RU2807992C2 - Antigenic polypeptides based on the respiratory syncytial virus sequence - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: antigenic polypeptide of the respiratory syncytial virus (RSV), as well as a ferritin particle and a composition containing it. A nucleic acid encoding the above polypeptide is also disclosed.

EFFECT: inducing an immune response to RSV in a subject.

32 cl, 23 dwg, 1 tbl

Description

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/652199, filed April 3, 2018, which is incorporated herein by reference in its entirety.

[0002] This application contains a sequence listing that was filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy of the file in question, created on March 27, 2019, is named 2019-03-27_01121-0031-00PCT_SL_ST25.txt and is 187354 bytes in size.

[0003] Even with numerous advances in the field of vaccinology, new discoveries are needed to protect people from many life-threatening infectious diseases. Many currently licensed vaccines rely on decades-old technologies to produce live attenuated or inactivated killed pathogens, which carry safety concerns and in many cases stimulate only short-term, weak immune responses that require multiple doses. Although advances in genetic and biochemical engineering have made it possible to develop therapeutics for intractable diseases, these avenues of application in the field of vaccinology have not been fully realized. Recombinant protein technologies now enable the development of optimal antigens. In addition, nanoparticles are increasingly showing potential for optimal antigen presentation and targeted drug delivery. Nanoparticles with multiple attached antigens have been shown to have increased binding avidity provided by the multivalent display of their molecular cargo and the ability to more effectively cross biological barriers due to their nanoscopic size. Ferritin nanoparticlesHelicobacter pylori (H. pylori) fused to the influenza virus hemagglutinin (HA) protein has improved antigen stability and immunogenicity in mouse models of influenza (see Kanekiyo et al., Nature 499:102-106 (2013)). This fusion protein self-assembled into an octahedrally symmetrical nanoparticle with 8 trimeric HA spikes to produce robust immune responses in various preclinical models when administered with an adjuvant.

[0004] Respiratory syncytial virus (RSV) is a leading cause of severe respiratory illness in infants and a leading cause of respiratory illness in the elderly. Despite decades of research, the need for vaccines remains unmet. Although the need for a vaccine is clear, RSV vaccine development stalled in the 1960s when clinical trials using formalin-inactivated RSV in infants resulted in more severe illness following RSV infection. See Hurwitz (2011) Expert Rev Vaccines 10(10): 1415-1433. More recently, clinical programs using the RSV F antigen in its postfusion conformation have failed to achieve sufficient efficacy in adults. See Faloon et al. (2017) JID 216:1362–1370. However, RSV F antigens stabilized in a prefusion conformation can elicit a neutralizing response superior to that of postfusion antigens that have not shown clinical benefit.

[0005] Presented herein are a number of novel polypeptides, nanoparticles, compositions, methods and uses that utilize RSV polypeptides. New RSV F polypeptides have been created, including polypeptides in which an epitope of the RSV polypeptide that is common to prefusion RSV F and postfusion RSV F is blocked, for example by an N-glycan at a glycosylation site added by mutation. Antigenic polypeptides and nanoparticles containing these new RSV polypeptides and ferritin have also been generated. Ferritin-based antigenic polypeptides containing RSV G polypeptides have also been generated. In addition, self-adjuvant antigenic polypeptides containing RSV polypeptides and ferritin have been developed, in which immunostimulatory functional groups, such as adjuvants, are directly chemically attached to the antigenic polypeptide. Direct conjugation of the immunostimulatory moiety to the antigenic polypeptide allows for targeted co-delivery of the immunostimulatory moiety and the RSV polypeptide as a single macromolecular compound, which can significantly reduce the potential for systemic toxicity that is feared with traditional vaccines that contain antigens and immunostimulatory molecules such as adjuvants, in the form of individual molecules. Co-delivery of immunostimulatory functional groups along with RSV polypeptides as a macromolecular compound and their multivalent presentation may also reduce the total dose required to provide protection, reducing manufacturing costs and costs.

SHORT DESCRIPTION

[0006] The purpose of the present disclosure is to provide compositions, kits, methods and uses that are capable of providing one or more of the benefits discussed above, or at least providing the public with a useful choice. Accordingly, the following embodiments are disclosed herein.

[0007] Embodiment 1 is an RSV sequence-based antigenic polypeptide comprising an RSV F polypeptide, wherein an epitope of the RSV polypeptide that is common to prefusion RSV F and postfusion RSV F polypeptide is blocked.

[0008] Embodiment 2 is an RSV sequence-based antigenic polypeptide comprising an RSV F polypeptide, wherein the RSV F polypeptide contains amino acid residues 62-69 and 196-209 of SEQ ID NO: 26 and an asparagine corresponding to position 328, 348 or 507 from SEQ ID NO: 26.

[0009] Embodiment 3 is an antigenic polypeptide based on the RSV sequence of Embodiment 2, wherein an epitope of the RSV polypeptide that is common to prefusion RSV F and postfusion RSV F is blocked.

[0010] Embodiment 3b is an antigenic polypeptide based on the RSV sequence of embodiment 1 or 3, wherein the blocked epitope is an RSV antigenic site 1 F epitope.

[0011] Embodiment 3c is an antigenic polypeptide based on the RSV sequence of embodiment 1 or 3-3b, where two or more epitopes common to prefusion RSV F and postfusion RSV F are blocked.

[0012] Embodiment 3d is an antigenic polypeptide based on the RSV sequence of embodiment 1 or 3-3c, where two or more epitopes of the RSV antigenic site 1 F are blocked.

[0013] Embodiment 3e is an antigenic polypeptide based on the RSV sequence of embodiment 1 or 3-3d, wherein one, more, or all of the epitopes that topologically overlap with the blocked epitope are also blocked.

[0014] Embodiment 3f is an antigenic polypeptide based on the RSV sequence of embodiment 3e, wherein the blocked epitope is an RSV antigenic site 1 F epitope.

[0015] Embodiment 4 is an antigenic polypeptide based on the RSV sequence of any of the previous embodiments, comprising RSV F prior to fusion.

[0016] Embodiment 5 is an antigenic polypeptide based on the RSV sequence of any of the previous embodiments that is recognized by an antibody specific for prefusion RSV F selected from D25 or AM14.

[0017] Embodiment 6 is an antigenic polypeptide based on the RSV sequence of Embodiment 4 or 5, wherein the prefusion RSV F contains an epitope not found on the postfusion RSV F.

[0018] Embodiment 7 is an RSV sequence-based antigenic polypeptide according to any of Embodiments 1-3, comprising RSV F after fusion.

[0019] Embodiment 8 is an antigenic polypeptide based on the RSV sequence of any of embodiments 1 or 3-6, wherein the epitope is blocked by an N-glycan attached to asparagine.

[0020] Embodiment 9 is an antigenic polypeptide based on the RSV sequence of Embodiment 7, wherein asparagine corresponds to a non-asparagine residue in the wild-type RSV F sequence (SEQ ID NO: 26), wherein optionally the non-asparagine residue is corresponds to position 328, 348 or 507 of SEQ ID NO: 26.

[0021] Embodiment 10 is an antigenic polypeptide based on the RSV sequence of any of the previous embodiments, further comprising a ferritin protein.

[0022] Embodiment 11 is an antigenic polypeptide based on the RSV sequence of Embodiment 11, wherein ferritin contains a mutation to replace the surface-exposed amino acid with cysteine.

[0023] Embodiment 12 is an RSV sequence-based antigenic polypeptide comprising an RSV F polypeptide and a ferritin protein, wherein the ferritin protein contains a mutation to replace a surface-exposed amino acid with cysteine.

[0024] Embodiment 13 is an antigenic polypeptide based on the RSV sequence of any of embodiments 11-12, wherein the ferritin contains one or more of the E12C, S26C, S72C, A75C, K79C, S100C, and S111C ferritin mutations from H. pylori or one or more relevant ferritin mutations from an organism other than H. pylori , as determined by pairwise or structural alignment.

[0025] Embodiment 14 is an antigenic polypeptide based on the RSV sequence of any of embodiments 10-13, comprising one or more immunostimulatory functional groups linked to ferritin via a surface exposed amino acid, wherein optionally the surface exposed amino acid is cysteine, resulting from mutation.

[0026] Embodiment 15 is an antigenic polypeptide based on the RSV sequence of any one of embodiments 10-14, wherein the ferritin contains a mutation to replace surface-exposed asparagine with a non-asparagine amino acid, wherein optionally asparagine is at position 19 of ferritin from H. pylori or a similar ferritin position from an organism other than H. pylori , as determined by pairwise or structural alignment.

[0027] Embodiment 16 is an antigenic polypeptide based on the RSV sequence of any one of embodiments 10-15, wherein the ferritin contains a mutation to replace the internal cysteine with an amino acid other than cysteine, wherein optionally the internal cysteine is at position 31 of ferritin from H. pylori or a position that corresponds to position 31 of ferritin from H. pylori , as determined by pairwise or structural alignment.

[0028] Embodiment 17 is an antigenic polypeptide based on an RSV sequence according to any of embodiments 12-16, wherein the RSV F polypeptide contains an epitope not found on the fusion RSV F, which is a site 0 epitope, wherein optionally the site 0 epitope contains amino acid residues 62-69 and 196-209 from SEQ ID NO: 26.

[0029] Embodiment 18 is an antigenic polypeptide based on the RSV sequence of any of the previous embodiments, wherein the RSV F polypeptide contains an asparagine at a position corresponding to position 328 of SEQ ID NO: 26.

[0030] Embodiment 19 is an antigenic polypeptide based on the RSV sequence of any of the previous embodiments, wherein the RSV F polypeptide contains an asparagine at a position corresponding to position 348 of SEQ ID NO: 26.

[0031] Embodiment 20 is an antigenic polypeptide based on the RSV sequence of any of the previous embodiments, wherein the RSV F polypeptide contains an asparagine at a position corresponding to position 507 of SEQ ID NO: 26.

[0032] Embodiment 21 is an antigenic polypeptide based on the RSV sequence of any of the previous embodiments, wherein the RSV F polypeptide contains a leucine at a position corresponding to the position of lysine 498 of SEQ ID NO: 26.

[0033] Embodiment 22 is an antigenic polypeptide based on the RSV sequence of any of the previous embodiments, wherein the RSV F polypeptide contains a proline at a position corresponding to the position of isoleucine 217 of SEQ ID NO: 26.

[0034] Embodiment 23 is an antigenic polypeptide based on the RSV sequence of any of the previous embodiments, wherein the RSV F polypeptide contains an amino acid other than cysteine at a position corresponding to position 155 of SEQ ID NO: 26, and/or an amino acid other than cysteine, at a position corresponding to position 290 of SEQ ID NO: 26.

[0035] Embodiment 24 is an antigenic polypeptide based on the RSV sequence of any of the previous embodiments, comprising a serine at a position corresponding to position 155 of SEQ ID NO: 26 and/or a serine at a position corresponding to position 290 of SEQ ID NO: : 26.

[0036] Embodiment 25 is an antigenic polypeptide based on the RSV sequence of any of the previous embodiments, wherein the RSV F polypeptide does not contain a furin cleavage site, wherein optionally a linker is present instead of a furin cleavage site.

[0037] Embodiment 26 is an RSV sequence-based antigenic polypeptide of any of the preceding embodiments, wherein the RSV F polypeptide comprises a sequence characterized by at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identical to amino acids 1-478 of SEQ ID NO: 17.

[0038] Embodiment 27 is an RSV sequence-based antigenic polypeptide of any of the preceding embodiments, wherein the RSV F polypeptide comprises a sequence characterized by at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 17.

[0039] Embodiment 28 is an antigenic polypeptide based on the RSV sequence of Embodiment 20 or 21 comprising amino acids 1-478 of SEQ ID NO: 17.

[0040] Embodiment 29 is an RSV sequence-based antigenic polypeptide according to any of embodiments 1-19, wherein the RSV F polypeptide comprises a sequence characterized by at least 85%, 90%, 95%, 97%, 98%, 99 % or 99.5% identity to amino acids 1-478 of SEQ ID NO: 23.

[0041] Embodiment 30 is an RSV sequence-based antigenic polypeptide according to any of Embodiments 1-19 or 23, wherein the RSV F polypeptide comprises a sequence characterized by at least 85%, 90%, 95%, 97%, 98% , 99% or 99.5% sequence identity to SEQ ID NO: 23.

[0042] Embodiment 31 is an RSV sequence-based antigenic polypeptide of Embodiment 23 or 24 comprising amino acids 1-478 of SEQ ID NO: 23.

[0043] Embodiment 32 is an antigenic polypeptide based on the RSV sequence of any of the previous embodiments, comprising the sequence of any of SEQ ID NO: 3-23.

[0044] Embodiment 32a is an RSV sequence-based antigenic polypeptide of Embodiment 32 containing the sequence of SEQ ID NO: 17.

[0045] Embodiment 32b is an RSV sequence-based antigenic polypeptide of Embodiment 32 containing the sequence of SEQ ID NO: 23.

[0046] Embodiment 33 is a ferritin particle containing an antigenic polypeptide based on an RSV sequence according to any of embodiments 10-32b.

[0047] Embodiment 34 is a composition comprising an RSV sequence-based antigenic polypeptide, or a ferritin particle according to any of the previous embodiments, and an RSV G polypeptide.

[0048] Embodiment 34b is the composition of Embodiment 34, wherein the composition comprises a ferritin particle, and the ferritin particle contains an RSV G polypeptide, where optionally the RSV G polypeptide is chemically conjugated to the ferritin particle.

[0049] Embodiment 34c is a composition according to embodiment 34 or 34b, wherein the RSV G polypeptide is not glycosylated.

[0050] Embodiment 35 is a composition comprising an RSV sequence-based antigenic polypeptide or ferritin particle according to any of embodiments 1-33, or a composition according to any of embodiments 34-34c, further comprising a pharmaceutically acceptable carrier.

[0051] Embodiment 36 is an RSV sequence-based antigenic polypeptide, ferritin particle, or composition according to any of embodiments 1-35 for use in a method of eliciting an immune response to RSV or in protecting a subject from RSV infection.

[0052] Embodiment 37 is a method of generating an immune response to RSV or protecting a subject from RSV infection, comprising administering any one or more of an RSV sequence-based antigenic polypeptide, a ferritin particle, or a composition according to any of embodiments 1-36 subject.

[0053] Embodiment 38 is an RSV sequence-based antigenic polypeptide, ferritin particle, composition, or method according to any of embodiments 36-37, wherein the subject is a human.

[0054] Embodiment 39 is a nucleic acid encoding an antigenic polypeptide based on an RSV sequence according to any of embodiments 1-32b, wherein optionally the nucleic acid is mRNA.

[0055] Embodiment 39b is a composition or kit comprising a nucleic acid according to embodiment 39 and a nucleic acid encoding an RSV G polypeptide, where optionally one or both nucleic acids are mRNA.

[0056] Additional objects and advantages will be set forth in part in the description that follows, and in part will be apparent from the description, or may be ascertainable from practice. The objects and advantages will be realized and achieved through the elements and combinations specifically set forth in the appended claims.

[0057] It should be understood that both the foregoing general description and the following detailed description are illustrative and explanatory only and do not limit the claims.

[0058] The accompanying drawings, which are included in and constitute a part of this specification, illustrate several embodiments and, together with the description, serve to explain the principles described herein.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0059] In FIG. 1A-1D show the structure of an exemplary RSV Pre-F-NP polypeptide. (Figure 1A) Line diagram showing the residue numbers corresponding to the N-terminus of each segment. Numbering corresponds to SEQ ID NO: 26. Domains 1-3 are designated DI, DII and DIII, respectively, and heptad repeat region A (HRA) and heptad repeat region B (HRB) are also designated. C-terminal ferritin (ferritin nanoparticle) is marked. The F1 and F2 fragments of the functional group representing RSV F are labeled below the figure. The region between fragments F1 and F2, where the fusion peptide (FP) peptide 27 (p27) fragment and the furin cleavage sites (furin sites) have been removed and replaced with a flexible linker to form single-stranded F constructs, is depicted as a line and marked above drawing. The asterisks above the diagram indicate the approximate locations of the engineered glycosylation sites E328N, S348N, and R507N. (Figure 1B) Structural model of the functional group representing prefusion RSV F, indicating the key epitopes to be neutralized by antibodies (Nabs) D25, AM14, 101F, and palivizumab. The approximate region of structural epitopes common to the prefusion and postfusion conformations is indicated by a white triangle. The positions of exemplary engineered glycosylation sites E328N, S348N, and R507N are indicated. The designed glycosylation sites correspond to regions that are structurally common to the prefusion and postfusion conformations and are away from key neutralizing epitopes recognized by antibodies such as D25, AM14, 101F, and palivizumab. As such, constructs containing these engineered glycan sites retain the ability to bind to the above neutralizing antibodies (data not shown). (Figure 1C) Structural model of the prefusion RSV F protein nanoparticle (Pre-F-NP) with the HRA and HRB regions shaded in darker color. The resulting folded Pre-F-NP constructs can form 24-mers that expose the key epitopes listed in FIG. 1B. (Figure 1D) Class averages of 2D electron micrographs of the RF8085 Pre-F-NP RSV construct (SEQ ID NO: 1) showing the symmetry of the arrangement of the trimeric F RSV functional groups on the 24-mer ferritin nanoparticle.

[0060] In FIG. 2 shows the fine-scale expression of several Pre-F-NP constructs expressed in the conditioned medium of 293 cells, as measured by Western blotting using antibody D25. RF8090 is SEQ ID NO: 2, which is a cloning variant used when expressed in CHO, having the same sequence as RF8085, i.e. SEQ ID NO:1. RF8085 and RF8090 are Pre-F-NP constructs carrying disulfide and DS-CAV cavity-filling mutations with deletions and the single-stranded linker described in FIG. 1A fused to the N-terminus of ferritin. RF8100-RF8105 and RF8108-RF8112 have sequences under SEQ ID NO: 3-8 and 11-14, respectively. scF-pFerr=fusion protein based on RSV F polypeptide and ferritin. Mutations found to provide improved expression of the construct compared to the RF8090 reference are indicated below the Western blot results. Noteworthy mutations include the addition of glycan sites through the E328N, S348N and R507N mutations and the I327P mutation. capping the central helix, which resulted in increased expression and secretion of RSV F nanoparticles in conditioned media, as measured by Western blotting.

[0061] In FIG. 3 shows the expression of RF8085 (SEQ ID NO: 1; control construct) and RF8106 (SEQ ID NO: 9; containing the I217P mutation as in RF8108 and not containing the disulfide bond (DS) mutation in DS-CAV1), as measured by analysis by Western blotting in conditioned medium when expressed at 293. Replacement of DS with the I217P mutation, which caps the central helix, provided a significant increase in the expression level. Replacement of DS with a mutation that caps the central helix did not affect the binding of the construct to antibodies D25 and AM14, which are specific for the protein in the prefusion conformation.

[0062] In FIG. 4 shows the results of purification of construct RF8106 using size exclusion chromatography (SEQ ID NO: 9). The RF8106 nanoparticle retention volume of approximately 65 mL on an SEC Superose 6 preparative column corresponds to a folded 24-mer nanoparticle, indicating that mutations in RF8106 do not interfere with nanoparticle formation.

[0063] Onfig. 5A-5B shows dynamic light scattering (DLS) analysis results for unrefurbished (5A) and refurbished (5B) RF8106. Like SEC analysis, DLS demonstrates that RSV Pre-F-NP formed the expected folded nanoparticle. The data for the reduced protein also show that the particle was not destroyed by the reduction performed prior to conjugation of the adjuvant to the surface-exposed cysteine introduced into ferritin by mutation (see Fig. fig. 6).

[0064] In FIG. Figure 6 shows the results of Coomassie SDS-PAGE gel analysis for RF8106 with and without TLR9 agonist CpG conjugation. The increase in gel shift for the CpG-treated nanoparticle demonstrated that up to approximately 40-50% CpG adjuvant could be added to the RSV F nanoparticle. Conjugation of CpGs or other immunostimulatory functional groups, such as the TLR7/8 agonist SM7/8, did not inhibit the ability of the particle to bind antibodies D25 and AM14, which are specific for the protein in the prefusion conformation.

[0065] In FIG. 7 shows Western blot results of nanoparticles containing RSV F with additional glycans (RF8117, SEQ ID NO: 17) and without additional glycans (RF8085, SEQ ID NO: 1; and RF8113, SEQ ID NO: 16). RF8113 is similar to RF8106, but the surface exposed cysteine S111C (using ferritin residue numbering, i.e., corresponding to the ferritin sequence positions SEQ ID NO: 208) from RF8106 was replaced by the surface exposed cysteine K79C (also using ferritin residue numbering), to place the conjugation site further away from the functional group representing Pre-F. Like RF8106, RF8113 maintains improved expression compared to the reference molecule RF8085. RF8117 is similar to RF8113 but additionally contains the three glycosylation-promoting mutations identified in FIG. 2, i.e., E328N, S348N, and R507N, to further improve expression and block non-neutralization epitopes common to F in the prefusion conformation and F in the postfusion conformation, as described in FIG. 1B.

[0066] In FIG. Figure 8 shows the expression of RSV F constructs with different substitutions at potential trypsin-like protease cleavage sites. When RF8090 was expressed in the CHO cell line (the same protein sequence as RF8085, but with a different DNA sequence adapted to the expression vector in CHO), it was observed that the region between the functional groups representing F and ferritin was excised in the polypeptide, which led to a decrease in expression levels. Based on the obtained mass values of the functional group representing F, it was determined that proteolysis could occur near the HRB, the bullfrog linker region of the Pre-F-NP construct. Mutations of lysine and arginine residues in this region (residues ∼450–550) were examined to eliminate potential trypsin-type proteolysis of the construct. Mutations in RF8122 (SEQ ID NO: 18) compared to RF8117 (K498L and K508Q) provided improved expression in 293 cells and may reduce or eliminate proteolysis in CHO cells. Alternative mutations limited expression.

[0067] FIG. 9A-B . Expression of RF8090, RF8117, and RF8140 in stably transfected CHO cells. Expression yield of RF8090 (SEQ ID NO: 2) was observed at low levels. Disulfide substitution mutations in DS-CAV1 and linker mutations between the F functional group and the ferritin functional group to eliminate potential trypsin cleavage sites were introduced as described above in construct RF8117 (SEQ ID NO: 17) and RF8140 (SEQ ID NO: 23), which were cloned into stably expressing CHO cells. (Figure 9A) Expression of RF8117 and RF8140 from three and four pools of CHO cells, respectively, in CHO conditioned medium was compared with the yields of RF8090 in CHO conditioned medium by Western blot analysis using D25. All three CHO pools for RF8117 and all four CHO pools for RF8140 produced expression at higher yields than RF8090. (Figure 9B) Expression of RF8117 in CHO conditioned medium measured using the prefusion F specific antibody D25 using Octet. The left panel shows a plot of the response for known concentrations of RF8140 purified from medium 293 versus the response to binding to D25 on the Protein A tip, providing a standard curve. Individual dots represent responses to binding of RF8117 from CHO conditioned medium by D25. The right panel shows the calculated yield of RF8117 or RF8140 in pooled CHO conditioned medium based on the D25 binding response. Both RF8117 and RF8140 were expressed in the medium, as measured by D25 and AM14 binding, demonstrating that like 293 cells, CHO cells are able to express Pre-F-NP in a folded form, allowing the prefusion trimeric structure of F to be maintained.

[0068] FIG. 10A-B . Neutralizing antibody response to Pre-F-NP RF8117. (Fig. 10A) Comparison of RSV neutralizing antibody titers obtained by immunization with high dose (1 μg) and low dose (0.1 μg) of DS-CAV1 (Pre-F trimer, SEQ ID NO: 25), post-fusion trimer F (Post-F trimer; SEQ ID NO: 24) or Pre-F-NP with glycosylation by design (Pre-F-NP; RF8117, SEQ ID NO: 17), were measured using the VERO cell assay. All RSV polypeptides were administered with AF03 adjuvant as described herein. In all cases, unless otherwise stated, AF03 was administered with, but not conjugated to, RSV polypeptide or nanoparticle. RSV polypeptides and doses are indicated under the x-axis. Statistical analysis of responses at high doses relative to Pre-F-NP immunization is shown. (Figure 10B) Comparison of RSV neutralizing antibody titers obtained by immunization with high dose (1 μg) and low dose (0.1 μg) DS-CAV1 (Pre-F trimer), Pre-F-NP without glycosylation by engineering ( RF8113, SEQ ID NO: 16) or Pre-F-NP with glycosylation by design (RF8117, SEQ ID NO: 17), as measured by the VERO cell-based assay. All RSV polypeptides were administered with AF03 adjuvant (not conjugated to any polypeptide or nanoparticle) as described herein. RSV polypeptides and doses are indicated under the x-axis.

[0069] FIG. 11A-D . Comparison of prefusion RSV F trimer binding antibody (DS-CAV1) and RSV neutralizing antibodies induced by immunization with postfusion F trimer (SEQ ID NO: 24) or Pre-F-NP (RF8140 SEQ ID NO: 23) in a mouse model or a non-human primate model. (FIG. 11A) Comparison of the prefusion F trimer binding antibody response induced in mice by immunization with postfusion F and Pre-F-NP (RF8140, SEQ ID NO: 23). (FIG. 11B) Shown are the neutralizing antibody responses elicited in mice by immunization with Ffusion F and Pre-F-NP (RF8140, SEQ ID NO: 23). (Fig. 11C) Comparison of prefusion F trimer binding antibody responses induced in non-human primates by Pre-F-NP with or without adjuvant (AF03, indicated in parentheses below). (Figure 11D) Comparison of RSV neutralizing antibody titers obtained by immunization with Pre-F-NP (RF8140, SEQ ID NO: 23) with and without AF03 adjuvant. In mice, Pre-F-NP elicits a prefusion F-binding antibody response and a higher level of RSV neutralizing antibody response than the postfusion trimer. In nonhuman primates, Pre-F-NP elicits a potent neutralizing response.

[0070] In FIG. 12A-12B show that the engineered glycosylation sites block epitopes present in the postfusion conformation. (Figure 12A) Shown is the anti-F prefusion antibody response (DS-CAV1) induced by immunization with Pre-F-NP without glycosylation by design (RF8113) or Pre-F-NP with glycosylation by design (particle with engineered Gly) at high (1 μg) and low (0.1 μg) dose, as measured by Octet. (Figure 12B) Shows the anti-fusion trimer antibody response induced by immunization with Pre-F-NP without engineered glycosylation (RF8113) or Pre-F-NP with engineered glycosylation (RF8117) at high (1 μg ) and low (0.1 μg) dose, as measured by Octet. As stated above, all RSV polypeptides were mixed with AF03 at the time of immunization. Although both RF8113 and RF8117 elicit robust prefusion anti-F antibody responses, the postfusion anti-F antibody response induced by RF8117 is significantly reduced. This is due to the presence of engineered glycans that map to epitopes common to the prefusion and postfusion conformations ( Figure 2B ).

[0071] Onfig. 13A-C blocking of epitopes that cannot be neutralized using engineered glycosylation sites is shown. (Fig. 13A) Comparison of neutralizing antibody titers to RSV obtained by immunization with pre-F-NP with wild-type glycosylation sites ("Wt glycan particle"; RF8113, SEQ ID NO: 16) and pre-F-NP with added engineered glycosylation sites ( "particle+glycan"; RF8117, SEQ ID NO: 17) at a dose of 0.1 μg in mouse studies, as measured by the VERO cell-based assay. (FIG. 13B) Comparison of RSV fusion trimer F binding antibody responses induced by immunization with Wt glycan particle (RF8113, SEQ ID NO: 16) and +glycan particle (RF8117, SEQ ID NO: 17) at a dose of 0.1 mcg in mouse studies. (Figure 13C) Ratio of measured neutralizing antibody titers to binding antibody titers from panels A and B, demonstrating that the engineered glycans did not reduce functional neutralizing antibody responses, but did reduce the level of non-neutralizing antibodies produced in response to epitopes. common to the prefusion/postfusion conformation (Figure 1B), thereby providing an improvement in the neutralizing/binding antibody ratio.

[0072]Fig. 14A-D. Characterization of RSV central G domain peptide (Gcc) conjugated to a ferritin nanoparticle. (Figure 14A) Coomassie-stained SDS-PAGE gel showing click conjugation of the central G domain of RSV (SEQ ID NO: 29) to a ferritin nanoparticle to form the Gcc-NP antigen. (Figure 14B) Structural model of Gcc-NP. (Figure 14C) Comparison of Gcc binding antibody responses induced by immunization with Gcc peptide alone (Gcc peptide, SEQ ID NO. 29) and Gcc peptide conjugated to a nanoparticle (Gcc-NP) in mouse studies. Representative responses in unexposed serum samples are shown with a white box, whereas responses from the second immunization are shown with light gray boxes, and responses from third immunization procedures are shown with dark gray boxes. (FIG. 14D) Comparison of RSV neutralizing antibody titers obtained by immunization with Gcc peptide (SEQ ID NO. 29) and Gcc-NP in mouse studies after the third injection, as measured by the HAE cell-based assay. Serum samples from naive animals and serum samples from animals immunized with Gcc peptide were pooled and titers are shown as bars.

[0073] FIG. 15A-C . Coadministration of RSV Pre-F-NP (RF8140) and Gcc-NP induces a neutralizing response. Mice were immunized with Pre-F-NP (RF8140) alone, Gcc-NP alone, or Pre-F-NP and Gcc-NP combined at a dose of 1 μg per antigen. All immunization procedures were performed using AF03 adjuvant as shown above. (Figure 15A) Immunization of mice with RF8140 alone (Pre-F-NP) or RF8140 and Gcc-NP (Pre-F-NP+Gcc-NP) generated antibodies that bind the prefusion F trimer. (Figure 15B) Immunization of mice with Gcc-NP alone (Gcc-NP) or RF8140 and Gcc-NP (Pre-F-NO+Gcc-NP) produced antibodies that bind the Gcc peptide. (FIG 15C) In animals, immunization with Pre-F-NP alone, Gcc-NP alone, or coadministration of Pre-F-NP and Gcc-NP produces a neutralizing response after the second and after the third immunization, as measured by a cell-based neutralization assay HAE. Coadministration of Pre-F-NP+Gcc-NP produced a neutralizing response superior to that caused by immunization with Pre-F-NP alone.

[0074] FIG. 16A-B . Coadministration of Pre-F-NP and Gcc-NP has no effect on mediating the formation of antibodies that bind the prefusion F trimer or the Gcc-nanoparticle. Neutralizing antibody titers measured using the F-sensitive VERO cell-based assay are shown on the left in FIG. 16A, while neutralizing antibody titers measured using the F- and G-sensitive HAE cell-based assay are shown on the right in FIG. 16B. Animal immunization procedures were the same as in FIG. 15. RSV polypeptides used in immunization are shown below the horizontal axis. Black bars represent serum samples collected from the immunization groups described in FIG. 15, and are marked in the same way. Serum samples from unexposed animals are also shown as black bars and labeled for comparison. Trimer F-depleted prefusion serum samples are shown in white, immediately to the right of the corresponding black bar. Ectodomain G-depleted serum samples are shown as diagonally shaded bars immediately to the right of the corresponding black bar. Serum samples depleted of trimer F before fusion followed by depletion of ectodomain G are shown as vertically shaded bars. (Figure 16A) Neutralizing antibody titers were observed in VERO cell-based assays for serum samples obtained from immunization with RF8140 and co-administration of RF8140+Gcc-NP, but not for serum samples from naïve animals or serum samples. , obtained by immunization with Gcc-NP alone. Depletion of serum samples from the RF8140- or RF8140+Gcc-NP-treated groups of prefusion trimer F provided a reduction in measurable neutralizing antibody titers. (Figure 16B) Neutralizing antibody titers were observed in HAE cell-based assays for serum samples from animals immunized with RF8140, Gcc-NP, or RF8140 co-administered with Gcc-NP. Serum samples from unexposed animals did not demonstrate a neutralizing response. Serum from animals immunized with RF8140 depleted of the prefusion F trimer exhibited a decrease in measurable neutralizing antibody titer. Serum from animals immunized with Gcc-NPs depleted of ectodomain G is characterized by a decrease in measurable neutralizing antibody titer. Serum from animals immunized by coadministration of RF8140 and Gcc-NP does not show a decrease in measurable neutralizing antibody titers when depleted of prefusion F trimer alone, but does show a decrease in measurable neutralizing antibody titers when depleted of both prefusion trimer F and and across the ectodomain of G. Together, these data suggest that coadministration of Pre-F-NP and Gcc-NP does not affect the respective abilities of the antigens to generate neutralizing antibodies to prefusion F or G.

[0075] FIG. 17A-B . Administration of RF8117 or RF8140 adjuvanted with AF03, SPA09, or alum produced a superior neutralizing response in mice compared with RF8117 without adjuvant. (FIG. 17A) Shown are serum neutralizing antibody titers from mice immunized with RF8117, either unadjuvanted (no adjuvant), alum adjuvanted, or AF03 adjuvant, as measured by the assay. based on VERO cells. (Figure 17B) Shown are serum neutralizing antibody titers from mice immunized with RF8117 either unadjuvanted (no adjuvant), RF8117 adjuvanted SPA09, or RF8140 adjuvanted AF03, as measured by the assay. based on VERO cells. In all cases, RF8117 or RF8140 in the adjuvanted groups of naïve mice produced higher neutralizing antibody titers than the non-adjuvanted groups.

[0076] FIG. 18A-B . Administration of RF8140 with adjuvant AF03 or SPA09 produced a superior neutralizing response in non-human primates (NHP) compared to that of immunization procedures with RF8140 without adjuvant. (Figure 18A) Pre-fusion trimer F responses measured in serum from NHPs following immunization with RF8140 without adjuvant (no adjuvant), with either AF03 adjuvant or SPA09 adjuvant (using two doses of SPA09 as indicated below) as measured by ELISA. At all time points, adjuvant AF03 or SPA09 produced a superior neutralizing response. (Fig. 18B) Neutralizing antibody titers in serum samples from NHPs immunized with RF8140 without adjuvant (no adjuvant), or with AF03 adjuvant, or with SPA09 adjuvant (two doses of SPA09 were used as indicated below), as measured using the VERO cell-based assay. In all cases, immunization with adjuvanted RF8140 resulted in higher neutralizing antibody titers than the unadjuvanted group at all time points.

[0077] FIG. 19A-B . Conjugation of RF8140 to the TLR7/8 agonist SM7/8 or TLR9 CpG agonist results in superior titers of prefusion F binding antibodies compared to RF8140 alone without adjuvant. (FIG. 19A) Shows the prefusion F trimer binding antibody response measured in serum from naïve mice, mice immunized with RF8140 without adjuvant, mice immunized with RF8140 conjugated with SM7 adjuvant. 8, RF8140, adjuvanted with 130 ng SM7/8, or RF8140, adjuvanted with 20 μg SM7/8. RF8140 conjugated to SM7/8 produced a higher titer of prefusion trimer F binding antibodies than the unadjuvanted or SM7/8 adjuvanted groups. (Figure 19B) Shows the prefusion F trimer binding antibody response measured in serum samples from naïve mice, mice immunized with RF8140 without adjuvant, mice immunized with RF8140 conjugated to a CpG adjuvant. , RF8140 adjuvanted with 680 ng CpG or RF8140 adjuvanted with 20 μg SM7/8. RF8140 conjugated to SM7/8 produced a higher titer of prefusion trimer F binding antibodies than the unadjuvanted or SM7/8 adjuvanted groups.

[0078] FIG. 20A-G . F-subunit vaccine candidates induce neutralizing antibodies directed against pre-F and a Th1 CD4 ⁺ T cell response in the MIMIC system. (Figure 20A) Anti-pre-F antibody titers in the MIMIC system were measured by AF after priming with each Ag at a molar equivalent concentration of F with 10 ng/ml pre-F-NP (n=48-49 donors per group). (Fig. 20B) Microneutralization titers were measured and presented in international units/ml (IU/ml). (Figure 20C) A ratio of pre-F antibodies to anti-post-F antibodies of >1 corresponds to a higher level of pre-F binding antibody compared to post-F binding antibody, while a ratio value of < 1 corresponds to a higher level of response in the form of Ab to post-F formation. (Figure 20D) TNFα production in activated CD154 ⁺ /CD4 ⁺ T cells restimulated with F protein-loaded target cells was measured using flow cytometry, n=48. Statistical significance was determined by Tukey-Kramer-HSD multiple comparison. (Figure 20E) The titer of pre-existing antibodies in human subjects (serostatus) was highly correlated with the magnitude of the immune response to RSV in the MIMIC system. A linear regression plot showing the relationship between the level of anti-pre-F IgG in serum samples from each donor and the overall response in the form of anti-pre-F IgG production was generated using software or an algorithm, and the p -value for the overall slope was analyzed by statistical method ( n=50). The Y axis represents anti-pre-F IgG levels obtained after priming with RSV. (Fig. 20 F) As in Fig. 20E, linear regression plot showing the relationship between the level of pre-F IgG in serum from each donor and the total level of pre-F IgG after priming with F-subunit vaccine candidates (post-F - squares, pre-F-NP - circles and DC-Cav1 - diamonds). Previous circulating pre-F IgG titers ranged from 199800 to 3037600000. Each dot represents the IgG value of each individual donor. (Figure 20G) Comparison of Gcc binding antibody responses induced by treatment with Gcc peptide alone (Gcc peptide) with those of Gcc peptide conjugated to a nanoparticle (Gcc-NP) in human B cells. The no-treatment group is shown for comparison as above.

[0079] FIG. 21A-C. Neutralizing antibody titers obtained with a low dose (0.5 μg) of Gcc RSV-ferritin nanoparticles (“Gcc-NP”). Shown are neutralizing antibody titers to RSV A strain in HAE elicited by immunization with Gcc RSV-NPs containing the Gcc RSV A2 sequence (formulated with AF03) from serum samples collected two weeks after the second immunization (2wp2) (Fig. 21A ) or two weeks after the third immunization (2wp3) (Fig. 21B), with unexposed and hyperimmune serum samples as negative and positive controls. Also shown is the titer of neutralizing antibodies to RSV strain B in HAE induced by immunization with Gcc RSV-NPs containing the Gcc RSV A2 sequence (formulated with AF03) from serum samples collected two weeks after the third immunization (2wp3) (Fig. 21C).

[0080] FIG. 22A-B . RSV strain A2 antigen-binding antibody responses induced by RSV-NP Gcc. (Figure 22A) Gcc binding antibody responses induced to Gcc strain A2 measured two weeks after the second injection (light gray boxes) and two weeks after the third injection (dark gray boxes) induced by the high dose (5 μg) Gcc RSV-NP. Serum response from a naïve mouse is shown as a negative control. (Figure 22B) Gcc-binding antibody responses induced to the Gcc A2 strain measured two weeks after the second injection (light gray boxes) and two weeks after the third injection (dark gray boxes) induced by the low dose (0.5 μg) Gcc RSV-NP.

[0081] FIG. 23A-B . RSV strain B1 antigen-binding antibody responses induced by RSV-NP Gcc. (Figure 23A) Gcc binding antibody responses induced to the Gcc B1 strain measured two weeks after the second injection (light gray boxes) and two weeks after the third injection (dark gray boxes) induced by the high dose (5 μg) Gcc RSV-NP. Serum response from a naïve mouse is shown as a negative control. (Figure 23B) Gcc-binding antibody responses induced to the Gcc B1 strain measured two weeks after the second injection (light gray boxes) and two weeks after the third injection (dark gray boxes) induced by the low dose (0.5 μg) Gcc RSV-NP.

DETAILED DESCRIPTION

[0082] RSV polypeptides are provided that can be antigenic when administered alone, adjuvanted as a single molecule, and/or as part of a nanoparticle (eg, a ferritin particle or lumazine synthase particle) that can be self-adjuvant. In some embodiments, the RSV sequence-based antigenic polypeptides comprise an RSV F polypeptide and ferritin and/or an RSV F polypeptide in which an epitope of the RSV polypeptide that is common to prefusion RSV F and postfusion RSV F polypeptide is blocked. RSV F polypeptides that drive the production of antibodies against RSV F in the prefusion conformation induced a stronger in vivo antibody response to prefusion RSV F compared with postfusion RSV F. Also disclosed herein are RSV G polypeptides containing all or part of RSV G and which may additionally contain ferritin. The RSV G and RSV F proteins are required for RSV attachment to and fusion with host cells.

[0083] RSV F exists in two conformational states, prefusion and postfusion conformations. In its native state before fusion F, RSV is a trimer consisting of 3 protomers. Thus, immunization with RSV F polypeptides in the prefusion conformation may have improved properties. In some embodiments, the RSV F polypeptide is designed to induce immunity against RSV F in a prefusion conformation. RSV G is an attachment protein responsible for the association of RSV with human airway epithelial cells.

Definitions

[0084] The term “antigenic site 0” or “site 0 epitope” as used herein refers to the site located at the top of the prefusion RSV F trimer containing amino acid residues 62-69 and 196-209 of wild-type RSV F (SEQ ID NO: 26). The site 0 epitope is a binding site for antibodies that have specificity for prefusion RSV F, such as D25 and AM14, and antibody binding to the site 0 epitope blocks RSV attachment to the cell surface (see McLellan et al., Science 340(6136):1113–1117 (2013)).

[0085] The term "antigen stability" as used herein refers to the stability of an antigen over time or in solution.

[0086] The term “cavity-filling substitutions” as used herein refers to engineered hydrophobic substitutions to fill cavities present in the prefusion RSV F trimer.

[0087] The term “F protein” or “RSV F protein” refers to the RSV protein responsible for fusion of the viral envelope with the host cell membrane during viral entry.

[0088] The term “RSV F polypeptide” or “F polypeptide” refers to a polypeptide containing at least one F protein epitope.

[0089] The term "glycan addition" as used herein refers to the addition of mutations that introduce glycosylation sites not present in wild-type RSV F, which can be engineered to increase the expression level of the construct, increase the stability of the construct, or block epitopes shared by for prefusion and postfusion conformations. A modified protein involving the addition of glycan will have a higher level of glycosylation and, therefore, a higher molecular weight. The addition of a glycan may reduce the extent to which the RSV F polypeptide produces antibodies against RSV F in the postfusion conformation.

[0090] The term “G protein” or “RSV G protein” as used herein refers to the attachment protein responsible for the association of RSV with human airway epithelial cells. An exemplary amino acid sequence of wild-type RSV G is provided as SEQ ID NO: 27. The RSV G protein contains an ectodomain (approximately amino acids 66-297 of RSV G (SEQ ID NO: 27)) that is found outside the cell. Within the RSV G ectodomain is a central conserved region (Gcc or CCR, approximately amino acids 151-193 of SEQ ID NO: 27). The CCR in RSV G contains the CX3C motif. The CX3C motif mediates the binding of the G protein to the CX3CR1 receptor.

[0091] The term “PRO helix capping” or “proline helix capping” as used herein refers to the case where the helix cap includes a proline, which can stabilize helix formation.

[0092] The term “intraprotomer stabilization substitutions” as used herein describes amino acid substitutions in RSV F that stabilize the prefusion conformation by stabilizing interactions within the protomer of the RSV F trimer.

[0093] The term “interprotomer stabilization substitutions” as used herein describes amino acid substitutions in RSV F that stabilize the prefusion conformation by stabilizing the interaction of the protomers of the RSV F trimer with each other.

[0094] The term “protease cleavage” as used herein refers to the proteolysis (sometimes referred to in the art as “excision”) of sensitive residues (eg, lysine or arginine) in a polypeptide sequence.

[0095] The term “post-fusion” as used herein in relation to RSV F refers to the stable conformation of RSV F that occurs after fusion of the virus and cell membranes.

[0096] The term "prefusion" as used herein in relation to RSV F refers to the conformation of RSV F that exists before the virus interacts with the cell.

[0097] The term "protomer" as used herein refers to the structural unit of an oligomeric protein. In the case of F RSV, the individual unit of the F RSV trimer is the protomer.

[0098] As used herein, the terms “ferritin” or “ferritin protein” refer to a protein with detectable sequence identity to ferritin from H. pylori (SEQ ID NO: 208 or 209) or other ferritin discussed herein, such as ferritin P. furiosus , Trichoplusia ni ferritin or human ferritin, which serves to store iron, for example intracellularly or in tissues, or to transport iron in the bloodstream. Such exemplary ferritins, including those that occur as two polypeptide chains known as heavy and light chains (eg, T. ni ferritin and human ferritin), are discussed in detail below. In some embodiments, ferritin includes a sequence of at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% identity to the ferritin sequence disclosed herein, for example, in Table 1 (sequence table). Ferritin may be a fragment of a full-length naturally occurring sequence.

[0099] As used herein, the term “wild-type ferritin” refers to ferritin whose sequence consists of a naturally occurring sequence. Ferritins also include full-length ferritin or a fragment of ferritin with one or more differences in its amino acid sequence from wild-type ferritin.

[00100] As used herein, the term “ferritin monomer” refers to one ferritin molecule (or, if applicable, one ferritin heavy or light chain) that has not been assembled with other ferritin molecules. A "ferritin multimer" contains several associated ferritin monomers. "Ferritin protein" includes monomeric ferritin and multimeric ferritin.

[00101] As used herein, the term “ferritin particle” refers to ferritin that has self-assembled into a globular form. Ferritin particles are sometimes called "ferritin nanoparticles" or simply "nanoparticles". In some embodiments, the ferritin particle contains 24 ferritin monomers (or, if applicable, 24 total heavy and light chains).

[00102] As used herein, the term “hybrid ferritin” refers to a ferritin comprising ferritin from H. pylori with an amino terminal extension from bullfrog ferritin. An exemplary sequence used as an amino-terminal extension from bullfrog ferritin is shown as SEQ ID NO: 217. In a hybrid ferritin, the amino-terminal extension from bullfrog ferritin can be fused to ferritin from H. pylori such that the attachment sites of the immunostimulatory moieties are evenly distributed distributed on the surface of the ferritin particle. As used herein, the term “bullfrog linker” is a linker containing the sequence of SEQ ID NO: 217. Hybrid ferritin is also sometimes referred to as “bfpFerr” or “bfp-ferritin”. Any of the constructs comprising a bullfrog sequence may be provided without the bullfrog sequence, eg, without a linker or with an alternative linker. Exemplary bullfrog linker sequences are provided in Table 1. If a bullfrog linker is shown in Table 1, the same construct can be made without the linker or with an alternative linker.

[00103] As used herein, the term “N-glycan” refers to a saccharide chain attached to a protein at the amide nitrogen of the N (asparagine) residue of the protein. Thus, N-glycan is formed through the process of N-glycosylation. This glycan may be a polysaccharide.

[00104] As used herein, the term "glycosylation" refers to the addition of a saccharide unit to a protein.

[00105] As used herein, the term “immune response” refers to the response of an immune system cell, such as a B cell, T cell, dendritic cell, macrophage, or polymorphonuclear leukocyte, to a stimulus, such as an antigen or vaccine. The immune response may include any cell of the body involved in the host defense response, including, for example, an epithelial cell that secretes interferon or a cytokine. The immune response includes, but is not limited to, the innate and/or adaptive immune response. As used herein, the term “protective immune response” refers to an immune response that protects a subject from infection (eg, prevents infection or prevents the development of disease associated with infection). Methods for measuring immune responses are well known in the art and include, for example, measuring the proliferation and/or activity of lymphocytes (such as B or T cells), secretion of cytokines or chemokines, inflammation, antibody formation, etc. Antibody" is an immune response in which antibodies are produced.

[00106] As used herein, the term "antigen" refers to an agent that elicits an immune response and/or an agent that is bound by a T cell receptor (eg, when presented by an MHC molecule) or an antibody (eg, produced by a B-cell receptor). cell) when exposed or introduced into the body. In some embodiments, the antigen induces a humoral response (eg, including the formation of antigen-specific antibodies) in the body. Alternatively or additionally, in some embodiments, the antigen elicits a cellular response (eg, involving T cells whose receptors specifically interact with the antigen) in the body. A particular antigen may elicit an immune response in one or more members of the target organism (eg, mice, rabbits, primates, humans), but not all members of the target organism's species. In some embodiments, the antigen elicits an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% representatives of the target organism species. In some embodiments, the antigen binds to an antibody and/or a T cell receptor and may or may not induce a specific physiological response in the body. In some embodiments, for example, an antigen can bind to an antibody and/or a T cell receptor in vitro, regardless of whether such interaction occurs in vivo. In some embodiments, the antigen reacts with specific humoral or cellular immune products, including inducible heterologous immunogens. Antigens include ferritin sequence-based antigenic proteins containing a ferritin polypeptide (eg, containing one or more mutations), and a non-ferritin protein polypeptide (eg, an RSV polypeptide), as described herein.

[00107] As used herein, the term "immunostimulatory fragment" refers to a fragment that is covalently attached to ferritin or an antigenic polypeptide based on a ferritin sequence and that is capable of activating a component of the immune system (either alone or when attached to ferritin or an antigenic polypeptide based on a ferritin sequence ferritin). Exemplary immunostimulatory moieties include toll-like receptor (TLR) agonists, such as TLR 4, 7, 8, or 9. In some embodiments, the immunostimulatory moiety is an adjuvant.

[00108] As used herein, the term “adjuvant” refers to a substance or carrier medium that nonspecifically enhances the immune response to an antigen. Adjuvants may include, but are not limited to, a suspension of minerals (eg, aluminum alum, aluminum hydroxide, or phosphate) onto which the antigen is adsorbed; a water-in-oil or oil-in-water emulsion in which a solution of the antigen is emulsified in mineral oil or water (for example, Freund's incomplete adjuvant). Sometimes killed mycobacteria are included (eg, Freund's complete adjuvant) to further enhance antigenicity. Immunostimulatory oligonucleotides can also be used as adjuvants (for example, see US Patent Nos. 6194388, 6207646, 6214806, 6218371, 6239116, 6339068, 6406705 and 6429199). Adjuvants may also include biological molecules such as Toll-like receptor (TLR) agonists and costimulatory molecules. The adjuvant can be administered as a separate molecule in the composition or covalently linked (conjugated) to ferritin or an antigenic polypeptide based on the ferritin sequence.

[00109] The term “RSV sequence-based antigenic polypeptide” is used herein to refer to a polypeptide containing all or part of the amino acid sequence of RSV, or a portion thereof, of sufficient length for the molecule to be antigenic to RSV. The antigenicity may be a feature of the RSV sequence as part of a construct further containing a heterologous sequence such as ferritin and/or an immunostimulatory functional group. That is, if the RSV sequence is part of a construct additionally containing a heterologous sequence, then it is sufficient that the construct can serve as an antigen that produces antibodies against RSV, regardless of whether the RSV sequence without the heterologous sequence can do so.

[00110] The terms “ferritin-based antigenic polypeptide” and “ferritin-based antigenic protein” are used interchangeably herein to refer to a polypeptide containing ferritin and a non-ferritin polypeptide (e.g., RSV polypeptide) of sufficient length such that the molecule is antigenic in regarding a polypeptide other than ferritin. The ferritin sequence-based antigenic polypeptide may further comprise an immunostimulatory moiety. The antigenicity may be a feature of a sequence other than the ferritin sequence as part of a larger construct. That is, it is sufficient that the construct can serve as an antigen against a non-ferritin polypeptide, regardless of whether the non-ferritin polypeptide can do so without ferritin (and the immunostimulatory moiety, if applicable). In some embodiments, the non-ferritin polypeptide is an RSV polypeptide, in which case the ferritin sequence-based antigenic polypeptide is also an "RSV sequence-based antigenic polypeptide." However, for clarity, it should be noted that an antigenic polypeptide based on the RSV sequence does not necessarily contain ferritin. The term “antigenic polypeptide” is used herein to refer to a polypeptide that is either of a ferritin sequence-based antigenic polypeptide and an RSV sequence-based antigenic polypeptide, or both.

[00111] As used herein, the term “self-adjuvant” refers to a composition or polypeptide comprising ferritin and an immunostimulatory moiety directly conjugated to ferritin such that the ferritin and the immunostimulatory moiety are contained within a single molecular entity. An antigenic polypeptide based on the ferritin sequence, including a polypeptide other than ferritin, can be conjugated to an immunostimulatory moiety to form a self-adjuvant polypeptide.

[00112] As used herein, the term "surface exposed" amino acid refers to an amino acid residue in a protein (eg, ferritin) with a side chain that is capable of contact with solvent molecules when the protein is in its native three-dimensional conformation after multimerization, if applicable. Thus, for example, in the case of ferritin that forms a 24-mer, the surface exposed amino acid residue is one whose side chain is capable of contacting a solvent when the ferritin is assembled as a 24-mer, eg, as a ferritin multimer or ferritin particle.

[00113] As used herein, the term “subject” refers to any member of the animal kingdom. In some embodiments, “subject” refers to people. In some embodiments, “subject” refers to non-human animals. In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In certain embodiments, the non-human subject is a mammal (e.g. rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cattle, primate and/or pig). In some embodiments, the subject may be a transgenic animal, a genetically engineered animal, and/or a clone. In certain embodiments of the present invention, the subject is an adult, adolescent, or infant. In some embodiments, the terms “individual” or “patient” are used and are intended to be interchangeable with the term “subject.”

[00114] As used herein, the terms “vaccinate” or “vaccinate” refer to the administration of a composition designed to generate an immune response, for example, to a disease-causing factor. Vaccination can be administered before, during, and/or after exposure to a disease-causing agent and/or development of one or more symptoms, and, in some embodiments, before, during, and/or shortly after exposure to the agent. In some embodiments, vaccination includes repeated administration of the vaccination composition at appropriate intervals.

[00115] The present invention describes nucleic acid sequences and amino acid sequences that have a certain degree of identity to a given nucleic acid sequence or amino acid sequence, respectively (reference sequence).

[00116] "Sequence identity" of two nucleic acid sequences indicates the percentage of nucleotides that are identical in the two sequences. The "sequence identity" of two amino acid sequences indicates the percentage of amino acids that are identical in the two sequences.

[00117] The terms "% identical", "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids that are identical when optimally aligned in the compared sequences. The percentages stated are purely statistical and the differences between two sequences may, but need not, be randomly distributed over the entire length of the sequences being compared. Comparison of two sequences is typically accomplished by comparing said sequences after optimal alignment with respect to a segment or “window of comparison” to identify local regions of the corresponding sequences. Optimal alignment for comparison can be done manually or using the local homology search algorithm according to Smith and Waterman, 1981, Ads App. Math. 2, 482, using the local homology search algorithm according to Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, using the matching algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or by computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Drive, Madison, WI).

[00118] Percent identity is obtained by determining the number of identical positions at which the compared sequences match, dividing this number by the number of compared positions (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[00119] In some embodiments, the degree of identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or approximately 100% of the full length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, then the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides , in some embodiments, for continuous nucleotides. In some embodiments, the degree of identity is given for the full length of the reference sequence.

[00120] Nucleic acid sequences or amino acid sequences that have a certain degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, may have at least one functional property of that specified sequence, for example, and in some cases are functionally equivalent to that specified sequence. One important property includes the ability to act as a cytokine, particularly when administered to a subject. In some embodiments, a nucleic acid sequence or amino acid sequence having a certain degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to the specified sequence.

[00121] As used herein, the term “kit” refers to a packaged collection of associated components, such as one or more compounds or compositions and one or more associated materials, such as solvents, solutions, buffers, instructions, or drying agents.

RSV sequence-based antigenic polypeptides containing an RSV F polypeptide containing one or more asparagine residues at specific positions

[00122] Provided herein are RSV sequence-based antigenic polypeptides comprising an RSV F polypeptide. The RSV F polypeptide may comprise the entire RSV F sequence or a portion of the RSV F sequence. In some embodiments, an epitope of an RSV polypeptide that is common to prefusion RSV F and postfusion RSV F is blocked. Epitope blocking reduces or eliminates the formation of antibodies against the epitope when an antigenic polypeptide based on the RSV sequence is administered to a subject. This may provide an increase in the proportion of antibodies targeting an epitope specific for a particular F conformation, such as the prefusion conformation. Because F has a prefusion conformation in viruses that have not yet entered cells, an increased proportion of antibodies targeting prefusion F may provide a greater degree of neutralization (eg, expressed as the ratio of neutralization to binding, as described herein). Blocking can be achieved by designing a bulky functional group, such as an N-glycan, near a common epitope. For example, an N-glycosylation site not present in wild-type F may be added, for example by mutating the corresponding residue to asparagine. In some embodiments, the blocked epitope is an RSV F antigenic site 1 epitope. In some embodiments, two or more epitopes common to prefusion RSV F and postfusion RSV F are blocked. In some embodiments, two or more epitopes of the RSV 1 F antigenic site are blocked. In some embodiments, one, or more, or all epitopes that topologically overlap with the blocked epitope are also blocked, wherein the blocked epitope is not necessarily an RSV 1 F antigenic site epitope.

[00123] In some embodiments, the RSV F polypeptide contains an asparagine corresponding to position 328, 348, or 507 of SEQ ID NO: 26. In some embodiments, the polypeptide contains asparagine residues that correspond to at least two of positions 328, 348, or 507 of SEQ ID NO: 26. In some embodiments, the polypeptide contains asparagine residues that correspond to positions 328, 348, or 507 of SEQ ID NO: 26. As described in the Examples, it has been discovered that such asparagine residues can function as glycosylation sites. Additionally, without being limited by any particular theory, when a polypeptide is administered to a subject, the presence of glycans at these sites may inhibit the development of antibodies against nearby epitopes, which include epitopes common to the pre- and postfusion RSV F protein. In some embodiments, glycosylation of the asparagine corresponding to position 328, 348, or 507 of SEQ ID NO: 26 blocks at least one epitope common to prefusion RSV F and postfusion RSV F, such as an antigenic site 1 epitope. Developmental Inhibition antibodies against epitopes common to the pre- and post-fusion RSV F protein may be beneficial as it may provide direction towards the development of antibodies against epitopes specific to the pre-fusion RSV F protein, such as the site 0 epitope, which may be more effective neutralizing activity compared to antibodies against other RSV F epitopes. The site 0 epitope comprises amino acid residues 62-69 and 196-209 of SEQ ID NO: 26. Therefore, in some embodiments, the RSV F polypeptide comprises amino acid residues 62-69 and 196-209 of SEQ ID NO: 26.

[00124] It should be noted that the constructs described herein may have deletions or substitutions of varying lengths relative to wild-type RSV F. For example, in the construct at SEQ ID NO: 23 and others, positions 98-144 of the wild type sequence (SEQ ID NO: 26) are replaced with GSGNVGL (positions 98-104 of SEQ ID NO: 23; also SEQ ID NO: 31), resulting in to a total of 40 amino acids removed such that positions 328, 348, or 507 of SEQ ID NO: 26 correspond to positions 288, 308, and 467 of SEQ ID NO: 23. In general, positions in the constructs described herein may be mapped to the wild-type sequence from SEQ ID NO: 26 by pairwise alignment, for example, using the Needleman-Wunsch algorithm with standard parameters (EBLOSUM62 matrix, gap penalty 10, gap extension penalty 0.5). see also a discussion of structural alignment presented in this document as an alternative approach to identifying relevant provisions.

[00125] In some embodiments, the RSV F polypeptide contains mutations that add glycans to block epitopes on the prefusion antigen that are structurally similar to those on the postfusion RSV F surface. In some embodiments, glycans are added to specifically block epitopes that may be present on RSV F in the postfusion conformation. In some embodiments, glycans are added that block epitopes that may be present in the RSV F in the postfusion conformation, but have no effect on one or more epitopes present in the RSV F in the prefusion conformation, such as the site 0 epitope.

[00126] In some embodiments, glycans added at one or more of the glycosylation sites discussed above provide increased levels of secretion in expression systems, such as mammalian cells, compared to other constructs.

[00127] In some embodiments, the RSV F polypeptide comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identity to amino acids 1-478 of SEQ ID NO: 17 In some embodiments, the RSV F polypeptide comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 17. In some embodiments, The RSV F polypeptide contains amino acids 1-478 of SEQ ID NO: 17. In some embodiments, the RSV F polypeptide contains the sequence of SEQ ID NO: 17.

[00128] In some embodiments, the RSV F polypeptide comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identity to amino acids 1-478 of SEQ ID NO: 23 In some embodiments, the RSV F polypeptide comprises a sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 23. In some embodiments, The RSV F polypeptide contains amino acids 1-478 of SEQ ID NO: 23. In some embodiments, the RSV F polypeptide contains the sequence of SEQ ID NO: 23.

[00129] In some embodiments, the RSV F polypeptide comprises a DS-CAV1 sequence (described, for example, in McLellan, JS, et al., Science 342(6158):592-598 (2013)) (SEQ ID NO: 25), wherein further modifications are made including at least one, two or three of the asparagine residues described above.

[00130] In some embodiments, the polypeptide further comprises ferritin protein. The ferritin protein may additionally have any of the features described below in the section regarding ferritin, or a combination thereof.

[00131] The RSV F polypeptide may further have any of the additional features presented in the following discussion, or any possible combination of such features.

Single-stranded designs

[00132] In some embodiments, the RSV polypeptide is a single chain construct, such as an RSV polypeptide that does not contain furin cleavage sites. In some embodiments, RSV F does not contain one or more furin cleavage sites. Constructs that do not contain furin cleavage sites are expressed as complete polypeptides that are not cleaved into biological F1/F2 fragments of the native F protein.

Amino acid substitutions

[00133] In some embodiments, RSV F contains one amino acid substitution relative to the wild type sequence. In some embodiments, RSV F contains more than one amino acid substitution, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 substitutions, relative to the wild-type sequence. An exemplary wild-type sequence is SEQ ID NO: 26.

[00134] In some embodiments, the amino acid substitution or pair of amino acid substitutions is the substitution(s) that provide interprotomer stabilization. Exemplary substitutions that may provide interprotomer stabilization are V207L; N228F; I217V and E218F; I221L and E222M; or Q224A and Q225L, when applying the position numbering from SEQ ID NO: 26.

[00135] In some embodiments, the amino acid substitution or pair of amino acid substitutions are substitutions that provide intraprotomer stabilization. Exemplary replacements that may provide intraprotomer stabilization are V220I, as well as A74L and Q81L, when applying the position numbering of SEQ ID NO: 26.

[00136] In some embodiments, the amino acid substitution provides helix stabilization, i.e. it is predicted to mediate stabilization of the helical F domain of RSV. Stabilization of the helical domain may contribute to the stability of the site 0 epitope and overall RSV F in the prefusion conformation. Exemplary replacements that may provide helix stabilization are N216P or I217P, when applying the position numbering of SEQ ID NO: 26.

[00137] In some embodiments, the amino acid substitution is a helix capping substitution. In some embodiments, the amino acid substitution is a substitution that provides PRO helix capping. Helix capping is based on the observation, using biophysical methods, that while the presence of a mutation at the site of a proline residue in an alpha helix can disrupt helix formation, the presence of a proline at the N-terminus of the helical region can help induce helix formation by stabilizing bond angles PHI/PSI. Exemplary replacements that may provide helix capping are N216P or I217P, when applying the position numbering of SEQ ID NO: 26.

[00138] In some embodiments, the amino acid substitution replaces the disulfide mutation of DS-CAV1. In some embodiments, the engineered disulfide in DS-CAV1 reverts to wild type (mutations C69S and/or C212S in DS-CAV1, using the position numbering of SEQ ID NO: 26). In some embodiments, one or more C residues in DS-CAV1 are replaced with an S residue to eliminate the disulfide bond. In some embodiments, the C69S or C212S substitution, when applying the position numbering of SEQ ID NO: 26, eliminates the disulfide bond. In some embodiments, the RSV F polypeptide contains both C69S and C212S, using the position numbering of SEQ ID NO: 26. In some embodiments, substitution of such cysteine residues, thereby eliminating the disulfide bond, provides reduction blocking (i.e., electron gain from the reducing agent) RSV F polypeptide. In some embodiments, the I217P substitution, using the position numbering of SEQ ID NO: 26, is included in the antigen in lieu of the C69 and/or C212 substitution. Position 217 in SEQ ID NO: 26 corresponds to position 177 in SEQ ID NO: 23.

[00139] In some embodiments, the amino acid substitution prevents proteolysis by trypsin or trypsin-like proteases. In some embodiments, the amino acid substitution that prevents such proteolysis is in the heptad repeat B region (HRB) of RSV F. The appearance of proteolysis-permissive fragments in the RSV-ferritin F construct, which contained the wild-type HRB region, suggests that the lysine or arginine in this region was a target for proteolysis. An amino acid substitution that removes a K or R residue may be called a knockout (KO). In some embodiments, K or R is replaced by L or Q. In some embodiments, K is replaced by L or Q. In some embodiments, the RSV F polypeptide comprises K498L and/or K508Q, when applying the position numbering of SEQ ID NO: 26. Respectively the positions in SEQ ID NO: 23 are 458 and 468, respectively. In some embodiments, the RSV F polypeptide contains both K498L and K508Q.

[00140] In some embodiments, the amino acid substitution provides the addition of glycans. In some embodiments, the amino acid substitution provides an increase in the level of glycosylation by adding glycans to RSV F polypeptides. Substitutions that provide added glycans can also be referred to as glycosylation by design versus natural glycosylation (without additional glycans).

[00141] In some embodiments, the amino acid substitution for adding glycans is an N substitution. In some embodiments, the N amino acid substitution allows for N-linked glycosylation. In some embodiments, a substitution with N is accompanied by a substitution with T or S of the amino acid in the second position from N toward the C terminus, thereby forming the NxT/S glycosylation motif. In some embodiments, N is exposed on the surface. As shown in the examples below, mutations that provide an increase in the level of glycosylation can provide an increase in the level of expression of a polypeptide containing the RSV F polypeptide.

Changes in the properties of the RSV F polypeptide depending on modifications

[00142] Modifications to the amino acid sequence of RSV F may alter the properties of the RSV F polypeptide. An RSV F polypeptide property may include any structural or functional characteristic of an RSV F polypeptide.

[00143] In some embodiments, a single amino acid sequence modification alters multiple properties of the RSV F polypeptide. In some embodiments, the RSV F polypeptide may contain multiple modifications that alter various properties of the RSV F polypeptide. In some embodiments, multiple modifications cause a greater change in the properties of the RSV F polypeptide.

[00144] In some embodiments, multiple modifications may have an additive effect on a particular property. For example, two amino acid substitutions to add glycans can cause a greater increase in glycosylation of the RSV F polypeptide compared to any single amino acid substitution.

[00145] In some embodiments, multiple modifications have an effect on different properties of the RSV F polypeptide. For example, one or more amino acid substitutions to increase the level of glycosylation may be made together with one or more amino acid substitutions to block reduction.

[00146] In some embodiments, modifications to the CSV F polypeptide provide stabilization of the prefusion conformation.

[00147] In some embodiments, modifications provide stabilization of the site 0 epitope (also known as antigenic site 0) of RSV F prior to fusion, as described, for example, in McLellan et al., Science 340(6136):1113-1117 (2013). In some embodiments, the modification that provides stabilization of the site 0 epitope is a modification that provides interprotomer stabilization. In some embodiments, the modification that stabilizes the site 0 epitope stabilizes prefusion F as measured by binding to site 0 and site V as measured by binding to D25 or AM14 antibodies, respectively.

[00148] In some embodiments, modifications provide increased expression levels of RSV F in expression systems. In some embodiments, the modifications provide an increase in the level of RSV F secretion in expression systems. In some embodiments, the modifications provide increased stability of the recombinant RSV F after expression. This change can occur in any type of expression system, such as a bacterial, fungal, insect or mammalian based expression system.

[00149] In some embodiments, amino acid substitutions that introduce proline provide increased expression levels compared to other constructs. In some embodiments, amino acid substitutions that add glycans provide increased expression levels compared to other constructs. In some embodiments, amino acid substitutions that replace K or R with other amino acids provide increased expression levels compared to other constructs. The observed increase in expression level may be due to any mechanism that increases the yield of a fermentation or other production process, including appropriate inhibition of protease cleavage or degradation and/or increased stability in the host cell or extracellular environment. In some embodiments, amino acid substitutions that replace one or more K residues in the HRB F region of RSV with different amino acids provide increased expression compared to other constructs.

[00150] In some embodiments, amino acid substitutions that replace K with other amino acids provide increased stability of RSV F polypeptides. In some embodiments, amino acid substitutions that replace one or more K residues in the HRB region of RSV F with other amino acids provide increased stability of RSV F polypeptides. In some embodiments, such increased stability is due to reduced levels of protease degradation.

[00151] In some embodiments, RSV F contains mutation(s) that provide disulfide removal, for example to prevent conjugation after reduction. In some embodiments, the I217P substitution blocks the reduction of the RSV F polypeptide. In some embodiments, amino acid substitutions that replace K with other amino acids block the reduction of RSV F polypeptide in the presence of a reducing agent.

[00152] In some embodiments, single-strand constructs provide increased expression levels compared to other constructs.

[00153] In some embodiments, the RSV F polypeptide contains the sequence DS-CAV1 (SEQ ID NO: 25) (as described in McLellan, JS, et al., Science 342(6158):592-598 (2013)). In some embodiments, the RSV F polypeptide comprises a DS-CAV1 sequence that is further modified, for example, including at least one, two, or three asparagine residues described above.

RSV G polypeptides

[00154] As used herein, an RSV G polypeptide may comprise the entire RSV G sequence or a portion of RSV G. The RSV G polypeptide may contain modifications compared to the wild type sequence. In some embodiments, the RSV G polypeptide is RSV G modified from wild-type RSV G (SEQ ID NO: 27).

[00155] In some embodiments, such modifications are amino acid changes of the RSV G polypeptide compared to wild-type RSV G.

[00156] In some embodiments, the RSV G polypeptide comprises all or a portion of the RSV G ectodomain (SEQ ID NO: 28 or positions corresponding thereto). In some embodiments, the RSV G polypeptide comprises all or a portion of the Gcc region (amino acids 151-193 of RSV G (SEQ ID NO: 27)). In some embodiments, the RSV G polypeptide contains a CX3C motif. In some embodiments, RSV polypeptide G binds to the CX3CR1 receptor. The Gcc region is both conserved and immunogenic and can therefore be used to generate antibodies with broad activity against RSV strains. In some embodiments, the Gcc RSV of strain A is provided, as shown in SEQ ID NO: 32. In some embodiments, the Gcc RSV of strain B is provided, as shown in SEQ ID NO: 33.

[00157] In some embodiments, the RSV G polypeptide is not glycosylated. For example, an RSV G polypeptide may lack NXS/TX glycosylation sites, either due to truncation or mutation of N or S/T residues (eg, substitution of Q or A, respectively), or a combination thereof.

[00158] In some embodiments, the RSV G polypeptide is part of an antigenic polypeptide based on a ferritin sequence. For example, RSV polypeptide G can be conjugated to ferritin as described herein, for example through a surface exposed cysteine in ferritin. In some embodiments, such a ferritin nanoparticle is a fusion protein also comprising an RSV F polypeptide, such as any of the RSV F polypeptide and ferritin protein-containing polypeptides described above.

Antigenic polypeptides based on the RSV sequence containing ferritin

[00159] Also provided herein is an RSV sequence-based antigenic polypeptide containing ferritin and an RSV polypeptide. The RSV polypeptide may be an RSV F polypeptide, such as any of the RSV F polypeptides described herein. The RSV F polypeptide may comprise the entire RSV F sequence or a portion of the RSV F sequence. The RSV F polypeptide may contain one or more modifications (eg, amino acid substitution) compared to the wild type sequence. The RSV polypeptide may be an RSV G polypeptide, such as any of the RSV G polypeptides described herein.

[00160] In some embodiments, the ferritin in the polypeptide is wild-type ferritin. In some embodiments, ferritin is derived from bacteria, insects, fungi, birds, or mammals. In some embodiments, the ferritin is human. In some embodiments, the ferritin is bacterial.

[00161] In some embodiments, the ferritin is a ferritin light chain and/or a ferritin heavy chain. In some embodiments, the ferritin is an insect ferritin, such as ferritin heavy chain from Trichoplusia ni (SEQ ID NO: 211) or ferritin light chain from Trichoplusia ni (SEQ ID NO: 212). In some embodiments, the ferritin is human ferritin, such as human ferritin heavy chain (SEQ ID NO: 214 or FTH1, GENE ID No: 2495) or human ferritin light chain (SEQ ID NO: 215 or FTL, GENE ID No: 2512 ). In some embodiments, the ferritin nanoparticle contains a total of 24 ferritin heavy chain and ferritin light chain subunits, such as in human or Trichoplusia ni ferritin nanoparticles. Ferritin nanoparticles from T. ni can contain 12 ferritin heavy chain subunits and 12 ferritin light chain subunits.

[00162] In some embodiments, the RSV sequence-based antigenic polypeptide comprises a ferritin light chain and an RSV polypeptide. In some embodiments, the RSV sequence-based antigenic polypeptide comprises a ferritin heavy chain and an RSV polypeptide. In some embodiments, an RSV sequence-based antigenic polypeptide comprising a ferritin light chain and an RSV polypeptide may assemble with a ferritin heavy chain that is not associated with the RSV polypeptide. In some embodiments, an RSV sequence-based antigenic polypeptide comprising a ferritin heavy chain and an RSV polypeptide may assemble with a ferritin light chain that is not associated with the RSV polypeptide. Ferritin not bound to an RSV polypeptide (or, more broadly, a non-ferritin polypeptide) may be referred to as "naked ferritin".

[00163] In some embodiments, an antigenic polypeptide comprising a ferritin heavy chain and a polypeptide can be assembled with an antigenic polypeptide comprising a ferritin light chain and an RSV polypeptide, allowing the two same or different non-ferritin polypeptides to be presented on a single ferritin nanoparticle. In some embodiments, the two different polypeptides other than ferritin are RSV polypeptides. In some embodiments, two different polypeptides other than ferritin are encoded by RSV and another infectious agent. In some embodiments, the other polypeptide, other than ferritin, from a different infectious agent is derived from a virus or bacterium.

[00164] In some embodiments, an antigenic polypeptide containing a ferritin heavy chain and a non-ferritin polypeptide can be combined with a polypeptide containing a ferritin light chain and a non-ferritin polypeptide to produce a divalent composition in which one or both polypeptides are different from ferritin are RSV polypeptides, such as RSV F or G polypeptides, such as RSV F or G polypeptide described herein.

[00165] In some embodiments, the ferritin is ferritin fromH. pylori (cm. SEQ ID NO: 208 or 209 for an exemplary ferritin sequence fromH. pylori), optionally with one or more mutations such as those described herein. In some embodiments, a lower degree of sequence homology between ferritin fromH. pylori (or other bacterial ferritin variants) and human ferritin may reduce the potential for autoimmunity when used as a vaccine platform (see Kanekiyo et al., Cell 162, 1090-1100 (2015)).

[00166] In some embodiments, the ferritin is ferritin from Pyrococcus furiosus ( NCBI seq WP_011011871.1) with one or more mutations described herein.

[00167] In some embodiments, ferritin includes a sequence characterized by greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 97%, greater than 98 % or greater than 99% identity with wild-type ferritin.

[00168] In some embodiments, a nanoparticle comprising an RSV sequence-based antigenic polypeptide disclosed herein is provided, comprising an RSV polypeptide and ferritin.

[00169] In some embodiments, another protein capable of forming a nanoparticle replaces ferritin. In some embodiments, the protein is lumazine synthase (see Ra et al., Clin Exp Vaccine Res. 3:227-234 (2014)). In some embodiments, the protein is a lumazine synthase serotype 1, 2, 3, 4, 5, 6, or 7. Exemplary lumazine synthase sequences are provided as SEQ ID NOs: 216 and 219. In some embodiments, the lumazine synthase comprises 80%, 85% sequence , 90%, 95%, 98% or 99% identity with SEQ ID NO: 216 or 219.

1. Cysteine for conjugation

[00170] In some embodiments, ferritin is mutated to provide a chemical basis for conjugation of an immunostimulatory functional group and/or RSV polypeptide. This can be achieved by mutation to replace a surface-exposed amino acid other than cysteine with cysteine. For the avoidance of doubt, language such as “replacing a surface-exposed amino acid with a cysteine” necessarily implies that the surface-exposed amino acid in the wild-type or premutation sequence is not a cysteine. Another approach to provide a chemical basis for conjugation of an RSV immunostimulatory functional group or polypeptide is to include an amino acid segment, such as a linker, at the N- or C-terminus of ferritin, wherein the amino acid segment contains cysteine. In some embodiments, this cysteine (whether it replaces a surface-exposed amino acid or in an N- or C-terminal linker) is unpaired, meaning that it does not have a suitable cysteine partner to form a disulfide bond. In some embodiments, this cysteine does not alter the secondary structure of ferritin. In some embodiments, this cysteine does not alter the tertiary structure of ferritin.

[00171] In some embodiments, this cysteine can be used to conjugate agents, such as immunostimulatory moieties, to ferritin. In some embodiments, this cysteine provides a free thiol group that is reactive. In some embodiments, agents conjugated to this cysteine on ferritin are exposed on the surface of the assembled ferritin particle. In some embodiments, the cysteine is capable of interacting with molecules and cells of the subject after administration during ferritin particle assembly.

[00172] In some embodiments, the presence of this cysteine enables conjugation of one or more immunostimulatory moieties, such as adjuvants. In some embodiments, conjugation of the immunostimulatory moiety would not occur in the absence of this cysteine.

[00173] In some embodiments, the non-cysteine amino acid that is replaced with cysteine is selected from E12, S72, A75, K79, S100, and S111 from H. pylori ferritin. Thus, in some embodiments, the surface-exposed amino acid that is replaced by a cysteine is an amino acid residue that corresponds to E12, S26, S72, A75, K79, S100, or S111 from H. pylori ferritin. Similar amino acids can be found in ferritin other than ferritin from H. pylori , through pairwise or structural alignment. In some embodiments, the non-cysteine amino acid that is replaced with cysteine is selected from an amino acid that corresponds to S3, S19, S33, I82, A86, A102, and A120 of the human ferritin light chain. In some embodiments, the surface exposed amino acid to be replaced with cysteine is selected based on the understanding that if the native amino acid were replaced with cysteine, it would be reactive in the assembled ferritin multimer or ferritin particle, and/or that the cysteine does not disrupt stability of the ferritin multimer or ferritin particle, and/or that this cysteine does not reduce ferritin expression levels.

[00174] In some embodiments, ferritin contains the E12C mutation. In some embodiments, the E12C residue may be used to conjugate agents (eg, immunostimulatory functional groups and/or RSV polypeptides) to ferritin. In some embodiments, the E12C moiety provides a free thiol group that is reactive. In some embodiments, agents conjugated to the E12C residue on ferritin monomers are expressed on the surface of the assembled ferritin multimer or ferritin particle. In some embodiments, twenty-four E12C residues (one from each monomer) are present on the surface of the ferritin multimer or ferritin particle.

[00175] In some embodiments, ferritin contains the S26C mutation. In some embodiments, the S26C residue may be used to conjugate agents (eg, immunostimulatory functional groups and/or RSV polypeptides) to ferritin. In some embodiments, the S26C residue provides a free thiol group that is reactive. In some embodiments, agents conjugated to the S26C residue on ferritin monomers are expressed on the surface of the assembled ferritin multimer or ferritin particle. In some embodiments, twenty-four S26C residues (one from each monomer) are present on the surface of the ferritin multimer or ferritin particle.

[00176] In some embodiments, ferritin contains the S72C mutation. In some embodiments, the S72C residue may be used to conjugate agents (eg, immunostimulatory functional groups and/or RSV polypeptides) to ferritin. In some embodiments, the S72C residue provides a free thiol group that is reactive. In some embodiments, agents conjugated to the S72C residue on ferritin monomers are expressed on the surface of the assembled ferritin multimer or ferritin particle. In some embodiments, twenty-four S72C residues (one from each monomer) are present on the surface of the ferritin multimer or ferritin particle.

[00177] In some embodiments, ferritin contains the A75C mutation. In some embodiments, the A75C residue may be used to conjugate agents (eg, immunostimulatory functional groups and/or RSV polypeptides) to ferritin. In some embodiments, the A75C residue provides a free thiol group that is reactive. In some embodiments, agents conjugated to the A75C residue on ferritin monomers are expressed on the surface of the assembled ferritin multimer or ferritin particle. In some embodiments, twenty-four A75C residues (one from each monomer) are present on the surface of the ferritin multimer or ferritin particle.

[00178] In some embodiments, ferritin contains the K79C mutation. In some embodiments, the K79C residue may be used to conjugate agents (eg, immunostimulatory functional groups and/or RSV polypeptides) to ferritin. In some embodiments, the K79C residue provides a free thiol group that is reactive. In some embodiments, agents conjugated to the K79C residue on ferritin monomers are expressed on the surface of the assembled ferritin multimer or ferritin particle. In some embodiments, twenty-four K79C residues (one from each monomer) are present on the surface of the ferritin multimer or ferritin particle.

[00179] In some embodiments, ferritin contains the S100C mutation. In some embodiments, the S100C residue may be used to conjugate agents (eg, immunostimulatory functional groups and/or RSV polypeptides) to ferritin. In some embodiments, the S100C residue provides a free thiol group that is reactive. In some embodiments, agents conjugated to the S100C residue on ferritin monomers are expressed on the surface of the assembled ferritin multimer or ferritin particle. In some embodiments, twenty-four S100C residues (one from each monomer) are present on the surface of the ferritin multimer or ferritin particle.

[00180] In some embodiments, ferritin contains the S111C mutation. In some embodiments, the S111C residue may be used to conjugate agents (eg, immunostimulatory functional groups and/or RSV polypeptides) to ferritin. In some embodiments, the S111C residue provides a free thiol group that is reactive. In some embodiments, agents conjugated to the S111C residue on ferritin monomers are expressed on the surface of the assembled ferritin multimer or ferritin particle. In some embodiments, twenty-four S111C residues (one from each monomer) are present on the surface of the ferritin multimer or ferritin particle.

2. Removal of internal cysteine

[00181] In some embodiments, ferritin contains a mutation that replaces the internal cysteine with an amino acid other than cysteine. Removal of the native internal cysteine residue can ensure that there is only one unpaired cysteine in the ferritin monomer, which avoids unwanted reactions such as disulfide bond formation and can lead to a more stable and effective outcome (e.g., adjuvant presentation). In some embodiments, C31 from H. pylori ferritin is replaced with an amino acid other than cysteine. In some embodiments, C31 from H. pylori ferritin is replaced by serine (C31S), although any residue other than cysteine, such as alanine, glycine, threonine, or asparagine, can be used. Similar amino acids can be found in ferritin other than H. pylori ferritin, through pairwise or structural alignment. Thus, in some embodiments, the internal cysteine that is replaced with a non-cysteine amino acid is an amino acid residue that aligns with C31 from H. pylori ferritin. Exemplary ferritin sequences demonstrating the C31S mutation are shown in SEQ ID NOs: 201-207. In some embodiments, when more than one internal cysteine is present in ferritin, two or more (eg, each) internal cysteines are replaced with an amino acid other than cysteine, such as serine, or an amino acid selected from serine, alanine, glycine, threonine, or asparagine.

3. Glycosylation

[00182] Glycosylation compatible with human glycosylation may contribute to the safety and efficacy of recombinant drug products. Regulatory approval may be contingent on demonstration of appropriate glycosylation as a critical quality attribute (see Zhang et al., Drug Discovery Today 21(5):740–765 (2016)). N-glycans can be formed by glycosylation of asparagine side chains and can vary in structure between humans and other organisms such as bacteria and yeast. Thus, it may be desirable to reduce or eliminate non-human glycosylation and/or N-glycan formation in ferritin in accordance with the present invention. In some embodiments, control of ferritin glycosylation improves the efficacy and/or safety of the composition, particularly when used for human vaccination.

[00183] In some embodiments, ferritin is mutated to inhibit N-glycan formation. In some embodiments, the mutated ferritin has reduced glycosylation compared to its corresponding wild-type ferritin.

[00184] In some embodiments, ferritin contains a mutation that replaces the surface-exposed asparagine with a non-asparagine amino acid. In some embodiments, the surface exposed asparagine is N19 of H. pylori ferritin or is at a position that corresponds to position 31 of H. pylori ferritin as determined by pairwise or structural alignment. In some embodiments, mutating such an asparagine, such as N19 from H. pylori ferritin, reduces the glycosylation of ferritin. In some embodiments, the mutation replaces asparagine with glutamine. In some embodiments, the ferritin is ferritin from H. pylori including the N19Q mutation. SEQ ID NOs: 201-207 are exemplary ferritin sequences including N19Q mutations.

[00185] A mammal exposed to a glycosylated protein produced in bacteria or yeast may generate an immune response to the glycosylated protein because the glycosylation pattern of the protein in the bacteria or yeast may be different from the glycosylation pattern of the same protein in mammals. Thus, some glycosylated therapeutic proteins may not be suitable for production in bacteria or yeast.

[00186] In some embodiments, reducing ferritin glycosylation by amino acid mutation facilitates protein production in bacteria or yeast. In some embodiments, reducing the glycosylation of ferritin reduces the potential for adverse effects to occur in mammals when administered with mutant ferritin that is expressed in bacteria or yeast. In some embodiments, the reactogenicity of the mutant ferritin produced in bacteria or yeast is lower in a human subject because glycosylation is reduced. In some embodiments, the incidence of hypersensitivity responses in human subjects is lower following treatment with reduced glycosylation mutant ferritin compared to wild-type ferritin.

[00187] In some embodiments, in a subject, degradation of a composition comprising a reduced glycosylation mutant ferritin occurs more slowly compared to a composition containing wild-type ferritin or a composition containing a corresponding wild-type ferritin with glycosylation. In some embodiments, a composition containing a reduced glycosylation mutant ferritin has reduced clearance in a subject compared to a composition containing wild-type ferritin or a composition containing a corresponding wild-type ferritin with glycosylation. In some embodiments, a composition comprising a reduced glycosylation mutant ferritin has a longer serum half-life compared to wild-type ferritin or a composition containing a corresponding wild-type ferritin with glycosylation.

4. Combinations of mutations

[00188] In some embodiments, ferritin includes more than one type of mutation described herein. In some embodiments, ferritin contains one or more mutations independently selected from a mutation to reduce glycosylation, a mutation to remove an internal cysteine, and a mutation to produce a surface-exposed cysteine. In some embodiments, ferritin contains a mutation to reduce glycosylation, a mutation to remove an internal cysteine, and a mutation to produce a surface-exposed cysteine.

[00189] In some embodiments, ferritin contains an N19Q mutation, a C31S mutation, and a mutation to produce a surface-exposed cysteine. In some embodiments, ferritin contains the N19Q mutation, the C31S mutation, and the E12C mutation. In some embodiments, ferritin contains the N19Q mutation, the C31S mutation, and the S72C mutation. In some embodiments, ferritin contains the N19Q mutation, the C31S mutation, and the A75C mutation. In some embodiments, ferritin contains the N19Q mutation, the C31S mutation, and the K79C mutation. In some embodiments, ferritin contains the N19Q mutation, the C31S mutation, and the S100C mutation. In some embodiments, ferritin contains the N19Q mutation, the C31S mutation, and the S111C mutation. In some embodiments, ferritin includes mutations corresponding to any of the above groups of mutations, wherein the corresponding mutations change N to Q, C to S, and a non-cysteine surface exposed amino acid to cysteine at positions determined by pairwise alignment of the ferritin amino acid sequence with the amino acid sequence ferritin from H. pylori (SEQ ID NO: 208 or 209).

[00190] Exemplary ferritins including more than one type of mutation are provided under SEQ ID NO: 201-207.

5. Structural alignment

[00191] As discussed herein, mutation positions corresponding to mutation positions described for a given polypeptide (eg, ferritin from H. pylori ) can be identified through pairwise or structural alignment. Structural alignment is suitable for large families of proteins, such as ferritin, where the proteins have similar structures despite significant sequence variations, and many members of this family have been structurally characterized and can be used to identify corresponding positions in different versions of other polypeptides described in this document, such as RSV polypeptides (eg RSV F or G). The Protein Databank (PDB) contains 3D structures of many ferritins, including those listed below with their accession numbers.

[00192] 2jd6, 2jd7 - PfFR - Pyrococcus furiosus. 2jd8 - PfFR+Zn. 3a68 - soFR from the SferH4 gene - soybean. 3a9q - soFR from the SferH4 gene (mutant). 3egm, 3bvf, 3bvi, 3bvk, 3bvl - HpFR - Heliobacter pylori. 5c6f - HpFR (mutant) + Fe. 1z4a, 1vlg - FR - Thermotoga maritime. 1s3q, 1sq3, 3kx9 - FR - Archaeoglubus fulgidus. 1krq - FR - Campylobacter jejuni. 1eum - EcFR - Escherichia coli. 4reu - EcFR+Fe. 4xgs - EcFR (mutant) + Fe2O2. 4ztt - EcFR (mutant) + Fe2O+Fe2+Fe+O2. 1qgh - LiFR - Listeria innocua. 3qz3 - VcFR - Vibrio cholerae. 3vnx - FR - Ulva pertusa. 4ism, 4isp, 4itt, 4itw, 4iwj, 4iwk, 4ixk, 3e6s - PnmFR - mixed genus of Pseudo-nitschia. 4zkh, 4zkw, 4zkx, 4zl5, 4zl6, 4zlw, 4zmc - PnmFR (mutant) + Fe. 1z6o - FR - Trichoplusia ni. 4cmy - FR+Fe - Chlorobaculum tepidum. Ferritin light chain (FTL). 1lb3, 1h96 - mFTL - mouse. 1rcc, 1rcd, 1rci - bFTL+tartrate+Mg. 1rce, 1rcg - bFTL+tartrate+Mn. 3noz, 3np0, 3np2, 3o7r - hoFTL (mutant) - horse. 3o7s, 3u90 - hoFTL. 4v1w - hoFTL - cryo EM. 3rav, 3rd0 - hoFTL+barbiturate. Ferritin light chains+ferritin heavy chains: 5gn8 - hFTH+Ca.

[00193] Structural alignment involves identifying corresponding residues in two (or more) polypeptide sequences by (i) modeling the structure of the first sequence using the known structure of a second sequence, or (ii) comparing the structures of the first and second sequences, where both are known, and identifying the residue in the first sequence located most similar to the residue of interest in the second sequence. Relevant residues are identified using some algorithms based on minimizing the distance between alpha carbons in overlapping structures (e.g., which set of paired alpha carbons provides the minimized standard deviation for the alignment). When identifying positions in ferritin other than ferritin fromH. pylori, corresponding to the provisions described in relation to ferritin fromH. pylori, ferritin fromH. pylori may represent a "second" sequence. If the ferritin of interest is other than the ferritin fromH. pylori, has no known structure available, but is closely related to another ferritin other than ferritin fromH. pylori, which has a known structure compared to ferritin fromH. pylori, then it may be most effective to model the ferritin of interest other than the ferritin fromH. pylori, using the known structure of closely related ferritin, different from ferritin fromH. pylori, and then compare this model with the structure of ferritin fromH. pylori to identify the required corresponding residue in the ferritin of interest. There is an extensive literature on structural modeling and alignment; illustrative disclosures include US 6,859,736; US 8738343; and disclosures cited in Aslam et al., Electronic Journal of Biotechnology 20 (2016) 9-13. For a discussion of modeling a structure based on a known related structure or structures, seeFor example, Bordoli et al., Nature Protocols 4 (2009) 1-13, and references cited therein.

6. Immunostimulatory functional groups, adjuvants, RSV-based conjugated polypeptides

[00194] In some embodiments, the RSV polypeptide and/or an immunostimulatory functional group, such as an adjuvant, is attached to a surface exposed amino acid. In some embodiments, the surface exposed amino acid is a cysteine, for example, resulting from the mutation discussed above. In some embodiments, the surface exposed amino acid is lysine, aspartate, or glutamate. Conjugation procedures using glutaraldehyde (to conjugate lysine to an amino-bearing linker or functionality) or carbodiimide (e.g., 1-cyclohexyl-3-(2-morpholin-4-yl-ethyl)carbodiimide or 1-ethyl-3-(3- dimethyl-aminopropyl)carbodiimide (EDC; EDAC) for the conjugation of aspartate or glutamate with an amino group-bearing linker or functional group, or lysine with a carboxyl group-bearing linker or functional group) are described, for example, in section 4 in Holtzhauer, M., Basic Methods for the Biochemical Lab, Springer 2006, ISBN 978-3-540-32785-1, available at www.springer.com.

[00195] In some embodiments, an immunostimulatory moiety, such as an adjuvant, is attached to a surface exposed amino acid in ferritin. In some embodiments, more than one immunostimulatory moiety, such as an adjuvant, is attached to a surface exposed amino acid in ferritin. In some embodiments, twenty-four immunostimulatory moieties are attached to a ferritin multimer or ferritin particle (eg, one moiety for each monomer in a H. pylori ferritin particle). In some embodiments, with multiple immunostimulatory moieties attached to a ferritin nanoparticle, all immunostimulatory moieties are identical. In some embodiments, with multiple immunostimulatory moieties attached to a ferritin nanoparticle, all immunostimulatory moieties are not identical.

Types of immunostimulating functional groups, adjuvants

[00196] Any immunostimulatory functional group that can be attached to a surface exposed amino acid (eg, cysteine) can be used in the ferritin variants of the present invention. In some embodiments, the immunostimulatory moiety is a B cell agonist.

[00197] In some embodiments, the immunostimulatory moiety is not hydrophobic. In some embodiments, the immunostimulatory moiety is hydrophilic. In some embodiments, the immunostimulatory moiety is polar. In some embodiments, the immunostimulatory moiety is capable of forming a hydrogen bond or an ionic bond, for example, contains a hydrogen bond donor, a hydrogen bond acceptor, a cationic moiety, or an anionic moiety. A moiety is considered cationic or anionic if it will ionize in aqueous solution at a physiologically relevant pH, such as pH 6, 7, 7.4, or 8.

[00198] In some embodiments, the immunostimulatory moiety is an adjuvant. In some embodiments, the adjuvant includes a pathogen-associated molecular pattern (PAMP). In some embodiments, the adjuvant is a Toll-like receptor (TLR) agonist or a stimulatory interferon gene (STING) agonist. In some embodiments, the adjuvant activates TLR signaling in B and/or T cells. In some embodiments, the adjuvant regulates the adaptive immune response.

(1) TLR2 agonists

[00199] In some embodiments, the immunostimulatory moiety is a TLR2 agonist. In some embodiments, the immunostimulatory moiety stimulates TLR2 signaling. In some embodiments, the immunostimulatory fragment is a synthetic small molecule TLR2 ligand. In some embodiments, the immunostimulatory fragment is a synthetic small molecule agonist of TLR2 signaling.

[00200] In some embodiments, the TLR2 agonist is PAM2CSK4, FSL-1, or PAM3CSK4.

(2) TLR7/8 agonists

[00201] In some embodiments, the immunostimulatory moiety is a TLR7 and/or TLR8 agonist (ie, an agonist of at least one of TLR7 and TLR8). In some embodiments, the immunostimulatory moiety stimulates TLR7 and/or TLR8 signaling. In some embodiments, the immunostimulatory moiety is a synthetic small molecule TLR7 and/or TLR8 ligand. In some embodiments, the immunostimulatory fragment is a synthetic small molecule agonist of TLR7 and/or TLR8 signaling.

[00202] In some embodiments, the TLR7 and/or TLR8 agonist is single-stranded RNA (ssRNA). In some embodiments, the TLR7 and/or TLR8 agonist is an imidazoquinoline. In some embodiments, the TLR7 and/or TLR8 agonist is a nucleoside analog.

[00203] In some embodiments, the TLR7 and/or TLR8 agonist is an imidazoquinolinamine Toll-like receptor (TLR) agonist, such as 3M-012 (3M Pharmaceuticals). The structure of free 3M-012 is:

. It should be understood that an immunostimulatory moiety, such as 3M-012 or any moiety discussed herein, can be conjugated to ferritin by replacing the corresponding peripheral atom of the moiety (e.g., hydrogen) with a bond to ferritin described herein, e.g. sulfur exposed on the surface of the cysteine or via a linker attached to such a sulfur atom. Thus, when conjugated with ferritin, the structure of the immunostimulating fragment will be slightly different from the structure of the free molecule.

[00204] In some embodiments, the TLR7 and/or TLR8 agonist is SM 7/8a. The structure of free SM 7/8a is as follows:

.

[00205] See, for example, Nat Biotechnol. 2015 Nov;33(11):1201-10. doi: 10.1038/nbt.3371.

(3) TLR9 agonists

[00206] In some embodiments, the immunostimulatory moiety is a TLR9 agonist. In some embodiments, the immunostimulatory moiety stimulates TLR9 signaling. In some embodiments, the immunostimulatory moiety is a synthetic small molecule TLR9 ligand. In some embodiments, the immunostimulatory fragment is a synthetic small molecule agonist of TLR9 signaling.

[00207] In some embodiments, the TLR9 agonist is a CpG oligodeoxynucleotide (ODN). In some embodiments, the TLR9 agonist is an unmethylated ODN CpG. In some embodiments, the ODN CpG includes a partial or full phosphorothioate (PS) backbone instead of the natural phosphodiester (PO) backbone found in regular DNA.

[00208] In some embodiments, the CpG ODN is a class B ODN that includes one or more 6-mer CpG motifs containing 5'-purine (Pu)-pyrimidine (Py)-C-G-Py-Pu-3'; has an entirely phosphorothioate (i.e., PS-modified) backbone and is 18–28 nucleotides in length. In some embodiments, the ODN CpG includes the sequence of SEQ ID NO: 210, optionally containing phosphorothioate linkages in the backbone.

[00209] In some embodiments, the TLR9 agonist includes an immunostimulatory sequence (ISS). In some embodiments, the TLR9 agonist is ISS-1018 (Dynavax) (SEQ ID NO: 210).

(4) STING agonists

[00210] In some embodiments, the immunostimulatory moiety is a STING (stimulator of interferon gene protein, also known as endoplasmic reticulum stimulatory IFN) agonist. In some embodiments, the immunostimulatory moiety stimulates STING signaling. In some embodiments, the immunostimulatory moiety is a synthetic small molecule STING ligand. In some embodiments, the immunostimulatory moiety is a synthetic small molecule STING signaling agonist.

[00211] In some embodiments, the STING agonist is a cyclic dinucleotide (CDN). See for example Danilchanka et al., Cell 154:962–970 (2013). Exemplary CDNs include cdA, cdG, cAMP-cGMP, and 2'-5',3'-5'-cGAMP (for structures, see in Danilchanka et al.). STING agonists also include synthetic agonists such as DMXAA

.

RSV-based conjugated polypeptides

[00212] In some embodiments, the RSV polypeptide is conjugated to a surface exposed ferritin amino acid. In some embodiments, the RSV polypeptide makes the ferritin protein antigenic. In some embodiments, the RSV polypeptide is antigenic by itself, while in some embodiments, the RSV polypeptide is antigenic due to its association with ferritin. In some embodiments, the RSV polypeptide is any of the RSV F or G polypeptides described herein.

Conjugation

[00213] In some embodiments, a surface exposed cysteine (e.g., resulting from a mutation described herein) or a cysteine in a peptide linker attached to ferritin (e.g., the N-terminus of ferritin) is used to conjugate an immunostimulatory functional group, such as an adjuvant, or an RSV polypeptide with ferritin. In some embodiments, the linker is conjugated to such a cysteine, wherein the linker can then be conjugated to an immunostimulatory functional group, such as an adjuvant, or an RSV polypeptide. In some embodiments, such cysteine provides the chemical basis for conjugation reactions involving the addition of an RSV adjuvant, linker, or polypeptide. In some embodiments, bioconjugates are prepared wherein an immunostimulatory functional group, such as an adjuvant, or RSV polypeptide binds to ferritin after reduction of such cysteine. In some embodiments, the cysteine is an unpaired surface exposed cysteine, i.e. in which there is no cysteine partner in a suitable position for the formation of a disulfide bond. In some embodiments, the cysteine is an unpaired cysteine that contains a free thiol side chain.

(1) Types of conjugation chemistry

[00214] Chemical reactions of any type can be used to conjugate an immunostimulatory functional group, such as an adjuvant, or an RSV polypeptide to ferritin, for example, by reacting with a surface exposed amino acid such as cysteine or another amino acid such as Lys, Glu or Asp.

[00215] In some embodiments, conjugation is performed using click chemistry. As used herein, the term “click chemistry” refers to a reaction between a pair of functional groups that react rapidly and selectively (ie, “click”) with each other. In some embodiments, click chemistry can be performed under mild aqueous conditions. In some embodiments, the click chemistry reaction takes advantage of a cysteine on the surface of ferritin, such as a cysteine resulting from mutation of a surface-exposed amino acid, to perform click chemistry using a functional group that is capable of reacting with the cysteine.

[00216] Various reactions that satisfy the criteria of click chemistry are known in the art, and one skilled in the art can employ any of a number of published techniques (see, for example, Hein et al., Pharm Res 25(10):2216-2230 (2008)). A wide range of commercially available click chemistry reagents can be used, such as those from Sigma Aldrich, Jena Bioscience or Lumiprobe. In some embodiments, conjugation is performed using click chemistry, as described in the examples below.

[00217] In some embodiments, the click chemistry reaction occurs after ferritin reduction.

[00218] In some embodiments, the click chemistry may be a 1-step click reaction. In some embodiments, the click chemistry may be a 2-step click reaction.

[00219] In some embodiments, the reaction(s) involve click chemistry without the participation of a metal. In some embodiments, the reaction(s) involve thiol-maleimide and/or disulfide exchange.

Click chemistry without metal

[00220] Metal-free click chemistry can be used for conjugation reactions to avoid potential protein oxidation. Metal-free click chemistry has been used to form antibody conjugates (see van Geel et al., Bioconjugate Chem. 2015, 26, 2233-2242).

[00221] In some embodiments, metal-free click chemistry is used in reactions to couple an adjuvant to ferritin. In some embodiments, copper-free conjugation is used in reactions to couple an adjuvant to ferritin. In some embodiments, metal-free click chemistry employs bicyclo[6.1.0]nonine (BCN). In some embodiments, metal-free click chemistry uses dibenzoazacyclooctine (DBCO). In some embodiments, BCN or DBCO is reacted with an azide group.

[00222] DBCO is characterized by high specificity for azide groups due to a voltage-stimulated click reaction in the absence of a catalyst, resulting in high yields of a stable triazole. In some embodiments, DBCO reacts with an azide in the absence of a copper catalyst.

[00223] In some embodiments, metal-free click chemistry is used in a 1-step click reaction. In some embodiments, metal-free click chemistry is used in a 2-step click reaction.

Thiol-maleimide and disulfide exchange

[00224] Ferritins described herein may include a cysteine containing thiol, also known as a sulfhydryl, which is available for reaction with sulfhydryl-reactive chemical groups (or which can be made available through reduction). Thus, cysteine provides a chemoselective modification with the addition of an immunostimulatory moiety, such as an adjuvant, to ferritin. Under basic conditions, the cysteine will be deprotonated to form a thiolate nucleophile, which is capable of reacting with soft electrophiles such as maleimides and iodoacetamides. The reaction of cysteine with maleimide or iodoacetamide results in the formation of a carbon-sulfur bond.

[00225] In some embodiments, the sulfhydryl-reactive chemical moiety reacts with a surface-exposed cysteine or cysteine in the ferritin linker. In some embodiments, the sulfhydryl-reactive chemical group is a haloacetyl, maleimide, aziridine, acryloyl, arylation agent, vinyl sulfone, pyridyl disulfide, or TNB-thiol.

[00226] In some embodiments, the sulfhydryl-reactive chemical group is conjugated to the cysteine sulfhydryl via alkylation (ie, formation of a thioether bond). In some embodiments, a sulfhydryl-reactive chemical group is conjugated to a cysteine sulfhydryl via disulfide exchange (ie, the formation of a disulfide bond).

[00227] In some embodiments, the reaction for conjugating an immunostimulatory moiety, such as an adjuvant, to ferritin is a thiol-maleimide reaction.

[00228] In some embodiments, the sulfhydryl-reactive chemical moiety is a maleimide. In some embodiments, the reaction of a maleimide with a cysteine results in the formation of a stable thioether bond, for example, which is irreversible. In some embodiments, maleimide does not react with tyrosines, histidines, or methionines in ferritin. In some embodiments, unreacted maleimides are quenched upon completion of the reaction by adding a free thiol, for example in excess.

[00229] In some embodiments, the reaction for conjugating an immunostimulatory moiety, such as an adjuvant, to ferritin is a thiol-disulfide exchange, also known as a disulfide exchange. In some embodiments, the reaction includes the formation of a mixed disulfide containing a portion of the original disulfide. In some embodiments, the parent disulfide is a cysteine incorporated into ferritin through mutation of a surface-exposed amino acid or addition of an N-terminal linker.

[00230] In some embodiments, the sulfhydryl-reactive chemical moiety is pyridyldithiol. In some embodiments, the sulfhydryl-reactive chemical group is a TNB-thiol group.

(2) Conjugated linkers

[00231] In some embodiments, an immunostimulatory moiety, such as an adjuvant, or RSV polypeptide is attached to ferritin through a linker that covalently binds to a surface exposed amino acid, such as cysteine. In some embodiments, the linker includes a polyethylene glycol, such as a PEG linker. In some embodiments, the polyethylene glycol (eg, PEG) linker enhances the aqueous solubility and ligation efficiency of ferritin bound to an immunostimulatory moiety, such as an adjuvant. The PEG linker is 2-18 PEG in length, for example PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, PEG15, PEG16, PEG17 and PEG18.

[00232] In some embodiments, the linker comprises a maleimide. In some embodiments, the linker contains components of an (ISM)-linker-maleimide immunostimulatory moiety. In some embodiments, the maleimide ISM linker is conjugated to ferritin in a 1-step click chemistry reaction by reacting the maleimide with the cysteine in the ferritin. In some embodiments, the ISM from the maleimide linker adjuvant is SM7/8a. In some embodiments, the ISM-linker-maleimide linker is PEG4. In some embodiments, the maleimide ISM linker is SM7/8a-PEG4 maleimide.

[00233] In some embodiments, a 2-step click chemistry protocol is used with a linker comprising a sulfhydryl-reactive chemical group at one end and an amine-reactive group at the other end. In such a 2-step click chemistry protocol, the sulfhydryl-reactive chemical group reacts with the cysteine in ferritin, while the amine-reactive group reacts with the reagent attached to the ISM. Thus, ISM is conjugated to ferritin using a set of 2 click chemistry reagents.

[00234] In some embodiments of the 2-step click chemistry protocol, the sulfhydryl-reactive chemical moiety is a maleimide. In some embodiments of the 2-step click chemistry protocol, maleimide reacts with a cysteine embedded in ferritin through mutation of a surface-exposed amino acid or addition of an N-terminal linker.

[00235] In some embodiments of the 2-step click chemistry protocol, the amine-reactive group is DBCO. In some embodiments of the 2-step click chemistry protocol, DBCO reacts with an azide group attached to the ISM.

[00236] In some embodiments, a maleimide linker-DBCO is used. In some embodiments, the maleimide linker-DBCO is conjugated to ferritin after ferritin has been reduced. In some embodiments, the maleimide linker reagent is conjugated to ferritin by reacting the maleimide with a cysteine in the ferritin in a first step. In some embodiments, DBCO is used to couple to an ISM attached to an azide. In some embodiments, the ISM bound to the azide is ISS-1018. In some embodiments, the azide-linked adjuvant is 3M-012 or CpG.

[00237] In some embodiments, a linker with a reactive group is added to the ISM. In some embodiments, the linker is a PEG4 azide linker or a PEG4 maleimide linker.

[00238] In some embodiments, the PEG4 azide linker is conjugated to 3M-012. An exemplary structure of 3M-012 conjugated to a PEG4 azide linker is:

[00239] In some embodiments, the PEG4 azide linker is conjugated to SM7/8a. An exemplary structure of SM7/8a conjugated to a PEG4 azide linker is:

.

[00240] In some embodiments, the PEG4 maleimide linker is conjugated to SM7/8a. An exemplary structure of SM7/8a conjugated to a PEG4-maleimide linker is:

[00241] In some embodiments, the azide group is conjugated to ISS-1018. An exemplary structure of ISS-1018 conjugated to an NHS ester azide linker is:

Linkers

[00242] In some embodiments, the linker separates the amino acid sequence of the RSV polypeptide from the amino acid sequence of ferritin. Any linker can be used. In some embodiments, the linker is a peptide linker that can facilitate expression of an antigenic polypeptide based on a ferritin sequence as a fusion protein (eg, from a single open reading frame). In some embodiments, the linker is a glycine-serine linker. In some embodiments, the glycine-serine linker is GS, GGGS (SEQ ID NO: 226), 2XGGGS (SEQ ID NO: 227) (i.e., GGGSGGGS (SEQ ID NO: 227)), or 5XGGGS (SEQ ID NO: 227) : 228). The linker may be N- or C-terminal to ferritin.

[00243] In some embodiments, the linker length is 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, the linker length is approximately 2-4, 2-6, 2-8, 2-10, 2-12, or 2-14 amino acids. In some embodiments, the linker length is at least 15 amino acids. In some embodiments, the linker length is at least 25 amino acids. In some embodiments, the linker length is at least 30 amino acids. In some embodiments, the linker length is at least 35 amino acids. In some embodiments, the linker length is at least 40 amino acids. In some embodiments, the linker length is less than 60 amino acids or equal to 60 amino acids. In some embodiments, the linker length is less than 50 amino acids or equal to 50 amino acids. In some embodiments, the linker length is approximately 16, 28, 40, 46, or 47 amino acids. In some embodiments, the linker is flexible. In some embodiments, the linker includes a cysteine, for example, for use as a site for conjugation of an immunostimulatory moiety (eg, an adjuvant); an exemplary linker including cysteine is provided as SEQ ID NO: 225. In some embodiments, the linker includes a sequence with at least 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO: 225 and further includes a cysteine , corresponding to cysteine in SEQ ID NO: 225. In some embodiments, the linker includes at least 25 amino acids (e.g., 25-60 amino acids), wherein the cysteine is located at a position ranging from the 8th amino acid from the N-terminus to the 8th amino acids from the C-terminus, or within 10 amino acids of the central residue or linker bond.

[00244] In some embodiments, the linker includes the amino acids glycine (G) and/or serine (S). In some embodiments, the linker includes or consists of the amino acids glycine (G), serine (S), asparagine (N), and/or alanine (A), and optionally cysteine, as discussed above. In some embodiments, the linker comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 222. In some embodiments, the linker comprises ggggsggggsggggsg (SEQ ID NO: 220), ggsgsgsnssassgassggasggsggsg (SEQ ID NO: 221), ggsgsgsgasasgssngsgsgsgsnssassgassggasggsggsg (SEQ ID NO: 222) or GS. In some embodiments, the linker comprises FR1 (SEQ ID NO: 223) or FR2 (SEQ ID NO: 224).

[00245] In some embodiments, ferritin includes ferritin fromH. pyloriwith an amino-terminal extension from bullfrog ferritin (which will be called hybrid ferritin). In some embodiments, this hybrid ferritin forms multimers with RSV polypeptide attachment sites evenly distributed over the surface (see Kanekyo 2015). In some embodiments, N-terminal ferritin fusion proteins cause the RSV polypeptide to be presented on the surface of a ferritin nanoparticle. In some embodiments, ferritin includes glutamate at a position corresponding to position 13 of SEQ ID NO: 208 (a hybrid ferritin that includes this glutamate), or at position 6 in SEQ ID NO: 209 (ferritin fromH. pylori wild type, in which position 6 contains isoleucine). In combination with the bullfrog linker, this glutamate is thought to retain the conserved salt bridge found in human and bullfrog ferritins (6R and 14E in both the human ferritin light chain and the bullfrog ferritins with fewer subunits). Cm. Kanekiyo et al., Cell 162, 1090-1100 (2015)).

[00246] In some embodiments, the RSV polypeptide binds to ferritin via a cysteine-thrombin-histidine linker. In some embodiments, this linker is used to directly conjugate a functional group (eg, an immunostimulatory functional group or RSV polypeptide) to ferritin via click chemistry. An exemplary sequence including the cysteine-thrombin-histidine linker is SEQ ID NO: 218 . Click chemistry suitable for conjugation reactions involving a cysteine-thrombin-histidine linker is discussed above.

[00247] In some embodiments, a linker containing a cysteine as a conjugation site for an immunostimulatory moiety, such as an adjuvant, is used in a construct containing a ferritin molecule that does not have an unpaired surface exposed cysteine, or in a construct containing a ferritin molecule containing an unpaired exposed cysteine. cysteine on the surface.

[00248] In some embodiments, the design does not include a linker. In some embodiments, the design includes a single linker. In some embodiments, the design includes two linkers or more than two linkers.

Compositions, uses and methods of vaccination

[00249] In some embodiments, the present invention provides methods for immunizing a subject against an RSV infection. In addition, the present invention provides methods for causing an immune response against RSV to be generated in a subject. In some embodiments, the methods of the present invention comprise administering to a subject an effective amount of a pharmaceutical composition described herein. In some embodiments, the methods of the present invention comprise administering to a subject an effective amount of an RSV sequence-based antigenic polypeptide, a ferritin sequence-based antigenic polypeptide, or a nanoparticle described herein.

[00250] In some embodiments, a composition is provided comprising any one or more of the polypeptides, nanoparticles or fusion proteins described herein and a pharmaceutically acceptable carrier vehicle, adjuvant or excipient.

[00251] In some embodiments, a polypeptide, nanoparticle, or composition described herein is administered to a subject, such as a human, for immunization against RSV infection. In some embodiments, a polypeptide or fusion protein described herein is administered to a subject, such as a human, to elicit a protective immune response to a future RSV infection. In some embodiments, any one or more of the polypeptides, nanoparticles, or compositions described herein are provided for use in immunization against RSV infection. In some embodiments, any one or more of the polypeptides, nanoparticles, or compositions described herein are provided for use in eliciting a protective immune response to RSV infection in the future. In some embodiments, the protective immune response provides a reduction in the incidence of RSV infection, pneumonia, bronchitis, or asthma.

[00252] In some embodiments, the composition comprises an RSV F polypeptide described herein. In some embodiments, the composition comprises an RSV G polypeptide described herein. In some embodiments, the composition comprises an RSV F polypeptide described herein and an RSV G polypeptide. In some embodiments, the composition comprises an RSV G polypeptide described herein and an RSV F polypeptide. In some embodiments, the composition comprises an RSV F polypeptide described herein and an RSV G polypeptide described herein.

[00253] In some embodiments, a composition comprising an RSV F polypeptide described herein produces a neutralizing response to RSV that is superior to that of immunization with a post-fusion RSV F polypeptide. In some embodiments, immunization with an RSV F polypeptide described herein (e.g., a polypeptide or nanoparticle comprising an RSV F polypeptide described herein) results in a higher titer of antibodies directed against prefusion RSV F polypeptide compared to that of immunization with RSV F after fusion. In some embodiments, immunization with the RSV F polypeptide described herein produces a lower titer of antibodies directed against post-fusion RSV F compared to that of immunization with post-fusion RSV F. In some embodiments, immunization with the RSV F polypeptide described herein produces a greater proportion of total antibody directed against prefusion RSV F compared to immunization with postfusion RSV F. Immunization with the RSV antigen described herein may provide better protection against RSV compared to immunization with RSV fusion F. Epitopes present in postfusion RSV F that are shared with prefusion F may represent epitopes that cannot be neutralized and have been suggested in some cases to mediate the production of antibodies that enhance RSV infection. In some embodiments, a composition comprising an RSV F polypeptide described herein induces a stronger neutralizing response to RSV while providing a reduction in the level of antibodies directed against RSV F after fusion. Thus, in some embodiments, a composition comprising an RSV F polypeptide described herein produces a higher titer of anti-RSV neutralizing antibodies in response to postfusion F binding.

[00254] In some embodiments, immunization with an RSV antigen described herein provides an improved safety profile compared to immunization with fusion RSV F. This improved safety profile may be due to the blocking of non-neutralizable or low-neutralizable epitopes present on the protein in the postfusion conformation. It has been reported that antibodies that bind the protein in the postfusion conformation may mediate enhanced RSV infection due to antibody-mediated viral infection. Thus, antibodies against a protein in the postfusion conformation that do not significantly neutralize RSV may enhance RSV infection, such as antibodies that recognize proteins in both the prefusion and postfusion conformations.

[00255] In some embodiments, a composition comprising an RSV G polypeptide described herein induces a neutralizing response to RSV.

[00256] In some embodiments, a composition comprising RSV F and RSV G polypeptides described herein induces a neutralizing response to RSV. In some embodiments, a composition containing RSV F and RSV G polypeptides described herein provides improved protection against RSV, such as a higher titer of neutralizing antibodies, than a composition that does not contain both antigens.

1. Subjects

[00257] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[00258] In some embodiments, the subject is an adult (18 years of age or older). In some embodiments, the subject is a child or adolescent (under 18 years of age). In some embodiments, the subject is an elderly person (over 60 years of age). In some embodiments, the subject is a non-elderly adult (aged 18 years or older to 60 years or older).

[00259] In some embodiments, the subject undergoes more than one administration of the composition. In some embodiments, booster administration improves the immune response.

[00260] In some embodiments, any one or more of the antigenic polypeptides or compositions described herein are for use in a mammal, such as a primate (e.g., a non-human primate such as a monkey (e.g., a macaque, such as a macaque - rhesus or cynomolgus macaque) or ape), rodent (for example, mouse or rat) or domesticated mammal (for example, dog, rabbit, cat, horse, sheep, cow, goat, camel or donkey). In some embodiments, any one or more of the antigenic polypeptides or compositions described herein are for use in birds, such as poultry (eg, chicken, turkey, duck, goose, guinea fowl, or swan).

2. Adjuvants

[00261] As described herein, adjuvants can be conjugated to ferritin via a surface exposed amino acid, such as cysteine. The unconjugated adjuvant can also be administered to a subject along with the ferritin sequence-based antigenic polypeptides described herein. In some embodiments, administration of an adjuvant together with a ferritin sequence-based antigenic polypeptide results in the production of a higher titer of antibodies against the RSV polypeptide in a subject compared to administration of the RSV polypeptide alone or the ferritin-based antigenic polypeptide alone without an adjuvant. An adjuvant may promote an earlier, stronger, or more persistent immune response to an antigenic polypeptide.

[00262] In some embodiments, the composition contains a single adjuvant. In some embodiments, the composition contains more than one adjuvant. In some embodiments, the composition does not contain an adjuvant.

[00263] In some embodiments, the adjuvant includes aluminum. In some embodiments, the adjuvant is aluminum phosphate. In some embodiments, the adjuvant is aluminum alum (Alyhydrogel '85 2%; Brenntag, catalog no. 21645-51-2).

[00264] In some embodiments, the adjuvant is an organic adjuvant. In some embodiments, the adjuvant is an oil-based adjuvant. In some embodiments, the adjuvant comprises an oil-in-water nanoemulsion.

[00265] In some embodiments, the adjuvant includes squalene. In some embodiments, the squalene-containing adjuvant is Ribi (Sigma adjuvant system, Cat. No. S6322-1vl), Addavax™ MF59, AS03, or AF03 (see US9703095). In some embodiments, the adjuvant comprising squalene is a nanoemulsion.

[00266] In some embodiments, the adjuvant includes a polyacrylic acid (PAA) polymer. In some embodiments, the adjuvant comprising the PAA is SPA09 (see WO 2017218819).

[00267] In some embodiments, the adjuvant includes non-metabolizable oils. In some embodiments, the adjuvant is Freund's incomplete adjuvant (IFA).

[00268] In some embodiments, the adjuvant includes non-metabolizable oils and killed Mycobacterium tuberculosis. In some embodiments, the adjuvant is Freund's complete adjuvant (CFA).

[00269] In some embodiments, the adjuvant is a lipopolysaccharide. In some embodiments, the adjuvant is monophosphoryl A (MPL or MPLA).

3. Pharmaceutical compositions

[00270] In various embodiments, a pharmaceutical composition is provided comprising a ferritin sequence-based antigenic polypeptide described herein and/or related entities. In some embodiments, the pharmaceutical composition is an immunogenic composition (eg, a vaccine) capable of inducing an immune response, such as a protective immune response against a pathogen.

[00271] For example, in some embodiments, the pharmaceutical compositions may comprise one or more of the following: (1) a ferritin sequence-based antigenic protein comprising (i) a mutation to replace a surface-exposed amino acid with cysteine, and (ii) an RSV polypeptide; (2) an antigenic protein based on the ferritin sequence containing (i) a mutation to replace a surface-exposed amino acid with cysteine and an immunostimulatory functional group associated with cysteine; and (ii) RSV polypeptide; (3) an antigenic protein based on the ferritin sequence, comprising (i) a surface-exposed cysteine, (ii) a peptide linker N-terminal to the ferritin protein, and (iii) an RSV polypeptide N-terminal to the peptide linker; (4) an antigenic protein based on the ferritin sequence, containing (i) a mutation to replace a surface-exposed amino acid with a cysteine and an immunostimulatory functional group associated with cysteine, (ii) a mutation to replace an internal cysteine at position 31 of ferritin from H. pylori , or a mutation of an internal cysteine at a position similar to that of ferritin 31 from an organism other than H. pylori , as determined by pairwise or structural alignment, with an amino acid other than cysteine, (iii) a mutation causing the replacement of a surface-exposed asparagine residue with an amino acid other than from asparagine, and (iv) RSV polypeptide; or (5) a ferritin particle containing any of the above ferritin proteins. In some embodiments, the pharmaceutical composition comprises an RSV sequence-based antigenic polypeptide comprising an RSV F polypeptide, wherein an epitope of the RSV polypeptide that is common to pre-fusion RSV F and post-fusion RSV F polypeptide is blocked, and/or an RSV sequence-based antigenic polypeptide comprising an RSV F polypeptide, wherein the RSV F polypeptide comprises amino acid residues 62-69 and 196-209 of SEQ ID NO: 26 and an asparagine corresponding to position 328, 348 or 507 of SEQ ID NO: 26, wherein the optionally antigenic polypeptide based on the sequence RSV additionally contains ferritin.

[00272] In some embodiments, the present invention provides pharmaceutical compositions containing antibodies or other agents related to the antigenic polypeptides described herein. In one embodiment, the pharmaceutical composition contains antibodies that bind to and/or compete with an antigenic polypeptide described herein. Alternatively, the antibodies may recognize viral particles containing the RSV polypeptide component of the antigenic polypeptide described herein.

[00273] In some embodiments, the pharmaceutical compositions described herein are administered alone or in combination with one or more agents to enhance the immune response, such as an adjuvant described above. In some embodiments, the pharmaceutical composition further comprises an adjuvant as described above.

[00274] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. As used herein, the term “carrier” refers to the diluent, adjuvant, excipient or carrier medium with which the pharmaceutical composition is administered. In illustrative embodiments, carriers may include sterile liquids, such as, for example, water and oils, including petroleum, animal, vegetable, or synthetic oils, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. etc. In some embodiments, the carriers are or include one or more solid components. Pharmaceutically acceptable carriers may also include, but are not limited to, saline, buffered saline, dextrose, glycerol, ethanol, and combinations thereof. As used herein, the term excipient refers to any non-therapeutic agent that can be included in a pharmaceutical composition, for example, to provide or help achieve a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, but are not limited to, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, glycol, water, ethanol, etc. In various embodiments, the pharmaceutical composition is sterile.

[00275] In some embodiments, the pharmaceutical composition contains minor amounts of wetting or emulsifying agents or pH buffering agents. In some embodiments, the pharmaceutical compositions may contain any of a variety of additives, such as stabilizers, buffers, or preservatives. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be included.

[00276] In various embodiments, the pharmaceutical composition can be formulated to suit any desired route of administration. For example, the pharmaceutical composition may be in the form of solutions, suspensions, emulsions, drops, tablets, pills, pellets, capsules, liquid capsules, gelatin capsules, powders, sustained release formulations, suppositories, emulsions, aerosols, sprays, suspensions, lyophilized powder , frozen suspension, dried powder or any other form suitable for use. General considerations for the formulation and manufacture of pharmaceuticals can be found, for example, in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Co., Easton, PA, 1995; incorporated herein by reference.

[00277] The pharmaceutical composition can be administered by any route of administration. Routes of administration include, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, transmucosal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, intratracheal instillation, bronchial instillation, inhalation, or topical. Administration may be local or systemic. In some embodiments, administration is orally. In another embodiment, administration is via parenteral injection. In some cases, administration results in the release of an antigenic polypeptide based on the ferritin sequence described herein into the bloodstream. The route of administration may be left to the discretion of the practitioner.

[00278] In some embodiments, the pharmaceutical composition is suitable for parenteral administration (eg, intravenous, intramuscular, intraperitoneal, and subcutaneous). Such compositions can be formulated, for example, in the form of solutions, suspensions, dispersions, emulsions, etc. They can also be formulated in the form of sterile solid compositions (for example, a lyophilized composition), which can be dissolved or suspended in a sterile injection medium directly before use. For example, parenteral administration can be accomplished by injection. In such embodiments, the injectable preparations are prepared in conventional forms, i.e., either as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in liquid prior to injection, or as emulsions. In some embodiments, injectable solutions and suspensions are prepared from sterile powders, lyophilized powders, or granules.

[00279] In a further embodiment, the pharmaceutical composition is formulated for delivery by inhalation (eg, for direct delivery to the lungs and respiratory system). For example, the composition may be in the form of a nasal spray or any other known aerosol formulation. In some embodiments, formulations for inhalation or aerosol delivery contain multiple particles. In some embodiments, such formulations may have an average particle size of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or about 13 microns. In some embodiments, the formulations for inhalation or aerosol delivery are formulated as a dry powder. In some embodiments, formulations for inhalation or aerosol delivery are formulated as a wet powder, for example, by including a wetting agent. In some embodiments, the wetting agent is selected from the group consisting of water, saline, or other liquid having a physiological pH value.

[00280] In some embodiments, the pharmaceutical composition in accordance with the present invention is administered as drops into the nasal or oral cavity. In some embodiments, the dose may comprise multiple drops (e.g., 1-100, 1-50, 1-20, 1-10, 1-5, etc.)

[00281] The pharmaceutical composition of the present invention can be administered in any dose suitable to achieve the desired result. In some embodiments, the desired result is the induction of a long-lasting adaptive immune response against the source of the RSV polypeptide present in the ferritin sequence-based antigenic polypeptide present in the composition. In some embodiments, the desired result is a reduction in the intensity, severity, frequency, and/or delay of onset of one or more symptoms of the infection. In some embodiments, the desired result is inhibition or prevention of infection. The required dosage will vary from subject to subject depending on the species, age, weight and general condition of the subject, the severity of the infection being prevented or treated, the particular composition used and the route of administration.

[00282] In some embodiments, the pharmaceutical compositions of the present invention are administered in one or more doses. In some embodiments, the pharmaceutical compositions are administered in multiple doses administered on different days (eg, prime-boost vaccination strategies). In some embodiments, the pharmaceutical composition is administered as part of a booster regimen.

[00283] In various embodiments, the pharmaceutical composition is administered in conjunction with one or more additional therapeutic agents. Coadministration does not require simultaneous administration of therapeutic agents if the timing of their administration is such that the pharmacological activities of the additional therapeutic agent and the active ingredient(s) in the pharmaceutical composition overlap in time, thereby producing a combined therapeutic effect. Typically, each agent will be administered at the dose and schedule established for that agent.

4. Nucleic acid/mRNA

[00284] Also provided is a nucleic acid encoding an antigenic polypeptide described herein. In some embodiments, the nucleic acid is mRNA. Any nucleic acid capable of undergoing translation to produce a polypeptide is considered to be mRNA for the purposes of the present invention.

5. Sets

[00285] Also provided herein are kits containing one or more of the antigenic polypeptides, nucleic acids, antigenic ferritin particles, antigenic lumazine synthase particles, compositions, or pharmaceutical compositions described herein. In some embodiments, the kit further contains one or more of a solvent, solution, buffer, instructions, or drying agent.

Table 1 (sequence table): Description of sequences

Description Subsequence SEQ ID NO RF8085 NIH DS-CAV1 with single-stranded linker SGSGS (SEQ ID NO: 229) on bullfrog ferritin (bf) hp N19Q_C31S_S111C (control) (same protein sequence as 2, expressed using cloning vector for transient transfection) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISN
IETVIEFQQKNNRLLEITREFSVNAGVTTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYG KTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSGSGSESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAY EHEQHISESINNIVDHAIKCKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 1 RF8090: NIH DS-CAV1 with single-stranded linker SGSGS (SEQ ID NO: 229) on bullfrog ferritin (bf) hp N19Q_C31S_S111C (control) (same protein sequence as 1, expressed using cloning vector used for production in CHO cell lines) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISN
IETVIEFQQKNNRLLEITREFSVNAGVTTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYG KTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSGSGSESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAY EHEQHISESINNIVDHAIKCKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 2 RF8100: addition of one T324N glycan site in RSV scF_SGSGS-bf-pFerr_N19Q_C31S_S111C] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISN
IETVIEFQQKNNRLLEITREFSVNAGVTTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTNNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYG KTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSGSGSESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAY EHEQHISESINNIVDHAIKCKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 3 RF8101: addition of one E328N glycan site to RSV scF_SGSGS-bf-pFerr_N19Q_C31S_S111C MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISN
IETVIEFQQKNNRLLEITREFSVNAGVTTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKNGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYG KTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSGSGSESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAY EHEQHISESINNIVDHAIKCKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 4 RF8102: addition of one K390T glycan site to RSV scF_SGSGS-bf-pFerr_N19Q_C31S_S111C MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISN
IETVIEFQQKNNRLLEITREFSVNAGVTTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPTYDCKIMTSKTDVSSSVITSLGAIVSCYGK TKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSGSGSESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYE HEQHISESINNIVDHAIKCKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 5 RF8103: Add one glycan site S348N to RSVscF_SGSGS-bf-pFerr_N19Q_C31S_S111C MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISN
IETVIEFQQKNNRLLEITREFSVNAGVTTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGNVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYG KTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSGSGSESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAY EHEQHISESINNIVDHAIKCKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 6 RF8104: addition of one Y478S glycan site to RSV scF_SGSGS-bf-pFerr_N19Q_C31S_S111C MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISN
IETVIEFQQKNNRLLEITREFSVNAGVTTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYG KTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFSDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSGSGSESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYE HEQHISESINNIVDHAIKCKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 7 RF8105: addition of one R507N glycan site to RSV scF_SGSGS-bf-pFerr_N19Q_C31S_S111C MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISN
IETVIEFQQKNNRLLEITREFSVNAGVTTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYG KTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFINKSDELLSGSGSESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAY EHEQHISESINNIVDHAIKCKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 8 RF8106: RF8108 with I217P, which provides an increase in expression level, and removal of the disulfide bond (DS) from DS-CAV1, resulting in an even greater increase in expression level compared to RF8085 (or RF8090) mellilkanaittiltavtfcfasgqniteefyqstcsavskgylsalrtgwytsvitielsnikenkcngtdakvklikqeldkyknavtelqllmgsgnvglggaiasgvavskvlhlegevnkiksallstnkavvslsngvsvltfkvldlknyidkqllpilnkqscsisnpetviefqqknnrlleit refsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkegsnicltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsrtlpsevnlcnvdifnpkydckimtsktdvsssvitsl gaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnkqegkslyvkgepiinfydplvfpsdefdasisqvnekinqslafirksdellsgsgsesqvrqqfskdiekllneqvnkemqssnlymsmsswsythsldgaglflfdhaaeeyehakkliiflnennvpvq ltsisapehkfegltqifqkayeheqhisesinnivdhaikckdhatfnflqwyvaeqheeevlfkdildkielignenhglyladqyvkgiaksrks 9 RF8107: proline substitution (I217P) in RF8108 and cavity-filling hydrophobic substitution in RF8111 (N228L) along with disulfide bond removal in DS-CAV1 mellilkanaittiltavtfcfasgqniteefyqstcsavskgylsalrtgwytsvitielsnikenkcngtdakvklikqeldkyknavtelqllmgsgnvglggaiasgvavskvlhlegevnkiksallstnkavvslsngvsvltfkvldlknyidkqllpilnkqscsisnpetviefqqknlrlle itrefsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkegsnicltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsrtlpsevnlcnvdifnpkydckimtsktdvsssvits lgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnkqegkslyvkgepiinfydplvfpsdefdasisqvnekinqslafirksdellsgsgsesqvrqqfskdiekllneqvnkemqssnlymsmsswsythsldgaglflfdhaaeeyehakkliiflnennvpv qltsisapehkfegltqifqkayeheqhisesinnivdhaikckdhatfnflqwyvaeqheeevlfkdildkielignenhglyladqyvkgiaksrks 10 RF8108: substitution with proline I217P to stabilize the central helix prior to fusion (in the presence of DS in DS-CAV1) and increase expression level MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISN
PETVIEFQQKNNRLLEITREFSVNAGVTTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYG KTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSGSGSESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAY EHEQHISESINNIVDHAIKCKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS eleven RF8109: hydrophobic Q224L cavity filling on RSV scF_SGSGS-bf-pFerr_N19Q_C31S_S111C MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISN
IETVIEFLQKNNRLLEITREFSVNAGVTTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYG KTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSGSGSESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAY EHEQHISESINNIVDHAIKCKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 12 RF8110: cavity-filling hydrophobic replacements Q224L and Q225V on RSV scF_SGSGS-bf-pFerr_N19Q_C31S_S111C MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISN
IETVIEFLVKNNRLLEITREFSVNAGVTTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGK TKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSGSGSESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYE HEQHISESINNIVDHAIKCKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 13 RF8111: hydrophobic replacement for N228L providing cavity filling on RSV scF_SGSGS-bf-pFerr_N19Q_C31S_S111C MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISN
IETVIEFQQKNLRLLEITREFSVNAGVTTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYG KTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSGSGSESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAY EHEQHISESINNIVDHAIKCKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 14 RF8112 Hydrophobic filling replacement of N228F on RSV scF_SGSGS-bf-pFerr_N19Q_C31S_S111C MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISN
IETVIEFQQKNFRLLEITREFSVNAGVTTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYG KTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSGSGSESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAY EHEQHISESINNIVDHAIKCKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 15 RF8113 (mutation I217P in RF8106 removing DS in DS-CAV1, and with Wt ferritin ser111 and engineered CYS K79C for conjugation) mellilkanaittiltavtfcfasgqniteefyqstcsavskgylsalrtgwytsvitielsnikenkcngtdakvklikqeldkyknavtelqllmgsgnvglggaiasgvavskvlhlegevnkiksallstnkavvslsngvsvltfkvldlknyidkqllpilnkqscsisnpetviefqqknnrlleit refsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkegsnicltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsrtlpsevnlcnvdifnpkydckimtsktdvsssvitsl gaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnkqegkslyvkgepiinfydplvfpsdefdasisqvnekinqslafirksdellsgsgsesqvrqqfskdiekllneqvnkemqssnlymsmsswsythsldgaglflfdhaaeeyehakkliiflnennvpvq ltsisapehcfegltqifqkayeheqhisesinnivdhaikskdhatfnflqwyvaeqheeevlfkdildkielignenhglyladqyvkgiaksrks 16 RF8117 (combinations of the above successful mutations providing improved expression/secretion levels (Fig. 2): no DS, I217P, E328N, S348N, R507N, K79C ferritin) mellilkanaittiltavtfcfasgqniteefyqstcsavskgylsalrtgwytsvitielsnikenkcngtdakvklikqeldkyknavtelqllmgsgnvglggaiasgvavskvlhlegevnkiksallstnkavvslsngvsvltfkvldlknyidkqllpilnkqscsisnpetviefqqknnrlleit refsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkngsnicltrtdrgwycdnagnvsffpqaetckvqsnrvfcdtmnsrtlpsevnlcnvdifnpkydckimtsktdvsssvits lgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnkqegkslyvkgepiinfydplvfpsdefdasisqvnekinqslafinksdellsgsgsesqvrqqfskdiekllneqvnkemqssnlymsmsswsythsldgaglflfdhaaeeyehakkliiflnennvpvq ltsisapehCfegltqifqkayeheqhisesinnivdhaikskdhatfnflqwyvaeqheeevlfkdildkielignenhglyladqyvkgiaksrks 17 RF8122 (above RF8117 with additional K498L and K508Q to eliminate protease cleavage (LYS based) and increase stability/expression level) mellilkanaittiltavtfcfasgqniteefyqstcsavskgylsalrtgwytsvitielsnikenkcngtdakvklikqeldkyknavtelqllmgsgnvglggaiasgvavskvlhlegevnkiksallstnkavvslsngvsvltfkvldlknyidkqllpilnkqscsisnpetviefqqknnrlleit refsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkngsnicltrtdrgwycdnagnvsffpqaetckvqsnrvfcdtmnsrtlpsevnlcnvdifnpkydckimtsktdvsssvits lgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnkqegkslyvkgepiinfydplvfpsdefdasisqvnelinqslafinqsdellsgsgsesqvrqqfskdiekllneqvnkemqssnlymsmsswsythsldgaglflfdhaaeeyehakkliiflnennvpv qltsisapehcfegltqifqkayeheqhisesinnivdhaikskdhatfnflqwyvaeqheeevlfkdildkielignenhglyladqyvkgiaksrks 18 RF8123 (RF8117 with C knockouts at positions 69 and 212 for specific conjugation to ferritin CYS: C69V, C212V) mellilkanaittiltavtfcfasgqniteefyqstcsavskgylsalrtgwytsvitielsnikenkvngtdakvklikqeldkyknavtelqllmgsgnvglggaiasgvavskvlhlegevnkiksallstnkavvslsngvsvltfkvldlknyidkqllpilnkqsvsisnpetviefqqknnrlle itrefsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkngsnicltrtdrgwycdnagnvsffpqaetckvqsnrvfcdtmnsrtlpsevnlcnvdifnpkydckimtsktdvsssvit slgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnkqegkslyvkgepiinfydplvfpsdefdasisqvnekinqslafinksdellsgsgsesqvrqqfskdiekllneqvnkemqssnlymsmsswsythsldgaglflfdhaaeeyehakkliiflnennvpv qltsisapehcfegltqifqkayeheqhisesinnivdhaikskdhatfnflqwyvaeqheeevlfkdildkielignenhglyladqyvkgiaksrks 19 RF8134 (RF8122, like the K528N and K532N mutations, to limit instability due to proteolysis) mellilkanaittiltavtfcfasgqniteefyqstcsavskgylsalrtgwytsvitielsnikenkcngtdakvklikqeldkyknavtelqllmgsgnvglggaiasgvavskvlhlegevnkiksallstnkavvslsngvsvltfkvldlknyidkqllpilnkqscsisnpetviefqqknnrlleit refsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkngsnicltrtdrgwycdnagnvsffpqaetckvqsnrvfcdtmnsrtlpsevnlcnvdifnpkydckimtsktdvsssvits lgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnkqegkslyvkgepiinfydplvfpsdefdasisqvnelinqslafinqsdellsgsgsesqvrqqfsndienllneqvnkemqssnlymsmsswsythsldgaglflfdhaaeeyehakkliiflnennvpv qltsisapehcfegltqifqkayeheqhisesinnivdhaikskdhatfnflqwyvaeqheeevlfkdildkielignenhglyladqyvkgiaksrks 20 RF8135: RF8122, like the K465N and K470N mutations, to limit instability due to proteolysis mellilkanaittiltavtfcfasgqniteefyqstcsavskgylsalrtgwytsvitielsnikenkcngtdakvklikqeldkyknavtelqllmgsgnvglggaiasgvavskvlhlegevnkiksallstnkavvslsngvsvltfkvldlknyidkqllpilnkqscsisnpetviefqqknnrlleit refsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkngsnicltrtdrgwycdnagnvsffpqaetckvqsnrvfcdtmnsrtlpsevnlcnvdifnpkydckimtsktdvsssvits lgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnkqegnslyvngepiinfydplvfpsdefdasisqvnelinqslafinqsdellsgsgsesqvrqqfskdiekllneqvnkemqssnlymsmsswsythsldgaglflfdhaaeeyehakkliiflnennvpv qltsisapehcfegltqifqkayeheqhisesinnivdhaikskdhatfnflqwyvaeqheeevlfkdildkielignenhglyladqyvkgiaksrks 21 RF8136: RF8122, like mutations K465N, K470N, K528N and K532N, to limit instability due to proteolysis mellilkanaittiltavtfcfasgqniteefyqstcsavskgylsalrtgwytsvitielsnikenkcngtdakvklikqeldkyknavtelqllmgsgnvglggaiasgvavskvlhlegevnkiksallstnkavvslsngvsvltfkvldlknyidkqllpilnkqscsisnpetviefqqknnrlleit refsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkngsnicltrtdrgwycdnagnvsffpqaetckvqsnrvfcdtmnsrtlpsevnlcnvdifnpkydckimtsktdvsssvits lgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnkqegnslyvngepiinfydplvfpsdefdasisqvnelinqslafinqsdellsgsgsesqvrqqfsndienllneqvnkemqssnlymsmsswsythsldgaglflfdhaaeeyehakkliiflnennvpv qltsisapehcfegltqifqkayeheqhisesinnivdhaikskdhatfnflqwyvaeqheeevlfkdildkielignenhglyladqyvkgiaksrks 22 RF8140: RF8122 with R523Q in the bullfrog linker mutated to prevent potential proteolysis in CHO cells MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNPETVIEFQQKNNRLLEITREFSVNA GVTTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKNGSNICLTRTDRGWYCDNAGNVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYV SNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNELINQSLAFINQSDELLSGSGSESQVQQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHCFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFN FLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 23 Post-F, reference control molecule MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLP IVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGA IVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLGLEVLFQGPHHHHHHHSAWSHPQFEK 24 DS-CAV1, positive control molecule mellilkanaittiltavtfcfasgqniteefyqstcsavskgylsalrtgwytsvitielsnikenkcngtdakvklikqeldkyknavtelqllmqstpatnnrarrelprfmnytlnnakktnvtlskkrkrrflgfllgvgsaiasgvavckvlhlegevnkiksallstnkavvslsngvsvltfk vldlknyidkqllpilnkqscsisnietviefqqknnrlleitrefsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimciikeevlayvvqlplygvidtpcwklhtsplcttntkegsnicltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsltl psevnlcnvdifnpkydckimtsktdvsssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnkqegkslyvkgepiinfydplvfpsdefdasisqvnekinqslafirksdellsggssgssggsdiikllneqvnkemqssnlymsmsswcy thsldgaglflfdhaaeeyehakkliiflnennvpvqltsisapehkfegltqifqkayeheqhisesinnivdhaikskdhatfnflqwyvaeqheeevlfkdildkielignenhglyladqyvkgiaksrksgs 25 Wild type, native F RSV (strain A2) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLK NYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTD VSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN 26 Native G RSV strain A, UniProtKB/Swiss-Prot: P27022.1 MSKNKDQRTAKTLERTWDTLNHLLFISSCLYKLNLKSVAQITLSILAMIISTSLIIVAIIFIASANHKIT
STTTIIQDATNQIKNTTPTYLTQNPQLGISPSNPSDITSLITTILDSTTPGVKSTLQSTTVGTKNTTTTTQ
AQPNKPTTKQRQNKPPSKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKRTTTKPTKKPTPKT
TKKGPKPQTTKSKEAPTTKPTEEPTINTTKTNIITTLLTSNTTRNPELTSQMETFHSTSSEGNPSPSQVS
ITSEYPSQPSSPPNTPR 27 RSV ectodomain G, residues 66-297 NHKVTLTTAIIQDATSQIKNTTPTYLTQDPQLGISFSNLSEITSQTTTILASTTPGVKSNLQPTTVKTKNTTTTQTQPSKPTTKQRQNKPPNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKKPTFKTTKKDHKPQTTKPKEVPTTKPTEEPTINTTKTNIITTLLTNNTTGNP KLTSQMETFHSTSSEGNLSPSQVSTTSEHPSQPSSPPNTTRQ 28 Peptide G RSV A2 for conjugation with the N-terminal azido linker and flanking glutamate residues (aa 151-193) Azido-PEG4-SGGSSGSSEEEGGSRQNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKEEE 29 Oligodeoxynucleotide CpG (asterisks indicate phosphorothioate bonds) T*G*A*C*T*G*T*G*A*A*C*G*T*T*C*G*A*G*A*T*G*A thirty Substitution sequence in RF8117, which replaces positions 98-144 from SEQ ID NO: 26 (wild type RSV F) GSGNVGL 31 Gcc residues 151-193 (central conserved region) of RSV strain A2 RQNKPPNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKK 32 Gcc (central conserved region) of RSV strain B1 RKNPPKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPNKK 33 Not used 34-200 bfp-ferritin-N19Q/C31S/S26C ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMCMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 201 bfp-ferritin-N19Q/C31S/S72C ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTCISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 202 bfp-ferritin-N19Q/C31S/A75C ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENVPVQLTSISCPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 203 bfp-ferritin-N19Q/C31S/K79C ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHCFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 204 bfp-ferritin-N19Q/C31S/S100C ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISECINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 205 bfp-ferritin-N19Q/C31S/S111C ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKCKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 206 bfp-ferritin-N19Q/C31S/E12C ESQVRQQFSKDIEKLLNCQVNKEMQSSNLYMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 207 Illustrative ferritin from H. pylori with bullfrog linker ESQVRQQFSKDIEKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 208 Exemplary ferritin from wild-type H. pylori (GenBank accession no. AAD06160.1) (no bullfrog linker or N-terminal Met) LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 209 CpG (ISS-1018) TGACTGTGAACGTTCGAGATGA 210 Trichoplusia ni ferritin heavy chain TQCNVNPVQIPKDWITMHRSCRNSMRQQIQMEVGASLQYLAMGAHFSKDVVNRPGFAQLFFDAASEEREHAMKLIEYLLMRGELTNDVSSLLQVRPPTRSSWKGGVEALEHALSMESDVTKSIRNVIKACEDDSEFNDYHLVDYLTGDFLEEQYKGQRDLAGKASTLKKLMDRHEALGEF
IFDKKLLGIDV 211 Trichoplusia ni ferritin light chain ADTCYNDVALDCGITSNSLALPRCNAVYGEYGSHGNVATELQAYAKLHLERSYDYLLSAAYFNNYQTNRAGFSKLFKKLSDEAWSKTIDIIKHVTKRGDKMNFDQHSTMKTERKNYTAENHELEALAKALDTQKELAERAFYIHREATRNSQHLHDPEIAQYLEEEFIEDHAEKIRTLAGHTSDLKKFITANNGHDLSLA LYVFDEYLQKTV 212 Ferritin from Pyrococcus furiosus MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNY
IYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFL
EWFINEQVEEEASVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE 213 Human ferritin heavy chain MTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVQQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDQES 214 Human ferritin light chain (signal peptide underlined) MDSKGSSQKGSRLLLLLVVSNLLLPQGVLASSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFER LTLKHD 215 Lumazine synthase from Aquifex aeolicus MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR 216 Bullfrog linker ESQVRQQF 217 Cysteine-thrombin-His linker (cysteine is shown with a double underline) CLVPRGSLEHHHHHH 218 6,7-Dimethyl-8-ribityllumazine synthase from E. coli MNIIEANVATPDARVAITIARFNNFINDSLLEGAIDALKRIGQVKDENITVVWVPGAYELPLAAGALAKTGKYDAVIALGTVIRGGTAHFEYVAGGASNGLAHVAQDSEIPVAFGVLTTESIEQAIERAGTKAGNKGAEAALTALEMINVLKAIKA 219 16 amino acid linker GGGGSGGGGSGGGGSG 220 28 amino acid linker GGSGSGSNSSSASSGASSGGASGGGSGGSG 221 46 amino acid linker GGSGSASSGASASGSSNGSGSGSGSNSSASSGASSGGASGGGSGGSG 222 FR1 GGSGSASAEAAAKEAAAKAGGGSGGSG 223 FR2 GGSGSASAEAAAKEAAAKEAAAKASGGSGGSG 224 47 amino acid linker including C for conjugation SGGGSGSASSGASASGSSCSGSGSGSSSASSGASSGGASGGGSGGSG 225

[00286] This description and illustrative embodiments are not to be construed as limiting. For purposes of this specification and the accompanying claims, unless otherwise indicated, all numbers expressing quantities, percentages or proportions and other numerical values used in the specification and claims are to be understood as being modified in all cases by the term "about" as used herein. the extent to which they have not already been modified. “Approximately” indicates a degree of variation that does not significantly affect the properties of the described claimed item, such as within 10%, 5%, 2% or 1%. Accordingly, unless otherwise indicated, the numerical parameters set forth in the following description and the accompanying claims are exemplary values that may vary depending on the desired properties to be obtained. As a last resort, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed by the number of significant figures reported and by the application of normal rounding techniques.

[00287] It should be noted that, as used in this specification and the accompanying claims, the singular form, as well as any use of any word in the singular, includes entities in the plural unless expressly and unambiguously limited to a single entity. As used herein, the term “include” and its grammatical variations are not intended to be limiting, such that listing items in a list does not exclude other similar items that may be replaced or added to the listed items.

EXAMPLES

[00288] The following examples are provided to illustrate certain embodiments disclosed and should not be construed as limiting the scope of this disclosure in any way.

1. Design and characterization of RSV F polypeptide modifications

[00289] Like other paramyxovirus F proteins, RSV F is expressed as a precursor protein with an N-terminal signal peptide and a C-terminal transmembrane region that anchors the protein to the viral surface. RSV-F undergoes intracellular cleavage by the protease furin to release a hydrophobic fusion peptide (“FP” in Fig. 1A), whose role is to adhere to the target cell during infection. The fusion peptide is adjacent to the heptad repeat region A (HRA), while the heptad repeat region B (HRB) is adjacent to the transmembrane domain.

[00290] Crystal structures of RSV F ectodomain trimers in their prefusion and postfusion conformations demonstrate that the HRA and HRB regions undergo significant rearrangements to mediate the control of the cell fusion event (Figure 1B) ( see Swanson, KA, et al., Proc Natl Acad Sci USA 108(23): p. 9619-24 (2011); McLellan, J. S., et al., Science 342(6158):592-598 (2013); McLellan, J. S., et al., J Virol 85 (15):7788-96 (2011); and McLellan, J. S., et al., Science 342(6158): p. 592-8 (2013)). In the prefusion conformation, the heptad repeat region A (HRA) is associated with the globular end group, and the tip of the fusion peptide is mostly embedded in the center of the protein. The prefusion conformation contains a number of helices and involves specific contacts between protomers to form the prefusion trimer.

[00291] A series of amino acid substitutions have been developed for interprotomer stabilization. Exemplary replacements include V207L; N228F; I217V and E218F; I221L and E222M; or Q224A and Q225L. When numbering all RSV F amino acid sequences in the examples, SEQ ID NO: 26 is used.

[00292] Amino acid substitutions were developed to stabilize the helix. As such, these substitutions are predicted to stabilize the helical F domain of RSV. Exemplary replacements include N216P or I217P.

[00293] Amino acid substitutions for intraprotomer stabilization were developed. Exemplary replacements include V220I; or A74L and Q81L.

[00294] Amino acid substitutions for helix capping were developed. Exemplary replacements include N216P or I217P.

[00295] Amino acid substitutions have been developed to reduce aggregation. Illustrative replacements include V192E and L61Q.

[00296] Other amino acid substitutions have been developed to fill the cavity by introducing hydrophobic amino acids such as N228F.

[00297] The amino acid substitutions E328N, S348N and R507N were designed to add glycosylation sites by replacing residues other than asparagine with asparagine. It has been suggested that the addition of non-native glycans could be used to block epitopes that are exposed in postfusion RSV F (Fig. 1B) to the surface of the prefusion F protein.

[00298] RSV F constructs of interest were generated as single chain (scF) proteins fused to a ferritin fusion containing an N-terminal bullfrog ferritin linker and H. pylori ferritin (pFerr) (Figure 1A). Ferritin contained a surface exposed cysteine resulting from the K79C or S111C mutation (ferritin sequence numbering corresponds to SEQ ID NO: 208).

[00299] The generation of various RSV Pre-F-NP and ferritin coding sequences was performed using standard cloning techniques known in the art. In general, DNA for RSV F constructs with the described substitutions was synthesized and cloned into a mammalian expression vector using Genscript. RSV DS-CAV1 F and postfusion F trimers were prepared similarly to previously published protocols (see McLellan, J.S., et al., Science 342(6158):592–598 (2013)). The DS-CAV1 construct retains the C-terminal trimerization domain of RSV F and combines it with hydrophobic substitutions to promote cavity filling. F RSV DS-CAV1 contains the disulfide mutation (DS) S155C-S290C and S190F-V207L (CAV1).

[00300] Vectors encoding RSV F-ferritin nanoparticles, naked ferritin (i.e., not coupled to RSV F), and RSV F trimers were transfected into 293EXPI cells and expression products were collected from conditioned media after 4 days. F RSV nanoparticles were purified by a series of Q anion column purifications (GE Healthcare, Cat. No. 17-1154-01) at pH 7.0 and 8.5, followed by purification with Superose 6 SEC in PBS (GE Healthcare, Cat. No. 90 -1000-42) using generally accepted chromatographic methods. Prefusion DS-CAV1 trimers and postfusion trimers were stored at −80°C, and RSV F nanoparticles were stored at 4°C.

[00301] Electron microscopy was performed to determine the conformation of RSV F nanoparticles. RSV F nanoparticle preparations (30 μg/mL in 25 mM Tris, 50 mM NaCl) were absorbed onto a 400-mesh carbon-coated grid (Electron Microscopy Sciences) and stained with 0.75% uranyl formate. A JEOL 1200EX microscope operating at 80 kV was used to analyze the samples. Photomicrographs were taken at 65,000× magnification and 2D class averages were obtained using standard methods in the art by the EM specialist Nanoimaging Services, INC (San Diego, CA) (Fig. 1D).

[00302] Expression and secretion of polypeptides containing these RSV F polypeptides and ferritin (SEQ ID NOs: 1-8 and 11-15) by transiently transfected 293 EXPI cells (Invitrogen) were assessed by Western blotting with anti-RSV F antibodies. All Western blots with anti-RSV F antibodies used site 0 specific antibody D25, described in McLellan et al., Science 340(6136):1113-1117 (2013) and US Pat. No. 8,562,996. As shown in the figure 2, many constructs were successfully expressed and secreted.

[00303] Polypeptide RF8085 (SEQ ID NO: 1) is a single chain mutant variant of the published RSV DS-CAV1 F (see McLellan, J.S., et al., Science 342(6158):592–598 (2013)) fused at the N-terminus to a ferritin nanoparticle. This construct contains a mutant variant (DS) of RSV F with two mutations, S155C-S290C, which retains antigenic site 0.

[00304] Polypeptide RF8106 (SEQ ID NO: 9) contains an I217P substitution in place of the 2 cysteine residues replaced in DS-CAV1. As shown in Figure 3, the RF8106 construct had significantly better expression in transiently transfected 293 EXPI cells, as assessed in conditioned medium after 4 days by Western blotting with anti-RSV F antibody.

[00305] Size exclusion chromatography (SEC) of RF8106 shows the major peak eluting at a retention time corresponding to the assembled ferritin particle fused to the RSV antigen corresponding to the fusion protein nanoparticle (Pre-F-NP, Figure 4). Dynamic light scattering (DLS) analysis of RF8106 was performed in reduced (Figure 5B) and unreduced states (Figure 5A). Recovery involved treatment with 2 mM TCEP. RF8106 had a radius of approximately 15 nm, consistent with inclusion in the nanoparticle (24-mer) in both reduced and unreduced states. The stability of the fusion protein in the presence of reducing agents facilitates the conjugation of adjuvants to the fusion proteins to form self-adjuvant nanoparticles, as described below.

[00306] The conjugation of the adjuvant to an RSV F polypeptide-ferritin fusion protein (Pre-F-NP) was then evaluated. We found that the free surface cysteine on ferritin could be used to add an additional functional group to the scF-pFerr fusion protein. Figure 6 shows the successful conjugation of a CpG oligodeoxynucleotide (ODN) to the sequence T*G*A*C*T*G*T*G*A*A*C*G*T*T*C*G*A*G*A *T*G*A (SEQ ID NO: 30; asterisks indicate phosphorothioate bonds) with RF8106, as evidenced by an increase in molecular weight as assessed on a Coomassie-stained SDS-PAGE gel.

[00307] The effect of adding glycosylation sites using substitutions E328N, S348N and R507N (RF8117, SEQ ID NO: 17) was assessed in 293EXPI cells transiently transfected with this construct as a ferritin fusion protein (i.e., as a Pre- F-NP). The RF8117 also contains a replacement I217P as in the RF8113. As shown in Figure 7, increased expression levels were observed for RF8117 compared to both the control construct RF8085 and construct RF8113 (SEQ ID NO: 16, which contains the I217P proline substitution but not the E328N, S348N, and R507N substitutions). RF8113 is similar to RF8106 described previously, except that the engineered ferritin cysteine is located on ferritin residue K79C rather than S111C. The RF8117 construct also showed an increase in molecular weight compared to RF8113 and RF8117, indicating successful addition of glycans.

[00308] Figure 8 briefly describes modifications to RSV F nanoparticles that provided increased proteolytic stability of Pre-F-NP. The original design was the RF8117 (above). When the earlier RF8085 construct was cloned into the CHO vector RF8090 and transfected into CHO cells, some material was observed to be excised between F and the ferritin functional group. It has been proposed that arginine or lysine residues in the HRB region or linker between F and the ferritin functional group are cut by trypsin-like proteases. Mutations of lysine and arginine residues within the region were tested for expression in 293 cells. In Figure 8, mutations K498L and R508Q (in RF8122, SEQ ID NO: 18) are identified as having no effect or increase in expression compared to RF8117. These R523Q mutations were combined with the RF8117 mutations mentioned herein to form construct RF8140 (SEQ ID NO: 23).

[00309] A greater improvement in expression (approximately 5-fold) was noted with a combination of single-chain and proline (I217P) modifications when expressed in 293 cells (exemplary constructs with these substitutions include RF8106 (SEQ ID NO: 9) and RF8113 (SEQ ID NO: 16)) with further improvement in expression and solubility resulting from modifications to add a glycosylation site to F RSV (illustrative constructs RF8117 (SEQ ID NO: 17) and RF8140 (SEQ ID NO: 23). In all of these constructs, the fusion peptide regions and p27 peptide (amino acids 98-144 SEQ ID NO: 26) are replaced by the sequence GSGNVGL (SEQ ID NO: 31).However, when RF8090 was expressed in CHO-producing cell lines, additional RSV F bands were observed in Western blots, suggesting that the construct was sensitive to proteolysis, possibly to trypsin-like cleavage at the arginine or lysine residue.

[00310] The potential role of protease sensitivity was also explored. Replacement of K residues (knockout or KO) in the HRB region and in the linker between the F functional group and the ferritin functional group was performed as these were predicted to be possible sites for the K-mediated cleavage initially observed in the producer cell line CHO. As shown in FIG. 9A and 9B, both RF8117 and RF8140 are expressed at high levels compared to RF8090 in the CHO production cell line, as measured by Western blotting with D25 or Octet assay with D25 and AM14.

[00311] These data indicate that single-chain amino acid designs and modifications to cap the helix, increase the level of glycosylation, and eliminate lysine residues or arginine residues sensitive to protease cleavage may provide improvements in the expression of RSV F polypeptides, including Pre-F-NP antigens RSV.

2. Characterization of fusion proteins based on RSV F and ferritin nanoparticles

[00312] Prior to animal studies, the concentration of DS-CAV1 and RSV F nanoparticles was analyzed by binding using Octet. Binding of prefusion antigens to the prefusion conformation-specific antibodies D25 and AM14 was also measured using the Octet device from FortéBio. All analyzes were performed in PBS at 30°C. Antibodies were loaded onto protein A (ProA) sensor tips (FortéBio, #18-5013) for 400 seconds to ensure capture levels close to saturation. The biosensor tips were then equilibrated for 90 seconds in PBS, followed by antigen binding at known concentrations in PBS for 300 seconds, followed by antigen dissociation in PBS. Data analysis and curve fitting, assuming a 1:1 interaction, were performed using Octet Data Analysis HT10.0 software using an external standard curve for binding of purified Pre-F-NP at a known concentration. The result of an exemplary assay for determining the concentration of Pre-F-NP in CHO conditioned medium is shown in FIG. 9B.

3. Characteristics of the immune response to RSV F polypeptides in vivo

[00313] For evaluationin vivoresponse to RSV antigens in mice, female BALBc mice were immunized intramuscularly with RSV antigens at specified doses at weeks 0, 3, and 6. Unless otherwise noted, RSV antigens (eg, in experiments in Figures 10A-B and 12A-B, among others) was used with AF03 adjuvant using a pre-use mixing strategy. That is, 50 μl of the corresponding protein solution was mixed with 50 μl of Sanofi AF03 adjuvant (squalene-based emulsion; see Fig. Klucker et al., J Pharm Sci. 2012 Dec;101(12):4490-500) immediately before injection of 50 µl into each hind paw. For the unadjuvanted groups, antigens were mixed as above, but AF03 was replaced with an equivalent volume of PBS. For antigens mixed with SPA09 or alum, the above procedure was performed with replacement of AF03 with an equivalent volume of SPA09 or alum, respectively. No adverse effects from immunization were observed for either formulation. Blood was collected 1 day before the first immunization and at least 2 weeks after each injection (i.e., weeks 2, 5, and 8). Unless otherwise noted, data are shown for 2 weeks after the third injection (week 8, also referred to as 2wp3). Typically, serum samples from preimmunization animals (designated as unexposed) were analyzed two weeks after the second injection (post 2 or 2wp2) or two weeks after the third injection (post 3 or 2wp3).

[00314] For the neutralization assay on Vero cells, blood serum was heat inactivated for 30 minutes at 56°C. A series of four-fold serial dilutions of inactivated serum was performed in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2% fetal bovine serum (FBS), 1% GlutaMAX, and 1% antimitotic antibiotic. RSV stock solutions were combined 1:1 with serum dilutions and incubated for 1.5 hours at 37°C. The virus/serum mixture was then added to 24-well plates containing confluent monolayers of Vero cells in an amount of 100 μl per well and incubated for 1.5 hours at 37°C, 5% CO ₂ . The inoculum was then coated with 1 ml per well of 0.75% methylcellulose in DMEM supplemented with 2% FBS, 2% GlutaMAX, and 2% antibiotic antimitotic. After 5 days of incubation at 37°C, 5% CO2, the top layer was removed and the monolayers were fixed with ice-cold methanol for 20 minutes.

[00315] The plates were then washed once with water and blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS) for 30 minutes at room temperature with gentle shaking. The blocking solution was then replaced at 200 μl per well with 2% milk powder in PBS containing a 1:2000 dilution of horseradish peroxidase-conjugated anti-RSV antibody (Abcam AB20686). After 3 hours of incubation at room temperature, the plates were washed 2 times with water, developed with TrueBlue HRP Substrate, rinsed twice more with water and air dried.

[00316] Stained plaques were counted using a dissecting microscope. Neutralizing antibody titers were determined at the end point of a 60% reduction relative to mock virus neutralization controls using the formula: Titer at which the number of plaques is reduced by 60% = (C/V x 0.4 - low)/(high - low) x ( HSD - LSD) + LSD where C/V=average number of RSV plaques in mock neutralization control wells, "low" and "high" represent the average number of RSV plaques in two dilutions that limit the value of C/V x 0, 4 for a blood serum sample, and HSD and LSD represent higher and lower dilutions of blood serum.

[00317] For the HAE neutralization assay, blood serum was heat inactivated for 30 minutes at 56°C. A series of four-fold serial dilutions of inactivated blood serum was performed in PneumaCult™-ALI basal medium (Stem Cell Technologies; 05002), supplemented with PneumaCult™-ALI 10X Supplement (Stem Cell Technologies; 05003) and 1% antibiotic/antifungal agent (hereinafter referred to as medium). RSV stock solutions were combined 1:1 with serum dilutions and incubated for 1.5 hours at 37°C. The virus-serum mixture was then added to 24-well plates containing fully differentiated HAE cells at 50 μl per well and incubated for 1.5 hours at 37°C, 5% CO ₂ . After incubation, the inoculum was removed, the wells were washed twice with medium to remove unbound virus, and incubated for an additional 20 hours at 37°C, 5% CO ₂ . Infection events in RSV-infected cultures expressing the mKate reporter (TagFP635) were counted under a fluorescence microscope.

[00318] To detect RSV infection that does not express the mKate reporter, pseudostratified epithelium was thoroughly washed with demucus medium, then fixed with 4% paraformaldehyde for 30 minutes at room temperature, permeabilized with 0.25% Triton X-100 for 30 min and blocked with DMEM supplemented with 2% FBS for 1 h at 37°C. The blocking solution was replaced at a rate of 100 μl per well with a mixture of mouse monoclonal Ab against RSV (Millipore; MAB 858-4) diluted 1:200 in DMEM supplemented with 2% FBS, and the plates were incubated at 37°C for 2 hours. The plates were then washed 3 times with PBS supplemented with 0.05% Tween 20. 100 μl of goat anti-mouse IgG (H+L) (Invitrogen; A11001) diluted 1:200 in DMEM supplemented with 2% was added to each well. FBS, and the plates were incubated overnight at 4°C. The next morning, plates were washed 3 times with PBS supplemented with 0.05% Tween 20, the fluorescence signal was stabilized with ProLong Gold AntiFade with DAPI (Thermo Fisher Scientific; P36935) and counted on a fluorescence microscope. Neutralizing antibody titers were determined at the endpoint of 60% reduction as above.

[00319] For anti-F antibody binding, either pre-fusion F (DS-CAV1) or post-fusion F was coupled to anti-HIS antibody-coated tips on Octet. Unless otherwise stated, all anti-F antibody binding refers to anti-F trimer prefusion antibody binding (DS-CAV1). His _6- tagged (SEQ ID NO: 230) RSV F trimer (DS-CAV1 or post-fusion F) was preloaded onto Anti-Penta-HIS (HIS1K) sensor tips (FortéBio, #18-5122) for 400 seconds to ensure level of capture , close to the saturation state. The biosensor tips were then equilibrated for 90 seconds in Octet wash buffer, followed by binding to diluted blood serum for 300 seconds. The final binding curve responses were measured using Octet Data Analysis HT10.0 software and the response was multiplied by a dilution factor (100 or 300) to obtain the final reportable response.

[00320] For anti-Gcc antibody binding, a trimerized Gcc peptide dimer with a C-terminal HIS tag was used on an Octet tip as described above. His _6- tagged (SEQ ID NO: 230) Gcc hexamer (strain A2) was preloaded onto Anti-Penta-HIS (HIS1K) sensor tips (FortéBio, #18-5122) for 400 seconds to ensure capture levels close to the saturation state . The biosensor tips were then equilibrated for 90 seconds in Octet wash buffer, followed by binding to diluted serum for 300 seconds. The final binding curve responses were measured using Octet Data Analysis HT10.0 software and the response was multiplied by the dilution factor (100 or 300) to obtain the final recorded response.

[00321] In non-human primate (NHP) studies, NHPs were pre-screened for response to RSV (baseline levels were below detection limits for all assays). NHPs were immunized with 50 μg of RF8140 with the designated adjuvant using a protocol similar to that described above for mice, but with large volume of adjuvant (Fig. 11C-D and Fig. 18).

[00322] In the non-human primate study, neutralization assays in VERO were performed as described above. Pre-F binding was assessed using the ELISA assay described below.

[00323] Serum samples from NHPs were serially diluted 2-fold (initial dilution 1:100) and incubated in plates coated with blocked soluble RSV F (Sinobiological, no. 11049-V08B) (1 μg/ml, 100 μl/well), for 1 hour at 37°C. RSV F-specific IgG was detected using horseradish peroxidase-conjugated anti-monkey IgG antibody (BioRad AAI42P, 1:10,000 dilution) for 90 minutes at 37°C. Plates were developed using 3,3',5,5'-tetramethylbenzidine (TMB Tebu-Bio) and stopped with 1N. hydrochloric acid (Prolabo, No. 30024290). Optical density (OD) was measured at 450–650 nm using a microplate reader (SpectraMax). sF RSV-specific IgG titers were calculated using SoftmaxPro software for an OD range of 0.2 to 3.0 based on the titration curve (standard mouse hyperimmune serum was applied to each plate).

[00324] The IgG titers of this standard, expressed in ELISA arbitrary units (EU), corresponded to the log10 dilution fold giving an OD of 1.0. The antibody detection threshold was 20 (1.3 log10) EU. All final titers were expressed in log10 for plotting. Each titer < 1.3 log10 was assigned an arbitrary titer of 1.0 log10.

[00325] To assess cellular immunity in the NHP study, the IFNγ/IL-2 FluoroSpot kit (FS-2122-10, Mabtech) was used according to the manufacturer's instructions. Briefly, IPFL plate membranes were pre-wetted with 35% ethanol and coated with immobilized antibodies (anti-IFNγ antibodies and anti-IL-2 antibodies) overnight at 4°C.

[00326] The plates were then blocked for 2 hours at 37°C at 200 μl/well with cell incubation medium containing 10% fetal calf serum (FCS). The medium was removed and stimuli were added to the wells: full-length F antigen (antigen-specific stimulation), anti-CD3 antibody (positive control), or cell culture medium (unstimulated control). Macaque peripheral blood mononuclear cells (PBMCs) were thawed and counted. 400,000 cells per well were added and incubated for 24 hours at 37°C in a humidified 5% CO2 incubator.

[00327] For detection, cells were removed and detection antibodies (anti-IFNγ conjugated antibodies and anti-IL-2 antibodies) were added and incubated for 2 hours at room temperature. Then reagents conjugated to a fluorophore were added and incubated for 1 hour at room temperature. The plates were emptied of their contents, dried and stored in the dark at room temperature until analysis. Anti-CD3 MAb was used as a positive control, and all samples had responses of >500 spotting events (SFC)/million PBMC, confirming acceptable sample quality. Spots detected in wells without stimulation (cell culture medium) were subtracted from values for cells stimulated with F antigen.

[00328] To analyze human cells (or B cells), experiments similar to the experiment mentioned in Dauner, et al. Vaccine 2017 Oct 4;35(41):5487-5494 fig. 20). Cells were either left untreated (treated with PBS) or treated with RSV F or RSV G polypeptides as indicated at 100 ng doses. F binding and G binding responses were analyzed using the Luminex assay described in the literature with beads coated with pre-F trimer (DS-CAV1) or G ectodomain, respectively.

[00329] RF8117 (SEQ ID NO: 17) contains engineered glycosylation sites at positions E328N, S348N and R507N, which, as mentioned above, do not prevent binding to D25 or AM14. To demonstrate that this prefusion protein nanoparticle elicits an immune response similar to that of other prefusion antigens (DS-CAV1), we immunized mice in groups of 5 with either the pre-F trimer (DS-CAV1) or F after fusion, or RF8117, at doses of 1 μg or 0.1 μg, all administered with AF03 adjuvant, three times with three-week intervals between injections. Serum samples were tested for neutralizing antibody titers two weeks after the third immunization using the VERO cell-based assay. RF8117 at the higher dose produced neutralizing antibody titers similar to those of the prefusion control and superior to those of the postfusion control. At a lower dose, RF8117 produced a higher titer of neutralizing antibodies than the prefusion control and postfusion control (Figure 10A).

[00330] RSV Pre-F-NP contains glycosylation sites designed to block epitopes common to the prefusion and postfusion conformations. The inhibition of the neutralizing response by these glycans was assessed. RF8117 with engineered glycans (SEQ ID NO: 17) was compared to RF8113 (similar to RF8117 but without engineered glycans; SEQ ID NO: 16) and a prefusion trimer control (DS-CAV1). Mice in groups of 5 were immunized with doses of 1 μg or 0.1 μg, all adjuvanted with AF03, three times with three-week intervals between injections. Serum samples were tested for neutralizing antibody titers two weeks after the third immunization using the VERO cell-based assay. There was no significant difference at either dose between constructs RF8113 and RF8117, as assessed by neutralizing antibody titers (Figure 10B).

[00331] To demonstrate that the lysine and arginine knockouts in RF8140 (SEQ ID NO: 23) mentioned herein do not impair the ability of the antigen to elicit a neutralizing response, we compared the immunogenicity of RF8140 (SEQ ID NO: 25) with the immunogenicity of trimer F after fusion (SEQ ID NO: 24) in mice (Fig. 11A and B). At a low dose (0.1 μg), RF8140 (SEQ ID NO: 25) produces a neutralizing antibody titer superior to that of the fusion trimer (SEQ ID NO: 24). To demonstrate that RF8140 (SEQ ID NO: 23) induces an immune response in NHPs, we immunized NHPs with RF8140 (SEQ ID NO: 25) with or without adjuvant (AF03). Figure 11C shows the RSV F binding response (ELISA titer) and Figure 11D compares the RSV neutralizing antibody titers obtained by immunization with Pre-F-NP (RF8140, SEQ ID NO: 23). Both unadjuvanted and adjuvanted, RF8140 (SEQ ID NO: 25) elicits an immune response in NHPs.

[00332] By demonstrating that the engineered glycosylation sites RF8117 (SEQ ID NO: 17) and RF8140 (SEQ ID NO: 23) do not prevent these antigens from eliciting a neutralizing response, we wished to demonstrate that they did indeed block epitopes that not subject to neutralization or subject to neutralization to a small extent, common to the pre-fusion and post-fusion conformations (Figure 12). An antibody response to prefusion F (DS-CAV1, SEQ ID NO: 25) was induced by immunization with Pre-F-NP without glycosylation by design (RF8113, SEQ ID NO: 16) or with Pre-F-NP with glycosylation by design (engineered Gly particle, RF8117 SEQ ID NO: 17) at high (1 μg) and low (0.1 μg) dose, as measured by Octet (Fig. 12A). Responses elicited by either Pre-F-NP were similar. An antibody response to the post-fusion trimer was induced by immunization with Pre-F-NP without glycosylation by design (RF8113, SEQ ID NO: 16) or with Pre-F-NP with glycosylation by design (RF8117 SEQ ID NO: 17 ) at high (1 μg) and low (0.1 μg) dose, as measured by Octet (Fig. 12B). Postfusion F binding responses induced by RF8117 (SEQ ID NO: 17) were significantly lower than those induced by RF8113 (SEQ ID NO: 16). Therefore, although both RF8113 and RF8117 elicit robust prefusion anti-F responses, the postfusion anti-F response induced by RF8117 is significantly suppressed. This is enabled by the presence of engineered glycans that map to epitopes common to the prefusion and postfusion conformations ( Figure 2B ).

[00333] To further demonstrate that the engineered glycosylation sites block epitopes that are not subject to neutralization, but provide a shift in the titer ratio of neutralizing antibodies to non-neutralizing antibodies, we analyzed the above data in a different way (Fig. 13). RSV neutralizing antibody titers measured using the VERO cell-based assay following immunization with pre-F-NP with wild-type glycosylation sites (Wt glycan particle; RF8113, SEQ ID NO: 16) versus Pre-F- NPs with added engineered glycosylation sites (particle+glycan; RF8117, SEQ ID NO: 17) in mouse studies were measured and showed no significant difference (Figure 13A). Comparison of F trimer-binding antibody response after RSV fusion induced by immunization with Wt glycan particle (RF8113, SEQ ID NO: 16) and with +glycan particle (RF8117, SEQ ID NO: 17) in studies in mice showed suppression of the F binding response after fusion in the case of Pre-F-NP with engineered glycans (Fig. 13B). To further demonstrate that the engineered glycans do not reduce the response of functional neutralizing antibodies, but do reduce the level of non-neutralizing antibodies produced in response to common pre-fusion/post-fusion epitopes, thereby providing an improvement in the ratio of neutralizing to of total antibodies generated by the engineered glycan constructs, the ratio of neutralizing antibody titer to F binding response was plotted (Figure 13C). Consequently, in mouse studies, Pre-F-NPs with engineered glycans provide a neutralizing antibody profile superior to that of binding antibodies.

[00334] To demonstrate the feasibility of using ferritin nanoparticles to improve the immunogenicity of the RSV central G domain antigen, we developed a method for chemically conjugating a Gcc peptide (SEQ ID NO: 29) to a ferritin nanoparticle. Ferritin carrying the S111C mutation described herein can be conjugated to a Gcc peptide (SEQ ID NO: 29) synthesized with a maleimide group on a PEG4 linker attached to the N-terminus by an NHS group. The N-terminal maleimide Gcc peptide was synthesized and purified by HPLC at Peptides International (Louisville, KY, USA). When maleimide-Gcc antigen is added to a ferritin particle with S111C, maleimide conjugates to free cysteine to form Gcc-NP, which can be observed by analysis on a Coomassie-stained SDS-PAGE gel (Figure 14A). Although the conjugation efficiency is typically between 50% and 90%, a model nanoparticle based on Gcc peptide and ferritin (100% conjugated) is shown in FIG. 14B.

[00335] To determine whether Gcc-NP elicits an immune response superior to that of Gcc peptide (SEQ ID NO: 29), 5 mice per group were immunized with either Gcc peptide or Gcc-NP (dose 1.3 μg, mixture 1 :1 with RIBI for each immunization). The Gcc (Octet) binding response two weeks after the second and two weeks after the third immunization treatments was compared with representative sera from a group of unexposed mice (Figure 14C). Also, the neutralizing response induced by immunization with Gcc peptide (SEQ ID NO. 29) was compared with Gcc-NP in mice after the third injection in neutralization assays with HAE cells (FIG. 14D). Gcc-NP elicits an immune response superior to that of Gcc peptide alone, as assessed by both Gcc binding and neutralization responses.

[00336] To demonstrate that coadministration of RSV Pre-F-NP (RF8140) and Gcc-NP does not affect the ability of either antigen to elicit an immune response, mice were immunized with either Pre-F-NP alone (RF8140, SEQ ID NO : 23), either Gcc-NP (ferritin conjugated to Gcc peptide SEQ ID NO: 29) or Pre-F-NP (RF8140, SEQ ID NO: 23) in combination with Gcc-NP (FIG 15A-C). All immunization procedures were carried out using AF03 adjuvant. Mice immunized with RF8140 alone (Pre-F-NP) or RF8140 and Gcc-NP (Pre-F-NP+Gcc-NP) produced antibodies that bind the prefusion F trimer (DS-CAV1, SEQ ID: 25), whereas mice immunized with Gcc-NPs did not. Mice immunized with Gcc-NP alone (Gcc-NP) or RF8140 and Gcc-NP produced antibodies that bind the Gcc peptide, whereas mice immunized with RF8140 alone did not. All animals immunized with Pre-F-NP alone, Gcc-NP alone, or Pre-F-NP and Gcc-NP coadministered produced a neutralizing response after the second and after the third immunization, as measured in a neutralization assay with HAE cells.

[00337] To determine whether co-administration of RSV Pre-F-NP and Gcc-NP affects the ability of either antigen to elicit neutralizing antibodies, both anti-F and anti-G neutralizing antibodies were examined in a depletion assay (Figure 16A-B) . To demonstrate that addition of Gcc-NP does not affect the ability of Pre-F-NP to elicit a neutralizing response, neutralizing antibody titers were measured using the F-sensitive VERO cell assay for the above groups (Figure 16A). Serum samples from unexposed animals were also tested to judge the quality of antigen depletion. In the VERO-based assay, serum samples from mice immunized with RF8140 (SEQ ID NO: 23) alone or RF8140 mixed with Gcc-NP elicited similar neutralizing responses, whereas Gcc-NP did not appear to elicit a neutralizing response in the assay based on anti-F antibody-sensitive VERO cells. When pooled serum samples from animals immunized with RF8140 (SEQ ID NO:23) alone or immunized with RF8140 (SEQ ID: 23) and Gcc-NP were antibody depleted, binding trimer prefusion (DS-CAV1, SEQ ID: 25), observed a decrease in measurable neutralizing antibody titers in a VERO cell-based assay. When neutralizing antibody titers were measured in the HAE cell-based assay in the above groups, all immunization groups were observed to mount a neutralizing response in the F and G susceptibility assay (FIG. 16B). Pooled serum samples from animals immunized with RF8140 (SEQ ID NO: 23) alone induced a neutralizing response in an assay based on HAE cells that could be depleted of the prefusion F trimer (DS-CAV1, SEQ ID NO: 25) . Pooled serum samples from animals immunized with Gcc-NP alone induced a neutralizing response in an assay based on HAE cells that could be depleted of ectodomain G (SEQ ID NO: 28). Pooled serum samples from animals immunized with both RF8140 (SEQ ID NO: 23) and Gcc-NP induced a neutralizing response in an assay based on HAE cells that were not completely depleted of DS-CAV1 (SEQ ID NO: 25), but were completely depleted by sequential depletion procedures for DS-CAV1 and then ectodomain G (SEQ ID NO: 28). Together, these data suggest that coadministration with Pre-F-NP and Gcc-NP does not affect the ability of either antigen to elicit neutralizing antibodies against prefusion F or G, respectively.

[00338] To demonstrate the effect of adjuvanted administration of RF8117 (SEQ ID NO: 17) or RF8140 (SEQ ID NO: 23), mice were dosed with these constructs mixed with AF03, SPA09 or alum. Figure 17A shows that mice were immunized with 10 μg of antigen mixed with an adjuvant, while Figure 17B shows that mice were immunized with 1 μg of antigen mixed with an adjuvant. Figure 17A shows neutralizing antibody titers measured using the VERO cell-based assay at a time point two weeks after the third immunization. Shown are serum samples from mice immunized with RF8117 (SEQ ID NO: 17) with either no adjuvant (no adjuvant), alum adjuvant, or AF03 adjuvant. Figure 17B shows that neutralizing antibody titers were measured using the VERO cell-based assay for serum samples from mice immunized with RF8117 (SEQ ID NO: 17) using AF03 adjuvant, RF8117 (SEQ ID NO: 17) using adjuvant SPA09, or RF8140 using adjuvant AF03. In all cases for RF8117 (SEQ ID NO: 17) or RF8140 (SEQ ID NO: 23), the adjuvanted groups of naïve mice had higher neutralizing antibody titers than the non-adjuvanted groups. Mice immunized with RF8117 (SEQ ID NO: 17) or RF8140 (SEQ ID NO: 23) mixed with AF03 obtained a similar neutralizing response, suggesting that the mutations that add lysine and arginine to RF8140 (SEQ ID NO: 23) do not affect the ability of Pre-F-NP to induce a neutralizing response.

[00339] To further examine the unadjuvanted effect of AF03 and SPA09, non-human primates (NHPs) were immunized with RF8140 unadjuvanted, adjuvanted with AF03, or adjuvanted with two doses of SPA09 (Figure 18A). NHPs were immunized with 50 μg of antigen mixed with the indicated adjuvant on days 0 and 29, and the immune response was measured by ELISA or neutralization response in VERO cells at the indicated time points. Prefusion trimer F responses by ELISA were measured in serum samples from NHPs following immunization with RF8140, unadjuvanted (no adjuvant), AF03 adjuvanted, or SPA09 adjuvanted (500 μg and 2000 μg SPA09 doses were used). At all time points, adjuvant AF03 or SPA09 produced a superior neutralizing response. Neutralizing antibody titers in serum samples for the above NHP groups were also measured using the VERO cell-based assay (FIG. 18B). In all cases, immunization with adjuvanted RF8140 resulted in higher neutralizing antibody titers than the unadjuvanted group at all time points.

[00340] The effect of direct conjugation of RF8140 (SEQ ID NO: 23) with the TLR7/8 agonist SM7/8 or the TLR9 CpG agonist was tested. The antigen was conjugated to small molecules SM7/8 or CpG and administered to mice as a 10 μg dose. RF8140 contains a mutation in its ferritin sequence that allows for the replacement of a surface-exposed amino acid with cysteine (K79C), which can be used for conjugation using click chemistry. For comparison, mice were dosed with RF8140 either without the use of an adjuvant (no adjuvant) or with the use of an adjuvant by mixing with a small molecule at a high or low dose (no conjugation), as shown in Fig. 19. Serum samples from animals after the second and after the third immunization were tested for binding to trimer F before fusion.

[00341] Figure 19A shows the result of measuring the prefusion F trimer binding response in serum samples from any of naïve mice, mice immunized with RF8140 (SEQ ID NO: 23) without adjuvant, mice immunized with using RF8140 (SEQ ID NO: 23) conjugated with SM7/8 adjuvant, RF8140 (SEQ ID NO: 23) with 130 ng SM7/8 adjuvant, or RF8140 (SEQ ID NO: 23) with 20 adjuvant µg SM7/8. RF8140 (SEQ ID NO: 23) conjugated to SM7/8 produced a higher titer of prefusion F trimer binding antibodies than the RF8140 unadjuvanted or SM7/8 adjuvanted groups.

[00342] Figure 19B also shows the result of measuring the prefusion trimer F binding response in serum samples from any of naïve mice, mice immunized with RF8140 (SEQ ID NO: 23) without adjuvant, mice immunized using RF8140 (SEQ ID NO: 23) conjugated with a CpG adjuvant, RF8140 (SEQ ID NO: 23) with 680 ng CpG adjuvant, or RF8140 (SEQ ID NO: 23) with 20 μg CpG adjuvant . RF8140 (SEQ ID NO: 23) conjugated to SM7/8 produced a higher titer of prefusion F trimer binding antibodies than the RF8140 unadjuvanted or SM7/8 adjuvanted groups.

[00343] To demonstrate the ability of Pre-F-NP antigen and Gcc-NP antigen to induce a response in human cells, experiments were performed with the MIMIC platform (Fig. 20A-D). The MIMIC platform consists exclusively of autologous human immune cells capable of rapidly and reproducibly generating antigen-specific innate and adaptive responses upon infection. Previous work has demonstrated the ability of the MIMIC system to recapitulate in vivo immune profiles against targets as diverse as HBV, tetanus toxoid, monoclonal antibodies, YF-VAX, and B cell responses to influenza virus. The RSV prefusion F trimer binding antibody responses induced by treatment with Pre-F-NP RF8140 (SEQ ID NO: 23) and postfusion F trimer (SEQ ID NO: 24) were compared in human B- cells, and a representative baseline response is shown for comparison (no treatment) (Figure 20A). Ratios of measured binding responses to prefusion F trimer (DS-CAV1, SEQ ID NO: 25) and postfusion F trimer (SEQ ID NO: 24) induced by treatment with Pre-F-NP (RF8140, SEQ ID NO: 23) versus postfusion F (SEQ ID NO: 24) in human B cells are shown in FIG. 20C. Antibodies from MIMIC produced by treatment with various F antigens were measured using a VERO cell-based assay (FIG. 20B). Neutralizing antibody titers obtained by treatment with pre-F-NP (RF8140, SEQ ID NO: 23) and post-fusion trimer F (SEQ ID NO: 24) in human B cells were compared with those in the untreated group , whereby it was shown that RF8140 (SEQ ID NO: 23) caused a superior neutralizing response in human cells.

[00344] The magnitude of the Ab response to RSV infection or the F subunit vaccine candidate was determined based on the serostatus of the human subjects studied in the MIMIC studies, which was assessed using linear regression analysis. Donors with higher pre-existing titers of circulating anti-pre-F IgG produced significantly more anti-pre-F IgG after treatment with RSV (Fig. 20E,p=0.0041) and after priming with post-F (Fig. 20F,p=0.0019). Although the correlation did not reach statistical significance, pre-F also showed a relationship between the level of induced Ab and the level of pre-existing Ab. Notably, in contrast to other treatments, pre-F-NP induced relatively high levels of anti-pre-F IgG in donors with low pre-existing anti-pre-F IgG, as well as in donors with high levels of pre-existing Ab (Figure 20F ). This indicates that pre-F-NP is able to effectively maintain (or enhance) the Ab response even in donors with low levels of pre-existing IgG.

[00345] To demonstrate that Gcc-NP elicits an anti-G antibody response superior to that of Gcc peptide (SEQ ID NO: 29) alone, human cells were treated with Gcc peptide alone (SEQ ID NO: 29) or Gcc peptide , conjugated to a nanoparticle (Gcc-NP) in human B cells. Gcc-NP produced a superior G-binding antibody response (Figure 20G). Taken together, these data suggest that Pre-F-NP and Gcc-NP will elicit immune responses upon human immunization.

4. Characteristics of the immune response to nanoparticles based on Gcc RSV-ferritin in vivo

[00346] Gcc RSV-NP was prepared as described above. To evaluate the answerin vivoon Gcc RSV-NP in mice, female BALBc mice were immunized intramuscularly with RSV antigens at defined doses on weeks 0, 3, and 6 with either a high dose (5 μg) or a low dose (0.5 μg) of antigen. Unless otherwise stated, Gcc RSV-NP was administered with AF03 adjuvant using a pre-use mixing strategy. That is, 50 μl of the protein solution was mixed with 50 μl of Sanofi AF03 adjuvant (squalene-based emulsion; see Fig. Klucker et al., J Pharm Sci. 2012 Dec;101(12):4490-500) immediately before injection of 50 µl into each hind paw. No side effects of immunization were observed. Blood was collected 1 day before the first immunization and at least 2 weeks after each injection (i.e., weeks 2, 5, and 8). Unless otherwise noted, data are shown for 2 weeks after the third injection (week 8, also referred to as 2wp3). Typically, serum samples from animals were analyzed before immunization (designated as unexposed), two weeks after the second injection (after 2 or 2wp2), or two weeks after the third injection (after 3 or 2wp3).

[00347] For the HAE neutralization assay, blood serum was heat inactivated for 30 minutes at 56°C. A series of four-fold serial dilutions of inactivated serum were performed in PneumaCult™-ALI Basic Medium (Stem Cell Technologies; 05002) supplemented with PneumaCult™-ALI 10X Supplement (Stem Cell Technologies; 05003) and 1% antibiotic/antifungal agent (hereinafter referred to as media). RSV stock solutions were combined 1:1 with serum dilutions and incubated for 1.5 hours at 37°C. The virus-serum mixture was then added to 24-well plates containing fully differentiated HAE cells at a rate of 50 μl per well and incubated for 1 hour at 37°C, 5% CO ₂ . After incubation, the inoculum was removed, the wells were washed twice with medium to remove unbound virus, and incubated for an additional 20 hours at 37°C, 5% CO ₂ . Infection events in RSV-infected cultures expressing the mKate reporter (TagFP635) were counted under a fluorescence microscope.

[00348] To identify RSV infections that do not express the mKate reporter (neutralizing RSV strain B), pseudostratified epithelial samples were thoroughly washed with demucus medium, then fixed with 4% paraformaldehyde for 30 minutes at room temperature, permeabilized with 0 .25% Triton X-100 for 30 minutes and blocked with DMEM supplemented with 2% FBS for 1 hour at 37°C. The blocking solution was replaced at a rate of 100 μl per well with a mixture of mouse monoclonal Abs against RSV (Millipore; MAB 858-4), diluted 1:200 in DMEM supplemented with 2% FBS, and the plates were incubated at 37°C for 2 hours. The plates were then washed 3 times with PBS supplemented with 0.05% Tween 20. 100 μl of goat anti-mouse IgG (H+L) (Invitrogen; A11001) diluted 1:200 in DMEM supplemented with 2% was added to each well. FBS, and the plates were incubated overnight at 4°C. The next morning, plates were washed 3 times with PBS supplemented with 0.05% Tween 20, the fluorescence signal was stabilized with ProLong Gold AntiFade with DAPI (Thermo Fisher Scientific; P36935) and counted on a fluorescence microscope. Neutralizing antibody titers were determined at the end point of 60% reduction.

[00349] To demonstrate that a higher degree of polyvalency improves the ability to elicit a neutralizing response with RSV G antigens, mice were immunized with RSV F antigens. All immunization procedures were carried out using AF03 adjuvant. Mice were immunized with Gcc RSV-NP formulated with AF03, and neutralizing antibody titers were measured 2 weeks after the second and 2 weeks after the third injection (Fig. 21A-C). Gcc RSV-NPs induced a neutralizing response compared to serum from a naïve mouse. Both 2 weeks after the second (Fig. 21A) and 2 weeks after the third (Fig. 21B) immunization, mice immunized with Gcc-NPs containing Gcc from strain A2 exhibited neutralizing responses to RSV strain A. 2 weeks after the third injection, Gcc-NP also induced a neutralizing response to RSV strain B1 (Figure 21C).

[00350] For anti-Gcc antibody binding, a trimerized Gcc peptide dimer with a C-terminal HIS tag was used on an Octet tip. His ₆ -tagged Gcc hexamer (strain A2) or His ₆ -tagged Gcc hexamer (strain B1) was preloaded onto Anti-Penta-HIS (HIS1K) sensor tips (FortéBio, #18-5122) for 400 seconds to ensure a level of capture close to the saturation state. The biosensor tips were then equilibrated for 90 seconds in Octet wash buffer, followed by binding to diluted serum for 300 seconds. The final binding curve responses were measured using Octet Data Analysis HT10.0 software and the response was multiplied by the dilution factor (100 or 300) to obtain the final recorded response.

[00351] To determine whether Gcc RSV-NP elicits a Gcc binding immune response, serum samples obtained from the immunization procedures described above were tested for their ability to bind the Gcc A2 hexamer or the Gcc B1 hexamer. The Gcc binding response at high dose (FIG. 22A and FIG. 23A) and low dose (FIG. 22B and FIG. 23B) was tested 2 weeks after the second and 2 weeks after the third immunization treatments. In both strain A2 (FIG. 22A-B) and strain B1 (FIG. 23A-B), Gcc RSV-NP produced a binding response in contrast to serum samples from naïve mice.

5. Response to Pre-F-NP and Gcc-NP in human cells

[00352] To demonstrate the ability of Pre-F-NP and Gcc-NP to induce a response in human cells, experiments were performed with the MIMIC platform. The MIMIC platform consists exclusively of autologous human immune cells capable of rapidly and reproducibly generating antigen-specific innate and adaptive responses upon infection. Previous work has demonstrated the ability of the MIMIC system to recapitulate in vivo immune profiles against targets as diverse as HBV, tetanus toxoid, monoclonal antibodies, YF-VAX, and B cell responses to influenza virus. RSV pre-fusion F trimer-binding antibody responses induced by treatment with Pre-F-NP RF8140 (SEQ ID NO: 23) and post-fusion F trimer (SEQ ID NO: 24) in human B cells were compared , and also compared with a representative original response. Ratios of measured binding responses to prefusion F trimer (DS-CAV1, SEQ ID NO: 25) and postfusion F trimer (SEQ ID NO: 24) induced by treatment with Pre-F-NP (RF8140, SEQ ID NO: 23) compared to postfusion F treatment (SEQ ID NO: 24) was determined in human B cells. Antibodies from MIMIC produced by treatment with various F antigens were measured using a VERO cell-based assay. Neutralizing antibody titers obtained by treatment with pre-F-NP (RF8140, SEQ ID NO: 23) and post-fusion trimer F (SEQ ID NO: 24) in human B cells were compared with those in the untreated group , whereby it was shown that RF8140 (SEQ ID NO: 23) caused a superior neutralizing response in human cells. To demonstrate that Gcc-NP elicits an anti-G antibody response superior to that of Gcc peptide (SEQ ID NO: 29) alone, human cells were treated with Gcc peptide alone (SEQ ID NO: 29) or Gcc peptide conjugated to nanoparticle (Gcc-NP) in human B cells. Gcc-NP induced a superior response in the form of G-binding antibody production. Thus, Pre-F-NP and Gcc-NP will elicit immune responses upon human immunization.

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LIST OF SEQUENCES

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RESPIRATORY SYNCYTIAL VIRUS

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Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Asn Asn Thr Lys Glu

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Cys Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 4

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 4

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Asn

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Cys Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 5

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 5

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Thr Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Cys Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 6

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 6

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Asn Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Cys Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 7

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 7

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Ser Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Cys Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 8

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 8

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Asn Lys Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Cys Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 9

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 9

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Pro Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Cys Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 10

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 10

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Pro Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Leu Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Cys Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 11

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 11

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Pro Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Cys Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 12

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 12

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Ile Glu Thr Val Ile Glu Phe Leu Gln Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Cys Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 13

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 13

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Ile Glu Thr Val Ile Glu Phe Leu Val Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Cys Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 14

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 14

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Leu Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Cys Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 15

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 15

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Phe Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Cys Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 16

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 16

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Pro Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Cys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 17

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 17

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Pro Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Asn

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Asn Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Asn Lys Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Cys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 18

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 18

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Pro Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Asn

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Asn Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Leu Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Asn Gln Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Cys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 19

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 19

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Val Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Val Ser Ile Ser Asn

165 170 175

Pro Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Asn

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Asn Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Asn Lys Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Cys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 20

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 20

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Pro Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Asn

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Asn Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Leu Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Asn Gln Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Asn Asp Ile Glu Asn Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Cys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 21

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 21

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Pro Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Asn

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Asn Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Asn Ser Leu Tyr Val Asn Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Leu Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Asn Gln Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Cys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 22

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 22

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Pro Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Asn

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Asn Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Asn Ser Leu Tyr Val Asn Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Leu Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Asn Gln Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Arg Gln Gln Phe Ser Asn Asp Ile Glu Asn Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Cys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 23

<211> 651

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 23

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gly Ser Gly Asn Val Gly Leu Gly Gly Ala Ile Ala Ser Gly Val

100 105 110

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

115 120 125

Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

130 135 140

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

145 150 155 160

Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn

165 170 175

Pro Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

180 185 190

Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser

195 200 205

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met

210 215 220

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile

225 230 235 240

Val Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val

245 250 255

Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro

260 265 270

Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Asn

275 280 285

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

290 295 300

Asn Ala Gly Asn Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

305 310 315 320

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu Pro

325 330 335

Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp

340 345 350

Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr

355 360 365

Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

370 375 380

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp

385 390 395 400

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

405 410 415

Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu

420 425 430

Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

435 440 445

Asp Ala Ser Ile Ser Gln Val Asn Glu Leu Ile Asn Gln Ser Leu Ala

450 455 460

Phe Ile Asn Gln Ser Asp Glu Leu Leu Ser Gly Ser Gly Ser Glu Ser

465 470 475 480

Gln Val Gln Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu

485 490 495

Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser

500 505 510

Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe

515 520 525

Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe

530 535 540

Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro

545 550 555 560

Glu His Cys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu

565 570 575

His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala

580 585 590

Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val

595 600 605

Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys

610 615 620

Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln

625 630 635 640

Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

645 650

<210> 24

<211> 530

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 24

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Ala Ile Ala Ser Gly Val Ala Val

130 135 140

Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser Ala

145 150 155 160

Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser

165 170 175

Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln

180 185 190

Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu

195 200 205

Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr

210 215 220

Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr Tyr

225 230 235 240

Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro Ile

245 250 255

Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val Arg

260 265 270

Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu Ala

275 280 285

Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp

290 295 300

Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser

305 310 315 320

Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala

325 330 335

Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser

340 345 350

Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser Glu

355 360 365

Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys

370 375 380

Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser Leu

385 390 395 400

Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn

405 410 415

Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val

420 425 430

Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr

435 440 445

Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile

450 455 460

Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala

465 470 475 480

Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile

485 490 495

Arg Lys Ser Asp Glu Leu Leu Gly Leu Glu Val Leu Phe Gln Gly Pro

500 505 510

His His His His His His His Ser Ala Trp Ser His Pro Gln Phe

515 520 525

Glu Lys

530

<210> 25

<211> 689

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 25

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Cys Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Cys Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Ser Gly Gly Ser Ser Gly Ser Ser Gly Gly Ser Asp Ile Ile Lys

515 520 525

Leu Leu Asn Glu Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr

530 535 540

Met Ser Met Ser Ser Trp Cys Tyr Thr His Ser Leu Asp Gly Ala Gly

545 550 555 560

Leu Phe Leu Phe Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys

565 570 575

Leu Ile Ile Phe Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser

580 585 590

Ile Ser Ala Pro Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln

595 600 605

Lys Ala Tyr Glu His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile

610 615 620

Val Asp His Ala Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu

625 630 635 640

Gln Trp Tyr Val Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp

645 650 655

Ile Leu Asp Lys Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr

660 665 670

Leu Ala Asp Gln Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser Gly

675 680 685

Ser

<210> 26

<211> 574

<212> PROTEIN

<213> Human respiratory syncytial virus

<400> 26

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1. 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Pro Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Ile Asn Leu Cys Asn Val

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Met Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn Ile Met Ile Thr

515 520 525

Thr Ile Ile Ile Val Ile Ile Val Ile Leu Leu Ser Leu Ile Ala Val

530 535 540

Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr Pro Val Thr Leu Ser

545 550 555 560

Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala Phe Ser Asn

565 570

<210> 27

<211> 297

<212> PROTEIN

<213> Human respiratory syncytial virus

<400> 27

Met Ser Lys Asn Lys Asp Gln Arg Thr Ala Lys Thr Leu Glu Arg Thr

1. 5 10 15

Trp Asp Thr Leu Asn His Leu Leu Phe Ile Ser Ser Cys Leu Tyr Lys

20 25 30

Leu Asn Leu Lys Ser Val Ala Gln Ile Thr Leu Ser Ile Leu Ala Met

35 40 45

Ile Ile Ser Thr Ser Leu Ile Ile Val Ala Ile Ile Phe Ile Ala Ser

50 55 60

Ala Asn His Lys Ile Thr Ser Thr Thr Thr Ile Ile Gln Asp Ala Thr

65 70 75 80

Asn Gln Ile Lys Asn Thr Thr Pro Thr Tyr Leu Thr Gln Asn Pro Gln

85 90 95

Leu Gly Ile Ser Pro Ser Asn Pro Ser Asp Ile Thr Ser Leu Ile Thr

100 105 110

Thr Ile Leu Asp Ser Thr Thr Pro Gly Val Lys Ser Thr Leu Gln Ser

115 120 125

Thr Thr Val Gly Thr Lys Asn Thr Thr Thr Thr Gln Ala Gln Pro Asn

130 135 140

Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro Pro Ser Lys Pro Asn

145 150 155 160

Asn Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Cys Ser Ile Cys

165 170 175

Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro Asn Lys

180 185 190

Lys Pro Gly Lys Arg Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Pro

195 200 205

Lys Thr Thr Lys Lys Gly Pro Lys Pro Gln Thr Thr Lys Ser Lys Glu

210 215 220

Ala Pro Thr Thr Lys Pro Thr Glu Glu Pro Thr Ile Asn Thr Thr Lys

225 230 235 240

Thr Asn Ile Ile Thr Thr Leu Leu Thr Ser Asn Thr Thr Arg Asn Pro

245 250 255

Glu Leu Thr Ser Gln Met Glu Thr Phe His Ser Thr Ser Ser Glu Gly

260 265 270

Asn Pro Ser Pro Ser Gln Val Ser Ile Thr Ser Glu Tyr Pro Ser Gln

275 280 285

Pro Ser Ser Pro Pro Asn Thr Pro Arg

290 295

<210> 28

<211> 233

<212> PROTEIN

<213> Human respiratory syncytial virus

<400> 28

Asn His Lys Val Thr Leu Thr Thr Ala Ile Ile Gln Asp Ala Thr Ser

1. 5 10 15

Gln Ile Lys Asn Thr Thr Pro Thr Tyr Leu Thr Gln Asp Pro Gln Leu

20 25 30

Gly Ile Ser Phe Ser Asn Leu Ser Glu Ile Thr Ser Gln Thr Thr Thr

35 40 45

Ile Leu Ala Ser Thr Thr Pro Gly Val Lys Ser Asn Leu Gln Pro Thr

50 55 60

Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gln Thr Gln Pro Ser Lys

65 70 75 80

Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro Pro Asn Lys Pro Asn Asn

85 90 95

Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Cys Ser Ile Cys Ser

100 105 110

Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro Asn Lys Lys

115 120 125

Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys

130 135 140

Thr Thr Lys Lys Asp His Lys Pro Gln Thr Thr Lys Pro Lys Glu Val

145 150 155 160

Pro Thr Thr Lys Pro Thr Glu Glu Pro Thr Ile Asn Thr Thr Lys Thr

165 170 175

Asn Ile Ile Thr Thr Leu Leu Thr Asn Asn Thr Thr Gly Asn Pro Lys

180 185 190

Leu Thr Ser Gln Met Glu Thr Phe His Ser Thr Ser Ser Glu Gly Asn

195 200 205

Leu Ser Pro Ser Gln Val Ser Thr Thr Ser Glu His Pro Ser Gln Pro

210 215 220

Ser Ser Pro Pro Asn Thr Thr Arg Gln

225 230

<210> 29

<211> 56

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 29

Ser Gly Gly Ser Ser Gly Ser Ser Glu Glu Glu Gly Gly Ser Arg Gln

1. 5 10 15

Asn Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val Pro

20 25 30

Cys Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg

35 40 45

Ile Pro Asn Lys Lys Glu Glu Glu

50 55

<210> 30

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 30

tgactgtgaa cgttcgagat ga 22

<210> 31

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 31

Gly Ser Gly Asn Val Gly Leu

15

<210> 32

<211> 43

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 32

Arg Gln Asn Lys Pro Pro Asn Lys Pro Asn Asn Asp Phe His Phe Glu

1. 5 10 15

Val Phe Asn Phe Val Pro Cys Ser Ile Cys Ser Asn Asn Pro Thr Cys

20 25 30

Trp Ala Ile Cys Lys Arg Ile Pro Asn Lys Lys

35 40

<210> 33

<211> 42

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 33

Arg Lys Asn Pro Pro Lys Lys Pro Lys Asp Asp Tyr His Phe Glu Val

1. 5 10 15

Phe Asn Phe Val Pro Cys Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys

20 25 30

Ser Ile Cys Lys Thr Ile Pro Asn Lys Lys

35 40

<210> 34

<400> 34

000

<210> 35

<400> 35

000

<210> 36

<400> 36

000

<210> 37

<400> 37

000

<210> 38

<400> 38

000

<210> 39

<400> 39

000

<210> 40

<400> 40

000

<210> 41

<400> 41

000

<210> 42

<400> 42

000

<210> 43

<400> 43

000

<210> 44

<400> 44

000

<210> 45

<400> 45

000

<210> 46

<400> 46

000

<210> 47

<400> 47

000

<210> 48

<400> 48

000

<210> 49

<400> 49

000

<210> 50

<400> 50

000

<210> 51

<400> 51

000

<210> 52

<400> 52

000

<210> 53

<400> 53

000

<210> 54

<400> 54

000

<210> 55

<400> 55

000

<210> 56

<400> 56

000

<210> 57

<400> 57

000

<210> 58

<400> 58

000

<210> 59

<400> 59

000

<210> 60

<400> 60

000

<210> 61

<400> 61

000

<210> 62

<400> 62

000

<210> 63

<400> 63

000

<210> 64

<400> 64

000

<210> 65

<400> 65

000

<210> 66

<400> 66

000

<210> 67

<400> 67

000

<210> 68

<400> 68

000

<210> 69

<400> 69

000

<210> 70

<400> 70

000

<210> 71

<400> 71

000

<210> 72

<400> 72

000

<210> 73

<400> 73

000

<210> 74

<400> 74

000

<210> 75

<400> 75

000

<210> 76

<400> 76

000

<210> 77

<400> 77

000

<210> 78

<400> 78

000

<210> 79

<400> 79

000

<210> 80

<400> 80

000

<210> 81

<400> 81

000

<210> 82

<400> 82

000

<210> 83

<400> 83

000

<210> 84

<400> 84

000

<210> 85

<400> 85

000

<210> 86

<400> 86

000

<210> 87

<400> 87

000

<210> 88

<400> 88

000

<210> 89

<400> 89

000

<210> 90

<400> 90

000

<210> 91

<400> 91

000

<210> 92

<400> 92

000

<210> 93

<400> 93

000

<210> 94

<400> 94

000

<210> 95

<400> 95

000

<210> 96

<400> 96

000

<210> 97

<400> 97

000

<210> 98

<400> 98

000

<210> 99

<400> 99

000

<210> 100

<400> 100

000

<210> 101

<400> 101

000

<210> 102

<400> 102

000

<210> 103

<400> 103

000

<210> 104

<400> 104

000

<210> 105

<400> 105

000

<210> 106

<400> 106

000

<210> 107

<400> 107

000

<210> 108

<400> 108

000

<210> 109

<400> 109

000

<210> 110

<400> 110

000

<210> 111

<400> 111

000

<210> 112

<400> 112

000

<210> 113

<400> 113

000

<210> 114

<400> 114

000

<210> 115

<400> 115

000

<210> 116

<400> 116

000

<210> 117

<400> 117

000

<210> 118

<400> 118

000

<210> 119

<400> 119

000

<210> 120

<400> 120

000

<210> 121

<400> 121

000

<210> 122

<400> 122

000

<210> 123

<400> 123

000

<210> 124

<400> 124

000

<210> 125

<400> 125

000

<210> 126

<400> 126

000

<210> 127

<400> 127

000

<210> 128

<400> 128

000

<210> 129

<400> 129

000

<210> 130

<400> 130

000

<210> 131

<400> 131

000

<210> 132

<400> 132

000

<210> 133

<400> 133

000

<210> 134

<400> 134

000

<210> 135

<400> 135

000

<210> 136

<400> 136

000

<210> 137

<400> 137

000

<210> 138

<400> 138

000

<210> 139

<400> 139

000

<210> 140

<400> 140

000

<210> 141

<400> 141

000

<210> 142

<400> 142

000

<210> 143

<400> 143

000

<210> 144

<400> 144

000

<210> 145

<400> 145

000

<210> 146

<400> 146

000

<210> 147

<400> 147

000

<210> 148

<400> 148

000

<210> 149

<400> 149

000

<210> 150

<400> 150

000

<210> 151

<400> 151

000

<210> 152

<400> 152

000

<210> 153

<400> 153

000

<210> 154

<400> 154

000

<210> 155

<400> 155

000

<210> 156

<400> 156

000

<210> 157

<400> 157

000

<210> 158

<400> 158

000

<210> 159

<400> 159

000

<210> 160

<400> 160

000

<210> 161

<400> 161

000

<210> 162

<400> 162

000

<210> 163

<400> 163

000

<210> 164

<400> 164

000

<210> 165

<400> 165

000

<210> 166

<400> 166

000

<210> 167

<400> 167

000

<210> 168

<400> 168

000

<210> 169

<400> 169

000

<210> 170

<400> 170

000

<210> 171

<400> 171

000

<210> 172

<400> 172

000

<210> 173

<400> 173

000

<210> 174

<400> 174

000

<210> 175

<400> 175

000

<210> 176

<400> 176

000

<210> 177

<400> 177

000

<210> 178

<400> 178

000

<210> 179

<400> 179

000

<210> 180

<400> 180

000

<210> 181

<400> 181

000

<210> 182

<400> 182

000

<210> 183

<400> 183

000

<210> 184

<400> 184

000

<210> 185

<400> 185

000

<210> 186

<400> 186

000

<210> 187

<400> 187

000

<210> 188

<400> 188

000

<210> 189

<400> 189

000

<210> 190

<400> 190

000

<210> 191

<400> 191

000

<210> 192

<400> 192

000

<210> 193

<400> 193

000

<210> 194

<400> 194

000

<210> 195

<400> 195

000

<210> 196

<400> 196

000

<210> 197

<400> 197

000

<210> 198

<400> 198

000

<210> 199

<400> 199

000

<210> 200

<400> 200

000

<210> 201

<211> 173

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 201

Glu Ser Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu

1. 5 10 15

Asn Glu Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Cys

20 25 30

Met Ser Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe

35 40 45

Leu Phe Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile

50 55 60

Ile Phe Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser

65 70 75 80

Ala Pro Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala

85 90 95

Tyr Glu His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp

100 105 110

His Ala Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp

115 120 125

Tyr Val Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu

130 135 140

Asp Lys Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala

145 150 155 160

Asp Gln Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

165 170

<210> 202

<211> 173

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 202

Glu Ser Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu

1. 5 10 15

Asn Glu Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser

20 25 30

Met Ser Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe

35 40 45

Leu Phe Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile

50 55 60

Ile Phe Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Cys Ile Ser

65 70 75 80

Ala Pro Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala

85 90 95

Tyr Glu His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp

100 105 110

His Ala Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp

115 120 125

Tyr Val Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu

130 135 140

Asp Lys Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala

145 150 155 160

Asp Gln Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

165 170

<210> 203

<211> 173

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 203

Glu Ser Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu

1. 5 10 15

Asn Glu Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser

20 25 30

Met Ser Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe

35 40 45

Leu Phe Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile

50 55 60

Ile Phe Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser

65 70 75 80

Cys Pro Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala

85 90 95

Tyr Glu His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp

100 105 110

His Ala Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp

115 120 125

Tyr Val Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu

130 135 140

Asp Lys Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala

145 150 155 160

Asp Gln Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

165 170

<210> 204

<211> 173

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 204

Glu Ser Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu

1. 5 10 15

Asn Glu Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser

20 25 30

Met Ser Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe

35 40 45

Leu Phe Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile

50 55 60

Ile Phe Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser

65 70 75 80

Ala Pro Glu His Cys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala

85 90 95

Tyr Glu His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp

100 105 110

His Ala Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp

115 120 125

Tyr Val Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu

130 135 140

Asp Lys Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala

145 150 155 160

Asp Gln Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

165 170

<210> 205

<211> 173

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 205

Glu Ser Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu

1. 5 10 15

Asn Glu Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser

20 25 30

Met Ser Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe

35 40 45

Leu Phe Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile

50 55 60

Ile Phe Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser

65 70 75 80

Ala Pro Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala

85 90 95

Tyr Glu His Glu Gln His Ile Ser Glu Cys Ile Asn Asn Ile Val Asp

100 105 110

His Ala Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp

115 120 125

Tyr Val Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu

130 135 140

Asp Lys Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala

145 150 155 160

Asp Gln Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

165 170

<210> 206

<211> 173

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 206

Glu Ser Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu

1. 5 10 15

Asn Glu Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser

20 25 30

Met Ser Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe

35 40 45

Leu Phe Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile

50 55 60

Ile Phe Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser

65 70 75 80

Ala Pro Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala

85 90 95

Tyr Glu His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp

100 105 110

His Ala Ile Lys Cys Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp

115 120 125

Tyr Val Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu

130 135 140

Asp Lys Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala

145 150 155 160

Asp Gln Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

165 170

<210> 207

<211> 173

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 207

Glu Ser Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu

1. 5 10 15

Asn Cys Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser

20 25 30

Met Ser Ser Trp Ser Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe

35 40 45

Leu Phe Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile

50 55 60

Ile Phe Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser

65 70 75 80

Ala Pro Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala

85 90 95

Tyr Glu His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp

100 105 110

His Ala Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp

115 120 125

Tyr Val Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu

130 135 140

Asp Lys Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala

145 150 155 160

Asp Gln Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

165 170

<210> 208

<211> 173

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 208

Glu Ser Gln Val Arg Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu

1. 5 10 15

Asn Glu Gln Val Asn Lys Glu Met Asn Ser Ser Asn Leu Tyr Met Ser

20 25 30

Met Ser Ser Trp Cys Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe

35 40 45

Leu Phe Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile

50 55 60

Ile Phe Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser

65 70 75 80

Ala Pro Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala

85 90 95

Tyr Glu His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp

100 105 110

His Ala Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp

115 120 125

Tyr Val Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu

130 135 140

Asp Lys Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala

145 150 155 160

Asp Gln Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser

165 170

<210> 209

<211> 166

<212> PROTEIN

<213> Helicobacter pylori

<400> 209

Leu Ser Lys Asp Ile Ile Lys Leu Leu Asn Glu Gln Val Asn Lys Glu

1. 5 10 15

Met Asn Ser Ser Asn Leu Tyr Met Ser Met Ser Ser Trp Cys Tyr Thr

20 25 30

His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe Asp His Ala Ala Glu

35 40 45

Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe Leu Asn Glu Asn Asn

50 55 60

Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro Glu His Lys Phe Glu

65 70 75 80

Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu His Glu Gln His Ile

85 90 95

Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala Ile Lys Ser Lys Asp

100 105 110

His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val Ala Glu Gln His Glu

115 120 125

Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys Ile Glu Leu Ile Gly

130 135 140

Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln Tyr Val Lys Gly Ile

145 150 155 160

Ala Lys Ser Arg Lys Ser

165

<210> 210

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 210

tgactgtgaa cgttcgagat ga 22

<210> 211

<211> 191

<212> PROTEIN

<213> Trichoplusia ni

<400> 211

Thr Gln Cys Asn Val Asn Pro Val Gln Ile Pro Lys Asp Trp Ile Thr

1. 5 10 15

Met His Arg Ser Cys Arg Asn Ser Met Arg Gln Gln Ile Gln Met Glu

20 25 30

Val Gly Ala Ser Leu Gln Tyr Leu Ala Met Gly Ala His Phe Ser Lys

35 40 45

Asp Val Val Asn Arg Pro Gly Phe Ala Gln Leu Phe Phe Asp Ala Ala

50 55 60

Ser Glu Glu Arg Glu His Ala Met Lys Leu Ile Glu Tyr Leu Leu Met

65 70 75 80

Arg Gly Glu Leu Thr Asn Asp Val Ser Ser Leu Leu Gln Val Arg Pro

85 90 95

Pro Thr Arg Ser Ser Trp Lys Gly Gly Val Glu Ala Leu Glu His Ala

100 105 110

Leu Ser Met Glu Ser Asp Val Thr Lys Ser Ile Arg Asn Val Ile Lys

115 120 125

Ala Cys Glu Asp Asp Ser Glu Phe Asn Asp Tyr His Leu Val Asp Tyr

130 135 140

Leu Thr Gly Asp Phe Leu Glu Glu Gln Tyr Lys Gly Gln Arg Asp Leu

145 150 155 160

Ala Gly Lys Ala Ser Thr Leu Lys Lys Leu Met Asp Arg His Glu Ala

165 170 175

Leu Gly Glu Phe Ile Phe Asp Lys Lys Leu Leu Gly Ile Asp Val

180 185 190

<210> 212

<211> 212

<212> PROTEIN

<213> Trichoplusia ni

<400> 212

Ala Asp Thr Cys Tyr Asn Asp Val Ala Leu Asp Cys Gly Ile Thr Ser

1. 5 10 15

Asn Ser Leu Ala Leu Pro Arg Cys Asn Ala Val Tyr Gly Glu Tyr Gly

20 25 30

Ser His Gly Asn Val Ala Thr Glu Leu Gln Ala Tyr Ala Lys Leu His

35 40 45

Leu Glu Arg Ser Tyr Asp Tyr Leu Leu Ser Ala Ala Tyr Phe Asn Asn

50 55 60

Tyr Gln Thr Asn Arg Ala Gly Phe Ser Lys Leu Phe Lys Lys Leu Ser

65 70 75 80

Asp Glu Ala Trp Ser Lys Thr Ile Asp Ile Ile Lys His Val Thr Lys

85 90 95

Arg Gly Asp Lys Met Asn Phe Asp Gln His Ser Thr Met Lys Thr Glu

100 105 110

Arg Lys Asn Tyr Thr Ala Glu Asn His Glu Leu Glu Ala Leu Ala Lys

115 120 125

Ala Leu Asp Thr Gln Lys Glu Leu Ala Glu Arg Ala Phe Tyr Ile His

130 135 140

Arg Glu Ala Thr Arg Asn Ser Gln His Leu His Asp Pro Glu Ile Ala

145 150 155 160

Gln Tyr Leu Glu Glu Glu Phe Ile Glu Asp His Ala Glu Lys Ile Arg

165 170 175

Thr Leu Ala Gly His Thr Ser Asp Leu Lys Lys Phe Ile Thr Ala Asn

180 185 190

Asn Gly His Asp Leu Ser Leu Ala Leu Tyr Val Phe Asp Glu Tyr Leu

195 200 205

Gln Lys Thr Val

210

<210> 213

<211> 174

<212> PROTEIN

<213> Pyrococcus furiosus

<400> 213

Met Leu Ser Glu Arg Met Leu Lys Ala Leu Asn Asp Gln Leu Asn Arg

1. 5 10 15

Glu Leu Tyr Ser Ala Tyr Leu Tyr Phe Ala Met Ala Ala Tyr Phe Glu

20 25 30

Asp Leu Gly Leu Glu Gly Phe Ala Asn Trp Met Lys Ala Gln Ala Glu

35 40 45

Glu Glu Ile Gly His Ala Leu Arg Phe Tyr Asn Tyr Ile Tyr Asp Arg

50 55 60

Asn Gly Arg Val Glu Leu Asp Glu Ile Pro Lys Pro Pro Lys Glu Trp

65 70 75 80

Glu Ser Pro Leu Lys Ala Phe Glu Ala Ala Tyr Glu His Glu Lys Phe

85 90 95

Ile Ser Lys Ser Ile Tyr Glu Leu Ala Ala Leu Ala Glu Glu Glu Lys

100 105 110

Asp Tyr Ser Thr Arg Ala Phe Leu Glu Trp Phe Ile Asn Glu Gln Val

115 120 125

Glu Glu Glu Ala Ser Val Lys Lys Ile Leu Asp Lys Leu Lys Phe Ala

130 135 140

Lys Asp Ser Pro Gln Ile Leu Phe Met Leu Asp Lys Glu Leu Ser Ala

145 150 155 160

Arg Ala Pro Lys Leu Pro Gly Leu Leu Met Gln Gly Gly Glu

165 170

<210> 214

<211> 183

<212> PROTEIN

<213> Homo sapiens

<400> 214

Met Thr Thr Ala Ser Thr Ser Gln Val Arg Gln Asn Tyr His Gln Asp

1. 5 10 15

Ser Glu Ala Ala Ile Asn Arg Gln Ile Asn Leu Glu Leu Tyr Ala Ser

20 25 30

Tyr Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala

35 40 45

Leu Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg

50 55 60

Glu His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg

65 70 75 80

Ile Phe Leu Gln Asp Ile Lys Lys Pro Asp Cys Asp Asp Trp Glu Ser

85 90 95

Gly Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Asn Val Gln

100 105 110

Gln Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro

115 120 125

His Leu Cys Asp Phe Ile Glu Thr His Tyr Leu Asn Glu Gln Val Lys

130 135 140

Ala Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg Lys Met Gly

145 150 155 160

Ala Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe Asp Lys His Thr Leu

165 170 175

Gly Asp Ser Asp Gln Glu Ser

180

<210> 215

<211> 204

<212> PROTEIN

<213> Homo sapiens

<400> 215

Met Asp Ser Lys Gly Ser Ser Gln Lys Gly Ser Arg Leu Leu Leu Leu

1. 5 10 15

Leu Val Val Ser Asn Leu Leu Leu Pro Gln Gly Val Leu Ala Ser Ser

20 25 30

Gln Ile Arg Gln Asn Tyr Ser Thr Asp Val Glu Ala Ala Val Asn Ser

35 40 45

Leu Val Asn Leu Tyr Leu Gln Ala Ser Tyr Thr Tyr Leu Ser Leu Gly

50 55 60

Phe Tyr Phe Asp Arg Asp Asp Val Ala Leu Glu Gly Val Ser His Phe

65 70 75 80

Phe Arg Glu Leu Ala Glu Glu Lys Arg Glu Gly Tyr Glu Arg Leu Leu

85 90 95

Lys Met Gln Asn Gln Arg Gly Gly Arg Ala Leu Phe Gln Asp Ile Lys

100 105 110

Lys Pro Ala Glu Asp Glu Trp Gly Lys Thr Pro Asp Ala Met Lys Ala

115 120 125

Ala Met Ala Leu Glu Lys Lys Leu Asn Gln Ala Leu Leu Asp Leu His

130 135 140

Ala Leu Gly Ser Ala Arg Thr Asp Pro His Leu Cys Asp Phe Leu Glu

145 150 155 160

Thr His Phe Leu Asp Glu Glu Val Lys Leu Ile Lys Lys Met Gly Asp

165 170 175

His Leu Thr Asn Leu His Arg Leu Gly Gly Pro Glu Ala Gly Leu Gly

180 185 190

Glu Tyr Leu Phe Glu Arg Leu Thr Leu Lys His Asp

195 200

<210> 216

<211> 154

<212> PROTEIN

<213> Aquifex aeolicus

<400> 216

Met Gln Ile Tyr Glu Gly Lys Leu Thr Ala Glu Gly Leu Arg Phe Gly

1. 5 10 15

Ile Val Ala Ser Arg Phe Asn His Ala Leu Val Asp Arg Leu Val Glu

20 25 30

Gly Ala Ile Asp Cys Ile Val Arg His Gly Gly Arg Glu Glu Asp Ile

35 40 45

Thr Leu Val Arg Val Pro Gly Ser Trp Glu Ile Pro Val Ala Ala Gly

50 55 60

Glu Leu Ala Arg Lys Glu Asp Ile Asp Ala Val Ile Ala Ile Gly Val

65 70 75 80

Leu Ile Arg Gly Ala Thr Pro His Phe Asp Tyr Ile Ala Ser Glu Val

85 90 95

Ser Lys Gly Leu Ala Asn Leu Ser Leu Glu Leu Arg Lys Pro Ile Thr

100 105 110

Phe Gly Val Ile Thr Ala Asp Thr Leu Glu Gln Ala Ile Glu Arg Ala

115 120 125

Gly Thr Lys His Gly Asn Lys Gly Trp Glu Ala Ala Leu Ser Ala Ile

130 135 140

Glu Met Ala Asn Leu Phe Lys Ser Leu Arg

145 150

<210> 217

<211> 8

<212> PROTEIN

<213> Unidentified

<220>

<223> Bullfrog linker sequence

<400> 217

Glu Ser Gln Val Arg Gln Gln Phe

15

<210> 218

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 218

Cys Leu Val Pro Arg Gly Ser Leu Glu His His His His His

1. 5 10 15

<210> 219

<211> 156

<212> PROTEIN

<213> Escherichia coli

<400> 219

Met Asn Ile Ile Glu Ala Asn Val Ala Thr Pro Asp Ala Arg Val Ala

1. 5 10 15

Ile Thr Ile Ala Arg Phe Asn Asn Phe Ile Asn Asp Ser Leu Leu Glu

20 25 30

Gly Ala Ile Asp Ala Leu Lys Arg Ile Gly Gln Val Lys Asp Glu Asn

35 40 45

Ile Thr Val Val Trp Val Pro Gly Ala Tyr Glu Leu Pro Leu Ala Ala

50 55 60

Gly Ala Leu Ala Lys Thr Gly Lys Tyr Asp Ala Val Ile Ala Leu Gly

65 70 75 80

Thr Val Ile Arg Gly Gly Thr Ala His Phe Glu Tyr Val Ala Gly Gly

85 90 95

Ala Ser Asn Gly Leu Ala His Val Ala Gln Asp Ser Glu Ile Pro Val

100 105 110

Ala Phe Gly Val Leu Thr Thr Glu Ser Ile Glu Gln Ala Ile Glu Arg

115 120 125

Ala Gly Thr Lys Ala Gly Asn Lys Gly Ala Glu Ala Ala Leu Thr Ala

130 135 140

Leu Glu Met Ile Asn Val Leu Lys Ala Ile Lys Ala

145 150 155

<210> 220

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 220

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1. 5 10 15

<210> 221

<211> 28

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 221

Gly Gly Ser Gly Ser Gly Ser Asn Ser Ser Ala Ser Ser Gly Ala Ser

1. 5 10 15

Ser Gly Gly Ala Ser Gly Gly Ser Gly Gly Ser Gly

20 25

<210> 222

<211> 46

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 222

Gly Gly Ser Gly Ser Ala Ser Ser Gly Ala Ser Ala Ser Gly Ser Ser

1. 5 10 15

Asn Gly Ser Gly Ser Gly Ser Gly Ser Asn Ser Ser Ala Ser Ser Gly

20 25 30

Ala Ser Ser Gly Gly Ala Ser Gly Gly Ser Gly Gly Ser Gly

35 40 45

<210> 223

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 223

Gly Gly Ser Gly Ser Ala Ser Ala Glu Ala Ala Ala Lys Glu Ala Ala

1. 5 10 15

Ala Lys Ala Gly Gly Ser Gly Gly Ser Gly

20 25

<210> 224

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 224

Gly Gly Ser Gly Ser Ala Ser Ala Glu Ala Ala Ala Lys Glu Ala Ala

1. 5 10 15

Ala Lys Glu Ala Ala Ala Lys Ala Ser Gly Gly Ser Gly Gly Ser Gly

20 25 30

<210> 225

<211> 47

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 225

Ser Gly Gly Gly Ser Gly Ser Ala Ser Ser Gly Ala Ser Ala Ser Gly

1. 5 10 15

Ser Ser Cys Ser Gly Ser Gly Ser Gly Ser Ser Ser Ala Ser Ser Gly

20 25 30

Ala Ser Ser Gly Gly Ala Ser Gly Gly Gly Ser Gly Gly Ser Gly

35 40 45

<210> 226

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 226

Gly Gly Gly Ser

1

<210> 227

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 227

Gly Gly Gly Ser Gly Gly Gly Ser

15

<210> 228

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 228

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1. 5 10 15

Gly Gly Gly Ser

20

<210> 229

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 229

Ser Gly Ser Gly Ser

15

<210> 230

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> 6xHis-mark

<400> 230

His His His His His

15

<---

Claims (32)

1. A respiratory syncytial virus (RSV) antigenic polypeptide containing a sequence having at least 90% identity to amino acids 1-478 SEQ ID NO: 23. 2. An RSV antigenic polypeptide comprising an RSV F polypeptide, wherein the RSV F polypeptide contains a sequence having at least 90% identity to amino acids 1-478 of SEQ ID NO: 23, and wherein the RSV F polypeptide retains asparagine at at least one of the positions 288, 308 or 467 SEQ ID NO: 23. 3. The RSV antigenic polypeptide of claim 2, wherein asparagine of the RSV polypeptide is glycosylated, thereby blocking an epitope of the RSV polypeptide that is common to prefusion F RSV and postfusion RSV F. 4. RSV antigenic polypeptide according to any one of paragraphs. 1-3 containing F RSV before merging. 5. RSV antigenic polypeptide according to any one of paragraphs. 1-4, which is recognized by an antibody specific for prefusion RSV F selected from D25 or AM14. 6. The RSV antigenic polypeptide of claim 4 or 5, wherein the prefusion RSV F contains an epitope not found on the postfusion RSV F. 7. RSV antigenic polypeptide according to any one of paragraphs. 1-3 containing F RSV after merging. 8. RSV antigenic polypeptide according to any one of claims. 1 or 3-6, where the epitope is blocked by an N-glycan attached to asparagine. 9. RSV antigenic polypeptide according to any one of paragraphs. 1-8, additionally containing ferritin. 10. The RSV antigenic polypeptide according to claim 9, wherein ferritin contains a mutation that ensures the replacement of a surface-exposed amino acid with cysteine. 11. The RSV antigenic polypeptide of claim 10, wherein the ferritin comprises one or more of the E12C, S26C, S72C, A75C, K79C, S100C, and S111C ferritin mutations from H. pylori or one or more corresponding ferritin mutations from a non- H. pylori , as determined by pairwise or structural alignment. 12. RSV antigenic polypeptide according to any one of paragraphs. 9-11 comprising one or more immunostimulatory functional groups linked to ferritin via a surface exposed amino acid, wherein optionally the surface exposed amino acid is a mutated cysteine. 13. RSV antigenic polypeptide according to any one of paragraphs. 9-12, wherein the ferritin contains a mutation to replace surface-exposed asparagine with a non-asparagine amino acid, wherein optionally the asparagine is at position 19 of ferritin from H. pylori or a similar position of ferritin from a non -H. pylori organism, as determined by pairwise or structural alignment. 14. RSV antigenic polypeptide according to any one of paragraphs. 9-13, wherein the ferritin contains a mutation that results in the replacement of the internal cysteine with an amino acid other than cysteine, wherein optionally the internal cysteine is at position 31 of H. pylori ferritin or at a position that corresponds to position 31 of H. pylori ferritin, as determined by pairwise or structural alignment. 15. RSV antigenic polypeptide according to any one of claims. 9-14, wherein the RSV F polypeptide contains an epitope not found on post-fusion RSV F, which is a site 0 epitope, wherein optionally the site 0 epitope contains amino acid residues 62-69 and 156-169 of SEQ ID NO: 23. 16. RSV antigenic polypeptide according to any one of paragraphs. 1-15, wherein the RSV F polypeptide contains an asparagine at a position corresponding to position 288 of SEQ ID NO: 23. 17. RSV antigenic polypeptide according to any one of paragraphs. 1-16, wherein the RSV F polypeptide contains an asparagine at a position corresponding to position 308 of SEQ ID NO: 23. 18. RSV antigenic polypeptide according to any one of paragraphs. 1-17, wherein the RSV F polypeptide contains an asparagine at a position corresponding to position 467 of SEQ ID NO: 23. 19. RSV antigenic polypeptide according to any one of paragraphs. 1-18, wherein the RSV F polypeptide contains a leucine at a position corresponding to lysine 458 of SEQ ID NO: 23. 20. RSV antigenic polypeptide according to any one of paragraphs. 1-19, wherein the RSV F polypeptide contains a proline at a position corresponding to the position of isoleucine 177 of SEQ ID NO: 23. 21. RSV antigenic polypeptide according to any one of paragraphs. 1-20, wherein the RSV F polypeptide does not contain a furin cleavage site, wherein a linker is optionally present instead of a furin cleavage site. 22. RSV antigenic polypeptide according to any one of paragraphs. 1-21, wherein the RSV F polypeptide contains a sequence having at least 95%, 97%, 98%, 99%, or 99.5% identity to amino acids 1-478 of SEQ ID NO: 23. 23. RSV antigenic polypeptide according to any one of paragraphs. 1-21, wherein the RSV F polypeptide contains a sequence having at least 95%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 23. 24. RSV antigenic polypeptide according to any one of paragraphs. 1-23, containing amino acids 1-478 from SEQ ID NO: 23. 25. RSV antigenic polypeptide according to any one of paragraphs. 1-21 or 23, containing the sequence SEQ ID NO: 23. 26. A ferritin particle for use in a method of inducing an immune response to RSV in a subject, comprising an RSV antigenic polypeptide according to any one of claims. 9-25. 27. A composition for use in a method of inducing an immune response to RSV in a subject, comprising an effective amount of an RSV antigenic polypeptide according to any one of claims. 1-25 and RSV polypeptide G. 28. A composition for use in a method of inducing an immune response to RSV in a subject, comprising an effective amount of an RSV antigenic polypeptide according to any one of claims. 1-25 and a pharmaceutically acceptable carrier. 29. RSV antigenic polypeptide according to any one of paragraphs. 1-25 for use in a method of inducing an immune response to RSV in a subject. 30. A method of inducing an immune response to RSV in a subject, comprising administering to the subject any one or more of the RSV antigenic polypeptides of any one of claims. 1-25. 31. RSV antigenic polypeptide according to any one of paragraphs. 1-25, where the subject is a person. 32. Nucleic acid encoding an RSV antigenic polypeptide according to any one of claims. 1-25, 29 or 31, wherein the RSV F polypeptide of the RSV antigenic polypeptide contains a sequence having at least 90%, 95%, 97%, 98%, 99% or 99.5% identity to amino acids 1-478 SEQ ID NO: 23, wherein the nucleic acid is optionally mRNA.

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