Respiratory Syncytial Virus (rsv) Polyanhydride Nanoparticle Vaccine

Abstract

Disclosed are compositions and methods for vaccinating susceptible individuals against infection by respiratory syncytial virus (RSV). The disclosed compositions include vaccine compositions comprising an effective amount of respiratory syncytial virus (RSV) F protein in a pre-fusion stabilized form and/or M protein incorporated into biodegradable polyanhydride polymer particles for inducing an immune response against RSV. The vaccine compositions also may include a suitable adjuvant.

Description

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] The present application claims the benefit of priority under 35 U.S.C. .sctn. 119(e) to U.S. Provisional Application No. 62/752,006, filed on Oct. 29, 2018, the content of which is incorporated herein by reference in its entirety.

BACKGROUND

[0003] The field of the invention relates to compositions and methods for inducing an immune response against respiratory syncytial virus (RSV). In particular, the field of the invention relates to compositions and methods for vaccinating susceptible individuals against infection by RSV by administering particulate vaccines comprising RSV structural components or variants thereof and optionally an adjuvant.

[0004] Respiratory syncytial virus (RSV) is the leading cause of lower respiratory infections in young children, resulting in 34 million new RSV infections each year, and approximately 125,000 hospitalizations annually in the United States alone. RSV reinfection is common in children, and even adults can be susceptible to repeated infection due to short-lived and incomplete protective immunity after natural infection. Especially vulnerable populations include premature babies, the elderly, people with heart or lung disease, and people with a very weak immune system. There are no effective vaccines for RSV, and work in the field has been slow since two children died during an RSV vaccine test in 1966.

[0005] RSV is an RNA virus with a genome that expresses eleven (11) known proteins. One of these, F protein, causes the virion membrane to fuse with the target cell's membrane. F proteins are targeted by antibodies after infection and are the major target for antiviral drug development. Fusion causes a confirmation change that obscures the major F antigenic site, site 0. As such, RSV vaccines that induce immune response against 0 and other antigens of RSV such as M protein are desirable.

SUMMARY

[0006] Disclosed are compositions and methods for vaccinating susceptible individuals against infection by respiratory syncytial virus (RSV). The disclosed compositions include vaccine compositions comprising an effective amount of respiratory syncytial virus (RSV) F protein in a pre-fusion stabilized form and/or M protein incorporated into biodegradable polyanhydride polymer particles for inducing an immune response against RSV. The vaccine compositions also may include a suitable adjuvant.

BRIEF DESCRIPTION OF THE FIGURES

[0007] FIG. 1. Prime/boost nanoparticle vaccination strategy with prefusion RSV F protects against RSV-induced disease. BALB/c mice were primed with 500 .mu.g of the indicated nanoparticle formulation i.n. on day 0, and boosted with 500 .mu.g i.n. on day 28. Control mice (labeled RSV) were administered PBS i.n. on both prime and boost days. On day 56, all mice were challenged with 4.8.times.10.sup.6 PFU RSV-A2 i.n. and assessed for (A) weight loss, (B) Penh, and (C) EF50. Data are represented as mean.+-.SEM of 3 independent experiments (n=1 mice for prefusionF+M+CpG, n=12 mice for RSV, CpG, M+CpG, and postfusion F+CpG, n=10 mice for prefusion F+CpG). Asterisks represent significance between RSV and prefusion F+M+CpG, pound symbols represent significance between RSV and prefusion F+CpG, and $ represents significance between RSV and postfusion F+CpG as determined by a 2-way ANOVA with a Dunnett's post hoc test. .sup.*/#/.dagger-dbl.p<0.05, .sup.**/##/.dagger-dbl..dagger-dbl.p<0.01, .sup.***/###/.dagger-dbl..dagger-dbl..dagger-dbl.p<0.001

[0008] FIG. 2. Prime/boost nanoparticle vaccination with prefusion RSV F reduces RSV N gene viral copy numbers. BALB/c mice were primed with 500 .mu.g of the indicated nanoparticle formulation i.n. on day 0, and boosted with 500 .mu.g i.n. on day 28. Control mice (labeled RSV) were administered PBS i.n. on both prime and boost days. On day 56, all mice were challenged with 4.8.times.10.sup.6 PFU RSV-A2 i.n. 4 days post-challenge lungs were harvested for RNA. RT-PCR was performed to determine RSV N gene copy numbers. Data represent mean.+-.SEM of 2 independent experiments (n=8 mice for postfusion F+CpG, M+CpG, and prefusion F+CpG, n=7 mice for RSV, n=6 mice for prefusion F+M+CpG, n=4 mice for CpG). Statistical significance was determined by a one-way ANOVA with a Tukey's post hoc test. *p<0.05, **p<0.01

[0009] FIG. 3. Initial priming vaccination with prefusion RSV F induces lung-resident CD4 and CD8 T cells. BALB/c mice were primed with 500 .mu.g of the indicated nanoparticle formulation or PBS (naive) i.n. on day 0. Lungs and spleen were harvested on day 8 and analyzed by flow cytometry. Frequency of (A) activated (CD11a.sup.hiCD49d.sup.+) CD4 T cells and (B) activated (CD11a.sup.hiCD810) CD8 T cells. Number of i.v..sup.- (C) CD4 and (D) CD8 T cells. Number of activated i.v..sup.- (E) CD103.sup.-CD69.sup.+CD4 T cells and (F) CD103.sup.+CD69.sup.+CD8 T cells. (G) Representative flow cytometry plots of germinal center B cells (CD19.sup.+CDB220.sup.+Fas.sup.+GL-7.sup.+). Data are represented as mean.+-.SEM from a single experiment (n=4 mice for naive, preF+CpG, preF+M+CpG, n=3 mice for CpG, PreF, PreF+M). Statistical significance was determined by a 2-way ANOVA with Tukey's post hoc test (A and B). *p<0.05, **p<0.01, p<0.001

[0010] FIG. 4. Prime/boost nanoparticle vaccination induces RSV-specific antibodies in serum. BALB/c mice were primed with 500 .mu.g of the indicated nanoparticle formulation i.n. on day 0, and boosted with 500 .mu.g i.n. on day 28. Control mice (labeled naive) were administered PBS i.n. on both prime and boost days. On day 14 and 56 serum was assessed for (A,D) total RSV-specific IgG, (B,E) IgG1, or (C,F) IgG2a. Data are represented as mean.+-.SEM from a single experiment (n=4 mice). Asterisks represent significance between naive and prefusion F+M+CpG, and .dagger-dbl. represents significance between naive and postfusion F+CpG as determined by a 2-way ANOVA with Dunnett's post hoc test. .sup.*/#/.dagger-dbl.p<0.05, .sup.**/##/.dagger-dbl..dagger-dbl.p<0.01, ***/###/.dagger-dbl..dagger-dbl..dagger-dbl.p<0.001

[0011] FIG. 5. Prime/boost nanoparticle vaccination with prefusion RSV F induces lung-resident B cells, CD4, and CD8 T cells. BALB/c mice were primed with 500 .mu.g of the indicated nanoparticle formulation or PBS (naive) i.n. on day 0, and boosted with 500 .mu.g i.n. on day 28. Lung and spleen were harvested on day 42 and analyzed by flow cytometry. (A) Number of i.v..sup.- B cells. Number of activated i.v..sup.- (B) CD103.sup.-CD69.sup.+CD4 T cells and (C) CD103.sup.+CD69.sup.+CD8 T cells. Number of activated (D) IFN-.gamma..sup.+CD4 T cells and (E) IL-5.sup.+CD4 T cells following stimulation with PMA/ionomycin. Data are represented as mean.+-.SEM from a single experiment (n=4 mice). Asterisks represent significance between naive and prefusion F+M+CpG, and .dagger-dbl. represents significance between naive and postfusion F+CpG as determined by a one-way ANOVA with Tukey's post hoc test. *p<0.05, **p<0.01, ***p<0.001.

[0012] FIG. 6. Prime/boost nanoparticle vaccination with prefusion RSV F protects against RSV-induced weight loss and pulmonary dysfunction. (A-B) BALB/c mice were primed with 500 .mu.g of the indicated nanoparticle formulation i.n. on day 0, and challenged with 4.8.times.10.sup.6 PFU RSV-A2 i.n. on day 28. No vaccine mice were administered PBS i.n. at the prime. Groups were monitored daily for (A) weight loss and (B) airway obstruction (Penh). (C-D) BALB/c mice were primed with 500 .mu.g of the indicated nanoparticle formulation i.n. on day 0, and boosted with 500 .mu.g i.n. on day 28. No vaccine mice were administered PBS i.n. on both prime and boost days. All mice were challenged with 4.8.times.10.sup.6 PFU RSV-A2 i.n. on (C-D) day 56 or (E-F) day 100 and assessed for weight loss and airway obstruction (Penh). Asterisks represent significance between no vaccine and preF+CpG and pound symbols represent significance between CpG and preF+CpG as determined by (A-B) Student t test, or (C-F) 2-way ANOVA with Dunnett's post hoc test. .sup.*/# p<0.05, .sup.**/## p<0.01, .sup.***/### p<0.001.

[0013] FIG. 7. Prime/boost nanoparticle vaccination with prefusion RSV F reduces infectious RSV particles. (A-B) BALB/c mice were primed with 500 .mu.g of the indicated nanoparticle formulation i.n. on day 0, and boosted with 500 .mu.g i.n. on day 28. No vaccine mice were administered PBS i.n. on both prime and boost days. RSV immune mice received 4.8.times.10.sup.6 PFU RSV-A2 i.n. at the prime and PBS i.n. at the boost. On (A) day 56 or (B) day 100, all mice were challenged with 4.8.times.10.sup.6 PFU RSV-A2 i.n and infectious plaque-forming units (pfu) were quantified in the lung on (A) day 2 or (A and B) day 4 by plaque assay. (C) BALB/c mice were primed with 500 .mu.g of the indicated nanoparticle formulation i.n. on day 0, and challenged with 4.8.times.10.sup.6 PFU RSV-A2 i.n. on day 28. No vaccine mice were administered PBS i.n., and RSV immune mice received 4.8.times.10.sup.6 PFU RSV-A2 i.n. at the prime. On day 28, all mice were challenged with 4.8.times.10.sup.6 PFU RSV-A2 i.n and infectious pfu were quantified in the lung on day 4 by plaque assay. Statistical significance was determined by (A) 2-way ANOVA or (B-C) one-way ANOVA with a Tukey's post hoc test. *p<0.05, **p<0.01, ***p<0.001.

[0014] FIG. 8. Prime/boost nanoparticle vaccination with prefusion RSV F induces activated T cells in the lungs. BALB/c mice were primed with 500 .mu.g of the indicated nanoparticle formulation i.n. on day 0, and boosted with 500 .mu.g i.n. on day 28. No vaccine mice were administered PBS i.n. on both prime and boost days. RSV immune mice received 4.8.times.10.sup.6 PFU RSV-A2 i.n. at the prime and PBS i.n. at the boost. Lungs and spleens were harvested on day 42 and analyzed by flow cytometry. Frequency of (A) activated (CD11a.sup.hiCD49d.sup.+) CD4 T cells and (B) activated (CD11a.sup.hiCD8.sup.lo) CD8 T cells. Statistical significance was determined by 2-way ANOVA with Tukey's post hoc test. **p<0.01, ***p<0.001.

[0015] FIG. 9. Prime/boost nanoparticle vaccination with prefusion RSV F induces tissue-resident CD4 and CD8 T cells. BALB/c mice were primed with 500 .mu.g of the indicated nanoparticle formulation i.n. on day 0, and boosted with 500 .mu.g i.n. on day 28. No vaccine mice were administered PBS i.n. on both prime and boost days. RSV immune mice received 4.8.times.10.sup.6 PFU RSV-A2 i.n. at the prime and PBS i.n. at the boost. Lungs were harvested on day 42 and analyzed by flow cytometry. Number of activated CD45 intravascular antibody (i.v..sup.-) (A) CD103.sup.-CD69.sup.+CD4 T cells and (B) CD103.sup.+CD69.sup.+CD8 T cells. Number of activated IFN-.gamma..sup.+ (C) CD4 and (D) CD8 T cells following stimulation with PMA/ionomycin. Number of (E) activated F.sub.85-93 tetramer.sup.+CD8 T cells and (F) i.v..sup.- activated F.sub.85-93 tetramer.sup.+CD8 T cells. Statistical significance was determined by one-way ANOVA (C-F) with Tukey's post hoc test. *p<0.05, **p<0.01, **p<0.001.

[0016] FIG. 10. BALB/c mice were primed with 500 .mu.g of the indicated nanoparticle formulation i.n. on day 0, and boosted with 500 .mu.g i.n. on day 28. No vaccine mice were administered PBS i.n. on both prime and boost days. RSV immune mice received 4.8.times.10.sup.6 PFU RSV-A2 i.n. at the prime and PBS i.n. at the boost. (A-B) On (A) day 56 or (B) day 100, all mice were challenged with 4.8.times.10.sup.6 PFU RSV-A2 i.n and monitored daily for the respiratory parameter EF50. (C) On day 56, all groups were challenged with 4.8.times.10.sup.6 PFU RSV-A2 i.n and RSV N gene copy numbers per lung on day 4 were determined by RT-PCR. Asterisks represent significance between no vaccine and preF+CpG and pound symbols represent significance between CpG and preF+CpG as determined by (A-B) 2-way ANOVA or (C) one-way ANOVA with Tukey's post hoc test. .sup.*/# p<0.05, .sup.**/## p<0.01, .sup.***/### p<0.001.

[0017] FIG. 11. Illustration of Pre-F protein triggering, fold-back, and facilitating membrane fusion between a target cell and a virion.

[0018] FIG. 12. Illustrative vaccination schedule.

DETAILED DESCRIPTION

[0019] The disclosed subject matter further may be described utilizing terms as defined below.

[0020] Unless otherwise specified or indicated by context, the terms "a", "an", and "the" mean "one or more." For example, "a protein" and "an adjuvant" should be interpreted to mean "one or more proteins" and "adjuvants," respectively.

[0021] As used herein, "about", "approximately," "substantially," and "significantly" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, "about" and "approximately" will mean plus or minus .ltoreq.10% of the particular term and "substantially" and "significantly" will mean plus or minus >10% of the particular term.

[0022] As used herein, the terms "include" and "including" have the same meaning as the terms "comprise" and "comprising." The terms "comprise" and "comprising" should be interpreted as being "open" transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms "consist" and "consisting of" should be interpreted as being "closed" transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term "consisting essentially of" should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0023] The terms "subject," "patient," and "individual" may be used interchangeably herein. A subject may be a human subject. A subject may refer to a human subject having or at risk for infection by respiratory syncytial virus (RSV). Human RSV (HRSV) is the leading cause of severe respiratory infections in neonates and children. HRSV belongs to the Orthopneumovirus genus within the Pneumoviridae family of viruses. HRSV is enveloped and has a negative-sense, single-stranded RNA genome of approximately 15 kb that encodes 11 viral proteins which include the F (fusion) protein that is a transmembrane protein of the virus and the M (matrix) protein that is a core protein of the virus. The HRSV-GNA435/11 strain has been sequenced and includes 15,277 bp encoding 11 viral proteins. (See Lee et al., Complete Genome Sequence of Human Respiratory Syncytial Virus Genotype A with a 72-Nucleotide Duplication in the Attachment Protein G Gene, J. Virol., December 2012, Vol. 86, No. 24, p. 13810-13811, the content of which is incorporated herein by reference in its entirety). The corresponding DNA sequence of the HRSV genome is provided herein as SEQ ID NO:1. The amino acid sequences of the NS1, NS2, N, M, P, G, F, SH, MS-1, MS-2, and L genes are provided herein as SEQ ID NOs: 2-12, respectively.

[0024] The disclosed compositions may include an effective amount of respiratory syncytial virus (RSV) F protein in a pre-fusion stabilized form incorporated into biodegradable polyanhydride polymer particles. The RSV F protein is a class I viral fusion glycoprotein that mediates membrane fusion between RSV and a host cell during viral entry. The F protein undergoes a conformation change from a "pre-fusion" to a "post-fusion" state during virus entry. (See FIG. 11). Some neutralizing antibodies against RSV have been shown to bind to a site called (O), which is present only in the pre-fusion form of F protein. Recombinant stabilized pre-fusion forms of F protein have been disclosed in the art and have been shown to be more effective at inducing neutralizing antibodies. (See Steff et al., Nat. Commun., 8:1085 (2017), pages 1-10; Blais et al., J. Virol. 91, e02437-16 (2017); McLellan et al., Science 342, 593-598 (2013); Krarup et al., Nat. Commun. 6, 8143 (2015); and Joyce et al., Nat. Struct. Mol. Biol. 23, 611-820 (2016); the contents of which are incorporated herein by reference in their entireties. Pre-fusion stabilized forms of F protein that are disclosed in the art include forms called DS-Cav1, DS-TriC, Cav-1-TriC, and DS-Cav1-TriC (see McLellan et al., Science 342, 593-598 (2013)), Pre-F-GCN4t (see Blais et al., J. Virol. 91, e02437-16 (2017)), and SC-DM, and SC-TM Krarup et al., Nat. Commun. 6, 8143 (2015)).

[0025] The disclosed compositions may include an effective amount of respiratory syncytial virus (RSV) M protein incorporated into biodegradable polyanhydride polymer particles. The M protein of RSV is a core protein that may be important for inducing a cell-mediated response against RSV infection.

[0026] Optionally, the disclosed compositions may include an effective amount of other respiratory syncytial virus (RSV) proteins incorporated into biodegradable polyanhydride polymer particles. Other proteins or RSV may include one or more of the RSV proteins selected from NS1, NS2, N, P, SH, G, M2-1, M2-2, L, or any combinations thereof.

[0027] As used herein, the phrase "effective amount" shall mean that dosage that provides the specific immunological response for which a composition comprising that effective amount is administered in a significant number of subjects. For example, an effective amount of an antigen may include that amount which when administered to a vaccinee induces an immune response in the vaccinee, preferably a protective immune response against the pathogen from which the antigen is derived. An effective amount of an antigen that is administered to a particular patient in a particular instance will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art.

[0028] The compositions disclosed herein may be formulated as vaccine compositions for administration to a subject in need thereof. Such compositions can be formulated and/or administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the route of administration.

[0029] The compositions may include pharmaceutical solutions comprising carriers, diluents, excipients, and surfactants as known in the art. Further, the compositions may include preservatives. The compositions also may include buffering agents.

[0030] The disclosed compositions typically include biodegradable particles. The biodegradable particles typically have an average effective diameter of less than about 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, or 0.01 .mu.M, or the biodegradable particles have an average effective diameter within a range bounded by any of these values (e.g., 0.1-2 .mu.M).

[0031] In some embodiments, the disclosed particles may be phagocytosed by antigen presenting cells, such as macrophage and dendritic cells, when the disclosed particles are administered as an immunogenic composition or vaccine formulation to a subject in need thereof. Preferably, the disclosed particles have an effective average diameter to permit phagocytosis by antigen presenting cells. Particles larger than about 5 microns are unlikely to be phagocytosed by antigen presenting cells and preferably the particles have an effective average diameter of less than about 4 microns or more preferably the particles have an effective average diameter of less than about 3 microns.

[0032] The disclosed particles typically are biodegradable as would be understood in the art. The term "biodegradable" describes a material that is capable of being degraded in a physiological environment into smaller basic components by biochemical reactions and/or physical reactions. The term "biodegradable" may be used herein interchangeably with the term "bioerodible." Preferably, the biodegradable particles are degraded (or eroded) into smaller basic components are innocuous. For example, a biodegradable polymer may be degraded into basic components that include, but are not limited to, water, carbon dioxide, sugars, organic acids (e.g., tricarboxylic or amino acids), and alcohols (e.g., glycerol or polyethylene glycol).

[0033] The disclosed compositions typically include biodegradable polyanhydride polymer particles which may include particles comprising or formed from homopolymers or copolymers. Biodegradable polyanhydride homopolymer and copolymer particles are known in the art. (See, e.g., U.S. Pat. Nos. 8,173,104 and 7,858,093, the contents of which are incorporated herein by reference in their entireties). In some embodiments, the biodegradable polyanhydride polymer particles comprise a polymer formed from a 1,.omega.-bis(p-carboxyphenoxy)(C.sub.2-C.sub.12)alkane, a 1,.omega.-bis(p-carboxyphenoxy)(C.sub.2-C.sub.12)dioxa-alkane, and a (C.sub.5-C.sub.20)alkanoic diacid. In particular, the biodegradable polyanhydride polymer particles may comprise a polymer formed from 1,6-bis(p-carboxyphenoxy)hexane (CPH), 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctance (CPTEG), and sebacic acid (SA). Where the biodegradable polyanhydride polymer particles are comprised of a polymer formed from CPH and CPTEG, the ratio of CPH and CPTEG may be modulated to prepare particles having a suitable release profile. In some embodiments, the biodegradable polyanhydride polymer particles comprise a polymer formed from CPH and CPTEG at a ratio CPH:CPTEG selected from 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 90:10, or within a range bounded by any of these ratios (e.g., 80-60:20-40 CPH:CPTEG). In some embodiments, the biodegradable polyanhydride polymer particles may be formulated to enhance uptake by and activation of dendritic cells. (See Carrillo-Conde et al., Acta Biomaterialia 8 (2012) 3618-3628; the content of which is incorporated herein by reference in its entirety)

[0034] Other biodegradable materials that may be utilized to prepare the particles contemplated herein may include materials disclosed in one or more of U.S. Pat. Nos. 7,470,283; 7,390,333; 7,128,755; 7,094,260; 6,830,747; 6,709,452; 6,699,272; 6,527,801; 5,980,551; 5,788,979; 5,766,710; 5,670,161; and 5,443,458; and U.S. Published Application Nos. 20090319041; 20090299465; 20090232863; 20090192588; 20090182415; 20090182404; 20090171455; 20090149568; 20090117039; 20090110713; 20090105352; 20090082853; 20090081270; 20090004243; 20080249633; 20080243240; 20080233169; 20080233168; 20080220048; 20080154351; 20080152690; 20080119927; 20080103583; 20080091262; 20080071357; 20080069858; 20080051880; 20080008735; 20070298066; 20070288088; 20070287987; 20070281117; 20070275033; 20070264307; 20070237803; 20070224247; 20070224244; 20070224234; 20070219626; 20070203564; 20070196423; 20070141100; 20070129793; 20070129790; 20070123973; 20070106371; 20070050018; 20070043434; 20070043433; 20070014831; 20070005130; 20060287710; 20060286138; 20060264531; 20060198868; 20060193892; 20060147491; 20060051394; 20060018948; 20060009839; 20060002979; 20050283224; 20050278015; 20050267565; 20050232971; 20050177246; 20050169968; 20050019404; 20050010280; 20040260386; 20040230316; 20030153972; 20030153971; 20030144730; 20030118692; 20030109647; 20030105518; 20030105245; 20030097173; 20030045924; 20030027940; 20020183830; 20020143388; 20020082610; and 0020019661; the contents of which are incorporated herein by reference in their entireties.

[0035] Typically, the biodegradable particles disclosed herein are degraded in vivo at a degradation rate such that the particles lose no more than about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of their initial mass (or within a range bounded by any of these values) after about 1, 2, 3, 4, 5, 6, 7, or 8 weeks or more post-administration (or within a range bounded by any of these values). The particles may comprise or may be formed from polymeric or non-polymeric biodegradable material. If the particles comprise polymeric material, typically the particles are degraded into biodegradable monomers. If the particles comprise non-polymeric material, typically the particles are degraded into biodegradable components.

[0036] The disclosed biodegradable particles may be prepared by methods known in the art including, but not limited to, spray-drying, precipitation, and grinding. In some embodiments, the biodegradable particles may be formed from a solution or suspension of a biodegradable material optionally in the presence of one or more additional agents such as adjuvants, apoptosis inhibitors, and/or antigens (e.g., by spray-drying the solution or suspension). As such, the biodegradable particles may comprise biodegradable material and optionally may comprise one or more additional agents such as adjuvants, apoptosis inhibitors, and/or antigens.

[0037] The disclosed biodegradable particles may be administered by various routes in order to induce a response in a subject. Routes of administration may include, but are not limited to, intranasal, pulmonary, oral, subcutaneous, intramuscular, and intravenous.

[0038] In some embodiments, the disclosed methods comprise administering a composition comprising biodegradable particles to induce an immune response in the subject. In other embodiments, the disclosed methods consist of administering a composition consisting of biodegradable particles to induce an immune response in the subject. The induced immune response may include an antibody response, a Th1 cell response and a CD8 CTL response.

[0039] The compositions disclosed herein optionally include an adjuvant. The term "adjuvant" refers to a compound or mixture that enhances an immune response. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response. Examples of adjuvants which may be utilized in the disclosed compositions include but are not limited to, co-polymer adjuvants (e.g., Pluronic L1219 brand poloxamer 401, CRL1005, or a low molecular weight co-polymer adjuvant such as Polygen.RTM. adjuvant), poly (I:C), R-848 (a Th1-like adjuvant), resiquimod, imiquimod, PAM3CYS, aluminum phosphates (e.g., AlPO.sub.4), loxoribine, potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum, CpG oligodeoxynucleotides (ODN), cholera toxin derived antigens (e.g., CTA1-DD), lipopolysaccharide adjuvants, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin (e.g., Quil-A), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions in water (e.g., MF59 available from Novartis Vaccines or Montanide ISA 720), keyhole limpet hemocyanins, and dinitrophenol.

[0040] The compositions disclosed herein may include pharmaceutical compositions that are administered as vaccines. Typically, the pharmaceutical composition comprises an effective amount or concentration of an antigen for inducing a protective or therapeutic immune response against a disease, which may include, but is not limited to infection by a pathogen such as RSV. Inducing a protective or therapeutic immune response may include inducing an antibody response, as well as a CD4 and/or CD8 T cell immune response to one or more epitopes of a protein associated with a pathogen (e.g., a protein associated with RSV). Inducing a protective or therapeutic immune response may include inducing an antibody response, as well as a Th1 response and/or a CD8 T cell response to one or more epitopes of a protein associated with the pathogen. As utilized herein, a Th1-response may be characterized by cytokine production such as interferons (e.g., IFN-.gamma.), tumor necrosis factor (e.g., TNF), and interleukins (e.g., IL-2). A Th1-response also may be characterized by increased killing efficiency of macrophages with respect to a pathogen and the proliferation of cytotoxic CD8.sup.+ cells against the pathogen. A Th1 response also may be characterized by the presence of opsonizing antibodies against the antigen.

[0041] Inducing a protective response may include inducing immunity against the pathogen, and in some embodiments, inducing protective immunity and/or sterilizing immunity against the pathogen. Inducing a therapeutic response may include reducing the pathogenic load of a subject, for example, as determined by measuring the amount of circulating pathogen before and after administering the composition. Inducing a therapeutic response may include reducing the degree or severity of at least one symptom of infection by the pathogen.

[0042] The presently disclosed methods may be utilized for inducing a protective or therapeutic immune response against disease by administering the pharmaceutical compositions disclosed herein (e.g., as immunogenic compositions or vaccines) to a subject in need thereof, which may include a human or non-human having or at risk for acquiring the disease. The methods may include administering a first pharmaceutical composition and optionally may include administering a second pharmaceutical composition to augment or boost an immunogenic response induced by the first pharmaceutical composition. The first and second pharmaceutical compositions may be the same or different. The optionally administered second pharmaceutical composition may be administered prior to, concurrently with, or after administering the first pharmaceutical composition. In some embodiments, the first composition is administered and then the second composition is administered after waiting at least about 1, 2, 3, 4, 5, or 6 weeks. The first composition (and the second composition) may be administered one or more times.

[0043] The presently disclosed compositions, kits, and methods may be utilized to protect against or treat infection by a pathogen. As used herein, a "pathogen" includes, but is not limited to a living microorganism such as bacteria, viruses, and fungi that cause disease in a host. As used herein, a "pathogen" includes respiratory syncytial virus (RSV).

[0044] The presently disclosed composition may be administered to potentiate or enhance an immune response. As used herein, "potentiating" or "enhancing" an immune response means increasing the magnitude and/or the breadth of the immune response. For example, the number of cells that recognize a particular epitope may be increased ("magnitude") and/or the numbers of epitopes that are recognized may be increased ("breadth"). Preferably, an enhanced antibody response as well as an enhancement in CD4 and/or CD8 T-cell responses may be obtained by administering the pharmaceutical composition disclosed herein.

ILLUSTRATIVE EMBODIMENTS

[0045] The following Embodiments are illustrative and should not be interpreted to limit the scope of the claimed subject matter.

Embodiment 1

[0046] A vaccine composition comprising an effective amount of respiratory syncytial virus (RSV) F protein in a pre-fusion stabilized form and/or M protein incorporated into biodegradable polyanhydride polymer particles for inducing an immune response against RSV.

Embodiment 2

[0047] The vaccine composition of embodiment 1, comprising an effective amount of RSV F protein in the pre-fusion stabilized form incorporated into the biodegradable polyanhydride polymer particles for inducing an immune response against RSV.

Embodiment 3

[0048] The vaccine composition of embodiment 1 or 2, wherein the F protein in a pre-fusion stabilized form is selected from the group consisting of DS-Cav1, DS-TriC, Vav-1-TriC, DX-Cav1-TriC, Pre-F-GCN4t, SC-DM, and SC-TM.

Embodiment 4

[0049] The vaccine composition of any of the foregoing embodiments, wherein the F protein in a pre-fusion stabilized form is DS-Cav1.

Embodiment 5

[0050] The vaccine composition of embodiment 1, comprising an effective amount of RSV M protein incorporated into the biodegradable polyanhydride polymer particles for inducing an immune response against RSV.

Embodiment 6

[0051] The vaccine composition of any of the foregoing embodiments, further comprising an adjuvant, optionally wherein the adjuvant is incorporated into the biodegradable polyanhydride polymer particles.

Embodiment 7

[0052] The vaccine composition of any of the foregoing embodiments, further comprising a CpG oligonucleotide, optionally wherein the CpG oligonucleotide is incorporated into the biodegradable polyanhydride polymer particles.

Embodiment 8

[0053] The vaccine composition of any of the foregoing embodiments, further comprising a CpG oligodeoxynucleotide (ODN), optionally wherein the CpG ODN is incorporated into the biodegradable polyanhydride polymer particles.

Embodiment 9

[0054] The vaccine composition of any of the foregoing embodiments, wherein the particles have an average effective diameter of less than about 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, or 0.01 .mu.M, or have an average effective diameter within a range bounded by any of these values (e.g., 2-0.1 .mu.M).

Embodiment 10

[0055] The vaccine composition of any of the foregoing embodiments, wherein the vaccine composition induces an antibody response as well as a CD4 T cell response (e.g., a Th1-type response), a CD8 T cell response, or a combination thereof.

Embodiment 11

[0056] The vaccine composition of any of the foregoing embodiments, wherein biodegradable polyanhydride polymer particles comprise a polymer formed from a 1,.omega.-bis(p-carboxyphenoxy)(C.sub.2-C.sub.12)alkane, a 1,.omega.-bis(p-carboxyphenoxy)(C.sub.2-C.sub.12)dioxa-alkane, and a (C.sub.5-C.sub.20)alkanoic diacid.

Embodiment 12

[0057] The vaccine composition of any of the foregoing embodiments, wherein the biodegradable polyanhydride polymer particles comprise a polymer formed from 1,6-bis(p-carboxyphenoxy)hexane (CPH), 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctance (CPTEG), and sebacic acid (SA).

Embodiment 13

[0058] The vaccine composition of any of the foregoing embodiments, wherein the biodegradable polyanhydride polymer particles comprise a polymer formed from 1,6-bis(p-carboxyphenoxy)hexane (CPH), 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctance (CPTEG), and sebacic acid (SA), wherein the polymer is formed from CPH and CPTEG at a ratio CPH:CPTEG selected from 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 90:10, or within a range bounded by any of these ratios (e.g., 80-60:20-40 CPH:CPTEG).

Embodiment 14

[0059] The vaccine composition of any of the foregoing embodiments, wherein the biodegradable polyanhydride polymer particles comprise a polymer formed from 1,6-bis(p-carboxyphenoxy)hexane (CPH), 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctance (CPTEG), and sebacic acid (SA), wherein the polymer is formed from CPH and CPTEG at a ratio CPH:CPTEG of about 80:20.

Embodiment 15

[0060] The vaccine composition of any of the foregoing embodiments, further comprising an RSV protein selected from NS1, NS2, N, P, SH, G, M2-1, M2-2, L, or any combinations thereof.

Embodiment 16

[0061] A method comprising administering any of the foregoing vaccine compositions to a subject who is at risk for infection by RSV.

Embodiment 17

[0062] The method of embodiment 16, wherein after the vaccine composition is administered to the subject, the subject is protected against infection by RSV.

Embodiment 18

[0063] The method of embodiment 16 or 17, wherein the vaccine composition is administered by a route selected from intranasal, pulmonary, oral, subcutaneous, intramuscular, or intravenous.

EXAMPLES

[0064] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.

[0065] Title--Evaluation of a Polyanhydride-Based Nanoparticle Vaccine Utilizing RSV M and/or Prefusion F

[0066] Reference is made to the poster presentation entitled "Evaluation of a polyanhydride-based nanoparticle vaccine utilizing RSV M and/or prefusion F," presented at the 11th International Respiratory Syncytial Virus Symposium, Oct. 31-Nov. 4, 2018, which is incorporated herein by reference in its entirety.

[0067] Abstract

[0068] Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in young children, resulting in 34 million new RSV infections each year, and approximately 125,000 hospitalizations annually in the United States alone. RSV reinfection is common in children, and even adults can be susceptible to repeated infection due to short-lived and incomplete protective immunity following natural infection. Despite the critical need, there is currently no licensed vaccine for RSV. Here we developed a polyanhydride nanoparticle-based vaccine utilizing the RSV matrix (M) protein and/or a prefusion-stabilized variant of the RSV fusion (F) protein (DS-Cav1). Our nanoparticle formulation offers several advantages over current vaccine strategies as it allows for continual, controlled release of the antigen and has been shown to induce robust T and B cell responses when paired with antigens from other pathogens. Inclusion of the prefusion conformation of RSV F within the vaccine allows for better exposure of the major RSV F antigenic site, termed site 0, while the RSV M protein contains epitopes that are known targets of both CD4 and CD8 T cell responses. The RSV nanovaccine is composed of 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxoctane (CPTEG):1,6-bis(p-carboxyphenoxy)hexane (CPH) copolymer nanoparticles encapsulating the M and DS-Cav1 prefusion F proteins with CpG 1668 ODN. Additional formulations include RSV F in its postfusion conformation with CpG, and DS-Cav1 protein and CpG. Controls consist of nanoparticles containing either pre-F or postfusion RSV F without CpG, and nanoparticles loaded with CpG alone. Here we evaluated the immunogenicity and the protective capacity of our nanoparticle vaccine strategy against a primary RSV infection.

[0069] Methods

[0070] BALB/c mice were vaccinated intranasally with 500 .mu.g of the nanoparticles in PBS administered at day 0 and subsequently boosted intranasally at day 28. (See FIG. 12). At day 56 the protective capacity of the nanoparticles was assessed by determining viral titers in the lungs of vaccinated mice following an RSV challenge. Using whole body plethysmography, we also measured airway function to determine the capacity of the RSV nanovaccine to protect against RSV-induced pulmonary injury. Lungs were harvested post-prime or post-boost to evaluate the T and B cell responses elicited by the nanoparticle vaccination.

[0071] Results

[0072] The inventors are using the M+prefusion RSV F nanoparticle vaccine to determine the optimal vaccine regimen to provide protection against RSV-induced disease. Additionally, the inventors are investigating the ideal mixture of humoral and cell-mediated immunity necessary to establish long-term protective immunity against RSV. Information from these studies will be critical to establish optimal thresholds for protective immunity against RSV.

[0073] The results in FIG. 1 illustrate that a prime/boost nanoparticle vaccination strategy with prefusion RSV F protects against RSV-induced disease. BALB/c mice were primed with 500 .mu.g of the indicated nanoparticle formulation i.n. on day 0, and boosted with 500 .mu.g i.n. on day 28. Control mice (labeled RSV) were administered PBS i.n. on both prime and boost days. On day 56, all mice were challenged with 4.8.times.10.sup.6 PFU RSV-A2 i.n. and assessed for weight loss, Penh, and EF50.

[0074] FIG. 2 illustrates that prime/boost nanoparticle vaccination with prefusion RSV F reduces RSV N gene viral copy numbers. BALB/c mice were primed with 500 .mu.g of the indicated nanoparticle formulation i.n. on day 0, and boosted with 500 .mu.g i.n. on day 28. Control mice (labeled RSV) were administered PBS i.n. on both prime and boost days. On day 56, all mice were challenged with 4.8.times.10.sup.6 PFU RSV-A2 i.n. 4 days post-challenge lungs were harvested for RNA. RT-PCR was performed to determine RSV N gene copy numbers.

[0075] FIG. 3 illustrates that initial priming vaccination with prefusion RSV F induces lung-resident CD4 and CD8 T cells. BALB/c mice were primed with 500 .mu.g of the indicated nanoparticle formulation or PBS (naive) i.n. on day 0. Lungs and spleen were harvested on day 8 and analyzed by flow cytometry. The frequency of activated (CD11a.sup.hiCD49d.sup.+) CD4 T cells and activated (CD11a.sup.hiCD8.sup.lo) CD8 T cells was assessed as indicated in FIGS. 3A and 3B. The number of i.v..sup.-CD4 and i.v..sup.-CD8 T cells was assessed as indicated in FIGS. 3C and 3D. The number of activated i.v..sup.- CD103.sup.-CD69.sup.+CD4 T cells and CD103.sup.+CD69.sup.+CD8 T cells was assessed as indicated on FIGS. 3E and 3F. The representative flow cytometry plots of germinal center B cells (CD19.sup.+CDB220.sup.+Fas.sup.+GL-7.sup.+) is illustrated in FIG. 3G.

[0076] FIG. 4 illustrates that a prime/boost nanoparticle vaccination induces RSV-specific antibodies in serum. BALB/c mice were primed with 500 .mu.g of the indicated nanoparticle formulation i.n. on day 0, and boosted with 500 .mu.g i.n. on day 28. Control mice (labeled naive) were administered PBS i.n. on both prime and boost days. On day 14 and 56 serum was assessed for total RSV-specific IgG, IgG1, or IgG2a.

[0077] FIG. 5 illustrates that a prime/boost nanoparticle vaccination with prefusion RSV F induces lung-resident B cells, CD4, and CD8 T cells. BALB/c mice were primed with 500 .mu.g of the indicated nanoparticle formulation or PBS (naive) i.n. on day 0, and boosted with 500 .mu.g i.n. on day 28. Lung and spleen were harvested on day 42 and analyzed by flow cytometry. We assessed the number of i.v..sup.- B cells, the number of activated i.v..sup.- CD103.sup.-CD69.sup.+CD4 T cells and CD103.sup.+CD69.sup.+CD8 T cells. We also assessed the number of activated IFN-.gamma..sup.+CD4 T cells and IL-5.sup.+CD4 T cells following stimulation with PMA/ionomycin.

CONCLUSION

[0078] Prime/boost nanoparticle vaccination with a prefusion RSV F nanoparticle-based vaccine reduced weight loss, pulmonary dysfunction, and viral copy numbers following an RSV challenge. Priming vaccination alone was shown to be sufficient to elicit i.v.sup.- tissue-resident CD4 and CD8 T cells. Prime/boost nanoparticle vaccination generated RSV-specific antibodies in serum, and prime/boost vaccination elicited IFN-.gamma..sup.+CD4 T cells and tissue-resident T and B cells.

[0079] There are numerous benefits to the RSV vaccine as prepared by the inventors. First, the inventors' nanoparticle formulation allows for continual, controlled release of the antigen(s). Second, the inventors' nanoparticle formulation includes the F protein in a pre-fusion stabilized form, which allows for better exposure of the major RSV F antigenic site (0) to a vaccinee's immune system. Third, the inventors' nanoparticle formulation induces robust B and T cell responses due to inclusion of both of the F protein in a pre-fusion stabilized form and the M protein.

Example 2--Protection Against RSV-Induced Disease by Nanoparticle Formulation Containing RSV Pre-F Protein and CpG Adjuvant

[0080] Our data demonstrate that a single prime only immunization with the nanoparticle formulation containing RSV pre-F+CpG adjuvant is sufficient to provide protection against RSV-induced disease including weight loss and airway dysfunction (e.g Penh) as compared to unimmunized control mice or mice immunized with nanoparticles containing the CpG adjuvant only (without the PreF antigen). (See FIG. 6). Prime only immunization is also sufficient to significantly reduce virus replication in the lung as compared to unimmunized mice or mice immunized with nanoparticles containing the CpG adjuvant only (without the PreF antigen). Mice immunized using a prime/boost immunization approach also exhibit significant protection against RSV-induced disease including weight loss and airway dysfunction (e.g Penh) as compared to unimmunized mice when challenged either at 56 days post-prime (see FIGS. 6C and 6D) or Day 100 post-prime (see FIGS. 6E and 6F).

[0081] Mice that received a prime/boost immunization exhibited significantly reduced virus replication in the lung as compared to unimmunized mice or mice immunized with nanoparticles containing the CpG adjuvant only (without the PreF antigen) as shown in FIG. 7A (day 56 challenge) or FIG. 7B (day 100 challenge).

[0082] Our data indicate that prime/boost vaccination with PreF+CpG nanoparticles elicits an increase in activated CD4 and CD8 T cells as compared to mice immunized with nanoparticles containing only CpG or unimmunized mice (FIGS. 8A and 8B). This data is consistent with the induction of a T cell response following the nanoparticle vaccination.

[0083] Additional data indicate that the activated CD4 and CD8 T cells exhibit phenotypic cell surface markers (CD69.sup.+ for CD4 T cells and CD69.sup.+CD103.sup.+ for CD8 T cells) that is consistent with a tissue-resident population (e.g. Trm) that may be important in providing protection (FIGS. 9A and 9B). In addition, the activated CD4 and CD8 T cells make IFN-.gamma. following stimulation indicating that they are of a Th1 and Tc1 phenotype, respectively (FIGS. 9C and 9D).

[0084] Using MHC-class I tetramers to identify RSV-specific CD8 T cells, we observed a significant increase in the total number of RSV tetramer-specific CD8 T cells specific to the F85-93 CD8 T cell epitope 2 weeks following the boost immunization (eg day 42 post-prime) as compared to unimmunized mice or mice immunized with nanoparticles containing the CpG adjuvant only (without the PreF antigen) as shown in FIG. 9E. FIG. 9F shows that these RSV tetramer-staining cells are located in the lung tissue.

[0085] FIG. 10 represents additional data demonstrating that mice immunized using a prime/boost immunization approach exhibit significant protection against RSV-induced airway dysfunction (e.g EF50) as compared to unimmunized mice when challenged either at 56 days post-prime (FIG. 5A) or at 100 days post-prime (FIG. 5B). FIG. 5C demonstrates additional data showing that mice immunized using a prime/boost immunization approach using PreF+CpG nanoparticles exhibit significant protection against RSV replication as measured by RT-PCR for the RSV N gene as compared to mice immunized with nanoparticles containing only CpG or unimmunized mice.

[0086] It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0087] Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.

Sequence CWU 1

1

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aaaacacaac aacaacccaa 5100atattaccta gcaaacccac cacaaaacaa cgccaaaata aaccacaaaa caaacccaac 5160aatgattttc actttgaagt gttcaatttt gtaccctgca gcatatgcag caacaatcca 5220acctgctggg ccatctgcaa gagaatacca aacaaaaaac ctggaaagaa aaccaccacc 5280aagcccacaa aaaaaccaac cctcaagaca accaaaaaag atcccaaacc tcaaaccaca 5340aaaccaaagg aagtactcac taccaagcct acaggaaagc caaccatcaa caccactaaa 5400acaaacatca gaactacact gctcacctcc aacaccaaag gaaatccaga acacacaagt 5460caagaggaaa ccctccactc aaccacctcc gaaggctatc taagcccatc ccaagtctat 5520acaacatccg gtcaagagga aaccctccac tcaaccacct ccgaaggcta tctaagccca 5580tcacaagtct atacaacatc cgagtaccta tcacaatctc tatcttcatc caacacaaca 5640aaatgatagt cattaaaaag cgtattgttg caaaaagcca tgaccaaatc aaacagaatc 5700aaaatcaact ctggggcaaa taacaatgga gttgccaatc ctcaaaacaa atgctattac 5760cacaatcctt gctgcagtca cactctgttt cgcttccagt caaaacatca ctgaagaatt 5820ttatcaatca acatgcagtg cagttagcaa aggctatctt agtgctctaa gaactggttg 5880gtatactagt gttataacta tagaattaag taatatcaag gaaaataagt gtaatggtac 5940agacgctaag gtaaaattaa taaaacaaga attagataaa tataaaaatg ctgtaacaga 6000attgcagttg ctcatgcaaa gcacaccagc agccaacagt cgagccagaa gagaactacc 6060aagatttatg aattatacac tcaacaatac caaaaacacc aatgtaacat taagtaagaa 6120aaggaaaaga agatttcttg gatttttgtt aggtgttgga tctgcaatcg ccagtggcat 6180tgccgtatcc aaggtcctgc acctagaagg ggaagtgaac aaaatcaaaa gtgctctact 6240atccacaaac aaggctgtag tcagcttatc taatggagtc agtgtcttaa ccagcaaggt 6300gttagacctc aaaaactata tagataaaca gttgttacct attgttaaca agcaaagctg 6360cagcatatca aacattgaaa ctgtgataga gttccaacaa aagaacaaca gactactaga 6420gattaccaga gaatttagtg ttaatgcagg tgtaactaca cctgtaagca cttatatgtt 6480aactaatagt gagttattat cattaatcaa tgatatgcct ataacaaatg atcagaaaaa 6540gttaatgtcc agcaatgttc aaatagttag acagcaaagt tactctatca tgtcaataat 6600aaaagaggaa gtcttagcat atgtagtaca attaccacta tatggtgtaa tagatactcc 6660ttgttggaaa ctacacacat ctcctctatg tacaaccaac acaaaggaag gatccaacat 6720ctgcttaaca agaaccgaca gaggatggta ctgtgacaat gcaggatcag tatccttttt 6780cccacaagct gaaacatgta aagttcaatc gaatcgggtg ttttgtgaca caatgaacag 6840tttaacatta ccaagtgagg taaatctctg caacattgac atattcaacc ccaaatatga 6900ttgcaaaatt atgacttcaa aaacagatgt aagcagctcc gttatcacat ctctaggagc 6960cattgtgtca tgctatggca aaaccaaatg tacagcatcc aataaaaatc gtgggatcat 7020aaagacattc tctaacgggt gtgattatgt atcaaataag ggggtggata ctgtgtctgt 7080aggtaataca ttatattatg taaataagca agaaggcaaa agtctctatg taaaaggtga 7140accaataata aatttctatg atccattagt gttcccctct gatgaatttg atgcatcaat 7200atctcaagtc aatgagaaaa ttaatcagag tctagcattt atccgtaaat cagatgaatt 7260attacataat gtaaatgctg gtaaatccac cacaaatatc atgataacta ccataattat 7320agtaattata gtaatattgt tagcattaat tgcagttgga ctgcttctat actgcaaggc 7380cagaagcaca ccagtcacat taggtaagga tcaactgagt ggtataaata atattgcatt 7440taataactga ataaaaatag cacctaatca tattcttaca atggttcgct atttgaccat 7500agataaccca tctatcatta gattatccta aaatttgaac ttcatcacaa ctttcatcta 7560taaaccatct cacttacact ttttaagtag attcctattt tatagttata taaaacaatt 7620gaataccaaa ttaacttact atttgtaaaa atgagaactg gggcaaatat gtcacgaagg 7680aatccttgca aattcgaaat tcgaggtcat tgcttgaatg gtaaaaggtg tcattttagt 7740cataattatt ttgaatggcc accccatgca ctgcttgtaa gacaaaactt tatgttaaac 7800agaatactta agtctatgga taaaagcata gatactttgt cagaaataag tggagctgca 7860gagttggaca gaacagaaga gtatgccctc ggtgtagttg gagtgctaga gagttatata 7920ggatcaataa ataatataac taaacaatca gcatgtgttg ccatgagcaa actccttact 7980gaactcaaca gcgatgacat caaaaaacta agggacaatg aagagccaaa ctcacccaaa 8040gtaagagtgt acaatactgt catatcatat attgaaagca acaggaagaa caataaacaa 8100actatccatc tgttaaaaag attgccagca gacgtattga agaaaaccat caaaaacaca 8160ttggatatcc acaagagcat aaccatcaat aacccaaaag aatcaactgt tagtgatacg 8220aacgaccatg ccaaaaataa tgatactacc tgacaaatat ccttgtagta taaattccat 8280actaataaca agtaattgta gagtcactat gtataatcaa aaaaacacac tatatatcaa 8340tcaaaacaac caaaataacc atatataccc accggatcaa ccattcaatg aaatccattg 8400gacctctcaa gacttgattg atgcaactca aaattttcta caacatctag gtattactga 8460tgatatatac acaatatata tattagtgtc ataatactca atcctaatac ttaccacatc 8520atcaaattat taactcaaac aattcaagct atgggacaaa atggatccca ttattagtgg 8580aaattctgct aatgtttatc taactgatag ttatttaaaa ggtgttattt ctttctcaga 8640atgtaacgct ttaggaagtt acatattcaa tggtccttat ctcaaaaatg attataccaa 8700cttaattagt agacaaaatc cattaataga acacataaat ctaaagaaac taaatataac 8760acagtcctta atatctaagt atcataaagg tgaaataaaa atagaagaac ctacttactt 8820tcagtcatta cttatgacat acaagagtat gacctcgtca gaacagacta ctactactaa 8880tttacttaaa aagataataa gaagagctat agaaatcagt gatgtcaaag tctatgctat 8940attgaataaa ctggggctca aagaaaaaga caagattaaa tccaataatg gacaagatga 9000agacaactca gtcattacta ccataatcaa agatgatata cttttagctg tcaaggataa 9060tcaatctcat cttaaagcag acaaaaatca atccacaaaa caaaaagata caatcaaaac 9120aacacttttg aagaaattaa tgtgttcaat gcaacatcct ccatcatggt taatacattg 9180gtttaattta tacacaaaat taaacagcat attaacacaa tatcgatcta gtgaggtaaa 9240aaaccatggt tttatattga tagataatca tactcttagt ggattccaat ttattttgaa 9300tcaatatggt tgtatagttt atcataagga actcaaaaga attactgtga caacttataa 9360tcaattcttg acatggaaag atattagcct tagtagatta aatgtttgtt tgattacatg 9420gattagtaac tgtttgaaca cattaaacaa aagcttaggc ttaagatgtg gattcaataa 9480tgttatcttg acacaattat tcctttatgg agattgtata ctaaaactat tccacaatga 9540ggggttctac ataataaaag agatagaggg atttattatg tctctaattt taaatataac 9600agaagaagat caattcagaa aacggtttta taatagtatg ctcaacaaca tcacagatgc 9660cgccaacaaa gctcaaaaaa atctgctatc aagagtatgt catacattat tagataagac 9720aatatcagat aatataataa atggcagatg gataattcta ttgagcaagt tcctaaaatt 9780aattaagctt gcaggtgaca ataacctcaa caatctgagt gaattatatt ttttgttcag 9840gatatttgga cacccaatgg tagatgaaag acaagccatg gatgctgtta aagttaattg 9900caacgagacc aaattttact tgttaagtag tttgagtatg ttaagaggag cttttatata 9960tagaattata aaagggtttg taaataatta caacagatgg cctactttaa gaaatgccat 10020tgtcttaccc ttaagatggt taacttacta taaactaaac acttatcctt ccttgttgga 10080acttacagaa agagatttga ttgttctatc aggactacgt ttctatcgag agtttcggtt 10140gcctaaaaaa gtggatcttg aaatgatcat aaatgataag gctatatcac ctcctaaaaa 10200tttaatatgg actagtttcc ctagaaatta tatgccgtca cacatacaaa attatataga 10260acatgaaaaa ttaaaattct ctgatagtga taaatcaaga agagtattag agtattattt 10320aagagataac aaattcaatg aatgtgattt acacaactgt gtagttaatc aaagttatct 10380taacaacccg aatcatgtgg tatcattgac aggcaaagaa agagaactca gtgtaggtag 10440aatgtttgca atgcaaccag gaatgttcag acaagttcaa atattagcag agaaaatgat 10500agcagaaaac atattacaat ttttccctga aagtcttaca agatatggtg atctagaact 10560acagaaaata ttagaattga aagcaggaat aagtaacaaa tcaaatcgtt acaatgataa 10620ttacaacaat tacattagta agtgctctat catcacagat ctcagcaaat tcaatcaagc 10680atttcgatat gaaacatcat gtatttgtag tgatgtactg gatgaactgc atggtgtaca 10740atctctattt tcctggttac atttaactat tcctcatgtc acaataatat gcacatatag 10800gcatgcaccc ccctatataa aggatcatat tgtagatctt aacaatgtag atgagcaaag 10860tggactatat agatatcata tgggtggtat cgaagggtgg tgtcaaaaac tatggactat 10920agaagctata tcactattag atctaatatc tctcaaaggg aaattctcaa ttactgcttt 10980aattaatggt gacaatcaat caatagatat aagtaaacca gtcagactca tggaaggtca 11040aactcatgct caagcagatt atttgctagc attaaatagt ctcaaattac tgtataaaga 11100gtatgcagga ataggccaca aattaaaagg aactgagact tatatatcga gagatatgca 11160atttatgagt aaaacgatcc aacataacgg tgtatattac ccagctagta taaagaaagt 11220cctaagagtg ggaccgtgga taaacactat acttgatgac ttcaaagtga gtctagaatc 11280tataggtagt ttgacacaag aattagaata tagaggtgaa agtctattat gcagtttaat 11340atttaggaat gtatggttat ataatcaaat tgcattacaa cttaaaaatc atgcattatg 11400taacaacaaa ttatatttgg atatattaaa agttctaaaa cacttaaaaa ccttttttaa 11460tcttgataac attgatacag cattaacatt gtatatgaat ttgcccatgt tatttggtgg 11520tggtgatccc aacttgttat atcgaagttt ctatagaaga actcctgatt tcctcacaga 11580ggctatagtt cactctgtgt tcatacttag ttattataca aaccatgatt taaaagataa 11640acttcaagat ctgtcagatg atagattgaa taagttctta acatgcataa tcacgtttga 11700caaaaacccc aatgctgaat tcgttacatt gatgagagat cctcaagctt taggatctga 11760gaggcaagct aaaattacta gcgaaatcaa tagactggca gttaccgagg ttttgagcac 11820agctccaaac aaaatatttt ccaaaagtgc acaacactat accactacag agatagatct 11880taatgatatt atgcaaaata tagaacctac atatcctcac gggttaagag ttgtttatga 11940aagtttaccc ttttataaag cagagaaaat agtaaatctt atatccggta caaaatctat 12000aactaacata ctggaaaaga cttctgccat agacttaaca gatattgata gagccactga 12060gatgatgagg aaaaacataa ctttgcttat aaggatatta ccattagatt gtaacagaga 12120taaaagagaa atattgagta tggaaaacct aagtattact gaattaagca aatacgttag 12180agaaagatct tggtctttat ccaatatagt tggtgttaca tcacccagta tcatgtatac 12240aatggacata aaatatacaa caagcactat agctagtggc ataatcatag agaaatataa 12300tgtcaacagt ttaacacgtg gtgagagagg acccactaaa ccatgggttg gttcatctac 12360acaagagaaa aagacaatgc cagtttataa tagacaagtt ttaaccaaaa aacagagaga 12420tcaaatagat ctattagcaa aattggattg ggtgtatgca tctatagata acaaggatga 12480atttatggag gaacttagca taggaactct tgggttaaca tatgagaagg ccaaaaaatt 12540attcccacaa tatttaagtg ttaactattt gcatcgtctt acagtcagta gtagaccatg 12600tgaattccct gcatctatac cagcttatag aactacaaat tatcactttg atactagccc 12660tattaatcgc atattaacag aaaagtatgg tgatgaagat attgatatag tattccaaaa 12720ctgtataagc tttggcctta gcttaatgtc tgtagtagaa caatttacta atgtgtgtcc 12780taacagaatt attctcatac ccaagcttaa tgagatacat ttgatgaaac ctcccatatt 12840cacaggtgat gttgatattc acaagttaaa acaagtgata caaaaacaac atatgttttt 12900accagacaaa ataagtttga ctcaatatgt ggaattattc ttaagtaata aaacactcaa 12960atctggatct aatgttaatt ctaatttaat attggcgcat aagatatctg actattttca 13020taatacttac attttaagta ctaatttagc tggacattgg attcttatta tacaacttat 13080gaaagattct aagggtattt ttgaaaaaga ttggggagag ggatatataa ctgatcatat 13140gttcattaat ttgaaagttt tcttcaatgc ttataagaca tatctcttgt gttttcataa 13200aggttacggc agagcaaagc tggagtgtga tatgaatact tcagatctcc tatgtgtatt 13260ggaattaata gacagtagtt attggaagtc tatgtctaag gtgtttttag aacaaaaagt 13320tatcaaatac attcttagcc aggatgcaag tttacataga gtaaaaggat gtcatagctt 13380caaactatgg tttcttaaac gtcttaatgt agcagaattc acggtttgcc cttgggttgt 13440taacatagat tatcatccaa cacatatgaa agcaatatta acttatattg atcttgttag 13500aatgggattg ataaatatag atagaatata cattaaaaat aaacacaagt tcaatgatga 13560gttttatact tctaatctgt tttacattaa ttataacttc tcagataata ctcatctatt 13620aactaaacat ataaggattg ctaattccga attagaaagt aattacaaca aattatatca 13680tcccacacca gaaaccctag aaaatatact aaccaatccg gttaaaagta atggaaaaaa 13740gacactgagt gactattgta taggtaaaaa tgttgactca ataatgttac catcgttatc 13800taataagaag cttattaaat cgtctacaat gattagaacc aattgcagca gacaagattt 13860gtataattta tttcctacgg ttgtgattga taaaattata gatcattcag gyaatacagc 13920caaatctaac caactttaca ctactacttc tcatcaaata tccttagtgc acaatagcac 13980atcactttat tgcatgcttc cttggcatca tattaataga ttcaattttg tatttagttc 14040tacaggttgt aaaattagta tagagtatat tttaaaagat cttaaaatta aggatcctaa 14100ttgtatagca ttcataggtg aaggagcagg gaatttatta ttgcgtacag tagtggaact 14160tcatcctgat ataagatata tttacagaag tctgaaagat tgcaatgatc atagtttacc 14220aattgagttt ttaaggctgt acaatggaca tatcaacatt gattatggtg aaaatttgac 14280cattcctgct acagatgcaa ccaacaacat tcattggtct tatttacata taaagtttgc 14340tgaacctatc agtctttttg tctgtgatgc tgaattgcct gtaacagtca actggagtaa 14400gattataata gagtggagca agcatgtaag aaaatgcaag tactgttctt cagttaataa 14460atgtacatta atagtaaaat atcatgctca agatgatatc gatttcaaat tagacaacat 14520aactatatta aaaacttatg tatgcttagg cagtaagtta aagggatctg aagtttactt 14580agtccttaca ataggtcctg caaatgtgtt cccagtattt aatgtagcac aaaatgctaa 14640attgatacta tcaagaacca aaaatttcat catgcctaaa aaagctgata aagagtctat 14700tgatgcaaat attaagagtt tgataccctt tctttgttac cctataacaa aaaaaggaat 14760taataccgca ttgtctaaat taaagagtgt tgttagtgga gatatactat catattctat 14820agctggacgt aatgaagttt tcagcaataa acttataaat cataagcata tgaacatctt 14880aaagtggttc aatcatgttt taaatttcag atcaacagaa ttaaactata atcatttata 14940tatggtagaa tctacttatc ctcatctaag tgaattgtta aacagcttga ctaccaatga 15000acttaaaaaa ctgattaaaa

tcacaggtag tttgttatac aacttttata atgaataatg 15060agcaaaaatc ttataacaaa aatagctaca cactaacatt gtattcaatt atagttattt 15120aaaattaata attatataat ttttaataac ttctagtgaa ctaatcctaa aattatcatt 15180ttgatctagg aagaataagt ttaaatccaa atctaattgg tttatatgta tattaactaa 15240attacgagat attagttttt gacacttttt ttctcgt 152772139PRTHuman Respiratory Syncytial Virus 2Met Gly Ser Asn Ser Leu Ser Met Ile Lys Val Arg Leu Gln Asn Leu1 5 10 15Phe Asp Asn Asp Glu Val Ala Leu Leu Lys Ile Thr Cys Tyr Thr Asp 20 25 30Lys Leu Ile Gln Leu Thr Asn Ala Leu Ala Lys Ala Val Ile His Thr 35 40 45Ile Lys Leu Asn Gly Ile Val Phe Val His Val Ile Thr Ser Ser Asp 50 55 60Ile Cys Pro Asn Asn Asn Ile Val Val Lys Ser Asn Phe Thr Thr Met65 70 75 80Pro Val Leu Gln Asn Gly Gly Tyr Ile Trp Glu Met Met Glu Leu Thr 85 90 95His Cys Ser Gln Pro Asn Gly Leu Ile Asp Asp Asn Cys Glu Ile Lys 100 105 110Phe Ser Lys Lys Leu Ser Asp Ser Thr Met Thr Asn Tyr Met Asn Gln 115 120 125Leu Ser Glu Leu Leu Gly Phe Asp Leu Asn Pro 130 1353124PRTHuman Respiratory Syncytial Virus 3Met Asp Thr Thr His Asn Asp Thr Thr Pro Gln Arg Leu Met Ile Thr1 5 10 15Asp Met Arg Pro Leu Ser Leu Glu Thr Ile Ile Thr Ser Leu Thr Arg 20 25 30Asp Ile Ile Thr His Lys Phe Ile Tyr Leu Ile Asn His Glu Cys Ile 35 40 45Val Arg Lys Leu Asp Glu Arg Gln Ala Thr Phe Thr Phe Leu Val Asn 50 55 60Tyr Glu Met Lys Leu Leu His Lys Val Gly Ser Thr Lys Tyr Lys Lys65 70 75 80Tyr Thr Glu Tyr Asn Thr Lys Tyr Gly Thr Phe Pro Met Pro Ile Phe 85 90 95Ile Asn His Asp Gly Phe Leu Glu Cys Ile Gly Ile Lys Pro Thr Lys 100 105 110His Thr Pro Ile Ile Tyr Lys Tyr Asp Leu Asn Pro 115 1204391PRTHuman Respiratory Syncytial Virus 4Met Ala Pro Ser Lys Val Lys Leu Asn Asp Thr Leu Asn Lys Asp Gln1 5 10 15Pro Pro Ser Ser Ser Lys Tyr Thr Ile Gln Arg Ser Thr Gly Asp Ser 20 25 30Thr Asp Thr Pro Asn Tyr Asp Val Gln Lys His Thr Asn Lys Leu Cys 35 40 45Gly Met Leu Leu Ile Thr Glu Asp Ala Asn His Lys Phe Thr Gly Leu 50 55 60Ile Gly Met Leu Tyr Ala Met Ser Arg Leu Gly Arg Glu Asp Thr Ile65 70 75 80Lys Ile Leu Lys Asp Ala Gly Tyr His Val Lys Ala Asn Gly Val Asp 85 90 95Val Thr Thr His Arg Gln Asp Ile Asn Gly Lys Glu Met Lys Phe Glu 100 105 110Val Leu Thr Leu Ala Ser Leu Thr Thr Glu Ile Gln Ile Asn Ile Glu 115 120 125Ile Glu Ser Arg Lys Ser Tyr Lys Lys Met Leu Lys Glu Met Gly Glu 130 135 140Val Ala Pro Glu Tyr Arg His Asp Ser Pro Asp Cys Gly Met Ile Ile145 150 155 160Leu Cys Ile Ala Ala Leu Val Ile Thr Lys Leu Ala Ala Gly Asp Arg 165 170 175Ser Gly Leu Thr Ala Val Ile Arg Arg Ala Asn Asn Val Leu Lys Asn 180 185 190Glu Met Lys Arg Tyr Lys Gly Leu Leu Pro Lys Asp Ile Ala Asn Ser 195 200 205Phe Tyr Glu Val Phe Glu Lys Tyr Pro His Phe Ile Asp Val Phe Val 210 215 220His Phe Gly Ile Ala Gln Ser Ser Thr Arg Gly Gly Ser Arg Val Glu225 230 235 240Gly Ile Phe Ala Gly Leu Phe Met Asn Ala Tyr Gly Ala Gly Gln Val 245 250 255Met Leu Arg Trp Gly Val Leu Ala Lys Ser Val Lys Asn Ile Met Leu 260 265 270Gly His Ala Ser Val Gln Ala Glu Met Glu Gln Val Val Glu Val Tyr 275 280 285Glu Tyr Ala Gln Lys Leu Gly Gly Glu Ala Gly Phe Tyr His Ile Leu 290 295 300Asn Asn Pro Lys Ala Ser Leu Leu Ser Leu Thr Gln Phe Pro His Phe305 310 315 320Ser Ser Val Val Leu Gly Asn Ala Ala Gly Leu Gly Ile Met Gly Glu 325 330 335Tyr Arg Gly Thr Pro Arg Asn Gln Asp Leu Tyr Asp Ala Ala Lys Ala 340 345 350Tyr Ala Glu Gln Leu Lys Glu Asn Gly Val Ile Asn Tyr Ser Val Leu 355 360 365Asp Leu Thr Ala Glu Glu Leu Glu Ala Ile Lys His Gln Leu Asn Pro 370 375 380Lys Asp Asn Asp Val Glu Leu385 3905241PRTHuman Respiratory Syncytial Virus 5Met Glu Lys Phe Ala Pro Glu Phe His Gly Glu Asp Ala Asn Asn Arg1 5 10 15Ala Thr Lys Phe Leu Glu Ser Ile Lys Gly Lys Phe Thr Ser Pro Lys 20 25 30Asp Pro Lys Lys Lys Asp Ser Ile Ile Ser Val Asn Ser Ile Asp Ile 35 40 45Glu Val Thr Lys Glu Ser Pro Ile Thr Ser Asn Ser Thr Ile Ile Asn 50 55 60Pro Ile Asn Glu Thr Asp Asp Thr Val Gly Asn Lys Pro Asn Tyr Gln65 70 75 80Arg Lys Pro Leu Val Ser Phe Lys Glu Asp Pro Thr Pro Ser Asp Asn 85 90 95Pro Phe Ser Lys Leu Tyr Lys Glu Thr Ile Glu Thr Phe Asp Asn Asn 100 105 110Glu Glu Glu Ser Ser Tyr Ser Tyr Glu Glu Ile Asn Asp Gln Thr Asn 115 120 125Asp Asn Ile Thr Ala Arg Leu Asp Arg Ile Asp Glu Lys Leu Ser Glu 130 135 140Ile Leu Gly Met Leu His Thr Leu Val Val Ala Ser Ala Gly Pro Thr145 150 155 160Ser Ala Arg Asp Gly Ile Arg Asp Ala Met Val Gly Leu Arg Glu Glu 165 170 175Met Ile Glu Lys Ile Arg Thr Glu Ala Leu Met Thr Asn Asp Arg Leu 180 185 190Glu Ala Met Ala Arg Leu Arg Asn Glu Glu Ser Glu Lys Met Ala Lys 195 200 205Asp Thr Ser Asp Glu Val Ser Leu Asn Pro Thr Ser Glu Lys Leu Asn 210 215 220Asn Leu Leu Glu Gly Asn Asp Ser Asp Asn Asp Leu Ser Leu Glu Asp225 230 235 240Phe6256PRTHuman Respiratory Syncytial Virus 6Met Glu Thr Tyr Val Asn Lys Leu His Glu Gly Ser Thr Tyr Thr Ala1 5 10 15Ala Val Gln Tyr Asn Val Leu Glu Lys Asp Asp Asp Pro Ala Ser Leu 20 25 30Thr Ile Trp Val Pro Met Phe Gln Ser Ser Met Pro Ala Asp Leu Leu 35 40 45Ile Lys Glu Leu Ala Asn Val Asn Ile Leu Val Lys Gln Ile Ser Thr 50 55 60Pro Lys Gly Pro Ser Leu Arg Val Met Ile Asn Ser Arg Ser Ala Val65 70 75 80Leu Ala Gln Met Pro Ser Lys Phe Thr Ile Cys Ala Asn Val Ser Leu 85 90 95Asp Glu Arg Ser Lys Leu Ala Tyr Asp Val Thr Thr Pro Cys Glu Ile 100 105 110Lys Ala Cys Ser Leu Thr Cys Leu Lys Ser Lys Asn Met Leu Thr Thr 115 120 125Val Lys Asp Leu Thr Met Lys Thr Leu Asn Pro Thr His Asp Ile Ile 130 135 140Ala Leu Cys Glu Phe Glu Asn Ile Val Thr Ser Lys Lys Val Ile Ile145 150 155 160Pro Thr Tyr Leu Arg Ser Ile Ser Val Arg Asn Lys Asp Leu Asn Thr 165 170 175Leu Glu Asn Ile Thr Thr Thr Glu Phe Lys Asn Ala Ile Thr Asn Ala 180 185 190Lys Ile Ile Pro Tyr Ser Gly Leu Leu Leu Val Ile Thr Val Thr Asp 195 200 205Asn Lys Gly Ala Phe Lys Tyr Ile Lys Pro Gln Ser Gln Phe Ile Val 210 215 220Asp Leu Gly Ala Tyr Leu Glu Lys Glu Ser Ile Tyr Tyr Val Thr Thr225 230 235 240Asn Trp Lys His Thr Ala Thr Arg Phe Ala Ile Lys Pro Met Glu Asp 245 250 255764PRTHuman Respiratory Syncytial Virus 7Met Glu Asn Thr Ser Ile Thr Ile Glu Phe Ser Ser Lys Phe Trp Pro1 5 10 15Tyr Phe Thr Leu Ile His Met Ile Thr Thr Ile Ile Ser Leu Ile Ile 20 25 30Ile Ile Ser Ile Met Ile Ala Ile Leu Asn Lys Leu Cys Glu Tyr Asn 35 40 45Val Phe His Asn Lys Thr Phe Glu Leu Pro Arg Ala Arg Val Asn Thr 50 55 608321PRTHuman Respiratory Syncytial Virus 8Met Ser Lys Thr Lys Asp Gln Arg Thr Ala Lys Thr Leu Glu Arg Thr1 5 10 15Trp Asp Thr Leu Asn His Leu Leu Phe Ile Ser Ser Cys Leu Tyr Lys 20 25 30Leu Asn Leu Lys Ser Ile Ala Gln Ile Thr Leu Ser Ile Leu Ala Met 35 40 45Ile Ile Ser Thr Ser Leu Ile Ile Ala Ala Ile Ile Phe Ile Ala Ser 50 55 60Ala Asn His Lys Val Thr Leu Thr Thr Ala Ile Ile Gln Asp Ala Thr65 70 75 80Asn Gln Ile Lys Asn Thr Thr Pro Thr Tyr Leu Thr Gln Asn Pro Gln 85 90 95Leu Gly Ile Ser Phe Ser Asn Leu Ser Gly Thr Thr Ser Gln Ser Thr 100 105 110Thr Ile Leu Ala Ser Thr Thr Pro Ser Ala Glu Ser Thr Pro Gln Ser 115 120 125Thr Thr Val Lys Ile Lys Asn Thr Thr Thr Thr Gln Ile Leu Pro Ser 130 135 140Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro Gln Asn Lys Pro Asn145 150 155 160Asn Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Cys Ser Ile Cys 165 170 175Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro Asn Lys 180 185 190Lys Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Leu 195 200 205Lys Thr Thr Lys Lys Asp Pro Lys Pro Gln Thr Thr Lys Pro Lys Glu 210 215 220Val Leu Thr Thr Lys Pro Thr Gly Lys Pro Thr Ile Asn Thr Thr Lys225 230 235 240Thr Asn Ile Arg Thr Thr Leu Leu Thr Ser Asn Thr Lys Gly Asn Pro 245 250 255Glu His Thr Ser Gln Glu Glu Thr Leu His Ser Thr Thr Ser Glu Gly 260 265 270Tyr Leu Ser Pro Ser Gln Val Tyr Thr Thr Ser Gly Gln Glu Glu Thr 275 280 285Leu His Ser Thr Thr Ser Glu Gly Tyr Leu Ser Pro Ser Gln Val Tyr 290 295 300Thr Thr Ser Glu Tyr Leu Ser Gln Ser Leu Ser Ser Ser Asn Thr Thr305 310 315 320Lys9574PRTHuman Respiratory Syncytial Virus 9Met Glu Leu Pro Ile Leu Lys Thr Asn Ala Ile Thr Thr Ile Leu Ala1 5 10 15Ala Val Thr Leu Cys Phe Ala Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu 35 40 45Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys65 70 75 80Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95Met Gln Ser Thr Pro Ala Ala Asn Ser Arg Ala Arg Arg Glu Leu Pro 100 105 110Arg Phe Met Asn Tyr Thr Leu Asn Asn Thr Lys Asn Thr Asn Val Thr 115 120 125Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu145 150 155 160Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys 165 170 175Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn 195 200 205Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn225 230 235 240Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro305 310 315 320Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Ile 370 375 380Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr385 390 395 400Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly 450 455 460Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro465 470 475 480Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu 500 505 510Leu His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn Ile Met Ile Thr 515 520 525Thr Ile Ile Ile Val Ile Ile Val Ile Leu Leu Ala Leu Ile Ala Val 530 535 540Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr Pro Val Thr Leu Gly545 550 555 560Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala Phe Asn Asn 565 57010194PRTHuman Respiratory Syncytial Virus 10Met Ser Arg Arg Asn Pro Cys Lys Phe Glu Ile Arg Gly His Cys Leu1 5 10 15Asn Gly Lys Arg Cys His Phe Ser His Asn Tyr Phe Glu Trp Pro Pro 20 25 30His Ala Leu Leu Val Arg Gln Asn Phe Met Leu Asn Arg Ile Leu Lys 35 40 45Ser Met Asp Lys Ser Ile Asp Thr Leu Ser Glu Ile Ser Gly Ala Ala 50 55 60Glu Leu Asp Arg Thr Glu Glu Tyr Ala Leu Gly Val Val Gly Val Leu65 70 75 80Glu Ser Tyr Ile Gly Ser Ile Asn Asn Ile Thr Lys Gln Ser Ala Cys 85 90 95Val Ala Met Ser Lys Leu Leu Thr Glu Leu Asn Ser Asp Asp Ile Lys 100 105 110Lys Leu Arg Asp Asn Glu Glu Pro Asn Ser Pro Lys Val Arg Val Tyr 115 120 125Asn Thr Val Ile Ser Tyr Ile Glu Ser Asn Arg Lys Asn Asn Lys Gln 130 135 140Thr Ile His Leu Leu Lys Arg Leu Pro Ala Asp Val Leu Lys Lys Thr145 150 155 160Ile Lys Asn Thr Leu Asp Ile His Lys Ser Ile Thr Ile Asn Asn Pro 165 170 175Lys Glu Ser Thr Val Ser Asp Thr Asn Asp His Ala Lys Asn Asn Asp 180 185 190Thr Thr1188PRTHuman Respiratory Syncytial Virus 11Met Pro Lys Ile Met Ile Leu Pro Asp Lys Tyr Pro Cys Ser Ile Asn1 5 10 15Ser Ile Leu Ile Thr Ser Asn Cys Arg Val Thr Met Tyr Asn Gln Lys 20 25 30Asn Thr Leu Tyr Ile Asn Gln Asn Asn Gln Asn Asn His Ile Tyr Pro 35 40 45Pro Asp Gln Pro Phe Asn Glu Ile His Trp Thr Ser Gln Asp Leu Ile 50 55 60Asp Ala Thr Gln Asn Phe Leu Gln His Leu Gly Ile Thr Asp Asp Ile65 70 75

80Tyr Thr Ile Tyr Ile Leu Val Ser 85122165PRTHuman Respiratory Syncytial Virus 12Met Asp Pro Ile Ile Ser Gly Asn Ser Ala Asn Val Tyr Leu Thr Asp1 5 10 15Ser Tyr Leu Lys Gly Val Ile Ser Phe Ser Glu Cys Asn Ala Leu Gly 20 25 30Ser Tyr Ile Phe Asn Gly Pro Tyr Leu Lys Asn Asp Tyr Thr Asn Leu 35 40 45Ile Ser Arg Gln Asn Pro Leu Ile Glu His Ile Asn Leu Lys Lys Leu 50 55 60Asn Ile Thr Gln Ser Leu Ile Ser Lys Tyr His Lys Gly Glu Ile Lys65 70 75 80Ile Glu Glu Pro Thr Tyr Phe Gln Ser Leu Leu Met Thr Tyr Lys Ser 85 90 95Met Thr Ser Ser Glu Gln Thr Thr Thr Thr Asn Leu Leu Lys Lys Ile 100 105 110Ile Arg Arg Ala Ile Glu Ile Ser Asp Val Lys Val Tyr Ala Ile Leu 115 120 125Asn Lys Leu Gly Leu Lys Glu Lys Asp Lys Ile Lys Ser Asn Asn Gly 130 135 140Gln Asp Glu Asp Asn Ser Val Ile Thr Thr Ile Ile Lys Asp Asp Ile145 150 155 160Leu Leu Ala Val Lys Asp Asn Gln Ser His Leu Lys Ala Asp Lys Asn 165 170 175Gln Ser Thr Lys Gln Lys Asp Thr Ile Lys Thr Thr Leu Leu Lys Lys 180 185 190Leu Met Cys Ser Met Gln His Pro Pro Ser Trp Leu Ile His Trp Phe 195 200 205Asn Leu Tyr Thr Lys Leu Asn Ser Ile Leu Thr Gln Tyr Arg Ser Ser 210 215 220Glu Val Lys Asn His Gly Phe Ile Leu Ile Asp Asn His Thr Leu Ser225 230 235 240Gly Phe Gln Phe Ile Leu Asn Gln Tyr Gly Cys Ile Val Tyr His Lys 245 250 255Glu Leu Lys Arg Ile Thr Val Thr Thr Tyr Asn Gln Phe Leu Thr Trp 260 265 270Lys Asp Ile Ser Leu Ser Arg Leu Asn Val Cys Leu Ile Thr Trp Ile 275 280 285Ser Asn Cys Leu Asn Thr Leu Asn Lys Ser Leu Gly Leu Arg Cys Gly 290 295 300Phe Asn Asn Val Ile Leu Thr Gln Leu Phe Leu Tyr Gly Asp Cys Ile305 310 315 320Leu Lys Leu Phe His Asn Glu Gly Phe Tyr Ile Ile Lys Glu Ile Glu 325 330 335Gly Phe Ile Met Ser Leu Ile Leu Asn Ile Thr Glu Glu Asp Gln Phe 340 345 350Arg Lys Arg Phe Tyr Asn Ser Met Leu Asn Asn Ile Thr Asp Ala Ala 355 360 365Asn Lys Ala Gln Lys Asn Leu Leu Ser Arg Val Cys His Thr Leu Leu 370 375 380Asp Lys Thr Ile Ser Asp Asn Ile Ile Asn Gly Arg Trp Ile Ile Leu385 390 395 400Leu Ser Lys Phe Leu Lys Leu Ile Lys Leu Ala Gly Asp Asn Asn Leu 405 410 415Asn Asn Leu Ser Glu Leu Tyr Phe Leu Phe Arg Ile Phe Gly His Pro 420 425 430Met Val Asp Glu Arg Gln Ala Met Asp Ala Val Lys Val Asn Cys Asn 435 440 445Glu Thr Lys Phe Tyr Leu Leu Ser Ser Leu Ser Met Leu Arg Gly Ala 450 455 460Phe Ile Tyr Arg Ile Ile Lys Gly Phe Val Asn Asn Tyr Asn Arg Trp465 470 475 480Pro Thr Leu Arg Asn Ala Ile Val Leu Pro Leu Arg Trp Leu Thr Tyr 485 490 495Tyr Lys Leu Asn Thr Tyr Pro Ser Leu Leu Glu Leu Thr Glu Arg Asp 500 505 510Leu Ile Val Leu Ser Gly Leu Arg Phe Tyr Arg Glu Phe Arg Leu Pro 515 520 525Lys Lys Val Asp Leu Glu Met Ile Ile Asn Asp Lys Ala Ile Ser Pro 530 535 540Pro Lys Asn Leu Ile Trp Thr Ser Phe Pro Arg Asn Tyr Met Pro Ser545 550 555 560His Ile Gln Asn Tyr Ile Glu His Glu Lys Leu Lys Phe Ser Asp Ser 565 570 575Asp Lys Ser Arg Arg Val Leu Glu Tyr Tyr Leu Arg Asp Asn Lys Phe 580 585 590Asn Glu Cys Asp Leu His Asn Cys Val Val Asn Gln Ser Tyr Leu Asn 595 600 605Asn Pro Asn His Val Val Ser Leu Thr Gly Lys Glu Arg Glu Leu Ser 610 615 620Val Gly Arg Met Phe Ala Met Gln Pro Gly Met Phe Arg Gln Val Gln625 630 635 640Ile Leu Ala Glu Lys Met Ile Ala Glu Asn Ile Leu Gln Phe Phe Pro 645 650 655Glu Ser Leu Thr Arg Tyr Gly Asp Leu Glu Leu Gln Lys Ile Leu Glu 660 665 670Leu Lys Ala Gly Ile Ser Asn Lys Ser Asn Arg Tyr Asn Asp Asn Tyr 675 680 685Asn Asn Tyr Ile Ser Lys Cys Ser Ile Ile Thr Asp Leu Ser Lys Phe 690 695 700Asn Gln Ala Phe Arg Tyr Glu Thr Ser Cys Ile Cys Ser Asp Val Leu705 710 715 720Asp Glu Leu His Gly Val Gln Ser Leu Phe Ser Trp Leu His Leu Thr 725 730 735Ile Pro His Val Thr Ile Ile Cys Thr Tyr Arg His Ala Pro Pro Tyr 740 745 750Ile Lys Asp His Ile Val Asp Leu Asn Asn Val Asp Glu Gln Ser Gly 755 760 765Leu Tyr Arg Tyr His Met Gly Gly Ile Glu Gly Trp Cys Gln Lys Leu 770 775 780Trp Thr Ile Glu Ala Ile Ser Leu Leu Asp Leu Ile Ser Leu Lys Gly785 790 795 800Lys Phe Ser Ile Thr Ala Leu Ile Asn Gly Asp Asn Gln Ser Ile Asp 805 810 815Ile Ser Lys Pro Val Arg Leu Met Glu Gly Gln Thr His Ala Gln Ala 820 825 830Asp Tyr Leu Leu Ala Leu Asn Ser Leu Lys Leu Leu Tyr Lys Glu Tyr 835 840 845Ala Gly Ile Gly His Lys Leu Lys Gly Thr Glu Thr Tyr Ile Ser Arg 850 855 860Asp Met Gln Phe Met Ser Lys Thr Ile Gln His Asn Gly Val Tyr Tyr865 870 875 880Pro Ala Ser Ile Lys Lys Val Leu Arg Val Gly Pro Trp Ile Asn Thr 885 890 895Ile Leu Asp Asp Phe Lys Val Ser Leu Glu Ser Ile Gly Ser Leu Thr 900 905 910Gln Glu Leu Glu Tyr Arg Gly Glu Ser Leu Leu Cys Ser Leu Ile Phe 915 920 925Arg Asn Val Trp Leu Tyr Asn Gln Ile Ala Leu Gln Leu Lys Asn His 930 935 940Ala Leu Cys Asn Asn Lys Leu Tyr Leu Asp Ile Leu Lys Val Leu Lys945 950 955 960His Leu Lys Thr Phe Phe Asn Leu Asp Asn Ile Asp Thr Ala Leu Thr 965 970 975Leu Tyr Met Asn Leu Pro Met Leu Phe Gly Gly Gly Asp Pro Asn Leu 980 985 990Leu Tyr Arg Ser Phe Tyr Arg Arg Thr Pro Asp Phe Leu Thr Glu Ala 995 1000 1005Ile Val His Ser Val Phe Ile Leu Ser Tyr Tyr Thr Asn His Asp 1010 1015 1020Leu Lys Asp Lys Leu Gln Asp Leu Ser Asp Asp Arg Leu Asn Lys 1025 1030 1035Phe Leu Thr Cys Ile Ile Thr Phe Asp Lys Asn Pro Asn Ala Glu 1040 1045 1050Phe Val Thr Leu Met Arg Asp Pro Gln Ala Leu Gly Ser Glu Arg 1055 1060 1065Gln Ala Lys Ile Thr Ser Glu Ile Asn Arg Leu Ala Val Thr Glu 1070 1075 1080Val Leu Ser Thr Ala Pro Asn Lys Ile Phe Ser Lys Ser Ala Gln 1085 1090 1095His Tyr Thr Thr Thr Glu Ile Asp Leu Asn Asp Ile Met Gln Asn 1100 1105 1110Ile Glu Pro Thr Tyr Pro His Gly Leu Arg Val Val Tyr Glu Ser 1115 1120 1125Leu Pro Phe Tyr Lys Ala Glu Lys Ile Val Asn Leu Ile Ser Gly 1130 1135 1140Thr Lys Ser Ile Thr Asn Ile Leu Glu Lys Thr Ser Ala Ile Asp 1145 1150 1155Leu Thr Asp Ile Asp Arg Ala Thr Glu Met Met Arg Lys Asn Ile 1160 1165 1170Thr Leu Leu Ile Arg Ile Leu Pro Leu Asp Cys Asn Arg Asp Lys 1175 1180 1185Arg Glu Ile Leu Ser Met Glu Asn Leu Ser Ile Thr Glu Leu Ser 1190 1195 1200Lys Tyr Val Arg Glu Arg Ser Trp Ser Leu Ser Asn Ile Val Gly 1205 1210 1215Val Thr Ser Pro Ser Ile Met Tyr Thr Met Asp Ile Lys Tyr Thr 1220 1225 1230Thr Ser Thr Ile Ala Ser Gly Ile Ile Ile Glu Lys Tyr Asn Val 1235 1240 1245Asn Ser Leu Thr Arg Gly Glu Arg Gly Pro Thr Lys Pro Trp Val 1250 1255 1260Gly Ser Ser Thr Gln Glu Lys Lys Thr Met Pro Val Tyr Asn Arg 1265 1270 1275Gln Val Leu Thr Lys Lys Gln Arg Asp Gln Ile Asp Leu Leu Ala 1280 1285 1290Lys Leu Asp Trp Val Tyr Ala Ser Ile Asp Asn Lys Asp Glu Phe 1295 1300 1305Met Glu Glu Leu Ser Ile Gly Thr Leu Gly Leu Thr Tyr Glu Lys 1310 1315 1320Ala Lys Lys Leu Phe Pro Gln Tyr Leu Ser Val Asn Tyr Leu His 1325 1330 1335Arg Leu Thr Val Ser Ser Arg Pro Cys Glu Phe Pro Ala Ser Ile 1340 1345 1350Pro Ala Tyr Arg Thr Thr Asn Tyr His Phe Asp Thr Ser Pro Ile 1355 1360 1365Asn Arg Ile Leu Thr Glu Lys Tyr Gly Asp Glu Asp Ile Asp Ile 1370 1375 1380Val Phe Gln Asn Cys Ile Ser Phe Gly Leu Ser Leu Met Ser Val 1385 1390 1395Val Glu Gln Phe Thr Asn Val Cys Pro Asn Arg Ile Ile Leu Ile 1400 1405 1410Pro Lys Leu Asn Glu Ile His Leu Met Lys Pro Pro Ile Phe Thr 1415 1420 1425Gly Asp Val Asp Ile His Lys Leu Lys Gln Val Ile Gln Lys Gln 1430 1435 1440His Met Phe Leu Pro Asp Lys Ile Ser Leu Thr Gln Tyr Val Glu 1445 1450 1455Leu Phe Leu Ser Asn Lys Thr Leu Lys Ser Gly Ser Asn Val Asn 1460 1465 1470Ser Asn Leu Ile Leu Ala His Lys Ile Ser Asp Tyr Phe His Asn 1475 1480 1485Thr Tyr Ile Leu Ser Thr Asn Leu Ala Gly His Trp Ile Leu Ile 1490 1495 1500Ile Gln Leu Met Lys Asp Ser Lys Gly Ile Phe Glu Lys Asp Trp 1505 1510 1515Gly Glu Gly Tyr Ile Thr Asp His Met Phe Ile Asn Leu Lys Val 1520 1525 1530Phe Phe Asn Ala Tyr Lys Thr Tyr Leu Leu Cys Phe His Lys Gly 1535 1540 1545Tyr Gly Arg Ala Lys Leu Glu Cys Asp Met Asn Thr Ser Asp Leu 1550 1555 1560Leu Cys Val Leu Glu Leu Ile Asp Ser Ser Tyr Trp Lys Ser Met 1565 1570 1575Ser Lys Val Phe Leu Glu Gln Lys Val Ile Lys Tyr Ile Leu Ser 1580 1585 1590Gln Asp Ala Ser Leu His Arg Val Lys Gly Cys His Ser Phe Lys 1595 1600 1605Leu Trp Phe Leu Lys Arg Leu Asn Val Ala Glu Phe Thr Val Cys 1610 1615 1620Pro Trp Val Val Asn Ile Asp Tyr His Pro Thr His Met Lys Ala 1625 1630 1635Ile Leu Thr Tyr Ile Asp Leu Val Arg Met Gly Leu Ile Asn Ile 1640 1645 1650Asp Arg Ile Tyr Ile Lys Asn Lys His Lys Phe Asn Asp Glu Phe 1655 1660 1665Tyr Thr Ser Asn Leu Phe Tyr Ile Asn Tyr Asn Phe Ser Asp Asn 1670 1675 1680Thr His Leu Leu Thr Lys His Ile Arg Ile Ala Asn Ser Glu Leu 1685 1690 1695Glu Ser Asn Tyr Asn Lys Leu Tyr His Pro Thr Pro Glu Thr Leu 1700 1705 1710Glu Asn Ile Leu Thr Asn Pro Val Lys Ser Asn Gly Lys Lys Thr 1715 1720 1725Leu Ser Asp Tyr Cys Ile Gly Lys Asn Val Asp Ser Ile Met Leu 1730 1735 1740Pro Ser Leu Ser Asn Lys Lys Leu Ile Lys Ser Ser Thr Met Ile 1745 1750 1755Arg Thr Asn Cys Ser Arg Gln Asp Leu Tyr Asn Leu Phe Pro Thr 1760 1765 1770Val Val Ile Asp Lys Ile Ile Asp His Ser Gly Asn Thr Ala Lys 1775 1780 1785Ser Asn Gln Leu Tyr Thr Thr Thr Ser His Gln Ile Ser Leu Val 1790 1795 1800His Asn Ser Thr Ser Leu Tyr Cys Met Leu Pro Trp His His Ile 1805 1810 1815Asn Arg Phe Asn Phe Val Phe Ser Ser Thr Gly Cys Lys Ile Ser 1820 1825 1830Ile Glu Tyr Ile Leu Lys Asp Leu Lys Ile Lys Asp Pro Asn Cys 1835 1840 1845Ile Ala Phe Ile Gly Glu Gly Ala Gly Asn Leu Leu Leu Arg Thr 1850 1855 1860Val Val Glu Leu His Pro Asp Ile Arg Tyr Ile Tyr Arg Ser Leu 1865 1870 1875Lys Asp Cys Asn Asp His Ser Leu Pro Ile Glu Phe Leu Arg Leu 1880 1885 1890Tyr Asn Gly His Ile Asn Ile Asp Tyr Gly Glu Asn Leu Thr Ile 1895 1900 1905Pro Ala Thr Asp Ala Thr Asn Asn Ile His Trp Ser Tyr Leu His 1910 1915 1920Ile Lys Phe Ala Glu Pro Ile Ser Leu Phe Val Cys Asp Ala Glu 1925 1930 1935Leu Pro Val Thr Val Asn Trp Ser Lys Ile Ile Ile Glu Trp Ser 1940 1945 1950Lys His Val Arg Lys Cys Lys Tyr Cys Ser Ser Val Asn Lys Cys 1955 1960 1965Thr Leu Ile Val Lys Tyr His Ala Gln Asp Asp Ile Asp Phe Lys 1970 1975 1980Leu Asp Asn Ile Thr Ile Leu Lys Thr Tyr Val Cys Leu Gly Ser 1985 1990 1995Lys Leu Lys Gly Ser Glu Val Tyr Leu Val Leu Thr Ile Gly Pro 2000 2005 2010Ala Asn Val Phe Pro Val Phe Asn Val Ala Gln Asn Ala Lys Leu 2015 2020 2025Ile Leu Ser Arg Thr Lys Asn Phe Ile Met Pro Lys Lys Ala Asp 2030 2035 2040Lys Glu Ser Ile Asp Ala Asn Ile Lys Ser Leu Ile Pro Phe Leu 2045 2050 2055Cys Tyr Pro Ile Thr Lys Lys Gly Ile Asn Thr Ala Leu Ser Lys 2060 2065 2070Leu Lys Ser Val Val Ser Gly Asp Ile Leu Ser Tyr Ser Ile Ala 2075 2080 2085Gly Arg Asn Glu Val Phe Ser Asn Lys Leu Ile Asn His Lys His 2090 2095 2100Met Asn Ile Leu Lys Trp Phe Asn His Val Leu Asn Phe Arg Ser 2105 2110 2115Thr Glu Leu Asn Tyr Asn His Leu Tyr Met Val Glu Ser Thr Tyr 2120 2125 2130Pro His Leu Ser Glu Leu Leu Asn Ser Leu Thr Thr Asn Glu Leu 2135 2140 2145Lys Lys Leu Ile Lys Ile Thr Gly Ser Leu Leu Tyr Asn Phe Tyr 2150 2155 2160Asn Glu 2165

Claims

1. A vaccine composition comprising an effective amount of respiratory syncytial virus (RSV) F protein in a pre-fusion stabilized form and/or M protein incorporated into biodegradable polyanhydride polymer particles for inducing an immune response against RSV.

2. The vaccine composition of claim 1, comprising an effective amount of RSV F protein in the pre-fusion stabilized form incorporated into the biodegradable polyanhydride polymer particles for inducing an immune response against RSV.

3. The vaccine composition of claim 1, wherein the F protein in a pre-fusion stabilized form is selected from the group consisting of DS-Cav1, DS-TriC, Vav-1-TriC, DX-Cav1-TriC, Pre-F-GCN4t, SC-DM, and SC-TM.

4. The vaccine composition of claim 1, wherein the F protein in a pre-fusion stabilized form is DS-Cav1.

5. The vaccine composition of claim 1, comprising an effective amount of RSV M protein incorporated into the biodegradable polyanhydride polymer particles for inducing an immune response against RSV.

6. The vaccine composition of claim 1, further comprising an adjuvant.

7. The vaccine composition of claim 6, wherein the adjuvant is incorporated into the biodegradable polyanhydride polymer particles.

8. The vaccine composition of claim 1, further comprising a CpG oligonucleotide.

9. The vaccine composition of claim 8, wherein the CpG oligonucleotide is incorporated into the biodegradable polyanhydride polymer particles.

10. The vaccine composition of claim 8, wherein the CpG oligonucleotide is a CpG oligodeoxynucleotide (ODN).

11. The vaccine composition of claim 10, wherein the CpG oligonucleotide is incorporated into the biodegradable polyanhydride polymer particles.

12. The vaccine composition of claim 1, wherein the biodegradable polyanhydride polymer particles have an average effective diameter of less than about 5 .mu.M.

13. The vaccine composition of claim 1, wherein the vaccine composition induces an antibody response as well as a CD4 T cell response when administered to a subject in need thereof.

14. The vaccine composition of claim 1, wherein the biodegradable polyanhydride polymer particles comprise a polymer formed from a 1,.omega.-bis(p-carboxyphenoxy)(C.sub.2-C.sub.12)alkane, a 1,.omega.-bis(p-carboxyphenoxy)(C.sub.2-C.sub.12)dioxa-alkane, and a (C.sub.5-C.sub.20)alkanoic diacid.

15. The vaccine composition of claim 1, wherein the biodegradable polyanhydride polymer particles comprise a polymer formed from 1,6-bis(p-carboxyphenoxy)hexane (CPH), 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctance (CPTEG), and sebacic acid (SA).

16. The vaccine composition of claim 1, wherein the biodegradable polyanhydride polymer particles comprise a polymer formed from 1,6-bis(p-carboxyphenoxy)hexane (CPH), 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctance (CPTEG), and sebacic acid (SA), wherein the polymer is formed from CPH and CPTEG at a ratio range of CPH:CPTEG of 80-60:20-40.

17. The vaccine composition of claim 1, further comprising an RSV protein selected from NS1, NS2, N, P, SH, G, M2-1, M2-2, L, or any combinations thereof.

18. A method comprising administering the vaccine of claim 1 to a subject who is at risk for infection by RSV.

19. The method of claim 18, wherein after the vaccine composition is administered to the subject, the subject is protected against infection by RSV.

20. The method of claim 18, wherein the vaccine composition is administered by a route selected from intranasal, pulmonary, oral, subcutaneous, intramuscular, or intravenous.

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