WO2012006180A1 - Hiv immunogens - Google Patents

Abstract

Disclosed are HIV immunogens. Also disclosed are nucleic acids encoding these immunogens and methods of producing these antigens. Methods for generating an immune response in a subject are also disclosed. In some embodiments, the method is a method for treating or preventing a human immunodeficiency type 1 (HIV-1) infection in a subject.

Description

HIV IMMUNOGENS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No: 61/443,550, filed February 16, 2011, and U.S. Provisional Application No:

61/359,738, filed June 29, 2010, which are hereby incorporated herein by reference in their entirety.

STATEMENT OF JOINT RESEARCH

The research work described herein was performed under a Collaboration Agreement between the U.S. Government and Purdue University.

FIELD

The present disclosure relates to immunogenic polypeptides, and specifically to polypeptides that can provoke an immune response to human immunodeficiency virus (HIV).

BACKGROUND

Over 30 million people are infected with HIV worldwide, and 2.5 to 3 million new infections have been estimated to occur yearly. Although effective antiretroviral therapies are available, millions succumb to AIDS every year, especially in sub-Saharan Africa, underscoring the need to develop measure to prevent the spread of this disease.

The primary immunologic abnormality resulting from infection by HIV is the progressive depletion and functional impairment of T lymphocytes expressing the CD4 cell surface glycoprotein. The loss of CD4 helper/inducer T cell function probably underlies the defects in cellular and humoral immunity leading to the opportunistic infections and malignancies characteristic of the acquired

immunodeficiency syndrome (AIDS) (Lane et al., Ann. Rev. Immunol. 3:477, 1985). Studies of HIV-1 infection of fractionated CD4 and CD8 T cells from normal donors and AIDS patients have revealed that depletion of CD4 T cells results from the ability of HIV-1 to selectively infect, replicate in, and ultimately destroy this T lymphocyte subset (Klatzmann et al., Science 225:59, 1984). The possibility that CD4 itself is an essential component of the cellular receptor for HIV-1 was first indicated by the observation that monoclonal antibodies directed against CD4 block HIV-1 infection and syncytia induction (Dalgleish et al., Nature 312:767,1984;

McDougal et al., J. Immunol. 135:3151, 1985). This hypothesis has been confirmed by the demonstration that a molecular complex forms between CD4 and the major envelope glycoprotein of HIV-1 (McDougal et al., Science 231:382, 1986).

Over the past few decades the development of agents to combat HIV infections has vastly increased the average lifespan of those infected throughout the world. However, in some instances HIV is able to acquire resistance to antivirals within a few replication cycles.

To combat the ever-changing landscape of HIV resistance to the current therapies, the standard course of action for pharmaceutical companies is to develop an ever- increasing array of small molecule therapeutic agents. As an alternative, vaccines have been developed which stimulate the body to fight an infection by eliciting antibody responses to the target pathogen(s). In some examples, these vaccines are polypeptide epitopes that induce an immune response to pathogens and can be referred to as immunogens. These immunogens can be introduced into a subject where they can elicit an antibody response to specific epitopes of the pathogen. For example, immunogens derived from the envelope protein of HIV have been used to produce an antibody response.

Initial attempts at generating neutralizing antibodies by vaccination with recombinant HIV gpl20 protein analogous to some highly effective vaccines have thus far proved unsuccessful in generating protective immunity. For example, in 2009, a large multicenter, double-blind, placebo-controlled clinical study revealed that priming immunization with ALVAC-HIV (a canary pox vector vaccine expressing HIV Env genes) followed by AIDSVAX B/E booster immunizations (recombinant HIV Env gpl20 vaccine) can reduce the risk of HIV infection amongst heterosexuals by 31 percent. However, the vaccine only induced short-term protection, and there has been speculation that short-lived antibody responses to HIV Env protein might neutralize or block HIV mucosal transmission. An effective HIV-1 vaccine will likely need to induce neutralizing antibodies (NAbs) that block HIV-1 entry into human cells. To be effective, vaccine induced antibodies will have to be active against most circulating strains of HIV-1. Thus, there is a need for immunogens that can be used to elicit an immune response to pathogens, such as HIV.

SUMMARY

Disclosed are immunogens that include a human immunodeficiency virus (HIV) gpl20 outer domain polypeptide, which has been substantially modified from the wild type amino acid sequence to focus the immune response to the CD4 biding site of gpl20. In some examples, the gpl20 outer domain polypeptide includes amino acids 213-492 of gpl20, amino acids 213-482 of gpl20, amino acids 252-492 of gpl20, or 252-482 of gpl20. In some examples, the gpl20 outer domain polypeptide is part of a larger peptide, such as a HIV-1 gpl40 or HIV-1 gpl20 peptide.

In some embodiments, one or more of the loops of the outer domain peptide (such as one or more of the VI, V2, V3, V4 or V5 loop) have been altered to enhance or focus immunogenicity and/or increase stability of the immunogen. In some embodiments, the β20/β21 bridging sheet of the gpl20 outer domain is removed or truncated.

In some embodiments, the immunogen includes one or more amino acid substitutions relative to the wild type sequence, for example to add or remove glycosylation sites, and/or to increase protein stability. In some embodiments, the isolated immunogen includes one or more of a foldon domain, a ferritin polypeptide, a hybrid of different ferritin polypeptides, a six-histidine residue tag, a secretion signal sequence and a transmembrane domain. In specific examples, the immunogen includes an amino acid sequence at least 95% identical to the amino acid sequence set forth as any one of SEQ ID NOs : 203, 58-77, 1-57, 79, or 80.

In some embodiments, the immunogen is part of a virus-like particle (VLP), such as a Chikungunya virus VPL. Also disclosed are nucleic acids molecules encoding the disclosed immunogen. In specific examples, such nucleic acids include the nucleotide sequences set forth as one of SEQ ID NOs: 81-161.

Methods of generating an immune response in a subject are disclosed, as are methods of treating, inhibiting or preventing a HIV-1 infection in a subject. In such methods a subject, such as a human subject, is administered and effective amount of a disclosed antigen.

Methods for detecting or isolating an HIV-1 binding antibody in a subject infected with HIV-1 are disclosed. In such methods, a disclosed immunogen is contacted with an amount of bodily fluid from a subject and the binding of the HIV- 1 binding antibody to the immunogen is detected, thereby detecting or isolating the HIV-1 binding antibody in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A and IB are schematic representations depicting the development of representative outer domain immunogens disclosed herein.

FIG. 2 includes a ribbon diagram of modeled HIV gpl20 OD structures (upper left). The V3 loops are highlighted and circled. Also shown is an amino acid sequence alignment of various V3 modifications (upper right) (GSL; SEQ ID NO: 172) (V3.1; SEQ ID NO: 173), (V3.2; SEQ ID NO: 174), (V3.3; SEQ ID NO: 175), V3.4; SEQ ID NO: 176). The symbol "y" indicates a potential glycosylation site. The graphs (lower left) show antibody binding profiles of V3 -modified ODs. The purified parental OD4.2F and V3-modified ODs v3.3 and v3.4 proteins were coated onto plates pre-coated with snowdrop lectin (GNA) and tested for binding abilities to VRCOl and b 12 monoclonal antibodies by ELISA. The EC₅₀ values (lower right) were calculated from the curve and represented as mg/ml. The constructs used were A2.6 T383F (OD4.2F), A2.6.2 V3.1 (v3.1), A2.6.2 V3.2 (v3.2), A2.6.2 V3.3 (v3.3) and A2.6.2 V3.4 (v3.4).

FIG. 3 includes a ribbon diagram of modeled HIV gpl20 OD structures (upper left). The V5 loops are circled. Also shown is an amino acid sequence alignment of various V5 modifications (upper right) (wild type; SEQ ID NO: 204) (v5.1; SEQ ID NO: 187) (v5.2; SEQ ID NO: 188) (v5.3; SEQ ID NO: 189). The symbol "y" indicates a potential glycosylation site. The graphs (lower left) show antibody binding profiles of V5-modified ODs. The purified parental OD4.2F and V5-modified ODs v5.2 and v5.3 proteins were coated onto plates pre-coated with snowdrop lectin (GNA) and tested for binding abilities to VRCOl and b 12 monoclonal antibodies by ELISA. The EC₅₀ values (lower right) were calculated from the curve and represented as mg/ml. The constructs used were A2.6 T383F (OD4.2F), A2.6.2 V5.1 (v5.1), A2.6.2 V5.2 (v5.2) and A2.6.2 V5.3 (v5.3).

FIG. 4 includes a ribbon diagram of modeled OD structures (upper left). The V3 and V5 modifications are circled. Brief descriptions of each modification are listed (upper right). The graphs show antibody binding profiles of OD4.2 and

OD4.2'. The purified parental OD4.2 and modified OD4.2' proteins were coated onto plates pre-coated with snowdrop lectin (GNA) and tested for binding abilities to VRCOl, bl2 and bl3 monoclonal antibodies by ELISA. The constructs used were A2.3 3ab (OD4.2), A2.6.3 N276D V3.3 V5.2 (OD4.2').

FIG. 5 shows a surface representation of modeled OD structures with artificially added glycans (upper). The modeled OD surface is colored in gray, and VRCOl -contacting regions are highlighted. The native glycans and artificially added glycans are modeled. The numbers indicate the positions that have been

glycosylated. The front (looking down to CD4BS) and back views are shown. The designed OD variants with various degrees and positions of glycosylation are also listed (bottom). The constructs used were A2.9a-j Glyc(295, 442) (HG. l), A2.9a-j Glyc(295, 442, 479) (HG.2), A2.9a-j Glyc(295, 442, 436*) (HG.3), A2.9a-j

Glyc(295, 442, 424*) (HG.4), A2.9a-j Glyc(295, 442, 398) (HG.5), A2.9a-j

Glyc(295, 442, 369) (HG.6), A2.9a-j Glyc(295, 442, 273, 377, 348) (HG.7), A2.9a-j Glyc(295, 442, 479, 273, 377, 436*, 398, 348, 369) (HG.8), A2.9a-j Glyc(295, 442, 479, 273, 377, 424*, 398, 348, 369) (HG.9) and A2.9a-j Glyc(295, 442, 479, 273, 377) (HG.10).

FIG. 6 is a series of graphs showing antibody binding profiles of the glycosylation variants of OD4.2'. The purified OD variant proteins were coated onto plates pre-coated with snowdrop lectin (GNA) and tested for binding abilities to VRCOl, bl2 and bl3 monoclonal antibodies by ELISA. The constructs used were A2.9a-j Glyc(295, 442) (HG. l), A2.9a-j Glyc(295, 442, 479) (HG.2), A2.9a-j Glyc(295, 442, 436*) (HG.3), A2.9a-j Glyc(295, 442, 424*) (HG.4), A2.9a-j Glyc(295, 442, 398) (HG.5), A2.9a-j Glyc(295, 442, 369) (HG.6), A2.9a-j

Glyc(295, 442, 273, 377, 348) (HG.7), A2.9a-j Glyc(295, 442, 479, 273, 377, 436*, 398, 348, 369) (HG.8) and A2.9a-j Glyc(295, 442, 479, 273, 377) (HG.10).

FIG. 7 depicts the identification of the bl2-binding knock out variant HG.3. The antibody binding profile of HG.3 is highlighted with a frame (upper). The binding of VRCOl, bl2 and bl3 monoclonal antibodies are plotted as squares, circles and triangles, respectively. Also shown is a surface representation of modeled HG.3 structure (bottom). The modeled HG.3 surface is colored in gray, and VRCOl -contacting regions are highlighted. The native and artificially added glycans are modeled. The constructs used were A2.9a-j Glyc(295, 442, 436*) (HG.3).

FIG. 8 includes a surface representation of modeled HG.3 structures with artificially added glycans (upper left). The VRCOl -contacting regions are highlighted. The native glycans and artificially added glycans in HG.3 and in HG.3 variants are modeled. The numbers indicated positions that have been glycosylated. Also listed are the designed HG.3 variants with various degrees and positions of glycosylation (upper right). The purified OD variant proteins were coated onto plates pre-coated with snowdrop lectin (GNA) and tested for binding abilities to VRCOl, bl2 and bl3 monoclonal antibodies by ELISA (lower panels). The constructs used were A2.9a-j Glyc(295, 442, 436*) (HG.3), A2.HG3 Glyc(479) (HG.3.1), A2.HG3 Glyc(479, 398) (HG.3.2), A2.HG3 Glyc(479, 348) (HG.3.3), A2.HG3 Glyc(479, 377) (HG.3.4) and A2.HG3 Glyc(479, 273) (HG.3.5).

FIG. 9 includes a surface representation of modeled HG3.2 structures with artificially added glycans (upper left). The modeled HG3.2 surface is colored in gray, and VRCOl -contacting regions are highlighted. The native glycans and artificially added glycans are modeled. The HG3.2 structure complex with VRCOl (bottom left), bl2 (bottom center) and bl3 (bottom right) were modeled. The added glycans may prevent binding of antibodies with unfavorable angle (less potent neutralizing antibodies like bl2 and bl3). FIG. 10 includes schematic representations of the insertion of a

representative immunogen into the genome of the CHIK, and the creation of virus like particles presenting the immunogen on the surface. OD4.0 was inserted between amino acid 205 and amino acid 206 E2. Corresponding E2 from Sindbis map were shown in the middle and right panel. The purified CHIK-OD was reconstructed by Cryoelectron microscopy.

FIG. 11 shows antibody titers of sera against RSC3 or delta RSC3 mutant that knockout CD4 binding domain from an immunized rabbit. The rabbits were immunized intramuscularly two times with CHIK-OD. The curve fit was calculated by Prism software.

FIG. 12 shows the results of immunized monkeys with one of the CHIKV- OD VLP. Significant amount of gpl40 binding antibodies were elicited after 3 times of immunization. Interestingly, in 2 out of 3 sera the elicitation of CD4 BS antibodies was seen, again based on the RSC3/ARSC3 ELISA assay.

FIG. 13 shows that a heavily glycosylated OD displayed on CHIK VLP reacts only with VRCOl and VRCPG04.

FIG. 14 is a flow chart showing the work flow of designing the OD-based vaccine immunogen.

FIG. 15 shows the construction of a ferritin nanoparticle expressing an outer domain immunogen.

FIG. 16 shows the immunogenicity of HIV OD-ferritin nanoparticles.

FIG. 17 shows the induction of CD4 BS antibodies by glycan modified RSC3: Y5.

FIG. 18 is schematic representation depicting the development of representative RSC3 immunogens disclosed herein.

SEQUENCES

The nucleic acid and amino acid sequences disclosed herein are shown using standard letter abbreviations for nucleotide bases, and one letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the

complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file in the form of the file named Sequence_Listing.txt, which was created on June 29, 2011, is 432,644 bytes, and is incorporated by reference herein. SEQ ID NOs: 1-77, 79, 80, 148, 149 and 203 are the amino acid sequences of exemplary HIV antigens.

SEQ ID NO: 78 is the amino acid sequence of the HXB2 core.

SEQ ID NOs: 81-147, 150-161 and 206-215 are the nucleic acid sequences encoding exemplary HIV antigens.

SEQ ID NO: 162 is the amino acid sequence of an exemplary modified

V1/V2 loop.

SEQ ID NO: 163 is the amino acid sequence of an exemplary VI wild type loop.

SEQ ID NOs: 164-165 are the amino acid sequence of exemplary modified VI loops.

SEQ ID NOs: 166-167 are the amino acid sequence of exemplary modified V2 loops.

SEQ ID NOs: 168-181 are the amino acid sequence of exemplary modified V3 loops.

SEQ ID NOs: 182-186 are the amino acid sequence of exemplary modified

V4 loops.

SEQ ID NOs: 187-194 and 202 are the amino acid sequence of exemplary modified V5 loops.

SEQ ID NO: 195 is the amino acid sequence of an exemplary fibritin foldon. SEQ ID NO: 196 is the amino acid sequence of an exemplary CD4 transmembrane domain.

SEQ ID NO: 197 is the amino acid sequence of an influenza transmembrane neuraminidase domain.

SEQ ID NO: 198 is the amino acid sequence of an influenza transmembrane hemegglutin domain.

SEQ ID NO: 199 is the amino acid sequence of a CD5 leader sequence. SEQ ID NO: 200 is the amino acid sequence of an IL-2 secretion signal sequence.

SEQ ID NO: 201 is the amino acid sequence of an exemplary peptide linker. SEQ ID NO: 204 is the amino acid sequence of an exemplary wild type V5 loop.

SEQ ID NO: 205 is the amino acid sequence of an exemplary wild type V3 loop.

DETAILED DESCRIPTION

/. Summary of Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 1999; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other similar references.

As used herein, the singular forms "a," "an," and "the," refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term "an antigen" includes single or plural antigens and can be considered equivalent to the phrase "at least one antigen"

As used herein, the term "comprises" means "includes." Thus, "comprising an antigen" means "including an antigen" without excluding other elements.

It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described below. In case of conflict, the present specification, including

explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various embodiments of the invention, the following explanations of terms are provided:

Adjuvant: A vehicle used to enhance antigenicity. Adjuvants include a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water-in-oil emulsion in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages). Immuno stimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants (for example see U.S. Patent No. 6,194,388; U.S. Patent No. 6,207,646; U.S. Patent No. 6,214,806; U.S. Patent No. 6,218,371; U.S. Patent No. 6,239,116; U.S. Patent No. 6,339,068; U.S. Patent No. 6,406,705; and U.S. Patent No. 6,429,199).

Adjuvants include biological molecules (a "biological adjuvant"), such as costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-a, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL. Adjuvants can be used in combination with the disclosed antigens.

Administration: The introduction of a composition into a subject by a chosen route. For example, if the chosen route is intravenous, the composition (such as a disclosed antigen) is administered by introducing the composition into a vein of the subject.

Amino acid substitutions: The replacement of one amino acid in an antigen with a different amino acid. In some examples, an amino acid in an antigen is substituted with an amino acid from a homologous antigen.

Amplification: A technique that increases the number of copies of a nucleic acid molecule (such as an RNA or DNA). An example of amplification is the polymerase chain reaction, in which a biological sample is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to a nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. The product of amplification can be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing using standard techniques. Other examples of amplification include strand displacement amplification, as disclosed in U.S. Patent No. 5,744,311;

transcription-free isothermal amplification, as disclosed in U.S. Patent No.

6,033,881; repair chain reaction amplification, as disclosed in WO 90/01069; ligase chain reaction amplification, as disclosed in EP-A-320 308; gap filling ligase chain reaction amplification, as disclosed in U.S. Patent No. 5,427,930; and NASBA™ RNA transcription-free amplification, as disclosed in U.S. Patent No. 6,025,134.

Animal: A living multi-cellular vertebrate or invertebrate organism, a category that includes, for example, mammals. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects, such as non-human primates. Thus, administration to a subject can include administration to a human subject. Particular examples of veterinary subjects include domesticated animals (such as cats and dogs), livestock (for example, cattle, horses, pigs, sheep, and goats), laboratory animals (for example, mice, rabbits, rats, gerbils, guinea pigs, and non-human primates).

Antibody: A polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an analyte (such as an antigen or immunogen) such as a gpl20, gpl40 polypeptide or antigenic fragment thereof, such as a gpl20 outer domain or an resurfaced gpl20, gpl40 polypeptide or antigenic fragment thereof.

Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.

Antibodies exist, for example as intact immunoglobulins and as a number of well characterized fragments produced by digestion with various peptidases. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bind to gpl20, or gpl40 would be gpl20-, or gpl40 -specific binding agents. This includes intact immunoglobulins and the variants and portions of them well known in the art, such as Fab' fragments, F(ab)'₂ fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical

Co., Rockford, IL); Kuby, J., Immunology, 3 rd Ed., W.H. Freeman & Co., New York, 1997.

Antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab')2, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. The term

"antibody," as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies.

Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains"). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework region and CDRs have been defined (see, Kabat et ah, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human

Services, 1991, which is hereby incorporated by reference). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a V_H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a V_L CDRl is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Light chain CDRs are sometimes referred to as CDR LI, CDR L2, and CDR L3. Heavy chain CDRs are sometimes referred to as CDR HI, CDR H2, and CDR H3.

References to "V_H" or "VH" refer to the variable region of an

immunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab. References to "V_L" or "VL" refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A "monoclonal antibody" is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. These fused cells and their progeny are termed "hybridomas." Monoclonal antibodies include humanized monoclonal antibodies.

Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens. "Epitope" or "antigenic determinant" refers to the region of an antigen to which B and/or T cells respond. In one embodiment, T cells respond to the epitope, when the epitope is presented in conjunction with an MHC molecule. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and nuclear magnetic resonance.

Examples of antigens include, but are not limited to, peptides, lipids, polysaccharides, and nucleic acids containing antigenic determinants, such as those recognized by an immune cell. In some examples, antigens include peptides derived from a pathogen of interest. Exemplary pathogens include bacteria, fungi, viruses and parasites. In specific examples, an antigen is derived from HIV, such as a gpl20, gpl40 polypeptide or antigenic fragment thereof, such as an outer domain of gpl20.

CD4: Cluster of differentiation factor 4 polypeptide, a T-cell surface protein that mediates interaction with the MHC class II molecule. CD4 also serves as the primary receptor site for HIV on T-cells during HIV-1 infection.

The known sequence of the CD4 precursor has a hydrophobic signal peptide, an extracellular region of approximately 370 amino acids, a highly hydrophobic stretch with significant identity to the membrane- spanning domain of the class II MHC beta chain, and a highly charged intracellular sequence of 40 residues

(Maddon, Cell 42:93, 1985).

The extracellular domain of CD4 consists of four contiguous

immunoglobulin-like regions (Dl, D2, D3, and D4, see Sakihama et ah, Proc. Natl. Acad. Sci. 92:6444, 1995; U.S. Patent No. 6,117,655), and amino acids 1 to 183 have been shown to be involved in gpl20 binding.

CD4BS antibodies: Antibodies that bind to or substantially overlap the CD4 binding surface of a gpl20, or gpl40, polypeptide. The antibodies interfere with or prevent CD4 from binding to a gpl20 polypeptide.

CD4i antibodies: Antibodies that bind to a conformation of gpl20 induced by CD4 binding.

CD8: Cluster of differentiation factor 8, a T cell surface protein that mediates interaction with the MHC Class I molecule. Cells that express CD8 are often cytotoxic T cells.

Contacting: Placement in direct physical association; includes both in solid and liquid form. Contacting includes contact between one molecule and another molecule, for example the amino acid on the surface of one polypeptide, such as an antigen, that contact another polypeptide, such as an antibody. Contacting also includes administration, such as administration of a disclosed antigen to a subject by a chosen route.

Degenerate variant and conservative variant: A polynucleotide encoding a polypeptide or an antibody that includes a sequence that is degenerate as a result of the genetic code. For example, a polynucleotide encoding a disclosed antigen or an antibody that specifically binds a disclosed antigen includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the antigen or antibody that binds the antigen encoded by the nucleotide sequence is unchanged. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified within a protein encoding sequence, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are "silent variations," which are one species of conservative variations. Each nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation.

One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each "silent variation" of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

One of ordinary skill will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (for instance less than 5%, in some embodiments less than 1%) in an encoded sequence are conservative variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid.

Conservative amino acid substitutions providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Not all residue positions within a protein will tolerate an otherwise

"conservative" substitution. For instance, if an amino acid residue is essential for a function of the protein, even an otherwise conservative substitution may disrupt that activity, for example the specific binding of an antibody to a target epitope may be disrupted by a conservative mutation in the target epitope. Expression: Translation of a nucleic acid into a protein. Proteins may be expressed and remain intracellular, become a component of the cell surface membrane, or be secreted into the extracellular matrix or medium.

Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

A promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter- dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included (see for example, Bitter et ah, Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (such as metallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences. A polynucleotide can be inserted into an expression vector that contains a promoter sequence, which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.

Ferritin: A protein that stores iron and releases it in a controlled fashion. The protein is produced by almost all living organisms. Ferritin assembles into a globular protein complex that in some cases consists of 24 protein subunits. In some examples, ferritin is used to form a particle (such as a virus-like particle) presenting antigens on its surface, for example HIV antigens, such as the disclosed gpl20, gpl40 antigens or immunogenic fragments thereof.

Foldon domain: An amino acid sequence that naturally forms a trimeric structure. In some examples, a foldon domain can be included in the amino acid sequence of a disclosed antigen so that the antigen will form a trimer. In one example, a foldon domain is the T4 foldon domain.

Glycoprotein (gp): A protein that contains oligosaccharide chains (glycans) covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation. In proteins that have segments extending extracellularly, the extracellular segments are often glycosylated. Glycoproteins are often important integral membrane proteins, where they play a role in cell-cell interactions. In some examples a glycoprotein is an HIV glycoprotein, such as a HIV gpl20, gpl40 or an immunogenic fragment thereof.

Glycosylation site: An amino acid sequence on the surface of a polypeptide, such as a protein, which accommodates the attachment of a glycan. An N-linked glycosylation site is triplet sequence of NXS/T in which N is asparagine, X is any residues except proline, S/T means serine or threonine. A glycan is a polysaccharide or oligosaccharide. Glycan may also be used to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan.

gpl20: An envelope protein from human immunodeficiency virus (HIV).

The envelope protein is initially synthesized as a longer precursor protein of 845- 870 amino acids in size, designated gpl60. gpl60 forms a homotrimer and undergoes glycosylation within the Golgi apparatus. It is then cleaved by a cellular protease into gpl20 and gp41. gpl20 contains most of the external, surface-exposed, domains of the envelope glycoprotein complex, and it is gpl20 that binds both to the cellular CD4 receptor and to the cellular chemokine receptors (such as CCR5).

The mature gpl20 wild-type polypeptides have about 500 amino acids in the primary sequence. The gpl20 is heavily N-glycosylated giving rise to an apparent molecular weight of 120 kD. The polypeptide is comprised of five conserved regions (C1-C5) and five regions of high variability (V1-V5). Exemplary sequences of wild-type gpl60 polypeptides are shown on GENBANK®, for example

Accession Nos. AAB05604 and AAD12142, which are incorporated herein by reference in their entirety as available on June 29, 2010. Exemplary sequences of gpl20 polypeptides from HIV-1 DU156 are shown on GENBANK®, for example Accession Nos. ABD83635, AAO50350 and AAT91997, which are incorporated herein by reference in their entirety as available on September 27, 2010. Exemplary sequences of gpl20 polypeptides from HIV-1 ZA012 are shown on GENBANK®, for example Accession No. ACF75939, which is incorporated herein by reference in its entirety as available on September 27, 2010.

The gpl20 core has a unique molecular structure, which comprises two domains: an "inner" domain (which faces gp41) and an "outer" domain (which is mostly exposed on the surface of the oligomeric envelope glycoprotein complex). The two gpl20 domains are separated by a "bridging sheet" that is not part of either of these domains. The gpl20 core comprises 25 beta strands, 5 alpha helices, and 10 defined loop segments.

The core gpl20 comprises 25 β-strands, 5 a-helices and 10 defined loop segments. The polypeptide chain of gpl20 is folded into two major domains, plus certain excursions that emanate from this body. The inner domain (inner with respect to the N and C termini) features a two-helix, two-strand bundle with a small five-stranded β -sandwich at its termini-proximal end and a projection at the distal end from which the V1/V2 stem emanates. The outer domain is a stacked double barrel that lies alongside the inner domain so that the outer barrel and inner bundle axes are approximately parallel. The bridging sheet (β3, β2, β21, β20) packs primarily over the inner domain, although some surface residues of the outer domain, such as Phenylalanine 382, reach in to form part of its hydrophobic core.

The numbering used in the gpl20 derived antigens disclosed herein is relative to the HXB2 numbering scheme as set forth in Numbering Positions in HIV Relative to HXB2CG Bette Korber et al, Human Retroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Korber et ah, Eds. Theoretical Biology and Biophysics Group, Los Alamos National

Laboratory, Los Alamos, NM, which is incorporated by reference herein in its entirety.

gpl40: An oligomeric form of HIV envelope protein, which contains all of gpl20 and the entire gp41 ectodomain.

Homologous proteins: Proteins from two or more species that have a similar structure and function in the two or more species. For example a gpl20 antigen from one species of lenti virus such as HIV-1 is a homologous antigen to a gpl20 antigen from a related species such as HIV-2 or SIV. Homologous proteins share the same protein fold and can be considered structural homologs.

Homologous proteins typically share a high degree of sequence conservation, such as at least 30% at least 40% at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence conservation. Homologous proteins can share a high degree of sequence identity, such as at least 30% at least 40% at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity.

Host cells: Cells in which a vector can be propagated and its DNA

expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used.

Immunogenic polypeptide: A protein or a portion thereof that is capable of inducing an immune response in a mammal, such as a mammal infected or at risk of infection with a pathogen. Administration of an immunogenic polypeptide derived from a pathogen of interest that inducing an immune response. Administration of an immunogenic polypeptide can lead to protective immunity against a pathogen of interest. In some examples, an immunogenic polypeptide is an antigen that is resurfaced to focus immunogenicity to a target epitope. An "immunogenic gpl20 or gpl40 polypeptide" is gpl20, or gpl40 molecule, a resurfaced gpl20, or gpl40 molecule, or a portion thereof, such as a gpl20 outer domain (OD) that is capable of inducing an immune response in a mammal, such as a mammal with or without an HIV infection. Administration of an immunogenic gpl20 or gpl40 polypeptide that induces an immune response can lead to protective immunity against HIV. In some examples, an immunogenic gpl20 or gpl40 polypeptide is a disclosed antigen that is resurfaced to focus immunogenicity to a target epitope.

Immunogenic surface: A surface of a molecule, for example a protein such as gpl20 or gpl40 protein or polypeptide, capable of eliciting an immune response. An immunogenic surface includes the defining features of that surface, for example the three-dimensional shape and the surface charge. In some examples, an immunogenic surface is defined by the amino acids on the surface of a protein or peptide that are in contact with an antibody, such as a neutralizing antibody, when the protein and the antibody are bound together. A target epitope includes an immunogenic surface. Immunogenic surface is synonymous with antigenic surface.

Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen- specific response"). In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies.

Immunogenic composition: A composition comprising an immunogenic peptide that induces a measurable CTL response against virus expressing the immunogenic peptide, or induces a measurable B cell response (such as production of antibodies) against the immunogenic peptide. In one example, an "immunogenic composition" is composition includes a disclosed antigen derived from a gpl20, or gpl40 peptide or an antigenic fragment thereof, such as a gpl20 outer domain (OD) peptide that induces a measurable CTL response against virus expressing gpl20 polypeptide, or induces a measurable B cell response (such as production of antibodies) against a gpl20, or gpl40 polypeptide. It further refers to isolated nucleic acids encoding an antigen, such as a nucleic acid that can be used to express the antigen (and thus be used to elicit an immune response against this polypeptide).

For in vitro use, an immunogenic composition may consist of the isolated protein, peptide epitope, or nucleic acid encoding the protein, or peptide epitope. For in vivo use, the immunogenic composition will typically include the protein, immunogenic peptide or nucleic acid in pharmaceutically acceptable carriers, and/or other agents. Any particular peptide, such as a disclosed antigen or a nucleic acid encoding the antigen, can be readily tested for its ability to induce a CTL or B cell response by art-recognized assays. Immunogenic compositions can include adjuvants, which are well known to one of skill in the art.

Inhibiting or treating a disease: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as acquired immune deficiency syndrome (AIDS), AIDS related conditions, HIV-1 infection, or combinations thereof. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term "ameliorating," with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of metastases, an improvement in the overall health or well- being of the subject, or by other parameters well known in the art that are specific to the particular disease. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.

Isolated: An "isolated" biological component (such as a protein, for example a disclosed antigen or nucleic acid encoding such an antigen) has been substantially separated or purified away from other biological components in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA, RNA, and proteins. Proteins, peptides and nucleic acids that have been "isolated" include proteins purified by standard purification methods. The term also embraces proteins or peptides prepared by recombinant expression in a host cell as well as chemically synthesized proteins, peptides and nucleic acid molecules.

Isolated does not require absolute purity, and can include protein, peptide, or nucleic acid molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.

Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule. Specific, non- limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. In some examples, a disclosed antigen is labeled with a detectable label. In some examples, label is attached to a disclosed antigen or nucleic acid encoding such an antigen.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be

synthesized, for example, using an automated DNA synthesizer. The term

"oligonucleotide" typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T."

"Nucleotide" includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

Conventional notation is used herein to describe nucleotide sequences: the left-hand end of a single- stranded nucleotide sequence is the 5'-end; the left-hand direction of a double-stranded nucleotide sequence is referred to as the 5'-direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand;" sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5'-end of the RNA transcript are referred to as "upstream

sequences;" sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences."

"cDNA" refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.

"Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (for example, rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. In some examples, a nucleic acid encodes a disclosed antigen. "Recombinant nucleic acid" refers to a nucleic acid having nucleotide sequences that are not naturally joined together. This includes nucleic acid vectors comprising an amplified or assembled nucleic acid which can be used to transform a suitable host cell. A host cell that comprises the recombinant nucleic acid is referred to as a "recombinant host cell." The gene is then expressed in the recombinant host cell to produce, such as a "recombinant polypeptide." A recombinant nucleic acid may serve a non-coding function (such as a promoter, origin of replication, ribosome-binding site, etc.) as well.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Peptide modifications: Peptides, such as the HIV immunogens disclosed herein can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a C -C ester, or converted to an amide of formula NR R₂ wherein Ri and R₂ are each independently H or CrC₁₆ alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring. Amino groups of the peptide, whether amino-terminal or side chain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HC1, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to CrC₁₆ alkyl or dialkyl amino or further converted to an amide.

Hydroxyl groups of the peptide side chains can be converted to C -C alkoxy or to a C -C ester using well-recognized techniques. Phenyl and phenolic rings of the peptide side chains can be substituted with one or more halogen atoms, such as F, CI, Br or I, or with C C^ alkyl, C C^ alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide side chains can be extended to homologous C₂-C4 alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the peptides of this disclosure to select and provide conformational constraints to the structure that result in enhanced stability. For example, a C- or N- terminal cysteine can be added to the peptide, so that when oxidized the peptide will contain a disulfide bond, generating a cyclic peptide. Other peptide cyclizing methods include the formation of thioethers and carboxyl- and amino-terminal amides and esters.

Peptide: Any compound composed of amino acids, amino acid analogs, chemically bound together. Peptide as used herein includes oligomers of amino acids, amino acid analog, or small and large peptides, including polypeptides or proteins. Any chain of amino acids, regardless of length or post-translational modification (such as glycosylation or phosphorylation). "Peptide" applies to amino acid polymers to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer as well as in which one or more amino acid residue is a non-natural amino acid, for example a artificial chemical mimetic of a

corresponding naturally occurring amino acid. A "residue" refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic. A peptide has an amino terminal (N-terminal) end and a carboxy terminal (C-terminal) end.

A "protein" is a peptide that folds into a specific three-dimensional structure. A protein can include surface-exposed amino acid resides and non-surface-exposed amino acid resides. "Surface-exposed amino acid residues" are those amino acids that have some degree of exposure on the surface of the protein, for example such that they can contact the solvent when the protein is in solution. In contrast, non-surface- exposed amino acids are those amino acid residues that are not exposed on the surface of the protein, such that they do not contact solution when the protein is in solution. In some examples, the non-surface-exposed amino acid residues are part of the protein core.

A "protein core" is the interior of a folded protein, which is substantially free of solvent exposure, such as solvent in the form of water molecules in solution.

Typically, the protein core is predominately composed of hydrophobic or apolar amino acids. In some examples, a protein core may contain charged amino acids, for example aspartic acid, glutamic acid, arginine, and/or lysine. The inclusion of uncompensated charged amino acids (a compensated charged amino can be in the form of a salt bridge) in the protein core can lead to a destabilized protein. That is, a protein with a lower T_m then a similar protein without an uncompensated charged amino acid in the protein core. In other examples, a protein core may have a cavity within the protein core. Cavities are essentially voids within a folded protein where amino acids or amino acid side chains are not present. Such cavities can also destabilize a protein relative to a similar protein without a cavity. Thus, when creating a stabilized form of a protein, it may be advantageous to substitute amino acid residues within the core in order to fill cavities present in the wild-type protein.

Amino acids in a peptide, polypeptide or protein generally are chemically bound together via amide linkages (CONH). Additionally, amino acids may be bound together by other chemical bonds. For example, linkages for amino acids or amino acid analogs can include CH₂NH-, -CH₂S-, -CH₂-CH₂ -, -CH=CH- (cis and trans), -COCH₂ -, -CH(OH)CH₂-, and -CHH₂SO- (These and others can be found in Spatola, in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci pp. 463-468, 1980; Hudson, et al., Int J Pept Prot Res 14: 177-185, 1979; Spatola et al. Life Sci 38: 1243-1249, 1986; Harm /. Chem. Soc Perkin Trans. 1307-314, 1982; Almquist et al. J. Med. Chem. 23: 1392- 1398, 1980; Jennings-White et al. Tetrahedron Lett 23:2533, 1982; Holladay et al. Tetrahedron. Lett 24:4401-4404, 1983; and Hruby Life Sci 31: 189-199, 1982. Pharmaceutical agent or drug: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of the proteins and other compositions herein disclosed.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions, powder, pill, tablet, or capsule forms, conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein is one in which the protein is more enriched than the protein is in its natural environment within a cell. Preferably, a preparation is purified such that the protein represents at least 50% of the protein content of the preparation.

The resurfaced immunogens disclosed herein, or antibodies that specifically bind the disclosed resurfaced immunogens, can be purified by any of the means known in the art. See for example Guide to Protein Purification, ed. Deutscher, Meth. Enzymol. 185, Academic Press, San Diego, 1990; and Scopes, Protein

Purification: Principles and Practice, Springer Verlag, New York, 1982. Substantial purification denotes purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95% or 98% pure. Thus, in one specific, non-limiting example, a substantially purified protein is 90% free of other proteins or cellular components.

Resurfaced antigen or resurfaced immunogen: A polypeptide immunogen derived from a wild-type antigen in which amino acid residues outside or exterior to a target epitope are mutated in a systematic way to focus the immunogenicity of the antigen to the selected target epitope. In some examples a resurfaced antigen is referred to as an antigenically-cloaked immunogen or antigenically-cloaked antigen.

Sequence identity/similarity: The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith &

Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988;

Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio.

24:307-31, 1994. Altschul et al, J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 nucleotides is 75.0 percent identical to the test sequence (1166÷1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer.

For sequence comparison of nucleic acid sequences and amino acids sequences, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are used. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity method of Pearson & Lipman, Proc. Nat' I. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see for example, Current Protocols in Molecular Biology (Ausubel et al., eds 1995 supplement)). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Blastn is used to compare nucleic acid sequences, while blastp is used to compare amino acid sequences. Additional information can be found at the NCBI web site.

Another example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and the BLAST 2.0 algorithm, which are described in Altschul et al., J. Mol. Biol. 215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977. Software for performing BLAST analyses is publicly available through the National Center for

Biotechnology Information (World Wide Web address ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLASTP program (for amino acid sequences) uses as defaults a word length (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915, 1989).

Another indicia of sequence similarity between two nucleic acids is the ability to hybridize. The more similar are the sequences of the two nucleic acids, the more stringent the conditions at which they will hybridize. The stringency of hybridization conditions are sequence-dependent and are different under different environmental parameters. Thus, hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na⁺ and/or Mg⁺⁺ concentration) of the hybridization buffer will determine the stringency of hybridization, though wash times also influence stringency. Generally, stringent conditions are selected to be about 5°C to 20°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.

Conditions for nucleic acid hybridization and calculation of stringencies can be found, for example, in Sambrook et ah, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; Tijssen, Hybridization With Nucleic Acid Probes, Part I: Theory and Nucleic Acid

Preparation, Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Ltd., NY, NY, 1993. and Ausubel et al. Short Protocols in

Molecular Biology, 4^th ed., John Wiley & Sons, Inc., 1999.

"Stringent conditions" encompass conditions under which hybridization will only occur if there is less than 25% mismatch between the hybridization molecule and the target sequence. "Stringent conditions" may be broken down into particular levels of stringency for more precise definition. Thus, as used herein, "moderate stringency" conditions are those under which molecules with more than 25% sequence mismatch will not hybridize; conditions of "medium stringency" are those under which molecules with more than 15% mismatch will not hybridize, and conditions of "high stringency" are those under which sequences with more than 10% mismatch will not hybridize. Conditions of "very high stringency" are those under which sequences with more than 6% mismatch will not hybridize. In contrast nucleic acids that hybridize under "low stringency conditions include those with much less sequence identity, or with sequence identity over only short subsequences of the nucleic acid.

T Cell: A white blood cell critical to the immune response. T cells include, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ T lymphocyte is an immune cell that carries a marker on its surface known as "cluster of differentiation 4" (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8⁺ T cells carry the "cluster of differentiation 8" (CD8) marker. In one embodiment, a CD8 T cells is a cytotoxic T lymphocytes. In another embodiment, a CD8 cell is a suppressor T cell.

Therapeutic agent: A chemical compound, small molecule, or other composition, such as nucleic acid molecule, capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.

Therapeutically effective amount or Effective amount: The amount of agent, such as nucleic acid vaccine or other therapeutic agent, that is sufficient to prevent, treat (including prophylaxis), reduce and/or ameliorate the symptoms and/or underlying causes of any of a disorder or disease, for example to prevent, inhibit, and/or treat HIV. In some embodiments, an "effective amount" is sufficient to reduce or eliminate a symptom of a disease, such as AIDS. For instance, this can be the amount necessary to inhibit viral replication or to measurably alter outward symptoms of the viral infection, such as increase of T cell counts in the case of an HIV-1 infection. In general, this amount will be sufficient to measurably inhibit virus (for example, HIV) replication or infectivity. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in lymphocytes) that has been shown to achieve in vitro inhibition of viral replication. An "anti- viral agent" or "anti-viral drug" is an agent that specifically inhibits a virus from replicating or infecting cells. Similarly, an "anti-retro viral agent" is an agent that specifically inhibits a retrovirus from replicating or infecting cells.

Transformed: A transformed cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors,

transformation with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

Vaccine: A pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine elicits an antigen- specific immune response to an antigen of a pathogen, for example a viral pathogen, or to a cellular constituent correlated with a pathological condition. A vaccine may include a polynucleotide (such as a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (such as a disclosed antigen), a virus, a cell or one or more cellular constituents.

Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant DNA vectors having at least some nucleic acid sequences derived from one or more viruses.

Virus: A virus consists essentially of a core of nucleic acid surrounded by a protein coat, and has the ability to replicate only inside a living cell. "Viral replication" is the production of additional virus by the occurrence of at least one viral life cycle. A virus may subvert the host cells' normal functions, causing the cell to behave in a manner determined by the virus. For example, a viral infection may result in a cell producing a cytokine, or responding to a cytokine, when the uninfected cell does not normally do so. In some examples, a virus is a pathogen.

"Retroviruses" are RNA viruses wherein the viral genome is RNA. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate which is integrated very efficiently into the chromosomal DNA of infected cells. The integrated DNA intermediate is referred to as a pro virus. The term "lentivirus" is used in its conventional sense to describe a genus of viruses containing reverse transcriptase. The lentiviruses include the "immunodeficiency viruses" which include human immunodeficiency virus (HIV) type 1 and type 2 (HIV-1 and HIV-2), simian immunodeficiency virus (SIV), and feline

immunodeficiency virus (FIV).

HIV-1 is a retrovirus that causes immunosuppression in humans (HIV disease), and leads to a disease complex known as the acquired immunodeficiency syndrome (AIDS). "HIV disease" refers to a well-recognized constellation of signs and symptoms (including the development of opportunistic infections) in persons who are infected by an HIV virus, as determined by antibody or western blot studies. Laboratory findings associated with this disease are a progressive decline in T cells.

Virus-like particle (VLP): A non-replicating, viral shell, derived from any of several viruses. VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for producing particular VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. See, for example, Baker et al. (1991) Biophys. J. 60: 1445-1456; and Hagensee et al. (1994) J. Virol. 68:4503- 4505. For example, VLPs can be isolated by density gradient centrifugation and/or identified by characteristic density banding. Alternatively, cryoelectron microscopy can be performed on vitrified aqueous samples of the VLP preparation in question, and images recorded under appropriate exposure conditions.

Wild-type antigen: An antigen that has not been modified by selective mutation to focus that antigenicity of the antigen to a target epitope.

//. Description of Several Embodiments

As the human immunodeficiency virus (HIV) pandemic continues to infect millions of people each year, the need for an effective vaccine increases. However the development of such a vaccine has been stymied due to the difficulty in developing an immunogen capable of eliciting broadly neutralizing antibodies. The current disclosure meets these needs.

Because many epitopes on HIV are derived from different and noncontiguous regions of the primary amino acid sequence of the antigen, it has proven difficult to synthetically reproduce these complex epitopes. Thus, an effective immunogen and vaccine will likely need to be derived from a wild type HIV antigen.

One of the major hurdles to the construction of such an antigen is focusing the immune response to regions of HIV proteins which mostly produce broadly neutralizing antibodies. As disclosed herein, the inventors have constructed a series of HIV immunogens based on the HIV glycoprotein gpl20 that focus the immune response to the CD4 binding site of gpl20 by a using combination of antigen resurfacing and/or paring back the structural elements of gpl20 that do not contribute to elicitation of broadly neutralizing antibodies. These appropriately constructed immunogens retain the ability to stimulate the production of neutralizing antibodies directed to a target epitope, while eliminating extraneous epitopes on gpl20 that do not contribute to the elicitation of neutralizing epitopes. Such molecules have utility as both potential vaccines for HIV and as diagnostic molecules (for example, to detect and quantify target antibodies in a polyclonal serum response). A. Antigens

Provided herein in various embodiments are antigens derived from HIV glycoproteins, which are useful to induce immunogenic responses in vertebrate animals (such as mammals, for example primates, such as humans) to HIV (for example HIV-1 and HIV-2). In some embodiments, the antigen is an HIV antigen, such is a modified gpl20, or gpl40 or an immunogenic fragment thereof. In specific embodiments, the disclosed antigen is HIV-1 gpl20 or an immunogenic fragment thereof, for example, the outer domain (OD) of gpl20.

The disclosed antigens have been substantially resurfaced from the wild type sequence, such that the surface of the antigen has been altered to focus the immune response to a particular feature, or epitope on the surface of gpl20 or gpl40. In some embodiments, the disclosed antigen is a resurfaced gpl20 antigen in which the one or more of the VI, V2, V3, V4 and/or V5 variable loops from gpl20 or gpl40 or an immunogenic fragment, thereof such as a gpl20 outer domain, are removed or truncated. In some examples, the disclosed antigens have been modified to substitute the surface-exposed amino acids located exterior to the target epitope to focus the antigenicity of the antigen to the target epitope. For example, the method can remove non-target epitopes that might interfere with specific binding of an antibody to the target epitope. In some examples, the amino acid substitutions result in the antigen not being bound by antibodies in a polyclonal serum that specifically bind surface-exposed amino acid residues of the wild-type antigen located exterior of the target epitope. In some embodiments, the amino acid substitutions alter antigenicity of the antigen in vivo as compared to the wild-type antigen (unsubstituted antigen), but do not introduce additional glycosylation sites as compared to the wild-type antigen. In some embodiments, that antigen is glycosylated. Examples of antigen resurfacing methods are given in PCT Publication No. WO 09/100376, which is specifically incorporated by reference in its entirety.

HIV-I can be classified into four groups: the "major" group M, the "outlier" group O, group N, and group P. Within group M, there are several genetically distinct clades (or subtypes) of HIV-I. The disclosed immunogens can be derived from any subtype of HIV, such as groups M, N, O, or P or clade A, B, C, D, F, G, H, J or K and the like. The nucleic acid sequence can encode an Env polypeptide from any group or clade of HIV. HIV gpl20 and gpl40 proteins from the different HIV clades, as well as nucleic acid sequences encoding such proteins and methods for the manipulation and insertion of such nucleic acid sequences into vectors, are known (see, e.g., HIV Sequence Compendium, Division of AIDS, National Institute of Allergy and Infectious Diseases (2003); HIV Sequence Database (hiv- web.lanl.gov/content/hiv-db/mainpage.html); Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N. Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene

Publishing Associates and John Wiley & Sons, New York, N. Y. (1994)).

In some examples, the disclosed antigen is a gpl20 from a HIV-1 Clade A virus. In some examples, the HIV-1 Clade A virus is KER2018, such that the disclosed immunogen is a gpl20 or immunogenic fragment thereof from HIV-1 KER2018. In some examples, the disclosed antigen is a gpl20 from a HIV-1 Clade C virus. In some examples, the HIV-1 Clade C virus is DU156, such that the disclosed immunogen is a gpl20 or immunogenic fragment thereof from HIV-1 DU156. In some examples, the HIV-1 Clade C virus is ZA012, such that the disclosed immunogen is a gpl20 or immunogenic fragment thereof from HIV-1 ZA012.

In some embodiments, a disclosed antigen includes the outer domain of a gpl20.

In some examples, the outer domain of gpl20 includes amino acid residues 252-482 of gpl20. In some examples, the outer domain of gpl20 includes amino acid residues 213-492 of gpl20. In some examples, the outer domain of gpl20 includes amino acid residues 213-482 of gpl20. In some examples, the outer domain of gpl20 includes amino acid residues 252-492 of gpl20. In specific embodiments, the disclosed antigen is HIV-1 gpl20. In other specific embodiments, the disclosed antigen is HIV-1 gpl40.

In some examples, V1/V2 and β20/β21 regions of gpl20 or gpl40 are modified to reduce the immunogenicity or at least alter the immunogenicity of those regions. In some examples, the β20/β21 bridging sheet of the gpl20 or gpl40 antigen or immunogenic fragment thereof is removed by replacing the amino acid residues between 1423 and Y435 with Gly-Gly. In some examples, the β20/β21 bridging sheet of the antigen is removed, for example by replacing residues 422-436 of gpl20 or gpl40 or the outer domain of gpl20 with Gly-Gly. In some examples, residues 128-194 of gpl20 in the VI, V2 loop region are replaced by Gly-Arg-Gly. In some examples, a modified V1/V2 is taken from a core gpl20 previously designed that has improved expression yields known as "new 9c" (see International Patent Publication NO. WO 2007/030518, which is incorporated herein by reference), and includes the insertion of VKLTPLAGATSVITQA (SEQ ID NO: 162) between C 119 and C205.

In some examples, the wild type VI loop (residues 131-157) includes the amino acid sequence CTDLRNATNTTSSSWETMEKGEIKNC (SEQ ID NO: 163). In some examples, the VI loop is replaced with the amino acid sequence

CTDLRGSGGGSGGGSEIKNC (SEQ ID NO: 164), which in some examples is designated AVI.1.

In some examples, the wild type V2 loop (residues 157-198) includes the amino acid sequence CSFNIT TSIRDKVQKEYALFYKLDVVPIDNDNTSYRLINC (SEQ ID NO: 165). In some examples, the V2 loop is replaced with the amino acid sequence CSFNIT TSIRDKVQKEYALFYKLDVVPIGGSGGSYRLINC (SEQ ID NO: 166), which in some examples is designated V2.1, or the amino acid sequence CSFNIT TSIRDKVQKEYALFYKLDVVPIGSGGGSGSYRLINC (SEQ ID NO: 167), which in some examples is designated V2.2.

In some embodiments, the V3 loop of a disclosed antigen is mutated and/or truncated as compared to a wild type gpl20, gpl40 or immunogenic fragment thereof. In some examples, the wild-type V3 loop (residues 296-331) includes the amino acid sequence CSRPNNNTRKSIPMGPGRAFYTTGQIIGDIRQAHC (SEQ ID NO: 168). In some examples, the V3 loop is shortened by 18 amino acids, for example by removing 9 amino acids from each end of the loop, which in certain examples is designated V3-9,9. In some examples, residues 302-323 of gpl20, part of the V3 loop, are replaced with a basic hexapeptide (NTRGRR; SEQ ID NO: 169). In some examples, residues 305-323 of gpl20 in the V3 loop are replaced by Gly- Arg-Arg. In some examples, the V3 loop is replaced with the m5 loop, having the amino acid sequence CSRPNNNTRGRRGSSGGSHC (SEQ ID NO: 170). In some examples, the V3 loop is replaced with the m4 loop, having the amino acid sequence CSRPNNGGSGSGGSSGGSHC (SEQ ID NO: 171). In some examples, the V3 loop is replaced with Gly-Val-Gly. In some examples, the V3 loop is at least partially replaced with Gly-Ser. In some examples, the V3 loop is at least partially replaced with Gly-Ser-Leu. In some examples, the V3 loop is truncated to an 11-mer. In some examples, the V3 loop is replaced with the amino acid sequence

CARPSNNTRGRRGDIRQAYC (SEQ ID NO: 172). In some examples, the V3 loop is replaced with the amino acid sequence CARPSNNTDIRQAYC (SEQ ID NO:

173), which in some examples is designated V3.1. In some examples, the V3 loop is replaced with the amino acid sequence CARPSNNTRQAYC (SEQ ID NO: 174), which in some examples is designated V3.2. In some examples, the V3 loop is replaced with the amino acid sequence CARPSNNTQYC (SEQ ID NO: 175), which is some examples is designated V3.3. In some examples, the V3 loop is replaced with the amino acid sequence CARGSGSGSYC (SEQ ID NO: 176), which in some examples is designated V3.4. In some examples, the V3 loop is replaced with the amino acid sequence CSRPNNNTRGRRGDIRQAHC (SEQ ID NO: 177), which in some examples is designated V3(GSL). In some examples, the V3 loop is replaced with the amino acid sequence CSRPNNNTRRQAHC (SEQ ID NO: 178), which in some examples is designated V3.2. In some examples, the V3 loop is replaced with the amino acid sequence CSRPNNGGSGQAHC (SEQ ID NO: 179), which in some examples is designated V3.2GS. In some examples, the wild-type V3 loop includes the amino acid sequence CTRPNNNTRKSIHIGPGQAFYATGDIIGDIRQAHC (SEQ ID NO: 205). In some examples, the V3 loop is replaced with the amino acid sequence CTRPNNGGSGSGGSSGGSHC (SEQ ID NO: 180), which in some examples is designated V3.4. In some examples, the V3 loop is replaced with the amino acid sequence CTRPNNNTRGRRGSSGGSHC (SEQ ID NO: 181), which in some examples is designated V3.5. In some examples, the V3 loop is replaced with a 15 mer with a native glycan at the tip. In some examples, the V3 loop is replaced with al5 mer with a slightly shifted glycan at the tip. In some embodiments, the V4 loop of a disclosed antigen is mutated and/or truncated as compared to a wild type gpl20, gpl40 or immunogenic fragment thereof. In some examples, the V4 loop is at least partially replaced with Gly-Ser. In some examples, 9 amino acids are removed from the V4 loop, which in some examples is designated V4.2.1. In some examples, the V4 loop is shortened to SIWNNGGGSGGGSGGGSDTIT (SEQ ID NO: 182), which in some examples is designated V4.GS. In some examples, the V4 loop is replaced with the V4 loop from strain Ker2018 (a clade A strain), which in some examples is designated V4.Ker A. In some examples, one of the two glycan sites is removed from the V4 loop from strain Ker2018, which in some examples is designated V4.Ker A/AG2, or V4.Ker A/AGl. In some examples, both of the two glycan sites are removed from the V4 loop from strain Ker2018r, which in some examples is designated V4.Ker A/AG2, or V4.Ker A/AG12. In some examples, the wild-type V4 loop includes the amino acid sequence STWFNSTWSTKGSNNTEGSDTIT (SEQ ID NO: 183). In some examples, the V4 loop is replaced with the amino acid sequence

STWFNGSGSGGSGTIT (SEQ ID NO: 184), which in some examples is designated V4.1. In some examples, the V4 loop is replaced with the amino acid sequence STWFNSTWSTKGSNNTEGSDTIT (SEQ ID NO: 185), which in some examples is designated V4.2. In some examples, the V4 loop is replaced with the amino acid sequence STWFQGSGSGGSGTIT (SEQ ID NO: 186), which in some examples is designated V4.3. In some examples, the glycan site at the N terminal end of the V4 is removed, which is designated the V4.7 loop. In some examples, the glycan site at the C terminal end of the V4 is removed, which is designated v4.8. In some examples, the glycan site at both the N and C terminal end is removed, which is designated v4.9. In some examples, the V4 loop is replaced with a Gly-Ser repeat, which is designated V4.x

In some embodiments, the V5 loop of a disclosed antigen is mutated and/or truncated as compared to a wild type gpl20, gpl40 or immunogenic fragment thereof. In some examples, the V5 loop is at least partially replaced with Gly-Ser. In some examples, a wild type V5 loop has the amino acid sequence GGNTGNNSRTC (SEQ ID NO: 202). In some examples, the V5 loop is truncated to a 7-mer. In some examples, the V5 loop is truncated to a 5-mer. In some examples, the V5 loop is replaced with the amino acid sequence GGNTNRTC (SEQ ID NO: 187), which in some examples is designated V5.1. In some examples, the V5 loop is replaced with the amino acid sequence GGSGSGTC (SEQ ID NO: 188), which in some examples is designated V5.2. In some examples, the V5 loop is replaced with the amino acid sequence GGSGSTC (SEQ ID NO: 189), which in some examples is designated V5.2. In some examples, the V5 loop is truncated to a 16-mer. In some examples, the V5 loop is truncated to NDSDGNETFR (SEQ ID NO: 190) for example from KDDNSRDGNETFR (SEQ ID NO: 191), which in some examples is designated V5.2.1. In some examples, the V5 loop is replaced with the amino acid sequence

SGGSGQETFR (SEQ ID NO: 192), which in some examples is designated V5.2GS. In some examples, the wild-type V5 loop includes the amino acid sequence

GGNDNNESEI (SEQ ID NO: 193). In some examples, the V5 loop is replaced with the amino acid sequence GGGSGSGEI (SEQ ID NO: 194), which in some examples is designated V5.1. In some examples, the V5 loop is replaced with an 8 amino acid Gly-Ser repeat.

In some embodiments, an immunogen is an outer domain of gpl20 and includes residues 252-482 of gpl20. In specific embodiments, the outer domain of gpl20, and thus the immunogen, includes the sequence set forth as SEQ ID NO: 58, or a variant thereof that retains the outer domain fold of gpl20. In some examples, the outer domain of gpl20 of includes additional mutations. In specific examples, the immunogen including the outer domain of gpl20 includes one or more of the following mutations made in the context of SEQ ID NO: 203 (OD 1.0): 273N, T283N, T339N, A341T, 360N, 362N, N363Q, P369N, 137 IT, 37 IN, T373N, T373G, 377N, A379T, F383T, N386Q, D392N, R419N, G421N, G424N, A431T, P437T, N465Q, W479N, N280C and G458, K358C and N465C, and/or V255C and M475C. The cysteine residues can be introduced to stabilize the polypeptide, for example by stabilizing the loops and/or core of the folded polypeptide. When the residue number is not proceeded by a residue but is followed by a N it is meant that an asparagine is introduced at that position regardless of the starting residue, for example to insert a glycosylation site at that position. In specific examples, the immunogen including the outer domain of gpl20 includes one or more of the following mutations made in the context of SEQ ID NO: 1 (A2.3_KER2018.11): 273N, T283N, T339N, A341T, 360N, 362N, N363Q, P369N, 137 IT, 37 IN, T373N, T373G, 377N, A379T, F383T, N386Q, D392N, R419N, G421N, G424N, A431T, P437T, N465Q, W479N, N280C and G458, K358C and N465C, and/or V255C and M475C. The cysteine residues can be introduced to stabilize the polypeptide, for example by stabilizing the loops and/or core of the folded polypeptide. In specific examples, the immunogen including the outer domain of gpl20 includes one or more of the following mutations made in the context of SEQ ID NO: 1: V257N, V272N, N276Q, N276D, N276E, T283V,

A297T, D368R, N362T, E363N, P364S, T373N, S375T, F382T, S388A, A388S, K389D, E398N, N406Q, N410Q, K421T, G422V, V427N, V442N, R444T, N478L, S48 IT, and/or E482S.

In specific examples, the immunogen including the outer domain of gpl20 includes one or more of the following mutations made in the context of SEQ ID NO: 78 (HXB2 core): T26N, K55N, A49S, D91T, T92N, D94T, D98N, M100T V103N, K113N, P118N, T207N, R209T, V257N, V272N, N276D, R421N, I423T, T424N, M426T, I434N, A436T, R487N, V489T, and/or R490N. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations made in the context of SEQ ID NO: 74: T26N, V257N, A49S, V103N, V272N. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations made in the context of SEQ ID NO: 74: T84N, A107S, K113N, V244N, V427N, and V442N.

In some examples, the immunogen includes the amino acid sequence set forth as anyone of SEQ ID NOs: 74-76. In some examples, the immunogen includes the amino acid sequence set forth as anyone of SEQ ID NOs: 74-76.

In some examples, glycosylation sites are mutational introduced into the

immunogen, for example as show in the table in FIG. 5, in which the numbers indicate the positions of glycosylation. In some examples, glycosylation sites are introduced at one or more of position 295, 442, 479, 272, 377, 436, 424, 398, 348, and/or 369. In some examples glycosylation sites are introduced positions 295 and 442, which is termed HG.l. In some examples glycosylation sites are introduced positions 295, 442, and 479, which is termed HG.2. In some examples glycosylation sites are introduced positions 295, 442, and 436, which is termed HG.3. In some examples glycosylation sites are introduced positions 295, 442, and 424, which is termed HG.4. In some examples glycosylation sites are introduced positions 295, 442, and 398, which is termed HG.5. In some examples glycosylation sites are introduced positions 295, 442, and 369, which is termed HG.6. In some examples glycosylation sites are introduced positions 295, 442, 273, 377, and 348, which is termed HG.7. In some examples glycosylation sites are introduced positions 295, 442, 479, 273, 377, 436*, 398, 348, and 369, which is termed HG.8. In some examples glycosylation sites are introduced positions 295, 442, 479, 273, 377, 424*, 398, 348, and 369, which is termed HG.9. In some examples glycosylation sites are introduced positions 295, 442, 479, 273, and 377, which is termed HG.10. In specific examples, the immunogen including the outer domain of gpl20 includes one or more of the following mutations V257N, V272N, N276Q, N276D, N276E, T283V, A297T, D368R, N362T, E363N, P364S, T373N, S375T, F382T, S388A, A388S, K389D, E398N, N406Q, N410Q, K421T, G422V, V427N, V442N, R444T, N478L, S481T, and/or E482S. Mutations that are recited outside of the gpl20 outer domain are made in the context of a larger HIV immunogen, such as a gpl20 or gpl40 immunogen.

In specific examples, the immunogen including the outer domain of gpl20 includes one or more of the following mutations V257N, V272N, N276Q, N276D, N276E, T283V, A297T, D368R, N362T, E363N, P364S, T373N, S375T, F382T, S388A, A388S, K389D, E398N, N406Q, N410Q, K421T, G422V, V427N, V442N, R444T, N478L, S48 IT, and/or E482S. Mutations that are recited outside of the gpl20 outer domain are made in the context of a larger HIV immunogen, such as a gpl20 or gpl40 immunogen. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations N363Q, K358C- N465C, T283N, N386Q, and N465Q. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations K358C- N465C, T283N, N386Q, and N465Q. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations V255C- M475C, T283N, D392N, T339N, and N465Q. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations N363Q, V255C-M475C, T283N, N465Q, D392N, T339N, A431T, and T373N. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations N363Q, V255C-M475C, T283N, N465Q, and R419N. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations V255C-M475C, T283N, N465Q, D392N, T339N, G424N, and P437T. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations N276D, T283N, A297T, E398N, V442N, R444T, N478L, W479N, S481T, E482S, N362T, E363N, and P364S. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations K358C-N465C, T283N, and N386Q. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations K358C-N465C, T283N, N386Q, and N465Q. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations K358C-N465C, T283N, N386Q, and N465Q. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations F383T, and N280C-G458C, In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations F383T, K358C-N465C, In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations F383T, V255C- M475C. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations D392N, T339N, and A341T, In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations D392N, T339N, A341T, and 386N, In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations P369N and 137 IT. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations 42 IN, 377N, and 479N. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations 421N, 377N, 479N, and 273N. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations 424N, 377N, 479N, 273N. In specific examples, the

immunogen including the outer domain of gpl20 includes all of the following mutations 424N, 377N, and 479N. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations 421N and 363N. In specific examples, the immunogen including the outer domain of gpl20 includes all of the following mutations 421N, 377N, 479N, and 363N.

In some embodiments the antigen is a multimer, such as a multimer of gpl20, gpl40 or an immunogenic fragment thereof, for example, a dimer, trimer, etc. of gpl20, gpl40 or an immunogenic fragment thereof, such as an outer domain of gpl20. Several exogenous oligomerization motifs have been successfully used to promote stable trimers of soluble recombinant proteins: the GCN4 leucine zipper (Harbury et al. 1993 Science 262: 1401-1407), the trimerization motif from the lung surfactant protein (Hoppe et al. 1994 FEBS Lett 344: 191-195), collagen (McAlinden et al. 2003 J Biol Chem 278:42200-42207), and the phage T4 fibritin foldon

(Miroshnikov et al. 1998 Protein Eng 11:329-414). Thus, in some examples, the disclosed antigen includes one or more of a foldon domain. In specific examples, the foldon domain is a T4 fibritin foldon domain such as the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTF (SEQ ID NO: 195), which adopts a β- propeller conformation, and can fold and trimerize in an autonomous way (Tao et al. 1997 Structure 5:789-798). In some specific examples, an immunogen including a T4 fibritin foldon domain includes the amino acid sequence set forth as one of SEQ ID NOs: 71-74.

In some embodiments, the disclosed antigen includes a ferritin polypeptide or hybrid of different ferritin polypeptides (for example, to induce multimerization). In specific examples, the ferritin polypeptide is E. coli ferritin, human light chain ferritin, bullfrog ferritin or a hybrid thereof, such as E. co/i-human hybrid ferritin, E. co/i-bullfrog hybrid ferritin, or human-bullfrog hybrid ferritin. Exemplary amino acid sequences of ferritin polypeptides and nucleic acid sequences encoding ferritin polypeptides for use in the disclosed antigens can be found in GENBANK®, for example at accession numbers ZP_03085328, ZP_06990637, AAA35832, NP_000137 AAA49532, AAA49525, AAA49524 and AAA49523, which are specifically incorporated by reference herein in their entirety as available June 29, 2010; and in the Protein Data Base (PDB), for example at PDB accession numbers leum, 2ffx and lrcc, which are specifically incorporated by reference herein in their entirety as available June 29, 2010.

Ferritin is the iron ion storage protein ubiquitously found in almost all living organisms, including bacteria, fungi, and higher plants, and animals. It forms an octahedron consisting of 24 subunits of -20 kDa protein. In some examples, a disclosed immunogen has been genetically fused to the amino terminus of engineered ferritin, such as eumrcc (a hybrid E coli and human ferritin), with a Ser- Gly linker. When the constructs have been made in HEK 293 Freestyle cells, the fusion proteins are secreted from the cells and self-assembled into octahedral particles. The particles can be purified by a few different chromatography procedures, e.g. Mono Q (anion exchange) followed by size exclusion

(SUPEROSE® 6) chromatography.

In some embodiments, a disclosed antigen includes a transmembrane domain, for example to anchor the antigen to the surface of a cell. In some examples, the transmembrane domain is a gp41 transmembrane domain. In some examples, the transmembrane domain is a CD4 transmembrane domain. In specific examples, a CD4 transmembrane domain is set forth as the amino acid sequence LrVLGGVAGLLLFIGLGI (SEQ ID NO: 196). In some embodiments, a transmembrane domain is an influenza transmembrane domain, such as a neuraminidase (NA) or hemegglutin (HA) amino acid sequence. In specific examples, the HA sequence includes the amino acid sequence

ILAIYSTVASSLVLLVSLGAISF (SEQ ID NO: 197). In some specific examples, an immunogen including a HA transmembrane domain includes the amino acid sequence set forth as one of SEQ ID NOs: 53-55. In specific examples, the NA sequence includes the amino acid sequence

MNPNQKIITIGSICMVVGIISLILQIGNIISIWVS (SEQ ID NO: 198). In some specific examples, an immunogen including a HA transmembrane domain includes the amino acid sequence set forth as one of SEQ ID NOs: 56-57. In some examples a disclosed antigen includes a secretion signal sequence, such as human CD5-derived secretion signal sequence or an IL-2 secretion signal sequence at the N-terminus so that the antigen is secreted from a cell, for example to aid in production and purification of the antigen. In some examples, the secretion signal sequence is a CD5 leader amino acid sequence, such as

MPMGSLQPLATLYLLGMLVASVLA (SEQ ID NO. 199). In some specific examples, an immunogen including a CD5 leader amino acid sequence includes the amino acid sequence set forth as one of SEQ ID NOs: 53-55 and 75-76. In other examples, the secretion signal sequence, is an IL-2 secretion signal sequence, such as an murine IL-2 amino acid sequence, for example MQLASCVTLTLVLLVNSAP (SEQ ID NO: 200). In some specific examples, an immunogen including a murine IL-2 amino acid sequence includes the amino acid sequence set forth as one of SEQ ID NOs: 58-70.

In some examples, the disclosed antigen is a part of a virus-like particle (VLP), such as a CHIKV VLP. In one non-limiting example, the disclosed antigen such as a gpl20, gpl40 or immunogenic fragment thereof, for example a gpl20 outer domain (OD), is inserted between Chikungunya virus (CHIKV) E2 205 amino acid (a.a.) and 206 a.a. on CHIKV VLP (strain 37997). Immunogens are typically presented multimerically (240 molecules per CHIIKV VLP particle) to immune cells such as B cells and antigen presenting cells. This results in effectively inducing immune responses against the immunogen, in particular, antibody responses. In specific embodiments, the antigen that is part of a CHIKV VLP includes the amino acid sequence set forth as one of SEQ ID NOs: 79-80.

In some embodiments, a disclosed antigen includes a six-histidine residue tag (for example, to induce oligomerization and/or aid in purification). In some examples the disclosed antigen includes a 3C protease cleavage site, for example so that a 6X His tag or other peptide fragment, such as those described herein can be cleaved from the antigen.

A disclosed antigen can be covalently linked to a carrier, which is an immunogenic macromolecule to which an antigenic molecule can be bound. When bound to a carrier, the bound polypeptide becomes more immunogenic. Carriers are chosen to increase the immunogenicity of the bound molecule and/or to elicit higher titers of antibodies against the carrier which are diagnostic ally, analytically, and/or therapeutically beneficial. Covalent linking of a molecule to a carrier can confer enhanced immunogenicity and T cell dependence (see Pozsgay et ah, PNAS 96:5194-97, 1999; Lee et al, J. Immunol. 116: 1711-18, 1976; Dintzis et al, PNAS 73:3671-75, 1976). Useful carriers include polymeric carriers, which can be natural (for example, polysaccharides, polypeptides or proteins from bacteria or viruses), semi- synthetic or synthetic materials containing one or more functional groups to which a reactant moiety can be attached. Bacterial products and viral proteins (such as hepatitis B surface antigen and core antigen) can also be used as carriers, as well as proteins from higher organisms such as keyhole limpet hemocyanin, horseshoe crab hemocyanin, edestin, mammalian serum albumins, and mammalian

immunoglobulins. Additional bacterial products for use as carriers include bacterial wall proteins and other products (for example, streptococcal or staphylococcal cell walls and lipopolysaccharide (LPS)).

In some embodiments, the disclosed antigen includes one or more peptide linkers, for example to attach the gpl20, gpl40 or immunogenic fragment thereof to one or more of a foldon domain, a ferritin polypeptide, a hybrid of different ferritin polypeptides a six-histidine residue tag and a transmembrane domain and the like. Linker peptides are typically a short amino acid sequence providing a flexible linker that permits attachment of an antigenic polypeptide, such as a gpl20 or a gpl40 antigen or an antigenic fragment thereof, without disruption of the structure, aggregation (multimerization) or activity of the self-aggregating polypeptide component. Typically, a linear linking peptide consists of between two and 25 amino acids. Usually, the linear linking peptide is between two and 15 amino acids in length, although in certain circumstances it can be only one, such as a single glycine residue. In one example, the linker polypeptide is two to three amino acids in length, such as a serine and an arginine, or two serine residues and an arginine residue, or two arginine residues and a serine residue, two glycines and a serine, two serines and a glycine or any combination thereof. In specific examples, a peptide linker includes the amino acid sequence set forth as (G₃S)₂G (SEQ ID NO: 201). In specific examples, a peptide linker includes the amino acid sequence set forth as GSG. In other examples, a linker includes a single G. In other examples, the linear linking peptide can be a short sequence of alternating glycines and prolines, such as the amino acid sequence glycine-proline- glycine-proline. A linking peptide can also consist of one or more repeats of the sequence glycine-glycine-serine. Alternatively, the linear linking peptide can be somewhat longer, such as the glycine(4)- serine spacer described by Chaudhary et ah, Nature 339:394-397,1989.

The antigens disclosed herein can be chemically synthesized by standard methods, or can be produced recombinantly, for example by expression of the antigen from a nucleic acid molecule that encodes the antigen (see Section C below). An exemplary process for polypeptide production is described in Lu et ah,

Federation of European Biochemical Societies Letters. 429:31-35, 1998. They can also be isolated by methods including preparative chromatography and

immunological separations.

Exemplary amino acid sequences of gpl20, gpl40 antigens or immunogenic fragments thereof are given below as SEQ ID NOs: 1-77, 79 and 80. In some embodiments, a disclosed gpl20, gpl40 antigen or immunogenic fragment thereof comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth as one of SEQ ID NOs: 1-80, such as at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or even 100% identical to the amino acid sequence set forth as one of SEQ

ID NOs: 1-77, 79 and 80. In some embodiments, the a disclosed gpl20, gpl40 antigen or immunogenic fragment thereof consists of the amino acid sequence set forth as one of SEQ ID NOs: 1-77, 79 and 80.

A2.3_KER2018.11 (SEQ ID NO: 1):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTRG RRGDIRQAYCVVNRTQWNDTLGQVAIQLRKHWNTTIIFNEPSGGDLEITTHS FNCGGEFTYCNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGV IRCQSNITGILLTRDGGNTGNNSRTNETFRPGGGDMRDNWRSE

A2.5_KER2018.11 (SEQ ID NO: 2):

KPVVSTQLLLNGSLAEKEIRIKSENISDCAKTIIVQLTKPVLINCARPSNNTRG RRGDIRQAYCVVNRTQWNDTLGQVAIQLRKHWNTTIIFNEPSGGDLEITTHS FNCGGEFTYCNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGV IRCQSNITGILLTRDCGNTGNNSRTNETFRPGGGDMRDNWRSE

A2.6_KER2018.11 (SEQ ID NO: 3):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTRG RRGDIRQAYCVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHS FNCGGEFTYCNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGV IRCQSNITGILLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE. A2.7_KER2018.11 (SEQ ID NO: 4):

KPVCSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTRG RRGDIRQAYCVVNRTQWNDTLGQVAIQLRKHWNTTIIFNEPSGGDLEITTHS FNCGGEFTYCNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGV IRCQSNITGILLTRDGGNTGNNSRTNETFRPGGGDCRDNWRSE.

A2.6.1_KER2018.11 (SEQ ID NO: 5):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKNIIVQLTKPVLINCARPSNNTRG RRGDIRQAYCVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHS FNCGGEFTYCNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGV IRCQSNITGILLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE.

A2.6.2_KER2018.11 (SEQ ID NO: 6):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTRG RRGDIRQAYCVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHS FNCGGEFFYCNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGV IRCQSNITGILLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE.

A2.6.3_KER2018.11 (SEQ ID NO: 7):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKNIIVQLTKPVLINCARPSNNTRG RRGDIRQAYCVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHS FNCGGEFFYCNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGV IRCQSNITGILLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE.

A2.6.5_KER2018.11 (SEQ ID NO: 8):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKTIIVQLTKPVLINCARPSNNTRG RRGDIRQAYCVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHS FNCGGEFFYCNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGV IRCQSNITGILLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE. A2.8a_KER2018.11 (SEQ ID NO: 9):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGVIRCQSNITGI LLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE. A2.8b_KER2018.11(SEQ ID NO: 10):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARGSGSGSY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGVIRCQSNITGI LLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE.

A2.8j_KER2018.11 (SEQ ID NO: 11):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTRG RRGDIRQAYCVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHS FNCGGEFFYCNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGV IRCQSNITGILLTRDGGSGSGTCETFRPGGGDMRDNWRSE.

A2.8k_KER2018.11 (SEQ ID NO: 12):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTRG RRGDIRQA YCVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHS FNCGGEFFYCNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGV IRCQSNITGILLTRDGGSGSTCETFRPGGGDMRDNWRSE.

A2.8af_KER2018.11 (SEQ ID NO: 13):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASIDGTESNDNITLPCRIKGSGAPPIQGVIRCQSNITGI LLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE. A2.8ag_KER2018.11 (SEQ ID NO: 14):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASINGTESNDDITLPCRIKGSGAPPIQGVIRCQSNITGI LLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE.

A2.8ah_KER2018.11 (SEQ ID NO: 15):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASIDGTESNDDITLPCRIKGSGAPPIQGVIRCQSNITGI LLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE.

A2.8ai_KER2018.11 (SEQ ID NO: 16):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWGSGSGSGSDGSGSGSDITLPCRIKGSGAPPIQGVIRCQSNITG ILLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE.

A2.8aj_KER2018.11 (SEQ ID NO: 17):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGVIRCQSNITGI LLTRDGGSGSGTCETFRPGGGDMRDNWRSE.

A2.8afj_KER2018.11 (SEQ ID NO: 18):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASIDGTESNDNITLPCRIKGSGAPPIQGVIRCQSNITGI LLTRDGGSGSGTCETFRPGGGDMRDNWRSE. A2.8agj_KER2018.11 (SEQ ID NO: 19):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASINGTESNDDITLPCRIKGSGAPPIQGVIRCQSNITGI LLTRDGGSGSGTCETFRPGGGDMRDNWRSE.

A2.8ahj_KER2018.11 (SEQ ID NO: 20):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASIDGTESNDDITLPCRIKGSGAPPIQGVIRCQSNITGI LLTRDGGSGSGTCETFRPGGGDMRDNWRSE.

A2.8aij_KER2018.11 (SEQ ID NO: 21):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWGSGSGSGSDGSGSGSDITLPCRIKGSGAPPIQGVIRCQSNITG ILLTRDGGSGSGTCETFRPGGGDMRDNWRSE.

A2.9a_KER2018.11 (SEQ ID NO: 22):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGVIRCQSNITGI LLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE.

A2.9af_KER2018.11 (SEQ ID NO: 23):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASIDGTESNDNITLPCRIKGSGAPPIQGVIRCQSNITGI LLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE. A2.9ag_KER2018.11 (SEQ ID NO: 24):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASINGTESNDDITLPCRIKGSGAPPIQGVIRCQSNITGI LLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE. A2.9ah_KER2018.11 (SEQ ID NO: 25):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASIDGTESNDDITLPCRIKGSGAPPIQGVIRCQSNITGI LLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE.

A2.9ai_KER2018.11 (SEQ ID NO: 26):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWGSGSGSGSDGSGSGSDITLPCRIKGSGAPPIQGVIRCQSNITG ILLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE.

A2.9aj_KER2018.11 (SEQ ID NO: 27):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEIT THSFNCGGEFFY CNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGVIRCQSNITGI LLTRDGGSGSGTCETFRPGGGDMRDNWRSE.

A2.9afj_KER2018.11 (SEQ ID NO: 28):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASIDGTESNDNITLPCRIKGSGAPPIQGVIRCQSNITGI LLTRDGGSGSGTCETFRPGGGDMRDNWRSE. A2.9agj_KER2018.11 (SEQ ID NO: 29):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASINGTESNDDITLPCRIKGSGAPPIQGVIRCQSNITGI LLTRDGGSGSGTCETFRPGGGDMRDNWRSE

A2.9ahj_KER2018.11 (SEQ ID NO: 30):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASIDGTESNDDITLPCRIKGSGAPPIQGVIRCQSNITGI LLTRDGGSGSGTCETFRPGGGDMRDNWRSE

A2.9aij_KER2018.11 (SEQ ID NO: 31):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCARPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWGSGSGSGSDGSGSGSDITLPCRIKGSGAPPIQGVIRCQSNITG ILLTRDGGSGSGTCETFRPGGGDMRDNWRSE

A2.6.2 V3.1 (OD4.2F v3.1) (SEQ ID NO: 32):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTDIR QAYCVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGG EFFYCNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGVIRCQS NrrGILLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE

A2.6.2 V3.2 (OD4.2F v3.2) (SEQ ID NO: 33):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTRQ AYCVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEF FYCNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGVIRCQSNIT GILLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSE A2.6.2 V5.1 (OD4.2F v5.1) (SEQ ID NO: 34):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTRG RRGDIRQAYCVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHS FNCGGEFFYCNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGV IRCQSNITGILLTRDGGNTNRTCETFRPGGGDMRDNWRSE

A2.HG.1 (OD4.2' HG.l) (SEQ ID NO: 35):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCTRPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGNITCQSNrrGI LLTRDGGSGSGTCETFRPGGGDMRDNWRSE

A2.HG.2 (OD4.2' HG.2) (SEQ ID NO: 36):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCTRPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGNITCQSNrfGI LLTRDGGSGSGTCETFRPGGGDMRDLNRTS

A2.HG.3 (OD4.2' HG.3) (SEQ ID NO: 37):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCTRPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASINGTESNDNITLPCRINGTIQGNITCQSNITGILLTR DGGSGSGTCETFRPGGGDMRDNWRSE

A2.HG.4 (OD4.2' HG.4) (SEQ ID NO: 38):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCTRPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSNATVIQGNITCQSNrrGI LLTRDGGSGSGTCETFRPGGGDMRDNWRSE A2.HG.5 (OD4.2' HG.5) (SEQ ID NO: 39):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCTRPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNINGTASINGTESNDNITLPCRIKGSGAPPIQGNITCQSNITGI LLTRDGGSGSGTCETFRPGGGDMRDNWRSE A2.HG.6 (OD4.2' HG.6) (SEQ ID NO: 40):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCTRPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDNETTTHSFNCGGEFF YCNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGNITCQSNIT GILLTRDGGSGSGTCETFRPGGGDMRDNWRSE

A2.HG.7 (OD4.2' HG.7) (SEQ ID NO: 41):

KPVVSTQLLLNGSLAEKEIRINSTDISDNAKNIIVQLTKPVLINCTRPSNNTQY CVVNRTQWNDTLGQVAINLTKHWNTCIIFNEPSGGDLEITTHSFNCTGEFFY CNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGNITCQSNITGI LLTRDGGSGSGTCETFRPGGGDMRDNWRSE

A2.HG.8 (OD4.2' HG.8) (SEQ ID NO: 42):

KPVVSTQLLLNGSLAEKEIRINSTDISDNAKNIIVQLTKPVLINCTRPSNNTQY CVVNRTQWNDTLGQVAINLTKHWNTCIIFNEPSGGDNETTTHSFNCTGEFF Y CNTSDLFNSTWNINGTASINGTESNDNITLPCRINGTIQGNITCQSNITGILLTR DGGSGSGTCETFRPGGGDMRDLNRTS

A2.HG.9 (OD4.2' HG.9) (SEQ ID NO: 43):

KPVVSTQLLLNGSLAEKEIRINSTDISDNAKNIIVQLTKPVLINCTRPSNNTQY CVVNRTQWNDTLGQVAINLTKHWNTCIIFNEPSGGDNETTTHSFNCTGEFFY CNTSDLFNSTWNINGTASINGTESNDNITLPCRIKGSNATVIQGNITCQSNITG ILLTRDGGSGSGTCETFRPGGGDMRDLNRTS A2.HG.10 (OD4.2' HG.10) (SEQ ID NO: 44):

KPVVSTQLLLNGSLAEKEIRINSTDISDNAKNIIVQLTKPVLINCTRPSNNTQY CVVNRTQWNDTLGQVAINLTKHWNTCIIFNEPSGGDLEITTHSFNCTGEFFY CNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGNITCQSNITGI LLTRDGGSGSGTCETFRPGGGDMRDLNRTS

A2.HG.3.1 (OD4.2' HG.3.1) (SEQ ID NO: 45):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCTRPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASINGTESNDNITLPCRINGTIQGNITCQSNITGILLTR DGGSGSGTCETFRPGGGDMRDLNRTS

A2.HG.3.2 (OD4.2' HG.3.2) (SEQ ID NO: 46):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCTRPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNINGTASINGTESNDNITLPCRINGTIQGNITCQSNITGILLTR DGGSGSGTCETFRPGGGDMRDLNRTS

A2.HG.3.3 (OD4.2' HG.3.3) (SEQ ID NO: 47):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCTRPSNNTQY CVVNRTQWNDTLGQVAINLTKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASINGTESNDNITLPCRINGTIQGNITCQSNITGILLTR DGGSGSGTCETFRPGGGDMRDLNRTS

A2.HG.3.4 (OD4.2' HG.3.4) (SEQ ID NO: 48):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCTRPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCTGEFFY CNTSDLFNSTWNIEGTASINGTESNDNITLPCRINGTIQGNITCQSNITGILLTR DGGSGSGTCETFRPGGGDMRDLNRTS A2.HG.3.5 (OD4.2' HG.3.5) (SEQ ID NO: 49):

KPVVSTQLLLNGSLAEKEIRINSTDISDNAKNIIVQLTKPVLINCTRPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASINGTESNDNITLPCRINGTIQGNITCQSNITGILLTR DGGSGSGTCETFRPGGGDMRDLNRTS

A2.HG.3.6 (OD4.2' HG.3.6) (SEQ ID NO: 50):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCTRPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFTNSSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNIEGTASINGTESNDNITLPCRINGTIQGNITCQSNITGILLTR DGGSGSGTCETFRPGGGDMRDLNRTS

A2.HG.3.2.1 (OD4.2' HG.3.2.1) (SEQ ID NO: 51):

KPVVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCTRPSNNTQY CVVNRTQWNDTLGQVAIQLRKHWNTCIIFTNSSGGDLEITTHSFNCGGEFFY CNTSDLFNSTWNINGTASINGTESNDNITLPCRINGTIQGNITCQSNITGILLTR DGGSGSGTCETFRPGGGDMRDLNRTS

A2.6_SG_eumrcc (SEQ ID NO: 52):

KPVVSTQLLLNGSLAEKEIRIKSENISDNAKTIIVQLTKPVLINCARPSNNTRG RRGDIRQAYCVVNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHS FNCGGEFTYCNTSDLFNSTWNIEGTASINGTESNDNITLPCRIKGSGAPPIQGV IRCQSNITGILLTRDGGNTGNNSRTCETFRPGGGDMRDNWRSESGESQVRQN FKPEMEEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEM THMQRLFDYLTDTGNLPRINTVESPFAEYSSLDELFQETYKHEQLITQKINEL AHAAMTNQDYPTFNFLQWYVSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDK ELSTLD

KER2018_OD4.2'_HG3.2_HATM (SEQ ID NO: 53):

MPMGSLQPLATLYLLGMLVASVLAKPVVSTQLLLNGSLAEKEIRIKSEDISD NAKNIIVQLTKPVLINCTRPSNNTQYCVVNRTQWNDTLGQVAIQLRKHWNT CIIFNEPSGGDLEITTHSFNCGGEFFYCNTSDLFNSTWNINGTASINGTESNDN ITLPCRINGTIQGNITCQSNITGILLTRDGGSGSGTCETFRPGGGDMRDLNRTS GSGILAIYSTVASSLVLLVSLGAISF KER2018_OD4.2'_HG3.2_HATM (SEQ ID NO: 54):

MPMGSLQPLATLYLLGMLVASVLAKPVVSTQLLLNGSLAEKEIRIKSEDISD NAKNIIVQLTKPVLINCTRPSNNTQYCVVNRTQWNDTLGQVAIQLRKHWNT CIIFNEPSGGDLEITTHSFNCGGEFFYCNTSDLFNSTWNINGTASINGTESNDN ITLPCRINGTIQGNITCQSNITGILLTRDGGSGSGTCETFRPGGGDMRDLNRTS GSGILAIYSTVASSLVLLVSLGAISF

KER2018_OD4.2'_HG3.2.1_HATM (SEQ ID NO: 55):

MPMGSLQPLATLYLLGMLVASVLAKPVVSTQLLLNGSLAEKEIRIKSEDISD NAKNIIVQLTKPVLINCTRPSNNTQYC VVNRTQWNDTLGQVAIQLRKHWNT CIIFTNSSGGDLEITTHSFNCGGEFFYCNTSDLFNSTWNINGTASINGTESNDN ITLPCRINGTIQGNITCQSNITGILLTRDGGSGSGTCETFRPGGGDMRDLNRTS GSGILAIYSTVASSLVLLVSLGAISF KER2018_OD4.2'_HG3.2_NATM (SEQ ID NO: 56):

MNPNQKIITIGSICMVVGIISLILQIGNIISIWVSGKPVVSTQLLLNGSLAEKEIR IKSEDISDNAKNIIVQLTKPVLINCTRPSNNTQYCVVNRTQWNDTLGQVAIQL RKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFYCNTSDLFNSTWNINGTASIN GTESNDNITLPCRINGTIQGNITCQSNITGILLTRDGGSGSGTCETFRPGGGDM RDLNRTS

KER2018_OD4.2'_HG3.2.1_NATM (SEQ ID NO: 57):

MNPNQKIITIGSICMVVGIISLILQIGNIISIWVSGKPVVSTQLLLNGSLAEKEIR IKSEDISDNAKNIIVQLTKPVLINCTRPSNNTQYCVVNRTQWNDTLGQVAIQL RKHWNTCIIFTNSSGGDLEITTHSFNCGGEFFYCNTSDLFNSTWNINGTASIN GTESNDNITLPCRINGTIQGNITCQSNITGILLTRDGGSGSGTCETFRPGGGDM RDLNRTS

R2 OD1.0 (parent sequence) (SEQ ID NO: 58):

MQLASCVTLTLVLLVNSAPRRPVVSTQLLLNGSLAEEEVVIRSENFTNNAKT IIVQLREPVKINCSRPNNNTRKSIPMGPGRAFYTTGQIIGDIRQAHCNISKTNW TNALKQVVEKLGEQFNKTKIVFTNSSGGNPEIVTHSFNCAGEFFYCNTTQLF DSrWNSENGTWNITRGLNNTGRNDTITLPCRIKQIINRWQEVGKAMYAPPIK GNISCSSNITGLLLTRDGGKDDNSRDGNETFRPGGGDMRDNWRSEGSHHHH HH

R2 OD1.0 (parent sequence) (SEQ ID NO: 203):

RRPVVSTQLLLNGSLAEEEVVIRSENFTNNAKTIIVQLREPVKINCSRPNNNT RKSIPMGPGRAFYTTGQIIGDIRQAHCNISKTNWTNALKQVVEKLGEQFNKT KIVFTNSSGGNPEIVTHSFNCAGEFFYCNTTQLFDSr NSENGTWNITRGLNN TGRNDTITLPCRIKQIINRWQEVGKAMYAPPIKGNISCSSNITGLLLTRDGGK DDNSRDGNETFRPGGGDMRDNWRSE

R2 0D1.1 (SEQ ID NO: 59):

MQLASCVTLTLVLLVNSAPRRPVVSTQLLLNGSLAEEEVVIRSENFTNNAKT IIVQLREPVKINCSRPNNNTRKSIPMGPGRAFYTTGQIIGDIRQAHCNISKTNW TNALKQVVEKLGEQFNKTKIVFTQSSGGNPEIVTHSFNCAGEFFYCNTTQLF DSIWNSENGTWNITRGLNNTGRNDTITLPCRIKQIINRWQEVGKAMYAPPIK GNISCSSNITGLLLTRDGGKDDNSRDGNETFRPGGGDMRDNWRSEGSHHHH HH

R2 OD2.2 (SEQ ID NO: 60):

MQLASCVTLTLVLLVNSAPRRPVVSTQLLLNGSLAEEEVVIRSENFTNNAKT IIVQLREPVKINCSRPNNNTRGRRGDIRQAHCNISKTNWTNALKQVVEKLGE QFNKTKIVFTQSSGGDPErVTHSFNCAGEFFYCNTTQLFDSrWNSENGTWNIT RGLNNTGRNDTITLPCRIKGSGAPPIKGNISCSSNITGLLLTRDGGKDDNSRD GNETFRPGGGDMRDNWRSEGSHHHHHH

R2 0D3.2 (SEQ ID NO: 61):

MQLASCVTLTLVLLVNSAPRRPVVSTQLLLNGSLAEEEVVIRSENFTNNAKT IIVQLREPVKINCSRPNNNTRGRRGDIRQAHCNISKTNWTNALKQVVEKLGE QFNKTKIVFTQSSGGDPErVTHSFNCAGEFTYCNTTQLFDSrWNSENGTWNIT RGLNNTGRNDTITLPCRIKGSGAPPIKGNISCSSNITGLLLTRDGGKDDNSRD GNETFRPGGGDMRDNWRSEGSHHHHHH R2 OD4.1 (SEQ ID NO: 62):

MYSMQLASCVTLTLVLLVNSAPRRPVVSTQLLLNGSLAEEEVVIRSENFTNC AKTIIVQLREPVKINCSRPNNNTRGRRGDIRQAHCNISKTNWTNALKQVVEK LGEQFNKTKIVFTQSSGGDPEIVTHSFNCAGEFTYCNTTQLFDSrWNSENGT WNrfRGLNNTGRNDTITLPCRIKGSGAPPIKGNISCSSNITGLLLTRDCGKDD NSRDGNETFRPGGGDMRDNWRSEGSLEVLFQGPGHHHHHH

R2 OD4.2 (SEQ ID NO: 63):

MQLASCVTLTLVLLVNSAPRRPVVSTQLLLNGSLAEEEVVIRSENFTNNAKT IIVQLREPVKINCSRPNNNTRGRRGDIRQAHCNISKTNWTNALKQVVEKLGE QFNKTCIVFTQSSGGDPErVTHSFNCAGEFTYCNTTQLFDSnVNSENGTWNrr RGLNNTGRNDTITLPCRIKGSGAPPIKGNISCSSNITGLLLTRDGGKDDNSRD GCETFRPGGGDMRDNWRSEGSHHHHHH

R2 OD4.2.2 (SEQ ID NO: 64):

MQLASCVTLTLVLLVNSAPRRPVVSTQLLLNGSLAEEEVVIRSENFTNNAKN IIVQLREPVKINCSRPNNNTRGRRGDIRQAHCNISKTNWTNALKQVVEKLGE QFNKTCIVFTQSSGGDPErVTHSFNCAGEFFYCQTTQLFDSnVNSENGTWNrr RGLNNTGRNDTITLPCRIKGSGAPPIKGNISCSSNITGLLLTRDGGKDDNSRD GCETFRPGGGDMRDNWRSEGSLEVLFQGPGHHHHHH

R2 OD4.2.2/V3.2 (SEQ ID NO: 65):

MQLASCVTLTLVLLVNSAPRRPVVSTQLLLNGSLAEEEVVIRSENFTNNAKN IIVQLREPVKINCSRPNNNTRRQAHCNISKTNWTNALKQVVEKLGEQFNKTC IVFTQSSGGDPEIVTHSFNCAGEFFYCQTTQLFDSTWNIEGTASINGTESNDQI TLPCRIKGSGAPPIKGNISCSSNITGLLLTRDGGSGGSGCETFRPGGGDMRDN WRSEGSLEVLFQGPGHHHHHH R2 OD4.2.2/V3.2/p (SEQ ID NO: 66):

MQLASCVTLTLVLLVNSAPRRPVVSTQLLLNGSLANESVVIRSENFTNNAKN IIVQLREPVKINCSRPNNNTRRQAHCNISKTNWTNALKQVVEKLGEQFNKTC IVFTQSSGGDPEIVTHSFNCAGEFFYCQTTQLFDSTWNIEGTASINGTESNDQI TLPCRIKGSGANATKGNISCSSNITGLLLTRDGGSGGSGCETFRPGGGDMRD NNRSEGSLEVLFQGPGHHHHHH

R2 OD4.3 (SEQ ID NO: 67):

MQLASC VTLTLVLLVNS APRRPVCSTQLLLNGSLAEEEVVIRSENFTNNAKT

irVQLREPVKINCSRPNNNTRGRRGDIRQAHCNISKTNWTNALKQVVEKLGE QFNKTKIVFTQSSGGDPErVTHSFNCAGEFTYCNTTQLFDSrWNSENGTWNIT RGLNNTGRNDTITLPCRIKGSGAPPIKGNISCSSNITGLLLTRDGGKDDNSRD GNETFRPGGGDCRDNWRSEGSHHHHHH

R2 OD4.3.2 (SEQ ID NO: 68):

MQLASCVTLTLVLLVNSAPRRPVCSTQLLLNGSLAEEEVVIRSENFTNNAKN

irVQLREPVKINCSRPNNNTRGRRGDIRQAHCNISKTNWTNALKQVVEKLGE QFNKTKIVFTQSSGGDPErVTHSFNCAGEFFYCQTTQLFDSrWNSENGTWNIT RGLNNTGRNDTITLPCRIKGSGAPPIKGNISCSSNITGLLLTRDGGKDDNSRD GQETFRPGGGDCRDNWRSEGSLEVLFQGPGHHHHHH

R2 OD4.3.2/V3.2/p (SEQ ID NO: 69):

MQLASCVTLTLVLLVNSAPRRPVCSTQLLLNGSLAEEEVVIRSENFTNNAKN IIVQLREPVKINCSRPNNNTRRQAHCNISKTNWTNALKQVVEKLGEQFNKTK IVFTQSSGGDPEIVTHSFNCAGEFFYCQTTQLFDSTWNIEGTASINGTESNDNI TLPCRIKGSGAPPIKGNISCSSNITGLLLTRDGGSGGSGQETFRPGGGDCRDN WRSEGSLEVLFQGPGHHHHHH R2 OD4.3.2/V4.2.1 R2/p (SEQ ID NO: 70):

MQLASCVTLTLVLLVNSAPRRPVCSTQLLLNGSLANESVVIRSENFTNNAKN IIVQLREPVKINCSRPNNNTRRQAHCNISKTNWTNALKQVVEKLGEQFNKTK IVFTQSSGGDPEIVTHSFNCAGEFFYCQTTQLFDSTWNIEGTASINGTESNDNI TLPCRIKGSGANATKGNISCSSNITGLLLTRDGGSGGSGQETFRPGGGDCRD NNRSEGSLEVLFQGPGHHHHHH

R2 gpl40(N363Q)(AV123/GSL)(Ap2021)(mFC)/Foldon/3C/His (SEQ ID NO: 71):

MRVKGIRRNYQHWWGWGTMLLGLLMICSATEKLWVTVYYGVPVWKEAT

TTLFCASDAKAYDTEAHNVWATHACVPTDPNPQEVELVNVTENFNMWKN NMVEQMHEDIISLWDQSLKPCVKLTPLCVGSGSCNTSVrrQACPKISFEPIPIH YCAPAGFAILKCNDKKFSGKGSCKNVSTVQCTHGIRPVVSTQLLLNGSLAEE EVVIRSENFTNNAKTIIVQLREPVKINCSRPNNNTRGRRGDIRQAHCNISKTN WTNALKQVVEKLGEQFNKTKIVFTQSSGGDPErVTHSFNCAGEFFYCNTTQL FDSIWNSENGTWNITRGLNNTGRNDTITLPCRIKGSGAPPIKGNISCSSNITGL LLTRDGGKDDNSRDGNETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQSEESATGLGATYIGYLGAAGSTMGAASVTLTVQARQLLSGIVQ QQSNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGK LICTTTVPWNASWSKNKTLEAIWNNMTWMQWDKEIDNYTSLIYSLIEESQI QQEKNEQELLELDKWANLWNWFDISNWLWGSGYIPEAPRDGQAYVRKDG EWVLLSTFLGLEVLFQGPGHHHHHH

R2 gpl40(N363Q))(AV13/GSL)(Ap2021)(mFC)/Foldon/3C/His (SEQ ID NO: 72): MRVKGIRRNYQHWWGWGTMLLGLLMICSATEKLWVTVYYGVPVWKEAT TTLFCASDAKAYDTEAHNVWATHACVPTDPNPQEVELVNVTENFNMWKN NMVEQMHEDIISLWDQSLKPCVKLTPLCVGSKNCSFNIATSIGDKMQKEYA LLYKLDIEPIDNDNTS YRLISCNTS VITQACPKISFEPIPIHYCAPAGFAILKCN DKKFSGKGSCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSENFTNNA KTIIVQLREPVKINCSRPNNNTRGRRGDIRQAHCNISKTNWTNALKQVVEKL GEQFNKTKIVFTQSSGGDPEIVTHSFNCAGEFFYCNTTQLFDSIWNSENGTW NITRGLNNTGRNDTITLPCRIKGSGAPPIKGNISCSSNITGLLLTRDGGKDDNS RDGNETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQSEES ATGLGATYIGYLGAAGSTMGAASVTLTVQARQLLSGIVQQQSNLLRAIEAQ QHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTTVPWNAS WSKNKTLEARVNNMTWMQWDKEIDNYTSLIYSLIEESQIQQEKNEQELLEL DKWANLWNWFDISNWLWGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGLE VLFQGPGHHHHHH

R2 _gpl40(N363Q))(AV123/m5)(Ap2021)(mFC)/Foldon/3C/His (SEQ ID NO: 73):

MRVKGIRRNYQHWWGWGTMLLGLLMICSATEKLWVTVYYGVPVWKEAT

TTLFCASDAKAYDTEAHNVWATHACVPTDPNPQEVELVNVTENFNMWKN NMVEQMHEDIISLWDQSLKPCVKLTPLCVGSGSCNTSVrrQACPKISFEPIPIH YCAPAGFAILKCNDKKFSGKGSCKNVSTVQCTHGIRPVVSTQLLLNGSLAEE EVVIRSENFTNNAKTIIVQLREPVKINCSRPNNNTRGRRGSSGGSHCNISKTN WTNALKQVVEKLGEQFNKTKIVFTQSSGGDPErVTHSFNCAGEFFYCNTTQL FDSIWNSENGTWNITRGLNNTGRNDTITLPCRIKGSGAPPIKGNISCSSNITGL LLTRDGGKDDNSRDGNETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQSEESATGLGATYIGYLGAAGSTMGAASVTLTVQARQLLSGIVQ QQSNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGRVGCSGK LICTTTVPWNASWSKNKTLEAr NNMTWMQWDKEIDNYTSLIYSLIEESQI QQEKNEQELLELDKWANLWNWFDISNWLWGSGYIPEAPRDGQAYVRKDG EWVLLSTFLGLEVLFQGPGHHHHHH

RS3 (SEQ ID NO: 74):

TTVTVNVTVTFDWCADDMVATMNTAICTLWKTSNDPCTKCPTVRFKPVPIR YCAPPGYAILKCNNRDFNGTGPCTNVSVVTCTDGIHPVVSSQLLLNGTLADE KVVIRSCNFSDNAKTIIVQLNTSVEINCTGQGHCNITRAKWNQTLKQIAEKL REQFGNNKTIIFRPSSGGDPEIVTHWFNCGGKFFYCNSTQLFNSTWFNSTWS TKGSNNTEGSDTITLPCRIRSrrGMVCTVGKMIYAPPVEGVITCSSNITGLLLT RDGGNDNNESEIFRPGGGDMRDNWRSELYKYRVVRLT RSC3(Y5) (SEQ ID NO: 75):

MPMGSLQPLATLYLLGMLVASVLATNVTVNVTVTFDWCADDMVATMNTS ICTLWKTSNDPCTKCPTVRFKPVPIRYCAPPGYAILKCNNRDFNGTGPCTNV SNVTCTDGIHPVVSSQLLLNGTLADEKVVIRSCNFSDNAKTIIVQLNTSVEIN CTGQGHCNITRAKWNQTLKQIAEKLREQFGNNKTIIFRPSSGGDPEIVTHWF NCGGKFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRIRSITGMNC TVGKMIYAPPVEGNITCSSNITGLLLTRDGGNDNNESEIFRPGGGDMRDNWR SELYKYRVVRLTGSHHHHHH. RSC3 Y6.1 His (SEQ ID NO: 76):

MPMGSLQPLATLYLLGMLVASVLATNVTVNVTVTFDWCADDMVATMNTS ICTLWNTSNDPCTKCPTVRFKPVPIRYCAPPGYAILKCNNRDFNGTGPCTNV SNVTCTDGIHPVVSSQLLLNGTLADEKVVIRSCNFSDNAKTIIVQLNTSVEIN CTGQGHCNITRAKWNQTLKQIAEKLREQFGNNKTIIFRPSSGGDPEIVTHWF NCGGKFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRIRSITGMNC TVGKMIYAPPVEGNITCSSNITGLLLTRDGGNDNNESEIFRP GGGDMRDNWRSELYKYRVVRLTGSHHHHHH

Mosaic 2.1/gpl45(AVl.lV2.1V3.5)( ACFI) (SEQ ID NO: 77):

LWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQ EVVLGNVTENFNMWKNNMVEQMHEDIISLWDESLKPCVKLTPLCVTLNCT DLRGSGGGSGGGSEIKNCSFNITTSIRDKVQKEYALFYKLDVVPIGGSGGSY RLINCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNDKKFNGTGPCKNVST VQCTHGIRPVVSTQLLLNGSLAEEEVVIRSENFTNNAKTIIVQLNESVEINCTR PNNNTRGRRGSSGGSHCNISRAKWNNTLKQIVIKLREQFGNKTIVFNQSSGG DPErVMHSFNCGGEFFYCNTTQLFNSTWNVNGTWNGTGSENITLPCRIKQIV NMWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGNNNSTNETFRPGGGDM RDNWRSELYKYKVVKIEPLGVAPTKAKLTVQARLLLSGIVQQQNNLLRAIE AQQHMLQLTVWGIKQLQARVLAVERYLKDQQLLERVNNMTWMEWEREID NYTGLIYTLIEESQNQQEKNEQELLELDKWASLWNWFDITNWLWYIKIFIMI VGGLIGLRIVFA VLS I

HXB2 core (SEQ ID NO: 78):

EVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVGAGSC NTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTH GIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTGAGHC NIARAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMY APPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYK VVKIE

CHIKV185_OD4.2'_HG3.2 (SEQ ID NO: 79)

MEFIPTQTFYNRRYQPRPWAPRPTIQVIRPRPRPQRQAGQLAQLISAVNKLT MRAVPQQKPRRNRKNKKQRQKKQAPQNDPKQKKQPPQKKPAQKKKKPGR RERMCMKIENDCIFEVKHEGKVMGYACLVGDKVMKPAHVKGTIDNADLA KLAFKRSSKYDLECAQIPVHMKSDASKFTHEKPEGYYNWHHGAVQYSGGR FTIPTGAGKPGDSGRPIFDNKGRVVAIVLGGANEGARTALSVVTWNKDIVTK ITPEGAEEWSLALPVLCLLANTTFPCSQPPCTPCCYEKEPESTLRMLEDNVM RPGYYQLLKASLTCSPHRQRRSTKDNFNVYKATRPYLAHCPDCGEGHSCHS PIALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDSHTPADAERAG LLVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHTCTHPFHHEPPVI GRERFHSRPQHGKELPCSTYVQSTAATAEEIEVHMPPDTPDRTLMTQQSGKP VVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCTRPSNNTQYCV VNRTQWNDTLGQVAIQLRKHWNTCIIFNEPSGGDLEITTHSFNCGGEFFYCN TSDLFNSTWNINGTASINGTESNDNITLPCRINGTIQGNITCQSNITGILLTRDG GSGSGTCETFRPGGGDMRDLNRTSGSGNVKITVNGQTVRYKCNCGGSNEG LTTTDKVINNCKIDQCHAAVTNHKNWQYNSPLVPRNAELGDRKGKIHIPFPL ANVTCRVPKARNPTVTYGKNQVTMLLYPDHPTLLSYRNMGQEPNYHEEW VTHKKEVTLTVPTEGLEVTWGNNEPYKYWPQMSTNGTAHGHPHEIILYYY ELYPTMTVVIVS VASFVLLSM VGTA VGMC VC ARRRCrrPYELTPGATVPFLL SLLCCVRTTKAATYYEAAAYLWNEQQPLFWLQALIPLAALIVLCNCLKLLP CCCKTLAFLAVMSIGAHTVSAYEHVTVIPNTVGVPYKTLVNRPGYSPMVLE MELQSVTLEPTLSLDYITCEYKTVIPSPYVKCCGTAECKDKSLPDYSCKVFT GVYPFMWGGAYCFCDAENTQLSEAHVEKSESCKTEFASAYRAHTASASAK LRVLYQGNNrrVAAYANGDHAVTVKDAKFVVGPMSSAWTPFDNKIVVYK GDVYNMDYPPFGAGRPGQFGDIQSRTPESKDVYANTQLVLQRPAAGTVHV P YS Q APSGFKYWLKERG AS LQHT APFGCQIATNP VRA VNC A VGNIPIS IDIPD AAFTRVVDAPSVTDMSCEVPACTHSSDFGGVAIIKYTASKKGKCAVHSMTN AVTIREADVEVEGNSQLQISFSTALASAEFRVQVCSTQVHCAAACHPPKDHI VNYPASHTTLGVQDISTTAMSWVQKITGGVGLIVAVAALILIVVLCVSFSRH

CHIKV185_OD4.2'_HG3.2.1 (SEQ ID NO: 80)

MEFIPTQTFYNRRYQPRPWAPRPTIQVIRPRPRPQRQAGQLAQLISAVNKLT MRAVPQQKPRRNRKNKKQRQKKQAPQNDPKQKKQPPQKKPAQKKKKPGR RERMCMKIENDCIFEVKHEGKVMGYACLVGDKVMKPAHVKGTIDNADLA KLAFKRSSKYDLECAQIPVHMKSDASKFTHEKPEGYYNWHHGAVQYSGGR FTIPTGAGKPGDSGRPIFDNKGRVVAIVLGGANEGARTALSVVTWNKDIVTK ITPEGAEEWSLALPVLCLLANTTFPCSQPPCTPCCYEKEPESTLRMLEDNVM RPGYYQLLKASLTCSPHRQRRSTKDNFNVYKATRPYLAHCPDCGEGHSCHS PIALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDSHTPADAERAG LLVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHTCTHPFHHEPPVI GRERFHSRPQHGKELPCSTYVQSTAATAEEIEVHMPPDTPDRTLMTQQSGKP VVSTQLLLNGSLAEKEIRIKSEDISDNAKNIIVQLTKPVLINCTRPSNNTQYCV VNRTQWNDTLGQVAIQLRKHWNTCIIFTNSSGGDLEITTHSFNCGGEFFYCN TSDLFNSTWNINGTASINGTESNDNITLPCRINGTIQGNITCQSNITGILLTRDG GSGSGTCETFRPGGGDMRDLNRTSGSGNVKITVNGQTVRYKCNCGGSNEG LTTTDKVINNCKIDQCHAAVTNHKNWQYNSPLVPRNAELGDRKGKIHIPFPL ANVTCRVPKARNPTVTYGKNQVTMLLYPDHPTLLSYRNMGQEPNYHEEW VTHKKEVTLTVPTEGLEVTWGNNEPYKYWPQMSTNGTAHGHPHEIILYYY ELYPTMTVVIVSVASFVLLSMVGTAVGMCVCARRRCrrPYELTPGATVPFLL SLLCCVRTTKAATYYEAAAYLWNEQQPLFWLQALIPLAALIVLCNCLKLLP CCCKTLAFLAVMSIGAHTVSAYEHVTVIPNTVGVPYKTLVNRPGYSPMVLE MELQSVTLEPTLSLDYITCEYKTVIPSPYVKCCGTAECKDKSLPDYSCKVFT GVYPFMWGGAYCFCDAENTQLSEAHVEKSESCKTEFASAYRAHTASASAK LRVLYQGNNITVAAYANGDHAVTVKDAKFVVGPMSSAWTPFDNKIVVYK GDVYNMDYPPFGAGRPGQFGDIQSRTPESKDVYANTQLVLQRPAAGTVHV P YS Q APSGFKYWLKERG AS LQHT APFGCQIATNP VRA VNC A VGNIPIS IDIPD AAFTRVVDAPSVTDMSCEVPACTHSSDFGGVAIIKYTASKKGKCAVHSMTN AVTIREADVEVEGNSQLQISFSTALASAEFRVQVCSTQVHCAAACHPPKDHI VNYPASHTTLGVQDISTTAMSWVQKITGGVGLIVAVAALILIVVLCVSFSRH

CHIKV-OD A2.6 (SEQ ID NO: 148):

MEFIPTQTFYNRRYQPRPWAPRPTIQVIRPRPRPQRQAGQLAQLISAVNKLT

MRAVPQQKPRRNRKNKKQRQKKQAPQNDPKQKKQPPQKKPAQKKKKPGR

RERMCMKIENDCIFEVKHEGKVMGYACLVGDKVMKPAHVKGTIDNADLA KLAFKRSSKYDLECAQIPVHMKSDASKFTHEKPEGYYNWHHGAVQYSGGR FTIPTGAGKPGDSGRPIFDNKGRVVAIVLGGANEGARTALSVVTWNKDIVTK ITPEGAEEWSLALPVLCLLANTTFPCSQPPCTPCCYEKEPESTLRMLEDNVM RPGYYQLLKASLTCSPHRQRRSTKDNFNVYKATRPYLAHCPDCGEGHSCHS PIALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDSHTPADAERAG LLVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHTCTHPFHHEPPVI GRERFHSRPQHGKELPCSTYVQSTAATAEEIEVHMPPDTPDRTLMTQQSGN VKITVNGQTVRYKCNCGGSGKPVVSTQLLLNGSLAEKEIRIKSENISDNAKTI IVQLTKPVLINCARPSNNTRGRRGDIRQAYCVVNRTQWNDTLGQVAIQLRK HWNTCIIFNEPSGGDLEITTHSFNCGGEFTYCNTSDLFNSTWNIEGTASINGTE SNDNITLPCRIKGSGAPPIQGVIRCQSNITGILLTRDGGNTGNNSRTCETFRPG GGDMRDNWRSEGSGSNEGLTTTDKVINNCKIDQCHAAVTNHKNWQYNSPL VPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVTMLLYPDHP TLLSYRNMGQEPNYHEEWVTHKKEVTLTVPTEGLEVTWGNNEPYKYWPQ MSTNGTAHGHPHEIILYYYELYPTMTVVIVSVASFVLLSMVGTAVGMCVCA RRRCrrPYELTPGATVPFLLSLLCCVRTTKAATYYEAAAYLWNEQQPLFWL QALIPLAALIVLCNCLKLLPCCCKTLAFLAVMSIGAHTVSAYEHVTVIPNTV GVPYKTLVNRPGYSPMVLEMELQSVTLEPTLSLDYITCEYKTVIPSPYVKCC GTAECKDKSLPDYSCKVFTGVYPFMWGGAYCFCDAENTQLSEAHVEKSES CKTEFASAYRAHTASASAKLRVLYQGNNITVAAYANGDHAVTVKDAKFVV GPMSSAWTPFDNKIVVYKGDVYNMDYPPFGAGRPGQFGDIQSRTPESKDVY ANTQLVLQRPAAGTVHVPYSQAPSGFKYWLKERGASLQHT APFGCQIATNP VRAVNCAVGNIPISIDIPDAAFTRVVDAPSVTDMSCEVPACTHSSDFGGVAII KYTASKKGKCAVHSMTNAVTIREADVEVEGNSQLQISFSTALASAEFRVQV CSTQVHCAAACHPPKDHIVNYPASHTTLGVQDISTTAMSWVQKITGGVGLI VAVAALILIVVLCVSFSRH

CHIKV-OD4.1 (SEQ ID NO: 149):

MEFIPTQTFYNRRYQPRPWAPRPTIQVIRPRPRPQRQAGQLAQLISAVNKLT MRAVPQQKPRRNRKNKKQRQKKQAPQNDPKQKKQPPQKKPAQKKKKPGR RERMCMKIENDCIFEVKHEGKVMGYACLVGDKVMKPAHVKGTIDNADLA KLAFKRSSKYDLECAQIPVHMKSDASKFTHEKPEGYYNWHHGAVQYSGGR FTIPTGAGKPGDSGRPIFDNKGRVVAIVLGGANEGARTALSVVTWNKDIVTK ITPEGAEEWSLALPVLCLLANTTFPCSQPPCTPCCYEKEPESTLRMLEDNVM RPGYYQLLKASLTCSPHRQRRSTKDNFNVYKATRPYLAHCPDCGEGHSCHS PIALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDSHTPADAERAG LLVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHTCTHPFHHEPPVI GRERFHSRPQHGKELPCSTYVQSTAATAEEIEVHMPPDTPDRTLMTQQSGN VKrrVNGQTVRYKCNCGGSGRPVVSTQLLLNGSLAEEEVVIRSENFTNCAKT IIVQLREPVKINCSRPNNNTRGRRGDIRQAHCNISKTNWTNALKQVVEKLGE QFNKTKIVFTQSSGGDPErVTHSFNCAGEFTYCNTTQLFDSrWNSENGTWNIT RGLNNTGRNDTITLPCRIKGSGAPPIKGNISCSSNITGLLLTRDCGKDDNSRD GNETFRPGGGDMRDNWRSEGSGSNEGLTTTDKVINNCKIDQCHAAVTNHK NWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVT MLLYPDHPTLLSYRNMGQEPNYHEEWVTHKKEVTLTVPTEGLEVTWGNNE PYKYWPQMSTNGTAHGHPHEIILYYYELYPTMTVVIVSVASFVLLSMVGTA VGMCVCARRRCITPYELTPGATVPFLLSLLCCVRTTKAATYYEAAAYLWNE QQPLFWLQALIPLAALIVLCNCLKLLPCCCKTLAFLAVMSIGAHTVSAYEHV TVIPNTVGVPYKTLVNRPGYSPMVLEMELQSVTLEPTLSLDYITCEYKTVIPS PYVKCCGTAECKDKSLPDYSCKVFTGVYPFMWGGAYCFCDAENTQLSEAH VEKSESCKTEFASAYRAHTASASAKLRVLYQGNNITVAAYANGDHAVTVK DAKFVVGPMSSAWTPFDNKIVVYKGDVYNMDYPPFGAGRPGQFGDIQSRT PES KD V Y ANTQLVLQRP A AGT VH VP YS Q APSGFKYWLKERG AS LQHT APF GCQIATNPVRAVNCAVGNIPISIDIPDAAFTRVVDAPSVTDMSCEVPACTHSS DFGGVAIIKYTASKKGKCAVHSMTNAVTIREADVEVEGNSQLQISFSTALAS AEFRVQVCSTQVHCAAACHPPKDHIVNYPASHTTLGVQDISTTAMSWVQKI TGGVGLIVAVAALILIVVLCVSFSRH

B. Polynucleotides Encoding Antigens

Polynucleotides encoding the antigens disclosed herein are also provided. These polynucleotides include DNA, cDNA and RNA sequences which encode the antigen.

Methods for the manipulation and insertion of the nucleic acids of this disclosure into vectors are well known in the art (see for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y., 1994).

A nucleic acid encoding an antigen can be cloned or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self- sustained sequence replication system (3SR) and the QP replicase amplification system (QB). For example, a polynucleotide encoding the protein can be isolated by polymerase chain reaction of cDNA using primers based on the DNA sequence of the molecule. A wide variety of cloning and in vitro amplification methodologies are well known to persons skilled in the art. PCR methods are described in, for example, U.S. Patent No. 4,683,195; Mullis et al, Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). Polynucleotides also can be isolated by screening genomic or cDNA libraries with probes selected from the sequences of the desired polynucleotide under stringent hybridization conditions.

The polynucleotides encoding an antigen include a recombinant DNA which is incorporated into a vector into an autonomously replicating plasmid or virus or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (such as a cDNA) independent of other sequences. The nucleotides of the invention can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double forms of DNA.

DNA sequences encoding the antigen can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.

Polynucleotide sequences encoding antigens can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.

Hosts can include microbial, yeast, insect and mammalian organisms.

Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Non-limiting examples of suitable host cells include bacteria, archea, insect, fungi (for example, yeast), plant, and animal cells (for example, mammalian cells, such as human). Exemplary cells of use include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella

typhimurium, SF9 cells, C129 cells, 293 cells, Neurospora, and immortalized mammalian myeloid and lymphoid cell lines. Techniques for the propagation of mammalian cells in culture are well-known (see, Jakoby and Pastan (eds), 1979, Cell Culture. Methods in Enzymology, volume 58, Academic Press, Inc., Harcourt Brace Jovanovich, N.Y.). Examples of commonly used mammalian host cell lines are VERO and HeLa cells, CHO cells, and WI38, BHK, and COS cell lines, although cell lines may be used, such as cells designed to provide higher expression desirable glycosylation patterns, or other features.

Transformation of a host cell with recombinant DNA can be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as, but not limited to, E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl₂ method using procedures well known in the art. Alternatively, MgCl₂ or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or viral vectors can be used. Eukaryotic cells can also be co-transformed with

polynucleotide sequences encoding a disclosed antigen, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

A number of viral vectors have been constructed, that can be used to express the disclosed antigens, including polyoma, i.e., SV40 (Madzak et ah, 1992, J. Gen. Virol, 73: 15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol. Immunol., 158:39-6; Berliner et al, 1988, Bio Techniques, 6:616-629; Gorziglia et al, 1992, J. Virol, 66:4407-4412; Quantin et al, 1992, Proc. Natl. Acad. Sci. USA, 89:2581- 2584; Rosenfeld et al, 1992, Cell, 68: 143-155; Wilkinson et al, 1992, Nucl. Acids Res., 20:2233-2239; Stratford-Perricaudet et al, 1990, Hum. Gene Then, 1:241- 256), vaccinia virus (Mackett et al, 1992, Biotechnology, 24:495-499), adeno- associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol, 158:91-123; On et al, 1990, Gene, 89:279-282), herpes viruses including HSV and EBV

(Margolskee, 1992, Curr. Top. Microbiol. Immunol, 158:67-90; Johnson et al, 1992, /. Virol, 66:29522965; Fink et al, 1992, Hum. Gene Ther. 3: 11-19;

Breakfield et al, 1987, Mol. NeurobioL, 1:337-371; Fresse et al, 1990, Biochem. Pharmacol, 40:2189-2199), Sindbis viruses (H. Herweijer et al, 1995, Human Gene Therapy 6: 1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S. Schlesinger, 1993, Trends Biotechnol. 11: 18-22; I. Frolov et al, 1996, Proc. Natl. Acad. Sci. USA 93: 11371-11377) and retroviruses of avian (Brandyopadhyay et al, 1984, Mol. Cell Biol, 4:749-754; Petropouplos et al, 1992, /. Virol, 66:3391- 3397), murine (Miller, 1992, Curr. Top. Microbiol. Immunol, 158: 1-24; Miller et al, 1985, Mol. Cell Biol, 5:431-437; Sorge et al, 1984, Mol. Cell Biol, 4: 1730- 1737; Mann et al, 1985, /. Virol, 54:401-407), and human origin (Page et al, 1990, /. Virol, 64:5370-5276; Buchschalcher et al, 1992, /. Virol, 66:2731-2739).

Baculovirus (Autographa calif ornica multinuclear polyhedrosis virus; AcMNPV) vectors are also known in the art, and may be obtained from commercial sources (such as PharMingen, San Diego, Calif.; Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).

In some embodiments, a nucleic acid molecule that encodes a disclosed antigen is a nucleic acid given below as any one of SEQ ID NOs: 81-147, 150-161 and 206-215. In some embodiments, a nucleic acid molecule that encodes a disclosed antigen comprises a nucleic acid sequence at least about 95% identical, such as about 95%, about 96%, about 97%, about 98%, about 99% or even 100% identical to the nucleic acid sequence according to one of SEQ ID NOs: 81-147,

150-161 and 206-215. In some embodiments, a nucleic acid molecule that encodes a disclosed antigen consists of a nucleic acid sequence according to one of SEQ ID NOs: 81-147, 150-161 and 206-215.

R2 gpl40(N363Q))(AV123/GSL)(Ap2021)(mFC)/Foldon/3C/His (SEQ ID NO: 116):

atgcgggtgaagggcatcagacggaactatcagcattggtggggctggggcaccatgctgctgggactgctgatgatct gtagcgccaccgagaaactgtgggtgaccgtgtactacggcgtgcctgtgtggaaagaggccaccaccaccctgttttg tgcctctgacgccaaggcctatgataccgaggcccacaatgtgtgggctactcatgcctgtgtgcccaccgatcccaatc ctcaggaagtggagctggtcaacgtgaccgagaacttcaacatgtggaagaacaacatggtggagcagatgcacgag gacatcatcagcctgtgggaccagtctctgaagccttgcgtgaagctgacacctctgtgcgtgggcagcggcagctgca acaccagcgtgatcacacaggcctgccccaagatcagcttcgagcctatccccatccactattgtgcccctgccggcttt gccatcctgaagtgcaacgacaagaagttcagcggcaagggcagctgcaagaacgtgagcaccgtgcagtgtacaca cggcatcagacctgtggtgtctacacagctgctgctgaatggctctctggccgaggaagaggtggtgatcagaagcgag aatttcaccaacaacgccaagaccatcatcgtgcagctgagggaacccgtgaagatcaactgcagccggcccaacaac aatacccggggcagaagaggagacatcagacaggcccactgcaacatcagcaagaccaactggaccaacgccctga aacaggtggtggagaagctgggcgagcagttcaacaagaccaagatcgtgttcacccagagcagcggcggagatcct gagatcgtgacccacagcttcaattgtgccggcgagttcttctactgcaataccacccagctgttcgacagcatctggaac agcgagaacggcacctggaatatcaccaggggcctgaacaacaccggcaggaacgataccatcaccctgccctgcag gatcaagggaagcggagcccctcccatcaagggcaatattagctgcagcagcaacatcacaggactgctgctgacaag agatggcggcaaggacgacaatagcagggacggcaacgagacattcagacctggcggcggagacatgagggacaa ttggcggagcgagctgtacaagtacaaggtggtgaagatcgagcccctgggcgtggctcccacaaaggccaagagaa gagtggtgcagagcgaggagagcgccaccggcctgggcgccacctacatcggctacctgggagccgccggaagca ccatgggcgctgccagcgtgacactgacagtgcaggctagacagctgctgtctggcattgtgcagcagcagagcaatct gctgagagccatcgaagcccagcagcacctgctgcagctgacagtgtggggcatcaaacagctgcaggctcgcattct ggccgtggagagatacctgaaggatcagcagctgctcggaatctggggctgtagcggcaagctgatctgtaccaccac cgtgccttggaatgccagctggtccaagaacaagaccctggaagccatctggaataacatgacctggatgcagtggga caaagagatcgacaactacaccagcctgatctacagcctgatcgaggaaagccagatccagcaggaaaagaacgagc aggaactgctggaactggacaagtgggccaacctgtggaactggttcgacatcagcaactggctgtggggatccggat acatccccgaggctccacgcgacggccaggcttacgtgcgcaaggacggcgagtgggtgctgctgagcaccttcctg ggcctggaggtgctgttccagggcccaggccaccaccaccaccaccactga

R2 gpl40(N363Q))(AV13/GSL)(Ap2021)(mFC)/Foldon/3C/His (SEQ ID NO: 117): atgcgggtgaagggcatcagacggaactatcagcattggtggggctggggcaccatgctgctgggactgctgatgatct gtagcgccaccgagaaactgtgggtgaccgtgtactacggcgtgcctgtgtggaaagaggccaccaccaccctgttttg tgcctctgacgccaaggcctatgataccgaggcccacaatgtgtgggctactcatgcctgtgtgcccaccgatcccaatc ctcaggaagtggagctggtcaacgtgaccgagaacttcaacatgtggaagaacaacatggtggagcagatgcacgag gacatcatcagcctgtgggaccagtctctgaagccttgcgtgaagctgacacctctgtgcgtgggaagcaagaactgca gcttcaacattgccacctccatcggcgacaagatgcagaaagagtacgccctgctgtacaagctggacatcgagcccat cgacaacgacaacaccagctacaggctgatcagctgcaacaccagcgtgatcacacaggcctgccccaagatcagctt cgagcctatccccatccactattgtgcccctgccggctttgccatcctgaagtgcaacgacaagaagttcagcggcaagg gcagctgcaagaacgtgagcaccgtgcagtgtacacacggcatcagacctgtggtgtctacacagctgctgctgaatgg ctctctggccgaggaagaggtggtgatcagaagcgagaatttcaccaacaacgccaagaccatcatcgtgcagctgag ggaacccgtgaagatcaactgcagccggcccaacaacaatacccggggcagaagaggagacatcagacaggccca ctgcaacatcagcaagaccaactggaccaacgccctgaaacaggtggtggagaagctgggcgagcagttcaacaaga ccaagatcgtgttcacccagagcagcggcggagatcctgagatcgtgacccacagcttcaattgtgccggcgagttcttc tactgcaataccacccagctgttcgacagcatctggaacagcgagaacggcacctggaatatcaccaggggcctgaac aacaccggcaggaacgataccatcaccctgccctgcaggatcaagggaagcggagcccctcccatcaagggcaatat tagctgcagcagcaacatcacaggactgctgctgacaagagatggcggcaaggacgacaatagcagggacggcaac gagacattcagacctggcggcggagacatgagggacaattggcggagcgagctgtacaagtacaaggtggtgaagat cgagcccctgggcgtggctcccacaaaggccaagagaagagtggtgcagagcgaggagagcgccaccggcctggg cgccacctacatcggctacctgggagccgccggaagcaccatgggcgctgccagcgtgacactgacagtgcaggcta gacagctgctgtctggcattgtgcagcagcagagcaatctgctgagagccatcgaagcccagcagcacctgctgcagc tgacagtgtggggcatcaaacagctgcaggctcgcattctggccgtggagagatacctgaaggatcagcagctgctcg gaatctggggctgtagcggcaagctgatctgtaccaccaccgtgccttggaatgccagctggtccaagaacaagaccct ggaagccatctggaataacatgacctggatgcagtgggacaaagagatcgacaactacaccagcctgatctacagcct gatcgaggaaagccagatccagcaggaaaagaacgagcaggaactgctggaactggacaagtgggccaacctgtgg aactggttcgacatcagcaactggctgtggggatccggatacatccccgaggctccacgcgacggccaggcttacgtg cgcaaggacggcgagtgggtgctgctgagcaccttcctgggcctggaggtgctgttccagggcccaggccaccacca ccaccaccactga

R2 gpl40(N363Q))(AV123/m5)(Ap2021)(mFC)/Foldon/3C/His (SEQ ID NO: 118): atgcgggtgaagggcatcagacggaactatcagcattggtggggctggggcaccatgctgctgggactgctgatgatct gtagcgccaccgagaaactgtgggtgaccgtgtactacggcgtgcctgtgtggaaagaggccaccaccaccctgttttg tgcctctgacgccaaggcctatgataccgaggcccacaatgtgtgggctactcatgcctgtgtgcccaccgatcccaatc ctcaggaagtggagctggtcaacgtgaccgagaacttcaacatgtggaagaacaacatggtggagcagatgcacgag gacatcatcagcctgtgggaccagtctctgaagccttgcgtgaagctgacacctctgtgcgtgggcagcggcagctgca acaccagcgtgatcacacaggcctgccccaagatcagcttcgagcctatccccatccactattgtgcccctgccggcttt gccatcctgaagtgcaacgacaagaagttcagcggcaagggcagctgcaagaacgtgagcaccgtgcagtgtacaca cggcatcagacctgtggtgtctacacagctgctgctgaatggctctctggccgaggaagaggtggtgatcagaagcgag aatttcaccaacaacgccaagaccatcatcgtgcagctgagggaacccgtgaagatcaactgcagccggcccaacaac aacacccggggccggcggggaagcagcggcggcagccactgcaacatcagcaagaccaactggaccaacgccctg aaacaggtggtggagaagctgggcgagcagttcaacaagaccaagatcgtgttcacccagagcagcggcggagatcc tgagatcgtgacccacagcttcaattgtgccggcgagttcttctactgcaataccacccagctgttcgacagcatctggaa cagcgagaacggcacctggaatatcaccaggggcctgaacaacaccggcaggaacgataccatcaccctgccctgca ggatcaagggaagcggagcccctcccatcaagggcaatattagctgcagcagcaacatcacaggactgctgctgacaa gagatggcggcaaggacgacaatagcagggacggcaacgagacattcagacctggcggcggagacatgagggaca attggcggagcgagctgtacaagtacaaggtggtgaagatcgagcccctgggcgtggctcccacaaaggccaagaga agagtggtgcagagcgaggagagcgccaccggcctgggcgccacctacatcggctacctgggagccgccggaagc accatgggcgctgccagcgtgacactgacagtgcaggctagacagctgctgtctggcattgtgcagcagcagagcaat ctgctgagagccatcgaagcccagcagcacctgctgcagctgacagtgtggggcatcaaacagctgcaggctcgcatt ctggccgtggagagatacctgaaggatcagcagctgctcggaatctggggctgtagcggcaagctgatctgtaccacc accgtgccttggaatgccagctggtccaagaacaagaccctggaagccatctggaataacatgacctggatgcagtgg gacaaagagatcgacaactacaccagcctgatctacagcctgatcgaggaaagccagatccagcaggaaaagaacga gcaggaactgctggaactggacaagtgggccaacctgtggaactggttcgacatcagcaactggctgtggggatccgg atacatccccgaggctccacgcgacggccaggcttacgtgcgcaaggacggcgagtgggtgctgctgagcaccttcct gggcctggaggtgctgttccagggcccaggccaccaccaccaccaccactga

CHIKV-OD A2.6 (SEQ ID NO: 140):

atggagttcatcccgacgcaaactttctataacagaaggtaccaaccccgaccctgggccccacgccctacaattcaagt aattagacctagaccacgtccacagaggcaggctgggcaactcgcccagctgatctccgcagtcaacaaattgaccatg cgcgcggtacctcaacagaagcctcgcagaaatcggaaaaacaagaagcaaaggcagaagaagcaggcgccgcaa aacgacccaaagcaaaagaagcaaccaccacaaaagaagccggctcaaaagaagaagaaaccaggccgtagggag agaatgtgcatgaaaattgaaaatgattgcatcttcgaagtcaagcatgaaggcaaagtgatgggctacgcatgcctggt gggggataaagtaatgaaaccagcacatgtgaagggaactatcgacaatgccgatctggctaaactggcctttaagcgg tcgtctaaatacgatcttgaatgtgcacagataccggtgcacatgaagtctgatgcctcgaagtttacccacgagaaaccc gaggggtactataactggcatcacggagcagtgcagtattcaggaggccggttcactatcccgacgggtgcaggcaag ccgggagacagcggcagaccgatcttcgacaacaaaggacgggtggtggccatcgtcctaggaggggccaacgaag gtgcccgcacggccctctccgtggtgacgtggaacaaagacatcgtcacaaaaattacccctgagggagccgaagagt ggagcctcgccctcccggtcttgtgcctgttggcaaacactacattcccctgctctcagccgccttgcacaccctgctgct acgaaaaggaaccggaaagcaccttgcgcatgcttgaggacaacgtgatgagacccggatactaccagctactaaaag catcgctgacttgctctccccaccgccaaagacgcagtactaaggacaattttaatgtctataaagccacaagaccatatct agctcattgtcctgactgcggagaagggcattcgtgccacagccctatcgcattggagcgcatcagaaatgaagcaacg gacggaacgctgaaaatccaggtctctttgcagatcgggataaagacagatgacagccacgattggaccaagctgcgct atatggatagccatacgccagcggacgcggagcgagccggattgcttgtaaggacttcagcaccgtgcacgatcaccg ggaccatgggacactttattctcgcccgatgcccgaaaggagagacgctgacagtgggatttacggacagcagaaaga tcagccacacatgcacacacccgttccatcatgaaccacctgtgataggtagggagaggttccactctcgaccacaacat ggtaaagagttaccttgcagcacgtacgtgcagagcaccgctgccactgctgaggagatagaggtgcatatgccccca gatactcctgaccgcacgctgatgacgcagcagtctggcaacgtgaagatcacagttaatgggcagacggtgcggtac aagtgcaactgcggtggctccggaaagcccgtggtgagcacccagctgctgctgaacggcagcctggccgagaagg agatcaggatcaagagcgagaacatcagcgacaacgccaagaccatcatcgtgcagctgaccaagcccgtgctgatc aactgcgccaggcccagcaacaacaccaggggcaggaggggcgacatcaggcaggcctactgcgtggtgaacagg acccagtggaacgacaccctgggccaggtggccatccagctgaggaagcactggaacacctgcatcatcttcaacgag cccagcggcggcgacctggagatcaccacccacagcttcaactgcggcggcgagttcacctactgcaacaccagcga cctgttcaacagcacctggaacatcgagggcaccgccagcatcaacggcaccgagagcaacgacaacatcaccctgc cctgcaggatcaagggcagcggcgcccctcccatccagggcgtgatcaggtgccagagcaacatcaccggcatcctg ctgaccagggacggcggcaacaccggcaacaacagcaggacctgcgagaccttcaggcccggcggcggcgacatg agggacaactggaggagcgagggctccggatcaaacgagggactgacaaccacagacaaagtgatcaataactgca aaattgatcagtgccatgctgcagtcactaatcacaagaattggcaatacaactcccctttagtcccgcgcaacgctgaac tcggggaccgtaaaggaaagatccacatcccattcccattggcaaacgtgacttgcagagtgccaaaagcaagaaacc ctacagtaacttacggaaaaaaccaagtcaccatgctgctgtatcctgaccatccgacactcttgtcttaccgtaacatggg acaggaaccaaattaccacgaggagtgggtgacacacaagaaggaggttaccttgaccgtgcctactgagggtctgga ggtcacttggggcaacaacgaaccatacaagtactggccgcagatgtctacgaacggtactgctcatggtcacccacat gagataatcttgtactattatgagctgtaccccactatgactgtagtcattgtgtcggtggcctcgttcgtgcttctgtcgatgg tgggcacagcagtgggaatgtgtgtgtgcgcacggcgcagatgcattacaccatatgaattaacaccaggagccactgt tcccttcctgctcagcctgctatgctgcgtcagaacgaccaaggcggccacatattacgaggctgcggcatatctatgga acgaacagcagcccctgttctggttgcaggctcttatcccgctggccgccttgatcgtcctgtgcaactgtctgaaactctt gccatgctgctgtaagaccctggcttttttagccgtaatgagcatcggtgcccacactgtgagcgcgtacgaacacgtaac agtgatcccgaacacggtgggagtaccgtataagactcttgtcaacagaccgggttacagccccatggtgttggagatg gagctacaatcagtcaccttggaaccaacactgtcacttgactacatcacgtgcgagtacaaaactgtcatcccctccccg tacgtgaagtgctgtggtacagcagagtgcaaggacaagagcctaccagactacagctgcaaggtctttactggagtcta cccatttatgtggggcggcgcctactgcttttgcgacgccgaaaatacgcaattgagcgaggcacatgtagagaaatctg aatcttgcaaaacagagtttgcatcggcctacagagcccacaccgcatcggcgtcggcgaagctccgcgtcctttacca aggaaacaacattaccgtagctgcctacgctaacggtgaccatgccgtcacagtaaaggacgccaagtttgtcgtgggc ccaatgtcctccgcctggacaccttttgacaacaaaatcgtggtgtacaaaggcgacgtctacaacatggactacccacct tttggcgcaggaagaccaggacaatttggtgacattcaaagtcgtacaccggaaagtaaagacgtttatgccaacactca gttggtactacagaggccagcagcaggcacggtacatgtaccatactctcaggcaccatctggcttcaagtattggctga aggaacgaggagcatcgctacagcacacggcaccgttcggttgccagattgcgacaaacccggtaagagctgtaaatt gcgctgtggggaacataccaatttccatcgacataccggatgcggcctttactagggttgtcgatgcaccctctgtaacgg acatgtcatgcgaagtaccagcctgcactcactcctccgactttgggggcgtcgccatcatcaaatacacagctagcaag aaaggtaaatgtgcagtacattcgatgaccaacgccgttaccattcgagaagccgacgtagaagtagaggggaactccc agctgcaaatatccttctcaacagccctggcaagcgccgagtttcgcgtgcaagtgtgctccacacaagtacactgcgca gccgcatgccaccctccaaaggaccacatagtcaattacccagcatcacacaccacccttggggtccaggatatatcca caacggcaatgtcttgggtgcagaagattacgggaggagtaggattaattgttgctgttgctgccttaattttaattgtggtg ctatgcgtgtcgtttagcaggcactaa

CHIKV-0D4.1 (SEQ ID NO: 141):

atggagttcatcccgacgcaaactttctataacagaaggtaccaaccccgaccctgggccccacgccctacaattcaagt aattagacctagaccacgtccacagaggcaggctgggcaactcgcccagctgatctccgcagtcaacaaattgaccatg cgcgcggtacctcaacagaagcctcgcagaaatcggaaaaacaagaagcaaaggcagaagaagcaggcgccgcaa aacgacccaaagcaaaagaagcaaccaccacaaaagaagccggctcaaaagaagaagaaaccaggccgtagggag agaatgtgcatgaaaattgaaaatgattgcatcttcgaagtcaagcatgaaggcaaagtgatgggctacgcatgcctggt gggggataaagtaatgaaaccagcacatgtgaagggaactatcgacaatgccgatctggctaaactggcctttaagcgg tcgtctaaatacgatcttgaatgtgcacagataccggtgcacatgaagtctgatgcctcgaagtttacccacgagaaaccc gaggggtactataactggcatcacggagcagtgcagtattcaggaggccggttcactatcccgacgggtgcaggcaag ccgggagacagcggcagaccgatcttcgacaacaaaggacgggtggtggccatcgtcctaggaggggccaacgaag gtgcccgcacggccctctccgtggtgacgtggaacaaagacatcgtcacaaaaattacccctgagggagccgaagagt ggagcctcgccctcccggtcttgtgcctgttggcaaacactacattcccctgctctcagccgccttgcacaccctgctgct acgaaaaggaaccggaaagcaccttgcgcatgcttgaggacaacgtgatgagacccggatactaccagctactaaaag catcgctgacttgctctccccaccgccaaagacgcagtactaaggacaattttaatgtctataaagccacaagaccatatct agctcattgtcctgactgcggagaagggcattcgtgccacagccctatcgcattggagcgcatcagaaatgaagcaacg gacggaacgctgaaaatccaggtctctttgcagatcgggataaagacagatgacagccacgattggaccaagctgcgct atatggatagccatacgccagcggacgcggagcgagccggattgcttgtaaggacttcagcaccgtgcacgatcaccg ggaccatgggacactttattctcgcccgatgcccgaaaggagagacgctgacagtgggatttacggacagcagaaaga tcagccacacatgcacacacccgttccatcatgaaccacctgtgataggtagggagaggttccactctcgaccacaacat ggtaaagagttaccttgcagcacgtacgtgcagagcaccgctgccactgctgaggagatagaggtgcatatgccccca gatactcctgaccgcacgctgatgacgcagcagtctggcaacgtgaagatcacagttaatgggcagacggtgcggtac aagtgcaactgcggtggctccggaagacctgtggtgtctacacagctgctgctgaatggctctctggccgaggaagagg tggtgatcagaagcgagaatttcaccaactgcgccaagaccatcatcgtgcagctgagggaacccgtgaagatcaactg cagccggcccaacaacaatacccggggcagaagaggagacatcagacaggcccactgcaacatcagcaagaccaa ctggaccaacgccctgaaacaggtggtggagaagctgggcgagcagttcaacaagaccaagatcgtgttcacccaga gcagcggcggagatcctgagatcgtgacccacagcttcaattgtgccggcgagttcacctactgcaataccacccagct gttcgacagcatctggaacagcgagaacggcacctggaatatcaccaggggcctgaacaacaccggcaggaacgata ccatcaccctgccctgcaggatcaagggaagcggagcccctcccatcaagggcaatattagctgcagcagcaacatca caggactgctgctgacaagagattgcggcaaggacgacaatagcagggacggcaacgagacattcagacctggcgg cggagacatgagggacaattggcggagcgagggctccggatcaaacgagggactgacaaccacagacaaagtgatc aataactgcaaaattgatcagtgccatgctgcagtcactaatcacaagaattggcaatacaactcccctttagtcccgcgca acgctgaactcggggaccgtaaaggaaagatccacatcccattcccattggcaaacgtgacttgcagagtgccaaaagc aagaaaccctacagtaacttacggaaaaaaccaagtcaccatgctgctgtatcctgaccatccgacactcttgtcttaccgt aacatgggacaggaaccaaattaccacgaggagtgggtgacacacaagaaggaggttaccttgaccgtgcctactgag ggtctggaggtcacttggggcaacaacgaaccatacaagtactggccgcagatgtctacgaacggtactgctcatggtc acccacatgagataatcttgtactattatgagctgtaccccactatgactgtagtcattgtgtcggtggcctcgttcgtgcttct gtcgatggtgggcacagcagtgggaatgtgtgtgtgcgcacggcgcagatgcattacaccatatgaattaacaccagga gccactgttcccttcctgctcagcctgctatgctgcgtcagaacgaccaaggcggccacatattacgaggctgcggcata tctatggaacgaacagcagcccctgttctggttgcaggctcttatcccgctggccgccttgatcgtcctgtgcaactgtctg aaactcttgccatgctgctgtaagaccctggcttttttagccgtaatgagcatcggtgcccacactgtgagcgcgtacgaa cacgtaacagtgatcccgaacacggtgggagtaccgtataagactcttgtcaacagaccgggttacagccccatggtgtt ggagatggagctacaatcagtcaccttggaaccaacactgtcacttgactacatcacgtgcgagtacaaaactgtcatcc cctccccgtacgtgaagtgctgtggtacagcagagtgcaaggacaagagcctaccagactacagctgcaaggtctttac tggagtctacccatttatgtggggcggcgcctactgcttttgcgacgccgaaaatacgcaattgagcgaggcacatgtag agaaatctgaatcttgcaaaacagagtttgcatcggcctacagagcccacaccgcatcggcgtcggcgaagctccgcgt cctttaccaaggaaacaacattaccgtagctgcctacgctaacggtgaccatgccgtcacagtaaaggacgccaagtttg tcgtgggcccaatgtcctccgcctggacaccttttgacaacaaaatcgtggtgtacaaaggcgacgtctacaacatggac tacccaccttttggcgcaggaagaccaggacaatttggtgacattcaaagtcgtacaccggaaagtaaagacgtttatgc caacactcagttggtactacagaggccagcagcaggcacggtacatgtaccatactctcaggcaccatctggcttcaagt attggctgaaggaacgaggagcatcgctacagcacacggcaccgttcggttgccagattgcgacaaacccggtaagag ctgtaaattgcgctgtggggaacataccaatttccatcgacataccggatgcggcctttactagggttgtcgatgcaccctc tgtaacggacatgtcatgcgaagtaccagcctgcactcactcctccgactttgggggcgtcgccatcatcaaatacacag ctagcaagaaaggtaaatgtgcagtacattcgatgaccaacgccgttaccattcgagaagccgacgtagaagtagaggg gaactcccagctgcaaatatccttctcaacagccctggcaagcgccgagtttcgcgtgcaagtgtgctccacacaagtac actgcgcagccgcatgccaccctccaaaggaccacatagtcaattacccagcatcacacaccacccttggggtccagg atatatccacaacggcaatgtcttgggtgcagaagattacgggaggagtaggattaattgttgctgttgctgccttaatttta attgtggtgctatgcgtgtcgtttagcaggcactaa

CMV/R RSC3 Y6.1 His (SEQ ID NO: 143):

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggat gccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatc agagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaga ttggctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttga cattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataact tacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaa cgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatca tatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatggga ctttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtgg atagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgg gactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataag cagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggacc gatccagcctccatcggctcgcatctctccttcacgcgcccgccgccttacctgaggccgccatccacgccggttgagtc gcgttctgccgcctcccgcctgtggtgcctcctgaactacgtccgccgtctaggtaagtttagagctcaggtcgagaccg ggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactct agttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcgcgcgccaccagacataatagctgacagac taacagactgttcctttccatgggtcttttctgcagtcaccgtcgtcgacacgtgtgatcagatatcgcggccgctctagaga tatcgccaccatgcctatgggatctctgcagcctctggccacactgtatctgctgggaatgctggtcgcttctgtgctggcc acaaacgtgaccgtgaatgtgaccgtgaccttcgattggtgcgccgatgatatggtggctacaatgaacaccagcatctg caccctgtggaacaccagcaacgacccctgcaccaagtgtcctaccgtgcggtttaagcccgtgcccatcagatattgtg cccctcctggctatgccatcctgaagtgcaacaaccgggactttaatggcaccggcccttgcacaaatgtgtccaacgtg acctgtacagatggcatccaccctgtggtgtctagtcagctgctgctgaatggcacactggccgatgagaaggtggtgat cagaagctgcaacttcagcgacaacgccaagaccatcatcgtgcagctgaacaccagcgtggagatcaattgtacagg ccagggccactgcaatatcacccgggccaagtggaatcagaccctgaagcagatcgccgagaagctgagagagcagt tcggcaacaacaagacaatcatcttcaggcctagctctggcggagatcctgagatcgtgacccactggttcaattgcggc ggcaagttcttctactgcaacagcacccagctgttcaacagcacctggttcaactctacttggagcaccaagggcagcaa caacaccgagggcagcgataccatcaccctgccctgcaggatcagatctatcaccggcatgaactgcacagtgggcaa gatgatctacgcccctcctgtggaaggcaacatcacctgcagcagcaacatcacaggcctgctgctgacaagagatgg cggcaacgacaacaacgagagcgagatctttagacctggcggcggagacatgagggacaattggcggagcgagctg tacaagtacagagtggtgcggctgaccggatcccatcatcatcatcatcattagtctggaagggcgaattgatccagatct gctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgt cctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggac agcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgggtacccaggtgctgaa gaattgacccggttcctcctgggccagaaagaagcaggcacatccccttctctgtgacacaccctgtccacgcccctggt tcttagttccagccccactcataggacactcatagctcaggagggctccgccttcaatcccacccgctaaagtacttggag cggtctctccctccctcatcagcccaccaaaccaaacctagcctccaagagtgggaagaaattaaagcaagataggctat taagtgcagagggagagaaaatgcctccaacatgtgaggaagtaatgagagaaatcatagaattttaaggccatcatgg ccttaatcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaag gcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccag gaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctca agtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctg ttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtag gtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgcctt atccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggatta gcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtattt ggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggt agcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggg gtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttt taaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgagg cacctatctcagcgatctgtctatttcgttcatccatagttgcctgactcggggggggggggcgctgaggtctgcctcgtg aagaaggtgttgctgactcataccaggcctgaatcgccccatcatccagccagaaagtgagggagccacggttgatga gagctttgttgtaggtggaccagttggtgattttgaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtg atctgatccttcaactcagcaaaagttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtg ttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaat accatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtat cggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatc accatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccatta cgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcg ctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcac ctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagt acggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattg gcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgatt gcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgt ttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatctt gtgcaatgtaacatcagagattttgagacacaacgtggctttccccccccccccattattgaagcatttatcagggttattgt ctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccac ctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc

CMV8x/R_KER2018_OD4.2'_HG3.2_HATM (SEQ ID NO: 144):

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggat gccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatc agagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaga ttggctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttga cattgattattgactagttattaatagtaatcaattacgggaacttccatagcccatatatggagttccgcgttacataacttac gggaatttccaaacctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggaacttccattgacgtcaatgggtggagtatttacggtaaactgcccacttgggaatttccaagtgtatcatatgc caagtacgccccctattgacgtcaatgacgggaacttccataagcttgcattatgcccagtacatgaccttatgggaatttc ctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatag cggtttgactcacgggaacttccaagtctccaccccattgacgtcaatgggagtttgttttgactcaccaaaatcaacggga attcccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagca gagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccg atccagcctccatcggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtc gcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagaccg ggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactct agttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcgcgcgccaccagacataatagctgacagac taacagactgttcctttccatgggtcttttctgcagtcaccgtcgtcgacacgtgtgatcagatatcgcggccgctctagaca ccatgcccatgggcagcctgcagcccctggccaccctgtacctgctgggcatgctggtggctagcgtgctggccaagc ccgtggtgagcacccagctgctgctgaacggcagcctggccgagaaggagatcaggatcaagagcgaggacatcag cgacaacgccaagaacatcatcgtgcagctgaccaagcccgtgctgatcaactgcaccaggcccagcaacaacaccc agtactgcgtggtgaacaggacccagtggaacgacaccctgggccaggtggccatccagctgaggaagcactggaac acctgcatcatcttcaacgagcccagcggcggcgacctggagatcaccacccacagcttcaactgcggcggcgagttc TTCtactgcaacaccagcgacctgttcaacagcacctggaacatcaacggcaccgccagcatcaacggcaccgaga gcaacgacaacatcaccctgccctgcaggatcaacggcaccatccagggcaacatcacctgccagagcaacatcacc ggcatcctgctgaccagggacggcggcagcggcagcggcacctgcgagaccttcaggcccggcggcggcgacatg agggacctgaacaggaccagcgGATCCGGCATCCTGGCCATCTACAGCACCGTGGCC AGCAGCCTGGTGCTGCTGGTGAGCCTGGGCGCCATCAGCTTCTAgatcctagc atcatcatcatcatcattagtctgaagggcgaattgatccagctgtgccttctagttgccagccatctgttgtttgcccctccc ccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagt aggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgc tggggatgcggtgggctctatgggtacccaggtgctgaagaattgacccggttcctcctgggccagaaagaagcaggc acatccccttctctgtgacacaccctgtccacgcccctggttcttagttccagccccactcataggacactcatagctcagg agggctccgccttcaatcccacccgctaaagtacttggagcggtctctccctccctcatcagcccaccaaaccaaaccta gcctccaagagtgggaagaaattaaagcaagataggctattaagtgcagagggagagaaaatgcctccaacatgtgag gaagtaatgagagaaatcatagaattttaaggccatgatttaaggccatcatggccttaatcttccgcttcctcgctcactga ctcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatca ggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctg gcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacag gactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacc tgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgct ccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaac ccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgct acagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagtta ccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagca gattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactc acgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatct aaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttc atccatagttgcctgactcggggggggggggcgctgaggtctgcctcgtgaagaaggtgttgctgactcataccaggcc tgaatcgccccatcatccagccagaaagtgagggagccacggttgatgagagctttgttgtaggtggaccagttggtgat tttgaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaaagttcgatt tattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaa actcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatga aggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaa tacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaa tggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaacca aaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatc gaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaat gctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagagg cataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaa ctctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttataccca tataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcataacaccccttgt attactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacaca acgtggctttccccccccccccattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttaga aaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacatt aacctataaaaataggcgtatcacgaggccctttcgtc

CMV8x/R_KER2018_OD4.2'_HG3.2.1_HATM (SEQ ID NO: 145):

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggat gccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatc agagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaga ttggctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttga cattgattattgactagttattaatagtaatcaattacgggaacttccatagcccatatatggagttccgcgttacataacttac gggaatttccaaacctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggaacttccattgacgtcaatgggtggagtatttacggtaaactgcccacttgggaatttccaagtgtatcatatgc caagtacgccccctattgacgtcaatgacgggaacttccataagcttgcattatgcccagtacatgaccttatgggaatttc ctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatag cggtttgactcacgggaacttccaagtctccaccccattgacgtcaatgggagtttgttttgactcaccaaaatcaacggga attcccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagca gagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccg atccagcctccatcggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtc gcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagaccg ggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactct agttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcgcgcgccaccagacataatagctgacagac taacagactgttcctttccatgggtcttttctgcagtcaccgtcgtcgacacgtgtgatcagatatcgcggccgctctagaca ccatgcccatgggcagcctgcagcccctggccaccctgtacctgctgggcatgctggtggctagcgtgctggccaagc ccgtggtgagcacccagctgctgctgaacggcagcctggccgagaaggagatcaggatcaagagcgaggacatcag cgacaacgccaagaacatcatcgtgcagctgaccaagcccgtgctgatcaactgcaccaggcccagcaacaacaccc agtactgcgtggtgaacaggacccagtggaacgacaccctgggccaggtggccatccagctgaggaagcactggaac acctgcatcatcttcaccaacagcagcggcggcgacctggagatcaccacccacagcttcaactgcggcggcgagttc TTCtactgcaacaccagcgacctgttcaacagcacctggaacatcaacggcaccgccagcatcaacggcaccgaga gcaacgacaacatcaccctgccctgcaggatcaacggcaccatccagggcaacatcacctgccagagcaacatcacc ggcatcctgctgaccagggacggcggcagcggcagcggcacctgcgagaccttcaggcccggcggcggcgacatg agggacctgaacaggaccagcgGATCCGGCATCCTGGCCATCTACAGCACCGTGGCC AGCAGCCTGGTGCTGCTGGTGAGCCTGGGCGCCATCAGCTTCTAgatcctagc atcatcatcatcatcattagtctgaagggcgaattgatccagctgtgccttctagttgccagccatctgttgtttgcccctccc ccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagt aggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgc tggggatgcggtgggctctatgggtacccaggtgctgaagaattgacccggttcctcctgggccagaaagaagcaggc acatccccttctctgtgacacaccctgtccacgcccctggttcttagttccagccccactcataggacactcatagctcagg agggctccgccttcaatcccacccgctaaagtacttggagcggtctctccctccctcatcagcccaccaaaccaaaccta gcctccaagagtgggaagaaattaaagcaagataggctattaagtgcagagggagagaaaatgcctccaacatgtgag gaagtaatgagagaaatcatagaattttaaggccatgatttaaggccatcatggccttaatcttccgcttcctcgctcactga ctcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatca ggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctg gcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacag gactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacc tgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgct ccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaac ccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgct acagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagtta ccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagca gattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactc acgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatct aaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttc atccatagttgcctgactcggggggggggggcgctgaggtctgcctcgtgaagaaggtgttgctgactcataccaggcc tgaatcgccccatcatccagccagaaagtgagggagccacggttgatgagagctttgttgtaggtggaccagttggtgat tttgaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaaagttcgatt tattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaa actcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatga aggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaa tacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaa tggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaacca aaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatc gaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaat gctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagagg cataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaa ctctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttataccca tataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcataacaccccttgt attactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacaca acgtggctttccccccccccccattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttaga aaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacatt aacctataaaaataggcgtatcacgaggccctttcgtc CMV8x/R_KER2018_OD4.2'_HG3.2_NATM (SEQ ID NO: 146):

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggat gccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatc agagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaga ttggctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttga cattgattattgactagttattaatagtaatcaattacgggaacttccatagcccatatatggagttccgcgttacataacttac gggaatttccaaacctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggaacttccattgacgtcaatgggtggagtatttacggtaaactgcccacttgggaatttccaagtgtatcatatgc caagtacgccccctattgacgtcaatgacgggaacttccataagcttgcattatgcccagtacatgaccttatgggaatttc ctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatag cggtttgactcacgggaacttccaagtctccaccccattgacgtcaatgggagtttgttttgactcaccaaaatcaacggga attcccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagca gagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccg atccagcctccatcggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtc gcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagaccg ggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactct agttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcgcgcgccaccagacataatagctgacagac taacagactgttcctttccatgggtcttttctgcagtcaccgtcgtcgacacgtgtgatcagatatcgcggccgctctagaca ccATGAACCCCAACCAGAAGATCATCACCATCGGCAGCATCTGCATGGTG GTGGGCATCATCAGCCTGATCCTGCAGATCGGCAACATCATCAGCATCT GGGTGTCCGGAaagcccgtggtgagcacccagctgctgctgaacggcagcctggccgagaaggagatcag gatcaagagcgaggacatcagcgacaacgccaagaacatcatcgtgcagctgaccaagcccgtgctgatcaactgca ccaggcccagcaacaacacccagtactgcgtggtgaacaggacccagtggaacgacaccctgggccaggtggccat ccagctgaggaagcactggaacacctgcatcatcttcaacgagcccagcggcggcgacctggagatcaccacccaca gcttcaactgcggcggcgagttcTTCtactgcaacaccagcgacctgttcaacagcacctggaacatcaacggcacc gccagcatcaacggcaccgagagcaacgacaacatcaccctgccctgcaggatcaacggcaccatccagggcaacat cacctgccagagcaacatcaccggcatcctgctgaccagggacggcggcagcggcagcggcacctgcgagaccttc aggcccggcggcggcgacatgagggacctgaacaggaccagcggatcctagcatcatcatcatcatcattagtctgaa gggcgaattgatccagctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaag gtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtg gggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgg gtacccaggtgctgaagaattgacccggttcctcctgggccagaaagaagcaggcacatccccttctctgtgacacacc ctgtccacgcccctggttcttagttccagccccactcataggacactcatagctcaggagggctccgccttcaatcccacc cgctaaagtacttggagcggtctctccctccctcatcagcccaccaaaccaaacctagcctccaagagtgggaagaaatt aaagcaagataggctattaagtgcagagggagagaaaatgcctccaacatgtgaggaagtaatgagagaaatcataga attttaaggccatgatttaaggccatcatggccttaatcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggct gcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacat gtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccc tgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttcc ccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagc gtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaac cccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgcca ctggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggccta actacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagct cttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggat ctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgag attatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggt ctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactcgggg ggggggggcgctgaggtctgcctcgtgaagaaggtgttgctgactcataccaggcctgaatcgccccatcatccagcc agaaagtgagggagccacggttgatgagagctttgttgtaggtggaccagttggtgattttgaacttttgctttgccacgga acggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaaagttcgatttattcaacaaagccgccgtccc gtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaa ctgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggca gttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgt caaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttcttt ccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgc gcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaa cactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcag tggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagttta gtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttccca tacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttgga atttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagaca gttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttccccccccccc cattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttc cgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtat cacgaggccctttcgtc CMV8x/R_KER2018_OD4.2'_HG3.2.1_NATM (SEQ ID NO: 147):

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggat gccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatc agagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaga ttggctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttga cattgattattgactagttattaatagtaatcaattacgggaacttccatagcccatatatggagttccgcgttacataacttac gggaatttccaaacctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggaacttccattgacgtcaatgggtggagtatttacggtaaactgcccacttgggaatttccaagtgtatcatatgc caagtacgccccctattgacgtcaatgacgggaacttccataagcttgcattatgcccagtacatgaccttatgggaatttc ctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatag cggtttgactcacgggaacttccaagtctccaccccattgacgtcaatgggagtttgttttgactcaccaaaatcaacggga attcccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagca gagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccg atccagcctccatcggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtc gcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagaccg ggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactct agttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcgcgcgccaccagacataatagctgacagac taacagactgttcctttccatgggtcttttctgcagtcaccgtcgtcgacacgtgtgatcagatatcgcggccgctctagaca ccATGAACCCCAACCAGAAGATCATCACCATCGGCAGCATCTGCATGGTG GTGGGCATCATCAGCCTGATCCTGCAGATCGGCAACATCATCAGCATCT GGGTGTCCGGAaagcccgtggtgagcacccagctgctgctgaacggcagcctggccgagaaggagatcag gatcaagagcgaggacatcagcgacaacgccaagaacatcatcgtgcagctgaccaagcccgtgctgatcaactgca ccaggcccagcaacaacacccagtactgcgtggtgaacaggacccagtggaacgacaccctgggccaggtggccat ccagctgaggaagcactggaacacctgcatcatcttcaccaacagcagcggcggcgacctggagatcaccacccaca gcttcaactgcggcggcgagttcTTCtactgcaacaccagcgacctgttcaacagcacctggaacatcaacggcacc gccagcatcaacggcaccgagagcaacgacaacatcaccctgccctgcaggatcaacggcaccatccagggcaacat cacctgccagagcaacatcaccggcatcctgctgaccagggacggcggcagcggcagcggcacctgcgagaccttc aggcccggcggcggcgacatgagggacctgaacaggaccagcggatcctagcatcatcatcatcatcattagtctgaa gggcgaattgatccagctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaag gtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtg gggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgg gtacccaggtgctgaagaattgacccggttcctcctgggccagaaagaagcaggcacatccccttctctgtgacacacc ctgtccacgcccctggttcttagttccagccccactcataggacactcatagctcaggagggctccgccttcaatcccacc cgctaaagtacttggagcggtctctccctccctcatcagcccaccaaaccaaacctagcctccaagagtgggaagaaatt aaagcaagataggctattaagtgcagagggagagaaaatgcctccaacatgtgaggaagtaatgagagaaatcataga attttaaggccatgatttaaggccatcatggccttaatcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggct gcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacat gtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccc tgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttcc ccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagc gtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaac cccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgcca ctggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggccta actacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagct cttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggat ctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgag attatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggt ctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactcgggg ggggggggcgctgaggtctgcctcgtgaagaaggtgttgctgactcataccaggcctgaatcgccccatcatccagcc agaaagtgagggagccacggttgatgagagctttgttgtaggtggaccagttggtgattttgaacttttgctttgccacgga acggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaaagttcgatttattcaacaaagccgccgtccc gtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaa ctgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggca gttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgt caaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttcttt ccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgc gcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaa cactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcag tggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagttta gtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttccca tacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttgga atttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagaca gttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttccccccccccc cattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttc cgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtat cacgaggccctttcgtc

0D4.1 (SEQ ID NO: 150):

Atgcagctggccagctgtgtgacactgacactggtgctgctggtgaattccgcccctaggagacctgtgtgctctacaca gctgctgctgaatggctctctggccgaggaagaggtggtgatcagaagcgagaatttcaccaacaacgccaagaacat catcgtgcagctgagggaacccgtgaagatcaactgcagccggcccaacaacaatacccggagacaggcccactgca acatcagcaagaccaactggaacaacaccctgaaacaggtggtggagaagctgggcgagcagttcaacaagaccaa gatcgtgttcacccagagcagcggcggagatcctgagatcgtgaacctgaccttcaattgtgccggcgagttcttctactg caataccacccagctgttcaacagcacctggaatatcgagggcaccgccagcatcaacggaaccgagagcaacgata acatcaccctgccctgcaggatcaagggaagcggagcccctcccatcaagggcaatattagctgcagcagcaacatca caggactgctgctgacaagagatggcggcagcggcggcagcggccaggagacattcagacctggcggcggagact gcagggacaattggcggagcgagggatccctggaggtgctgttccagggcccaggccaccaccaccaccaccactg atga CHIKV-OD4.1 (SEQ ID NO: 151):

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggat gccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatc agagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaga ttggctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttga cattgattattgactagttattaatagtaatcaattacgggaacttccatagcccatatatggagttccgcgttacataacttac gggaatttccaaacctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggaacttccattgacgtcaatgggtggagtatttacggtaaactgcccacttgggaatttccaagtgtatcatatgc caagtacgccccctattgacgtcaatgacgggaacttccataagcttgcattatgcccagtacatgaccttatgggaatttc ctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatag cggtttgactcacgggaacttccaagtctccaccccattgacgtcaatgggagtttgttttgactcaccaaaatcaacggga attcccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagca gagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccg atccagcctccatcggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtc gcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagaccg ggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactct agttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcgcgcgccaccagacataatagctgacagac taacagactgttcctttccatgggtcttttctgcagtcaccgtcgtcgacacgtgtgatcagatatcgcggccgctctagaca ccatgtatagcatgcagctggccagctgtgtgacactgacactggtgctgctggtgaattccgcccctaggagacctgtgt gctctacacagctgctgctgaatggctctctggccgaggaagaggtggtgatcagaagcgagaatttcaccaacaacgc caagaacatcatcgtgcagctgagggaacccgtgaagatcaactgcagccggcccaacaacaatacccggagacagg cccactgcaacatcagcaagaccaactggaacaacaccctgaaacaggtggtggagaagctgggcgagcagttcaac aagaccaagatcgtgttcacccagagcagcggcggagatcctgagatcgtgaacctgaccttcaattgtgccggcgagt tcttctactgcaataccacccagctgttcaacagcacctggaatatcgagggcaccgccagcatcaacggaaccgagag caacgataacatcaccctgccctgcaggatcaagggaagcggagcccctcccatcaagggcaatattagctgcagcag caacatcacaggactgctgctgacaagagatggcggcagcggcggcagcggccaggagacattcagacctggcggc ggagactgcagggacaattggcggagcgagggatccctggaggtgctgttccagggcccaggccaccaccaccacc accactgatgaagatcttcaagggcgaattctgcagatgatctgctgtgccttctagttgccagccatctgttgtttgcccct cccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctg agtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggca tgctggggatgcggtgggctctatgggtacccaggtgctgaagaattgacccggttcctcctgggccagaaagaagca ggcacatccccttctctgtgacacaccctgtccacgcccctggttcttagttccagccccactcataggacactcatagctc aggagggctccgccttcaatcccacccgctaaagtacttggagcggtctctccctccctcatcagcccaccaaaccaaac ctagcctccaagagtgggaagaaattaaagcaagataggctattaagtgcagagggagagaaaatgcctccaacatgtg aggaagtaatgagagaaatcatagaattttaaggccatgatttaaggccatcatggccttaatcttccgcttcctcgctcact gactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaat caggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgct ggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgaca ggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatac ctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcg ctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtcca acccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtg ctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagt taccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcag cagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaa ctcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatca atctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttc gttcatccatagttgcctgactcggggggggggggcgctgaggtctgcctcgtgaagaaggtgttgctgactcatacca ggcctgaatcgccccatcatccagccagaaagtgagggagccacggttgatgagagctttgttgtaggtggaccagttg gtgattttgaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaaagt tcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgatta gaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgt aatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaa catcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccgg tgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcat caaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaaca ggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacc tggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcgga agaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcaga aacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttat acccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcataacacc ccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgag acacaacgtggctttccccccccccccattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgta tttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcat gacattaacctataaaaataggcgtatcacgaggccctttcgtc

OD4.3.2Vmut_3G/424N (SEQ ID NO: 152):

atggagttcatcccgacgcaaactttctataacagaaggtaccaaccccgaccctgggccccacgccctacaattcaagt aattagacctagaccacgtccacagaggcaggctgggcaactcgcccagctgatctccgcagtcaacaaattgaccatg cgcgcggtacctcaacagaagcctcgcagaaatcggaaaaacaagaagcaaaggcagaagaagcaggcgccgcaa aacgacccaaagcaaaagaagcaaccaccacaaaagaagccggctcaaaagaagaagaaaccaggccgtagggag agaatgtgcatgaaaattgaaaatgattgcatcttcgaagtcaagcatgaaggcaaagtgatgggctacgcatgcctggt gggggataaagtaatgaaaccagcacatgtgaagggaactatcgacaatgccgatctggctaaactggcctttaagcgg tcgtctaaatacgatcttgaatgtgcacagataccggtgcacatgaagtctgatgcctcgaagtttacccacgagaaaccc gaggggtactataactggcatcacggagcagtgcagtattcaggaggccggttcactatcccgacgggtgcaggcaag ccgggagacagcggcagaccgatcttcgacaacaaaggacgggtggtggccatcgtcctaggaggggccaacgaag gtgcccgcacggccctctccgtggtgacgtggaacaaagacatcgtcacaaaaattacccctgagggagccgaagagt ggagcctcgccctcccggtcttgtgcctgttggcaaacactacattcccctgctctcagccgccttgcacaccctgctgct acgaaaaggaaccggaaagcaccttgcgcatgcttgaggacaacgtgatgagacccggatactaccagctactaaaag catcgctgacttgctctccccaccgccaaagacgcagtactaaggacaattttaatgtctataaagccacaagaccatatct agctcattgtcctgactgcggagaagggcattcgtgccacagccctatcgcattggagcgcatcagaaatgaagcaacg gacggaacgctgaaaatccaggtctctttgcagatcgggataaagacagatgacagccacgattggaccaagctgcgct atatggatagccatacgccagcggacgcggagcgagccggattgcttgtaaggacttcagcaccgtgcacgatcaccg ggaccatgggacactttattctcgcccgatgcccgaaaggagagacgctgacagtgggatttacggacagcagaaaga tcagccacacatgcacacacccgttccatcatgaaccacctgtgataggtagggagaggttccactctcgaccacaacat ggtaaagagttaccttgcagcacgtacgtgcagagcaccgctgccactgctgaggagatagaggtgcatatgccccca gatactcctgaccgcacgctgatgacgcagcagtctggcaacgtgaagatcacagttaatgggcagacggtgcggtac aagtgcaactgcggtggctccggaagacctgtgtgctctacacagctgctgctgaatggctctctggccgaggaagagg tggtgatcagaagcgagaatttcaccaacaacgccaagaacatcatcgtgcagctgagggaacccgtgaagatcaact gcagccggcccaacaacaatacccggagacaggcccactgcaacatcagcaagaccaactggaacaacaccctgaa acaggtggtggagaagctgggcgagcagttcaacaagaccaagatcgtgttcacccagagcagcggcggagatcctg agatcgtgacccacagcttcaattgtgccggcgagttcttctactgcaataccacccagctgttcaacagcacctggaatat cgagggcaccgccagcatcaacggaaccgagagcaacgataacatcaccctgccctgcaggatcaagggaagcaac gccaccgtgatcaagggcaatattagctgcagcagcaacatcacaggactgctgctgacaagagatggcggcagcgg cggcagcggccaggagacattcagacctggcggcggagactgcagggacaactggaggagcgagggctccggatc aaacgagggactgacaaccacagacaaagtgatcaataactgcaaaattgatcagtgccatgctgcagtcactaatcac aagaattggcaatacaactcccctttagtcccgcgcaacgctgaactcggggaccgtaaaggaaagatccacatcccatt cccattggcaaacgtgacttgcagagtgccaaaagcaagaaaccctacagtaacttacggaaaaaaccaagtcaccatg ctgctgtatcctgaccatccgacactcttgtcttaccgtaacatgggacaggaaccaaattaccacgaggagtgggtgac acacaagaaggaggttaccttgaccgtgcctactgagggtctggaggtcacttggggcaacaacgaaccatacaagtac tggccgcagatgtctacgaacggtactgctcatggtcacccacatgagataatcttgtactattatgagctgtaccccactat gactgtagtcattgtgtcggtggcctcgttcgtgcttctgtcgatggtgggcacagcagtgggaatgtgtgtgtgcgcacg gcgcagatgcattacaccatatgaattaacaccaggagccactgttcccttcctgctcagcctgctatgctgcgtcagaac gaccaaggcggccacatattacgaggctgcggcatatctatggaacgaacagcagcccctgttctggttgcaggctctta tcccgctggccgccttgatcgtcctgtgcaactgtctgaaactcttgccatgctgctgtaagaccctggcttttttagccgta atgagcatcggtgcccacactgtgagcgcgtacgaacacgtaacagtgatcccgaacacggtgggagtaccgtataag actcttgtcaacagaccgggttacagccccatggtgttggagatggagctacaatcagtcaccttggaaccaacactgtc acttgactacatcacgtgcgagtacaaaactgtcatcccctccccgtacgtgaagtgctgtggtacagcagagtgcaagg acaagagcctaccagactacagctgcaaggtctttactggagtctacccatttatgtggggcggcgcctactgcttttgcg acgccgaaaatacgcaattgagcgaggcacatgtagagaaatctgaatcttgcaaaacagagtttgcatcggcctacag agcccacaccgcatcggcgtcggcgaagctccgcgtcctttaccaaggaaacaacattaccgtagctgcctacgctaac ggtgaccatgccgtcacagtaaaggacgccaagtttgtcgtgggcccaatgtcctccgcctggacaccttttgacaacaa aatcgtggtgtacaaaggcgacgtctacaacatggactacccaccttttggcgcaggaagaccaggacaatttggtgac attcaaagtcgtacaccggaaagtaaagacgtttatgccaacactcagttggtactacagaggccagcagcaggcacgg tacatgtaccatactctcaggcaccatctggcttcaagtattggctgaaggaacgaggagcatcgctacagcacacggca ccgttcggttgccagattgcgacaaacccggtaagagctgtaaattgcgctgtggggaacataccaatttccatcgacata ccggatgcggcctttactagggttgtcgatgcaccctctgtaacggacatgtcatgcgaagtaccagcctgcactcactcc tccgactttgggggcgtcgccatcatcaaatacacagctagcaagaaaggtaaatgtgcagtacattcgatgaccaacgc cgttaccattcgagaagccgacgtagaagtagaggggaactcccagctgcaaatatccttctcaacagccctggcaagc gccgagtttcgcgtgcaagtgtgctccacacaagtacactgcgcagccgcatgccaccctccaaaggaccacatagtca attacccagcatcacacaccacccttggggtccaggatatatccacaacggcaatgtcttgggtgcagaagattacggga ggagtaggattaattgttgctgttgctgccttaattttaattgtggtgctatgcgtgtcgtttagcaggcactaa CHIKV-OD4.3.2Vmut_3G/424N (SEQ ID NO: 153):

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggat gccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatc agagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaga ttggctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttga cattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataact tacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaa cgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatca tatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatggga ctttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtgg atagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgg gactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataag cagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggacc gatccagcctccatcggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagt cgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagacc gggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactc tagttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcgcgcgccaccagacataatagctgacaga ctaacagactgttcctttccatgggtcttttctgcagtcaccgtcgtcgacacgtgtgatcagatatcgcggccgctctagac accatggagttcatcccgacgcaaactttctataacagaaggtaccaaccccgaccctgggccccacgccctacaattca agtaattagacctagaccacgtccacagaggcaggctgggcaactcgcccagctgatctccgcagtcaacaaattgacc atgcgcgcggtacctcaacagaagcctcgcagaaatcggaaaaacaagaagcaaaggcagaagaagcaggcgccg caaaacgacccaaagcaaaagaagcaaccaccacaaaagaagccggctcaaaagaagaagaaaccaggccgtagg gagagaatgtgcatgaaaattgaaaatgattgcatcttcgaagtcaagcatgaaggcaaagtgatgggctacgcatgcct ggtgggggataaagtaatgaaaccagcacatgtgaagggaactatcgacaatgccgatctggctaaactggcctttaag cggtcgtctaaatacgatcttgaatgtgcacagataccggtgcacatgaagtctgatgcctcgaagtttacccacgagaaa cccgaggggtactataactggcatcacggagcagtgcagtattcaggaggccggttcactatcccgacgggtgcaggc aagccgggagacagcggcagaccgatcttcgacaacaaaggacgggtggtggccatcgtcctaggaggggccaacg aaggtgcccgcacggccctctccgtggtgacgtggaacaaagacatcgtcacaaaaattacccctgagggagccgaa gagtggagcctcgccctcccggtcttgtgcctgttggcaaacactacattcccctgctctcagccgccttgcacaccctgc tgctacgaaaaggaaccggaaagcaccttgcgcatgcttgaggacaacgtgatgagacccggatactaccagctacta aaagcatcgctgacttgctctccccaccgccaaagacgcagtactaaggacaattttaatgtctataaagccacaagacc atatctagctcattgtcctgactgcggagaagggcattcgtgccacagccctatcgcattggagcgcatcagaaatgaag caacggacggaacgctgaaaatccaggtctctttgcagatcgggataaagacagatgacagccacgattggaccaagc tgcgctatatggatagccatacgccagcggacgcggagcgagccggattgcttgtaaggacttcagcaccgtgcacgat caccgggaccatgggacactttattctcgcccgatgcccgaaaggagagacgctgacagtgggatttacggacagcag aaagatcagccacacatgcacacacccgttccatcatgaaccacctgtgataggtagggagaggttccactctcgacca caacatggtaaagagttaccttgcagcacgtacgtgcagagcaccgctgccactgctgaggagatagaggtgcatatgc ccccagatactcctgaccgcacgctgatgacgcagcagtctggcaacgtgaagatcacagttaatgggcagacggtgc ggtacaagtgcaactgcggtggctccggaagacctgtgtgctctacacagctgctgctgaatggctctctggccgagga agaggtggtgatcagaagcgagaatttcaccaacaacgccaagaacatcatcgtgcagctgagggaacccgtgaagat caactgcagccggcccaacaacaatacccggagacaggcccactgcaacatcagcaagaccaactggaacaacacc ctgaaacaggtggtggagaagctgggcgagcagttcaacaagaccaagatcgtgttcacccagagcagcggcggag atcctgagatcgtgacccacagcttcaattgtgccggcgagttcttctactgcaataccacccagctgttcaacagcacctg gaatatcgagggcaccgccagcatcaacggaaccgagagcaacgataacatcaccctgccctgcaggatcaagggaa gcaacgccaccgtgatcaagggcaatattagctgcagcagcaacatcacaggactgctgctgacaagagatggcggc agcggcggcagcggccaggagacattcagacctggcggcggagactgcagggacaactggaggagcgagggctc cggatcaaacgagggactgacaaccacagacaaagtgatcaataactgcaaaattgatcagtgccatgctgcagtcact aatcacaagaattggcaatacaactcccctttagtcccgcgcaacgctgaactcggggaccgtaaaggaaagatccaca tcccattcccattggcaaacgtgacttgcagagtgccaaaagcaagaaaccctacagtaacttacggaaaaaaccaagtc accatgctgctgtatcctgaccatccgacactcttgtcttaccgtaacatgggacaggaaccaaattaccacgaggagtgg gtgacacacaagaaggaggttaccttgaccgtgcctactgagggtctggaggtcacttggggcaacaacgaaccatac aagtactggccgcagatgtctacgaacggtactgctcatggtcacccacatgagataatcttgtactattatgagctgtacc ccactatgactgtagtcattgtgtcggtggcctcgttcgtgcttctgtcgatggtgggcacagcagtgggaatgtgtgtgtg cgcacggcgcagatgcattacaccatatgaattaacaccaggagccactgttcccttcctgctcagcctgctatgctgcgt cagaacgaccaaggcggccacatattacgaggctgcggcatatctatggaacgaacagcagcccctgttctggttgcag gctcttatcccgctggccgccttgatcgtcctgtgcaactgtctgaaactcttgccatgctgctgtaagaccctggctttttta gccgtaatgagcatcggtgcccacactgtgagcgcgtacgaacacgtaacagtgatcccgaacacggtgggagtaccg tataagactcttgtcaacagaccgggttacagccccatggtgttggagatggagctacaatcagtcaccttggaaccaac actgtcacttgactacatcacgtgcgagtacaaaactgtcatcccctccccgtacgtgaagtgctgtggtacagcagagtg caaggacaagagcctaccagactacagctgcaaggtctttactggagtctacccatttatgtggggcggcgcctactgctt ttgcgacgccgaaaatacgcaattgagcgaggcacatgtagagaaatctgaatcttgcaaaacagagtttgcatcggcct acagagcccacaccgcatcggcgtcggcgaagctccgcgtcctttaccaaggaaacaacattaccgtagctgcctacg ctaacggtgaccatgccgtcacagtaaaggacgccaagtttgtcgtgggcccaatgtcctccgcctggacaccttttgac aacaaaatcgtggtgtacaaaggcgacgtctacaacatggactacccaccttttggcgcaggaagaccaggacaatttg gtgacattcaaagtcgtacaccggaaagtaaagacgtttatgccaacactcagttggtactacagaggccagcagcagg cacggtacatgtaccatactctcaggcaccatctggcttcaagtattggctgaaggaacgaggagcatcgctacagcaca cggcaccgttcggttgccagattgcgacaaacccggtaagagctgtaaattgcgctgtggggaacataccaatttccatc gacataccggatgcggcctttactagggttgtcgatgcaccctctgtaacggacatgtcatgcgaagtaccagcctgcact cactcctccgactttgggggcgtcgccatcatcaaatacacagctagcaagaaaggtaaatgtgcagtacattcgatgac caacgccgttaccattcgagaagccgacgtagaagtagaggggaactcccagctgcaaatatccttctcaacagccctg gcaagcgccgagtttcgcgtgcaagtgtgctccacacaagtacactgcgcagccgcatgccaccctccaaaggaccac atagtcaattacccagcatcacacaccacccttggggtccaggatatatccacaacggcaatgtcttgggtgcagaagatt acgggaggagtaggattaattgttgctgttgctgccttaattttaattgtggtgctatgcgtgtcgtttagcaggcactaatga ggatccagatctgctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgc cactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtgggg tggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgggtac ccaggtgctgaagaattgacccggttcctcctgggccagaaagaagcaggcacatccccttctctgtgacacaccctgtc cacgcccctggttcttagttccagccccactcataggacactcatagctcaggagggctccgccttcaatcccacccgcta aagtacttggagcggtctctccctccctcatcagcccaccaaaccaaacctagcctccaagagtgggaagaaattaaag caagataggctattaagtgcagagggagagaaaatgcctccaacatgtgaggaagtaatgagagaaatcatagaatttta aggccatgatttaaggccatcatggccttaatcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcgg cgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtga gcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgac gagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccct ggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtg gcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccc ccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactgg cagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaacta cggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttga tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctca agaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattat caaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctga cagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactcggggggg gggggcgctgaggtctgcctcgtgaagaaggtgttgctgactcataccaggcctgaatcgccccatcatccagccagaa agtgagggagccacggttgatgagagctttgttgtaggtggaccagttggtgattttgaacttttgctttgccacggaacgg tctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaaagttcgatttattcaacaaagccgccgtcccgtca agtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgc aatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttc cataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaa aataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccag acttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctg agcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactg ccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtg agtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctg accatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaa tcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaa tcgcggcctcgagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttat tgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttccccccccccccattatt gaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgc acatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacga ggccctttcgtc

OD 4.3.2Vmut 3G (SEQ ID NO: 154):

atggagttcatcccgacgcaaactttctataacagaaggtaccaaccccgaccctgggccccacgccctacaattcaagt aattagacctagaccacgtccacagaggcaggctgggcaactcgcccagctgatctccgcagtcaacaaattgaccatg cgcgcggtacctcaacagaagcctcgcagaaatcggaaaaacaagaagcaaaggcagaagaagcaggcgccgcaa aacgacccaaagcaaaagaagcaaccaccacaaaagaagccggctcaaaagaagaagaaaccaggccgtagggag agaatgtgcatgaaaattgaaaatgattgcatcttcgaagtcaagcatgaaggcaaagtgatgggctacgcatgcctggt gggggataaagtaatgaaaccagcacatgtgaagggaactatcgacaatgccgatctggctaaactggcctttaagcgg tcgtctaaatacgatcttgaatgtgcacagataccggtgcacatgaagtctgatgcctcgaagtttacccacgagaaaccc gaggggtactataactggcatcacggagcagtgcagtattcaggaggccggttcactatcccgacgggtgcaggcaag ccgggagacagcggcagaccgatcttcgacaacaaaggacgggtggtggccatcgtcctaggaggggccaacgaag gtgcccgcacggccctctccgtggtgacgtggaacaaagacatcgtcacaaaaattacccctgagggagccgaagagt ggagcctcgccctcccggtcttgtgcctgttggcaaacactacattcccctgctctcagccgccttgcacaccctgctgct acgaaaaggaaccggaaagcaccttgcgcatgcttgaggacaacgtgatgagacccggatactaccagctactaaaag catcgctgacttgctctccccaccgccaaagacgcagtactaaggacaattttaatgtctataaagccacaagaccatatct agctcattgtcctgactgcggagaagggcattcgtgccacagccctatcgcattggagcgcatcagaaatgaagcaacg gacggaacgctgaaaatccaggtctctttgcagatcgggataaagacagatgacagccacgattggaccaagctgcgct atatggatagccatacgccagcggacgcggagcgagccggattgcttgtaaggacttcagcaccgtgcacgatcaccg ggaccatgggacactttattctcgcccgatgcccgaaaggagagacgctgacagtgggatttacggacagcagaaaga tcagccacacatgcacacacccgttccatcatgaaccacctgtgataggtagggagaggttccactctcgaccacaacat ggtaaagagttaccttgcagcacgtacgtgcagagcaccgctgccactgctgaggagatagaggtgcatatgccccca gatactcctgaccgcacgctgatgacgcagcagtctggcaacgtgaagatcacagttaatgggcagacggtgcggtac aagtgcaactgcggtggctccggaagacctgtgtgctctacacagctgctgctgaatggctctctggccgaggaagagg tggtgatcagaagcgagaatttcaccaacaacgccaagaacatcatcgtgcagctgagggaacccgtgaagatcaact gcagccggcccaacaacaatacccggagacaggcccactgcaacatcagcaagaccaactggaacaacaccctgaa acaggtggtggagaagctgggcgagcagttcaacaagaccaagatcgtgttcacccagagcagcggcggagatcctg agatcgtgacccacagcttcaattgtgccggcgagttcttctactgcaataccacccagctgttcaacagcacctggaatat cgagggcaccgccagcatcaacggaaccgagagcaacgataacatcaccctgccctgcaggatcaagggaagcgga gcccctcccatcaagggcaatattagctgcagcagcaacatcacaggactgctgctgacaagagatggcggcagcgg cggcagcggccaggagacattcagacctggcggcggagactgcagggacaactggaggagcgagggctccggatc aaacgagggactgacaaccacagacaaagtgatcaataactgcaaaattgatcagtgccatgctgcagtcactaatcac aagaattggcaatacaactcccctttagtcccgcgcaacgctgaactcggggaccgtaaaggaaagatccacatcccatt cccattggcaaacgtgacttgcagagtgccaaaagcaagaaaccctacagtaacttacggaaaaaaccaagtcaccatg ctgctgtatcctgaccatccgacactcttgtcttaccgtaacatgggacaggaaccaaattaccacgaggagtgggtgac acacaagaaggaggttaccttgaccgtgcctactgagggtctggaggtcacttggggcaacaacgaaccatacaagtac tggccgcagatgtctacgaacggtactgctcatggtcacccacatgagataatcttgtactattatgagctgtaccccactat gactgtagtcattgtgtcggtggcctcgttcgtgcttctgtcgatggtgggcacagcagtgggaatgtgtgtgtgcgcacg gcgcagatgcattacaccatatgaattaacaccaggagccactgttcccttcctgctcagcctgctatgctgcgtcagaac gaccaaggcggccacatattacgaggctgcggcatatctatggaacgaacagcagcccctgttctggttgcaggctctta tcccgctggccgccttgatcgtcctgtgcaactgtctgaaactcttgccatgctgctgtaagaccctggcttttttagccgta atgagcatcggtgcccacactgtgagcgcgtacgaacacgtaacagtgatcccgaacacggtgggagtaccgtataag actcttgtcaacagaccgggttacagccccatggtgttggagatggagctacaatcagtcaccttggaaccaacactgtc acttgactacatcacgtgcgagtacaaaactgtcatcccctccccgtacgtgaagtgctgtggtacagcagagtgcaagg acaagagcctaccagactacagctgcaaggtctttactggagtctacccatttatgtggggcggcgcctactgcttttgcg acgccgaaaatacgcaattgagcgaggcacatgtagagaaatctgaatcttgcaaaacagagtttgcatcggcctacag agcccacaccgcatcggcgtcggcgaagctccgcgtcctttaccaaggaaacaacattaccgtagctgcctacgctaac ggtgaccatgccgtcacagtaaaggacgccaagtttgtcgtgggcccaatgtcctccgcctggacaccttttgacaacaa aatcgtggtgtacaaaggcgacgtctacaacatggactacccaccttttggcgcaggaagaccaggacaatttggtgac attcaaagtcgtacaccggaaagtaaagacgtttatgccaacactcagttggtactacagaggccagcagcaggcacgg tacatgtaccatactctcaggcaccatctggcttcaagtattggctgaaggaacgaggagcatcgctacagcacacggca ccgttcggttgccagattgcgacaaacccggtaagagctgtaaattgcgctgtggggaacataccaatttccatcgacata ccggatgcggcctttactagggttgtcgatgcaccctctgtaacggacatgtcatgcgaagtaccagcctgcactcactcc tccgactttgggggcgtcgccatcatcaaatacacagctagcaagaaaggtaaatgtgcagtacattcgatgaccaacgc cgttaccattcgagaagccgacgtagaagtagaggggaactcccagctgcaaatatccttctcaacagccctggcaagc gccgagtttcgcgtgcaagtgtgctccacacaagtacactgcgcagccgcatgccaccctccaaaggaccacatagtca attacccagcatcacacaccacccttggggtccaggatatatccacaacggcaatgtcttgggtgcagaagattacggga ggagtaggattaattgttgctgttgctgccttaattttaattgtggtgctatgcgtgtcgtttagcaggcactaa CHIKV OD 4.3.2Vmut 3G (SEQ ID NO: 155):

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggat gccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatc agagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaga ttggctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttga cattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataact tacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaa cgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatca tatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatggga ctttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtgg atagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgg gactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataag cagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggacc gatccagcctccatcggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagt cgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagacc gggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactc tagttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcgcgcgccaccagacataatagctgacaga ctaacagactgttcctttccatgggtcttttctgcagtcaccgtcgtcgacacgtgtgatcagatatcgcggccgctctagac accatggagttcatcccgacgcaaactttctataacagaaggtaccaaccccgaccctgggccccacgccctacaattca agtaattagacctagaccacgtccacagaggcaggctgggcaactcgcccagctgatctccgcagtcaacaaattgacc atgcgcgcggtacctcaacagaagcctcgcagaaatcggaaaaacaagaagcaaaggcagaagaagcaggcgccg caaaacgacccaaagcaaaagaagcaaccaccacaaaagaagccggctcaaaagaagaagaaaccaggccgtagg gagagaatgtgcatgaaaattgaaaatgattgcatcttcgaagtcaagcatgaaggcaaagtgatgggctacgcatgcct ggtgggggataaagtaatgaaaccagcacatgtgaagggaactatcgacaatgccgatctggctaaactggcctttaag cggtcgtctaaatacgatcttgaatgtgcacagataccggtgcacatgaagtctgatgcctcgaagtttacccacgagaaa cccgaggggtactataactggcatcacggagcagtgcagtattcaggaggccggttcactatcccgacgggtgcaggc aagccgggagacagcggcagaccgatcttcgacaacaaaggacgggtggtggccatcgtcctaggaggggccaacg aaggtgcccgcacggccctctccgtggtgacgtggaacaaagacatcgtcacaaaaattacccctgagggagccgaa gagtggagcctcgccctcccggtcttgtgcctgttggcaaacactacattcccctgctctcagccgccttgcacaccctgc tgctacgaaaaggaaccggaaagcaccttgcgcatgcttgaggacaacgtgatgagacccggatactaccagctacta aaagcatcgctgacttgctctccccaccgccaaagacgcagtactaaggacaattttaatgtctataaagccacaagacc atatctagctcattgtcctgactgcggagaagggcattcgtgccacagccctatcgcattggagcgcatcagaaatgaag caacggacggaacgctgaaaatccaggtctctttgcagatcgggataaagacagatgacagccacgattggaccaagc tgcgctatatggatagccatacgccagcggacgcggagcgagccggattgcttgtaaggacttcagcaccgtgcacgat caccgggaccatgggacactttattctcgcccgatgcccgaaaggagagacgctgacagtgggatttacggacagcag aaagatcagccacacatgcacacacccgttccatcatgaaccacctgtgataggtagggagaggttccactctcgacca caacatggtaaagagttaccttgcagcacgtacgtgcagagcaccgctgccactgctgaggagatagaggtgcatatgc ccccagatactcctgaccgcacgctgatgacgcagcagtctggcaacgtgaagatcacagttaatgggcagacggtgc ggtacaagtgcaactgcggtggctccggaagacctgtgtgctctacacagctgctgctgaatggctctctggccgagga agaggtggtgatcagaagcgagaatttcaccaacaacgccaagaacatcatcgtgcagctgagggaacccgtgaagat caactgcagccggcccaacaacaatacccggagacaggcccactgcaacatcagcaagaccaactggaacaacacc ctgaaacaggtggtggagaagctgggcgagcagttcaacaagaccaagatcgtgttcacccagagcagcggcggag atcctgagatcgtgacccacagcttcaattgtgccggcgagttcttctactgcaataccacccagctgttcaacagcacctg gaatatcgagggcaccgccagcatcaacggaaccgagagcaacgataacatcaccctgccctgcaggatcaagggaa gcggagcccctcccatcaagggcaatattagctgcagcagcaacatcacaggactgctgctgacaagagatggcggc agcggcggcagcggccaggagacattcagacctggcggcggagactgcagggacaactggaggagcgagggctc cggatcaaacgagggactgacaaccacagacaaagtgatcaataactgcaaaattgatcagtgccatgctgcagtcact aatcacaagaattggcaatacaactcccctttagtcccgcgcaacgctgaactcggggaccgtaaaggaaagatccaca tcccattcccattggcaaacgtgacttgcagagtgccaaaagcaagaaaccctacagtaacttacggaaaaaaccaagtc accatgctgctgtatcctgaccatccgacactcttgtcttaccgtaacatgggacaggaaccaaattaccacgaggagtgg gtgacacacaagaaggaggttaccttgaccgtgcctactgagggtctggaggtcacttggggcaacaacgaaccatac aagtactggccgcagatgtctacgaacggtactgctcatggtcacccacatgagataatcttgtactattatgagctgtacc ccactatgactgtagtcattgtgtcggtggcctcgttcgtgcttctgtcgatggtgggcacagcagtgggaatgtgtgtgtg cgcacggcgcagatgcattacaccatatgaattaacaccaggagccactgttcccttcctgctcagcctgctatgctgcgt cagaacgaccaaggcggccacatattacgaggctgcggcatatctatggaacgaacagcagcccctgttctggttgcag gctcttatcccgctggccgccttgatcgtcctgtgcaactgtctgaaactcttgccatgctgctgtaagaccctggctttttta gccgtaatgagcatcggtgcccacactgtgagcgcgtacgaacacgtaacagtgatcccgaacacggtgggagtaccg tataagactcttgtcaacagaccgggttacagccccatggtgttggagatggagctacaatcagtcaccttggaaccaac actgtcacttgactacatcacgtgcgagtacaaaactgtcatcccctccccgtacgtgaagtgctgtggtacagcagagtg caaggacaagagcctaccagactacagctgcaaggtctttactggagtctacccatttatgtggggcggcgcctactgctt ttgcgacgccgaaaatacgcaattgagcgaggcacatgtagagaaatctgaatcttgcaaaacagagtttgcatcggcct acagagcccacaccgcatcggcgtcggcgaagctccgcgtcctttaccaaggaaacaacattaccgtagctgcctacg ctaacggtgaccatgccgtcacagtaaaggacgccaagtttgtcgtgggcccaatgtcctccgcctggacaccttttgac aacaaaatcgtggtgtacaaaggcgacgtctacaacatggactacccaccttttggcgcaggaagaccaggacaatttg gtgacattcaaagtcgtacaccggaaagtaaagacgtttatgccaacactcagttggtactacagaggccagcagcagg cacggtacatgtaccatactctcaggcaccatctggcttcaagtattggctgaaggaacgaggagcatcgctacagcaca cggcaccgttcggttgccagattgcgacaaacccggtaagagctgtaaattgcgctgtggggaacataccaatttccatc gacataccggatgcggcctttactagggttgtcgatgcaccctctgtaacggacatgtcatgcgaagtaccagcctgcact cactcctccgactttgggggcgtcgccatcatcaaatacacagctagcaagaaaggtaaatgtgcagtacattcgatgac caacgccgttaccattcgagaagccgacgtagaagtagaggggaactcccagctgcaaatatccttctcaacagccctg gcaagcgccgagtttcgcgtgcaagtgtgctccacacaagtacactgcgcagccgcatgccaccctccaaaggaccac atagtcaattacccagcatcacacaccacccttggggtccaggatatatccacaacggcaatgtcttgggtgcagaagatt acgggaggagtaggattaattgttgctgttgctgccttaattttaattgtggtgctatgcgtgtcgtttagcaggcactaatga ggatccagatctgctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgc cactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtgggg tggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgggtac ccaggtgctgaagaattgacccggttcctcctgggccagaaagaagcaggcacatccccttctctgtgacacaccctgtc cacgcccctggttcttagttccagccccactcataggacactcatagctcaggagggctccgccttcaatcccacccgcta aagtacttggagcggtctctccctccctcatcagcccaccaaaccaaacctagcctccaagagtgggaagaaattaaag caagataggctattaagtgcagagggagagaaaatgcctccaacatgtgaggaagtaatgagagaaatcatagaatttta aggccatgatttaaggccatcatggccttaatcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcgg cgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtga gcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgac gagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccct ggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtg gcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccc ccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactgg cagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaacta cggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttga tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctca agaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattat caaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctga cagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactcggggggg gggggcgctgaggtctgcctcgtgaagaaggtgttgctgactcataccaggcctgaatcgccccatcatccagccagaa agtgagggagccacggttgatgagagctttgttgtaggtggaccagttggtgattttgaacttttgctttgccacggaacgg tctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaaagttcgatttattcaacaaagccgccgtcccgtca agtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgc aatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttc cataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaa aataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccag acttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctg agcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactg ccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtg agtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctg accatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaa tcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaa tcgcggcctcgagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttat tgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttccccccccccccattatt gaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgc acatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacga ggccctttcgtc OD4.3.2Vmut_3G/419N-3 CH (SEQ ID NO: 156):

atgcagctggccagctgtgtgacactgacactggtgctgctggtgaattccgcccctaggagacctgtgtgctctacaca gctgctgctgaatggctctctggccgaggaagaggtggtgatcagaagcgagaatttcaccaacaacgccaagaacat catcgtgcagctgagggaacccgtgaagatcaactgcagccggcccaacaacaatacccggagacaggcccactgca acatcagcaagaccaactggaacaacaccctgaaacaggtggtggagaagctgggcgagcagttcaacaagaccaa gatcgtgttcacccagagcagcggcggagatcctgagatcgtgacccacagcttcaattgtgccggcgagttcttctact gcaataccacccagctgttcaacagcacctggaatatcgagggcaccgccagcatcaacggaaccgagagcaacgat aacatcaccctgccctgcaacatcaccgtgagcggagcccctcccatcaagggcaatattagctgcagcagcaacatca caggactgctgctgacaagagatggcggcagcggcggcagcggccaggagacattcagacctggcggcggagact gcagggacaattggcggagcgagggatccctggaggtgctgttccagggcccaggccaccaccaccaccaccactg atga

CHIKV-OD4.3.2Vmut_3G/419N-3 CH (SEQ ID NO: 157):

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggat gccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatc agagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaga ttggctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttga cattgattattgactagttattaatagtaatcaattacgggaacttccatagcccatatatggagttccgcgttacataacttac gggaatttccaaacctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggaacttccattgacgtcaatgggtggagtatttacggtaaactgcccacttgggaatttccaagtgtatcatatgc caagtacgccccctattgacgtcaatgacgggaacttccataagcttgcattatgcccagtacatgaccttatgggaatttc ctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatag cggtttgactcacgggaacttccaagtctccaccccattgacgtcaatgggagtttgttttgactcaccaaaatcaacggga attcccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagca gagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccg atccagcctccatcggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtc gcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagaccg ggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactct agttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcgcgcgccaccagacataatagctgacagac taacagactgttcctttccatgggtcttttctgcagtcaccgtcgtcgacacgtgtgatcagatatcgcggccgctctagaca ccatgtatagcatgcagctggccagctgtgtgacactgacactggtgctgctggtgaattccgcccctaggagacctgtgt gctctacacagctgctgctgaatggctctctggccgaggaagaggtggtgatcagaagcgagaatttcaccaacaacgc caagaacatcatcgtgcagctgagggaacccgtgaagatcaactgcagccggcccaacaacaatacccggagacagg cccactgcaacatcagcaagaccaactggaacaacaccctgaaacaggtggtggagaagctgggcgagcagttcaac aagaccaagatcgtgttcacccagagcagcggcggagatcctgagatcgtgacccacagcttcaattgtgccggcgag ttcttctactgcaataccacccagctgttcaacagcacctggaatatcgagggcaccgccagcatcaacggaaccgaga gcaacgataacatcaccctgccctgcaacatcaccgtgagcggagcccctcccatcaagggcaatattagctgcagca gcaacatcacaggactgctgctgacaagagatggcggcagcggcggcagcggccaggagacattcagacctggcgg cggagactgcagggacaattggcggagcgagggatccctggaggtgctgttccagggcccaggccaccaccaccac caccactgatgaagatcttcaagggcgaattctgcagatgatctgctgtgccttctagttgccagccatctgttgtttgcccc tcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtct gagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggc atgctggggatgcggtgggctctatgggtacccaggtgctgaagaattgacccggttcctcctgggccagaaagaagca ggcacatccccttctctgtgacacaccctgtccacgcccctggttcttagttccagccccactcataggacactcatagctc aggagggctccgccttcaatcccacccgctaaagtacttggagcggtctctccctccctcatcagcccaccaaaccaaac ctagcctccaagagtgggaagaaattaaagcaagataggctattaagtgcagagggagagaaaatgcctccaacatgtg aggaagtaatgagagaaatcatagaattttaaggccatgatttaaggccatcatggccttaatcttccgcttcctcgctcact gactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaat caggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgct ggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgaca ggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatac ctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcg ctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtcca acccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtg ctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagt taccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcag cagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaa ctcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatca atctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttc gttcatccatagttgcctgactcggggggggggggcgctgaggtctgcctcgtgaagaaggtgttgctgactcatacca ggcctgaatcgccccatcatccagccagaaagtgagggagccacggttgatgagagctttgttgtaggtggaccagttg gtgattttgaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaaagt tcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgatta gaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgt aatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaa catcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccgg tgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcat caaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaaca ggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacc tggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcgga agaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcaga aacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttat acccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcataacacc ccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgag acacaacgtggctttccccccccccccattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgta tttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcat gacattaacctataaaaataggcgtatcacgaggccctttcgtc

OD4.3Vmut3G-3CH (SEQ ID NO: 158):

Atgcagctggccagctgtgtgacactgacactggtgctgctggtgaattccgcccctaggagacctgtgtgctctacaca gctgctgctgaatggctctctggccgaggaagaggtggtgatcagaagcgagaatttcaccaacaacgccaagaacat catcgtgcagctgagggaacccgtgaagatcaactgcagccggcccaacaacaatacccggagacaggcccactgca acatcagcaagaccaactggaacaacaccctgaaacaggtggtggagaagctgggcgagcagttcaacaagaccaa gatcgtgttcacccagagcagcggcggagatcctgagatcgtgacccacagcttcaattgtgccggcgagttcttctact gcaataccacccagctgttcaacagcacctggaatatcgagggcaccgccagcatcaacggaaccgagagcaacgat aacatcaccctgccctgcaggatcaagggaagcggagcccctcccatcaagggcaatattagctgcagcagcaacatc acaggactgctgctgacaagagatggcggcagcggcggcagcggccaggagacattcagacctggcggcggagac tgcagggacaattggcggagcgagggatccctggaggtgctgttccagggcccaggccaccaccaccaccaccactg atga

CHIKV-OD4.3Vmut3G-3CH (SEQ ID NO: 159):

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggat gccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatc agagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaga ttggctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttga cattgattattgactagttattaatagtaatcaattacgggaacttccatagcccatatatggagttccgcgttacataacttac gggaatttccaaacctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggaacttccattgacgtcaatgggtggagtatttacggtaaactgcccacttgggaatttccaagtgtatcatatgc caagtacgccccctattgacgtcaatgacgggaacttccataagcttgcattatgcccagtacatgaccttatgggaatttc ctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatag cggtttgactcacgggaacttccaagtctccaccccattgacgtcaatgggagtttgttttgactcaccaaaatcaacggga attcccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagca gagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccg atccagcctccatcggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtc gcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagaccg ggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactct agttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcgcgcgccaccagacataatagctgacagac taacagactgttcctttccatgggtcttttctgcagtcaccgtcgtcgacacgtgtgatcagatatcgcggccgctctagaca ccatgtatagcatgcagctggccagctgtgtgacactgacactggtgctgctggtgaattccgcccctaggagacctgtgt gctctacacagctgctgctgaatggctctctggccgaggaagaggtggtgatcagaagcgagaatttcaccaacaacgc caagaacatcatcgtgcagctgagggaacccgtgaagatcaactgcagccggcccaacaacaatacccggagacagg cccactgcaacatcagcaagaccaactggaacaacaccctgaaacaggtggtggagaagctgggcgagcagttcaac aagaccaagatcgtgttcacccagagcagcggcggagatcctgagatcgtgacccacagcttcaattgtgccggcgag ttcttctactgcaataccacccagctgttcaacagcacctggaatatcgagggcaccgccagcatcaacggaaccgaga gcaacgataacatcaccctgccctgcaggatcaagggaagcggagcccctcccatcaagggcaatattagctgcagca gcaacatcacaggactgctgctgacaagagatggcggcagcggcggcagcggccaggagacattcagacctggcgg cggagactgcagggacaattggcggagcgagggatccctggaggtgctgttccagggcccaggccaccaccaccac caccactgatgaagatcttcaagggcgaattctgcagatgatctgctgtgccttctagttgccagccatctgttgtttgcccc tcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtct gagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggc atgctggggatgcggtgggctctatgggtacccaggtgctgaagaattgacccggttcctcctgggccagaaagaagca ggcacatccccttctctgtgacacaccctgtccacgcccctggttcttagttccagccccactcataggacactcatagctc aggagggctccgccttcaatcccacccgctaaagtacttggagcggtctctccctccctcatcagcccaccaaaccaaac ctagcctccaagagtgggaagaaattaaagcaagataggctattaagtgcagagggagagaaaatgcctccaacatgtg aggaagtaatgagagaaatcatagaattttaaggccatgatttaaggccatcatggccttaatcttccgcttcctcgctcact gactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaat caggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgct ggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgaca ggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatac ctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcg ctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtcca acccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtg ctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagt taccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcag cagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaa ctcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatca atctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttc gttcatccatagttgcctgactcggggggggggggcgctgaggtctgcctcgtgaagaaggtgttgctgactcatacca ggcctgaatcgccccatcatccagccagaaagtgagggagccacggttgatgagagctttgttgtaggtggaccagttg gtgattttgaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaaagt tcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgatta gaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgt aatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaa catcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccgg tgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcat caaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaaca ggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacc tggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcgga agaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcaga aacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttat acccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcataacacc ccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgag acacaacgtggctttccccccccccccattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgta tttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcat gacattaacctataaaaataggcgtatcacgaggccctttcgtc

CMV/R_CHIKV185_OD4.2'_HG3.2 (SEQ ID NO: 160):

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggat gccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatc agagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaga ttggctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttga cattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataact tacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaa cgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatca tatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatggga ctttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtgg atagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgg gactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataag cagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggacc gatccagcctccatcggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagt cgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagacc gggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactc tagttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcgcgcgccaccagacataatagctgacaga ctaacagactgttcctttccatgggtcttttctgcagtcaccgtcgtcgacacgtgtgatcagatatcgcggccgctctagac accatggagttcatcccgacgcaaactttctataacagaaggtaccaaccccgaccctgggccccacgccctacaattca agtaattagacctagaccacgtccacagaggcaggctgggcaactcgcccagctgatctccgcagtcaacaaattgacc atgcgcgcggtacctcaacagaagcctcgcagaaatcggaaaaacaagaagcaaaggcagaagaagcaggcgccg caaaacgacccaaagcaaaagaagcaaccaccacaaaagaagccggctcaaaagaagaagaaaccaggccgtagg gagagaatgtgcatgaaaattgaaaatgattgcatcttcgaagtcaagcatgaaggcaaagtgatgggctacgcatgcct ggtgggggataaagtaatgaaaccagcacatgtgaagggaactatcgacaatgccgatctggctaaactggcctttaag cggtcgtctaaatacgatcttgaatgtgcacagataccggtgcacatgaagtctgatgcctcgaagtttacccacgagaaa cccgaggggtactataactggcatcacggagcagtgcagtattcaggaggccggttcactatcccgacgggtgcaggc aagccgggagacagcggcagaccgatcttcgacaacaaaggacgggtggtggccatcgtcctaggaggggccaacg aaggtgcccgcacggccctctccgtggtgacgtggaacaaagacatcgtcacaaaaattacccctgagggagccgaa gagtggagcctcgccctcccggtcttgtgcctgttggcaaacactacattcccctgctctcagccgccttgcacaccctgc tgctacgaaaaggaaccggaaagcaccttgcgcatgcttgaggacaacgtgatgagacccggatactaccagctacta aaagcatcgctgacttgctctccccaccgccaaagacgcagtactaaggacaattttaatgtctataaagccacaagacc atatctagctcattgtcctgactgcggagaagggcattcgtgccacagccctatcgcattggagcgcatcagaaatgaag caacggacggaacgctgaaaatccaggtctctttgcagatcgggataaagacagatgacagccacgattggaccaagc tgcgctatatggatagccatacgccagcggacgcggagcgagccggattgcttgtaaggacttcagcaccgtgcacgat caccgggaccatgggacactttattctcgcccgatgcccgaaaggagagacgctgacagtgggatttacggacagcag aaagatcagccacacatgcacacacccgttccatcatgaaccacctgtgataggtagggagaggttccactctcgacca caacatggtaaagagttaccttgcagcacgtacgtgcagagcaccgctgccactgctgaggagatagaggtgcatatgc ccccagatactcctgaccgcacgctgatgacgcagcagtccggaaagcccgtggtgagcacccagctgctgctgaac ggcagcctggccgagaaggagatcaggatcaagagcgaggacatcagcgacaacgccaagaacatcatcgtgcagc tgaccaagcccgtgctgatcaactgcaccaggcccagcaacaacacccagtactgcgtggtgaacaggacccagtgg aacgacaccctgggccaggtggccatccagctgaggaagcactggaacacctgcatcatcttcaacgagcccagcgg cggcgacctggagatcaccacccacagcttcaactgcggcggcgagttcTTCtactgcaacaccagcgacctgttca acagcacctggaacatcaacggcaccgccagcatcaacggcaccgagagcaacgacaacatcaccctgccctgcag gatcaacggcaccatccagggcaacatcacctgccagagcaacatcaccggcatcctgctgaccagggacggcggca gcggcagcggcacctgcgagaccttcaggcccggcggcggcgacatgagggacctgaacaggaccagcggctccg gaaacgtgaagatcacagttaatgggcagacggtgcggtacaagtgcaactgcggtggctcaaacgagggactgaca accacagacaaagtgatcaataactgcaaaattgatcagtgccatgctgcagtcactaatcacaagaattggcaatacaa ctcccctttagtcccgcgcaacgctgaactcggggaccgtaaaggaaagatccacatcccattcccattggcaaacgtga cttgcagagtgccaaaagcaagaaaccctacagtaacttacggaaaaaaccaagtcaccatgctgctgtatcctgaccat ccgacactcttgtcttaccgtaacatgggacaggaaccaaattaccacgaggagtgggtgacacacaagaaggaggtta ccttgaccgtgcctactgagggtctggaggtcacttggggcaacaacgaaccatacaagtactggccgcagatgtctac gaacggtactgctcatggtcacccacatgagataatcttgtactattatgagctgtaccccactatgactgtagtcattgtgtc ggtggcctcgttcgtgcttctgtcgatggtgggcacagcagtgggaatgtgtgtgtgcgcacggcgcagatgcattacac catatgaattaacaccaggagccactgttcccttcctgctcagcctgctatgctgcgtcagaacgaccaaggcggccaca tattacgaggctgcggcatatctatggaacgaacagcagcccctgttctggttgcaggctcttatcccgctggccgccttg atcgtcctgtgcaactgtctgaaactcttgccatgctgctgtaagaccctggcttttttagccgtaatgagcatcggtgccca cactgtgagcgcgtacgaacacgtaacagtgatcccgaacacggtgggagtaccgtataagactcttgtcaacagaccg ggttacagccccatggtgttggagatggagctacaatcagtcaccttggaaccaacactgtcacttgactacatcacgtgc gagtacaaaactgtcatcccctccccgtacgtgaagtgctgtggtacagcagagtgcaaggacaagagcctaccagact acagctgcaaggtctttactggagtctacccatttatgtggggcggcgcctactgcttttgcgacgccgaaaatacgcaatt gagcgaggcacatgtagagaaatctgaatcttgcaaaacagagtttgcatcggcctacagagcccacaccgcatcggc gtcggcgaagctccgcgtcctttaccaaggaaacaacattaccgtagctgcctacgctaacggtgaccatgccgtcaca gtaaaggacgccaagtttgtcgtgggcccaatgtcctccgcctggacaccttttgacaacaaaatcgtggtgtacaaagg cgacgtctacaacatggactacccaccttttggcgcaggaagaccaggacaatttggtgacattcaaagtcgtacaccgg aaagtaaagacgtttatgccaacactcagttggtactacagaggccagcagcaggcacggtacatgtaccatactctcag gcaccatctggcttcaagtattggctgaaggaacgaggagcatcgctacagcacacggcaccgttcggttgccagattg cgacaaacccggtaagagctgtaaattgcgctgtggggaacataccaatttccatcgacataccggatgcggcctttact agggttgtcgatgcaccctctgtaacggacatgtcatgcgaagtaccagcctgcactcactcctccgactttgggggcgt cgccatcatcaaatacacagctagcaagaaaggtaaatgtgcagtacattcgatgaccaacgccgttaccattcgagaag ccgacgtagaagtagaggggaactcccagctgcaaatatccttctcaacagccctggcaagcgccgagtttcgcgtgca agtgtgctccacacaagtacactgcgcagccgcatgccaccctccaaaggaccacatagtcaattacccagcatcacac accacccttggggtccaggatatatccacaacggcaatgtcttgggtgcagaagattacgggaggagtaggattaattgtt gctgttgctgccttaattttaattgtggtgctatgcgtgtcgtttagcaggcactaatgaggatccagatctgctgtgccttcta gttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaa atgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaaggggga ggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgggtacccaggtgctgaagaattgacccgg ttcctcctgggccagaaagaagcaggcacatccccttctctgtgacacaccctgtccacgcccctggttcttagttccagc cccactcataggacactcatagctcaggagggctccgccttcaatcccacccgctaaagtacttggagcggtctctccct ccctcatcagcccaccaaaccaaacctagcctccaagagtgggaagaaattaaagcaagataggctattaagtgcagag ggagagaaaatgcctccaacatgtgaggaagtaatgagagaaatcatagaattttaaggccatgatttaaggccatcatg gccttaatcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaa ggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggcca ggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctc aagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcct gttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgta ggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgcc ttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggatt agcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatt tggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctgg tagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacgg ggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatcct tttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgag gcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactcggggggggggggcgctgaggtctgcctcgt gaagaaggtgttgctgactcataccaggcctgaatcgccccatcatccagccagaaagtgagggagccacggttgatg agagctttgttgtaggtggaccagttggtgattttgaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgt gatctgatccttcaactcagcaaaagttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagt gttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaa taccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggta tcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaat caccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccatt acgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatc gctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttca cctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggag tacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattg gcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgatt gcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgt ttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatctt gtgcaatgtaacatcagagattttgagacacaacgtggctttccccccccccccattattgaagcatttatcagggttattgt ctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccac ctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc

CMV/R_CHIKV185_OD4.2'_HG3.2.1 (SEQ ID NO: 161):

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggat gccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatc agagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaga ttggctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttga cattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataact tacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaa cgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatca tatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatggga ctttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtgg atagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgg gactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataag cagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggacc gatccagcctccatcggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagt cgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagacc gggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactc tagttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcgcgcgccaccagacataatagctgacaga ctaacagactgttcctttccatgggtcttttctgcagtcaccgtcgtcgacacgtgtgatcagatatcgcggccgctctagac accatggagttcatcccgacgcaaactttctataacagaaggtaccaaccccgaccctgggccccacgccctacaattca agtaattagacctagaccacgtccacagaggcaggctgggcaactcgcccagctgatctccgcagtcaacaaattgacc atgcgcgcggtacctcaacagaagcctcgcagaaatcggaaaaacaagaagcaaaggcagaagaagcaggcgccg caaaacgacccaaagcaaaagaagcaaccaccacaaaagaagccggctcaaaagaagaagaaaccaggccgtagg gagagaatgtgcatgaaaattgaaaatgattgcatcttcgaagtcaagcatgaaggcaaagtgatgggctacgcatgcct ggtgggggataaagtaatgaaaccagcacatgtgaagggaactatcgacaatgccgatctggctaaactggcctttaag cggtcgtctaaatacgatcttgaatgtgcacagataccggtgcacatgaagtctgatgcctcgaagtttacccacgagaaa cccgaggggtactataactggcatcacggagcagtgcagtattcaggaggccggttcactatcccgacgggtgcaggc aagccgggagacagcggcagaccgatcttcgacaacaaaggacgggtggtggccatcgtcctaggaggggccaacg aaggtgcccgcacggccctctccgtggtgacgtggaacaaagacatcgtcacaaaaattacccctgagggagccgaa gagtggagcctcgccctcccggtcttgtgcctgttggcaaacactacattcccctgctctcagccgccttgcacaccctgc tgctacgaaaaggaaccggaaagcaccttgcgcatgcttgaggacaacgtgatgagacccggatactaccagctacta aaagcatcgctgacttgctctccccaccgccaaagacgcagtactaaggacaattttaatgtctataaagccacaagacc atatctagctcattgtcctgactgcggagaagggcattcgtgccacagccctatcgcattggagcgcatcagaaatgaag caacggacggaacgctgaaaatccaggtctctttgcagatcgggataaagacagatgacagccacgattggaccaagc tgcgctatatggatagccatacgccagcggacgcggagcgagccggattgcttgtaaggacttcagcaccgtgcacgat caccgggaccatgggacactttattctcgcccgatgcccgaaaggagagacgctgacagtgggatttacggacagcag aaagatcagccacacatgcacacacccgttccatcatgaaccacctgtgataggtagggagaggttccactctcgacca caacatggtaaagagttaccttgcagcacgtacgtgcagagcaccgctgccactgctgaggagatagaggtgcatatgc ccccagatactcctgaccgcacgctgatgacgcagcagtccggaaagcccgtggtgagcacccagctgctgctgaac ggcagcctggccgagaaggagatcaggatcaagagcgaggacatcagcgacaacgccaagaacatcatcgtgcagc tgaccaagcccgtgctgatcaactgcaccaggcccagcaacaacacccagtactgcgtggtgaacaggacccagtgg aacgacaccctgggccaggtggccatccagctgaggaagcactggaacacctgcatcatcttcaccaacagcagcgg cggcgacctggagatcaccacccacagcttcaactgcggcggcgagttcTTCtactgcaacaccagcgacctgttca acagcacctggaacatcaacggcaccgccagcatcaacggcaccgagagcaacgacaacatcaccctgccctgcag gatcaacggcaccatccagggcaacatcacctgccagagcaacatcaccggcatcctgctgaccagggacggcggca gcggcagcggcacctgcgagaccttcaggcccggcggcggcgacatgagggacctgaacaggaccagcggctccg gaaacgtgaagatcacagttaatgggcagacggtgcggtacaagtgcaactgcggtggctcaaacgagggactgaca accacagacaaagtgatcaataactgcaaaattgatcagtgccatgctgcagtcactaatcacaagaattggcaatacaa ctcccctttagtcccgcgcaacgctgaactcggggaccgtaaaggaaagatccacatcccattcccattggcaaacgtga cttgcagagtgccaaaagcaagaaaccctacagtaacttacggaaaaaaccaagtcaccatgctgctgtatcctgaccat ccgacactcttgtcttaccgtaacatgggacaggaaccaaattaccacgaggagtgggtgacacacaagaaggaggtta ccttgaccgtgcctactgagggtctggaggtcacttggggcaacaacgaaccatacaagtactggccgcagatgtctac gaacggtactgctcatggtcacccacatgagataatcttgtactattatgagctgtaccccactatgactgtagtcattgtgtc ggtggcctcgttcgtgcttctgtcgatggtgggcacagcagtgggaatgtgtgtgtgcgcacggcgcagatgcattacac catatgaattaacaccaggagccactgttcccttcctgctcagcctgctatgctgcgtcagaacgaccaaggcggccaca tattacgaggctgcggcatatctatggaacgaacagcagcccctgttctggttgcaggctcttatcccgctggccgccttg atcgtcctgtgcaactgtctgaaactcttgccatgctgctgtaagaccctggcttttttagccgtaatgagcatcggtgccca cactgtgagcgcgtacgaacacgtaacagtgatcccgaacacggtgggagtaccgtataagactcttgtcaacagaccg ggttacagccccatggtgttggagatggagctacaatcagtcaccttggaaccaacactgtcacttgactacatcacgtgc gagtacaaaactgtcatcccctccccgtacgtgaagtgctgtggtacagcagagtgcaaggacaagagcctaccagact acagctgcaaggtctttactggagtctacccatttatgtggggcggcgcctactgcttttgcgacgccgaaaatacgcaatt gagcgaggcacatgtagagaaatctgaatcttgcaaaacagagtttgcatcggcctacagagcccacaccgcatcggc gtcggcgaagctccgcgtcctttaccaaggaaacaacattaccgtagctgcctacgctaacggtgaccatgccgtcaca gtaaaggacgccaagtttgtcgtgggcccaatgtcctccgcctggacaccttttgacaacaaaatcgtggtgtacaaagg cgacgtctacaacatggactacccaccttttggcgcaggaagaccaggacaatttggtgacattcaaagtcgtacaccgg aaagtaaagacgtttatgccaacactcagttggtactacagaggccagcagcaggcacggtacatgtaccatactctcag gcaccatctggcttcaagtattggctgaaggaacgaggagcatcgctacagcacacggcaccgttcggttgccagattg cgacaaacccggtaagagctgtaaattgcgctgtggggaacataccaatttccatcgacataccggatgcggcctttact agggttgtcgatgcaccctctgtaacggacatgtcatgcgaagtaccagcctgcactcactcctccgactttgggggcgt cgccatcatcaaatacacagctagcaagaaaggtaaatgtgcagtacattcgatgaccaacgccgttaccattcgagaag ccgacgtagaagtagaggggaactcccagctgcaaatatccttctcaacagccctggcaagcgccgagtttcgcgtgca agtgtgctccacacaagtacactgcgcagccgcatgccaccctccaaaggaccacatagtcaattacccagcatcacac accacccttggggtccaggatatatccacaacggcaatgtcttgggtgcagaagattacgggaggagtaggattaattgtt gctgttgctgccttaattttaattgtggtgctatgcgtgtcgtttagcaggcactaatgaggatccagatctgctgtgccttcta gttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaa atgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaaggggga ggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgggtacccaggtgctgaagaattgacccgg ttcctcctgggccagaaagaagcaggcacatccccttctctgtgacacaccctgtccacgcccctggttcttagttccagc cccactcataggacactcatagctcaggagggctccgccttcaatcccacccgctaaagtacttggagcggtctctccct ccctcatcagcccaccaaaccaaacctagcctccaagagtgggaagaaattaaagcaagataggctattaagtgcagag ggagagaaaatgcctccaacatgtgaggaagtaatgagagaaatcatagaattttaaggccatgatttaaggccatcatg gccttaatcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaa ggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggcca ggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctc aagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcct gttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgta ggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgcc ttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggatt agcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatt tggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctgg tagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacgg ggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatcct tttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgag gcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactcggggggggggggcgctgaggtctgcctcgt gaagaaggtgttgctgactcataccaggcctgaatcgccccatcatccagccagaaagtgagggagccacggttgatg agagctttgttgtaggtggaccagttggtgattttgaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgt gatctgatccttcaactcagcaaaagttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagt gttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaa taccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggta tcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaat caccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccatt acgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatc gctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttca cctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggag tacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattg gcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgatt gcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgt ttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatctt gtgcaatgtaacatcagagattttgagacacaacgtggctttccccccccccccattattgaagcatttatcagggttattgt ctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccac ctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc

C. Therapeutic Methods and Pharmaceutical Compositions

The antigens as disclosed herein, or a nucleic acid molecule encoding the disclosed immunogen, can be administered to a subject in order to generate an immune response to a pathogen, such as HIV.

In exemplary applications, compositions are administered to a subject suffering from HIV infection or at risk of becoming infected from HIV. In other applications, the immunogens disclosed herein can be administered prophylactically, for example as part of an immunization regimen. The antigen is administered in an amount sufficient to raise an immune response against HIV.

The immunogen is administered in an amount sufficient to raise an immune response against HIV virus. Administration induces a sufficient immune response to treat the pathogenic infection, for example, to inhibit the infection and/or reduce the signs and/or symptoms of the infection. Amounts effective for this use will depend upon the severity of the disease, the general state of the subject's health, and the robustness of the subject's immune system. A therapeutically effective amount of the antigen is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.

An antigen can be administered by any means known to one of skill in the art (see Banga, A., "Parenteral Controlled Delivery of Therapeutic Peptides and

Proteins," in Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster, PA, 1995) either locally or systemically, such as by intramuscular, subcutaneous, or intravenous injection, but even oral, nasal, or anal administration is contemplated. In one embodiment, administration is by subcutaneous or

intramuscular injection. To extend the time during which the disclosed antigen is available to stimulate a response, the antigen can be provided as an implant, an oily injection, or as a particulate system. The particulate system can be a microparticle, a microcapsule, a microsphere, a nanocapsule, or similar particle, (see, e.g., Banga, supra). A particulate carrier based on a synthetic polymer has been shown to act as an adjuvant to enhance the immune response, in addition to providing a controlled release. Aluminum salts can also be used as adjuvants to produce an immune response.

Optionally, one or more cytokines, such as interleukin (IL)-2, IL-6, IL-12, IL-15, RANTES, granulocyte macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF) -a, interferon (IFN)-a or IFN-γ, one or more growth factors, such as GM-CSF or G-CSF, one or more costimulatory molecules, such as ICAM-1, LFA-3, CD72, B7-1, B7-2, or other B7 related molecules; one or more molecules such as OX-40L or 41 BBL, or combinations of these molecules, can be used as biological adjuvants (see, for example, Salgaller et al., 1998, J. Surg. Oncol. 68(2): 122-38; Lotze et al., 2000, Cancer J Sci. Am. 6(Suppl l):S61-6; Cao et al., 1998, Stem Cells 16(Suppl 1 J.-251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol. 465:381-90). These molecules can be administered systemically (or locally) to the host. In several examples, IL-2, RANTES, GM-CSF, TNF-a, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, B7-1 B.7-2, OX-40L, 41 BBL and ICAM-1 are administered.

A number of means for inducing cellular responses, both in vitro and in vivo, are known. Lipids have been identified as agents capable of assisting in priming CTL in vivo against various antigens. For example, as described in U.S. Patent No. 5,662,907, palmitic acid residues can be attached to the alpha and epsilon amino groups of a lysine residue and then linked (for example, via one or more linking residues, such as glycine, glycine-glycine, serine, serine-serine, or the like) to an immunogenic peptide. The lipidated peptide can then be injected directly in a micellar form, incorporated in a liposome, or emulsified in an adjuvant. As another example, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine can be used to prime tumor specific CTL when covalently attached to an appropriate peptide (see, Deres et al., Nature 342:561, 1989).

A pharmaceutical composition including an isolated immunogen is provided. In some embodiments, the immunogen is mixed with an adjuvant containing two or more of a stabilizing detergent, a micelle-forming agent, and an oil. Suitable stabilizing detergents, micelle-forming agents, and oils are detailed in U.S. Patent No. 5,585,103; U.S. Patent No. 5,709,860; U.S. Patent No. 5,270,202; and U.S. Patent No. 5,695,770. A stabilizing detergent is any detergent that allows the components of the emulsion to remain as a stable emulsion. Such detergents include polysorbate, 80 (TWEEN) (Sorbitan-mono-9-octadecenoate-poly(oxy-l,2- ethanediyl; manufactured by ICI Americas, Wilmington, DE), TWEEN 40™, TWEEN 20™, TWEEN 60™, ZWITTERGENT™ 3-12, TEEPOL HB7™, and SPAN 85™. These detergents are usually provided in an amount of approximately 0.05 to 0.5%, such as at about 0.2%. A micelle forming agent is an agent which is able to stabilize the emulsion formed with the other components such that a micellelike structure is formed. Such agents generally cause some irritation at the site of injection in order to recruit macrophages to enhance the cellular response. Examples of such agents include polymer surfactants described by BASF Wyandotte publications, e.g. , Schmolka, J. Am. Oil. Chem. Soc. 54: 110, 1977, and Hunter et al. , J. Immunol 129: 1244, 1981, PLURONIC™ L62LF, L101, and L64, PEG1000, and TETRONIC™ 1501, 150R1, 701, 901, 1301, and 130R1. The chemical structures of such agents are well known in the art. In one embodiment, the agent is chosen to have a hydrophile-lipophile balance (HLB) of between 0 and 2, as defined by Hunter and Bennett, J. Immun. 133:3167, 1984. The agent can be provided in an effective amount, for example between 0.5 and 10%, or in an amount between 1.25 and 5%.

The oil included in the composition is chosen to promote the retention of the antigen in oil-in-water emulsion, for example to provide a vehicle for the desired antigen, and preferably has a melting temperature of less than 65°C such that emulsion is formed either at room temperature (about 20°C to 25°C), or once the temperature of the emulsion is brought down to room temperature. Examples of such oils include squalene, Squalane, EICOSANE™, tetratetracontane, glycerol, and peanut oil or other vegetable oils. In one specific, non-limiting example, the oil is provided in an amount between 1 and 10%, or between 2.5 and 5%. The oil should be both biodegradable and biocompatible so that the body can break down the oil over time, and so that no adverse effects, such as granulomas, are evident upon use of the oil.

In one embodiment, the adjuvant is a mixture of stabilizing detergents, micelle-forming agent, and oil available under the name PRO VAX® (IDEC Pharmaceuticals, San Diego, CA). An adjuvant can also be an immunostimulatory nucleic acid, such as a nucleic acid including a CpG motif, or a biological adjuvant (see above).

Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems, see Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, 1995. Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μιη are generally referred to as nanoparticles, nanospheres, and

nanocapsules, respectively. Capillaries have a diameter of approximately 5 μιη so that only nanoparticles are administered intravenously. Microparticles are typically around 100 μιη in diameter and are administered subcutaneously or intramuscularly (see Kreuter, Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342, 1994; Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, 1992).

Polymers can be used for ion-controlled release. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:53 ^', 1993). For example, the block copolymer, polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et ah, Pharm. Res. 9:425, 1992; and Pec, /. Parent. Sci. Tech. 44(2):58, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et ah, Int. J. Pharm. 112:215, 1994). In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et ah, Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA, 1993). Numerous additional systems for controlled delivery of therapeutic proteins are known (e.g., U.S. Patent No. 5,055,303; U.S. Patent No. 5,188,837; U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; U.S. Patent No. 4,957,735; and U.S. Patent No. 5,019,369; U.S. Patent No. 5,055,303; U.S. Patent No. 5,514,670; U.S. Patent No. 5,413,797; U.S. Patent No. 5,268,164; U.S. Patent No. 5,004,697; U.S. Patent No. 4,902,505; U.S. Patent No. 5,506,206; U.S. Patent No. 5,271,961; U.S. Patent No. 5,254,342; and U.S. Patent No. 5,534,496).

In another embodiment, a pharmaceutical composition includes a nucleic acid encoding a disclosed immunogen. A therapeutically effective amount of the nucleic acid can be administered to a subject in order to generate an immune response. In one specific, non-limiting example, a therapeutically effective amount of a nucleic acid encoding a disclosed gpl20, gpl40 antigen, or immunogenic fragment thereof is administered to a subject to treat or prevent or inhibit HIV infection.

Optionally, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES,

GM-CSF, TNF-a, or IFN-γ, one or more growth factors, such as GM-CSF or G- CSF, one or more costimulatory molecules, such as ICAM-1, LFA-3, CD72, B7-1, B7-2, or other B7 related molecules; one or more molecules such as OX-40L or 41 BBL, or combinations of these molecules, can be used as biological adjuvants (see, for example, Salgaller et al. , 1998, J. Surg. Oncol. 68(2): 122-38; Lotze et al. , 2000, Cancer J Sci. Am. 6(Suppl l):S61-6; Cao et al, 1998, Stem Cells 16(Suppl 1):251- 60; Kuiper et al., 2000, Adv. Exp. Med. Biol. 465:381-90). These molecules can be administered systemically to the host. It should be noted that these molecules can be co-administered via insertion of a nucleic acid encoding the molecules into a vector, for example, a recombinant pox vector (see, for example, U.S. Pat. No. 6,045,802). In various embodiments, the nucleic acid encoding the biological adjuvant can be cloned into same vector as the disclosed antigen coding sequence, or the nucleic acid can be cloned into one or more separate vectors for co-administration. In addition, nonspecific immunomodulating factors such as Bacillus Cahnette-Guerin (BCG) and levamisole can be co-administered. One approach to administration of nucleic acids is direct immunization with plasmid DNA, such as with a mammalian expression plasmid. As described above, the nucleotide sequence encoding the disclosed antigen can be placed under the control of a promoter to increase expression of the molecule.

Immunization by nucleic acid constructs is well known in the art and taught, for example, in U.S. Patent No. 5,643,578 (which describes methods of immunizing vertebrates by introducing DNA encoding a desired antigen to elicit a cell-mediated or a humoral response), and U.S. Patent No. 5,593,972 and U.S. Patent No.

5,817,637 (which describe operably linking a nucleic acid sequence encoding an antigen to regulatory sequences enabling expression). U.S. Patent No. 5,880,103 describes several methods of delivery of nucleic acids encoding immunogenic peptides or other antigens to an organism. The methods include liposomal delivery of the nucleic acids (or of the synthetic peptides themselves), and immune- stimulating constructs, or ISCOMS™, negatively charged cage-like structures of 30- 40 nm in size formed spontaneously on mixing cholesterol and Quil A™ (saponin). Protective immunity has been generated in a variety of experimental models of infection, including toxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMS™ as the delivery vehicle for antigens (Mowat and Donachie, Immunol. Today 12:383, 1991). Doses of antigen as low as 1 μg encapsulated in ISCOMS™ have been found to produce Class I mediated CTL responses (Takahashi et ah, Nature 344:873, 1990).

In another approach to using nucleic acids for immunization, a disclosed antigen can also be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, cytogmeglo virus or other viral vectors can be used to express the peptide or protein, thereby eliciting a CTL response. For example, vaccinia vectors and methods useful in immunization protocols are described in U.S. Patent No.

4,722,848. BCG (Bacillus Calmette Guerin) provides another vector for expression of the peptides (see Stover, Nature 351:456-460, 1991).

Simultaneous production of an immunostimulatory molecule and the disclosed antigen enhances the generation of specific effectors. Without being bound by theory, dependent upon the specific immunostimulatory molecules, different mechanisms might be responsible for the enhanced immunogenicity: augmentation of help signal (IL-2), recruitment of professional APC (GM-CSF), increase in CTL frequency (IL-2), effect on antigen processing pathway and MHC expression (IFNy and TNFa) and the like. For example, IL-2, IL-6, interferon, tumor necrosis factor, or a nucleic acid encoding these molecules, can be administered in conjunction with a disclosed antigen, or a nucleic acid encoding a disclosed antigen. The co- expression of a disclosed antigen together with at least one immunostimulatory molecule can be effective in an animal model to show anti-pathogen effects.

In one embodiment, a nucleic acid encoding a disclosed antigen is introduced directly into cells. For example, the nucleic acid can be loaded onto gold

microspheres by standard methods and introduced into the skin by a device such as Bio-Rad's HELIOS™ Gene Gun. The nucleic acids can be "naked," consisting of plasmids under control of a strong promoter. Typically, the DNA is injected into muscle, although it can also be injected directly into other sites, including tissues in proximity to metastases. Dosages for injection are usually around 0.5 g/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see, e.g., U.S. Patent No. 5,589,466).

In one specific, non-limiting example, a pharmaceutical composition for intravenous administration would include about 0.1 μg to 10 mg of a disclosed antigen per subject per day. Dosages from 0.1 up to about 100 mg per subject per day can be used, particularly if the agent is administered to a secluded site and not into the circulatory or lymph system, such as into a body cavity or into a lumen of an organ. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remingtons Pharmaceutical Sciences, 19^th Ed., Mack Publishing Company, Easton, Pennsylvania, 1995.

Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the subject. In one embodiment, the dosage is administered once as a bolus, but in another embodiment can be applied periodically until a therapeutic result is achieved. Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the subject. Systemic or local

administration can be utilized.

The immunogenic compositions of this disclosure can be employed to generate antibodies that recognize the antigens disclosed herein and the antigen from which the disclosed antigen was derived. The methods include administering to a subject an immunogenic composition including a disclosed antigen or administering to the subject a polynucleotide encoding a disclosed antigen to generate antibodies that recognize the disclosed antigen. The subject employed in this embodiment is one typically employed for antibody production. Mammals, such as, rodents, rabbits, goats, sheep, etc., are preferred.

The antibodies generated can be either polyclonal or monoclonal antibodies. Polyclonal antibodies are raised by injecting (for example subcutaneous or intramuscular injection) antigenic polypeptides into a suitable animal (for example, a mouse or a rabbit). The antibodies are then obtained from blood samples taken from the animal. The techniques used to produce polyclonal antibodies are extensively described in the literature. Polyclonal antibodies produced by the subjects can be further purified, for example, by binding to and elution from a matrix that is bound with the polypeptide against which the antibodies were raised. Those of skill in the art will know of various standard techniques for purification and/or concentration of polyclonal, as well as monoclonal, antibodies. Monoclonal antibodies can also be generated using techniques known in the art.

Any of the disclosed immunogens and nucleic acid molecules encoding such immunogens can be used to elicit an immune response (immunogenic compositions) to gpl20 such as to a gpl20 expressing virus, for example to reduce HIV-1 infection or a symptom of HIV-1 infection. Following administration of a therapeutically effective amount of the disclosed therapeutic compositions, the subject can be monitored for HIV-1 infection, symptoms associated with HIV-1 infection, or both. Disclosed herein are methods of administering the therapeutic molecules disclosed herein (such as gpl20, gpl40 antigens or immunogenic fragments thereof and nucleic acids encoding such antigens) to reduce HIV-1 infection. Immunogenic compositions can be administered for therapeutic treatments. In therapeutic applications, a therapeutically effective amount of the immunogenic composition is administered to a subject suffering from a disease, such as HIV-1 infection or AIDS.

In therapeutic applications, a therapeutically effective amount of the composition is administered to a subject prior to or following exposure to or infection by HIV. When administered prior to exposure, the therapeutic application can be referred to as a prophylactic administration (such as in the form of a vaccine). Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the subject. In one embodiment, the dosage is administered once as a bolus, but in another embodiment can be applied periodically until a therapeutic result, such as a protective immune response, is achieved. Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the subject. Systemic or local administration can be utilized.

It may be advantageous to administer the immunogenic compositions disclosed herein with other agents such as proteins, peptides, antibodies, and other antiviral agents, such as anti-HIV agents. Examples of such anti-HIV therapeutic agents include nucleoside reverse transcriptase inhibitors, such as abacavir, AZT, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, zalcitabine, zidovudine, and the like, non-nucleoside reverse transcriptase inhibitors, such as delavirdine, efavirenz, nevirapine, protease inhibitors such as amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, osamprenavir, ritonavir, saquinavir, tipranavir, and the like, and fusion protein inhibitors such as enfuvirtide and the like. In certain

embodiments, immunogenic compositions are administered concurrently with other anti-HIV therapeutic agents. In some examples, the disclosed antigens are administered with T-helper cells, such as exogenous T-helper cells. Exemplary methods for the producing and administering T-helper cells can be found in

International Patent Publication WO 03/020904, which is incorporated herein by reference. In certain embodiments, the immunogenic compositions are administered sequentially with other anti-HIV therapeutic agents, such as before or after the other agent. One of ordinary skill in the art would know that sequential administration can mean immediately following or after an appropriate period of time, such as hours, days, weeks, months, or even years later.

The gpl20, gpl40 antigens or immunogenic fragments thereof and nucleic acids encoding these antigens can be used in a multistep immunization regime. In some examples, the regime includes administering to a subject a therapeutically effective amount of a first gpl20, gpl40 antigens or immunogenic fragments thereof as disclosed herein (the prime) and boosting the immunogenic response with one or more additional gpl20, gpl40 antigens or immunogenic fragments thereof after an appropriate period of time. The method of eliciting such an immune reaction is what is known as "prime-boost." In this method, the antibody response to the selected immunogenic surface is focused by giving the subject's immune system a chance to "see" the antigenic surface in multiple contexts. In other words, the use of multiple gpl20, gpl40 antigens or immunogenic fragments thereof with an antigenic surface in common selects for antibodies that bind the antigen's surface in common.

Different dosages can be used in a series of sequential inoculations. Thus, a practitioner may administer a relatively large dose in a primary inoculation and then boost with relatively smaller doses of the boost. The immune response against the gpl20, gpl40 antigens or immunogenic fragments thereof can be generated by one or more inoculations of a subject with a disclosed immunogenic composition.

In some examples, the gpl20, gpl40 antigens or immunogenic fragments thereof and nucleic acids encoding these antigens can are administered in "prime- boost" immunization regimes with stabilized gpl40 trimer (see for example Yang et al. J Virol. 76(9):4634-42, 2002), and/or stabilized gpl20 polypeptides (such as those described in WO 07/030518). In some examples of this method, gpl20, gpl40 antigens or immunogenic fragments thereof is initially administered to a subject and at periodic times thereafter stabilized gpl40 trimer boosts are administered. In other examples of this method stabilized gpl40 trimer is initially administered to a subject and at periodic times thereafter one or gpl20, gpl40 antigens or immunogenic fragments thereof are administered. Examples of stabilized gpl40 or gpl20 trimers can be found for example in U.S. Patent No. 6,911,205 which is incorporated herein in its entirety.

One can also use cocktails containing a variety of different HIV strains to prime and boost with trimers from a variety of different HIV strains or with trimers that are a mixture of multiple HIV strains. For example, the first prime could be with a gpl20, gpl40 antigens or immunogenic fragments thereof from one primary HIV isolate, with subsequent boosts different primary isolates.

In one embodiment, a suitable immunization regimen includes at least three separate inoculations with one or more immunogenic compositions of the invention, with a second inoculation being administered more than about two, about three to eight, or about four, weeks following the first inoculation. Generally, the third inoculation is administered several months after the second inoculation, and in specific embodiments, more than about five months after the first inoculation, more than about six months to about two years after the first inoculation, or about eight months to about one year after the first inoculation. Periodic inoculations beyond the third are also desirable to enhance the subject's "immune memory." The adequacy of the vaccination parameters chosen, e.g. , formulation, dose, regimen and the like, can be determined by taking aliquots of serum from the subject and assaying antibody titers during the course of the immunization program. Alternatively, the T cell populations can be monitored by conventional methods. In addition, the clinical condition of the subject can be monitored for the desired effect, e.g. , prevention of HIV- 1 infection or progression to AIDS, improvement in disease state (e.g., reduction in viral load), or reduction in transmission frequency to an uninfected partner. If such monitoring indicates that vaccination is sub-optimal, the subject can be boosted with an additional dose of immunogenic composition, and the vaccination parameters can be modified in a fashion expected to potentiate the immune response. Thus, for example, the dose of the chimeric non-HIV polypeptide or polynucleotide and/or adjuvant can be increased or the route of administration can be changed. It is contemplated that there can be several boosts, and that each boost can be a different gpl20, gpl40 antigen or immunogenic fragment thereof. It is also contemplated that in some examples that the boost may be the same disclosed antigen as another boost, or the prime.

The prime can be administered as a single dose or multiple doses, for example two doses, three doses, four doses, five doses, six doses or more can be administered to a subject over days, weeks or months. The boost can be

administered as a single dose or multiple doses, for example two to six doses, or more can be administered to a subject over a day, a week or months. Multiple boosts can also be given, such one to five, or more. Different dosages can be used in a series of sequential inoculations. For example a relatively large dose in a primary inoculation and then a boost with relatively smaller doses. The immune response against the selected antigenic surface can be generated by one or more inoculations of a subject with an immunogenic composition disclosed herein.

D. Immunodiagnostic Reagents and Kits

In addition to the therapeutic methods provided above, any of the disclosed antigens disclosed herein can be utilized to produce antigen specific

immunodiagnostic reagents, for example, for sero surveillance. Immunodiagnostic reagents can be designed from any of the antigenic polypeptide described herein. For example, in the case of the gpl20 and/or gpl40 antigens, the presence of serum antibodies to HIV is monitored using the isolated gpl20 and/or gpl40 antigens disclosed herein, such as to detect an HIV infection and/or the presence of antibodies that specifically bind the gpl20 and/or gpl40 antigens.

Generally, the method includes contacting a sample from a subject, such as, but not limited to a blood, serum, plasma, urine or sputum sample from the subject with one or more of the disclosed antigens disclosed herein (including a polymeric form thereof) and detecting binding of antibodies in the sample to the disclosed antigens. The binding can be detected by any means known to one of skill in the art, including the use of labeled secondary antibodies that specifically bind the antibodies from the sample. Labels include radiolabels, enzymatic labels, and fluorescent labels.

Any such immunodiagnostic reagents can be provided as components of a kit. Optionally, such a kit includes additional components including packaging, instructions and various other reagents, such as buffers, substrates, antibodies or ligands, such as control antibodies or ligands, and detection reagents

Methods are further provided for a diagnostic assay to monitor HIV-1 induced disease in a subject and/or to monitor the response of the subject to immunization by an HIV vaccine. By "HIV-1 induced disease" is intended any disease caused, directly or indirectly, by HIV. An example of an HIV-1 induced disease is acquired immunodeficiency syndrome (AIDS). The method includes contacting a disclosed gpl20 and/or gpl40 antigen with a sample of bodily fluid from the subject, and detecting binding of antibodies in the sample to the disclosed antigens. In addition, the detection of the HIV-1 binding antibody also allows the response of the subject to immunization by a HIV vaccine to be monitored. In still other embodiments, the titer of the HIV-1 binding antibodies is determined. The binding can be detected by any means known to one of skill in the art, including the use of labeled secondary antibodies that specifically bind the antibodies from the sample. Labels include radiolabels, enzymatic labels, and fluorescent labels. In other embodiments, a disclosed gpl20 and/or gpl40 antigen is used to isolate antibodies present in a subject or biological sample obtained from a subject.

EXAMPLES

Example 1

This example describes exemplary procedures for the production of antigens and immunization of animals with the antigens.

In some examples nucleic acid molecules encoding the disclosed antigens are cloned into expression vector CMV/R. Expression vectors are then transfected into

293F cells using 293Fectin (Invitrogen, Carlsbad, CA). Five days after transfection, cell culture supernatant is harvested and concentrated/buffer-exchanged to 500mM

NaCl/50 mM Tris pH8.0. The protein initially is purified using HiTrap IMAC HP Column (GE, Piscataway, NJ), and subsequent gel-filtration using SUPERDEX™ 200 (GE). In some examples the 6x His tag is cleaved off using 3C protease

(Novagen, Madison, WI).

For vaccinations with the disclosed antigens 3-4 months old rabbits

(NZW)(Covance, Princeton, NJ ) are immunized using the Sigma Adjuvant System (Sigma, St. Louis, MO) according to manufacture's protocol. Specifically, three rabbits in each group are vaccinated with 50 μg of protein in 300μ1 PBS emulsified with 300μ1 of adjuvant intramuscularly (both legs, 300μ1 each leg) for example at week 0, 4, 8, 12, 16. Sera are collected for example at week 6 (Post-1), 10 (Post-2), 14 (Post-3), and 18 (Post-4), and subsequently analyzed for their neutralization activities against a panel of HIV-1 strains, and the profile of antibodies that mediate the neutralization.

The disclosed antigens are also tested for antigenic profiling using well- characterized human monoclonal antibodies. For example:

1) potent neutralizing CD4 binding site (CD4BS) antibody (IgGibl2, the bl2 neutralizing antibody);

2) the non-neutralizing CD4 binding site antibodies ( IgGibl3 and F105,);

3) the non-neutralizing CD4 induced (CD4i) antibodies ( 17b, 48d, m6); and

4) the broad neutralizing antibody 2G12 which binds to glycan motifs on the outer domain, serving as a control for structural integrity of the resurfaced protein.

In some examples, the disclosed antigens are used to coat ELISA plates (4μg/ml in PBS). An amount of the different antibodies is added to the wells, and incubated at room temperature for one hour. The plates are washed six times with PBS+0.05% TWEEN®20, and followed by incubation with HRP conjugated goat anti-human IgG (1:5000) for another hour. The plates are washed again six times with

PBS+0.05% TWEEN®20 and developed by adding OPN substrate for 30 minutes at room temperature. The results are evaluated by plotting the readout at OD490 vs. the antibody concentrations.

The antigens are also used to probe for rabbit anti-sera for existence of CD4BS antibodies in the anti-sera. Example 2

Treatment of HIV in a Subject

This example describes exemplary methods for treating or inhibiting an HIV infection in a subject, such as a human subject by administration of one or more of the antigens disclosed herein. Although particular methods, dosages and modes of administrations are provided, one skilled in the art will appreciate that variations can be made without substantially affecting the treatment.

HIV, such as HIV type 1 (HIV-1) or HIV type 2 (HIV-2), is treated by administering a therapeutically effective amount of a disclosed antigen that induces an immune response to HIV, for example by inducing an immune response, such as a neutralizing antibody response to a protein present on the surface of HIV, for example a gpl20 peptide.

Briefly, the method includes screening subjects to determine if they have HIV, such as HIV-1 or HIV-2. Subjects having HIV are selected for further treatment. In one example, subjects are selected who have increased levels of HIV antibodies in their blood, as detected with an enzyme-linked immunosorbent assay, Western blot, immunofluorescence assay or nucleic acid testing, including viral RNA or proviral DNA amplification methods. In one example, half of the subjects follow the established protocol for treatment of HIV (such as a highly active antiretro viral therapy). The other half follow the established protocol for treatment of HIV (such as treatment with highly active antiretroviral compounds) in combination with administration of the agents including a therapeutically effective amount of a disclosed gpl20 antigen that induces an immune response to HIV. In another example, half of the subjects follow the established protocol for treatment of HIV (such as a highly active antiretroviral therapy). The other subjects receive a therapeutically effective amount of a disclosed resurfaced gpl20 antigen that induces an immune response to HIV, such as a neutralizing antibody response.

- I l l - Screening subjects

In particular examples, the subject is first screened to determine if the subject has HIV. Examples of methods that can be used to screen for HIV include measuring a subject's CD4+ T cell count and the level of HIV in serum blood levels.

In some examples, HIV testing consists of initial screening with an enzyme- linked immunosorbent assay (ELISA) to detect antibodies to HIV, such as to HIV-1. Specimens with a nonreactive result from the initial ELISA are considered HIV- negative unless new exposure to an infected partner or partner of unknown HIV status has occurred. Specimens with a reactive ELISA result are retested in duplicate. If the result of either duplicate test is reactive, the specimen is reported as repeatedly reactive and undergoes confirmatory testing with a more specific supplemental test (for example, Western blot or an immunofluorescence assay (IFA)). Specimens that are repeatedly reactive by ELISA and positive by IFA or reactive by Western blot are considered HIV-positive and indicative of HIV infection. Specimens that are repeatedly ELISA-reactive occasionally provide an indeterminate Western blot result, which may be either an incomplete antibody response to HIV in an infected person or nonspecific reactions in an uninfected person. IFA can be used to confirm infection in these ambiguous cases. In some instances, a second specimen will be collected more than a month later and retested for subjects with indeterminate Western blot results. In additional examples, nucleic acid testing (for example, viral RNA or proviral DNA amplification method) can also help diagnosis in certain situations.

The detection of HIV in a subject's blood is indicative that the subject has HIV and is a candidate for receiving the therapeutic compositions disclosed herein. Moreover, detection of a CD4+ T cell count below 350 per microliter, such as 200 cells per microliter, is also indicative that the subject is likely to have HIV.

Pre- screening is not required prior to administration of the therapeutic compositions disclosed herein. Pre-treatment of subjects

In particular examples, the subject is treated prior to diagnosis of AIDS with the administration of a therapeutically effective amount of a disclosed antigen that induces an immune response to HIV. In some examples, the subject is treated with an established protocol for treatment of AIDS (such as a highly active antiretroviral therapy) prior to treatment with the administration of a therapeutic agent that includes one or more of the disclosed antigens that induces an immune response to HIV. However, such pre-treatment is not always required and can be determined by a skilled clinician.

Administration of therapeutic compositions

Following selection, a therapeutic effective dose of a therapeutically effective amount of a disclosed antigen that induces an immune response to HIV is administered to the subject (such as an adult human or a newborn infant either at risk for contracting HIV or known to be infected with HIV). Additional agents, such as anti-viral agents, can also be administered to the subject simultaneously or prior to or following administration of the disclosed agents. Administration can be achieved by any method known in the art, such as oral administration, inhalation, intravenous, intramuscular, intraperitoneal or subcutaneous.

The amount of the composition administered to prevent, reduce, inhibit, and/or treat HIV or a condition associated with it depends on the subject being treated, the severity of the disorder and the manner of administration of the therapeutic composition. Ideally, a therapeutically effective amount of an agent is the amount sufficient to prevent, reduce, and/or inhibit, and/or treat the condition (for example, HIV) in a subject without causing a substantial cytotoxic effect in the subject. An effective amount can be readily determined by one skilled in the art, for example using routine trials establishing dose response curves. In addition, particular exemplary dosages are provided above. The therapeutic compositions can be administered in a single dose delivery, via continuous delivery over an extended time period, in a repeated administration protocol (for example, by a daily, weekly or monthly repeated administration protocol). In one example, a therapeutically effective amount of a disclosed antigen that induces an immune response to HIV administered intravenously to a human. As such, these compositions may be formulated with an inert diluent or with a pharmaceutically acceptable carrier.

Therapeutic compositions can be taken long term (for example over a period of months or years).

Assessment

Following the administration of one or more therapies, subjects having HIV (for example, HIV-1 or HIV-2) can be monitored for reductions in HIV levels, increases in a subjects CD4+ T cell count or reductions in one or more clinical symptoms associated with HIV infection. In particular examples, subjects are analyzed one or more times, starting 7 days following treatment. Subjects can be monitored using any method known in the art. For example, biological samples from the subject, including blood, can be obtained and alterations in HIV or CD4+ T cell levels evaluated.

Additional treatments

In particular examples, if subjects are stable or have a minor, mixed or partial response to treatment, they can be re-treated after re-evaluation with the same schedule and preparation of agents that they previously received for the desired amount of time, including the duration of a subject's lifetime. A partial response is a reduction, such as at least a 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 70% reduction of HIV viral load, HIV replication or combination thereof. A partial response may also be an increase in CD4+ T cell count such as at least 350 T cells per microliter.

Example 3

Treatment of Subjects

This example describes methods that can be used to treat a subject that has or is at risk of having an infection from HIV that can be treated by eliciting an immune response, such as a neutralizing antibody response to the pathogen of interest. In particular examples, the method includes screening a subject having, thought to have or at risk of having a HIV infection. Subjects of an unknown infection status can be examined to determine if they have an infection, for example using serological tests, physical examination, enzyme-linked immunosorbent assay (ELISA), radiological screening or other diagnostic technique known to those of skill in the art. In some examples, subjects are screened to identify a HIV infection, with a serological test, or with a nucleic acid probe specific for a HIV. Subjects found to (or known to) have a HIV infection can be administered a disclosed antigen that cam elicit an antibody response to HIV. Subjects may also be selected who are at risk of developing HIV for example, subjects exposed to HIV.

Subjects selected for treatment can be administered a therapeutic amount of the disclosed antigen. The disclosed antigen can be administered at doses of 1 μg/kg body weight to about 1 mg/kg body weight per dose, such as 1 μg/kg body weight - 100 μg/kg body weight per dose, 100 μg/kg body weight - 500 μg/kg body weight per dose, or 500 μg/kg body weight - 1000 μg/kg body weight per dose. However, the particular dose can be determined by a skilled clinician. The disclosed antigen can be administered in one or several doses, for example continuously, daily, weekly, or monthly. When administered sequentially the time separating the administration of the disclosed antigen can be seconds, minutes, hours, days, or even weeks.

The mode of administration can be any used in the art. The amount of agent administered to the subject can be determined by a clinician, and may depend on the particular subject treated. Specific exemplary amounts are provided herein (but the disclosure is not limited to such doses).

Example 4

Identification of Immunogenic Fragments of gpl20

This example describes the selection of immunogenic fragments of the disclosed antigens.

A nucleic acid molecule encoding a disclosed antigen is expressed in a host using standard techniques (see Sambrook et ah, Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1989). Preferable gpl20 antigen fragment is expressed such that the antigen can be isolated or purified in sufficient quantity. The antigens that are expressed are analyzed by various techniques known in the art, such as immunoblot, and ELISA, and for binding to known neutralizing antibodies of HIV, for example the bl2 antibody.

To determine the antigenic antigen fragments, subjects such as mice, rabbits or other suitable subjects are immunized with resurfaced gpl20 antigen fragments. Sera from such immunized subjects are tested for antibody activity for example by ELISA with the expressed polypeptide. They are also tested in a CD4 binding assay, for example by qualitative biacore, and the binding of neutralizing antibodies, for example, by using the bl2 antibody. Thus antigenic fragments of gpl20 are selected to achieve broadly reactive neutralizing antibody responses.

Example 5

HIV Immunogens

The class of immunogens disclosed herein is aimed at eliciting anti-HIV- 1 neutralization antibodies with special emphasis on induction of neutralization antibodies targeting the CD4 binding site (BS), the primary receptor for HIV-1 entry. The outer domain (OD) of HIV-1 envelope gpl20 is an engineered subunit protein containing residues 252 to 482. The V3 and β20-21 loops are truncated and replaced by short linkers. The OD retains a functional CD4-binding site (CD4BS) which represents a potential target for vaccine design. The same region is also targeted by potent anti-HIV neutralization antibodies VRCOl. The ODs described in this example retain the antigenic area for eliciting potent anti-CD4BS antibody, but with much more selective exposure of this region to the immune system. The ODs described herein contain various modifications in the one or more of the VI, V2, V3, V4 and V5 loops for improved protein conformation and stability and various additions of glycosylation sites for covering exposed immunodominant sites other than CD4BS. Biosynthesis of outer domain proteins

Genes encoding the outer domain of HIV-1 gpl20 were synthesized by PCR- based accurate synthesis (PAS). All genes were ligated with human CD5 secretion signal sequence and cloned into CMV/R expression vector. For protein production, the plasmids were transfected to 293-F cells by 293fectin (Invitrogen) and the supernatants were harvested 4 days later. The OD proteins were purified from the supernatants by cobalt- (TALON, Clontech) or nickel-coupled resin (ProPur IMAC, Nunc), or by covalently cross-linked bl2-sepharose 6 affinity column followed by gel filtration on Superdex 200 16/60 (GE Healthcare).

Enzyme-linked immunosorbent assay (ELISA)

ELISA was conducted with Maxisorp plates (Nunc). The plates were first coated with snowdrop lectin (GNA, Sigma) and blocked with 1% BSA in PBS. After washing the wells, the plates were incubated with the purified proteins (200 ng/well). The plates were then washed and incubated with serial dilutions of monoclonal antibodies. After washing the wells, the bound antibodies were detected by HRP-labeled anti-human IgG (Southern Biotech). The wells were developed by the substrates (OPD, Sigma), stopped by sulfuric acid and the absorbance was read at 490 nm. The curve fits and the EC₅₀ values were calculated by using Prism 5 software (Graphpad Software).

While this disclosure has been described with an emphasis upon particular embodiments, it will be obvious to those of ordinary skill in the art that variations of the particular embodiments may be used and it is intended that the disclosure may be practiced otherwise than as specifically described herein. Features, characteristics, compounds, chemical moieties or examples described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example of the invention.

Accordingly, this disclosure includes all modifications encompassed within the spirit and scope of the disclosure as defined by the following claims.

Claims

We Claim:

1. An isolated immunogen, comprising a human immunodeficiency virus (HIV) gpl20 outer domain polypeptide, wherein the gpl20 outer domain polypeptide, comprises amino acids 252-482 of gpl20, and wherein the outer domain comprises the amino acid sequence according to SEQ ID NO: 203, SEQ ID NO: 1 or SEQ ID NO: 74 and wherein the immunogen optionally further comprises one or more mutations selected from the following groups, a-h:

a: wherein the β20/β21 bridging sheet of the gpl20 outer domain is removed or truncated by replacing the amino acid residues between residue number 423 and residue number 435 in gpl20 with Gly-Gly or by replacing amino acid residues 421 through 436 of gpl20 with Gly-Gly;

b: wherein the VI and V2 loops of the gpl20 outer domain are removed or truncated by replacing amino acid residues 128 through 194 of gpl20 with GAG or by replacing the amino acid residues between residue number CI 19 and residue number C205 in gpl20 with VKLTPLAGATSVITQA (SEQ ID NO: 162);

c: wherein the VI loop of the gpl20 outer domain is removed or truncated by replacing amino acid residues 131-157 with the amino acid sequence CTDLRGSGGGSGGGSEIKNC (SEQ ID NO: 164);

d: wherein the V2 loop of the gp 120 outer domain is removed or truncated by replacing amino acid residues 157-198 in gpl20 with the amino acid sequence CSFNITTSIRDKVQKEYALFYKLDVVPIDNDNTSYRLINC (SEQ ID NO: 165), the amino acid sequence

CSFNITTSIRDKVQKEYALFYKLDVVPIGGSGGSYRLINC (SEQ ID NO: 166), or the amino acid sequence

CSFNITTSIRDKVQKEYALFYKLDVVPIGSGGGSGSYRLINC (SEQ ID NO: 167);

e: wherein the V3 loop of the gpl20 outer domain is removed or truncated by replacing amino acid residues 302-323 of gpl20 with the amino acid sequence NTRGRR (SEQ ID NO: 169), by replacing amino acid residues 302-323 of gpl20, with the amino acid sequence GRR, by replacing amino acid residues 296- 331 of gpl20 with the amino acid sequence CSRPNNNTRGRRGSSGGSHC (SEQ ID NO: 170), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence CSRPNNGGSGSGGSSGGSHC (SEQ ID NO: 171), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence

CARPSNNTRGRRGDIRQAYC (SEQ ID NO: 172), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence CARPSNNTDIRQAYC (SEQ ID NO: 173), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence CARPSNNTRQAYC (SEQ ID NO: 174), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence CARPSNNTQYC (SEQ ID NO: 175), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence CARGSGSGSYC (SEQ ID NO: 176), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence

CSRPNNNTRGRRGDIRQAHC (SEQ ID NO: 177), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence CSRPNNNTRRQAHC (SEQ ID NO: 178), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence CSRPNNGGSGQAHC (SEQ ID NO: 179), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence

CTRPNNGGSGSGGSSGGSHC (SEQ ID NO: 180), or by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence

CTRPNNNTRGRRGSSGGSHC (SEQ ID NO: 181);

f: wherein the V4 loop of the gpl20 outer domain is removed or truncated by replacing the V4 loop with the amino acid sequence

SrWNNGGGSGGGSGGGSDTIT (SEQ ID NO: 182), the amino acid sequence STWFNGSGSGGSGTIT (SEQ ID NO: 184), the amino acid sequence

STWFNSTWSTKGSNNTEGSDTIT (SEQ ID NO: 185), the amino acid sequence STWFQGSGSGGSGTIT (SEQ ID NO: 186), by at least partially replacing the V4 loop with Gly-Ser, by truncating the V4 loop by removing 9 amino acids from the V4 loop, or replacing the V4 loop with a V4 loop from HIV strain Ker2018;

g: wherein the V5 loop of the gpl20 outer domain is removed or truncated by replacing the V5 loop with the amino acid sequence GGNTNRTC

(SEQ ID NO: 187), the amino acid sequence GGSGSGTC (SEQ ID NO: 188), the amino acid sequence GGSGSTC (SEQ ID NO: 189), the amino acid sequence SGGSGQETFR (SEQ ID NO: 192), the amino acid sequence GGGSGSGEI (SEQ ID NO: 194), by truncating the V5 loop to NDSDGNETFR (SEQ ID NO: 190) or by replacing the V5 loop with an 8 amino acid Gly-Ser repeat; and

h: wherein the immunogen comprises one or more amino acid substitutions corresponding to: 273N, T283N, T339N, A341T, 360N, 362N, N363Q, P369N, 137 IT, 37 IN, T373N, T373G, 377N, A379T, F383T, N386Q, D392N, R419N, G421N, G424N, A431T, P437T, N465Q, W479N, N280C and G458, K358C and N465C, V255C and M475C, V257N, V272N, N276Q, N276D, N276E, T283V, A297T, D368R, N362T, E363N, P364S, T373N, S375T, F382T, S388A, A388S, K389D, E398N, N406Q, N410Q, K421T, G422V, V427N, V442N, R444T, N478L, S481T, E482S, V257N, V272N, N276D, A388S, K421S, R421N, I423T, T424N, M426T, I434N, A436T, R487N, V489T, and/or R490N.

2. The isolated immunogen of claim 1, wherein the gpl20 outer domain polypeptide comprises or consists of amino acids 213-492 of gpl20, amino acids 213-482 of gpl20, amino acids 252-492 of gpl20, or 252-482 of gpl20.

3. The isolated immunogen of any one of claims 1 or 2, wherein the immunogen comprises or consists of amino acids 213-492 of gpl20, amino acids

213-482 of gpl20, amino acids 252-492 of gpl20, or 252-482 of gpl20.

4. The isolated immunogen of any one of claims 1 or 2, wherein the immunogen comprises or consists of HIV-1 gpl40.

5. The isolated immunogen of any one of claims 1 or 2, wherein the immunogen comprises or consists of HIV-1 gpl20.

6. The isolated immunogen of any one of claims 4 or 5, wherein the immunogen comprises one or more amino acid substitutions corresponding to: T26N, K55N, A49S, D91T, T92N, D94T, D98N, M100T V103N, K113N, P118N, T207N, R209T.

7. The isolated immunogen of any one of claims 1-6, wherein the β20/β21 bridging sheet of the gpl20 outer domain is removed or truncated by replacing the amino acid residues between residue number 423 and residue number 435 in gpl20 with Gly-Gly or by replacing amino acid residues 421 through 436 of gpl20 with Gly-Gly.

8. The isolated immunogen of any one of claims 1-7, wherein the VI and V2 loops of the gpl20 outer domain are removed or truncated by replacing amino acid residues 128 through 194 of gpl20 with GAG or by replacing the amino acid residues between residue number CI 19 and residue number C205 in gpl20 with VKLTPLAGATSVITQA (SEQ ID NO: 162).

9. The isolated immunogen of any one of claims 1-7, wherein the wherein the VI loop of the gpl20 outer domain is removed or truncated by replacing amino acid residues 131-157 with the amino acid sequence

CTDLRGSGGGSGGGSEIKNC (SEQ ID NO: 164).

10. The isolated immunogen of any one of claims 1-7 or 9, wherein the V2 loop of the gpl20 outer domain is removed or truncated by replacing amino acid residues 157-198 in gpl20 with the amino acid sequence

CSFNITTSIRDKVQKEYALFYKLDVVPIDNDNTSYRLINC (SEQ ID NO: 165), the amino acid sequence

CSFNITTSIRDKVQKEYALFYKLDVVPIGGSGGSYRLINC (SEQ ID NO: 166), or the amino acid sequence

CSFNIT TSIRDKVQKEYALFYKLDVVPIGSGGGSGSYRLINC (SEQ ID NO: 167).

11. The isolated immunogen of any one of claims 1-10, wherein the V3 loop of the gpl20 outer domain is removed or truncated by replacing amino acid residues 302-323 of gpl20 with the amino acid sequence NTRGRR (SEQ ID NO: 169), by replacing amino acid residues 302-323 of gpl20, with the amino acid sequence GRR, by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence CSRPNNNTRGRRGSSGGSHC (SEQ ID NO: 170), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence

CSRPNNGGSGSGGSSGGSHC (SEQ ID NO: 171), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence

CARPSNNTRGRRGDIRQAYC (SEQ ID NO: 172), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence CARPSNNTDIRQAYC (SEQ ID NO: 173), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence CARPSNNTRQAYC (SEQ ID NO: 174), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence CARPSNNTQYC (SEQ ID NO: 175), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence CARGSGSGSYC (SEQ ID NO: 176), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence

CSRPNNNTRGRRGDIRQAHC (SEQ ID NO: 177), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence CSRPNNNTRRQAHC (SEQ ID NO: 178), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence CSRPNNGGSGQAHC (SEQ ID NO: 179), by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence

CTRPNNGGSGSGGSSGGSHC (SEQ ID NO: 180), or by replacing amino acid residues 296-331 of gpl20 with the amino acid sequence

CTRPNNNTRGRRGSSGGSHC (SEQ ID NO: 181).

12. The isolated immunogen of any one of claims 1-11, wherein the V4 loop of the gpl20 outer domain is removed or truncated by replacing the V4 loop with the amino acid sequence SIWNNGGGSGGGSGGGSDTIT (SEQ ID NO: 182), the amino acid sequence STWFNGSGSGGSGTIT (SEQ ID NO: 184), , the amino acid sequence STWFNSTWSTKGSNNTEGSDTIT (SEQ ID NO: 185), the amino acid sequence STWFQGSGSGGSGTIT (SEQ ID NO: 186), by at least partially replacing the V4 loop with Gly-Ser, by truncating the V4 loop by removing 9 amino acids from the V4 loop, or replacing the V4 loop with a V4 loop from HIV strain Ker2018.

13. The isolated immunogen of any one of claims 1-12, wherein the V5 loop of the gpl20 outer domain is removed or truncated by replacing the V5 loop with the amino acid sequence GGNTNRTC (SEQ ID NO: 187), the amino acid sequence GGSGSGTC (SEQ ID NO: 188), the amino acid sequence GGSGSTC (SEQ ID NO: 189), the amino acid sequence SGGSGQETFR (SEQ ID NO: 192), the amino acid sequence GGGSGSGEI (SEQ ID NO: 194), by truncating the V5 loop to NDSDGNETFR (SEQ ID NO: 190) or by replacing the V5 loop with a 8 amino acid Gly-Ser repeat.

14. The isolated immunogen of any one of claims 1-13, wherein the immunogen comprises one or more amino acid substitutions corresponding to:

273N, T283N, T339N, A341T, 360N, 362N, N363Q, P369N, 137 IT, 37 IN, T373N, T373G, 377N, A379T, F383T, N386Q, D392N, R419N, G421N, G424N, A431T, P437T, N465Q, W479N, N280C and G458, K358C and N465C, V255C and M475C, V257N, V272N, N276Q, N276D, N276E, T283V, A297T, D368R, N362T, E363N, P364S, T373N, S375T, F382T, S388A, A388S, K389D, E398N, N406Q, N410Q, K421T, G422V, V427N, V442N, R444T, N478L, S481T, E482S, T26N, K55N, A49S, D91T, T92N, D94T, D98N, M100T V103N, K113N, P118N, T207N, R209T, V257N, V272N, N276D, A388S, K421S, R421N, I423T, T424N, M426T, I434N, A436T, R487N, V489T, and/or R490N.

15. The isolated immunogen of any one of claims 1-14, wherein the immunogen comprises one or more of a foldon domain, a ferritin polypeptide, a hybrid of different ferritin polypeptides, a six-histidine residue tag, a secretion signal sequence and a transmembrane domain .

16. The isolated immunogen of claim 15, wherein the immunogen comprises a T4 fibritin foldon comprising the amino acid sequence

GYIPEAPRDGQAYVRKDGEWVLLSTF (SEQ ID NO: 195).

17. The isolated immunogen of any one of claims 15 or 16, wherein the immunogen comprises a gp41 transmembrane domain, a CD4 transmembrane domain or an influenza transmembrane domain

18. The isolated immunogen of claim 17, wherein the CD4

transmembrane domain comprises the amino acid sequence

LIVLGGVAGLLLFIGLGI (SEQ ID NO: 196).

19. The isolated immunogen of claim 17, wherein the influenza transmembrane domain comprises a neuraminidase (NA) transmembrane domain comprising the amino acid sequence

MNPNQKIITIGSICMVVGIISLILQIGNIISIWVS (SEQ ID NO: 198) or a hemegglutin (HA) transmembrane domain comprising the amino acid sequence ILAIYSTVASSLVLLVSLGAISF (SEQ ID NO: 197).

20. The isolated immunogen of any one of claims 15-19, wherein secretion signal sequence is a CD5 secretion signal sequence comprising the amino acid sequence MPMGSLQPLATLYLLGMLVASVLA (SEQ ID NO. 199) or a IL-2 secretion signal sequence comprising the amino acid sequence

MQLASCVTLTLVLLVNSAP (SEQ ID NO: 200).

21. The isolated immunogen of any one of claims 1-20, wherein the immunogen comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth as any one of SEQ ID NOs: 203, 58-77, 1-57, 79, or 80 148, or 149.

22. A virus-like particle (VLP) comprising the isolated immunogen of any one of claims 1-21.

23. The VLP of claim 22, wherein the VLP comprises a Chikungunya virus peptide covalently linked to the immunogen.

24. An isolated nucleic acid molecule encoding the antigen of any one of claims 1-21 or a fusion protein comprising a Chikungunya virus peptide covalently linked to the immunogen of any one of claims 22 and 22.

25. The isolated nucleic acid molecule of claim 24, wherein the nucleotide sequence comprises the nucleic acid sequence set forth as one of SEQ ID NOs: 81-147, 150-161 and 206-215.

26. The isolated nucleic acid molecule of any one of claims 24 or

25operably linked to a promoter.

27. A vector comprising the nucleic acid molecule of claim 26.

28. An isolated host cell comprising the vector of claim 27.

29. A pharmaceutical composition comprising an effective amount of the isolated immunogen of any one of claims 1-21, an isolated nucleic acid molecule encoding the isolated immunogen of any one of claims 1-21, or a VLP comprising the isolated immunogen of any one of claims 22 or 23, and a pharmaceutically acceptable carrier.

30. A method for generating an immune response in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 29, thereby generating the immune response.

31. A method for treating or preventing a human immunodeficiency type 1 (HIV-1) infection in a subject, comprising administering to the subject a therapeutically effective amount of a first isolated immunogen, wherein the first isolated immunogen comprises the isolated immunogen of any one of claims 1-21, an isolated nucleic acid molecule encoding the isolated immunogen, or a VLP comprising the first isolated immunogen, and wherein the first isolated immunogen comprises a target epitope, thereby treating the subject or preventing infection of the subject.

32. The method of claim 31, further comprising administering a therapeutically effective amount of at least one additional isolated immunogen of any one of claims 1-20, an isolated nucleic acid molecule encoding the isolated immunogen, or a VLP comprising the first isolated immunogen, wherein a target epitope of the additional immunogen is identical to the first immunogen, and wherein surface-exposed amino acid residues located exterior to the target epitope of the first immunogen are not identical to surface-exposed amino acid residues located exterior to the target epitope on the additional isolated immunogen.

33. The method of any one of claims 31-32, further comprising administering a therapeutically effective amount of a polypeptide comprising:

a) a monomeric or trimeric gpl40 polypeptide;

b) an monomeric or trimeric wild-type gpl20 polypeptide;

c) a wild-type outer domain gpl20 polypeptide;

d) a nucleic acid molecule expressing the polypeptide of a-c; or e) any combination of a-d, above.

34. The method of any one of claims 31-33, further comprising administering to the subject a therapeutically effective amount of an anti- viral agent.

35. A method for detecting or isolating an HIV-1 binding antibody in a subject infected with HIV-1, comprising: providing the immunogen of any one of claims 1-21;

contacting the immunogenic composition with an amount of bodily fluid from the subject; and

detecting binding of the HIV- 1 binding antibody to the immunogen, thereby detecting or isolating the HIV-1 binding antibody in a subject.