacgcct attaatttag tgcgtgatct 2880

ccctcagggt ttttcggctt tagaaccatt ggtagatttg ccaataggta ttaacatcac 2940

taggtttcaa actttacttg ctttacatag aagttatttg actcctggtg attcttcttc 3000

aggttggaca gctggtgctg cagcttatta tgtgggttat cttcaaccta ggacttttct 3060

attaaaatat aatgaaaatg gaaccattac agatgctgta gactgtgcac ttgaccctct 3120

ctcagaaaca aagtgtacgt tgaaatcctt cactgtagaa aaaggaatct atcaaacttc 3180

taactttaga gtccaaccaa cagaatctat tgttagattt cctaatatta caaacttgtg 3240

cccttttggt gaagttttta acgccaccag atttgcatct gtttatgctt ggaacaggaa 3300

gagaatcagc aactgtgttg ctgattattc tgtcctatat aattccgcat cattttccac 3360

ttttaagtgt tatggagtgt ctcctactaa attaaatgat ctctgcttta ctaatgtcta 3420

tgcagattca tttgtaatta gaggtgatga agtcagacaa atcgctccag ggcaaactgg 3480

aaagattgct gattataatt ataaattacc agatgatttt acaggctgcg ttatagcttg 3540

gaattctaac aatcttgatt ctaaggttgg tggtaattat aattacctgt atagattgtt 3600

taggaagtct aatctcaaac cttttgagag agatatttca actgaaatct atcaggccgg 3660

tagcacacct tgtaatggtg ttgaaggttt taattgttac tttcctttac aatcatatgg 3720

tttccaaccc actaatggtg ttggttacca accatacaga gtagtagtac tttcttttga 3780

acttctacat gcaccagcaa ctgtttgtgg acctaaaaag tctactaatt tggttaaaaa 3840

caaatgtgtc aatttcaact tcaatggttt aacaggcaca ggtgttctta ctgagtctaa 3900

caaaaagttt ctgcctttcc aacaatttgg cagagacatt gctgacacta ctgatgctgt 3960

ccgtgatcca cagacacttg agattcttga cattacacca tgttcttttg gtggtgtcag 4020

tgttataaca ccaggaacaa atacttctaa ccaggttgct gttctttatc aggatgttaa 4080

ctgcacagaa gtccctgttg ctattcatgc agatcaactt actcctactt ggcgtgttta 4140

ttctacaggt tctaatgttt ttcaaacacg tgcaggctgt ttaatagggg ctgaacatgt 4200

caacaactca tatgagtgtg acatacccat tggtgcaggt atatgcgcta gttatcagac 4260

tcagactaat tctcctggca gcgccagcag tgtagctagt caatccatca ttgcctacac 4320

tatgtcactt ggtgcagaaa attcagttgc ttactctaat aactctattg ccatacccac 4380

aaattttact attagtgtta ccacagaaat tctaccagtg tctatgacca agacatcagt 4440

agattgtaca atgtacattt gtggtgattc aactgaatgc agcaatcttt tgttgcaata 4500

tggcagtttt tgtacacaat taaaccgtgc tttaactggg atagctgttg aacaagacaa 4560

aaacacccaa gaagtttttg cacaagtcaa acaaatttac aaaacaccac caattaaaga 4620

ttttggtggt tttaattttt cacaaatatt accagatcca tcaaaaccaa gcaagaggtc 4680

atttattgaa gatctacttt tcaacaaagt gacacttgca gatgctggct tcatcaaaca 4740

atatggtgat tgccttggtg atattgctgc tagggacctc atttgtgcac aaaagtttaa 4800

cggccttact gttttgccac ctttgctcac agatgaaatg attgctcaat acacttctgc 4860

actgttagcg ggtacaatca cttctggttg gacctttggt gcaggtgctg cattacaaat 4920

accatttgct atgcaaatgg cttataggtt taatggtatt ggagttacac agaatgttct 4980

ctatgagaac caaaaattga ttgccaacca atttaatagt gccattggca aaattcaaga 5040

ctcactttct tccacagcaa gtgcacttgg aaaacttcaa gatgtggtca accaaaatgc 5100

acaagcttta aacacgcttg ttaaacaact tagctccaat tttggtgcaa tttcaagtgt 5160

tttaaatgat atcctttcac gtcttgaccc tcccgaggct gaagtgcaaa ttgataggtt 5220

gatcacaggc agacttcaaa gtttgcagac atatgtgact caacaattaa ttagagctgc 5280

agaaatcaga gcttctgcta atcttgctgc tactaaaatg tcagagtgtg tacttggaca 5340

atcaaaaaga gttgattttt gtggaaaggg ctatcatctt atgtccttcc ctcagtcagc 5400

acctcatggt gtagtcttct tgcatgtgac ttatgtccct gcacaagaaa agaacttcac 5460

aactgctcct gccatttgtc atgatggaaa agcacacttt cctcgtgaag gtgtctttgt 5520

ttcaaatggc acacactggt ttgtaacaca aaggaatttt tatgaaccac aaatcattac 5580

tacagacaac acatttgtgt ctggtaactg tgatgttgta ataggaattg tcaacaacac 5640

agtttatgat cctttgcaac ctgaattaga ctcattcaag gaggagttag ataaatattt 5700

taagaatcat acatcaccag atgttgattt aggtgacatc tctggcatta atgcttcagt 5760

tgtaaacatt caaaaagaaa ttgaccgcct caatgaggtt gccaagaatt taaatgaatc 5820

tctcatcgat ctccaagaac ttggaaagta tgagcagtat ataaaatggc catggtacat 5880

ttggctaggt tttatagctg gcttgattgc catagtaatg gtgacaatta tgctttgctg 5940

tatgaccagt tgctgtagtt gtctcaaggg ctgttgttct tgtggatcct gctgcaaatt 6000

tgatgaagac gactctgagc cagtgctcaa aggagtcaaa ttacattaca cataaaaaaa 6060

acaaaaaaca aaacggctat tatgcgttac cggcgagacg ctacggactt aaataattga 6120

gccttaaaga agaaattctt taagtggatg ctctcaaact cagggaaacc taaatctagt 6180

tatagacaag gcaatcctga gccaagccga agtagtaatt agtaagacca gtggacaatc 6240

gacggataac agcatatcta gctgggcctc atgggccttc ctttcactgc ccgctttcca 6300

gtcgggaaac ctgtcgtgcc agctgcatta acatggtcat agctgtttcc ttgcgtattg 6360

ggcgctctcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggg taaagcctgg 6420

ggtgcctaat gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 6480

gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 6540

aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 6600

gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 6660

ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 6720

cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 6780

ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 6840

actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 6900

tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 6960

gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 7020

ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 7080

cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 7140

ttggtcatg 7149

<210> 21

<211> 3822

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 21

atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60

agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120

aaagttttca gatcctcagt tttacattca actcaggatt tgttcttacc tttcttttcc 180

aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240

aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300

ataagaggct ggatctttgg tactacttta gattcgaaga cccagtccct acttattgtt 360

aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420

ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480

tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540

ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600

tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660

tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720

ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780

ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840

gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900

tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020

gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320

cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440

aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500

aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620

ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680

cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740

acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800

ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860

cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920

aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980

gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040

cctggcagcg ccagcagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100

gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160

agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220

tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280

acacaattaa accgtgcttt aactgggata gctgttgaac aagacaaaaa cacccaagaa 2340

gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400

aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460

ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520

cttggtgata ttgctgctag ggacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580

ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640

acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700

caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760

aaattgattg ccaaccaatt taatagtgcc attggcaaaa ttcaagactc actttcttcc 2820

acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880

acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940

ctttcacgtc ttgaccctcc cgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000

cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060

tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120

gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180

gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240

atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300

cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360

tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420

ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480

tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540

aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600

caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660

atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc 3720

tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac 3780

tctgagccag tgctcaaagg agtcaaatta cattacacat aa 3822

<210> 22

<211> 7038

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 22

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 60

atctaaagta tatatgagta aacttggtct gacagttatt agaaaaattc atccagcaga 120

cgataaaacg caatacgctg gctatccggt gccgcaatgc catacagcac cagaaaacga 180

tccgcccatt cgccgcccag ttcttccgca atatcacggg tggccagcgc aatatcctga 240

taacgatccg ccacgcccag acggccgcaa tcaataaagc cgctaaaacg gccattttcc 300

accataatgt tcggcaggca cgcatcacca tgggtcacca ccagatcttc gccatccggc 360

atgctcgctt tcagacgcgc aaacagctct gccggtgcca ggccctgatg ttcttcatcc 420

agatcatcct gatccaccag gcccgcttcc atacgggtac gcgcacgttc aatacgatgt 480

ttcgcctgat gatcaaacgg acaggtcgcc gggtccaggg tatgcagacg acgcatggca 540

tccgccataa tgctcacttt ttctgccggc gccagatggc tagacagcag atcctgaccc 600

ggcacttcgc ccagcagcag ccaatcacgg cccgcttcgg tcaccacatc cagcaccgcc 660

gcacacggaa caccggtggt ggccagccag ctcagacgcg ccgcttcatc ctgcagctcg 720

ttcagcgcac cgctcagatc ggttttcaca aacagcaccg gacgaccctg cgcgctcaga 780

cgaaacaccg ccgcatcaga gcagccaatg gtctgctgcg cccaatcata gccaaacaga 840

cgttccaccc acgctgccgg gctacccgca tgcaggccat cctgttcaat catactcttc 900

ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 960

gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 1020

cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct 1080

cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg 1140

agatagggtt gagtggccgc tacagggcgc tcccattcgc cattcaggct gcgcaactgt 1200

tgggaagggc gtttcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg 1260

tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac 1320

gacggccagt gagcgcgacg taatacgact cactataggg cgaattgaag gaaggccgtc 1380

aaggccgcat gggaagccct cgaccgtcga ttgtccactg gtcaacaata gatgacttac 1440

aactaatcgg aaggtgcaga gactcgacgg gagctaccct aacgtcaaga cgagggtaaa 1500

gagagagtcc aattctcaaa gccaataggc agtagcgaaa gctgcaagag aatgaaaatc 1560

cgttgacctt aaacggtcgt gtgggttcaa gtccctccac ccccacgccg gaaacgcaat 1620

agccgaaaaa caaaaaacaa aaaaaacaaa aaaaaaacca aaaaaacaaa acacaacgtt 1680

actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc 1740

atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc 1800

attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag 1860

gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg 1920

cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat 1980

acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga 2040

gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc 2100

cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg 2160

ttaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat 2220

gataatagcc accatgtttg tttttcttgt tttattgcca ctagtctcta gtcagtgtgt 2280

taatcttaca accagaactc aattaccccc tgcatacact aattctttca cacgtggtgt 2340

ttattaccct gacaaagttt tcagatcctc agttttacat tcaactcagg atttgttctt 2400

acctttcttt tccaatgtta cttggttcca tgctatacat gtctctggga ccaatggtac 2460

taagaggttt gataaccctg tcctaccatt taatgatggt gtttattttg cttccactga 2520

gaagtctaac ataataagag gctggatctt tggtactact ttagattcga agacccagtc 2580

cctacttatt gttaataacg ctactaatgt tgttattaaa gtctgtgaat ttcaattttg 2640

taatgatcca tttttgggtg tttattacca caaaaacaac aaaagttgga tggaaagtga 2700

gttcagagtt tattctagtg cgaataattg cacttttgaa tatgtctctc agccttttct 2760

tatggacctt gaaggaaaac agggtaattt caaaaatctt agggaatttg tgtttaagaa 2820

tattgatggt tattttaaaa tatattctaa gcacacgcct attaatttag tgcgtgatct 2880

ccctcagggt ttttcggctt tagaaccatt ggtagatttg ccaataggta ttaacatcac 2940

taggtttcaa actttacttg ctttacatag aagttatttg actcctggtg attcttcttc 3000

aggttggaca gctggtgctg cagcttatta tgtgggttat cttcaaccta ggacttttct 3060

attaaaatat aatgaaaatg gaaccattac agatgctgta gactgtgcac ttgaccctct 3120

ctcagaaaca aagtgtacgt tgaaatcctt cactgtagaa aaaggaatct atcaaacttc 3180

taactttaga gtccaaccaa cagaatctat tgttagattt cctaatatta caaacttgtg 3240

cccttttggt gaagttttta acgccaccag atttgcatct gtttatgctt ggaacaggaa 3300

gagaatcagc aactgtgttg ctgattattc tgtcctatat aattccgcat cattttccac 3360

ttttaagtgt tatggagtgt ctcctactaa attaaatgat ctctgcttta ctaatgtcta 3420

tgcagattca tttgtaatta gaggtgatga agtcagacaa atcgctccag ggcaaactgg 3480

aaagattgct gattataatt ataaattacc agatgatttt acaggctgcg ttatagcttg 3540

gaattctaac aatcttgatt ctaaggttgg tggtaattat aattacctgt atagattgtt 3600

taggaagtct aatctcaaac cttttgagag agatatttca actgaaatct atcaggccgg 3660

tagcacacct tgtaatggtg ttgaaggttt taattgttac tttcctttac aatcatatgg 3720

tttccaaccc actaatggtg ttggttacca accatacaga gtagtagtac tttcttttga 3780

acttctacat gcaccagcaa ctgtttgtgg acctaaaaag tctactaatt tggttaaaaa 3840

caaatgtgtc aatttcaact tcaatggttt aacaggcaca ggtgttctta ctgagtctaa 3900

caaaaagttt ctgcctttcc aacaatttgg cagagacatt gctgacacta ctgatgctgt 3960

ccgtgatcca cagacacttg agattcttga cattacacca tgttcttttg gtggtgtcag 4020

tgttataaca ccaggaacaa atacttctaa ccaggttgct gttctttatc aggatgttaa 4080

ctgcacagaa gtccctgttg ctattcatgc agatcaactt actcctactt ggcgtgttta 4140

ttctacaggt tctaatgttt ttcaaacacg tgcaggctgt ttaatagggg ctgaacatgt 4200

caacaactca tatgagtgtg acatacccat tggtgcaggt atatgcgcta gttatcagac 4260

tcagactaat tctcctggca gcgccagcag tgtagctagt caatccatca ttgcctacac 4320

tatgtcactt ggtgcagaaa attcagttgc ttactctaat aactctattg ccatacccac 4380

aaattttact attagtgtta ccacagaaat tctaccagtg tctatgacca agacatcagt 4440

agattgtaca atgtacattt gtggtgattc aactgaatgc agcaatcttt tgttgcaata 4500

tggcagtttt tgtacacaat taaaccgtgc tttaactggg atagctgttg aacaagacaa 4560

aaacacccaa gaagtttttg cacaagtcaa acaaatttac aaaacaccac caattaaaga 4620

ttttggtggt tttaattttt cacaaatatt accagatcca tcaaaaccaa gcaagaggtc 4680

atttattgaa gatctacttt tcaacaaagt gacacttgca gatgctggct tcatcaaaca 4740

atatggtgat tgccttggtg atattgctgc tagggacctc atttgtgcac aaaagtttaa 4800

cggccttact gttttgccac ctttgctcac agatgaaatg attgctcaat acacttctgc 4860

actgttagcg ggtacaatca cttctggttg gacctttggt gcaggtgctg cattacaaat 4920

accatttgct atgcaaatgg cttataggtt taatggtatt ggagttacac agaatgttct 4980

ctatgagaac caaaaattga ttgccaacca atttaatagt gccattggca aaattcaaga 5040

ctcactttct tccacagcaa gtgcacttgg aaaacttcaa gatgtggtca accaaaatgc 5100

acaagcttta aacacgcttg ttaaacaact tagctccaat tttggtgcaa tttcaagtgt 5160

tttaaatgat atcctttcac gtcttgaccc tcccgaggct gaagtgcaaa ttgataggtt 5220

gatcacaggc agacttcaaa gtttgcagac atatgtgact caacaattaa ttagagctgc 5280

agaaatcaga gcttctgcta atcttgctgc tactaaaatg tcagagtgtg tacttggaca 5340

atcaaaaaga gttgattttt gtggaaaggg ctatcatctt atgtccttcc ctcagtcagc 5400

acctcatggt gtagtcttct tgcatgtgac ttatgtccct gcacaagaaa agaacttcac 5460

aactgctcct gccatttgtc atgatggaaa agcacacttt cctcgtgaag gtgtctttgt 5520

ttcaaatggc acacactggt ttgtaacaca aaggaatttt tatgaaccac aaatcattac 5580

tacagacaac acatttgtgt ctggtaactg tgatgttgta ataggaattg tcaacaacac 5640

agtttatgat cctttgcaac ctgaattaga ctcattcaag gaggagttag ataaatattt 5700

taagaatcat acatcaccag atgttgattt aggtgacatc tctggcatta atgcttcagt 5760

tgtaaacatt caaaaagaaa ttgaccgcct caatgaggtt gccaagaatt taaatgaatc 5820

tctcatcgat ctccaagaac ttggaaagta tgagcagtat ataaaatggc catggtacat 5880

ttggctaggt tttatagctg gcttgattgc catagtaatg gtgacaatta tgctttgctg 5940

ttaaaaaaaa caaaaaacaa aacggctatt atgcgttacc ggcgagacgc tacggactta 6000

aataattgag ccttaaagaa gaaattcttt aagtggatgc tctcaaactc agggaaacct 6060

aaatctagtt atagacaagg caatcctgag ccaagccgaa gtagtaatta gtaagaccag 6120

tggacaatcg acggataaca gcatatctag ctgggcctca tgggccttcc tttcactgcc 6180

cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa catggtcata gctgtttcct 6240

tgcgtattgg gcgctctccg cttcctcgct cactgactcg ctgcgctcgg tcgttcgggt 6300

aaagcctggg gtgcctaatg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 6360

gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 6420

tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 6480

agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 6540

ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 6600

taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 6660

gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 6720

gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 6780

ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 6840

ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 6900

gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 6960

caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 7020

taagggattt tggtcatg 7038

<210> 23

<211> 3711

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 23

atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60

agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120

aaagttttca gatcctcagt tttacattca actcaggatt tgttcttacc tttcttttcc 180

aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240

aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300

ataagaggct ggatctttgg tactacttta gattcgaaga cccagtccct acttattgtt 360

aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420

ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480

tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540

ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600

tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660

tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720

ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780

ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840

gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900

tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020

gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320

cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440

aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500

aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620

ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680

cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740

acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800

ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860

cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920

aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980

gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040

cctggcagcg ccagcagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100

gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160

agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220

tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280

acacaattaa accgtgcttt aactgggata gctgttgaac aagacaaaaa cacccaagaa 2340

gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400

aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460

ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520

cttggtgata ttgctgctag ggacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580

ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640

acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700

caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760

aaattgattg ccaaccaatt taatagtgcc attggcaaaa ttcaagactc actttcttcc 2820

acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880

acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940

ctttcacgtc ttgaccctcc cgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000

cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060

tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120

gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180

gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240

atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300

cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360

tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420

ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480

tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540

aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600

caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660

atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtta a 3711

<210> 24

<211> 4044

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 24

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 60

atctaaagta tatatgagta aacttggtct gacagttatt agaaaaattc atccagcaga 120

cgataaaacg caatacgctg gctatccggt gccgcaatgc catacagcac cagaaaacga 180

tccgcccatt cgccgcccag ttcttccgca atatcacggg tggccagcgc aatatcctga 240

taacgatccg ccacgcccag acggccgcaa tcaataaagc cgctaaaacg gccattttcc 300

accataatgt tcggcaggca cgcatcacca tgggtcacca ccagatcttc gccatccggc 360

atgctcgctt tcagacgcgc aaacagctct gccggtgcca ggccctgatg ttcttcatcc 420

agatcatcct gatccaccag gcccgcttcc atacgggtac gcgcacgttc aatacgatgt 480

ttcgcctgat gatcaaacgg acaggtcgcc gggtccaggg tatgcagacg acgcatggca 540

tccgccataa tgctcacttt ttctgccggc gccagatggc tagacagcag atcctgaccc 600

ggcacttcgc ccagcagcag ccaatcacgg cccgcttcgg tcaccacatc cagcaccgcc 660

gcacacggaa caccggtggt ggccagccag ctcagacgcg ccgcttcatc ctgcagctcg 720

ttcagcgcac cgctcagatc ggttttcaca aacagcaccg gacgaccctg cgcgctcaga 780

cgaaacaccg ccgcatcaga gcagccaatg gtctgctgcg cccaatcata gccaaacaga 840

cgttccaccc acgctgccgg gctacccgca tgcaggccat cctgttcaat catactcttc 900

ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 960

gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 1020

cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct 1080

cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg 1140

agatagggtt gagtggccgc tacagggcgc tcccattcgc cattcaggct gcgcaactgt 1200

tgggaagggc gtttcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg 1260

tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac 1320

gacggccagt gagcgcgacg taatacgact cactataggg cgaattgaag gaaggccgtc 1380

aaggccgcat gggaagccct cgaccgtcga ttgtccactg gtcaacaata gatgacttac 1440

aactaatcgg aaggtgcaga gactcgacgg gagctaccct aacgtcaaga cgagggtaaa 1500

gagagagtcc aattctcaaa gccaataggc agtagcgaaa gctgcaagag aatgaaaatc 1560

cgttgacctt aaacggtcgt gtgggttcaa gtccctccac ccccacgccg gaaacgcaat 1620

agccgaaaaa caaaaaacaa aaaaaacaaa aaaaaaacca aaaaaacaaa acacaacgtt 1680

actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc 1740

atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc 1800

attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag 1860

gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg 1920

cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat 1980

acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga 2040

gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc 2100

cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg 2160

ttaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat 2220

gataatagcc accatgtttg tttttcttgt tttattgcca ctagtctcta gtcagtgtag 2280

agtccaacca acagaatcta ttgttagatt tcctaatatt acaaacttgt gcccttttgg 2340

tgaagttttt aacgccacca gatttgcatc tgtttatgct tggaacagga agagaatcag 2400

caactgtgtt gctgattatt ctgtcctata taattccgca tcattttcca cttttaagtg 2460

ttatggagtg tctcctacta aattaaatga tctctgcttt actaatgtct atgcagattc 2520

atttgtaatt agaggtgatg aagtcagaca aatcgctcca gggcaaactg gaaagattgc 2580

tgattataat tataaattac cagatgattt tacaggctgc gttatagctt ggaattctaa 2640

caatcttgat tctaaggttg gtggtaatta taattacctg tatagattgt ttaggaagtc 2700

taatctcaaa ccttttgaga gagatatttc aactgaaatc tatcaggccg gtagcacacc 2760

ttgtaatggt gttgaaggtt ttaattgtta ctttccttta caatcatatg gtttccaacc 2820

cactaatggt gttggttacc aaccatacag agtagtagta ctttcttttg aacttctaca 2880

tgcaccagca actgtttgtg gacctaaaaa gtctactaat ttggttaaaa acaaatgtgt 2940

caatttctaa aaaaaacaaa aaacaaaacg gctattatgc gttaccggcg agacgctacg 3000

gacttaaata attgagcctt aaagaagaaa ttctttaagt ggatgctctc aaactcaggg 3060

aaacctaaat ctagttatag acaaggcaat cctgagccaa gccgaagtag taattagtaa 3120

gaccagtgga caatcgacgg ataacagcat atctagctgg gcctcatggg ccttcctttc 3180

actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaacatg gtcatagctg 3240

tttccttgcg tattgggcgc tctccgcttc ctcgctcact gactcgctgc gctcggtcgt 3300

tcgggtaaag cctggggtgc ctaatgagca aaaggccagc aaaaggccag gaaccgtaaa 3360

aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 3420

cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 3480

cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 3540

gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 3600

tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 3660

cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 3720

ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 3780

gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 3840

gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 3900

accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 3960

ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 4020

tcacgttaag ggattttggt catg 4044

<210> 25

<211> 717

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 25

atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtagagt ccaaccaaca 60

gaatctattg ttagatttcc taatattaca aacttgtgcc cttttggtga agtttttaac 120

gccaccagat ttgcatctgt ttatgcttgg aacaggaaga gaatcagcaa ctgtgttgct 180

gattattctg tcctatataa ttccgcatca ttttccactt ttaagtgtta tggagtgtct 240

cctactaaat taaatgatct ctgctttact aatgtctatg cagattcatt tgtaattaga 300

ggtgatgaag tcagacaaat cgctccaggg caaactggaa agattgctga ttataattat 360

aaattaccag atgattttac aggctgcgtt atagcttgga attctaacaa tcttgattct 420

aaggttggtg gtaattataa ttacctgtat agattgttta ggaagtctaa tctcaaacct 480

tttgagagag atatttcaac tgaaatctat caggccggta gcacaccttg taatggtgtt 540

gaaggtttta attgttactt tcctttacaa tcatatggtt tccaacccac taatggtgtt 600

ggttaccaac catacagagt agtagtactt tcttttgaac ttctacatgc accagcaact 660

gtttgtggac ctaaaaagtc tactaatttg gttaaaaaca aatgtgtcaa tttctaa 717

<210> 26

<211> 717

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 26

atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtagagt ccaaccaaca 60

gaatctattg ttagatttcc taatattaca aacttgtgcc cttttggtga agtttttaac 120

gccaccagat ttgcatccgt gtatgcttgg aacaggaaga gaatcagcaa ctgtgttgct 180

gattattctg tcctatataa ttccgcatca ttttccactt ttaagtgtta tggagtgtct 240

cctactaaat taaatgatct ctgctttact aatgtctatg cagattcatt tgtaattaga 300

ggtgatgaag tcagacaaat cgctccaggg caaactggaa agattgctga ttataattat 360

aaattaccag atgattttac aggctgcgtt atagcttgga attctaacaa tcttgattct 420

aaggttggtg gtaattataa ttacctgtat agattgttta ggaagtctaa tctcaaacct 480

tttgagagag atatttcaac tgaaatctat caggccggta gcacaccttg taatggtgtt 540

gaaggtttta attgttactt tcctttacaa tcatatggtt tccaacccac taatggtgtt 600

ggttaccaac catacagagt agtagtactt tcttttgaac ttctacatgc accagcaact 660

gtttgtggac ctaaaaagtc tactaatttg gttaaaaaca aatgtgtcaa tttctaa 717

<210> 27

<211> 3822

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 27

atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60

agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120

aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180

aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240

aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300

ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360

aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420

ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480

tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540

ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600

tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660

tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720

ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780

ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840

gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900

tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020

gtttttaacg ccaccagatt tgcatccgtg tatgcttgga acaggaagag aatcagcaac 1080

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320

cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440

aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500

aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620

ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680

cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740

acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800

ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860

cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920

aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980

gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040

cctggcagcg ccagcagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100

gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160

agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220

tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280

acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 2340

gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400

aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460

ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520

cttggtgata ttgctgctag ggacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580

ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640

acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700

caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760

aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 2820

acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880

acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940

ctttcacgtc ttgaccctcc cgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000

cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060

tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120

gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180

gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240

atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300

cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360

tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420

ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480

tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540

aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600

caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660

atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc 3720

tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac 3780

tctgagccag tgctcaaagg agtcaaatta cattacacat aa 3822

<210> 28

<211> 3822

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 28

atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60

agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120

aaagttttca gatcctcagt tttacattca actcaggatt tgttcttacc tttcttttcc 180

aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240

aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300

ataagaggct ggatctttgg tactacttta gattcgaaga cccagtccct acttattgtt 360

aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420

ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480

tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540

ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600

tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660

tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720

ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780

ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840

gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900

tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020

gtttttaacg ccaccagatt tgcatccgtg tatgcttgga acaggaagag aatcagcaac 1080

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320

cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440

aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500

aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620

ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680

cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740

acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800

ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860

cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920

aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980

gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040

cctggcagcg ccagcagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100

gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160

agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220

tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280

acacaattaa accgtgcttt aactgggata gctgttgaac aagacaaaaa cacccaagaa 2340

gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400

aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460

ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520

cttggtgata ttgctgctag ggacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580

ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640

acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700

caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760

aaattgattg ccaaccaatt taatagtgcc attggcaaaa ttcaagactc actttcttcc 2820

acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880

acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940

ctttcacgtc ttgaccctcc cgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000

cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060

tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120

gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180

gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240

atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300

cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360

tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420

ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480

tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540

aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600

caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660

atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc 3720

tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac 3780

tctgagccag tgctcaaagg agtcaaatta cattacacat aa 3822

<210> 29

<211> 3822

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 29

atgttcgttt tccttgtttt actgcccctc gtgtcttcac agtgtgtgaa cctcaccact 60

cgcacacagc tacctcccgc gtacactaat tcatttacca ggggcgtcta ttatcctgat 120

aaggtgttcc ggagttcagt gttgcatagc actcaagacc tgttcctgcc cttcttctcc 180

aatgtcactt ggtttcatgc gatacatgtg tctggtacca acggaacgaa gagatttgat 240

aaccccgtac tgccattcaa tgatggcgta tactttgctt cgactgaaaa atccaacatc 300

atcaggggct ggatttttgg tacaacgctt gattccaaga cccagtccct ccttattgtg 360

aacaatgcga ccaacgtcgt gataaaggtc tgtgagtttc agttctgcaa tgacccattc 420

cttggagtgt attatcacaa gaacaacaaa tcgtggatgg agtcagagtt tagggtgtac 480

agcagcgcta acaactgtac atttgagtat gtgagtcagc cgtttctgat ggatcttgag 540

ggcaaacagg gcaattttaa gaatctcaga gaatttgtgt tcaagaacat tgatggttac 600

tttaagatct atagcaaaca tacgccaatc aacttggttc gtgatctgcc acagggattt 660

agcgcactgg aacctctcgt tgacttgccc ataggtatta acatcaccag gttccagacg 720

ctcttggcat tacaccgtag ttatctgacc cccggggact ccagttccgg atggactgca 780

ggagccgctg cctactatgt gggttacctc cagcccagga cctttctttt gaaatataac 840

gagaacggca caatcactga tgctgtggac tgcgcattgg atcctttgtc agagactaag 900

tgcactctga agtcattcac agtcgagaaa ggcatttacc agacgtctaa cttcagggtt 960

cagcctactg agtccatcgt gagattccca aacatcacaa atctttgtcc cttcggtgag 1020

gtattcaatg cgacacgatt tgcctcagtg tacgcgtgga atcggaagag gatctccaat 1080

tgcgtggccg actactccgt cttatacaac tcagctagct tttcaacatt caagtgctat 1140

ggcgtgagcc ctaccaagct caatgacctg tgcttcacta atgtgtatgc cgactctttt 1200

gtcattcgcg gcgacgaggt ccgacaaatc gcaccgggcc aaaccggtaa aattgccgac 1260

tacaactaca agctgcctga cgacttcacc ggctgcgtaa tcgcctggaa cagcaataac 1320

ctggatagca aagtgggcgg aaactacaac tacctgtacc ggctctttag aaagtccaac 1380

ctgaaaccat tcgagcgcga tatctcgacc gaaatctacc aggcgggcag caccccctgt 1440

aatggtgtag aagggttcaa ttgttacttt ccactccaga gttatgggtt ccagccgacc 1500

aatggcgtcg gttatcaacc atatcgcgtt gtggtgttgt cctttgagct gctacacgcc 1560

ccagctacag tgtgcgggcc aaagaaaagc acaaatcttg tcaagaacaa atgcgttaac 1620

tttaatttta atggactcac aggtacagga gtcctgaccg aatctaataa gaagttcctg 1680

ccctttcaac agttcggacg agacattgcc gacaccaccg atgccgttcg ggacccacag 1740

accttagaaa ttctggatat tactccatgt agttttgggg gagtgtctgt catcacccct 1800

ggcactaata catctaacca ggttgcagtc ctctaccagg atgtgaactg taccgaagtg 1860

ccggtcgcta ttcacgcaga ccagctcact cctacctggc gggtgtactc cacaggctct 1920

aacgtgtttc agacacgtgc agggtgccta atcggcgcag agcatgtaaa taactcctat 1980

gagtgtgata tccccatcgg agccgggatc tgcgcttcct accagacaca aacgaatagt 2040

cccggatctg cctcaagcgt ggcatctcaa tccattatag catatacgat gtcccttgga 2100

gctgaaaaca gcgttgcgta ttcaaacaat agtatcgcta ttccaaccaa ttttacaatt 2160

agcgtgacca cagaaatact ccctgtgagc atgaccaaga ccagtgtaga ctgtactatg 2220

tacatctgcg gcgacagtac tgagtgtagc aatctgctgc tacagtatgg gtccttctgt 2280

actcagctta atcgggctct caccggaatc gctgtagagc aagataaaaa cacacaagaa 2340

gtgtttgctc aagtgaagca gatctataag acacctccca tcaaggattt cggtgggttc 2400

aactttagcc agattctgcc cgatccgtct aaaccgtcca agcgaagttt catcgaagac 2460

ctgcttttca ataaggtcac gctggcagat gctggattta tcaaacagta cggcgactgt 2520

ctgggcgata tcgccgcaag agacttgata tgcgcccaaa agtttaatgg gttaaccgtc 2580

cttccaccgc tcctgacaga cgagatgatc gcccagtata caagtgcctt attagctggg 2640

accattacta gtggatggac atttggcgcc ggggctgctc tacagatacc cttcgccatg 2700

cagatggctt accgcttcaa cggaatcgga gttacccaga acgtactgta cgaaaatcag 2760

aaactcatag ctaatcaatt taactctgcc atcgggaaga ttcaggattc cctgtcgtct 2820

acagcgtccg ccttggggaa actgcaagat gtagtgaacc agaacgccca ggccttaaat 2880

actctggtca agcagttatc ttcaaatttc ggagcaatta gctctgtgtt gaacgatatt 2940

ctttccaggc tggaccctcc agaagccgaa gtgcaaatag accggctcat cacggggcgc 3000

ttgcaaagcc tgcaaaccta tgtcacccag caactgattc gagcagccga gatccgggcc 3060

agtgctaatc tggccgccac aaaaatgagc gagtgcgtcc tcgggcagag caaacgcgta 3120

gacttctgcg gtaaaggcta tcacctgatg agcttccctc agagcgcacc ccacggggtg 3180

gtcttcctcc acgttaccta cgtccctgcg caggagaaga acttcactac ggcccctgca 3240

atttgccacg atggcaaggc ccactttccc agggaggggg tcttcgtttc caacgggact 3300

cattggttcg tgactcagag aaatttttat gaacctcaga tcattaccac tgataataca 3360

ttcgtgtctg gcaactgtga tgtggttatt gggatagtta ataatacggt atacgaccca 3420

ctccagcccg agctggactc cttcaaagag gagctggaca agtactttaa aaatcacacc 3480

tcacctgatg tggacctagg tgacatatct ggcataaatg ctagcgtggt taacattcag 3540

aaggaaatcg acagactcaa cgaggtggcc aaaaatctga acgagagtct gatcgacctg 3600

caggagttgg gaaaatatga acagtacatc aaatggccat ggtacatctg gctgggcttc 3660

atagcaggcc tgatcgccat cgtcatggtg actattatgc tgtgctgcat gacatcctgt 3720

tgtagctgtt tgaaggggtg ttgctcctgc ggctcatgct gcaaattcga cgaggacgat 3780

tcagaacctg tgctgaaggg agtgaagctg cattacacat aa 3822

<210> 30

<211> 717

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 30

atgttcgttt tccttgtttt actgcccctc gtgtcttcac agtgtagggt tcagcctact 60

gagtccatcg tgagattccc aaacatcaca aatctttgtc ccttcggtga ggtattcaat 120

gcgacacgat ttgcctcagt gtacgcgtgg aatcggaaga ggatctccaa ttgcgtggcc 180

gactactccg tcttatacaa ctcagctagc ttttcaacat tcaagtgcta tggcgtgagc 240

cctaccaagc tcaatgacct gtgcttcact aatgtgtatg ccgactcttt tgtcattcgc 300

ggcgacgagg tccgacaaat cgcaccgggc caaaccggta aaattgccga ctacaactac 360

aagctgcctg acgacttcac cggctgcgta atcgcctgga acagcaataa cctggatagc 420

aaagtgggcg gaaactacaa ctacctgtac cggctcttta gaaagtccaa cctgaaacca 480

ttcgagcgcg atatctcgac cgaaatctac caggcgggca gcaccccctg taatggtgta 540

gaagggttca attgttactt tccactccag agttatgggt tccagccgac caatggcgtc 600

ggttatcaac catatcgcgt tgtggtgttg tcctttgagc tgctacacgc cccagctaca 660

gtgtgcgggc caaagaaaag cacaaatctt gtcaagaaca aatgcgttaa cttttaa 717

<210> 31

<211> 552

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 31

acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt 60

ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga 120

cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg 180

tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt 240

gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat 300

aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg 360

aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg 420

taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt 480

cgaggttaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac 540

acgatgataa ta 552

<210> 32

<211> 37

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 32

actcttctgg tccccacaga ctcagagaga acccacc 37

<210> 33

<211> 110

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 33

gctggagcct cggtagccgt tcctcctgcc cgctgggcct cccaacgggc cctcctcccc 60

tccttgcacc ggcccttcct ggtctttgaa taaagtctga gtgggcagca 110

<210> 34

<211> 330

<212> PRT

<213> Chile person

<400> 34

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 35

<211> 326

<212> PRT

<213> Chile person

<400> 35

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 36

<211> 377

<212> PRT

<213> Chile person

<400> 36

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro

100 105 110

Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg

115 120 125

Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys

130 135 140

Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

145 150 155 160

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

165 170 175

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

180 185 190

Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr

195 200 205

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

210 215 220

Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His

225 230 235 240

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

245 250 255

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln

260 265 270

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

275 280 285

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

290 295 300

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

305 310 315 320

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

325 330 335

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

340 345 350

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

355 360 365

Lys Ser Leu Ser Leu Ser Pro Gly Lys

370 375

<210> 37

<211> 327

<212> PRT

<213> Chile person

<400> 37

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 38

<211> 428

<212> PRT

<213> Chile person

<400> 38

Ala Ser Thr Gln Ser Pro Ser Val Phe Pro Leu Thr Arg Cys Cys Lys

1 5 10 15

Asn Ile Pro Ser Asn Ala Thr Ser Val Thr Leu Gly Cys Leu Ala Thr

20 25 30

Gly Tyr Phe Pro Glu Pro Val Met Val Thr Trp Asp Thr Gly Ser Leu

35 40 45

Asn Gly Thr Thr Met Thr Leu Pro Ala Thr Thr Leu Thr Leu Ser Gly

50 55 60

His Tyr Ala Thr Ile Ser Leu Leu Thr Val Ser Gly Ala Trp Ala Lys

65 70 75 80

Gln Met Phe Thr Cys Arg Val Ala His Thr Pro Ser Ser Thr Asp Trp

85 90 95

Val Asp Asn Lys Thr Phe Ser Val Cys Ser Arg Asp Phe Thr Pro Pro

100 105 110

Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly His Phe Pro

115 120 125

Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr

130 135 140

Ile Asn Ile Thr Trp Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu

145 150 155 160

Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser

165 170 175

Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp Arg Thr Tyr Thr

180 185 190

Cys Gln Val Thr Tyr Gln Gly His Thr Phe Glu Asp Ser Thr Lys Lys

195 200 205

Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro

210 215 220

Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr Cys Leu

225 230 235 240

Val Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu Thr Trp Ser

245 250 255

Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys Glu Glu Lys

260 265 270

Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr Leu Pro Val Gly Thr

275 280 285

Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val Thr His Pro

290 295 300

His Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr Ser Gly Pro

305 310 315 320

Arg Ala Ala Pro Glu Val Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly

325 330 335

Ser Arg Asp Lys Arg Thr Leu Ala Cys Leu Ile Gln Asn Phe Met Pro

340 345 350

Glu Asp Ile Ser Val Gln Trp Leu His Asn Glu Val Gln Leu Pro Asp

355 360 365

Ala Arg His Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly Ser Gly Phe

370 375 380

Phe Val Phe Ser Arg Leu Glu Val Thr Arg Ala Glu Trp Glu Gln Lys

385 390 395 400

Asp Glu Phe Ile Cys Arg Ala Val His Glu Ala Ala Ser Pro Ser Gln

405 410 415

Thr Val Gln Arg Ala Val Ser Val Asn Pro Gly Lys

420 425

<210> 39

<211> 353

<212> PRT

<213> Chile person

<400> 39

Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Cys Ser Thr

1 5 10 15

Gln Pro Asp Gly Asn Val Val Ile Ala Cys Leu Val Gln Gly Phe Phe

20 25 30

Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln Gly Val

35 40 45

Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp Leu Tyr

50 55 60

Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Leu Ala Gly

65 70 75 80

Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Pro Ser Gln Asp

85 90 95

Val Thr Val Pro Cys Pro Val Pro Ser Thr Pro Pro Thr Pro Ser Pro

100 105 110

Ser Thr Pro Pro Thr Pro Ser Pro Ser Cys Cys His Pro Arg Leu Ser

115 120 125

Leu His Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser Glu Ala Asn

130 135 140

Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly Val Thr Phe

145 150 155 160

Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly Pro Pro Glu

165 170 175

Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser Val Leu Pro Gly Cys

180 185 190

Ala Glu Pro Trp Asn His Gly Lys Thr Phe Thr Cys Thr Ala Ala Tyr

195 200 205

Pro Glu Ser Lys Thr Pro Leu Thr Ala Thr Leu Ser Lys Ser Gly Asn

210 215 220

Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser Glu Glu Leu

225 230 235 240

Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg Gly Phe Ser

245 250 255

Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln Glu Leu Pro

260 265 270

Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro Ser Gln Gly

275 280 285

Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala Ala Glu Asp

290 295 300

Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val Gly His Glu Ala Leu

305 310 315 320

Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Leu Ala Gly Lys Pro

325 330 335

Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp Gly Thr Cys

340 345 350

Tyr

<210> 40

<211> 340

<212> PRT

<213> Chile person

<400> 40

Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Asp Ser Thr

1 5 10 15

Pro Gln Asp Gly Asn Val Val Val Ala Cys Leu Val Gln Gly Phe Phe

20 25 30

Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln Asn Val

35 40 45

Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp Leu Tyr

50 55 60

Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Pro Asp Gly

65 70 75 80

Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Ser Ser Gln Asp

85 90 95

Val Thr Val Pro Cys Arg Val Pro Pro Pro Pro Pro Cys Cys His Pro

100 105 110

Arg Leu Ser Leu His Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser

115 120 125

Glu Ala Asn Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly

130 135 140

Ala Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly

145 150 155 160

Pro Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser Val Leu

165 170 175

Pro Gly Cys Ala Gln Pro Trp Asn His Gly Glu Thr Phe Thr Cys Thr

180 185 190

Ala Ala His Pro Glu Leu Lys Thr Pro Leu Thr Ala Asn Ile Thr Lys

195 200 205

Ser Gly Asn Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser

210 215 220

Glu Glu Leu Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg

225 230 235 240

Gly Phe Ser Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln

245 250 255

Glu Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro

260 265 270

Ser Gln Gly Thr Thr Thr Tyr Ala Val Thr Ser Ile Leu Arg Val Ala

275 280 285

Ala Glu Asp Trp Lys Lys Gly Glu Thr Phe Ser Cys Met Val Gly His

290 295 300

Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Met Ala

305 310 315 320

Gly Lys Pro Thr His Ile Asn Val Ser Val Val Met Ala Glu Ala Asp

325 330 335

Gly Thr Cys Tyr

340

<210> 41

<211> 453

<212> PRT

<213> Chile person

<400> 41

Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn

1 5 10 15

Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp

20 25 30

Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser

35 40 45

Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys

50 55 60

Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln

65 70 75 80

Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn

85 90 95

Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys

100 105 110

Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg

115 120 125

Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile

130 135 140

Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr

145 150 155 160

Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr

165 170 175

Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Gly Gln

180 185 190

Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln

195 200 205

Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val

210 215 220

Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr

225 230 235 240

Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr

245 250 255

Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn

260 265 270

Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala

275 280 285

Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr

290 295 300

Val Thr His Thr Asp Leu Pro Ser Pro Leu Lys Gln Thr Ile Ser Arg

305 310 315 320

Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro

325 330 335

Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu

340 345 350

Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg

355 360 365

Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro

370 375 380

Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val

385 390 395 400

Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala

405 410 415

His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser

420 425 430

Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr

435 440 445

Ala Gly Thr Cys Tyr

450

<210> 42

<211> 384

<212> PRT

<213> Chile person

<400> 42

Ala Pro Thr Lys Ala Pro Asp Val Phe Pro Ile Ile Ser Gly Cys Arg

1 5 10 15

His Pro Lys Asp Asn Ser Pro Val Val Leu Ala Cys Leu Ile Thr Gly

20 25 30

Tyr His Pro Thr Ser Val Thr Val Thr Trp Tyr Met Gly Thr Gln Ser

35 40 45

Gln Pro Gln Arg Thr Phe Pro Glu Ile Gln Arg Arg Asp Ser Tyr Tyr

50 55 60

Met Thr Ser Ser Gln Leu Ser Thr Pro Leu Gln Gln Trp Arg Gln Gly

65 70 75 80

Glu Tyr Lys Cys Val Val Gln His Thr Ala Ser Lys Ser Lys Lys Glu

85 90 95

Ile Phe Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro

100 105 110

Thr Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala

115 120 125

Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys

130 135 140

Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu

145 150 155 160

Cys Pro Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala

165 170 175

Val Gln Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val

180 185 190

Val Gly Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly

195 200 205

Lys Val Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser

210 215 220

Asn Gly Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu

225 230 235 240

Trp Asn Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu

245 250 255

Pro Pro Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro

260 265 270

Val Lys Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala

275 280 285

Ala Ser Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile

290 295 300

Leu Leu Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe

305 310 315 320

Ala Pro Ala Arg Pro Pro Pro Gln Pro Arg Ser Thr Thr Phe Trp Ala

325 330 335

Trp Ser Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr

340 345 350

Tyr Thr Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala

355 360 365

Ser Arg Ser Leu Glu Val Ser Tyr Val Thr Asp His Gly Pro Met Lys

370 375 380

<210> 43

<211> 106

<212> PRT

<213> Chile person

<400> 43

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

1 5 10 15

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

20 25 30

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

35 40 45

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

65 70 75 80

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

85 90 95

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 44

<211> 106

<212> PRT

<213> Chile person

<400> 44

Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro

35 40 45

Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

65 70 75 80

Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

85 90 95

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 45

<211> 741

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 45

ttaaaacagc ctgtgggttg atcccaccca caggcccatt gggcgctagc actctggtat 60

cacggtacct ttgtgcgcct gttttatacc ccctccccca actgtaactt agaagtaaca 120

cacaccgatc aacagtcagc gtggcacacc agccacgttt tgatcaagca cttctgttac 180

cccggactga gtatcaatag actgctcacg cggttgaagg agaaagcgtt cgttatccgg 240

ccaactactt cgaaaaacct agtaacaccg tggaagttgc agagtgtttc gctcagcact 300

accccagtgt agatcaggtc gatgagtcac cgcattcccc acgggcgacc gtggcggtgg 360

ctgcgttggc ggcctgccca tggggaaacc catgggacgc tctaatacag acatggtgcg 420

aagagtctat tgagctagtt ggtagtcctc cggcccctga atgcggctaa tcctaactgc 480

ggagcacaca ccctcaagcc agagggcagt gtgtcgtaac gggcaactct gcagcggaac 540

cgactacttt gggtgtccgt gtttcatttt attcctatac tggctgctta tggtgacaat 600

tgagagatcg ttaccatata gctattggat tggccatccg gtgactaata gagctattat 660

atatcccttt gttgggttta taccacttag cttgaaagag gttaaaacat tacaattcat 720

tgttaagttg aatacagcaa a 741

<210> 46

<211> 1229

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 46

cgcggatcct aatacgactc actataggga atagccgaaa aacaaaaaac aaaaaaaaca 60

aaaaaaaaac caaaaaaaca aaacacaacg ttactggccg aagccgcttg gaataaggcc 120

ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg 180

cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca 240

aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa 300

gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt 360

gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt 420

gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca 480

acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc 540

ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa cgtctaggcc ccccgaacca 600

cggggacgtg gttttccttt gaaaaacacg atgataatag ccaccatggg agtcaaagtt 660

ctgtttgccc tgatctgcat cgctgtggcc gaggccaagc ccaccgagaa caacgaagac 720

ttcaacatcg tggccgtggc cagcaacttc gcgaccacgg atctcgatgc tgaccgcggg 780

aagttgcccg gcaagaagct gccgctggag gtgctcaaag agatggaagc caatgcccgg 840

aaagctggct gcaccagggg ctgtctgatc tgcctgtccc acatcaagtg cacgcccaag 900

atgaagaagt tcatcccagg acgctgccac acctacgaag gcgacaaaga gtccgcacag 960

ggcggcatag gcgaggcgat cgtcgacatt cctgagattc ctgggttcaa ggacttggag 1020

cccatggagc agttcatcgc acaggtcgat ctgtgtgtgg actgcacaac tggctgcctc 1080

aaagggcttg ccaacgtgca gtgttctgac ctgctcaaga agtggctgcc gcaacgctgt 1140

gcgacctttg ccagcaagat ccagggccag gtggacaaga tcaagggggc cggtggtgac 1200

taaaaaaaac aaaaaacaaa acggctatt 1229

<210> 47

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 47

gtttttcggc tattcccaat agccgttttg 30

<210> 48

<211> 732

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 48

atgtaccgga tgcagttgtt gtcctgtata gctctttccc ttgcattggt cactaattct 60

agagtccaac caacagaatc tattgttaga tttcctaata ttacaaactt gtgccctttt 120

ggtgaagttt ttaacgccac cagatttgca tccgtgtatg cttggaacag gaagagaatc 180

agcaactgtg ttgctgatta ttctgtccta tataattccg catcattttc cacttttaag 240

tgttatggag tgtctcctac taaattaaat gatctctgct ttactaatgt ctatgcagat 300

tcatttgtaa ttagaggtga tgaagtcaga caaatcgctc cagggcaaac tggaaagatt 360

gctgattata attataaatt accagatgat tttacaggct gcgttatagc ttggaattct 420

aacaatcttg attctaaggt tggtggtaat tataattacc tgtatagatt gtttaggaag 480

tctaatctca aaccttttga gagagatatt tcaactgaaa tctatcaggc cggtagcaca 540

ccttgtaatg gtgttgaagg ttttaattgt tactttcctt tacaatcata tggtttccaa 600

cccactaatg gtgttggtta ccaaccatac agagtagtag tactttcttt tgaacttcta 660

catgcaccag caactgtttg tggacctaaa aagtctacta atttggttaa aaacaaatgt 720

gtcaatttct aa 732

<210> 49

<211> 726

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 49

atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagagagtc 60

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 120

gtttttaacg ccaccagatt tgcatccgtg tatgcttgga acaggaagag aatcagcaac 180

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 240

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 300

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 360

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 420

cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 480

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 540

aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 600

aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 660

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 720

ttctaa 726

<210> 50

<211> 60

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 50

atgtaccgga tgcagttgtt gtcctgtata gctctttccc ttgcattggt cactaattct 60

<210> 51

<211> 54

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 51

atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caag 54

<210> 52

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<220>

<221> feature not yet classified

<222> (2)..(2)

<223> Xaa is Val or Ile

<220>

<221> feature not yet classified

<222> (4)..(4)

<223> Xaa can be any naturally occurring amino acid

<400> 52

Asp Xaa Glu Xaa Asn Pro Gly Pro

1 5

<210> 53

<211> 1273

<212> PRT

<213> Severe acute respiratory syndrome coronavirus 2

<400> 53

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

675 680 685

Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

705 710 715 720

Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Val Gln

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

1025 1030 1035

Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

1055 1060 1065

Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn

1085 1090 1095

Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln

1100 1105 1110

Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

1115 1120 1125

Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro

1130 1135 1140

Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1145 1150 1155

His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn

1160 1165 1170

Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu

1175 1180 1185

Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu

1190 1195 1200

Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu

1205 1210 1215

Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met

1220 1225 1230

Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys

1235 1240 1245

Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro

1250 1255 1260

Val Leu Lys Gly Val Lys Leu His Tyr Thr

1265 1270

<210> 54

<211> 3819

<212> DNA

<213> Severe acute respiratory syndrome coronavirus 2

<400> 54

atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60

agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120

aaagttttca gatcctcagt tttacattca actcaggatt tgttcttacc tttcttttcc 180

aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240

aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300

ataagaggct ggatctttgg tactacttta gattcgaaga cccagtccct acttattgtt 360

aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420

ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480

tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540

ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600

tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660

tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720

ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780

ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840

gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900

tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020

gtttttaacg ccaccagatt tgcatccgtg tatgcttgga acaggaagag aatcagcaac 1080

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320

cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440

aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500

aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620

ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680

cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740

acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800

ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860

cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920

aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980

gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040

cctcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100

gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160

agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220

tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280

acacaattaa accgtgcttt aactgggata gctgttgaac aagacaaaaa cacccaagaa 2340

gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400

aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460

ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520

cttggtgata ttgctgctag ggacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580

ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640

acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700

caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760

aaattgattg ccaaccaatt taatagtgcc attggcaaaa ttcaagactc actttcttcc 2820

acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880

acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940

ctttcacgtc ttgaccctcc cgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000

cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060

tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120

gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180

gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240

atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300

cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360

tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420

ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480

tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540

aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600

caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660

atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc 3720

tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac 3780

tctgagccag tgctcaaagg agtcaaatta cattacaca 3819

<210> 55

<211> 241

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 55

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala Lys Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile

20 25 30

Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala

35 40 45

Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp

50 55 60

Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr

65 70 75 80

Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr

85 90 95

Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro

100 105 110

Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp

115 120 125

Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys

130 135 140

Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn

145 150 155 160

Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly

165 170 175

Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu

180 185 190

Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr

195 200 205

Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val

210 215 220

Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn

225 230 235 240

Phe

<210> 56

<211> 723

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 56

atgggagtca aagttctgtt tgccctgatc tgcattgctg tggccgaggc caagagagtc 60

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 120

gtttttaacg ccaccagatt tgcatccgtg tatgcttgga acaggaagag aatcagcaac 180

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 240

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 300

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 360

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 420

cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 480

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 540

aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 600

aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 660

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 720

ttc 723

<210> 57

<211> 185

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 57

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala

20 25 30

Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro

35 40 45

Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Met Glu Ala Asn Ala

50 55 60

Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile

65 70 75 80

Lys Cys Thr Pro Lys Met Lys Lys Phe Ile Pro Gly Arg Cys His Thr

85 90 95

Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile

100 105 110

Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu

115 120 125

Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys

130 135 140

Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp

145 150 155 160

Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val

165 170 175

Asp Lys Ile Lys Gly Ala Gly Gly Asp

180 185

<210> 58

<211> 558

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 58

atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagcccacc 60

gagaacaacg aagacttcaa catcgtggcc gtggccagca acttcgcgac cacggatctc 120

gatgctgacc gcgggaagtt gcccggcaag aagctgccgc tggaggtgct caaagagatg 180

gaagccaatg cccggaaagc tggctgcacc aggggctgtc tgatctgcct gtcccacatc 240

aagtgcacgc ccaagatgaa gaagttcatc ccaggacgct gccacaccta cgaaggcgac 300

aaagagtccg cacagggcgg cataggcgag gcgatcgtcg acattcctga gattcctggg 360

ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 420

acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 480

ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 540

ggggccggtg gtgactaa 558

<210> 59

<211> 566

<212> PRT

<213> influenza A Virus

<400> 59

Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn

1 5 10 15

Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr

20 25 30

Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn

35 40 45

Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val

50 55 60

Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly

65 70 75 80

Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile

85 90 95

Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe

100 105 110

Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe

115 120 125

Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp

130 135 140

Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser

145 150 155 160

Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro

165 170 175

Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val

180 185 190

Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu

195 200 205

Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser

210 215 220

Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln

225 230 235 240

Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys

245 250 255

Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe

260 265 270

Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro

275 280 285

Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn

290 295 300

Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys

305 310 315 320

Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg

325 330 335

Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly

340 345 350

Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr

355 360 365

His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser

370 375 380

Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile

385 390 395 400

Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His

405 410 415

Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe

420 425 430

Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn

435 440 445

Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu

450 455 460

Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly

465 470 475 480

Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val

485 490 495

Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu

500 505 510

Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr

515 520 525

Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Val

530 535 540

Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu

545 550 555 560

Gln Cys Arg Ile Cys Ile

565

<210> 60

<211> 1698

<212> DNA

<213> influenza A Virus

<400> 60

atgaaagcaa tactagtagt tcttctatat acatttgcaa ccgctaacgc tgatacattg 60

tgtataggat atcacgcgaa caactccacc gatacagtag atacagtact agagaagaac 120

gtaacagtaa cacattctgt taatcttcta gaagacaagc ataacggcaa actgtgcaaa 180

ctaagaggtg tagccccatt gcatctagga aagtgtaata tagctggctg gattttggga 240

aatccagagt gtgaatcatt aagtacagca agctcctggt cctatatagt ggaaacacct 300

agtagtgata acggaacgtg ttacccagga gattttatag attacgagga gctaagagag 360

cagctgtcgt cagtatcatc atttgaaagg tttgaaattt tcccgaaaac atcctcctgg 420

cccaatcacg atagtaacaa aggagtaaca gcagcctgtc ctcacgctgg agcaaaaagc 480

ttctataaaa atttaatctg gctagtgaag aagggaaatt catatccaaa gctaagtaaa 540

agttatataa acgataaggg caaggaagta ctcgtactgt ggggcattca tcatccatct 600

actagtgctg atcaacaaag tttatatcaa aacgcagacg catacgtttt tgtggggtca 660

agtagatata gcaagaaatt taaaccagaa atagcaataa gacctaaagt aagggatcaa 720

gaaggcagaa tgaactatta ttggacacta gtagaaccgg gagataaaat aacttttgaa 780

gcaacaggaa atctagtggt tcccaggtac gcatttgcaa tggaaagaaa cgctggatca 840

ggcatcatta tatctgatac accagtccac gattgtaata caacttgtca aacacctaaa 900

ggagctataa acaccagctt accatttcaa aatattcatc ctatcacaat tggaaagtgt 960

ccaaaatacg taaaaagtac aaaattgaga ttggccacag gattacgaaa tattccatca 1020

attcaatcta gaggactttt tggtgcaatt gcaggtttca tagaaggagg ctggactggg 1080

atggtagacg gctggtacgg ttatcatcat caaaacgaac agggaagtgg atacgcagct 1140

gatcttaaaa gtacacaaaa cgcaattgac gagattacta ataaagtaaa ttctgtaatt 1200

gaaaaaatga atactcagtt tacagcagta gggaaagagt ttaaccacct ggaaaaaaga 1260

atagaaaatt taaataaaaa agtagacgac ggatttcttg acatttggac ttataacgcc 1320

gaactattgg tattactaga aaacgaaaga actctagatt atcacgattc aaacgtaaaa 1380

aatttatacg aaaaagtaag aagccaactt aaaaataacg caaaagaaat aggaaacggc 1440

tgttttgaat tttatcacaa gtgtgataat acctgcatgg aaagtgttaa aaacgggaca 1500

tacgattatc caaaatactc agaagaagca aaattaaata gagaagaaat agacggcgta 1560

aaattagaat caacaaggat atatcaaata ttagcaatat attcaactgt cgcttcttca 1620

ttggtactgg tagtttctct aggtgcaata tcattttgga tgtgctctaa cggctcccta 1680

cagtgtagaa tttgtata 1698

<210> 61

<211> 96

<212> RNA

<213> hepatitis delta Virus

<400> 61

ggcucaucuc gacaagaggc ggcaguccuc aguacucuua cucuuuucug uaaagaggag 60

acugcuggac ucgccgccca aguucgagca ugagcc 96

<210> 62

<211> 74

<212> RNA

<213> hepatitis delta Virus

<400> 62

ggcuagaggc ggcaguccuc aguacucuua cucuuuucug uaaagaggag acugcuggac 60

ucgccgcccg agcc 74

Claims (37)

1. An immunogenic composition comprising a cyclic polyribonucleotide comprising a sequence that encodes a coronavirus antigen.

2. An immunogenic composition comprising a cyclic polyribonucleotide comprising a sequence that encodes a coronavirus antigen, wherein said coronavirus antigen comprises a sequence that has at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to a coronavirus antigen selected from any one of SEQ ID NOs 1-10, 13, 15, 17, 19, 21, 23, 25-30, 48 and 49, or said cyclic polyribonucleotide comprises a sequence that has at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to a cyclic polyribonucleotide selected from SEQ ID NOs 12, 14, 16, 18, 20, 22 and 24.

3. The immunogenic composition of any one of the preceding claims, further comprising the coronavirus antigen.

4. The immunogenic composition of any one of the preceding claims, wherein the coronavirus antigen is from a beta coronavirus or fragment thereof, or from a sabcomeae virus or fragment thereof.

5. The immunogenic composition of any one of the preceding claims, wherein the coronavirus antigen is from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a fragment thereof, severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) or a fragment thereof, or middle east respiratory syndrome coronavirus (MERS-CoV) or a fragment thereof.

6. The immunogenic composition of any one of the preceding claims, wherein the coronavirus antigen is a membrane protein or variant or fragment thereof, an envelope protein or variant or fragment thereof, a spike protein or variant or fragment thereof, a nucleocapsid protein or variant or fragment thereof, a helper protein or variant or fragment thereof.

7. The immunogenic composition of any one of the preceding claims, wherein the coronavirus antigen is a receptor binding domain of a spike protein or variant or fragment thereof.

8. The immunogenic composition of claim 7, wherein the spike protein lacks a cleavage site.

9. The immunogenic composition of any one of the preceding claims, wherein the helper protein of the coronavirus is selected from the group consisting of ORF3a, ORF7b, ORF8, ORF10, or any variant or fragment thereof.

10. The immunogenic composition of any one of the preceding claims, wherein the cyclic polyribonucleotide comprises a plurality of sequences, each sequence encoding an antigen, and at least one sequence encoding a coronavirus antigen.

11. The immunogenic composition of any one of the preceding claims, wherein the cyclic polyribonucleotide comprises two or more ORFs.

12. The immunogenic composition of any one of the preceding claims, wherein the cyclic polyribonucleotides comprise sequences encoding antigens from at least two different microorganisms, and at least one microorganism is a coronavirus.

13. The immunogenic composition of any one of the preceding claims, wherein the coronavirus antigen comprises an epitope.

14. The immunogenic composition of any one of the preceding claims, wherein the coronavirus antigen comprises an epitope recognized by B cells.

15. The immunogenic composition of any one of the preceding claims, further comprising a second cyclic polyribonucleotide comprising a sequence that encodes a second antigen.

16. The immunogenic composition of any one of the preceding claims, further comprising a second cyclic polyribonucleotide comprising a second ORF.

17. The immunogenic composition of any one of the preceding claims, further comprising a third, fourth or fifth cyclic polyribonucleotide comprising a sequence that encodes a third, fourth or fifth antigen.

18. The immunogenic composition of any one of the preceding claims, wherein the first antigen, second antigen, third antigen, fourth antigen and fifth antigen are different antigens.

19. The immunogenic composition of any one of the preceding claims, wherein the immunogenic composition further comprises a pharmaceutically acceptable carrier or excipient.

20. The immunogenic composition of any one of the preceding claims, wherein the immunogenic composition further comprises a pharmaceutically acceptable excipient and is free of any carrier.

21. An immunogenic composition comprising a linear polyribonucleotide comprising a sequence selected from any of SEQ ID NOs 13, 15 and 12.

22. The immunogenic composition of claim 21, wherein the linear polyribonucleotides comprise sequences that encode two or more antigens, and at least one antigen is the coronavirus antigen.

23. The immunogenic composition of claim 21 or claim 22, wherein the linear polyribonucleotides comprise a sequence that encodes at least 2, 3, 4 or 5 antigens, and at least one antigen is a coronavirus antigen encoded by the sequences of SEQ ID NOs 13, 15 and 12.

24. A method of delivering an immunogenic composition to a human subject, the method comprising: a) Administering to the human subject the immunogenic composition of any one of the preceding claims.

25. A method of inducing an immune response against a coronavirus antigen in a non-human animal or human subject, the method comprising: a) Administering the immunogenic composition of any one of the preceding claims to the non-human animal or human subject.

26. A method of delivering an immunogenic composition to a human subject, the method comprising: a) Administering the immunogenic composition of any one of the preceding claims to the human subject, and b) collecting antibodies against the coronavirus antigen from the non-human animal or human subject.

27. A method of inducing an immune response against a coronavirus antigen in a non-human animal or human subject, the method comprising: a) Administering the immunogenic composition of any one of the preceding claims to the non-human animal or human subject, and b) collecting antibodies to the coronavirus antigen from the non-human animal or human subject.

28. The method of any one of the preceding claims, further comprising administering an adjuvant to the non-human animal or human subject.

29. The method of claim 28, wherein the adjuvant is co-formulated and co-administered with the immunogenic composition or is formulated and administered separately from the immunogenic composition.

30. The method of any one of the preceding claims, further comprising formulating the immunogenic composition with a carrier.

31. The method of any one of the preceding claims, further comprising administering or immunizing the non-human animal or human subject with the cyclic polyribonucleotide at least twice.

32. The method of any one of the preceding claims, further comprising administering or immunizing a vaccine to the non-human animal or human subject.

33. The method of claim 32, wherein the vaccine is a pneumococcal polysaccharide vaccine.

34. The method of claim 32, wherein the vaccine is for bacterial infection.

35. The method of any one of the preceding claims, wherein the non-human animal or human subject is vaccinated with the cyclic polyribonucleotide by injection immunization.

36. The method of any one of the preceding claims, wherein one of the polyclonal antibodies specifically binds to the coronavirus antigen.

37. The method of any one of the preceding claims, wherein one of the polyclonal antibodies is a humanized antibody or a fully human antibody.

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