HK1106723B - Vaccines against japanese encephalitis virus and west nile virus - Google Patents

Description

Vaccine against Japanese encephalitis virus and West Nile virus

Technical Field

The present invention relates to vaccines against Japanese encephalitis virus (Japanese encephalitis virus) and West Nile virus (West Nile virus).

Background

The flaviviridae family of flaviviridae includes about 70 viruses, most of which are arboviruses, many of which are the major human pathogens such as Yellow Fever (YF), Dengue (DEN), Japanese Encephalitis (JE), and tick-borne encephalitis (TBE) viruses (rev. in Burke and Monath, Fields Virology, 4)^thEd.: 1043-1126, 2001). For example, in asia, japanese encephalitis is the major cause of viral encephalitis, with 30,000 to 50,000 new cases reported each year. As another example, since the first case was diagnosed in the new york area in 1999, west nile virus has spread rapidly in north america. The risk of this virus migrating to south america and becoming an epidemic in underdeveloped countries is very high. There is a need for effective methods to prevent infection by these viruses, and vaccination is most appropriateAnd (4) calculating measures.

Flavivirus particles comprise a nucleocapsid composed of viral RNA and capsid protein C. The nucleocapsid is surrounded by an envelope containing the envelope glycoprotein E (50-60kDa) and a small membrane protein M (7-8 kDa). The genomic RNA is translated to generate a polyprotein precursor that is cleaved by cellular and viral proteases to form the viral proteins: c, prM/M, E, NS1, NS2A, NS2B, NS3, NS4A, 2K, NS4B, and NS5, wherein C to E are structural components of virions, and NS1 to NS5 are nonstructural proteins required for replication (Lindenbach and Rice, Fields Virology, 4)^thEd.: 991-1041, 2001). The prM protein (. about.25 kDa) is an intracellular precursor of M. Immature virions containing prM are produced by budding (budding) into the lumen of the endoplasmic reticulum and then transported to the cell surface via the exocytosis pathway. prM lysis occurs shortly before the particles are released in the post-golgi vesicles. Mature extracellular virus contains mainly M protein, but a small fraction of uncleaved prM will also be present.

The E protein is the main functional and antigenic surface component of the virion. The molecular structure of the extracellular domain of E has been resolved by cryoelectron microscopy, which forms homodimers on the surface of mature virions under pH neutral conditions (Rey et al, Nature 375: 291-298, 1995) and has been matched (fitito) into the electron density map of virions (Kuhn et al, Cell 108: 717-725, 2002). During infection, the E protein functions as a class II fusion protein (Modis et al, Nature 427: 313-319, 2004). After the virus binds to the cellular receptor and internalizes, the acidic pH in the resulting endosomes promotes dissociation of the dimer, thereby exposing the previously hidden hydrophobic fusion loop of each monomer outward. At the same time, these loops insert into the cell (endosomal) membrane and the monomers rearrange to form elongated trimers. Further refolding of the trimer brings the cell membrane into close proximity with the viral membrane and fuses them, releasing the contents of the virion into the cytoplasm. Previous studies showed that some substitutions on the E proteins of DEN and JE, selected in mouse brain cells and serial passages of cultured monkey kidney and mosquito cells, have been localized to specific sites of the 3D structure of the protein and reportedly associated with changes in viral fusion function. These studies indicate that the fusion PH threshold of some attenuated vaccines is reduced by 0.6 to 1 PH unit compared to the corresponding parental viral isolate. Some of the alterations in the six residues (residues 54, 191, 202, 266, 268 and 277) of the DEN3E protein are located in the region of domain II. This region was proposed as a focus of low pH-mediated conformational changes required to surface-expose conserved hydrophobic cd fusion loops (Lee et al, Virology 232: 28l-290, 1997).

There is no evidence that the small (mature) M protein plays a role or has any other significant function in events leading to virus internalization from endosomes, but its intracellular precursor prM is known to be important for morphogenesis and trafficking of progeny virions. The prM protein also facilitates correct folding of E (Lorenz et al, J.Virol.76: 5480-5491, 2002) and acts to avoid pre-maturation conformational rearrangements of the E protein dimer at the stage of export of new particles to the cell surface via the acidic secretory compartment (Guirakho et al, J.Gen.Virol.72: 1323-1329, 1991; Guirakho et al, Virology 191: 921-931, 1992).

ChimeriVax has already been published^TMThe technology is applied to the generation of attenuated live vaccine candidates against flaviviruses of medical importance. It uses YF17D Vaccine virus as a vector in which the prM-E gene is replaced by the prM-E gene of a heterologous flavivirus such as JE, dengue, West Nile or St.Louis encephalitis virus (Monath et al, Vaccine 17: 1869-. Previously, ChimeriVax comprising the prM-E gene from SA14-14-2 virus (a live attenuated JE vaccine used in China)^TMJE vaccine virus was propagated to high titers in Vero cells cultured supplemented with fetal bovine kidney cellsSerum (FBS) (Monath et al, Biologicals 33: 131; 144, 2005). It was successfully tested in preclinical and phase I, II clinical trials (Monath et al, Vaccine 20: 1004-. Similarly, ChimeriVax has been used^TMSuccessful phase I clinical trials were conducted with WN vaccine candidates that contained the prM-E sequence of West Nile Virus (strain NY99) and incorporated three specific amino acid changes in the E protein to increase attenuation (Arroyo et al, J.Virol.78: 12497) -12507, 2004).

Summary of The Invention

The present invention provides recombinant flaviviruses comprising one or more membrane (M) protein mutations (e.g., substitutions, deletions, or insertions), such as mutations that attenuate the flavivirus (e.g., mutations that reduce the viscerotropism/viremia of the flavivirus), mutations that increase the genetic stability of the flavivirus when propagated in cell culture (e.g., prepared in serum-free media), and/or mutations that increase vaccine virus production. The flavivirus of the present invention can be a chimeric flavivirus, e.g., comprising the capsid and non-structural proteins of a first flavivirus (e.g., a yellow fever virus such as YF17D) and the membrane and/or envelope proteins of a second flavivirus (e.g., japanese encephalitis virus, west nile virus, dengue virus (dengue-1, dengue-2, dengue-3, or dengue-4), st.

In the flavivirus of the present invention, the mutation (e.g., substitution) may be in the transmembrane or extracellular domain of membrane protein M. For example, the mutation may be within the region of amino acids 40-75 of the predicted membrane helix (membrane helix) of the flavivirus membrane protein M. For example, the mutation can be a substitution, the substitution in flavivirus (such as Japanese encephalitis virus) membrane protein in the 60 th amino acid (such as Japanese encephalitis virus M arginine to cysteine), or in another flavivirus corresponding amino acid. As is well known to those skilled in the art, standard amino acid sequence alignments can be used to determine which amino acid in a given flavivirus "corresponds to" an amino acid in another flavivirus. As another example, the mutation may be a substitution in amino acid 66 of a membrane protein of a flavivirus, such as West Nile virus (e.g., a substitution of proline for leucine in the M protein of West Nile virus), or in the corresponding amino acid of another flavivirus. In another example, the mutation is in another membrane-anchored amino acid, such as selected from the group consisting of amino acids flanking residue M66, including positions 60, 61, 62, 63, 64, 65, and 66 of japanese encephalitis virus or west nile virus (or the corresponding amino acids of other flaviviruses) or other amino acid residues of the transmembrane region.

We also provide for the first time evidence that the extracellular domain of the M protein is of significant functional significance, since the change from glutamine to proline at M5 residue raises the pH threshold of infection. It is therefore now contemplated that not only the C-terminal hydrophobic anchor (anchor) may be amino acid altered or various deletions or insertions introduced into the amino-terminal extracellular domain or surface portion of the M protein to achieve flavivirus attenuation. Thus in other examples, the virus of the invention comprises one or more mutations in the extracellular domain (residues 1-40) of the M protein as described herein. This result is quite unexpected if it deprives the mature M protein of flavivirus of any known function.

In addition to the membrane protein mutations described above, in the case of a chimeric flavivirus comprising the membrane and envelope proteins of west nile virus, the virus of the invention may comprise one or more envelope protein mutations at amino acids selected from the group consisting of amino acids 107, 138, 176, 177, 224, 264, 280, 316, and 440. In other flaviviruses, the mutations may be at the amino acids corresponding to these amino acids. As a specific example, the flavivirus may comprise mutations corresponding to a mutation at amino acid 66 of the West Nile M protein and mutations in the E protein at amino acids corresponding to amino acids 107,316, and 440 of West Nile Virus. In addition to the mutations described above, the flavivirus of the present invention may also comprise one or more mutations in the hydrophobic pocket of the hinge region of the envelope protein as described elsewhere herein. Further mutations that may be included in the viruses of the invention are mutations in the 3' UTR, capsid protein, or other envelope protein regions as described below.

The present invention also provides vaccine compositions comprising a flavivirus as described above and elsewhere herein, and a pharmaceutically acceptable carrier or diluent, and methods of inducing an immune response to a flavivirus in a patient by administering such vaccine compositions. Patients treated according to this method include those who are not yet but at risk of being infected with a flavivirus, as well as patients infected with a flavivirus. Further, the invention includes the use of the flaviviruses described herein in the methods of prevention and treatment described herein, as well as the use in the preparation of medicaments for these purposes.

The present invention also provides methods of making vaccines comprising the flaviviruses described herein, which involve introducing mutations into the membrane proteins of the flaviviruses that reduce viscerotropism/viremia and/or increase genetic stability/yield. Further, the present invention provides nucleic acid molecules (RNA or DNA) corresponding to the genome of the flavivirus (or the complement thereof) described herein, as well as methods of using these nucleic acid molecules to make the viruses of the present invention.

The flaviviruses of the present invention are advantageous because they have reduced virulence (as shown, for example, by reduced viscerotropism/viremia) and they provide a higher level of safety when administered to patients relative to their unmutated counterparts (counterparts). Another benefit is that some mutations are as in ChimeriVax^TMThe M-60 mutation in JE, eliminates the accumulation of undesirable mutations that would compromise safety in vaccine preparation in the absence of them, and increases production yield. Other benefits of these viruses are derived from their ability to contain the yellow fever YF17D sequence (e.g., encoded byThe sequence of the code capsid and non-structural proteins), YF17D (i) has been confirmed to be safe for more than 60 years, at which stage more than 3.5 billion doses have been administered to humans, (ii) induces long-term immunity after a single dose, and (iii) rapidly induces immunity within a few days of vaccination. In addition, the vaccine virus of the present invention actively infects the treated patient. Since the cytokine milieu (mileu) and innate immune responses of immunized individuals are similar to those in natural infections, antigenic material (mass) spreads in the host (expand), correctly folded conformational epitopes are efficiently processed, adaptive immune responses are stronger, and memory is established.

Given that the mature M protein of flavivirus loses any known function, the advantage of mutations in the M protein in vaccine safety and preparation in cell culture would be new and unexpected.

Other features and advantages of the invention will be apparent from the following detailed description, the accompanying drawings, and the claims.

Brief Description of Drawings

FIG. 1A is a diagram of the 3 'untranslated region of yellow fever virus, showing the domains within this region (repeat (RS), conserved sequence CS2, CS1, and stem-loop structure at the 3' -end), as well as examples of mutations contained in the virus of the invention (e.g., deletions of dA, dB, dC, dD, d7, d14, CS2d5, and CS2d 16).

FIG. 1B is a schematic representation of the sequence of the 3' untranslated region of the yellow fever 17D virus from the middle of the third RS element to the end of the UTR and the published secondary structure prediction (Proutski et al, J.Gen.Virol.78: 1543-1549, 1999) (SEQ ID NO: 31).

FIG. 1C is a schematic representation of the most reasonable prediction of the secondary structure of the YF17D 3' UTR generated using the Zuker RNA folding algorithm (SEQ ID NO: 32).

FIG. 1D is a schematic representation of the effect of 3' UTR deletion (showing dC deletion; Zuker method) on the most reasonable YF17D structure (compare with FIG. 1C) (SEQ ID NO: 33).

Figure 2A illustrates the sequence of capsid proteins of tick-borne encephalitis virus, and the sequence of the capsid proteins in Kofler et al, j.virol.76: 3534 and 3543, 2002 (SEQ ID NO: 34).

Fig. 2B illustrates the sequence of the capsid protein of YF17D virus. Therein, it is pointed out that in some ChimeriVax^TMThe region of WN virus predicted by computer analysis to have the alpha-helical secondary structure (alpha-helix I-IV), as well as the hydrophobic region (solid bar) and deletions introduced into this protein (e.g., deletions C1 and C2; boxed) (SEQ ID NO: 35).

FIG. 3 shows the growth of the indicated viruses (WN01, WN02P5, large plaques (plaque), small plaques, and YF/17D) in HepG2 cells.

FIG. 4 shows the growth of the indicated viruses (WN01, WN02P5, large plaques, small plaques, and YF/17D) in THLE-3 cells.

FIG. 5 shows viremia in hamsters induced by the indicated viruses (WN02P 5; mixed plaques), small plaques (PMS, P10), and large plaques (PMS, P10).

FIG. 6 illustrates SF ChimeriVax^TMPassage of JE virus samples (g.s., laboratory passage to study genetic stability).

FIG. 7 shows SF ChimeriVax of the present invention^TMGrowth curves of JE viruses (unclosed P2, P3MS (E-107), P4PS (E-107), P5g.s. (M-60), and P5VB (E-107)) at indicated times post-infection, showing the growth curves containing M-60[ arginine (R) → cysteine (C) and E-107 phenylalanine (F) → leucine (L))]The mutant virus samples had higher growth rates in SF cultures than the non-mutant virus (P2).

FIG. 8A is a graph showing that the vaccine batches were tested at a range of acidic pH values compared to the P5 uncloned vaccine (vaccine bulk) and clone I (E-107 mutant), non-mutant (clone A), and M-60 mutant (clone C)Post-treatment M-5ChimeriVax^TMInfectivity of JE mutant (clone E). Of interest is the appearance of a ramp and the loss of infectious pH of the virus, rather than diluting the original titer of the sample (e.g., at pH 6.8).

FIG. 8B is ChimeriVax^TMJE vaccine (1.9 log determined by back titration against cocca)₁₀PFU/dose) and ChimeriVax^TMJE M5 mutant (1.4 log as determined by reverse titration of the inoculum)₁₀PFU/dose) survival profile in 3-4 day old suckling mice vaccinated via the intracerebral route.

FIG. 8C is ChimeriVax^TMJE M5 mutant Virus (1.4 log determined by reverse titration of the inoculum)₁₀PFU/dose) with(0.9 log as determined by reverse titration of the inoculum)₁₀PFU/dose) survival profile in 3-4 day old suckling mice vaccinated via the intracerebral route.

FIG. 8D shows the results of an Indirect Fusion assay (Indirect Fusion assay), which compares ChimeriVax^TMP7 and P10 of DEN1-4 virus. The virus yield (output) of each assay was determined using a standard plaque assay. A, ChimeriVax^TM-DEN1PMS P7 (triangle) and P10 (diamond); b, ChimeriVax^TM-DEN2PMS P7 (triangle) and P10 (diamond); c, ChimeriVax^TM-DEN3PMS P7 (triangle) and P10 (diamond); d, ChimeriVax^TMDEN4PMS P7 (triangle) and P10 (diamond).

FIG. 8E shows ChimeriVax^TMResults of indirect fusion assay of DEN3, which compares PMS (P7) vaccine with vaccine lot (lot) (P10) and P15 viruses. Standard plaque assays were used to determine the virus yield of each assay. ChimeriVax^TMDEN3PMS P7 (triangle), P10 (diamond), and P15 (square).

FIG. 8F shows ChimeriVax^TMThe structure of DEN 1E-protein dimer (amino acids 1 to 394) of DEN1 virus (Guirakho et al, J.Vir)ol.78: 9998-10008, 2004). (A) The position of the positively charged lysine (K) at residue 204 (PMS, 204K) of the P7 virus is shown by the CPK (sphere showing the size of van der Waal radius). The three domains are shown in black (domain I), light grey (domain II), and dark grey (domain III). (B) An enlarged view of a portion of the labeled area in fig. a. (C) The E protein model from mutant DEN1 virus is the same region as in panel B (P10, 204R shown in black). The selected amino acids are shown in the schematic stick diagrams in FIGS. B and C. Middle ash (Medium gray), carbon; dark gray, nitrogen; black, oxygen; light ash, sulfur.

FIG. 9A shows ChimeriVax^TMThe penetration efficiency of the JE virus M60 mutant (clone C), the E107 mutant (clone I), and the non-mutant (clone A) at the indicated times. These results indicate that the M60 mutation clearly favours penetration into SF vero cells at the 5 and 10 minute time points. Extracellular virus was inactivated by infecting SF African green monkey kidney cells with the appropriate diluted virus (clones A, C, and I in serum-free medium) for 5, 10, 20, or 60 minutes, followed by treatment with a solution of 0.1M glycine, 0.1M NaCl, pH3.0 for 3 minutes. The wells were washed twice with PBS, then the monolayers were covered with methylcellulose, after which the plaques were stained with crystal violet on day 5. Penetration efficiency is shown as the percentage of plaques observed after glycine treatment that occupied PBS rather than glycine treated control infected wells.

FIG. 9B is a schematic representation of the positions of E-107, M-5, and M-60 amino acid residues in envelope proteins E and M, showing the putative effect of M-5 residues on fusion. The dashed sequence (stretch) at the top of domain II of the E protein containing residues E-107 represents the fusion peptide (c-d loop) inserted into the cell membrane (Rey et al, Nature 375: 291-298, 1995). The M-5 residue is in the N-terminal portion of the extracellular domain of the M protein. The E protein monomers rearrange into a trimeric complex, forcing the fusion of the cell and viral membrane by its folding (Modis et al, Nature427 (6972): 313-319, 2004). The M protein may be a functional component in a complex, for example, to facilitate fusion of the viral membrane to the cell membrane through interaction with the E protein. The M-60 residue is between the two C-terminal transmembrane sequences of M and may be involved in the interaction of the cell with the viral membrane in fusion.

Detailed description of the invention

The present invention provides vaccines and methods for preventing and treating flavivirus (e.g., Japanese Encephalitis (JE) or West Nile (WN) virus) infection. The methods of the invention generally involve immunizing a subject with a live attenuated chimeric flavivirus that consists of a first flavivirus (e.g., yellow fever virus) in which one or more structural proteins (e.g., membrane and/or envelope proteins) have been replaced with those of a second flavivirus (e.g., Japanese Encephalitis (JE) and/or West Nile (WN) virus; see also below). As described further below, the membrane protein of the chimeras of the invention comprise one or more mutations. As also described below, other flavivirus structural proteins such as membrane and/or envelope proteins can be used to replace the chimeric virus of Japanese encephalitis virus or West Nile virus in those. Further, the membrane protein mutations of the present invention may also be used with intact non-chimeric flaviviruses (e.g., any of those listed herein), excluding any substitutions to structural proteins, and optionally one or more additional mutations, such as those described herein.

A specific example of a chimeric virus that can be included in a vaccine of the invention is the human yellow fever vaccine strain, YF17D (e.g., YF17D-204 (R) (R))，Sanofi-Pasteur，Swiftwater，PA，USA； ，Sanofi-Pasteur，Marcy-L’Etoile，France；ARILVAX^TM，Chiron，Speke，Liverpool，UK；Berna Biotech, Bern, Switzerland); YF17D-204France (X15067, X15062); YF17D-204, 234US (Rice et al Science 229: 726-The white has been replaced by membrane and envelope proteins of japanese encephalitis virus (including M protein mutations, such as the substitution in M60, as described herein). In another example, the membrane and envelope proteins of YF17D were replaced by those of west nile virus (including M protein mutations, such as substitutions in M66, as described herein).

In other examples, another flavivirus, such as dengue virus (serotype 1, 2, 3, or 4), st-lewy encephalitis virus, Murray valley encephalitis virus, yellow fever virus, including YF17D strain, or any other flavivirus, can provide the membrane and/or envelope proteins in this chimeric virus. Other flaviviruses that may be attenuated according to the present invention, whether as intact non-chimeric viruses or as a source of membrane and/or envelope proteins in chimeras, include any other mosquito-borne (mosquito-borne) flaviviruses such as Kunjin, Rocio encephalitis and brazil (iheus) virus; tick-borne flaviviruses, such as central european encephalitis (central european encephalitis), Siberian encephalitis (Siberian encephalitis), Russian Spring-Summer encephalitis (Russian Spring-Summer encephalitis), quarantin forest disease (Kyasanur forest disease), eastern (ompk) hemorrhagic fever, skip disease (Louping ill), Powassan, Negishi, Absettarov, Hansalova, Apoi, and Hypr virus; and viruses from the Hepacivirus genus (e.g., hepatitis c virus). Other yellow fever Virus strains, such as YF17DD (GenBank accession No. U17066), YF17D-213(GenBank accession No. U17067; dos Santos et al, Virus Res.35: 35-41, 1995), and Galler et al, Vaccines16 (9/10): the 17DD strain of yellow fever virus described in 1024-1028, 1998 can also be used as a backbone virus (backbone virus) into which a heterologous structural protein can be inserted according to the present invention.

Each of the above-listed viruses has some propensity to infect internal organs. The viscerotropism of these viruses can cause dysfunction of viable internal organs, as observed in YF vaccine-related adverse disease events, only infrequently. Viral replication in these organs can also cause viremia and thus contribute to invasion of the central nervous system. Thus, reduction of the viscerotropic phenomena of these viruses by mutagenesis in accordance with the present invention can reduce the ability of the viruses to cause adverse viscerotropic disease and/or to invade the brain and cause encephalitis.

The mutations of the invention have beneficial effects on the virus, including, for example, attenuation, stability, and/or increased replication. The mutations are present in the membrane proteins, for example in the transmembrane or extracellular domain of the membrane proteins. For example, the mutation may be in amino acid 60 or 66 of the membrane protein and/or in other amino acids of the predicted transmembrane region (e.g., any one or more of amino acids 40-75), or in the N-terminal extracellular domain of the M protein (e.g., M-5). As a specific example, the membrane protein of the 60 th amino acid (in wild type Japanese encephalitis virus arginine) can be replaced by another amino acid such as cysteine. In ChimeriVax^TMSubstitution of the M-60 position of the JE virus from arginine to cysteine significantly reduced viremia of the virus in humans in clinical trials (viscerotropism), in which vaccine variants with and without the M-60 mutation were examined (tables 11A and 11B). In addition to cysteine, other amino acids such as serine, threonine, glycine, methionine, etc. may be substituted for the wild-type amino acid at position 60 of the membrane protein. In another example, amino acid 66 of the membrane protein (leucine in wild-type west nile virus) may be replaced by another amino acid such as proline. In addition to proline, other hydrophobic amino acids, such as isoleucine, methionine or valine, or small amino acids, such as alanine or glycine, may be substituted for the wild-type amino acid at position 66 of the membrane protein. These mutations can also be present in the corresponding amino acids of other flaviviruses, as described herein.

Other examples of substitutions that may be made in the membrane protein sequence, the amino acids at positions 61, 62, 63, and/or 64 may be replaced by mutations at position 60, mutation at position 66, and/or other mutations, either alone or in combination with one another. Alternative examples of such positions in the west nile virus membrane protein sequence include: valine to alanine at position 61, valine to glutamic acid or methionine at position 62, phenylalanine to serine at position 63, and valine to isoleucine at position 64. These mutations can also be present in the corresponding amino acids of other flaviviruses, as described herein.

Examples of substitutions at or around these positions in the JE virus membrane protein sequence include any of the remaining 20 amino acids that would be expected to achieve the desired effect on viscerotropism and/or replication/stability of the vaccine virus in cell culture during manufacture. Other examples in chimeric or non-chimeric flaviviruses include substitutions in the N-terminal extracellular domain of the M protein by any amino acid in residues 1-40 of the protein, alone or in combination, as well as deletions of various sizes (e.g., 1, 2, 3,4, 5, etc. amino acids long) introduced into the extracellular domain and/or transmembrane domain of the M protein.

In addition to one or more of the above-described mutations in the membrane protein, the viruses of the present invention may also comprise one or more additional mutations. For example, for west nile virus, such other mutations may be in the region at position 107 (e.g., L to F), 316 (e.g., a to V), or 440 (e.g., K to R) of the envelope protein of west nile virus (or a combination thereof). Thus, the mutations may be, for example, in one or more of amino acids 102-112, 138 (e.g., E-to-K), 176 (e.g., Y-to-V), 177 (e.g., T-to-A), 244 (e.g., E-to-G), 264 (e.g., Q-to-H), 280 (e.g., K-to-M), 311-321, and/or 435-445 of the West Nile envelope protein. As a specific example, using the sequence of West Nile strain NY 99-flamingo 382-99 (GenBank accession AF196835) as a reference, the lysine at position 107 may be replaced by phenylalanine, the alanine at position 316 may be replaced by valine, and/or the lysine at position 440 may be replaced by arginine. Examples of other combinations of amino acids that can be mutated include the following: 176. 177, and 280; 176. 177, 244, 264, and 280; and 138, 176, 177, and 280. Further, as described herein, these mutations may also be present at corresponding amino acids of other flaviviruses.

ChimeriVax^TMThe JE vaccine already contains all of the above-mentioned SA14-14-2 specific mutations, since it contains the SA 14-14-2-specific JE envelope. Also hasChanges to other amino acids in the E protein can be selected and introduced for additional attenuation based on knowledge of the structure/function of the E protein (e.g., as described below). These mutations may also be present in the corresponding amino acids of other flaviviruses, as described herein.

In addition to the amino acids described above, other amino acids, such as those that produce conservative changes as described above, may be substituted. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, and leucine; aspartic acid, glutamic acid, asparagine, and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.

The viruses of the invention (e.g., Japanese encephalitis and West Nile viruses, as well as chimeric flaviviruses comprising membrane and envelope proteins from these or other flaviviruses) may comprise, in addition to the mutations discussed above (e.g., membrane protein mutations), one or more mutations in the hinge region or hydrophobic pocket of the envelope protein, as these mutations have been shown to result in a reduction in viscerotropism (Monath et al, J.Virol.76: 1932-1943, 2002; WO03/103571A 2; WO 05/082020; Guirakho et al, J.Virol.78 (18): 9998-10008, 2004). The polypeptide chain of the envelope protein folds into three distinct domains: a central domain (domain I), a dimerization domain (domain II), and an immunoglobulin-like regulatory domain (domain III). The hinge region is present between domains I and II and upon exposure to acidic pH, undergoes a conformational change in the formation of an envelope protein trimer (hence the name "hinge") which is involved in the fusion of the viral and endosomal (endosomal) membranes following uptake of the virus by receptor-mediated endocytosis. Prior to the conformational change, the protein exists as a dimer.

A large number of envelope amino acids are present in the hinge region, including, for example, amino acids 48-61, 127-131, and 196-283 of the yellow fever virus (Rey et al, Nature 375: 291-298, 1995). Any of these amino acids, or the immediately adjacent amino acids (and the corresponding amino acids in other flavivirus envelope proteins), can be mutated in accordance with the present invention and tested for attenuation. Of particular interest are amino acids within the hydrophobic pocket of the hinge region. As a specific example, in a chimeric flavivirus comprising the sequence of the dengue-1 envelope protein inserted into a yellow fever virus vector, an attenuation has been shown to occur in place of amino acid 204 (K to R) of the envelope protein within the hydrophobic pocket of the hinge region (Guirakho et al, J.Virol.78: 9998-. This replacement causes a structural change in the envelope protein, disrupting intermolecular hydrogen bonding between one and the other envelope monomer of the wild-type protein, and replacing with a new intramolecular interaction within the monomer. This observation suggests that the attenuation resulting from this alternative is due to these new interactions that alter the protein structure in the pre-fusion conformation, likely by altering the pH threshold required to fuse the viral membrane to the host cell, and provides the basis for designing further attenuating mutants in which other alternatives can be used to enhance intramolecular interactions in the hydrophobic pocket, leading to attenuation. Examples of such mutations/substitutions that may be made within the hydrophobic pocket and included within the viruses of the present invention include substitutions in E202K, E204K, E252V, E253L, E257E, E258G, and E261H (and corresponding substitutions in other flaviviruses). Any amino acid changes in the corresponding regions of the E proteins of the JE and WN viruses can be designed and incorporated based on knowledge of the structure of the homologous proteins.

The E gene contains a functional domain in which amino acid changes may affect function to reduce virulence as described by Hurrelbrink and McMinn (adv. virus dis.60: 1-42, 2003). Where the functional region of the E protein, where mutations can be inserted, together with the membrane deletions/mutations described herein, a suitably attenuated vaccine can be obtained comprising: a) a known receptor binding region on the outer surface of domain III, b) a molecular hinge region between domains I and II that determines an acid-dependent conformational change of the E protein in the endosome and reduces the effectiveness of viral internalization; c) the interface of prM and E protein, i.e. the region of E protein that interfaces with prM after rearrangement from dimer to trimer upon exposure to low pH in endosomes; d) the apical end of the fusion domain of domain II, which is involved in fusion with the membrane of the endosome during internalization; and E) stem-anchor region, it also in acid induced fusion process involved in E protein conformation changes play a role.

Other attenuating mutations in the viruses of the invention, including mutations in the 3' untranslated region of the yellow fever virus backbone, may be combined with one or more membrane protein mutations. FIG. 1A shows the organization of the 3' UTR of yellow fever virus vaccine strain YF17D, which is all ChimeriVax^TMCommon to viruses. It contains, in order from the 3 ' end, (i) a 3 ' -terminal stem-and-loop structure, which has been hypothesized to function as a promoter for negative strand RNA synthesis and is conserved in all flaviviruses, (ii) two conserved sequence elements, CS1 and CS2, which share a high degree of nucleotide sequence homology with all mosquito-borne flaviviruses, and (iii) three copies of the repeat sequence element (RS) located in the upstream portion of the 3 ' UTR, characteristic of the YF17D vaccine virus (Chambers et al, Annu. Rev. Microbiol. 44: 649-. The 3' UTR also contains multiple stem-loop-structures, such as those of non-conserved regions downstream of the RS element, as shown in figure 1B.

The 3' UTR mutations included in the viruses of the invention are typically short attenuating deletions, e.g., less than 30 nucleotides (e.g., 1, 2, 3, etc., and up to 29 (e.g., 2-25, 3-20, 4-15, 5-10, or 6-8 nucleotides in length); U.S. patent applications 60/674,546 and 60/674,415). In some instances, short 3 'UTR deletions are designed to destabilize the secondary structure of one or more stalk structures in the 3' UTR. In addition to deletions, mutations in these structures also include substitutions that also destabilize the stem structure. In certain examples, mutated stem-loop-structures are present in non-conserved regions of the 3' UTR or in conserved regions that are able to tolerate these mutations (e.g., in CS 2). For example, a stem-destabilizing mutation can be present in any one or more of the predicted stem structures shown in FIG. 1B, four examples of these deletions (dA, dB, dC, and dD) are shown in FIG. 1B. Thus, in addition to these specific examples, other examples of mutations in the 3 ' UTR in the yellow fever virus include mutations of 1-2, 3-8, 4-7, or 5-6 nucleotides comprising, for example, the following stem sequence, which are shown in FIG. 1B from 5 ' to 3 ': TGGAG, CTCCA, GACAG, TTGTC, AGTTT, GGCTG, CAGCC, AACCTGG, TTCTGGG, CTACCACC, GGTGGTAG, GGGGTCT, AGACCCT, AGTGG, and TTGACG. These mutations can also be present at the corresponding amino acids of other flaviviruses, as described herein.

In addition to stem-destabilizing mutations, other shortages in the 3' UTR may be included in the viruses of the invention along with one or more membrane (and possibly other) mutations. For example, the previously described Δ 30 mutations (Men et al, J.Virol.70: 3930-. Thus, for example, the invention encompasses any operable deletion within this region of 1, 2, 3, etc., and up to 29 (e.g., 1-25, 2-20, 3-15, 4-14, 5-13, 6-12, 7-11, 8-10, or 9) nucleotides in length. As a specific example, the virus of the invention may comprise deletion d7, wherein the following nucleotides in this region of YF17D are deleted: nucleotides 345-. The invention also encompasses mutations that include deletions from the 3 'or 5' end of this sequence, e.g., 1, 2, 3,4, or 5 additional nucleotides. In other examples, the invention includes short deletions in the conserved sequences CS1 and CS 2. Such mutations may include deletions of, for example, 1-29, 2-25, 3-20, 4-15, 5-10, or 6-8 nucleotides of these sequences. As two specific examples, the deletion of nucleotides 360-364 (GGTTA; CS2d 5; FIG. 1A) and/or nucleotides 360-375 (GGTTAGAGGAGACCCT; (SEQ ID NO: 17); CS2d 16; FIG. 1A) from CS2 of YF 17D-specific 3' UTR. Mutations comprising deletions from the 3 'or 5' end of this sequence, e.g., 1, 2, 3,4, or 5 additional nucleotides, can also be used. Similar mutations can be made to the 3 ' UTR of other flaviviruses based on the secondary structure of the 3 ' UTR's. Predictions of the secondary structure of the 3' UTR of other flaviviruses such as dengue, Kunjin and TBE (see, e.g., Proutski et al, Virus Res.64: 107-123, 1999) and HCV (see, e.g., Kolykhalov et al, J.Virol.70: 3363-3371, 1996) have been published. Further, multiple 3' UTR nucleotide sequences of many flavivirus strains representing all four major serum complexes (YF, JE, dengue, and TBE) are available from GenBank. The sequences of the other strains can be determined by virus sequencing. The secondary structure of these sequences can be predicted simply by standard software (e.g., mfold or RNAfold programs) to reveal potential stem-loop structures that can be subjected to mutagenesis.

It should be noted that the true secondary structure of the 3' UTR of flaviviruses, including YF17D virus, is unknown, since there is no way to experimentally confirm their presence in the complete viral context, and therefore the predictions that have been published, such as those made by Proutski and cooperators (fig. 1B), for YF17D may be incorrect. Many alternative structures can be predicted to form in relatively long RNA molecules (Zuker et al, N.A.R.19: 2707-. The actual structure can be influenced by the formation of various pseudoknot (pseudokinot) interactions (Olsthoorn et al, RNA 7: 1370-. To complicate the interpretation of published results of theoretical computer predictions, manual manipulations are often used, such as initial folding of partial sequences, followed by entry of the initially predicted structure into the structure of longer RNA sequences, artificial application of N's in the initial folding step, and subjective selection of preferred structural elements (e.g., Mutebi et al, J.Virol.78: 9652. sup. 9665, 2004). Therefore, we used the commonly used Zuker's prediction algorithm to fold the 3' UTR RNA sequence of YF 17D. Fig. 1C shows the predicted optimal structure, which is different from the proputsky prediction shown in fig. 1B. Importantly, the small deletions dA, dB, dC, dD, d7, and d14 in fig. 1A and 1B generally destabilize the predicted optimal (fig. 1C) and suboptimal (suboptimal) structures of native YF 17D. FIG. 1D shows an example of the optimal structure for such an alteration (for dC mutants). In contrast, the deletions of CS2d5 and CS2d16 (fig. 1A and 1B) did not significantly alter the optimal native structure, suggesting that by altering CS2 itself rather than the 3' UTR junctionStructural sequences or alternatively by altering some suboptimal structure, these deletions possibly attenuating the virus (in ChimeriVax)^TM-attenuation in hamster model of WN). Thus, even though some of the deletions were designed based on proptski structural prediction (fig. 1B), their true role may be due to destabilizing the different structural elements in fig. 1B rather than the predicted stem-loop.

Other mutations comprised by the virus of the invention, together with membrane protein (and possibly other) mutations, are loss-of-shortage mutations (e.g., 1, 2, 3, or 4 amino acid mutations) within the capsid protein. Examples of such mutations include the possible deletions affecting the protein helix I, provided reference is made to the YF17D viral capsid protein (see fig. 2A). A specific example of such a mutation is the mutation C2, which comprises the deletion of the amino acid PSR in helix I (fig. 2A). The feasibility (viability) and attenuation of other short mutations in this region (and corresponding mutations in other flavivirus sequences) can be tested and also used in the present invention. Capsid protein sequences have been disclosed for other flaviviruses such as TBE, WN, Kunjin, JE, and dengue virus (e.g., Pletnev et al, Virology 174: 250-263, 1990).

The following are specific examples of chimeric flaviviruses that are deposited under the terms of the budapest treaty in the American Type Culture Collection (ATCC) Manassas, Virginia, u.s.a, and which are useful for preparing the viruses of the present invention given a deposit date of 6 days 1 month 1998: chimeric yellow fever 17D/type 2 dengue virus (YF/DEN-2; ATCC accession number ATCC VR-2593) and chimeric yellow fever 17D/Japanese encephalitis SA14-14-2 virus (YF/JE A1.3; ATCC accession number ATCC VR-2594). Details of the preparation of chimeric viruses useful in the present invention are disclosed in, for example, U.S. Pat. nos. 6,696,281B 1; international applications PCT/US98/03894(WO98/37911) and PCT/US00/32821(WO 01/39802); and Chambers et al, j.virol.73: 3095-. These methods are improved for use in the present invention by including the step of introducing one or more of the mutations described herein into the sequence to be inserted (e.g., membrane protein or other sequence of japanese encephalitis virus or west nile virus). Methods useful for preparing the viruses of the present invention are also disclosed in PCT/US03/01319(WO03/060088A 2; see also below).

The viruses of the invention can be mutated by standard methods such as site-directed mutagenesis. One example of the type of mutation present in the virus of the invention is a substitution, but other types of mutations such as deletions and insertions may also be used. In addition, as noted above, the mutations may be present alone or within one or more other mutations, whether within the membrane protein itself or in any combination of sequences such as the 3' UTR, capsid, or envelope.

The viruses of the invention (including chimeras) can be prepared by methods standard in the art. For example, RNA molecules corresponding to the viral genome can be introduced into primary cells (primary cells), chicken embryos, or diploid cell lines, from which (or supernatant from which) progeny viruses can then be purified. Other methods that can be used to prepare the virus employ heteroploid cells such as African green monkey kidney cells (Yasumura et al, Nihon Rinsho 21: 1201-1215, 1963). In this method, a nucleic acid molecule (e.g., an RNA molecule) corresponding to the genome of a virus is introduced into a heteroploid cell, the virus is harvested from the medium in which the cell has been cultured, and a nuclease (e.g., an endonuclease that degrades DNA and RNA such as Benzonase) is used^TM(ii) a U.S. Pat. No. 5,173,418) processes the harvested virus. In Benzonase^TMIn the case of (2), the nucleic acid can be digested with 15 units/mL and the conditioned medium is frozen at 2-8 ℃ for about 16 hours or more. The nuclease treated virus is then concentrated (e.g., by ultrafiltration using a filter with a molecular weight threshold of, for example, 500kDa (e.g., Pellicon-2Mini unfilter cassette)), diafiltered (diafiltered) with phenol red or FBS free MEME, formulated by addition of lactose, and filtered into sterile containers. Details of this method are disclosed in WO03/060088A 2. Further, as described below, the cells used for propagation of the virus of the present invention may be grown in serum-free medium.

The viruses of the invention may be administered to those patients at risk of infection as primary prophylactic agents (primary therapeutic agents) or may be used as secondary agents for treating infected patients. Because the viruses are attenuated, they are particularly suitable for administration to "at-risk individuals" such as the elderly, children, or HIV-infected individuals. The vaccine may also be used in veterinary contexts, for example to vaccinate horses against west nile virus infection, or domestic pets (e.g. cats, dogs and birds (birds)), livestock (e.g. sheep, cattle, pigs, birds and goats), and valuable animals such as rare birds. Further, the vaccines of the present invention can comprise a virus, such as a chimeric virus, that comprises a particular mutation (e.g., M5, M60, and/or M66 mutation) and is mixed with a virus that lacks such a mutation.

The viruses of the present invention can be formulated using standard methods in the art. A variety of pharmaceutically acceptable solutions for vaccine formulation are well known and can be readily adapted for use in the present invention by those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences (18)^thedition), ed.a.gennaro, 1990, Mack Publishing co., Easton, PA). In two specific examples, the virus was formulated in minimal essential medium Earle's Salt (MEME) containing 7.5% lactose and 2.5% human serum albumin or MEME containing 10% sorbitol. However, the virus may simply be diluted in a physiologically acceptable solution such as sterile physiological saline or sterile buffered saline. In another example, the virus may be administered and formulated, for example, as a clear suspension of infected chicken embryonic tissue, or a liquid harvested from a cell culture infected with the chimeric yellow fever virus, in the same manner as the yellow fever 17D vaccine.

The vaccines of the present invention can be administered by methods well known in the art, and the appropriate amount of vaccine to be administered can be readily determined by one skilled in the art. The appropriate amount of virus to be administered can be determined by considering factors such as, for example, the size and overall health of the subject to which the virus is to be administered. For example, the virus of the invention may be formulated to comprise 10 in a dose volume of 0.1 to 1.0ml to be administered, for example, by the intramuscular, subcutaneous or intradermal routes²To 10⁸E.g. 10³To 10⁷Or 10⁴To 10⁶Sterile aqueous solution of infectious units (e.g. plaque-forming units or tissue culture infectious dose). In addition, since flaviviruses may infect human hosts via mucosal routes, such as The oral route (Gresikova et al, "Tick-borne Encephalitis," In The Arboviruses, Ecology and Epidemiology, Monath (ed.), CRC Press, Boca Raton, Florida, 1988, Volume IV, 177-203), The viruses may also be administered via mucosal (e.g., oral) routes. Further, the vaccine of the invention may be administered in a single dose, or alternatively, administration may involve the use of a priming dose followed by one or more booster doses administered, as determined by one of skill in the art, as appropriate, e.g., after 2-6 months.

Alternatively, adjuvants known to those skilled in the art may be used in administering the viruses of the present invention. Adjuvants that may be used to increase the immunogenicity of the virus include, for example, liposomal formulations, synthetic adjuvants such as (e.g., QS21), muramyl dipeptide, monophosphoryl lipid A, or polyphosphazine. Although these adjuvants are commonly used to enhance the immune response to inactivated vaccines, they may also be used with live vaccines. In the case of viruses delivered via the mucosal route, adjuvants such as, for example, oral or mucosal adjuvants such as heat Labile Toxin (LT) of e. In addition, a gene encoding a cytokine having adjuvant activity may be inserted into the virus. Thus, genes encoding cytokines (e.g., GM-CSF, IL-2, IL-12, IL-13, or IL-5) can be inserted with foreign antigen genes to prepare vaccines that will enhance the immune response or modulate immunity more specifically to cellular, humoral, or mucosal responses. Other adjuvants that may be optionally employed in the present invention include toll-like receptor (TLR) modulators.

In the case of dengue viruses and/or chimeric flaviviruses comprising the membrane and envelope proteins of dengue viruses, optimal immunization against them may involve inducing immunity against all four dengue serotypes, and the viruses of the invention may be used in the formulation of tetravalent vaccines. As described herein, any or all of the viruses used in these tetravalent formulations may comprise one or more mutations that reduce the entropic phenomenon. The viruses may be mixed at any point in the formulation to form a tetravalent preparation, or the viruses may be administered continuously. In the case of tetravalent vaccines, equal amounts of each virus may be used. Alternatively, the amount of each different virus in the vaccine administered may vary (WO03/101397A 2).

The invention also encompasses nucleic acid molecules (e.g., RNA or DNA (e.g., cDNA) molecules) or complements thereof corresponding to the viral genomes of the invention described herein. For example, these nucleic acid molecules can be used in the method for producing the virus of the present invention. In these methods, a nucleic acid molecule corresponding to the viral genome is introduced into a cell that can produce and replicate the virus (e.g., a primary cell, chicken embryo, diploid cell line, or an isoploid cell line (e.g., vero cell), from which (or supernatant from which) progeny virus can then be purified. These methods further comprise a purification step of the virus, as known in the art.

As noted above, details of the preparation of chimeric viruses useful in the present invention are disclosed in, for example, U.S. patent nos. 6,696,281B 1; international applications PCT/US98/03894(WO98/37911) and PCT/US00/32821(WO 01/39802); and Chambers et al, j.virol.73: 3095-3101, 1999. Details for the construction of chimeric flaviviruses comprising the pre-membrane and envelope proteins of japanese encephalitis virus (or west nile virus), as well as the capsid and non-structural proteins of yellow fever virus are provided below. Those skilled in the art can readily adapt these methods to construct chimeras containing the above mutations as well as chimeras containing other precursor-membrane and envelope sequences.

Briefly, derivatization of the YF/JE chimera may involve the following. The YF genomic sequence was replicated in two plasmids (YF5 '3' IV and YFM5.2) encoding nucleotides 1-2,276 and 8 of The YF sequence, 279-10,861(YF5 '3' IV) and 1,373-8,704(YFM5.2) (Rice et al, The New biology 1: 285-296, 1989). Full-length cDNA templates were generated by ligating appropriate restriction fragments derived from these plasmids. The YF sequence within the YF5 '3' IV and YFM5.2 plasmids was then replaced with the corresponding JE sequence from the start of the prM protein (nucleotide 478, amino acid 128) to the E/NS1 cleavage site (nucleotide 2,452, amino acid 817).

Preparing a clone of a true JE structural protein gene from a JE SA14-14-2 strain, wherein the JE SA14-14-2 strain is a live attenuated vaccine strain of the JE; it is available from the Centers for disease control (center for disease control), Fort Collins, Colorado and yarrowia virus research group (the yarn antiviral research Unit), yarn University, New Haven, connection, both of which are the world health organization-designated insect virus Reference Centers (references Centers) in the united states). JE SA14-14-2 virus was obtained at the PDK-5 passage level and was used in LLC-MK₂Passaging in cells to obtain sufficient amounts of virus for cDNA cloning. The strategy used involved cloning the structural regions in two fragments that overlapped at the NheI site (nucleotides 1,125 of JE), which could then be used for in vitro ligation.

From infected LLC-MK₂RNA was extracted from a monolayer of cells and first strand (first strand) synthesis of negative sense cDNA was performed using reverse transcriptase and a negative sense primer (JE nucleotide sequence 2,456-71) containing nested XbaI and NarI restriction sites for cloning first into pBluescript II KS (+) and then YFM5.2(NarI), respectively. After first strand cDNA synthesis, PCR amplification of nucleotide 1,108-2,471 of the JE sequence was performed using the same negative and positive sense primers (JE nucleotide sequence 1,108-1,130) containing the nested XbaI and NsiI restriction sites for cloning into pBluescript and YFM5.2(NarI), respectively. JE sequences were confirmed by restriction enzyme digestion and nucleotide sequencing. Nucleotides 1 to 1,130 of the JE nucleotide sequence were derived by PCR amplification of minus-strand JEcDNA using a negative-sense primer corresponding to nucleotides 1,116 to 1,130 of JE and a positive-sense primer corresponding to nucleotides 1 to 18 of JE, both primers containing an EcoRI restriction site. The PCR fragment was cloned into pBluescript and the JE sequence was confirmed by nucleotide sequencing. Taken together, this indicates that nucleotides 1-2,471 (amino acids 1-792) of the JE sequence have been cloned.

To insert the C-terminus of the JE envelope protein at the YF E/NS1 cleavage site, a unique NarI restriction site was introduced into the YFM5.2 plasmid to generate YFM5.2(NarI) by oligonucleotide-directed mutagenesis of the signal peptidase sequence at the E/NS1 cleavage site (nucleotide 2,447-2,452, amino acid 816-817 of YF). The infectivity of transcripts derived from the template incorporating this alteration was tested and produced a specific infectivity similar to the parental template (approximately 100 plaque-forming units/250 ng transcript). Nucleotides 1,108 to 2,471 of the JE sequence were subcloned from multiple independent PCR-derived pBluescript/JE clones into YFM5.2(NarI) using unique NsiI and NarI restriction sites. YF5 ' 3 ' IV/JE clone containing the YF5 ' untranslated region (nucleotides 1-118) adjacent to the JE prM-E region was derived by PCR amplification.

To derive the sequence of the linker containing the YF capsid and JE prM, a PCR fragment was prepared using a negative sense chimeric primer spanning this region with a sense primer corresponding to nucleotide 6,625-6,639 of YF5 '3' IV, and this PCR fragment was then used as a negative sense PCR primer to amplify nucleotide 477 of the JE sequence (encoded in the reverse direction in the pBluescript vector) (N-terminus of the prM protein) to the NheI site at nucleotide 1,125 in combination with a sense primer complementary to the pBluescript vector sequence upstream of the EcoRI site. The resulting PCR fragment was inserted into the YF5 '3' IV plasmid using NotI and EcoRI restriction sites. This construct comprised the SP6 promoter followed by YF 5' -untranslated region, followed by the sequence: YF (C) JE (prM-E), and contains the NheI site (nucleotide 1,125 of JE) required for in vitro ligation.

To use the NheI site within the JE envelope sequence as the 5' in vitro ligation site, the excess NheI site (nucleotide 5,459) in the YFM5.2 plasmid was removed. This was achieved by silent mutation of the YF sequence at nucleotide 5,461 (TC; alanine, amino acid 1820). This site was incorporated into YFM5.2 by ligation with appropriate restriction fragments and YFM5.2(NarI)/JE was introduced by exchange with a NsiI/NarI fragment encoding the chimeric YF/JE sequence.

To generate a unique 3 ' restriction site for in vitro ligation, a BspEI site downstream of the AatII site was engineered, which was typically used to generate full-length templates from YF5 ' 3 ' IV and YFM 5.2. (multiple AatII sites are present in the JE structural sequence, precluding the use of this site for in vitro ligation). The BspEI site was generated by silent mutation of YF at nucleotide 8,581 (AC; serine, amino acid 2,860) and YFM5.2 was introduced by exchange with the appropriate restriction fragment. This unique site was incorporated into YFM5.2/JE by the exchange of XbaI/SphI fragments and YF5 '3' IV/JE (prM-E) plasmid by tripartite (three-piece) ligation of appropriate restriction fragments from these parental plasmids and from YFM5.2 derivatives (BspEI) that deleted the YF sequence between the EcoRI sites at nucleotides 1 to 6,912.

The cDNA from a clone of the JE Nakayama strain, which has been deeply characterized in expression experiments for its ability to induce protective immunity (see, e.g., McIda et al, Virology 158: 348-. The Nakayama cDNA was inserted into the YF/JE chimeric plasmid using the existing restriction sites (HindIII to PvuII and BpmI to MunI) to replace the entire prM-E region in the two plasmid system, but leaving only one amino acid intact, a serine at position 49, which was used to take advantage of the NheI site for in vitro ligation.

Methods for generating full-length cDNA templates are essentially as described by Rice et al, (The New Biologist 1: 285-96, 1989). In the case of the chimeric template, plasmids YF5 '3' IV/JE (prM-E) and YFM5.2/JE were digested with NheI/BspEI and ligated in vitro with 300 ng of the purified fragment in the presence of T4DNA ligase. The ligation products were linearized with XhoI to allow for run-off transcription. Synthesis of SP6 transcript with 50 ng of purified template by incorporation³H-UTP was quantified and RNA integrity was confirmed by non-denaturing agarose gel electrophoresis. Yields using this method ranged from 5 to 10 micrograms of RNA per reaction, which was mostly present as full-length transcripts. E.g., Rice et al, supra, in the presence of cationic liposomesTransfection of the RNA transcript of YF17D was performed to prepare chimeric viruses.

In cases where the chimeric flavivirus comprises West Nile virus and yellow fever virus sequences, the two-plasmid system described above may also be used. In one example, the used West Nile (WN) virus prM and E genes were cloned from WNV flamingo isolate 383-99, sequence GenBank accession AF 196835. Viral prME cDNA was obtained by RT-PCR (XL-PCR Kit, Perkin Elmer). The 5 'end of the WN prM gene was cloned precisely to the 3' end of the YF17D capsid gene by overlap-extension PCR with Pwo polymerase (Roche). The 3 'end of the E gene was also cloned exactly to the 5' end of the YF NS1 coding sequence by overlap-extension PCR. Silent mutations were introduced into the sequences of prM and E, resulting in unique restriction sites Bsp EI and Eag I. The DNA fragments generated by digesting the two plasmids with these enzymes were gel purified and ligated in vitro to prepare full-length chimeric cDNAs. The cDNA was linearized with Xho I to facilitate in vitro transcription with SP6 polymerase (Epicentre). The RNA product is introduced into a eukaryotic cell line that allows viral RNA translation and viral replication. For the YF/JE chimera, the mutations of the present invention can be introduced into the YF/WN chimera as described above using standard methods, as described above.

Other flavivirus chimeras can be engineered using similar strategies, with natural or engineered restriction sites, and oligonucleotide primers such as those shown in table 14.

The invention is based in part on the experimental results described in the examples below.

Examples

Example 1: ChimeriVax^TM-WN

Test results

Background and summary

Chimeric yellow fever-West Nile (YE-WN) virus ChimeriVax^TMWN is determined byThe precursor-membrane (prM) and envelope (E) genes of WN virus (NY99) were inserted into YF17D backbone. This virus was prepared in vero cells (passage 5, P5) under serum-free conditions, evaluated for safety, immunogenicity, and efficacy in preclinical models, and tested in phase I studies in humans. Additional attenuation of the vaccine virus (P5) was determined by three SA 14-14-2-specific mutations (residues 107,316, and 440) in the E protein. The vaccine virus has a neurotoxicity ratio when tested in mice and monkeys vaccinated by the IC route and mice, hamsters and monkeys protected by a single vaccinationWeak (Arroyo et al, J.Virol.78: 12497-. The vaccine virus contains a mixed population (population) of viruses (exhibiting both small (S) and large (L) plaque phenotypes) that differ by a single amino acid residue at position 66 (M66) of the M protein. This mutation did not affect the neurovirulence of the virus in 8-day old suckling mice (Arroyo et al, J.Virol.78: 12497-12507, 2004). In the present invention, we describe that the M66 mutation reduces viral hematopathy in the host and thus can be used to improve existing vaccines (ChimeriVax)^TMWN02, P5, mixed populations of parental and mutant viruses) or large plaque variant (non-mutant) viruses.

P5ChimeriVax produced in vero cells under serum-free conditions^TMNucleotide heterogeneity (-50%) of T and C (CTA/CCA) was observed in consensus sequences of west nile vaccine viruses. This mutation results in the presence of a virus containing the amino acid proline (mutant) or leucine (parent wild type) at residue 66 of the membrane (M) protein (designated herein as the M66 mutation). ChimeriVax is provided in the attached sequence appendix^TMWN02 and ChimeriVax^TMThe sequence of the variant WN02M66, which also contains the alignment of the amino acid sequences of these proteins.

The M protein of west nile virus contains 75 amino acids. By submitting the protein sequence tohttp://www.predictprotein.orgThe website to predict the structure of this protein and compare it with the M protein structures of JE SA14(AAA67174), Kunjin (AAP78942), MVE (CAA27184), SLE MSI (AAP44973), and SLE CORAN (AAP 44972). In all predicted structures, the first 40 amino acids (SLTVQTHGESTLANKKGAWMDSTKATRYLVKTESW ILRN; (SEQ ID NO: 18) of the M protein are predicted to be non-membrane regions, while the remaining 35 amino acids (40-75) (PGYALVAAVIGWMLGSNTMQRVVFVVLLLLVAPAYS; (SEQ ID NO: 19) are predicted to be within the membrane region of the virus. in addition, there are 9-10 charged amino acids (3-4 acidic, E or D) and 6 basic (R or K) within the first 40 amino acid residues, while all 5 flaviviruses described herein (WNV, SLE, MVE, JE, and Kunin) have only one charged amino acid (basic) at residue 60.

Plaque morphology of P5 vaccine virus showed a mixed population of L and S plaque size phenotypes. Sequencing of the P2, P3, P4, and P5 viruses showed that mutations were first present at P4 (10% of the overall population) and reached-50% at P5. Sequencing of S and L plaque isolates of vaccine viruses showed that the mutations caused a change in plaque size from L to S. S and L virus variants (prepared as study viruses) did not differ significantly in their neurovirulence against 8-day-old suckling mice (p ═ 0.0001).

From ChimeriVax^TMWN02(P5), prepr-Master Seed (PMS, P10) stocks of L and S viruses were prepared in Vero cells by 3 rounds of direct plaque to plaque purification and 2 rounds of virus amplification in "clean laboratory conditions". Sequencing of P10S and L viruses revealed a single amino acid difference at residue M66 (S virus contains proline at residue M66, while L virus contains leucine at this site). The M66 mutation appears to be stable under large scale manufacturing conditions. When S-plaque virus (P10, PMS) was inoculated into hamsters by subcutaneous inoculation, it induced very low levels of viremia compared to either vaccine virus (P5) or L-plaque virus variant (P10, PMS). After inoculation of ChimeriVax^TMIn monkey and human sera of the-WN P5 virus (containing S and L plaque variants at-50: 50), the majority of the virus was the L plaque size phenotype. In addition, S plaques were shown to grow to lower titers in human liver cancer cell lines than L plaques. This data indicates S-plaque virus (ChimeriVax with M66 mutation)^TMWN02) may induce ChimeriVax in humans^TMLow levels of viremia of WN02 (without M66 mutation) and may therefore be suitable (safe) WN vaccine candidates for "at-risk individuals", such as elderly, children or HIV-infected individuals. Additional mutations in the M region were found by sequencing individual plaques isolated from P10 to P12 or inoculated with ChimeriVax^TMLarge scale preparation of passages (e.g. M62, M63 and M64) of PMS S plaques of monkeys of WN02 vaccine (e.g. M60, M61, and M63). These mutations can be used in the construction of the viruses of the present invention.

Preparation of PMS of S and L plaque viruses in Vero cells

Make ChimeriVax^TM-WN02 vaccine material (P5) grown in serum-free vero cells; 10 spots identified as "small" (S) and 10 spots identified as "large" (L) were picked. Each isolate was then passaged on serum-free Vero cells and one plaque was picked from each isolate. This procedure was repeated once more for a total of three cycles of plaque purification. T25cm in serum-free vero cells²The plaque purified isolate (P8) was expanded in a flask (and grown in Serum Free (SF) medium) and then harvested and stored at-80 ℃. Isolates were sequenced to find PMS candidates free of spurious (spirious) mutations. Two isolates were identified as no expression (non-silent) mutations: one isolate was identified as a small plaque (M66 proline) (table 1) and the other contained the wt sequence (M66 leucine) (table 2). These two isolates were then grown in large flasks, aliquoted (aliquote), and submitted to QC catalogue (inventoryy) as LP and SP PMS (P10) viruses.

Genetic stability of large-scale produced SP viruses

To determine whether the S plaque phenotype was stable during large scale preparation, the small plaque PMS virus was passaged twice by infecting vero cells in a bioreactor and growing them under serum-free conditions to produce P12 virus. The P12 virus was harvested and plaques (plaque) were formed in 6-well plates. Most plaques are small in size. The twenty largest plaques that were present were picked, amplified on O-Vero (One passage) and the prME region transcribed/amplified by Titan One-Tube RT-PCR kit (Roche). The cDNA fragments containing the M region were sequenced and the morphology of the isolates was confirmed by immunostaining with WN-specific monoclonal antibodies. Thirteen of the 20 plaques contained only M66 (the genetic marker leading to SP morphology), and 5 isolates contained other mutations besides M66. Isolate #4 contained M63(LP phenotype), while isolate #16 contained a mixed population of wt and M66. These data indicate that despite the fact that some plaques exhibit large sizes, they still contain the M66 mutation and were amplified to demonstrate S size. Only one plaque (#4) of 20 showed L size, apparently due to the mutation from L to P at M63. Plaque #16 showed the production of a mixed population of large and small plaque size viruses containing wtL and the mutated P amino acid at position M66 (table 3).

ChimeriVax^TMGrowth of WN virus variants in hepatocytes

ChimeriVax with MOI0.005^TMWN01 (wild-type prME), ChimeriVax^TMWN02P5 (mutations in mixed L/P amino acids at E107, E313, E316, E440, M66, mixed S and L plaques), ChimeriVax^TMWN LP (E107, E313, E316, and E440, WNL), and ChimeriVax^TMWN SP (E107, E313, E316, E440, and M66P, WNS) infected human hepatoma cell lines HepG2 and THLE-3 cells. Supernatants were collected daily and titrated on O-African green monkey kidney cells using a standard red double-layered agarose overlay (neutral red double agarose overlay).

In HepG2 cells (fig. 3), WN01 (wild-type prME) was observed to have the highest viral growth at day 5 (7x 10)⁶PFU/ml), followed by LP on day 5 (2.7x 10)⁶PFU/ml)。By usingIn the case of (2), the peak of the virus was reached on day 3 (1.17X 10)⁶PFU/ml), followed by WN02 mixed vaccine virus reached a viral peak at day 4 (6.4x 10)⁵PFU/ml). SP virus was found to grow minimally (peak titer 6.1X10 on day 4)⁵PFU/ml) comprising a single amino acid substitution at M66 (L to P). In THLE-3 cells (FIG. 4), the same pattern as that of HepG2 cells was observed, except thatThe titer of (c) was slightly higher than that of the LP virus. Again the highest titer of WN01 was seen (1.3X 10)⁵PFU/ml, day 4), followed by LP (5.7X 10)⁴PFU/ml, day 7),(8.8x10⁴PFU/ml, day 4), and mixed P5 virus (1.8X 10)⁴PFU/ml, day 4). The lowest titer of SP virus was again observed (9.2X 10)³PFU/ml, day 4).

The induction of cytopathic effect (CPE) by each virus was recorded daily (table 4). CPE for WN01 and LP virus was first observed on day 5 and was complete after 2 days (100%), while SP or mixed plaque populations induced CPE at an earlier time point (day 3) and completely destroyed the cell monolayer 1 day earlier than WN01 or LP (day 6). First observed on day 3CPE induction and complete destruction of monolayer cells at day 6 post inoculation. CPE induction in HepG2 cells was probably due to the apoptotic activity of the M protein as shown by the wild-type dengue virus (Catteau et al, J.Gen.Virol.84: 2781-2793, 2003). These data show that SP virus variants grow to lower titers than mixed or LP viruses, suggesting that the M66 mutation may reduce viral liver tropism in humans.

In-use mixed (SP and LP virus) P5 vaccineChimeriVax could not be detected after virus inoculation of monkeys^TMWN, SP Virus

Giving a total of 8 first experiment cynomolgus monkeys that lack detectable antibodies to flaviviruses such as WN, JE, and YF viruses (cynomolgus monkeys) (determined by the Plaque Reduction Neutralization Test (PRNT) were inoculated subcutaneously with ChimeriVax^TM-WN02(P5) (n ═ 4) or(n-4). The purpose of this experiment was to evaluate ChimeriVax^TMViremia, biodistribution and possible toxicity of the WN02 vaccine over a 3-day observation period. For ChimeriVax^TMWN02 andthe inoculation dose is respectively 1.25x10⁵PFU/0.5mL and 5.5X10⁴PFU/mL. Animals were bled daily and sacrificed on day 4 post inoculation. Viremia levels were determined by standard plaque assays on vero cells using blood, and collected tissues were either snap frozen for virus analysis or stored for histopathological evaluation.

Monkey sera collected from day 1 (pre-inoculation) to day 4 (pre-euthanasia) were evaluated for viremia. Viremia levels were determined by staining with agarose bilayer coverings and neutral red (to isolate and sequence individual plaques) or by methylcellulose coverings and crystal violet staining, as described by Monath et al, j.virol.74 (4): 1742 the experiments were carried out as described in 1751, 2000. After inoculation of ChimeriVax^TMMagnitude (magnitude) and duration of viremia in monkeys of WN02 aboveThe condition of (1) (table 5). For theIn particular, the highest titer of viremia was 200PFU/mL (animal MF21157, day 4). For ChimeriVax^TMThe highest titer of viremia for WN P5 virus was 1000PFU/mL (animal MF21191F, day 4). Inoculated with ChimeriVax^TMAll animals of WN02 virus (4/4) were viremic 3 days after inoculation, whereas only 2/4 was inoculatedThe animals became viremic (only 2 days) (table 5).

Because of inoculation of ChimeriVax^TMWN02 virus animals have received a mixture of SP and LP viruses, requiring isolation of multiple SP and LP viruses from serum to identify viral variants (S or L) that cause high levels of viremia. Sera of all 4 monkeys obtained from day 2 to day 4 post-inoculation were diluted to 1:2 and 1:10 and used to inoculate duplicate wells of 6-well plates seeded with vero cells. After addition of a second agarose overlay containing neutral red, individual plaques (4S and 3L plaques) were picked and directly sequenced to identify the presence of the M66 mutant virus (Table 6). None of these isolated plaques contained the M66 mutation (L to P substitution), indicating that the M66 mutant virus did not cause the high levels of viremia detected in these animals. Interestingly, 3 other mutations were observed in the M region (M60, M61, and M63). These viral variants may already be present in small amounts in ChimeriVax^TMWN02 vaccine viruses (cannot be detected by consensus sequencing), or they are produced in vivo (monkeys) by mutations within the LP virus variant genome.

After inoculation of ChimeriVax^TMViremia and neutralizing antibody responses in-WN SP (PMS, P10), LP (PMS, P10), or mixed (P5, SP, and LP) viral hamsters

Throughout the experiment, the animals used in this experiment were housed (maintained) in a micro-separator (microsparator) under BL2 and treated according to IACUC approved animal protocols. Three ChimeriVax strains were used^TMWN02 virus (SP, PMS, P10; LP, PMS, P10, and mixed SP and LP vaccine virus, P5) to infect 7 week old female Golden Syrian hamsters (Golden hamsters) from Harlan Sprague-Dawley, each virus was injected subcutaneously into the inguinal region of a group of 15 hamsters. The infection dose is 10⁵pfu, inoculation volume of 100. mu.l. Another group of 5 animals was similarly injected with 100 μ l of virus dilution as a sham control. Blood samples were collected by retro-orbital bleeding of all animals except the sham control group on the day of viral infection (day 0) and each subsequent day up to 5 days post infection. Animals were anesthetized by isoflurane inhalation prior to blood collection and inoculation. The virus concentration in the test samples was determined by direct plaque formation in duplicate wells of vero cell cultures grown in 12-well plates from 0.1mL of a 1:10 diluted serum sample (figure 5).

As shown in fig. 5, peak viremia was observed at a higher level (3logs of pfu on average) in serum samples collected from hamsters infected with LP virus, whereas very low levels (<10pfu) viremia was seen in blood samples from hamsters vaccinated with SP virus. When the ratio of SP virus in the inoculum increased (to 50% for mixed plaque virus), the peak viremia titer dropped to about half of the LP virus-induced viremia level. Additionally, the time to peak viremia after infection is delayed by at least 1 to 4 days.

These data indicate that ChimeriVax is derived from the same parental virus^TMWN02 the isolated LP and SP variants have different biological properties. LP virus proliferated to a higher level in hamsters at a faster rate than SP virus. In addition, mixing the SP virus with the LP (P5 virus) clearly negates some of the properties of the LP virus. This is shown in hamster infection experiments, where the presence of virus in blood is reduced to lower levels and the kinetics of virus replication in hamsters infected with mixed viruses is reduced. In summary, the presence of the M66 mutation (L to P) in the SP variant virus significantly reduced viremia in hamsters.

Example 2: ChimeriVax^TMJE and ChimeriVax^TM-DEN1-4

Background and summary

In the experiments described below, we prepared and characterized novel ChimeriVax using Vero cells grown in serum-free (SF) medium^TMJE virus (seed virus) to eliminate any concern about possible contamination of the vaccine by prion factors (agent) of bovine transmissible encephalopathy (bovine transmissible encephalopathy). During propagation in SF cultures, the uncloneable virus accumulated mutations not seen before in serum-containing medium, which showed adaptation to SF growth conditions, i.e. increased virus replication rate. These mutations appear in the E or M protein (F to L for E-107 or R to C for M-60) and suggest a functional significance of the M protein during viral replication, which becomes apparent during growth of the virus under SF conditions (see amino acid R at position 60 of the M-protein shown in example 1 (ChimeriVax)^TM-WN). Mutations in the M protein are defined (ChimeriVax)^TMM60, M5 in JE) or mutations in the E protein (ChimeriVax)^TME-107, ChimeriVax in JE^TME202/204 in-DEN 1 and-DEN 3, and ChimeriVax^TME251 in DEN 2) on the biological properties of the vaccine. All of these chimeric viruses have been tested in clinical trials.

Materials and methods

Cells and culture media

Vero cells (ATCC; Manassas, VA; CCL 81; Vero cells) were originally obtained from the American Type Culture Collection. These cells were adapted to growth in SF medium and were obtained as passages 133 from Baxter (Orth, Austria) and then used directly by seeding into flasks or seeded from cell banks at passage 136. In all experiments, noneThe passage level of vero cells did not exceed 149 th passage. At 7.5% CO₂Conditions of 36 ℃ allow cell and virus growth. Cells were propagated under SF conditions.

ChimeriVax^TM-JE variant

Initiation of (initial) Virus (P1 passages) by electroporation of SF African Green monkey kidney cells with in vitro RNA transcripts (stored at-80 ℃) that were previously used to prepare non-SF ChimeriVax cells to be tested in preclinical and clinical trials^TMJE Vaccine candidates were identical (Monath et al, Vaccine 20: 1004-. Passage for amplification is usually done at an MOI of 0.001 pfu/cell and the viral harvest is collected 3-4 days after infection (when CPE is-10%), clarified by slow centrifugation, supplemented with 10% sorbitol and stored at-80 ℃. Cloned variants were prepared in Baxter African Green monkey kidney cells by three sequential plaque purifications with standard agar-neutral Red overlay method in the presence of gamma-irradiated FBS (HyClone; FBS was used because the cells did not form plaques in agar prepared with SF medium) followed by amplification under SF conditions. Plaque assays to determine viral titer in a given specimen were performed using the single layer methylcellulose cover method and plaques were visualized by crystal violet on day 5 post infection.

ChimeriVax^TM-DEN virus

ChimeriVax was prepared by electroporation of Vero cells with RNA transcripts prepared from viral cDNA^TMDEN1-4 vaccine virus. Progeny virus was subjected to three rounds of plaque purification to generate Pre-Master Seed (PMS) virus at passage 7 (P7). Bulk vaccine (vaccine lot) (P10 virus) was prepared by three additional passages using current food Manufacturing Practices (cGMP) in the United states. After multiple passages in Vero cells, some mutations were shown in the chimera E gene (Guirakho et al, J.Virol.78: 4761-4775, 2004). One of these mutations (ChimeriVax)^TME204 in DEN 1) significantly reduced the viscerotropism of the virus in non-human primates (guirakho et al, j.viro 1.78: 9998-10008, 2004).

Consensus sequencing

Consensus sequencing of indicated viral samples was performed as described above (Pugachev et al, Vaccine 20: 996-999, 2003). Briefly, virion RNA extracted with TRIZOL LS reagent (Life Technologies-Gibco BRL) was amplified in 5 overlapping cDNA amplicons 2-3kb long using Titan One-Tube RT-PCR kit (Roche). Amplicons were sequenced with pools of JE-and YF-specific oligonucleotide primers in forward and reverse directions (collection) and CEQ Dye Terminator cycle sequencing kit (Beckman). The reaction products were sequenced using a CEQ2000XL automated sequencer (Beckman Coulter) for resolution (resolve). The data were aligned and analyzed using Sequencher4.1.4(GeneCodes) software. Nucleotide heterogeneity was recorded when a heterogeneous signal was observed in all chromatograms (chromatograms) reflecting positive and negative strand sequencing reactions. For some viruses, only the first of the five cDNA amplicons (Fragment I) was sequenced and shown to include the structural gene.

Neurovirulence in suckling mice

The breeding and care of mice conforms to the guidelines for humane use of laboratory animals by the National Institutes of health (National Institutes of health). Pregnant, cross-bred (outbred) ICR female mice were purchased from Taconic Farms (Germantown, NY). Newborn mice were bred and mixed into new groups 6 days before inoculation. Groups of 8-day-old suckling mice were inoculated with 0.02ml of the indicated virus sample by the Intracerebral (IC) route. Virus used for inoculation was serially diluted 1:10 in MEM-10% FBS. Undiluted inoculum was back-titrated and the exact dose for each dilution was calculated. Mortality was recorded over 21 days. The YF17D control virus was reconstituted from a purchased vaccine vial(Aventis Pasteur，Swiftwater，PA)。

Safety and efficacy testing in monkeys

Experiment 1 study of the New clone C (M-60) ChimeriVax according to the GLP criteria in cynomolgus monkeys^TMStock of JE Vaccine Master Viral Bank (MVB; P11) and Production Viral Bank (PVB; P12) withNeurovirulence/virulence profile compared to control (YF17D vaccine virus). Thirty-three (33) were first used in the experiment (experimental-) And cynomolgus monkeys negative for flavivirus-serum response (determined by HAI assay) were assigned to the treatment groups shown in table 9. All monkeys were dosed by a single IC injection on day 1, observed for 30 days, then euthanized and necropsied. Monkeys were evaluated for clinical signs (twice daily), and for changes in food intake (daily), body weight (weekly), and clinical pathology indices (pathology). Clinical scores were assessed according to the clinical scoring system, according to the requirements of the World Health Organization (WHO) for yellow fever vaccines (WHO, Technical Report Series, No.872, 1998). Blood samples were collected for clinical pathology analysis (serum chemistry and hematology parameters) before inoculation on day 1 and on days 3, 5, 7, 15 and 31. Additional blood samples were collected on day 1 (pre-dose) and days 2-11 for quantitative viremia assays, and on day 1 (pre-dose) and day 31 for neutralizing antibody titer analysis. Complete autopsies were made on day 31 and tissues collected for storage. The prepared tissue can be used for histopathology of liver, spleen, heart, kidney and adrenal gland. Histopathology of the brain and spinal cord was performed according to the method described by Levenbook et al (J.biol.Stand.15: 305, 1987), and the WHO requirement for a yellow fever vaccine was introduced (WHO, 1998).

Experiment 2. this experiment was performed to compare ChimeriVax^TMJE vaccine [ original unclosed vaccine P5, previously it was in LS5 Vero cells in the presence of FBS (BB-IND #9167, Serial #000)Preparation which is free of mutations other than the L to F alteration of E491 at the hydrophobic tail of the E protein]And novel clone C (M-60 mutant) ChimeriVax^TM-viremia, immune response and safety in a period of 30 days after a single Subcutaneous (SC) administration to cynomolgus monkeys according to GLP standard of JE purified bulk vaccine preparation (P13). Eighteen (18) naive, flavivirus-seronegative (as determined by the HAI assay) cynomolgus monkeys were assigned to the treatment groups as shown in table 10. All monkeys were given one dose by SC injection at a single location in one arm on day 1. Monkeys were evaluated for clinical signs of toxicity (twice daily), body weight (weekly), and changes in serum chemistry, hematology, and coagulation parameters (coagulation parameter). Blood samples were collected on day 1 (pre-inoculation) and on days 4, 7, 15 and 31 for analysis of serum chemistry, hematology and coagulation parameters. Other blood samples were collected on day 1 (pre-inoculation) and days 2-11 for quantitative viremia analysis, and on day 1 (pre-inoculation) and on day 31 for japanese encephalitis virus-specific serum antibody titer analysis.

pH threshold for Virus inactivation (Indirect fusion test)

One of the consequences of exposure of flavivirus to low pH (in the absence of cell membranes) is the induction of irreversible conformational changes in the E protein and inactivation (loss of potency) of the virus. In the presence of cell membranes, these conformational changes are necessary for the fusion of the viral membrane and those cell membranes, resulting in the release of the viral genome into the host cell. The pH threshold for fusion of mosquito-borne viruses such as WN, DEN, YF and JE can be measured by fusion from within FFWI using the mosquito cell line C6/36 (Guirakho et al, Virology169 (1): 90-99, 1989). However, all of our ChimeriVax^TMThe viruses did not show any FFWI, probably due to lack of sufficient growth of these viruses in mosquitoes and mosquito cell lines (Johnson et al, am.J.trop.Med.Hyg.70 (1): 89-97, 2004). Therefore, we attempted to measure the loss of viral potency after exposure to different pH levels in the assay designated herein as the "indirect fusion assay". This assay indirectly measures the pH threshold at which viral membranes fuse with those cell membranes.

At pH7.0, 6.8, 6.6, 6.4, 6.2, 6.0, 5.8, 5.6, 5.4 and 5.0, with a solution supplemented with 2 mML-glutamine, 2.7% sodium bicarbonate, 10% HI FBS and 1% antibiotic/antifungal solution [ (100U/ml penicillin, 0.1mg/ml streptomycin, 0.25. mu.g/ml Amphotericin (Sigma)]And fusion was performed with 1 XMEM adjusted to an appropriate pH with MES (Sigma). Equal aliquots of each virus were aliquoted at 1 × 10⁴Plaque Forming Unit (PFU)/ml dilution in Each pH Medium (10)^-1Dilution). After 10 minutes exposure to each pH value, 50% heat-inactivated (HI) FBS was added to each vial and the pH of each solution was neutralized with sodium bicarbonate. African green monkey kidney cell monolayers (at 9X 10) were infected with each virus at each pH value in a volume of 100. mu.l⁵Density of cells/well plated in 6-well plates) to determine their titer. Infection was performed in duplicate to generate 50 PFU/well; two wells of uninfected cells were kept per plate and used as negative controls. Samples at pH7.0 and 6.8 were taken as controls. Titers were analyzed using standard plaque assays. In this assay, serial dilutions of the virus were used (10)^-1To 10^-6) Duplicate wells infected with vero cells. After infection, a solution supplemented with 2 mML-glutamine, 2.7% sodium bicarbonate, 5% HI FBS, 1% antibiotic/antifungal solution [100U/ml penicillin, 0.1mg/ml streptomycin, 0.25. mu.g/ml Amphotericin (Sigma)]And 44% 1 XMEM (Sigma) in 0.6% agarose (Sigma) covering African green monkey kidney monolayer cells. At 37 ℃ 5% CO₂Cells were incubated for 4 days and then covered with a second cover containing 1 XMEM supplemented with 2mM L-glutamine, 2.6% sodium bicarbonate, 2% HI FBS, 1% antibiotic/antifungal solution, 44% 0.6% agarose, and 3% Neutral red solution (Sigma). Plaques were counted 24 hours after the addition of the second overlay to determine viral titer as defined by Plaque Forming Units (PFU) per ml.

Virus penetration test according to Vlaycheva et al (J.Virol.76: 6172-6184, 2002)

To demonstrate that the M-60 mutation (and the E-107 mutation) facilitated penetration into SF African green monkey kidney cells, the extracellular virus was inactivated by infecting SF African green monkey kidney cells with the appropriate dilutions of clones A, C, and I virus in SF medium for 5, 10, 20, or 60 minutes, followed by treatment with 0.1M glycine, 0.1M NaCl, pH3.0 for 3 minutes. The wells were washed twice with PBS, then the monolayers were covered with methylcellulose, after which the plaques were stained with crystal violet on day 5. Penetration effectiveness is shown by the percentage of plaques observed after glycine treatment that occupied PBS versus glycine treated control infected wells.

ChimeriVax^TMClinical trials of JE

Clinical studies were performed (protocol H-040-003). Vaccines administered to healthy adult male and female subjects have native sequences at M60 (arginine). Healthy adult subjects/groups received ChimeriVax^TMSubcutaneous doses of graded doses of JE vaccine, including multiple control groups. Between 11 and 33 subjects were tested per dose group. Viremia was determined daily by plaque assay in vero cells monolayers. The same experiment and laboratory determined the viremia levels in both experiments.

Safety assessments include the recording of negative events (adorse events), body temperature, physical examination, and laboratory tests including the determination of viremia levels. Acceptance of ChimeriVax in large part^TMViremia was seen in subjects with JE.

A second study (procedure H-040-007) was conducted in healthy adult male and female subjects, in which 31 or 32 subjects per group received ChimeriVax containing the M60 cysteine mutation^TMFractionated subcutaneous doses of JE (3, 4, or 5 log)₁₀PFU). Dose ranges received 2.8, 3.8, and 4.8log from previous studies₁₀Subjects with PFU had similar dose ranges.

Results

In the uncloneable SF ChimeriVax^TM-adaptive mutations in JE viruses, and preparation of cloned variants

A graph of the virus samples prepared in this study is shown in figure 6. Samples (Pre-Master Seed candidates; PMS) of the original uncloneable passage 2 (P2) were obtained in SF cultures by transfection of cells with in vitro RNA transcripts which have been used for the preparation of vaccines in FBS-containing medium for use in previous studies (Monath et al, Vaccine 20: 1004-. The whole genome of this virus was sequenced and showed no detectable mutations (Table 7) (note that the consensus sequencing method did not detect a minority subpopulation (minor mutation); limit of detection of mutations was-10%). Small scale passaging from this P2 virus to the P10 level was performed in T25 flasks to analyze its genetic stability (g.s.) on prolonged propagation in SF cultures (FIG. 6; g.s. passage). The whole genome sequence of the g.s.P5 and g.s.P10 passages had a nucleotide change from C to T at nucleotide 935, resulting in an amino acid substitution from R to C at residue M-60 (Table 7). This mutation was first detected as heterogeneity at the g.s.p4 passage rather than g.s.p3.

Regardless of the results of small scale genetic stability analysis, when three large scale SF passage preparations were performed in roller bottles from uncloneable P2PMS to generate candidate uncloneable Master seeds (Master Seed) (P3) and Production Seed (Production Seed) (P4), followed by preparation of a batch vaccine (P5) in a 100L bioreactor, different mutations were accumulated at 50: the 50% heterogeneity was observed to result in F to L amino acid changes at residues E-107 due to T to C changes at nucleotide 1301 (Table 7). This is an unacceptable mutation because it is a back mutation from the SA14-14-2 sequence to the wild type JE sequence at the key attenuating residue (Arroyo et al, J.Virol.75: 934-942, 2001), and thus may reduce the safety of the vaccine.

Based on the considerations mentioned below, cloned PMS candidates were then generated by plaque purification, thereby stabilizing the SF vaccine and avoiding the accumulation of unwanted mutations such as E-107. Plaque purification removes random mutations introduced by in vitro transcription in uncloneable viruses characterized by lower fidelity of RNA synthesis than that of viral RNA synthesis with YF 17D-specific RNA polymerase (Pugachev et al, J.Virol.78: 1032-1038, 2004). Starting from the uncloned P2PMS virus, the biological clone at P7, clone a virus, designated non-mutant P7 clone a PMS, was obtained by three successive plaque purifications followed by two passages of amplification in SF medium without any amino acid substitution. Its genome contained two silent nucleotide changes at nucleotides 6952 and 7147 (table 7). These changes are acceptable because they do not alter the amino acid sequence of viral proteins and are located outside of the cis-acting RNA elements required for efficient viral replication. Clone C P10 virus containing the M-60 mutation (designated M-60P10 clone CPMS variant) was prepared similarly starting from the P5g.s. virus (fig. 6). Except for the desired M-60 mutation, it contained only a silent nucleotide change at nucleotide 3616 (Table 7). In addition, study grade clone I and clone E viruses were later isolated from the uncloneable P5 bulk vaccine virus by a single plaque purification (selection of large plaques) and one passage of expansion in Vero cells. Clone I contained a single amino acid change at residues E-107, which is a back mutation from amino acid F to amino acid L to the wild type. Thus, clone I represents a pure population of E-107 revertants. Clone E contained a single amino acid mutation at the N-terminus of the M protein, with an amino acid change from Q to P at residue M-5.

To confirm the genetic stability of cloned PMS variants, a rather large scale g.s. passage mock preparation was performed in SF culture (fig. 6) (serial passages designated S were prepared in T-225 flasks and passages designated F were prepared in bioreactors of 5 or 15L, where african green monkey kidney cells were grown on Cytodex I microcarrier beads). Sequencing of only the prM-E region (fragment I of cDNA) was performed on SSS and SSF samples of both candidates (obtained by three static (static) passages, or two static and one Fermenter (Fermenter) passages, respectively) and FFF samples of the M-60 variant. None of the triplicate g.s. samples had any detectable mutation in the prM or E proteins of the virus, except for the M-60 mutation in clone C. There was no trace of the E-107 mutation (Table 7). This indicates that an acceptable level of genetic stability was achieved by plaque purification. High genetic stability of the M-60 variant was subsequently confirmed during the preparation of new Master (P11) and Production Virus (P12) Seeds prepared in the cell factory and of the final batch vaccine (P13) prepared in a 50L bioreactor, which both retained the M-60 mutation but no other changes were detected in their complete genome by consensus sequencing.

Effect of M-60 and E-107 mutations on viral growth in SF African Green monkey Kidney cells

To compare the growth kinetics of non-mutant, M-60 mutant, and E-107 mutant viruses in SF cultures, seed viruses were prepared at an MOI of 0.001pfu/ml (confirmed by back titration) with either unclosed P2PMS, unclosed P5g.s. samples (M-60 mutant), or unclosed P5 bulk vaccine variants (containing the E-107 mutation), and unclosed P3 main seed and P4, which also contained a proportion of the E-107 mutation, to infect cells. Equal daily aliquots of virus-containing medium were harvested and titrated by plaque assay. As shown in figure 7, M-60 virus grew faster than non-mutant P2 virus and produced significantly higher (more than 10-fold) titers at day 3 and 4 post-infection. Similar to the M-60 mutation, the E-107 mutation also increased viral replication. Thus, the M-60 and E-107 mutations clearly confer a growth advantage in SF cultures. To support this conclusion, samples of S, SSS, and SSF g.s. passage from non-mutant clone A and M-60 mutant clone C virus were collected daily (see FIG. 6) and growth kinetics were analyzed by titration, with the result that the M-60 mutant always produced peak titers up to 10-fold higher than the non-mutant (approaching 8log of 8 log)₁₀pfu/ml). In addition, this conclusion was confirmed by comparing the growth curves of clones A, C, and I viruses in small-scale SF cultures, since clones C (M-60) and I (E-107) always grow to higher titers than clone A (non-mutant).

M-60 and E-107 mutations for ChimeriVax^TMEffect of JE on neurovirulence in suckling mice

ChimeriVa has been ensured by a mouse neurovirulence testx^TMThe vaccine candidate was not more neurovirulent than YF17D vector. After IC vaccination, YF17D vaccine was lethal to mice of all ages. In contrast, ChimeriVax^TMThe vaccine is significantly attenuated. Since mature mice are generally insensitive to detecting subtle differences in neurovirulence, e.g., due to single amino acid changes, a more sensitive suckling mouse model using survival assays can be used for this purpose (Guirakho et al, Virology 257: 363-.

Eight-day-old suckling mice IC were inoculated with clone A P7 virus, clone C P10 virus (M-60 mutation), unclosed P5 batch vaccine (E-107 mutation), and previously prepared FBS-containing control ChimeriVax^TMJE virus (Quality Control reference Standard virus P5; no mutation), YF17D positive Control: (JE virus)) Or simulating inoculation with a diluent. Mortality at 21 days, median IC 50% lethal dose value (LD)₅₀) And the Average Survival Time (AST) of dead mice are shown in table 8. As is to be expected, the number of,has high neurotoxicity. Inoculation 2.4log₁₀PFU of this virus resulted in 100% mortality and 8.8 days of short AST. The P7 non-mutant and P10M-60 mutant clones were highly attenuated as the original FBS-containing chimeras, their LD₅₀Value of>5log₁₀PFU and AST are longer. Thus, the M-60 mutation does not alter the highly attenuated phenotype of the vaccine in this animal model. The unclosed P5 bulk vaccine virus was significantly more virulent than the clone, IC LD₅₀Is 3.1log₁₀PFU, but it is greater thanHas low toxicity. Subsequently, cloned M-60 vaccines were examined in this experiment under GLP conditionsPreparation of (2) passage products (manufacturing passages) (Master Seed), Production Seed (Production Seed), and batch vaccines) gave similar results. This confirms the high genetic/phenotypic stability obtained by plaque purification and the use of the M-60 mutation.

Analysis of safety and efficacy in non-human primates

Test 1

In this test, the test was carried out usingVirus as a control, clones C (M-60 mutant), ChimeriVax were compared after IC administration to cynomolgus monkeys^TMNeurovirulence of JE Vaccine Master Viral Bank (MVB) and Production Viral Bank (PVB) (Table 9).

No vaccine-related clinical indications or changes in food intake, body weight, or serum chemistry were observed, and hematological parameters were observed. Lymphoid hyperplasia (Lymphoid hyperplasia) was seen in 9, 4 and 8, respectively, of 11 monkeys in groups 1-3, which was manifested by an increase in the volume and number of lymph nodes in the spleen. Although this finding is a common background (background) finding in cynomolgus monkeys, the group incidence of these monkeys was higher than normal and this was considered secondary to the expected immune response induced by the vaccine. Noteworthy in ChimeriVax^TM-JE treatment group andsimilar changes occurred in the control reference group. [ some monkeys in all three groups developed low-level post-inoculation viremia of short duration, which was within acceptable range, and all animals had seroconverted (seroconverted) to the virus used for inoculation. On day 31, in the LNI testTiter of yellow fever virus-specific neutralizing antibodies of treated monkeys ranging from 2.07 to>6.13 and in PRNT₅₀In the testNone of the treated monkeys had cross-reactive antibodies to the JE virus. All ChimeriVax^TMJE MVB vaccine-treated monkeys all had ≧ 320 (from 320 to 320)>20480) JE neutralizing antibody titers of (a) and no cross-reactive antibodies to YF virus in the LNI assay. All ChimeriVax^TMJE PVB vaccine-treated monkeys all had ≥ 160 (from 160 to 160)>20480) JE neutralizing antibody titers of (a) and no cross-reactive antibodies to YF virus. There was no discernable relationship between the magnitude or duration of detectable viremia and the magnitude of induction of JE-neutralizing antibody titers]。

ChimeriVax in this experiment^TMThe JE MVB and PVB preparations showed the weakest neurovirulence. In monkey neurovirulence tests for flavivirus vaccines, the most complex neurovirulence assay is the combined group mean lesion score (combined group mean score), which represents the mean of the mean target area and mean discriminatory score (average of the mean target area and mean discriminatory score). The target areas of cynomolgus monkeys are the cervical and lumbar enlargement of the substantia nigra (substantia nigra) and spinal cord, and represent Central Nervous System (CNS) areas that are damaged by all flaviviruses. The discrimination regions are globus pallidus (globus pallidus), putamen (putamen), anterior-medial thalamic nucleus, and lateral thalamic nucleus, and represent regions of the CNS selectively damaged by YF17D strain (and presumably other flaviviruses) with different virulence characteristics and distinguishing the reference strain from strains with increased neurovirulence. Using ChimeriVax^TMMean damage score for the combination of JE MVB and PVB article-treated monkeys was significantly lower thanReference control group (p)<0.05). Using ChimeriVax^TMThe mean resolution center score (mean distinguisher center score) of the JE MVB and PVB treated groups of monkeys was also significantly lower than that of the two groups of monkeysReference control group case (p)<0.05) (table 9). Has accepted ChimeriVax^TMMean scores of two groups of monkeys of the JE vaccine preparation were not statistically significantly different, and both preparations showed similar low neurovirulence in the monkey neurovirulence test.

Thus, monkey neurovirulence test results showed a satisfactory safety profile for the novel (M60, clone C) plaque-purified MVB and PVB. The test articles (test articles) showed no clinical toxicity and the lesion scores resolved and combined in the neuropathic study were significantly lower than the reference controls. By quantitative determination of viremia, the test substance is compared with reference controlThere is no difference in visceral tropism.

Test 2

This experiment was performed to compare the original unclosed P5ChimeriVax after a single Subcutaneous (SC) administration in cynomolgus monkeys^TMJE Vaccine [ previously prepared in Vero cells in the presence of FBS, without mutation except the L to F change of E491 in the hydrophobic tail of the E protein, it appeared to be an innocuous mutation in biological phenotype and it had been tested in clinical trials (Monath et al, J.Infect.Dis.188: 1213. sup. 1230, 2003; Monath et al, Vaccine 20: 1004. sup. 1018, 2002)]And novel clone C (M-60 mutant) ChimeriVax^TMViremia, immune response and safety of JE purified bulk vaccine (P13). Inoculating the initial unclosed P5ChimeriVax^TMChimeriVax was detected in 5 (100%) of 5 seronegative monkeys of JE vaccine^TM-JE virus. The duration of viremia ranged from 2-5 days with titers ranging from 20 to 790 PFU/mL. The mean peak viremia (+ -SD) was 244 (+ -310) PFU/mL and the mean number of days a viremia occurred was 3.4 (+ -1.34) (Table 10).

ChimeriVax was detected in 4 (100%) of 4 seronegative monkeys vaccinated with the new purified P13JE batch vaccine^TM-JE virus. The duration of viremia ranged from 2-5 days, with titers ranging from 50 to 290 PFU/mL. The mean peak viremia (+ -SD) was 160 (+ -123) PFU/mL, and the mean day of viremia was 3.75 (+ -1.26) days (Table 10). Mean peak viremia and days of viremia did not differ significantly in both treatment groups (p-values of 0.6290 and 0.7016, respectively; ANOVA).

Using the original unclosed P5ChimeriVax^TMAll seronegative monkeys seroconverted following treatment with either JE vaccine or purified P13JE bulk vaccine (table 10). On day 31, titers of JE virus neutralizing antibodies ranged from 640 to 5120 (geometric mean titer 1689) in sera of 5 (100%) of 5 monkeys vaccinated with the uncloned P5 vaccine. After inoculation with P13ChimeriVax^TMThe titer of JE virus neutralizing antibodies ranged from 320 to 2560 (geometric mean titer 761) in the serum of 4 monkeys (100%) of the JE purified batch vaccine. Antibody titers did not differ significantly between treatment groups (p-0.2986, ANOVA).

Thus, the new M-60 vaccine was combined with the original unclosed ChimeriVax^TMJE vaccine (no mutation except E491) was compared in safety (viremia) and immunogenicity. The new vaccine is slightly less viscerotropic (desirable feature), but still highly immunogenic. The differences in the magnitude of viremia and immunogenicity were not statistically significant.

Effect of M-5, M-60, and E-107 mutations on the pH threshold for viral infectivity

ChimeriVax was prepared by inserting the prM and E genes of SA14-14-2 strain of JE virus into YF17D virus backbone^TM-a JE vaccine. Envelope of SA14-14-2 virus (in ChimeriVax)^TM-present in JE) differs from its parent SA14 virus by 10 amino acids: E107L to F, E138E to K, E176I to V, E177T to A, E227P to S, E244E to G, E264Q to H, E279K to M, E315A to V, and E439K to RChanges (Guirakho et al, Virology 257: 363-. Some of these residues were shown to be involved in ChimeriVax by site-directed mutagenesis^TM-attenuation of JE virus. Selecting ChimeriVax^TMMutants or revertants of JE to identify whether the mutation has altered the pH threshold of these viruses. To determine whether M-60, E-107 or M-5 mutations affect viral infectivity in a pH-dependent manner, a standard test of pH threshold for infectivity was performed as described in the materials and methods section. The following viruses were tested: (1) ChimeriVax^TMJE non-mutant (clone A, P7 containing all 10 SA14-14-2 mutations in the E protein); (2) ChimeriVax^TMRevertants of JE E107F to L (clone I containing 9 mutations of the E protein, P6); (3) ChimeriVax^TMMutants of JE M60R to C (clone C, P10, containing all 10 mutations of the E protein), and (4) mutants of M-5Q to P (clone E, P6, containing all 10 mutations of the E protein) (Table 12).

Non-mutant clone A P7 virus, M-60 mutant clone CP10 virus, M-5 mutant clone E, and unclosed P5 virus containing the E-107 mutation were treated with a series of reduced pH's followed by titration for residual virus infectivity. The infectivity of the three viruses (clone A control virus, clone C M60 mutant, and clone IE-107 mutant) began to decrease uniformly after pH6.0 and lost infectivity (pH threshold 5.9) at pH5.8, with the exception of M5 mutant clone E virus. The pH threshold (pH6.3) of the M-5 mutant was significantly higher than all other viruses (pH5.9) (fig. 8A). This is the first direct evidence that the extracellular domain of the M protein plays a substantial role in the process of flavivirus infection of cells. Thus, in fusions triggered by low pH in the endosome following uptake and internalization of the virus, the N-terminus of the M protein may play a role that was previously thought to be due solely to the envelope E protein.

ChimeriVax^TMThe fusion pH threshold of JE viruses is 5.9, which is lower than that described for other wild-type (wt) flaviviruses (Guirakho et al, J.Gen.Virol.72: 1323. 1329, 1991) and may be related to attenuation of the virus.

These data certificatesMing, ChimeriVax^TME-107 mutations in the E region of JE did not change the pH threshold of the fusion. In general, a low pH threshold means that the conformational change to be present in the E-protein requires more protonation of a particular amino acid, which is necessary for the transition from dimer to trimer. It is possible that one or more of the SA14-14-2 specific mutations (except the E107 mutation, which is located within a conserved fusion peptide) results in a low pH threshold (pH5.9) for the retention of the fusion, thereby retaining the attenuated phenotype of the virus for the host in question. M-5 mutations clearly increased this threshold from 5.9 to 6.3, which is closer to the wt flavivirus threshold (Guirakho et al, Virology: 169 (1): 90-99, 1989; Guirakho et al, J.Gen.Virol.72: 1323-. An increase in the pH threshold of the fusion should theoretically reduce the attenuated phenotype of the virus, since the virus can fuse at higher pH and requires less protonation to transition to the fusion active state. This appears to be correct because of the 1.4log₁₀The M5 virus, which was inoculated intracerebrally to 3-4 days old suckling mice by PFU, was significantly more virulent than at 1.7log₁₀PFU inoculated control virus (ChimeriVax without M5 mutation)^TMJE vaccine virus) (p ═ 0.056) (fig. 8B). However, M5 mutant virus (at 1.4 log) in 3-4 day old suckling mice₁₀Dose of PFU) still significantly less than neurovirulence(at 0.9 log)₁₀Dose of PFU) (fig. 8C), indicating that the SA14-14-2 mutation within the envelope protein of the vaccine virus still provides sufficient levels of attenuation of the virus.

Mutations in other chimeras affecting fusion pH threshold

With each ChimeriVax^TMTwo groups of DEN vaccine viruses were subjected to an indirect fusion assay: ChimeriVax without E protein mutation^TMDEN1-4P7 and ChimeriVax containing a single mutation in the E protein^TMDEN1-4P10, except ChimeriVax^TM-DEN4P 10. The virus was incubated with media of different pH for 10 minutes at room temperature. Titers were determined after returning the pH to neutral pH using standard plaque assays. ChimeriVax as shown in Table 13^TMThe virus inactivation (fusion) thresholds for P7 and P10 of DEN2 and DEN4 viruses were similar (pH 6.4). In contrast, ChimeriVax^TMpH threshold ratio ChimeriVax of DEN1P10^TMDEN1P7 virus was 0.4 units lower (pH6.0 and pH6.4). For ChimeriVax^TMDEN3P10 virus, the pH threshold of which is not very different (pH6.4 from pH 6.2).

For ChimeriVax^TMAll P7 of DEN virus showed maximal viral inactivation at pH6.2, only ChimeriVax^TMDEN4 was slightly lower (pH 6.0). ChimeriVax is shown^TMDEN1P10 required a significantly lower pH to be completely inactivated (pH 5.6). ChimeriVax^TMBoth the-DEN 1 and-DEN 3 viruses contained amino acid substitutions from K to R in E-204 (the E-protein of DEN3 is 2 amino acids less than the other 3 serotypes, so the E-202 residues in this virus are homologous to E-204 in DEN 1). For the DEN3 chimera, the less significant difference in fusion thresholds was probably due to the presence of wt (K) and mutated R amino acids (E204K/R) in the P10 virus stock as shown by co-sequencing (K: R ═ 50:50) (Pugachev et al, j.virol.78: 1032-1038, 2004). Despite the presence of the E251 mutation, since no change in the threshold for viral inactivation was observed with the DEN2P10 chimera, it can be concluded that the mutation at this residue was not involved in the viral fusion process (fig. 8D).

To determine in ChimeriVax^TMWhether the presence of K/R heterogeneity in P10 of DEN3 would cause an insignificant change in the pH threshold of the fusion, indirect fusion assays were performed with P7 (no mutation, E202K), P10 (50% mutation, E202K/R), and P15 (full mutation, E202R) viruses. ChimeriVax as shown in FIG. 8E^TMThe pH threshold for inactivation (fusion) of DEN3P10 is at pH6.2, between ChimeriVax^TMDEN3P7(pH6.4) and ChimeriVax^TMDEN3P15(pH6.0) virus pH threshold. Since the mutation of E202K to R is the only amino acid substitution detected in the E-protein of these chimeras, it is likely that this mutation causes a shift in pH of 0.4 for the pH threshold of the P15 viral fusion.

As described above, the mutation of E204K to R, which occurs in the cell culture for vaccine preparation, lowers the fusion pH threshold by 0.4pH units. The mutation of E204K to R showed the creation of new intramolecular H bonds and new salt bridges, which may have a significant impact on the dissociation of the E dimer. ChimeriVax was given based on the atomic coordinates of residue 394 of the DEN 2E-protein extracellular domain (strain S1) determined in the presence of the detergent n-octyl-B-D-glucoside^TMDEN1(PMS, P7) structural modeling of the E protein (mode1 line) (Modis et al, Proc. Natl. Acad. Sci. U.S.A.100: 6986-6991, 2003). Changing the K residue at position 204 to R to mimic mutant virus, and repeated modeling to represent ChimeriVax^TMThe E-protein structure of DEN1(VL, P10) virus (Guirakho et al, J.Virol.78: 9998-10008, 2004). The K residue at position 204 (204K) is located in a short loop inside a hydrophobic pocket lined by a residue that has been shown to affect the pH threshold of neurovirulence or fusion (Lee et al, Virology 232: 281-]1, 991-; monath et al, J.Virol.76: 1932-1943, 2002). The homology model for the E-homodimer structure of the vaccine virus (204R) and PMS (204K) viruses is compared in FIG. 8F. The side chains of 204K and 261H of one of the E monomers were shown to form H bonds with the backbone atoms of the 252V and 253L residues, respectively, on the opposite monomer. At position 204, it is predicted that R in the E protein of the vaccine virus (VL P10) will reorient itself (reorient) and will thus lose these hydrogen (H) bonds. However, the side chain of mutant R is adjacent to 261H and 257E, resulting in a new intramolecular H bond between 204R and 261H and possibly a new salt bridge between 204R and 257E. Since the pk of histidine may be about 6.0, slightly below the fusion threshold (pH6.4), Guirakho et al, (J.Virol.78: 9998-. This theory appears to be correct because the experiments described herein show ChimeriVax^TMThe fusion threshold of DEN1 was about 6.0, 0.4pH units lower than that of its P7 virus (pH 6.4). The introduction of a new intermolecular bond at residue 204 via R significantly strengthens the E-dimer linkage (association), thus a shift to low pH requires a suitable pHMore protonation of the residue (e.g., H261). Lower fusion thresholds affect the viscerotropism of the virus in monkeys and reduce neurovirulence in suckers vaccinated via the i.c. route (Guirakho et al, J.Virol.78: 9998-.

In ChimeriVax^TMReplacement of E202K to R in the E-protein of DEN3P10 vaccine with ChimeriVax^TMHomology of the E204 mutation in the DEN1P10 vaccine. And ChimeriVax^TMChimeriVax, like DEN1P10^TMDEN3P10 (heterogeneous at residue 202 containing K and R residues) showed a lower fusion pH threshold (. about.0.2 pH units) than P7. When the mutation is fixed in ChimeriVax^TMP15 of DEN3, the pH threshold for fusion was further lowered (0.4 pH units, similar to ChimeriVax)^TMDEN1P 10). This data shows that residue 202/204 can be a universal determinant of attenuation in all dengue viruses. Now, ChimeriVax^TMThe P10 vaccine viruses of-DEN 3 and-DEN 4 did not contain this mutation, and both viruses induced higher viremia levels in monkeys vaccinated with the tetravalent vaccine formulation (Guirakhoo et al, J.Virol.78: 4761-4775, 2004). Still need to be observed in ChimeriVax^TMDEN3 or ChimeriVax^TMWhether the K to R mutation in DEN4 reduces their entropic phenomenon in their host.

The pH threshold of WT-JE fusion was previously reported to be 6.4(Guirakho et al, J.Gen.Virol.72: 1323-. In this study, ChimeriVax^TMThe pH threshold for all variants of JE was 5.9. The low pH threshold observed in these experiments may be due to ChimeriVax^TM-the presence of one or more of 10 attenuating mutations in the JE envelope protein. This mutation may strengthen the attachment of the E-protein dimer, thereby requiring a lower pH for dissociation and conversion to a trimeric structure and subsequent fusion. The data here show that neither the mutation from E107F to L (located within the cd-loop of domain II of the E-protein) nor the mutation from E279M to K (located within the hydrophobic pocket of domain II) causes a decrease in pH threshold. It is possible that other mutations in the JE E protein will affect the pH threshold of the fusion. Crystal structure on TBE virus E protein closely resembling JE E proteinCan help predict residues that, upon alteration, will alter the pH threshold of the fusion. Based on this model, it may be that mutations in residues E244G and/or E264H result in ChimeriVax^TMThe pH threshold for JE virus fusion is lower than for WT JE.

Effect of M-60 and E-107 mutations on the effectiveness of viral penetration

The influence of M-60 (clone C virus) and E-107 (clone I virus) mutations on the penetration of the virus into SF African green monkey kidney cells was examined by the method of Chambers (Vlaycheva et al, J.Virol.76: 6172-6184, 2002). In this assay, SF vero cells were infected with virus (to generate-50 plaques/well at each time point) at appropriate dilutions for 5, 10, 20, or 60 minutes. The non-internalized virus was inactivated by addition of acidic glycine solution (precipitation), while the control parallel wells were treated with PBS (neutral pH). Cells were washed with PBS and covered with a methylcellulose cover, followed by visual inspection and counting of plaques on day 5. The effectiveness of penetration is expressed as the average number of plaques in glycine-treated wells as a percentage of the number of plaques in the control (PBS-treated wells). The results of the preliminary penetration test are shown in fig. 9A. Importantly, at the 5 and 10 minute time points, where the effect of the mutation on penetration is more likely to be detected, the percentage of non-mutant clone a virus was high for clone C and clone I virus that penetrated. The results were not statistically significant as shown by the standard deviation bars and required confirmation in additional replicates. However, this experiment suggests that both M-60 and E-107 mutations may enhance ChimeriVax^TMEffectiveness of membrane fusion of JE virus with cells grown under SF conditions. A possible mechanism by which the M-60 and E-107 residues contribute to the membrane fusion process is shown in FIG. 9B. The M-60 residue is located in the viral membrane, while the E-107 residue is inserted into the cell membrane, and the two membranes are forced to fuse after a low pH-dependent rearrangement of the E protein (which is facilitated by the extracellular domain of the M protein according to our data). More suitable amino acids at either of these two residues may facilitate membrane fusion.

Since our data for the first time found that both the extracellular domain of the M protein and its transmembrane domain had functional significance, it is now believed that the intact M protein is an attractive target for mutagenesis to attenuate flaviviruses for the development of new live attenuated vaccines. For example, random or specific (upon further analysis of the protein structure) amino acid changes, or deletions of, for example, 1, 2, 3,4, 5 amino acids, etc., of increasing length, can be incorporated into the entire protein, with the expectation that the biological phenotype of the virus will change, thereby resulting in significant attenuation.

Results of clinical trials

ChimeriVax with arginine and cysteine M60 residues obtained from the above clinical trial is compared in tables 11A and B^TM-viremia profile of JE. And 29-50% received ChimeriVax^TMChimeriVax was present in subjects receiving JE M60 cysteine^TMOf the subjects with JEM60 arginine, 67-100% of subjects were viremic on one or more days. Has accepted ChimeriVax^TMThe mean highest viremia levels for subjects with JE M60 arginine ranged from 13 to 40PFU/ml, and ChimeriVax^TMIn the case of JE M60 cysteine, the mean maximum viremia level was 3.5-6.3 PFU/ml. In ChimeriVax^TMIn the case of JE M60 arginine, the duration of viremia was significantly longer.

These data demonstrate that the level of viremia is significantly lower in the case of vaccines containing the M60 mutation. Viremia is an indicator of the viscerotropism (virulence) of vaccine viruses. Vaccines with hypoviremia are considered safer because of the reduced cellular damage and dysfunction of the organs that support viral replication and cause viremia, as well as the reduced likelihood that the virus will cross the blood brain barrier and invade the central nervous system. In other experiments, the M60 mutant was shown to be highly immunogenic to humans as well as non-mutants.

TABLE 1 consensus sequence for small plaques (P10PMS) (P/N IT-0116; L/N I020504A) (plaques purified from the P5Run1 vaccine lot (lot)).

Position of	Amino acid changes	NT position	NT alteration
				M(66)	Leucine → proline	954	CTA→CCA
E(313)	Glycine → arginine	1919	GGG→AGG
					Asparagine (silence)	2926	AAC→AAT
	Glycine (Silent)	7126	GGA→GGG

TABLE 2 consensus sequence of large plaque PMS (P10, PMS) (P/N IT-0117; L/N I030804A) (from P5Run1 vaccine lot).

Position of	Amino acid changes	NT position	NT alteration
				E(313)	Glycine → arginine	1919	GGG→AGG

Glycine (Silent)

7126

GGA→GGG

TABLE 3 sequence of isolated large plaques after an additional 2 passages of S plaque PMS (p10) in vero cells under serum-free conditions.

Table 4 CPE observed for HepG 2.

Days after infection

0

1

2

3

4

5

6

7

8

WN01

0％

0％

0％

0％

0％

30％

90％

～100％

100％

WN02P5

0％

0％

0％

5％

30％

50％

～100％

100％

WNL

0％

0％

0％

0％

0％

30％

90％

～100％

100％

WNS

0％

0％

0％

5％

30％

50％

～100％

100％

YF/17D

0％

0％

0％

20％

50％

70％

～100％

100％

TABLE 5 inoculation of ChimeriVax^TMWN02 vaccine orThe viremia of monkeys.

^*Viremia was expressed as pfu/mL

^**Day 1: day 1 of study, monkeys were vaccinated on day 1 of study

0PFU/mL is below the detection limit, and the theoretical test cut-off (assay cutoff) is 10PFU/mL

Table 6. M region sequence of YF-WN chimera obtained directly from plaque isolates from virally vaccinated monkeys with WN02 vaccine virus.

Monkey #	Days of viremia	Plaque isolate #	Visible plaque morphology (when picking)	M66 present?	Other M mutations
						21205	4	#4	SP	Whether or not	Is free of
2808	3	#8	SP	Whether or not	Is free of
						2808	3	#9	LP	Whether or not	M60(R to G)
21191	2	#10	LP	Whether or not	Is free of
						21191	1	#14	SP	Whether or not	M61(V to A)
21191	1	#15	SP	Whether or not	Is free of
						21191	1	#16	LP	Whether or not	M63(F to S)

TABLE 7 unclosed and cloned SF ChimeriVax^TMNucleotide and amino acid sequences of JE samples (see Table 6).

^a: from the beginning of the genome^b: from the N-terminus of the protein of interest

TABLE 8 clone A P7, clone C P10, unclosed P5, FBS-containing standards, andneurovirulence of the virus in 8-day-old suckling mice.

TABLE 9M-60 (clone C) Master seed and seed preparation andcontrol comparisons were made for neurovirulence in cynomolgus monkeys.

¹PFU ═ plaque-forming unit

²Groups 1, 2 and 3 had 4 of 11, 2 of 11 and 1 of 11 animals, respectively, excluded the calculation of the score because it was found in a retrospective PRNT50 trial on day 1 (pre-inoculation)These were JE-seropositive, with the PRNT50 test being more sensitive than the HAI test used for the pre-screening.

TABLE 10 comparison of the magnitude of viremia and immunogenicity of cynomolgus monkeys SC inoculated with otherwise unclosed P5ChimeriVax produced in FBS-containing media^TMJE vaccine (no mutation except E491) and a new clone C P13 purified batch vaccine (M-60 mutant).

¹2 of 6,1 of 6,2 of 6, and 2 of 6 animals in groups 1, 2 and 3, respectively, were excluded from the calculation of values because they were found to be JE-seropositive in a retrospective PRNT50 test on day 1 (pre-inoculation), the PRNT50 test being more sensitive than the HAI test used for pre-screening.

TABLE 11A. viremia profiles of subjects participating in test H-040-003, in which ChimeriVax with M60 arginine was administered to the subjects^TM-JE. The bolded dose range was similar to that given in another trial (H-040-007) in which the mutant M-60 cysteine vaccine was administered.

TABLE 11B viremia profile of a subject involved in the test H-040-007, to which ChimeriVax with M60 cysteine was administered^TM-JE。

TABLE 12 passage of each ChimeriVax^TMFusion pH threshold found in fusion test for JE vaccine

TABLE 13. by each couple of ChimeriVax^TMFusion pH threshold found in indirect fusion assay of DEN P7 and P10

Virus	pH threshold for fusion
		ChimeriVaxTM-DEN1PMS P7	6.4
ChimeriVaxTM-DEN1VL P10	6.0
		ChimeriVaxTM-DEN2PMS P7	6.4
ChimeriVaxTM-DEN2VL P10	6.4
		ChimeriVaxTM-DEN3PMS P7	6.4
ChimeriVaxTM-DEN3VL P10	6.2
		ChimeriVaxTM-DEN4PMS P7	6.4
ChimeriVaxTM-DEN4VL P10	6.4

TABLE 14

Transformation of YF/flavivirus chimeras

Viral chimeric C/prM conjugation¹Chimeric E/NS1 junctions²5 '3' removal or

Connection of³Connection of⁴Raw) site 5

YF/WN X-cactgggagagcttgaaggtcaaagccagttgcagccgcggtttaa AatII NsiI

(SEQ ID NO：1) (SEQ ID NO：2)

YF/DEN-1 X-aaggtagactggtgggctcccgatcctcagtaccaaccgcggtttaa AatII SphI DEN SphI

(SEQ ID NO：3) (SEQ ID NO：4)

YF/DEN-2 X-aaggtagattggtgtgcattgaaccctcagtaccacccgcggtttaa AatII SphI

(SEQ ID NO：5) (SEQ ID NO：6)

YF/DEN-3 X-aaggtgaattgaagtgctctaacccccagcaccacccgcggtttaa AatII SphI DEN XhoI

(SEQ ID NO: 7) (SEQ ID NO: 8) (SphI in DEN)

YF/DEN-4 X-aaaaggaacagttgttctctaacccgaagtgtcaaccgcggtttaa AatII NsiI

(SEQ ID NO：9) (SEQ ID NO：10)

YF/SLE X-aacgtgaatagttggatagtcaccgttggtcgcacccgcggtttaa AatII SphI SLE AatII

(SEQ ID NO：11) (SEQ ID NO：12)

YF/MVE X-aatttcgaaaggtggaaggtcgaccggtgtttacagccgcggtttaa AatII AgeI (AgeI in Y)

(SEQ ID NO：13) (SEQ ID NO：14)

YF/TBE X-tactgcgaacgacgttgccacactgggaacctcacccgcggtttaa AatII AgeI (AgeI in YF)

(SEQ ID NO：15) (SEQ ID NO：16)

1,2: the two columns show the oligonucleotides used to generate chimeric YF/flavivirus primers corresponding to C/prM or E/NS1 junctions. (see text). X ═ YF capsid carboxy-terminal coding sequence. The underlined region corresponds to the targeted heterologous sequence immediately upstream of the NarI site (antisense-ccgcgg). This site allows the insertion of the PCR product into the Yfm5.2(NarI) plasmid required to generate the full-length cDNA template. Other nucleotides are specific for heterologous viruses. The oligonucleotide primers are listed in the 5 'to 3' direction.

3,4: unique restriction sites are enumerated for the generation of restriction fragments that can be isolated and ligated in vitro to generate a full-length chimeric cDNA template. Since some sequences do not contain convenient sites, appropriate site modifications are sometimes required (footer 5).

5: in parentheses are restriction enzyme sites that must be generated in the YF backbone or heterologous virus to allow efficient in vitro ligation. Sites not in brackets must be removed. All of these modifications were made by cDNA silent mutagenesis of individual clones. Blank intervals indicated that no modification was required for the cDNA clones.

ChimerivaxWN02 was changed to bottling the final product (Run1) L/N #02H 01; P/N # FP-0008

[ chain ]

SEQ ID NOS: 20 and 21

1 NGTAAATCCT GTGTGCTAAT TGAGGTGCAT TGGTCTGCAA

41 ATCGAGTTGC TAGGCAATAA ACACATTTGG ATTAATTTTA

81 ATCGTTCGTT GAGCGATTAG CAGAGAACTG ACCAGAACAT

M

121 GTCTGGTCGT AAAGCTCAGG GAAAAACCCT GGGCGTCAAT

S G R K A Q G K T L G V N

161 ATGGTACGAC GAGGAGTTCG CTCCTTGTCA AACAAAATAA

M V R R G V R S L S N K I

201 AACAAAAAAC AAAACAATT GGAAACAGAC CTGGACCTTC

K Q K T K Q I G N R P G P S

241 AAGAGGTGTT CAAGGATTTA TCTTTTTCTT TTTGTTCAAC

R G V Q G F I F F F L F N

281 ATTTTGACTG GAAAAAAGAT CACAGCCCAC CTAAAGAGGT

I L T G K K I T A H L K R

321 TGTGGAAAAT GCTGGACCCA AGACAAGGCT TGGCTGTTCT

L W K M L D P R Q G L A V L

361 AAGGAAAGTC AAGAGAGTGG TGGCCAGTTT GATGAGAGGA

R K V K R V V A S L M R G

401 TTGTCCTCAA GGAAACGCCG TTCCCATGAT GTTCTGACTG

L S S R K R R S H D V L T

441 TGCAATTCCT AATTTTGGGA ATGCTGTTGA TGACGGGTGG

V Q F L I L G M L L M T G G

481 AGTTACCCTC TCTAACTTCC AAGGGAAGGT GATGATGACG

V T L S N F Q G K V M M T

521 GTAAATGCTA CTGACGTCAC AGATGTCATC ACGATTCCAA

V N A T D V T D V I T I P

561 CAGCTGCTGG AAAGAACCTA TGCATTGTCA GAGCAATGGA

T A A G K N L C I V R A M D

601 TGTGGGATAC ATGTGCGATG ATACTATCAC TTATGAATGC

V G Y M C D D T I T Y E C

641 CCAGTGCTGT CGGCTGGTAA TGATCCAGAA GACATCGACT

P V L S A G N D P E D I D

ChimerivaxWN02 was changed to bottling the final product (Run1) L/N #02H 01; P/N # FP-0008

[ chain ]

681 GTTGGTGCAC AAAGTCAGCA GTCTACGTCA GGTATGGAAG

C W C T K S A V Y V R Y G R

721 ATGCACCAAG ACACGCCACT CAAGACGCAG TCGGAGGTCA

C T K T R H S R R S R R S

761 CTGACAGTGC AGACACACGG AGAAAGCACT CTAGCGAACA

L T V Q T H G E S T L A N

801 AGAAGGGGGC TTGGATGGAC AGCACCAAGG CCACAAGGTA

K K G A W M D S T K A T R Y

841 TTTGGTAAAA ACAGAATCAT GGATCTTGAG GAACCCTGGA

L V K T E S W I L R N P G

881 TATGCCCTGG TGGCAGCCGT CATTGGTTGG ATGCTTGGGA

Y A L V A A V I G W M L G

921 GCAACACCAT GCAGAGAGTT GTGTTTGTCG TGCTATTGCT

S N T M Q R V V F V V L L L

961 TTTGGTGGCC CCAGCTTACA GCTTCAACTG CCTTGGAATG

L V A P A Y S F N C L G M

1001 AGCAACAGAG ACTTCTTGGA AGGAGTGTCT GGAGCAACAT

S N R D F L E G V S G A T

1041 GGGTGGATTT GGTTCTCGAA GGCGACAGCT GCGTGACTAT

W V D L V L E G D S C V T I

1081 CATGTCTAAG GACAAGCCTA CCATCGACGT CAAGATGATG

M S K D K P T I D V K M M

1121 AATATGGAGG CGGCCAACCT GGCAGAGGTC CGCAGTTATT

N M E A A N L A E V R S Y

1161 GCTATTTGGC TACCGTCAGC GATCTCTCCA CCAAAGCTGC

C Y L A T V S D L S T K A A

1201 ATGCCCGACC ATGGGAGAAG CTCACAATGA CAAACGTGCT

C P T M G E A H N D K R A

1241 GACCCAGCTT TTGTGTGCAG ACAAGGAGTG GTGGACAGGG

D P A F V C R Q G V V D R

1281 GCTGGGGCAA CGGCTGCGGA TTTTTTGGCA AAGGATCCAT

G W G N G C G F F G K G S I

1321 TGACACATGC GCCAAATTTG CCTGCTCTAC CAAGGCAATA

D T C A K F A C S T K A I

ChimerivaxWN02 was changed to bottling the final product (Run1) L/N #02H 01; P/N # FP-0008

[ chain ]

1361 GGAAGAACCA TCTTGAAAGA GAATATCAAG TACGAAGTGG

G R T I L K E N I K Y E V

1401 CCATTTTTGT CCATGGACCA ACTACTGTGG AGTCGCACGG

A I F V H G P T T V E S H G

1441 AAATTACTCC ACACAGGTTG GAGCCACTCA GGCCGGCCGA

N Y S T Q V G A T Q A G R

1481 TTCAGCATCA CTCCTGCTGC GCCTTCATAC ACACTAAAGC

F S I T P A A P S Y T L K

1521 TTGGAGAATA TGGAGAGGTG ACAGTGGACT GTGAACCACG

L G E Y G E V T V D C E P R

1561 GTCAGGGATT GACACCAATG CATACTACGT GATGACTGTT

S G I D T N A Y Y V M T V

1601 GGAACAAAGA CGTTCTTGGT CCATCGTGAG TGGTTCATGG

G T K T F L V H R E W F M

1641 ACCTCAACCT CCCTTGGAGC AGTGCTGGAA GTACTGTGTG

D L N L P W S S A G S T V W

1681 GAGGAACAGA GAGACGTTAA TGGAGTTTGA GGAACCACAC

R N R E T L M E F E E P H

1721 GCCACGAAGC AGTCTGTGAT AGCATTGGGC TCACAAGAGG

A T K Q S V I A L G S Q E

1761 GAGCTCTGCA TCAAGCTTTG GCTGGAGCCA TTCCTGTGGA

G A L H Q A L A G A I P V E

1801 ATTTTCAAGC AACACTGTCA AGTTGACGTC GGGTCATTTG

F S S N T V K L T S G H L

1841 AAGTGTAGAG TGAAGATGGA AAAATTGCAG TTGAAGGGAA

K C R V K M E K L Q L K G

1881 CAACCTATGG CGTCTGTTCA AAGGCTTTCA AGTTTCTTAG

T T Y G V C S K A F K F L R

1921 GACTCCCGTG GACACCGGTC ACGGCACTGT GGTGTTGGAA

T P V D T G H G T V V L E

1961 TTGCAGTACA CTGGCACGGA TGGACCTTGC AAAGTTCCTA

L Q Y T G T D G P C K V P

2001 TCTCGTCAGT GGCTTCATTG AACGACCTAA CGCCAGTGGG

I S S V A S L N D L T P V G

ChimerivaxWN02 was changed to bottling the final product (Run1) L/N #02H 01; P/N # FP-0008

[ chain ]

2041 CAGATTGGTC ACTGTCAACC CTTTTGTTTC AGTGGCCACG

R L V T V N P F V S V A T

2081 GCCAACGCTA AGGTCCTGAT TGAATTGGAA CCACCCTTTG

A N A K V L I E L E P P F

2121 GAGACTCATA CATAGTGGTG GGCAGAGGAG AACAACAGAT

G D S Y I V V G R G E Q Q I

2161 CAATCACCAT TGGCACAAGT CTGGAAGCAG CATTGGCAAA

N H H W H K S G S S I G K

2201 GCCTTTACAA CCACCCTCAA AGGAGCGCAG AGACTAGCCG

A F T T T L K G A Q R L A

2241 CTCTAGGAGACACAGCTTGG GACTTTGGGAT CAGTTGGAGG

A L G D T A W D F G S V G G

2281 GGTGTTCACTAGTGTTCGGC GGGCTGTCCA TCAAGTGTTC

V F T S V G R A V H Q V F

2321 GGAGGAGCAT TCCGCTCACT GTTCGGAGGC ATGTCCTGGA

G G A F R S L F G G M S W

2361 TAACGCAAGG ATTGCTGGGG GCTCTCCTGT TGTGGATGG

I T Q G L L G A L L L W M G

2401 CATCAATGCT CGTGATAGGT CCATAGCTCT CACGTTTCTC

I N A R D R S I A L T F L

2441 GCAGTTGGAG GAGTTCTGCT CTTCCTCTDC GTGAACGTGG

A V G G V L L F L S V N V

2481 GCGCCGATCA AGGATGCGCC ATCAACTTTG GCAAGAGAGA

G A D Q G C A I N F G K R E

2521 GCTCAAGTGC GGAGATGGTA TCTTCATATT TAGAGACTCT

L K C G D G I F I F R D S

2561 GATGACTGCC TGAACAAGTA CTCATACTAT CCAGAAGATC

D D W L N K Y S Y Y P E D

2601 CTGTGAAGCT TGCATCAATA GTGAAAGCCT CTTTTGAAGA

P V K L A S I V K A S F E E

2641 AGGGAAGTGT GGCCTAAATT CAGTTGACTC CCTTGAGCAT

G K C G L N S V D S L E H

2681 GAGATGTGGA GAAGCAGGGC AGATGAGATC AATGCCATTT

E M W R S R A D E I N A I

ChimerivaxWN02 was changed to bottling the final product (Run1) L/N #02H 01; P/N # FP-0008

[ chain ]

2721 TTGAGGAAAA CGAGGTGGAC ATTTCTGTTG TCGTGCAGGA

F E E N E V D I S V V V Q D

2761 TCCAAAGAAT GTTTACCAGA GAGGAACTCA TCCATTTTCC

P K N V Y Q R G T H P F S

2801 AGAATTCGGG ATGGTCTGCA GTATGGTTGG AAGACTTGGG

R I R D G L Q Y G W K T W

2841 GTAAGAACCT TGTGTTCTCC CCAGGGAGGA AGAATGGAAG

G K N L V F S P G R K N G S

2881 CTTCATCATA GATGGAAAGT CCAGGAAAGA ATGCCCGTTT

F I I D G I S R K E C P F

2921 TCAAACCGGG TCTGGAATTC TTTCCAGATA GAGGAGTTTG

S N R V W N S F Q I E E F

2961 GGACGGGAGT GTTCACCACA CGCGTGTACA TGGACGCAGT

G T G V F T T R V Y M D A V

3001 CTTTGAATAC ACCATAGACT GCGATGGATC TATCTTGGGT

F E Y T I D C D G S I L G

3041 GCAGCGGTGA ACGGAAAAAA GAGTGCCCAT GGCTCTCCAA

A A V N G K K S A H G S P

3081 CATTTTGGAT GGGAAGTCAT GAAGTAAATG GGACATGGAT

T F W M G S H E V N G T W M

3121 GATCCACACC TTGGAGGCAT TAGATTACAA GGAGTGTGAG

I H T L E A L D Y K E C E

3161 TGGCCACTGA CACATACGAT TGGAACATCA GTTGAAGAGA

W P L T H T I G T S V E E

3201 GTGAAATGTT CATGCCGAGA TCAATCGGAG GCCCAGTTAG

S E M F M P R S I G G P V S

3241 CTCTCACAAT CATATCCCTG GATACAAGGT TCAGACGAAC

S H N H I P G Y K V Q T N

3281 GGACCTTGGA TGCAGGTACC ACTAGAAGTG AAGAGAGAAG

G P W M Q V P L E V K R E

3321 CTTGCCCAGG GACTAGCGTG ATCATTGATG GCAACTGTGA

A C P G T S V I I D G N C D

3361 TGGACGGGGA AAATCAACCA GATCCACCAC GGATAGCGGG

G R G K S T R S T T D S G

ChimerivaxWN02 was changed to bottling the final product (Run1) L/N #02H 01; P/N # FP-0008

[ chain ]

3401 AAAGTTATTC CTGAATGGTG TTGCCGCTCC TGCACAATGC

K V I P E W C C R S C T M

3441 CGCCTGTGAG CTTCCATGGT AGTGATGGGT GTTGGTATCC

P P V S F H G S D G C W Y P

3481 CATGGAAATT AGGCCAAGGA AAACGCATGA AAGCCATCTG

M E I R P R K T H E S H L

3521 GTGCGCTCCT GGGTTACAGC TGGAGAAATA CATGCTGTCC

V R S W V T A G E I H A V

3561 CTTTTGGTTT GGTGAGCATG ATGATAGCAA TGGAAGTGGT

P F G L V S M M I A M E V V

3601 CCTAAGGAAA AGACAGGGAC CAAAGCAAAT GTTGGTTGGA

L R K R Q G P K Q M L V G

3641 GGAGTAGTGC TCTTGGGAGC AATGCTGGTC GGGCAAGTAA

G V V L L G A M L V G Q V

3681 CTCTCCTTGA TTTGCTGAAA CTCACAGTGG CTGTGGGATT

T L L D L L K L T V A V G L

3721 GCATTTCCAT GAGATGAACA ATGGAGGAGA CGCCATGTAT

H F H E M N N G G D A M Y

3761 ATGGCGTTGA TTGCTGCCTT TTCAATCAGA CCAGGGCTGC

M A L I A A F S I R P G L

3801 TCATCGGCTT TGGGCTCAGG ACCCTATGGA GCCCTCGGGA

L I G F G L R T L W S P R E

3841 ACGCCTTGTG CTGACCCTAG GAGCAGCCAT GGTGGAGATT

R L V L T L G A A M V E I

3881 GCCTTGGGTG GCGTGATGGG CGGCCTGTGG AAGTATCTAA

A L G G V M G G L W K Y L

3921 ATGCAGTTTC TCTCTGCATC CTGACAATAA ATGCTGTTGC

N A V S L C I L T I N A V A

3961 TTCTAGGAAA GCATCAAATA CCATCTTGCC CCTCATGGCT

S R K A S N T I L P L M A

4001 CTGTTGACAC CTGTCACTAT GGCTGAGGTG AGACTTGCCG

L L T P V T M A E V R L A

4041 CAATGTTCTT TTGTGCCATG GTTATCATAG GGGTCCTTCA

A M F F C A M V I I G V L H

ChimerivaxWN02 was changed to bottling the final product (Run1) L/N #02H 01; P/N # FP-0008

[ chain ]

4081 CCAGAATTTC AAGGACACCT CCATGCAGAA GACTATACCT

Q N F K D T S M Q K T I P

4121 CTGGTGGCCC TCACACTCAC ATCTTACCTG GGCTTGACAC

L V A L T L T S Y L G L T

4161 AACCTTTTTT GGGCCTGTGT GCATTTCTGG CAACCCGCAT

Q P F L G L C A F L A T R I

4201 ATTTGGGCGA AGGAGTATCC CAGTGAATGA GGCACTCGCA

F G R R S I P V N E A L A

4241 GCAGCTGGTC TAGTGGGAGT GCTGGCAGGA CTGGCTTTTC

A A G L V G V L A G L A F

4281 AGGAGATGGA GAACTTCCTT GGTCCGATTG CAGTTGGAGG

Q E M E N F L G P I A V G G

4321 ACTCCTGATG ATGCTGGTTA GCGTGGCTGG GAGGGTGGAT

L L M M L V S V A G R V D

4361 GGGCTAGAGC TCAAGAAGCT TGGTGAAGTT TCATGGGAAG

G L E L K K L G E V S W E

4401 AGGAGGCGGA GATCAGCGGG AGTTCCGCCC GCTATGATGT

E E A E I S G S S A R Y D V

4441 GGCACTCAGT GAACAAGGGG AGTTCAAGCT GCTTTCTGAA

A L S E Q G E F K L L S E

4481 GAGAAAGTGC CATGGGACCA GGTTGTGATG ACCTCGCTGG

E K V P W D Q V V M T S L

4521 CCTTGGTTGG GGCTGCCCTC CATCCATTTG CTCTTCTGCT

A L V G A A L H P F A L L L

4561 GGTCCTTGCT GGGTGGCTGT TTCATGTCAG GGGAGCTAGG

V L A G W L F H V R G A R

4601 AGAAGTGGGG ATGTCTTGTG GGATATTCCC ACTCCTAAGA

R S G D V L W D I P T P K

4641 TCATCGAGGA ATGTGAACAT CTGGAGGATG GGATTTATGG

I I E E C E H L E D G I Y G

4681 CATATTCCAG TCAACCTTCT TGGGGGCCTC CCAGCGAGGA

I F Q S T F L G A S Q R G

4721 GTGGGAGTGG CACAGGGAGG GGTGTTCCAC ACAATGTGGC

V G V A Q G G V F H T M W

ChimerivaxWN02 was changed to bottling the final product (Run1) L/N #02H 01; P/N # FP-0008

[ chain ]

4761 ATGTCACAAG AGGAGCTTTC CTTGTCAGGA ATGGCAAGAA

H V T R G A F L V R N G K K

4801 GTTGATTCCA TCTTGGGCTT CAGTAAAGGA AGACCTTGTC

L I P S W A S V K E D L V

4841 GCCTATGGTG GCTCATGGAA GTTGGAAGGC AGATGGGATG

A Y G G S W K L E G R W D

4881 GAGAGGAAGA GGTCCAGTTG ATCGCGGCTG TTCCAGGAAA

G E E E V Q L I A A V P G K

4921 GAACGTGGTC AACGTCCAGA CAAAACCGAG CTTGTTCAAA

N V V N V Q T K P S L F K

4961 GTGAGGAATG GGGGAGAAAT CGGGGCTGTC GCTCTTGACT

V R N G G E I G A V A L D

5001 ATCCGAGTGG CACTTCAGGA TCTCCTATTG TTAACAGGAA

Y P S G T S G S P I V N R N

5041 CGGAGAGGGTG ATTGGGCTGT ACGGCAATGG CATCCTTGTC

G E V I G L Y G N G I L V

5081 GGTGACAACT CCTTCGTGTC CGCCATATCC CAGACTGAGG

G D N S F V S A I S Q T E

5121 TGAAGGAAGA AGGAAAGGAG GAGCTCCAAG AGATCCCGAC

V K E E G K E E L Q E I P T

5161 AATGCTAAAG AAAGGAATGA CAACTGTCCT TGATTTTCAT

M L K K G M T T V L D F H

5201 CCTGGAGCTG GGAAGACAAG ACGTTTCCTC CCACAGATCT

P G A G K T R R F L P Q I

5241 TGGCCGAGTG CGCACGGAGA CGCTTGCGCA CTCTTGTGTT

L A E C A R R R L R T L V L

5281 GGCCCCCACC AGGGTTGTTC TTTCTGAAAT GAAGGAGGCT

A P T R V V L S E M K E A

5321 TTTCACGGCC TGGACGTGAA ATTCCACACA CAGGCTTTTT

F H G L D V K F H T Q A F

5361 CCGCTCACGG CAGCGGGAGA GAAGTCATTG ATGCCATGTG

S A H G S G R E V I D A M C

5401 CCATGCCACC CTAACTTACA GGATGTTGGA ACCAACTAGG

H A T L T Y R M L E P T R

ChimerivaxWN02 was changed to bottling the final product (Run1) L/N #02H 01; P/N # FP-0008

[ chain ]

5441 GTTGTTAACT GGGAAGTGAT CATTATGGAT GAAGCCCATT

V V N W E V I I M D E A H

5481 TTTTGGATCC AGCCAGCATA GCCGCTAGAG GTTGGGCAGC

F L D P A S I A A R G W A A

5521 GCACAGAGCT AGGGCAAATG AAAGTGCAAC AATCTTGATG

H R A R A N E S A T I L M

5561 ACAGCCACAC CGCCTGGGAC TAGTGATGAA TTTCCACATT

T A T P P G T S D E F P H

5601 CAAATGGTGA AATAGAAGAT GTTCAAACGG ACATACCCAG

S N G E I E D V Q T D I P S

5641 TGAGCCCTGG AACACAGGGC ATGACTGGAT CCTGGCTGAC

E P W N T G H D W I L A D

5681 AAAAGGCCCA CGGCATGGTT CCTTCCATCC ATCAGAGCIG

K R P T A W F L P S I R A

5721 CAAATGTCAT GGCTGCCTCT TTGCGTAAGG CTGGAAAGAG

A N V M A A S L R K A G K S

5761 TGTGGTGGTC CTGAACAGGA AAACCTTTGA GAGAGAATAC

V V V L N R K T F E R E Y

5801 CCCACGATAA AGCAGAAGAA ACCTGAGTTT ATATTGGCCA

P T I K Q K K P D F I L A

5841 CTGACATAGC TGAAATGGGA GCCAACCTTT GCGTGGAGCG

T D I A E M G A N L C V E R

5881 AGTGCTGGAT TGCAGGACGG CTTTTAAGCC TGTGCTTGTG

V L D C R T A F K P V L V

5921 GATGAAGGGA GGAAGGTGGC AATAAAAGGG CCACTTCGTA

D E G R K V A I K G P L R

5961 TCTCCGCATC CTCTGCTGCT CAAAGGAGGG GGCGCATTGG

I S A S S A A Q R R G R I G

6001 GAGAAATCCC AACAGAGATG GAGACTCATA CTACTATTCT

R N P N R D G D S Y Y Y S

6041 GAGCCTACAA GTGAAAATAA TGCCCACCAC GTCTGCTGGT

E P T S E N N A H H V C W

6081 TGGAGGCCTC AATGCTCTTG GACAACATGG AGGTGAGGGG

L E A S M L L D N M E V R G

ChimerivaxWN02 was changed to bottling the final product (Run1) L/N #02H 01; P/N # FP-0008

[ chain ]

6121 TGGAATGGTC GCCCCACTCT ATGGCGTTGA AGGAACTAAA

G M V A P L Y G V E G T K

6161 ACACCAGTTT CCCCTGGTGA AATGAGACTG AGGGATGACC

T P V S P G E M R L R D D

6201 AGAGGAAAGT CTTCAGAGAA CTAGTGAGGA ATTGTGACCT

Q R K V F R E L V R N C D L

6241 GCCCGTTTGG CTTTCGTGGC AAGTGGCCAA GGCTGGTTTG

P V W L S W Q V A K A G L

6281 AAGACGAATG ATCGTAAGTG GTGTTTTGAA GGCCCTGAGG

K T N D R K W C F E G P E

6321 AACATGAGAT CTTGAATGAC AGCGGTGAAA CAGTGAAGTG

E H E I L N D S G E T V K C

6361 CAGGGCTCCT GGAGGAGCAA AGAAGCCTCT GCGCCCAAGG

R A P G G A K K P L R P R

6401 TGGTGTGATG AAAGGGTGTC ATCTGACCAG AGTGCGCTGT

W C D E R V S S D Q S A L

6441 CTGAATTTAT TAAGTTTGCT GAAGGTAGGA GGGGAGCTGC

S E F I K F A E G R R G A A

6481 TGAAGTGCTA GTTGTGCTGA GTGAACTCCC TGATTTCCTG

E V L V V L S E L P D F L

6521 GCTAAAAAAG GTGGAGAGGC AATGGATACC ATCAGTGTGT

A K K G G E A M D T I S V

6561 TCCTCCACTC TGAGGAAGGC TCTAGGGCTT ACCGCAATGC

F L H S E E G S R A Y R N A

6601 ACTATCAATG ATGCCTGAGG CAATGACAAT AGTCATGCTG

L S M M P E A M T I V M L

6641 TTTATACTGG CTGGACTACT GACATCGGGA ATGGTCATCT

F I L A G L L T S G M V I

6681 TTTTCATGTC TCCCAAAGGC ATCAGTAGAA TGTCTATGGC

F F M S P K G I S R M S M A

6721 GATGGGCACA ATGGCCGGCT GTGGATATCT CATGTTCCTT

M G T M A G C G Y L M F L

6761 GGAGGCGTCA AACCCACTCA CATCTCCTAT GTCATGCTCA

G G V K P T H I S Y V M L

ChimeriVaxWN02 was changed to bottling the final product (Run1) L/N #02H 01; P/N # FP-0008

[ chain ]

6801 TATTCTTTGT CCTGATGGTG GTTGTGATCC CCGAGCCAGG

I F F V L M V V V I P E P G

6841 GCAACAAAGG TCCATCCAAG ACAACCAAGT GGCATACCTC

Q Q R S I Q D N Q V A Y L

6881 ATTATTGGCA TCCTGACGCT GGTTTCAGCG GTGGCAGCCA

I I G I L T L V S A V A A

6921 ACGAGCTAGG CATGCTGGAG AAAACCAAAG AGGACCTCTT

N E L G M L E K T K E D L F

6961 TGGGAAGAAG AACTTAATTC CATCTAGTGC TTCACCCTGG

G K K N L I P S S A S P W

7001 AGTTGGCCGG ATCTTGACCT GAAGCCAGGA GCTGCCTGGA

S W P D L D L K P G A A W

7041 CAGTGTACGT TGGCATTGTT ACAATGCTCT CTCCAATGTT

T V Y V G I V T M L S P M L

7081 GCACCACTGG ATCAAAGTCG AATATGGCAA CCTGTCTCTG

H H W I K V E Y G N L S L

7121 TCTGGAATAG CCCAGTCAGC CTCAGTCCTT TCTTTCATGG

S G I A Q S A S V L S F M

7161 ACAAGGGGAT ACCATTCATG AAGATGAATA TCTCGGTCAT

D K G I P F M K M N I S V I

7201 AATGCTGCTG GTCAGTGGCT GGAATTCAAT AACAGTGATG

M L L V S G W N S I T V M

7241 CCTCTGCTCT GTGGCATAGG GTGCGCCATG CTCCACTGGT

P L L C G I G C A M L H W

7281 CTCTCATTTT ACCTGGAATC AAAGCGCAGC AGTCAAAGCT

S L I L P G I K A Q Q S K L

7321 TGCACAGAGA AGGGTGTTCC ATGGCGTTGC CAAGAACCCT

A Q R R V F H G V A K N P

7361 GTGGTTGATG GGAATCCAAC AGTTGACATT GAGAAGCTC

V V D G N P T V D I E E A

7401 CTGAAATGCC TGCCCTTTAT GAGAAGAAAC TGGCTCTATA

P E M P A L Y E K K L A L Y

7441 TCTCCTTCTT GCTCTCAGCC TAGCTTCTGT TGCCATGTGC

L L L A L S L A S V A M C

ChimerivaxWN02 was changed to bottling the final product (Run1) L/N #02H 01; P/N # FP-0008

[ chain ]

7481 AGAACGCCCT TTTCATTGGC TGAAGGCATT GTCCTAGCAT

R T P F S L A E G I V L A

7S21 CAGCTGCCTT AGGGCCGCTC ATAGAGGGAA ACACCAGCCT

S A A L G P L I E G N T S L

7561 TCTTTGGAAT GGACCCATGG CTGTCTCCAT GACAGGAGTC

L W N G P M A V S M T G V

7601 ATGAGGGGGA ATCACTATGC TTTTGTGGGA GTCATGTACA

M R G N H Y A F V G V M Y

7641 ATCTATGGAA GATGAAAACT GGACGCCGGG GGAGCGCGAA

N L W K M K T G R R G S A N

7681 TGGAAAAACT TTGGGTGAAG TCTGGAAGAG GGAACTGAAT

G K T L G E V W K R E L N

7721 CTGTTGGACA AGCGACAGT TGAGTTGTAT AAAAGGACCG

L L D K R Q F E L Y K R T

7761 ACATTGTGGA GGTGGATCGT GATACGGCAC GCAGGCATTT

D I V E V D R D T A R R H L

7801 GGCCGAAGGG AAGGTGGACA CCGGGGTGGC GGTCTCCAGG

A E G K V D T G V A V S R

7841 GGGACCGCAA AGTTAAGGTG GTTCCATGAG CGTGGCTATG

G T A K L R W F H E R G Y

7881 TCAAGCTGGA AGGTAGGGTG ATTGACCTGG GGTGTGGCCG

V K L E G R V I D L G C G R

7921 CGGAGGCTGG TGTTACTACG CTGCTGCGCA AAAGGAAGTG

G G W C Y Y A A A Q K E V

7961 AGTGGGGTCA AAGGATTTAC TCTTGGAAGA GACGGCCATG

S G V K G F T L G R D G H

8001 AGAAACCCAT GAATGTGCAA AGTCTGGGAT GGAACATCAT

E K P M N V Q S L G W N I I

8041 CACCTTCAAG GACAAAACTG ATATCCACCG CCTAGAACCA

T F K D K T D I H R L E P

8081 GTGAAATGTG ACACCCTTTT GTGTGACATT GGAGAGTCAT

V K C D T L L C D I G E S

8121 CATCGTCATC GGTCACAGAG GGGGAAAGGA CCGTGAGAGT

S S S S V T E G E R T V R V

ChimerivaxWN02 was changed to bottling the final product (Run1) L/N #02H 01; P/N # FP-0008

[ chain ]

8161 TCTTGATACT GTAGAAAAAT GGCTGGCTTG TGGGGTTGAC

L D T V E K W L A C G V D

8201 AACTTCTGTG TGAAGGTGTT AGCTCCATAC ATGCCAGATG

N F C V K V L A P Y M P D

8241 TTCTTGAGAA ACTGGAATTG CTCCAAAGGA GGTTTGGCGG

V L E K L E L L Q R R F G G

8281 AACAGTGATC AGGAACCCTC TCTCCAGGAA TTCCACTCAT

T V I R N P L S R N S T H

8321 GAAATGTACT ACGTGTCTGG AGCCCGCAGC AATGTCACAT

E M Y Y V S G A R S N V T

8361 TTACTGTGAA CCAAACATCC CGCCTCCTGA TGAGGAGAAT

F T V N Q T S R L L M R R M

8401 GAGGCGTCCA ACTGGAAAAG TGACCCTGGA GGCTGACGTC

R R P T G K V T L E A D V

8441 ATCCTCCCAA TTGGGACACG CAGTGTTGAG ACAGACAAGG

I L P I G T R S V E T D K

8481 GACCCCTGGA CAAAGAGGCC ATAGAAGAAA GGGTTGAGAG

G P L D K E A I E E R V E R

8521 GATAAAATCT GAGTACATGA CCTCTTGGTT TTATGACAAT

I K S E Y M T S W F Y D N

8561 GACAACCCCT ACAGGACCTG GCACTACTGT GGCTCCTATG

D N P Y R T W H Y C G S Y

8601 TCACAAAAAC CTCCGGAAGT GCGGCGAGCA TGGTAAATGG

V T K T S G S A A S M V N G

8641 TGTTATTAAA ATTCTGACAT ATCCATGGGA CAGGATAGAG

V I K I L T Y P W D R I E

8681 GAGGTCACAA GAATGGCAAT GACTGACACA ACCCCTTTTG

E V T R M A M T D T T P F

8721 GACAGCAAAG AGTGTTTAAA GAAAAAGTTG ACACCAGAGC

G Q Q R V F K E K V D T R A

8761 AAAGGATCCA CCAGCGGGAA CTAGGAAGAT CATGAAAGTT

K D P P A G T R K I M K V

8801 GTCAACAGGT GGCTGTTCCG CCACCTGGCC AGAGAAAAGA

V N R W L F R H L A R E K

ChimerivaxWN02 was changed to bottling the final product (Run1) L/N #02H 01; P/N # FP-0008

[ chain ]

8841 ACCCCAGACT GTGCACAAAG GAAGAATTTA TTGCAAAAGT

N P R L C T K E E F I A K V

8881 CCGAAGTCAT GCAGCCATTG GAGCTTACCT GGAAGAACAA

R S H A A I G A Y L E E Q

8921 GAACAGTGGA AGACTGCCAA TGAGGCTGTC CAAGACCCAA

E Q W K T A N E A V Q D P

8961 AGTTCTGGGA ACTGGGGAT GAAGAAAGGA AGCTGCACCA

K F W E L V D E E R K L H Q

9001 ACAAGGCAGG TGTCGGACTT GTGTGTACAA CATGATGGGG

Q G R C R T C V Y N M M G

9041 AAAAGAGAGA AGAAGCTGTC AGAGTTTGGG AAAGCAAAGG

K R E K K L S E F G K A K

9081 GAAGCCGTGC CATATGGTAT ATGTGGCTGG GAGCGCGGTA

G S R A I W Y M W L G A R Y

9121 TCTTGAGTTT GAGGCCCTGG GATTCCTGAA TGAGGACCAT

L E F E A L G F L N E D H

9161 TGGGCTTCCA GGGAAAACTC AGGAGGAGGA GTGGAAGGCA

W A S R E N S G G G V E G

9201 TTGGCTTACA ATACCTAGGA TATGTGATCA GAGACCTGGC

I G L Q Y L G Y V I R D L A

9241 TGCAATGGAT GGTGGTGGAT TCTACGCGGA TGACACCGCT

A M D G G G F Y A D D T A

9281 GGATGGGACA CGCGCATCAC AGAGGCAGAC CTTGATGATG

G W D T R I T E A D L D D

9321 AACAGGAGAT CTTGACTAC ATGAGCCCAC ATCACAAAAA

E Q E I L N Y M S P H H K K

9361 ACTGGCACAA GCAGTGATGG AAATGACATA CAAGAACAAA

L A Q A V M E M T Y K N K

9401 GTGGTGAAAG TGTTGAGACC AGCCCCAGGA GGGAAAGCCT

V V K V L R P A P G G K A

9441 ACATGGATGT CATAAGTCGA CGAGACCAGA GAGGATCCGG

Y M D V I S R R D Q R G S G

9481 GCAGGTAGTG ACTTATGCTC TGAACACCAT CACAACTTG

Q V V T Y A L N T I T N L

ChimerivaxWN02 was changed to bottling the final product (Run1) L/N #02H 01; P/N # FP-0008

[ chain ]

9521 AAAGTCCAAT TGATCAGAAT GGCAGAAGCA GAGATGGTGA

K V Q L I R M A E A E M V

9561 TACATCACCA ACATGTTCAA GATTGTGATG AATCAGTTCT

I H H Q H V Q D C D E S V L

9601 GACCAGGCTG GAGGCATGGC TCACTGAGCA CGGATGTGAC

T R L E A W L T E H G C D

9641 AGACTGAAGA GGATGGCGGT GAGTGGAGAC GACTGTGTGG

R L K R M A V S G D D C V

9681 TCCGGCCCAT CGATGACAGG TTCGGCCTGG CCCTGTCCCA

V R P I D D R F G L A L S H

9721 TCTCAACGCC ATGTCCAAGG TTAGAAGGA CATATCTGAA

L N A M S K V R K D I S E

9761 TGGCAGCCAT CAAAAGGGTG GAATGATTGG GAGAATGTGC

W Q P S K G W N D W E N V

9801 CCTTCTGTTC CCACCACTTC CATGAACTAC AGCTGAAGGA

P F C S H H F H E L Q L K D

9841 TGGCAGGAGG ATTGTGGTGC CTTGCCGAGA ACAGGACGAG

G R R I V V P C R E Q D E

9881 CTCATTGGGA GAGGAAGKGGT GTCTCCAGGA AACGGCTGGA

L I G R G R V S P G N G W

9921 TGATCAAGGA AACAGCTTGC CTCAGCAAAG CCTATGCCAA

M I K E T A C L S K A Y A N

9961 CATGTGGTCA CTGATGTATT TTCACAAAAG GGACATGAGG

M W S L M Y F H K R D M R

10001 CTACTGTCAT TGGCTGTTTC CTCAGCTGTT CCCACCTCAT

L L S L A V S S A V P T S

10041 GGGTTCCACA AGGACGCACA ACATGGTCGA TTCATGGGAA

W V P Q G R T T W S I H G K

10081A AGGGGAGTGG ATGACCACGG AAGACATGCT TGAGGTGTGG

G E W M T T E D M L E V W

10121A AACAGAGTAT GGATAACCAA CAACCCACAC ATGCAGGACA

N R V W I T N N P H M Q D

10161A AGACAATGGT GAAAAAATGG AGAGATGTCC CTTATCTAAC

K T M V K K W R D V P Y L T

ChimerivaxWN02 was changed to bottling the final product (Run1) L/N #02H 01; P/N # FP-0008

[ chain ]

10201 CAAGAGACAA GACAAGCTGT GCGGATCACT GATTGGAATG

K R Q D K L C G S L I G M

10241 ACCAATAGGG CCACCTGGGC CTCCCACATC CATTTAGTCA

T N R A T L A S H I H L V

10281 TCCATCGTAT CCGAACGCTG ATTG3ACAGG AGAAATACAC

I H R I R T L I G Q E K Y T

10321 TGACTACCTA ACAGTCATGG ACAGGTATTC TGTGGATGCT

D Y L T V M D R Y S V D A

10361 GACCTGCAAC TGGGTGAGCT TATCTGAAAC AC1TCTAAC

D L Q L G E L I

10401 AGGAATAACC GGGATACAAA CCACGGGTGG AGAACCGGAC

10441 TCCCCACAAC CTGAAACCGG GATATAAACC ACGGCTGGAG

10481 AACCGGACTC CGCACTTAAA ATGAAACAGA AACCGGGATA

20521 AAAACTACGG ATGGAGAACC GGACTCCACA CATTGAGACA

10561 GAAGAAGTTG TCAGCCCAGA ACCCCACACG AGTTTTGCCA

10601 CTGCTAAGCT GTGAGGCAGT GCAGGCTGGG ACAGCCGACC

10641 TCCAGGTTGC GAAAAACCTG GTTTCTGGGA CCTCCCACCC

20681 CAGAGTAAAA AGAACGGAGC CTCCGCTACC ACCCTCCCAC

10721 GTGGTGGTAG AAAGACGGGG TCTAGAGGTT AGAGGAGACC

10761 CTCCAGGGAA CAAATAGTGG GACCATATTG ACGCCAGGGA

10801 AAGACCGGAG TGGTTCTCTG CTTTTCCTCC AGAGGTCTGT

10841 GAGCACAGTT TGCTCAAGAA TAAGCAGACC TTTGGATGAC

10881 AAACACAAAA CCACAA

Chimerivax WN02M66 variant

[ chain ]

SEQ ID NOS: 22 and 23

1 NGTAAATCCT GTGTGCTAAT TGAGGTGCAT TGGTCTGCAA

41 ATCGAGTTGC TAGGCAATAA ACACATTTGG ATTAATTTTA

81 ATCGTTCGTT GAGCGATTAG CAGAGAACTG ACCAGAACAT

M

121 GTCTGGTCGT AAAGCTCAGG GAAAAACCCT GGGCGTCAAT

S G R K A Q G K T L G V N

161 ATGGTACGAC GAGGAGTTCG CTCCTTGTCA AACAAAATAA

M V R R G V R S L S N K I

201 AACAAAAAAC AAAACAAATT GGAAACAGAC CTGGACCTTC

K Q K T K Q I G N R P G P S

241 AAGAGGTGTT CAAGGATTTA TCTTTTTCTT TTTGTTCAAC

R G V Q G F I F F F L F N

281 ATTTTGACTG GAAAAAAGAT CACAGCCCAC CTAAAGAGGT

I L T G K K I T A H L K R

321 TGTGGAAAAT GCTGGACCCA AGACAAGGCT TGGCTGTTCT

L W K M L D P R Q G L A V L

361 AAGGAAAGTC AAGAGAGTGG TGGCCAGTTT GATGAGAGGA

R K V K R V V A S L M R G

401 TTGTCCTCAA GGAAACGCCG TTCCCATGAT GTTCTGACTG

L S S R K R R S H D V L T

441 TGCAATTCCT AATTTTGGGA ATGCTGTTGA TGACGGGTGG

V Q F L I L G M L L M T G G

481 AGTTACCCTC TCTAACTTCC AAGGGAAGGT GATGATGACG

V T L S N F Q G K V M M T

521 GTAAATGCTA CTGACGTCAC AGATGTCATC ACGATTCCAA

V N A T D V T D V I T I P

561 CAGCTGCTGG AAAGAACCTA TGCATTGTCA GAGCAATGGA

T A A G K N L C I V R A M D

601 TGTGGGATAC ATGTGCGATG ATACTATCAC TTATGAATGC

V G Y M C D D T I T Y E C

641 CCAGTGCTGT CGGCTGGTAA TGATCCAGAA GACATCGACT

P V L S A G N D P E D I D

Chimerivax WN02M66 variant

[ chain ]

681 GTTGGTGCAC AAAGTCAGCA GTCTACGTCA GGTATGGAAG

C W C T K S A V Y V R Y G R

721 ATGCACCAAG ACACCCACT CAAGACGCCAG TCGGAGGTCA

C T K T R H S R R S R R S

761 CTGACAGTGC AGACACACGG AGAAAGCACT CTAGCGAACA

L T V Q T H G E S T L A N

801 AGAAGGGGGC TTGGATGGAC AGCACCAAGG CCACAAGGTA

K K G A W M D S T K A T R Y

841 TTTGGTAAA ACAGAATCAT GGATCTTGAG GAACCCTGGA

L V K T E S W I L R N P G

881 TATGCCCTGG TGGCAGCCGT CATTGGTTGG ATGCTTGGGA

Y A L V A A V I G W M L G

921 GCAACACCAT GCAGAGAGTT GTGTTTGTCG TGCCATTGCT

S N T M Q R V V F V V P L L

961 TTTGGTGGCC CCAGCTTACA GCTTCAACTG CCTTGGAATG

L V A P A Y S F N C L G M

1001 AGCAACAGAG ACTTCTTGGA AGGAGTGTCT GGAGCAACAT

S N R D F L E G V S G A T

1041 GGGTGGATTT GGTTCTCGAA GGCGACAGCT GCGTGACTST

W V D L V L E G D S C V T I

1081 CATGTCTAAG GACAAGCCTA CCATCGACGT CAAGATGATG

M S K D K P T I D V K M M

1121 AATATGGAGG CGGCCAACCT GGCAGAGGTC CGCAGTTATT

N M E A A N L A E V R S Y

1161 GCTATTTGGC TACCGTCAGC GATCTCTCCA CCAAAGCTGC

C Y L A T V S D L S T K A A

1201 ATGCCCGACC ATGGGAGAAG CTCACAATGA CAAACGTGCT

C P T M G E A H N D K R A

1241 GACCCAGCTT TTGTGTGCAG ACAAGGAGTG GTGGACAGGG

D P A F V C R Q G V V D R

1281 GCTGGGGCAA CGGCTGCGGA TTTTTTGGCA AAGGATCCAT

G W G N G C G F F G K G S I

1321 TGACACATGC GCCAAATTTG CCTGCTCTAC CAAGGCAATA

D T C A K F A C S T K A I

Chimerivax WNO2M66 variant

[ chain ]

1361 GGAAGAACCA TCTTGAAAGA GAATATCAAG TACGAAGTGG

G R T I L K E N I K Y E V

1401 CCATTTTTGT CCATGGACCA ACTACTGTGG AGTCGCACGG

A I F V H G P T T V E S H G

1441 AAATTACTCC ACACAGGTTG GAGCCACTCA GGCCGGCCGA

N Y S T Q V G A T Q A G R

1481 TTCAGCATCA CTCCTGCTGC GCCTTCATAC ACACTAAAGC

F S I T P A A P S Y T L K

1521 TTGGAGAATA TGGAGAGGTG ACAGTGGACT GTGAACCACG

L G E Y G E V T V D C E P R

1561 GTCAGGGATT GACACCAATG CATACTACGT GATGACTGTT

S G I D T N A Y Y V M T V

1601 GGAACAAAGA CGTTCTTGGT CCATCGTGAG TGGTTCATGG

G T K T F L V H R E W F M

1641 ACCTCAACCT CCCTTGGAGC AGTGCTGGAA GTACTGTGTG

D L N L P W S S A G S T V W

1681 GAGGAACA GAGAGACGTTAA TGGAGTTTGA GGAACCACAC

R N R E T L M E F E E P H

1721 GCCACGAAGC AGTCTGTGAT AGCATTGGGC TCACAAGAGG

A T K Q S V I A L G S Q E

1761 GAGCTCTGCA TCAAGCTTTG GCTGGAGCCA TTCCTGTGGA

G A L H Q A L A G A I P V E

1801 ATTTTCAAGC AACACTGTCA AGTTGACGTC GGGTCATTTG

F S S N T V K L T S G H L

1841 AAGTGTAGAG TGAAGATGGA AAAATTGCAG TTGAAGGGAA

K C R V K I E K L Q L K G

1881 CAACCTATGG CGTCTGTTCA AAGGCTTTCA AGTTTCTTAG

T T Y G V C S K A F K F L R

1921 GACTCCCGTG GACACCGGTC ACGGCACTGT GGTGTTGGAA

T P V D T G H G T V V L E

1961 TTGCAGTACA CTGGCACGGA TGGACCTTGC AAAGTTCCTA

L Q Y T G T D G P C K V P

2001 TCTCGTCAGT GGCTTCATTG AACGACCTAA CGCCAGTGGG

I S S V A S L N D L T P V G

Chimerivax WNO2M66 variant

[ chain ]

2041 CAGATTGGTC ACTGTCAACC CTTTGTTTC AGTGGCCACG

R L V T V N P F V S V A T

2081 GCCAACGCTA AGGTCCTGAT TGAATTGGAA CCACCCTTTG

A N A K V L I E L E P P F

2121 GAGACTCATA CATAGTGGTG GGCAGAGGAG AACAACAGAT

G D S Y I V V G R G E Q Q I

2161 CAATCACCAT TGGCACAAGT CTGGAAGCAG CATTGGCAAA

N H H W H K S G S S I G K

2201 GCCTTTACAA CCACCCTCAA AGGAGCGCAG AGACTAGCCG

A F T T T L K G A Q R L A

2241 CTCTAGGAGA CACAGCTTGG GACTTTGGAT CAGTTGGAGG

A L G D T A W D F G S V G G

2281 GGTGTTCACT AGTGTTGGGC GGGCTGTCCA TCAAGTGTTC

V F T S V G R A V H Q V F

2321 GGAGGAGCAT TCCGCTCACT GTTCGGAGGC ATGTCCTGGA

G G A F R S L F G G M S W

2361 TAACGCAAGG ATTGCTGGGG GCTCTCCTGT TGTGGATGGG

I T Q G L L G A L L L W M G

2401 CATCAATGCT CGTGATAGGT CCATAGCTCT CACCTTTCTC

I N A R D R S I A L T F L

2441 GCAGTTGGAG GAGTTCTGCT CITCCICTCC GTGAACGTGG

A V G G V L L F L S V N V

2481 GCGCCGATCA AGGATGCGCC ATCAACTTTG GCAAGAGAGA

G A D Q G C A I N F G K R E

2521 GCTCAAGTGC GGAGATGGTA TCTTCATATT TAGAGACTCI

L K C G D G I F I F R D S

2562 GATGACTGGC TGAACAAGIA CICATACTAT CCAGAAGAIC

D D W L N K Y S Y Y P E D

2601 CTGTGAAGCT TACATCAATA GIGAAAGCCT CTTTTGAAGA

P V K L A S I V K A S F E E

2641 AGGGAAGTGT GGCCTAAATT CAGTTGACTC CCTTGAGCAT

C K C G L N S V D S L E H

2681 GAGATGTGGA GAAGCAGGGC AGATGAGATC AATGCCATTT

E M W R S R A D E I N A I

ChimerivaxWN02M66 variant

[ chain ]

2721 TTGAGGAAAA CGAGGTGGAC ATTTCTGTTG TCGTGCAGGA

F E E N E V D I S V V V Q D

2761 TCCAAAGAAT GTTTACCAGA GAGGAACTCA TCCATTTTCC

P K N V Y Q R G T H P F S

2801 AGAATTCGGG ATGGTCTGCA GTATGGTTGG AAGACTTGGG

R I R D G L Q Y G W K T W

2841 GTAAGAACCT TGTGTTCTCC CCAGGGAGGA AGAATGGAAG

G K N L V F S P G R K N G S

2881 CTTCATCATA GATGGAAAGT CCAGGAAAGA ATGCCCGTTT

F I I D G K S R K E C P F

2921 TCAAACCGGG TCTGGAATTC TTTCCAGATA GAGGAGTTTG

S N R V W N S F Q I E E F

2961 GGACGGGAGT GTTCACCACA CGCGTGTACA TGGACGCAGT

G T G V F T T R V Y M D A V

3001 CTTTGAATAC ACCATAGACT GCGATGGATC TATCTTGGGT

F E Y T I D C D G S I L G

3041 GCAGCGGTGA ACGGAAAAAA GAGTGCCCAT GGCTCTCCAA

A A V N G K K S A H G S P

3081 CATTTTGGAT GGGAAGTCAT GAAGTAAATG GGACATGGAT

T F W M G S H E V N G T W M

3121 GATCCACACC TTGGAGGCAT TAGATTACAA GGAGTGTGAG

I H T L E A L D Y K E C E

3161 TGGCCACTGA CACATACGAT TGGAACATCA GTTGAAGAGA

W P L T H T I G T S V E E

3201 GTGAAATGTT CATGCCGAGA TCAATCGGAG GCCCAGTTAG

S E M F M P R S I G G P V S

3241 CTCTCACAAT CATATCCCTG GATACAAGGT TCAGACGAAC

S H N H I P G Y K V Q T N

3281 GGACCTTGGA TGCAGGTACC ACTAGAAGTG AAGAGAGAAG

G P W M Q V P L E V K R E

3321 CTTGCCCAGG GACTAGCGTG ATCATTGATG GCAACTGTGA

A C P G T S V I I D G N C D

3361 TGGACGGGGA AAATCAACCA GATCCACCAC GGATAGCGGG

G R G K S T R S T T D S G

Chimerivax WN02M66 variant

[ chain ]

3401 AAAGTTATTC CTGAATGGTG TTGCCGCTCC TGCACAATGC

K V I P E W C C R S C T M

3441 CGCCTGTGAG CTTCCATGGT AGTGATGGGT GTTGGTATCC

P P V S F H G S D G C W Y P

3481 CATGGAAATT AGGCCAAGGA AAACGCATGA AAGCCATCTG

M E I R P R K T H E S H L

3521 GTGCGCTCCT GGGTTACAGC TGGAGAAATA CATGCTGTCC

V R S W V T A G E I H A V

3561 CTTTTGGTTT GGTGAGCATG ATGATAGCAA TGGAAGTGGT

P P G L V S M M I A M E V V

3601 CCTAAGGAAA AGACAGGGAC CAAAGCAAAT GTTGGTTGGA

L R K R Q G P K Q M L V G

3641 GGAGTAGTGC TCTTGGGAGC AATGCTGGTC GGGCAAGTAA

G V V L L G A M L V G Q V

3681 CTCTCCTTGA TTTGCTGAAA CTCACAGTGG CTGTGGGATT

T L L D L L K L T V A V G L

3721 GCATTTCCAT GAGATGAACA ATGGAGGAGA CGCCATGTAT

H F H E M N N G G D A M Y

3761 ATGGCGTTGA TTGCTGCCTT TTCAATCAGA CCAGGGCTGC

M A L I A A F S I R P G L

3802 TCATCGGCTT TGGGCTCAGG ACCCTATGGA GCCCTCGGGA

L I G F G L R T L W S P R E

3841 ACGCCTTGTG CTGACCCTAG GAGCAGCCAT GGTGGAGATT

R L V L T L G A A M V E I

3881 GCCTTGGGTG GCGTGATGGG CGGCCTGTGG AAGTATCTAA

A L G G V M G G L W K Y L

3921 ATGCAGTTTC TCTCTGCATC CTGACAATAA ATGCTGTTGC

N A V S L C I L T I N A V A

3961 TTCTAGGAAA GCATCAAATA CCATCTTGCC CCTCATGGCT

S R K A S N T I L P L M A

4001 CTGTTGACAC CTGTCACTAT GGCTGAGGTG AGACTTGCCG

L L T P V T M A E V R L A

4041 CAATGTTCTT TTGTGCCATG GTTATCATAG GGGTCCTTCA

A M F F C A M V I I G V L H

Chimerivax WN02M66 variant

[ chain ]

4081 CCAGAATTTC AAGGACACCT CCATGCAGAA GACTATACCT

Q N F K D T S M Q K T I P

4121 CTGGTGGCCC TCACACTCAC ATCTTACCTG GGCTTGACAC

L V A L T L T S Y L G L T

4161 AACCTTTTTT GGGCCTGTGT GCATTTCTGG CAACCCGCAT

Q P F L G L C A F L A T R I

4201 ATTTGGGCGA AGGAGTATCC CAGTGAATGA GGCACTCGCA

F G R R S I P V N E A L A

4241 GCAGCTGGTC TAGTGGGAGT GCTGGCAGGA CTGGCTTTTC

A A G L V G V L A G L A F

4281 AGGAGATGGA GAACTTCCTT GGTCCGATTG CAGTTGGAGG

Q E M E N F L G P I A V G G

4321 ACTCCTGATG ATGCTGGTTA GCGTGGCTGG GAGGGTGGAT

L L M M L V S V A G R V D

4361 GGGCTAGAGC TCAAGAAGCT TGGTGAAGTT TCATGGGAAG

G L E L K K L G E V S W E

4401 AGGAGGCGGA GATCAGCGGG AGTTCCGCCC GCTATGATGT

E E A E I S G S S A R Y D V

4441 GGCACTCAGT GAACAAGGGG AGTTCAAGCT GCTTTCTGAA

A L S E Q G E F K L L S E

4481 GAGAAAGTGC CATCGGACCA GGTTGTGATG ACCTCGCTGG

E K V P V D Q V V M T S L

4521 CCTTGGTTGG GGCTGCCCTC CATCCATTTG CTCTTCTGCT

A L V G A A L H P F A L L L

4561 GGTCCTTGCT GGGTGGCTGT TTCATGTCAG GGGAGCTAGG

V L A G W L F H V R G A R

4601 AGAAGTGGGG ATGTCTTGTG GGATATTCCC ACTCCTAAGA

R S G D V L W D I P T P K

4641 TCATCGAGGA ATGTGAACAT CTGGAGGATG GGATTTATGG

I I E E C E H L E D G I Y G

4681 CATATTCCAG TCAACCTTCT TGGGGGCCTC CCAGCGAGGA

I F Q S T F L G A S Q R G

4721 GTGGGAGTGG CACAGGGAGG GGTGTTCCAC ACAATGTGGC

V G V A Q G G V F H T M W

Chimerivax WN02M66 variant

[ chain ]

4761 ATGTCACAAG AGGAGCTTTC CTTGTCAGGA ATGGCAAGAA

H V T R G A F L V R N G K K

4801 GTTGATTCCA TCTTGGGCTT CAGTAAAGGA AGACCTTGTC

L I P S W A S V K E D L V

4841 GCCTATGGTG GCTCATGGAA GTTGGAAGGC AGATGGATG

A Y G G S W K L E G R W D

4881 GAGAGGAAGA GGTCCAGTTG ATCGCGGCTG TTCCAGGAAA

G E E E V Q L I A A V P G K

4922 GAACGTGGTC AACGTCCAGA CAAAACCGAG CTTGTTCAAA

N V V N V Q T K P S L F K

4961 GTGAGGAATG GGGGAGAAAT CGGGGCTGTC GCTCTTGACT

V R N G G E I G A V A L D

5001 ATCCGAGTGG CACTTCAGGA TCTCCTATTG TTAACAGGAA

Y P S G T S G S P I V N R N

5041 CGGAGAGGTG ATTGGGCTGT ACGGCAATGG CATCCTTGTC

G E V I G L Y G N G I L V

5081 GGTGACAACT CCTTCGTGTC CGCCATATCC CAGACTGAGG

G D N S F V S A I S Q T E

5121 TGAAGGAAGA AGGAAAGGAG GAGCTCCAAG AGATCCCGAC

V K E E G K E E L Q E I P T

5161 AATGCTAAAG AAAGGAATGA CAACTGTCCT TGATTTTCAT

M L K K G M T T V L D F H

5201 CCTGGAGCTG GGAAGACAAG ACGTTTCCTC CCACAGATCT

P G A G K T R R F L P Q I

5241 TGGCCGAGTG CGCACGGAGA CGCTTGCGCA CTCTTGTGTT

L A E C A R R R L R T L V L

5281 GGCCCCCACC AGGGTTGTTC TTTCTGAAAT GAAGGAGGCT

A P T R V V L S E M K E A

5321 TTTCACGGCC TGGACGTGAA ATTCCACACA CAGGCTTTTT

F H G L D V K F H T Q A F

5361 CCGCTCACGG CAGCGGGAGA GAAGTCATTG ATGCCATGTG

S A H G S G R E V I D A M C

5401 CCATGCCACC CTAACTTACA GGATGTTGGA ACCAACTAGG

H A T L T Y R M L E P T R

Chimerivax WN02M66 variant

[ chain ]

5441 GTTGTTAACT GGGAAGTGAT CATTATGGAT GAAGCCCATT

V V N W E V I I M D E A H

5481 TTTTGGATCC AGCCAGCATA GCCGCTAGAG GTTGGGCAGC

F L D P A S I A A R G W A A

5521 GCACAGAGCT AGGGCAAATG AAAGTGCAAC AATCTTGATG

H R A R A N E S A T I L M

5561 ACAGCCACAC CGCCTGGGAC TAGTGATGAA TTTCCACATT

T A T P P G T S D E F P H

5601 CAAATGGTGA AATAGAAGAT GTTCAAACGG ACATACCCAG

S N G E I E D V Q T D I P S

5641 TGAGCCCTGG AACACAGGGC ATGACTGGAT CCTGGCTGAC

E P W N T G H D W I L A D

5681 AAAAGGCCCA CGGCATGGTT CCTTCCATCC ATCAGAGCTG

K R P T A W F L P S I R A

5721 CAAATGTCAT GGCTGCCTCT TTGCGTAAGG CTGGAAAGAG

A N V M A A S L R K A G K S

5761 TGTGGTGGTC CTGAACAGGA AAACCTTTGA GAGAGAATAC

V V V L N R K T F E R E Y

5801 CCCACGATAA AGCAGAAGAA ACCTGACTTT ATATTGGCCA

P T I K Q K K P D F I L A

5841 CTGACATAGC TGAAATGGGA GCCAACCTTT GCGTGGAGCG

T D I A E M G A N L C V E R

5881 AGTGCTGGAT TGCAGGACGG CTTTTAAGCC TGTGCTTGTG

V L D C R T A F K P V L V

5921 GATGAAGGGA GGAAGGTGGC AATAAAAGGG CCACTTCGTA

D E G R K V A I K G P L R

5961 TCTCCGCATC CTCTGCTGCT CAAAGGAGGG GGCGCATTGG

I S A S S A A Q R R G R I G

6001 GAGAAATCCC AACAGAGATG GAGACTCATA CTACTATTCT

R N P N R D G D S Y Y Y S

6041 GAGCCTACAA GTGAAAATAA TGCCCACCAC GTCTGCTGGT

E P T S E N N A H H V C W

6081 TGGAGGCCTC AATGCTCTTG GACAACATGG AGGTGAGGGG

L E A S M L L D N M E V R G

Chimerivax WN02M66 variant

[ chain ]

6121 TGGAATGGTC GCCCCACTCT ATGGCGTTGA AGGAACTAAA

G M V A P L Y G V E G T K

6161 ACACCAGTTT CCCCTGGTGA AATGAGACTG AGGGATGACC

T P V S P G E M R L R D D

6201 AGAGGAAAGT CTTCAGAGAA CTAGTGAGGA ATTGTGACCT

Q R K V F R E L V R N C D L

6241 GCCCGTTTGG CTTTCGTGGC AAGTGGCCAA GGCTGGTTTG

P V W L S W Q V A K A G L

6281 AAGACGAATG ATCGTAAGTG GTGTTTTGAA GGCCCTGAGG

K T N D R K W C F E G P E

6321 AACATGAGAT CTTGAATGAC AGCGGTGAAA CAGTGAAGTG

E H E I L N D S G E T V K C

6361 CAGGGCTCCT GGAGGAGCAA AGAAGCCTCT GCGCCCAAGG

R A P G G A K K P L R P R

6401 TGGTGTGATG AAAGGGTGTC ATCTGACCAG AGTGCGCTGT

W C D E R V S S D Q S A L

6441 CTGAATTTAT TAAGTTTGCT GAAGGTAGGA GGGGAGCTGC

S E F I K F A E G R R G A A

6481 TGAAGTGCTA GTTGTGCTGA GTGAACTCCC TGATTTCCTG

E V L V V L S E L P D F L

6521 GCTAAAAAAG GTGGAGAGGC AATGGATACC ATCAGTGTGT

A K K G G E A M D T I S V

6561 TCCTCCACTC TGAGGAAGGC TCTAGGGCTT ACCGCAATGC

F L H S E E G S R A Y R N A

6601 ACTATCAATG ATGCCTGAGG CAATGACAAT AGTCATGCTG

L S M M P E A M T I V M L

6641 TTTATACTGG CTGGACTACT GACATCGGGA ATGGTCATCT

F I L A G L L T S G M V I

6681 TTTTCATGTC TCCCAAAGGC ATCAGTAGAA TGTCTATGGC

F F M S P K G I S R M S M A

6721 GATGGGCACA ATGGCCGGCT GTGGATATCT CATGTTCCTT

M G T M A G C G Y L M F L

6761 GGAGGCGTCA AACCCACTCA CATCTCCTAT GTCATGCTCA

G G V K P T H I S Y V M L

Chimerivax WN02M66 variant

[ chain ]

6801 TATTCTTTGT CCTGATGGTG GTTGTGATCC CCGAGCCAGG

I F F V L M V V V I P E P G

6841 GCAACAAAGG TCCATCCAAG ACAACCAAGT GGCATACCTC

Q Q R S I Q D N Q V A Y L

6881 ATTATTGGCA TCCTGACGCT GGTTTCAGCG GTGGCAGCCA

I I G I L T L V S A V A A

6921 ACGAGCTAGG CATGCTGGAG AAAACCAAAG AGGACCTCTT

N E L G M L E K T K E D L F

6961 TGGGAAGAAG AACTTAATTC CATCTAGTGC TTCACCCTGG

G K K N L I P S S A S P W

7001 AGTTGGCCGG ATCTTGACCT GAAGCCAGGA GCTGCCTGGA

S W P D L D L K P G A A W

7041 CAGTGTACGT TGGCATTGTT ACAATGCTCT CTCCAATGTT

T V Y V G I V T M L S P M L

7081 GCACCACTGG ATCAAAGTCG AATATGGCAA CCTGTCTCTG

H H W I K V E Y G N L S L

7121 TCTGGAATAG CCCAGTCAGC CTCAGTCCTT TCTTTCATGG

S G I A Q S A S V L S F M

7161 ACAAGGGGAT ACCATTCATG AAGATGAATA TCTCGGTCAT

D K G I P F M K M N I S V I

7201 AATGCTGCTG GTCAGTGGCT GGAATTCAAT AACAGTGATG

M L L V S G W N S I T V M

7241 CCTCTGCTCT GTGGCATAGG GTGCGCCATG CTCCACTGGT

P L L C G I G C A M L H W

7281 CTCTCATTTT ACCTGGAATC AAAGCGCAGC AGTCAAAGCT

S L I L P G I K A Q Q S K L

7321 TGCACAGAGA AGGGTGTTCC ATGGCGTTGC CAAGAACCCT

A Q R R V F H G V A K N P

7361 GTGGTTGATG GGAATCCAAC AGTTGACATT GAGGAAGCTC

V V D G N P T V D I E E A

7401 CTGAAATGCC TGCCCTTTAT GAGAAGAAAC TGGCTCTATA

P E M P A L Y E K K L A L Y

7441 TCTCCTTCTT GCTCTCAGCC TAGCTTCTGT TGCCATGTGC

L L L A L S L A S V A M C

Chimerivax WN02M66 variant

[ chain ]

7481 AGAACGCCCT TTTCATTGGC TGAAGGCATT GTCCTAGCAT

R T P F S L A E G I V L A

7521 CAGCTGCCTT AGGGCCGCTC ATAGAGGGAA ACACCAGCCT

S A A L G P L I E G N T S L

7561 TCTTTGGAAT GGACCCATGG CTGTCTCCAT GACAGGAGTC

L W N G P M A V S M T G V

7601 ATGAGGGGGA ATCACTATGC TTTTGTGGGA GTCATGTACA

M R G N H Y A F V G V M Y

7641 ATCTATGGAA GATGAAAACT GGACGCCGGG GGAGCGCGAA

N L W K M K T G R R G S A N

7681 TGGAAAAACT TTGGGTGAAG TCTGGAAGAG GGAACTGAAT

G K T L G E V W K R E L N

7721 CTGTTGGACA AGCCACAGTT TGAGTTGTAT AAAAGGACCG

L L D K R Q F E L Y K R T

7761 ACATTGTGGA GGTGGATCGT GATACGGCAC GCAGGCATTT

D I V E V D R D T A R R H L

7801 GGCCGAACGG AACGTGGACA CCGGGGTGGC GGTCTCCAGG

A E G K V D T G V A V S R

7841 GGGACCGCAA AGTTAAGGTG GTTCCATGAG CGTGGCTATG

G T A K L R W F H E R G Y

7881 TCAAGCTGGA AGGTAGGGTG ATTGACCTGG GGTGTGGCCG

V K L E G R V I D L G C G R

7921 CGGAGGCTGG TGTTACTACG CTGCTGCGCA AAAGGAAGTG

G G W C Y Y A A A Q K E V

7961 AGTGGGGTCA AAGGATTTAC TCTTGGAAGA GACGGCCATG

S G V K G F T L G R D G H

8001 AGAAACCCAT GAATGTGCAA AGICIGGGAT GGAACATCAT

E K P M N V Q S L G W N I I

8041 CACCTTCAAG GACAAAACTG ATATCCACCG CCTAGAACCA

T F K D K T D I H R L E P

8081 GTGAAATGTG ACACCCTTTT GTGTGACATT GGAGAGTCAT

V K C D T L L C D I G E S

8121 CATCGTCATC GGTCACAGAG GGGGAAAGGA CCGTGAGAGT

S S S S V T E G E R T V R V

Chimerivax WN02M66 variant

[ chain ]

8161 TCTTGATACT GTAGAAAAAT GGCTGGCTTG TGGGGTTGAC

L D T V E K W L A C G V D

8201 AACTTCTGTG TGAAGGTGTT AGCTCCATAC ATGCCAGATG

N F C V K V L A P Y M P D

8241 TTCTTGAGAA ACTGGAATTG CTCCAAAGGA GGTTTGGCGG

V L E K L E L L Q R R F G G

8281 AACAGTGATC AGGAACCCTC TCTCCAGGAA TTCCACTCAT

T V I R N P L S R N S T H

8321 GAAATGTACT ACGTGTCTGG AGCCCGCAGC AATGTCACAT

E M Y Y V S G A R S N V T

8361 TTACTGTGAA CCAAACATCC CGCCTCCTGA TGAGGAGAAT

F T V N Q T S R L L M R R M

8401 GAGGCGTCCA ACTGGAAAAG TGACCCTGGA GGCTGACGTC

R R P T G K V T L E A D V

8441 ATCCTCCCAA TTGGGACACG CAGTGTTGAG ACAGACAAGG

I L P I G T R S V E T D K

8481 GACCCCTGGA CAAAGAGGCC ATAGAAGAAA GGGTTGAGAG

G P L D K E A I E E R V E R

8521 GATAAAATCT GAGTACATGA CCTCTTGGTT TTATGACAAT

I K S E Y M T S W F Y D N

8561 GACAACCCCT ACAGGACCTG GCACTACTGT GGCTCCTATG

D N P Y R T W H Y C G S Y

8601 TCACAAAAAC CTCCGGAAGT GCGGCGAGCA TGGTAAATGG

V T K T S G S A A S M V N G

8641 TGTTATTAAA ATTCTGACAT ATCCATGGGA CAGGATAGAG

V I K I L T Y P W D R I E

8681 GAGGTCACAA GAATGGCAAT GACTGACACA ACCCCTTTTG

E V T R M A M T D T T P F

8721 GACAGCAAAG AGTGTTTAAA GAAAAAGTTG ACACCAGAGC

G Q Q R V F K E K V D T R A

8761 AAAGGATCCA CCAGCGGGAA CTAGGAAGAT CATGAAAGTT

K D P P A G T R K I M K V

8801 GTCAACAGGT GGCTGTTCCG CCACCTGGCC AGAGAAAAGA

V N R W L F R H L A R E K

Chimerivax WN02M66 variant

[ chain ]

8841 ACCCCAGACT GTGCACAAAG GAAGAATTTA TTGCAAAAGT

N P R L C T K E E F I A K V

8881 CCGAAGTCAT GCAGCCATTG GAGCTTACCT GGAAGAACAA

R S H A A I G A Y L E E Q

8921 GAACAGTGGA AGACTGCCAA TGAGGCTGTC CAAGACCCAA

E Q W K T A N E A V Q D P

8961 AGTTCTGGGA ACTGGTGGAT GAAGAAAGGA AGCTGCACCA

K F W E L V D E E R K L H Q

9001 ACAAGGCAGG TGTCGGACTT GTGTGTACAA CATGATGGGG

Q G R C R T C V Y N M M G

9041 AAAAGAGAGA AGAAGCTGIC AGAGTTTGGG AAAGCAAAGG

K R E K K L S E F G K A K

9081 GAAGCCGTGC CATATGGTAT ATGTGGCTGG GAGCGCGGTA

G S R A I W Y M W L G A R Y

9121 TCTTGAGTTT GAGGCCCTGG GATTCCTGAA TGAGGACCAT

L E F E A L G F L N E D H

9161 TGGGCTTCCA GGGAAAACTC AGGAGGAGGA GTGGAAGGCA

W A S R E N S G G G V E G

9201 TTGGCTTACA ATACCTAGGA TATGTGATCA GAGACCTGGC

I G L Q Y L G Y V I R D L A

9241 TGCAATGGAT GGTGGTGGAT TCTACGCGGA TGACACCGCT

A M D G G G F Y A D D T A

9281 GGATGGGACA CGCGCATCAC AGAGGCAGAC CTTGATGATG

G W D T R I T E A D L D D

9321 AACAGGAGAT CTTGAACTAC ATGAGCCCAC ATCACAAAAA

E Q E I L N Y M S P H H K K

9362 ACTGGCACAA GCAGTGATGG AAATGACATA CAAGAACAAA

L A Q A V M E M T Y K N K

9401 GTGGTGAAAG TGTTGAGACC AGCCCCAGGA GGGAAAGCCT

V V K V L R P A P G G K A

9441 ACATGGATGT CATAAGTCGA CGAGACCAGA GAGGATCCGG

Y M D V I S R R D Q R G S G

9481 GCAGGTAGTG ACTTATGCTC TGAACACCAT CACCAACTTG

Q V V T Y A L N T I T N L

Chimerivax WN02M66 variant

[ chain ]

9521 AAAGTCCAAT TGATCAGAAT GGCAGAAGCA GAGATGGTGA

K V Q L I R M A E A E M V

9561 TACATCACCA ACATGTTCAA GATTGTGATG AATCAGTTCT

I H H Q H V Q D C D E S V L

9601 GACCAGGCTG GAGGCATGGC TCACTGAGCA CGGATGTGAC

T R L E A W L T E H G C D

9641 AGACTGAAGA GGATGGCCGT GAGTGGAGAC GACTGTGTGG

R L K R M A V S G D D C V

9681 TCCGGCCCAT CGATGACAGG TTCGGCCTGG CCCTGTCCCA

V R P I D D R F G L A L S H

9721 TCTCAACGCC ATGTCCAAGG TTAGAAAGGA CATATCTGAA

L N A M S K V R K D I S E

9761 TGGCAGCCAT CAAAAGGGTG GAATGATTGG GAGAATGTGC

W Q P S K G W N D W E N V

9801 CCTTCTGTTC CCACCACTTC CATGAACTAC AGCTGAAGGA

P F C S H H F H E L Q L K D

9841 TGGCAGGAGG ATTGTGGTGC CTTGCCGAGA ACAGGACGAG

G R R I V V P C R E Q D E

9881 CTCATTGGGA GAGGAAGGGT GTCTCCAGGA AACGGCTGGA

L I G R G R V S P G N G W

9921 TGATCAAGGA AACAGCTTGC CTCAGCAAAG CCTATGCCAA

M I K E T A C L S K A Y A N

9961 CATGTGGTCA CTGATGTATT TTCACAAAAG GGAGATGAGG

M W S L M Y F H K R D M R

10001 CTACTGTCAT TGGCTGTTTC CTCAGCTGTT CCCACCTCAT

L L S L A V S S A V P T S

10041 GGGTTCCACA AGGACGCACA ACATGGTCGA TTCATGGGAA

W V P Q G R T T W S I H G K

10081 AGGGCAGTCG ATGACCACGG AAGACATGCT TGAGGTGTGG

G E W M T T E D M L E V W

10121 AACAGAGTAT GGATAACCAA CAACCCACAC ATGCAGGACA

N R V W I T N N P H M Q D

10161 AGACAATGGT GAAAAAATGG AGAGATGTCC CTTATCTAAC

K T M V K K W R D V P Y L T

Chimerivax WN02M66 variant

[ chain ]

10201 CAAGAGACAA GACAAGCTGT GCGGATCACT GATTGGAATG

K R Q D K L C G S L I G M

10241 ACCAATAGGG CCACCTGGGC CTCCCACATC CATTTAGTCA

T N R A T W A S H I H L V

10281 TCCATCGTAT CCGAACGCTG ATTGGACAGG AGAAATACAC

I H R I R T L I G Q E K Y T

10321 TGACTACCTA ACAGTCATGG ACAGGTATTC TGTGGATGCT

D Y L T V M D R Y S V D A

10361 GACCTGCAAC TGGGTGAGCT TATCTGAAAC ACCATCTAAC

D L Q L G E L I

10401 AGGAATAACC GGGATACAAA CCACGGGTGG AGAACCGGAC

10441 TCCCCACAAC CTGAAACCGG GATATAAACC ACGGCTGGAG

10481 AACCGGACTC CGCACTTAAA ATAAAACAGA AACCGGGATA

10521 AAAACTACGG ATGGAGAACC GGACTCCACA CATTGAGACA

10561 GAAGAAGTTG TCAGCCCAGA ACCCCACACG AGTTTTGCCA

10601 CTGCTAAGCT GTGAGGCAGT GCAGGCTGGG ACAGCCGACC

10641 TCCAGGTTGC GAAAAACCTG GTTTCTGGGA CCTCCCACCC

10681 CAGAGTAAAA AGAACGGAGC CTCCGCTACC ACCCTCCCAC

10721 GTGGTGGTAG AAAGACGGGG TCTAGAGGTT AGAGGAGACC

10761 CTCCAGGGAA CAAATAGTGG GACCATATTG ACGCCAGGGA

10801 AAGACCGGAG TGGTTCTCTG CTTTTCCTCC AGAGGTCTGT

10841 GAGCACAGTT TGCTCAAGAA TAAGCAGACC TTTGGATGAC

10881 AAACACAAAA CCACAA

###DNA Strider^TM13f7 ###

WN02x M66 variantDNA alignment

DNA sequence 10896 bp^＊G taaatcctgt.. ACAAAACCACAA linear SEQ ID NOS: 36 and 37

DNA sequence 10896 bp^＊G taaatcctgt.. ACAAAACCACAA linear SEQ ID NOS: 36 and 37

Layout： Compacted

Metbod： Blooks(Martimez)

Mismatch penalty： Smaller(1)

Gap penalty： Mediom(2)

Transformation: off SEQ ID NOS: 24 and 38

1^＊*G TAAATCCTGTGTGCTAATTGAGGTGCATTGGTCTGCAAATCGAGTTGCTAGGCAATAAACACATTTGGATTAATTTTA 80

1 ................................................................................ 80

81 ATCGTTCGTTGAGCGATTAGCAGAGAACTGACCAGACATGTCTGGTCGTAAAGCTCAGGGAAAAACCCTGGGCGTCAAT 160

81 ................................................................................ 160

161 ATCGTACGACGAGGAGTTCGCTCCTTGTCAAACAAAATAAAACAAAAAACAAAACAAATTGGAAACAGACCTGGACCTTC 240

161 ................................................................................ 240

241 AAGAGGTTGTCAAGGAATTTATCTTTTTCTTTTTGTTCAACATTTTGACTGGAAAAAAGATCACAGCCCACCTAAAGAGGT 320

241 ................................................................................ 320

321 TGTGGAAAATGCTGGGACCCAAGACAAGGCTTGGCTGTTCTAAGGAAAGTCAAGAGAGTGGTGGCCAGTTTGATGAGAGGA 400

321 ................................................................................ 400

401 TTGTCCTCAAGGAAACGCCGTTCCCATGATGTTCTGACTGTGCAATTCCTAATTTTGGGAATGCTGTTGATGACGGTGG 480

401 ................................................................................ 480

481 AGTTACCCTCTCTAACTTCCAAGGGAAGGTGATGATGACGGTAAATGCTACTGAGTCACAGATGTCATCACGATTCCAA 560

481 ................................................................................ 560

561 CAGCTGCTGGAAAGAACCTATGCATTGTCAGAGCAATGGATGTGGGATACATGTGCGATGATACTATCACTTATGAATGC 640

561 ................................................................................ 640

641 CCAGTGCTGTCGGCTGGTAATGATCCAGAAGACATCGACTGTTGGTGCACAAAGTCAGCAGTCTACGTCAGGTATGGAAG 720

641 ................................................................................ 720

721 ATGCACCAAGACACGCCACTCAGACGCAGTCGGAGGTCACTGACAGTGCAGACACACGGAGAAAGCACTCTAGCGAACA 800

721 ................................................................................ 800

801 AGAAGGGGGCTTGGATGGACAGCACCAAGGCCACAAGGTATTTGGTAAAAACAGAATCATGGATCTTGAGGAACCCTGGA 880

801 ................................................................................ 880

881 TATGCCCTGGTGGCAGCCGTCATTGGTTGGATGCTTGGGAGCAACACCATGCAGAGAGTTGTHTTTGTCGTGCTATTGCT 960

881 .........................................................................C...... 960

961 TTTGGTGGCCCCAGCTTACAGCTTCAACTGCCTTGGAATGAGCAACAGAGACTTCTTGGAAGGAGTGTCGGAGCAACAT 1040

961 ................................................................................ 1040

1041 GGGTGGATTTGGTTCTCGAAGGCGACAGCTGCGTGACTATCATGTCTAAGGACAAGCCTACCATCGACGTCAAGATGATG 1120

1041 ................................................................................ 1120

1121 AATATGGAGGCGGCCAACCTGGCAGAGGTCCGCAGTTATTGCTATTTGGCTACCGTCAGCGATCTCTCCACCAAAGCTGC 1200

1121 ............................................................................... 1200

1201 ATGCCCGACCATGGGAGAAGCTCACAATGACAAACGTGCTGACCCAGTTTTGTGTGCAGACAAGGAGTGGTGGACAGGG 1280

1201 ............................................................................... 1280

1281 GCTGGGCAACGGCTGCGGATTTTTTGGCAAAGGATCCATTGACACATGCGCCAAATTTGCCTGCTCTACCAAGGCAATA 1360

1281 ............................................................................... 1360

1361 GGAAGAACCATCTTGAAAGAGAATATCAAGTACGAAGTGGCCATTTTTGTCCATGGACCACTACTGTGGAGTCGCACGG 1440

1361 ............................................................................... 1440

WN02xM66 variantsDNA alignment

1441 AAATTACTCCACACAGGTTGGAGCCACTCAGGCCGGCCGATTCAGCATCACTCCTGCTGCGCCTTCATACACACTAAAGC 1520

1441 ................................................................................ 1520

1521 TTGGAGAATATGGAGAGGTGACAGTGGACTGTGAACCACGGTCAGGGATTGACACCAATGCATACTACGTGATGACTGTT 1600

1521 ................................................................................ 1600

1601 GGAACAAAGACGTTCTTGGTCCATCGTGAGTGGTTCATGGACCTCAACCTCCCTTGGAGCAGTGCTGGAAGTACTGTGTG 1680

1601 ................................................................................ 1680

1681 GAGGAACAGAGAGACGTTAATGGAGTTTGAGGAACCACACGCCACGAAGCAGTCTGTGATAGCATTGGGCTCACAAGAGG 1760

1681 ................................................................................ 1760

1761 GAGCTCTGCATCAAGCTTTGGCTGGAGCCATTCCTGTGGAATTTTCAAGCAACACTGTCAAGTTGACGTCGGGTCATTTG 1840

1761 ................................................................................ 1840

1841 AAGTGTAGAGTGAAGATGGAAAAATTGCAGTTGAAGGGAACAACCTATGGCGTCTGTTCAAAGGCTTTCAAGTTTCTTAG 1920

1841 ................................................................................ 1920

1921 GACTCCCGTGGACACCGGTCACGGCACTGTGGTGTTGGAATTGCAGTACACTGGCACGGATGGACCTTGCAAAGTTCCTA 2000

1921 ................................................................................ 2000

2001 TCTCGTCAGTGGCTTCATTGAACGACCTAACGCCAGTGGGCAGATTGGTCACTGTCAACCCTTTTGTTTCAGTGGCCACG 2080

2001 ................................................................................ 2080

2081 GCCAACGCTAAGGTCCTGATTGAATTGGAACCACCCTTTGGAGACTCATACATAGTGGTGGGCAGAGGAGAACAACAGAT 2160

2081 ................................................................................ 2160

2161 CAATCACCATTGGCACAAGTCTGGAAGCAGCATTGGCAAAGCCTTTACAACCACCCTCAAAGGAGCGCAGAGACTAGCCG 2240

2161 ................................................................................ 2240

2241 CTCTAGGAGACACAGCTTGGGACTTTGGATCAGTTGGAGGGGTGTTCACTAGTGTTGGGCGGGCTGTCCATCAAGTGTTC 2320

2241 ................................................................................ 2320

2321 GGAGGAGCATTCCGCTCACTGTTCGGAGGCATGTCCTGGATAACGCAAGGATTGCTGGGGGCTCTCCTGTTGTGGATGGG 2400

2321 ................................................................................ 2400

2401 CATCAATGCTCGTGATAGGTCCATAGCTCTCACGTTTCTCGCAGTTGGAGGAGTTCTGCTCTTCCTCTCCGTGAACGTGG 2480

2401 ................................................................................ 2480

2481 GCGCCGATCAAGGATGCGCCATCAACTTTGGCAAGAGAGAGCTCAAGTGCGGAGATGGTATCTTCATATTTAGAGACTCT 2560

2481 ................................................................................ 2560

2561 GATGACTGGCTGAACAAGTACTCATACTATCCAGAAGATCCTGTGAAGCTTGCATCAATAGTGAAAGCCTCTTTTGAAGA 2640

2561 ................................................................................ 2640

2641 AGGGAAGTGTGGCCTAAATTCAGTTGACTCCTTGAGCATGAGATGTGGAGAAGCAAGGGCAGATGAGATCAATGCCATTT 2720

2641 ................................................................................ 2720

2721 TTGAGGAAAACGAGGTGGACATTTCTGTTGTCGTGGAGGATCCAAAGAATGTTTACCAGAGAGGAACTCATCCATTTTCC 2800

2721 ................................................................................ 2800

2801 AGAATTCGGGATGGTCTGCAGTATGGTTGGAAGACTTGGGGTAAGAACCTTGTGTTCTCCCCAGGGAGGAAGAATGGAAG 2880

2801 ................................................................................ 2880

2881 CTTCATCATAAGATGGAAAGTCCAGGAAAGAATGCCCGTTTTCAAACCGGGTCTGGAATTCTTTCCAGATAGAGGAGTTG 2960

2881 ................................................................................ 2960

2961 GGACGGGAGTGTTCACCACACGCGTGTACATGGACCCAGTCTTTGAATACACCATAGACTGCGATGGATCTATCTTGGGT 3040

2961 ................................................................................ 3040

3041 GCAGCGGTGAACGGAAAAAAGAGTGCCCATGGCTCTCCAACATTTTGGATGGGAAGTCATGAAGTAAATGGGACATGGAT 3120

3041 ................................................................................ 3120

3121 GATCCACACCTTGGAGGCATTAGATTACAAGGAGTGTGAGTGGCCACTGACACATACGATTGGAACATCAGTTGAAGAGA 3200

3121 ................................................................................ 3200

WN02x M66 variantDNA alignment

3201 GTGAAATGTTCATGCCGAGATCAATCGGAGGCCCAGTTAGCTCTCACAATCATATCCCTGGATACAAGGTTCAGACGAAC 3280

3201 ................................................................................ 3280

32B1 GGACCTTGGATGCAGGTACCACTAGAAGTGAAGAGAGAAGCTTGCCCAGGGACTAGCGTGATCATTGATGGCAACTGTGA 3360

281 ................................................................................ 3360

3361 TGGACGGGGAAAATCAACCAGATCCACCACGGATAGCGGGAAAGTTATTCCTGAATGGTGTTGCCGCTCCTGCACAATGC 3440

3361 ................................................................................ 3440

3441 CGCCTGTGAGCTTCCATGGTAGTGATGGGTGTTGGTATCCCATGGAAATTAGGCCAAGGAAAACGCATGAAAGCCATCTG 3520

3441 ................................................................................ 3520

3521 GTGCGCTCCTGGGTTACACCTGGAGAAATACATGCTGTCCCTTTTGGTTTGGTGAGCATGATGATAGCAATGGAAGTGGT 3600

3521 ................................................................................ 3600

3601 CCTAAGGAAAAGACAGGGACCAAAGCAAATGTTGGTTGGAGGAGTAGTGCTCTTGGGAGCAATGCTGGTCGGGCAAGTAA 3680

3601 ................................................................................ 3680

36B1 CTCTCCCTTGATTTGCTGAAACTCACAGTGGCTGTGGGATTGCATTCCATGAGATGAACAATGGAGGAGACGCCATGTAT 3760

3681 ................................................................................ 3760

3761 ATGGCGTTGATTGCTGCCTTTTCAATCAGACCAGGGCTGCTCATCGGCTTTGGGCTCAGGACCCTATGGAGCCCTCGGGA 3840

3761 ................................................................................ 3840

3841 ACGCCTTGTGCTGACCCTAGGAGCAGCCATGGTGGAGATTGCCTTGGGTGGCGTGATGGGCGGCCTGTGGAAGTATCTAA 3920

3841 ................................................................................ 3920

3921 ATGCAGTTTCTCTCTGCATCCTGACAATAAATGCTGTTGCTTCTAGGAAAGCATCAAATACCATCTTGCCCCTCATGGCT 4000

3921 ................................................................................ 4000

4001 CTGTTGACACCTGGTCACTATGGCTGAGGTTGAGCTGCCGCAATGTTCTTTTGTGCCATGGTTATCATAGGGTTCCTTCA 4080

4001 ................................................................................. 4080

4081 CCAGAATTTCAAGGACACCTCCATGCAGAAGACTATACCTCTGGTGGCCCTCACACTCACATCTTACCTGGGCTTGACAC 4160

4081 ................................................................................ 4160

4161 AACCTTTTTTGGGCCTGTGTGCATTTCTGGCAACCGCATATTTGGGCGAAGGAGTATCCCAGTGAATGAAGGCACTCGCA 4240

4161 ................................................................................ 4240

4241 GCAGCTGGTCTAGTGGGAGTGCTGGCAGGACTGGCTTTTCAGGAGATGGAGAACTTCCTTGGTCCGATTGCAGTTGGAGG 4320

4241 ................................................................................ 4320

4321 ACTCCTGATGATGCTGGTTAGCGTGGCTGGGAGGGTGGATGGGCTAGAGCTCAAGAAGCTTGGTGAAGTTTCATGGGAAG 4400

4321 ................................................................................ 4400

4401 AGGAGGCGGAGATCAGCGGGAGTTCCGCCCGCTATGATGTGGCACTCAGTGAACAAGGGGAGTTCAAGCTGCTTTCTGAA 4480

4401 ................................................................................ 4480

4481 GAGAAAGTGCCATGGGACCAGGTTGTGATGACCTCGCTGGCCTTGGTTGGGGCTGCCCTCCATCCATTTGCTCTTCTGCT 4560

4481 ................................................................................ 4560

4561 GGTCCTTGCTGGGTGGCTGTTTCATGTCAGGGGAGCTAGGAGAAGTGGGGATGTCTTGTGGGATATTCCCACTCCTAAGA 4640

4561 ................................................................................ 4640

4641 TCATCGAGGAATGTGAACATCTGGAGGATGGGATTTATGGCATATTCCAGTCAACCTTCTTGGGGGCCTCCCAGCGAGGA 4720

4641 ................................................................................ 4720

4721 GTGGGAGTGGCACAGGGAGGGGTGTTCCACACAATGTGGCATGTCACAAGAGGAGCTTTCCTTGTCAGGAATGGCAAGAA 4800

4721 ................................................................................ 4800

4801 GTTGATTCCATCTTGGGCTTCAGTAAAGGAAGACCTTGTCGCCTATGGTGGCTCATGGAAGTTGGAAGGCAGATGGGGATG 4880

4801 ................................................................................ 4880

4881 GAGAGGAAGAGGTCCAGTTGATCGCGGCTGTTCCAGGAAAGAACGTGGTCAACGTCCAGACAAAACCGAGCTTGTTCAAA 4960

4881 ................................................................................ 4960

WN02x M66 variantDNA alignment

4961 GTGAGGAATGGGGGAGAAATCGGGGCTGTCGCTCTTGACTATCCGAGTGGCACTTCAGGATCTCCTATTGTTAACAGGAA 5040

4961 ................................................................................ 5040

5041 CGGAGAGGTGATTGGGCTGTACGGCAATGGCATCCTTGTCGGTGACAACTCCTTCGTGTCCGCCATATCCCAGACTGAGG 5120

5041 ................................................................................ 5120

5122 TGAAGGAAGAAGGAAAGGAGGAGCTCCAAGAGATCCCGACAATGCTAAAGAAAGGAATGACAACTGTCCTTGATTTTCGT 5200

5121 ................................................................................ 5200

5201 CCTGGAGCTGGGAAGACAAGACGTTTCCTCCCACAGATCTTGGCCGAGTGCGCACGGAGACGCTTGCGCACTCTTGTGTT 5280

5201 ............................................................................... 5280

5281 GGCCCCCACCAGGGTTGTTCTTTCTGAAATGAAGGAGGCTTTTCACGGCCTGGACGTGAAATTCCACACACAGGCTTTTT 5360

5281 ................................................................................ 5360

5361 CCGCTCACGGCAGCGGGAGAGAAGTCATTTGATGCCATGTGCCATGCCACCCTAACTTACAGATGTTGGAACCAACTAGG 5440

5361 ................................................................................ 5440

5441 GTTGTTAACTGGGAAGTGATCATTATGGATGAAGCCCATTTTTTGGATCCAGCCAGCATAGCCGCTAGAGGTTGGGCAGC 5520

5441 ................................................................................ 5520

5521 GCACAGAGCTAGGGCAAATGAAAGTGCAACAATCTTGATGACAGCCACACCGCCTGGGACTAGTGATGAATTTCCACATT 5600

5521 ................................................................................ 5600

5601 CAAATGGTGAAATAGAAGATGTTCAAACGGACATACCCAGTGAGCCCTGGAACACAGGGCATGACTGCATCCTGGCTGAC 5680

5601 ................................................................................ 5680

5681 AAAAGGCCCACGGCATGGTTCCTTCCATCCATCAGAGCTGCAAATGTCATGGCTGCCTCTTTGCGTAAGGCTGGAAAGAG 5760

5681 ................................................................................ 5760

5761 TGTGGTGGTCCTGAACAGGAAAACCTTTGAGAGAGAATACCCCACGATAAAGCAGAAGAAACCTGACTTTATATTGGCCA 5840

5761 ................................................................................ 5840

5841 CTGACATAAGCTGAAATGGGAGCCAACCTTTGCGTGGAGCGAGTGCTGGATTGCAGGACGCTTTTAAGCCTGTGCTTGTG 5920

5841 ................................................................................ 5920

5921 GATGAAGGGAGGAAGGTGGCAATAAAAGGGCCACTTCGTATCTCCGCATCCTCTGCTGCTCAAAGGAGGGGGCGCATTGG 6000

5921 ................................................................................ 6000

6001 GAGAAATCCCAACAGAGATGGAGACTCATACTACTATTCTGAGCCTACAAGTGAAAATAATGCCCAACCAGTCTGCTGGT 6080

6001 ................................................................................ 6080

6081 TGGAGGCCTCAATGCTCTTGGACAACATGGAGGTGAGGGGTGGAATGGTCGCCCCACTCTATGGCGTTGAAGGAACTAAA 6160

6081 ................................................................................ 6160

6161 ACACCAGTTTCCCCTGGTGAAATGAGACTGAGGGACGACCAGAGGAAAGTCTTCAGAGAACTAGTGAGGAATTGTGACCT 6240

6161 ................................................................................ 6240

6241 GCCCGTTTGGCTTTCGTGGCAAGTGGCCAAGGCTGGTTTGAAGACGAATGATCGTAAGTGGTGTTTTGAAGGCCCTGAGG 6320

6241 ................................................................................ 6320

6321 AACATGAGATCTTGAATGACAGCGGTGAAACAGTGAAGTGCAGGGCTCCTGGAGGAGCAAAGAAGCCTCTGCGCCCAAGG 6400

6321 ................................................................................ 6400

6401 TGGTGTGATGAAAGGGTGTCATCTGACCAGAGTGCGCTGTCTGAATTTATTAAGTTTGCTGAAGGTAGGAGGGGAGCTGC 6480

5401 ................................................................................ 6480

6481 TGAAGTGCTAGTTGTGCTGAGTGAACTCCTGATTTCCTGGCTAAAAAAGGTGGAGAGGCAAATGGATACCATCAGTGTGT 6560

6481 ................................................................................ 6560

6561 TCCTCCACTCTGAGGAAGGCTCTAGGGCTTACCGCAATGCACTATCAATGATGCCTGAGGCAATGACAATAGTCATGCTG 6640

6561 ................................................................................ 6640

6641 TTTATACTGGCTGGACTACTGACATCGGGAATGGTCATCTTTTTCATGTCTCCCAAAGGCATCAGTAGAATGTCTATGGC 6720

6641................................................................................ 6720

WN02x M66 variantDNA alignment

6721 GATGGGCACAATGGCCGGCTGTGGATATCTCATGTTCCTTGGAGGCGTCAAACCCACTCACATCTCCTATGTCATGCTCA 6800

6721 ................................................................................ 6800

6801 TATTCTTTGTCCTGATGGTGGTTGTGATCCCCGAGCCAGGGCAACAAAGGTCCATCCAAGACAACCAAGTGGCATACCTC 6880

6801 ................................................................................ 6880

6881 ATTATTGGCATCCTGACGCTGGTTTCAGCGGTGGCAGCCAACGAGCTAGGCATGCTGGAGAAAACCAAAGAGGACCTCTT 6960

6881 ................................................................................ 6960

6961 TGGGAAGAAGAACTTAATTCCATCTAGTGCTTCACCCTGGAGTTGGCCGGATCTTGACCTGAAGCCAGGAGCTGCCTGGA 7040

6961 ................................................................................ 7040

7041 CAGTGTACGTTGGCATTGTTACAATGCTCTCTCCAATGTTGCACCACTGGATCAAAGTCGAATATGGCAACCTGTCTCTG 7120

7041 ................................................................................ 7120

7121 TCTGGAATAGCCCAGTCAGCCTCAGTCCTTTCTTTCATGGACAAGGGGATACCATTCATGAAGATGAATATCTCGGTCAT 7200

7121 ................................................................................ 7200

7201 AATGCTGCTGGTCAGTGGCTGGAATTCAATAACAGTGATGCCTCTGCTCTGTGGCATAGGGTGCGCCATGCTCCACTGGT 7280

7201 ................................................................................ 7280

7281 CTCTCATTTTACCTGGAATCAAAGCGCAGCAGTCAAAGCTTGCACAGAGAAGGGTGTTCCATGGCGTTGCCAAGAACCCT 7360

7281 ................................................................................ 7360

7361 GTGGTTGATGGGAATCCAACAGTTGACATTGAGGAAGCTCCTGAAATGCCTGCCCTTTATGAGAAGAAACTGGCTCTATA 7440

7361 ................................................................................ 7440

7441 TCTCCTTCTTGCTCTCAGCCTAGCTTCTGTTGCCATGTGCAGAACGCCCTTTTCATTGGCTGAAGGCATTGTCCTAGCAT 7520

7441 ................................................................................ 7520

7521 CAGCTTGCCTTAGGGCCGCTCATAGAGGGAAACACCAGCCTTCTTTGGAATGGACCATGGCTGTCTCCATGACAGGAGTC 7600

7521 ................................................................................ 7600

7601 ATGAGGGGGAATCACTATGCTTTTGTGGGAGTCATGTACAATCTATGGAAGATGAAAACTGGACGCCGGGGGAGCGCGAA 7680

7601 ................................................................................ 7680

7681 TGGAAAACTTTGGGTGAAGTCTGGAAGAGGGAACTGAATCTGTTGGACAAGCTGACAGTTTGCAGTTGTATAAAGGACCG 7760

7681 ................................................................................ 7760

7761 ACATTGTGGAGGTGGATCGTGATACGGCACGCAGGCATTTGGCCGAAGGGAAGGTGGACACCGGGGTGGCGGTCTCCAGG 7840

7761 ................................................................................ 7840

7841 GGGACCGCAAAGTTAAGGTGGTTCCATGAGCGTGGCTATGTCAAGCTGGAAGGTAGGGTGATTGACCTGGGGTGTGGCCG 7920

7841 ................................................................................ 7920

7921 CGGAGGCTGGTGTTACTACGCTGCTGCGCAAAAGGAAGTGAGTGGGGTCAAAGGATTTACTCTTTGGAGAGACGGCCATG 8000

7921 ................................................................................ 8000

8001 AGAAACCCATGAATGTGCAAAGTCTGGGATGGAACATCATCACCTTCAAGGACAAAACTGATATCCACCGCCTAGAACCA 8080

8001 ................................................................................ 8080

8081 GTGAAATGTGACACCCTTTTGTGTGACATTGGAGAGTCATCATCGTCATCGGTCACAGAGGGGGAAAGGACCGTGAGAGT 8160

8081 ................................................................................ 8160

8161 TCTTGATACTGTAGAAAAATGGCTGGCTTGTGGGGTTGACAACTTCTGTGTGAAGGTGTTAGCTCCATACATGCCAGATG 8240

8161 ................................................................................ 8240

8241 TTCTTGAGAAACTGGAATTGCTCCAAAGGAGGTTTGGCGGAACAGTGATCAGGAACCCTCTCTCCAGGAATTCCACTCAT 8320

8241 ................................................................................ 8320

8321 GAAATGTACTACGTGTCTGGAGCCCGCAGCAATGTCACATTTACTGTGAACCAAACATCCCGCCTCCTGATGAGGAGAAT 8400

8321 ................................................................................ 8400

8401 GAGGCGTCCAACTGGAAAAGTGACCCTGGAGGCTGACGTCATCCTCCCAATTGGGACACGCAGTGTTGAGACAGACAAGG 8480

8401 ................................................................................ 8480

WN02x M66 variantCNA alignment

8481 GACCCCTGGACAAAGAGGCCATAGAAGAAAGGGTTGAGAGGATAAAATCTGAGTACATGACCTCTTGGTTTTATGACAAT 8560

8481 ................................................................................ 8560

8561 GACAACCCCTACAGGACCTGGCACTACTGTGGCTCCTATGTCACAAAAACCTCCGGAAGTGCGGCGAGCATGGTAAATGG 8640

8561 ............................................................................... 8640

8641 TGT1TATTAAAATTTCCTGACATATCCATGGGACAGGATAGAGGAGGTCACAAGAATGGCAATGACTACAACCCCTTTTG 8720

8641 ................................................................................ 8720

8721 GACAGCAAAGAGTGTTTAAAGAAAAAGTTGACACCAGAGCAAAGGATCCACCAGCGGGAACTAGGAAGATCATGAAAGTT 8800

8721 ................................................................................ 8800

8801 GTCAACAGGTGGCTGTTCCGCCACCTGGCCAGAGAAAAGAACCCCAGACTGTGCACAAAGGAAGAATTTATTGCAAAAGT 8880

8801 ................................................................................ 8880

8881 CCGAAGTCATGCAGCCATTGGAGCTTACCTGGAAGAACAAGAACAGTGGAAGACTGCCAATGAGGCTGTCCAAGACCCAA 8960

8881 ................................................................................ 8960

8961 AGTTCTGGGAACTGGTGGATGAAGAAAGGAAGCTGCACCAACAAGGCAGGTGTCGGACTTGTGTGTACAACATGATGGGG 9040

8961 ................................................................................ 9040

9041 AAAAGAGAGAAGAAGCTGTCAGAGTTTGGGAAAGCAAAGGGAAGCCGTGCCATATGGTATATCTGGCTGGGAGCGCGGTA 9120

9041 ................................................................................ 9120

9121 TCTTGAGTTTGAGGCCCTGGGATTCCTGAATGAGGACCATTGGGCTTCCAGGGAAAACTCAGGAGGAGGAGTGGAAGGCA 9200

9121 ................................................................................ 9200

9201 TTGGCTTACAATACCTAGGATATGTGATCAGAGACCTGGCTGCAATGGATGGTGGTGGATTCTACGCGGATGACACCGCT 9280

9201 ................................................................................ 9280

9281 GGATGGGACACGCGCATCACAGAGGCAGACCTTGATGATGAACAGGAGATCTTGAACTACATGAGCCCACATCACAAAAA 9360

9281 ................................................................................ 9350

9361 ACTGGCACAAGCAGTGATGGAAATGACATACAAGAACAAAGTGGTGAAAGTGTTGAGACCAGCCCCAGGAGGGAAAGCCT 9440

9361 ................................................................................ 9440

9441 ACATGGATGTCATAAGTCGACGAGACCAGAGAGGATCCGGGCAGGTAGTGACTTATGCTCTGAACACCATCACCAACTTG 9520

9441 ................................................................................ 9520

9521 AAAGTCCAATTGATCAGAATGGCAGAAGCAGAGATCGTGATACATCACCAACATGTTCAAGATTGTGATGAATCAGTTCT 9600

9521 ............................................................................... 9600

9601 GACCAGGCTGGAGGCATGGCTCACTGAGCACGGATGTGACAGACTGAAGAGGATGGCGGTGAGTGGAGACGACTGTGTGG 9680

9601 ................................................................................ 9680

9681 TCCGGCCCATCGATGACAGGTTCGGCCTGGCCCTGTCCCATCTCAACGCCATGTCCAAGGTTAGAAAGGACATATCTGAA 9760

9681 ................................................................................ 9760

9761 TGGCAGCCATCAAAAGGGTGGAATGATTGGGAGAATGTGCCCTTCTGTTCCCACCACTTCCATGAACTACAGCTGAAGGA 9840

9761 ................................................................................ 9840

9841 TGGCAGGAGGATTGTGGTGCCTTGCCGAGAACAGGACGAGCTCATTGGGAGAGGAAGGGTGTCTCCAGGAAACGGCTGGA 9920

9841 ................................................................................ 9920

9921 TGATCAAGGAAACAGCTTGCCTCAGCAAAGCCTATGCCAACATGTGGTCACTGATGTATTTTCACAAAAGGGACATGAGG 10000

9921 ................................................................................ 10000

10001 CTACTGTCATTGGCTGTTTCCTCAGCTGTTCCCACCTCATGGGTTCCACAAGGACCCACAACATGGTCGATTCATGGGAA 20080

10001 ................................................................................ 10080

10081 AGGGGAGTGGATGACCACGGAAGACATGCTTGAGGTGTGGAACAGAGTATGGATAACCAACAACCCACACATGCAGGACA 10160

10081 ................................................................................ 10160

10161 AGACAATGGTGAAAAAATGGAGAGATGTCCCTTATCTAACCAAGAGACAAGACAAGCTGTGCGGATCACTGATTGGAATG 10240

10161 ................................................................................ 10240

### DNA Strider^TM13f7# # # Thursday, 10/21/2004 3:10:16PM

WN02M Prot.x M66 M Prot.Protein alignment

Protein sequence 75 aa sltvqthgbstl.. VVLLLLVAPAY S SEQ ID NOS: 25 and 26

Protein sequence 75 aa sltvqthgksil.. VVPLLLVAPAY S SEQ ID NOS: 25 and 27

Layout： Standard

Hetthod： Singla Block

Blocck Length s： 6-aa

Mismatch Penalty：Smaller(1)

Gap penalty： Medium(2) SEQ ID NOS：28-30

Weighting； BLOSOH62

. 20 . 40 . 60 .

1 SLTVQTHGESTLANKKGAWMIDSTKATRYLVKTESWILLRNPGYALAAVIGWMLGSNIMQRVVFVVLLLVAPAYS 75

SLTVQTHGESTLANKKGAWMDSTKATRYLVKTESWILRNPGYALVAAVIGWMLGSNTMQRVVFVV LLLVAPAYS

1 SLTVQTEGESTLANNKKGAWMDSTKATRYLVKTESWILRNPGYALVAAVIGWMLGSNTMQVVFVVPLLLVAPAYS 75

. 20 . 40 . 60 .

% identity =98.7(74/75)

Sequence listing

<110> Acambis Inc. (Acambis Inc.)

<120> vaccine against Japanese encephalitis virus and West Nile virus

<130>06132/099WO3

<140>PCT/US05/37369

<141>2005-10-19

<150>US 60/718,923

<151>2005-09-19

<150>US 60/674,546

<151>2005-04-25

<150>US 60/674,415

<151>2005-04-24

<150>US 60/620,948

<151>2004-10-21

<150>US 60/620,466

<151>2004-10-20

<160>38

<170>PatentIn version 3.3

<210>1

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> derived from Yellow Fever virus (Yellow river virus) and West Nile virus (West Nile virus)

<400>1

cactgggaga gcttgaaggt c 21

<210>2

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> derived from yellow fever virus and West Nile virus

<400>2

aaagccagtt gcagccgcgg tttaa 25

<210>3

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> derived from yellow fever virus and Dengue virus type 1 (Dengue-1 virus)

<400>3

aaggtagact ggtgggctcc c 21

<210>4

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> is derived from yellow fever virus and dengue type 1 virus

<400>4

gatcctcagt accaaccgcg gtttaa 26

<210>5

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> derived from yellow fever virus and Dengue type 2 virus (Dengue-2 virus)

<400>5

aaggtagatt ggtgtgcatt g 21

<210>6

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> is derived from yellow fever virus and dengue type 2 virus

<400>6

aaccctcagt accacccgcg gtttaa 26

<210>7

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> derived from yellow fever virus and Dengue virus type 3 (Dengue-3 virus)

<400>7

aaggtgaatt gaagtgctct a 21

<210>8

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> is derived from yellow fever virus and dengue virus type 3

<400>8

acccccagca ccacccgcgg tttaa 25

<210>9

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> derived from yellow fever virus and Dengue virus type 4 (Dengue-4 virus)

<400>9

aaaaggaaca gttgttctct a 21

<210>10

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> is derived from yellow fever virus and dengue type 4 virus

<400>10

acccgaagtg tcaaccgcgg tttaa 25

<210>11

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> is derived from yellow fever virus and St.Louis Encephalitis virus

<400>11

aacgtgaatag ttggatagt c 21

<210>12

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> derived from yellow fever virus and St.Louis encephalitis virus

<400>12

accgttggtc gcacccgcgg tttaa 25

<210>13

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> is derived from yellow fever virus and Murray Valley Encephalitis virus

<400>13

aatttcgaaa ggtggaaggt c 21

<210>14

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> derived from yellow fever virus and Murray Valley encephalitis virus

<400>14

gaccggtgtt tacagccgcg gtttaa 26

<210>15

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> derived from yellow fever virus and Tick-Borne Encephalitis virus (Tick-Borne Encephalitis virus)

<400>15

tactgcgaac gacgttgcca c 21

<210>16

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> derived from yellow fever virus and tick-borne encephalitis virus

<400>16

actgggaacc tcacccgcgg tttaa 25

<210>17

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> derived from yellow fever virus

<400>17

ggttagagga gaccct 16

<210>18

<211>39

<212>PRT

<213> Artificial sequence

<220>

<223> is derived from West Nile Virus

<400>18

Ser Leu Thr Val Gln Thr His Gly Glu Ser Thr Leu Ala Asn Lys Lys

1 5 10 15

Gly Ala Trp Met Asp Ser Thr Lys Ala Thr Arg Tyr Leu Val Lys Thr

20 25 30

Glu Ser Trp Ile Leu Arg Asn

35

<210>19

<211>36

<212>PRT

<213> Artificial sequence

<220>

<223> is derived from West Nile Virus

<400>19

Pro Gly Tyr Ala Leu Val Ala Ala Val Ile Gly Trp Met Leu Gly Ser

1 5 10 15

Asn Thr Met Gln Arg Val Val Phe Val Val Leu Leu Leu Leu Val Ala

20 25 30

Pro Ala Tyr Ser

35

<210>20

<211>10896

<212>DNA

<213> Artificial sequence

<220>

<223> derived from yellow fever virus and West Nile virus

<220>

<221>misc_feature

<222>(1)..(1)

<223> n is a, c, g, or t

<220>

<221>CDS

<222>(119)..(10384)

<400>20

ngtaaatcct gtgtgctaat tgaggtgcat tggtctgcaa atcgagttgc taggcaataa 60

acacatttgg attaatttta atcgttcgtt gagcgattag cagagaactg accagaac 118

atg tct ggt cgt aaa gct cag gga aaa acc ctg ggc gtc aat atg gta 166

Met Ser Gly Arg Lys Ala Gln Gly Lys Thr Leu Gly Val Asn Met Val

1 5 10 15

cga cga gga gtt cgc tcc ttg tca aac aaa ata aaa caa aaa aca aaa 214

Arg Arg Gly Val Arg Ser Leu Ser Asn Lys Ile Lys Gln Lys Thr Lys

20 25 30

caa att gga aac aga cct gga cct tca aga ggt gtt caa gga ttt atc 262

Gln Ile Gly Asn Arg Pro Gly Pro Ser Arg Gly Val Gln Gly Phe Ile

35 40 45

ttt ttc ttt ttg ttc aac att ttg act gga aaa aag atc aca gcc cac 310

Phe Phe Phe Leu Phe Asn Ile Leu Thr Gly Lys Lys Ile Thr Ala His

50 55 60

cta aag agg ttg tgg aaa atg ctg gac cca aga caa ggc ttg gct gtt 358

Leu Lys Arg Leu Trp Lys Met Leu Asp Pro Arg Gln Gly Leu Ala Val

65 70 75 80

cta agg aaa gtc aag aga gtg gtg gcc agt ttg atg aga gga ttg tcc 406

Leu Arg Lys Val Lys Arg Val Val Ala Ser Leu Met Arg Gly Leu Ser

85 90 95

tca agg aaa cgc cgt tcc cat gat gtt ctg act gtg caa ttc cta att 454

Ser Arg Lys Arg Arg Ser His Asp Val Leu Thr Val Gln Phe Leu Ile

100 105 110

ttg gga atg ctg ttg atg acg ggt gga gtt acc ctc tct aac ttc caa 502

Leu Gly Met Leu Leu Met Thr Gly Gly Val Thr Leu Ser Asn Phe Gln

115 120 125

ggg aag gtg atg atg acg gta aat gct act gac gtc aca gat gtc atc 550

Gly Lys Val Met Met Thr Val Asn Ala Thr Asp Val Thr Asp Val Ile

130 135 140

acg att cca aca gct gct gga aag aac cta tgc att gtc aga gca atg 598

Thr Ile Pro Thr Ala Ala Gly Lys Asn Leu Cys Ile Val Arg Ala Met

145 150 155 160

gat gtg gga tac atg tgc gat gat act atc act tat gaa tgc cca gtg 646

Asp Val Gly Tyr Met Cys Asp Asp Thr Ile Thr Tyr Glu Cys Pro Val

165 170 175

ctg tcg gct ggt aat gat cca gaa gac atc gac tgt tgg tgc aca aag 694

Leu Ser Ala Gly Asn Asp Pro Glu Asp Ile Asp Cys Trp Cys Thr Lys

180 185 190

tca gca gtc tac gtc agg tat gga aga tgc acc aag aca cgc cac tca 742

Ser Ala Val Tyr Val Arg Tyr Gly Arg Cys Thr Lys Thr Arg His Ser

195 200 205

aga cgc agt cgg agg tca ctg aca gtg cag aca cac gga gaa agc act 790

Arg Arg Ser Arg Arg Ser Leu Thr Val Gln Thr His Gly Glu Ser Thr

210 215 220

cta gcg aac aag aag ggg gct tgg atg gac agc acc aag gcc aca agg 838

Leu Ala Asn Lys Lys Gly Ala Trp Met Asp Ser Thr Lys Ala Thr Arg

225 230 235 240

tat ttg gta aaa aca gaa tca tgg atc ttg agg aac cct gga tat gcc 886

Tyr Leu Val Lys Thr Glu Ser Trp Ile Leu Arg Asn Pro Gly Tyr Ala

245 250 255

ctg gtg gca gcc gtc att ggt tgg atg ctt ggg agc aac acc atg cag 934

Leu Val Ala Ala Val Ile Gly Trp Met Leu Gly Ser Asn Thr Met Gln

260 265 270

aga gtt gtg ttt gtc gtg cta ttg ctt ttg gtg gcc cca gct tac agc 982

Arg Val Val Phe Val Val Leu Leu Leu Leu Val Ala Pro Ala Tyr Ser

275 280 285

ttc aac tgc ctt gga atg agc aac aga gac ttc ttg gaa gga gtg tct 1030

Phe Asn Cys Leu Gly Met Ser Asn Arg Asp Phe Leu Glu Gly Val Ser

290 295 300

gga gca aca tgg gtg gat ttg gtt ctc gaa ggc gac agc tgc gtg act 1078

Gly Ala Thr Trp Val Asp Leu Val Leu Glu Gly Asp Ser Cys Val Thr

305 310 315 320

atc atg tct aag gac aag cct acc atc gac gtc aag atg atg aat atg 1126

Ile Met Ser Lys Asp Lys Pro Thr Ile Asp Val Lys Met Met Asn Met

325 330 335

gag gcg gcc aac ctg gca gag gtc cgc agt tat tgc tat ttg gct acc 1174

Glu Ala Ala Asn Leu Ala Glu Val Arg Ser Tyr Cys Tyr Leu Ala Thr

340 345 350

gtc agc gat ctc tcc acc aaa gct gca tgc ccg acc atg gga gaa gct 1222

Val Ser Asp Leu Ser Thr Lys Ala Ala Cys Pro Thr Met Gly Glu Ala

355 360 365

cac aat gac aaa cgt gct gac cca gct ttt gtg tgc aga caa gga gtg 1270

His Asn Asp Lys Arg Ala Asp Pro Ala Phe Val Cys Arg Gln Gly Val

370 375 380

gtg gac agg ggc tgg ggc aac ggc tgc gga ttt ttt ggc aaa gga tcc 1318

Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Phe Phe Gly Lys Gly Ser

385 390 395 400

att gac aca tgc gcc aaa ttt gcc tgc tct acc aag gca ata gga aga 1366

Ile Asp Thr Cys Ala Lys Phe Ala Cys Ser Thr Lys Ala Ile Gly Arg

405 410 415

acc atc ttg aaa gag aat atc aag tac gaa gtg gcc att ttt gtc cat 1414

Thr Ile Leu Lys Glu Asn Ile Lys Tyr Glu Val Ala Ile Phe Val His

420 425 430

gga cca act act gtg gag tcg cac gga aat tac tcc aca cag gtt gga 1462

Gly Pro Thr Thr Val Glu Ser His Gly Asn Tyr Ser Thr Gln Val Gly

435 440 445

gcc act cag gcc ggc cga ttc agc atc act cct gct gcg cct tca tac 1510

Ala Thr Gln Ala Gly Arg Phe Ser Ile Thr Pro Ala Ala Pro Ser Tyr

450 455 460

aca cta aag ctt gga gaa tat gga gag gtg aca gtg gac tgt gaa cca 1558

Thr Leu Lys Leu Gly Glu Tyr Gly Glu Val Thr Val Asp Cys Glu Pro

465 470 475 480

cgg tca ggg att gac acc aat gca tac tac gtg atg act gtt gga aca 1606

Arg Ser Gly Ile Asp Thr Asn Ala Tyr Tyr Val Met Thr Val Gly Thr

485 490 495

aag acg ttc ttg gtc cat cgt gag tgg ttc atg gac ctc aac ctc cct 1654

Lys Thr Phe Leu Val His Arg Glu Trp Phe Met Asp Leu Asn Leu Pro

500 505 510

tgg agc agt gct gga agt act gtg tgg agg aac aga gag acg tta atg 1702

Trp Ser Ser Ala Gly Ser Thr Val Trp Arg Asn Arg Glu Thr Leu Met

515 520 525

gag ttt gag gaa cca cac gcc acg aag cag tct gtg ata gca ttg ggc 1750

Glu Phe Glu Glu Pro His Ala Thr Lys Gln Ser Val Ile Ala Leu Gly

530 535 540

tca caa gag gga gct ctg cat caa gct ttg gct gga gcc att cct gtg 1798

Ser Gln Glu Gly Ala Leu His Gln Ala Leu Ala Gly Ala Ile Pro Val

545 550 555 560

gaa ttt tca agc aac act gtc aag ttg acg tcg ggt cat ttg aag tgt 1846

Glu Phe Ser Ser Asn Thr Val Lys Leu Thr Ser Gly His Leu Lys Cys

565 570 575

aga gtg aag atg gaa aaa ttg cag ttg aag gga aca acc tat ggc gtc 1894

Arg Val Lys Met Glu Lys Leu Gln Leu Lys Gly Thr Thr Tyr Gly Val

580 585 590

tgt tca aag gct ttc aag ttt ctt agg act ccc gtg gac acc ggt cac 1942

Cys Ser Lys Ala Phe Lys Phe Leu Arg Thr Pro Val Asp Thr Gly His

595 600 605

ggc act gtg gtg ttg gaa ttg cag tac act ggc acg gat gga cct tgc 1990

Gly Thr Val Val Leu Glu Leu Gln Tyr Thr Gly Thr Asp Gly Pro Cys

610 615 620

aaa gtt cct atc tcg tca gtg gct tca ttg aac gac cta acg cca gtg 2038

Lys Val Pro Ile Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val

625 630 635 640

ggc aga ttg gtc act gtc aac cct ttt gtt tca gtg gcc acg gcc aac 2086

Gly Arg Leu Val Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn

645 650 655

gct aag gtc ctg att gaa ttg gaa cca ccc ttt gga gac tca tac ata 2134

Ala Lys Val Leu Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile

660 665 670

gtg gtg ggc aga gga gaa caa cag atc aat cac cat tgg cac aag tct 2182

Val Val Gly Arg Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser

675 680 685

gga agc agc att ggc aaa gcc ttt aca acc acc ctc aaa gga gcg cag 2230

Gly Ser Ser Ile Gly Lys Ala Phe Thr Thr Thr Leu Lys Gly Ala Gln

690 695 700

aga cta gcc gct cta gga gac aca gct tgg gac ttt gga tca gtt gga 2278

Arg Leu Ala Ala Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val Gly

705 710 715 720

ggg gtg ttc act agt gtt ggg cgg gct gtc cat caa gtg ttc gga gga 2326

Gly Val Phe Thr Ser Val Gly Arg Ala Val His Gln Val Phe Gly Gly

725 730 735

gca ttc cgc tca ctg ttc gga ggc atg tcc tgg ata acg caa gga ttg 2374

Ala Phe Arg Ser Leu Phe Gly Gly Met Ser Trp Ile Thr Gln Gly Leu

740 745 750

ctg ggg gct ctc ctg ttg tgg atg ggc atc aat gct cgt gat agg tcc 2422

Leu Gly Ala Leu Leu Leu Trp Met Gly Ile Asn Ala Arg Asp Arg Ser

755 760 765

ata gct ctc acg ttt ctc gca gtt gga gga gtt ctg ctc ttc ctc tcc 2470

Ile Ala Leu Thr Phe Leu Ala Val Gly Gly Val Leu Leu Phe Leu Ser

770 775 780

gtg aac gtg ggc gcc gat caa gga tgc gcc atc aac ttt ggc aag aga 2518

Val Asn Val Gly Ala Asp Gln Gly Cys Ala Ile Asn Phe Gly Lys Arg

785 790 795 800

gag ctc aag tgc gga gat ggt atc ttc ata ttt aga gac tct gat gac 2566

Glu Leu Lys Cys Gly Asp Gly Ile Phe Ile Phe Arg Asp Ser Asp Asp

805 810 815

tgg ctg aac aag tac tca tac tat cca gaa gat cct gtg aag ctt gca 2614

Trp Leu Asn Lys Tyr Ser Tyr Tyr Pro Glu Asp Pro Val Lys Leu Ala

820 825 830

tca ata gtg aaa gcc tct ttt gaa gaa ggg aag tgt ggc cta aat tca 2662

Ser Ile Val Lys Ala Ser Phe Glu Glu Gly Lys Cys Gly Leu Asn Ser

835 840 845

gtt gac tcc ctt gag cat gag atg tgg aga agc agg gca gat gag atc 2710

Val Asp Ser Leu Glu His Glu Met Trp Arg Ser Arg Ala Asp Glu Ile

850 855 860

aat gcc att ttt gag gaa aac gag gtg gac att tct gtt gtc gtg cag 2758

Asn Ala Ile Phe Glu Glu Asn Glu Val Asp Ile Ser Val Val Val Gln

865 870 875 880

gat cca aag aat gtt tac cag aga gga act cat cca ttt tcc aga att 2806

Asp Pro Lys Asn Val Tyr Gln Arg Gly Thr His Pro Phe Ser Arg Ile

885 890 895

cgg gat ggt ctg cag tat ggt tgg aag act tgg ggt aag aac ctt gtg 2854

Arg Asp Gly Leu Gln Tyr Gly Trp Lys Thr Trp Gly Lys Asn Leu Val

900 905 910

ttc tcc cca ggg agg aag aat gga agc ttc atc ata gat gga aag tcc 2902

Phe Ser Pro Gly Arg Lys Asn Gly Ser Phe Ile Ile Asp Gly Lys Ser

915 920 925

agg aaa gaa tgc ccg ttt tca aac cgg gtc tgg aat tct ttc cag ata 2950

Arg Lys Glu Cys Pro Phe Ser Asn Arg Val Trp Asn Ser Phe Gln Ile

930 935 940

gag gag ttt ggg acg gga gtg ttc acc aca cgc gtg tac atg gac gca 2998

Glu Glu Phe Gly Thr Gly Val Phe Thr Thr Arg Val Tyr Met Asp Ala

945 950 955 960

gtc ttt gaa tac acc ata gac tgc gat gga tct atc ttg ggt gca gcg 3046

Val Phe Glu Tyr Thr Ile Asp Cys Asp Gly Ser Ile Leu Gly Ala Ala

965 970 975

gtg aac gga aaa aag agt gcc cat ggc tct cca aca ttt tgg atg gga 3094

Val Asn Gly Lys Lys Ser Ala His Gly Ser Pro Thr Phe Trp Met Gly

980 985 990

agt cat gaa gta aat ggg aca tgg atg atc cac acc ttg gag gca tta 3142

Ser His Glu Val Asn Gly Thr Trp Met Ile His Thr Leu Glu Ala Leu

995 1000 1005

gat tac aag gag tgt gag tgg cca ctg aca cat acg att gga aca 3187

Asp Tyr Lys Glu Cys Glu Trp Pro Leu Thr His Thr Ile Gly Thr

1010 1015 1020

tca gtt gaa gag agt gaa atg ttc atg ccg aga tca atc gga ggc 3232

Ser Val Glu Glu Ser Glu Met Phe Met Pro Arg Ser Ile Gly Gly

1025 1030 1035

cca gtt agc tct cac aat cat atc cct gga tac aag gtt cag acg 3277

Pro Val Ser Ser His Asn His Ile Pro Gly Tyr Lys Val Gln Thr

1040 1045 1050

aac gga cct tgg atg cag gta cca cta gaa gtg aag aga gaa gct 3322

Asn Gly Pro Trp Met Gln Val Pro Leu Glu Val Lys Arg Glu Ala

1055 1060 1065

tgc cca ggg act agc gtg atc att gat ggc aac tgt gat gga cgg 3367

Cys Pro Gly Thr Ser Val Ile Ile Asp Gly Asn Cys Asp Gly Arg

1070 1075 1080

gga aaa tca acc aga tcc acc acg gat agc ggg aaa gtt att cct 3412

Gly Lys Ser Thr Arg Ser Thr Thr Asp Ser Gly Lys Val Ile Pro

1085 1090 1095

gaa tgg tgt tgc cgc tcc tgc aca atg ccg cct gtg agc ttc cat 3457

Glu Trp Cys Cys Arg Ser Cys Thr Met Pro Pro Val Ser Phe His

1100 1105 1110

ggt agt gat ggg tgt tgg tat ccc atg gaa att agg cca agg aaa 3502

Gly Ser Asp Gly Cys Trp Tyr Pro Met Glu Ile Arg Pro Arg Lys

1115 1120 1125

acg cat gaa agc cat ctg gtg cgc tcc tgg gtt aca gct gga gaa 3547

Thr His Glu Ser His Leu Val Arg Ser Trp Val Thr Ala Gly Glu

1130 1135 1140

ata cat gct gtc cct ttt ggt ttg gtg agc atg atg ata gca atg 3592

Ile His Ala Val Pro Phe Gly Leu Val Ser Met Met Ile Ala Met

1145 1150 1155

gaa gtg gtc cta agg aaa aga cag gga cca aag caa atg ttg gtt 3637

Glu Val Val Leu Arg Lys Arg Gln Gly Pro Lys Gln Met Leu Val

1160 1165 1170

gga gga gta gtg ctc ttg gga gca atg ctg gtc ggg caa gta act 3682

Gly Gly Val Val Leu Leu Gly Ala Met Leu Val Gly Gln Val Thr

1175 1180 1185

ctc ctt gat ttg ctg aaa ctc aca gtg gct gtg gga ttg cat ttc 3727

Leu Leu Asp Leu Leu Lys Leu Thr Val Ala Val Gly Leu His Phe

1190 1195 1200

cat gag atg aac aat gga gga gac gcc atg tat atg gcg ttg att 3772

His Glu Met Asn Asn Gly Gly Asp Ala Met Tyr Met Ala Leu Ile

1205 1210 1215

gct gcc ttt tca atc aga cca ggg ctg ctc atc ggc ttt ggg ctc 3817

Ala Ala Phe Ser Ile Arg Pro Gly Leu Leu Ile Gly Phe Gly Leu

1220 1225 1230

agg acc cta tgg agc cct cgg gaa cgc ctt gtg ctg acc cta gga 3862

Arg Thr Leu Trp Ser Pro Arg Glu Arg Leu Val Leu Thr Leu Gly

1235 1240 1245

gca gcc atg gtg gag att gcc ttg ggt ggc gtg atg ggc ggc ctg 3907

Ala Ala Met Val Glu Ile Ala Leu Gly Gly Val Met Gly Gly Leu

1250 1255 1260

tgg aag tat cta aat gca gtt tct ctc tgc atc ctg aca ata aat 3952

Trp Lys Tyr Leu Asn Ala Val Ser Leu Cys Ile Leu Thr Ile Asn

1265 1270 1275

gct gtt gct tct agg aaa gca tca aat acc atc ttg ccc ctc atg 3997

Ala Val Ala Ser Arg Lys Ala Ser Asn Thr Ile Leu Pro Leu Met

1280 1285 1290

gct ctg ttg aca cct gtc act atg gct gag gtg aga ctt gcc gca 4042

Ala Leu Leu Thr Pro Val Thr Met Ala Glu Val Arg Leu Ala Ala

1295 1300 1305

atg ttc ttt tgt gcc atg gtt atc ata ggg gtc ctt cac cag aat 4087

Met Phe Phe Cys Ala Met Val Ile Ile Gly Val Leu His Gln Asn

1310 1315 1320

ttc aag gac acc tcc atg cag aag act ata cct ctg gtg gcc ctc 4132

Phe Lys Asp Thr Ser Met Gln Lys Thr Ile Pro Leu Val Ala Leu

1325 1330 1335

aca ctc aca tct tac ctg ggc ttg aca caa cct ttt ttg ggc ctg 4177

Thr Leu Thr Ser Tyr Leu Gly Leu Thr Gln Pro Phe Leu Gly Leu

1340 1345 1350

tgt gca ttt ctg gca acc cgc ata ttt ggg cga agg agt atc cca 4222

Cys Ala Phe Leu Ala Thr Arg Ile Phe Gly Arg Arg Ser Ile Pro

1355 1360 1365

gtg aat gag gca ctc gca gca gct ggt cta gtg gga gtg ctg gca 4267

Val Asn Glu Ala Leu Ala Ala Ala Gly Leu Val Gly Val Leu Ala

1370 1375 1380

gga ctg gct ttt cag gag atg gag aac ttc ctt ggt ccg att gca 4312

Gly Leu Ala Phe Gln Glu Met Glu Asn Phe Leu Gly Pro Ile Ala

1385 1390 1395

gtt gga gga ctc ctg atg atg ctg gtt agc gtg gct ggg agg gtg 4357

Val Gly Gly Leu Leu Met Met Leu Val Ser Val Ala Gly Arg Val

1400 1405 1410

gat ggg cta gag ctc aag aag ctt ggt gaa gtt tca tgg gaa gag 4402

Asp Gly Leu Glu Leu Lys Lys Leu Gly Glu Val Ser Trp Glu Glu

1415 1420 1425

gag gcg gag atc agc ggg agt tcc gcc cgc tat gat gtg gca ctc 4447

Glu Ala Glu Ile Ser Gly Ser Ser Ala Arg Tyr Asp Val Ala Leu

1430 1435 1440

agt gaa caa ggg gag ttc aag ctg ctt tct gaa gag aaa gtg cca 4492

Ser Glu Gln Gly Glu Phe Lys Leu Leu Ser Glu Glu Lys Val Pro

1445 1450 1455

tgg gac cag gtt gtg atg acc tcg ctg gcc ttg gtt ggg gct gcc 4537

Trp Asp Gln Val Val Met Thr Ser Leu Ala Leu Val Gly Ala Ala

1460 1465 1470

ctc cat cca ttt gct ctt ctg ctg gtc ctt gct ggg tgg ctg ttt 4582

Leu His Pro Phe Ala Leu Leu Leu Val Leu Ala Gly Trp Leu Phe

1475 1480 1485

cat gtc agg gga gct agg aga agt ggg gat gtc ttg tgg gat att 4627

His Val Arg Gly Ala Arg Arg Ser Gly Asp Val Leu Trp Asp Ile

1490 1495 1500

ccc act cct aag atc atc gag gaa tgt gaa cat ctg gag gat ggg 4672

Pro Thr Pro Lys Ile Ile Glu Glu Cys Glu His Leu Glu Asp Gly

1505 1510 1515

att tat ggc ata ttc cag tca acc ttc ttg ggg gcc tcc cag cga 4717

Ile Tyr Gly Ile Phe Gln Ser Thr Phe Leu Gly Ala Ser Gln Arg

1520 1525 1530

gga gtg gga gtg gca cag gga ggg gtg ttc cac aca atg tgg cat 4762

Gly Val Gly Val Ala Gln Gly Gly Val Phe His Thr Met Trp His

1535 1540 1545

gtc aca aga gga gct ttc ctt gtc agg aat ggc aag aag ttg att 4807

Val Thr Arg Gly Ala Phe Leu Val Arg Asn Gly Lys Lys Leu Ile

1550 1555 1560

cca tct tgg gct tca gta aag gaa gac ctt gtc gcc tat ggt ggc 4852

Pro Ser Trp Ala Ser Val Lys Glu Asp Leu Val Ala Tyr Gly Gly

1565 1570 1575

tca tgg aag ttg gaa ggc aga tgg gat gga gag gaa gag gtc cag 4897

Ser Trp Lys Leu Glu Gly Arg Trp Asp Gly Glu Glu Glu Val Gln

1580 1585 1590

ttg atc gcg gct gtt cca gga aag aac gtg gtc aac gtc cag aca 4942

Leu Ile Ala Ala Val Pro Gly Lys Asn Val Val Asn Val Gln Thr

1595 1600 1605

aaa ccg agc ttg ttc aaa gtg agg aat ggg gga gaa atc ggg gct 4987

Lys Pro Ser Leu Phe Lys Val Arg Asn Gly Gly Glu Ile Gly Ala

1610 1615 1620

gtc gct ctt gac tat ccg agt ggc act tca gga tct cct att gtt 5032

Val Ala Leu Asp Tyr Pro Ser Gly Thr Ser Gly Ser Pro Ile Val

1625 1630 1635

aac agg aac gga gag gtg att ggg ctg tac ggc aat ggc atc ctt 5077

Asn Arg Asn Gly Glu Val Ile Gly Leu Tyr Gly Asn Gly Ile Leu

1640 1645 1650

gtc ggt gac aac tcc ttc gtg tcc gcc ata tcc cag act gag gtg 5122

Val Gly Asp Asn Ser Phe Val Ser Ala Ile Ser Gln Thr Glu Val

1655 1660 1665

aag gaa gaa gga aag gag gag ctc caa gag atc ccg aca atg cta 5167

Lys Glu Glu Gly Lys Glu Glu Leu Gln Glu Ile Pro Thr Met Leu

1670 1675 1680

aag aaa gga atg aca act gtc ctt gat ttt cat cct gga gct ggg 5212

Lys Lys Gly Met Thr Thr Val Leu Asp Phe His Pro Gly Ala Gly

1685 1690 1695

aag aca aga cgt ttc ctc cca cag atc ttg gcc gag tgc gca cgg 5257

Lys Thr Arg Arg Phe Leu Pro Gln Ile Leu Ala Glu Cys Ala Arg

1700 1705 1710

aga cgc ttg cgc act ctt gtg ttg gcc ccc acc agg gtt gtt ctt 5302

Arg Arg Leu Arg Thr Leu Val Leu Ala Pro Thr Arg Val Val Leu

1715 1720 1725

tct gaa atg aag gag gct ttt cac ggc ctg gac gtg aaa ttc cac 5347

Ser Glu Met Lys Glu Ala Phe His Gly Leu Asp Val Lys Phe His

1730 1735 1740

aca cag gct ttt tcc gct cac ggc agc ggg aga gaa gtc att gat 5392

Thr Gln Ala Phe Ser Ala His Gly Ser Gly Arg Glu Val Ile Asp

1745 1750 1755

gcc atg tgc cat gcc acc cta act tac agg atg ttg gaa cca act 5437

Ala Met Cys His Ala Thr Leu Thr Tyr Arg Met Leu Glu Pro Thr

1760 1765 1770

agg gtt gtt aac tgg gaa gtg atc att atg gat gaa gcc cat ttt 5482

Arg Val Val Asn Trp Glu Val Ile Ile Met Asp Glu Ala His Phe

1775 1780 1785

ttg gat cca gec agc ata gcc gct aga ggt tgg gca gcg cac aga 5527

Leu Asp Pro Ala Ser Ile Ala Ala Arg Gly Trp Ala Ala His Arg

1790 1795 1800

gct agg gca aat gaa agt gca aca atc ttg atg aca gcc aca ccg 5572

Ala Arg Ala Asn Glu Ser Ala Thr Ile Leu Met Thr Ala Thr Pro

1805 1810 1815

cct ggg act agt gat gaa ttt cca cat tca aat ggt gaa ata gaa 5617

Pro Gly Thr Ser Asp Glu Phe Pro His Ser Asn Gly Glu Ile Glu

1820 1825 1830

gat gtt caa acg gac ata ccc agt gag ccc tgg aae aca ggg cat 5662

Asp Val Gln Thr Asp Ile Pro Ser Glu Pro Trp Asn Thr Gly His

1835 1840 1845

gac tgg atc ctg gct gac aaa agg ccc acg gca tgg ttc ctt cca 5707

Asp Trp Ile Leu Ala Asp Lys Arg Pro Thr Ala Trp Phe Leu Pro

1850 1855 1860

tcc atc aga gct gca aat gtc atg gct gcc tct ttg cgt aag gct 5752

Ser Ile Arg Ala Ala Asn Val Met Ala Ala Ser Leu Arg Lys Ala

1865 1870 1875

gga aag agt gtg gtg gtc ctg aac agg aaa acc ttt gag aga gaa 5797

Gly Lys Ser Val Val Val Leu Asn Arg Lys Thr Phe Glu Arg Glu

1880 1885 1890

tac ccc acg ata aag cag aag aaa cct gac ttt ata ttg gcc act 5842

Tyr Pro Thr Ile Lys Gln Lys Lys Pro Asp Phe Ile Leu Ala Thr

1895 1900 1905

gac ata gct gaa atg gga gcc aac ctt tgc gtg gag cga gtg ctg 5887

Asp Ile Ala Glu Met Gly Ala Asn Leu Cys Val Glu Arg Val Leu

1910 1915 1920

gat tgc agg acg gct ttt aag cct gtg ctt gtg gat gaa ggg agg 5932

Asp Cys Arg Thr Ala Phe Lys Pro Val Leu Val Asp Glu Gly Arg

1925 1930 1935

aag gtg gca ata aaa ggg cca ctt cgt atc tcc gca tcc tct gct 5977

Lys Val Ala Ile Lys Gly Pro Leu Arg Ile Ser Ala Ser Ser Ala

1940 1945 1950

gct caa agg agg ggg cgc att ggg aga aat ccc aac aga gat gga 6022

Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn Pro Asn Arg Asp Gly

1955 1960 1965

gac tca tac tac tat tct gag cct aca agt gaa aat aat gcc cac 6067

Asp Ser Tyr Tyr Tyr Ser Glu Pro Thr Ser Glu Asn Asn Ala His

1970 1975 1980

cac gtc tgc tgg ttg gag gcc tca atg ctc ttg gac aac atg gag 6112

His Val Cys Trp Leu Glu Ala Ser Met Leu Leu Asp Asn Met Glu

1985 1990 1995

gtg agg ggt gga atg gtc gcc cca ctc tat ggc gtt gaa gga act 6157

Val Arg Gly Gly Met Val Ala Pro Leu Tyr Gly Val Glu Gly Thr

2000 2005 2010

aaa aca cca gtt tcc cct ggt gaa atg aga ctg agg gat gac cag 6202

Lys Thr Pro Val Ser Pro Gly Glu Met Arg Leu Arg Asp Asp Gln

2015 2020 2025

agg aaa gtc ttc aga gaa cta gtg agg aat tgt gac ctg ccc gtt 6247

Arg Lys Val Phe Arg Glu Leu Val Arg Asn Cys Asp Leu Pro Val

2030 2035 2040

tgg ctt tcg tgg caa gtg gcc aag gct ggt ttg aag acg aat gat 6292

Trp Leu Ser Trp Gln Val Ala Lys Ala Gly Leu Lys Thr Asn Asp

2045 2050 2055

cgt aag tgg tgt ttt gaa ggc cct gag gaa cat gag atc ttg aat 6337

Arg Lys Trp Cys Phe Glu Gly Pro Glu Glu His Glu Ile Leu Asn

2060 2065 2070

gac agc ggt gaa aca gtg aag tgc agg gct cct gga gga gca aag 6382

Asp Ser Gly Glu Thr Val Lys Cys Arg Ala Pro Gly Gly Ala Lys

2075 2080 2085

aag cct ctg cgc cca agg tgg tgt gat gaa agg gtg tca tct gac 6427

Lys Pro Leu Arg Pro Arg Trp Cys Asp Glu Arg Val Ser Ser Asp

2090 2095 2100

cag agt gcg ctg tct gaa ttt att aag ttt gct gaa ggt agg agg 6472

Gln Ser Ala Leu Ser Glu Phe Ile Lys Phe Ala Glu Gly Arg Arg

2105 2110 2115

gga gct gct gaa gtg cta gtt gtg ctg agt gaa ctc cct gat ttc 6517

Gly Ala Ala Glu Val Leu Val Val Leu Ser Glu Leu Pro Asp Phe

2120 2125 2130

ctg gct aaa aaa ggt gga gag gca atg gat acc atc agt gtg ttc 6562

Leu Ala Lys Lys Gly Gly Glu Ala Met Asp Thr Ile Ser Val Phe

2135 2140 2145

ctc cac tct gag gaa ggc tct agg gct tac cgc aat gca cta tca 6607

Leu His Ser Glu Glu Gly Ser Arg Ala Tyr Arg Asn Ala Leu Ser

2150 2155 2160

atg atg cct gag gca atg aca ata gtc atg ctg ttt ata ctg gct 6652

Met Met Pro Glu Ala Met Thr Ile Val Met Leu Phe Ile Leu Ala

2165 2170 2175

gga cta ctg aca tcg gga atg gtc atc ttt ttc atg tct ccc aaa 6697

Gly Leu Leu Thr Ser Gly Met Val Ile Phe Phe Met Ser Pro Lys

2180 2185 2190

ggc atc agt aga atg tct atg gcg atg ggc aca atg gcc ggc tgt 6742

Gly Ile Ser Arg Met Ser Met Ala Met Gly Thr Met Ala Gly Cys

2195 2200 2205

gga tat ctc atg ttc ctt gga ggc gtc aaa ccc act cac atc tcc 6787

Gly Tyr Leu Met Phe Leu Gly Gly Val Lys Pro Thr His Ile Ser

2210 2215 2220

tat gtc atg ctc ata ttc ttt gtc ctg atg gtg gtt gtg atc ccc 6832

Tyr Val Met Leu Ile Phe Phe Val Leu Met Val Val Val Ile Pro

2225 2230 2235

gag cca ggg caa caa agg tcc atc caa gac aac caa gtg gca tac 6877

Glu Pro Gly Gln Gln Arg Ser Ile Gln Asp Asn Gln Val Ala Tyr

2240 2245 2250

ctc att att ggc atc ctg acg ctg gtt tca gcg gtg gca gcc aac 6922

Leu Ile Ile Gly Ile Leu Thr Leu Val Ser Ala Val Ala Ala Asn

2255 2260 2265

gag cta ggc atg ctg gag aaa acc aaa gag gac ctc ttt ggg aag 6967

Glu Leu Gly Met Leu Glu Lys Thr Lys Glu Asp Leu Phe Gly Lys

2270 2275 2280

aag aac tta att cca tct agt gct tca ccc tgg agt tgg ccg gat 7012

Lys Asn Leu Ile Pro Ser Ser Ala Ser Pro Trp Ser Trp Pro Asp

2285 2290 2295

ctt gac ctg aag cca gga gct gcc tgg aca gtg tac gtt ggc att 7057

Leu Asp Leu Lys Pro Gly Ala Ala Trp Thr Val Tyr Val Gly Ile

2300 2305 2310

gtt aca atg ctc tct cca atg ttg cac cac tgg atc aaa gtc gaa 7102

Val Thr Met Leu Ser Pro Met Leu His His Trp Ile Lys Val Glu

2315 2320 2325

tat ggc aac ctg tct ctg tct gga ata gcc cag tca gcc tca gtc 7147

Tyr Gly Asn Leu Ser Leu Ser Gly Ile Ala Gln Ser Ala Ser Val

2330 2335 2340

ctt tct ttc atg gac aag ggg ata cca ttc atg aag atg aat atc 7192

Leu Ser Phe Met Asp Lys Gly Ile Pro Phe Met Lys Met Asn Ile

2345 2350 2355

tcg gtc ata atg ctg ctg gtc agt ggc tgg aat tca ata aca gtg 7237

Ser Val Ile Met Leu Leu Val Ser Gly Trp Asn Ser Ile Thr Val

2360 2365 2370

atg cct ctg ctc tgt ggc ata ggg tgc gcc atg ctc cac tgg tct 7282

Met Pro Leu Leu Cys Gly Ile Gly Cys Ala Met Leu His Trp Ser

2375 2380 2385

ctc att tta cct gga atc aaa gcg cag cag tca aag ctt gca cag 7327

Leu Ile Leu Pro Gly Ile Lys Ala Gln Gln Ser Lys Leu Ala Gln

2390 2395 2400

aga agg gtg ttc cat ggc gtt gcc aag aac cct gtg gtt gat ggg 7372

Arg Arg Val Phe His Gly Val Ala Lys Asn Pro Val Val Asp Gly

2405 2410 2415

aat cca aca gtt gac att gag gaa gct cct gaa atg cct gcc ctt 7417

Asn Pro Thr Val Asp Ile Glu Glu Ala Pro Glu Met Pro Ala Leu

2420 2425 2430

tat gag aag aaa ctg gct cta tat ctc ctt ctt gct ctc agc cta 7462

Tyr Glu Lys Lys Leu Ala Leu Tyr Leu Leu Leu Ala Leu Ser Leu

2435 2440 2445

gct tct gtt gcc atg tgc aga acg ccc ttt tca ttg gct gaa ggc 7507

Ala Ser Val Ala Met Cys Arg Thr Pro Phe Ser Leu Ala Glu Gly

2450 2455 2460

att gtc cta gca tca gct gcc tta ggg ccg ctc ata gag gga aac 7552

Ile Val Leu Ala Ser Ala Ala Leu Gly Pro Leu Ile Glu Gly Asn

2465 2470 2475

acc agc ctt ctt tgg aat gga ccc atg gct gtc tcc atg aca gga 7597

Thr Ser Leu Leu Trp Asn Gly Pro Met Ala Val Ser Met Thr Gly

2480 2485 2490

gtc atg agg ggg aat cac tat gct ttt gtg gga gtc atg tac aat 7642

Val Met Arg Gly Asn His Tyr Ala Phe Val Gly Val Met Tyr Asn

2495 2500 2505

cta tgg aag atg aaa act gga cgc cgg ggg agc gcg aat gga aaa 7687

Leu Trp Lys Met Lys Thr Gly Arg Arg Gly Ser Ala Asn Gly Lys

2510 2515 2520

act ttg ggt gaa gtc tgg aag agg gaa ctg aat ctg ttg gac aag 7732

Thr Leu Gly Glu Val Trp Lys Arg Glu Leu Asn Leu Leu Asp Lys

2525 2530 2535

cga cag ttt gag ttg tat aaa agg acc gac att gtg gag gtg gat 7777

Arg Gln Phe Glu Leu Tyr Lys Arg Thr Asp Ile Val Glu Val Asp

2540 2545 2550

cgt gat acg gca cgc agg cat ttg gcc gaa ggg aag gtg gac acc 7822

Arg Asp Thr Ala Arg Arg His Leu Ala Glu Gly Lys Val Asp Thr

2555 2560 2565

ggg gtg gcg gtc tcc agg ggg acc gca aag tta agg tgg ttc cat 7867

Gly Val Ala Val Ser Arg Gly Thr Ala Lys Leu Arg Trp Phe His

2570 2575 2580

gag cgt ggc tat gtc aag ctg gaa ggt agg gtg att gac ctg ggg 7912

Glu Arg Gly Tyr Val Lys Leu Glu Gly Arg Val Ile Asp Leu Gly

2585 2590 2595

tgt ggc cgc gga ggc tgg tgt tac tac gct gct gcg caa aag gaa 7957

Cys Gly Arg Gly Gly Trp Cys Tyr Tyr Ala Ala Ala Gln Lys Glu

2600 2605 2610

gtg agt ggg gtc aaa gga ttt act ctt gga aga gac ggc cat gag 8002

Val Ser Gly Val Lys Gly Phe Thr Leu Gly Arg Asp Gly His Glu

2615 2620 2625

aaa ccc atg aat gtg caa agt ctg gga tgg aac atc atc acc ttc 8047

Lys Pro Met Asn Val Gln Ser Leu Gly Trp Asn Ile Ile Thr Phe

2630 2635 2640

aag gac aaa act gat atc cac cgc cta gaa cca gtg aaa tgt gac 8092

Lys Asp Lys Thr Asp Ile His Arg Leu Glu Pro Val Lys Cys Asp

2645 2650 2655

acc ctt ttg tgt gac att gga gag tca tca tcg tca tcg gtc aca 8137

Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Ser Ser Ser Val Thr

2660 2665 2670

gag ggg gaa agg acc gtg aga gtt ctt gat act gta gaa aaa tgg 8182

Glu Gly Glu Arg Thr Val Arg Val Leu Asp Thr Val Glu Lys Trp

2675 2680 2685

ctg gct tgt ggg gtt gac aac ttc tgt gtg aag gtg tta gct cca 8227

Leu Ala Cys Gly Val Asp Asn Phe Cys Val Lys Val Leu Ala Pro

2690 2695 2700

tac atg cca gat gtt ctt gag aaa ctg gaa ttg ctc caa agg agg 8272

Tyr Met Pro Asp Val Leu Glu Lys Leu Glu Leu Leu Gln Arg Arg

2705 2710 2715

ttt ggc gga aca gtg atc agg aac cct ctc tcc agg aat tcc act 8317

Phe Gly Gly Thr Val Ile Arg Asn Pro Leu Ser Arg Asn Ser Thr

2720 2725 2730

cat gaa atg tac tac gtg tct gga gcc cgc agc aat gtc aca ttt 8362

His Glu Met Tyr Tyr Val Ser Gly Ala Arg Ser Asn Val Thr Phe

2735 2740 2745

act gtg aac caa aca tcc cgc ctc ctg atg agg aga atg agg egt 8407

Thr Val Asn Gln Thr Ser Arg Leu Leu Met Arg Arg Met Arg Arg

2750 2755 2760

cca act gga aaa gtg acc ctg gag gct gac gtc atc ctc cca att 8452

Pro Thr Gly Lys Val Thr Leu Glu Ala Asp Val Ile Leu Pro Ile

2765 2770 2775

ggg aca cgc agt gtt gag aca gac aag gga ccc ctg gac aaa gag 8497

Gly Thr Arg Ser Val Glu Thr Asp Lys Gly Pro Leu Asp Lys Glu

2780 2785 2790

gcc ata gaa gaa agg gtt gag agg ata aaa tct gag tac atg acc 8542

Ala Ile Glu Glu Arg Val Glu Arg Ile Lys Ser Glu Tyr Met Thr

2795 2800 2805

tct tgg ttt tat gac aat gac aac ccc tac agg acc tgg cac tac 8587

Ser Trp Phe Tyr Asp Asn Asp Asn Pro Tyr Arg Thr Trp His Tyr

2810 2815 2820

tgt ggc tcc tat gtc aca aaa acc tcc gga agt gcg gcg agc atg 8632

Cys Gly Ser Tyr Val Thr Lys Thr Ser Gly Ser Ala Ala Ser Met

2825 2830 2835

gta aat ggt gtt att aaa att ctg aca tat cca tgg gac agg ata 8677

Val Asn Gly Val Ile Lys Ile Leu Thr Tyr Pro Trp Asp Arg Ile

2840 2845 2850

gag gag gtc aca aga atg gca atg act gac aca acc cct ttt gga 8722

Glu Glu Val Thr Arg Met Ala Met Thr Asp Thr Thr Pro Phe Gly

2855 2860 2865

cag caa aga gtg ttt aaa gaa aaa gtt gac acc aga gca aag gat 8767

Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg Ala Lys Asp

2870 2875 2880

cca cca gcg gga act agg aag atc atg aaa gtt gtc aac agg tgg 8812

Pro Pro Ala Gly Thr Arg Lys Ile Met Lys Val Val Asn Arg Trp

2885 2890 2895

ctg ttc cgc cac ctg gcc aga gaa aag aac ccc aga ctg tgc aca 8857

Leu Phe Arg His Leu Ala Arg Glu Lys Asn Pro Arg Leu Cys Thr

2900 2905 2910

aag gaa gaa ttt att gca aaa gtc cga agt cat gca gcc att gga 8902

Lys Glu Glu Phe Ile Ala Lys Val Arg Ser His Ala Ala Ile Gly

2915 2920 2925

gct tac ctg gaa gaa caa gaa cag tgg aag act gcc aat gag gct 8947

Ala Tyr Leu Glu Glu Gln Glu Gln Trp Lys Thr Ala Asn Glu Ala

2930 2935 2940

gtc caa gac cca aag ttc tgg gaa ctg gtg gat gaa gaa agg aag 8992

Val Gln Asp Pro Lys Phe Trp Glu Leu Val Asp Glu Glu Arg Lys

2945 2950 2955

ctg cac caa caa ggc agg tgt cgg act tgt gtg tac aac atg atg 9037

Leu His Gln Gln Gly Arg Cys Arg Thr Cys Val Tyr Asn Met Met

2960 2965 2970

ggg aaa aga gag aag aag ctg tca gag ttt ggg aaa gca aag gga 9082

Gly Lys Arg Glu Lys Lys Leu Ser Glu Phe Gly Lys Ala Lys Gly

2975 2980 2985

agc cgt gcc ata tgg tat atg tgg ctg gga gcg cgg tat ctt gag 9127

Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Tyr Leu Glu

2990 2995 3000

ttt gag gcc ctg gga ttc ctg aat gag gac cat tgg gct tcc agg 9172

Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Ala Ser Arg

3005 3010 3015

gaa aac tca gga gga gga gtg gaa ggc att ggc tta caa tac cta 9217

Glu Asn Ser Gly Gly Gly Val Glu Gly Ile Gly Leu Gln Tyr Leu

3020 3025 3030

gga tat gtg atc aga gac ctg gct gca atg gat ggt ggt gga ttc 9262

Gly Tyr Val Ile Arg Asp Leu Ala Ala Met Asp Gly Gly Gly Phe

3035 3040 3045

tac gcg gat gac acc gct gga tgg gac acg cgc atc aca gag gca 9307

Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Ala

3050 3055 3060

gac ctt gat gat gaa cag gag atc ttg aac tac atg agc cca cat 9352

Asp Leu Asp Asp Glu Gln Glu Ile Leu Asn Tyr Met Ser Pro His

3065 3070 3075

cac aaa aaa ctg gca caa gca gtg atg gaa atg aca tac aag aac 9397

His Lys Lys Leu Ala Gln Ala Val Met Glu Met Thr Tyr Lys Asn

3080 3085 3090

aaa gtg gtg aaa gtg ttg aga cca gcc cca gga ggg aaa gcc tac 9442

Lys Val Val Lys Val Leu Arg Pro Ala Pro Gly Gly Lys Ala Tyr

3095 3100 3105

atg gat gtc ata agt cga cga gac cag aga gga tcc ggg cag gta 9487

Mct Asp Val Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly Gln Val

3110 3115 3120

gtg act tat gct ctg aac acc atc acc aac ttg aaa gtc caa ttg 9532

Val Thr Tyr Ala Leu Asn Thr Ile Thr Asn Leu Lys Val Gln Leu

3125 3130 3135

atc aga atg gca gaa gca gag atg gtg ata cat cac caa cat gtt 9577

Ile Arg Met Ala Glu Ala Glu Met Val Ile His His Gln His Val

3140 3145 3150

caa gat tgt gat gaa tca gtt ctg acc agg ctg gag gca tgg ctc 9622

Gln Asp Cys Asp Glu Ser Val Leu Thr Arg Leu Glu Ala Trp Leu

3155 3160 3165

act gag cac gga tgt gac aga ctg aag agg atg gcg gtg agt gga 9667

Thr Glu His Gly Cys Asp Arg Leu Lys Arg Met Ala Val Ser Gly

3170 3175 3180

gac gac tgt gtg gtc cgg ccc atc gat gac agg ttc ggc ctg gcc 9712

Asp Asp Cys Val Val Arg Pro Ile Asp Asp Arg Phe Gly Leu Ala

3185 3190 3195

ctg tcc cat ctc aac gcc atg tcc aag gtt aga aag gac ata tct 9757

Lcu Ser His Leu Asn Ala Met Ser Lys Val Arg Lys Asp Ile Ser

3200 3205 3210

gaa tgg cag cca tca aaa ggg tgg aat gat tgg gag aat gtg ccc 9802

Glu Trp Gln Pro Ser Lys Gly Trp Asn Asp Trp Glu Asn Val Pro

3215 3220 3225

ttc tgt tcc cac cac ttc cat gaa cta cag ctg aag gat ggc agg 9847

Phe Cys Ser His His Phe His Glu Leu Gln Leu Lys Asp Gly Arg

3230 3235 3240

agg att gtg gtg cct tgc cga gaa cag gac gag ctc att ggg aga 9892

Arg Ile Val Val Pro Cys Arg Glu Gln Asp Glu Leu Ile Gly Arg

3245 3250 3255

gga agg gtg tct cca gga aac ggc tgg atg atc aag gaa aca gct 9937

Gly Arg Val Ser Pro Gly Asn Gly Trp Met Ile Lys Glu Thr Ala

3260 3265 3270

tgc ctc agc aaa gcc tat gcc aac atg tgg tca ctg atg tat ttt 9982

Cys Leu Ser Lys Ala Tyr Ala Asn Met Trp Ser Leu Met Tyr Phe

3275 3280 3285

cac aaa agg gac atg agg cta ctg tca ttg gct gtt tcc tca gct 10027

His Lys Arg Asp Met Arg Leu Leu Ser Leu Ala Val Ser Ser Ala

3290 3295 3300

gtt ccc acc tca tgg gtt cca caa gga cgc aca aca tgg tcg att 10072

Val Pro Thr Ser Trp Val Pro Gln Gly Arg Thr Thr Trp Ser Ile

3305 3310 3315

cat ggg aaa ggg gag tgg atg acc acg gaa gac atg ctt gag gtg 10117

His Gly Lys Gly Glu Trp Met Thr Thr Glu Asp Met Leu Glu Val

3320 3325 3330

tgg aac aga gta tgg ata acc aac aac cca cac atg cag gac aag 10162

Trp Asn Arg Val Trp Ile Thr Asn Asn Pro His Met Gln Asp Lys

3335 3340 3345

aca atg gtg aaa aaa tgg aga gat gtc cct tat cta acc aag aga 10207

Thr Met Val Lys Lys Trp Arg Asp Val Pro Tyr Leu Thr Lys Arg

3350 3355 3360

caa gac aag ctg tgc gga tca ctg att gga atg acc aat agg gcc 10252

Gln Asp Lys Leu Cys Gly Ser Leu Ile Gly Met Thr Asn Arg Ala

3365 3370 3375

acc tgg gcc tcc cac atc cat tta gtc atc cat cgt atc cga acg 10297

Thr Trp Ala Ser His Ile His Leu Val Ile His Arg Ile Arg Thr

3380 3385 3390

ctg att gga cag gag aaa tac act gac tac cta aca gtc atg gac 10342

Leu Ile Gly Gln Glu Lys Tyr Thr Asp Tyr Leu Thr Val Met Asp

3395 3400 3405

agg tat tct gtg gat gct gac ctg caa ctg ggt gag ctt atc 10384

Arg Tyr Ser Val Asp Ala Asp Leu Gln Leu Gly Glu Leu Ile

3410 3415 3420

tgaaacacca tctaacagga ataaccggga tacaaaccac gggtggagaa ccggactccc 10444

cacaacctga aaccgggata taaaccacgg ctggagaacc ggactccgca cttaaaatga 10504

aacagaaacc gggataaaaa ctacggatgg agaaccggac tccacacatt gagacagaag 10564

aagttgtcag cccagaaccc cacacgagtt ttgccactgc taagctgtga ggcagtgcag 10624

gctgggacag ccgacctcca ggttgcgaaa aacctggttt ctgggacctc ccaccccaga 10684

gtaaaaagaa cggagcctcc gctaccaccc tcccacgtgg tggtagaaag acggggtcta 10744

gaggttagag gagaccctcc agggaacaaa tagtgggacc atattgacgc cagggaaaga 10804

ccggagtggt tctctgcttt tcctccagag gtctgtgagc acagtttgct casgaataag 10864

cagacctttg gatgacaaac acaaaaaccac aa 10896

<210>21

<211>3422

<212>PRT

<213> Artificial

<220>

<223> synthetic constructs

<400>21

Met Ser Gly Arg Lys Ala Gln Gly Lys Thr Leu Gly Val Asn Met Val

1 5 10 15

Arg Arg Gly Val Arg Ser Leu Ser Asn Lys Ile Lys Gln Lys Thr Lys

20 25 30

Gln Ile Gly Asn Arg Pro Gly Pro Ser Arg Gly Val Gln Gly Phe Ile

35 40 45

Phe Phe Phe Leu Phe Asn Ile Leu Thr Gly Lys Lys Ile Thr Ala His

50 55 60

Leu Lys Arg Leu Trp Lys Met Leu Asp Pro Arg Gln Gly Leu Ala Val

65 70 75 80

Leu Arg Lys Val Lys Arg Val Val Ala Ser Leu Met Arg Gly Leu Ser

85 90 95

Ser Arg Lys Arg Arg Ser His Asp Val Leu Thr Val Gln Phe Leu Ile

100 105 110

Leu Gly Met Leu Leu Met Thr Gly Gly Val Thr Leu Ser Asn Phe Gln

115 120 125

Gly Lys Val Met Met Thr Val Asn Ala Thr Asp Val Thr Asp Val Ile

130 135 140

Thr Ile Pro Thr Ala Ala Gly Lys Asn Leu Cys Ile Val Arg Ala Met

145 150 155 160

Asp Val Gly Tyr Met Cys Asp Asp Thr Ile Thr Tyr Glu Cys Pro Val

165 170 175

Leu Ser Ala Gly Asn Asp Pro Glu Asp Ile Asp Cys Trp Cys Thr Lys

180 185 190

Ser Ala Val Tyr Val Arg Tyr Gly Arg Cys Thr Lys Thr Arg His Ser

195 200 205

Arg Arg Ser Arg Arg Ser Leu Thr Val Gln Thr His Gly Glu Ser Thr

210 215 220

Leu Ala Asn Lys Lys Gly Ala Trp Met Asp Ser Thr Lys Ala Thr Arg

225 230 235 240

Tyr Leu Val Lys Thr Glu Ser Trp Ile Leu Arg Asn Pro Gly Tyr Ala

245 250 255

Leu Val Ala Ala Val Ile Gly Trp Met Leu Gly Ser Asn Thr Met Gln

260 265 270

Arg Val Val Phe Val Val Leu Leu Leu Leu Val Ala Pro Ala Tyr Ser

275 280 285

Phe Asn Cys Leu Gly Met Ser Asn Arg Asp Phe Leu Glu Gly Val Ser

290 295 300

Gly Ala Thr Trp Val Asp Leu Val Leu Glu Gly Asp Ser Cys Val Thr

305 310 315 320

Ile Met Ser Lys Asp Lys Pro Thr Ile Asp Val Lys Met Met Asn Met

325 330 335

Glu Ala Ala Asn Leu Ala Glu Val Arg Ser Tyr Cys Tyr Leu Ala Thr

340 345 350

Val Ser Asp Leu Ser Thr Lys Ala Ala Cys Pro Thr Met Gly Glu Ala

355 360 365

His Asn Asp Lys Arg Ala Asp Pro Ala Phe Val Cys Arg Gln Gly Val

370 375 380

Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Phe Phe Gly Lys Gly Ser

385 390 395 400

Ile Asp Thr Cys Ala Lys Phe Ala Cys Ser Thr Lys Ala Ile Gly Arg

405 410 415

Thr Ile Leu Lys Glu Asn Ile Lys Tyr Glu Val Ala Ile Phe Val His

420 425 430

Gly Pro Thr Thr Val Glu Ser His Gly Asn Tyr Ser Thr Gln Val Gly

435 440 445

Ala Thr Gln Ala Gly Arg Phe Ser Ile Thr Pro Ala Ala Pro Ser Tyr

450 455 460

Thr Leu Lys Leu Gly Glu Tyr Gly Glu Val Thr Val Asp Cys Glu Pro

465 470 475 480

Arg Ser Gly Ile Asp Thr Asn Ala Tyr Tyr Val Met Thr Val Gly Thr

485 490 495

Lys Thr Phe Leu Val His Arg Glu Trp Phe Met Asp Leu Asn Leu Pro

500 505 510

Trp Ser Ser Ala Gly Ser Thr Val Trp Arg Asn Arg Glu Thr Leu Met

515 520 525

Glu Phe Glu Glu Pro His Ala Thr Lys Gln Ser Val Ile Ala Leu Gly

530 535 540

Ser Gln Glu Gly Ala Leu His Gln Ala Leu Ala Gly Ala Ile Pro Val

545 550 555 560

Glu Phe Ser Ser Asn Thr Val Lys Leu Thr Ser Gly His Leu Lys Cys

565 570 575

Arg Val Lys Met Glu Lys Leu Gln Leu Lys Gly Thr Thr Tyr Gly Val

580 585 590

Cys Ser Lys Ala Phe Lys Phe Leu Arg Thr Pro Val Asp Thr Gly His

595 600 605

Gly Thr Val Val Leu Glu Leu Gln Tyr Thr Gly Thr Asp Gly Pro Cys

610 615 620

Lys Val Pro Ile Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val

625 630 635 640

Gly Arg Leu Val Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn

645 650 655

Ala Lys Val Leu Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile

660 665 670

Val Val Gly Arg Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser

675 680 685

Gly Ser Ser Ile Gly Lys Ala Phe Thr Thr Thr Leu Lys Gly Ala Gln

690 695 700

Arg Leu Ala Ala Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val Gly

705 710 715 720

Gly Val Phe Thr Ser Val Gly Arg Ala Val His Gln Val Phe Gly Gly

725 730 735

Ala Phe Arg Ser Leu Phe Gly Gly Met Ser Trp Ile Thr Gln Gly Leu

740 745 750

Leu Gly Ala Leu Leu Leu Trp Met Gly Ile Asn Ala Arg Asp Arg Ser

755 760 765

Ile Ala Leu Thr Phe Leu Ala Val Gly Gly Val Leu Leu Phe Leu Ser

770 775 780

Val Asn Val Gly Ala Asp Gln Gly Cys Ala Ile Asn Phe Gly Lys Arg

785 790 795 800

Glu Leu Lys Cys Gly Asp Gly Ile Phe Ile Phe Arg Asp Ser Asp Asp

805 810 815

Trp Leu Asn Lys Tyr Ser Tyr Tyr Pro Glu Asp Pro Val Lys Leu Ala

820 825 830

Ser Ile Val Lys Ala Ser Phe Glu Glu Gly Lys Cys Gly Leu Asn Ser

835 840 845

Val Asp Ser Leu Glu His Glu Met Trp Arg Ser Arg Ala Asp Glu Ile

850 855 860

Asn Ala Ile Phe Glu Glu Asn Glu Val Asp Ile Ser Val Val Val Gln

865 870 875 880

Asp Pro Lys Asn Val Tyr Gln Arg Gly Thr His Pro Phe Ser Arg Ile

885 890 895

Arg Asp Gly Leu Gln Tyr Gly Trp Lys Thr Trp Gly Lys Asn Leu Val

900 905 910

Phe Ser Pro Gly Arg Lys Asn Gly Ser Phe Ile Ile Asp Gly Lys Ser

915 920 925

Arg Lys Glu Cys Pro Phe Ser Asn Arg Val Trp Asn Ser Phe Gln Ile

930 935 940

Glu Glu Phe Gly Thr Gly Val Phe Thr Thr Arg Val Tyr Met Asp Ala

945 950 955 960

Val Phe Glu Tyr Thr Ile Asp Cys Asp Gly Ser Ile Leu Gly Ala Ala

965 970 975

Val Asn Gly Lys Lys Ser Ala His Gly Ser Pro Thr Phe Trp Met Gly

980 985 990

Ser His Glu Val Asn Gly Thr Trp Met Ile His Thr Leu Glu Ala Leu

995 1000 1005

Asp Tyr Lys Glu Cys Glu Trp Pro Leu Thr His Thr Ile Gly Thr

1010 1015 1020

Ser Val Glu Glu Ser Glu Met Phe Met Pro Arg Ser Ile Gly Gly

1025 1030 1035

Pro Val Ser Ser His Asn His Ile Pro Gly Tyr Lys Val Gln Thr

1040 1045 1050

Asn Gly Pro Trp Met Gln Val Pro Leu Glu Val Lys Arg Glu Ala

1055 1060 1065

Cys Pro Gly Thr Ser Val Ile Ile Asp Gly Asn Cys Asp Gly Arg

1070 1075 1080

Gly Lys Ser Thr Arg Ser Thr Thr Asp Ser Gly Lys Val Ile Pro

1085 1090 1095

Glu Trp Cys Cys Arg Ser Cys Thr Met Pro Pro Val Ser Phe His

1100 1105 1110

Gly Ser Asp Gly Cys Trp Tyr Pro Met Glu Ile Arg Pro Arg Lys

1115 1120 1125

Thr His Glu Ser His Leu Val Arg Ser Trp Val Thr Ala Gly Glu

1130 1135 1140

Ile His Ala Val Pro Phe Gly Leu Val Ser Met Met Ile Ala Met

1145 1150 1155

Glu Val Val Leu Arg Lys Arg Gln Gly Pro Lys Gln Met Leu Val

1160 1165 1170

Gly Gly Val Val Leu Leu Gly Ala Met Leu Val Gly Gln Val Thr

1175 1180 1185

Leu Leu Asn Leu Leu Lys Leu Thr Val Ala Val Gly Leu His Phe

1190 1195 1200

His Glu Met Asn Asn Gly Gly Asp Ala Met Tyr Met Ala Leu Ile

1205 1210 1215

Ala Ala Phe Ser Ile Arg Pro Gly Leu Leu Ile Gly Phe Gly Leu

1220 1225 1230

Arg Thr Leu Trn Ser Pro Arg Glu Arg Leu Val Leu Thr Leu Gly

1235 1240 1245

Ala Ala Met Val Glu Ile Ala Leu Gly Gly Val Met Gly Gly Leu

1250 1255 1260

Trp Lys Tyr Leu Asn Ala Val Ser Leu Cys Ile Leu Thr Ile Asn

1265 1270 1275

Ala Val Ala Ser Arg Lys Ala Ser Asn Thr Ile Leu Pro Leu Met

1280 1285 1290

Ala Leu Leu Thr Pro Val Thr Met Ala Glu Val Arg Leu Ala Ala

1295 1300 1305

Met Phe Phe Cys Ala Met Val Ile Ile Gly Val Leu His Gln Asn

1310 1315 1320

Phe Lys Asn Thr Ser Met Gln Lys Thr Ile Pro Leu Val Ala Leu

1325 1330 1335

Thr Leu Thr Ser Tyr Leu Gly Leu Thr Gln Pro Phe Leu Gly Leu

1340 1345 1350

Cys Ala Phe Leu Ala Thr Arg Ile Phe Gly Arg Arg Ser Ile Pro

1355 1360 1365

Val Asn Glu Ala Leu Ala Ala Ala Gly Leu Val Gly Val Leu Ala

1370 1375 1380

Gly Leu Ala Phe Gln Glu Met Glu Asn Phe Leu Gly Pro Ile Ala

1385 1390 1395

Val Gly Gly Leu Leu Met Met Leu Val Ser Val Ala Gly Arg Val

1400 1405 1410

Asp Gly Leu Glu Leu Lys Lys Leu Gly Glu Val Ser Trp Glu Glu

1415 1420 1425

Glu Ala Glu Ile Ser Gly Ser Ser Ala Arg Tyr Asp Val Ala Leu

1430 1435 1440

Ser Glu Gln Gly Glu Phe Lys Leu Leu Ser Glu Glu Lys Val Pro

1445 1450 1455

Trp Asp Gln Val Val Met Thr Ser Leu Ala Leu Val Gly Ala Ala

1460 1465 1470

Leu His Pro Phe Ala Leu Leu Leu Val Leu Ala Gly Trp Leu Phe

1475 1480 1485

His Val Arg Gly Ala Arg Arg Ser Gly Asp Val Leu Trp Asp Ile

1490 1495 1500

Pro Thr Pro Lys Ile Ile Glu Glu Cys Glu His Leu Glu Asp Gly

1505 1510 1515

Ile Tyr Gly Ile Phe Gln Ser Thr Phe Leu Gly Ala Ser Gln Arg

1520 1525 1530

Gly Val Gly Val Ala Gln Gly Gly Val Phe His Thr Met Trp His

1535 1540 1545

Val Thr Arg Gly Ala Phe Leu Val Arg Asn Gly Lys Lys Leu Ile

1550 1555 1560

Pro Ser Trp Ala Ser Val Lys Glu Asp Leu Val Ala Tyr Gly Gly

1565 1570 1575

Ser Trp Lys Leu Glu Gly Arg Trp Asp Gly Glu Glu Glu Val Gln

1580 1585 1590

Leu Ile Ala Ala Val Pro Gly Lys Asn Val Val Asn Val Gln Thr

1595 1600 1605

Lys Pro Ser Leu Phe Lys Val Arg Asn Gly Gly Glu Ile Gly Ala

1610 1615 1620

Val Ala Leu Asp Tyr Pro Ser Gly Thr Ser Gly Ser Pro Ile Val

1625 1630 1635

Asn Arg Asn Gly Glu Val Ile Gly Leu Tyr Gly Asn Gly Ile Leu

1640 1645 1650

Val Gly Asp Asn Ser Phe Val Ser Ala Ile Ser Gln Thr Glu Val

1655 1660 1665

Lys Glu Glu Gly Lys Glu Glu Leu Gln Glu Ile Pro Thr Met Leu

1670 1675 1680

Lys Lys Gly Met Thr Thr Val Leu Asp Phe His Pro Gly Ala Gly

1685 1690 1695

Lys Thr Arg Arg Phe Leu Pro Gln Ile Leu Ala Glu Cys Ala Arg

1700 1705 1710

Arg Arg Leu Arg Thr Leu Val Leu Ala Pro Thr Arg Val Val Leu

1715 1720 1725

Ser Glu Met Lys Glu Ala Phe His Gly Leu Asp Val Lys Phe His

1730 1735 1740

Thr Gln Ala Phe Ser Ala His Gly Ser Gly Arg Glu Val Ile Asp

1745 1750 1755

Ala Met Cys His Ala Thr Leu Thr Tyr Arg Met Leu Glu Pro Thr

1760 1765 1770

Arg Val Val Asn Trp Glu Val Ile Ile Met Asp Glu Ala His Phe

1775 1780 1785

Leu Asp Pro Ala Ser Ile Ala Ala Arg Gly Trp Ala Ala His Arg

1790 1795 1800

Ala Arg Ala Asn Glu Ser Ala Thr Ile Leu Met Thr Ala Thr Pro

1805 1810 1815

Pro Gly Thr Ser Asp Glu Phe Pro His Ser Asn Gly Glu Ile Glu

1820 1825 1830

Asp Val Gln Thr Asp Ile Pro Ser Glu Pro Trp Asn Thr Gly His

1835 1840 1845

Asp Trp Ile Leu Ala Asp Lys Arg Pro Thr Ala Trp Phe Leu Pro

1850 1855 1860

Ser Ile Arg Ala Ala Asn Val Met Ala Ala Ser Leu Arg Lys Ala

1865 1870 1875

Gly Lys Ser Val Val Val Leu Asn Arg Lys Thr Phe Glu Arg Glu

1880 1885 1890

Tyr Pro Thr Ile Lys Gln Lys Lys Pro Asp Phe Ile Leu Ala Thr

1895 1900 1905

Asp Ile Ala Glu Met Gly Ala Asn Leu Cys Val Glu Arg Val Leu

1910 1915 1920

Asp Cys Arg Thr Ala Phe Lys Pro Val Leu Val Asp Glu Gly Arg

1925 1930 1935

Lys Val Ala Ile Lys Gly Pro Leu Arg Ile Ser Ala Ser Ser Ala

1940 1945 1950

Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn Pro Asn Arg Asp Gly

1955 1960 1965

Asp Ser Tyr Tyr Tyr Ser Glu Pro Thr Ser Glu Asn Asn Ala His

1970 1975 1980

His Val Cys Trp Leu Glu Ala Ser Met Leu Leu Asp Asn Met Glu

1985 1990 1995

Val Arg Gly Gly Met Val Ala Pro Leu Tyr Gly Val Glu Gly Thr

2000 2005 2010

Lys Thr Pro Val Ser Pro Gly Glu Met Arg Leu Arg Asp Asp Gln

2015 2020 2025

Arg Lys Val Phe Arg Glu Leu Val Arg Asn Cys Asp Leu Pro Val

2030 2035 2040

Trp Leu Ser Trp Gln Val Ala Lys Ala Gly Leu Lys Thr Asn Asp

2045 2050 2055

Arg Lys Trp Cys Phe Glu Gly Pro Glu Glu His Glu Ile Leu Asn

2060 2065 2070

Asp Ser Gly Glu Thr Val Lys Cys Arg Ala Pro Gly Gly Ala Lys

2075 2080 2085

Lys Pro Leu Arg Pro Arg Trp Cys Asp Glu Arg Val Ser Ser Asp

2090 2095 2100

Gln Ser Ala Leu Ser Glu Phe Ile Lys Phe Ala Glu Gly Arg Arg

2105 2110 2115

Gly Ala Ala Glu Val Leu Val Val Leu Ser Glu Leu Pro Asp Phe

2120 2125 2130

Leu Ala Lys Lys Gly Gly Glu Ala Met Asp Thr Ile Ser Val Phe

2135 2140 2145

Leu His Ser Glu Glu Gly Ser Arg Ala Tyr Arg Asn Ala Leu Ser

2150 2155 2160

Met Met Pro Glu Ala Met Thr Ile Val Met Leu Phe Ile Leu Ala

2165 2170 2175

Gly Leu Leu Thr Ser Gly Met Val Ile Phe Phe Met Ser Pro Lys

2180 2185 2190

Gly Ile Ser Arg Met Ser Met Ala Met Gly Thr Met Ala Gly Cys

2195 2200 2205

Gly Tyr Leu Met Phe Leu Gly Gly Val Lys Pro Thr His Ile Ser

2210 2215 2220

Tyr Val Met Leu Ile Phe Phe Val Leu Met Val Val Val Ile Pro

2225 2230 2235

Glu Pro Gly Gln Gln Arg Ser Ile Gln Asp Asn Gln Val Ala Tyr

2240 2245 2250

Leu Ile Ile Gly Ile Leu Thr Leu Val Ser Ala Val Ala Ala Asn

2255 2260 2265

Glu Leu Gly Met Leu Glu Lys Thr Lys Glu Asp Leu Phe Gly Lys

2270 2275 2280

Lys Asn Leu Ile Pro Ser Ser Ala Ser Pro Trp Ser Trp Pro Asp

2285 2290 2295

Leu Asp Leu Lys Pro Gly Ala Ala Trp Thr Val Tyr Val Gly Ile

2300 2305 2310

Val Thr Met Leu Ser Pro Met Leu His His Trp Ile Lys Val Glu

2315 2320 2325

Tyr Gly Asn Leu Ser Leu Ser Gly Ile Ala Gln Ser Ala Ser Val

2330 2335 2340

Leu Ser Phe Met Asp Lys Gly Ile Pro Phe Met Lys Met Asn Ile

2345 2350 2355

Ser Val Ile Met Leu Leu Val Ser Gly Trp Asn Ser Ile Thr Val

2360 2365 2370

Met Pro Leu Leu Cys Gly Ile Gly Cys Ala Met Leu His Trp Ser

2375 2380 2385

Leu Ile Leu Pro Gly Ile Lys Ala Gln Gln Ser Lys Leu Ala Gln

2390 2395 2400

Arg Arg Val Phe His Gly Val Ala Lys Asn Pro Val Val Asp Gly

2405 2410 2415

Asn Pro Thr Val Asp Ile Glu Glu Ala Pro Glu Met Pro Ala Leu

2420 2425 2430

Tyr Glu Lys Lys Leu Ala Leu Tyr Leu Leu Leu Ala Leu Ser Leu

2435 2440 2445

Ala Ser Val Ala Met Cys Arg Thr Pro Phe Ser Leu Ala Glu Gly

2450 2455 2460

Ile Val Leu Ala Ser Ala Ala Leu Gly Pro Leu Ile Glu Gly Asn

2465 2470 2475

Thr Ser Leu Leu Trp Asn Gly Pro Met Ala Val Ser Met Thr Gly

2480 2485 2490

Val Met Arg Gly Asn His Tyr Ala Phe Val Gly Val Met Tyr Asn

2495 2500 2505

Leu Trp Lys Met Lys Thr Gly Arg Arg Gly Ser Ala Asn Gly Lys

2510 2515 2520

Thr Leu Gly Glu Val Trp Lys Arg Glu Leu Asn Leu Leu Asp Lys

2525 2530 2535

Arg Gln Phe Glu Leu Tyr Lys Arg Thr Asp Ile Val Glu Val Asp

2540 2545 2550

Arg Asp Thr Ala Arg Arg His Leu Ala Glu Gly Lys Val Asp Thr

2555 2560 2565

Gly Val Ala Val Ser Arg Gly Thr Ala Lys Leu Arg Trp Phe His

2570 2575 2580

Glu Arg Gly Tyr Val Lys Leu Glu Gly Arg Val Ile Asp Leu Gly

2585 2590 2595

Cys Gly Arg Gly Gly Trp Cys Tyr Tyr Ala Ala Ala Gln Lys Glu

2600 2605 2610

Val Ser Gly Val Lys Gly Phe Thr Leu Gly Arg Asp Gly His Glu

2615 2620 2625

Lys Pro Met Asn Val Gln Ser Leu Gly Trn Asn Ile Ile Thr Phe

2630 2635 2640

Lys Asp Lys Thr Asp Ile His Arg Leu Glu Pro Val Lys Cys Asp

2645 2650 2655

Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Ser Ser Ser Val Thr

2660 2665 2670

Glu Gly Glu Arg Thr Val Arg Val Leu Asp Thr Val Glu Lys Trp

2675 2680 2685

Leu Ala Cys Gly Val Asp Asn Phe Cys Val Lys Val Leu Ala Pro

2690 2695 2700

Tyr Met Pro Asp Val Leu Glu Lys Leu Glu Leu Leu Gln Arg Arg

2705 2710 2715

Phe Gly Gly Thr Val Ile Arg Asn Pro Leu Ser Arg Asn Ser Thr

2720 2725 2730

His Glu Met Tyr Tyr Val Ser Gly Ala Arg Ser Asn Val Thr Phe

2735 2740 2745

Thr Val Asn Gln Thr Ser Arg Leu Leu Met Arg Arg Met Arg Arg

2750 2755 2760

Pro Thr Gly Lys Val Thr Leu Glu Ala Asp Val Ile Leu Pro Ile

2765 2770 2775

Gly Thr Arg Ser Val Glu Thr Asn Lys Gly Pro Leu Asn Lys Glu

2780 2785 2790

Ala Ile Glu Glu Arg Val Glu Arg Lle Lys Ser Glu Tyr Met Thr

2795 2800 2805

Ser Trp Phe Tyr Asp Asn Asp Asn Pro Tyr Arg Thr Trp His Tyr

2810 2815 2820

Cys Gly Ser Tyr Val Thr Lys Thr Ser Gly Ser Ala Ala Ser Met

2825 2830 2835

Val Asn Gly Val Ile Lys Ile Leu Thr Tyr Pro Trn Asn Arg Ile

2840 2845 2850

Glu Glu Val Thr Arg Met Ala Met Thr Asp Thr Thr Pro Phe Gly

2855 2860 2865

Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg Ala Lys Asp

2870 2875 2880

Pro Pro Ala Gly Thr Arg Lys Ile Met Lys Val Val Asn Arg Trp

2885 2890 2895

Leu Phe Arg His Leu Ala Arg Glu Lys Asn Pro Arg Leu Cys Thr

2900 2905 2910

Lys Glu Glu Phe Ile Ala Lys Val Arg Ser His Ala Ala Ile Gly

2915 2920 2925

Ala Tyr Leu Glu Glu Gln Glu Gln Trp Lys Thr Ala Asn Glu Ala

2930 2935 2940

Val Gln Asp Pro Lys Phe Trp Glu Leu Val Asp Glu Glu Arg Lys

2945 2950 2955

Leu His Gln Gln Gly Arg Cys Arg Thr Cys Val Tyr Asn Met Met

2960 2965 2970

Gly Lys Arg Glu Lys Lys Leu Ser Glu Phe Gly Lys Ala Lys Gly

2975 2980 2985

Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Tyr Leu Glu

2990 2995 3000

Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Ala Ser Arg

3005 3010 3015

Glu Asn Ser Gly Gly Gly Val Glu Gly Ile Gly Leu Gln Tyr Leu

3020 3025 3030

Gly Tyr Val Ile Arg Asp Leu Ala Ala Met Asp Gly Gly Gly Phe

3035 3040 3045

Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Ala

3050 3055 3060

Asp Leu Asp Asp Glu Gln Glu Ile Leu Asn Tyr Met Ser Pro His

3065 3070 3075

His Lys Lys Leu Ala Gln Ala Val Met Glu Met Thr Tyr Lys Asn

3080 3085 3090

Lys Val Val Lys Val Leu Arg Pro Ala Pro Gly Gly Lys Ala Tyr

3095 3100 3105

Met Asp Val Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly Gln Val

3110 3115 3120

Val Thr Tyr Ala Leu Asn Thr Ile Thr Asn Leu Lys Val Gln Leu

3125 3130 3135

Ile Arg Met Ala Glu Ala Glu Met Val Ile His His Gln His Val

3140 3145 3150

Gln Asp Cys Asp Glu Ser Val Leu Thr Arg Leu Glu Ala Trp Leu

3155 3160 3165

Thr Glu His Gly Cys Asp Arg Leu Lys Arg Met Ala Val Ser Gly

3170 3175 3180

Asp Asp Cys Val Val Arg Pro Ile Asp Asp Arg Phe Gly Leu Ala

3185 3190 3195

Leu Ser His Leu Asn Ala Met Ser Lys Val Arg Lys Asp Ile Ser

3200 3205 3210

Glu Trp Gln Pro Ser Lys Gly Trp Asn Asp Trp Glu Asn Val Pro

3215 3220 3225

Phe Cys Ser His His Phe His Glu Leu Gln Leu Lys Asp Gly Arg

3230 3235 3240

Arg Ile Val Val Pro Cys Arg Glu Gln Asn Glu Leu Ile Gly Arg

3245 3250 3255

Gly Arg Val Ser Pro Gly Asn Gly Trp Met Ile Lys Glu Thr Ala

3260 3265 3270

Cys Leu Ser Lys Ala Tyr Ala Asn Met Trp Ser Leu Met Tyr Phe

3275 3280 3285

His Lys Arg Asp Met Arg Leu Leu Ser Leu Ala Val Ser Ser Ala

3290 3295 3300

Val Pro Thr Ser Trp Val Pro Gln Gly Arg Thr Thr Trp Ser Ile

3305 3310 3315

His Gly Lys Gly Glu Trp Met Thr Thr Glu Asp Met Leu Glu Val

3320 3325 3330

Trp Asn Arg Val Trp Ile Thr Asn Asn Pro His Met Gln Asp Lys

3335 3340 3345

Thr Met Val Lys Lys Trp Arg Asp Val Pro Tyr Leu Thr Lys Arg

3350 3355 3360

Gln Asp Lys Leu Cys Gly Ser Leu Ile Gly Met Thr Asn Arg Ala

3365 3370 3375

Thr Trp Ala Ser His Ile His Leu Val Ile His Arg Ile Arg Thr

3380 3385 3390

Leu Ile Gly Gln Glu Lys Tyr Thr Asp Tyr Leu Thr Val Met Asp

3395 3400 3405

Arg Tyr Ser Val Asp Ala Asp Leu Gln Leu Gly Glu Leu Ile

3410 3415 3420

<210>22

<211>10896

<212>DNA

<213> Artificial

<220>

<223> derived from yellow fever virus and West Nile virus

<220>

<221>misc_feature

<222>(1)..(1)

<223> n is a, c, g, or t

<220>

<221>CDS

<222>(119)..(10384)

<400>22

ngtaaatcct gtgtgctaat tgaggtgcat tggtctgcaa atcgagttgc taggcaataa 60

acacatttgg attaatttta atcgttcgtt gagcgattag cagagaactg accagaac 118

atg tct ggt cgt aaa gct cag gga aaa acc ctg ggc gtc aat atg gta 166

Met Ser Gly Arg Lys Ala Gln Gly Lys Thr Leu Gly Val Asn Met Val

1 5 10 15

cga cga gga gtt cgc tcc ttg tca aac aaa ata aaa caa aaa aea aaa 214

Arg Arg Gly Val Arg Ser Leu Ser Asn Lys Ile Lys Gln Lys Thr Lys

20 25 30

caa att gga aac aga cct gga cct tca aga ggt gtt caa gga ttt atc 262

Gln Ile Gly Asn Arg Pro Gly Pro Ser Arg Gly Val Gln Gly Phe Ile

35 40 45

ttt ttc ttt ttg ttc aac att ttg act gga aaa aag atc aca gcc cac 310

Phe Phe Phe Leu Phe Asn Ile Leu Thr Gly Lys Lys Ile Thr Ala His

50 55 60

cta aag agg ttg tgg aaa atg ctg gac cca aga caa ggc ttg gct gtt 358

Leu Lys Arg Leu Trp Lys Met Leu Asp Pro Arg Gln Gly Leu Ala Val

65 70 75 80

cta agg aaa gtc aag aga gtg gtg gcc agt ttg atg aga gga ttg tcc 406

Leu Arg Lys Val Lys Arg Val Val Ala Ser Leu Met Arg Gly Leu Ser

85 90 95

tca agg aaa cgc cgt tcc cat gat gtt ctg act gtg caa ttc cta att 454

Ser Arg Lys Arg Arg Ser His Asp Val Leu Thr Val Gln Phe Leu Ile

100 105 110

ttg gga atg ctg ttg atg acg ggt gga gtt acc ctc tct aac ttc caa 502

Leu Gly Met Leu Leu Met Thr Gly Gly Val Thr Leu Ser Asn Phe Gln

115 120 125

ggg aag gtg atg atg acg gta aat gct act gac gtc aca gat gtc atc 550

Gly Lys Val Met Met Thr Val Asn Ala Thr Asp Val Thr Asp Val Ile

130 135 140

acg att cca aca gct gct gga aag aac cta tgc att gtc aga gca atg 598

Thr Ile Pro Thr Ala Ala Gly Lys Asn Leu Cys Ile Val Arg Ala Met

145 150 155 160

gat gtg gga tac atg tgc gat gat act atc act tat gaa tgc cca gtg 646

Asp Val Gly Tyr Met Cys Asp Asp Thr Ile Thr Tyr Glu Cys Pro Val

165 170 175

ctg tcg gct ggt aat gat cca gaa gac atc gac tgt tgg tgc aca aag 694

Leu Ser Ala Gly Asn Asp Pro Glu Asp Ile Asp Cys Trp Cys Thr Lys

180 185 190

tca gca gtc tac gtc agg tat gga aga tgc acc aag aca cgc cac tca 742

Ser Ala Val Tyr Val Arg Tyr Gly Arg Cys Thr Lys Thr Arg His Ser

195 200 205

aga cgc agt cgg agg tca ctg aca gtg cag aca cac gga gaa agc act 790

Arg Arg Ser Arg Arg Ser Leu Thr Val Gln Thr His Gly Glu Ser Thr

210 215 220

cta gcg aac aag aag ggg gct tgg atg gac agc acc aag gcc aca agg 838

Leu Ala Asn Lys Lys Gly Ala Trp Met Asp Ser Thr Lys Ala Thr Arg

225 230 235 240

tat ttg gta aaa aca gaa tca tgg atc ttg agg aac cct gga tat gcc 886

Tyr Leu Val Lys Thr Glu Ser Trp Ile Leu Arg Asn Pro Gly Tyr Ala

245 250 255

ctg gtg gca gcc gtc att ggt tgg atg ctt ggg agc aac acc atg cag 934

Leu Val Ala Ala Val Ile Gly Trp Met Leu Gly Ser Asn Thr Met Gln

260 265 270

aga gtt gtg ttt gtc gtg cca ttg ctt ttg gtg gcc cca gct tac agc 982

Arg Val Val Phe Val Val Pro Leu Leu Leu Val Ala Pro Ala Tyr Ser

275 280 285

ttc aac tgc ctt gga atg agc aac aga gac ttc ttg gaa gga gtg tct 1030

Phe Asn Cys Leu Gly Met Ser Asn Arg Asp Phe Leu Glu Gly Val Ser

290 295 300

gga gca aca tgg gtg gat ttg gtt ctc gaa ggc gac agc tgc gtg act 1078

Gly Ala Thr Trp Val Asp Leu Val Leu Glu Gly Asp Ser Cys Val Thr

305 310 315 320

atc atg tct aag gac aag cct acc atc gac gtc aag atg atg aat atg 1126

Ile Met Ser Lys Asp Lys Pro Thr Ile Asp Val Lys Met Met Asn Met

325 330 335

gag gcg gcc aac ctg gca gag gtc cgc agt tat tgc tat ttg gct acc 1174

Glu Ala Ala Asn Leu Ala Glu Val Arg Ser Tyr Cys Tyr Leu Ala Thr

340 345 350

gtc agc gat ctc tcc acc aaa gct gca tgc ccg acc atg gga gaa gct 1222

Val Ser Asp Leu Ser Thr Lys Ala Ala Cys Pro Thr Met Gly Glu Ala

355 360 365

cac aat gac aaa cgt gct gac cca gct ttt gtg tgc aga caa gga gtg 1270

His Asn Asp Lys Arg Ala Asp Pro Ala Phe Val Cys Arg Gln Gly Val

370 375 380

gtg gac agg ggc tgg ggc aac ggc tgc gga ttt ttt ggc aaa gga tcc 1318

Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Phe Phe Gly Lys Gly Ser

385 390 395 400

att gac aca tgc gcc aaa ttt gcc tgc tct acc aag gca ata gga aga 1366

Ile Asp Thr Cys Ala Lys Phe Ala Cys Ser Thr Lys Ala Ile Gly Arg

405 410 415

acc atc ttg aaa gag aat atc aag tac gaa gtg gcc att ttt gtc cat 1414

Thr Ile Leu Lys Glu Asn Ile Lys Tyr Glu Val Ala Ile Phe Val His

420 425 430

gga cca act act gtg gag tcg cac gga aat tac tcc aca cag gtt gga 1462

Gly Pro Thr Thr Val Glu Ser His Gly Asn Tyr Ser Thr Gln Val Gly

435 440 445

gcc act cag gcc ggc cga ttc agc atc act cct gct gcg cct tca tac 1510

Ala Thr Gln Ala Gly Arg Phe Ser Ile Thr Pro Ala Ala Pro Ser Tyr

450 455 460

aca cta aag ctt gga gaa tat gga gag gtg aca gtg gac tgt gaa cca 1558

Thr Leu Lys Leu Gly Glu Tyr Gly Glu Val Thr Val Asp Cys Glu Pro

465 470 475 480

cgg tca ggg att gac acc aat gca tac tac gtg atg act gtt gga aca 1606

Arg Ser Gly Ile Asp Thr Asn Ala Tyr Tyr Val Met Thr Val Gly Thr

485 490 495

aag acg ttc ttg gtc cat cgt gag tgg ttc atg gac ctc aac ctc cct 1654

Lys Thr Phe Leu Val His Arg Glu Trp Phe Met Asp Leu Asn Leu Pro

500 505 510

tgg agc agt gct gga agt act gtg tgg agg aac aga gag acg tta atg 1702

Trp Ser Ser Ala Gly Ser Thr Val Trp Arg Asn Arg Glu Thr Leu Met

515 520 525

gag ttt gag gaa cca cac gcc acg aag cag tct gtg ata gca ttg ggc 1750

Glu Phe Glu Glu Pro His Ala Thr Lys Gln Ser Val Ile Ala Leu Gly

530 535 540

tca caa gag gga gct ctg cat caa gct ttg gct gga gcc att cct gtg 1798

Ser Gln Glu Gly Ala Leu His Gln Ala Leu Ala Gly Ala Ile Pro Val

545 550 555 560

gaa ttt tca agc aac act gtc aag ttg acg tcg ggt cat ttg aag tgt 1846

Glu Phe Ser Ser Asn Thr Val Lys Leu Thr Ser Gly His Leu Lys Cys

565 570 575

aga gtg aag atg gaa aaa ttg cag ttg aag gga aca acc tat ggc gtc 1894

Arg Val Lys Met Glu Lys Leu Gln Leu Lys Gly Thr Thr Tyr Gly Val

580 585 590

tgt tca aag gct ttc aag ttt ctt agg act ccc gtg gac acc ggt cac 1942

Cys Ser Lys Ala Phe Lys Phe Leu Arg Thr Pro Val Asp Thr Gly His

595 600 605

ggc act gtg gtg ttg gaa ttg cag tac act ggc acg gat gga cct tgc 1990

Gly Thr Val Val Leu Glu Leu Gln Tyr Thr Gly Thr Asp Gly Pro Cys

610 615 620

aaa gtt cct atc tcg tca gtg gct tca ttg aac gac cta acg cca gtg 2038

Lys Val Pro Ile Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val

625 630 635 640

ggc aga ttg gtc act gtc aac cct ttt gtt tca gtg gcc acg gcc aac 2086

Gly Arg Leu Val Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn

645 650 655

gct aag gtc ctg att gaa ttg gaa cca ccc ttt gga gac tca tac ata 2134

Ala Lys Val Leu Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile

660 665 670

gtg gtg ggc aga gga gaa caa cag atc aat cac cat tgg cac aag tct 2182

Val Val Gly Arg Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser

675 680 685

gga agc agc att ggc aaa gcc ttt aca acc acc ctc aaa gga gcg cag 2230

Gly Ser Ser Ile Gly Lys Ala Phe Thr Thr Thr Leu Lys Gly Ala Gln

690 695 700

aga cta gcc gct cta gga gac aca gct tgg gac ttt gga tca gtt gga 2278

Arg Leu Ala Ala Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val Gly

705 710 715 720

ggg gtg ttc act agt gtt ggg cgg gct gtc cat caa gtg ttc gga gga 2326

Gly Val Phe Thr Ser Val Gly Arg Ala Val His Gln Val Phe Gly Gly

725 730 735

gca ttc cgc tca ctg ttc gga ggc atg tcc tgg ata acg caa gga ttg 2374

Ala Phe Arg Ser Leu Phe Gly Gly Met Ser Trp Ile Thr Gln Gly Leu

740 745 750

ctg ggg gct ctc ctg ttg tgg atg ggc atc aat gct cgt gat agg tcc 2422

Leu Gly Ala Leu Leu Leu Trp Met Gly Ile Asn Ala Arg Asp Arg Ser

755 760 765

ata gct ctc acg ttt ctc gca gtt gga gga gtt ctg ctc ttc ctc tcc 2470

Ile Ala Leu Thr Phe Leu Ala Val Gly Gly Val Leu Leu Phe Leu Ser

770 775 780

gtg aac gtg ggc gcc gat caa gga tgc gcc atc aac ttt ggc aag aga 2518

Val Asn Val Gly Ala Asp Gln Gly Cys Ala Ile Asn Phe Gly Lys Arg

785 790 795 800

gag ctc aag tgc gga gat ggt atc ttc ata ttt aga gac tct gat gac 2566

Glu Leu Lys Cys Gly Asp Gly Ile Phe Ile Phe Arg Asp Ser Asp Asp

805 810 815

tgg ctg aac aag tac tca tac tat cca gaa gat cct gtg aag ctt gca 2614

Trp Leu Asn Lys Tyr Ser Tyr Tyr Pro Glu Asp Pro Val Lys Leu Ala

820 825 830

tca ata gtg aaa gcc tct ttt gaa gaa ggg aag tgt ggc cta aat tca 2662

Ser Ile Val Lys Ala Ser Phe Glu Glu Gly Lys Cys Gly Leu Asn Ser

835 840 845

gtt gac tcc ctt gag cat gag atg tgg aga agc agg gca gat gag atc 2710

Val Asp Ser Leu Glu His Glu Met Trp Arg Ser Arg Ala Asp Glu Ile

850 855 860

aat gcc att ttt gag gaa aac gag gtg gac att tct gtt gtc gtg cag 2758

Asn Ala Ile Phe Glu Glu Asn Glu Val Asp Ile Ser Val Val Val Gln

865 870 875 880

gat cca aag aat gtt tac cag aga gga act cat cca ttt tcc aga att 2806

Asp Pro Lys Asn Val Tyr Gln Arg Gly Thr His Pro Phe Ser Arg Ile

885 890 895

cgg gat ggt ctg cag tat ggt tgg aag act tgg ggt aag aac ctt gtg 2854

Arg Asp Gly Leu Gln Tyr Gly Trp Lys Thr Trp Gly Lys Asn Leu Val

900 905 910

ttc tcc cca ggg agg aag aat gga agc ttc atc ata gat gga aag tcc 2902

Phe Ser Pro Gly Arg Lys Asn Gly Ser Phe Ile Ile Asp Gly Lys Ser

915 920 925

agg aaa gaa tgc ccg ttt tca aac cgg gtc tgg aat tct ttc cag ata 2950

Arg Lys Glu Cys Pro Phe Ser Asn Arg Val Trp Asn Ser Phe Gln Ile

930 935 940

gag gag ttt ggg acg gga gtg ttc acc aca cgc gtg tac atg gac gca 2998

Glu Glu Phe Gly Thr Gly Val Phe Thr Thr Arg Val Tyr Met Asp Ala

945 950 955 960

gtc ttt gaa tac acc ata gac tgc gat gga tct atc ttg ggt gca gcg 3046

Val Phe Glu Tyr Thr Ile Asp Cys Asp Gly Ser Ile Leu Gly Ala Ala

965 970 975

gtg aac gga aaa aag agt gcc cat ggc tct cca aca ttt tgg atg gga 3094

Val Asn Gly Lys Lys Ser Ala His Gly Ser Pro Thr Phe Trp Met Gly

980 985 990

agt cat gaa gta aat ggg aca tgg atg atc cac acc ttg gag gca tta 3142

Ser His Glu Val Asn Gly Thr Trp Met Ile His Thr Leu Glu Ala Leu

995 1000 1005

gat tac aag gag tgt gag tgg cca ctg aca cat acg att gga aca 3187

Asp Tyr Lys Glu Cys Glu Trp Pro Leu Thr His Thr Ile Gly Thr

1010 1015 1020

tca gtt gaa gag agt gaa atg ttc atg ccg aga tca atc gga ggc 3232

Ser Val Glu Glu Ser Glu Met Phe Met Pro Arg Ser Ile Gly Gly

1025 1030 1035

cca gtt agc tct cac aat cat atc cct gga tac aag gtt cag acg 3277

Pro Val Ser Ser His Asn His Ile Pro Gly Tyr Lys Val Gln Thr

1040 1045 1050

aac gga cct tgg atg cag gta cca cta gaa gtg aag aga gaa gct 3322

Asn Gly Pro Trp Met Gln Val Pro Leu Glu Val Lys Arg Glu Ala

1055 1060 1065

tgc cca ggg act agc gtg atc att gat ggc aac tgt gat gga cgg 3367

Cys Pro Gly Thr Ser Val Ile Ile Asp Gly Asn Cys Asp Gly Arg

1070 1075 1080

gga aaa tca acc aga tcc acc acg gat agc ggg aaa gtt att cct 3412

Gly Lys Ser Thr Arg Ser Thr Thr Asp Ser Gly Lys Val Ile Pro

1085 1090 1095

gaa tgg tgt tgc cgc tcc tgc aca atg ccg cct gtg agc ttc cat 3457

Glu Trp Cys Cys Arg Ser Cys Thr Met Pro Pro Val Ser Phe His

1100 1105 1110

ggt agt gat ggg tgt tgg tat ccc atg gaa att agg cca agg aaa 3502

Gly Ser Asp Gly Cys Trp Tyr Pro Met Glu Ile Arg Pro Arg Lys

1115 1120 1125

acg cat gaa agc cat ctg gtg cgc tcc tgg gtt aca gct gga gaa 3547

Thr His Glu Ser His Leu Val Arg Ser Trp Val Thr Ala Gly Glu

1130 1135 1140

ata cat gct gtc cct ttt ggt ttg gtg agc atg atg ata gca atg 3592

Ile His Ala Val Pro Phe Gly Leu Val Ser Met Met Ile Ala Met

1145 1150 1155

gaa gtg gtc cta agg aaa aga cag gga cca aag caa atg ttg gtt 3637

Glu Val Val Leu Arg Lys Arg Gln Gly Pro Lys Gln Met Leu Val

1160 1165 1170

gga gga gta gtg ctc ttg gga gca atg ctg gtc ggg caa gta act 3682

Gly Gly Val Val Leu Leu Gly Ala Met Leu Val Gly Gln Val Thr

1175 1180 1185

ctc ctt gat ttg ctg aaa ctc aca gtg gct gtg gga ttg cat ttc 3727

Leu Leu Asp Leu Leu Lys Leu Thr Val Ala Val Gly Leu His Phe

1190 1195 1200

cat gag atg aac aat gga gga gac gcc atg tat atg gcg ttg att 3772

His Glu Met Asn Asn Gly Gly Asp Ala Met Tyr Met Ala Leu Ile

1205 1210 1215

gct gcc ttt tca atc aga cca ggg ctg ctc atc ggc ttt ggg ctc 3817

Ala Ala Phe Ser Ile Arg Pro Gly Leu Leu Ile Gly Phe Gly Leu

1220 1225 1230

agg acc cta tgg agc cct cgg gaa cgc ctt gtg ctg acc cta gga 3862

Arg Thr Leu Trp Ser Pro Arg Glu Arg Leu Val Leu Thr Leu Gly

1235 1240 1245

gca gcc atg gtg gag att gcc ttg ggt ggc gtg atg ggc ggc ctg 3907

Ala Ala Met Val Glu Ile Ala Leu Gly Gly Val Met Gly Gly Leu

1250 1255 1260

tgg aag tat cta aat gca gtt tct ctc tgc atc ctg aca ata aat 3952

Trp Lys Tyr Leu Asn Ala Val Ser Leu Cys Ile Leu Thr Ile Asn

1265 1270 1275

gct gtt gct tct agg aaa gca tca aat acc atc ttg ccc ctc atg 3997

Ala Val Ala Ser Arg Lys Ala Ser Asn Thr Ile Leu Pro Leu Met

1280 1285 1290

gct ctg ttg aca cct gtc act atg gct gag gtg aga ctt gcc gca 4042

Ala Leu Leu Thr Pro Val Thr Met Ala Glu Val Arg Leu Ala Ala

1295 1300 1305

atg ttc ttt tgt gcc atg gtt atc ata ggg gtc ctt cac cag aat 4087

Met Phe Phe Cys Ala Met Val Ile Ile Gly Val Leu His Gln Asn

1310 1315 1320

ttc aag gac acc tcc atg cag aag act ata cct ctg gtg gcc ctc 4132

Phe Lys Asp Thr Ser Met Gln Lys Thr Ile Pro Leu Val Ala Leu

1325 1330 1335

aca ctc aca tct tac ctg ggc ttg aca caa cct ttt ttg ggc ctg 4177

Thr Leu Thr Ser Tyr Leu Gly Leu Thr Gln Pro Phe Leu Gly Leu

1340 1345 1350

tgt gca ttt ctg gca acc cgc ata ttt ggg cga agg agt atc cca 4222

Cys Ala Phe Leu Ala Thr Arg Ile Phe Gly Arg Arg Ser Ile Pro

1355 1360 1365

gtg aat gag gca ctc gca gca gct ggt cta gtg gga gtg ctg gca 4267

Val Asn Glu Ala Leu Ala Ala Ala Gly Leu Val Gly Val Leu Ala

1370 1375 1380

gga ctg gct ttt cag gag atg gag aac ttc ctt ggt ccg att gca 4312

Gly Leu Ala Phe Gln Glu Met Glu Asn Phe Leu Gly Pro Ile Ala

1385 1390 1395

gtt gga gga ctc ctg atg atg ctg gtt agc gtg gct ggg agg gtg 4357

Val Gly Gly Leu Leu Met Met Leu Val Ser Val Ala Gly Arg Val

1400 1405 1410

gat ggg cta gag ctc aag aag ctt ggt gaa gtt tca tgg gaa gag 4402

Asp Gly Leu Glu Leu Lys Lys Leu Gly Glu Val Ser Trp Glu Glu

1415 1420 1425

gag gcg gag atc agc ggg agt tcc gcc cgc tat gat gtg gca ctc 4447

Glu Ala Glu Ile Ser Gly Ser Ser Ala Arg Tyr Asp Val Ala Leu

1430 1435 1440

agt gaa caa ggg gag ttc aag ctg ctt tct gaa gag aaa gtg cca 4492

Ser Glu Gln Gly Glu Phe Lys Leu Leu Ser Glu Glu Lys Val Pro

1445 1450 1455

tgg gac cag gtt gtg atg acc tcg ctg gcc ttg gtt ggg gct gcc 4537

Trp Asp Gln Val Val Met Thr Ser Leu Ala Leu Val Gly Ala Ala

1460 1465 1470

ctc cat cca ttt gct ctt ctg ctg gtc ctt gct ggg tgg ctg ttt 4582

Leu His Pro Phe Ala Leu Leu Leu Val Leu Ala Gly Trp Leu Phe

1475 1480 1485

cat gtc agg gga gct agg aga agt ggg gat gtc ttg tgg gat att 4627

His Val Arg Gly Ala Arg Arg Ser Gly Asp Val Leu Trp Asp Ile

1490 1495 1500

ccc act cct aag atc atc gag gaa tgt gaa cat ctg gag gat ggg 4672

Pro Thr Pro Lys Ile Ile Glu Glu Cys Glu His Leu Glu Asp Gly

1505 1510 1515

att tat ggc ata ttc cag tca acc ttc ttg ggg gcc tcc cag cga 4717

Ile Tyr Gly Ile Phe Gln Ser Thr Phe Leu Gly Ala Ser Gln Arg

1520 1525 1530

gga gtg gga gtg gca cag gga ggg gtg ttc cac aca atg tgg cat 4762

Gly Val Gly Val Ala Gln Gly Gly Val Phe His Thr Met Trp His

1535 1540 1545

gtc aca aga gga gct ttc ctt gtc agg aat ggc aag aag ttg att 4807

Val Thr Arg Gly Ala Phe Leu Val Arg Asn Gly Lys Lys Leu Ile

1550 1555 1560

cca tct tgg gct tca gta aag gaa gac ctt gtc gcc tat ggt ggc 4852

Pro Ser Trp Ala Ser Val Lys Glu Asp Leu Val Ala Tyr Gly Gly

1565 1570 1575

tca tgg aag ttg gaa ggc aga tgg gat gga gag gaa gag gtc cag 4897

Ser Trp Lys Leu Glu Gly Arg Trp Asp Gly Glu Glu Glu Val Gln

1580 1585 1590

ttg atc gcg gct gtt cca gga aag aac gtg gtc aac gtc cag aca 4942

Leu Ile Ala Ala Val Pro Gly Lys Asn Val Val Asn Val Gln Thr

1595 1600 1605

aaa ccg agc ttg ttc aaa gtg agg aat ggg gga gaa atc ggg gct 4987

Lys Pro Ser Leu Phe Lys Val Arg Asn Gly Gly Glu Ile Gly Ala

1610 1615 1620

gtc gct ctt gac tat ccg agt ggc act tca gga tct cct att gtt 5032

Val Ala Leu Asp Tyr Pro Ser Gly Thr Ser Gly Ser Pro Ile Val

1625 1630 1635

aac agg aac gga gag gtg att ggg ctg tac ggc aat ggc atc ctt 5077

Asn Arg Asn Gly Glu Val Ile Gly Leu Tyr Gly Asn Gly Ile Leu

1640 1645 1650

gtc ggt gac aac tcc ttc gtg tcc gcc ata tcc cag act gag gtg 5122

Val Gly Asp Asn Ser Phe Val Ser Ala Ile Ser Gln Thr Glu Val

1655 1660 1665

aag gaa gaa gga aag gag gag ctc caa gag atc ccg aca atg cta 5167

Lys Glu Glu Gly Lys Glu Glu Leu Gln Glu Ile Pro Thr Met Leu

1670 1675 1680

aag aaa gga atg aca act gtc ctt gat ttt cat cct gga gct ggg 5212

Lys Lys Gly Met Thr Thr Val Leu Asp Phe His Pro Gly Ala Gly

1685 1690 1695

aag aca aga cgt ttc ctc cca cag atc ttg gcc gag tgc gca cgg 5257

Lys Thr Arg Arg Phe Leu Pro Gln Ile Leu Ala Glu Cys Ala Arg

1700 1705 1710

aga cgc ttg cgc act ctt gtg ttg gcc ccc acc agg gtt gtt ctt 5302

Arg Arg Leu Arg Thr Leu Val Leu Ala Pro Thr Arg Val Val Leu

1715 1720 1725

tct gaa atg aag gag gct ttt cac ggc ctg gac gtg aaa ttc cac 5347

Ser Glu Met Lys Glu Ala Phe His Gly Leu Asp Val Lys Phe His

1730 1735 1740

aca cag gct ttt tcc gct cac ggc agc ggg aga gaa gtc att gat 5392

Thr Gln Ala Phe Ser Ala His Gly Ser Gly Arg Glu Val Ile Asp

1745 1750 1755

gcc atg tgc cat gcc acc cta act tac agg atg ttg gaa cca act 5437

Ala Met Cys His Ala Thr Leu Thr Tyr Arg Met Leu Glu Pro Thr

1760 1765 1770

agg gtt gtt aac tgg gaa gtg atc att atg gat gaa gcc cat ttt 5482

Arg Val Val Asn Trp Glu Val Ile Ile Met Asp Glu Ala His Phe

1775 1780 1785

ttg gat cca gcc agc ata gcc gct aga ggt tgg gca gcg cac aga 5527

Leu Asp Pro Ala Ser Ile Ala Ala Arg Gly Trp Ala Ala His Arg

1790 1795 1800

gct agg gca aat gaa agt gca aca atc ttg atg aca gcc aca ccg 5572

Ala Arg Ala Asn Glu Ser Ala Thr Ile Leu Met Thr Ala Thr Pro

1805 1810 1815

cct ggg act agt gat gaa ttt cca cat tca aat ggt gaa ata gaa 5617

Pro Gly Thr Ser Asp Glu Phe Pro His Ser Asn Gly Glu Ile Glu

1820 1825 1830

gat gtt caa acg gac ata ccc agt gag ccc tgg aac aca ggg cat 5662

Asp Val Gln Thr Asp Ile Pro Ser Glu Pro Trp Asn Thr Gly His

1835 1840 1845

gac tgg atc ctg gct gac aaa agg ccc acg gca tgg ttc ctt cca 5707

Asp Trp Ile Leu Ala Asp Lys Arg Pro Thr Ala Trp Phe Leu Pro

1850 1855 1860

tcc atc aga gct gca aat gtc atg gct gcc tct ttg cgt aag gct 5752

Ser Ile Arg Ala Ala Asn Val Met Ala Ala Ser Leu Arg Lys Ala

1865 1870 1875

gga aag agt gtg gtg gtc ctg aac agg aaa acc ttt gag aga gaa 5797

Gly Lys Ser Val Val Val Leu Asn Arg Lys Thr Phe Glu Arg Glu

1880 1885 1890

tac ccc acg ata aag cag aag aaa cct gac ttt ata ttg gcc act 5842

Tyr Pro Thr Ile Lys Gln Lys Lys Pro Asp Phe Ile Leu Ala Thr

1895 1900 1905

gac ata gct gaa atg gga gcc aac ctt tgc gtg gag cga gtg ctg 5887

Asp Ile Ala Glu Met Gly Ala Asn Leu Cys Val Glu Arg Val Leu

1910 1915 1920

gat tgc agg acg gct ttt aag cct gtg ctt gtg gat gaa ggg agg 5932

Asp Cys Arg Thr Ala Phe Lys Pro Val Leu Val Asp Glu Gly Arg

1925 1930 1935

aag gtg gca ata aaa ggg cca ctt cgt atc tcc gca tcc tct gct 5977

Lys Val Ala Ile Lys Gly Pro Leu Arg Ile Ser Ala Ser Ser Ala

1940 1945 1950

gct caa agg agg ggg cgc att ggg aga aat ccc aac aga gat gga 6022

Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn Pro Asn Arg Asp Gly

1955 1960 1965

gac tca tac tac tat tct gag cct aca agt gaa aat aat gcc cac 6067

Asp Ser Tyr Tyr Tyr Ser Glu Pro Thr Ser Glu Asn Asn Ala His

1970 1975 1980

cac gtc tgc tgg ttg gag gcc tca atg ctc ttg gac aac atg gag 6112

His Val Cys Trp Leu Glu Ala Ser Met Leu Leu Asp Asn Met Glu

1985 1990 1995

gtg agg ggt gga atg gtc gcc cca ctc tat ggc gtt gaa gga act 6157

Val Arg Gly Gly Met Val Ala Pro Leu Tyr Gly Val Glu Gly Thr

2000 2005 2010

aaa aca cca gtt tcc cct ggt gaa atg aga ctg agg gat gac cag 6202

Lys Thr Pro Val Ser Pro Gly Glu Met Arg Leu Arg Asp Asp Gln

2015 2020 2025

agg aaa gtc ttc aga gaa cta gtg agg aat tgt gac ctg ccc gtt 6247

Arg Lys Val Phe Arg Glu Leu Val Arg Asn Cys Asp Leu Pro Val

2030 2035 2040

tgg ctt tcg tgg caa gtg gcc aag gct ggt ttg aag acg aat gat 6292

Trp Leu Ser Trp Gln Val Ala Lys Ala Gly Leu Lys Thr Asn Asp

2045 2050 2055

cgt aag tgg tgt ttt gaa ggc cct gag gaa cat gag atc ttg aat 6337

Arg Lys Trp Cys Phe Glu Gly Pro Glu Glu His Glu Ile Leu Asn

2060 2065 2070

gac agc ggt gaa aca gtg aag tgc agg gct cct gga gga gca aag 6382

Asp Ser Gly Glu Thr Val Lys Cys Arg Ala Pro Gly Gly Ala Lys

2075 2080 2085

aag cct ctg cgc cca agg tgg tgt gat gaa agg gtg tca tct gac 6427

Lys Pro Leu Arg Pro Arg Trp Cys Asp Glu Arg Val Ser Ser Asp

2090 2095 2100

cag agt gcg ctg tct gaa ttt att aag ttt gct gaa ggt agg agg 6472

Gln Ser Ala Leu Ser Glu Phe Ile Lys Phe Ala Glu Gly Arg Arg

2105 2110 2115

gga gct gct gaa gtg cta gtt gtg ctg agt gaa ctc cct gat ttc 6517

Gly Ala Ala Glu Val Leu Val Val Leu Ser Glu Leu Pro Asp Phe

2120 2125 2130

ctg gct aaa aaa ggt gga gag gca atg gat acc atc agt gtg ttc 6562

Leu Ala Lys Lys Gly Gly Glu Ala Met Asp Thr Ile Ser Val Phe

2135 2140 2145

ctc cac tct gag gaa ggc tct agg gct tac cgc aat gca cta tca 6607

Leu His Ser Glu Glu Gly Ser Arg Ala Tyr Arg Asn Ala Leu Ser

2150 2155 2160

atg atg cct gag gca atg aca ata gtc atg ctg ttt ata ctg gct 6652

Met Met Pro Glu Ala Met Thr Ile Val Met Leu Phe Ile Leu Ala

2165 2170 2175

gga cta ctg aca tcg gga atg gtc atc ttt ttc atg tct ccc aaa 6697

Gly Leu Leu Thr Ser Gly Met Val Ile Phe Phe Met Ser Pro Lys

2180 2185 2190

ggc atc agt aga atg tct atg gcg atg ggc aca atg gcc ggc tgt 6742

Gly Ile Ser Arg Met Ser Met Ala Met Gly Thr Met Ala Gly Cys

2195 2200 2205

gga tat ctc atg ttc ctt gga ggc gtc aaa ccc act cac atc tcc 6787

Gly Tyr Leu Met Phe Leu Gly Gly Val Lys Pro Thr His Ile Ser

2210 2215 2220

tat gtc atg ctc ata ttc ttt gtc ctg atg gtg gtt gtg atc ccc 6832

Tyr Val Met Leu Ile Phe Phe Val Leu Met Val Val Val Ile Pro

2225 2230 2235

gag cca ggg caa caa agg tcc atc caa gac aac caa gtg gca tac 6877

Glu Pro Gly Gln Gln Arg Ser Ile Gln Asp Asn Gln Val Ala Tyr

2240 2245 2250

ctc att att ggc atc ctg acg ctg gtt tca gcg gtg gca gcc aac 6922

Leu Ile Ile Gly Ile Leu Thr Leu Val Ser Ala Val Ala Ala Asn

2255 2260 2265

gag cta ggc atg ctg gag aaa acc aaa gag gac ctc ttt ggg aag 6967

Glu Leu Gly Met Leu Glu Lys Thr Lys Glu Asp Leu Phe Gly Lys

2270 2275 2280

aag aac tta att cca tct agt gct tca ccc tgg agt tgg ccg gat 7012

Lys Asn Leu Ile Pro Ser Ser Ala Ser Pro Trp Ser Trp Pro Asp

2285 2290 2295

ctt gac ctg aag cca gga gct gcc tgg aca gtg tac gtt ggc att 7057

Leu Asp Leu Lys Pro Gly Ala Ala Trp Thr Val Tyr Val Gly Ile

2300 2305 2310

gtt aca atg ctc tct cca atg ttg cac cac tgg atc aaa gtc gaa 7102

Val Thr Met Leu Ser Pro Met Leu His His Trp Ile Lys Val Glu

2315 2320 2325

tat ggc aac ctg tct ctg tct gga ata gcc cag tca gcc tca gtc 7147

Tyr Gly Asn Leu Ser Leu Ser Gly Ile Ala Gln Ser Ala Ser Val

2330 2335 2340

ctt tct ttc atg gac aag ggg ata cca ttc atg aag atg aat atc 7192

Leu Ser Phe Met Asp Lys Gly Ile Pro Phe Met Lys Met Asn Ile

2345 2350 2355

tcg gtc ata atg ctg ctg gtc agt ggc tgg aat tca ata aca gtg 7237

Ser Val Ile Met Leu Leu Val Ser Gly Trp Asn Ser Ile Thr Val

2360 2365 2370

atg cct ctg ctc tgt ggc ata ggg tgc gcc atg ctc cac tgg tct 7282

Met Pro Leu Leu Cys Gly Ile Gly Cys Ala Met Leu His Trp Ser

2375 2380 2385

ctc att tta cct gga atc aaa gcg cag cag tca aag ctt gca cag 7327

Leu Ile Leu Pro Gly Ile Lys Ala Gln Gln Ser Lys Leu Ala Gln

2390 2395 2400

aga agg gtg ttc cat ggc gtt gcc aag aac cct gtg gtt gat ggg 7372

Arg Arg Val Phe His Gly Val Ala Lys Asn Pro Val Val Asp Gly

2405 2410 2415

aat cca aca gtt gac att gag gaa gct cct gaa atg cct gcc ctt 7417

Asn Pro Thr Val Asp Ile Glu Glu Ala Pro Glu Met Pro Ala Leu

2420 2425 2430

tat gag aag aaa ctg gct cta tat ctc ctt ctt gct ctc agc cta 7462

Tyr Glu Lys Lys Leu Ala Leu Tyr Leu Leu Leu Ala Leu Ser Leu

2435 2440 2445

gct tct gtt gcc atg tgc aga acg ccc ttt tca ttg gct gaa ggc 7507

Ala Ser Val Ala Met Cys Arg Thr Pro Phe Ser Leu Ala Glu Gly

2450 2455 2460

att gtc cta gca tca gct gcc tta ggg ccg ctc ata gag gga aae 7552

Ile Val Leu Ala Ser Ala Ala Leu Gly Pro Leu Ile Glu Gly Asn

2465 2470 2475

acc agc ctt ctt tgg aat gga ccc atg gct gtc tcc atg aca gga 7597

Thr Ser Leu Leu Trp Asn Gly Pro Met Ala Val Ser Met Thr Gly

2480 2485 2490

gtc atg agg ggg aat cac tat gct ttt gtg gga gtc atg tac aat 7642

Val Met Arg Gly Asn His Tyr Ala Phe Val Gly Val Met Tyr Asn

2495 2500 2505

cta tgg aag atg aaa act gga cgc cgg ggg agc gcg aat gga aaa 7687

Leu Trp Lys Met Lys Thr Gly Arg Arg Gly Ser Ala Asn Gly Lys

2510 2515 2520

act ttg ggt gaa gtc tgg aag agg gaa ctg aat ctg ttg gac aag 7732

Thr Leu Gly Glu Val Trp Lys Arg Glu Leu Asn Leu Leu Asn Lys

2525 2530 2535

cga cag ttt gag ttg tat aaa agg acc gac att gtg gag gtg gat 7777

Arg Gln Phe Glu Leu Tyr Lys Arg Thr Asp Ile Val Glu Val Asp

2540 2545 2550

cgt gat acg gca cgc agg cat ttg gcc gaa ggg aag gtg gac acc 7822

Arg Asp Thr Ala Arg Arg His Leu Ala Glu Gly Lys Val Asp Thr

2555 2560 2565

ggg gtg gcg gtc tcc agg ggg acc gca aag tta agg tgg ttc cat 7867

Gly Val Ala Val Ser Arg Gly Thr Ala Lys Leu Arg Trp Phe His

2570 2575 2580

gag cgt ggc tat gtc aag ctg gaa ggt agg gtg att gac ctg ggg 7912

Glu Arg Gly Tyr Val Lys Leu Glu Gly Arg Val Ile Asp Leu Gly

2585 2590 2595

tgt ggc cgc gga ggc tgg tgt tac tac gct gct gcg caa aag gaa 7957

Cys Gly Arg Gly Gly Trp Cys Tyr Tyr Ala Ala Ala Gln Lys Glu

2600 2605 2610

gtg agt ggg gtc aaa gga ttt act ctt gga aga gac ggc cat gag 8002

Val Ser Gly Val Lys Gly Phe Thr Leu Gly Arg Asp Gly His Glu

2615 2620 2625

aaa ccc atg aat gtg caa agt ctg gga tgg aac atc atc acc ttc 8047

Lys Pro Met Asn Val Gln Ser Leu Gly Trp Asn Ile Ile Thr Phe

2630 2635 2640

aag gac aaa act gat atc cac cgc cta gaa cca gtg aaa tgt gac 8092

Lys Asp Lys Thr Asp Ile His Arg Leu Glu Pro Val Lys Cys Asp

2645 2650 2655

acc ctt ttg tgt gac att gga gag tca tca tcg tca tcg gtc aca 8137

Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Ser Ser Ser Val Thr

2660 2665 2670

gag ggg gaa agg acc gtg aga gtt ctt gat act gta gaa aaa tgg 8182

Glu Gly Glu Arg Thr Val Arg Val Leu Asp Thr Val Glu Lys Trp

2675 2680 2685

ctg gct tgt ggg gtt gac aac ttc tgt gtg aag gtg tta gct cca 8227

Leu Ala Cys Gly Val Asp Asn Phe Cys Val Lys Val Leu Ala Pro

2690 2695 2700

tac atg cca gat gtt ctt gag aaa ctg gaa ttg ctc caa agg agg 8272

Tyr Met Pro Asp Val Leu Glu Lys Leu Glu Leu Leu Gln Arg Arg

2705 2710 2715

ttt ggc gga aca gtg atc agg aac cct ctc tcc agg aat tcc act 8317

Phe Gly Gly Thr Val Ile Arg Asn Pro Leu Ser Arg Asn Ser Thr

2720 2725 2730

cat gaa atg tac tac gtg tct gga gcc cgc agc aat gtc aca ttt 8362

His Glu Met Tyr Tyr Val Ser Gly Ala Arg Ser Asn Val Thr Phe

2735 2740 2745

act gtg aac caa aca tcc cgc ctc ctg atg agg aga atg agg cgt 8407

Thr Val Asn Gln Thr Ser Arg Leu Leu Met Arg Arg Met Arg Arg

2750 2755 2760

cca act gga aaa gtg acc ctg gag gct gac gtc atc ctc cca att 8452

Pro Thr Gly Lys Val Thr Leu Glu Ala Asp Val Ile Leu Pro Ile

2765 2770 2775

ggg aca cgc agt gtt gag aca gac aag gga ccc ctg gac aaa gag 8497

Gly Thr Arg Ser Val Glu Thr Asp Lys Gly Pro Leu Asp Lys Glu

2780 2785 2790

gcc ata gaa gaa agg gtt gag agg ata aaa tct gag tac atg acc 8542

Ala Ile Glu Glu Arg Val Glu Arg Ile Lys Ser Glu Tyr Met Thr

2795 2800 2805

tct tgg ttt tat gac aat gac aac ccc tac agg acc tgg cac tac 8587

Ser Trp Phe Tyr Asp Asn Asp Asn Pro Tyr Arg Thr Trp His Tyr

2810 2815 2820

tgt ggc tcc tat gtc aca aaa acc tcc gga agt gcg gcg agc atg 8632

Cys Gly Ser Tyr Val Thr Lys Thr Ser Gly Ser Ala Ala Ser Met

2825 2830 2835

gta aat ggt gtt att aaa att ctg aca tat cca tgg gac agg ata 8677

Val Asn Gly Val Ile Lys Ile Leu Thr Tyr Pro Trp Asp Arg Ile

2840 2845 2850

gag gag gtc aca aga atg gca atg act gac aca acc cct ttt gga 8722

Glu Glu Val Thr Arg Met Ala Met Thr Asp Thr Thr Pro Phe Gly

2855 2860 2865

cag caa aga gtg ttt aaa gaa aaa gtt gac acc aga gca aag gat 8767

Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg Ala Lys Asp

2870 2875 2880

cca cca gcg gga act agg aag atc atg aaa gtt gtc aac agg tgg 8812

Pro Pro Ala Gly Thr Arg Lys Ile Met Lys Val Val Asn Arg Trp

2885 2890 2895

ctg ttc cgc cac ctg gcc aga gaa aag aac ccc aga ctg tgc aca 8857

Leu Phe Arg His Leu Ala Arg Glu Lys Asn Pro Arg Leu Cys Thr

2900 2905 2910

aag gaa gaa ttt att gca aaa gtc cga agt cat gca gcc att gga 8902

Lys Glu Glu Phe Ile Ala Lys Val Arg Ser His Ala Ala Ile Gly

2915 2920 2925

gct tac ctg gaa gaa caa gaa cag tgg aag act gcc aat gag gct 8947

Ala Tyr Leu Glu Glu Gln Glu Gln Trp Lys Thr Ala Asn Glu Ala

2930 2935 2940

gtc caa gac cca aag ttc tgg gaa ctg gtg gat gaa gaa agg aag 8992

Val Gln Asp Pro Lys Phe Trp Glu Leu Val Asp Glu Glu Arg Lys

2945 2950 2955

ctg cac caa caa ggc agg tgt cgg act tgt gtg tac aac atg atg 9037

Leu His Gln Gln Gly Arg Cys Arg Thr Cys Val Tyr Asn Met Met

2960 2965 2970

ggg aaa aga gag aag aag ctg tca gag ttt ggg aaa gca aag gga 9082

Gly Lys Arg Glu Lys Lys Leu Ser Glu Phe Gly Lys Ala Lys Gly

2975 2980 2985

agc cgt gcc ata tgg tat atg tgg ctg gga gcg cgg tat ctt gag 9127

Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Tyr Leu Glu

2990 2995 3000

ttt gag gcc ctg gga ttc ctg aat gag gac cat tgg gct tcc agg 9172

Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Ala Ser Arg

3005 3010 3015

gaa aac tca gga gga gga gtg gaa ggc att ggc tta caa tac cta 9217

Glu Asn Ser Gly Gly Gly Val Glu Gly Ile Gly Leu Gln Tyr Leu

3020 3025 3030

gga tat gtg atc aga gac ctg gct gca atg gat ggt ggt gga ttc 9262

Gly Tyr Val Ile Arg Asp Leu Ala Ala Met Asp Gly Gly Gly Phe

3035 3040 3045

tac gcg gat gac acc gct gga tgg gac acg cgc atc aca gag gca 9307

Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Ala

3050 3055 3060

gac ctt gat gat gaa cag gag atc ttg aac tac atg agc cca cat 9352

Asp Leu Asp Asp Glu Gln Glu Ile Leu Asn Tyr Met Ser Pro His

3065 3070 3075

cac aaa aaa ctg gca caa gca gtg atg gaa atg aca tac aag aac 9397

His Lys Lys Leu Ala Gln Ala Val Met Glu Met Thr Tyr Lys Asn

3080 3085 3090

aaa gtg gtg aaa gtg ttg aga cca gcc cca gga ggg aaa gcc tac 9442

Lys Val Val Lys Val Leu Arg Pro Ala Pro Gly Gly Lys Ala Tyr

3095 3100 3105

atg gat gtc ata agt cga cga gac cag aga gga tcc ggg cag gta 9487

Met Asp Val Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly Gln Val

3110 3115 3120

gtg act tat gct ctg aac acc atc acc aac ttg aaa gtc caa ttg 9532

Val Thr Tyr Ala Leu Asn Thr Ile Thr Asn Leu Lys Val Gln Leu

3125 3130 3135

atc aga atg gca gaa gca gag atg gtg ata cat cac caa cat gtt 9577

Ile Arg Met Ala Glu Ala Glu Met Val Ile His His Gln His Val

3140 3145 3150

caa gat tgt gat gaa tca gtt ctg acc agg ctg gag gca tgg ctc 9622

Gln Asp Cys Asp Glu Ser Val Leu Thr Arg Leu Glu Ala Trp Leu

3155 3160 3165

act gag cac gga tgt gac aga ctg aag agg atg gcg gtg agt gga 9667

Thr Glu His Gly Cys Asp Arg Leu Lys Arg Met Ala Val Ser Gly

3170 3175 3180

gac gac tgt gtg gtc cgg ccc atc gat gac agg ttc ggc ctg gcc 9712

Asp Asp Cys Val Val Arg Pro Ile Asp Asp Arg Phe Gly Leu Ala

3185 3190 3195

ctg tcc cat ctc aac gcc atg tcc aag gtt aga aag gac ata tct 9757

Leu Ser His Leu Asn Ala Met Ser Lys Val Arg Lys Asp Ile Ser

3200 3205 3210

gaa tgg cag cca tca aaa ggg tgg aat gat tgg gag aat gtg ccc 9802

Glu Trp Gln Pro Ser Lys Gly Trp Asn Asp Trp Glu Asn Val Pro

3215 3220 3225

ttc tgt tcc cac cac ttc cat gaa cta cag ctg aag gat ggc agg 9847

Phe Cys Ser His His Phe His Glu Leu Gln Leu Lys Asp Gly Arg

3230 3235 3240

agg att gtg gtg cct tgc cga gaa cag gac gag ctc att ggg aga 9892

Arg Ile Val Val Pro Cys Arg Glu Gln Asp Glu Leu Ile Gly Arg

3245 3250 3255

gga agg gtg tct cca gga aac ggc tgg atg atc aag gaa aca gct 9937

Gly Arg Val Ser Pro Gly Asn Gly Trp Met Ile Lys Glu Thr Ala

3260 3265 3270

tgc ctc agc aaa gcc tat gcc aac atg tgg tca ctg atg tat ttt 9982

Cys Leu Ser Lys Ala Tyr Ala Asn Met Trp Ser Leu Met Tyr Phe

3275 3280 3285

cac aaa agg gac atg agg cta ctg tca ttg gct gtt tcc tca gct 10027

His Lys Arg Asp Met Arg Leu Leu Ser Leu Ala Val Ser Ser Ala

3290 3295 3300

gtt ccc acc tca tgg gtt cca caa gga cgc aca aca tgg tcg att 10072

Val Pro Thr Ser Trp Val Pro Gln Gly Arg Thr Thr Trp Ser Ile

3305 3310 3315

cat ggg aaa ggg gag tgg atg acc acg gaa gac atg ctt gag gtg 10117

His Gly Lys Gly Glu Trp Met Thr Thr Glu Asp Met Leu Glu Val

3320 3325 3330

tgg aac aga gta tgg ata acc aac aac cca cac atg cag gac aag 10162

Trp Asn Arg Val Trp Ile Thr Asn Asn Pro His Met Gln Asp Lys

3335 3340 3345

aca atg gtg aaa aaa tgg aga gat gtc cct tat cta acc aag aga 10207

Thr Met Val Lys Lys Trp Arg Asp Val Pro Tyr Leu Thr Lys Arg

3350 3355 3360

caa gac aag ctg tgc gga tca ctg att gga atg acc aat agg gcc 10252

Gln Asp Lys Leu Cys Gly Ser Leu Ile Gly Met Thr Asn Arg Ala

3365 3370 3375

acc tgg gcc tcc cac atc cat tta gtc atc cat cgt atc cga acg 10297

Thr Trp Ala Ser His Ile His Leu Val Ile His Arg Ile Arg Thr

3380 3385 3390

ctg att gga cag gag aaa tac act gac tac cta aca gtc atg gac 10342

Leu Ile Gly Gln Glu Lys Tyr Thr Asp Tyr Leu Thr Val Met Asp

3395 3400 3405

agg tat tct gtg gat gct gac ctg caa ctg ggt gag ctt atc 10384

Arg Tyr Ser Val Asp Ala Asp Leu Gln Leu Gly Glu Leu Ile

3410 3415 3420

tgaaacacca tctaacagga ataaccggga tacaaaccac gggtggagaa ccggactccc 10444

cacaacctga aaccgggata taaaccacgg ctggagaacc ggactccgca cttaaaatga 10504

aacagaaacc gggataaaaa ctacggatgg agaaccggac tccacacatt gagacagaag 10564

aagttgtcag cccagaaccc cacacgagtt ttgccactgc taagctgtga ggcagtgcag 10624

gctgggacag ccgacctcca ggttgcgaaa aacctggttt ctgggacctc ccaccccaga 10684

gtaaaaagaa cggagcctcc gctaccaccc tcccacgtgg tggtagaaag acggggtcta 10744

gaggttagag gagaccctcc agggaacaaa tagtgggacc atattgacgc cagggaaaga 10804

ccggagtggt tctctgcttt tcctccagag gtctgtgagc acagtttgct caagaataag 10864

cagacctttg gatgacaaac acaaaaccac aa 10896

<210>23

<211>3422

<212>PRT

<213> Artificial

<220>

<223> synthetic constructs

<400>23

Met Ser Gly Arg Lys Ala Gln Gly Lys Thr Leu Gly Val Asn Met Val

1 5 10 15

Arg Arg Gly Val Arg Ser Leu Ser Asn Lys Ile Lys Gln Lys Thr Lys

20 25 30

Gln Ile Gly Asn Arg Pro Gly Pro Ser Arg Gly Val Gln Gly Phe Ile

35 40 45

Phe Phe Phe Leu Phe Asn Ile Leu Thr Gly Lys Lys Ile Thr Ala His

50 55 60

Leu Lys Arg Leu Trp Lys Met Leu Asp Pro Arg Gln Gly Leu Ala Val

65 70 75 80

Leu Arg Lys Val Lys Arg Val Val Ala Ser Leu Met Arg Gly Leu Ser

85 90 95

Ser Arg Lys Arg Arg Ser His Asp Val Leu Thr Val Gln Phe Leu Ile

100 105 110

Leu Gly Met Leu Leu Met Thr Gly Gly Val Thr Leu Ser Asn Phe Gln

115 120 125

Gly Lys Val Met Met Thr Val Asn Ala Thr Asp Val Thr Asp Val Ile

130 135 140

Thr Ile Pro Thr Ala Ala Gly Lys Asn Leu Cys Ile Val Arg Ala Met

145 150 155 160

Asp Val Gly Tyr Met Cys Asp Asp Thr Ile Thr Tyr Glu Cys Pro Val

165 170 175

Leu Ser Ala Gly Asn Asp Pro Glu Asp Ile Asp Cys Trp Cys Thr Lys

180 185 190

Ser Ala Val Tyr Val Arg Tyr Gly Arg Cys Thr Lys Thr Arg His Ser

195 200 205

Arg Arg Ser Arg Arg Ser Leu Thr Val Gln Thr His Gly Glu Ser Thr

210 215 220

Leu Ala Asn Lys Lys Gly Ala Trp Met Asp Ser Thr Lys Ala Thr Arg

225 230 235 240

Tyr Leu Val Lys Thr Glu Ser Trp Ile Leu Arg Asn Pro Gly Tyr Ala

245 250 255

Leu Val Ala Ala Val Ile Gly Trp Met Leu Gly Ser Asn Thr Met Gln

260 265 270

Arg Val Val Phe Val Val Pro Leu Leu Leu Val Ala Pro Ala Tyr Ser

275 280 285

Phe Asn Cys Leu Gly Met Ser Asn Arg Asp Phe Leu Glu Gly Val Ser

290 295 300

Gly Ala Thr Trp Val Asp Leu Val Leu Glu Gly Asp Ser Cys Val Thr

305 310 315 320

Ile Met Ser Lys Asp Lys Pro Thr Ile Asp Val Lys Met Met Asn Met

325 330 335

Glu Ala Ala Asn Leu Ala Glu Val Arg Ser Tyr Cys Tyr Leu Ala Thr

340 345 350

Val Ser Asp Leu Ser Thr Lys Ala Ala Cys Pro Thr Met Gly Glu Ala

355 360 365

His Asn Asp Lys Arg Ala Asp Pro Ala Phe Val Cys Arg Gln Gly Val

370 375 380

Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Phe Phe Gly Lys Gly Ser

385 390 395 400

Ile Asp Thr Cys Ala Lys Phe Ala Cys Ser Thr Lys Ala Ile Gly Arg

405 410 415

Thr Ile Leu Lys Glu Asn Ile Lys Tyr Glu Val Ala Ile Phe Val His

420 425 430

Gly Pro Thr Thr Val Glu Ser His Gly Asn Tyr Ser Thr Gln Val Gly

435 440 445

Ala Thr Gln Ala Gly Arg Phe Ser Ile Thr Pro Ala Ala Pro Ser Tyr

450 455 460

Thr Leu Lys Leu Gly Glu Tyr Gly Glu Val Thr Val Asp Cys Glu Pro

465 470 475 480

Arg Ser Gly Ile Asp Thr Asn Ala Tyr Tyr Val Met Thr Val Gly Thr

485 490 495

Lys Thr Phe Leu Val His Arg Glu Trp Phe Met Asp Leu Asn Leu Pro

500 505 510

Trp Ser Ser Ala Gly Ser Thr Val Trp Arg Asn Arg Glu Thr Leu Met

515 520 525

Glu Phe Glu Glu Pro His Ala Thr Lys Gln Ser Val Ile Ala Leu Gly

530 535 540

Ser Gln Glu Gly Ala Leu His Gln Ala Leu Ala Gly Ala Ile Pro Val

545 550 555 560

Glu Phe Ser Ser Asn Thr Val Lys Leu Thr Ser Gly His Leu Lys Cys

565 570 575

Arg Val Lys Met Glu Lys Leu Gln Leu Lys Gly Thr Thr Tyr Gly Val

580 585 590

Cys Ser Lys Ala Phe Lys Phe Leu Arg Thr Pro Val Asp Thr Gly His

595 600 605

Gly Thr Val Val Leu Glu Leu Gln Tyr Thr Gly Thr Asp Gly Pro Cys

610 615 620

Lys Val Pro Ile Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val

625 630 635 640

Gly Arg Leu Val Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn

645 650 655

Ala Lys Val Leu Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile

660 665 670

Val Val Gly Arg Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser

675 680 685

Gly Ser Ser Ile Gly Lys Ala Phe Thr Thr Thr Leu Lys Gly Ala Gln

690 695 700

Arg Leu Ala Ala Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val Gly

705 710 715 720

Gly Val Phe Thr Ser Val Gly Arg Ala Val His Gln Val Phe Gly Gly

725 730 735

Ala Phe Arg Ser Leu Phe Gly Gly Met Ser Trp Ile Thr Gln Gly Leu

740 745 750

Leu Gly Ala Leu Leu Leu Trp Met Gly Ile Asn Ala Arg Asp Arg Ser

755 760 765

Ile Ala Leu Thr Phe Leu Ala Val Gly Gly Val Leu Leu Phe Leu Ser

770 775 780

Val Asn Val Gly Ala Asp Gln Gly Cys Ala Ile Asn Phe Gly Lys Arg

785 790 795 800

Glu Leu Lys Cys Gly Asp Gly Ile Phe Ile Phe Arg Asp Ser Asp Asp

805 810 815

Trp Leu Asn Lys Tyr Ser Tyr Tyr Pro Glu Asp Pro Val Lys Leu Ala

820 825 830

Ser Ile Val Lys Ala Ser Phe Glu Glu Gly Lys Cys Gly Leu Asn Ser

835 840 845

Val Asp Ser Leu Glu His Glu Met Trp Arg Ser Arg Ala Asp Glu Ile

850 855 860

Asn Ala Ile Phe Glu Glu Asn Glu Val Asp Ile Ser Val Val Val Gln

865 870 875 880

Asp Pro Lys Asn Val Tyr Gln Arg Gly Thr His Pro Phe Ser Arg Ile

885 890 895

Arg Asp Gly Leu Gln Tyr Gly Trp Lys Thr Trp Gly Lys Asn Leu Val

900 905 910

Phe Ser Pro Gly Arg Lys Asn Gly Ser Phe Ile Ile Asp Gly Lys Ser

915 920 925

Arg Lys Glu Cys Pro Phe Ser Asn Arg Val Trp Asn Ser Phe Gln Ile

930 935 940

Glu Glu Phe Gly Thr Gly Val Phe Thr Thr Arg Val Tyr Met Asp Ala

945 950 955 960

Val Phe Glu Tyr Thr Ile Asp Cys Asp Gly Ser Ile Leu Gly Ala Ala

965 970 975

Val Asn Gly Lys Lys Ser Ala His Gly Ser Pro Thr Phe Trp Met Gly

980 985 990

Ser His Glu Val Asn Gly Thr Trp Met Ile His Thr Leu Glu Ala Leu

995 1000 1005

Asp Tyr Lys Glu Cys Glu Trp Pro Leu Thr His Thr Ile Gly Thr

1010 1015 1020

Ser Val Glu Glu Ser Glu Met Phe Met Pro Arg Ser Ile Gly Gly

1025 1030 1035

Pro Val Ser Ser His Asn His Ile Pro Gly Tyr Lys Val Gln Thr

1040 1045 1050

Asn Gly Pro Trp Met Gln Val Pro Leu Glu Val Lys Arg Glu Ala

1055 1060 1065

Cys Pro Gly Thr Ser Val Ile Ile Asp Gly Asn Cys Asp Gly Arg

1070 1075 1080

Gly Lys Ser Thr Arg Ser Thr Thr Asp Ser Gly Lys Val Ile Pro

1085 1090 1095

Glu Trp Cys Cys Arg Ser Cys Thr Met Pro Pro Val Ser Phe His

1100 1105 1110

Gly Ser Asp Gly Cys Trp Tyr Pro Met Glu Ile Arg Pro Arg Lys

1115 1120 1125

Thr His Glu Ser His Leu Val Arg Ser Trp Val Thr Ala Gly Glu

1130 1135 1140

Ile His Ala Val Pro Phe Gly Leu Val Ser Met Met Ile Ala Met

1145 1150 1155

Glu Val Val Leu Arg Lys Arg Gln Gly Pro Lys Gln Met Leu Val

1160 1165 1170

Gly Gly Val Val Leu Leu Gly Ala Met Leu Val Gly Gln Val Thr

1175 1180 1185

Leu Leu Asp Leu Leu Lys Leu Thr Val Ala Val Gly Leu His Phe

1190 1195 1200

His Glu Met Asn Asn Gly Gly Asp Ala Met Tyr Met Ala Leu Ile

1205 1210 1215

Ala Ala Phe Ser Ile Arg Pro Gly Leu Leu Ile Gly Phe Gly Leu

1220 1225 1230

Arg Thr Leu Trp Ser Pro Arg Glu Arg Leu Val Leu Thr Leu Gly

1235 1240 1245

Ala Ala Met Val Glu Ile Ala Leu Gly Gly Val Met Gly Gly Leu

1250 1255 1260

Trp Lys Tyr Leu Asn Ala Val Ser Leu Cys Ile Leu Thr Ile Asn

1265 1270 1275

Ala Val Ala Ser Arg Lys Ala Ser Asn Thr Ile Leu Pro Leu Met

1280 1285 1290

Ala Leu Leu Thr Pro Val Thr Met Ala Glu Val Arg Leu Ala Ala

1295 1300 1305

Met Phe Phe Cys Ala Met Val Ile Ile Gly Val Leu His Gln Asn

1310 1315 1320

Phe Lys Asp Thr Ser Met Gln Lys Thr Ile Pro Leu Val Ala Leu

1325 1330 1335

Thr Leu Thr Ser Tyr Leu Gly Leu Thr Gln Pro Phe Leu Gly Leu

1340 1345 1350

Cys Ala Phe Leu Ala Thr Arg Ile Phe Gly Arg Arg Ser Ile Pro

1355 1360 1365

Val Asn Glu Ala Leu Ala Ala Ala Gly Leu Val Gly Val Leu Ala

1370 1375 1380

Gly Leu Ala Phe Gln Glu Met Glu Asn Phe Leu Gly Pro Ile Ala

1385 1390 1395

Val Gly Gly Leu Leu Met Met Leu Val Ser Val Ala Gly Arg Val

1400 1405 1410

Asp Gly Leu Glu Leu Lys Lys Leu Gly Glu Val Ser Trp Glu Glu

1415 1420 1425

Glu Ala Glu Ile Ser Gly Ser Ser Ala Arg Tyr Asp Val Ala Leu

1430 1435 1440

Ser Glu Gln Gly Glu Phe Lys Leu Leu Ser Glu Glu Lys Val Pro

1445 1450 1455

Trp Asp Gln Val Val Met Thr Ser Leu Ala Leu Val Gly Ala Ala

1460 1465 1470

Leu His Pro Phe Ala Leu Leu Leu Val Leu Ala Gly Trp Leu Phe

1475 1480 1485

His Val Arg Gly Ala Arg Arg Ser Gly Asp Val Leu Trp Asp Ile

1490 1495 1500

Pro Thr Pro Lys Ile Ile Glu Glu Cys Glu His Leu Glu Asp Gly

1505 1510 1515

Ile Tyr Gly Ile Phe Gln Ser Thr Phe Leu Gly Ala Ser Gln Arg

1520 1525 1530

Gly Val Gly Val Ala Gln Gly Gly Val Phe His Thr Met Trp His

1535 1540 1545

Val Thr Arg Gly Ala Phe Leu Val Arg Asn Gly Lys Lys Leu Ile

1550 1555 1560

Pro Ser Trp Ala Ser Val Lys Glu Asp Leu Val Ala Tyr Gly Gly

1565 1570 1575

Ser Trp Lys Leu Glu Gly Arg Trp Asp Gly Glu Glu Glu Val Gln

1580 1585 1590

Leu Ile Ala Ala Val Pro Gly Lys Asn Val Val Asn Val Gln Thr

1595 1600 1605

Lys Pro Ser Leu Phe Lys Val Arg Asn Gly Gly Glu Ile Gly Ala

1610 1615 1620

Val Ala Leu Asp Tyr Pro Ser Gly Thr Ser Gly Ser Pro Ile Val

1625 1630 1635

Asn Arg Asn Gly Glu Val Ile Gly Leu Tyr Gly Asn Gly Ile Leu

1640 1645 1650

Val Gly Asp Asn Ser Phe Val Ser Ala Ile Ser Gln Thr Glu Val

1655 1660 1665

Lys Glu Glu Gly Lys Glu Glu Leu Gln Glu Ile Pro Thr Met Leu

1670 1675 1680

Lys Lys Gly Met Thr Thr Val Leu Asp Phe His Pro Gly Ala Gly

1685 1690 1695

Lys Thr Arg Arg Phe Leu Pro Gln Ile Leu Ala Glu Cys Ala Arg

1700 1705 1710

Arg Arg Leu Arg Thr Leu Val Leu Ala Pro Thr Arg Val Val Leu

1715 1720 1725

Ser Glu Met Lys Glu Ala Phe His Gly Leu Asp Val Lys Phe His

1730 1735 1740

Thr Gln Ala Phe Ser Ala His Gly Ser Gly Arg Glu Val Ile Asp

1745 1750 1755

Ala Met Cys His Ala Thr Leu Thr Tyr Arg Met Leu Glu Pro Thr

1760 1765 1770

Arg Val Val Asn Trp Glu Val Ile Ile Met Asp Glu Ala His Phe

1775 1780 1785

Leu Asp Pro Ala Ser Ile Ala Ala Arg Gly Trp Ala Ala His Arg

1790 1795 1800

Ala Arg Ala Asn Glu Ser Ala Thr Ile Leu Met Thr Ala Thr Pro

1805 1810 1815

Pro Gly Thr Ser Asp Glu Phe Pro His Ser Asn Gly Glu Ile Glu

1820 1825 1830

Asp Val Gln Thr Asp Ile Pro Ser Glu Pro Trp Asn Thr Gly His

1835 1840 1845

Asp Trp Ile Leu Ala Asp Lys Arg Pro Thr Ala Trp Phe Leu Pro

1850 1855 1860

Ser Ile Arg Ala Ala Asn Val Met Ala Ala Ser Leu Arg Lys Ala

1865 1870 1875

Gly Lys Ser Val Val Val Leu Asn Arg Lys Thr Phe Glu Arg Glu

1880 1885 1890

Tyr Pro Thr Ile Lys Gln Lys Lys Pro Asp Phe Ile Leu Ala Thr

1895 1900 1905

Asp Ile Ala Glu Met Gly Ala Asn Leu Cys Val Glu Arg Val Leu

1910 1915 1920

Asp Cys Arg Thr Ala Phe Lys Pro Val Leu Val Asp Glu Gly Arg

1925 1930 1935

Lys Val Ala Ile Lys Gly Pro Leu Arg Ile Ser Ala Ser Ser Ala

1940 1945 1950

Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn Pro Asn Arg Asp Gly

1955 1960 1965

Asp Ser Tyr Tyr Tyr Ser Glu Pro Thr Ser Glu Asn Asn Ala His

1970 1975 1980

His Val Cys Trp Leu Glu Ala Ser Met Leu Leu Asp Asn Met Glu

1985 1990 1995

Val Arg Gly Gly Met Val Ala Pro Leu Tyr Gly Val Glu Gly Thr

2000 2005 2010

Lys Thr Pro Val Ser Pro Gly Glu Met Arg Leu Arg Asp Asp Gln

2015 2020 2025

Arg Lys Val Phe Arg Glu Leu Val Arg Asn Cys Asp Leu Pro Val

2030 2035 2040

Trp Leu Ser Trp Gln Val Ala Lys Ala Gly Leu Lys Thr Asn Asp

2045 2050 2055

Arg Lys Trp Cys Phe Glu Gly Pro Glu Glu His Glu Ile Leu Asn

2060 2065 2070

Asp Ser Gly Glu Thr Val Lys Cys Arg Ala Pro Gly Gly Ala Lys

2075 2080 2085

Lys Pro Leu Arg Pro Arg Trp Cys Asp Glu Arg Val Ser Ser Asp

2090 2095 2100

Gln Ser Ala Leu Ser Glu Phe Ile Lys Phe Ala Glu Gly Arg Arg

2105 2110 2115

Gly Ala Ala Glu Val Leu Val Val Leu Ser Glu Leu Pro Asp Phe

2120 2125 2130

Leu Ala Lys Lys Gly Gly Glu Ala Met Asp Thr Ile Ser Val Phe

2135 2140 2145

Leu His Ser Glu Glu Gly Ser Arg Ala Tyr Arg Asn Ala Leu Ser

2150 2155 2160

Met Met Pro Glu Ala Met Thr Ile Val Met Leu Phe Ile Leu Ala

2165 2170 2175

Gly Leu Leu Thr Ser Gly Met Val Ile Phe Phe Met Ser Pro Lys

2180 2185 2190

Gly Ile Ser Arg Met Ser Met Ala Met Gly Thr Met Ala Gly Cys

2195 2200 2205

Gly Tyr Leu Met Phe Leu Gly Gly Val Lys Pro Thr His Ile Ser

2210 2215 2220

Tyr Val Met Leu Ile Phe Phe Val Leu Met Val Val Val Ile Pro

2225 2230 2235

Glu Pro Gly Gln Gln Arg Ser Ile Gln Asp Asn Gln Val Ala Tyr

2240 2245 2250

Leu Ile Ile Gly Ile Leu Thr Leu Val Ser Ala Val Ala Ala Asn

2255 2260 2265

Glu Leu Gly Met Leu Glu Lys Thr Lys Glu Asp Leu Phe Gly Lys

2270 2275 2280

Lys Asn Leu Ile Pro Ser Ser Ala Ser Pro Trp Ser Trp Pro Asp

2285 2290 2295

Leu Asp Leu Lys Pro Gly Ala Ala Trp Thr Val Tyr Val Gly Ile

2300 2305 2310

Val Thr Met Leu Ser Pro Met Leu His His Trp Ile Lys Val Glu

2315 2320 2325

Tyr Gly Asn Leu Ser Leu Ser Gly Ile Ala Gln Ser Ala Ser Val

2330 2335 2340

Leu Ser Phe Met Asp Lys Gly Ile Pro Phe Met Lys Met Asn Ile

2345 2350 2355

Ser Val Ile Met Leu Leu Val Ser Gly Trp Asn Ser Ile Thr Val

2360 2365 2370

Met Pro Leu Leu Cys Gly Ile Gly Cys Ala Met Leu His Trp Ser

2375 2380 2385

Leu Ile Leu Pro Gly Ile Lys Ala Gln Gln Ser Lys Leu Ala Gln

2390 2395 2400

Arg Arg Val Phe His Gly Val Ala Lys Asn Pro Val Val Asp Gly

2405 2410 2415

Asn Pro Thr Val Asp Ile Glu Glu Ala Pro Glu Met Pro Ala Leu

2420 2425 2430

Tyr Glu Lys Lys Leu Ala Leu Tyr Leu Leu Leu Ala Leu Ser Leu

2435 2440 2445

Ala Ser Val Ala Met Cys Arg Thr Pro Phe Ser Leu Ala Glu Gly

2450 2455 2460

Ile Val Leu Ala Ser Ala Ala Leu Gly Pro Leu Ile Glu Gly Asn

2465 2470 2475

Thr Ser Leu Leu Trp Asn Gly Pro Met Ala Val Ser Met Thr Gly

2480 2485 2490

Val Met Arg Gly Asn His Tyr Ala Phe Val Gly Val Met Tyr Asn

2495 2500 2505

Leu Trp Lys Met Lys Thr Gly Arg Arg Gly Ser Ala Asn Gly Lys

2510 2515 2520

Thr Leu Gly Glu Val Trp Lys Arg Glu Leu Asn Leu Leu Asp Lys

2525 2530 2535

Arg Gln Phe Glu Leu Tyr Lys Arg Thr Asp Ile Val Glu Val Asp

2540 2545 2550

Arg Asp Thr Ala Arg Arg His Leu Ala Glu Gly Lys Val Asp Thr

2555 2560 2565

Gly Val Ala Val Ser Arg Gly Thr Ala Lys Leu Arg Trp Phe His

2570 2575 2580

Glu Arg Gly Tyr Val Lys Leu Glu Gly Arg Val Ile Asp Leu Gly

2585 2590 2595

Cys Gly Arg Gly Gly Trp Cys Tyr Tyr Ala Ala Ala Gln Lys Glu

2600 2605 2610

Val Ser Gly Val Lys Gly Phe Thr Leu Gly Arg Asp Gly His Glu

2615 2620 2625

Lys Pro Met Asn Val Gln Ser Leu Gly Trp Asn Ile Ile Thr Phe

2630 2635 2640

Lys Asp Lys Thr Asp Ile His Arg Leu Glu Pro Val Lys Cys Asp

2645 2650 2655

Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Ser Ser Ser Val Thr

2660 2665 2670

Glu Gly Glu Arg Thr Val Arg Val Leu Asp Thr Val Glu Lys Trp

2675 2680 2685

Leu Ala Cys Gly Val Asp Asn Phe Cys Val Lys Val Leu Ala Pro

2690 2695 2700

Tyr Met Pro Asp Val Leu Glu Lys Leu Glu Leu Leu Gln Arg Arg

2705 2710 2715

Phe Gly Gly Thr Val Ile Arg Asn Pro Leu Ser Arg Asn Ser Thr

2720 2725 2730

His Glu Met Tyr Tyr Val Ser Gly Ala Arg Ser Asn Val Thr Phe

2735 2740 2745

Thr Val Asn Gln Thr Ser Arg Leu Leu Met Arg Arg Met Arg Arg

2750 2755 2760

Pro Thr Gly Lys Val Thr Leu Glu Ala Asp Val Ile Leu Pro Ile

2765 2770 2775

Gly Thr Arg Ser Val Glu Thr Asp Lys Gly Pro Leu Asp Lys Glu

2780 2785 2790

Ala Ile Glu Glu Arg Val Glu Arg Ile Lys Ser Glu Tyr Met Thr

2795 2800 2805

Ser Trp Phe Tyr Asp Asn Asp Asn Pro Tyr Arg Thr Trp His Tyr

2810 2815 2820

Cys Gly Ser Tyr Val Thr Lys Thr Ser Gly Ser Ala Ala Ser Met

2825 2830 2835

Val Asn Gly Val Ile Lys Ile Leu Thr Tyr Pro Trp Asp Arg Ile

2840 2845 2850

Glu Glu Val Thr Arg Met Ala Met Thr Asp Thr Thr Pro Phe Gly

2855 2860 2865

Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg Ala Lys Asp

2870 2875 2880

Pro Pro Ala Gly Thr Arg Lys Ile Met Lys Val Val Asn Arg Trp

2885 2890 2895

Leu Phe Arg His Leu Ala Arg Glu Lys Asn Pro Arg Leu Cys Thr

2900 2905 2910

Lys Glu Glu Phe Ile Ala Lys Val Arg Ser His Ala Ala Ile Gly

2915 2920 2925

Ala Tyr Leu Glu Glu Gln Glu Gln Trp Lys Thr Ala Asn Glu Ala

2930 2935 2940

Val Gln Asp Pro Lys Phe Trp Glu Leu Val Asp Glu Glu Arg Lys

2945 2950 2955

Leu His Gln Gln Gly Arg Cys Arg Thr Cys Val Tyr Asn Met Met

2960 2965 2970

Gly Lys Arg Glu Lys Lys Leu Ser Glu Phe Gly Lys Ala Lys Gly

2975 2980 2985

Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Tyr Leu Glu

2990 2995 3000

Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Ala Ser Arg

3005 3010 3015

Glu Asn Ser Gly Gly Gly Val Glu Gly Ile Gly Leu Gln Tyr Leu

3020 3025 3030

Gly Tyr Val Ile Arg Asp Leu Ala Ala Met Asp Gly Gly Gly Phe

3035 3040 3045

Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Ala

3050 3055 3060

Asp Leu Asp Asp Glu Gln Glu Ile Leu Asn Tyr Met Ser Pro His

3065 3070 3075

His Lys Lys Leu Ala Gln Ala Val Met Glu Met Thr Tyr Lys Asn

3080 3085 3090

Lys Val Val Lys Val Leu Arg Pro Ala Pro Gly Gly Lys Ala Tyr

3095 3100 3105

Met Asp Val Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly Gln Val

3110 3115 3120

Val Thr Tyr Ala Leu Asn Thr Ile Thr Asn Leu Lys Val Gln Leu

3125 3130 3135

Ile Arg Met Ala Glu Ala Glu Met Val Ile His His Gln His Val

3140 3145 3150

Gln Asp Cys Asp Glu Ser Val Leu Thr Arg Leu Glu Ala Trp Leu

3155 3160 3165

Thr Glu His Gly Cys Asp Arg Leu Lys Arg Met Ala Val Ser Gly

3170 3175 3180

Asp Asp Cys Val Val Arg Pro Ile Asp Asp Arg Phe Gly Leu Ala

3185 3190 3195

Leu Ser His Leu Asn Ala Met Ser Lys Val Arg Lys Asp Ile Ser

3200 3205 3210

Glu Trp Gln Pro Ser Lys Gly Trp Asn Asp Trp Glu Asn Val Pro

3215 3220 3225

Phe Cys Ser His His Phe His Glu Leu Gln Leu Lys Asp Gly Arg

3230 3235 3240

Arg Ile Val Val Pro Cys Arg Glu Gln Asp Glu Leu Ile Gly Arg

3245 3250 3255

Gly Arg Val Ser Pro Gly Asn Gly Trp Met Ile Lys Glu Thr Ala

3260 3265 3270

Cys Leu Ser Lys Ala Tyr Ala Asn Met Trp Ser Leu Met Tyr Phe

3275 3280 3285

His Lys Arg Asp Met Arg Leu Leu Ser Leu Ala Val Ser Ser Ala

3290 3295 3300

Val Pro Thr Ser Trp Val Pro Gln Gly Arg Thr Thr Trp Ser Ile

3305 3310 3315

His Gly Lys Gly Glu Trp Met Thr Thr Glu Asp Met Leu Glu Val

3320 3325 3330

Trp Asn Arg Val Trp Ile Thr Asn Asn Pro His Met Gln Asp Lys

3335 3340 3345

Thr Met Val Lys Lys Trp Arg Asp Val Pro Tyr Leu Thr Lys Arg

3350 3355 3360

Gln Asp Lys Leu Cys Gly Ser Leu Ile Gly Met Thr Asn Arg Ala

3365 3370 3375

Thr Trp Ala Ser His Ile His Leu Val Ile His Arg Ile Arg Thr

3380 3385 3390

Leu Ile Gly Gln Glu Lys Tyr Thr Asp Tyr Leu Thr Val Met Asp

3395 3400 3405

Arg Tyr Ser Val Asp Ala Asp Leu Gln Leu Gly Glu Leu Ile

3410 3415 3420

<210>24

<211>10239

<212>DNA

<213> Artificial sequence

<220>

<223> derived from yellow fever virus and West Nile virus

<400>24

gtaaatcctg tgtgctaatt gaggtgcatt ggtctgcaaa tcgagttgct aggcaataaa 60

cacatttgga ttaattttaa tcgttcgttg agcgattagc agagaactga ccagaacatg 120

tctggtcgta aagctcaggg aaaaaccctg ggcgtcaata tggtacgacg aggagttcgc 180

tccttgtcaa acaaaataaa acaaaaaaca aaacaaattg gaaacagacc tggaccttca 240

agaggtgttc aaggatttat ctttttcttt ttgttcaaca ttttgactgg aaaaaagatc 300

acagcccacc taaagaggtt gtggaaaatg ctggacccaa gacaaggctt ggctgttcta 360

aggaaagtca agagagtggt ggccagtttg atgagaggat tgtcctcaag gaaacgccgt 420

tcccatgatg ttctgactgt gcaattccta attttgggaa tgctgttgat gacgggtgga 480

gttaccctct ctaacttcca agggaaggtg atgatgacgg taaatgctac tgacgtcaca 540

gatgtcatca cgattccaac agctgctgga aagaacctat gcattgtcag agcaatggat 600

gtgggataca tgtgcgatga tactatcact tatgaatgcc cagtgctgtc ggctggtaat 660

gatccagaag acatcgactg ttggtgcaca aagtcagcag tctacgtcag gtatggaaga 720

tgcaccaaga cacgccactc aagacgcagt cggaggtcac tgacagtgca gacacacgga 780

gaaagcactc tagcgaacaa gaagggggct tggatggaca gcaccaaggc cacaaggtat 840

ttggtaaaaa cagaatcatg gatcttgagg aaccctggat atgccctggt ggcagccgtc 900

attggttgga tgcttgggag caacaccatg cagagagttg tgtttgtcgt gctattgctt 960

ttggtggccc cagcttacag cttcaactgc cttggaatga gcaacagaga cttcttggaa 1020

ggagtgtctg gagcaacatg ggtggatttg gttctcgaag gcgacagctg cgtgactatc 1080

atgtctaagg acaagcctac catcgacgtc aagatgatga atatggaggc ggccaacctg 1140

gcagaggtcc gcagttattg ctatttggct accgtcagcg atctctccac caaagctgca 1200

tgcccgacca tgggagaagc tcacaatgac aaacgtgctg acccagcttt tgtgtgcaga 1260

caaggagtgg tggacagggg ctggggcaac ggctgcggat tttttggcaa aggatccatt 1320

gacacatgcg ccaaatttgc ctgctctacc aaggcaatag gaagaaccat cttgaaagag 1380

aatatcaagt acgaagtggc catttttgtc catggaccaa ctactgtgga gtcgcacgga 1440

aattactcca cacaggttgg agccactcag gccggccgat tcagcatcac tcctgctgcg 1500

ccttcataca cactaaagct tggagaatat ggagaggtga cagtggactg tgaaccacgg 1560

tcagggattg acaccaatgc atactacgtg atgactgttg gaacaaagac gttcttggtc 1620

catcgtgagt ggttcatgga cctcaacctc ccttggagca gtgctggaag tactgtgtgg 1680

aggaacagag agacgttaat ggagtttgag gaaccacacg ccacgaagca gtctgtgata 1740

gcattgggct cacaagaggg agctctgcat caagctttgg ctggagccat tcctgtggaa 1800

ttttcaagca acactgtcaa gttgacgtcg ggtcatttga agtgtagagt gaagatggaa 1860

aaattgcagt tgaagggaac aacctatggc gtctgttcaa aggctttcaa gtttcttagg 1920

actcccgtgg acaccggtca cggcactgtg gtgttggaat tgcagtacac tggcacggat 1980

ggaccttgca aagttcctat ctcgtcagtg gcttcattga acgacctaac gccagtgggc 2040

agattggtca ctgtcaaccc ttttgtttca gtggccacgg ccaacgctaa ggtcctgatt 2100

gaattggaac caccctttgg agactcatac atagtggtgg gcagaggaga acaacagatc 2160

aatcaccatt ggcacaagtc tggaagcagc attggcaaag cctttacaac caccctcaaa 2220

ggagcgcaga gactagccgc tctaggagac acagcttggg actttggatc agttggaggg 2280

gtgttcacta gtgttgggcg ggctgtccat caagtgttcg gaggagcatt ccgctcactg 2340

ttcggaggca tgtcctggat aacgcaagga ttgctggggg ctctcctgtt gtggatgggc 2400

atcaatgctc gtgataggtc catagctctc acgtttctcg cagttggagg agttctgctc 2460

ttcctctccg tgaacgtggg cgccgatcaa ggatgcgcca tcaactttgg caagagagag 2520

ctcaagtgcg gagatggtat cttcatattt agagactctg atgactggct gaacaagtac 2580

tcatactatc cagaagatcc tgtgaagctt gcatcaatag tgaaagcctc ttttgaagaa 2640

gggaagtgtg gcctaaattc agttgactcc cttgagcatg agatgtggag aagcagggca 2700

gatgagatca atgccatttt tgaggaaaac gaggtggaca tttctgttgt cgtgcaggat 2760

ccaaagaatg tttaccagag aggaactcat ccattttcca gaattcggga tggtctgcag 2820

tatggttgga agacttgggg taagaacctt gtgttctccc cagggaggaa gaatggaagc 2880

ttcatcatag atggaaagtc caggaaagaa tgcccgtttt caaaccgggt ctggaattct 2940

ttccagatag aggagtttgg gacgggagtg ttcaccacac gcgtgtacat ggacgcagtc 3000

tttgaataca ccatagactg cgatggatct atcttgggtg cagcggtgaa cggaaaaaag 3060

agtgcccatg gctctccaac attttggatg ggaagtcatg aagtaaatgg gacatggatg 3120

atccacacct tggaggcatt agattacaag gagtgtgagt ggccactgac acatacgatt 3180

ggaacatcag ttgaagagag tgaaatgttc atgccgagat caatcggagg cccagttagc 3240

tctcacaatc atatccctgg atacaaggtt cagacgaacg gaccttggat gcaggtacca 3300

ctagaagtga agagagaagc ttgcccaggg actagcgtga tcattgatgg caactgtgat 3360

ggacggggaa aatcaaccag atccaccacg gatagcggga aagttattcc tgaatggtgt 3420

tgccgctcct gcacaatgcc gcctgtgagc ttccatggta gtgatgggtg ttggtatccc 3480

atggaaatta ggccaaggaa aacgcatgaa agccatctgg tgcgctcctg ggttacagct 3540

ggagaaatac atgctgtccc ttttggtttg gtgagcatga tgatagcaat ggaagtggtc 3600

ctaaggaaaa gacagggacc aaagcaaatg ttggttggag gagtagtgct cttgggagca 3660

atgctggtcg ggcaagtaac tctccttgat ttgctgaaac tcacagtggc tgtgggattg 3720

catttccatg agatgaacaa tggaggagac gccatgtata tggcgttgat tgctgccttt 3780

tcaatcagac cagggctgct catcggcttt gggctcagga ccctatggag ccctcgggaa 3840

cgccttgtgc tgaccctagg agcagccatg gtggagattg ccttgggtgg cgtgatgggc 3900

ggcctgtgga agtatctaaa tgcagtttct ctctgcatcc tgacaataaa tgctgttgct 3960

tctaggaaag catcaaatac catcttgccc ctcatggctc tgttgacacc tgtcactatg 4020

gctgaggtga gacttgccgc aatgttcttt tgtgccatgg ttatcatagg ggtccttcac 4080

cagaatttca aggacacctc catgcagaag actatacctc tggtggccct cacactcaca 4140

tcttacctgg gcttgacaca accttttttg ggcctgtgtg catttctggc aacccgcata 4200

tttgggcgaa ggagtatccc agtgaatgag gcactcgcag cagctggtct agtgggagtg 4260

ctggcaggac tggcttttca ggagatggag aacttccttg gtccgattgc agttggagga 4320

ctcctgatga tgctggttag cgtggctggg agggtggatg ggctagagct caagaagctt 4380

ggtgaagttt catgggaaga ggaggcggag atcagcggga gttccgcccg ctatgatgtg 4440

gcactcagtg aacaagggga gttcaagctg ctttctgaag agaaagtgcc atgggaccag 4500

gttgtgatga cctcgctggc cttggttggg gctgccctcc atccatttgc tcttctgctg 4560

gtccttgctg ggtggctgtt tcatgtcagg ggagctagga gaagtgggga tgtcttgtgg 4620

gatattccca ctcctaagat catcgaggaa tgtgaacatc tggaggatgg gatttatggc 4680

atattccagt caaccttctt gggggcctcc cagcgaggag tgggagtggc acagggaggg 4740

gtgttccaca caatgtggca tgtcacaaga ggagctttcc ttgtcaggaa tggcaagaag 4800

ttgattccat cttgggcttc agtaaaggaa gaccttgtcg cctatggtgg ctcatggaag 4860

ttggaaggca gatgggatgg agaggaagag gtccagttga tcgcggctgt tccaggaaag 4920

aacgtggtca acgtccagac aaaaccgagc ttgttcaaag tgaggaatgg gggagaaatc 4980

ggggctgtcg ctcttgacta tccgagtggc acttcaggat ctcctattgt taacaggaac 5040

ggagaggtga ttgggctgta cggcaatggc atccttgtcg gtgacaactc cttcgtgtcc 5100

gccatatccc agactgaggt gaaggaagaa ggaaaggagg agctccaaga gatcccgaca 5160

atgctaaaga aaggaatgac aactgtcctt gattttcatc ctggagctgg gaagacaaga 5220

cgtttcctcc cacagatctt ggccgagtgc gcacggagac gcttgcgcac tcttgtgttg 5280

gcccccacca gggttgttct ttctgaaatg aaggaggctt ttcacggcct ggacgtgaaa 5340

ttccacacac aggctttttc cgctcacggc agcgggagag aagtcattga tgccatgtgc 5400

catgccaccc taacttacag gatgttggaa ccaactaggg ttgttaactg ggaagtgatc 5460

attatggatg aagcccattt tttggatcca gccagcatag ccgctagagg ttgggcagcg 5520

cacagagcta gggcaaatga aagtgcaaca atcttgatga cagccacacc gcctgggact 5580

agtgatgaat ttccacattc aaatggtgaa atagaagatg ttcaaacgga catacccagt 5640

gagccctgga acacagggca tgactggatc ctggctgaca aaaggcccac ggcatggttc 5700

cttccatcca tcagagctgc aaatgtcatg gctgcctctt tgcgtaaggc tggaaagagt 5760

gtggtggtcc tgaacaggaa aacctttgag agagaatacc ccacgataaa gcagaagaaa 5820

cctgacttta tattggccac tgacatagct gaaatgggag ccaacctttg cgtggagcga 5880

gtgctggatt gcaggacggc ttttaagcct gtgcttgtgg atgaagggag gaaggtggca 5940

ataaaagggc cacttcgtat ctccgcatcc tctgctgctc aaaggagggg gcgcattggg 6000

agaaatccca acagagatgg agactcatac tactattctg agcctacaag tgaaaataat 6060

gcccaccacg tctgctggtt ggaggcctca atgctcttgg acaacatgga ggtgaggggt 6120

ggaatggtcg ccccactcta tggcgttgaa ggaactaaaa caccagtttc ccctggtgaa 6180

atgagactga gggatgacca gaggaaagtc ttcagagaac tagtgaggaa ttgtgacctg 6240

cccgtttggc tttcgtggca agtggccaag gctggtttga agacgaatga tcgtaagtgg 6300

tgttttgaag gccctgagga acatgagatc ttgaatgaca gcggtgaaac agtgaagtgc 6360

agggctcctg gaggagcaaa gaagcctctg cgcccaaggt ggtgtgatga aagggtgtca 6420

tctgaccaga gtgcgctgtc tgaatttatt aagtttgctg aaggtaggag gggagctgct 6480

gaagtgctag ttgtgctgag tgaactccct gatttcctgg ctaaaaaagg tggagaggca 6540

atggatacca tcagtgtgtt cctccactct gaggaaggct ctagggctta ccgcaatgca 6600

ctatcaatga tgcctgaggc aatgacaata gtcatgctgt ttatactggc tggactactg 6660

acatcgggaa tggtcatctt tttcatgtct cccaaaggca tcagtagaat gtctatggcg 6720

atgggcacaa tggccggctg tggatatctc atgttccttg gaggcgtcaa acccactcac 6780

atctcctatg tcatgctcat attctttgtc ctgatggtgg ttgtgatccc cgagccaggg 6840

caacaaaggt ccatccaaga caaccaagtg gcatacctca ttattggcat cctgacgctg 6900

gtttcagcgg tggcagccaa cgagctaggc atgctggaga aaaccaaaga ggacctcttt 6960

gggaagaaga acttaattcc atctagtgct tcaccctgga gttggccgga tcttgacctg 7020

aagccaggag ctgcctggac agtgtacgtt ggcattgtta caatgctctc tccaatgttg 7080

caccactgga tcaaagtcga atatggcaac ctgtctctgt ctggaatagc ccagtcagcc 7140

tcagtccttt ctttcatgga caaggggata ccattcatga agatgaatat ctcggtcata 7200

atgctgctgg tcagtggctg gaattcaata acagtgatgc ctctgctctg tggcataggg 7260

tgcgccatgc tccactggtc tctcatttta cctggaatca aagcgcagca gtcaaagctt 7320

gcacagagaa gggtgttcca tggcgttgcc aagaaccctg tggttgatgg gaatccaaca 7380

gttgacattg aggaagctcc tgaaatgcct gccctttatg agaagaaact ggctctatat 7440

ctccttcttg ctctcagcct agcttctgtt gccatgtgca gaacgccctt ttcattggct 7500

gaaggcattg tcctagcatc agctgcctta gggccgctca tagagggaaa caccagcctt 7560

ctttggaatg gacccatggc tgtctccatg acaggagtca tgagggggaa tcactatgct 7620

tttgtgggag tcatgtacaa tctatggaag atgaaaactg gacgccgggg gagcgcgaat 7680

ggaaaaactt tgggtgaagt ctggaagagg gaactgaatc tgttggacaa gcgacagttt 7740

gagttgtata aaaggaccga cattgtggag gtggatcgtg atacggcacg caggcatttg 7800

gccgaaggga aggtggacac cggggtggcg gtctccaggg ggaccgcaaa gttaaggtgg 7860

ttccatgagc gtggctatgt caagctggaa ggtagggtga ttgacctggg gtgtggccgc 7920

ggaggctggt gttactacgc tgctgcgcaa aaggaagtga gtggggtcaa aggatttact 7980

cttggaagag acggccatga gaaacccatg aatgtgcaaa gtctgggatg gaacatcatc 8040

accttcaagg acaaaactga tatccaccgc ctagaaccag tgaaatgtga cacccttttg 8100

tgtgacattg gagagtcatc atcgtcatcg gtcacagagg gggaaaggac cgtgagagtt 8160

cttgatactg tagaaaaatg gctggcttgt ggggttgaca acttctgtgt gaaggtgtta 8220

gctccataca tgccagatgt tcttgagaaa ctggaattgc tccaaaggag gtttggcgga 8280

acagtgatca ggaaccctct ctccaggaat tccactcatg aaatgtacta cgtgtctgga 8340

gcccgcagca atgtcacatt tactgtgaac caaacatccc gcctcctgat gaggagaatg 8400

aggcgtccaa ctggaaaagt gaccctggag gctgacgtca tcctcccaat tgggacacgc 8460

agtgttgaga cagacaaggg acccctggac aaagaggcca tagaagaaag ggttgagagg 8520

ataaaatctg agtacatgac ctcttggttt tatgacaatg acaaccccta caggacctgg 8580

cactactgtg gctcctatgt cacaaaaacc tccggaagtg cggcgagcat ggtaaatggt 8640

gttattaaaa ttctgacata tccatgggac aggatagagg aggtcacaag aatggcaatg 8700

actgacacaa ccccttttgg acagcaaaga gtgtttaaag aaaaagttga caccagagca 8760

aaggatccac cagcgggaac taggaagatc atgaaagttg tcaacaggtg gctgttccgc 8820

cacctggcca gagaaaagaa ccccagactg tgcacaaagg aagaatttat tgcaaaagtc 8880

cgaagtcatg cagccattgg agcttacctg gaagaacaag aacagtggaa gactgccaat 8940

gaggctgtcc aagacccaaa gttctgggaa ctggtggatg aagaaaggaa gctgcaccaa 9000

caaggcaggt gtcggacttg tgtgtacaac atgatgggga aaagagagaa gaagctgtca 9060

gagtttggga aagcaaaggg aagccgtgcc atatggtata tgtggctggg agcgcggtat 9120

cttgagtttg aggccctggg attcctgaat gaggaccatt gggcttccag ggaaaactca 9180

ggaggaggag tggaaggcat tggcttacaa tacctaggat atgtgatcag agacctggct 9240

gcaatggatg gtggtggatt ctacgcggat gacaccgctg gatgggacac gcgcatcaca 9300

gaggcagacc ttgatgatga acaggagatc ttgaactaca tgagcccaca tcacaaaaaa 9360

ctggcacaag cagtgatgga aatgacatac aagaacaaag tggtgaaagt gttgagacca 9420

gccccaggag ggaaagccta catggatgtc ataagtcgac gagaccagag aggatccggg 9480

caggtagtga cttatgctct gaacaccatc accaacttga aagtccaatt gatcagaatg 9540

gcagaagcag agatggtgat acatcaccaa catgttcaag attgtgatga atcagttctg 9600

accaggctgg aggcatggct cactgagcac ggatgtgaca gactgaagag gatggcggtg 9660

agtggagacg actgtgtggt ccggcccatc gatgacaggt tcggcctggc cctgtcccat 9720

ctcaacgcca tgtccaaggt tagaaaggac atatctgaat ggcagccatc aaaagggtgg 9780

aatgattggg agaatgtgcc cttctgttcc caccacttcc atgaactaca gctgaaggat 9840

ggcaggagga ttgtggtgcc ttgccgagaa caggacgagc tcattgggag aggaagggtg 9900

tctccaggaa acggctggat gatcaaggaa acagcttgcc tcagcaaagc ctatgccaac 9960

atgtggtcac tgatgtattt tcacaaaagg gacatgaggc tactgtcatt ggctgtttcc 10020

tcagctgttc ccacctcatg ggttccacaa ggacgcacaa catggtcgat tcatgggaaa 10080

ggggagtgga tgaccacgga agacatgctt gaggtgtgga acagagtatg gataaccaac 10140

aacccacaca tgcaggacaa gacaatggtg aaaaaatgga gagatgtccc ttatctaacc 10200

aagagacaag acaagctgtg cggatcactg attggaatg 10239

<210>25

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> is derived from West Nile Virus

<400>25

Ser Leu Thr Val Gln Thr His Gly Glu Ser Thr Leu

1 5 10

<210>26

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> is derived from West Nile Virus

<400>26

Val Val Leu Leu Leu Leu Val Ala Pro Ala Tyr Ser

1 5 10

<210>27

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> is derived from West Nile Virus

<400>27

Val Val Pro Leu Leu Leu Val Ala Pro Ala Tyr Ser

1 5 10

<210>28

<211>75

<212>PRT

<213> Artificial sequence

<220>

<223> is derived from West Nile Virus

<400>28

Ser Leu Thr Val Gln Thr His Gly Glu Ser Thr Leu Ala Asn Lys Lys

1 5 10 15

Gly Ala Trp Met Asp Ser Thr Lys Ala Thr Arg Tyr Leu Val Lys Thr

20 25 30

Glu Ser Trp Ile Leu Arg Asn Pro Gly Tyr Ala Leu Val Ala Ala Val

35 40 45

Ile Gly Trp Met Leu Gly Ser Asn Thr Met Gln Arg Val Val Phe Val

50 55 60

Val Leu Leu Leu Leu Val Ala Pro Ala Tyr Ser

65 70 75

<210>29

<211>74

<212>PRT

<213> Artificial sequence

<220>

<223> is derived from West Nile Virus

<400>29

Ser Leu Thr Val Gln Thr His Gly Glu Ser Thr Leu Ala Asn Lys Lys

1 5 10 15

Gly Ala Trp Met Asp Ser Thr Lys Ala Thr Arg Tyr Leu Val Lys Thr

20 25 30

Glu Ser Trp Ile Leu Arg Asn Pro Gly Tyr Ala Leu Val Ala Ala Val

35 40 45

Ile Gly Trp Met Leu Gly Ser Asn Thr Met Gln Arg Val Val Phe Val

50 55 60

Val Leu Leu Leu Val Ala Pro Ala Tyr Ser

65 70

<210>30

<211>75

<212>PRT

<213> Artificial sequence

<220>

<223> is derived from West Nile Virus

<400>30

Ser Leu Thr Val Gln Thr His Gly Glu Ser Thr Leu Ala Asn Lys Lys

1 5 10 15

Gly Ala Trp Met Asp Ser Thr Lys Ala Thr Arg Tyr Leu Val Lys Thr

20 25 30

Glu Ser Trp Ile Leu Arg Asn Pro Gly Tyr Ala Leu Val Ala Ala Val

35 40 45

Ile Gly Trp Met Leu Gly Ser Asn Thr Met Gln Arg Val Val Phe Val

50 55 60

Val Pro Leu Leu Leu Val Ala Pro Ala Tyr Ser

65 70 75

<210>31

<211>377

<212>DNA

<213> Artificial sequence

<220>

<223> derived from yellow fever virus

<400>31

taaaaactac ggatggagaa ccggactcca cacattgaga cagaagaagt tgtcagccca 60

gaaccccaca cgagttttgc cactgctaag ctgtgaggca gtgcaggctg ggacagccga 120

cctccaggtt gcgataaacc tggtttctgg gacctcccac cccagagtaa aaagaacgga 180

gcctccgcta ccaccttccc acgtggtggt agaaagacgg ggtctagagg ttagaggaga 240

ccctccaggg aacaaatagt gggaccatat tgacgccagg gaaagaccgg agtggttctc 300

tgcttttcct ccagaggtct gtgagcacag tttgctcaag aataagcaga cctttggatg 360

acaaacacaa aaccact 377

<210>32

<211>343

<212>RNA

<213> Artificial sequence

<220>

<223> derived from yellow fever virus

<400>32

uugagacaga agaaguuguc agcccagaac cccacacgag uuuugccacu gcuaagcugu 60

gaggcagugc aggcugggac agccgaccuc cagguugcga aaaaccuggu uucugggacc 120

ucccacccca gaguaaaaag aacggagccu ccgcuaccac ccucccacgu ggugguagaa 180

agacgggguc uagagguuag aggagacccu ccagggaaca aauaguggga ccauauugac 240

gccagggaaa gaccggagug guucucugcu uuuccuccag aggucuguga gcacaguuug 300

cucaagaaua agcagaccuu uggaugacaa acacaaaacc acu 343

<210>33

<211>338

<212>RNA

<213> Artificial sequence

<220>

<223> derived from yellow fever virus

<400>33

uugagacaga agaaguuguc agcccagaac cccacacgag uuuugccacu gcuaagcugu 60

gaggcagugc aggcugggac agccgaccuc cagguugcga aaaaccuggu uucugggacc 120

ucccaccgua aaaagaaggg agccucggcu accacccucc cacguggugg uagaaagacg 180

gggucuagag guuagaggag acccuccagg gaacaaauag ugggaccaua uugaggccag 240

ggaaagaccg gagugguucu cugcuuuucc uccagagguc ugugagcaca guuugcucaa 300

gaauaagcag accuuuggau gacaaacaga aaaccacu 338

<210>34

<211>116

<212>PRT

<213> Artificial sequence

<220>

<223> derived from tick-borne encephalitis virus

<400>34

Met Val Lys Lys Ala Ile Leu Lys Gly Lys Gly Gly Gly Pro Pro Arg

1 5 10 15

Arg Val Ser Lys Glu Thr Ala Thr Lys Thr Arg Gln Pro Arg Val Gln

20 25 30

Met Pro Asn Gly Leu Val Leu Met Arg Met Met Gly Ile Leu Trp His

35 40 45

Ala Val Ala Gly Thr Ala Arg Asn Pro Val Leu Lys Ala Phe Trp Asn

50 55 60

Ser Val Pro Leu Lys Gln Ala Thr Ala Ala Leu Arg Lys Ile Lys Arg

65 70 75 80

Thr Val Ser Ala Leu Met Val Gly Leu Gln Lys Arg Gly Lys Arg Arg

85 90 95

Ser Ala Thr Asp Trp Met Ser Trp Leu Leu Val Ile Thr Leu Leu Gly

100 105 110

Met Thr Leu Ala

115

<210>35

<211>121

<212>PRT

<213> Artificial sequence

<220>

<223> derived from yellow fever virus

<400>35

Met Ser Gly Arg Lys Ala Gln Gly Lys Thr Leu Gly Val Asn Met Val

1 5 10 15

Arg Arg Gly Val Arg Ser Leu Ser Asn Lys Ile Lys Gln Lys Thr Lys

20 25 30

Gln Ile Gly Asn Arg Pro Gly Pro Ser Arg Gly Val Gln Gly Phe Ile

35 40 45

Phe Phe Phe Leu Phe Asn Ile Leu Thr Gly Lys Lys Ile Thr Ala His

50 55 60

Leu Lys Arg Leu Trp Lys Met Leu Asp Pro Arg Gln Gly Leu Ala Val

65 70 75 80

Leu Arg Lys Val Lys Arg Val Val Ala Ser Leu Met Arg Gly Leu Ser

85 90 95

Ser Arg Lys Arg Arg Ser His Asp Val Leu Thr Val Gln Phe Leu Ile

100 105 110

Leu Gly Met Leu Leu Met Thr Gly Gly

115 120

<210>36

<211>11

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>36

gtaaatcctg t 11

<210>37

<211>12

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>37

acaaaaccac aa 12

<210>38

<211>10239

<212>DNA

<213> Artificial sequence

<220>

<223> derived from yellow fever virus and West Nile virus

<400>38

gtaaatcctg tgtgctaatt gaggtgcatt ggtctgcaaa tcgagttgct aggcaataaa 60

cacatttgga ttaattttaa tcgttcgttg agcgattagc agagaactga ccagaacatg 120

tctggtcgta aagctcaggg aaaaaccctg ggcgtcaata tggtacgacg aggagttcgc 180

tccttgtcaa acaaaataaa acaaaaaaca aaacaaattg gaaacagacc tggaccttca 240

agaggtgttc aaggatttat ctttttcttt ttgttcaaca ttttgactgg aaaaaagatc 300

acagcccacc taaagaggtt gtggaaaatg ctggacccaa gacaaggctt ggctgttcta 360

aggaaagtca agagagtggt ggccagtttg atgagaggat tgtcctcaag gaaacgccgt 420

tcccatgatg ttctgactgt gcaattccta attttgggaa tgctgttgat gacgggtgga 480

gttaccctct ctaacttcca agggaaggtg atgatgacgg taaatgctac tgacgtcaca 540

gatgtcatca cgattccaac agctgctgga aagaacctat gcattgtcag agcaatggat 600

gtgggataca tgtgcgatga tactatcact tatgaatgcc cagtgctgtc ggctggtaat 660

gatccagaag acatcgactg ttggtgcaca aagtcagcag tctacgtcag gtatggaaga 720

tgcaccaaga cacgccactc aagacgcagt cggaggtcac tgacagtgca gacacacgga 780

gaaagcactc tagcgaacaa gaagggggct tggatggaca gcaccaaggc cacaaggtat 840

ttggtaaaaa cagaatcatg gatcttgagg aaccctggat atgccctggt ggcagccgtc 900

attggttgga tgcttgggag caacaccatg cagagagttg tgtttgtcgt gccattgctt 960

ttggtggccc cagcttacag cttcaactgc cttggaatga gcaacagaga cttcttggaa 1020

ggagtgtctg gagcaacatg ggtggatttg gttctcgaag gcgacagctg cgtgactatc 1080

atgtctaagg acaagcctac catcgacgtc aagatgatga atatggaggc ggccaacctg 1140

gcagaggtcc gcagttattg ctatttggct accgtcagcg atctctccac caaagctgca 1200

tgcccgacca tgggagaagc tcacaatgac aaacgtgctg acccagcttt tgtgtgcaga 1260

caaggagtgg tggacagggg ctggggcaac ggctgcggat tttttggcaa aggatccatt 1320

gacacatgcg ccaaatttgc ctgctctacc aaggcaatag gaagaaccat cttgaaagag 1380

aatatcaagt acgaagtggc catttttgtc catggaccaa ctactgtgga gtcgcacgga 1440

aattactcca cacaggttgg agccactcag gccggccgat tcagcatcac tcctgctgcg 1500

ccttcataca cactaaagct tggagaatat ggagaggtga cagtggactg tgaaccacgg 1560

tcagggattg acaccaatgc atactacgtg atgactgttg gaacaaagac gttcttggtc 1620

catcgtgagt ggttcatgga cctcaacctc ccttggagca gtgctggaag tactgtgtgg 1680

aggaacagag agacgttaat ggagtttgag gaaccacacg ccacgaagca gtctgtgata 1740

gcattgggct cacaagaggg agctctgcat caagctttgg ctggagccat tcctgtggaa 1800

ttttcaagca acactgtcaa gttgacgtcg ggtcatttga agtgtagagt gaagatggaa 1860

aaattgcagt tgaagggaac aacctatggc gtctgttcaa aggctttcaa gtttcttagg 1920

actcccgtgg acaccggtca cggcactgtg gtgttggaat tgcagtacac tggcacggat 1980

ggaccttgca aagttcctat ctcgtcagtg gcttcattga acgacctaac gccagtgggc 2040

agattggtca ctgtcaaccc ttttgtttca gtggccacgg ccaacgctaa ggtcctgatt 2100

gaattggaac caccctttgg agactcatac atagtggtgg gcagaggaga acaacagatc 2160

aatcaccatt ggcacaagtc tggaagcagc attggcaaag cctttacaac caccctcaaa 2220

ggagcgcaga gactagccgc tctaggagac acagcttggg actttggatc agttggaggg 2280

gtgttcacta gtgttgggcg ggctgtccat caagtgttcg gaggagcatt ccgctcactg 2340

ttcggaggca tgtcctggat aacgcaagga ttgctggggg ctctcctgtt gtggatgggc 2400

atcaatgctc gtgataggtc catagctctc acgtttctcg cagttggagg agttctgctc 2460

ttcctctccg tgaacgtggg cgccgatcaa ggatgcgcca tcaactttgg caagagagag 2520

ctcaagtgcg gagatggtat cttcatattt agagactctg atgactggct gaacaagtac 2580

tcatactatc cagaagatcc tgtgaagctt gcatcaatag tgaaagcctc ttttgaagaa 2640

gggaagtgtg gcctaaattc agttgactcc cttgagcatg agatgtggag aagcagggca 2700

gatgagatca atgccatttt tgaggaaaac gaggtggaca tttctgttgt cgtgcaggat 2760

ccaaagaatg tttaccagag aggaactcat ccattttcca gaattcggga tggtctgcag 2820

tatggttgga agacttgggg taagaacctt gtgttctccc cagggaggaa gaatggaagc 2880

ttcatcatag atggaaagtc caggaaagaa tgcccgtttt caaaccgggt ctggaattct 2940

ttccagatag aggagtttgg gacgggagtg ttcaccacac gcgtgtacat ggacgcagtc 3000

tttgaataca ccatagactg cgatggatct atcttgggtg cagcggtgaa cggaaaaaag 3060

agtgcccatg gctctccaac attttggatg ggaagtcatg aagtaaatgg gacatggatg 3120

atccacacct tggaggcatt agattacaag gagtgtgagt ggccactgac acatacgatt 3180

ggaacatcag ttgaagagag tgaaatgttc atgccgagat caatcggagg cccagttagc 3240

tctcacaatc atatccctgg atacaaggtt cagacgaacg gaccttggat gcaggtacca 3300

ctagaagtga agagagaagc ttgcccaggg actagcgtga tcattgatgg caactgtgat 3360

ggacggggaa aatcaaccag atccaccacg gatagcggga aagttattcc tgaatggtgt 3420

tgccgctcct gcacaatgcc gcctgtgagc ttccatggta gtgatgggtg ttggtatccc 3480

atggaaatta ggccaaggaa aacgcatgaa agccatctgg tgcgctcctg ggttacagct 3540

ggagaaatac atgctgtccc ttttggtttg gtgagcatga tgatagcaat ggaagtggtc 3600

ctaaggaaaa gacagggacc aaagcaaatg ttggttggag gagtagtgct cttgggagca 3660

atgctggtcg ggcaagtaac tctccttgat ttgctgaaac tcacagtggc tgtgggattg 3720

catttccatg agatgaacaa tggaggagac gccatgtata tggcgttgat tgctgccttt 3780

tcaatcagac cagggctgct catcggcttt gggctcagga ccctatggag ccctcgggaa 3840

cgccttgtgc tgaccctagg agcagccatg gtggagattg ccttgggtgg cgtgatgggc 3900

ggcctgtgga agtatctaaa tgcagtttct ctctgcatcc tgacaataaa tgctgttgct 3960

tctaggaaag catcaaatac catcttgccc ctcatggctc tgttgacacc tgtcactatg 4020

gctgaggtga gacttgccgc aatgttcttt tgtgccatgg ttatcatagg ggtccttcac 4080

cagaatttca aggacacctc catgcagaag actatacctc tggtggccct cacactcaca 4140

tcttacctgg gcttgacaca accttttttg ggcctgtgtg catttctggc aacccgcata 4200

tttgggcgaa ggagtatccc agtgaatgag gcactcgcag cagctggtct agtgggagtg 4260

ctggcaggac tggcttttca ggagatggag aacttccttg gtccgattgc agttggagga 4320

ctcctgatga tgctggttag cgtggctggg agggtggatg ggctagagct caagaagctt 4380

ggtgaagttt catgggaaga ggaggcggag atcagcggga gttccgcccg ctatgatgtg 4440

gcactcagtg aacaagggga gttcaagctg ctttctgaag agaaagtgcc atgggaccag 4500

gttgtgatga cctcgctggc cttggttggg gctgccctcc atccatttgc tcttctgctg 4560

gtccttgctg ggtggctgtt tcatgtcagg ggagctagga gaagtgggga tgtcttgtgg 4620

gatattccca ctcctaagat catcgaggaa tgtgaacatc tggaggatgg gatttatggc 4680

atattccagt caaccttctt gggggcctcc cagcgaggag tgggagtggc acagggaggg 4740

gtgttccaca caatgtggca tgtcacaaga ggagctttcc ttgtcaggaa tggcaagaag 4800

ttgattccat cttgggcttc agtaaaggaa gaccttgtcg cctatggtgg ctcatggaag 4860

ttggaaggca gatgggatgg agaggaagag gtccagttga tcgcggctgt tccaggaaag 4920

aacgtggtca acgtccagac aaaaccgagc ttgttcaaag tgaggaatgg gggagaaatc 4980

ggggctgtcg ctcttgacta tccgagtggc acttcaggat ctcctattgt taacaggaac 5040

ggagaggtga ttgggctgta cggcaatggc atccttgtcg gtgacaactc cttcgtgtcc 5100

gccatatccc agactgaggt gaaggaagaa ggaaaggagg agctccaaga gatcccgaca 5160

atgctaaaga aaggaatgac aactgtcctt gattttcatc ctggagctgg gaagacaaga 5220

cgtttcctcc cacagatctt ggccgagtgc gcacggagac gcttgcgcac tcttgtgttg 5280

gcccccacca gggttgttct ttctgaaatg aaggaggctt ttcacggcct ggacgtgaaa 5340

ttccacacac aggctttttc cgctcacggc agcgggagag aagtcattga tgccatgtgc 5400

catgccaccc taacttacag gatgttggaa ccaactaggg ttgttaactg ggaagtgatc 5460

attatggatg aagcccattt tttggatcca gccagcatag ccgctagagg ttgggcagcg 5520

cacagagcta gggcaaatga aagtgcaaca atcttgatga cagccacacc gcctgggact 5580

agtgatgaat ttccacattc aaatggtgaa atagaagatg ttcaaacgga catacccagt 5640

gagccctgga acacagggca tgactggatc ctggctgaca aaaggcccac ggcatggttc 5700

cttccatcca tcagagctgc aaatgtcatg gctgcctctt tgcgtaaggc tggaaagagt 5760

gtggtggtcc tgaacaggaa aacctttgag agagaatacc ccacgataaa gcagaagaaa 5820

cctgacttta tattggccac tgacatagct gaaatgggag ccaacctttg cgtggagcga 5880

gtgctggatt gcaggacggc ttttaagcct gtgcttgtgg atgaagggag gaaggtggca 5940

ataaaagggc cacttcgtat ctccgcatcc tctgctgctc aaaggagggg gcgcattggg 6000

agaaatccca acagagatgg agactcatac tactattctg agcctacaag tgaaaataat 6060

gcccaccacg tctgctggtt ggaggcctca atgctcttgg acaacatgga ggtgaggggt 6120

ggaatggtcg ccccactcta tggcgttgaa ggaactaaaa caccagtttc ccctggtgaa 6180

atgagactga gggatgacca gaggaaagtc ttcagagaac tagtgaggaa ttgtgacctg 6240

cccgtttggc tttcgtggca agtggccaag gctggtttga agacgaatga tcgtaagtgg 6300

tgttttgaag gccctgagga acatgagatc ttgaatgaca gcggtgaaac agtgaagtgc 6360

agggctcctg gaggagcaaa gaagcctctg cgcccaaggt ggtgtgatga aagggtgtca 6420

tctgaccaga gtgcgctgtc tgaatttatt aagtttgctg aaggtaggag gggagctgct 6480

gaagtgctag ttgtgctgag tgaactccct gatttcctgg ctaaaaaagg tggagaggca 6540

atggatacca tcagtgtgtt cctccactct gaggaaggct ctagggctta ccgcaatgca 6600

ctatcaatga tgcctgaggc aatgacaata gtcatgctgt ttatactggc tggactactg 6660

acatcgggaa tggtcatctt tttcatgtct cccaaaggca tcagtagaat gtctatggcg 6720

atgggcacaa tggccggctg tggatatctc atgttccttg gaggcgtcaa acccactcac 6780

atctcctatg tcatgctcat attctttgtc ctgatggtgg ttgtgatccc cgagccaggg 6840

caacaaaggt ccatccaaga caaccaagtg gcatacctca ttattggcat cctgacgctg 6900

gtttcagcgg tggcagccaa cgagctaggc atgctggaga aaaccaaaga ggacctcttt 6960

gggaagaaga acttaattcc atctagtgct tcaccctgga gttggccgga tcttgacctg 7020

aagccaggag ctgcctggac agtgtacgtt ggcattgtta caatgctctc tccaatgttg 7080

caccactgga tcaaagtcga atatggcaac ctgtctctgt ctggaatagc ccagtcagcc 7140

tcagtccttt ctttcatgga caaggggata ccattcatga agatgaatat ctcggtcata 7200

atgctgctgg tcagtggctg gaattcaata acagtgatgc ctctgctctg tggcataggg 7260

tgcgccatgc tccactggtc tctcatttta cctggaatca aagcgcagca gtcaaagctt 7320

gcacagagaa gggtgttcca tggcgttgcc aagaaccctg tggttgatgg gaatccaaca 7380

gttgacattg aggaagctcc tgaaatgcct gccctttatg agaagaaact ggctctatat 7440

ctccttcttg ctctcagcct agcttctgtt gccatgtgca gaacgccctt ttcattggct 7500

gaaggcattg tcctagcatc agctgcctta gggccgctca tagagggaaa caccagcctt 7560

ctttggaatg gacccatggc tgtctccatg acaggagtca tgagggggaa tcactatgct 7620

tttgtgggag tcatgtacaa tctatggaag atgaaaactg gacgccgggg gagcgcgaat 7680

ggaaaaactt tgggtgaagt ctggaagagg gaactgaatc tgttggacaa gcgacagttt 7740

gagttgtata aaaggaccga cattgtggag gtggatcgtg atacggcacg caggcatttg 7800

gccgaaggga aggtggacac cggggtggcg gtctccaggg ggaccgcaaa gttaaggtgg 7860

ttccatgagc gtggctatgt caagctggaa ggtagggtga ttgacctggg gtgtggccgc 7920

ggaggctggt gttactacgc tgctgcgcaa aaggaagtga gtggggtcaa aggatttact 7980

cttggaagag acggccatga gaaacccatg aatgtgcaaa gtctgggatg gaacatcatc 8040

accttcaagg acaaaactga tatccaccgc ctagaaccag tgaaatgtga cacccttttg 8100

tgtgacattg gagagtcatc atcgtcatcg gtcacagagg gggaaaggac cgtgagagtt 8160

cttgatactg tagaaaaatg gctggcttgt ggggttgaca acttctgtgt gaaggtgtta 8220

gctccataca tgccagatgt tcttgagaaa ctggaattgc tccaaaggag gtttggcgga 8280

acagtgatca ggaaccctct ctccaggaat tccactcatg aaatgtacta cgtgtctgga 8340

gcccgcagca atgtcacatt tactgtgaac caaacatccc gcctcctgat gaggagaatg 8400

aggcgtccaa ctggaaaagt gaccctggag gctgacgtca tcctcccaat tgggacacgc 8460

agtgttgaga cagacaaggg acccctggac aaagaggcca tagaagaaag ggttgagagg 8520

ataaaatctg agtacatgac ctcttggttt tatgacaatg acaaccccta caggacctgg 8580

cactactgtg gctcctatgt cacaaaaacc tccggaagtg cggcgagcat ggtaaatggt 8640

gttattaaaa ttctgacata tccatgggac aggatagagg aggtcacaag aatggcaatg 8700

actgacacaa ccccttttgg acagcaaaga gtgtttaaag aaaaagttga caccagagca 8760

aaggatccac cagcgggaac taggaagatc atgaaagttg tcaacaggtg gctgttccgc 8820

cacctggcca gagaaaagaa ccccagactg tgcacaaagg aagaatttat tgcaaaagtc 8880

cgaagtcatg cagccattgg agcttacctg gaagaacaag aacagtggaa gactgccaat 8940

gaggctgtcc aagacccaaa gttctgggaa ctggtggatg aagaaaggaa gctgcaccaa 9000

caaggcaggt gtcggacttg tgtgtacaac atgatgggga aaagagagaa gaagctgtca 9060

gagtttggga aagcaaaggg aagccgtgcc atatggtata tgtggctggg agcgcggtat 9120

cttgagtttg aggccctggg attcctgaat gaggaccatt gggcttccag ggaaaactca 9180

ggaggaggag tggaaggcat tggcttacaa tacctaggat atgtgatcag agacctggct 9240

gcaatggatg gtggtggatt ctacgcggat gacaccgctg gatgggacac gcgcatcaca 9300

gaggcagacc ttgatgatga acaggagatc ttgaactaca tgagcccaca tcacaaaaaa 9360

ctggcacaag cagtgatgga aatgacatac aagaacaaag tggtgaaagt gttgagacca 9420

gccccaggag ggaaagccta catggatgtc ataagtcgac gagaccagag aggatccggg 9480

caggtagtga cttatgctct gaacaccatc accaacttga aagtccaatt gatcagaatg 9540

gcagaagcag agatggtgat acatcaccaa catgttcaag attgtgatga atcagttctg 9600

accaggctgg aggcatggct cactgagcac ggatgtgaca gactgaagag gatggcggtg 9660

agtggagacg actgtgtggt ccggcccatc gatgacaggt tcggcctggc cctgtcccat 9720

ctcaacgcca tgtccaaggt tagaaaggac atatctgaat ggcagccatc aaaagggtgg 9780

aatgattggg agaatgtgcc cttctgttcc caccacttcc atgaactaca gctgaaggat 9840

ggcaggagga ttgtggtgcc ttgccgagaa caggacgagc tcattgggag aggaagggtg 9900

tctccaggaa acggctggat gatcaaggaa acagcttgcc tcagcaaagc ctatgccaac 9960

atgtggtcac tgatgtattt tcacaaaagg gacatgaggc tactgtcatt ggctgtttcc 10020

tcagctgttc ccacctcatg ggttccacaa ggacgcacaa catggtcgat tcatgggaaa 10080

ggggagtgga tgaccacgga agacatgctt gaggtgtgga acagagtatg gataaccaac 10140

aacccacaca tgcaggacaa gacaatggtg aaaaaatgga gagatgtccc ttatctaacc 10200

aagagacaag acaagctgtg cggatcactg attggaatg 10239

Claims (12)

1. An attenuated chimeric flavivirus, comprising a yellow fever virus, wherein the membrane and envelope proteins have been replaced with membrane and envelope proteins of a japanese encephalitis virus or a west nile virus, wherein the chimeric flavivirus, when replaced with membrane and envelope proteins of a japanese encephalitis virus, comprises a mutation within amino acid 60 of the chimeric flavivirus membrane protein, the mutation replacing the arginine at amino acid 60 of the membrane protein with a cysteine; or in the use of West Nile virus membrane and envelope protein substitution contains chimeric flavivirus membrane protein within amino acid 66 mutation, the mutation in the membrane protein of the 66 th amino acid leucine by proline replacement, wherein the Japanese encephalitis virus is SA14-14-2 strain, West Nile virus is NY99 strain.

2. The flavivirus of claim 1, wherein the mutation reduces viscerotropism/viremia of the flavivirus.

3. The flavivirus of claim 1, wherein the mutation results in increased viral replication in a cell relative to a corresponding flavivirus lacking the mutation.

4. The flavivirus of claim 1, wherein the mutation attenuates the flavivirus.

5. The flavivirus of any of claims 1-4, wherein the yellow fever virus is YF-17D.

6. The flavivirus of any of claims 1-5, wherein the membrane and envelope proteins of the yellow fever virus have been replaced with membrane and envelope proteins of Japanese encephalitis virus.

7. The flavivirus of any of claims 1-5, wherein the membrane and envelope proteins of the yellow fever virus have been replaced with the membrane and envelope proteins of West Nile virus.

8. The flavivirus of claim 7, wherein the flavivirus comprises mutations at residues corresponding to positions 107,316, and 440 of the envelope protein of West Nile virus, and wherein the lysine at position 107 is replaced with phenylalanine, the alanine at position 316 is replaced with valine, and the lysine at position 440 is replaced with arginine.

9. A method of making the chimeric flavivirus of claim 1, comprising introducing the mutation.

10. The method of claim 9, wherein said mutation attenuates said flavivirus relative to a corresponding flavivirus lacking said mutation.

11. The method of claim 9, wherein said mutation causes increased replication of the flavivirus relative to a corresponding flavivirus lacking said mutation.

12. A nucleic acid molecule corresponding to the genome of the flavivirus of any of claims 1-8 or the complement thereof.