RU2795459C2 - Stabilized f rsv proteins and their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to obtaining a trimer of the F protein of the respiratory syncytial virus (RSV), and can be used in medicine to induce an immune response to the RSV antigen. The F RSV protein is obtained recombinantly, containing a flexible linker instead of a protease cleavage site and mutations in the amino acid sequence, conferring greater stability to the protein in the “before fusion” conformation.

EFFECT: invention makes it possible to obtain a trimer of the F RSV protein, which is more stable in the “before fusion” conformation.

25 cl, 19 dwg, 4 tbl, 6 ex

Description

The field of technology to which the invention belongs

The present invention relates to stable F RSV proteins and immunogenic compositions containing them, as well as methods of using immunogenic compositions and compositions containing F RSV proteins.

Cross-reference to related applications

This application claims priority to U.S. Provisional Application No. 62/623,184, filed January 29, 2018, the contents of which are hereby incorporated by reference in their entirety.

Submission of a sequence listing filed electronically

The sequence listing in this application is submitted electronically via EFS-Web as an ASCII sequence listing with the file name "24566WOPCT-SEQLIST-18JAN2019.TXT", created on January 18, 2019, and 167 KB in size. This sequence listing, provided via EFS-Web, is part of the application and is incorporated herein by reference in its entirety.

State of the art

Respiratory syncytial virus (RSV) is a member of the pneumovirus family. RSV infection is a major cause of lower respiratory tract infection in young children and the elderly (> 65 years of age). There is currently no licensed vaccine and therapeutic options are limited.

The RSV envelope contains three surface glycoproteins: F, G, and SH. Proteins G and F are protective antigens and targets of neutralizing antibodies. However, the F protein is more conserved among strains and types of RSV (A and B). F RSV is a type I viral fusion protein that rearranges from a metastable pre-fusion form to a highly stable post-fusion form. Although targets for neutralizing monoclonal antibodies are in the post-fusion F protein conformation, the response of neutralizing Abs primarily targets the pre-fusion F protein conformation in humans naturally infected with RSV (Magro M et al., Proc Natl Acad Sci USA 109(8): 3089-94, 2012 Ngwuta J O et al Sci Transl Med 2015 7(309) 309ra162 Therefore, the engineered RSV F protein, stabilized in its pre-fusion conformation, is an attractive strategy for the development of vaccine F RSV antigens. For example, the recombinant RSV F trimer containing the "DS-Cav1" substitutions (155C, 290C, 190F, and 207L) has previously been shown to induce a neutralizing immune response in animal models that is stronger than that observed for the F protein in the " after fusion" based on F RSV immunogens (McLellan et al., Science, 342: 592-598, 2013). New RSV antigens are described here that are even more stable in the pre-fusion form of interest.

The essence of the invention

The present invention relates to a recombinant respiratory syncytial virus (RSV) F trimer comprising: three recombinant RSV F peptides, each containing an amino acid deletion at positions 98-146 of F RSV wild-type and an eight to fourteen amino acid linker between amino acid positions 97 and 147 F RSV wild type, wherein the recombinant F peptides contain the following modifications to stabilize the recombinant RSV F trimer in a pre-fusion conformation: (i) amino acid substitutions 190F and 207L, (ii) amino acid substitutions 155C and 290C, and one (or more) ) from: (a) amino acid substitutions 486C and 490C; (b) amino acid substitutions 180C and 186C; (c) amino acid substitutions 486C and 489C; (d) amino acid substitutions 512C and 513C; (e) amino acid substitution 505C; and (f) deletions of amino acids 482-513 F of wild-type RSV. In one embodiment, each F RSV peptide further comprises a C-terminal deletion of the transmembrane domain and a wild-type F RSV cystoplasmic domain (eg, contains a C-terminal deletion of wild-type F RSV amino acids 525-574). In a further embodiment, each F RSV peptide contains a deletion of wild-type F RSV amino acids 514-574.

In one embodiment of the recombinant RSV F trimer, each of the recombinant F peptides further comprises a foldon sequence at the C-terminus of each peptide. In a further embodiment, the sequence of the foldon domain starts at the C-terminus of the peptide and replaces the transmembrane domain and the cytoplasmic F RSV domain of the wild type (eg, the foldon domain replaces amino acids 525-574 of F RSV). In a further embodiment, the foldon domain sequence begins after amino acid position 513 F of wild-type RSV (ie, the foldon sequence replaces amino acids 514-574 F of wild-type RSV). In yet another embodiment, when the RSV F peptides contain an additional deletion of wild-type F RSV amino acids 482-513, then the foldon domain sequence begins after wild-type F RSV amino acid position 481 (see, for example, SEQ ID NO: 44). In some embodiments, the foldon sequence contains SEQ ID NO: 8.

In one embodiment of the F RSV trimer, each of the recombinant F peptides contains amino acid substitutions 190F and 207L, amino acid substitutions 155C and 290C, amino acid substitutions 486C and 490C, and a deletion of wild-type F RSV amino acids 514-574. In one embodiment, each recombinant F-peptide further comprises a non-native intrapeptide disulfide bond between cysteines introduced by the 155C and 290C amino acid substitutions. In yet another embodiment, the F RSV trimer contains one or more non-native interpeptide disulfide bonds between cysteines introduced by the 486C and 490C amino acid substitutions. In a further embodiment, the C-terminus of each of the recombinant RSV F peptides contains a foldon domain sequence. In yet another embodiment, the foldon domain sequence comprises SEQ ID NO: 8.

In one embodiment of the F RSV trimer, each of the recombinant F peptides contains amino acid substitutions 190F and 207L, amino acid substitutions 155C and 290C, amino acid substitutions 180C and 186C, and a deletion of amino acids 514-574 F RSV wild type. In one embodiment, each recombinant RSV F peptide further comprises a non-native intrapeptide disulfide bond between cysteines introduced at 155C and 290C amino acid substitutions and/or a non-native intrapeptide disulfide bond between cysteines introduced at 190C and 186C amino acid substitutions. In a further embodiment, the C-terminus of each of the recombinant RSV F peptides contains a foldon domain sequence. In yet another embodiment, the foldon domain sequence comprises SEQ ID NO: 8.

In one embodiment of the recombinant RSV F trimer, each of the recombinant F peptides contains amino acid substitutions 190F and 207L, amino acid substitutions 155C and 290C, amino acid substitutions 486C and 489C, and a deletion of amino acids 514-574 from wild-type F RSV. In one embodiment, each recombinant RSV F peptide further comprises a non-native intrapeptide disulfide bond between cysteines introduced by the 155C and 290C amino acid substitutions. In yet another embodiment, the F RSV trimer contains a non-native interpeptide disulfide bond between cysteines introduced by amino acid substitutions 486C and 489C. In a further embodiment, the C-terminus of each of the recombinant RSV F peptides contains a foldon domain sequence. In yet another embodiment, the foldon domain sequence comprises SEQ ID NO: 8.

In one embodiment of the RSV trimer, each of the recombinant F peptides contains amino acid substitutions 190F and 207L, amino acid substitutions 155C and 290C, amino acid substitutions 512C and 513C, and a deletion of wild-type RSV amino acids 514-574 F. In one embodiment, each recombinant RSV F peptide further comprises a non-native intrapeptide disulfide bond between cysteines introduced by the 155C and 290C amino acid substitutions. In yet another embodiment, the F RSV trimer contains a non-native interpeptide disulfide bond between cysteines introduced by the 512C and 513C amino acid substitutions. In a further embodiment, the C-terminus of each of the recombinant RSV F peptides contains a foldon domain sequence. In yet another embodiment, the foldon domain sequence comprises SEQ ID NO: 8.

In one embodiment of the recombinant RSV F trimer, each of the recombinant F peptides comprises amino acid substitutions 190F and 207L, amino acid substitutions 155C and 290C, amino acid substitution 505C, and deletion of amino acids 514-574 F RSV wild type. In yet another embodiment, each recombinant RSV F peptide further comprises a non-native intrapeptide disulfide bond between cysteines introduced by the 155C and 290C amino acid substitutions. In a further embodiment, the C-terminus of each of the recombinant RSV F peptides contains a foldon domain sequence. In yet another embodiment, the foldon domain sequence comprises SEQ ID NO: 8.

In one embodiment of the recombinant RSV F trimer, each of the recombinant F peptides comprises amino acid substitutions 190F and 207L, amino acid substitutions 155C and 290C, and a deletion of wild-type F RSV amino acids 482-513, as well as a deletion of wild-type F RSV amino acids 514-574. In one embodiment, each recombinant RSV F peptide further comprises a non-native intrapeptide disulfide bond between cysteines introduced by the 155C and 290C amino acid substitutions. In a further embodiment, the C-terminus of each of the recombinant RSV F peptides contains a foldon domain sequence. In yet another embodiment, the foldon domain sequence comprises SEQ ID NO: 8.

In one embodiment, the linker is eight (8), ten (10), twelve (12) or fourteen (14) amino acids long. In one embodiment, the linker contains the amino acid sequence shown in SEQ ID NO: 46. In yet another embodiment, the linker has the amino acid sequence shown in any of SEQ ID NO: 1, 2, 3, or 4.

In one embodiment, each of the recombinant F peptides of an F RSV trimer comprises, consists essentially of, or consists of the mature amino acid sequence shown in SEQ ID NO: 22. In one embodiment, the recombinant F peptides of each F RSV trimer comprise, consist of essentially from or consist of the mature amino acid sequence set forth in SEQ ID NO: 24. In one embodiment, the recombinant F peptides of each F RSV trimer comprise, consist essentially of, or consist of the mature amino acid sequence set forth in SEQ ID NO: 26. B in one embodiment, the recombinant F peptides of each F RSV trimer comprise, consist essentially of, or consist of the mature amino acid sequence shown in SEQ ID NO 28. In one embodiment, the recombinant F peptides of each F RSV trimer comprise, consist essentially of, or consist of the mature amino acid sequence shown in SEQ ID NO: 30. In one embodiment, the recombinant F peptides of each RSV F trimer comprise, consist essentially of, or consist of the mature amino acid sequence shown in SEQ ID NO: 44.

The present invention also relates to an RSV immunogenic composition comprising a recombinant respiratory syncytial virus (RSV) F trimer comprising three recombinant RSV F peptides, each containing a deletion of amino acids at positions 98-146 of wild-type F RSV and an eight to fourteen amino acid linker between amino acid positions 97 and 147 of wild-type F RSV, wherein the recombinant F peptides include the following modifications to stabilize the recombinant RSV F trimer in a pre-fusion conformation: (i) amino acid substitutions 190F and 207L, (ii) amino acid substitutions 155C and 290C, and one (or more) of (a) amino acid substitutions 486C and 490C; (b) amino acid substitutions 180C and 186C; (c) amino acid substitutions 486C and 489C; (d) amino acid substitutions 512C and 513C; (e) amino acid substitution 505C; and (f) deletions of amino acids 482-513 F of wild-type RSV. In one embodiment, each F RSV peptide further comprises a C-terminal deletion of the transmembrane domain and a wild-type F RSV cystoplasmic domain (eg, contains a C-terminal deletion of wild-type F RSV amino acids 525-574). In a further embodiment, each F RSV peptide contains a deletion of wild-type F RSV amino acids 514-574.

In one embodiment of the immunogenic composition, each of the recombinant F-peptides further comprises a foldon sequence at the C-terminus of each peptide. In a further embodiment, the foldon domain sequence begins after amino acid position 513 F of wild-type RSV (ie, the foldon sequence replaces amino acids 514-574 F of wild-type RSV). In yet another embodiment, when the RSV F peptides contain an additional deletion of wild-type F RSV amino acids 482-513, then the foldon domain sequence begins after wild-type F RSV amino acid position 481 (see, for example, SEQ ID NO: 44). In some embodiments, the foldon sequence contains SEQ ID NO: 8.

In one embodiment of the immunogenic composition, the F RSV trimer is any of the RSV trimers described herein. In another embodiment of the immunogenic composition, the recombinant F peptides of each RSV F trimer comprise, consist essentially of, or consist of the mature amino acid sequence shown in any of SEQ ID NOs: 22, 24, 26, 28, 30, and 44.

The present invention also relates to an RSV peptide that includes a deletion of amino acids at positions 98-146 of wild-type F RSV and an eight-to-fourteen amino acid linker between positions 97 and 147 of wild-type F RSV, and further modifications to stabilize a recombinant RSV F trimer containing three such a recombinant RSV peptide in a "pre-fusion" conformation. Such additional modifications in the RSV single chain peptide include: (i) amino acid substitutions 190F and 207L, (ii) amino acid substitutions 155C and 290C, and one (or more) of (a) amino acid substitutions 486C and 490C; (b) amino acid substitutions 180C and 186C; (c) amino acid substitutions 486C and 489C; (d) amino acid substitutions 512C and 513C; (e) amino acid substitution 505C; and (f) deletion of amino acids 482-513 F of wild-type RSV. In one embodiment, the F RSV peptide further comprises a C-terminal deletion of the transmembrane domain and a wild-type F RSV cystoplasmic domain (eg, contains a C-terminal deletion of wild-type F RSV amino acids 525-574). In a further embodiment, the F RSV peptide comprises a deletion of wild-type F RSV amino acids 514-574.

In one embodiment, the F RSV peptide further comprises a foldon sequence at the C-terminus. In a further embodiment, the foldon domain sequence begins after amino acid position 513 F of wild-type RSV (ie, the foldon sequence replaces amino acids 514-574 of wild-type RSV-F). In yet another embodiment, when the RSV F peptide contains an additional deletion of wild-type F RSV amino acids 482-513, then the foldon domain sequence begins after wild-type F RSV amino acid position 482 (see, for example, SEQ ID NO: 44). In some embodiments, the foldon sequence contains SEQ ID NO: 8.

In one embodiment, the RSV F peptide comprises amino acid substitutions 190F and 207L, amino acid substitutions 155C and 290C, amino acid substitutions 486C and 490C, and deletion of amino acids 514-574 F RSV wild type. In yet another embodiment, the RSV F peptide further comprises a non-native intrapeptide disulfide bond between cysteines introduced by the 155C and 290C amino acid substitutions. In a further embodiment, the C-terminus of the F RSV peptide contains a foldon domain sequence. In yet another embodiment, the foldon domain sequence comprises SEQ ID NO: 8.

In one embodiment, the F RSV peptide comprises amino acid substitutions 190F and 207L, amino acid substitutions 155C and 290C, amino acid substitutions 180C and 186C, and deletion of amino acids 514-574 F RSV wild type. In yet another embodiment, the RSV F peptide further comprises a non-native intrapeptide disulfide bond between cysteines introduced at 155C and 290C amino acid substitutions and/or a non-native intrapeptide disulfide bond between cysteines introduced at 190C and 186C amino acid substitutions. In a further embodiment, the C-terminus of the F RSV peptide contains a foldon domain sequence. In yet another embodiment, the foldon domain sequence comprises SEQ ID NO: 8.

In one embodiment, the F RSV peptide comprises amino acid substitutions 190F and 207L, amino acid substitutions 155C and 290C, amino acid substitutions 486C and 489C, and deletion of amino acids 514-574 F RSV wild type. In yet another embodiment, the RSV F peptide further comprises a non-native intrapeptide disulfide bond between cysteines introduced by amino acid substitutions at 155°C and 290°C. In a further embodiment, the C-terminus of the F RSV peptide contains a foldon domain sequence. In yet another embodiment, the foldon domain sequence comprises SEQ ID NO: 8.

In one embodiment, the RSV F peptide comprises amino acid substitutions 190F and 207L, amino acid substitutions 155C and 290C, amino acid substitutions 512C and 513C, and deletion of amino acids 514-574 F RSV wild type. In yet another embodiment, the RSV F peptide further comprises a non-native intrapeptide disulfide bond between cysteines introduced by amino acid substitutions at 155°C and 290°C. In a further embodiment, the C-terminus of the F RSV peptide contains a foldon domain sequence. In yet another embodiment, the foldon domain sequence comprises SEQ ID NO: 8.

In one embodiment, the RSV F peptide comprises amino acid substitutions 190F and 207L, amino acid substitutions 155C and 290C, amino acid substitution 505C, and deletion of amino acids 514-574 F RSV wild type. In yet another embodiment, the RSV F peptide further comprises a non-native intrapeptide disulfide bond between cysteines introduced by the 155C and 290C amino acid substitutions. In a further embodiment, the C-terminus of the F RSV peptide contains a foldon domain sequence. In yet another embodiment, the foldon domain sequence comprises SEQ ID NO: 8.

In one embodiment, the RSV F peptide comprises amino acid substitutions 190F and 207L, amino acid substitutions 155C and 290C, and a deletion of wild-type F RSV amino acids 482-513, as well as a deletion of wild-type F RSV amino acids 514-574. In one embodiment, the F RSV peptide further comprises a non-native intrapeptide disulfide bond between cysteines introduced by the 155C and 290C amino acid substitutions. In a further embodiment, each C-terminus of each F RSV peptide contains a foldon domain sequence. In yet another embodiment, the foldon domain sequence comprises SEQ ID NO: 8.

In one embodiment, the linker is eight (8), ten (10), twelve (12) or fourteen (14) amino acids long. In one embodiment, the linker contains the amino acid sequence shown in SEQ ID NO: 46. In yet another embodiment, the linker has the amino acid sequence shown in any of SEQ ID NO: 1, 2, 3, or 4.

In one embodiment, the RSV F peptide comprises, consists essentially of, or consists of the mature amino acid sequence shown in SEQ ID NO: 22. In one embodiment, the RSV F peptide contains, consists essentially of, or consists of the mature amino acid sequence shown in SEQ ID NO: 24. In one embodiment, the RSV F peptide comprises, consists essentially of, or consists of the mature amino acid sequence shown in SEQ ID NO: 26. In one embodiment, the RSV F peptide contains, consists essentially of, or consists of the mature the amino acid sequence shown in SEQ ID NO: 28. In one embodiment, the RSV F peptide comprises, consists essentially of, or consists of the mature amino acid sequence shown in SEQ ID NO: 30. In one embodiment, the RSV F peptide comprises, consists essentially of from or consists of the mature amino acid sequence shown in SEQ ID NO: 44.

In one embodiment, the RSV peptide upon receipt recombinantly forms the trimer F RSV, as described here.

The present invention also provides an isolated nucleic acid molecule encoding a single stranded RSV peptide as described herein. In one embodiment, the isolated nucleic acid molecule is a DNA molecule. The present invention further provides a vector containing said nucleic acid molecule.

The present invention also provides a method for producing a recombinant respiratory syncytial virus (RSV) F trimer as described herein, wherein said method comprises (i) expressing a nucleic acid molecule or vector, each of which is described above, and (ii) purifying the recombinant RSV F trimer derived from them.

The present invention also relates to antibody molecules, including full-length antibodies and antibody derivatives directed against the F RSV trimer described here, or against the F RSV peptides described here.

In some embodiments, the immunogenic composition is formulated with an adjuvant. In one embodiment, the adjuvant is an aluminum compound adjuvant. In some embodiments, the aluminum compound adjuvant is MAA or MAPA.

In some embodiments, the recombinant RSV F trimer or RSV F peptides, each described herein, is formulated as a lipid nanoparticle (LNP) containing a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid.

Some embodiments of the present invention provide methods for inducing an F RSV-specific immune response in a subject, comprising administering to the subject any of the immunogenic compositions described herein, or the recombinant F RSV trimers described herein, in an amount effective to elicit an F RSV-specific immune response. In some embodiments, the antigen-specific immune response comprises a T-cell response or a B-cell response.

In some embodiments, the method includes administering to the subject a single dose (no booster dose) of an immunogenic composition or F RSV trimer, as described herein. In yet another embodiment, the method further comprises administering to the subject a second (booster) dose of the immunogenic composition or F RSV trimer as described herein. In yet another embodiment, the method further comprises administering at least one booster dose of RSV F trimer or RSV immunogenic composition. Additional doses may be administered.

In some embodiments, the RSV immunogenic composition or RSV F trimer is administered to a subject by intradermal injection, intramuscular injection, or intranasal administration. In some embodiments, the RSV vaccine is administered to the subject by intramuscular injection.

In some embodiments, the F RSV trimer or immunogenic composition confers immunity to the subject against RSV for 1 or 2 years. In some embodiments, the F RSV trimer or immunogenic composition confers immunity to the subject against RSV for more than 2 years, more than 3 years, more than 4 years, or for 5-10 years. In one embodiment, the immunogenic composition is administered as a vaccine annually.

In some embodiments, the subject is about 5 years old or younger. For example, the subject may be from about 1 year to about 5 years of age (e.g., about 1, 2, 3, 4, or 5 years) or from about 6 months to about 1 year (e.g., about 6, 7, 8 , 9, 10, 11 or 12 months). In some embodiments, the subject is about 12 months old or younger (eg, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 months, or 1 month). In some embodiments, the subject is about 6 months old or younger.

In some embodiments, the subject was born at term (eg, at about 37-42 weeks of gestation). In some embodiments, the subject was born prematurely, such as at or before about 36 weeks of gestation (eg, at about 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, or 25 weeks). For example, the subject may have been born at or before 32 weeks of gestation. In some embodiments, the subject was born prematurely between about 32 and about 36 weeks of gestation. Such subjects may be vaccinated at an older age, eg, from about 6 months to about 5 years of age or older.

In some embodiments, the subject is a young adult between about 20 years old and about 50 years old (eg, about 20, 25, 30, 35, 40, 45, or 50 years old).

In some embodiments, the subject is an elderly subject aged about 50-60 years old, 60 years old, about 70 years old or older, 80 years old or older, 90 years old or older (e.g., aged about 60, 65, 70, 75, 80 , 85 or 90 years). In some embodiments, the subject is immunocompromised (eg, has an immune disorder or an autoimmune disease).

In some embodiments, the female subject is pregnant when the RSV immunogenic composition is administered to her. In some embodiments, the subject has a chronic lung disease, such as chronic obstructive pulmonary disease (COPD) or asthma.

In some embodiments, the subject has been exposed to RSV, has (has) an RSV infection, or is at risk of RSV infection.

Details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and claims.

Brief description of the figures

The above and other objects, features and advantages will be apparent from the following description of specific embodiments of the invention as shown in the accompanying figures, in which the same reference features refer to the same parts in different views. The figures are not necessarily drawn to scale; instead, emphasis is placed on illustrating the principles of various embodiments of the invention.

Figures 1A and 1B show the binding of freshly harvested cell culture supernatants (supe) at day 7 after transfection with LZF40 (DS-Cav1 with an 8 amino acid linker) (Figure 1A) or those stored at 4°C for 8 days ( Fig. 1B) with monoclonal antibodies D25 and Synagis® (palivizumab).

Figures 2A-2F show binding of freshly harvested cell culture supernatants (supe) at day 3 after transfection with LZF55(F55), LZF56(F56), and LZF57(F57) (DS-Cav1 with a 10, 12, or 14 amino acid linker, respectively; FIG. .2A, 2B and 2C respectively) or those stored at 4°C for 7 days (FIGS. 2D, 2E and 2F respectively) with D25 and Synagis® (palivizumab).

Figure 3 shows the results of Western blotting with anti-F RSV serum for single-stranded RSV mutants LZF55, LZF56 and LZF57 in comparison with DS-Cav1.

4A-4C show binding of freshly harvested cell culture supernatants DS-Cav1 (FIG. 4A), LZF57 (FIG. 4B) or LZF111 (FIG. 4C) to monoclonal antibodies D25 and 4D7 as determined by ELISA. In FIG. 4D shows purified DS-Cav1, F57 and F111 analyzed by SDS-PAGE under reducing (R) and non-reducing (NR) conditions.

On figa and 5B shows the CD spectra of LZF111 and DS-Cav1, respectively.

In FIG. 6 shows the results of secondary structure analysis by reconstructing the CD spectra (185 to 260 nm) of DS-Cav1 and LZF111 using a trained artificial neural network for CD spectra of 3D structure resolved proteins.

Figure 7 shows the thermal stability of purified LZF111 and DS-Cav1 analyzed by differential scanning fluorometry (DSF).

8A-8E show the long-term stability of purified DS-Cav1 or LZF111 protein stored frozen or at 4°C for 1 (Fig. 8A-8C), 2 (Fig. 8D), or 3 (Fig. 8E) months, as measured by binding to D25, 4D7 and Synagis® (palivizumab) in an ELISA binding assay.

Figures 9A and 9B show ED10 ELISA titers of sera from PD2 mice against F-protein in pre-fusion conformation (FIG. 9A) and neutralization titers of sera from PD2 mice against RSV Long strain (FIG. 9B). The horizontal dotted line indicates the detection limit. The data show that LZF111 induced similar levels of neutralizing antibodies with DS-Cav1 at different doses.

Figure 10 is a schematic diagram of the LZF111 (top), DS-Cav1 (center) and LZF57 (bottom).

Figure 11 shows serum neutralizing antibody titers (individual NT ₅₀ and GMT values with 95% confidence intervals) against RSV A induced in cotton rats by mRNA vaccines and control formulations.

Figure 12 shows serum antibody titers in a competitive ELISA (individual IT50 and GMT values with 95% confidence intervals) for D25 (site Ø), palivizumab (site II) and 4D7 (site I) measured on day 56 (4 weeks PD2 ).

Figure 13 shows the content of RSV in the lungs and nasal cavity after infection of cotton rats with RSV A.

Figure 14 shows serum neutralizing antibody titers (individual NT ₅₀ and GMT values at 95% confidence intervals) against RSV A induced in African green monkeys by mRNA vaccines and control formulations.

Figure 15 shows serum antibody titers in competitive ELISA (individual IT ₅₀ and GMT values with 95% confidence intervals) for D25 (site Ø), palivizumab (site II) and 4D7 (site I) measured at week 10 (2 weeks PD3).

On figa-16C shows the content of RSV in bronchoalveolar lavage (BAL) after infection with AGM. In FIG. 16A shows a high dose immunization. In FIG. 16B shows low dose immunization. In FIG. 16C shows the area under the curve.

On figa-17C shows the content of RSV in swabs from the nasal cavity after infection with AGM. In FIG. 17A shows a high dose immunization. In FIG. 17B shows low dose immunization. In FIG. 17C shows the area under the curve.

Figure 18 shows serum neutralizing antibody titers (individual NT ₅₀ and GMT values with 95% confidence intervals) against RSV A induced in experimental African green monkeys by mRNA vaccines and control formulations.

Figure 19 shows serum antibody titers in a competitive ELISA (individual IT ₅₀ and GMT values with 95% confidence intervals) for D25 (site Ø), palivizumab (site II) and 4D7 (site I) measured at week 10 (2 weeks PD3).

Detailed description of the invention

The RSV F protein is a type I fusion glycoprotein that is fairly conserved among clinical isolates, including the RSV-A and RSV-B antigenic subgroups. The F protein transitions between a pre-fusion state and a more stable post-fusion state, thereby facilitating entry into target cells. The RSV F glycoprotein is initially synthesized as the F0 precursor protein. F RSV0 is converted to a trimer that is activated by furin cleavage to a mature pre-fusion protein containing F1 and F2 subunits (Bolt, et al., Virus Res., 68:25, 2000). The F RSV protein stabilized in the pre-fusion conformation elicits a higher neutralizing immune response in animal models than that seen with the F RSV protein stabilized in the post-fusion conformation (McLellan et al., Science, 342:592- 598, 2013). Thus, pre-fusion stabilized RSV F proteins are good candidates for inclusion in an RSV vaccine. Soluble RSV ectodomains stabilized in a "pre-fusion" conformation have been prepared previously, including "DS-Cav1" substitutions. See WO 2014/160463A1 and WO 2017/172890A1, the content of each of which is incorporated here by reference.

It has previously been shown that a stabilized pre-fusion F RSV construct, DS-Cav1, undergoes conformational changes and forms intermediate structures upon long-term storage at 4°C (Flynn JA et al., PLoS ONE, 2016; 11 (10) : e0164789). Long-term stability at 4° C. or higher is a desirable property for the RSV F subunit vaccine antigen. Additional structure-based modifications are described herein to further improve the stability of the F RSV trimer in the pre-fusion conformation. Such constructs have improved stability at 4°C compared to DS-Cav1 while maintaining immunogenicity.

The terms "RSV fusion protein" and "RSV F protein" as used herein refer to the RSV envelope glycoprotein that facilitates the fusion of viral and cellular membranes. In nature, the RSV F protein is synthesized as a single polypeptide precursor, designated F0, which includes a signal peptide that directs localization to the endoplasmic reticulum, where the signal peptide is cleaved off. The remaining F0 residues oligomerize to form a trimer and undergo proteolytic processing by a protease with two conserved furin cleavage sequences to form two fragments, F ₁ and F ₂ , linked by a disulfide bond. In nature, the three F ₂ -F ₁ peptides oligomerize into a trimer to form a mature F protein that adopts a metastable pre-fusion conformation and can undergo a conformational change to a post-fusion conformation.

The "RSV F transmembrane domain" corresponds to the wild-type F RSV transmembrane domain (ie, amino acids 525-550 of SEQ ID NO: 10).

The "cytoplasmic domain F RSV" or "cytoplasmic tail F RSV" corresponds to the cytoplasmic tail domain F RSV wild-type (ie, amino acids 551-574 of SEQ ID NO: 10).

"Antibody D25" or "D25" in the context of the present description describes a neutralizing antibody that specifically binds to RSV F peptides in a pre-fusion conformation. This antibody is described in US Patent Application Publication No. 2010/0239593, the entire content of which is incorporated here by reference.

"Single-stranded RSV mutants" refers to the RSV F protein that has been modified to not include furin cleavage sites so that when a single-stranded RSV mutant is produced in cells, the F0 peptide is not cleaved into separate F ₁ and F ₂ chains. A non-limiting example of a single stranded RSV mutant includes position 97 of the F ₂ polypeptide linked to position 97-147 of the F ₁ peptide by a flexible linker to generate a single stranded RSV mutant.

Replacements "DS-CAV1"; "DS-Cav1", each of which is used herein, refers to genetic modifications of the RSV F protein, which contains the "DS" substitutions 155C and 290C, to introduce a non-native disulfide bond between cysteines introduced by the substitutions (such as substitutions S155C and S290C ) and "Cav1" substitutions, which include amino acid substitutions that fill the 190F and 207L cavity (such as S190F and V207L). DS-Cav1 is described in WO 2014/160463, the full content of which is incorporated here by reference.

The terms "foldon domain" or "foldon", each of which is used here, refers to the fibrin trimerization domain of bacteriophage T4, which contains an amino acid sequence that naturally forms a trimeric structure. In some examples, the RSV F protein sequence is modified to contain a foldon domain. In other examples, RSV single strand mutants contain a foldon domain. An example of trimerization of the foldon domain includes the amino acid sequence shown in SEQ ID NO: 8.

In this context, "signal peptide" or "signal sequence" are short amino acid sequences that direct newly synthesized secretory or membrane proteins to and through membranes and thus universally control the entry of most proteins in both eukaryotes and prokaryotes into the secretory path. As a result of processing in the endoplasmic reticulum, mature proteins are formed, in which the signal peptide is cleaved from precursor proteins. As indicated here, the "mature amino acid sequence" does not contain a signal peptide. The mature amino acid sequence of single-stranded RSV mutants does not contain a signal peptide. In addition, the RSV trimer, which consists of three mature RSV single chain mutants, does not contain a signal peptide sequence.

As used herein, the term "substitution", "amino acid substitution" or "substitution variant" in relation to polypeptides means those that have at least one amino acid residue in the native or original sequence removed and replaced by another amino acid inserted in its place in the same position. The substitutions may be single, where only one amino acid in the molecule has been changed, or they may be multiple, where two or more (eg, 3, 4, or 5) amino acids have been changed in the same molecule. For example, as used herein, reference to a "155C" substitution in an F RSV protein or a single-stranded F RSV mutant refers to a single-stranded F RSV protein having a cysteine residue at position 155, where that cysteine residue has been replaced with the corresponding native residue at position 155 in the protein FRSV. By way of reference, SEQ ID NO: 10 is the reference sequence for the position of the substitution. For example, the replacement "S155C" represents the replacement of S at position 155 of SEQ ID NO: 10 with C.

"Isolated" polypeptides or polynucleotides are at least partially free of other biological molecules from the cells or cell cultures in which they are produced. Such biological molecules include other nucleic acids, proteins, lipids, carbohydrates, or other material such as cell debris and culture media. In addition, they may at least partially be free of components of the expression system, such as biological molecules from the host cell or its culture medium. In general, the term "isolated" is not intended to mean the complete absence of such biological molecules, or the absence of water, buffers, or salts, or components of a pharmaceutical formulation that includes polypeptides or polynucleotides.

A "polypeptide variant" is a molecule that differs in its amino acid sequence from a native sequence or a reference sequence. Amino acid sequence variants may have substitutions, deletions, insertions, or a combination of any two or three of the foregoing at specific positions in the amino acid sequence as compared to the native sequence or reference sequence. Typically, variants share at least 50% identity with respect to the native sequence or reference sequence. In some embodiments, the variants have at least 80% identity or at least 90% identity with the native sequence or reference sequence.

By "analogues" is meant polypeptide variants that differ in one or more amino acid changes, such as substitutions, additions, or deletions of amino acid residues, that still retain one or more properties of the parent or parent polypeptide.

The present invention provides several types of compositions based on polynucleotides or polypeptides, including variants and derivatives. These include, for example, substitution, insertion, deletion and covalent variants and derivatives. The term "derivative" is synonymous with the term "variant" and generally refers to a molecule that has been modified and/or altered in any way from a reference molecule or parent molecule.

As such, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and/or additions, deletions and covalent modifications relative to reference sequences, in particular the polypeptide sequences disclosed herein, are included in the scope of this disclosure. For example, tag sequences or amino acids, such as one or more lysines, can be added to peptide sequences (eg, at the N-terminus or C-terminus). Label sequences can be used to detect, purify, or localize peptides. Lysines can be used to increase the solubility of peptides or to provide biotinylation. Alternatively, amino acid residues located at the carboxy- and amino-terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted to provide truncated sequences. Some amino acids (eg C-terminal residues or N-terminal residues) may alternatively be deleted depending on the use of the sequence, eg expression of the sequence as part of a larger sequence that is soluble or bound to a solid support.

As used here, the term "conservative amino acid substitution" refers to an amino acid substitution that is normally present in a sequence with another amino acid of similar size, charge, or polarity. Examples of conservative substitutions include replacing a non-polar (hydrophobic) residue, such as an isoleucine, valine, and leucine residue, with another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another, such as the one chosen between arginine and lysine, between glutamine and asparagine, and between glycine and serine. In addition, substitution of a basic residue such as lysine, arginine or histidine with another basic one, or substitution of one acidic residue such as aspartic acid or glutamic acid with another acidic residue are further examples of conservative substitutions. Examples of non-conservative substitutions include replacing a non-polar (hydrophobic) amino acid residue, such as an isoleucine, valine, leucine, alanine, methionine residue, with a polar (hydrophilic) residue, such as a cysteine, glutamine, glutamic acid, or lysine residue, and/or a polar residue with non-polar residue.

As used herein, the term "domain" refers to a polypeptide motif having one or more identifiable structural or functional characteristics or properties (eg, a binding ability that serves as a site for protein-protein interactions).

As used herein, the term "site" in relation to polypeptides as it relates to amino acid-based embodiments is used as a synonym for "amino acid residue" and "amino acid side chain". As used here, the term "site" in relation to polynucleotides as it refers to variants of implementation based on nucleotides, is used as a synonym for "nucleotide". A site is a position in a peptide or polypeptide or polynucleotide that can be modified, manipulated, altered, derivatized or varied in the polypeptide-based or polynucleotide-based molecules.

As used here, the terms "ends" or "end" when referring to polypeptides or polynucleotides refer to the end of the polypeptide or polynucleotide, respectively. Such an end is not limited to the first or final site of a polypeptide or polynucleotide, but may include additional amino acids or nucleotides at the end regions. Polypeptide-based molecules can be characterized as having both an N-terminus (ending in an amino acid with a free amino group (NH2)) and a C-terminus (ending in an amino acid with a free carboxyl group (COOH)). In some cases, proteins consist of many polypeptide chains connected by disulfide bonds or non-covalent forces (multimers, oligomers). These proteins have multiple N- and C-termini. Alternatively, the ends of the polypeptides may be modified to begin or end, as the case may be, with a non-polypeptide group, such as an organic conjugate.

Those skilled in the art will appreciate that protein fragments, functional protein domains, and homologous proteins are also considered as bulk polypeptides of interest. For example, any protein fragment (meaning a polypeptide sequence that is at least one amino acid residue shorter than a reference polypeptide sequence, but otherwise identical) of a reference protein having a length of 10, 20, 30, 40, 50, 60 is provided here. , 70, 80, 90, 100 or more than 100 amino acids. In another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 (adjacent) amino acids, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any of the sequences described here can be used in accordance with the invention. In some embodiments, the implementation of the polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, as shown in any of the sequences presented here or mentioned here. In yet another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 amino acids that is more than 80%, 90%, 95%, or 100% identical to any of the sequences described herein, where the protein has a stretch of 5, 10, 15, 20, 25, or 30 amino acids that is less than 80%, 75%, 70%, 65% to 60% identical to any of the sequences described herein may be used in accordance with the disclosure.

The polypeptide or polynucleotide molecules of the present invention may have a certain degree of similarity or sequence identity to reference molecules (e.g., reference polypeptides or reference polynucleotides), e.g., molecules described in the art (e.g., genetically engineered or engineered or wild-type molecules) . The term "identity", as known in this field, refers to the relationship between the sequences of two or more polypeptides or polynucleotides, which is determined by comparing the sequences. In the art, identity also refers to the degree of relatedness between two sequences, as determined by the number of matches between regions of two or more amino acid or nucleic acid residues. Identity determines the percentage of identical matches between the smaller of two or more sequences by gap alignment (if any), using a particular mathematical model or computer program (eg, "algorithms"). The identity of related peptides can be easily calculated by known methods. The term "% identity" in relation to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid sequence or nucleic acid sequence that are identical to residues in the amino acid sequence or nucleic acid sequence of the second sequence after sequence alignment. and the introduction of gaps, if necessary, to achieve the maximum percentage of identity. Methods and computer programs for alignment are well known in the art. Identity depends on the identity percentage calculation, but may vary in value due to gaps and penalties introduced into the calculation. Typically, variants of a particular polynucleotide or polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity with a specific reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art in this field of technology. Such alignment tools include those from the BLAST algorithm (Stephen F. Altschul et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402). Another popular local alignment method is based on the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981), "Identification of common molecular subsequences", J. Mol. Biol. 147:195-197). A general method for global alignment based on dynamic programming is the Needleman, SB & Wunsch, CD (1970), "A general method applicable to the search for similarities in the amino acid sequences of two proteins", J. Mol Biol., 48:443-453). More recently, a Fast Optimal Global Sequence Alignment (FOGSAA) algorithm has been developed that purportedly performs global nucleotide and protein sequence alignment faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm. Other tools are described here, in particular, under the definition of the term "identity" below.

As used here, the term "homology" refers to a common relationship between polymeric molecules, for example, between nucleic acid molecules (eg, DNA molecules) and/or between polypeptide molecules. Polymeric molecules (eg, nucleic acid molecules (eg, DNA molecules) and/or polypeptide molecules) that have a threshold level of similarity or identity, determined by the alignment of matching residues, are said to be homologous. Homology is a qualitative term that describes relationships between molecules and can be based on quantitative similarity or identity. Similarity or identity is a quantitative term that defines the degree of sequence matching between two compared sequences. In some embodiments, polymer molecules are considered "homologous" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical or similar. The term "homologous" necessarily refers to a comparison of at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered homologous if the polypeptides they encode comprise at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% of at least one region of at least of 20 amino acids. In some embodiments, the homologous polynucleotide sequences are characterized by the ability to encode a region of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a region of at least 4-5 uniquely specified amino acids. Two protein sequences are considered homologous if the proteins are at least 50%, 60%, 70%, 80%, or 90% identical in at least one region of at least 20 amino acids.

Homology implies that the compared sequences diverged in the evolutionary process from a common source of origin. The term "homolog" refers to a first amino acid sequence or nucleic acid sequence (e.g., a gene sequence (DNA or RNA) or a protein sequence) that is related to a second amino acid or nucleic acid sequence in derivation from a common ancestral sequence. The term "homolog" can be applied to the relationship between genes and/or proteins separated by speciation, or to the relationship between genes and/or proteins separated by genetic duplication. "Orthologs" represent genes (or proteins) across species that have evolved from a common ancestral gene (or protein) through speciation. As a rule, orthologs retain the same function during evolution. "Paralogs" are genes (or proteins) linked by duplication in the genome. Orthologues retain the same function through evolution, while paralogs develop new functions, even if they are related to the original one.

The term "identity" refers to a common relationship between polymeric molecules, for example, between polynucleotide molecules (eg DNA molecules) and/or between polypeptide molecules. For example, the calculation of percent identity between two polynucleic acid sequences can be performed by aligning the two sequences for optimal comparison (e.g., gaps can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In some embodiments, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the length of the reference sequence. The nucleotides are then compared at the corresponding nucleotide positions. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that must be entered to optimally align the two sequences. Sequence comparison and determination of percent identity between two sequences can be performed using a mathematical algorithm. For example, percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology , Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology , von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each source is incorporated here by reference. For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which was included in the ALIGN program (version 2.0) using the PAM120 residual weight table, length penalty a gap of 12 and a gap penalty of 4. The percent identity between two nucleic acid sequences can alternatively be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix. Methods commonly used to determine percent identity between sequences include, but are not limited to, those disclosed in Carillo H. and Lipman D., SIAM J. Applied Math., 48: 1073 (1988); which is incorporated here by reference. The determination methods are systematized in publicly available computer programs. Examples of computer software for determining homology between two sequences include, but are not limited to, the GCG software package, Devereux, J. et al., Nucleic Acids Research, 12, 387 (1984)), BLASTP, BLASTN and FASTA Altschul, SF et al ., J. Molec. Biol., 215, 403 (1990)).

Lipid nanoparticles

As used herein, the term "lipid nanoparticle" or "LNP" refers to any lipid composition that can be used to deliver a product, including, but not limited to, liposomes or vesicles, where the water volume is encapsulated by amphipathic lipid bilayers (e.g., single, unilamellar, or multiple, multi-layered) or, in other embodiments, where the lipids coat the interior cavity containing the prophylactic product, or lipid aggregates or micelles, where the lipid-encapsulated therapeutic product is in a relatively random lipid mixture. Except where noted, there is no need to include an antigenic polypeptide in a lipid nanoparticle and it can be used to deliver a product in the same composition.

As used here, the term "polyamine" means compounds having two or more amino groups. Examples include putrescine, cadaverine, spermidine and spermine.

Unless otherwise indicated, mole % refers to mole percent of total lipids. Typically, the LNP compositions of the invention are composed of one or more cationic lipids (including ionizable cationic lipids) and one or more poly(ethylene glycol) lipids (PEG lipid). In certain embodiments, the LNPs further comprise one or more non-cationic lipids. One or more non-cationic lipids may include a phospholipid, a phospholipid derivative, a sterol, a fatty acid, or a combination thereof.

Cationic lipids and ionizable cationic lipids suitable for LNP are described here. Ionizable cationic lipids are characterized by a weak basicity of their lipid head groups, which affects the surface charge of the lipid in a pH dependent manner, making them positively charged at acidic pH but close to neutral charge at physiological pH. Cationic lipids are characterized by a monovalent or polyvalent cationic charge on their head groups, making them positively charged at neutral pH. In some embodiments, the cationic and ionizable lipid is capable of complexing with hydrophilic biologically active molecules to form a hydrophobic complex that separates into the organic phase of a biphasic aqueous-organic system. It is believed that both monovalent and polyvalent cationic lipids can be used to form hydrophobic complexes with biologically active molecules.

In some embodiments, cationic and ionizable cationic lipids for use in LNP formation include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTAP"); 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane ("DOTAP"); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Chol"); diheptadecylamidoglycylspermidine ("DHGS") and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"). In addition, there are a number of commercial preparations of cationic lipids, as well as other components that can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic lipid nanoparticles containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine ("DOPE") manufactured by GIBCOBRL, Grand Island, N.Y., USA); and LIPOFECTAMINE® (commercially available cationic lipid nanoparticles containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate ("DOSPA") and ( "DOPE") manufactured by (GIBCOBRL).The following lipids are cationic and have a positive charge below physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 4-(2,2- diocta)-9,12-dienyl-[1,3]dioxolan-4-ylmethyl)dimethylamine, DLinKDMA (WO 2009/132131 A1), DLin-K-C2-DMA (WO2010/042877), DLin-M-C3- DMA (WO2010/146740 and/or WO2010/105209), DLin-MC3-DMA (heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate; Jayaraman et al., 2012, Angew. Chem. Int. Ed. Engl. 51: 8529-8533), 2-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-N, N-dimethyl-3-[(9Z, 12Z)-octadeca-9,12-dienyloxyl]propane-1-amine) (CLinDMA) and the like. Other cationic lipids suitable for use in the invention include, for example, the cationic lipids described in US Pat. Other cationic lipids suitable for use in the invention include, for example, lipids E0001-E0118 or E0119-E0180 as disclosed in Table 6 (pages 112-139) of International Patent Application Publication No. WO2011/076807 (which also discloses production methods and methods use of these cationic lipids).

In some embodiments, the implementation of the cationic lipid includes any compound from DLinDMA; DlinKC2DMA; Dlin-MC3-DMA; CLinDMA; S-octyl CLinDMA;

(2S)-1-{7-[(3β)-cholest-5-en-3-yloxy]heptyloxy}-3-[(4Z)-dec-4-en-1-yloxy]-N, N dimethylpropan- 2-amine;

(2R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-[(4Z)-dec-4-en-1-yloxy]-N, N dimethylpropan- 2-amine;

1-[(2R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)propan-2-yl]guanidine;

1-[(2R)-1-{7-[(3β)-cholest-5-en-3-yloxy]heptyloxy}-N, N-dimethyl-3-[(9Z,12Z)-octadeca-9,12 -dien-1-yloxy]propan-2-amine;

1-[(2R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-N, N-dimethyl-3-[(9Z,12Z)-octadeca-9.12 -dien-1-yloxy]propan-2-amine;

(2S)-1-({6-[(3β))-cholest-5-en-3-yloxy]hexyl}oxy)-N, N-dimethyl-3-[(9Z)-octadeca-9-en- 1-yloxy]propan-2-amine;

(3β)-3-[6-{[(2S)-3-[(9Z)-octadeca-9-en-1-yloxy]-2-(pyrrolidin-1-yl)propyl]oxy}hexyl)oxy] cholest-5-ene;

(2R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)propan-2-amine;

(2R)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-(pentyloxy)propan-2-amine;

(2R)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-3-(heptyloxy)-N,N-dimethylpropan-2-amine;

(2R)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N, N-dimethyl-3-[(2Z)pent-2-en-1- yloxy]propan-2-amine;

(2S)-1-butoxy-3-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethylpropan-2-amine;

(2S-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-3-[2,2,3,3,4,4,5,5,6, 6,7,7,8,8,9,9-hexadecafluoronyl)oxy]-N,N-dimethylpropan-2-amine;

2-amino-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1,3-diol;

2-amino-3-((9-(((3S,10R,13R)-10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9, 10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)nonyl)oxy)-2-((((9Z,12Z)-octadeca- 9,12-dien-1-yl)oxy)methyl)propan-1-ol;

2-amino-3-((6-(((3S,10R,13R)-10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9, 10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)hexyl)oxy)-2-((((Z)-octadec-9- en-1-yl)oxy)methyl)propan-1-ol;

(20Z,23Z)-N,N-dimethylnonacose-20,23-diene-10-amine;

(17Z,20Z)-N,N-dimethylhexacose-17,20-diene-9-amine;

(16Z,19Z)-N,N-dimethylpentacose-16,19-diene-8-amine;

(13Z,16Z)-N,N-dimethyldocose-13,16-diene-5-amine;

(12Z,15Z)-N,N-dimethylgenicose-12,15-diene-4-amine;

(14Z,17Z)-N,N-dimethyltricose-14,17-diene-6-amine;

(15Z,18Z)-N,N-dimethyltetracose-15,18-diene-7-amine;

(18Z,21Z)-N,N-dimethylheptacose-18,21-diene-10-amine;

(15Z,18Z)-N,N-dimethyltetracose-15,18-diene-5-amine;

(14Z,17Z)-N,N-dimethyltricose-14,17-diene-4-amine;

(19Z,22Z)-N,N-dimethyloctacose-19,22-diene-9-amine;

(18Z,21Z)-N,N-dimethylheptacose-18,21-diene-8-amine;

(17Z,20Z)-N,N-dimethylhexacose-17,20-diene-7-amine;

(16Z,19Z)-N,N-dimethylpentacose-16,19-diene-6-amine;

(22Z,25Z)-N,N-dimethylhentriaconta-22,25-diene-10-amine;

(21Z,24Z)-N,N-dimethyltriaconta-21,24-diene-9-amine;

(18Z)-N,N-dimethylheptacose-18-ene-10-amine;

(17Z)-N,N-dimethylhexacose-17-ene-9-amine;

(19Z,22Z)-N,N-dimethyloctacose-19,22-diene-7-amine;

N,N-dimethylheptacosan-10-amine;

(20Z,23Z)-N-ethyl-N-methylnonacose-20,23-diene-10-amine;

1-[(11Z,14Z)-1-nonylicose-11,14-dien-1-yl]-pyrrolidine;

(20Z)-N,N-dimethylheptacose-20-ene-10-amine;

(15Z)-N,N-dimethylheptacose-15-en-10-amine;

(14Z)-N,N-dimethylnonacose-14-en-10-amine;

(17Z)-N,N-dimethylnonacose-17-ene-10-amine;

(24Z)-N,N-dimethyltritriacont-24-ene-10-amine;

(20Z)-N,N-dimethylnonacose-20-en-10-amine;

(22Z)-N,N-dimethylgentriacont-22-en-10-amine;

(16Z)-N,N-dimethylpentacose-16-en-8-amine;

(12Z,15Z)-N,N-dimethyl-2-nonylgenicose-12,15-diene-1-amine;

(13Z,16Z)-N,N-dimethyl-3-nonyldocose-13,16-diene-1-amine;

N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine;

1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine;

N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine;

N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]genicosan-10-amine;

N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine;

N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine;

N,N-dimethyl-1-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine;

N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine;

1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine;

1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine;

N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine; And

(11E,20Z,23Z)-N,N-dimethylnonacose-11,20,23-trien-10-amine;

or a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing.

In some aspects of this embodiment, the LNPs comprise one or more of the following ionizable cationic lipids: DLinDMA, DlinKC2DMA, DLin-MC3-DMA, CLinDMA, or S-Octyl CLinDMA (see International Patent Application Publication No. WO2010/021865). In still other aspects of this embodiment, the LNPs comprise one or more of the following ionizable cationic lipids: (13Z,16Z)-N,N-dimethyl-3-nonyldocose-13,16-dien-1-amine or N,N-dimethyl -1-[(1S,2R)-2-octylcyclopropyl]hexadecane-8-amine, each of which is described in PCT/US2011/0523238, published as WO 2012/040184, which is incorporated herein by reference in its entirety.

In some aspects of this embodiment, the ionizable cationic lipid may include the lipid described in International Patent Application WO 2017/049245, which is incorporated herein by reference in its entirety.

In certain aspects of this embodiment, the LNPs include one or more of the ionizable cationic lipids described in International Patent Application Publication No. WO2011/022460 A1, or any pharmaceutically acceptable salt or stereoisomer of any of the compounds or salts described therein.

When structures of the same structure differ in the spatial arrangement of certain atoms or groups, then they are stereoisomers, and the considerations that are important in the analysis of their relationships are topological. If the relationship between two stereoisomers is the same as that of an object and its non-overlapping mirror image, then the two structures are enantiomeric, and each structure is said to be chiral. Stereoisomers also include diastereomers, cis-trans isomers, and conformational isomers. Diastereoisomers may be chiral or achiral and are not mirror images of each other. Cis-trans isomers differ only in the positions of the atoms relative to the specified plane in cases where these atoms are or are considered as such, as if they were part of a rigid structure. Conformational isomers are isomers that can be converted into each other by rotation around formally single bonds. Examples of such conformational isomers include cyclohexane conformations with chair and boat conformers, carbohydrates, linear alkanes with hindered, occluded conformers and gauche conformers, etc. See J. Org. Chem. 35, 2849 (1970).

Many organic compounds are in optically active forms capable of rotating the plane of plane polarized light. When describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule with respect to its chiral center(s). The prefixes d and l or (+) and (-) are used to indicate the direction of rotation of a plane-polarized light connection, where (-) or l means that the connection is left-handed. A connection with a (+) or d prefix is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are non-superimposable mirror images of each other. A mixture of enantiomers is often referred to as an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture. Many of the cationic lipids described herein may have one or more chiral centers and therefore may exist in different enantiomeric forms. If desired, a chiral carbon may be designated with an asterisk (*). When bonds to a chiral carbon are depicted in claims as straight lines, it is understood that both configurations (R) and (S) of the chiral carbon, and hence enantiomers and mixtures thereof, are included in the formula. As used in the art when an absolute configuration relative to a chiral carbon is required, one of the bonds to the chiral carbon may be drawn as a wedge (bonds to atoms above the plane) and the other may be drawn as a series or wedge of short parallel lines (bonds with atoms below the plane). The Kahn-Ingold-Prelog system can be used to assign an (R) or (S) configuration to a chiral carbon.

When the cationic lipids of the present invention contain a single chiral center, the compounds are in two enantiomeric forms, and the present invention includes both enantiomers and mixtures of enantiomers, such as a specific 50:50 mixture called a racemic mixture. Enantiomers can be separated by methods known to those skilled in the art, such as obtaining diastereomeric salts, which can be separated, for example, by crystallization (see "CRC Handbook of Optical Resolutions via Diastereomeric Salt Formation", David Kozma (CRC Press, 2001)); obtaining diastereomeric derivatives or complexes that can be separated, for example, by crystallization, gas-liquid or liquid chromatography; selective interaction of one enantiomer with an enantiomer-specific reagent, such as enzymatic esterification; or gas-liquid or liquid chromatography in a chiral medium, for example, on a chiral carrier, for example, silicon dioxide with an associated chiral ligand, or in the presence of a chiral solvent. It should be understood that when the desired enantiomer is converted to another chemical molecule using one of the separation procedures described above, then an additional step is required to release the desired enantiomeric form. Alternatively, particular enantiomers can be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts, or solvents, or converted from one enantiomer to another by asymmetric conversion.

The designation of a specific absolute configuration on the chiral carbon of the cationic lipids described herein is understood to mean that the indicated enantiomeric form of the compounds is in an enantiomeric excess (ee) or, in other words, essentially does not contain the other enantiomer. For example, the "R" forms of the compounds are substantially free of the "S" forms of the compounds and thus are in enantiomeric excess of the "S" forms. In contrast, the "S" forms of the compounds essentially do not contain the "R" forms of the compounds and thus are in enantiomeric excess of the "R" forms. As used here, the term "enantiomeric excess" represents the presence of a particular enantiomer in an amount of more than 50%. In a specific embodiment, when a specific absolute configuration is indicated, the enantiomeric excess of the depicted compounds is at least about 90%.

When a cationic lipid as described herein contains two or more chiral carbon atoms, it may have more than two optical isomers and may exist in diastereomeric forms. For example, when there are two chiral carbon atoms, then the compound can have up to 4 optical isomers and 2 enantiomeric pairs (((S, S)/(R, R) and (R, S)/(S, R)). (e.g. (S, S)/(R, R)) are mirror image stereoisomers of each other. Stereoisomers that are not mirror images (e.g. (S, S) and (R, S)) are diastereomers. can be separated by methods known to those skilled in the art, such as chromatography or crystallization, and the individual enantiomers in each pair can be separated as described above.The LNPs described herein include each diastereoisomer of such cationic lipids and mixtures thereof.

LNPs may also contain any combination of two or more of the cationic lipids described here. In certain aspects, the cationic lipid typically comprises from about 0.1 to about 99.9 mole % of the total lipid present in said particle. In some aspects, the cationic lipid may be from about 80 to about 99.9% by mole. In still other aspects, the cationic lipid is from about 2% to about 70%, from about 5% to about 50%, from about 10% to about 45%, from about 20% to about 99.8%, from about 30% to about 70%, about 34% to about 59%, about 20% to about 40%, or about 30% to about 40% (in mol%) of the total amount of lipids present in said particle.

The LNPs described herein may further comprise a non-cationic lipid, which may be any of a variety of neutral, uncharged, zwitterionic, or anionic lipids capable of forming a stable complex. Preferably they are neutral, although they may be negatively charged. Examples of non-cationic lipids used herein include phospholipid-related substances such as natural phospholipids, synthetic derivatives of phospholipids, fatty acids, sterols, and combinations thereof. Natural phospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (phosphatidate) (PA), dipalmitoylphosphatidylcholine, monoacylphosphatidylcholine (lyso PC), 1-palmitoyl-2 -oleoyl-sn-glycero-3-phosphocholine (POPC), N-acyl-PE, phosphoinositides and phosphosphingolipids. Phospholipid derivatives include phosphatidic acid (DMPA, DPPA, DSPA), phosphatidylcholine (DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC), phosphatidylglycerol (DMPG, DPPG, DSPG, POPG), phosphatidylethanolamine (DMPE, DPPE, DSPE , DOPE) and phosphatidylserine (DOPS). Fatty acids include palmitic acid (C14:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3) and arachidonic acid (C20:4) , C20:0, C22:0 and lecithin.

In some LPN embodiments described herein, the non-cationic lipid is selected from lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, diacetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), di palmitoylphosphatidylcholine ( DPPC), dioleylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleylphosphatidylcholine (POPC), palmitoyloleylphosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (DOPE-mal ). Non-cationic lipids also include sterols such as cholesterol, stigmasterol or stigmastanol. Cholesterol is known in the art. See U.S. Patent Application Publications: 2006/0240554 and 2008/0020058. In certain embodiments, the LNP comprises a combination of a phospholipid and a sterol.

The non-cationic lipid, if present, is typically about 0.1% to about 65%, about 2% to about 65%, about 10% to about 65%, or about 25% to about 65%, when expressed as molar percentage of the total lipid present in the LNP. The LNPs described herein further include a polyethylene glycol (PEG)-lipid ("PEG-lipid") conjugate, which is used as a bilayer stabilizing component. The lipid component of a PEG lipid may be any non-cationic lipid described above, including naturally occurring phospholipids, synthetic derivatives of phospholipids, fatty acids, sterols, and combinations thereof. In some embodiments of the LNPs described herein, the PEG lipids include PEG linked to dialkyloxypropyls (PEG-DAA) as described, for example, in International Patent Application Publication No. WO 05/026372, PEG linked to diacylglycerol (PEG-DAG), described, for example, in US Patent Application Publication Nos. 20030077829 and 2005008689; PEG linked to phosphatidylethanolamine (PE) (PEG-PE), or PEG conjugated to 1,2-di-O-hexadecyl-sn-glyceride (PEG-DSG), or any mixture thereof (see e.g. US Pat. No. 5885613).

In one embodiment, the PEG-DAG conjugate is dilaurylglycerol (C12)-PEG conjugate, PEG-dimyristylglycerol (C14) conjugate, PEG-dipalmitoylglycerol (C16) conjugate, PEG-dilaurylglycamide (C12) conjugate, PEG-dimiritylglycamide (C14) conjugate, conjugate of PEG-dipalmitoylglycamide (C16) or PEG-disterylglycamide (C18). Those skilled in the art will readily appreciate that other diacylglycerols may be used in PEG-DAG conjugates.

In some embodiments, PEG lipids include, but are not limited to, PEG-dimyristolglycerol (PEG-DMG), PEG-disterylglycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerol (PEG-DAG ), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE) and PEG-1,2-dimyristyloxylpropyl-3-amine (PEG-c-DMA).

In some embodiments, the PEG lipid is dimyristoylglycerol-linked PEG (PEG-DMG), for example, as described in Abrams et al. and US 2008/0020058 including, for example, 2KPEG/PEG200-DMG.

In some embodiments, a PEG lipid such as PEG-DAG, PEG-cholesterol, PEG-DMB comprises a polyethylene glycol having an average molecular weight ranging from about 500 Daltons to about 10,000 Daltons, from about 750 Daltons to about 5000 Daltons, from about 1000 Daltons to about 5000 Daltons, from about 1500 Daltons to about 3000 Daltons or about 2000 Daltons. In some embodiments, the PEG lipid comprises PEG400, PEG1500, PEG2000, or PEG5000.

The acyl groups in any of the lipids described above are preferably acyl groups derived from fatty acids having from about 10 to about 24 carbon atoms. In one embodiment, the acyl group is lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

The PEG-lipid conjugate is typically about 0.1% to about 15%, about 0.5% to about 20%, about 1.5% to about 18%, about 4% to about 15%, about 5% to about 12%, from about 1% to about 4%, or about 2%, when expressed in mol.% of the total amount of lipid present in the specified particle.

In some embodiments, the LNPs comprise one or more cationic lipids, cholesterol, and 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (PEG-DMG).

In some embodiments, the LNPs comprise one or more cationic lipids, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (PEG-DMG).

In some embodiments, the LNPs contain lipid compounds combined in the following molar ratios:

cationic lipid (20-99.8 mole%)

non-cationic lipid (0.1-65 mol.%) AND

PEG-DMG (0.1-20 mole %).

In some embodiments, the LNPs contain lipid compounds combined in the following molar ratios:

cationic lipid (30-70 mol.%)

non-cationic lipid (20-65 mol.%) and

PEG-DMG (1-15 mole %).

In some aspects of this embodiment, the non-cationic lipid is cholesterol.

Typical LNPs may include cationic lipid/cholesterol/PEG-DMG in the following molar ratios: 58/30/10.

In some aspects of this embodiment, the non-cationic lipid is cholesterol and DSPC. Typical LNPs may include cationic lipid/cholesterol/DSPC/PEG-DMG in the following molar ratios: 59/30/10/1; 58/30/10/2; 43/41/15/1; 42/41/15/2; 40/48/10/2; 39/41/19/1; 38/41/19/2; 34/41/24/1 and 33/41/24/2.

Cooking LNP

LNP can be obtained, for example, by a rapid precipitation process that involves micromixing lipid components dissolved in ethanol with an aqueous solution using a volume-limited mixing apparatus, such as a volume-limited T-micromixer, a multi-nozzle vortex mixer (MIVM) , or microfluidic mixers as described below. The lipid solution contains one or more cationic lipids, one or more non-cationic lipids (eg DSPC), PEG-DMG and optionally cholesterol in specific molar ratios in ethanol. The aqueous solution consists of buffered saline sodium citrate or sodium acetate with a pH in the range of 2-6, preferably 3.5-5.5. The two solutions are heated to a temperature in the range of 25°C-45°C, preferably 30°C-40°C, and then mixed in a limited volume mixer, thereby instantly obtaining LNP. When a volume limited T-Mixer is used, the Micro-T-Mixer has an internal diameter (ID) in the range of 0.25 to 1.0 mm. Alcoholic and aqueous solutions enter the T-micromixer input from the used programmable syringe pumps and with a total flow rate of 10-600 ml/min. The alcoholic and aqueous solutions are combined in a limited volume mixer in a ratio of 1:1 to 1:3 v/v, but preferably 1:1.1 to 1:2.3. The combination of the ethanol volume fraction, the flow rates of the reagent solution, and the ID of the T-micromixer nozzles used in this mixing step allows the LNP particle size to be controlled from 30 to 300 nm. The resulting LNP slurry is diluted twice with a higher pH buffer in the range of 6-8 in a serial multi-stage in-line mixing process. For the first dilution, the LNP suspension is mixed with a buffered solution at a higher pH (pH 6-7.5) with a mixing ratio ranging from 1:1 to 1:3 v/v, but preferably 1:2 v/v. This buffered solution has a temperature in the range of 15-40°C, preferably 30-40°C. The resulting LNP suspension is further mixed with a buffered solution at a higher pH, for example 6-8, and with a mixing ratio ranging from 1:1 to 1:3 v/v, but preferably 1:2 v/v. This later buffered solution has a temperature in the range of 15-40°C, preferably 16-25°C. The mixed LNPs are held for 30 minutes to 2 hours before the anion exchange filtration step. The temperature during the incubation period is in the range of 15-40°C, preferably 30-40°C. After incubation, the LNP suspension is filtered through a 0.8 µm filter, including an anion exchange separation step. This process uses tubes with an ID of 1 mm to 5 mm and a flow rate of 10 to 2000 ml/min. The LNP is concentrated and diafiltered through an ultrafiltration process where the alcohol is removed and the buffer is replaced with a final buffer such as phosphate buffered saline or a buffer system suitable for cryopreservation (eg containing sucrose, trehalose, or combinations thereof). The ultrafiltration process uses the tangential flow filtration format (TFF). This process uses a membrane with a nominal molecular weight cutoff range of 30 to 500 kDa, preferably 100 kDa. The membrane format may be a hollow fiber cassette or a flat sheet. TFF processes with proper molecular weight cutoff keep the LNP in the retentate and the filtrate or permeate contains the alcohol and final buffer waste. The TFF process is a multi-step process with initial concentration up to a lipid concentration of 20-30 mg/ml. After concentration, the LNP suspension is diafiltered against a final buffer (e.g. phosphate buffered saline (PBS) pH 7-8, 10 mM Tris, 140 mM NaCl pH 7-8 or 10 mM Tris, 70 mM NaCl, 5 wt.%. sucrose, pH 7-8) with 5-20 volumes to remove alcohol and replace with a buffer. The material is then further concentrated 1-3 times by ultrafiltration. The final steps in the LNP production process are the sterilizing filtration of the concentrated LNP solution into a suitable container under aseptic conditions. Sterilizing filtration is performed by passing the LNP solution through a pre-filter (Acropak 500 PES 0.45/0.8 µm capsule) and a bioburden reduction filter (Acropak 500 PES 0.2/0.8 µm capsule). After filtration, the bottled LNP product is stored under suitable storage conditions (2°C-8°C or -20°C if the formulation is to be frozen).

In some embodiments, the LNPs in the compositions provided herein have a geometric mean diameter that is less than 1000 nm. In some embodiments, the LNPs have a geometric mean diameter that is greater than 50 nm but less than 500 nm. In some embodiments, the LNP population geometric mean diameter is about 60 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm or 475 nm. In some embodiments, the geometric mean diameter is 100-400 nm, 100-300 nm, 100-250 nm, or 100-200 nm. In some embodiments, the geometric mean diameter is 60-400 nm, 60-350 nm, 60-300 nm, 60-250 nm, or 60-200 nm. In some embodiments, the implementation of the average geometric diameter is 75-250 nm. In some embodiments, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the LNPs from the LNP population are less than 500 nm in diameter. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the LNPs from the LNP population have a diameter greater than 50 nm but less than 500 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the LNPs from the LNP population have a diameter of about 60 nm, 75 nm, 100 nm, 125 nm , 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm or 475 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the LNPs from the LNP population have a diameter of 100-400 nm, 100-300 nm, 100- 250 nm or 100-200 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the LNPs from the LNP population have a diameter that is in the range of 60-400 nm, 60- 350 nm, 60-300 nm, 60-250 nm or 60-200 nm.

In a specific embodiment, the size of the LNP is from about 1 to 1000 nm, preferably from about 10 to 500 nm, more preferably from about 100 to 300 nm, and preferably 100 nm.

Nucleic acids/polynucleotides

The DNA of the present invention is, in some embodiments, codon-optimized. Methods for codon optimization are known in the art and can be used as described here. Codon optimization, in some embodiments, can be used to match codon frequencies in target organisms and host organisms to ensure proper folding; shifting GC content to increase mRNA stability or reduce the formation of secondary structures; minimizing tandem repeats of codons or bases that can interfere with the construction or expression of genes; modification of transcriptional and translational control regions; insertions or deletions of protein transport sequences; deletions/additions of post-translational modification sites in the encoded protein (eg, glycosylation sites); additions, deletions, or permutations of protein domains; insertions or deletions of restriction sites; modification of ribosome binding sites and mRNA degradation sites; translation rate adjustments to allow different protein domains to fold correctly; or reduce or eliminate the problem of formation of secondary structures within the polynucleotide. Tools, algorithms and kits for codon optimization are known in the art. Non-limiting examples include GeneArt kits (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.

In some embodiments, the codon-optimized sequence has less than 95% sequence identity, less than 90% sequence identity, less than 85% sequence identity, less than 80% sequence identity, or less than 75% sequence identity with a naturally occurring or wild-type sequence. type.

In some embodiments, the codon-optimized sequence has 65% to 85% (e.g., about 67% to about 85%, or about 67% to about 80%) sequence identity with a naturally occurring or wild-type sequence. In some embodiments, the codon-optimized sequence has 65% to 75% or about 80% sequence identity with a naturally occurring or wild-type sequence.

Methods of treatment

Provided herein are compositions (eg, pharmaceutical compositions), methods, kits, and reagents for the prevention and/or treatment of RSV in humans and other mammals. RSV vaccines can be used as therapeutic or prophylactic agents. They can be used in medicine for the prevention and/or treatment of infectious diseases. In exemplary aspects, the RSV immunogenic compositions of the present invention are used as vaccines to provide prophylactic protection against the RSV virus. Prophylactic protection against the RSV virus can be achieved after administration of the RSV vaccine of the present invention. Vaccines can be administered one, two, three, four or more times. It is possible, although less desirable, to administer the vaccine to an infected person in order to achieve a therapeutic response. In this case, it may be necessary to adjust the dosage accordingly.

In some embodiments, the RSV immunogenic compositions of the present invention can be used as a method of preventing an RSV infection in a subject, wherein the method comprises administering to said subject at least one RSV immunogenic composition described herein. In some embodiments, the RSV immunogenic compositions of the present invention can be used as a method of treating an RSV infection in a subject, wherein the method comprises administering to said subject at least one RSV immunogenic composition described herein. In some embodiments, the RSV immunogenic compositions of the present invention can be used as a method of reducing the incidence of RSV in a subject, wherein the method comprises administering to said subject at least one RSV immunogenic composition described herein. In some embodiments, the RSV immunogenic composition of the present invention can be used as a method for inhibiting the spread of RSV from a first subject infected with RSV to a second subject not infected with RSV, wherein the method comprises administering to at least one of said first subject and said second subject at least one RSV immunogenic composition described herein.

In aspects of the invention, a method is provided for inducing an immune response in a subject against RSV. The method includes administering to a subject an RSV immunogenic composition as described herein, thereby inducing an RSV-specific immune response in the subject.

A prophylactically effective dose is a therapeutically effective dose that prevents viral infection at a clinically acceptable level. In some embodiments, the therapeutically effective dose is the dose indicated on the vaccine package insert.

Therapeutic and prophylactic compositions

Provided herein are compositions (eg, pharmaceutical compositions), methods, kits, and reagents for the prevention, treatment, or diagnosis of RSV, eg, in humans and other mammals. RSV immunogenic compositions, including vaccines, can be used as therapeutic or prophylactic agents. They can be used in medicine for the prevention and/or treatment of infectious diseases. In some embodiments, the RSV immunogenic compositions of the present invention can be used to treat RSV.

RSV immunogenic compositions, including RSV vaccines, can be administered prophylactically or therapeutically as part of an active immunization regimen in healthy individuals or in the early stages of infection during the incubation period or during active infection after the onset of symptoms. In some embodiments, the amount of the vaccine of the present invention provided to a cell, tissue, or subject may be an amount effective for immunoprophylaxis.

RSV immunogenic compositions, including RSV vaccines, can be administered with other prophylactic or therapeutic compounds. As a non-limiting example, the prophylactic or therapeutic compound may be an adjuvant or booster component. As used here, in relation to a prophylactic composition, such as a vaccine, the term "booster component" refers to the additional administration of a prophylactic (vaccine) composition. The booster (or booster component) may be administered after the earlier administration of the prophylactic composition. The administration time between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes. , 20 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 1 day, 36 h, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months , 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 16 years old, 17 years old, 18 years old, 19 years old, 20 years old, 25 years old, 30 years old, 35 years old, 40 years old, 45 years old, 50 years old, 55 years old, 60 years old, 65 years old, 70 years old, 75 years old, 80 years old, 85 years old, 90 years old, 95 years old or over 99 years old. In some embodiments, the administration time between initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year.

In some embodiments, RSV immunogenic compositions, including RSV vaccines, can be administered intramuscularly, intradermally, or intranasally, similar to the administration of inactivated vaccines known in the art. In some embodiments, RSV immunogenic compositions, including RSV vaccines, are administered intramuscularly.

RSV immunogenic compositions, including RSV vaccines, can be used in a variety of situations depending on the prevalence of the infection or the degree or level of unmet medical need. Vaccines are superior in that they produce much higher antibody titers and cause reactions earlier than commercially available antiviral agents/compositions.

Provided herein are pharmaceutical compositions comprising RSV immunogenic compositions, optionally in combination with one or more pharmaceutically acceptable excipients.

RSV immunogenic compositions that include RSV vaccines may be formulated or administered alone or in combination with one or more other components. For example, such compositions may contain other components, including, but not limited to, adjuvants.

In some embodiments, RSV immunogenic compositions do not include an adjuvant (ie, they do not contain an adjuvant).

It has long been established that aluminum compounds stimulate an immune response against co-administered antigens. The vaccine formulations of the present invention are adsorbed on an aluminum adjuvant. Preferably, the aluminum adjuvant in the compositions of the present invention is not in the form of an aluminum precipitate. Aluminum deposited vaccines can enhance the immune response to the target antigen, but are highly heterogeneous formulations and do not provide comparable results (see Lindblad E. B. Immunology and Cell Biology 82:497-505 (2004)). In contrast, aluminum adsorbed vaccines can be standardized, which is an important feature of vaccine formulations intended for human administration. In addition, the physical adsorption of the desired antigen to the aluminum adjuvant is believed to be essential to the functioning of the adjuvant, probably due to slower spread from the injection site or more efficient uptake of the antigen by the antigen presenting cells.

The aluminum compound adjuvant of the present invention may be in the form of aluminum hydroxide (Al(OH) ₃ ), aluminum phosphate (AlPO ₄ ), aluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate (AAHS) or so-called "alum" (KA1(S0 ₄ )⋅12H ₂ 0) (see Klein et al., Klein et al., Analysis of aluminum hydroxyphosphate vaccine adjuvants by (27)A1 MAS NMR., J. Pharm. Sci. 89(3): 311-21 (2000) ). In exemplary embodiments, the aluminum-based adjuvant is aluminum hydroxyphosphate or AAHS. The ratio of phosphate to aluminum in the aluminum adjuvant can be from 0 to 1.3. In preferred embodiments of this aspect of the invention, the ratio of phosphate to aluminum is in the range of 0.1 to 0.70. In particularly preferred embodiments, the ratio of phosphate to aluminum is in the range of 0.2 to 0.50. MAPA is an aqueous suspension of aluminum hydroxyphosphate. MAPA is prepared by mixing aluminum chloride and sodium phosphate in a 1:1 volume ratio to precipitate aluminum hydroxyphosphate. After the mixing process, the material is reduced in size using a high shear mixer to achieve a target aggregate particle size in the range of 2-8 µm. The product is then diafiltered against saline and steam sterilized. See, for example, International Patent Application Publication No. WO2013/078102.

In some embodiments, the aluminum compound adjuvant is in the form of AAHS (refers herein interchangeably to "Merck aluminum adjuvant (MAA)"). MAA carries a zero charge at neutral pH, while AlOH carries an overall positive charge, while AlPO ₄ generally carries a net negative charge at neutral pH.

One skilled in the art can determine the optimal dose of an aluminum compound adjuvant that is both safe and effective in enhancing the immune response to the target antigenic polypeptides. For the safety profile of aluminum, as well as the amount of aluminum included in FDA-licensed vaccines, see Baylor et al., Vaccine 20: S18-S23 (2002). Generally, an effective and safe dose of aluminum adjuvant ranges from 150 to 600 µg/dose (concentration 300 to 1200 µg/mL). In specific embodiments, the formulations and compositions of the present invention contain 200-300 micrograms of aluminum adjuvant per vaccine dose. In alternative embodiments of the formulations and compositions of the present invention, the amount of aluminum adjuvant is 300-500 micrograms per vaccine dose.

RSV immunogenic compositions, including RSV vaccines, may be formulated or administered in combination with one or more pharmaceutically acceptable excipients. In some embodiments, the compositions contain at least one additional active agent, such as, for example, a therapeutically active agent, a prophylactically active agent, or a combination thereof. The compositions may be sterile, pyrogen-free, or both sterile and pyrogen-free. General principles for the formulation and/or manufacture of pharmaceutical agents such as vaccine compositions can be found, for example, in Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated here in its entirety by links). In some embodiments, RSV immunogenic compositions, including RSV vaccines, are administered to humans, human patients, or subjects.

The formulations of the RSV immunogenic compositions described herein can be prepared by any method known or developed in the future in the field of pharmacology. Typically, such preparations include the step of bringing the active ingredient (e.g., polypeptide or polynucleotide) into association with the excipient and/or one or more other auxiliary ingredients, and then, if necessary and/or if desired, separating, shaping and/or packaging the product. into the desired single or multi-dose unit.

The relative amounts of active ingredient, pharmaceutically acceptable excipient and/or any additional ingredients in a pharmaceutical composition in accordance with the present invention will vary depending on the identity, size and/or condition of the subject being treated and further depending on the route in which composition should be introduced. For example, the composition may contain from 0.1 to 100%, for example, from 0.5 to 50%, from 1 to 30%, from 5 to 80%, at least 80% (w/w) active ingredient .

Ways of administering vaccines

RSV immunogenic compositions, including RSV vaccines, can be administered by any route that results in a therapeutically effective result. These include, but are not limited to, intradermal, intramuscular, intranasal and/or subcutaneous administration. The present disclosure provides methods including administering the compositions to a subject in need thereof. The exact amount required will vary from subject to subject depending on the species, age and general health of the subject, the severity of the disease, the particular composition, its mode of administration, its mechanism of action, and the like. RSV immunogenic compositions are typically formulated in unit dosage form for ease of administration and dosage uniformity. It is understood, however, that the decision on the total daily use of the vaccine compositions will be made by the attending physician based on the results of a thorough clinical assessment. The particular therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the particular compound used; specific composition used; age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the particular compound used; duration of treatment; drugs used in combination or simultaneously with the specific compound used; and similar factors well known in medicine.

In some embodiments, RSV immunogenic compositions may be administered at doses sufficient to deliver 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005 mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 to 0.5 mg/kg, 0.01 to 50 mg/kg, 0.1 to 40 mg/kg, 0.5 up to 30 mg/kg, 0.01 mg/kg. up to 10 mg/kg, from 0.1 mg/kg to 10 mg/kg, or from 1 mg/kg to 25 mg/kg of the subject's body weight per day, one or more times a day, a week, a month, etc. to obtain the desired therapeutic, diagnostic, prophylactic or visual effect. In exemplary embodiments, RSV vaccine immunogenic compositions may be administered at doses sufficient to deliver from 0.0005 to 0.01 mg/kg, e.g., about 0.0005 to about 0.0075 mg/kg, e.g., about 0 .0005 mg/kg, about 0.001 mg/kg, about 0.002 mg/kg, about 0.003 mg/kg, about 0.004 mg/kg, or about 0.005 mg/kg.

In some embodiments, RSV immunogenic compositions may be administered one or two (or more) times at doses sufficient to deliver 0.025 mg/kg to 0.250 mg/kg, 0.025 mg/kg to 0.500 mg/kg, 0.025 mg/kg up to 0.750 mg/kg or from 0.025 to 1.0 mg/kg.

The immunogenic RSV pharmaceutical composition described herein may be formulated into a dosage form as described herein, such as intranasal, intratracheal, or injection dosage form (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, intranasal, and subcutaneous introductions).

The present invention is not limited in its application to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and may be practiced or carried out in various ways. In addition, the phraseology and terminology used in this document is for the purposes of description and should not be construed as limiting. The use of "comprising," "comprising," or "having," "comprising," "involving," and variations thereof in this document is intended to include the elements listed after and their equivalents, as well as additional elements.

Examples

Example 1: Preparation of single-strand mutants containing F1 and F2 fragments connected by a peptide linker

The wild-type RSV F polypeptide is cleaved into F1 and F2 fragments by the host furin protease after translation. To evaluate RSV single-strand mutants that are not cleaved by furin and that remain as a single polypeptide, WT RSV amino acids 98-146 (where these amino acids include furin cleavage sites, the p27 peptide, and part of the fusion peptide) were replaced with a flexible amino acid linker of various lengths (8-14 amino acids) against the background of stabilizing pre-fusion mutations in DS-Cav1 (S155C, S190F, V207L and S290C) (McLellan et al. 2013). In these mutants, the T4 fabritin trimerization domain (foldon) was fused to the C-terminus of the F RSV ectodomain, followed by a protease cleavage site and a purification label. Mutant F RSV sequences were codon-optimized for mammalian codon usage, cloned into an expression vector, and transiently transfected into Expi293 cell suspension culture (Life Technologies). Cell culture supernatants were harvested 3-7 days after plasmid transfection to evaluate the binding of these mutants to various anti-F RSV antibodies. Briefly, Ni-NTA coated 96-well plates (Thermo Scientific) were coated with cell culture supernatants for 1 hour at room temperature. Unbound sites were blocked by the addition of 2% (v/v) bovine serum albumin (BSA) in PBS and incubation for 1 h at room temperature. Plates were washed with PBS containing 0.05% (v/v) Tween™ 20 (polysorbate 20) (PBS-T) and incubated with serial dilutions of antibodies (D25 or Synagis® (palivizumab)) at room temperature for 1 h. The plates were washed again with PBS-T and incubated for 1 h at room temperature with a secondary goat anti-human IgG antibody conjugated to HRG (Thermo Fisher) at a dilution of 1:2000. After an additional wash with PBS-T and a quick rinse with ddH ₂ O, Super AquaBlue ELISA substrate (eBiosience) was added and the plate was immediately analyzed on a reader at 405 nm for 5 min. mOD/min was calculated for each well.

In FIG. 1A and 1B show binding of freshly harvested cell culture supernatants after transfection on day 7 to construct 40a (DS-Cav1 with linker from 8 aa) (Fig. 1A) or supernatant samples that were stored at 4°C for 8 days (Fig. 1B ), with monoclonal antibodies D25 and Synagis® (palivizumab). The LZF40a mutant showed low specific binding to the pre-fusion form specific D25 mAb. Binding to palivizumab (which reacts with both pre-fusion F and post-fusion F forms) was high, indicating that the mutant was well expressed. In FIG. 2A and 2B show the binding of freshly harvested cell culture supernatants to LZF55a, LZF56a, and LZF57a at day 3 post-transfection (DS-Cav1 with a 10, 12, or 14 amino acid linker, respectively; FIGS. 2A, 2B, 2C, respectively) or samples stored at 4°C for 7 days (FIGS. 2D, 2E and 2F respectively), with D25 and palivizumab. All three mutants expressed well based on reactivity with palivizumab. Among these mutants, LZF57a (DS-Cav1 with a 14 amino acid linker) showed the highest pre-fusion form-specific binding to mAb D25 and retained binding to D25 after 7 days of storage at 4°C.

In addition to ELISA, cell culture supernatants collected at various time points post-transfection were analyzed by Western blotting with anti-F RSV antisera. Supernatants were treated with reductant SDS loading buffer (Life Technologies), loaded onto an electrophoresis gel and then electrotransferred to nitrocellulose membranes (Life Technologies). The membranes were blocked overnight at 4° C. with blotting blocker (BioRad) in 1X TBST (Tris-buffered saline + Tween). The membranes were incubated with polyclonal guinea pig sera against wild-type RSV F ectodomain protein (Sino Biological), and then with AP-conjugated secondary goat anti-guinea pig IgG (Santa Cruz Biotechnology). Single-stranded RSV mutants LZF55a, LZF56a and LZF57a showed significantly higher levels of expression compared to the original DS-Cav1 (figure 3). DS-Cav1 appeared on the gel as two bands: the upper band represents the uncleaved F protein and the lower band represents the F1 fragment cleaved with furin. RSV single strand mutants LZF55, LZF56, and LZF57 appeared as a single predominant band on the gel representing uncleaved F.

Example 2: additional stabilizing mutations

The monoclonal antibody 4D7, which reacts with the antigenic site I on the F protein in the post-fusion RSV conformation, can be used to assess the stability of the pre-fusion F structure (Flynn et al. 2016). Freshly harvested cell culture supernatants with DS-Cav1 or LZF57a constructs were evaluated by ELISA for binding to D25 and 4D7 (Fig. 4C). Although the LZF57a construct retained the ability to bind to D25, indicating its pre-fusion conformation, it appears to show increased binding to 4D7 compared to the original DS-Cav1 construct, indicating that when the introduction of a single-stranded linker may result in subtle conformational changes. To further stabilize the RSV LZF57a single strand mutant construct, structure-based design was performed to generate variants with disulfide bond mutations; cavity filling mutations; and/or "post-fusion" conformation destabilization mutations. In particular, the D486C/D489C disulfide mutant model, based on the pre-fusion F protein crystal structure, suggests that an intermolecular disulfide bond within 3.8 Å may help further stabilize the pre-fusion conformation (data not shown). Selected mutations were combined with LZF57a mutations and evaluated using pre-fusion form-specific mAbs D25 and 4D7 as described above. The LZF 111a mutant (which corresponds to LZF57a with the D486C/D489C mutation) showed reduced binding to 4D7 compared to the LZF57a construct, suggesting that it may adopt a greater pre-fusion conformation (FIGS. 4B, 4C and 4D). In FIG. 10 is a diagram of constructs LZF111 and LZF57.

To further characterize the LZF111 mutant, RSV F proteins (DS-Cav1 and LZF111a) were purified from culture supernatants using a modified method described previously (McLellan 2013). Briefly, His-tagged proteins were purified using Ni-sepharose chromatography (GE Healthcare). Labels were removed by overnight digestion with thrombin. Cleavage was performed during dialysis to reduce the concentration of imidazole. To remove coeluting impurities and uncleaved F-protein, the samples were subjected to a second stage chromatography on Ni-Sepharose. F proteins were further purified by size exclusion chromatography (Superdex 200, GE Healthcare) and stored in 50 mM HEPES, pH 7.5, 300 mM NaCl buffer. SDS-PAGE analysis was performed under reducing and non-reducing conditions to assess the formation of disulfide bonds (FIG. 4D). Briefly, purified protein samples were treated with SDS loading buffer with or without reductant (Life Technologies), heated at 95° C. for 2-3 min, and loaded onto NuPAGE electrophoresis gel (Invitrogen). The gels were stained with Gel Code Blue (Pierce) staining solution and the excess stain was washed off with water. Under reducing conditions, DS-Cav1 appeared as two bands on the gel representing F1 and F2 cleavages, as expected. Under non-reducing conditions, DS-Cav1 appeared primarily as a band corresponding to about 50 kDa, representing one cleaved F1 fragment and one F2 fragment held together by native disulfide bonds. As expected, the RSV single strand mutant, LZF57, appeared on the gel primarily as a band corresponding to the uncleaved F0 monomer under any electrophoresis condition. The change in mobility of LZF57 under reducing and non-reducing conditions is most likely the result of loss of compactness under reducing conditions, resulting in an increase in apparent molecular weight. The RSV single strand mutant, LZF111, also appeared primarily as a band of uncleaved F0 monomer on the gel under reducing conditions. Under non-reducing conditions, although there were two subdominant lower bands corresponding to 50 kDa and 100 kDa, consistent with the monomeric and dimeric forms, the dominant LZF111 band shifted upwards to about 150 kDa, consistent with the molecular weight of the trimer. Since the D486C/D489C mutations were the only difference between the LZF57 and LZF111 constructs, these data indicate that the engineered intermolecular disulfide bond is indeed formed in most LZF111 molecules.

Example 3: LZF111 Stability Studies

To analyze the secondary structures of the purified RSV F proteins, circular dichroism ("CD") spectra were obtained on a Chirascan spectrometer (Applied Photophysics Ltd, UK). Samples were analyzed after buffer exchange with 10 mM Na ₂ HPO ₄ using Zeba spin columns (Pierce) and subsequent 1:2 dilution in 10 mM Na ₂ HPO ₄ resulting in a final protein concentration of 3.9 μM and 4.4 μM for DsCav-1 and LZF111 respectively. CD spectra were recorded undiluted using a quartz cuvette with a path length of 0.5 mm. The temperature control was set to 20°C. The bandwidth was set to 1 nm. Data points between 185 and 260 nm were obtained at 1 nm intervals with a sampling time of 5 s per time point. Spectra of sample and buffer were obtained after 10 min of temperature equilibration using three technical repetitions, respectively. The average spectra of the buffer were subtracted from the spectra of the samples. The resulting data points were smoothed using the Savitzky-Golay algorithm (polynomial order 2, two data points left and right) using the Origin Pro 7.5 SR7 software package (Origin Lab Corporation). The CD spectra of DS-Cav1 and LZF 111 were almost identical (FIGS. 5A and 5B). Secondary structures were further analyzed by reconstruction of CD spectra (from 185 to 260 nm) using a trained artificial neural network for protein CD spectra with 3D structure resolved (CDNN software: Circular Dichroism Neural Network) (Fig. 6), indicating that that the modification of LZF111 did not lead to significant changes in the secondary structures of the F-protein in the “pre-fusion” conformation.

Differential scanning fluorimetric analysis (DSF) was performed with DS-Cav1 and LZF111 proteins to assess their stability. Solutions of DS-Cav1 protein (0.27-35 μM) in 50 mM HEPES, 300 mM NaCl buffer at pH 7.5 were prepared by serial dilution. The fluorescence signal of each protein sample in a volume of 85 µl in a microquartz cuvette with an optical path length of 3×3 mm (Thermo Fisher) was recorded using a Cary Eclipse fluorometer equipped with a Cary temperature controller (Agilent Technologies, CA). The intrinsic fluorescence of the protein was recorded at 330 nm and 350 nm. The excitation wavelength was set to 280 nm with a slit width of 10 nm. The emission slit width was set to 2.5 nm. The photomultiplier voltage was adjusted before each measurement to values between 500V and 800V to maximize the fluorescence signal. Experiments on thermal unfolding of proteins were performed using a temperature change of 1°C/min from 20°C to 95°C in steps of 0.5°C. The sample was equilibrated at initial temperature for 1 min and the fluorescence signals were averaged for each data point over 1.5 s. The multi-cell holder made it possible to analyze up to 4 samples simultaneously. The raw data was exported for further processing with Origin Pro®7.5 SR7 to obtain melt curves of fluorescence intensity as a function of temperature. Melting curves were smoothed (polynomial order=1, number of points=12) and the peak centers of the first derivative with a ratio between 350 nm and 330 nm were used as melting points (Tm). The data was normalized to the highest signal intensity to compare different protein samples or protein concentrations. DS-Cav1 has two transition midpoints (60.85±1.98°C and 80.7±0.93°C) which are presented as the mean value obtained from all protein concentrations analyzed for the same type sample (Flynn et al. 2016) (Fig. 7). For LZF111, the reported Tm (~81° C.) was obtained from a single concentration of 15 μM (FIG. 7). The absence of a lower Tm at ~60.85°C and a narrower peak for a higher Tm at ~81°C indicates that LZF111 has improved stability compared to DS-Cav1.

Long-term stability at or above 4°C is a desirable feature for a subunit vaccine antigen. To evaluate the long-term stability of DS-Cav1, binding assays with D25 and 4D7 antibodies, as well as biophysical assays, have previously been used. The obtained data showed that during long-term storage at 4°C, DS-Cav1 undergoes conformational changes, adopting alternative structures that acquire the ability to bind to 4D7 (Flynn et al. 2016). To assess the long-term stability of LZF111, the purified DS-Cav1 protein or LZF111 construct was stored frozen or at 4°C for 1, 2 or 3 months and evaluated using D25, 4D7 and palivizumab in an ELISA binding assay. Briefly, the purified proteins were diluted to 1 μg/ml in PBS and loaded into a 96-well ELISA plate (NUNC) overnight at 4°C. Unbound sites were blocked by the addition of 2% (v/v) bovine serum albumin (BSA) in PBS and incubation for 1 h at room temperature. Plates were washed with PBS containing 0.05% (v/v) Tween® 20 (polysorbate 20) (PBS-T) and incubated with serial dilutions of antibodies (D25 or palivizumab) at room temperature for 1 hour. Plates again washed with PBS-T and incubated for 1 h at room temperature with secondary goat anti-human IgG (for D25 and palivizumab) or secondary anti-mouse IgG HRP conjugated (Thermo Fisher) at a dilution of 1:2000. After an additional wash with PBS-T and a quick rinse with ddH ₂ O, Super AquaBlue ELISA substrate (eBiosience) was added and the plate was immediately analyzed on a reader at 405 nm for 5 min. mOD/min was calculated for each well. In FIG. 8A, 8B and 8C show that after 1 month storage at 4°C, while D25 and palivizumab reactivity was maintained for both constructs, a significant increase in 4D7 binding to DS-Cav1 was found, but not to improved LZF111. In addition, no increased binding of 4D7 to LZF111 was observed after storage for 2 and 3 months at 4°C (FIGS. 8D and 8E), indicating that the long-term stability of LZF111 is superior to that of DS-Cav1.

Example 4 Immunogenicity Test

An immunogenicity study in mice was performed to compare the immunogenicity of the DS-Cav1 and LZF111 subunit vaccines. The test animals were female BALB/c mice obtained from Charles River Laboratories. 10 mice per group were immunized twice with three different doses (2 μg, 0.4 μg and 0.2 μg) of purified DS-Cav1 or LZF 111a proteins with aluminum-based adjuvants at week 0 and 3. Blood samples were taken 2 weeks after each immunizations and sera were analyzed. To determine the titers of antibodies binding to the F-protein in the pre-fusion conformation, immulon 12HB (NUNC) microtiter plates were coated at 2 μg/ml with purified recombinant F protein RSV DS-Cav1 and incubated at 4°C overnight. The plates were then washed and blocked for 1 h with PBS-T containing 3% skimmed milk (blocking buffer) at room temperature. 4-fold serial dilutions of test samples were prepared in blocking buffer (starting at a dilution of 1:50), transferred to F RSV-coated plates, and incubated for 2 hours at room temperature. After three washes with PBS-T, anti-mouse IgG secondary antibody conjugated to HRP (Invitrogen) at a 1:3000 dilution in blocking buffer was added to the plates and incubated for another 1 h. The plates were washed again and stained with SuperBlu Turbo TMB (Virolabs) In the dark. The reaction was stopped after 5 min and the absorbance was measured at 450 nm on a VersaMax ELISA plate reader (Molecular Devices). ED10 titers in the ELISA, which indicated an effective dilution of the serum sample giving 10% of the maximum signal, were determined by four parameter curve fitting using GraphPad Prism 7 software. FIG. 9A shows pre-confluence ED10 ELISA titers for F-protein after dose 2. The lower horizontal dotted line indicates the limit of detection. The data obtained showed that LZF111 induced similar levels of pre-fusion anti-F protein antibodies compared to DS-Cav1 at different doses.

Neutralization assays were also performed on mouse serum that had been treated at 56° C. for 30 minutes to inactivate complement prior to testing. Prepared two-fold serial dilutions of serum samples in EMEM containing 2% FBS, starting with a dilution of 1:4. The diluted serum was added in duplicate to 96-well plates and mixed with the RSV Long strain (100 pfu/ml) in a total volume of 100 μl. A mixture of virus and serum samples was incubated for 1 h at 37°C in an atmosphere with 5% CO ₂ . After incubation, Hep-2 cells were added at a concentration of 1.5×10 ⁴ cells per well. The plates were incubated for 3 days at 37°C with 5% CO ₂ . The cells were then washed and fixed with 80% acetone for 15 min. The cells infected with RSV were then immunostained. Briefly, F RSV- and N-specific monoclonal antibodies were added to the fixed cell test plates and incubated for 1 hour at room temperature. After washing, biotinylated goat anti-mouse IgG antibodies were added and incubated for 1 hour. The plates were washed again and developed using a dual-channel near infrared (NID) detection system. Infrared dye-streptavidin for detection of RSV-specific signal and two cell dyes for normalization of the analysis were added to 96-well plates and incubated for 1 h in the dark. The plates were washed, dried in the dark for 20 min, and analyzed on a Licor Aerius1 automated imaging system using a 700 channel laser to normalize cells and an 800 channel laser to detect RSV-specific signal. Ratios of 800/700 were calculated and serum neutralizing antibody titers (IC50) were determined using a four-parameter curve fitting with GraphPad Prism 7 software. Serum PD2 neutralization titers of serum against RSV Long strain are shown in FIG. 9B. The lower horizontal dotted line indicates the detection limit. The data showed that LZF111 induced similar levels of neutralizing antibodies with DS-Cav1 at different doses.

Example 5: Immunogenicity study in cotton rats

In this example, experiments were performed to test the immunogenicity and efficacy of mRNA/LNP vaccines in an RSV-infected cotton rat model. More specifically, female Sigmodon hispidus cotton rats were used and immunization started at 6-7 weeks of age. Used mRNA vaccines were prepared and formulated as lipid nanoparticles. The mRNA vaccines tested in this study included:

MRK-04 membrane-bound DS-Cav1 (pre-fusion stabilized F-protein)

MRK-04_nopolyA_3mut membrane-bound DS-Cav1 (pre-fusion stabilized F-protein)

mVRC-1 (v2) membrane-bound single-chain sc9 mDS-Cav1, A149C, Y458C (pre-fusion stabilized F-protein)

MLZF-111 membrane bound single chain mDS-Cav1, D486C, D489C (pre-fusion stabilized F protein)

Groups of 8 cotton rats were immunized intramuscularly with 100 μl of the vaccine, injecting 50 μl into each quadriceps muscle of the thigh. Groups were vaccinated with the following vaccines:

Group Vaccine Concentration (µg/ml) Dose (mcg) 1 None not applicable not applicable 2 MRK-04, in / m 250 25 3 MRK-04_nopolyA_3mut, w/m 250 25 4 MRK-04_nopolyA_3mut, w/m 50 5 5 MRK-04_nopolyA_3mut, w/m 10 1 6 mVRC-1 (v2), i/m 250 25 7 mVRC-1 (v2), i/m 50 5 8 mVRC-1 (v2), i/m 10 1 9 mLZF-111, w/m 250 25 10 mLZF-111, w/m 50 5 eleven mLZF-111, w/m 10 1

Animals were immunized on day 0 and day 28 of the experiment. On days 28 and 56, blood samples were taken from each animal and used for serological analyses. On day 56 cotton rats were injected intranasally with 1×10 ^5.5 PFU RSV A2. Four days after inoculation, the animals were euthanized by CO ₂ inhalation and the lungs (left lobes) and turbinates were removed and homogenized in 10 volumes of Hank's balanced salt solution (Lonza) containing SPG on wet ice. Samples were clarified by centrifugation at 2000 rpm for 10 min, aliquoted, flash frozen and immediately stored frozen at -70°C.

RSV Neutralization Assay:

Cotton rat sera from each animal were assessed for neutralization of RSV-A (Long strain) using the following procedures:

1. All serum samples were heat inactivated by placing them in a dry thermostatic bath set at 56°C for 30 min. The test and control sera were then diluted 1:3 with virus diluent (2% FBS in EMEM) and duplicate samples were added to the assay plate and serial dilutions were made.

2. Stock solution of RSV-Long virus was removed from the freezer and quickly thawed in a water bath at 37°C. Viruses were diluted to 2000 pfu/ml with virus diluent.

3. 50 µl of diluted virus was added to each well of a 96-well plate, except for one row of cells, which was used as a "no virus" control.

4. HEp-2 cells were trypsinized, washed, resuspended at 1.5×10 ⁵ cells/ml with virus diluent, and 100 ml of the suspended cells were added to each well of a 96-well plate. Then the tablets were incubated for 72 h at 37°C, in an atmosphere with 5% CO ₂ .

5. After 72 hours of incubation, cells were washed with PBS and fixed using 80% acetone dissolved in PBS for 10-20 minutes at 16-24°C. The fixative was removed and the plates were air dried.

6. The plates were then thoroughly washed with PBS + 0.05% Tween. Detection monoclonal antibodies, 143-F3-1B8 and 34C9, were diluted to a concentration of 2.5 μg/ml with assay diluent (1% BSA-PBS-0.1% Tween) and 50 μl of the diluted antibodies were added to each well of 96 plates . Then the plates were incubated in a humid chamber at 16-24°C for 60-75 min on a laboratory shaker.

7. After incubation, the plates were thoroughly washed.

8. Biotinylated horse anti-mouse IgG antibodies were diluted 1:200 with assay diluent and added to each well of a 96-well plate. The plates were incubated as above and washed.

9. A mixture of streptavidin IRDye 800CW (1:1000 final dilution), Sapphire 700 (1:1000 dilution) and 5 mM DRAQ5 solution (1:10000 dilution) was prepared in assay diluent and 50 ml of the mixture was added to each well of a 96-well tablet. The plates were incubated as described above in the dark, washed and allowed to air dry.

10. The plates were then analyzed using the Aerius Imager. Serum neutralizing antibody titers were then calculated using a 4-parameter curve using Graphpad Prism.

The titers measured after dose 1 (day 28) and after dose 2 (day 56) are shown in FIG. 11. Neutralizing antibody titers were found to be dose dependent for all mRNA vaccines. All mRNA vaccines resulted in increased titers after the second dose, regardless of the dose tested. Both mVRC-1 (v2) and mLZF111 induced higher titers than MRK-04 and MRK-04_nopolyA_3mut, showing high or similar serum neutralizing antibody titers at 5-fold lower dose.

alphaLISA competitive analysis

The immune response to specific epitopes of the RSV F protein for neutralizing antibodies was characterized. Antigenic site II is the binding site for palivizumab, a monoclonal antibody designed to prevent RSV lower respiratory tract infection in infants and young children at increased risk of infection. The Ø antigenic site is the binding site for the stronger neutralizing antibodies that are induced by natural RSV infection. In addition, an antibody (4D7) was created that targets the site I epitope of the antibody (4D7) absent in the pre-fusion conformation. Therefore, in contrast to D25, the detection of 4D7-competitive antibodies may indicate the generation of F-like proteins in the post-fusion conformation in vivo. The alphaLISA competitive assay was developed to characterize the response of antigenic site Ø, antigenic site I, and antigenic site II to various mRNA-based vaccines.

To measure competitive antibody titers, 10 µl of samples serially diluted in HiBlock buffer (PerkinElmer) were placed in a 384-well alphaLISA plate. The diluted samples were mixed with 5 µl of AlphaLISA acceptor beads (100 µg/ml) that had previously been conjugated with stabilized F RSV protein in pre-fusion conformation (DS-Cav1) or F RSV protein in post-fusion conformation (F RSV wt ). After a 30 min incubation at room temperature, 10 µl of biotinylated D25 antibody, palivizumab, or 4D7 diluted in HiBlock buffer was added to each well. After an additional 30 min incubation, 25 µl of donor beads coated with streptavidin (20 µg/ml) in HiBlock buffer were added to each well and incubated for 30 min in the dark. The plate was then analyzed for EnVision_Alpha (detection at 615 nm).

Palivizumab competitive antibody titers, D25 and 4D7, measured on day 56 (4 weeks PD2) are shown in FIG. 12. Competitive analysis data revealed that mVRC-1(v2) induced lower levels of competitive 4D7 antibodies for the post-fusion F protein, while D25 pre-fusion and palivizumab titers were unchanged. This different competition profile correlates with mVRC-1 (v2) mRNA expressing a more stable pre-fusion protein than MRK-04_nopolyA_3mut and mLZF-111.

C. Results of infestation of cotton rats

Procedures for measuring RSV titers in lung and nasal homogenates from cotton rats are described below. Lung and nasal homogenates were clarified by centrifugation and diluted 1:10 and 1:100 in EMEM. Confluent HEp-2 monolayers were infected in duplicate at 50 μl per well, starting with undiluted (pure) samples and then diluted homogenates in 24-well plates. After incubation for one hour at 37°C in a 5% CO ₂ incubator, the wells were covered with 0.75% methylcellulose medium and the plates were incubated at 37°C. After 4 days of incubation, the cover layer was removed and the cells were fixed with 0.1% crystal violet dye for one hour, then washed and air dried. The number of plaques was counted and virus titers were expressed in plaque-forming units per gram of tissue.

To assess vaccine-mediated protection, viral titers were measured in the lungs and nasal cavity 5 days after infection. All mRNA vaccines provided complete lung protection, and mVRC-1 (v2) and mLZF111 showed improved nasal protection showing high or similar efficacy to MRK-04 and MRK-04_nopolyA_3mut at a 5-fold lower dose (Fig. 13) .

Example 6: Evaluation of Immunogenicity and Efficacy in African Green Monkeys

In this example, assays were performed to evaluate the immunogenicity and efficacy of mRNA/LNP vaccines in an African green monkey RSV challenge model.

More specifically, adult male and female African green monkeys weighing between 1.6 and 2.65 kg were used, which were confirmed by neutralizing antibody titer to be RSV-negative. Used mRNA vaccines were prepared and formulated as lipid nanoparticles. The mRNA vaccines tested in this study included:

MRK-04_nopolyA_3mut membrane-bound DS-Cav1 (pre-fusion stabilized F-protein)

mVRC-1 (v2) membrane-bound single-chain sc9 mDS-Cav1, A149C, Y458C (pre-fusion stabilized F-protein)

MLZF-111 membrane bound single chain mDS-Cav1, D486C, D489C (pre-fusion stabilized F protein)

Groups of 4 African green monkeys were immunized intramuscularly with 500 μl of the vaccine in one quadriceps femoris muscle. The groups were vaccinated with the following vaccines as shown in Table 1.

Table 1
Vaccine formulations tested for immunogenicity in African green monkeys Group Vaccine Concentration mcg/ml) Dose (mcg) 1 MRK-04_nopolyA_3mut, w/m 50 25 2 MRK-04_nopolyA_3mut, w/m 10 5 3 mVRC-1 (v2) 50 25 4 mVRC-1 (v2) 10 5 5 mLZF-111 50 25 6 mLZF-111 10 5 7 RSV A2 5.5log10pfu, w/m not applicable not applicable 8 Without a vaccine not applicable not applicable

Animals were immunized on day 0, day 28 and day 56 of the experiment. On days 0, 14, 28, 42, 56 and 70, blood samples were taken from each animal and used for serological analyses. Per day, 70 African green monkeys were intranasally injected with 1×10 ^5.5 PFU RSV A2. Nasopharyngeal swabs were collected on days 1-14 post infection, and bronchoalveolar lavage samples were collected on days 3, 5, 7, 9, 12 and 14 post infection to test for viral replication.

A. RSV Neutralization Assay:

Monkey sera from each animal were assessed for neutralization of RSV-A (Long strain) as described above. The NT ₅₀ titers determined after dose 1 and after dose 2 are shown in FIG. 14. It can be seen that the titers increased after each dose in all groups receiving mRNA vaccines. The GMTs obtained with the mRNA vaccines at week 10 (2 weeks post-dose 3) were 1-2 orders of magnitude higher than RSV A2-treated animals, depending on the dose and mRNA tested. Serum samples from animals immunized with mVRC-1 (v2) showed the highest neutralization titers, showing 5-fold higher activity compared to MRK-04_nopolyA_3mut.

B. Competitive ELISA

Competitive ELISA titers were determined for palivizumab, D25 and 4D7 to characterize the response of antigenic site Ø, antigenic site I and antigenic site II to various mRNA-based vaccines as described above.

Palivizumab competitive antibody titers, D25 and 4D7, measured at week 10 (2 weeks PD3) are shown in FIG. Competitive analysis data revealed that mVRC-1(v2) induced lower levels of competitive 4D7 antibodies to the post-fusion F-protein, while pre-fusion D25 protein titers and palivizumab titers did not change. This different competition profile correlates with mVRC-1 (v2) mRNA expressing a more stabilized pre-fusion protein than MRK-04_nopolyA_3mut and mLZF-111.

C. African green monkey infection outcomes

As described above, African green monkeys were intranasally injected with 1 x 10 ^5.5 PFU of RSV A2 on day 70 post-vaccination to evaluate vaccine efficacy, and nasopharyngeal swabs and bronchoalveolar lavage were collected post-challenge to determine the presence of virus.

To measure RSV titers in bronchoalveolar lavage samples from African green monkeys, a viral plaque assay was used to measure virus titers as described below. Briefly, samples were diluted and plated in duplicate in 24-well plates containing confluent monolayers of HEp-2 cells. The plates were incubated at 37°C for one hour. After incubation for one hour, the sample inoculum was aspirated and 1 ml of a cover layer containing 0.75% methylcellulose was added. The plates were incubated at 37°C for 5 days. After a 5 day incubation, the cells were fixed and stained with a crystal violet/glutaraldehyde solution. The number of plaques was counted and the titers were expressed in PFU/ml. Analysis of the virus content in bronchoalveolar lavage fluid (BAL) (FIGS. 16A-16C) showed that only mVRC-1 (v2) (25 μg) provided complete protection in the lungs and provided better protection at a lower dose (5 μg).

To measure RSV titers in African green monkey nasopharyngeal swabs, RSV analysis was performed using RT-qPCR to detect RSV A as follows:

1) Equipment and materials:

A. Equipment

1. Stratagene Mx3005P real-time PCR system and MxPro software

2. Centrifuge Jouan GR422 or equivalent

3. Jouan Plate media or equivalent

B. Reagents

1. Quantitect® Probe Rt-PCR kit (1000), part number 204445

2. Molecular Biology DNase and Protease Free Ultrapure Water, 5 Prime, P/N 2900136

3. Buffer TE, 10 mM Tris, 1 mM EDTA, pH 8.0, Fisher Bioreagents, catalog number BP2473-100

4. Viral primers: forward and reverse primers RSV A, Sigma custom, purified by HPLC. Primer stocks were reconstituted to 100 µM with ultrapure water and stored at -20°C.

5. RSV dual label probe, Sigma custom, HPLC cleaned. Probe stocks were reconstituted to 100 μM with TE buffer and stored at -20°C protected from light.

6. Standard RSV received internally and stored at -20°C. Standards for analysis were generated by designing primer pairs for the RSV A N gene. The product length for the RSV A standard is 885 bp. QIAGEN OneStep RT-PCR was used to generate this standard.

Primers Sequences RSV A F N gene 5' CTC AAT TTC CTC ACT TCT CCA GTG T (SEQ ID NO: 46) RSV A R N gene 5' CTT GAT TCC TCG GTG TAC CTC TGT (SEQ ID NO: 47) RSV A FAM N gene 5'FAM-TCC CAT TAT GCC TAG GCC AGC AGC A (BHQ1) (SEQ ID NO: 48)

7. Promega Total Viral Nucleic Acid Purification Kit, Maxwell® 16 (Part # AS1150)

C. Supplies

1. Stratagene Optical Caps for Reaction Tubes in strips of 8, p/n 401425

2. Stratagene Mx3000P 96-well rimmed plates, p/n 401334

3. Tips with ART filter

2) RT-PCR reactions and setup

A. Preparation of the Complete Master Mix for PCR

1. Prepare the complete master mix following the instructions below for a final reaction volume of 50 µl. The following table shows the volume per well. The final primer concentration is 300 nM and the final probe concentration is 200 nM.

Reagent µl 2X Master Mix 25 RSV A F 100 µM 0.2 RSV A R 100 µM 0.2 RSV A FAM 100 µM 0.1 enzyme blend for OT 0.5 water 19

2. Add 45 µl of the complete master mix to each well. Cover the plate with a lid and wrap with aluminum foil to protect from light.

B. Building a Standard Curve

1. The standard is removed from the freezer at -20°C.

2. Dilute the standards to final concentrations from 1ed copies/5 µl to 1 copy/5 µl using 10-fold dilutions.

C. Sample preparation

1. Nasopharyngeal and bronchoalveolar lavage swab specimens are prepared for RT-PCR reaction using the Maxwell® 16 Total Viral Nucleic Acid Purification Kit (Promega, item number AS1150).

2. 200 µl of sample is removed according to the manufacturer's protocol and eluted in 50 µl for use in PCR reactions.

D. Sample additions

1. Add 5 µl of selected samples to the appropriate wells. After adding the samples, the sample wells are carefully capped before adding the standards to plot the curves.

2. Add 5 µl of the diluted standard to the appropriate wells and cover with a lid.

3. Add 5 µl of ultrapure water to the wells of the control without matrix (NTC).

4. Wrap the plates in aluminum foil and transfer the plates to a centrifuge.

5. The plates are centrifuged for 2 minutes at 100 rpm to completely pellet the samples or master mix, including debris that may be on the sides of the well.

6. The plates are wrapped in aluminum foil and transferred to the Stratagene instrument.

E. thermal cycler: Stratagene MX 3005P

1. The plates are placed in the Stratagene Mx3005P and the thermal conditions profile is set:

Stage Time Temperature reverse transcription 30 min 50 Initial PCR activation step 15 minutes 95 2 stage cycle: Denaturation 16 s 94 Combined annealing/elongation 60 s 62 Number of cycles 40

2. Analysis of results using Stratagene Mx3005p software.

The average number of RNA copies found in nasal samples is shown in FIG. 17A-17C. The protective effect of all mRNA-based vaccines was less pronounced in the nasal cavity, but again mVRC-1(v2) showed a 5-fold higher efficacy compared to MRK-04_nopolyA_3mut.

Example 6: Immunogenicity in African green monkeys infected with RSV

The immunogenicity of mRNA vaccines formulated in LNP was tested in African green monkeys infected with RSV.

Healthy adult African green monkeys of both sexes (n=4 or 5 per group) weighing between 2.85 and 4.65 kg were selected for the study and were confirmed by ELISA and neutralizing antibody titers to be seropositive for RSV. The pool of animals selected for this study were experimentally challenged with RSV in previous experiments and assigned to treatment groups based on their RSV neutralization titers prior to the present study so that all groups had similar GMT values at baseline. Experimental animals challenged with RSV provide a model of immune memory responses to vaccination that may reflect the responses expected in seropositive adult humans, with clarification that the antibody response in AGM after exposure to RSV is more biased towards "pre-fusion" F-protein epitopes than in immune responses. human repertoire.

A single dose of the vaccine was administered to each animal at week 0 intramuscularly (IV). The study design also included a control group receiving only PBS. The vaccines were administered as shown in Table 2. After vaccination, the animals were observed daily for any changes in the site of inoculation or other changes in activity or feeding habits that might indicate an adverse reaction to the vaccine, but nothing was noted. Serum samples were collected to determine RSV neutralizing antibody titers as well as palivizumab (Site II), D25 (Site Ø) and 4D7 (Site I) competitive antibodies.

table 2
Vaccine formulations tested for immunogenicity in RSV-seropositive African green monkeys Group Vaccine Concentration mcg/ml) Dose mcg) 1 MRK-04_nopolyA_3mut, w/m 10 5 2 mVRC-1 (v2), i/m 10 5 3 mVRC-1 (v2), i/m 2 1 4 mLZF-111 10 5 5 mLZF-111 2 1 6 No Vaccine (PBS) not applicable not applicable

NT ₅₀ titers from individual animals were measured from serum samples collected at the start of the trial and 2 weeks post-vaccination using the methods described above, and the results are shown in FIG. 18. All vaccines were found to be highly immunogenic, as demonstrated by increased levels of serum antibodies binding F RSV proteins (on F RSV both "pre-fusion" and "post-fusion", data not shown) and increased levels of serum neutralizing antibodies. . mVRC-1 (v2) induced the highest increase in neutralizing antibody titers (>100-fold at the highest dose) and exhibited similar efficacy at a 5-fold lower dose compared to MRK-04_nopolyA_3mut. Similarly, mLZF-111 also showed increased activity compared to MRK-04_nopolyA_3mut. No increase in titers was observed in the PBS control group.

To evaluate the quality of booster responses in vaccinated animals, titers of competitive antibodies palivizumab (site II), D25 (site Ø) and 4D7 (site I) were determined in serum collected 2 weeks after vaccination (Fig. 19). As described above, the antigenic site II is a neutralization epitope found in both the pre-fusion and post-fusion conformations of the F protein, the Ø site is the pre-fusion specific neutralization epitope, and 4D7 is the epitope specific to the "after merge" form. Due to the initial immune bias to the post-fusion conformation, RSV-infected AGMs lack detectable competitive D25 antibody titers prior to immunization. However, all three antigen mRNAs induced high titers of competitive D25 antibodies, demonstrating that AGM does induce an immune response to the Ø antigenic site after RSV challenge, which can be enhanced by immunization. The rise in competitive D25 antibody titers following mVRC-1(v2) immunization was the highest (>450-fold) and again showed similar efficacy at a 5-fold lower dose than MRK-04_nopolyA_3mut. In contrast to naive animals (cotton rats and AGM), high growth of 4D7/F-specific antibodies to protein in post-fusion conformation was observed in RSM-infected AGMs in the mVRC-1(v2) group, demonstrating that the initial pool of antibodies can determine the profile of epitope-specific antibodies after immunization. Since the pool of memory B cells is thought to be strongly biased towards the pre-fusion conformation as a result of natural RSV infection in humans, it is hypothesized that immunization with mVRC-1(v2) in humans would enhance production of predominantly antibodies against these epitopes, which are known to , have a stronger neutralizing activity, which leads to an increase in efficiency compared to MRK-04_nopolyA_3mut.

Table 3
Amino acid linker sequence, TEV cleavage site, thrombin cleavage site, and Strep tag Name Subsequence SEQID NO: 8 amino acid linker GGGSGGGS 1 10 amino acid linker GGGSGGGSGG 2 12 amino acid linker GGGSGGGSGGGS 3 14 amino acid linker GGGSGGGSGGGSGS 4 Amino acid linker GGGS 49 TEV cleavage site ENLYFQS 5 Strep label WSHPQFEK 6 Thrombin cleavage site LVPRGS 7 Foldon SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL 8 Signal sequence MELLILKANAITTILTAVTFCFASG 9

Table 4
Antigenic polypeptide sequences Name Subsequence SEQID NO: Reference F RSV protein sequence MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLP IVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSC YGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN 10

The sequence of the F RSV WT reference protein with the foldon domain (highlighted in bold), replacing amino acids 514-574 F RSV WT GYIPEAPRDGQAYVRKDGEWVLLSTFL eleven F RSV WT ectodomain sequence (sequence contains a foldon in bold followed by a TEV cleavage site, strep tag, linker (underlined), strep tag, another linker (underlined) and His tag (in italics)) GYIPEAPRDGQAYVRKDGEWVLLSTFL ENLYFQSWSHPQFEK GGGSGGGSGGGS WSHPQFEK GSGSGS HHHHHHHH 12 DS-Cav1 peptide sequence (foldon sequence in bold) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQL LPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGA IVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL 13 LZF40:
the sequence contains an 8 amino acid linker (in bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F and V207L) and a C-terminal foldon (in bold) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREVNAG VTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVS VGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL 14 LZF 40(a):
sequence contains an 8 amino acid linker (bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F, and V207L), C-terminal foldon (bold), GG linker (underlined), thrombin cleavage site, His- tag, SA linker (underlined), and strep tag MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSF FPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL GGLVPRGSHHHHHHSAWSHPQFEK 15 LZF 55:
the sequence contains a 10 amino acid linker (in bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F and V207L) and a C-terminal foldon (in bold) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGG AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREV NAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGV DTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL 16 LZF 55a:
sequence contains a 10 amino acid linker (bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F, and V207L), C-terminal foldon (bold), GG linker (underlined), thrombin cleavage site, His- tag, SA linker (underlined), and strep tag MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGG AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSF FPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL GGLVPRGSHHHHHHSAWSHPQFEK 17 LZF 56:
the sequence contains a 12 amino acid linker (in bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F and V207L) and a C-terminal foldon (in bold) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGGGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFS VNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSVITSLGAIVSCYGKTKCTASNKNRGIVSIKTFSNGCDYNKG VDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL 18 LZF 56a:
sequence contains a 12 amino acid linker (bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F, and V207L), C-terminal foldon (bold), GG linker (underlined), thrombin cleavage site, His- tag, SA linker (underlined), and strep tag MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGGGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSF FPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL GGLVPRGSHHHHHHSAWSHPQFEK 19 LZF57:
the sequence contains a 14 amino acid linker (in bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F and V207L) and a C-terminal foldon (in bold) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGGGSGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITRE FSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNK GVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL 20 LZF 57a:
sequence contains a 14 amino acid linker (bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F, and V207L), C-terminal foldon (bold), GG linker (underlined), thrombin cleavage site, His- tag, SA linker (underlined), and strep tag MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGGGSGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSF FPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL GGLVPRGSHHHHHHSAWSHPQFEK 21 LZF 109:
the sequence contains a 14 amino acid linker (bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F and V207L), additional S180C and S186C mutations (bold, italic and underlined), and a C-terminal foldon (bold font) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGGGSGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVV C LSNGV C VLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRL LEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCD YVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL 22 LZF 109a:
the sequence contains a 14 amino acid linker (bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F and V207L), additional S180C and S186C mutations (bold, italic and underlined), and a C-terminal foldon (bold font), and GG linker (underlined), thrombin cleavage site, His tag, SA linker (underlined), and strep tag MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGGGSGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVV C LSNGV C VLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDI FNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL GGLVPRGSHHHHHHSAWSHPQFEK 23 LZF 110:
the sequence contains a 14 amino acid linker (bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F, and V207L), additional D486C and A490C mutations (bold, italic, and underlined), and a C-terminal foldon (bold font) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGGGSGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITRE FSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNK GVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPS C EFD C SISQVNEKINQSLAFIRKSDELL SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL 24 LZF 110a:
sequence contains a 14 amino acid linker (bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F and V207L), additional D486C and A490C mutations (bold, italic and underlined), C-terminal foldon (bold font), and GG linker (underlined), thrombin cleavage site, His tag, SA linker (underlined) and strep tag MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGGGSGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSF FPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPS C EFD C SISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL GGLVPRGSHHHHHHSAWSHPQFEK 25 LZF 111:
the sequence contains a 14 amino acid linker (bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F and V207L), additional D486C and D489C mutations (bold, italic and underlined) and a C-terminal foldon (bold font) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGGGSGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITRE FSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNK GVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPS C EF C ASISQVNEKINQSLAFIRKSDELL SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL 26 LZF 111a:
sequence contains a 14 amino acid linker (bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F and V207L), additional D486C and D489C mutations (bold, italic and underlined), C-terminal foldon (bold font), GG linker (underlined), thrombin cleavage site, His tag, SA linker (underlined), and strep tag MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGGGSGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSF FPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPS C EF C ASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL GGLVPRGSHHHHHHSAWSHPQFEK 27 LZF 112:
the sequence contains a 14 amino acid linker (bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F and V207L), additional L512C and L513C mutations (bold, italic and underlined) and a C-terminal foldon (bold font) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGGGSGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITRE FSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNK GVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDE CC SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL 28 LZF 112a:
sequence contains a 14 amino acid linker (bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F and V207L), additional L512C and L513C mutations (bold, italic and underlined), C-terminal foldon (bold font), and GG linker (underlined), thrombin cleavage site, His tag, SA linker (underlined) and strep tag MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGGGSGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSF FPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDE CC SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL GGLVPRGSHHHHHHSAWSHPQFEK 29 LZF 113:
the sequence contains a 14 amino acid linker (in bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F and V207L), an additional F505C mutation (in bold, italics and underline) and a c-terminal foldon (in bold) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGGGSGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITRE FSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNK GVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA C IRKSDELL SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL thirty LZF 113a:
the sequence contains a 14 amino acid linker (bold and underlined), DS-Cav1 substitutions (S155C, S290C, S190F and V207L), an additional F505C mutation (bold, italic and underlined), C-terminal foldon (bold) , GG linker (underlined), thrombin cleavage site, His tag, SA linker (underlined), and strep tag MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGGGSGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSF FPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA C IRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL GGLVPRGSHHHHHHSAWSHPQFEK 31 LZF 123:
sequence contains DS-Cav1 substitutions (S155C, S290C, S190F and V207L) and additional S180C and S186C substitutions (in bold, italics and underline) and C-terminal foldon (in bold) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVV C LSNGV C VLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDI FNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL 32 LZF 123a:
sequence contains DS-Cav1 substitutions (S155C, S290C, S190F and V207L) and additional S180C and S186C substitutions (in bold, italics and underline), C-terminal foldon (in bold), GG linker (underlined), thrombin cleavage site , His tag, SA linker (underlined) and strep tag MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVV C LSNGV C VLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDI FNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL GGLVPRGSHHHHHHSAWSHPQFEK 33 LZF124:
sequence contains DS-Cav1 substitutions (S155C, S290C, S190F and V207L) and additional substitutions D486C and A490C (bold, italic and underlined) and C-terminal foldon (bold) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQL LPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGA IVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPS C EFD C SISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL 34 LZF 124a:
sequence contains DS-Cav1 substitutions (S155C, S290C, S190F and V207L), additional D486C and A490C substitutions (bold, italic and underlined) and C-terminal foldon (bold), GG linker (underlined), thrombin cleavage site , His tag, SA linker (underlined) and strep tag MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQL LPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGA IVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPS C EFD C SISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL GGLVPRGSHHHHHHSAWSHPQFEK 35 LZF 125:
sequence contains DS-Cav1 substitutions (S155C, S290C, S190F and V207L) and additional substitutions D486C and D489C (in bold, italics and underline) and C-terminal foldon (in bold) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQL LPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGA IVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPS C EF C ASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL 36 LZF 125a:
sequence contains DS-Cav1 substitutions (S155C, S290C, S190F and V207L), additional D486C and D489C substitutions (bold, italic and underlined) and C-terminal foldon (bold), GG linker (underlined), thrombin cleavage site , His tag, SA linker (underlined) and strep tag MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQL LPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGA IVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPS C EF C ASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL GGLVPRGSHHHHHHSAWSHPQFEK 37 LZF 126:
sequence contains DS-Cav1 substitutions (S155C, S290C, S190F and V207L) and additional substitutions L512C and L513 (in bold, italics and underline) and c-terminal foldon (in bold) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQL LPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGA IVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDE CC SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL 38 LZF 126a:
sequence contains DS-Cav1 substitutions (S155C, S290C, S190F and V207L), additional substitutions L512C and L513C (bold, italic and underlined), C-terminal foldon (bold), GG linker (underlined), thrombin cleavage site , His tag, SA linker (underlined) and strep tag MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQL LPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGA IVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDE CC SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL GGLVPRGSHHHHHHSAWSHPQFEK 39 LZF 127:
the sequence contains the DS-Cav1 substitutions (S155C, S290C, S190F and V207L) and the additional F505C substitution (bold, italic and underlined) and C-terminal foldon (bold) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQL LPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGA IVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA C IRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL 40 LZF 127a:
sequence contains DS-Cav1 substitutions (S155C, S290C, S190F and V207L), F505C additional substitution (in bold, italics and underline), C-terminal foldon (in bold), linker (underlined), thrombin cleavage site, His- label, linker (underlined) and strep label MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQL LPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGA IVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA C IRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL GGLVPRGSHHHHHHSAWSHPQFEK 41 LZF 128:
the sequence contains the DS-Cav1 substitutions (S155C, S290C, S190F and V207L) and an additional deletion of the HRB domain corresponding to amino acids 482-513 and a C-terminal foldon (in bold) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQL LPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGA IVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPL SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL 42 LZF 128a:
the sequence contains the DS-Cav1 substitutions (S155C, S290C, S190F and V207L), an additional deletion of the HRB domain corresponding to amino acids 482-513, a C-terminal foldon (highlighted in bold), a linker (underlined), a thrombin cleavage site, a His-tag, linker (underlined) and strep tag MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQL LPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGA IVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL GGLVPRGSHHHHHHSAWSHPQFEK 43 LZF 129 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGGGSGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITRE FSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNK GVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPL SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL 44 LZF 129a MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM GGGSGGGSGGGSGS AIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITRE FSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNK GVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPL SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL GG LVPRGSHHHHHH SA WSHPQFEK 45

Primers Sequences RSV A F N gene 5’ CTC AAT TTC CTC ACT TCT CCA GTG T (SEQ ID NO: 46) RSV A R N gene 5’ CTT GAT TCC TCG GTG TAC CTC TGT (SEQ ID NO: 47) RSV A FAM N gene 5'FAM-TCC CAT TAT GCC TAG GCC AGC AGC A (BHQ1) (SEQ ID NO: 48)

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be covered by the following claims.

All references, including patent documents disclosed herein, are incorporated herein by reference in their entirety.

--->

Sequence list

<110> Merck Sharp & Dohme Corp.

Zhang, Lan

Friedman, Arthur

Durr, Eberhard

Bett, Andrew

<120> Stabilized RSV F proteins and their uses

<130> 24566

<150> 62/623.184

<151> 2018-01-29

<160> 49

<170>PatentIn version 3.5

<210> 1

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 1

Gly Gly Gly Ser Gly Gly Gly Ser

15

<210> 2

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 2

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly

1 5 10

<210> 3

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 3

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10

<210> 4

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 4

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser

1 5 10

<210> 5

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TEV Cleavage Site

<400> 5

Glu Asn Leu Tyr Phe Gln Ser

15

<210> 6

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Strep Tag

<400> 6

Trp Ser His Pro Gln Phe Glu Lys

15

<210> 7

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Thrombin cleavage site

<400> 7

Leu Val Pro Arg Gly Ser

15

<210> 8

<211> 31

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Foldon

<400> 8

Ser Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala

1 5 10 15

Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

20 25 30

<210> 9

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Signal sequence

<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

20 25

<210> 10

<211> 574

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Reference sequence RSV F

<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 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> 11

<211> 556

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<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 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 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 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 His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn Ile Met Ile Thr

515 520 525

Thr Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg

530 535 540

Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

545 550 555

<210> 12

<211> 605

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<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 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 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 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 His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn Ile Met Ile Thr

515 520 525

Thr Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg

530 535 540

Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Glu Asn Leu Tyr

545 550 555 560

Phe Gln Ser Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly

565 570 575

Gly Gly Ser Gly Gly Gly Ser Trp Ser His Pro Gln Phe Glu Lys Gly

580 585 590

Ser Gly Ser Gly Ser His His His His His His His His His

595 600 605

<210> 13

<211> 544

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<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 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 Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

515 520 525

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

530 535 540

<210> 14

<211> 503

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<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 Gly Gly Ser Gly Gly Gly Ser Ala Ile Ala Ser Gly Val Ala

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Ile Arg Lys Ser Asp Glu Leu Leu Ser Ala Ile Gly Gly Tyr Ile Pro

465 470 475 480

Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp

485 490 495

Val Leu Leu Ser Thr Phe Leu

500

<210> 15

<211> 527

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<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 Gly Gly Ser Gly Gly Gly Ser Ala Ile Ala Ser Gly Val Ala

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Ile Arg Lys Ser Asp Glu Leu Leu Ser Ala Ile Gly Gly Tyr Ile Pro

465 470 475 480

Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp

485 490 495

Val Leu Leu Ser Thr Phe Leu Gly Gly Leu Val Pro Arg Gly Ser His

500 505 510

His His His His His Ser Ala Trp Ser His Pro Gln Phe Glu Lys

515 520 525

<210> 16

<211> 505

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<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 Gly Gly Ser Gly Gly Gly Ser Gly Gly Ala Ile Ala Ser Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly

485 490 495

Glu Trp Val Leu Leu Ser Thr Phe Leu

500 505

<210> 17

<211> 529

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<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 Gly Gly Ser Gly Gly Gly Ser Gly Gly Ala Ile Ala Ser Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly

485 490 495

Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly Leu Val Pro Arg Gly

500 505 510

Ser His His His His His His Ser Ala Trp Ser His Pro Gln Phe Glu

515 520 525

Lys

<210> 18

<211> 507

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<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 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Ala Ile Ala

100 105 110

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

115 120 125

Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile

210 215 220

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

225 230 235 240

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

245 250 255

Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile

260 265 270

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

275 280 285

Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp

290 295 300

Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys

485 490 495

Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

500 505

<210> 19

<211> 531

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<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 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 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Ala Ile Ala

100 105 110

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

115 120 125

Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile

210 215 220

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

225 230 235 240

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

245 250 255

Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile

260 265 270

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

275 280 285

Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp

290 295 300

Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys

485 490 495

Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly Leu Val Pro

500 505 510

Arg Gly Ser His His His His His His Ser Ala Trp Ser His Pro Gln

515 520 525

Phe Glu Lys

530

<210> 20

<211> 509

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<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 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Ala

100 105 110

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

115 120 125

Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val

130 135 140

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

145 150 155 160

Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser

165 170 175

Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn

180 185 190

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

195 200 205

Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser

210 215 220

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

225 230 235 240

Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile

245 250 255

Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly

260 265 270

Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr

275 280 285

Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg

290 295 300

Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala

305 310 315 320

Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn

325 330 335

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

340 345 350

Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe

435 440 445

Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile

450 455 460

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

465 470 475 480

Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val

485 490 495

Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

500 505

<210> 21

<211> 533

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<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 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Ala

100 105 110

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

115 120 125

Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val

130 135 140

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

145 150 155 160

Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser

165 170 175

Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn

180 185 190

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

195 200 205

Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser

210 215 220

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

225 230 235 240

Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile

245 250 255

Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly

260 265 270

Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr

275 280 285

Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg

290 295 300

Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala

305 310 315 320

Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn

325 330 335

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

340 345 350

Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe

435 440 445

Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile

450 455 460

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

465 470 475 480

Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val

485 490 495

Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly Leu

500 505 510

Val Pro Arg Gly Ser His His His His His His Ser Ala Trp Ser His

515 520 525

Pro Gln Phe Glu Lys

530

<210> 22

<211> 509

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<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 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Ala

100 105 110

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

115 120 125

Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val

130 135 140

Cys Leu Ser Asn Gly Val Cys Val Leu Thr Phe Lys Val Leu Asp Leu

145 150 155 160

Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser

165 170 175

Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn

180 185 190

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

195 200 205

Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser

210 215 220

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

225 230 235 240

Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile

245 250 255

Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly

260 265 270

Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr

275 280 285

Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg

290 295 300

Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala

305 310 315 320

Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn

325 330 335

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

340 345 350

Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe

435 440 445

Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile

450 455 460

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

465 470 475 480

Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val

485 490 495

Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

500 505

<210> 23

<211> 533

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<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 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Ala

100 105 110

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

115 120 125

Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val

130 135 140

Cys Leu Ser Asn Gly Val Cys Val Leu Thr Phe Lys Val Leu Asp Leu

145 150 155 160

Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser

165 170 175

Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn

180 185 190

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

195 200 205

Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser

210 215 220

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

225 230 235 240

Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile

245 250 255

Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly

260 265 270

Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr

275 280 285

Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg

290 295 300

Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala

305 310 315 320

Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn

325 330 335

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

340 345 350

Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe

435 440 445

Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile

450 455 460

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

465 470 475 480

Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val

485 490 495

Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly Leu

500 505 510

Val Pro Arg Gly Ser His His His His His His Ser Ala Trp Ser His

515 520 525

Pro Gln Phe Glu Lys

530

<210> 24

<211> 509

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<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 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Ala

100 105 110

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

115 120 125

Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val

130 135 140

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

145 150 155 160

Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser

165 170 175

Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn

180 185 190

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

195 200 205

Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser

210 215 220

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

225 230 235 240

Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile

245 250 255

Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly

260 265 270

Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr

275 280 285

Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg

290 295 300

Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala

305 310 315 320

Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn

325 330 335

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

340 345 350

Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe

435 440 445

Pro Ser Cys Glu Phe Asp Cys Ser Ile Ser Gln Val Asn Glu Lys Ile

450 455 460

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

465 470 475 480

Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val

485 490 495

Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

500 505

<210> 25

<211> 533

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<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 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Ala

100 105 110

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

115 120 125

Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val

130 135 140

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

145 150 155 160

Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser

165 170 175

Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn

180 185 190

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

195 200 205

Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser

210 215 220

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

225 230 235 240

Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile

245 250 255

Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly

260 265 270

Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr

275 280 285

Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg

290 295 300

Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala

305 310 315 320

Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn

325 330 335

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

340 345 350

Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe

435 440 445

Pro Ser Cys Glu Phe Asp Cys Ser Ile Ser Gln Val Asn Glu Lys Ile

450 455 460

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

465 470 475 480

Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val

485 490 495

Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly Leu

500 505 510

Val Pro Arg Gly Ser His His His His His His Ser Ala Trp Ser His

515 520 525

Pro Gln Phe Glu Lys

530

<210> 26

<211> 509

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<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 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Ala

100 105 110

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

115 120 125

Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val

130 135 140

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

145 150 155 160

Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser

165 170 175

Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn

180 185 190

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

195 200 205

Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser

210 215 220

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

225 230 235 240

Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile

245 250 255

Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly

260 265 270

Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr

275 280 285

Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg

290 295 300

Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala

305 310 315 320

Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn

325 330 335

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

340 345 350

Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe

435 440 445

Pro Ser Cys Glu Phe Cys Ala Ser Ile Ser Gln Val Asn Glu Lys Ile

450 455 460

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

465 470 475 480

Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val

485 490 495

Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

500 505

<210> 27

<211> 533

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 27

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 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Ala

100 105 110

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

115 120 125

Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val

130 135 140

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

145 150 155 160

Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser

165 170 175

Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn

180 185 190

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

195 200 205

Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser

210 215 220

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

225 230 235 240

Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile

245 250 255

Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly

260 265 270

Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr

275 280 285

Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg

290 295 300

Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala

305 310 315 320

Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn

325 330 335

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

340 345 350

Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe

435 440 445

Pro Ser Cys Glu Phe Cys Ala Ser Ile Ser Gln Val Asn Glu Lys Ile

450 455 460

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

465 470 475 480

Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val

485 490 495

Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly Leu

500 505 510

Val Pro Arg Gly Ser His His His His His His Ser Ala Trp Ser His

515 520 525

Pro Gln Phe Glu Lys

530

<210> 28

<211> 509

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 28

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 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Ala

100 105 110

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

115 120 125

Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val

130 135 140

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

145 150 155 160

Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser

165 170 175

Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn

180 185 190

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

195 200 205

Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser

210 215 220

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

225 230 235 240

Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile

245 250 255

Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly

260 265 270

Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr

275 280 285

Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg

290 295 300

Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala

305 310 315 320

Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn

325 330 335

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

340 345 350

Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe

435 440 445

Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile

450 455 460

Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Cys Cys Ser Ala

465 470 475 480

Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val

485 490 495

Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

500 505

<210> 29

<211> 533

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 29

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 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Ala

100 105 110

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

115 120 125

Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val

130 135 140

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

145 150 155 160

Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser

165 170 175

Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn

180 185 190

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

195 200 205

Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser

210 215 220

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

225 230 235 240

Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile

245 250 255

Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly

260 265 270

Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr

275 280 285

Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg

290 295 300

Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala

305 310 315 320

Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn

325 330 335

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

340 345 350

Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe

435 440 445

Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile

450 455 460

Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Cys Cys Ser Ala

465 470 475 480

Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val

485 490 495

Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly Leu

500 505 510

Val Pro Arg Gly Ser His His His His His His Ser Ala Trp Ser His

515 520 525

Pro Gln Phe Glu Lys

530

<210> 30

<211> 509

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 30

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 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Ala

100 105 110

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

115 120 125

Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val

130 135 140

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

145 150 155 160

Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser

165 170 175

Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn

180 185 190

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

195 200 205

Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser

210 215 220

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

225 230 235 240

Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile

245 250 255

Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly

260 265 270

Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr

275 280 285

Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg

290 295 300

Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala

305 310 315 320

Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn

325 330 335

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

340 345 350

Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe

435 440 445

Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile

450 455 460

Asn Gln Ser Leu Ala Cys Ile Arg Lys Ser Asp Glu Leu Leu Ser Ala

465 470 475 480

Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val

485 490 495

Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

500 505

<210> 31

<211> 533

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 31

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 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Ala

100 105 110

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

115 120 125

Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val

130 135 140

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

145 150 155 160

Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser

165 170 175

Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn

180 185 190

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

195 200 205

Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser

210 215 220

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

225 230 235 240

Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile

245 250 255

Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly

260 265 270

Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr

275 280 285

Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg

290 295 300

Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala

305 310 315 320

Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn

325 330 335

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

340 345 350

Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe

435 440 445

Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile

450 455 460

Asn Gln Ser Leu Ala Cys Ile Arg Lys Ser Asp Glu Leu Leu Ser Ala

465 470 475 480

Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val

485 490 495

Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly Leu

500 505 510

Val Pro Arg Gly Ser His His His His His His Ser Ala Trp Ser His

515 520 525

Pro Gln Phe Glu Lys

530

<210> 32

<211> 544

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 32

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 Cys Leu Ser Asn Gly Val Cys 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 Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

515 520 525

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

530 535 540

<210> 33

<211> 568

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 33

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 Cys Leu Ser Asn Gly Val Cys 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 Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

515 520 525

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

530 535 540

Gly Gly Leu Val Pro Arg Gly Ser His His His His His His Ser Ala

545 550 555 560

Trp Ser His Pro Gln Phe Glu Lys

565

<210> 34

<211> 544

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 34

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 Cys Glu Phe Asp Cys 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 Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

515 520 525

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

530 535 540

<210> 35

<211> 568

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 35

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 Cys Glu Phe Asp Cys 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 Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

515 520 525

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

530 535 540

Gly Gly Leu Val Pro Arg Gly Ser His His His His His His Ser Ala

545 550 555 560

Trp Ser His Pro Gln Phe Glu Lys

565

<210> 36

<211> 544

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 36

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 Cys Glu Phe Cys 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 Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

515 520 525

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

530 535 540

<210> 37

<211> 568

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 37

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 Cys Glu Phe Cys 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 Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

515 520 525

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

530 535 540

Gly Gly Leu Val Pro Arg Gly Ser His His His His His His Ser Ala

545 550 555 560

Trp Ser His Pro Gln Phe Glu Lys

565

<210> 38

<211> 544

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 38

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 Cys

500 505 510

Cys Ser Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

515 520 525

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

530 535 540

<210> 39

<211> 568

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 39

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 Cys

500 505 510

Cys Ser Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

515 520 525

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

530 535 540

Gly Gly Leu Val Pro Arg Gly Ser His His His His His His Ser Ala

545 550 555 560

Trp Ser His Pro Gln Phe Glu Lys

565

<210> 40

<211> 544

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 40

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 Cys Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Ser Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

515 520 525

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

530 535 540

<210> 41

<211> 568

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 41

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 Cys Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Ser Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

515 520 525

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

530 535 540

Gly Gly Leu Val Pro Arg Gly Ser His His His His His His Ser Ala

545 550 555 560

Trp Ser His Pro Gln Phe Glu Lys

565

<210> 42

<211> 512

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 42

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 Ser Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

485 490 495

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

500 505 510

<210> 43

<211> 536

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 43

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 Ser Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

485 490 495

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

500 505 510

Gly Gly Leu Val Pro Arg Gly Ser His His His His His His Ser Ala

515 520 525

Trp Ser His Pro Gln Phe Glu Lys

530 535

<210> 44

<211> 477

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 44

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 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Ala

100 105 110

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

115 120 125

Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val

130 135 140

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

145 150 155 160

Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser

165 170 175

Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn

180 185 190

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

195 200 205

Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser

210 215 220

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

225 230 235 240

Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile

245 250 255

Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly

260 265 270

Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr

275 280 285

Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg

290 295 300

Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala

305 310 315 320

Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn

325 330 335

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

340 345 350

Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Ser Ala

435 440 445

Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val

450 455 460

Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

465 470 475

<210> 45

<211> 501

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide construct

<400> 45

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 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Ala

100 105 110

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

115 120 125

Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val

130 135 140

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

145 150 155 160

Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser

165 170 175

Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn

180 185 190

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

195 200 205

Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser

210 215 220

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

225 230 235 240

Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile

245 250 255

Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly

260 265 270

Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr

275 280 285

Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg

290 295 300

Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala

305 310 315 320

Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn

325 330 335

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

340 345 350

Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Ser Ala

435 440 445

Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val

450 455 460

Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly Leu

465 470 475 480

Val Pro Arg Gly Ser His His His His His His Ser Ala Trp Ser His

485 490 495

Pro Gln Phe Glu Lys

500

<210> 46

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 46

ctcaatttcc tcacttctcc agtgt 25

<210> 47

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 47

cttgattcct cggtgtacct ctgt 24

<210> 48

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 48

tcccattatg cctaggccag cagca 25

<210> 49

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid linker

<400> 1

Gly Gly Gly Ser

1

<---

Claims (86)

1. Recombinant respiratory syncytial virus (RSV) F protein trimer for generating an immune response to RSV F protein in a subject, comprising the amino acid sequence shown in any of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 44 or 45. 2. An immunogenic RSV composition for inducing an immune response to an RSV antigen, comprising a recombinant RSV F protein trimer according to claim 1 in an effective amount and a pharmaceutically acceptable excipient. 3. An immunogenic composition according to claim 2, formulated to induce an antigen-specific immune response directed against a recombinant RSV F trimer or a recombinant RSV F peptide. 4. An immunogenic composition according to any one of claims 2 or 3 which is formulated with an adjuvant. 5. The immunogenic composition of claim 4 wherein the adjuvant is an aluminum compound adjuvant. 6. The immunogenic composition of claim 5 wherein the aluminum compound adjuvant is amorphous aluminum hydroxyphosphate sulfate or an aqueous suspension of aluminum hydroxyphosphate. 7. The immunogenic composition of claim 2 further comprising a lipid nanoparticle comprising one or more cationic lipids and a poly(ethylene glycol) lipid (PEG lipid). 8. The immunogenic composition of claim 7 wherein the one or more cationic lipids are ionizable cationic lipids. 9. The immunogenic composition according to claim 8, where the ionizable cationic lipids are selected from DLinDMA; DlinKC2DMA; Dlin-MC3-DMA; CLinDMA; S-Octyl CLinDMA; (2S)-1-{7-[(3β)-cholest-5-en-3-yloxy]heptyloxy}-3-[(4Z)-dec-4-en-1-yloxy]-N, N dimethylpropan- 2-amines; (2R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-[(4Z)-dec-4-en-1-yloxy]-N, N dimethylpropan- 2-amines; 1-[(2R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)propan-2-yl]guanidine; 1-[(2R)-1-{7-[(3β)-cholest-5-en-3-yloxy]heptyloxy}-N, N-dimethyl-3-[(9Z,12Z)-octadeca-9,12 -dien-1-yloxy]propan-2-amine; 1-[(2R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-N, N-dimethyl-3-[(9Z,12Z)-octadeca-9.12 -dien-1-yloxy]propan-2-amine; (2S)-1-({6-[(3β))-cholest-5-en-3-yloxy]hexyl}oxy)-N, N-dimethyl-3-[(9Z)-octadec-9-en- 1-yloxy]propan-2-amine; (3β)-3-[6-{[(2S)-3-[(9Z)-octadec-9-en-1-yloxy]-2-(pyrrolidin-1-yl)propyl]oxy}hexyl)oxy] cholest-5-ene; (2R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)propan-2-amine; (2R)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-(pentyloxy)propan-2-amine; (2R)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-3-(heptyloxy)-N,N-dimethylpropan-2-amine; (2R)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N, N-dimethyl-3-[(2Z)pent-2-en-1- yloxy]propane-2-amine; (2S)-1-butoxy-3-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethylpropan-2-amine; (2S-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-3-[2,2,3,3,4,4,5,5,6, 6,7,7,8,8,9,9-hexadecafluoronyl)oxy]-N,N-dimethylpropan-2-amine; 2-amino-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1,3-diol; 2-amino-3-((9-(((3S,10R,13R)-10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9, 10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)nonyl)oxy)-2 -((((9Z,12Z)-octadeca- 9,12-dien-1-yl)oxy)methyl)propan-1-ol; 2-amino-3-((6-(((3S,10R,13R)-10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9, 10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)hexyl)oxy)-2-((((Z)-octadec-9- en-1-yl)oxy)methyl)propan-1-ol; (20Z,23Z)-N,N-dimethylnonacose-20,23-diene-10-amine; (17Z,20Z)-N,N-dimethylhexacose-17,20-diene-9-amine; (16Z,19Z)-N,N-dimethylpentacose-16,19-diene-8-amine; (13Z,16Z)-N,N-dimethyldocose-13,16-diene-5-amine; (12Z,15Z)-N,N-dimethylgenicose-12,15-diene-4-amine; (14Z,17Z)-N,N-dimethyltricose-14,17-diene-6-amine; (15Z,18Z)-N,N-dimethyltetracose-15,18-diene-7-amine; (18Z,21Z)-N,N-dimethylheptacose-18,21-diene-10-amine; (15Z,18Z)-N,N-dimethyltetracose-15,18-diene-5-amine; (14Z,17Z)-N,N-dimethyltricose-14,17-diene-4-amine; (19Z,22Z)-N,N-dimethyloctacose-19,22-diene-9-amine; (18Z,21Z)-N,N-dimethylheptacose-18,21-diene-8-amine; (17Z,20Z)-N,N-dimethylhexacose-17,20-diene-7-amine; (16Z,19Z)-N,N-dimethylpentacose-16,19-diene-6-amine; (22Z,25Z)-N,N-dimethylhentriaconta-22,25-diene-10-amine; (21Z,24Z)-N,N-dimethyltriaconta-21,24-diene-9-amine; (18Z)-N,N-dimethylheptacose-18-ene-10-amine; (17Z)-N,N-dimethylhexacose-17-ene-9-amine; (19Z,22Z)-N,N-dimethyloctacose-19,22-diene-7-amine; N,N-dimethylheptacosan-10-amine; (20Z,23Z)-N-ethyl-N-methylnonacose-20,23-diene-10-amine; 1-[(11Z,14Z)-1-nonylicose-11,14-dien-1-yl]-pyrrolidine; (20Z)-N,N-dimethylheptacose-20-en-10-amine; (15Z)-N,N-dimethylheptacose-15-ene-10-amine; (14Z)-N,N-dimethylnonacose-14-ene-10-amine; (17Z)-N,N-dimethylnonacose-17-ene-10-amine; (24Z)-N,N-dimethyltritriacont-24-ene-10-amine; (20Z)-N,N-dimethylnonacose-20-ene-10-amine; (22Z)-N,N-dimethylgentriacont-22-ene-10-amine; (16Z)-N,N-dimethylpentacose-16-en-8-amine; (12Z,15Z)-N,N-dimethyl-2-nonylgenicose-12,15-diene-1-amine; (13Z,16Z)-N,N-dimethyl-3-nonyldocose-13,16-diene-1-amine; N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine; 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine; N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine; N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]genicosan-10-amine; N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine; N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine; N,N-dimethyl-1-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine; N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl} dodecan-1-amine; 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine; 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine; N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine; And (11E,20Z,23Z)-N,N-dimethylnonacose-11,20,23-trien-10-amine; or a pharmaceutically acceptable salt, or a stereoisomer of any of the above. 10. The immunogenic composition of claim 9 wherein the ionizable cationic lipids are selected from the group consisting of (2S)-1-({6-[(3β))-cholest-5-en-3-yloxy]hexyl}oxy)- N,N-dimethyl-3-[(9Z)-octadec-9-en-1-yloxy]propan-2-amine; (13Z,16Z)-N,N-dimethyl-3-nonyldocose-13,16-dien-1-amine and N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine ; or a pharmaceutically acceptable salt, or a stereoisomer of any of the above. 11. An immunogenic composition according to claim 10, which contains 30-75 mol.% ionizable cationic lipid and 0.1-20 mol.% PEG lipid. 12. An immunogenic composition according to any one of claims 7-11, further comprising one or more non-cationic lipids selected from a phospholipid, a phospholipid derivative, a fatty acid, a sterol, or a combination thereof. 13. The immunogenic composition of claim 12 wherein the sterol is cholesterol, stigmasterol or stigmastanol. 14. The immunogenic composition according to claim 12, wherein the phospholipid is selected from phosphatidylserine, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dilauroylphosphatidylcholine (DLPC), 1,2-dieucosenoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). 15. An immunogenic composition according to any one of claims 8 to 14, wherein the PEG lipid is 1,2-dimyristoyl-sn-glycerol-methoxypolyethylene glycol (PEG-DMG), PEG-disterylglycerol (PEG-DSG), PEG-dipalmetoleyl, PEG- dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE) or PEG-1,2-dimyristyloxylpropyl-3-amine (PEG-c-DMA). 16. An immunogenic composition according to any one of claims 12-15, which contains 20-99.8 mole% ionizable cationic lipids, 0.1-65 mole% non-cationic lipids and 0.1-20 mole% PEG lipid. 17. The immunogenic composition of claim 16 wherein the non-cationic lipids comprise a mixture of cholesterol and DSPC. 18. An immunogenic composition according to claim 12, which contains 34-59 mole % of ionizable cationic lipids selected from the group consisting of (2S)-1-({6-[(3β))-cholest-5-en-3 -yloxy]hexyl}oxy)-N,N-dimethyl-3-[(9Z)-octadec-9-en-1-yloxy]propan-2-amine; (13Z,16Z)-N,N-dimethyl-3-nonyldocose-13,16-diene-1-amine; and N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine, 30-48 mol% cholesterol, 10-24% DSPC and 1-2 mol% PEG-DMG. 19. A method of producing an immune response to an RSV F protein in a subject, wherein the method comprises administering to the subject an effective amount of the RSV F protein trimer of claim 1. 20. The method of claim 19 wherein the subject is administered a single dose of RSV F trimer protein. 21. The method of claim 19 wherein the subject is administered at least one booster dose of RSV F trimer protein. 22. The method of any one of claims 19-21, wherein the RSV F protein trimer is administered to the subject by intradermal injection or intramuscular injection. 23. The method of any one of claims 19-22, wherein the subject has been exposed to RSV, where the subject is infected with RSV, or where the subject is at risk of being infected with RSV. 24. Use of the RSV F protein trimer of claim 1 in a method of inducing an immune response to the RSV F protein in a subject, wherein the method comprises administering to the subject the RSV F protein trimer in an amount effective to elicit an antigen-specific immune response in the subject. 25. The use of the RSV F protein trimer of claim 1 in the manufacture of a medicament for use in a method of inducing an immune response to the RSV F protein in a subject, wherein the method comprises administering to the subject the RSV F protein trimer in an amount effective to produce an antigen-specific immune response in the subject.

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