RSV mRNA VaccinesPRIORITY CLAIMS AND RELATED PATENT APPLICATIONS
This application claims the benefit of priority to PCT International application No. PCT/CN2023/077722, filed February 22, 2023, the entire content of which is incorporated herein by reference for all purposes.
FIELD OF THE INVENTIONThe present disclosure relates to a human respiratory syncytial virus vaccine comprising RNA polynucleotide that comprises an open reading frame encoding the recombinant RSV F protein.
BACKGROUNDRespiratory syncytial virus (RSV) is a single-stranded, negative-sense RNA pleomorphic enveloped Pneumovirus of the Paramyxoviruidae family. Its genome consists of an RNA molecule that encodes 11 proteins, including nine structural proteins (three glycoproteins and six internal proteins) and two non-structural proteins. The structural proteins include three transmembrane surface glycoproteins: the attachment protein (G protein) , fusion protein (F protein) , and the small hydrophobic protein (SH protein) , respectively.
One of the primary antigens explored for RSV subunit vaccines is the F protein. It was evidenced that the F protein of RSV as the target of vaccine antigen was better than the G protein of RSV. The RSV F protein trimer mediates fusion between the virion membrane and the host cellular membrane and promotes the formation of syncytia. In the virion prior to fusion with the membrane of the host cell, the largest population of F molecules forms a lollipop-shaped structure, with the TM domain anchored in the viral envelope (Dormitzer, P.R., Grandi, G., Rappuoli, R., Nature Reviews Microbiol, 10, 807, 2012. ) . During RSV entry into cells, the F protein rearranges from the prefusion state (which may be referred to herein as “pre-F” ) , through an intermediate extended structure, to a postfusion state ( “post-F” ) . During this rearrangement, the C-terminal coiled-coil of the prefusion molecule dissociates into its three constituent strands, which then wrap around the globular head and join three additional helices to form the postfusion six-helix bundle.
There is a need for immunogens derived from an RSV F protein that have improved properties, such as enhanced immunogenicity or improved stability, as compared with the corresponding native RSV F protein, as well as compositions comprising such an immunogen, such as a vaccine.
SUMMARYIn one aspect, the present disclosure provides a recombinant RSV F protein, comprising one or more modifications, relative to wild-type RSV F protein, selected from the group consisting of: (i) cysteine substitutions to introduce a non-native disulfide bond; (ii) a 210P amino acid substitution; (iii) a 211P amino acid substitution; (iv) a 488W cavity filling amino acid substitution; (v) a deletion of the cytoplasmic tail comprising amino acids at positions 554-574 or amino acids at positions 551-574; (vi) a substitution of amino acids 104-144 with a linker molecule, wherein the cysteine substitutions comprises 1) 35C and 471C; 2) 62C and 99C; 3) 145C and 370C; 4) 157C and 181C; or 5) 392C and 491C.
In some embodiments, the recombinant RSV F protein comprising a combination of modifications, relative to wild-type RSV F protein, wherein the combination of modifications is selected from the group consisting of:
(1) 35C, 471C, 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(2) 35C, 471C, 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, a substitution of amino acids 104-144 with a linker molecule, and a deletion of amino acids at positions 1-25;
(3) 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(4) 62C, 99C, 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(5) 145C, 370C, 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(6) 157C, 181C, 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(7) 392C, 491C, 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(8) 35C, 471C, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(9) 35C, 471C, 210P, 211P, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(10) 35C, 471C, 210P, 211P, 488W, and a substitution of amino acids 104-144 with a linker molecule;
(11) 35C, 471C, 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574;
(12) 35C, 471C, 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 551-574, and a substitution of amino acids 104-144 with a linker molecule;
(13) 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(14) 35C and 471C; or
(15) 210P and 211P.
In one aspect, the present disclosure provides a recombinant RSV F protein, comprising a variant of SEQ ID NO: 1, wherein the variant comprises one or more modifications, relative to wild-type RSV F protein, selected from the group consisting of: (i) cysteine substitutions to introduce a non-native disulfide bond; (ii) a Q210P amino acid substitution; (iii) an S211P amino acid substitution; (iv) an F488W cavity filling amino acid substitution; (v) a deletion of the cytoplasmic tail comprising amino acids at positions 554-574 or amino acids at positions 551-574; (vi) a substitution of amino acids 104-144 with a linker molecule, wherein the cysteine substitutions comprises 1) 35C and 471C; 2) 62C and 99C; 3) 145C and 370C; 4) 157C and 181C; or 5) 392C and 491C.
In one aspect, the disclosure is to provide a recombinant protein, comprising a variant of SEQ ID NO: 1, wherein the variant comprises: Q210P and S211P substitutions; and preferably, the variant further comprises one or more modifications, or a combination of modifications, relative to wild-type RSV F protein, wherein the combination of modifications is selected from the group consisting of: (i) cysteine substitutions to introduce a non-native disulfide bond; (ii) an F488W cavity filling amino acid substitution; (iii) a deletion of the cytoplasmic tail comprising amino acids at positions 554-574 or amino acids at positions 551-574; (iv) a substitution of amino acids 104-144 with a linker molecule, wherein the cysteine substitutions comprises 1) 35C and 471C; 2) 62C and 99C; 3) 145C and 370C; 4) 157C and 181C; or 5) 392C and 491C.
In some embodiments, the present disclosure provides a recombinant RSV F protein, comprising a variant of SEQ ID NO: 1, wherein the variant comprises a combination of modifications, relative to the amino acid sequence of the wild-type RSV F protein, wherein the combination of modifications is selected from the group consisting of:
(1) S35C, G471C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(2) S35C, G471C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(3) Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(4) S62C, I99C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(5) G145C, M370C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(6) V157C, L181C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(7) D392C, S491C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(8) S35C, G471C, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(9) S35C, G471C, Q210P, S211P, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(10) S35C, G471C, Q210P, S211P, F488W, and a substitution of amino acids 104-144 with a linker molecule;
(11) S35C, G471C, Q210P, S211P, F488W, and a deletion of the cytoplasmic tail comprising amino acids at positions 554-574;
(12) S35C, G471C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 551-574, and a substitution of amino acids 104-144 with a linker molecule;
(13) F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(14) S35C and G471C; or
(15) Q210P and S211P.
In some embodiments, the variant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-15, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to the amino acid sequence of SEQ ID NOs: 2-15.
In some embodiments, the variant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7 and 13, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 3, 7 and 13.
In some embodiments, the linker molecule is a heterologous glycine-serine peptide linker, preferably comprising (GGGGS) n, n is an integer greater than or equal to 1.
In some embodiments, the recombinant RSV F protein in present disclosure further optionally comprises a signal peptide optionally at N-terminal, preferably comprising a sequence of SEQ ID NO: 16.
In some embodiments, the recombinant RSV F protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 45-58, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 2 and 45-58.
In some embodiments, the recombinant RSV F protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 50 and 56, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 46, 50 and 56.
In some embodiments, the recombinant RSV F protein comprises a tag sequence optionally at the N terminal or C terminal.
In some aspects, the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence that encodes the recombinant RSV F protein.
In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 18-43, and a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 18-43.
In some embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 22, 30, 36 and 40, and a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 22, 30, 36 and 40.
In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 60-85, and a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 60-85.
In some embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 64, 72, 78 and 82, and a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 64, 72, 78 and 82,
In some aspects, the present disclosure provides an RNA polynucleotide encoding the recombinant RSV F protein.
In some embodiments, the RNA polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 87-112, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 87-112.
In some embodiments, the RNA polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 91, 99, 105 and 109, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 91, 99, 105 and 109.
In some embodiments, the RNA polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 114-139, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 114-139.
In some embodiments, the RNA polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 118, 126, 132 and 136, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 118, 126, 132 and 136.
In some aspects, the present disclosure provides an RSV vaccine, comprising RNA polynucleotide that comprises at least one open reading frame (ORF) encoding the recombinant RSV F protein.
In some embodiments, the RNA polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 87-112, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to the sequence of SEQ ID NOs: 87-112.
In some embodiments, the RNA polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 91, 99, 105 and 109, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 91, 99, 105 and 109.
In some embodiments, the RNA polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 87 and114-139, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 87 and 114-139.
In some embodiments, the RNA polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 118, 126, 132 and 136, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 118, 126, 132 and 136.
In some embodiments, the RNA comprises messenger RNA (mRNA) .
In some embodiments, the RNA further comprises a 5′UTR. Preferably, the 5′UTR comprises a sequence of SEQ ID NO: 168.
In some embodiments, the RNA further comprises a 3′UTR. Preferably, the 3′UTR comprises a sequence of SEQ ID NO: 169.
In some embodiments, the RNA is unmodified.
In some embodiments, the RNA comprises at least one modified nucleotide.
In some embodiments, the open reading frame is codon-optimized.
In some embodiments, the RNA polynucleotide further comprises at least one 5′terminal cap, a 3′polyA tail, and/or a Kozak sequence.
In some embodiments, the RNA polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 140-167, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to the sequence of SEQ ID NOs: 140-167.
In some embodiments, the RNA polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 146, 154, 160 and 164, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to the sequence of SEQ ID NOs: 146, 154, 160 and 164.
In some embodiments, the vaccine is formulated in a lipid nanoparticle.
In some aspects, the present disclosure provides a pharmaceutical composition comprising (i) the recombinant RSV F protein, the nucleic acid molecule, the vaccine in present disclosure, or the RNA polynucleotide; and (ii) a pharmaceutically acceptable carrier.
In some aspects, the present disclosure provides a method of inducing an antigen specific immune response in a subject, comprising administering to the subject an effective amount of the recombinant RSV F protein, the nucleic acid molecule, the RNA polynucleotide, the vaccine, or the pharmaceutical composition in present disclosure to produce an antigen specific immune response.
In some embodiments, the antigen specific immune response comprises a T cell response.
In some embodiments, the antigen specific immune response comprises a B cell response.
In some aspects, the present disclosure provides a method of preventing RSV infection in a subject, comprising administering to the subject an effective amount of the recombinant RSV F protein, the nucleic acid molecule, the RNA polynucleotide, the vaccine, or the pharmaceutical composition in present disclosure.
A method of eliciting an immune response against RSV in a subject, comprising administering to the subject an effective amount of the recombinant RSV F protein, the nucleic acid molecule, the RNA polynucleotide, the vaccine, or the pharmaceutical composition in present disclosure.
In some embodiments, the subject is human.
BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1 shows ELISA analysis of selected prefusion F variants using two different monoclonal antibodies (mAbs) D25 and 131-2A.
FIG. 2 shows western blot analysis of selected prefusion F variant under reducing conditions using two different monoclonal antibodies (mAbs) 2F7 and Palivizumab.
FIG. 3 shows western blot analysis of selected prefusion F variant under reducing conditions using monoclonal antibody (mAbs) 2F7.
FIG. 4 shows in vitro screening of mRNAs having different features in HEK293T cells 24 hours post-transfection. AM14, a monoclonal antibody specific for the prefusion form of RSV F glycoprotein, was used for the flow cytometric analysis.
FIG. 5 shows in vitro screening of mRNAs having different mutations (FIG. 5A) and codon optimization (FIG. 5B) in Vero and HEK293T cells 24 hours post-transfection. AM14 and D25, two monoclonal antibodies specific for the prefusion form of RSV F glycoprotein, were used for the flow cytometric analysis.
FIG. 6 shows in vivo screening of mRNAs having different features in mice. Following two doses of the mRNAs, the titer of IgG against prefusion form RSV F (FIG. 6A) and post fusion form RSV F (FIG. 6B) were measured.
FIG. 7 shows in vivo screening of mRNAs having different features in mice. Three weeks post the second dose of the mRNAs, mouse sera were harvested, prefusion (FIG. 7A 7B) and postfusion (FIG. 7C and 7D) RSV F protein-specific IgG levels were measured.
FIG. 8 shows the RSV A2 neutralizing titer of serum samples 42-days post first dose immunization. Different mutants (FIG. 8A) and different optimized sequences (FIG. 8B) were compared.
FIG. 9 shows the RSV neutralizing titer of individual serum samples of RSV10 on Day 28 and Day 56.
FIG. 10 shows the cellular immune responses elicited by RSV10 in ELISPOT study.
FIG. 11 shows the results of a mouse immunogenicity study . RSV A2 neutralizing titers of serum samples from Day 42 (FIG. 5A) and cellular immune responses in CD4+ (FIG. 5B) and CD8+ (FIG. 5C) T cell immune responses were compared.
DETAILED DESCRIPTIONThe present disclosure relates to RSV F protein variants, immunogenic compositions comprising the recombinant RSV F protein, methods for producing the recombinant RSV F protein, compositions comprising the RSV F recombinant protein, nucleic acids that encode the recombinant RSV F protein, the vaccine comprising an RNA polynucleotide that comprises at least one open reading frame (ORF) encoding the recombinant RSV F protein, and pharmaceutical composition thereof. The present disclosure also provides a method of preventing RSV infection or eliciting an immune response against RSV in the subject.
RSV F PROTEIN VARIANTS
RSV F protein is a type I fusion glycoprotein that is well conserved between clinical isolates, including between the RSV-A and RSV-B antigenic subgroups. The F protein transitions between prefusion and more stable post-fusion states, thereby facilitating entry into target cells. “RSV F protein” , as used herein, refers to an RSV envelope glycoprotein that facilitates the fusion of viral and cellular membranes. The present disclosure is not limited by a particular strain of RSV. The strain of RSV used in a vaccine may be any strain of RSV.
Antigens are proteins capable of inducing an immune response (e.g., causing an immune system to produce antibodies against the antigens) . Herein, use of the term antigen encompasses immunogenic proteins and immunogenic fragments (an immunogenic fragment that induces (or is capable of inducing) an immune response to RSV) , unless otherwise stated. It should be understood that the term “protein” encompasses peptides and the term “antigen” encompasses antigenic fragments.
The recombinant RSV F protein in present disclosure comprises one or more modifications relative to wild-type RSV F protein, wherein the sequence of wild-type RSV F protein is an RSV strain according to Uniprot Nos. P03420, O36634, P12568, P12568, P11209, P03420, P13843, Q89535, R9TCY6, X4YBB7, R9TFV6, V9VGR9, Q8JQU3, V5N7I1, G8EJ09, A0A0U2LT33, W8RJF9, O09720, A0A109QF37, A0A088SBE5, Q84850, or Q4KRW4.
Exemplary RSV protein comprises one or more modifications, relative to wild-type RSV F protein, selected from the group consisting of: (i) cysteine substitutions to introduce a non-native disulfide bond; (ii) a 210P amino acid substitution; (iii) a 211P amino acid substitution; (iv) a 488W cavity filling amino acid substitution; (v) a deletion of the cytoplasmic tail comprising amino acids at positions 554-574 or amino acids at positions 551-574; (vi) a substitution of amino acids 104-144 with a linker molecule, wherein the cysteine substitutions comprises 1) 35C and 471C; 2) 62C and 99C; 3) 145C and 370C; 4) 157C and 181C; or 5) 392C and 491C.
Exemplary RSV protein comprises a combination of modifications, relative to wild-type RSV F protein, wherein the combination of modifications is selected from the group consisting of:
(1) 35C, 471C, 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(2) 35C, 471C, 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, a substitution of amino acids 104-144 with a linker molecule, and a deletion of amino acids at positions 1-25;
(3) 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(4) 62C, 99C, 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(5) 145C, 370C, 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(6) 157C, 181C, 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(7) 392C, 491C, 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(8) 35C, 471C, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(9) 35C, 471C, 210P, 211P, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(10) 35C, 471C, 210P, 211P, 488W, and a substitution of amino acids 104-144 with a linker molecule;
(11) 35C, 471C, 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574;
(12) 35C, 471C, 210P, 211P, 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 551-574, and a substitution of amino acids 104-144 with a linker molecule;
(13) 488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(14) 35C and 471C; or
(15) 210P and 211P.
Exemplary RSV protein comprises a variant of SEQ ID NO: 1, and the variant one or more modifications, relative to wild-type RSV F protein, selected from the group consisting of: (i) S35C and G471C substitutions to introduce a non-native disulfide bond; (ii) a Q210P amino acid substitution; (iii) an S211P amino acid substitution; (iv) an F488W cavity filling amino acid substitution; (v) a deletion of the cytoplasmic tail comprising amino acids at positions 554-574 or amino acids at positions 551-574; (vi) a substitution of amino acids 104-144 with a linker molecule. Amino acid positions are numbered according to wild-type RSV F protein (Uniprot: P03420) .
In one aspect, the disclosure is to provide an RSV F protein, comprising a variant of SEQ ID NO: 1, wherein the variant comprises: Q210P and S211P substitutions; and preferably, the variant further comprises one or more modifications, or a combination of modifications, relative to wild-type RSV F protein, wherein the combination of modifications is selected from the group consisting of:(i) S35C and G471C substitutions to introduce a non-native disulfide bond; (ii) an F488W cavity filling amino acid substitution; (iii) a deletion of the cytoplasmic tail comprising amino acids at positions 554-574 or amino acids at positions 551-574; (iv) a substitution of amino acids 104-144 with a linker molecule.
In some embodiments, the present disclosure provides an RSV F protein, comprising a variant of SEQ ID NO: 1, wherein the variant comprises a combination of modifications, relative to wild-type RSV F protein, selected from the group consisting of:
(1) S35C, G471C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(2) S35C, G471C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(3) Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(4) S62C, I99C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(5) G145C, M370C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(6) V157C, L181C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(7) D392C, S491C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(8) S35C, G471C, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(9) S35C, G471C, Q210P, S211P, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(10) S35C, G471C, Q210P, S211P, F488W, and a substitution of amino acids 104-144 with a linker molecule;
(11) S35C, G471C, Q210P, S211P, F488W, and a deletion of the cytoplasmic tail comprising amino acids at positions 554-574;
(12) S35C, G471C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 551-574, and a substitution of amino acids 104-144 with a linker molecule;
(13) F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule;
(14) S35C and G471C; or
(15) Q210P and S211P.
Exemplary RSV antigens are provided in the Sequence Listing. For example, the antigens may comprise or consist of any of sequences set forth in SEQ ID NOs: 2-15 and 45-58, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to the amino acid sequence of SEQ ID NOs: 2-15 and 45-58.
In some embodiments, the variant of SEQ ID NO: 1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-15, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to the amino acid sequence of SEQ ID NOs: 2-15.
In some embodiments, the linker molecule is a heterologous glycine-serine peptide linker, preferably comprising (GGGGS) n, n is an integer greater than or equal to 1.
In some embodiments, the recombinant RSV F protein in present disclosure further optionally comprises a signal peptide optionally at N-terminal, preferably comprising a sequence of SEQ ID NO: 16.
In some embodiments, the recombinant RSV F protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 45-58, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 2 and 45-58.
In some embodiments, the recombinant RSV F protein comprises a tag sequence optionally at the N or C terminal.
In some embodiments, the combination of modifications is: S35C, G471C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 2; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 2. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 47; preferably, the variant comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to the amino acid sequence of SEQ ID NO: 47.
In some embodiments, the combination of modifications is: S35C, G471C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, a substitution of amino acids 104-144 with a linker molecule. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 2; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 2. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 48; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 48. More preferably, and the variant comprises a deletion of amino acids at positions 1-25.
In some embodiments, the combination of modifications is: Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule; preferably, the variant comprises an amino acid sequence of SEQ ID NO: 3; preferably, the variant comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 3. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 46; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 46.
In some embodiments, the combination of modifications is: S62C, I99C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 4; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 4. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 47; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 47.
In some embodiments, the combination of modifications is: G145C, M370C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 5; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 5. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 48; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 48.
In some embodiments, the combination of modifications is: V157C, L181C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 6; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 6. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 49; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 49.
In some embodiments, the combination of modifications is: D392C, S491C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule. preferably, the variant comprises an amino acid sequence of SEQ ID NO: 7; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 7. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 50; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 50.
In some embodiments, the combination of modifications is: S35C, G471C, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 8; preferably, the variant comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to the amino acid sequence of SEQ ID NO: 8. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 51; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 51.
In some embodiments, the combination of modifications is: S35C, G471C, Q210P, S211P, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 9; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 9. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 52; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 52.
In some embodiments, the combination of modifications is: S35C, G471C, Q210P, S211P, F488W, and a substitution of amino acids 104-144 with a linker molecule. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 10; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 10. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 53; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 53.
In some embodiments, the combination of modifications is: S35C, G471C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 11; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 11. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 54; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 54.
In some embodiments, the combination of modifications is: S35C, G471C, Q210P, S211P, F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 551-574, and a substitution of amino acids 104-144 with a linker molecule. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 12; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 12. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 55; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 55.
In some embodiments, the combination of modifications is: F488W, a deletion of the cytoplasmic tail comprising amino acids at positions 554-574, and a substitution of amino acids 104-144 with a linker molecule. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 13; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 13. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 56; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 56.
In some embodiments, the combination of modifications is: S35C and G471C. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 14; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 14. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 57; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 57.
In some embodiments, the combination of modifications is: Q210P and S211P. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 15; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 15. Preferably, the variant comprises an amino acid sequence of SEQ ID NO: 58; preferably, the variant comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NO: 58.
NUCLEIC ACIDS
In some aspects, the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence that encodes the recombinant RSV F protein.
In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 18-43, and a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 18-43.
In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 60-85, and a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 60-85.
In some aspects, the present disclosure provides an RNA polynucleotide encoding the recombinant RSV F protein.
In some embodiments, the RNA polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 87-112, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 87-112.
In some embodiments, the RNA polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 114-139, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 114-139.
In some embodiments, the RNA comprises messenger RNA (mRNA) . Messenger RNA (mRNA) is any ribonucleic acid that encodes a (at least one) protein (anaturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded protein in vitro, in vivo, in situ or ex vivo. The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application may recite “T” sin a representative DNA sequence but where the sequence represents RNA (e.g., mRNA) , the “T” swould be substituted for “U” . Thus, any of the DNAs disclosed and identified by a particular sequence identification number herein also disclose the corresponding RNA (e.g., mRNA) sequence complementary to the DNA, where each “T” of the DNA sequence is substituted with “U. ” Likewise, any of the RNAs disclosed and identified by a particular sequence identification number herein also disclose the corresponding DNA sequence complementary to the RNA, where each “U” of the RNA sequence is substituted with “T. ”
RSV VACCINE
In some aspects, the present disclosure provides an RSV vaccine, comprising RNA polynucleotide that comprises at least one open reading frame (ORF) encoding the recombinant RSV F protein.
In some embodiments, the RSV vaccine comprises an RNA polynucleotide further comprising a 5’ untranslated region (UTR) , a 3’ UTR, a 5’ cap and a poly-A tail.
An open reading frame (ORF) is a continuous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG or AUG) ) and ending with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA) . An ORF typically encodes a protein. It will be understood that the sequences disclosed herein may further comprise additional elements, e.g., 5′and 3′untranslated regions (UTRs) , but that those elements, unlike the ORF, need not necessarily be present in a vaccine of the present disclosure. In mRNA, the 5 'UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3'UTR starts immediately following the stop codon and continues until the transcriptional termination signal. While not wishing to be bound by theory, UTRs may have a role in the stability and translation of the nucleic acid molecule. Variants of UTRs may be utilized where one or more nucleotides (e.g., A, T/U, C or G) are added or removed to the termini, of the UTR.
In some embodiments, the UTRs of the polynucleotide of the nucleic acid vaccine may range independently from 15-1,000 nucleotides in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides) .
The UTR from any gene may be incorporated into the regions of the polynucleotides of the nucleic acid vaccines. Alternatively, artificial UTRs, which are not variants of wild type regions, may also be used in the polynucleotides of the nucleic acid vaccines. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. As a non-limiting example, a 5'or 3'UTR may be inverted, shortened, lengthened, or made with one or more other 5'UTRs or 3'UTRs from a different parental sequence.
In some embodiments, the RNA polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 87-112, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to the sequence of SEQ ID NOs: 87-112.
In some embodiments, the RNA polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 87 and114-139, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to SEQ ID NOs: 87 and 114-139.
In some embodiments, the RNA comprises messenger RNA (mRNA) .
In some embodiments, the RNA further comprises a 5′UTR. Preferably, the 5′UTR comprises a sequence of SEQ ID NO: 168.
In some embodiments, the RNA further comprises a 3′UTR. Preferably, the 3′UTR comprises a sequence of SEQ ID NO: 169.
In some embodiments, the RNA is unmodified.
In some embodiments, the RNA comprises at least one modified nucleotide. The terms “modification” or, as appropriate, “modified” refer to modification with respect to A, G, U or C ribonucleotides. Generally, herein, these terms are not intended to refer to the ribonucleotide modifications in naturally occurring 5′-terminal mRNA cap moieties. In a polypeptide, the term “modification” refers to a modification as compared to the canonical set of 20 amino acids.
The modifications may be various distinct modifications. In some embodiments, the coding region (s) , the untranslated region (s) , the flanking region (s) , and/or the terminal or tailing regions may contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, nucleic acid vaccines of the present disclosure comprise one or more modifications which render the nucleic acid molecules, when introduced to a cell, more resistant to degradation in the cell and/or more stable in the cell as compared to unmodified polynucleotides.
In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil selected from pseudouridine (ψ) , pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U) , 4-thio-uridine (s4U) , 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U) , 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine) , 3-methyl-uridine (m3U) , 5-methoxy-uridine (mo5U) , uridine 5-oxyacetic acid (cmo5U) , uridine 5-oxyacetic acid methyl ester (mcmo5U) , 5-carboxymethyl-uridine (cm5U) , 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U) , 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U) , 5-methoxycarbonylmethyl-uridine (mcm5U) , 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U) , 5-aminomethyl-2-thio-uridine (nm5s2U) , 5-methylaminomethyl-uridine (mnm5U) , 5-methylaminomethyl-2-thio-uridine (mnm5s2U) , 5-methylaminomethyl-2-seleno-uridine (mnm5se2U) , 5-carbamoylmethyl-uridine (ncm5U) , 5-carboxymethylaminomethyl-uridine (cmnm5U) , 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U) , 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U) , 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U) , 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine) , 1-methylpseudouridine (m1ψ) , 5-methyl-2-thio-uridine (m5s2U) , 1-methyl-4-thio-pseudouridine (m1s4ψ) , 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ) , 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D) , dihydropseudouridine, 5, 6-dihydrouridine, 5-methyl-dihydrouridine (m5D) , 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine (also known as 1-methylpseudouridine (m1ψ) ) , 3- (3-amino-3-carboxypropyl) uridine (acp3U) , 1-methyl-3- (3-amino-3-carboxypropyl) pseudouridine (acp3 ψ) , 5- (isopentenylaminomethyl) uridine (inm5U) , 5-(isopentenylaminomethyl) -2-thio-uridine (inm5s2U) , α-thio-uridine, 2′-O-methyl-uridine (Um) , 5,2′-O-dimethyl-uridine (m5Um) , 2′-O-methyl-pseudouridine (ψm) , 2-thio-2′-O-methyl-uridine (s2Um) , 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm5Um) , 5-carbamoylmethyl-2′-O-methyl-uridine (ncm5Um) , 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm5Um) , 3, 2′-O-dimethyl-uridine (m3Um) , 5- (isopentenylaminomethyl) -2′-O-methyl-uridine (inm5Um) , 1-thio-uridine, deoxythymidine, 2'‐F‐ara‐uridine, 2'‐F‐uridine, 2'‐OH‐ara‐uridine, 5‐ (2‐carbomethoxyvinyl) uridine, and 5‐ [3‐ (1‐E‐propenylamino) uridine.
In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C) , N4-acetyl-cytidine (ac4C) , 5-formyl-cytidine (f5C) , N4-methyl-cytidine (m4C) , 5-methyl-cytidine (m5C) , 5-halo-cytidine (e.g., 5-iodo-cytidine) , 5-hydroxymethyl-cytidine (hm5C) , 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C) , 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C) , α-thio-cytidine, 2′-O-methyl-cytidine (Cm) , 5, 2′-O-dimethyl-cytidine (m5Cm) , N4-acetyl-2′-O-methyl-cytidine (ac4Cm) , N4, 2′-O-dimethyl-cytidine (m4Cm) , 5-formyl-2′-O-methyl-cytidine (f5Cm) , N4, N4, 2′-O-trimethyl-cytidine (m42Cm) , 1-thio-cytidine, 2'‐F‐ara‐cytidine, 2'‐F‐cytidine, and 2'‐OH‐ara‐cytidine.
In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine) , 6-halo-purine (e.g., 6-chloro-purine) , 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyl-adenosine (m1A) , 2-methyl-adenine (m2A) , N6-methyl-adenosine (m6A) , 2-methylthio-N6-methyl-adenosine (ms2m6A) , N6-isopentenyl-adenosine (i6A) , 2-methylthio-N6-isopentenyl-adenosine (ms2i6A) , N6- (cis-hydroxyisopentenyl) adenosine (io6A) , 2-methylthio-N6- (cis-hydroxyisopentenyl) adenosine (ms2io6A) , N6-glycinylcarbamoyl-adenosine (g6A) , N6-threonylcarbamoyl-adenosine (t6A) , N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A) , 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A) , N6, N6-dimethyl-adenosine (m62A) , N6-hydroxynorvalylcarbamoyl-adenosine (hn6A) , 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A) , N6-acetyl-adenosine (ac6A) , 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am) , N6, 2′-O-dimethyl-adenosine (m6Am) , N6, N6, 2′-O-trimethyl-adenosine (m62Am) , 1, 2′-O-dimethyl-adenosine (m1Am) , 2′-O-ribosyladenosine (phosphate) (Ar (p) ) , 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'‐F‐ara‐adenosine, 2'‐F‐adenosine, 2'‐OH‐ara‐adenosine, and N6‐ (19‐amino‐pentaoxanonadecyl) -adenosine.
In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I) , 1-methyl-inosine (m1I) , wyosine (imG) , methylwyosine (mimG) , 4-demethyl-wyosine (imG-14) , isowyosine (imG2) , wybutosine (yW) , peroxywybutosine (o2yW) , hydroxywybutosine (OHyW) , undermodified hydroxywybutosine (OHyW*) , 7-deaza-guanosine, queuosine (Q) , epoxyqueuosine (oQ) , galactosyl-queuosine (galQ) , mannosyl-queuosine (manQ) , 7-cyano-7-deaza-guanosine (preQ0) , 7-aminomethyl-7-deaza-guanosine (preQ1) , archaeosine (G+) , 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G) , 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m1G) , N2-methyl-guanosine (m2G) , N2, N2-dimethyl-guanosine (m22G) , N2, 7-dimethyl-guanosine (m2, 7G) , N2, N2, 7-trimethyl-guanosine (m2, 2, 7G) , 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2, N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2′-O-methyl-guanosine (Gm) , N2-methyl-2′-O-methyl-guanosine (m2Gm) , N2, N2-dimethyl-2′-O-methyl-guanosine (m22Gm) , 1-methyl-2′-O-methyl-guanosine (m1Gm) , N2, 7-dimethyl-2′-O-methyl-guanosine (m2, 7Gm) , 2′-O-methyl-inosine (Im) , 1, 2′-O-dimethyl-inosine (m1Im) , and 2′-O-ribosylguanosine (phosphate) (Gr (p) ) .
In one embodiment of the disclosure, the polynucleotide of the nucleic acid vaccine is modified to comprise N1-methyl-pseudouridine nucleotides.
In some embodiments, the open reading frame is codon-optimized.
In some embodiments, the RNA polynucleotide further comprises at least one 5′terminal cap, a 3′polyA tail, and/or a Kozak sequence.
In some embodiments, the 5’ terminal capping region of the polynucleotide of the nucleic acid vaccine may comprise a single cap or a series of nucleotides forming the cap. The capping region may be from 1 to 10, e.g., 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In some examples, the capping region may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In some embodiments, the cap is absent.
In some embodiments, cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs may be used in the nucleic acid vaccines. Cap analogs, which may be chemically (e.g., non-enzymatically) or enzymatically synthesized, differ from natural (e.g., endogenous, wild-type or physiological) 5'-caps in their chemical structure, but they retain cap function. For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5′-5′-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3′-O-methyl group (i.e., N7, 3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m7G-3′mppp-G; which may equivalently be designated 3′O-Me-m7G (5′) ppp (5′) G) . The 3′-O atom of the other, unmodified, guanine becomes linked to the 5′-terminal nucleotide of the capped nucleic acid molecule (e.g., an mRNA) . The N7-and 3′-O-methlyated guanine provide the terminal moiety of the capped nucleic acid molecule (e.g., mRNA) .
In some embodiments, the 5'terminal caps of the polynucleotides of the nucleic acid vaccines may include endogenous caps or cap analogs.
In exemplary aspects of the present disclosure, polynucleotides, e.g., mRNAs, can be capped post-transcriptionally, using enzymes. For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2′-O-methyltransferase enzyme can create a canonical 5′-5′-triphosphate linkage between the 5′-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5′-terminal nucleotide of the mRNA contains a 2′-O-methyl. Such a structure is termed the Cap 1 structure. In some embodiments, the Cap 1 structure provides a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5′cap analog structures known in the art. Cap structures include 7mG (5′) ppp (5′) N, pN2p (Cap 0) , 7mG (5′) ppp (5′) N1mpNp (Cap 1) , and 7mG(5′) -ppp (5′) N1mpN2mp (Cap 2) .
In one embodiment, the polynucleotide of the nucleic acid vaccine described herein comprises a Cap 1 structure.
The 3′-poly (A) tail is typically a stretch of adenine nucleotides added to the 3′-end of the transcribed mRNA. It can, in some instances, comprise up to about 400 adenine nucleotides. In some embodiments, the length of the 3′-poly (A) tail may be an essential element with respect to the stability of the individual mRNA. In some embodiments, a vaccine includes at least one RNA polynucleotide comprising an open reading frame encoding at least one antigenic polypeptide comprising at least one modification, and at least one 5′terminal cap. In some embodiments, RSV RNA vaccines may or may not contain an enhancer and/or promoter sequence, which may be modified or unmodified or which may be activated or inactivated.
In some embodiments, poly-A tails may also be added after the construct is exported from the nucleus. In some embodiments, a long chain of adenine nucleotides (poly-A tail) may be added to a polynucleotide such as an mRNA molecule during RNA processing in order to increase stability. Immediately after transcription, the 3'end of the transcript may be cleaved to free a 3'hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the RNA.
In some embodiments, an RSV vaccine comprises an RNA comprising an ORF that encodes a signal peptide fused to the RSV antigen. Signal peptides, comprising the N-terminal 15-60 amino acids of proteins, are typically needed for the translocation across the membrane on the secretory pathway and, thus, universally control the entry of most proteins both in eukaryotes and prokaryotes to the secretory pathway. In eukaryotes, the signal peptide of a nascent precursor protein (pre-protein) directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across it for processing. ER processing produces mature proteins, wherein the signal peptide is cleaved from precursor proteins, typically by an ER-resident signal peptidase of the host cell, or they remain uncleaved and function as a membrane anchor. A signal peptide may also facilitate the targeting of the protein to the cell membrane.
In some embodiments, the RNA polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 140-167, and a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to the sequence of SEQ ID NOs: 140-167.
In some embodiments, the nucleic acid vaccine is vectorized after codon optimization. Non-limiting examples of vectors include, but are not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
In some embodiments, the vaccine is formulated in a lipid nanoparticle.
mRNA SYNTHESES
cDNA encoding the polynucleotides of the nucleic acid vaccines described herein may be transcribed using an in vitro transcription (IVT) system. The system typically comprises a transcription buffer, nucleotide triphosphates (NTPs) , an RNase inhibitor and a polymerase. The NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs. The polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and polymerase variants.
Any number of RNA polymerases or variants may be used in the synthesis of the polynucleotides of the nucleic acid vaccine described herein. RNA polymerases may be modified by inserting or deleting amino acids of the RNA polymerase sequence.
In some embodiments, mRNA is subjected to enzymatic treatment, HPLC purification, ultrafiltration and diafiltration to remove residual DNA, DNA template, and dsRNA.
In some embodiments, polynucleotides of the nucleic acid vaccines described herein may be manufactured in whole or in part using solid phase techniques. Solid-phase chemical synthesis of polynucleotides or nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution. Impurities and excess reagents are washed away and no purification is required after each step. The automation of the process is amenable on a computer-controlled solid-phase synthesizer. Solid-phase synthesis allows rapid production of polynucleotides or nucleic acids in a relatively large scale that leads to the commercial availability of some polynucleotides or nucleic acids.
In some embodiments, synthesis of polynucleotides of the nucleic acid vaccines described herein by the sequential addition of monomer building blocks may be carried out in a liquid phase.
In some embodiments, the polynucleotide may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis) .
PHARMACEUTICAL COMPOSITION
In some aspects, the present disclosure provides a pharmaceutical composition comprising (i) the recombinant RSV F protein, the nucleic acid molecule, or the vaccine in present disclosure, or the RNA polynucleotide; and (ii) a pharmaceutically acceptable carrier.
In some embodiments, compositions are administered to humans, human patients or subjects.
Provided herein are nucleic acid vaccines and pharmaceutical compositions thereof which may be used in combination with one or more pharmaceutically acceptable excipients.
In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated in a lipid nanoparticle (LNP) . In general, LNPs can be characterized as small solid or semi-solid particles possessing an exterior lipid layer with a hydrophilic exterior surface that is exposed to the non-LNP environment, an interior space which may aqueous (vesicle like) or non-aqueous (micelle like) , and at least one hydrophobic inter-membrane space. LNP membranes may be lamellar or non-lamellar and may be comprised of 1, 2, 3, 4, 5 or more layers. In some embodiments, LNPs may comprise a cargo or a payload into their interior space, into the inter membrane space, onto their exterior surface, or any combination thereof.
LNPs useful herein are known in the art and generally comprise cholesterol, a phospholipid, a polyethylene glycol (PEG) derivative, and an ionizable lipid. In some embodiments a lipid nanoparticle may be comprised of at least one cationic lipid, at least one non-cationic lipid, at least one sterol, at least one additional LNP functional component, or any combination thereof.
In some embodiments, the LNP comprises a PEG derivative. As a non-limiting example, the PEG derivative may be a lipid-anchored such as PEG is C14-PEG2000, C14-PEG1000, C14-PEG3000, C14-PEG5000, C12-PEG1000, C12-PEG2000, C12-PEG3000, C12-PEG5000, C16-PEG1000, C16-PEG2000, C16-PEG3000, C16-PEG5000, C18-PEG1000, C18-PEG2000, C18-PEG3000, or C18-PEG5000. In some embodiments, the average molecular weight of the polymer moiety (e.g., PEG) may be between 500 and 20,000 daltons..
In some embodiments, at least one PEG-lipid conjugate may be selected from, but is not limited to at least one of Siglec-1L-PEG-DSPE, (R) -2, 3-bis (octadecyloxy) propyl-1-(methoxypoly (ethyleneglycol) 2000) propylcarbamate, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG-PAA, PEG-OH DSPE C18, PEG-DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG-DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide C16, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-C11, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG (2k) -DMG, PEG DSPE C18, PEG DMPE C14, PEG DLPE C12, PEG Click DMG C14, PEG Click C12, PEG Click C10, N (Carbonyl-methoxypolyethyleneglycol-2000) -l, 2-distearoyl-sn-glycero3-phosphoethanolamine, Myrj52, mPEG-PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, l, 2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG-DSPE, DSPE-PEGMA 500, DSPE-PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE-PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG-PEGMA, DOPE-PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE-mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol-polyethyleneglycol, Cl8PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14: 0-PEG2KPE, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, (R) -2, 3-bis (octadecyloxy) propyl-1- (methoxypoly (ethyleneglycol) 2000) propylcarbamate, (PEG) -C-DOMG, PEG-C-DMA, and DSPE-PEG-X.
In some embodiments, the LNP comprises at least one cationic lipid in an amount of about 0.1 to 100 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of about 20 to 60 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of about 50 to 85 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of less than about 20 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of more than about 60 mol%or about 85 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of about 95 mol%or less..
In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of about 0.1 to 100 mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of about 5 to 35 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of about 5 to 25 mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of less than about 5 mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of more than about 25 mol%or about 35 mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of about 95 mol%or less..
In some embodiments, the LNP comprises at least one sterol in an amount of about 0.1 to 100 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of about 20 to 45 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of about 25 to 55 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of less than about 20 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of more than about 45 mol%or about 55 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of about 95 mol%or less..
In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of about 0.1 to 100 mol%. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of about 0.5 to 15 mol%. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of about 15 to 40 mol%. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of less than about 0.1 mol%. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of more than about 15 mol%or about 40 mol%. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of about 95 mol%or less.
In some embodiments, the LNP includes about 30-60 mol%of at least one cationic lipid, about 0-30 mol%of at least one non-cationic lipid (e.g., a phospholipid) , about 18.5-48.5 mol%of at least one sterol (e.g., cholesterol) , and about 0-10 mol%of at least one additional LNP functional component (e.g., a PEGylated lipid) .
In some embodiments, the LNPs can be characterized by their size. In some embodiments, the size of an LNP can be defined as the diameter of its largest circular cross section, referred to herein simply as its diameter. In some embodiments, the LNPs may have a diameter between 30 nm to about 150 nm.
Uniformity may be expressed in some embodiments as the polydispersity index (PDI) of the population. In some embodiments, uniformity may be expressed in some embodiments as the disparityof the population. The terms “polydispersity index” and “disparity” are understood herein to be equivalent and may be used interchangeably. In some embodiments, a population of LNPs resulting from a given formulation will have a PI of between about 0.1 and 1. In some embodiments, a population of LNPs resulting from a giving formulation will have a PI of less than about 1, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1.
In some embodiments, the LNP may fully or partially encapsulate a cargo, such as nucleic acid constructs of the present disclosure. In some embodiments, essentially 0%of the cargo present in the final formulation is exposed to the environment outside of the LNP (i.e., the cargo is fully encapsulated) . In some embodiments, the cargo is associated with the LNP but is at least partially exposed to the environment outside of the LNP. In some embodiments, the LNP may be characterized by the %of the cargo not exposed to the environment outside of the LNP, e.g., the encapsulation efficiency. For the sake of clarity, an encapsulation efficiency of about 100%refers to an LNP formulation where essentially all the cargo is fully encapsulated by the LNP, while an encapsulation rate of about 0%refers to an LNP where essentially none of the cargo is encapsulated in the LNP, such as with an LNP where the cargo is bound to the external surface of the LNP.
In some embodiments, the lipid nanoparticles may have a diameter from about 10 to 500 nm.
TREATMENT
In some aspects, the present disclosure provides a method of inducing an antigen specific immune response in a subject, comprising administering to the subject an effective amount of the recombinant RSV F protein, the nucleic acid molecule, the RNA polynucleotide, the vaccine, or the pharmaceutical composition in present disclosure to produce an antigen specific immune response.
In some embodiments, the antigen specific immune response comprises a T cell response.
In some embodiments, the antigen specific immune response comprises a B cell response.
In some aspects, the present disclosure provides a method of preventing RSV infection in a subject, comprising administering to the subject an effective amount of the recombinant RSV F protein, the nucleic acid molecule, the RNA polynucleotide, the vaccine, or the pharmaceutical composition in present disclosure.
A method of eliciting an immune response against RSV in a subject, comprising administering to the subject an effective amount of the recombinant RSV F protein, the nucleic acid molecule, the RNA polynucleotide, the vaccine, or the pharmaceutical composition in present disclosure.
In some embodiments, the subject is human.
Definitions
As used herein, the singular form "a" , "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term “a substitution" or “at least one substitution " may include a plurality of substitutions.
As used herein, the term “about” means within an acceptable error range for the particular value which is known to one of ordinary skill in the art.
As used herein, the terms "comprises” , “comprising” , mean "including but not limited to" . For example, “a variant comprises a substitution” may include further substitutions.
As used herein, the term “modification” refers to deletion, addition, or substitution of amino acid residues in the amino acid sequence of a protein or polypeptide as compared to the amino acid sequence of a reference protein or polypeptide. Throughout the specification and claims, the substitution of an amino acid at one particular location in the protein sequence is referred to using a notation “ (amino acid residue in wild type protein) (amino acid position) ” . For example, a notation Y75A refers to a substitution of a tyrosine (Y) residue at the 75th position of the amino acid sequence of the reference protein by an alanine (A) residue (in a variant of the reference protein) . In cases where there is variation in the amino acid residue at the same position among different wild-type sequences, the amino acid code preceding the position number may be omitted in the notation, such as “75A” .
As used herein, the term “identical” or percent “identity, ” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence. Methods of alignment of sequences for comparison are well known in the art. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence) , followed by multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166÷1554*100=75.0) .
As used herein, the term “vaccine” refers to a pharmaceutical composition comprising an immunogen that can elicit a prophylactic or therapeutic immune response in a subject. Typically, a vaccine elicits an antigen-specific immune response to an antigen of a pathogen, for example, a viral pathogen.
RSV vaccines, as provided herein, comprise at least one (one or more) ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one RSV antigenic polypeptide. The term “nucleic acid, ” in its broadest sense, includes any compound and/or substance that comprises a polymer of nucleotides. These polymers are referred to as polynucleotides.
As used herein, the term “Messenger RNA” (mRNA) refers to any polynucleotide that encodes a (at least one) polypeptide (anaturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo. The skilled artisan will appreciate that, except where otherwise noted, polynucleotide sequences set forth in the instant application will recite “T” s in a representative DNA sequence but where the sequence represents RNA (e.g., mRNA) , the “T” s would be substituted for “U” s. Thus, any of the RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each “T” of the DNA sequence is substituted with “U. ”
The basic components of an mRNA molecule typically include at least one coding region, a 5′untranslated region (UTR) , a 3′UTR, a 5′cap and a poly-A tail. Polynucleotides of the present disclosure may function as mRNA but can be distinguished from wild-type mRNA in their functional and/or structural design features, which serve to overcome existing problems of effective polypeptide expression using nucleic-acid based therapeutics.
Polynucleotides (e.g., mRNAs) of the present disclosure, in some embodiments, are codon optimized. Codon optimization methods are known in the art and may be used as provided herein. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites) ; add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art-non-limiting examples include services from GeneArt (Life Technologies) , DNA2.0 (Menlo Park Calif. ) and/or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.
In some embodiments, a codon optimized sequence shares less than 95%sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide) ) . In some embodiments, a codon optimized sequence shares less than 90%sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide) ) . In some embodiments, a codon optimized sequence shares less than 85%sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide) ) . In some embodiments, a codon optimized sequence shares less than 80%sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide) ) . In some embodiments, a codon optimized sequence shares less than 75%sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide) ) .
In some embodiments, an in vitro transcription template encodes a 5′untranslated (UTR) region, contains an open reading frame, and encodes a 3′UTR and a polyA tail. The particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the mRNA encoded by the template.
As used herein, the term “5′untranslated region” (UTR) refers to a region of an mRNA that is directly upstream (i.e., 5′) from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide.
As used herein, the term “3′untranslated region” (UTR) refers to a region of an mRNA that is directly downstream (i.e., 3′) from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide.
As used herein, the term “open reading frame” is a continuous stretch of DNA beginning with a start codon (e.g., methionine (ATG) ) , and ending with a stop codon (e.g., TAA, TAG or TGA) and encodes a polypeptide.
As used herein, the term “polyA tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3′) , from the 3′UTR that contains multiple, consecutive adenosine monophosphates. A polyA tail may contain 10 to 300 adenosine monophosphates. For example, a polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a polyA tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo) the poly (A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, and/or export of the mRNA from the nucleus and translation.
As used herein, the term “pharmaceutically acceptable carriers” refers to a material or composition which, when combined with an active ingredient, is compatible with the active ingredient and does not cause toxic or otherwise unwanted reactions when administered to a subject, particularly a mammal. Examples of pharmaceutically acceptable carriers include solvents, surfactants, suspending agents, buffering agents, lubricating agents, emulsifiers, absorbents, dispersion media, coatings, and stabilizers.
As used herein, the term “immune response” refers to any detectable response of a cell or cells of the immune system of a host mammal to a stimulus (such as an immunogen) , including, but not limited to, innate immune responses (e.g., activation of Toll receptor signaling cascade) , cell-mediated immune responses (e.g., responses mediated by T cells, such as antigen-specific T cells, and non-specific cells of the immune system) , and humoral immune responses (e.g., responses mediated by B cells, such as generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids) . Examples of immune responses include an alteration (e.g., increase) in Toll-like receptor activation, lymphokine (e.g., cytokine (e.g., Th1, Th2 or Th17 type cytokines) or chemokine) expression or secretion, macrophage activation, dendritic cell activation, T cell (e.g., CD4+ or CD8+ T cell) activation, NK cell activation, B cell activation (e.g., antibody generation and/or secretion) , binding of an immunogen (e.g., antigen (e.g., immunogenic polypeptide) ) to an MHC molecule, induction of a cytotoxic T lymphocyte ( “CTL” ) response, induction of a B cell response (e.g., antibody production) , and, expansion (e.g., growth of a population of cells) of cells of the immune system (e.g., T cells and B cells) , and increased processing and presentation of antigen by antigen presenting cells. The term “immune response” also encompasses any detectable response to a particular substance (such as an antigen or immunogen) by one or more components of the immune system of a vertebrate animal in vitro.
As used herein, the term “effective amount” refers to an amount of agent that is sufficient to generate a desired response. For instance, this can be the amount necessary to inhibit viral replication or to measurably alter outward symptoms of the viral infection.
As used herein, the term “subject” refers to either a human or a non-human mammal. The term “mammal” refers to any animal species of the Mammalia class. Examples of mammals include: humans; non-human primates such as monkeys; laboratory animals such as rats, mice, and guinea pigs; domestic animals such as cats, dogs, rabbits, cattle, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, elephants, and the like.
As used herein, the term “administration” refers to the introduction of a substance or composition into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intramuscular, the composition (such as a composition including a disclosed immunogen) is administered by introducing the composition into a muscle of the subject.
Examples
The disclosure is further described by the following illustrative examples. The examples do not limit the disclosure in any way. They merely serve to clarify the disclosure.
Example 1: Design and Preparation of RSV F Protein Variants
A panel of mRNA antigens, containing multiple structure stabilizing features (5′UTR, 3′UTR, 3′polyA tail, and a Kozak sequence) , were designed for comparing the immunogenicity of different forms of the recombinant RSV F protein, and their encoding sequences of RSV F protein variants and sequences of mRNA constructs were shown in the Table 2-4, respectively. The specific information for modifications, relative to wildtype of RSV F protein, were described in Table 1. The designed constructs were expected to express a higher level of RSV prefusion F and lower RSV post-fusion F. Membrane-anchored DS-Cav1 (mDS-Cav1, mRNA-1777, see SEQ ID NO: 7 of WO2017/070622) , membrane-anchored VRC1 (mVRC1, see Table 7 of WO2019148101A1, see SEQ ID NO: 21) were set as the references. Wildtype of RSV F was designed as a reference construct, in which the encoding sequence of RSV F protein is derived from the wildtype of RSV F protein (Uniprot: P03420) with P102A, I379V, and M447V substitutions.
Table 1. RSV F protein designed for evaluation
Note: Amino acid positions are numbered according to wild-type RSV F protein (Uniprot: P03420) .
Table 2. Amino acid sequences and CDS sequences expressing forms of RSV F protein without a signaling peptide
Table 3. Amino acid sequences and CDS sequences expressing forms of RSV F protein with a signaling peptide
Table 4. encoding mRNA sequences of RSV F protein
Table 5. mRNA sequences of construct with supporting untranslated regions, polyA tail, and a signal peptide sequence attached to the coding sequence
Example 2: Expression of RSV F Protein Variants detected by ELISA
293T cells were transfected mRNA constructs (Table 5) using the MessengerMax Transfection reagent protocol. Briefly, MessengerMax reagent was incubated with OptiMEM I Reduced serum medium for 5 min at room temperature before mixing with mRNA. After 10 min incubation at room temperature, mRNA/MessengerMax mix was added to 293T cells incubated in 24-well plates. Cells were incubated at 37℃ for 24 h. At that time, cells were harvested and cell lysates were prepared with RIPA lysis buffer following the manufacture’s instruction.
Cell lysates were analyzed following standard ELISA protocols. Briefly, 96-well flat bottom plates (MaxiSorp ELISA plates, Nunc) were incubated with Palivizumab antibody in PBS (1 μg/mL, 100 μL/well) at 4℃ overnight. Plates were washed once with PBST using a plate washer and incubated with Block buffer (3%BSA in PBS) at room temperature for 1 h. After five washes with PBST, thawed cell lysates were added to the wells at 1/5 dilutions (100 μL/well) in Block buffer. Purified recombinant prefusion or post-fusion F proteins were added to control column in 2-fold dilution from 250 ng/well. After 2 h room temperature incubation, plates were washed five times with PBST and incubated with biotinylated D25 or131-2a antibodies. After 1 h room temperature incubation, plates were washed five times with PBST and incubated with Streptavidin-HRP (Pierce) following manufacturer’s recommendation. After 30 min incubation, plates were washed 5 times with PBST and incubated with TMB substrate. Color development was stopped after 15 min with stop solution and plates were read in a plate reader at a 450 nm wavelength.
RSV pre and post F protein concentrations in cell lysates were shown in FIG. 1. Construct RSV09, RSV10, RSV11, RSV14 and RSV15 showed higher prefusion F expression compared to mDS-Cav1 and RSV06.
Example 3: Expression of RSV F Protein Variants detected by Western blot
Cell lysates were prepared as described above. After thawing, the concentration of total protein in each cell lysate sample was determined by Bradford assay (Thermo Fisher) . Cell lysates were adjusted to the same concentration as PBS. Western samples were prepared by mixing cell lysates with 5× SDS loading buffer. The mixtures were heated at 100℃ for 5 min. The mixtures were then loaded onto 4-12%gradient SDS-PAGE (Thermo Fisher) and ran under 150V for 60 min. After running, proteins were transferred to a PVDF membrane with iBlot 2 system (Invitrogen) . Membranes were then blocked with Block buffer (5%Non-fat milk in TBST) at room temperature for 1 h. After blocking, membranes were incubated with 1: 1000 diluted Palivizumab or 2F7 in Block buffer. The membranes were then incubated with the secondary antibody, washed, and developed.
In western blot, two antibodies were used, Palivizumab (capture antibody) targeting RSV F site II and 2F7 targeting RSV fusion peptide. The results were shown in FIG. 2. The designed constructs, RSV09, RSV10, RSV11, RSV14 and RSV15 showed higher prefusion F expression compared to mDS-Cav1 and mVRC1.
Another Western Blot analysis was performed to examine the level of transgene expression provided by the different mRNAs, encoding RSV F protein variants (Table 5) , wildtype F, mVRC1 (SEQ ID NO: 21, WO2019148101A1) and Ref-mRNA (SEQ ID NO: 15, WO2021155243A1) . HEK293 cells plated in 6 well plates were transfected with the mRNAs using Lipofectamine MessengerMax (ThermoFisher) . The transfected cells were harvested and lysed, and the production of RSV F protein was analyzed by SDS gel followed by Western Blot analysis. Equivalent quantities of total protein were loaded on reducing SDS gel; after electrophoresis separation, the proteins were transferred to a PVDF membrane to be probed with an anti-RSV F mouse monoclonal antibody (clone 2F7 catalog no: sc-101362, Santa Cruz) . After the incubation with primary antibody, the membrane was washed and then incubated with anti-mouse HRP conjugate secondary antibody. Finally, the membrane was developed by electrochemiluminescence using standard techniques (ECL detection reagents, Pierce catalog no 34580) . The Western Blot results are shown in FIG. 3. A band of about 65 kD was revealed, which corresponds to the expected size of F protein. Among the constructs with mutations or truncations, RSV20 produced highest expression (Fig. 3A) . When comparing with its corresponding non-optimized constructs, the codon optimization constructs of RSV09 and RSV15 showed increased F protein expression (Fig. 3B) .
Example 4: Expression of RSV F Protein Variants detected by Flow cytometry
After transfection, 293T cells were pelleted in a V-bottom 96-well plate and resuspended in PBS. Live/Dead Fixable Cell stain (ThermoFisher) was added to each well (1 μL/well) and incubated at room temperature in the dark for 30 min. Cells were pelleted, washed twice with Stain buffer (10%FBS in PBS) and added to a V-bottom 96-well plate. Cells were resuspended in Stain buffer with AM14-FITC and 131-2A-PE and incubated for 15 min on ice in the dark. Cells were then pelleted and washed twice with Stain Buffer, resuspended in the Stain Buffer, and analyzed in a Fortessa flow cytometer (BD Science) .
The surface expression of RSV prefusion F protein is highly correlated with immunogenicity. The cell surface expression of our constructs on Day 1 post cell transfection was measured. The results were shown in FIG. 4.
AM14, targeting to RSV prefusion F trimer, was used to detect the RSV prefusion F protein expression.
RSV09 and RSV15 expressed more membrane-Anchored RSV Prefusion F protein compared to wildtype F protein.
In another experiment, different mRNAs encoding RSV F protein variants (Table 5) , wildtype F, mVRC1 (SEQ ID NO: 21, WO2019148101A1) and reference mRNA ( “Ref-mRNA” , SEQ ID NO: 15, WO2021155243A1) were tested by flow cytometry. HEK293T or Vero cells were transfected with different mRNAs. One day post transfection, cell surface prefusion RSV F protein was detected using antibodies specific to prefusion RSV F protein (D25) and prefusion RSV F trimer (AM14) . The results were shown in FIG. 5. When detected with AM14, RSV20 generated the highest level of cell surface prefusion RSV F protein among constructs with mutations or truncations. Similar results were observed when detected with D25. The codon optimization also enhanced the cell surface prefusion RSV F protein expression, which was in consistency with western blot results.
Example 5: IgG titer of RSV F Protein Variants
Based on the results of in vitro characterization, RSV07, RSV08, RSV09, RSV10, RSV11 and RSV15 were chosen for animal study.
Groups of 10 female BALB/c mice (CRL) aged 5–7 weeks were immunized twice at a 2-week interval with 1 μg of candidate mRNA vaccines in a lipid nanoparticle formulation. Two weeks following the second immunization, blood was drawn for serological assays.
ELISAs to evaluate binding antibody titers against prefusion or post-fusion F protein were performed. Ninety-six-well ELISA plates (NUNC) were coated with 2 μg/mL purified recombinant RSV prefusion or post-fusion F protein, respectively, and incubated at 4 ℃ overnight. IgG standard curves were prepared as followed. ELISA plates were coated with serially 2 times diluted mouse IgG from 800 ng/mL (100 μL/well) in PBS at 4℃ overnight. The plates were then washed and blocked for 1 h at room temperature with 3%BSA dissolved in PBS-T. Sera from mice was diluted with blocking buffer, transferred to the RSV F coated plates, and incubated for 2 h at room temperature. The plates were then washed three times with PBS-T. Following the plate wash, HRP conjugated goat anti-mouse/rat IgG secondary antibody (Invitrogen) , diluted at 1: 3000in blocking buffer, was added to the plates, and incubated for an additional 1 h. Plates were washed again and developed with TMB substrate (BioLegend) . The reaction was stopped after 5 min, and absorbance was read at 450 nm on an M3 ELISA microplate reader (Molecular Devices) . Antibody titers were calculated against IgG standard curve. The results were shown in FIG. 6.
Example 6: Mouse immunogenicity study 1
Selected constructs were then evaluated in vivo. Groups of 8 female BALB/c mice (CRL) aged 5–7 weeks were immunized twice at a 3-week interval with 1 μg of candidate mRNA vaccines in a lipid nanoparticle formulation (Table 6) . Two weeks following the second immunization, sera were collected for serological assays.
Table 6. Study of mouse immunogenicity study 1
Serum antibody titers against prefusion and postfusion F were determined with an ELISA. Briefly, 96-well ELISA plates (NUNC) were coated with 2 μg/mL purified recombinant RSV prefusion or postfusion F protein (AcroBiosystems) at 4 ℃ overnight. IgG standard curves were prepared as follows: ELISA plates were coated with serially 2 times diluted mouse IgG from 800 ng/mL (100 μL/well) in PBS at 4℃ overnight. After coating, the plates were washed with PBS-T and blocked for 1 h at room temperature with 3%BSA dissolved in PBS. Mouse sera were diluted with the blocking buffer, transferred to the RSV F coated plates, and incubated for 2 h at room temperature. The plates were then washed three times with PBS-T. Following the plate wash, HRP conjugated goat anti-mouse/rat IgG secondary antibody (Invitrogen) , diluted at 1: 3000 in blocking buffer, was added to the plates and incubated for 1 h at room temperature. Plates were washed again as before and developed with TMB substrate (BioLegend) . The reaction was stopped after 5 min reaction and absorbance of 450 nm was determined with an M3 ELISA microplate reader (Molecular Devices) . Antibody titers were calculated with the IgG standard curve. RSV09-C4, RSV15-C1 and RSV15-C4 elicited prefusion F-specific titers higher than other candidates (FIG. 7A and 7B) . On the contrary, all candidates showed lower postfusion F-specific antibody titers (FIG. 7C and 7D) . The data indicated that the designed mutations enhanced the stability of prefusion conformation.
The neutralization titer against RSV A2 was also determined using a microneutralization assay. In brief, sera were heat inactivated at 56℃ for 30 min and serially diluted with DMEM in 96-well plates. Fifty microliter of diluted sera were mixed with 50 μL of RSV A2 at a final inoculum of 1,000 PFU/well and incubated at 37 ℃ for 2 hours. One hundred microliter of HEp-2 cell suspension were added to each well (25,000 cells/well) and the plates were incubated at 37 ℃ for 5 days. Following the 5 day incubation, cell viability was determined by CCK-8 (Dojindo Molecular Technologies) . Plates were read with a M3 microplate reader (Molecular Device) . The neutralizing titers were determined using a four-parameter logistic curve fit in GraphPad 10 software.
The serum neutralizing titers were shown in FIG. 8A and 8B. According to the results, RSV09-C1, RSV15, RSV15-C4 and RSV22 elicited higher neutralizing titers compared to Ref-mRNA.
Example 7: Microneutralization assay
Sera were heat inactivated at 56℃ for 30 min and serially diluted into a 96-well plate. The sera were combined with RSV A2 at a final concentration of 200 TCID50. HEp-2 cells were added to each well and the plates were incubated at 37 ℃ for 96 h. Cells were then washed and fixed with acetone. Each well was then incubated with anti-RSV nucleocapsid protein antibody, followed by biotinylated goat anti-mouse monoclonal antibody. The signal was developed by adding TMB substrate. Plates were read on an M3 microplate reader (Molecular Device) . Titers were calculated by four-parameter curve fit using GraphPad 7 software.
Neutralization titers from animals immunized with RSV10 were the highest compared to other constructs on Day 28 and Day 56. (FIG. 9) . The neutralizing titer elicited by RSV 10 maintained highly on Day 56 compared to that on Day 28.
Example 8: ELISPOT to evaluate cellular immune responses
Spleens were placed on a 70-μm cell strainer and ground with a syringe. Strainers were then washed with 5 mL of 5%FBS RPMI-1640 to collect cells into a 50 mL tube. Cells were pelleted by centrifugation and resuspended with 5 mL of 1×RBC Lysis buffer for 5 min. After that, 35 mL of 5%FBS RPMI-1640 was added. Cells were pelleted and washed once with 15 mL RPMI-1640 with 10%FBS. Splenocytes were added to the plate at 4×105 cells/well, and the cells were stimulated with peptide pools for RSV-F for 36-48 hours. After stimulation, anti-IFN-γ-biotin antibody was added. Plates were incubated for 2 hours at room temperature. Then, diluted Streptavidin-HRP were added to the plates. Plates were washed and developed with BCIP/NBT substrate. The spots were then counted with AID ELISpot machine.
The data showed that RSV10 elicited high cellular immune responses (FIG. 10) .
Example 9: Mouse immunogenicity study 2
The immunogenicity of selected mRNA constructs was evaluated at low dose (1 μg per immunization) and high dose levels (4 μg per immunization) in vivo. Groups of 6 female BALB/c mice (CRL) aged 5–7 weeks were immunized twice at a 3-week interval with candidate mRNAs or Ref-mRNA vaccine in a lipid nanoparticle formulation. Serum samples were collected 42 days after the first immunization for serological assays (Table 7) . Splenocytes were harvested for intracellular cytokine (ICS) staining studies.
Table 7. Study design of mouse immunogenicity study 2
Neutralizing assay was performed as previously described. The RSV neutralizing antibody titers were shown in FIG. 11A. On Day 42, 4 μg RSV15 elicited higher neutralizing antibody titers than that of the Ref-mRNA at the same dose level. 1 μg RSV15-C4 elicited higher neutralizing antibody titers than the Ref-mRNA at the same dose level. RSV09-C1 also elicited higher levels of neutralizing antibodies than Ref-mRNA although no significant difference can be observed.
ICS study was performed as below. Spleens were placed on a 70-μm cell strainer and ground with a syringe. Strainers were then washed with 5 mL of 5%FBS RPMI-1640 to collect cells into a 50 mL tube. Cells were pelleted by centrifugation and resuspended in 5 mL of 1×RBC Lysis buffer (eBioscience) for 5 min. After that, 35 mL of 5%FBS RPMI-1640 was added to stop the reaction. Cells were pelleted and washed once with 15 mL RPMI-1640 with 10%FBS. Splenocytes were seeded to the U-bottom plates at 1×106 cells/well and they were stimulated with peptide pool of fusion protein from RSV A2 strain for 24 hours in the presence of protein transport inhibitor cocktail (eBioscience) . After stimulation, cells were washed once with PBS and stained with a LIVE/DEADTM Fixable Violet Stain (Invitrogen) for 15 min. Cells were then washed and stained using the Cytofix/Cytoperm kit (BD Bioscience) following the manufacturer’s instructions. Antibodies used to stain cells were as below: CD3-BV510, CD4-BV605, CD8-R718, IFN-γ-PE-CF594, IL-4-FITC, IL-5-APC (BD Bioscience) , TNF-α-PerCP-eFluor 710 and IL-2-PE-Cy7 (eBioscience) . The fluorescent signals were analyzed by an LSRFortessa flow cytometer (BD Biosciences) and analyzed by FlowJo software (Tree Star, Inc) . The results were shown in FIG. 11B and FIG. 11C.
According to the results, 4 μg RSV09-C1 elicited higher CD4+IL-2 T cell responses compared to the Ref-mRNA at the same dose level. 1 μg RSV15-C4 elicited higher frequency of IFN-γ, IL-2 and TNF-α producing CD4+ T cells when comparing with Ref-mRNA vaccine at the same dose level. RSV09-C1 and RSV15 also elicited comparable CD8+ T cell responses.
Example 10: In vitro transcription of mRNAs
Plasmids containing RSV F genes were linearized with specific restriction enzymes. The mRNA was synthesized in vitro using T7 RNA polymerase-mediated transcription. mRNAs were transcribed to contain 100 nucleotide-long poly (A) tails. N1-methyl-pseudouridine-5’ -triphosphate instead of UTP was used to generate modified nucleoside-containing mRNA. Capping of the mRNAs was completed co-transcriptionally using the trinucleotide cap1 analog. mRNAs were purified by chromatographic methods to remove impurities. The integrity and purity of mRNAs were examined by capillary electrophoresis, agarose gel electrophoresis and HPLC.
Example 11: Formulation of LNP encapsulated mRNA vaccine
LNPs were formulated using a microfluidic or T-mix method. Lipids were dissolved in ethanol containing an ionizable lipid, a helper lipid (DSPC or DOPE) , cholesterol and PEG-lipid with a molar ratio of 50: 10: 38.5: 1.5. mRNAs were prepared in an acetate buffer (pH4.0) . LNPs and mRNAs were mixed with a flow rate of 1: 3 to form LNPs. Formulated LNPs were ultra-filtrated/diafiltrated against 1x PBS (pH 7.4) and concentrated through a centrifugal filtration membrane or tangential flow filtration membrane. Concentrated LNPs were resuspended in a Tris-buffer containing sucrose as a cryoprotectant, filtered through a 0.22 μm membrane, and stored at -70℃till use.
Example 12: Characterization and quality control of LNP encapsulated mRNA vaccine
Average size and polydispersity index (PDI) of formulated LNPs were measured using dynamic light scattering (DLS) with a Malvern Zetasizer. LNP samples were diluted 50-100x with 1x PBS before measurements. Zeta potential of LNPs were measured on a Malvern Zetasizer. LNP samples were diluted 100x with distilled water before measurements. Encapsulation efficiency of LNPs was determined with a modified RiboGreen assay. Total mRNAs were quantified by RiboGreen after lysed with Triton X-100. The encapsulation efficiency was calculated as % [1-(Free form) / (Total form) ] .
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