VACCINES AND METHODS OF MAKING AND USING VACCINES FOR PREVENTION OF RESPIRATORY SYNCYTIAL VIRUS (RSV) INFECTIONS

Abstract

Disclosed herein are vaccines, immunogenic compositions, and methods of using the same to treat and prevent respiratory syncytial virus (RSV). Specifically, disclosed are immunogenic compositions wherein a protein or immunogenic fragment of RSV is delivered to a subject in a recombinant viral vector platform, such as vesicular stomatis virus (rVSV).

Claims

1. A composition comprising one or more recombinant viral vectors and one or more respiratory syncytial virus (RSV) proteins.

2. The composition of claim 1, wherein the composition comprises at least two RSV proteins expressed in the same recombinant viral vector.

3. The composition of claim 1, wherein the one or more recombinant viral vector is recombinant vesicular stomatitis virus (rVSV).

4. The composition of claim 3, wherein the composition comprises two or more rVSV vectors.

5. The composition of claim 1, wherein the RSV protein is G protein.

6. The composition of claim 1, wherein the RSV protein is mG protein (codon-optimized, membrane bound G protein).

7. The composition of claim 1, wherein the RSV G protein is codon-optimized.

8. The composition of claim 7, wherein the codon-optimized RSV G protein is encoded by a nucleic acid comprising SEQ ID NO: 1.

9. The composition of claim 1, wherein the RSV protein is F protein.

10. The composition of claim 9, wherein the F-protein is codon-optimized F protein, pre-fusion conformation stabilized F-protein, or post-fusion F protein.

11. The composition of claim 1, wherein the RSV protein is tandem-expressed F and G proteins.

12. The composition of claim 1, wherein the RSV protein is M2-1 protein.

13. The composition of claim 1, wherein the RSV protein is N protein.

14. The composition of claim 2, further wherein RSV G protein is one of the RSV proteins.

15. The composition of claim 2, wherein mG protein is one of the RSV proteins.

16. The composition of claim 6, wherein the composition comprises at least one additional RSV protein selected from the group comprising F, M2-1, and N proteins.

17. The composition of claim 1, further comprising an adjuvant.

18. The composition of claim 17, wherein the adjuvant is Hsp70.

19. A vaccine comprising a composition of claim 1 in a carrier.

20. A method of eliciting an immune response against RSV in a subject, the method comprising administering to the subject a composition of claim 1 or the vaccine of claim 19.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1 shows a schematic representation of the VSV vector (Indiana strain; sequence listed as the last sequence in the list of sequences) with the location for cloning of the RSV genes.

[0007] FIGS. 2A, 2B, and 2C show clearance of challenge virus (a and b) and VN antibody titers (c) in the rVSV-GF immunized cotton rats. Cotton rats (n=4 per group) were immunized with indicated dose and combination of the rVSV candidates and challenged with RSV-A2 four weeks after immunization and euthanized four days after challenge. Virus titration was done using lung and nasal homogenates collected on the day of euthanization and VN antibody levels were determined from the serum samples collected on the day of challenge. Statistical analysis was done by one-way ANOVA and statistically significant difference (at P<0.05) between indicated group representing bars is indicated by asterisk (*) symbol.

[0008] FIGS. 3A, 3B, and 3C show clearance of challenge virus (a and b) and VN antibody titers (c) in the rVSV-GF immunized cotton rats. Cotton rats (n=4 per group) were immunized with indicated dose, interval and combination of the rVSV candidates and challenged with RSV-A2 three weeks after booster dose and euthanized four days after challenge. Virus titration was done using lung and nasal homogenates and VN antibody levels were determined from the serum samples collected on the day of booster immunization (day 21) and RSV challenge (day 42). Statistical analysis was done by one-way ANOVA and statistically significant difference (at P<0.05) between indicated groups representing bars is indicated by asterisk (*) symbol.

[0009] FIGS. 4A, 4B, and 4C show clearance of challenge virus (a and b) and VN antibody titers (c) in the indicated rVSV-G+F+rVSV-Hsp70 immunized cotton rats. Cotton rats (n=4 per group) were immunized with indicated dose, interval and combination of the rVSV candidates and challenged with RSV-A2 three weeks after booster dose and euthanized four days after challenge. Virus titration was done using lung and nasal homogenates and VN antibody levels were determined from the serum samples collected on the day of booster immunization (day 21) and RSV challenge (day 42). Statistical analysis was done by one-way ANOVA and statistically significant difference (at P<0.05) between indicated groups representing bars is indicated by asterisk (*) symbol.

[0010] FIGS. 5A, 5B, 5C show clearance of challenge virus (a and b) and VN antibody titers (c) in the indicated variant of RSV G expressing rVSV immunized cotton rats. Cotton rats (n=4 per group) were immunized with indicated dose, interval and combination of the rVSV candidates and challenged with RSV-A2 three weeks after booster dose and euthanized four days after challenge. Virus titration was done using lung and nasal homogenates and VN antibody levels were determined from the serum samples collected on the day of booster immunization (day 21) and RSV challenge (day 42). Statistical analysis was done by one-way ANOVA and statistically significant difference (at P<0.05) between indicated groups representing bars is indicated by asterisk (*) symbol.

[0011] FIGS. 6A, 6B, and 6C show clearance of challenge virus (a and b) and VN antibody titers (c) in the rVSV-G variants immunized cotton rats. Cotton rats (n=4 per group) were immunized with indicated dose and combination of the rVSV candidates and challenged with RSV-A2 after four weeks and euthanized four days after challenge. Virus titration was done using lung and nasal homogenates and VN antibody levels were determined from the serum samples collected on the day of challenge. Statistical analysis was done by one-way ANOVA and statistically significant difference (at P<0.05) between indicated group representing bars is indicated by asterisk (*) symbol.

[0012] FIG. 7 shows a schematic representation of the ectodomain of the RSV F gene with details of the mutations and substitutions included to stabilize F protein in perfusion conformation (Pre-F).

[0013] FIG. 8 shows a schematic representation of RSV N gene and segments of the gene selected for expression in rVSVs vectors as detailed in Table. 3.

DETAILED DESCRIPTION

[0014] The present invention can be understood more readily by reference to the following detailed description of the invention and the Examples included therein.

All patents, patent applications, and publications cited herein, whether supra or infra, are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0015] Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

Definitions

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description of the embodiments herein is for describing particular embodiments only and is not intended to be limiting of the embodiments disclosed. As used in the description, the singular forms a, an, and the are intended to include the plural forms as well, unless the context clearly indicates otherwise. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0017] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in this disclosure are to be understood as being modified in all instances by the term about. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this disclosure are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0018] Ranges can be expressed herein as from about one particular value, and/or to about another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent about, it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values described herein, and that each value is also herein disclosed as about that particular value in addition to the value itself. For example, if the value 10 is disclosed, then about 10 is also disclosed. It is also understood that when a value is disclosed that less than or equal to the value, greater than or equal to the value and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value 10 is disclosed the less than or equal to 10 as well as greater than or equal to 10 is also disclosed. It is also understood that throughout the application, data are provided in a number of different formats, and that these data, represent endpoints, starting points, and ranges for any combination of the data points. For example, if a particular data point 10 and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0019] As used herein, the term amino acid sequence refers to a list of abbreviations, letters, characters or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; C, cysteine; D aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine.

[0020] Polypeptide as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids. The term polypeptide encompasses naturally occurring or synthetic molecules. The terms polypeptide, peptide, and protein can be used interchangeably.

[0021] In addition, as used herein, the term polypeptide refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc. and may contain modified amino acids other than the 20 gene-encoded amino acids. The polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can have many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. (See ProteinsStructure and Molecular Properties 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)).

[0022] As used herein, isolated polypeptide or purified polypeptide is meant to mean a polypeptide (or a fragment thereof) that is substantially free from the materials with which the polypeptide is normally associated in nature. The polypeptides of the invention, or fragments thereof, can be obtained, for example, by extraction from a natural source (for example, a mammalian cell), by expression of a recombinant nucleic acid encoding the polypeptide (for example, in a cell or in a cell-free translation system), or by chemically synthesizing the polypeptide. In addition, polypeptide fragments may be obtained by any of these methods, or by cleaving full length proteins and/or polypeptides.

[0023] The phrase nucleic acid as used herein refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing. Nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof.

[0024] As used herein, isolated nucleic acid or purified nucleic acid is meant to mean DNA that is free of the genes that, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, such as an autonomously replicating plasmid or virus; or incorporated into the genomic DNA of a prokaryote or eukaryote (e.g., a transgene); or which exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR, restriction endonuclease digestion, or chemical or in vitro synthesis). It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. The term isolated nucleic acid also refers to RNA, e.g., an mRNA molecule that is encoded by an isolated DNA molecule, or that is chemically synthesized, or that is separated or substantially free from at least some cellular components, for example, other types of RNA molecules or polypeptide molecules.

[0025] As used herein, sample is meant to mean an animal; a tissue or organ from an animal; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (e.g. a polypeptide or nucleic acid), which is assayed as described herein. A sample can also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells or cell components.

[0026] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term comprises means includes. Thus, unless the context requires otherwise, the word comprises, and variations such as comprise and comprising will be understood to imply the inclusion of a stated compound or composition (e.g., nucleic acid, polypeptide, antigen) or step, or group of compounds or steps, but not to the exclusion of any other compounds, composition, steps, or groups thereof.

[0027] An immunogenic composition is a composition of matter suitable for administration to a human or animal subject (e.g., in an experimental setting) that is capable of eliciting a specific immune response, e.g., against a pathogen, such as RSV. As such, an immunogenic composition includes one or more antigens (for example, whole purified virus or antigenic subunits, e.g., polypeptides, thereof) or antigenic epitopes. An immunogenic composition can also include one or more additional components capable of eliciting or enhancing an immune response, such as an excipient, carrier, and/or adjuvant. In certain instances, immunogenic compositions are administered to elicit an immune response that protects the subject against symptoms or conditions induced by a pathogen. In some cases, symptoms or disease caused by a pathogen is prevented (or treated, e.g., reduced or ameliorated) by inhibiting replication of the pathogen following exposure of the subject to the pathogen. In the context of this disclosure, the term immunogenic composition will be understood to encompass compositions that are intended for administration to a subject or population of subjects for the purpose of eliciting a protective or palliative immune response against the virus (that is, vaccine compositions or vaccines).

[0028] The term purification (e.g., with respect to a pathogen or a composition containing a pathogen) refers to the process of removing components from a composition, the presence of which is not desired. Purification is a relative term, and does not require that all traces of the undesirable component be removed from the composition. In the context of vaccine production, purification includes such processes as centrifugation, dialization, ion-exchange chromatography, and size-exclusion chromatography, affinity-purification or precipitation. Thus, the term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified virus preparation is one in which the virus is more enriched than it is in its generative environment, for instance within a cell or population of cells in which it is replicated naturally or in an artificial environment. A preparation of substantially pure viruses can be purified such that the desired virus or viral component represents at least 50% of the total protein content of the preparation. In certain embodiments, a substantially pure virus will represent at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% or more of the total protein content of the preparation.

[0029] An isolated biological component (such as a virus, nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell and/or organism in which the component occurs or is produced. Viruses and viral components, e.g., proteins, which have been isolated include viruses, and proteins, purified by standard purification methods. The term also embraces viruses and viral components (such as viral proteins) prepared by recombinant expression in a host cell.

[0030] An antigen is a compound, composition, or substance that can stimulate the production of antibodies and/or a T cell response in an animal, including compositions that are injected, absorbed or otherwise introduced into an animal. The term antigen includes all related antigenic epitopes. The term epitope or antigenic determinant refers to a site on an antigen to which B and/or T cells respond. The dominant antigenic epitopes or dominant epitope are those epitopes to which a functionally significant host immune response, e.g., an antibody response or a T-cell response, is made. Thus, with respect to a protective immune response against a pathogen, the dominant antigenic epitopes are those antigenic moieties that when recognized by the host immune system result in protection from disease caused by the pathogen. The term T-cell epitope refers to an epitope that when bound to an appropriate MHC molecule is specifically bound by a T cell (via a T cell receptor). A B-cell epitope is an epitope that is specifically bound by an antibody (or B cell receptor molecule). An antigen can also affect the innate immune response.

[0031] An immune response is a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. An immune response can be a B cell response, which results in the production of specific antibodies, such as antigen specific neutralizing antibodies. An immune response can also be a T cell response, such as a CD4+ response or a CD8+ response. In some cases, the response is specific for a particular antigen (that is, an antigen-specific response). An immune response can also include the innate response. If the antigen is derived from a pathogen, the antigen-specific response is a pathogen-specific response. A protective immune response is an immune response that inhibits a detrimental function or activity of a pathogen, reduces infection by a pathogen, or decreases symptoms (including death) that result from infection by the pathogen. A protective immune response can be measured, for example, by the inhibition of viral replication or plaque formation in a plaque reduction assay or ELISA-neutralization assay, or by measuring resistance to pathogen challenge in vivo.

The immunogenic compositions disclosed herein are suitable for preventing, ameliorating and/or treating disease caused by infection of the virus.

[0032] By reduce or other forms of the word, such as reducing or reduction, is meant lowering of an event or characteristic (e.g., viral infection). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, reduces viral infection means decreasing the amount of virus relative to a standard or a control.

[0033] By prevent or other forms of the word, such as preventing or prevention, is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

[0034] As used herein, treatment refers to obtaining beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, any one or more of: alleviation of one or more symptoms (such as infection), diminishment of extent of infection, stabilized (i.e., not worsening) state of infection, preventing or delaying spread of the infection, preventing or delaying occurrence or recurrence of infection, and delay or slowing of infection progression.

[0035] The term patient preferably refers to a human in need of treatment with an antibiotic or treatment for any purpose, and more preferably a human in need of such a treatment to treat viral infection. However, the term patient can also refer to non-human animals, preferably mammals such as dogs, cats, horses, cows, pigs, sheep and non-human primates, among others, that are in need of treatment with antibiotics.

[0036] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

In addition, where features or aspects of the inventions are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

General Description

[0037] RSV has four major structural proteins (glycoprotein [G], fusion [F] protein, Nucleoprotein [N] and M.sub.2-1) which are responsible for induction of humoral and cell mediated immune responses in the infected individual. Humoral (or antibody mediated) immunity is required for neutralizing/limiting the virus spread, whereas, cell mediated immunity is required for clearance of the virus from the body of the infected individual. G and F are surface proteins and induce both neutralizing antibodies and T cell mediated immune responses. N and M.sub.2-1 are internal proteins and contribute in induction of T cell response.

[0038] Four types of recombinant VSVs have been developed, each individually expressing one of the four above mentioned antigenic structural proteins (modified or unmodified) between glycoprotein (G) and polymerase (L) protein genes of the rVSV vector (FIG. 1). For expression of G protein, in addition to the cloning of wildtype G protein gene in the rVSV, a codon optimized version of the gene has been cloned. Codon optimization of a gene enables higher expression of the vaccine antigen (G protein, in this case). Therefore, from the same dose of the vaccine, a codon optimized gene expressing VSV produces significantly higher levels of the antigen protein resulting in dose amplification, so that the required dose of the rVSV can be significantly reduced. Further, in the context of RSV infection, G protein is produced in two forms (membrane bound [mG] and secretory [sG] forms). rVSVs expressing both forms have been produced. Further, RSV-G protein has been cloned with (Table 1) and pre-clinical in vivo efficacy studies have been conducted in the cotton rat animal model.

[0039] It is noted that viruses other than RSV can be used with the rVSV platforms disclosed herein. Examples of other viruses are known to those of skill in the art and include other respiratory (human and animal) viruses such as, human metapneumo virus, influenza, and bRSV.

TABLE-US-00001 TABLE 1 S. Name of the Characteristic of the RSV Rationale for expression in the No. recombinant G protein rVSV vector 1 rVSV-G Wildtype RSV-G protein G protein is the receptor binding ligand of the RSV and immunogenic because of presence of antigenic epitopes 2 rVSV-cG Codon-optimized RSV- G Codon optimization enhances protein (full length with 298 expression of the G protein resulting amino acid [AA] length). in dose sparing/amplification effect. 3 rVSV-mG Codon-optimized RSV- G Membrane bound G protein is more protein stabilized to express immunogenic than secretory G only membrane bound form protein. by mutating second start codon at amino acid (AA) position 48, from Methionine to Isoleucine (M48I). 4 rVSV-G Codon-optimized RSV- G Expected to be attenuated because of (C186S) protein with disrupted the disrupted receptor binding receptor binding domain, domain and possibly increasing the CX3C motif, in the cysteine immunogenicity of the G protein. noose of the central conserved domain of the G protein. 5 rVSV- SecG Codon-optimized 250 AA To elucidate the purported role of sized mRSV- G (starting Sec-G as decoy antigen from second start codon at AA position 48/with truncated cytoplasmic tail and part of the transmembrane domain) 6 rVSV-GNg Codon optimized RSV- G Few reports have shown that protein with deletion of five unglycosylated/prokaryotically putative N-glycosylation expressed G protein was more sites by mutation of immunogenic than glycosylated form. Aspargine residue to alanine. 7 rVSV- Codon optimized membrane We predicted that membrane-bound mGNg bound RSV- G protein (as partially unglycosylated G is more in rVSV-mG) with deletion immunogenic than rVSV-GNg of five putative N- glycosylation sites by mutation of Aspargine residue to alanine. 8 rVSV-G 28 AA long peptide Shown to be immunogenic in other (aa163-190) comprising of central expression systems as it compasses conserved domain of the G the most conserved region of the G protein protein including receptor binding CX3C motif 9 rVSV-G 101 AA long peptide Shown to be immunogenic in other (aa130-230) comprising of central expression systems as it compasses conserved domain of the G the most conserved region of the G protein protein including receptor binding CX3C motif

[0040] RSV F protein is involved in the fusion of the virus to the cell membrane of the infected cell and has a higher number of neutralizing epitopes, antigenic sites and T-cell epitopes than G protein, thus, making it an attractive vaccine candidate. F protein exists in two different structural conformations, pre-fusion and post-fusion (Pre-F and Post-F), and Pre-F has been shown to be more immunogenic than Post-F. Therefore, wildtype F and Pre-F genes have been cloned in rVSV (Table 2). The codon-optimized F gene in rVSV can also be cloned. Disclosed herein are various formats of F-protein, including codon-optimized F protein, pre-fusion conformation stabilized F-protein, and post-fusion F protein. The F protein can be wildtype or codon-optimized.

TABLE-US-00002 TABLE 2 S. Name of the Characteristic of the RSV Rationale for expression in the No. recombinant F protein rVSV vector 1 rVSV-F Wildtype RSV-F protein F protein is responsible for the fusion of the virus with host cell and has more number of neutralizing and CTL epitopes. 2 rVSV-Pre-F- Codon-optimized RSV- F Codon optimization enhances Foldon protein with mutations in the expression of the F protein resulting F gene leading to stabilizing in dose sparing/amplification effect. the protein in Pre-F Further, stabilization of the conformation. conformation in pre-fusion state enables it to induce highly protective immune response. 3 rVSV-Pre-F Codon-optimized full-length Codon optimization enhances RSV- F protein with expression of the F protein resulting mutations in the F gene in dose sparing/amplification effect. leading to stabilizing the Further, stabilization of the protein in Pre-F conformation in pre-fusion state conformation. enables it to induce highly protective immune response. 4 rVSV-Post F Codon-optimized RSV- F Post-fusion F protein is shown to protein ectodomain induce protective immunity in few conformation. studies. 5 rVSV-HEK- Codon-optimized full-length Codon optimization enhances Pre-F RSV- F protein with expression of the F protein resulting mutations in the F gene in dose sparing/amplification effect. leading to stabilizing the Further, stabilization of the protein in Pre-F conformation in pre-fusion state conformation with HEK enables it to induce highly protective assignments. immune response.

[0041] Further, N and M.sub.2-1 proteins have been shown to contain several putative sites of T-cell epitopes inducing cell mediated immunity, which is responsible for clearance of the infective RSV virus from the body. Therefore, rVSVs expressing M.sub.2-1 and different segments of the N gene have been cloned and recovered (Table 3).

TABLE-US-00003 TABLE 3 S. Name of the Characteristic of the RSV Rationale for expression in the No. recombinant N or M2-1 protein rVSV vector 1 rVSV-N3 238 AA length amino terminal Comprises of two putative T-cell domain (NTD) of the N protein epitopes 2 rVSV-N3-l 254 AA length NTD and 16 AA Comprises of five putative T-cell of the carboxylic terminal epitopes domain (CTD) downstream of the NTD and CTD junction of the N protein 3 rVSV-N- 71 AA region of CTD Comprises of two putative T-cell CTL-2 epitopes 4 rVSV-N- 38 AA region of NTD and Comprises of four putative T-cell CTL-4 CTD junction epitopes 5 rVSV-M.sub.2-1 Full-length wild type RSV- Shown to possess CTL epitopes M.sub.2-1 protein

[0042] When a human or non-human animal is challenged by a foreign organism/pathogen the challenged individual responds by launching an immune response which may be protective. This immune response is characterized by the coordinated interaction of the innate and acquired immune response systems.

The innate immune response forms the first line of defense against a foreign organism/pathogen. An innate immune response can be triggered within minutes of infection in an antigen-independent, but pathogen-dependent, manner. The innate, and indeed the adaptive, immune system can be triggered by the recognition of pathogen associated molecular patterns unique to microorganisms by pattern recognition receptors present on most host cells. Once triggered the innate system generates an inflammatory response that activates the cellular and humoral adaptive immune response systems.

[0043] The adaptive immune response becomes effective over days or weeks and provides the antigen specific responses needed to control and usually eliminate the foreign organism/pathogen. The adaptive response is mediated by T cells (cell mediated immunity) and B cells (antibody mediated or humoral immunity) that have developed specificity for the pathogen. Once activated these cells have a long lasting memory for the same pathogen.

[0044] The ability of an individual to generate immunity to foreign organisms/pathogens, thereby preventing or at least reducing the chance of infection by the foreign organism/pathogen, is a powerful tool in disease control and is the principle behind vaccination.

[0045] Vaccines function by preparing the immune system to mount a response to a pathogen. Typically, a vaccine comprises an antigen, which is a foreign organism/pathogen or a toxin produced by an organism/pathogen, or a portion thereof, that is introduced into the body of a subject to be vaccinated in a non-toxic, and/or non-pathogenic form. The antigen in the vaccine causes the subject's immune system to be primed or sensitized to the organism/pathogen from which the antigen is derived. Subsequent exposure of the immune system of the subject to the organism/pathogen or toxin results in a rapid and robust specific immune response, that controls or destroys the organism/pathogen or toxin before it can multiply and infect or damage enough cells in the host organism to cause disease symptoms.

Compositions

[0046] Disclosed herein are multiple rVSVs expressing one of the four different antigenic proteins (in natural or modified conformation) of RSV, which have been shown to be efficacious in a cotton rat animal model, with or without combining with an adjuvant expressing rVSV (rVSV-Hsp70). It has been demonstrated that when delivered intranasally, rVSVs expressing RSV proteins induce protective immunity in vaccinated cotton rats against wildtype RSV challenge.

[0047] Specifically, disclosed herein are compositions comprising a recombinant viral vector and one or more respiratory syncytial virus (RSV) proteins. The recombinant viral vector can be selected from recombinant viral vectors known to those of skill in the art. Non-limiting examples of vectors that can be used include viral-based vectors, such as those described in Lundstrom et al. (Vaccines 2016, 4, 39), hereby incorporated by reference in its entirety for its teaching concerning viral vectors (e.g., retrovirus, adenovirus, adeno-associated virus, lentivirus, HMPV, PIV). Examples of rVSV that can be used include, but are not limited to the expression of G and F in one vector, G and N sequences or an expression of an RSV gene and HSP as adjuvant. HSP can be human or other.

[0048] As mentioned above and in Example 1, there are four categories of RSV proteins which can be used in the compositions disclosed herein. It is noted that RSV can be from any source, such as human, bovine, etc. The RSV proteins include the G protein, the F protein, the M2-1 protein, and the N protein. Further, the G protein is present in two forms, the membrane bound (mG) and secretory (sG) forms. Either form can be used with the compositions and methods disclosed herein. These proteins can be used alone in the composition, or can be presented together to increase the antigenic response. For example, the G protein can be coupled with N, M2-1, or F proteins. The mG protein can be coupled with N, M2-1, or F proteins. Any of these proteins can be combined in any possible permutation for use in an immunogenic composition or vaccine. The RSV proteins used in the compositions and vaccines disclosed herein can be full length, or can be functional immunogenic fragments that retain their immunogenicity when administered to a subject. One of skill in the art will readily understand how to obtain immunogenic fragments of an RSV protein.

[0049] Furthermore, the proteins disclosed herein can be codon optimized. For example, the codon optimization of G and pre-fusion conformation stabilized F leads to higher and more stable expression of these proteins. Sequences are listed in the sequences listing. Codon optimization is defined as modifying a nucleic acid sequence for enhanced expression in the cells of the vertebrate of interest, e.g. human, by replacing at least one, more than one, or a significant number, of codons of the native sequence with codons that are more frequently or most frequently used in the genes of that vertebrate. Various species exhibit particular bias for certain codons of a particular amino acid.

[0050] The composition disclosed herein can also comprise one or more adjuvants. As used herein, adjuvant is understood as an aid or contributor to increase the efficacy or potency of a vaccine or in the prevention, amelioration, or cure of disease by increasing the efficacy or potency of a therapeutic agent as compared to a vaccine or agent administered without the adjuvant. An increase in the efficacy or potency can include a decrease in the amount of vaccine or agent to be administered, a decrease in the frequency and/or number of doses to be administered, or a more rapid or robust response to the agent or vaccine (i.e., higher antibody titer). The adjuvant can be HSP70 (see FIG. 4), but may also include alumn, detoxified monophosphoryl lipid A (MPLA), detoxified saponin derivative QS-21 or other pattern recognition receptor agonists including NLP and TLR agonists. Other variants of HSP70 will have a similar effect, whether they are from a different species or mutated as long as the binding domain is intact.

[0051] Described herein are vaccines comprising a composition of this invention in a carrier wherein the vaccine is protective against RSV infection. The term immunogenic carrier as used herein can refer to a first polypeptide or fragment, variant, or derivative thereof which enhances the immunogenicity of a second polypeptide or fragment, variant, or derivative thereof. An immunogenic carrier can be fused, to or conjugated/coupled to the desired polypeptide or fragment thereof. See, e.g., European Patent No. EP 0385610 B1, which is incorporated herein by reference in its entirety for its teaching of fusing, conjugating or coupling a polypeptide to a carrier. An example of an immunogenic carrier is PLGA.

[0052] The vaccine composition of the present invention may also be co-administered with antigens from other pathogens as a multivalent vaccine.

Methods of Use and Administration

[0053] Also disclosed herein are methods of using the immunogenic compositions and vaccines disclosed herein. For example, disclosed are methods of eliciting an immune response against RSV in a subject, the method comprising administering to the subject a composition or vaccine as disclosed herein. The immune response can be protective against RSV, for example.

[0054] Also disclosed is a method of reducing symptoms or duration of RSV in a subject, the method comprising the steps of: (a) providing a composition of any of claims 1 to 15 or the vaccine of claim 16; and (b) administering said composition or vaccine to the subject, thereby reducing symptoms or duration of RSV.

[0055] Further disclosed is a method of stimulating an immune response in a subject, the method comprising: administering to said subject a composition or vaccine as disclosed herein.

[0056] The vaccines disclosed herein can be administered in a variety of ways, and at a variety of doses. For example, intranasal route, orally, intramuscular route, intradermal and subcutaneous injection as well as application by ocular, vaginal and anal route.

[0057] In one example, a single dose of the immunogenic composition or vaccine can be given, wherein the composition comprises about 110.sup.5 or more particles (which also are referred to as particle units (pu)) of the composition, e.g., about 110.sup.6 or more particles, about 110 or more particles, about 110.sup.8 or more particles, about 110.sup.9 or more particles, or about 310.sup.8 or more particles of the composition. Alternatively, or in addition, a single dose of the composition comprises about 310.sup.14 particles or less of the immunogenic composition, e.g., about 110.sup.13 particles or less, about 110.sup.12 particles or less, about 310.sup.11 particles or less, about 110.sup.11 particles or less, about 110.sup.10 particles or less, or about 110.sup.9 particles or less of the immunogenic composition. Thus, a single dose of immunogenic composition can comprise a quantity of particles of the immunogenic composition in a range defined by any two of the aforementioned values. For example, a single dose of immunogenic composition can comprise 110.sup.5-110.sup.14 particles, 110.sup.7-110.sup.12 particles, 110.sup.8-110.sup.11 particles, 310.sup.8-310 particles, 110.sup.9-110.sup.12 particles, 110.sup.9-110.sup.11 particles, 110.sup.9-110.sup.10 particles, or 110.sup.10-110.sup.12 particles, of the immunogenic composition. In other words, a single dose of immunogenic composition can comprise, for example, about 110.sup.6 pu, 210.sup.6 pu, 410.sup.6 pu, 110.sup.7 pu, 210.sup.7 pu, 410.sup.7 pu, 110.sup.8 pu, 210.sup.8 pu, 310.sup.8 pu, 410.sup.8 pu, 110.sup.9 pu, 210.sup.9 pu, 310.sup.9 pu, 410.sup.9 pu, 110.sup.10 pu, 210.sup.10 pu, 310.sup.10 pu, 410.sup.10 pu, 110.sup.11 pu, 210.sup.11 pu, 310.sup.11 pu, 410.sup.11 pu, 110.sup.12 pu, 210.sup.12 pu, 310.sup.12 pu, or 410.sup.12 pu of the adenoviral vector.

[0058] The vaccine can be given in single doses, or two doses which are separated. For example, when two doses are given, they can be given 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days apart. The vaccine can be administered in a variety of ways known to those of skill in the art, such as intranasally.

EXAMPLES

Example 1: 10.SUP.7 .Pfu/Dose/Animal of the rVSVs Expressing Wild Type G and F Proteins Induced Protective Immunity in Cotton Rats

[0059] Since 10.sup.5 TCID.sub.50/dose of the RSV induced protective immunity in cotton rats (n=4 per group), therefore, for relative comparison of rVSVs with the RSV immune efficacy of the rVSV-G and rVSV-F recombinants, 10.sup.5 pfu (plaque forming unit)/dose as the starting dose and also immunized with higher/10 fold incremental doses (10.sup.6 pfu/animal or 10.sup.7 pfu/animal) were evaluated. Cotton rats were immunized with either individual rVSV-G or rVSV-F recombinant or in combination (rVSV-G+F). The hypothesis was that rVSV induced protective effect is dose dependent and further, enhanced effect is possible by combining both G and F expressing rVSVs. Immunized animals were challenged with wild type RSV strain A2 (dose: 10.sup.5 TCID.sub.50) four weeks after vaccination and euthanized the animals four days after challenge. Clearance of the challenge virus was evaluated (by titrating the amount of virus using a cell culture cytopathic effect based assay) from the lower and upper respiratory tract (LRT and URT) represented by homogenates of the lungs and nasal passage respectively (collected on the day of euthanization) and virus neutralizing (VN) antibody levels (by cell culture based virus neutralization test) in the serum sample collected on the day of challenge. These studies demonstrated that non-invasive mucosal delivery of the rVSV-G or F by intranasal route was more effective than parental (by subcutaneous) route of administration. Therefore, for all subsequent studies, intranasal immunization method was employed. Further, it was also shown that 10.sup.5 pfu/animal of either rVSV-G or rVSV-F was effective in clearance of challenge virus from the LRT but not URT along with lower VN antibody levels. Therefore, the objective of this study was to extend the protection to URT and enhance the VN antibody levels by employing higher dose and combined vaccination strategy.

[0060] The results indicated that, higher (each rVSV at 10.sup.7 pfu/dose/cotton rat[CR]) and combined (rVSV-G+F) immunization strategy was effective in inducing protective immunity which could clear the challenge RSV from both LRT and URT (FIGS. 2a&b) along with higher levels of VN antibody levels (FIG. 2c).

[0061] These results and the comparison of VSV expressing either G or F with immunization results through immunization with purified G and post-fusion F protein (Table 4) demonstrate that VSV vectors deliver a better immune response.

Example 2: Prime-Boost Immunization Regimen of rVSVs Expressing Wild Type G and F Proteins Induced Protective Immunity in Cotton Rats Along with Enhanced VN Titers

[0062] Though 10.sup.7 pfu dose of rVSV-G and rVSV-F combination was adequate to protect the immunized cotton rats from the challenge virus, virus neutralization (VN) antibody titers were still lower than RSV-A2 immunized animals (which showed higher VN titer titers, 2.sup.8). Therefore, to enhance the VN titers in rVSV immunized groups, it was hypothesized that by following prime-boost regimen of immunization strategy, VN titers can be significantly enhanced with high (10.sup.7 pfu) and possibly with low dose (10.sup.5 pfu) immunization as well. Therefore, cotton rats were immunized with either high dose or low dose of rVSVs, individually or in combination, and the booster dose was administered three weeks after primary immunization and the immunized cotton rats were challenged three weeks after booster immunization.

[0063] The results indicated that, at low dose immunization, neither individual nor combined rVSVs induced protective immunity in URT, and VN titers were also not considerably improved upon booster immunization. Whereas, in higher dose immunization groups, in all three groups VN antibodies were enhanced after booster immunization (FIG. 3c) along with complete protection of URT in rVSV-G and rVSV-G+F immunized groups (FIGS. 3a&b). Prime-boost regimen was effective in enhancing the VN titers by up to 40% after booster immunization. Thus, it was evident from this study that, prime-boost immunization enhanced protective immunity in the immunized animals (and possibly indicating an extended longevity [i. e., memory immune response] of the protection).

[0064] Immunization can also be improved through the use of a VSV expressing HSP70 which functions as an adjuvant (FIG. 4).

Example 3: Coupling of an Adjuvant Expressing rVSV Along with Prime-Boost Immunization Regimen of rVSVs Expressing Wild Type G and F Proteins Induced Enhanced Protective Immunity in Cotton Rats

[0065] Though prime-boost immunization with rVSV-G+F enhanced the VN titers (titer: 2.sup.6), however, the VN titers in RSV-A2 immunized animals were significantly higher (titer: >2.sup.8). Therefore, with an objective to further enhancing the protective immunity in the rVSV-G+F immunized animals and to explore a possibility of extending the longevity of the protection, the vaccine rVSV candidates were combined with Hsp70 expressing rVSV (rVSV-Hsp-70). It has been demonstrated that rVSV-Hsp70 enhanced adjuvanticity of the vaccine antigen co-expressing rVSV (Ma, et al., 2014) resulting in enhanced mucosal immunity. Further the safe dose of rVSV-Hsp70 (i. e., 10.sup.7 pfu/dose/CR) has been shown in cotton rats. Therefore, in the present study, with an objective to identify the appropriate dose of rVSV-Hsp70 along with rVSV-G+F, cotton rats were immunized (following prime-boost regimen) with either high dose or low dose combination of rVSV-G+F and combined with one of the three doses (10.sup.5, 10.sup.6, or 10.sup.7 pfu/dose/CR) of the rVSV-Hsp70.

[0066] The results indicated that, 10.sup.5 pfu dose of the rVSV-Hsp70 was an appropriate dose along with high dose of the rVSV-G+F as there was complete protection of both LRT and URT (FIGS. 4a&b) along with enhanced VN titers by 33% (FIG. 4c).

[0067] It is clearly evident from the above studies that, prime-boost immunization of the 10.sup.7 pfu dose of each of rVSV-G and rVSV-F combination induced enhanced protective immunity in the cotton rat model. Further, efficacy of the combination (and possibly longevity of the protection) can be further enhanced by inclusion of the adjuvant expressing rVSV-Hsp70.

Example 4: Codon-Optimized or Membrane-Bound Codon Optimized RSV G Protein Expressing rVSVs (rVSV-cG or rVSV-mG) were More Effective than Wild-Type G (rVSV-G) in Inducing Protective Immunity in the URT Along with Enhanced VN Titers

[0068] In order to identify an effective G protein candidate, several modifications were made to the G protein to enhance its immunogenicity as explained in table 1 (S. No. 2-9) and expressed the indicated G variant in the VSV vector and tested the efficacy in the cotton rats. Cotton rats were immunized with each of the seven recovered rVSV G variants, following the previously established strategy for the rVSV-G+F immunization studies (i. e., high dose [10.sup.7 pfu/dose/CR] and prime-boost immunization).

[0069] The results clearly indicated that among all the tested G variants, two recombinants (rVSV-cG and rVSV-mG) were successful in inducing protective immunity in the in the URT (FIGS. 5a&b) as well as enhanced VN titers compared to rVSV-G (FIG. 5c). These results demonstrated that, either expressing the codon-optimized G protein (which produces higher levels of both membrane bound as well as secretory forms) at higher level endogenously or expressing it exclusively in the membrane bound form (by excluding the decoy effect of the secretory G), it is possible to induce protective immunity by RSV G protein alone. Further, single dose immunization with 10.sup.5, 10.sup.6, or 10.sup.7 dose of rVSV-cG or rVSV-mG was tested for effectiveness in eliciting protective immunity. The results demonstrated that higher dose (10.sup.7 pfu) of the either recombinant was effective in either completely protecting the URT (rVSV-cG) or reducing the challenge virus titer (rVSV-mG) (FIG. 6b). However, the VN titers in all the groups were low and comparable (FIG. 6c). These results demonstrated that, even though modified G recombinants (cG and mG) alone were effective in protecting both LRT and URT, however, to induce enhanced VN antibodies, prime-boost immunization regimen is essential.

TABLE-US-00004 TABLE 4 Immunization with G and F protein. G and post-fusion F protein were expressed eucaryotically in 293F cells. Cotton rats were immunized with 5 ug of purified protein in 200 ul alumn subcutaneously. Four weeks later, blood was drawn to determine neutralizing antibody titers and animals were challenged with 10.sup.5 TCID.sub.50 RSV. Four days later, virus titers were determined from lung and nasal tissue. Post-fusion F is currently tested in clinical trials. Neutralizing Nose Lung antibody Nave animals 3.8 0.2 3.4 0.3 2.sup.0 Immunized with G 3.9 0.4 3.9 0.4 2.sup.0 protein Immunized with 0 0 2.sup.4.5 post-F protein

TABLE-US-00005 SEQUENCES SequencesofRSVgenesexpressedintheVSVexpressionsystem SEQIDNO:1:RSV-G(Size:897nts) ATGTCCAAAAACAAGGACCAACGCACCGCTAAGACATTAGAAAGGACCTGG GACACTCTCAATCATTTATTATTCATATCATCGTGCTTATATAAGTTAAATCTTAAAT CTGTAGCACAAATCACATTATCCATTCTGGCAATGATAATCTCAACTTCACTTATAA TTGCAGCCATCATATTCATAGCCTCGGCAAACCACAAAGTCACACCAACAACTGCA ATCATACAAGATGCAACAAGCCAGATCAAGAACACAACCCCAACATACCTCACCCA GAATCCTCAGCTTGGAATCAGTCCCTCTAATCCGTCTGAAATTACATCACAAATCAC CACCATACTAGCTTCAACAACACCAGGAGTCAAGTCAACCCTGCAATCCACAACAG TCAAGACCAAAAACACAACAACAACTCAAACACAACCCAGCAAGCCCACCACAAA ACAACGCCAAAACAAACCACCAAGCAAACCCAATAATGATTTTCACTTTGAAGTGT TCAACTTTGTACCCTGCAGCATATGCAGCAACAATCCAACCTGCTGGGCTATCTGCA AAAGAATACCAAACAAAAAACCAGGAAAGAAAACCACTACCAAGCCCACAAAAAA ACCAACCCTCAAGACAACCAAAAAAGATCCCAAACCTCAAACCACTAAATCAAAGG AAGTACCCACCACCAAGCCCACAGAAGAGCCAACCATCAACACCACCAAAACAAAC ATCATAACTACACTACTCACCTCCAACACCACAGGAAATCCAGAACTCACAAGTCA AATGGAAACCTTCCACTCAACTTCCTCCGAAGGCAATCCAAGCCCTTCTCAAGTCTC TACAACATCCGAGTACCCATCACAACCTTCATCTCCACCCAACACACCACGCCAGTA G SEQIDNO:2:RSV-cGIcodonoptimizedG](size:897nts) ATGAGCAAGAACAAGGACCAGCGGACCGCCAAGACCCTGGAGCGGACCTGG GACACCCTGAACCACCTGCTGTTCATCAGCAGCTGCCTGTACAAGCTGAACCTGAAG AGCGTGGCCCAGATCACCCTGAGCATCCTGGCCATGATCATCAGCACCAGCCTGATC ATCGCCGCCATCATCTTCATCGCCAGCGCCAACCACAAGGTGACCCCCACCACCGCC ATCATCCAGGACGCCACCAGCCAGATCAAGAACACCACCCCCACCTACCTGACCCA GAACCCCCAGCTGGGCATCAGCCCCAGCAACCCCAGCGAGATCACCAGCCAGATCA CCACCATCCTGGCCAGCACCACCCCCGGCGTGAAGAGCACCCTGCAGAGCACCACC GTGAAGACCAAGAACACCACCACCACCCAGACCCAGCCCAGCAAGCCCACCACCAA GCAGCGGCAGAACAAGCCTCCCAGCAAGCCCAACAACGACTTCCACTTCGAGGTGT TCAACTTCGTGCCCTGCAGCATCTGCAGCAACAACCCCACCTGCTGGGCCATCTGCA AGCGGATTCCCAACAAGAAGCCCGGCAAGAAGACCACCACCAAGCCCACCAAGAA GCCCACCCTGAAGACCACCAAGAAGGACCCCAAGCCCCAGACCACCAAGAGCAAG GAGGTGCCCACCACCAAGCCCACCGAGGAGCCCACCATCAACACCACCAAGACCAA CATCATCACCACCCTGCTGACCAGCAACACCACCGGCAACCCCGAGCTGACCAGCC AGATGGAGACCTTCCACAGCACCAGCAGCGAGGGCAACCCCAGCCCCAGCCAGGTG AGCACCACCAGCGAGTACCCCAGCCAGCCCAGCAGCCCTCCCAACACCCCTCGGCA GTAG SEQIDNO:3:RSV-cmG[codonoptimizedmembraneboundG] (size:897nts) ATGAGCAAGAACAAGGACCAGCGGACCGCCAAGACCCTGGAGCGGACCTGG GACACCCTGAACCACCTGCTGTTCATCAGCAGCTGCCTGTACAAGCTGAACCTGAAG AGCGTGGCCCAGATCACCCTGAGCATCCTGGCCATTATCATCAGCACCAGCCTGATC ATCGCCGCCATCATCTTCATCGCCAGCGCCAACCACAAGGTGACCCCCACCACCGCC ATCATCCAGGACGCCACCAGCCAGATCAAGAACACCACCCCCACCTACCTGACCCA GAACCCCCAGCTGGGCATCAGCCCCAGCAACCCCAGCGAGATCACCAGCCAGATCA CCACCATCCTGGCCAGCACCACCCCCGGCGTGAAGAGCACCCTGCAGAGCACCACC GTGAAGACCAAGAACACCACCACCACCCAGACCCAGCCCAGCAAGCCCACCACCAA GCAGCGGCAGAACAAGCCTCCCAGCAAGCCCAACAACGACTTCCACTTCGAGGTGT TCAACTTCGTGCCCTGCAGCATCTGCAGCAACAACCCCACCTGCTGGGCCATCTGCA AGCGGATTCCCAACAAGAAGCCCGGCAAGAAGACCACCACCAAGCCCACCAAGAA GCCCACCCTGAAGACCACCAAGAAGGACCCCAAGCCCCAGACCACCAAGAGCAAG GAGGTGCCCACCACCAAGCCCACCGAGGAGCCCACCATCAACACCACCAAGACCAA CATCATCACCACCCTGCTGACCAGCAACACCACCGGCAACCCCGAGCTGACCAGCC AGATGGAGACCTTCCACAGCACCAGCAGCGAGGGCAACCCCAGCCCCAGCCAGGTG AGCACCACCAGCGAGTACCCCAGCCAGCCCAGCAGCCCTCCCAACACCCCTCGGCA GTAG SEQIDNO:4:RSV-G(C1865)(Size:897nts) ATGAGCAAGAACAAGGACCAGCGGACCGCCAAGACCCTGGAGCGGACCTGG GACACCCTGAACCACCTGCTGTTCATCAGCAGCTGCCTGTACAAGCTGAACCTGAAG AGCGTGGCCCAGATCACCCTGAGCATCCTGGCCATGATCATCAGCACCAGCCTGATC ATCGCCGCCATCATCTTCATCGCCAGCGCCAACCACAAGGTGACCCCCACCACCGCC ATCATCCAGGACGCCACCAGCCAGATCAAGAACACCACCCCCACCTACCTGACCCA GAACCCCCAGCTGGGCATCAGCCCCAGCAACCCCAGCGAGATCACCAGCCAGATCA CCACCATCCTGGCCAGCACCACCCCCGGCGTGAAGAGCACCCTGCAGAGCACCACC GTGAAGACCAAGAACACCACCACCACCCAGACCCAGCCCAGCAAGCCCACCACCAA GCAGCGGCAGAACAAGCCTCCCAGCAAGCCCAACAACGACTTCCACTTCGAGGTGT TCAACTTCGTGCCCTGCAGCATCTGCAGCAACAACCCCACCTGCTGGGCCATCTCCA AGCGGATTCCCAACAAGAAGCCCGGCAAGAAGACCACCACCAAGCCCACCAAGAA GCCCACCCTGAAGACCACCAAGAAGGACCCCAAGCCCCAGACCACCAAGAGCAAG GAGGTGCCCACCACCAAGCCCACCGAGGAGCCCACCATCAACACCACCAAGACCAA CATCATCACCACCCTGCTGACCAGCAACACCACCGGCAACCCCGAGCTGACCAGCC AGATGGAGACCTTCCACAGCACCAGCAGCGAGGGCAACCCCAGCCCCAGCCAGGTG AGCACCACCAGCGAGTACCCCAGCCAGCCCAGCAGCCCTCCCAACACCCCTCGGCA GTAG SEQIDNO:5:RSV-SecG(756nts) ATGATCATCAGCACCAGCCTGATCATCGCCGCCATCATCTTCATCGCCAGCGC CAACCACAAGGTGACCCCCACCACCGCCATCATCCAGGACGCCACCAGCCAGATCA AGAACACCACCCCCACCTACCTGACCCAGAACCCCCAGCTGGGCATCAGCCCCAGC AACCCCAGCGAGATCACCAGCCAGATCACCACCATCCTGGCCAGCACCACCCCCGG CGTGAAGAGCACCCTGCAGAGCACCACCGTGAAGACCAAGAACACCACCACCACCC AGACCCAGCCCAGCAAGCCCACCACCAAGCAGCGGCAGAACAAGCCTCCCAGCAA GCCCAACAACGACTTCCACTTCGAGGTGTTCAACTTCGTGCCCTGCAGCATCTGCAG CAACAACCCCACCTGCTGGGCCATCTGCAAGCGGATTCCCAACAAGAAGCCCGGCA AGAAGACCACCACCAAGCCCACCAAGAAGCCCACCCTGAAGACCACCAAGAAGGA CCCCAAGCCCCAGACCACCAAGAGCAAGGAGGTGCCCACCACCAAGCCCACCGAGG AGCCCACCATCAACACCACCAAGACCAACATCATCACCACCCTGCTGACCAGCAAC ACCACCGGCAACCCCGAGCTGACCAGCCAGATGGAGACCTTCCACAGCACCAGCAG CGAGGGCAACCCCAGCCCCAGCCAGGTGAGCACCACCAGCGAGTACCCCAGCCAGC CCAGCAGCCCTCCCAACACCCCTCGGCAGTAG SEQIDNO:6:RSV-GNg(897nts) ATGTCTAAAAACAAGGATCAGCGAACCGCCAAAACCCTGGAGCGTACATGG GATACACTCAACCACCTTCTGTTCATATCTAGCTGCCTTTACAAACTTAATCTCAAAA GCGTCGCCCAGATTACCCTCTCAATACTGGCTATGATAATCTCCACCTCTTTGATAAT AGCCGCTATCATTTTCATAGCTTCTGCAAACCACAAAGTAACTCCAACCACAGCTAT AATACAAGATGCCACCTCTCAGATTAAAAATACCACACCCACATATCTTACTCAGAA TCCTCAATTGGGAATCAGCCCATCTAAgCCATCCGAGATTACTTCACAGATCACCAC AATACTCGCATCCACAACACCAGGGGTCAAATCCACCCTGCAATCAACTACCGTGA AAACTAAAAAgACCACTACAACACAGACTCAACCCAGCAAGCCTACAACAAAGCAA CGCCAGAATAAGCCACCTTCTAAGCCAAACAATGATTTCCATTTTGAGGTCTTTAAT TTCGTGCCTTGCTCTATATGTTCCAACAAgCCAACTTGCTGGGCCATTTGCAAACGCA TCCCAAATAAGAAACCCGGTAAGAAAACCACAACCAAGCCAACTAAAAAGCCAACT TTGAAGACTACCAAAAAGGACCCTAAGCCCCAGACAACTAAATCAAAAGAAGTCCC AACTACTAAGCCAACTGAGGAACCAACAATAAAgACTACAAAAACCAACATCATCA CAACCCTTCTTACTAGCAAgACTACTGGTAACCCCGAGCTGACAAGCCAGATGGAGA CATTCCACAGTACAAGCAGCGAAGGAAACCCAAGCCCTAGTCAAGTGTCCACTACC TCAGAATACCCCAGCCAGCCTTCCTCACCTCCTAACACACCCCGGCAATAG SEQIDNO:7:RSV-mGNg(897nts) cagcaatctcgagATGTCTAAAAACAAGGATCAGCGAACCGCCAAAACCCTGGAGC GTACATGGGATACACTCAACCACCTTCTGTTCATATCTAGCTGCCTTTACAAACTTA ATCTCAAAAGCGTCGCCCAGATTACCCTCTCAATACTGGCTATTATAATCTCCACCTC TTTGATAATAGCCGCTATCATTTTCATAGCTTCTGCAAACCACAAAGTAACTCCAAC CACAGCTATAATACAAGATGCCACCTCTCAGATTAAAAATACCACACCCACATATCT TACTCAGAATCCTCAATTGGGAATCAGCCCATCTAAgCCATCCGAGATTACTTCACA GATCACCACAATACTCGCATCCACAACACCAGGGGTCAAATCCACCCTGCAATCAA CTACCGTGAAAACTAAAAAgACCACTACAACACAGACTCAACCCAGCAAGCCTACA ACAAAGCAACGCCAGAATAAGCCACCTTCTAAGCCAAACAATGATTTCCATTTTGA GGTCTTTAATTTCGTGCCTTGCTCTATATGTTCCAACAAgCCAACTTGCTGGGCCATT TGCAAACGCATCCCAAATAAGAAACCCGGTAAGAAAACCACAACCAAGCCAACTAA AAAGCCAACTTTGAAGACTACCAAAAAGGACCCTAAGCCCCAGACAACTAAATCAA AAGAAGTCCCAACTACTAAGCCAACTGAGGAACCAACAATAAAgACTACAAAAACC AACATCATCACAACCCTTCTTACTAGCAAgACTACTGGTAACCCCGAGCTGACAAGC CAGATGGAGACATTCCACAGTACAAGCAGCGAAGGAAACCCAAGCCCTAGTCAAGT GTCCACTACCTCAGAATACCCCAGCCAGCCTTCCTCACCTCCTAACACACCCCGGCA ATAGcccgggttcat SEQIDNO:8:RSV-G(aa163-190)(84nts) TTCCACTTCGAGGTGTTCAACTTCGTGCCCTGCAGCATCTGCAGCAACAACCC CACCTGCTGGGCCATCTGCAAGCGGATTCCC SEQIDNO:9:RSV-G(aa130-230)(303nts) ACCGTGAAGACCAAGAACACCACCACCACCCAGACCCAGCCCAGCAAGCCC ACCACCAAGCAGCGGCAGAACAAGCCTCCCAGCAAGCCCAACAACGACTTCCACTT CGAGGTGTTCAACTTCGTGCCCTGCAGCATCTGCAGCAACAACCCCACCTGCTGGGC CATCTGCAAGCGGATTCCCAACAAGAAGCCCGGCAAGAAGACCACCACCAAGCCCA CCAAGAAGCCCACCCTGAAGACCACCAAGAAGGACCCCAAGCCCCAGACCACCAA GAGCAAGGAGGTGCCCACCACCAAGCCC SEQIDNO:10:RSV-F(size:1725nts) ATGGAGTTGCTAATCCTCAAAGCAAATGCAATTACCACAATCCTCACTGCAG TCACATTTTGTTTTGCTTCTGGTCAAAACATCACTGAAGAATTTTATCAATCAACATG CAGTGCAGTTAGCAAAGGCTATCTTAGTGCTCTGAGAACTGGTTGGTATACCAGTGT TATAACTATAGAATTAAGTAATATCAAGAAAAATAAGTGTAATGGAACAGATGCTA AGGTAAAATTGATAAAACAAGAATTAGATAAATATAAAAATGCTGTAACAGAATTG CAGTTGCTCATGCAAAGCACACAAGCAACAAACAATCGAGCCAGAAGAGAACTACC AAGGTTTATGAATTATACACTCAACAATGCCAAAAAAACCAATGTAACATTAAGCA AGAAAAGGAAAAGAAGATTTCTTGGTTTTTTGTTAGGTGTTGGATCTGCAATCGCCA GTGGCGTTGCTGTATCTAAGGTCCTGCACCTAGAAGGGGAAGTGAACAAGATCAAA AGTGCTCTACTATCCACAAACAAGGCTGTAGTCAGCTTATCAAATGGAGTTAGTGTT TTAACCAGCAAAGTGTTAGACCTCAAAAACTATATAGATAAACAATTGTTACCTATT GTGAACAAGCAAAGCTGCAGCATATCAAATATAGAAACTGTGATAGAGTTCCAACA AAAGAACAACAGACTACTAGAGATTACCAGGGAATTTAGTGTTAATGCAGGCGTAA CTACACCTGTAAGCACTTACATGTTAACTAATAGTGAATTATTGTCATTAATCAATG ATATGCCTATAACAAATGATCAGAAAAAGTTAATGTCCAACAATGTTCAAATAGTTA GACAGCAAAGTTACTCTATCATGTCCATAATAAAAGAGGAAGTCTTAGCATATGTA GTACAATTACCACTATATGGTGTTATAGATACACCCTGTTGGAAACTACACACATCC CCTCTATGTACAACCAACACAAAAGAAGGGTCCAACATCTGTTTAACAAGAACTGA CAGAGGATGGTACTGTGACAATGCAGGATCAGTATCTTTCTTCCCACAAGCTGAAAC ATGTAAAGTTCAATCAAATCGAGTATTTTGTGACACAATGAACAGTTTAACATTACC AAGTGAAGTAAATCTCTGCAATGTTGACATATTCAACCCCAAATATGATTGTAAAAT TATGACTTCAAAAACAGATGTAAGCAGCTCCGTTATCACATCTCTAGGAGCCATTGT GTCATGCTATGGCAAAACTAAATGTACAGCATCCAATAAAAATCGTGGAATCATAA AGACATTTTCTAACGGGTGCGATTATGTATCAAATAAAGGGGTGGACACTGTGTCTG TAGGTAACACATTATATTATGTAAATAAGCAAGAAGGTAAAAGTCTCTATGTAAAA GGTGAACCAATAATAAATTTCTATGACCCATTAGTATTCCCCTCTGATGAATTTGAT GCATCAATATCTCAAGTCAACGAGAAGATTAACCAGAGCCTAGCATTTATTCGTAAA TCCGATGAATTATTACATAATGTAAATGCTGGTAAATCCACCACAAATATCATGATA ACTACTATAATTATAGTGATTATAGTAATATTGTTATCATTAATTGCTGTTGGACTGC TCTTATACTGTAAGGCCAGAAGCACACCAGTCACACTAAGCAAAGATCAACTGAGT GGTATAAATAATATTGCATTTAGTAACTAA SEQIDNO:11:RSV-Pre-F-Foldon(1941nts) ATGGAGCTGCTCATCCTGAAGGCCAACGCCATCACCACCATCCTCACCGCCG TGACCTTCTGCTTCGCCAGCGGCCAGAATATCACAGAGGAATTTTATCAGTCTACTT GTAGTGCCGTCAGTAAAGGATATCTGAGCGCTCTCAGAACAGGATGGTACACTAGT GTTATTACAATAGAATTGTCAAATATCAAGAAAAATAAGTGCAACGGTACTGACGC TAAGGTTAAGCTCATCAAACAGGAACTTGATAAATATAAGAACGCAGTTACAGAAC TTCAGCTTCTTATGCAGTCCACACAAGCCACCAATAATAAAGCTAAAAAAGAACTCC CACGGTTCATGAACTACACACTGAACAATGCAAAAAAAACCAACGTAACCCTTAGC AAGAAAAAGAAAAAAAAGTTCCTTGGCTTCCTCCTCGGAGTAGGCAGCGCTATTGC AAGTGGGGTAGCCGTGTGTAAGGTTTTGCATCTCGAAGGAGAAGTGAATAAAATAA AGAGCGCCTTGCTGTCCACTAATAAGGCCGTAGTCAGCCTTAGCAATGGCGTATCCG TTCTGACCTTTAAAGTACTGGATTTGAAGAACTACATCGATAAACAGCTTCTCCCCA TTTTGAATAAGCAATCATGTTCTATCAGTAACATAGAAACCGTCATCGAATTCCAAC AAAAAAACAATCGGCTTTTGGAAATAACTCGTGAATTTTCTGTAAACGCAGGCGTG ACAACTCCCGTATCAACCTACATGTTGACCAATAGCGAACTGCTGTCACTCATTAAC GACATGCCAATCACTAACGACCAGAAAAAACTTATGAGCAATAATGTACAGATTGT AAGACAGCAAAGTTACAGCATAATGTGCATTATTAAGGAAGAAGTTTTGGCATACG TTGTCCAACTCCCCCTTTATGGGGTCATTGATACCCCCTGCTGGAAGCTGCATACTA GCCCATTGTGTACTACCAACACCAAAGAGGGTAGTAACATATGCCTCACCAGAACT GACCGAGGCTGGTACTGTGATAATGCTGGAAGTGTCAGTTTCTTTCCTCAAGCAGAG ACCTGCAAAGTTCAGTCCAACCGCGTGTTCTGTGATACAATGAACTCCCTGACACTC CCTAGCGAAGTCAACCTTTGTAACGTCGATATATTTAATCCTAAATACGATTGTAAG ATCATGACTTCAAAAACTGACGTATCCTCTTCCGTTATTACTTCTTTGGGTGCCATAG TTAGTTGCTACGGCAAAACAAAATGCACCGCATCTAATAAAAACAGAGGAATTATT AAGACATTTTCAAATGGTTGCGACTACGTTAGTAATAAAGGTGTAGATACAGTAAGT GTTGGTAACACCCTCTATTACGTGAACAAACAGGAAGGGAAAAGCCTGTACGTGAA AGGGGAGCCCATAATCAACTTCTACGACCCCCTTGTATTTCCTAGTGATGAATTTGA CGCCTCCATCAGTCAAGTGAACGAAAAGATCAACCAAAGCCTTGCTTTCATCCGCAA ATCCGATGAGTTGCTCCACAATATTAAAGGCTCGGGATATATACCGGAGGCCCCGC GAGATGGTCAAGCTTATGTGCGCAAAGACGGTGAGTGGGTCTTGTTATCTACATTTT TGGGTAACACTAATAGTGGAGGTAGCACGACGACAATTACTAATAATAACTCGGGA ACTAACTCAAGCTCCACTACCTACACTGTCAAATCTGGTGATACATTGTGGGGCATA AGTCAAAGATATGGTATTTCAGTAGCCCAAATTCAATCGGCGAATAATTTAAAGAG CACAATAATTTACATAGGCCAGAAGCTCGTCCTGACAGGTTCCGCCTCGTCAACCAA TAGCGGAGGCAGCAACAACAGTGCTTCAACGACACCCACCACCTCGGTTACTCCTG CTAAGCCAACAAGTCAAACAACT SEQIDNO:12:hCdn.RSV-Pre-F(1725nts) ATGGAACTTCTTATATTGAAGGCAAACGCAATCACCACCATTTTGACTGCGGT TACATTCTGTTTCGCCTCAGGTCAAAATATTACAGAAGAATTCTACCAGAGCACATG CTCAGCGGTATCAAAGGGTTACTTGTCAGCCCTTAGGACCGGATGGTATACCTCTGT AATAACAATAGAACTTTCAAACATTAAAAAAAATAAGTGCAACGGGACCGATGCAA AAGTTAAACTGATCAAGCAAGAACTGGACAAGTATAAAAACGCAGTCACTGAACTT CAACTTCTTATGCAGTCCACGCAAGCCACTAATAATAAGGCTAAGAAAGAACTGCC AAGGTTTATGAACTATACCCTGAACAACGCGAAGAAGACTAATGTCACGTTGTCAA AAAAGAAAAAGAAAAAATTCCTGGGGTTCCTGCTCGGAGTAGGCAGTGCAATCGCG TCTGGAGTAGCCGTATGTAAAGTATTGCACCTTGAAGGAGAAGTAAACAAAATAAA GAGCGCTCTGCTCTCTACGAACAAAGCTGTTGTAAGTCTGAGCAATGGCGTCTCAGT CCTGACATTTAAAGTTCTTGATTTGAAAAATTATATTGACAAACAACTCCTCCCTATC CTCAACAAACAGTCTTGCTCTATTTCAAATATTGAGACAGTTATCGAATTTCAGCAA AAAAACAATAGGCTCCTTGAAATCACACGAGAATTTTCTGTAAACGCTGGAGTCAC AACACCAGTATCTACGTATATGCTCACCAATTCCGAACTTCTTTCATTGATAAATGA TATGCCCATAACAAACGACCAGAAAAAATTGATGTCCAATAATGTCCAAATCGTTC GCCAACAGAGCTATTCTATCATGTGTATAATAAAAGAGGAAGTTCTCGCTTACGTTG TCCAACTGCCGCTGTACGGGGTGATTGACACACCTTGCTGGAAACTTCATACTAGCC CTCTGTGCACGACTAACACCAAGGAAGGATCAAATATCTGCCTCACGCGAACTGAC AGGGGTTGGTACTGTGATAACGCTGGTTCCGTGTCATTTTTTCCTCAAGCTGAGACG TGTAAAGTACAGTCCAATCGAGTTTTCTGCGATACTATGAACTCACTCACCTTGCCG TCAGAGGTGAACCTCTGTAACGTAGATATATTTAACCCGAAATACGACTGTAAGATT ATGACTTCAAAGACCGATGTGTCAAGCTCCGTCATTACCTCCTTGGGAGCAATTGTT TCTTGCTATGGTAAGACGAAGTGCACTGCGAGCAACAAGAATCGCGGTATCATCAA GACGTTCTCCAACGGATGCGATTATGTAAGTAACAAGGGAGTTGACACGGTGAGTG TAGGGAACACGTTGTACTATGTAAACAAGCAGGAGGGGAAGTCCTTGTATGTCAAG GGCGAACCTATTATCAACTTCTACGACCCATTGGTGTTCCCTAGTGACGAGTTTGAT GCTAGTATTTCCCAGGTCAACGAGAAGATAAACCAAAGTTTGGCTTTCATTAGGAAG AGCGATGAGCTTCTCCACAATGTGAACGCCGGGAAGAGTACGACTAATATTATGAT CACAACCATCATAATCGTCATTATCGTTATTTTGCTCTCACTGATTGCAGTCGGACTT CTGCTGTACTGCAAAGCTCGCAGTACCCCAGTCACGCTTTCCAAGGACCAACTTTCA GGCATTAATAACATCGCATTTTCTAATTAA SEQIDNO:13:hCdn.RSV-Post-F(1509nts) ATGGAACTTTTGATACTGAAGGCGAACGCCATAACGACGATCCTGACAGCTG TAACTTTTTGCTTCGCGAGCGGTCAAAACATAACCGAGGAATTTTATCAGTCAACGT GCTCTGCTGTTAGCAAAGGATATCTCAGCGCACTCAGGACGGGCTGGTACACGTCA GTCATAACGATTGAGCTGTCTAATATCAAGAAGAACAAATGCAACGGAACGGACGC CAAAGTCAAGCTCATAAAACAAGAATTGGACAAGTACAAGAATGCTGTGACGGAGC TTCAGCTCTTGATGCAGTCCACCCAAGCGACGAATAATAGAGCGAGGAGAGAGCTC CCAAGATTTATGAACTATACACTGAACAATGCAAAGAAGACTAATGTGACCCTTAG CAAGAAAAGAAAAAGAAGAGCGATTGCAAGTGGAGTGGCTGTGTCAAAGGTCCTG CACCTTGAAGGTGAGGTGAACAAGATTAAATCCGCGCTGCTTTCTACGAACAAAGC TGTCGTTAGTTTGTCCAATGGCGTTTCAGTGCTCACTTCCAAGGTATTGGATTTGAAG AATTATATTGACAAACAGCTCCTTCCGATTGTTAATAAACAGAGTTGCTCAATTTCT AACATCGAAACTGTCATAGAGTTTCAGCAGAAGAACAATCGGCTCTTGGAAATAAC AAGGGAGTTTTCAGTCAACGCCGGGGTAACAACACCCGTGTCCACATACATGCTGA CAAACTCCGAGTTGCTCTCTCTTATCAACGACATGCCAATTACAAACGACCAGAAGA AATTGATGTCCAACAACGTCCAAATCGTACGACAGCAGTCTTATTCCATTATGAGTA TTATTAAGGAAGAGGTATTGGCTTATGTAGTACAACTCCCCTTGTACGGGGTAATAG ACACCCCCTGTTGGAAACTGCATACGAGTCCCCTGTGTACAACCAATACGAAGGAG GGCTCCAATATATGTTTGACAAGAACTGACCGCGGCTGGTACTGTGATAATGCTGGT AGTGTTAGCTTCTTTCCACAAGCGGAGACTTGCAAGGTACAATCTAATCGGGTTTTC TGCGATACGATGAACTCTCTGACTCTGCCGAGTGAGGTCAACCTGTGCAACGTGGAC ATATTCAATCCGAAGTACGATTGTAAAATTATGACATCCAAGACAGATGTAAGCAG CTCTGTTATTACGTCACTGGGCGCTATTGTGTCATGCTACGGTAAGACTAAATGTAC CGCATCCAATAAAAACAGGGGGATTATTAAAACCTTCAGCAACGGATGCGATTATG TCAGCAATAAGGGCGTGGATACCGTATCCGTTGGCAATACTCTCTATTACGTAAATA AACAGGAAGGCAAATCTCTCTATGTTAAAGGCGAACCTATAATCAATTTTTACGATC CGCTTGTATTCCCTTCCGATGAATTCGATGCCTCTATCTCTCAAGTTAACGAAAAAAT CAATCAATCTCTGGCATTTATTAGGAAGTCAGATGAACTCCTA SEQIDNO:14:hCdn.RSV-HEK-Pre-F(1725nts) ATGGAATTGCTCATTTTGAAAGCTAATGCTATAACAACAATACTCACGGCTGT AACTTTTTGCTTTGCCTCTGGTCAAAACATAACGGAAGAGTTTTATCAGTCAACGTG TTCAGCCGTATCAAAAGGGTATCTTAGCGCACTGCGCACTGGATGGTACACGTCTGT GATTACCATTGAACTCAGTAATATCAAGGAAAATAAATGCAACGGCACTGATGCAA AAGTCAAGCTCATAAAACAGGAGCTTGACAAGTACAAAAATGCGGTTACAGAACTC CAGCTCCTTATGCAATCTACCCCAGCAACCAACAACAAAGCCAAGAAGGAGCTGCC CAGGTTTATGAACTATACACTTAACAACGCGAAGAAAACCAATGTCACTCTCAGTA AAAAGAAAAAAAAGAAGTTCTTGGGGTTCCTTCTCGGTGTTGGAAGCGCCATTGCA AGCGGTGTAGCAGTTTGCAAAGTTCTCCACCTTGAGGGGGAGGTGAACAAAATTAA ATCTGCCCTCCTCTCAACTAACAAAGCCGTCGTCAGCTTGAGTAACGGCGTAAGCGT ACTCACTTTCAAAGTTCTCGATCTGAAGAACTATATTGATAAACAGCTGCTCCCAAT ACTGAACAAGCAGTCATGCAGCATCAGCAACATTGAAACCGTGATAGAGTTCCAGC AGAAAAATAATAGGCTTTTGGAGATAACTCGGGAGTTTTCAGTCAACGCGGGTGTA ACAACGCCAGTTTCCACGTATATGCTGACAAACAGTGAGCTCCTGAGCCTGATAAAT GATATGCCAATCACAAACGATCAGAAAAAACTCATGTCCAATAACGTTCAGATAGT ACGGCAACAGAGTTACAGCATAATGTGCATAATTAAAGAGGAGGTCCTGGCTTATG TTGTCCAGCTTCCACTGTACGGGGTTATAGATACCCCATGTTGGAAGCTCCATACAT CTCCCCTGTGTACTACTAACACCAAGGAGGGAAGCAATATATGTTTGACTCGCACTG ACAGGGGTTGGTACTGTGATAATGCCGGGTCCGTGAGCTTTTTTCCGCAGGCTGAAA CTTGCAAGGTGCAATCTAACCGAGTGTTCTGTGACACTATGAATTCTCTGACTCTCC CGTCAGAAGTAAACTTGTGTAATGTCGACATATTTAACCCTAAATACGATTGTAAGA TCATGACAAGCAAAACAGACGTCTCAAGTTCTGTCATAACAAGCTTGGGCGCGATT GTGTCCTGTTATGGTAAAACCAAATGCACGGCGTCCAACAAAAATAGGGGCATTAT TAAAACTTTTTCCAACGGCTGTGATTACGTCTCCAATAAAGGAGTGGATACGGTCTC AGTTGGGAATACTCTGTACTATGTTAACAAACAAGAGGGCAAGTCTCTTTATGTGAA AGGGGAACCGATTATAAACTTTTACGACCCGCTTGTGTTCCCGTCCGATGAGTTCGA TGCGAGTATTTCCCAAGTCAACGAGAAGATAAACCAGTCCCTCGCGTTTATCCGCAA AAGTGACGAGCTCCTTCATAACGTTAATGCTGGTAAGTCCACTACGAACATCATGAT CACAACAATTATCATAGTCATTATTGTTATACTGCTTAGCCTGATCGCTGTAGGGTTG CTCTTGTACTGTAAAGCGAGGTCTACCCCAGTTACCCTTAGTAAAGACCAATTGAGT GGGATCAACAACATTGCGTTTTCCAATTGA SEQIDNO:15:RSV-N3(714nts) CAACTTCTGTCATCCAGCAAATACACCATCCAACGGAGCACAGGAGATAGTA TTGATACTCCTAATTATGATGTGCAGAAACACATCAATAAGTTATGTGGCATGTTAT TAATCACAGAAGATGCTAATCATAAATTCACTGGGTTAATAGGTATGTTATATGCGA TGTCTAGGTTAGGAAGAGAAGACACCATAAAAATACTCAGAGATGCGGGATATCAT GTAAAAGCAAATGGAGTAGATGTAACAACACATCGTCAAGACATTAATGGAAAAGA AATGAAATTTGAAGTGTTAACATTGGCAAGCTTAACAACTGAAATTCAAATCAACAT TGAGATAGAATCTAGAAAATCCTACAAAAAAATGCTAAAAGAAATGGGAGAGGTA GCTCCAGAATACAGGCATGACTCTCCTGATTGTGGGATGATAATATTATGTATAGCA GCATTAGTAATAACTAAATTAGCAGCAGGGGACAGATCTGGTCTTACAGCCGTGATT AGGAGAGCTAATAATGTCCTAAAAAATGAAATGAAACGTTACAAAGGCTTACTACC CAAGGACATAGCCAACAGCTTCTATGAAGTGTTTGAAAAACATCCCCACTTTATAGA TGTTTTTGTTCATTTTGGTATAGCACAATCTTCTACCAGAGGTGGCAGTAGAGTTGA AGGGATTTTTGCAGGATTGTTTATGAATGCCTATGGTGCA SEQIDNO:16:RSV-N3-1(762nts) CAACTTCTGTCATCCAGCAAATACACCATCCAACGGAGCACAGGAGATAGTA TTGATACTCCTAATTATGATGTGCAGAAACACATCAATAAGTTATGTGGCATGTTAT TAATCACAGAAGATGCTAATCATAAATTCACTGGGTTAATAGGTATGTTATATGCGA TGTCTAGGTTAGGAAGAGAAGACACCATAAAAATACTCAGAGATGCGGGATATCAT GTAAAAGCAAATGGAGTAGATGTAACAACACATCGTCAAGACATTAATGGAAAAGA AATGAAATTTGAAGTGTTAACATTGGCAAGCTTAACAACTGAAATTCAAATCAACAT TGAGATAGAATCTAGAAAATCCTACAAAAAAATGCTAAAAGAAATGGGAGAGGTA GCTCCAGAATACAGGCATGACTCTCCTGATTGTGGGATGATAATATTATGTATAGCA GCATTAGTAATAACTAAATTAGCAGCAGGGGACAGATCTGGTCTTACAGCCGTGATT AGGAGAGCTAATAATGTCCTAAAAAATGAAATGAAACGTTACAAAGGCTTACTACC CAAGGACATAGCCAACAGCTTCTATGAAGTGTTTGAAAAACATCCCCACTTTATAGA TGTTTTTGTTCATTTTGGTATAGCACAATCTTCTACCAGAGGTGGCAGTAGAGTTGA AGGGATTTTTGCAGGATTGTTTATGAATGCCTATGGTGCAGGGCAAGTGATGTTACG GTGGGGAGTCTTAGCAAAATCAGTTAAAAAT SEQIDNO:17:RSV-CTL-2(213nts) GCAGGATTCTACCATATATTGAACAACCCAAAAGCATCATTATTATCTTTGAC TCAATTTCCTCACTTCTCCAGTGTAGTATTAGGCAATGCTGCTGGCCTAGGCATAAT GGGAGAGTACAGAGGTACACCGAGGAATCAAGATCTATATGATGCAGCAAAGGCAT ATGCTGAACAACTCAAAGAAAATGGTGTGATTAACTACAGTGTACTA SEQIDNO:18:RSV-N-CTL-4(114nts) TCTACCAGAGGTGGCAGTAGAGTTGAAGGGATTTTTGCAGGATTGTTTATGA ATGCCTATGGTGCAGGGCAAGTGATGTTACGGTGGGGAGTCTTAGCAAAATCAGTT AAAAAT SEQIDNO:19:RSV-M2-1(585nts) ATGTCACGAAGGAATCCTTGCAAATTTGAAATTCGAGGTCATTGCTTAAATG GTAAGAGGTGTCATTTTAGTCATAATTATTTTGAATGGCCACCCCATGCACTGCTTGT AAGACAAAACTTTATGTTAAACAGAATACTTAAGTCTATGGATAAAAGTATAGATA CCTTATCAGAAATAAGTGGAGCTGCAGAGTTGGACAGAACAGAAGAGTATGCTCTT GGTGTAGTTGGAGTGCTAGAGAGTTATATAGGATCAATAAACAATATAACTAAACA ATCAGCATGTGTTGCCATGAGCAAACTCCTCACTGAACTCAATAGTGATGATATCAA AAAGCTGAGGGACAATGAAGAGCTAAATTCACCCAAGATAAGAGTGTACAATACTG TCATATCATATATTGAAAGCAACAGGAAAAACAATAAACAAACTATCCATCTGTTA AAAAGATTGCCAGCAGACGTATTGAAGAAAACCATCAAAAACACATTGGATATCCA TAAGAGCATAACCATCAACAACCCAAAAGAATCAACTGTTAGTGATACAAATGACC ATGCCAAAAATAATGATACTACCTGA SEQIDNO:20:HumanHSP-70(1926ntsor642aa) ATGGCCAAAGCCGCGGCAGTCGGCATCGACCTGGGCACCACCTACTCCTGCG TGGGGGTGTTCCAACACGGCAAGGTGGAGATCATCGCCAACGACCAGGGCAACCGC ACCACCCCCAGCTACGTGGCCTTCACGGACACCGAGCGGCTCATCGGGGATGCGGC CAAGAACCAGGTGGCGCTGAACCCGCAGAACACCGTGTTTGACGCGAAGCGCCTGA TTGGCCGCAAGTTCGGCGACCCGGTGGTGCAGTCGGACATGAAGCACTGGCCTTTCC AGGTGATCAACGACGGAGACAAGCCCAAGGTGCAGGTGAGCTACAAGGGGGAGAC CAAGGCATTCTACCCCGAGGAGATCTCGTCCATGGTGCTGACCAAGATGAAGGAGA TCGCCGAGGCGTACCTGGGCTACCCGGTGACCAACGCGGTGATCACCGTGCCGGCC TACTTCAACGACTCGCAGCGCCAGGCCACCAAGGATGCGGGTGTGATCGCGGGGCT CAACGTGCTGCGGATCATCAACGAGCCCACGGCCGCCGCCATCGCCTACGGCCTGG ACAGAACGGGCAAGGGGGAGCGCAACGTGCTCATCTTTGACCTGGGCGGGGGCACC TTCGACGTGTCCATCCTGACGATCGACGACGGCATCTTCGAGGTGAAGGCCACGGCC GGGGACACCCACCTGGGTGGGGAGGACTTTGACAACAGGCTGGTGAACCACTTCGT GGAGGAGTTCAAGAGAAAACACAAGAAGGACATCAGCCAGAACAAGCGAGCCGTG AGGCGGCTGCGCACCGCCTGCGAGAGGGCCAAGAGGACCCTGTCGTCCAGCACCCA GGCCAGCCTGGAGATCGACTCCCTGTTTGAGGGCATCGACTTCTACACGTCCATCAC CAGGGCGAGGTTCGAGGAGCTGTGCTCCGACCTGTTCCGAAGCACCCTGGAGCCCG TGGAGAAGGCTCTGCGCGACGCCAAGCTGGACAAGGCCCAGATTCACGACCTGGTC CTGGTCGGGGGCTCCACCCGCATCCCCAAGGTGCAGAAGCTGCTGCAGGACTTCTTC AACGGGCGCGACCTGAACAAGAGCATCAACCCCGACGAGGCTGTGGCCTACGGGGC GGCGGTGCAGGCGGCCATCCTGATGGGGGACAAGTCCGAGAACGTGCAGGACCTGC TGCTGCTGGACGTGGCTCCCCTGTCGCTGGGGCTGGAGACGGCCGGAGGCGTGATG ACTGCCCTGATCAAGCGCAACTCCACCATCCCCACCAAGCAGACGCAGATCTTCACC ACCTACTCCGACAACCAACCCGGGGTGCTGATCCAGGTGTACGAGGGCGAGAGGGC CATGACGAAAGACAACAATCTGTTGGGGCGCTTCGAGCTGAGCGGCATCCCTCCGG CCCCCAGGGGCGTGCCCCAGATCGAGGTGACCTTCGACATCGATGCCAACGGCATC CTGAACGTCACGGCCACGGACAAGAGCACCGGCAAGGCCAACAAGATCACCATCAC CAACGACAAGGGCCGCCTGAGCAAGGAGGAGATCGAGCGCATGGTGCAGGAGGCG GAGAAGTACAAAGCGGAGGACGAGGTGCAGCGCGAGAGGGTGTCAGCCAAGAACG CCCTGGAGTCCTACGCCTTCAACATGAAGAGCGCCGTGGAGGATGAGGGGCTCAAG GGCAAGATCAGCGAGGCGGACAAGAAGAAGGTGCTGGACAAGTGTCAAGAGGTCA TCTCGTGGCTGGACGCCAACACCTTGGCCGAGAAGGACGAGTTTGAGCACAAGAGG AAGGAGCTGGAGCAGGTGTGTAACCCCATCATCAGCGGACTGTACCAGGGTGCCGG TGGTCCCGGGCCTGGGGGCTTCGGGGCTCAGGGTCCCAAGGGAGGGTCTGGGTCAG GCCCCACCATTGAGGAGGTAGATTAG SequencetoexpressRSV-GandFgenesintandem SEQIDNO:21:hCdn.RSVG-2A-F(2682nts)(GandFgenes separatedby2Apeptidesequence) ATGTCCAAAAACAAGGATCAACGAACGGCTAAAACACTGGAAAGAACTTGG GATACTCTTAATCACCTTCTTTTCATCAGCTCCTGTTTGTATAAGTTGAACTTGAAAA GTGTAGCACAAATTACCTTGTCAATTCTGGCTATGATTATTTCCACTAGTTTGATCAT TGCTGCGATTATATTTATTGCTTCTGCAAATCATAAGGTAACCCCGACTACAGCGAT CATTCAGGACGCTACAAGTCAAATAAAGAACACCACACCGACGTACTTGACCCAGA ATCCCCAGCTTGGCATCAGTCCTTCTAACCCTTCTGAAATCACCTCCCAAATCACCA CTATCCTTGCGTCTACCACACCTGGAGTAAAGAGTACATTGCAGTCTACTACCGTTA AGACCAAGAACACAACCACAACTCAAACGCAGCCATCTAAGCCAACTACCAAACAG CGGCAAAATAAACCTCCATCTAAACCGAATAACGATTTTCACTTTGAAGTATTCAAC TTTGTTCCCTGCTCAATTTGCAGCAATAATCCGACCTGCTGGGCTATATGTAAGCGG ATACCAAATAAAAAGCCAGGAAAGAAAACTACAACAAAACCTACGAAGAAGCCTA CACTGAAGACCACAAAAAAAGACCCAAAACCCCAGACAACCAAGTCCAAGGAAGT TCCCACTACTAAGCCCACTGAAGAGCCTACCATAAATACCACCAAGACAAACATCA TAACCACCTTGCTCACCTCTAATACTACCGGAAACCCTGAGCTCACTTCCCAAATGG AAACGTTCCATTCAACTAGTAGTGAGGGCAACCCGAGTCCCAGCCAGGTCTCTACA ACCTCAGAATACCCCTCCCAACCTAGTTCACCCCCAAATACTCCACGGCAGGGATCC GGAGAGGGAAGAGGAAGTTTGCTGACATGTGGAGATGTGGAGGAAAATCCCGGTCC AATGGAGCTTCTGATCCTGAAAGCTAACGCTATTACTACTATACTTACCGCCGTAAC ATTCTGCTTCGCCTCCGGACAAAACATCACAGAAGAGTTCTATCAATCCACGTGCAG CGCTGTGTCTAAGGGCTATCTGAGCGCATTGAGAACGGGGTGGTATACTTCCGTAAT TACTATAGAGCTGTCAAACATTAAGAAAAACAAGTGTAACGGTACCGACGCTAAAG TAAAGCTCATCAAGCAGGAGCTGGATAAATACAAAAATGCTGTCACTGAACTCCAG CTTCTTATGCAATCTACCCAAGCAACCAACAACCGGGCTAGGCGCGAATTGCCCAG GTTCATGAATTATACATTGAACAACGCCAAAAAGACTAATGTAACCCTCAGCAAGA AACGCAAGAGGCGGTTCCTGGGATTTCTTCTCGGAGTAGGTTCCGCTATAGCGTCCG GAGTAGCGGTCTCAAAAGTATTGCATCTGGAAGGCGAAGTTAACAAAATTAAGAGC GCGCTCCTCAGCACCAACAAGGCGGTAGTCAGCCTCAGCAACGGCGTATCTGTTCTC ACATCTAAAGTTTTGGACCTGAAAAACTATATAGACAAGCAGTTGCTTCCGATAGTA AATAAGCAATCATGTTCCATTTCAAACATAGAAACGGTTATCGAGTTTCAACAGAAA AATAATAGATTGCTTGAGATCACAAGAGAGTTCTCTGTCAATGCAGGTGTGACTACG CCGGTCAGCACATATATGCTCACGAATAGTGAACTGCTGAGTCTTATAAATGATATG CCGATTACTAATGACCAAAAAAAGCTCATGAGCAACAATGTCCAAATCGTTCGACA ACAAAGTTACTCTATCATGAGCATCATCAAAGAGGAGGTTCTCGCATATGTCGTGCA GCTTCCGTTGTATGGTGTAATAGATACCCCGTGCTGGAAGCTGCACACCTCTCCACT GTGCACAACCAATACTAAAGAGGGGTCTAATATCTGTCTCACGAGAACGGATCGAG GATGGTACTGCGATAACGCCGGTAGTGTGAGCTTCTTCCCCCAGGCTGAAACCTGTA AGGTACAGAGTAACAGGGTATTCTGTGACACTATGAACTCACTCACACTGCCAAGT GAAGTGAACCTTTGTAACGTTGACATATTTAATCCCAAGTACGACTGCAAAATCATG ACAAGCAAAACCGACGTTTCCTCAAGCGTCATAACGAGTTTGGGTGCTATAGTAAGT TGCTATGGGAAAACCAAGTGCACGGCATCCAATAAGAACAGAGGGATCATAAAAAC GTTCTCCAACGGATGTGACTATGTGTCAAACAAGGGGGTTGATACGGTATCAGTTGG AAATACCCTTTATTATGTCAACAAGCAGGAAGGAAAGAGCCTCTATGTAAAAGGCG AACCCATAATCAATTTTTATGACCCACTCGTATTCCCTAGTGATGAGTTCGATGCCTC TATTAGCCAGGTAAATGAGAAGATCAACCAGAGTTTGGCCTTTATCCGCAAATCTGA CGAGCTGCTCCATAATGTCAATGCAGGGAAAAGTACGACTAATATCATGATTACTAC GATTATTATCGTCATCATCGTCATCCTCTTGAGTCTTATAGCGGTAGGGCTCCTGCTC TACTGTAAAGCGCGCTCTACCCCTGTGACGCTGTCCAAAGATCAACTTTCTGGCATA AACAACATTGCCTTTAGTAATTAA SEQIDNO:22:VSV(Indianastrain) ACGAAGACAAACAAACCATTATTATCATTAAAAGGCTCAGGAGAAACTTTAA CAGTAATCAAAATGTCTGTTACAGTCAAGAGAATCATTGACAACACAGTCATAGTTC CAAAACTTCCTGCAAATGAGGATCCAGTGGAATACCCGGCAGATTACTTCAGAAAA TCAAAGGAGATTCCTCTTTACATCAATACTACAAAAAGTTTGTCAGATCTAAGAGGA TATGTCTACCAAGGCCTCAAATCCGGAAATGTATCAATCATACATGTCAACAGCTAC TTGTATGGAGCATTAAAGGACATCCGGGGTAAGTTGGATAAAGATTGGTCAAGTTTC GGAATAAACATCGGGAAAGCAGGGGATACAATCGGAATATTTGACCTTGTATCCTT GAAAGCCCTGGACGGCGTACTTCCAGATGGAGTATCGGATGCTTCCAGAACCAGCG CAGATGACAAATGGTTGCCTTTGTATCTACTTGGCTTATACAGAGTGGGCAGAACAC AAATGCCTGAATACAGAAAAAAGCTCATGGATGGGCTGACAAATCAATGCAAAATG ATCAATGAACAGTTTGAACCTCTTGTGCCAGAAGGTCGTGACATTTTTGATGTGTGG GGAAATGACAGTAATTACACAAAAATTGTCGCTGCAGTGGACATGTTCTTCCACATG TTCAAAAAACATGAATGTGCCTCGTTCAGATACGGAACTATTGTTTCCAGATTCAAA GATTGTGCTGCATTGGCAACATTTGGACACCTCTGCAAAATAACCGGAATGTCTACA GAAGATGTAACGACCTGGATCTTGAACCGAGAAGTTGCAGATGAAATGGTCCAAAT GATGCTTCCAGGCCAAGAAATTGACAAGGCCGATTCATACATGCCTTATTTGATCGA CTTTGGATTGTCTTCTAAGTCTCCATATTCTTCCGTCAAAAACCCTGCCTTCCACTTC TGGGGGCAATTGACAGCTCTTCTGCTCAGATCCACCAGAGCAAGGAATGCCCGACA GCCTGATGACATTGAGTATACATCTCTTACTACAGCAGGTTTGTTGTACGCTTATGC AGTAGGATCCTCTGCCGACTTGGCACAACAGTTTTGTGTTGGAGATAACAAATACAC TCCAGATGATAGTACCGGAGGATTGACGACTAATGCACCGCCACAAGGCAGAGATG TGGTCGAATGGCTCGGATGGTTTGAAGATCAAAACAGAAAACCGACTCCTGATATG ATGCAGTATGCGAAAAGAGCAGTCATGTCACTGCAAGGCCTAAGAGAGAAGACAAT TGGCAAGTATGCTAAGTCAGAATTTGACAAATGACCCTATAATTCTCAGATCACCTA TTATATATTATGCTACATATGAAAAAAACTAACAGATATCATGGATAATCTCACAAA AGTTCGTGAGTATCTCAAGTCCTACTCTCGTCTAGATCAGGCGGTAGGAGAGATAGA TGAGATCGAAGCACAACGAGCTGAAAAGTCCAATTATGAGTTGTTCCAAGAGGACG GAGTGGAAGAGCATACTAGGCCCTCTTATTTTCAGGCAGCAGATGATTCTGACACAG AATCTGAACCAGAAATTGAAGACAATCAAGGCTTGTATGTACCAGATCCGGAAGCT GAGCAAGTTGAAGGCTTTATACAGGGGCCTTTAGATGACTATGCAGATGAGGACGT GGATGTTGTATTCACTTCGGACTGGAAACAGCCTGAGCTTGAATCCGACGAGCATGG AAAGACCTTACGGTTGACATTGCCAGAGGGTTTAAGTGGAGAGCAGAAATCCCAGT GGCTTTTGACGATTAAAGCAGTCGTTCAAAGTGCCAAACACTGGAATCTGGCAGAG TGCACATTTGAAGCATCGGGAGAAGGGGTCATCATAAAAAAGCGCCAGATAACTCC GGATGTATATAAGGTCACTCCAGTGATGAACACACATCCGTACCAATCAGAAGCCG TATCAGATGTTTGGTCTCTCTCAAAGACATCCATGACTTTCCAACCCAAGAAAGCAA GTCTTCAGCCTCTCACCATATCCTTGGATGAATTGTTCTCATCTAGAGGAGAATTCAT CTCTGTCGGAGGTAACGGACGAATGTCTCATAAAGAGGCCATCCTGCTCGGTCTGAG GTACAAAAAGTTGTACAATCAGGCGAGAGTCAAATATTCTCTGTAGACTATGAAAA AAAGTAACAGATATCACAATCTAAGTGTTATCCCAATCCATTCATCATGAGTTCCTT AAAGAAGATTCTCGGTCTGAAGGGGAAAGGTAAGAAATCTAAGAAATTAGGGATCG CACCACCCCCTTATGAAGAGGACACTAGCATGGAGTATGCTCCGAGCGCTCCAATTG ACAAATCCTATTTTGGAGTTGACGAGATGGACACCTATGATCCGAATCAATTAAGAT ATGAGAAATTCTTCTTTACAGTGAAAATGACGGTTAGATCTAATCGTCCGTTCAGAA CATACTCAGATGTGGCAGCCGCTGTATCCCATTGGGATCACATGTACATCGGAATGG CAGGGAAACGTCCCTTCTACAAAATCTTGGCTTTTTTGGGTTCTTCTAATCTAAAGGC CACTCCAGCGGTATTGGCAGATCAAGGTCAACCAGAGTATCACGCTCACTGCGAAG GCAGGGCTTATTTGCCACATAGGATGGGGAAGACCCCTCCCATGCTCAATGTACCAG AGCACTTCAGAAGACCATTCAATATAGGTCTTTACAAGGGAACGATTGAGCTCACA ATGACCATCTACGATGATGAGTCACTGGAAGCAGCTCCTATGATCTGGGATCATTTC AATTCTTCCAAATTTTCTGATTTCAGAGAGAAGGCCTTAATGTTTGGCCTGATTGTCG AGAAAAAGGCATCTGGAGCGTGGGTCCTGGATTCTATCAGCCACTTCAAATGAGCT AGTCTAGCTTCCAGCTTCTGAACAATCCCCGGTTTACTCAGTCTCTCCTAATTCCAGC CTTTCGAACAACTAATATCCTGTCTTTTCTATCCCTATGAAAAAAACTAACAGAGAT CGATCTGTTTCCTTGACACCATGAAGTGCCTTTTGTACTTAGCTTTTTTATTCATCGG GGTGAATTGCAAGTTCACCATAGTTTTTCCACACAACCGAAAAGGAAACTGGAAAA ATGTTCCTTCCAATTACCATTATTGCCCGTCAAGCTCAGATTTAAATTGGCATAATGA CTTAATAGGCACAGCCTTACAAGTCAAAATGCCCAAGAGTCACAAGGCTATTCAAG CAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGTGATTTCCGCTGGT ACGGACCGGAGTATATAACACATTCCATCCGATCCTTCACTCCATCTGTAGAACAAT GCAAGGAAAGCATTGAACAAACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCT CCTCAAAGTTGTGGATATGCAACTGTGACGGATGCTGAAGCAGCGATTGTCCAGGT GACTCCTCACCATGTGCTTGTTGATGAATACACAGGAGAATGGGTTGATTCACAGTT CATCAACGGAAAATGCAGCAATGACATATGCCCCACTGTCCATAACTCCACAACCT GGCATTCCGACTATAAGGTCAAAGGGCTATGTGATTCTAACCTCATTTCCATGGACA TCACCTTCTTCTCAGAGGACGGAGAGCTATCATCCCTAGGAAAGGAGGGCACAGGG TTCAGAAGTAACTACTTTGCTTATGAAACTGGAGACAAGGCCTGCAAAATGCAGTA CTGCAAGCATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGATAA GGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGC TCCATCTCAGACCTCAGTGGATGTAAGTCTCATTCAGGACGTTGAGAGGATCTTGGA TTATTCCCTCTGCCAAGAAACCTGGAGCAAAATCAGAGCGGGTCTTCCCATCTCTCC AGTGGATCTCAGCTATCTTGCTCCTAAAAACCCAGGAACCGGTCCTGTCTTTACCAT AATCAATGGTACCCTAAAATACTTTGAGACCAGATACATCAGAGTCGATATTGCTGC TCCAATCCTCTCAAGAATGGTCGGAATGATCAGTGGAACTACCACAGAAAGGGAAC TGTGGGATGACTGGGCTCCATATGAAGACGTGGAAATTGGACCCAATGGAGTTCTG AGGACCAGTTCAGGATATAAGTTTCCTTTATATATGATTGGACATGGTATGTTGGAC TCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTTGAACATCCTCACATTCAAGAC GCTGCTTCGCAGCTTCCTGATGATGAGACTTTATTTTTTGGTGATACTGGGCTATCCA AAAATCCAATCGAGTTTGTAGAAGGTTGGTTCAGTAGTTGGAAGAGCTCTATTGCCT CTTTTTGCTTTATCATAGGGTTAATCATTGGACTATTCTTGGTTCTCCGAGTTGGTAT TTATCTTTGCATTAAATTAAAGCACACCAAGAAAAGACAGATTTATACAGACATAG AGATGAACCGACTTGGAAAGTAACTCAAATCCTGCACAACAGATTCTTCATGTTTGA ACCAAATCAACTTGTGATATCATGCTCAAAGAGGCCTTAATTATATTTTAATTTTTAA TTTTTATGAAAAAAACTAACAGCAATCATGGAAGTCCACGATTTTGAGACCGACGA GTTCAATGATTTCAATGAAGATGACTATGCCACAAGAGAATTCCTGAATCCCGATGA GCGCATGACGTACTTGAATCATGCTGATTACAATTTGAATTCTCCTCTAATTAGTGAT GATATTGACAATTTGATCAGGAAATTCAATTCTCTTCCGATTCCCTCGATGTGGGAT AGTAAGAACTGGGATGGAGTTCTTGAGATGTTAACATCATGTCAAGCCAATCCCATC TCAACATCTCAGATGCATAAATGGATGGGAAGTTGGTTAATGTCTGATAATCATGAT GCCAGTCAAGGGTATAGTTTTTTACATGAAGTGGACAAAGAGGCAGAAATAACATT TGACGTGGTGGAGACCTTCATCCGCGGCTGGGGCAACAAACCAATTGAATACATCA AAAAGGAAAGATGGACTGACTCATTCAAAATTCTCGCTTATTTGTGTCAAAAGTTTT TGGACTTACACAAGTTGACATTAATCTTAAATGCTGTCTCTGAGGTGGAATTGCTCA ACTTGGCGAGGACTTTCAAAGGCAAAGTCAGAAGAAGTTCTCATGGAACGAACATA TGCAGGATTAGGGTTCCCAGCTTGGGTCCTACTTTTATTTCAGAAGGATGGGCTTAC TTCAAGAAACTTGATATTCTAATGGACCGAAACTTTCTGTTAATGGTCAAAGATGTG ATTATAGGGAGGATGCAAACGGTGCTATCCATGGTATGTAGAATAGACAACCTGTT CTCAGAGCAAGACATCTTCTCCCTTCTAAATATCTACAGAATTGGAGATAAAATTGT GGAGAGGCAGGGAAATTTTTCTTATGACTTGATTAAAATGGTGGAACCGATATGCA ACTTGAAGCTGATGAAATTAGCAAGAGAATCAAGGCCTTTAGTCCCACAATTCCCTC ATTTTGAAAATCATATCAAGACTTCTGTTGATGAAGGGGCAAAAATTGACCGAGGT ATAAGATTCCTCCATGATCAGATAATGAGTGTGAAAACAGTGGATCTCACACTGGTG ATTTATGGATCGTTCAGACATTGGGGTCATCCTTTTATAGATTATTACACTGGACTAG AAAAATTACATTCCCAAGTAACCATGAAGAAAGATATTGATGTGTCATATGCAAAA GCACTTGCAAGTGATTTAGCTCGGATTGTTCTATTTCAACAGTTCAATGATCATAAA AAGTGGTTCGTGAATGGAGACTTGCTCCCTCATGATCATCCCTTTAAAAGTCATGTT AAAGAAAATACATGGCCCACAGCTGCTCAAGTTCAAGATTTTGGAGATAAATGGCA TGAACTTCCGCTGATTAAATGTTTTGAAATACCCGACTTACTAGACCCATCGATAAT ATACTCTGACAAAAGTCATTCAATGAATAGGTCAGAGGTGTTGAAACATGTCCGAA TGAATCCGAACACTCCTATCCCTAGTAAAAAGGTGTTGCAGACTATGTTGGACACAA AGGCTACCAATTGGAAAGAATTTCTTAAAGAGATTGATGAGAAGGGCTTAGATGAT GATGATCTAATTATTGGTCTTAAAGGAAAGGAGAGGGAACTGAAGTTGGCAGGTAG ATTTTTCTCCCTAATGTCTTGGAAATTGCGAGAATACTTTGTAATTACCGAATATTTG ATAAAGACTCATTTCGTCCCTATGTTTAAAGGCCTGACAATGGCGGACGATCTAACT GCAGTCATTAAAAAGATGTTAGATTCCTCATCCGGCCAAGGATTGAAGTCATATGAG GCAATTTGCATAGCCAATCACATTGATTACGAAAAATGGAATAACCACCAAAGGAA GTTATCAAACGGCCCAGTGTTCCGAGTTATGGGCCAGTTCTTAGGTTATCCATCCTT AATCGAGAGAACTCATGAATTTTTTGAGAAAAGTCTTATATACTACAATGGAAGACC AGACTTGATGCGTGTTCACAACAACACACTGATCAATTCAACCTCCCAACGAGTTTG TTGGCAAGGACAAGAGGGTGGACTGGAAGGTCTACGGCAAAAAGGATGGAGTATC CTCAATCTACTGGTTATTCAAAGAGAGGCTAAAATCAGAAACACTGCTGTCAAAGTC TTGGCACAAGGTGATAATCAAGTTATTTGCACACAGTATAAAACGAAGAAATCGAG AAACGTTGTAGAATTACAGGGTGCTCTCAATCAAATGGTTTCTAATAATGAGAAAAT TATGACTGCAATCAAAATAGGGACAGGGAAGTTAGGACTTTTGATAAATGACGATG AGACTATGCAATCTGCAGATTACTTGAATTATGGAAAAATACCGATTTTCCGTGGAG TGATTAGAGGGTTAGAGACCAAGAGATGGTCACGAGTGACTTGTGTCACCAATGAC CAAATACCCACTTGTGCTAATATAATGAGCTCAGTTTCCACAAATGCTCTCACCGTA GCTCATTTTGCTGAGAACCCAATCAATGCCATGATACAGTACAATTATTTTGGGACA TTTGCTAGACTCTTGTTGATGATGCATGATCCTGCTCTTCGTCAATCATTGTATGAAG TTCAAGATAAGATACCGGGCTTGCACAGTTCTACTTTCAAATACGCCATGTTGTATT TGGACCCTTCCATTGGAGGAGTGTCGGGCATGTCTTTGTCCAGGTTTTTGATTAGAG CCTTCCCAGATCCCGTAACAGAAAGTCTCTCATTCTGGAGATTCATCCATGTACATG CTCGAAGTGAGCATCTGAAGGAGATGAGTGCAGTATTTGGAAACCCCGAGATAGCC AAGTTTCGAATAACTCACATAGACAAGCTAGTAGAAGATCCAACCTCTCTGAACATC GCTATGGGAATGAGTCCAGCGAACTTGTTAAAGACTGAGGTTAAAAAATGCTTAAT CGAATCAAGACAAACCATCAGGAACCAGGTGATTAAGGATGCAACCATATATTTGT ATCATGAAGAGGATCGGCTCAGAAGTTTCTTATGGTCAATAAATCCTCTGTTCCCTA GATTTTTAAGTGAATTCAAATCAGGCACTTTTTTGGGAGTCGCAGACGGGCTCATCA GTCTATTTCAAAATTCTCGTACTATTCGGAACTCCTTTAAGAAAAAGTATCATAGGG AATTGGATGATTTGATTGTGAGGAGTGAGGTATCCTCTTTGACACATTTAGGGAAAC TTCATTTGAGAAGGGGATCATGTAAAATGTGGACATGTTCAGCTACTCATGCTGACA CATTAAGATACAAATCCTGGGGCCGTACAGTTATTGGGACAACTGTACCCCATCCAT TAGAAATGTTGGGTCCACAACATCGAAAAGAGACTCCTTGTGCACCATGTAACACA TCAGGGTTCAATTATGTTTCTGTGCATTGTCCAGACGGGATCCATGACGTCTTTAGTT CACGGGGACCATTGCCTGCTTATCTAGGGTCTAAAACATCTGAATCTACATCTATTT TGCAGCCTTGGGAAAGGGAAAGCAAAGTCCCACTGATTAAAAGAGCTACACGTCTT AGAGATGCTATCTCTTGGTTTGTTGAACCCGACTCTAAACTAGCAATGACTATACTT TCTAACATCCACTCTTTAACAGGCGAAGAATGGACCAAAAGGCAGCATGGGTTCAA AAGAACAGGGTCTGCCCTTCATAGGTTTTCGACATCTCGGATGAGCCATGGTGGGTT CGCATCTCAGAGCACTGCAGCATTGACCAGGTTGATGGCAACTACAGACACCATGA GGGATCTGGGAGATCAGAATTTCGACTTTTTATTCCAAGCAACGTTGCTCTATGCTC AAATTACCACCACTGTTGCAAGAGACGGATGGATCACCAGTTGTACAGATCATTATC ATATTGCCTGTAAGTCCTGTTTGAGACCCATAGAAGAGATCACCCTGGACTCAAGTA TGGACTACACGCCCCCAGATGTATCCCATGTGCTGAAGACATGGAGGAATGGGGAA GGTTCGTGGGGACAAGAGATAAAACAGATCTATCCTTTAGAAGGGAATTGGAAGAA TTTAGCACCTGCTGAGCAATCCTATCAAGTCGGCAGATGTATAGGTTTTCTATATGG AGACTTGGCGTATAGAAAATCTACTCATGCCGAGGACAGTTCTCTATTTCCTCTATC TATACAAGGTCGTATTAGAGGTCGAGGTTTCTTAAAAGGGTTGCTAGACGGATTAAT GAGAGCAAGTTGCTGCCAAGTAATACACCGGAGAAGTCTGGCTCATTTGAAGAGGC CGGCCAACGCAGTGTACGGAGGTTTGATTTACTTGATTGATAAATTGAGTGTATCAC CTCCATTCCTTTCTCTTACTAGATCAGGACCTATTAGAGACGAATTAGAAACGATTC CCCACAAGATCCCAACCTCCTATCCGACAAGCAACCGTGATATGGGGGTGATTGTCA GAAATTACTTCAAATACCAATGCCGTCTAATTGAAAAGGGAAAATACAGATCACAT TATTCACAATTATGGTTATTCTCAGATGTCTTATCCATAGACTTCATTGGACCATTCT CTATTTCCACCACCCTCTTGCAAATCCTATACAAGCCATTTTTATCTGGGAAAGATA AGAATGAGTTGAGAGAGCTGGCAAATCTTTCTTCATTGCTAAGATCAGGAGAGGGG TGGGAAGACATACATGTGAAATTCTTCACCAAGGACATATTATTGTGTCCAGAGGA AATCAGACATGCTTGCAAGTTCGGGATTGCTAAGGATAATAATAAAGACATGAGCT ATCCCCCTTGGGGAAGGGAATCCAGAGGGACAATTACAACAATCCCTGTTTATTATA CGACCACCCCTTACCCAAAGATGCTAGAGATGCCTCCAAGAATCCAAAATCCCCTGC TGTCCGGAATCAGGTTGGGCCAATTACCAACTGGCGCTCATTATAAAATTCGGAGTA TATTACATGGAATGGGAATCCATTACAGGGACTTCTTGAGTTGTGGAGACGGCTCCG GAGGGATGACTGCTGCATTACTACGAGAAAATGTGCATAGCAGAGGAATATTCAAT AGTCTGTTAGAATTATCAGGGTCAGTCATGCGAGGCGCCTCTCCTGAGCCCCCCAGT GCCCTAGAAACTTTAGGAGGAGATAAATCGAGATGTGTAAATGGTGAAACATGTTG GGAATATCCATCTGACTTATGTGACCCAAGGACTTGGGACTATTTCCTCCGACTCAA AGCAGGCTTGGGGCTTCAAATTGATTTAATTGTAATGGATATGGAAGTTCGGGATTC TTCTACTAGCCTGAAAATTGAGACGAATGTTAGAAATTATGTGCACCGGATTTTGGA TGAGCAAGGAGTTTTAATCTACAAGACTTATGGAACATATATTTGTGAGAGCGAAA AGAATGCAGTAACAATCCTTGGTCCCATGTTCAAGACGGTCGACTTAGTTCAAACAG AATTTAGTAGTTCTCAAACGTCTGAAGTATATATGGTATGTAAAGGTTTGAAGAAAT TAATCGATGAACCCAATCCCGATTGGTCTTCCATCAATGAATCCTGGAAAAACCTGT ACGCATTCCAGTCATCAGAACAGGAATTTGCCAGAGCAAAGAAGGTTAGTACATAC TTTACCTTGACAGGTATTCCCTCCCAATTCATTCCTGATCCTTTTGTAAACATTGAGA CTATGCTACAAATATTCGGAGTACCCACGGGTGTGTCTCATGCGGCTGCCTTAAAAT CATCTGATAGACCTGCAGATTTATTGACCATTAGCCTTTTTTATATGGCGATTATATC GTATTATAACATCAATCATATCAGAGTAGGACCGATACCTCCGAACCCCCCATCAGA TGGAATTGCACAAAATGTGGGGATCGCTATAACTGGTATAAGCTTTTGGCTGAGTTT GATGGAGAAAGACATTCCACTATATCAACAGTGTTTGGCAGTTATCCAGCAATCATT TCCGATTAGGTGGGAGGCTATTTCAGTAAAAGGAGGATACAAGCAGAAGTGGAGTA CTAGAGGTGATGGGCTCCCAAAAGATACCCGAATTTCAGACTCCTTGGCCCCAATCG GGAACTGGATCAGATCTTTGGAATTGGTCCGAAACCAAGTTCGTCTAAATCCATTCA ATAAGATCTTGTTCAATCAGCTATGTCGTACAGTGGATAATCATTTGAAGTGGTCAA ATTTGCGAAAAAACACAGGAATGATTGAATGGATCAATGGGCGAATTTCAAAAGAA GACCGGTCTATACTGATGTTGAAGAGTGACCTACATGAGGAAAACTCTTGGAGAGA TTAAAAAATCAGGAGGAGACTCCAAACTTTAAGTATGAAAAAAACTTTGATCCTTA AGACCCTCTTGTGGTTTTTATTTTTTTATCTGGTTTTGTGGTCTTCGT