mRNA PRIMING VACCINE COMPOSITION AND METHOD OF USE THEREOF

Abstract

A method for immunizing a subject against a pathogen, comprising the steps of: (1) administering to the subject an effective amount of a prime vaccine, wherein the prime vaccine comprises one or more mRNA constructs encoding one or more immunogens from the pathogen; and (2) administering to the subject an effective amount of a boost vaccine, wherein the booster vaccine comprises a non-mRNA vaccine modality, such as a recombinant protein, against the pathogen, wherein the boost vaccine comprises the immunogen or immunogens encoded in one or more mRNA constructs in the prime vaccine, or wherein the boost vaccine comprises the immunogen or immunogens that are not the same as encoded in one or more mRNA constructs in the prime vaccine, wherein the boost vaccine is administered after the administration of the prime vaccine.

Description

DOCKET NO: 2028-011 PCT TITLE mRNA PRIMING VACCINE COMPOSITION AND METHOD OF USE THEREOF

[0001] The present application claims priority of US Provisional Patent application No.63 / 623,446, filed January 22, 2024, which is incorporated herein by reference in its entirety. FIELD

[0002] The present disclosure relates generally to vaccine development and, in particular, to compositions and methods for vaccination against HIV-1. BACKGROUND

[0003] Nucleic acid vaccines have emerged as promising alternatives to conventional vaccine approaches.

[0004] The mRNA vaccine field is developing extremely rapidly due to the improvement of stability and translation of mRNA, and in vivo delivery efficiency, which highlighted by the approval of SARS-Cov-2 vaccine from Pfizer / BioNtech, Moderna, and others.

[0005] mRNA vaccine has many advantages such as high potency, capacity for rapid development and potential for low-cost manufacture and relatively safe administration.

[0006] However, the immunogenicity of mRNA vaccines is far from ideal, such as the short durability of immune responses induced by mRNA vaccines.

[0007] Also heterologous prime-boost vaccination has emerged as the more powerful strategy for future vaccine development and how to apply such concept to mRNA vaccine usage is the key discovery of the current application. SUMMARY

[0008] One aspect of the present application relates to a method for immunizing a subject against a pathogen, comprising the steps of: (1) administering to the subject an effective amount of a prime vaccine, wherein the prime vaccine comprises one or more mRNA constructs encoding one or more immunogens and / or modified immunogens from the pathogen; and (2) administering to the subject an effective amount of a boost vaccine, WAS:164427.1wherein the booster vaccine comprises a non-mRNA vaccine modality against the pathogen, wherein the boost vaccine is administered after the administration of the prime vaccine. In some embodiments, the pathogen is HIV-1 and the method comprises the steps of (1) administering to the subject an effective amount of a prime vaccine, wherein the prime vaccine comprises one or more mRNA constructs encoding one or more HIV-1 envelope immunogens and / or modified HIV-1 envelope immunogens; and (2) administering to the subject an effective amount of a boost vaccine (in any non-mRNA vaccine modality), wherein the booster vaccine comprises one or more HIV-1 envelope immunogens and / or modified HIV-1 envelope immunogens that are encoded in the one or more mRNA constructs in the prime vaccine, or wherein the booster vaccine comprises one or more HIV-1 envelope immunogens and / or modified HIV-1 envelope immunogens that are not the same (such as from different viral variants) as encoded in the one or more mRNA constructs in the prime vaccine, wherein the boost vaccine is administered after the administration of the prime vaccine. Examples of the HIV-1 envelope proteins include, but are not limited to, gp120, gp140, gp160 or trimeric HIV-1 envelope glycoprotein.

[0009] Another aspect of the present application relates to a method for introducing an immune response against HIV-1 in a subject, comprising the steps of: (1) administering to the subject an effective amount of a prime vaccine, wherein the prime vaccine comprises one or more mRNA constructs encoding one or more HIV-1 env immunogens and / or modified HIV-1 env immunogens ; and (2) administering to the subject an effective amount of a boost vaccine (in any non-mRNA vaccine modality), wherein the booster vaccine comprises one or more HIV-1 env immunogens and / or modified HIV-1 env immunogens that are encoded in the one or more mRNA constructs in the prime vaccine, or wherein the booster vaccine comprises one or more HIV-1 envelope immunogens and / or modified HIV-1 envelope immunogens that are not the same (such as from different viral variants) as encoded in the one or more mRNA constructs in the prime vaccine, wherein the boost vaccine is administered after the administration of the prime vaccine. The prime vaccine and / or the boost vaccine may be monovalent or polyvalent. The HIV-1 env immunogens and modified HIV-1 env immunogens may comprise HIV-1 gp120, gp140, gp160, trimeric HIV-1 envelope glycoprotein and other forms of HIV-1 envelope immunogens.

[0010] Another aspect of the present application relates to an mRNA priming HIV-1 vaccine formulation, comprising: (1) a prime component comprising a mRNA construct encoding an HIV-1 env immunogen or a modified HIV-1 env immunogen, such as in the form of gp120, gp140, gp160, trimeric HIV-1 envelope glycoprotein, or other modified env 2 WAS:164427.1immunogens and (2) a boost component (in any non-mRNA vaccine modality) comprising the immunogen encoded in the mRNA construct or immunogen that is not the same (such as from different viral variants) as encoded in the mRNA, wherein the prime component and boost components are formulated for separate administrations.

[0011] One of ordinary skill will understand that the differing embodiments disclosed in this application can all be used either independently or in combination with each other and there is no limitation implied on such combinations by the order or manner in which embodiments are disclosed. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG.1 shows the elements of the heterologous prime-boost vaccine regime. Panel A, prime vaccine. Panel B, boost vaccine.

[0013] FIG.2 shows the design of an exemplary HIV-1 conventional mRNA vaccine.

[0014] FIG.3 shows the design of an exemplary HIV-1 self-amplified RNA (SaRNA) vaccine.

[0015] FIG.4 shows distributions of binding antibody V1V2 heterologous breadth scores in each regimen. The box plots show the distribution of IgG3 binding antibody V1V2 heterologous breadth scores across participants in a given regimen, where each dot represents one participant. The horizontal line in each box represents the median regimen-specific breadth score. The number of participants (n) in each regimen is provided in the top row. Within a given regimen, the V1V2 heterologous breadth score was defined as the geometric mean of the binding antibody responses to the 3 heterologous V1V2 antigens with the highest median responses among all participants in that regimen.

[0016] While the present disclosure will now be described in detail, and it is done so in connection with the illustrative embodiments, it is not limited by the particular embodiments illustrated in the figures and the appended numbered paragraphs. DETAILED DESCRIPTION

[0017] REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0018] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on March 20, 2025, is named “2028-011 PCT.xml” and is 113,968 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.

[0019] The disclosure will now be discussed to enable one skilled in the art to practice the invention described herein. The skilled artisan will understand, however, that the 3 WAS:164427.1embodiments described below can be practiced without employing every specific detail, or that they can be used for purposes other than those described herein. Indeed, they can be modified and can be used in conjunction with products and techniques known to those of skill in the art considering the present disclosure. The descriptions are intended to be exemplary of various aspects of the disclosure and are not intended to narrow the scope of the appended claims.

[0020] It will be appreciated that reference throughout this specification to aspects, features, advantages, or similar language does not imply that all the aspects and advantages may be realized with the present disclosure or realized are in any single embodiment of the disclosure. Rather, language referring to the aspects and advantages should be understood to mean that a specific aspect, feature, advantage, or characteristic described in connection with an embodiment is included in at least one embodiment of the present disclosure. Thus, discussion of the aspects and advantages, and similar language, throughout this specification may, but does not necessarily, refer to the same embodiment.

[0021] The described aspects, features, advantages, and characteristics of the disclosure may be combined in any suitable manner in one or more further embodiments. Furthermore, one skilled in the relevant art will recognize that the embodiments may be practiced without one or more of the specific aspects or advantages of a particular embodiment. In other instances, additional aspects, features, and advantages may be recognized and claimed in certain embodiments that may not be present in all embodiments of the disclosure.

[0022] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any nucleic acid or protein encoded thereby; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0023] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and 4 WAS:164427.1configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, formulations, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, formulations, and / or methods, if such features, systems, articles, materials, formulations, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0024] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. I. Definitions

[0025] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0026] The terms “a”, “an,” or “the” as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean “at least one” or “one or more,” unless the content clearly dictates otherwise.

[0027] The phrase “and / or,” as used herein in the specification and claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. 5 WAS:164427.1

[0028] As used herein in the specification and claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0029] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. Further, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

[0030] Where ranges are given, endpoints are included. Further, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific subrange within the stated ranges in different embodiments of the invention, or any subrange defined by any pair of integers within a stated range, unless the context clearly dictates otherwise.

[0031] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. 6 WAS:164427.1

[0032] The term “heterologous prime-boost vaccination”, as used herein, refers to two or more vaccine modalities administrated sequentially.

[0033] The term “single immunogen”, as used herein, refers to one immunogen from the pathogen.

[0034] The term “multiple immunogens”, as used herein, refers to two or more immunogens from the same pathogen.

[0035] The term “monovalent”, as used herein, refers to one variant of an immunogen from the pathogen.

[0036] The term “polyvalent”, as used herein, refers to multiple variants of the same immunogen from the pathogen.

[0037] The term "vaccine" refers to a composition that induces an immune response in the recipient or host of the vaccine. A vaccine may induce a humoral (e.g., antibody, neutralizing antibody, other functional antibodies etc.) response to one or more immunogens, cell-mediated immune response (e.g., helper T lymphocyte, or cytotoxic T lymphocyte (CTL)) response against one or more immunogens, or both in a recipient so as to provide partial or complete protection against e.g., current or subsequent microbial infections or disease conditions characterized by the presence of e.g., one or more neoantigens or cancer antigens in the recipient.

[0038] The term "vaccine", as used herein, refers to a composition comprising at least one immunologically active component (i.e, an immunogen) that induces an immune response in the recipient or host of the vaccine and may possibly, but not necessarily, include one or more additional components that enhance the immunological activity of the active component. Examples of vaccines include, but are not limited to, live attenuated vaccines, inactivated vaccines, recombinant protein vaccines, viral particle vaccines, sugar-based vaccines (conjugated or natural), bacterial vector vaccines, viral vector vaccines, and nucleic acid vaccines such as mRNA vaccines and DNA vaccines.

[0039] A vaccine of the present application can induce antibody responses and / or cell-mediated immune response to a target pathogen in the recipient as protection against current or future target pathogen infections, including protection against infection upon subsequent challenge with target pathogen. Antibodies , CD4+ T cell or CD8+ T cell generated in the vaccinated host can provide this protection.

[0040] The term “vaccine modality” refers to the mode that an immunogen or immunogens is present in a vaccine. Examples of vaccine modalities include, but are not limited to, live attenuated vaccine, inactivated vaccine, recombinant protein vaccine, viral 7 WAS:164427.1like particle vaccine, viral vector vaccine, polysaccharide conjugate vaccine, toxoid vaccine, and nucleic acid vaccine such as mRNA vaccine and DNA vaccine.

[0041] The term "vaccination" refers to the administration of antigenic material to stimulate an individual's immune system to develop adaptive immunity to a pathogen. Vaccination can prevent or ameliorate of one or more symptoms associated with microbial infection or antigen- or epitope-specific cell associated with a disease, such as cancer; and / or lessening of the severity or frequency of one or more symptoms associated with the foregoing disease conditions.

[0042] The term “single-modality vaccination” refers to a vaccination process using vaccine or vaccines of the same modality (e.g., vaccination with inactivated vaccines).

[0043] The term “multiple-modality vaccination” refers to a vaccination process using vaccines of two or more modalities. (e.g., vaccination with a DNA prime vaccine first, followed by a protein boost vaccine).

[0044] The terms “immunogen” is referred to a substance or molecule from the pathogen capable of eliciting an immune response and generating specific antibodies (humoral response) or cell-mediated response against it. As such, the immunogen is capable of being recognized by components of the immune system, such as lymphocytes. An immunogen can be as small as a single epitope, or larger, and can include multiple epitopes. As such, the size of an immunogen can be as small as about 5-12 amino acids (e.g., a peptide) and as large as: a partial protein, a full-length protein, including a multimer and fusion protein, or a chimeric protein. In addition, immunogens can be polynucleotides, such as mRNAs and DNAs, or carbohydrates or attenuated / killed pathogen. An immunogen may be from a bacterium, virus, protozoan or fungus.

[0045] The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, or other binding molecule, domain, or protein. Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. Where an immunogen is or comprises a peptide or protein, the epitope can be comprised of consecutive amino acids (e.g., a linear epitope), or can be comprised of amino acids from different parts or regions of the protein that are brought into proximity by protein folding (e.g., a discontinuous or conformational epitope), or non-contiguous amino acids that are in close proximity irrespective of protein folding. 8 WAS:164427.1

[0046] As used herein, the term “multi-immunogen vaccine” refers to a vaccine modality that includes more than one immunogen.

[0047] As used herein, the term “DNA vaccine” refers to a type of nucleic acid vaccines that introduces a specific immunogen-coding DNA sequence into the cells of a host as a mechanism deliver one or more immunogens for the purpose of inducing immunogen specific immune responses in the host. The immunogen-coding DNA, when expressed in a subject, is translated into an antigenic protein or polypeptide that induces an immune response.

[0048] As used herein, the term “mRNA vaccine” refers to a type of nucleic acid vaccines that uses messenger RNA (mRNA) to produce an immunogen specific immune response. The vaccine delivers molecules of immunogen-encoding mRNA into host cells, which use the designed mRNA as a blueprint to build foreign protein that would normally be produced by a pathogen (such as a virus). These protein molecules stimulate an adaptive immune response that teaches the body to identify and destroy the corresponding pathogen.

[0049] As used herein, the term “live attenuated vaccine,” refers to a vaccine created by reducing the virulence of a pathogen, but still keeping it viable (or "live"). Attenuation takes an infectious agent and alters it so that it becomes harmless or less virulent. These vaccines contrast to those produced by "killing" the pathogen (inactivated vaccine).

[0050] As used herein, the term “inactivated vaccine,” refers to a vaccine that uses the killed version of a pathogen (e.g., a bacterium or virus) without causing a disease.

[0051] As used herein, the term “recombinant protein vaccine,” refers a vaccine that uses a recombinant protein as an immunogen.

[0052] As used herein, the term “viral vector vaccine,’ refers to a vaccine that uses a recombinant virus as a vector to deliver an immunogen.

[0053] As used herein, the term “viral like particle vaccine (VLP),” refers to a vaccine that uses molecules that closely resemble viruses, but are non-infectious because they can not replicate in the host. Viral like particles contain repetitive, high density displays of viral surface proteins that present conformational immunogen epitopes that can elicit immunogen specific T cell and B cell immune responses. Since VLPs cannot replicate, they provide a safer alternative to attenuated viruses.

[0054] As used herein, the term “polysaccharide conjugate vaccine,” refers to a vaccine that conjugate a polysaccharide immunogen from bacteria to a protein carrier, thereby improving the immunogenicity of the polysaccharide immunogen. 9 WAS:164427.1

[0055] As used herein, the term “toxoid vaccine,” refers to a vaccine that is made of detoxified toxin of a pathogen that no longer causes a disease. The vaccine creates immunity to the toxin in order to achieve the control of a pathogen.

[0056] As used herein, the term “heterologous prime-boost vaccination” refers to a vaccination process in which a prime vaccine (e.g., a DNA vaccine) is administered first (once or multiple times), followed with one or more administrations of a boost vaccine (e.g., a recombinant protein vaccine). This is different from the “homologous prime-boost vaccination” in which the prime vaccine and boost vaccine use the same vaccine modality. The prime vaccine, or the boost vaccine, or both can be monovalent or polyvalent, no matter whether it is the “homologous prime-boost vaccination” or the “heterologous prime-boost vaccination”.

[0057] As used herein, the term “adjuvant” refers to a compound or mixture that enhances an immune response. The term "adjuvant" refers to an agent that when administered concurrently with the vaccine composition of the present application, accelerates, prolongs, enhances and / or boosts the immune response thereto. Adjuvants can enhance an immune response by several mechanisms including, e.g., lymphocyte recruitment, stimulation of B and / or T cells, stimulation of dendritic cells and / or stimulation of macrophages.

[0058] As used herein, the term “adjuvanted recombinant protein or polypeptide” refers to a protein immunogen or polypeptide immunogen that is mixed with, or formed a complex with, an adjuvant.

[0059] As used herein, the term “polynucleotide” or “nucleic acid” refers to both DNA and mRNA molecules. In other words, a polynucleotide can be a DNA molecule or an mRNA molecule.

[0060] The terms “codon optimized” and “codon optimization” refer to a process for modifying a nucleic acid sequence according to one or more of the following: (1) to match codon frequencies in a host target; (2) to promote increased expression; (3) to ensure proper folding; (4) to provide a GC content suitable for increasing mRNA stability or reducing secondary structures; (5) to minimize tandem repeat codons or base runs that may impair gene construction or expression; (6) to customize transcriptional and translational control regions; (7) to insert or remove protein trafficking sequences; (8) to remove / add post translation modification sites in an encoded protein (e.g. glycosylation sites); (9) to add, remove or shuffle protein domains; (10) to insert or delete restriction sites; (11) modify ribosome binding sites and mRNA degradation sites; (12) to adjust translational rates to allow the various domains of the protein to fold properly; or (13) to reduce or eliminate problem 10 WAS:164427.1secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art—non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and / or proprietary methods.

[0061] The term "expression vector" refers to a gene construct that is capable of expressing a polypeptide (e.g., a mRNA, a guide RNA, shRNA or miRNA) in a cell. Expression vectors may be, for example, plasmids, viral vectors, and bacterial vectors.

[0062] An expression vector typically comprises (1) a polynucleotide coding sequence encoding the polypeptide to be expressed and (2) multiple regulatory sequences operably linked to the coding sequence. The regulatory sequence is capable of effecting the expression of the coding sequence in a suitable host. Such regulatory sequences may include promoters, enhancers, mRNA ribosome binding sites, introns, internal ribosome binding sequences, and sequences which control termination of transcription and translation. Once transfected into a suitable host, the expression vector may replicate and function independently of the host genome.

[0063] The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Unlinked" means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.

[0064] As used herein, the term "wild type,” “prototype” and “original” are used with reference to the original target isolate, including polynucleotides and proteins thereof

[0065] As used herein, the phrases “primary viral isolate" and "primary isolate" in reference to nucleic acid or amino acid sequences refer to a nucleic acid or amino acid sequence present in cells of individuals naturally infected with a virus, such as HIV-1. A primary viral isolate is a viral isolate that has been expanded and maintained only in primary cells, and has not been expanded and maintained in cell lines. Thus, a primary isolate differs from what is referred to as a "laboratory strain.".

[0066] As used herein, the phrases “variant virus”, “variant strain” and “variant of a virus” are used interchangeably and refer to a virus strain that differs from the original virus strain by one or more nucleotide in the viral genome. Variants of the original strain (such as in the case of HIV-1) may be divided into Groups, such as M group and within the M group, 11 WAS:164427.1further divided into clades (subtypes), such as clade A, B, C, D, A / E. Within each clade, there are a vast variety of strains that are slightly different from each other.

[0067] As used herein, the phrase “variant of an immunogen” refers to different versions or subtypes / strains of an immunogen. For example, the envelope glycoprotein gp120 (gp120) of different HIV-1 subtypes / strains, such as HIV-1 gp120-A / 92UG037.1, HIV-1 gp120-B / JRFL or 92US715.6, HIV-1 gp120-C / 93MW965.26, HIV-1 gp120- D / 92UG021.16, and HIV-1 gp120 A / E / consensus are variants of HIV-1 gp120. As used herein, the phrase “modified immunogen” or “modified forms of an immunogen” refers an immunogen with one or more modifications from its original / native immunogen sequences. Modified forms of an immunogen include fragments and polymers of the immunogen. For example, modified forms of HIV-1 gp120 include HIV-1 gp120 with substitutions, deletions and additions in its amnio acid sequence, fragments of HIV-1 gp120, and trimers of HIV-1 gp120. Modified immunogens include immunogens (such as viral envelope proteins) with optimized structural designs. Optimized structural designs is a well- accepted technical concept and is crucial for developing effective vaccines. Different strategies can be applied based on different targets and applications such as retaining the prefusion state for certain envelope glycoprotein.

[0068] The term “HIV-1 env immunogens,” as used herein, refers to all immunogenic forms of HIV-1 envelope proteins and modified forms thereof. Examples of HIV-1 envelope proteins include, but are not limited to, HIV-1 gp120, HIV-1 gp140, HIV-1 gp160, and HIV- 1 trimeric envelope glycoprotein.

[0069] The phrase "effective amount" of an agent, as used herein, is that amount sufficient to affect beneficial or desired results, for example, clinical results, and, as such, an "effective amount" depends upon the context in which it is being applied. For example, in the context of administering a vaccine composition, an effective amount refers to an amount sufficient to induce an immune response or provide immunity against HIV-1, as compared to responses (or lack thereof) obtained without administration of the agent. In general, an effective amount of the DNA / RNA and / or protein vaccines provide an induced or boosted immune response in a subject.

[0070] As used herein, the term "homology" refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be "homologous" to one another if their sequences are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% identical or similar. The term 12 WAS:164427.1"homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). In accordance with the present invention, two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 80%, 90%, 95%, or even 99% to one another. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids.

[0071] As used herein, the term "identity" or “sequence identity” refers to the overall relatedness between polymeric molecules, e.g., between oligonucleotide molecules (e.g. DNA molecules and / or mRNA molecules) and / or between polypeptide molecules. For example, a peptide that has 90% sequence identity to

[0072] Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. The percent identity between two nucleotide sequences can be determined using conventional methods known in the art at the time the present invention was made.

[0073] The term “pharmaceutically acceptable carrier” refers to those compounds, materials, compositions that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Examples of pharmaceutically acceptable carrier include, but are not limited to, solvents, solubilizers, fillers, diluents, stabilizers, surfactants, binders, absorbents, bases, buffering agents, excipients, emulsifying agents, encapsulating materials, humectants, 13 WAS:164427.1lubricants, gels, dispersion media, coatings, isotonic and absorption delaying agents. The use of such carriers and agents for pharmaceutically active substances is well-known in the art.

[0074] As used herein, the term "preventing" refers to partially or completely prevention of an infection (such as HIV infection), disease, disorder and / or condition thereof; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of an infection, disease, disorder, and / or condition thereof; partially or completely delaying progression from HIV infection, disease, disorder and / or condition thereof; and / or decreasing the risk of developing pathology associated with a HIV infection, disease, disorder, and / or condition thereof.

[0075] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. In certain embodiments, the phrase “substantially full length” refers to a nucleic acid or protein that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% in length relative to a reference nucleic acid encoding an open reading frame or a reference protein. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. II. Methods of vaccination

[0076] One aspect of the present application relates to a method for immunizing a subject against a pathogen, comprising the steps of: (1) administering to the subject an effective amount of a prime vaccine, wherein the prime vaccine comprises one or more mRNA constructs encoding one or more immunogens from the pathogen; and (2) administering to the subject an effective amount of a boost vaccine, wherein the booster vaccine comprises a non-mRNA vaccine modality against the pathogen, wherein the boost vaccine is administered after the administration of the prime vaccine.

[0077] Another aspect of the present application relates to a method for immunizing a subject against a pathogen, comprising the steps of: (1) administering to the subject an effective amount of a prime vaccine, wherein the prime vaccine comprises one or more mRNA constructs encoding one or more prime immunogens, wherein the one or more prime immunogens are selected from the group consisting of immunogens from the pathogen and modified same immunogens; and (2) administering to the subject an effective amount of a boost vaccine, wherein the booster vaccine comprises one or more booster immunogens, 14 WAS:164427.1wherein at least one of the booster immunogens has an amino acid sequence identity of 75% or greater to at least one of the prime immunogens, wherein the boost vaccine is in a non- mRNA vaccine modality, and wherein the boost vaccine is administered after the administration of the prime vaccine. In some embodiments, the pathogen is HIV-1 and the method comprises the steps of (1) administering to the subject an effective amount of a prime vaccine, wherein the prime vaccine comprises one or more mRNA constructs encoding one or more HIV-1 envelope immunogens and / or modified HIV-1 envelope immunogens, such as envelope proteins with optimized structural designs; and (2) administering to the subject an effective amount of a boost vaccine (in any non-mRNA vaccine modality), wherein the booster vaccine comprises one or more HIV-1 envelope immunogens, modified HIV-1 envelope immunogens and / or fragments thereof that are encoded in the one or more mRNA constructs in the prime vaccine, or wherein the booster vaccine comprises one or more HIV-1 envelope immunogens, modified HIV-1 envelope immunogens and / or fragments thereof that are not the same (such as from different viral variants) as encoded in the one or more mRNA constructs in the prime vaccine, wherein the boost vaccine is administered after the administration of the prime vaccine. Examples of the HIV-1 envelope proteins include, but are not limited to, gp120, gp140, gp160, and trimeric envelop glycoprotein.

[0078] Another aspect of the present application relates to a method for introducing an immune response against HIV-1 in a subject, comprising the steps of: (1) administering to the subject an effective amount of a prime vaccine, wherein the prime vaccine comprises one or more mRNA constructs encoding one or more prime immunogens, wherein the one or more prime immunogens are selected from the group consisting of HIV-1 env immunogens and modified HIV-1 env immunogens, such as envelope proteins with optimized structural designs; and (2) administering to the subject an effective amount of a boost vaccine, wherein the booster vaccine comprises one or more booster immunogens, wherein at least one of the booster immunogens has an amino acid sequence identity of 75% or greater to at least one of the prime immunogens, wherein the boost vaccine is in a non-mRNA vaccine modality, and wherein the boost vaccine is administered after the administration of the prime vaccine.

[0079] Another aspect of the present application relates to a method for introducing an immune response against HIV-1 in a subject, comprising the steps of: (1) administering to the subject an effective amount of a prime vaccine, wherein the prime vaccine comprises one or more mRNA constructs encoding one or more HIV-1 env immunogens and / or modified HIV-1 env immunogens, such as envelope proteins with optimized structural designs; and (2) 15 WAS:164427.1administering to the subject an effective amount of a boost vaccine (in any non-mRNA vaccine modality), wherein the booster vaccine comprises one or more HIV-1 env immunogens and / or modified HIV-1 env immunogens that are encoded in the one or more mRNA constructs in the prime vaccine, or wherein the booster vaccine comprises one or more HIV-1 envelope immunogen and / or modified HIV-1 envelope immunogen that are not the same (such as from different viral variants) as encoded in the one or more mRNA constructs in the prime vaccine, wherein the boost vaccine is administered after the administration of the prime vaccine. The prime vaccine and / or the boost vaccine may be monovalent or polyvalent. The HIV-1 env immunogens may comprise HIV-1 gp120, HIV-1 gp140, HIV-1 gp160, trimeric envelope glycoprotein and other forms of HIV-1 env immunogens.

[0080] Another aspect of the present application relates to a method for introducing an immune response against HIV-1 in a subject, comprising the steps of: (1) administering to the subject an effective amount of a prime vaccine, wherein the prime vaccine comprises one or more mRNA constructs encoding HIV-1 gp120 and / or modified HIV-1 gp120 and / or, envelope proteins with optimized structural designs; and (2) administering to the subject an effective amount of a boost vaccine (in any non-mRNA vaccine modality), wherein the booster vaccine comprises one or more HIV-1 gp120 and / or modified HIV-1 gp120 that are encoded in the one or more mRNA constructs in the prime vaccine, or wherein the booster vaccine comprises on or more HIV-1 gp120 and / or modified HIV-1 gp120 that are not the same (such as from different viral variants) as encoded in the one or more mRNA constructs in the prime vaccine, wherein the boost vaccine is administered after the administration of the prime vaccine. Subject

[0081] The subject can be any subject capable of generating an immune response against the pathogen. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human subject. Pathogen

[0082] The pathogen can be any microorganism that is capable of inducing an infectious disease in a subject. In some embodiments, the pathogen is an infectious microorganism selected from the group consisting of bacteria, fungi, viruses and parasites. In some embodiments, the pathogen is a virus selected from the group consisting of human immunodeficiency viruses (HIV), influenza A viruses including subtype H1N1, H3N2, H7N9, or H5N1, influenza B viruses, influenza C viruses, rotavirus A, rotavirus B, rotavirus 16 WAS:164427.1C, rotavirus D, rotavirus E, human coronaviruses, SARS coronaviruses, MERS coronaviruses, human adenoviruses, human papillomaviruses (HPV), parvoviruses, molluscum contagiosum viruses, JC viruses (JCV), BK viruses, Merkel cell polyomaviruses, coxsackie A viruses, noroviruses, Rubella viruses, lymphocytic choriomeningitis viruses (LCMV), Dengue viruses, Zika viruses, chikungunya, Eastern equine encephalitis viruses (EEEV), Western equine encephalitis viruses (WEEV), Venezuelan equine encephalitis viruses (VEEV), Ross River viruses, Barmah Forest viruses, yellow fever viruses, measles viruses, mumps viruses, respiratory syncytial viruses, rinderpest viruses, California encephalitis viruses, hantaviruses, rabies viruses, ebola viruses, marburg viruses, herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), varicella zoster viruses (VZV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV), herpes lymphotropic viruses, roseoloviruses, Kaposi's sarcoma-associated herpesviruses, hepatitis A viruses, hepatitis B viruses, hepatitis C viruses, hepatitis D viruses, hepatitis E viruses, human T-lymphotropic viruses, Friend spleen focus-forming viruses (SFFV), Xenotropic MuLVRelated Viruses (XMRV). In some embodiments, the pathogen is HIV. Prime vaccine

[0083] The prime vaccine comprises one or more mRNA constructs encoding one or more immunogens from the pathogen. In some embodiments, the prime vaccine comprises a conventional mRNA-based vaccine. As used herein, the term “conventional mRNA-based vaccine” refers to a mRNA vaccine construct that is not capable of self-replication when introduced into a target cell. In some embodiments, the conventional mRNA-based vaccine comprises a mRNA sequence that contains a coding sequence encoding an immunogen of interest and untranslated regions (UTRs) that flank the coding sequence at the 5’ and 3’ ends of the coding sequence. In some embodiments, the conventional mRNA-based vaccine further comprises a 5’- Cap and a 3’-poly A tail. FIG.2, shows an exemplary structure of a conventional mRNA-vaccine. The prime vaccine may be monovalent or polyvalent.

[0084] In some embodiments, the prime vaccine comprises a self-amplifying mRNA- based vaccine. As used herein, the term “self-amplifying mRNA-based vaccine” refers to a mRNA vaccine construct that is capable of self-replication when introduced into a target cell.

[0085] In some embodiments, the self-amplifying mRNA-based vaccine additionally contains viral 5′- and 3′-CSEs and the non-structure protein 1-4 (nsP1–nsP4) gene comparing to conventional mRNA. The immunogen is placed under control of the subgenomic promoter (SGP) replacing the viral structural proteins. Positive-sense alphavirus 17 WAS:164427.1genomes that have been commonly used for saRNA vaccine design include the Venezuelan equine encephalitis virus (VEE), and Semliki forest virus (SFV). Following in situ translation, the nsP1-4 proteins form an RdRP complex which recognizes flanking CSE sequences and amplifies vaccine-encoding transcripts. This results in an accumulation of the immunogen within the cell. In some embodiments, the RNA sequence further comprises a 5’- Cap and a 3’-poly A tail. FIG.3, shows an exemplary structure of a self-amplifying mRNA-construct. As a result of their self-replicative activity, self-amplifying RNA vaccines can be delivered at lower concentrations than conventional mRNA vaccines to achieve comparable immunogen expression.

[0086] In some embodiments, the pathogen is HIV-1 and the prime vaccine comprises a mRNA construct encoding a HIV-1 envelope immunogen. In some embodiments, the prime vaccine comprises mRNA construct(s) encoding one or more HIV-1 gp120, HIV-1 gp140, HIV-1 gp160, trimeric envelope glycoprotein, or modified HIV-1 gp120, gp140, HIV-1 gp160, trimeric envelope glycoprotein, wherein the boost vaccine (in any non-mRNA vaccine modality) comprises one or more HIV-1 gp120 , gp140, HIV-1 gp160, trimeric envelope glycoprotein, or modified HIV-1 gp120, gp140, HIV-1 gp160, trimeric envelope glycoprotein encoded in the prime vaccine, or wherein the boost vaccine comprises one or more HIV-1 gp120 , gp140, HIV-1 gp160, trimeric envelope glycoprotein or modified HIV-1 gp120, gp140, HIV-1 gp160, trimeric envelope glycoprotein that are not the same (such as from different viral variants) as encoded in the prime vaccine.

[0087] In some embodiments, the pathogen is HIV-1 and the prime vaccine comprises a mRNA construct encoding a HIV-1 envelope immunogen. In some embodiments, the prime vaccine comprises mRNA construct / constructs encoding one or more HIV-1 gp120 and / or modified HIV-1 gp120, and wherein the boost vaccine (in any non-mRNA vaccine modality) comprises the one or more HIV-1 gp120 and / or modified HIV-1 gp120 encoded in the prime vaccine, or wherein the boost vaccine comprises the one or more HIV-1 gp120, or modified HIV-1 gp120 that are not the same (such as from different viral variants) as encoded in the prime vaccine.

[0088] In some embodiments, the prime vaccine comprises a mRNA construct encoding an HIV-1 envelope protein or a modified HIV-1 envelope protein. In some embodiments, the prime vaccine comprises a mRNA construct encoding a HIV-1 gp120 or a fragment thereof. In some embodiments, the prime vaccine comprises a mRNA construct encoding a HIV-1 gp120 or a modified HIV-1 gp120, wherein the HIV-1 gp120 is selected 18 WAS:164427.1from the group consisting of HIV-1 gp120-A, HIV-1 gp120-B, HIV-1 gp120-C, HIV-1 gp120-D and HIV-1 gp120-A / E. In some embodiments, the prime vaccine comprises a mRNA construct encoding a HIV-1 gp120 or a fragment thereof, wherein the HIV gp120 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:1, 4, 7, 10, 13 and 16.

[0089] In some embodiments, the mRNA sequence encoding HIV-1 gp120 or a modified HIV-1 gp120 is codon optimized for expression in eukaryotic host cell. Enhanced expression is useful for producing large quantities of polypeptide immunogens of the present application, and for inducing immune responses to the polypeptide immunogen in a host, e.g., a human. To enhance expression of immunogen(s), other factors concerning the efficiency with which sequences can be expressed should be considered, including the presence of features that may negatively affect expression in a cell. For example, sequences that result in RNAs predicted to have a high degree of secondary structure should be avoided. AT- and GC-rich sequences that interfere with DNA synthesis should also be avoided. Other motifs that can be detrimental to expression include internal TATA boxes, chi-sites, ribosomal entry sites, prokaryotic inhibitory motifs, cryptic splice donor and acceptor sites, and branch points. These features can be identified manually or by computer software and they can be excluded from the optimized sequences. Codon preferences for abundantly expressed proteins have been determined in a number of species, and can provide guidelines for codon substitution. Synthesis of codon-optimized sequences can be achieved by substitution of optimal codons in cloned sequences using conventional recombinant DNA methodologies known to those skilled in the art.

[0090] Generally, mRNA vaccines are produced by in vitro synthesis through an enzymatic process. Plasmid DNA (pDNA), used as an in vitro transcription template can be first constructed which usually contains the following basic elements: an upstream promoter exclusively recognized by T7, SP6 or T3 RNA polymerase; 5' UTR , codon-optimized immunogen sequence, 3' UTR, a downstream poly A-tail; and a unique cleavage site downstream of the poly A-tail. The pDNA is first linearized with the selected unique restriction site enzyme. Following linearization, in vitro transcription and capping is performed in a mixed solution of recombinant RNA polymerase (T7, T3 or SP6) and nucleoside triphosphates, plus a cap analog such as CleanCap®Reagent or ARCA (Anti- Reverse Cap Analog). Finally, when the length of the poly A tail of the template pDNA is insufficient (up to 150 bases), it can be extended by use of poly A enzyme. Further purification will remove the linearized pDNA template and other contaminants. 19 WAS:164427.1

[0091] In some embodiments, the optimized DNA sequence encoding HIV-1 gp120 or a fragment thereof comprises an nucleotide sequence selected from the group consisting of SEQ ID NOS: 2, 5, 8, 11, 14 and 17.

[0092] In some embodiments, the optimized mRNA sequence encoding HIV-1 gp120 or a fragment thereof comprises an nucleotide sequence selected from the group consisting of SEQ ID NOS: 3, 6, 9, 12, 15 and 18.

[0093] In some embodiments, the prime vaccine further comprises an adjuvant. Examples of adjuvants include, but are not limited to GLA-SE, MPL, ALUM, QS-21, liposome, 3M-052.

[0094] In some embodiments, the prime vaccine further comprises a pharmaceutically acceptable carrier.

[0095] The polynucleotides in the prime vaccines described herein can be administered to an individual as naked nucleic acid molecules (e.g., naked mRNA) in physiologically compatible solution such as water, saline, Tris-EDTA (TE) buffer, or in phosphate buffered saline (PBS). They can also be administered in the presence of substances (e.g., lipid nanoparticles (LNPs) and other delivery carriers, facilitating agents and adjuvants described above) that have the capability of promoting nucleic acid uptake, increase the stability and / or recruiting immune system cells to the site of inoculation.

[0096] In some embodiments, the prime vaccine of the present application is administered intradermally (i.d.), intramuscularly (i.m.) or subcutaneously (s.c.). The prime vaccine can be administered by LNP formulation, needle injection, needle-free / needleless jet injection or gene gun.

[0097] In some embodiments, the prime vaccine of the present application is administered by intranasal, intratracheal, ocular, sublingual, transcutaneous, oral, vaginal, or rectal routes.

[0098] Suitable doses of the prime vaccine of the present application for humans can range from 1 µg per injection per person to 500 ug per injection per person of total RNA. "total RNA" refers to a pool of conventional mRNA constructs and / or self-amplifying RNA constructs encoding distinct immunogens. Boost vaccine

[0099] The boost vaccine of the present application in a non-mRNA based vaccine. In some embodiments, the boost vaccine comprises a vaccine modality selected from any of the following: live attenuated vaccine, inactivated vaccine, recombinant protein vaccine, viral vector vaccine, viral like particle vaccine, polysaccharide conjugate vaccine, toxoid vaccine 20 WAS:164427.1and DNA vaccine. In some embodiments, the boost vaccine comprises one or more immunogens of the pathogen or fragments thereof. In some embodiments, the boost vaccine further comprises an adjuvant.

[0100] In some embodiments, the pathogen is HIV-1, the prime vaccine comprises an mRNA construct encoding an HIV-1 env immunogen or a modified env immunogen and the boost vaccine (in any non-mRNA vaccine modality) comprises the HIV- 1 env immunogen or a modified env immunogen encoded in the prime vaccine.

[0101] In some embodiments, the booster vaccine comprises an immunogen that is not the same (such as from different viral variants) as encoded in the mRNA construct in the prime vaccine. In some embodiments, the HIV-1 env immunogen or a modified env immunogen in the boost vaccine is adjuvanted or is displayed on nanoparticles.

[0102] In some embodiments, the booster vaccine comprises, or encodes, an immunogen having an amino acid sequence identity of 98% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater to an immunogen that is present, or is encoded, in the prime vaccine. In some embodiments, the prime vaccine comprises an mRNA construct encoding an HIV-1 gp120 or a modified HIV-1 gp120. In some embodiments, the HIV-1 gp120 is selected from the group consisting of HIV-1 gp120-A, HIV-1 gp120-B, HIV-1 gp120-C, HIV-1 gp120-D and HIV-1 gp120- A / E. In some embodiments, the boost vaccine comprises recombinant HIV-1 gp120 protein, wherein the HIV-1 gp120 protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:1, 4, 7, 10, 13 and 16.

[0103] In some embodiments, the boost vaccine further comprises an adjuvant. Examples of adjuvants include, but are not limited to GLA-SE, MPL, ALUM, QS-21, liposome, 3M-052.

[0104] In some embodiments, the boost vaccine further comprises a pharmaceutically acceptable carrier.

[0105] In some embodiments, the boost vaccine of the present application is administered intradermally (ID), intramuscularly (IM) or subcutaneously (SC) by needle injection.

[0106] Suitable dose of the boost vaccine of the present application for humans can range from 2x1010to 8x1010total viral particle (viral vector vaccine) per dose or 10-500 ug total protein (recombinant protein) per dose. “total " refers to a pool of viral particle encoding distinct immunogens or distinct recombinant proteins. Multiple doses of the boost vaccine may be administered. 21 WAS:164427.1Prime-boost vaccination schedule

[0107] The prime vaccine and boost vaccine of the present application can be administered at the same route or different route, and at the same or different location. Exemplary heterologous prime-boost vaccine schedule and routes are shown in Tables 1 and 2 below.

[0108] Once the prime vaccine is delivered, the mRNA molecules are taken up into host cells, which then express the encoded immunogen thereof. Once expressed, the immunogen thereof is processed and presented in the context of self-major histocompatibility (MHC) class I and class II molecules. In the case that the RNA-based prime vaccine encodes a HIV protein or fragment thereof, the HIV protein or fragment thereof is expressed and processed after the RNA construct in the RNA-based vaccine entering the host cells and is presented in the context of major histocompatibility (MHC) class I and class II molecules. The host then develops an immune response against the HIV protein. The administration of the boost vaccine improves the immune response. The timing between the priming step and the boosting step varies and is adjusted for each vaccine.

[0109] In some embodiments, the prime vaccine is administered in one or more doses subcutaneously or intramuscularly with an interval of 2-8 weeks between the doses of the prime vaccine, followed by one or more doses of the boost vaccine through the subcutaneous or intramuscular route 2-24 weeks after the completion of the prime vaccination with an interval of 3-12 weeks between the doses of the boost vaccine.

[0110] In some embodiments, the prime vaccine is administered in one dose subcutaneously or intramuscularly, followed by a single dose of the boost vaccine through the subcutaneous or intramuscular route 2-24 weeks after the administration of the prime vaccine. 22 WAS:164427.1

[0111] In some embodiments, the prime vaccine is administered in one dose subcutaneously or intramuscularly, followed by a single dose of the boost vaccine through the subcutaneous or intramuscular route 2-6 weeks after the administration of the prime vaccine.

[0112] In some embodiments, the prime vaccine is administered in one dose subcutaneously or intramuscularly, followed by a single dose of the boost vaccine through the subcutaneous or intramuscular route 4 weeks after the administration of the prime vaccine.

[0113] In general, the vaccine compositions of the present application are administered to a subject in need of such treatment for a time and under conditions sufficient to induce a protective immune response to the pathogen. A protective immune response may be manifested by at least one of the following: preventing the pathogen infection and complications resulting from the infection of the pathogen; aiding, improving, enhancing, or stimulating recovery of the host from infection of the pathogen; and generating immunological memory that will prevent or limit a subsequent incidence of infection by the pathogen.

[0114] The vaccine compositions and methods of the present application may be applied to any pathogen where an immune response or vaccination is desired or needed. For prophylactic applications, the vaccine compositions of the present application are administered in advance of any symptom or infection caused by the pathogen. Prophylactic administration of the immunogenic compositions serves to prevent or ameliorate any subsequent infection.

[0115] Determination of effective dosages is additionally guided with animal model studies followed up by human clinical trials and further guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject. Additional factors may include the age of the subject, and other personal attributes of the specific subject (e.g., the general state of the subject's health and the robustness of the subject's immune system).

[0116] Another aspect of the present application relates to a method for immunizing a human subject against HIV-1 infection which will lead to AIDS. The method comprises the steps of (1) administering to the subject an effective amount of the prime vaccine of the preset application; and (2) administering to the subject an effective amount of the boost vaccine of the present application, wherein the boost vaccine is administered within 2-24 weeks of the administration of the prime vaccine.

[0117] Another aspect of the present application relates to a method for eliciting protective immunity against HIV-1 in a human subject. The method comprises the steps of 23 WAS:164427.1(1) administering intradermally (i.d.), intramuscularly (i.m.) or subcutaneously (s.c.) to the human subject one or more dose of a prime vaccine of the present application, and (2) administering to the subject an effective amount of a boost vaccine of the present application, wherein the administration of the prime and boost vaccines results in an immune response against HIV-1. III. Vaccine compositions

[0118] Another aspect of the present application relates to an mRNA-primed HIV-1 vaccine, comprising: (1) one or more mRNA constructs encoding one or more HIV-1 env immunogens or modified HIV-1 env immunogens; and (2) a pharmaceutically acceptable carrier. In some embodiments, the mRNA-primed HIV-1 vaccine is a monovalent vaccine. In some embodiments, the mRNA-primed HIV-1 vaccine is a polyvalent vaccine

[0119] In some embodiments, the vaccine comprises (1) a mRNA construct encoding HIV-1 gp120 or a modified HIV-1 gp120, and (2) a pharmaceutically acceptable carrier.

[0120] In some embodiments, the vaccine composition of the present application further comprises, or is administered in combination with, one or more adjuvants.

[0121] In some embodiments, the mRNA construct is a conventional mRNA construct comprising a coding region flanked by a 5’-untranslated region (UTR) and a 3’UTR. In some embodiments, the coding region comprises a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOS:1, 4, 7, 10, 13 and 16. In some embodiments, the coding region comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:3, 6, 9, 12, 15 and 18.

[0122] In some embodiments, the mRNA construct is a self-amplifying mRNA construct comprising sequences encoding 5′ and 3′ conserved sequence element (CSE) sequences, the non-structure proteins 1-4 (nsP1-4) , a subgenomic promoter from either Venezuelan equine encephalitis virus (VEEV) or Semlike Forest virus (SFV) and a coding sequence comprises a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOS:1, 4, 7, 10, 13 and 16. In some embodiments, the coding region comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:3, 6, 9, 12, 15 and 18. Codon optimization

[0123] In some embodiments, the mRNA construct of the prime vaccine composition of the present application is codon optimized for expression in humans. In some embodiments, the codon-optimized polynucleotide is prepared by replacing the codons of the 24 WAS:164427.1polynucleotide encoding the HIV protein or fragment thereof with e.g., "humanized" codons (e.g., codons that appear frequently in highly expressed human genes. Codon optimization methods are known in the art and may be used as provided herein. In some embodiments, the open reading frame (ORF) sequence in the polynucleotide is optimized using optimization algorithms as described herein and known in the art.

[0124] In some embodiments, the codon optimized mRNA sequence shares less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55% or less than 50% sequence identity to a naturally- occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding the HIV gp120 protein or a fragment thereof.

[0125] In some embodiments, a codon optimized mRNA sequence shares between 50% and 95%, between 50% and 90%, between 50% and 85%, between 50% and 80%, between 50% and 75%, between 50% and 70%, between 50% and 65%, between 50% and 60%, between 50% and 55%, between 55% and 95%, between 55% and 90%, between 55% and 85%, between 55% and 80%, between 55% and 75%, between 55% and 70%, between 55% and 65%, between 55% and 60%, between 60% and 95%, between 60% and 90%, between 60% and 85%, between 60% and 80%, between 60% and 75%, between 60% and 70%, between 60% and 65%, between 65% and 95%, between 65% and 90%, between 65% and 85%, between 65% and 80%, between 65% and 75%, between 65% and 70%, between 70% and 95%, between 70% and 90%, between 70% and 85%, between 70% and 80%, between 70% and 75%, between 75% and 95%, between 75% and 90%, between 75% and 85%, between 75% and 80%, between 80% and 95%, between 80% and 90%, between 80% and 85%, between 85% and 95%, between 85% and 90%, or between 90% and 95% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild- type mRNA sequence encoding the HIV gp120 protein or a fragment thereof. In some embodiments, the coding codon optimized mRNA sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:3, 6, 9, 12, 15 and 18. Pharmaceutically acceptable carrier

[0126] Suitable pharmaceutically acceptable carriers include sterile water, saline, dextrose, glucose, or other buffered solutions (e.g., phosphate buffered saline). Included in the pharmaceutically acceptable carrier can be physiologically acceptable preservatives, stabilizers, diluents, emulsifying agents, pH buffering agents, viscosity enhancing agents, colors, etc. For example, solutions or suspensions used for administration can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, 25 WAS:164427.1polyethylene glycols, glycerine; propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose, pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Adjuvants

[0127] In some embodiments, the vaccine composition of the present application further comprises, or is administered in combination with, one or more adjuvants. Adjuvants may be used in combination with the vaccine composition of the present application to e.g., improve stability and uptake, improve immune induction, enhance an immune response, but is not antigenic itself when administered in the absence of an antigen. Additionally, an adjuvant may decrease the dosage required to induce an effective immune response.

[0128] Exemplary adjuvants include, but are not limited to, mineral salts, complete and incomplete Freund's adjuvant, squalene based adjuvants, saponins based adjuvants, toll- like receptor (TLR) ligands, microbial derivatives, and cytokines. In some embodiments, vaccine composition of the present application comprises an adjuvant selected from the group consisting of GLA-SE, MPL, ALUM, QS-21, liposome and 3M-052.

[0129] It is especially advantageous to formulate the vaccine composition of the present application in dosage unit form for ease of administration and uniformity of dosage.

[0130] Another aspect of the present application relates to a HIV-1 vaccine formulation. In some embodiments, the HIV-1 vaccine formulation comprises: (1) a prime component comprising a mRNA construct encoding an HIV-1 env immunogen or a modified HIV-1 env immunogen, such as in the form of gp120, gp140, gp160, trimeric envelope glycoprotein or other modified env immunogens and (2) a boost component (in any non- mRNA vaccine modality) comprising the immunogen encoded in the mRNA construct or a boost component comprising the immunogen that are not the same (such as from different viral variants) as encoded in the mRNA construct, wherein the prime component and boost components are formulated for separate administration.

[0131] In some embodiments, the formulation comprises one or more other elements including: instructions for use; other reagents, e.g., a diluent, an adjuvant, devices or other materials for preparing the prime component and boost component for administration.

[0132] The present disclosure is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published 26 WAS:164427.1patent applications cited throughout this application, as well as the Figures and Tables, are incorporated herein by reference. 27 WAS:164427.1EXAMPLES Example 1: Polyvalent mRNA prime-protein boost HIV-1 vaccine formulations (mRNA and protein employing same immunogens)

[0133] A heterologous prime boost regimen is administered using an mRNA vaccine as the prime vaccination to the host, and then the host is boosted by another type of vaccine (such as in the form of recombinant protein but may be in any other types of vaccines) of the same / similar immunogen(s) as included in the prime mRNA vaccine. This method is applicable against a diverse range of human and animal pathogens.

[0134] The formulations below show exemplary design of a polyvalent HIV-1 protein vaccine (boost vaccine) that comprises the same polyvalent HIV-1 gp120 as encoded by an mRNA vaccine, which is used as the prime vaccine.

[0135] The regimen is adjuvanted with GLA-SE or other adjuvants such as MPL, ALUM, QS-21, liposome, 3M-052. Formulation 1 HIV-1 gp120-A (92UG037.1) HIV-1 gp120 –B (JRFL) HIV-1 gp120-C (93MW965.26) HIV-1 gp 120-D (92UG021.16) HIV-1 gp120-A / E (Consensus) Formulation 2 HIV-1 gp120-A (92UG037.1) HIV-1 gp120 –B (JRFL) HIV-1 gp120-C (93MW965.26) HIV-1 gp120-A / E (Consensus) Formulation 3 HIV-1 gp120-A (92UG037.1) HIV-1 gp120-B (92US715.6 ) HIV-1 gp120-C (93MW965.26) HIV-1 gp 120-D (92UG021.16) HIV-1 gp120-A / E (Consensus) Formulation 4 HIV-1 gp120-A (92UG037.1) HIV-1 Gp120-B (92US715.6 ) HIV-1 gp120-C (93MW965.26) 28 WAS:164427.1HIV-1 gp120-A / E (Consensus) Example 2: Importance of well-matched protein sequence between prime vaccine and boost vaccine in a prime-boost immunization regimen

[0136] A DNA prime + protein boost regimen used in HVTN 124 trial achieved robust immunogenicity including the best anti-V1V2 antibody responses by a large margin over other HVTN trials that also use prime boost regimen (FIG.4). Protein sequences analysis finds a low degree of sequence matching percentage (~70%) between prime and boost vaccines for other HVTN trials vs a 100% match in HVTN124 trial (Table 1). The immunogenicity results observed in HVTN 124 trial lend further credence to the value of matching prime–boost antigens approach. Table 1. Amino Acid Homologous analysis among multi-HVTN clinical trials including HVTN124 Vaccines Amino Acid HomologousTrialsPrime Boostbetween Prime and Boost Insert Platform Protein adjuvantsVaccines RV144, HVTN097 E: 92TH023gp120, B:gp41 Canarypox B: MNgp120 71%E: A244gp120 Alum92%HVTN100, HVTN702, VTN120 C: 96Z C: TV1.21gp120MF59, Alum,78%HVTN107, H M651gp120, B:gp41 CanarypoxC: 1086gp120AS0177%HVTN096, HVTN105 C: 96ZM651gp140 Vaccinia, DNA B: MNgp120 71%E: A244gp120 Alum71%HVTN108, HVTN111 C: 96ZM651gp140 DNA C: TV1.21gp120 78%C: 1086gp120 MF59, AS0177%HVTN705 Mos1 gp140Ad26 C: C97ZAgp14 73%Mos2Sgp160 (trunc.) 0 Alum78%A: 92UG037gp120 A: 92UG037gp120 100%HVTN124 B: JRFLgp120DNA B: JRFLgp120GLA-SE 100%C: 93MW965gp120 C: 93MW965gp120 100%AE: Consensusgp120 AE: Consensusgp120 100%

[0137] While various embodiments have been described above, it should be understood that such disclosures have been presented by way of example only and are not limiting. Thus, the breadth and scope of the subject compositions and methods should not be limited by any of the above-described exemplary embodiments. The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations can be included within the scope of the present application as defined by the embodiments described herein.

[0138] The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and it is not intended to detail all those 29 WAS:164427.1obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention, which is defined by the following claims. The claims are intended to cover the components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary. 30 WAS:164427.1List of Sequences SEQ ID NO:1 HIV-1 gp120-A (Strain 92UG037.1) Amino acid sequence (ACCESSION: 6ULC_A) LWVTVYYGVPVWKDAETTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIYMENV TEEFNMWKNNMVEQMHTDIISLWDQSLKPCVQLTPLCVTLDCSYNITNNITNSITNSSVN MREEIKNCSFNMTTELRDKNRKVYSLFYKLDVVQINNGNNSSNLYRLINCNTSALTQAC PKVTFEPIPIRYCAPAGYAILKCNDKEFNGTGLCKNVSTVQCTHGIRPVVSTQLLLNGSL AEGKVMIRSENITNNVKNIIVQLNETVTINCTRPNNNTRKSVRIGPGQTFYATGDIIGDIR QAHCNVSGSQWNRALHQVVGQLREYWNTTIIFKNSSGGDLEITTHSFNCGGEFFYCNTS GLFNSNWTHNDTASMKPNDTITLPCRIKQIINMWQRVGQAIYAPPIQGVIRCESNITGLIL TRDGGGNINESQIFRPGGGDMRDNWRSELYKYKV VRIEPLGVAPTKAKRRVVE SEQ ID NO:2 HIV-1 gp120-A (Strain 92UG037.1) Codon optimized DNA sequence CTGTGGGTGACCGTGTACTACGGCGTGCCCGTGTGGAAGGACGCCGAGACCACCCT GTTCTGCGCCAGCGACGCCAAGGCCTACGACACCGAGGTGCACAACGTGTGGGCCA CCCACGCCTGCGTGCCCACCGACCCCAACCCCCAGGAGATCTACATGGAGAACGTG ACCGAGGAGTTCAACATGTGGAAGAACAACATGGTGGAGCAGATGCACACCGACAT CATCAGCCTGTGGGACCAGAGCCTGAAGCCCTGCGTGCAGCTGACCCCCCTGTGCGT GACCCTGGACTGCAGCTACAACATCACCAACAACATCACCAACAGCATCACCAACA GCAGCGTGAACATGCGCGAGGAGATCAAGAACTGCAGCTTCAACATGACCACCGAG CTGCGCGACAAGAACCGCAAGGTGTACAGCCTGTTCTACAAGCTGGACGTGGTGCA GATCAACAACGGCAACAACAGCAGCAACCTGTACCGCCTGATCAACTGCAACACCA GCGCCCTGACCCAGGCCTGCCCCAAGGTGACCTTCGAGCCCATCCCCATCCGCTACT GCGCCCCCGCCGGCTACGCCATCCTGAAGTGCAACGACAAGGAGTTCAACGGCACC GGCCTGTGCAAGAACGTGAGCACCGTGCAGTGCACCCACGGCATCCGCCCCGTGGT GAGCACCCAGCTGCTGCTGAACGGCAGCCTGGCCGAGGGCAAGGTGATGATCCGCA GCGAGAACATCACCAACAACGTGAAGAACATCATCGTGCAGCTGAACGAGACCGTG ACCATCAACTGCACCCGCCCCAACAACAACACCCGCAAGAGCGTGCGCATCGGCCC CGGCCAGACCTTCTACGCCACCGGCGACATCATCGGCGACATCCGCCAGGCCCACT GCAACGTGAGCGGCAGCCAGTGGAACCGCGCCCTGCACCAGGTGGTGGGCCAGCTG CGCGAGTACTGGAACACCACCATCATCTTCAAGAACAGCAGCGGCGGCGACCTGGA GATCACCACCCACAGCTTCAACTGCGGCGGCGAGTTCTTCTACTGCAACACCAGCGG CCTGTTCAACAGCAACTGGACCCACAACGACACCGCCAGCATGAAGCCCAACGACA CCATCACCCTGCCCTGCCGCATCAAGCAGATCATCAACATGTGGCAGCGCGTGGGCC AGGCCATCTACGCCCCTCCCATCCAGGGCGTGATCCGCTGCGAGAGCAACATCACC GGCCTGATCCTGACCCGCGACGGCGGCGGCAACATCAACGAGAGCCAGATCTTCCG CCCCGGCGGCGGCGACATGCGCGACAACTGGCGCAGCGAGCTGTACAAGTACAAGG TGGTGCGCATCGAGCCCCTGGGCGTGGCCCCCACCAAGGCCAAGCGCCGCGTGGTG GAG WAS:164427.1SEQ ID NO:3 HIV-1 gp120-A (Strain 92UG037.1) Codon optimized mRNA sequence CUGUGGGUGACCGUGUACUACGGCGUGCCCGUGUGGAAGGACGCCGAGACCACCC UGUUCUGCGCCAGCGACGCCAAGGCCUACGACACCGAGGUGCACAACGUGUGGGC CACCCACGCCUGCGUGCCCACCGACCCCAACCCCCAGGAGAUCUACAUGGAGAAC GUGACCGAGGAGUUCAACAUGUGGAAGAACAACAUGGUGGAGCAGAUGCACACC GACAUCAUCAGCCUGUGGGACCAGAGCCUGAAGCCCUGCGUGCAGCUGACCCCCC UGUGCGUGACCCUGGACUGCAGCUACAACAUCACCAACAACAUCACCAACAGCAU CACCAACAGCAGCGUGAACAUGCGCGAGGAGAUCAAGAACUGCAGCUUCAACAUG ACCACCGAGCUGCGCGACAAGAACCGCAAGGUGUACAGCCUGUUCUACAAGCUGG ACGUGGUGCAGAUCAACAACGGCAACAACAGCAGCAACCUGUACCGCCUGAUCAA CUGCAACACCAGCGCCCUGACCCAGGCCUGCCCCAAGGUGACCUUCGAGCCCAUC CCCAUCCGCUACUGCGCCCCCGCCGGCUACGCCAUCCUGAAGUGCAACGACAAGG AGUUCAACGGCACCGGCCUGUGCAAGAACGUGAGCACCGUGCAGUGCACCCACGG CAUCCGCCCCGUGGUGAGCACCCAGCUGCUGCUGAACGGCAGCCUGGCCGAGGGC AAGGUGAUGAUCCGCAGCGAGAACAUCACCAACAACGUGAAGAACAUCAUCGUGC AGCUGAACGAGACCGUGACCAUCAACUGCACCCGCCCCAACAACAACACCCGCAA GAGCGUGCGCAUCGGCCCCGGCCAGACCUUCUACGCCACCGGCGACAUCAUCGGC GACAUCCGCCAGGCCCACUGCAACGUGAGCGGCAGCCAGUGGAACCGCGCCCUGC ACCAGGUGGUGGGCCAGCUGCGCGAGUACUGGAACACCACCAUCAUCUUCAAGAA CAGCAGCGGCGGCGACCUGGAGAUCACCACCCACAGCUUCAACUGCGGCGGCGAG UUCUUCUACUGCAACACCAGCGGCCUGUUCAACAGCAACUGGACCCACAACGACA CCGCCAGCAUGAAGCCCAACGACACCAUCACCCUGCCCUGCCGCAUCAAGCAGAU CAUCAACAUGUGGCAGCGCGUGGGCCAGGCCAUCUACGCCCCUCCCAUCCAGGGC GUGAUCCGCUGCGAGAGCAACAUCACCGGCCUGAUCCUGACCCGCGACGGCGGCG GCAACAUCAACGAGAGCCAGAUCUUCCGCCCCGGCGGCGGCGACAUGCGCGACAA CUGGCGCAGCGAGCUGUACAAGUACAAGGUGGUGCGCAUCGAGCCCCUGGGCGUG GCCCCCACCAAGGCCAAGCGCCGCGUGGUGGAG SEQ ID NO:4 HIV-1 gp120-B (Strain JRFL) Amino acid sequence (ACCESSION: AAB05604) VEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVL ENVTEHFNMWKNNMVEQMQEDIISLWDQSLKPCVKLTPLCVTLNCKDVNATNTTNDS EGTMERGEIKNCSFNITTSIRDEVQKEYALFYKLDVVPIDNNNTSYRLISCDTSVITQACP KISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAE EEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAH CNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLF NSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLLLT RDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV SEQ ID NO:5 HIV-1 gp120-B (Strain JRFL) Codon optimized DNA sequence 32 WAS:164427.1GTGGAGAAGCTGTGGGTGACTGTATACTATGGGGTGCCTGTGTGGAAGGAGGCCAC CACCACCCTGTTCTGTGCCTCTGATGCCAAGGCCTATGACACTGAGGTCCACAATGT CTGGGCCACCCATGCCTGTGTGCCCACTGACCCCAACCCTCAGGAGGTGGTGCTGGA GAATGTGACTGAGCACTTCAACATGTGGAAGAACAACATGGTGGAGCAGATGCAGG AGGACATCATCAGCCTGTGGGACCAGAGCCTGAAGCCCTGTGTGAAGCTGACCCCC CTGTGTGTGACCCTGAACTGCAAGGATGTGAATGCCACCAACACCACCAATGACTCT GAGGGCACTATGGAGAGGGGTGAGATCAAGAACTGCAGCTTCAACATCACCACCAG CATCAGGGATGAGGTGCAGAAGGAGTATGCCCTGTTCTACAAGCTGGATGTGGTG CCCATTGACAACAACAACACCAGCTACAGGCTGATCAGCTGTGACACCTCTGTGATC ACCCAGGCCTGCCCCAAGATCAGCTTTGAGCCCATCCCCATCCACTACTGTGCCCCT GCTGGCTTTGCCATCCTGAAGTGCAATGACAAGACCTTCAATGGCAAAGGCCCTTGC AAGAATGTGAGCACTGTGCAGTGCACTCATGGCATCAGGCCTGTGGTGAGCACCCA GCTGCTGCTGAATGGCAGCCTGGCTGAGGAGGAGGTGGTGATCAGGTCTGACAACT TCACCAACAATGCCAAGACCATCATTGTGCAGCTGAAGGAGTCTGTGGAGATCAAC TGCACCAGGCCCAACAACAACACCAGGAAGAGCATTCACATTGGCCCTGGCAGGGC CTTCTACACCACTGGGGAGATCATTGGGGACATCAGGCAGGCCCACTGCAACATC AGCAGGGCCAAGTGGAATGACACCCTGAAGCAGATTGTGATCAAGCTGAGGGAGCA GTTTGAGAACAAGACCATTGTGTTCAATCACAGCTCTGGTGGTGATCCTGAGATTGT GATGCACAGCTTCAACTGTGGTGGTGAGTTCTTCTACTGCAACAGCACCCAGCTGTT CAACAGCACCTGGAACAACAACACTGAGGGCAGCAACAACACTGAGGGCAACACC ATCACCCTGCCTTGCAGGATCAAGCAGATCATCAACATGTGGCAGGAGGTGGGCAA GGCCATGTATGCTCCTCCCATCAGGGGCCAGATCAGGTGCAGCAGCAACATCACTG GCCTGCTGCTGACCAGGGATGGTGGCATCAATGAGAATGGCACTGAGATTTTCAGG CCTGGTGGTGGGGACATGAGGGACAACTGGAGGTCTGAGCTGTACAAGTACAAGGT GGTGAAGATTGAGCCCCTTGGTGTGGCTCCCACCAAGGCTAAGCGCAGGGTGGTGC AGAGGGAGAAGCGCGCTGTG SEQ ID NO:6 HIV-1 gp120-B (Strain JRFL) Codon optimized mRNA sequence GUGGAGAAGCUGUGGGUGACUGUAUACUAUGGGGUGCCUGUGUGGAAGGAGGCC ACCACCACCCUGUUCUGUGCCUCUGAUGCCAAGGCCUAUGACACUGAGGUCCACA AUGUCUGGGCCACCCAUGCCUGUGUGCCCACUGACCCCAACCCUCAGGAGGUGGU GCUGGAGAAUGUGACUGAGCACUUCAACAUGUGGAAGAACAACAUGGUGGAGCA GAUGCAGGAGGACAUCAUCAGCCUGUGGGACCAGAGCCUGAAGCCCUGUGUG AAGCUGACCCCCCUGUGUGUGACCCUGAACUGCAAGGAUGUGAAUGCCACCAACA CCACCAAUGACUCUGAGGGCACUAUGGAGAGGGGUGAGAUCAAGAACUGCAGCU UCAACAUCACCACCAGCAUCAGGGAUGAGGUGCAGAAGGAGUAUGCCCUGUUCUA CAAGCUGGAUGUGGUGCCCAUUGACAACAACAACACCAGCUACAGGCUGAUCAGC UGUGACACCUCUGUGAUCACCCAGGCCUGCCCCAAGAUCAGCUUUGAGCCCAUCC CCAUCCACUACUGUGCCCCUGCUGGCUUUGCCAUCCUGAAGUGCAAUGACAAGAC CUUCAAUGGCAAAGGCCCUUGCAAGAAUGUGAGCACUGUGCAGUGCACUCAUGGC AUCAGGCCUGUGGUGAGCACCCAGCUGCUGCUGAAUGGCAGCCUGGCUGAGGAGG AGGUGGUGAUCAGGUCUGACAACUUCACCAACAAUGCCAAGACCAUCAUUGUGCA GCUGAAGGAGUCUGUGGAGAUCAACUGCACCAGGCCCAACAACAACACCAGGAAG AGCAUUCACAUUGGCCCUGGCAGGGCCUUCUACACCACUGGGGAGAUCAUUGGGG 33 WAS:164427.1ACAUCAGGCAGGCCCACUGCAACAUCAGCAGGGCCAAGUGGAAUGACACCCUGAA GCAGAUUGUGAUCAAGCUGAGGGAGCAGUUUGAGAACAAGACCAUUGUGUUCAA UCACAGCUCUGGUGGUGAUCCUGAGAUUGUGAUGCACAGCUUCAACUGUGGUGG UGAGUUCUUCUACUGCAACAGCACCCAGCUGUUCAACAGCACCUGGAACAACAAC ACUGAGGGCAGCAACAACACUGAGGGCAACACCAUCACCCUGCCUUGCAGGAUCA AGCAGAUCAUCAACAUGUGGCAGGAGGUGGGCAAGGCCAUGUAUGCUCCUCCCAU CAGGGGCCAGAUCAGGUGCAGCAGCAACAUCACUGGCCUGCUGCUGACCAGGGAU GGUGGCAUCAAUGAGAAUGGCACUGAGAUUUUCAGGCCUGGUGGUGGGGACAUG AGGGACAACUGGAGGUCUGAGCUGUACAAGUACAAGGUGGUGAAGAUUGAGCCC CUUGGUGUGGCUCCCACCAAGGCUAAGCGCAGGGUGGUGCAGAGGGAGAAGCGC GCUGUG SEQ ID NO:7 HIV-1 gp120-B (Strain 92US715.6 ) Amino acid sequence (ACCESSION:AAB04079 ) LWVTVYYGVPVWKEANTTLFCASDAKAYDTEVHNVWATHACVPTDPDPQEVELENV TENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTNLRNDTNTTRNATNT TSSETMMEEGEIKNCSFNITTSIRDKVQKEFALFYKLDVVPIENDTTSYRLISCNTSVLTQ ACPKVSFEPIPIHFCAPAGFAILKCKDKKFNGTGPCTNVSTVQCTHGIKPVVSTQLLLNGS LAEEEVVIRSANLSDNAKTIIVQLNESVQMNCTRPNNNTRKSIHIGPGRAFYTTGEIIGDIR QAHCNLSRTKWNETLKRIVIKLREQYENKTIVFNQSSGGDPEIVMLSFNCGGEFFYCNST KLFNSTWNGTESNNTGDDPIVLPCRIKQVINMWQEVGKAMYAPPIRGQIRCSSNITGLLL TRDGGNSNETNTTEIFRPGGGNMKDNWRSELY KYKVVRIEPLGIAPTRAKRRVVQ SEQ ID NO:8 HIV-1 gp120-B (Strain 92US715.6 ) Codon optimized DNA sequence CTGTGGGTGACCGTCTACTATGGGGTGCCTGTGTGGAAGGAGGCCAACACCACTCTG TTCTGCGCTTCTGACGCTAAGGCCTACGATACCGAGGTGCACAATGTGTGGGCCACC CACGCCTGTGTGCCCACCGACCCCGACCCTCAGGAGGTGGAGCTGGAGAACGTGAC CGAAAACTTCAACATGTGGAAGAATAACATGGTGGAGCAGATGCATGAGGATATCA TTAGCCTGTGGGACCAGAGCCTAAAGCCCTGCGTGAAGCTGACCCCCCTGTGTGTGA CTCTGAACTGCACCAACCTGAGGAATGATACTAACACCACCAGGAACGCCACTAAT ACGACCAGCAGCGAGACCATGATGGAGGAGGGCGAGATCAAGAACTGCTCTTTCAA CATCACCACGAGCATCAGAGACAAGGTGCAGAAGGAGTTTGCCCTTTTCTATAAA CTTGATGTGGTGCCTATCGAGAATGACACTACTAGCTACAGGCTGATCAGCTGCAAC ACCAGCGTCCTGACACAGGCCTGCCCCAAGGTGTCCTTCGAGCCAATTCCCATCCAC TTTTGTGCCCCGGCTGGTTTCGCCATTCTAAAGTGCAAGGATAAGAAGTTCAACGGC ACCGGTCCTTGTACCAATGTCAGCACCGTACAATGCACCCACGGCATTAAGCCCGTG GTGAGCACTCAGCTGCTGCTGAACGGCAGCCTGGCCGAGGAAGAGGTGGTGATTCG CTCCGCCAACCTCTCTGACAATGCTAAGACCATAATCGTGCAGCTGAACGAGTCTGT GCAGATGAACTGCACGAGGCCCAACAACAATACCAGGAAGAGTATCCATATCGGTC CCGGCAGGGCATTCTATACCACCGGCGAGATCATCGGCGACATCAGGCAGGCCCAC TGTAACCTTAGCAGGACAAAGTGGAACGAGACTCTGAAGAGGATCGTGATCAAGCT GAGGGAGCAGTACGAGAACAAGACCATCGTCTTTAATCAATCCAGCGGCGGGGACC 34 WAS:164427.1CTGAGATTGTGATGCTGAGCTTCAACTGCGGTGGGGAGTTCTTCTACTGTAACTCAA CCAAGCTGTTTAATAGCACTTGGAACGGCACTGAGTCTAACAACACCGGTGATGACC CCATCGTGCTGCCATGCAGGATCAAGCAGGTGATCAACATGTGGCAGGAAGTGGGC AAGGCCATGTATGCCCCTCCCATCAGGGGTCAGATTAGGTGCAGCAGCAATATTACC GGCCTGCTACTGACCCGCGACGGCGGTAACAGCAACGAGACCAACACCACCGAGAT CTTCAGGCCTGGGGGCGGCAACATGAAGGACAATTGGAGGAGCGAGTTATACAAAT ATAAGGTGGTGAGGATTGAGCCTCTGGGTATCGCCCCCACCAGGGCCAAGAGGAGG GTGGTGCAG SEQ ID NO:9 HIV-1 gp120-B (Strain 92US715.6 ) Codon optimized mRNA sequence CUGUGGGUGACCGUCUACUAUGGGGUGCCUGUGUGGAAGGAGGCCAACACCACU UGUUCUGCGCUUCUGACGCUAAGGCCUACGAUACCGAGGUGCACAAUGUGUGGGC CACCCACGCCUGUGUGCCCACCGACCCCGACCCUCAGGAGGUGGAGCUGGAGAAC GUGACCGAAAACUUCAACAUGUGGAAGAAUAACAUGGUGGAGCAGAUGCAUGAG GAUAUCAUUAGCCUGUGGGACCAGAGCCUAAAGCCCUGCGUGAAGCUGACC CCCCUGUGUGUGACUCUGAACUGCACCAACCUGAGGAAUGAUACUAACACCACCA GGAACGCCACUAAUACGACCAGCAGCGAGACCAUGAUGGAGGAGGGCGAGAUCA AGAACUGCUCUUUCAACAUCACCACGAGCAUCAGAGACAAGGUGCAGAAGGAGU UUGCCCUUUUCUAUAAACUUGAUGUGGUGCCUAUCGAGAAUGACACUACUAGCU ACAGGCUGAUCAGCUGCAACACCAGCGUCCUGACACAGGCCUGCCCCAAGGUG UCCUUCGAGCCAAUUCCCAUCCACUUUUGUGCCCCGGCUGGUUUCGCCAUUCUAA AGUGCAAGGAUAAGAAGUUCAACGGCACCGGUCCUUGUACCAAUGUCAGCACCGU ACAAUGCACCCACGGCAUUAAGCCCGUGGUGAGCACUCAGCUGCUGCUGAACGGC AGCCUGGCCGAGGAAGAGGUGGUGAUUCGCUCCGCCAACCUCUCUGACAAUGCUA AGACCAUAAUCGUGCAGCUGAACGAGUCUGUGCAGAUGAACUGCACGAGG CCCAACAACAAUACCAGGAAGAGUAUCCAUAUCGGUCCCGGCAGGGCAUUCUAUA CCACCGGCGAGAUCAUCGGCGACAUCAGGCAGGCCCACUGUAACCUUAGCAGGAC AAAGUGGAACGAGACUCUGAAGAGGAUCGUGAUCAAGCUGAGGGAGCAGUACGA GAACAAGACCAUCGUCUUUAAUCAAUCCAGCGGCGGGGACCCUGAGAUUGUGAU GCUGAGCUUCAACUGCGGUGGGGAGUUCUUCUACUGUAACUCAACCAAGCUG UUUAAUAGCACUUGGAACGGCACUGAGUCUAACAACACCGGUGAUGACCCCAUCG UGCUGCCAUGCAGGAUCAAGCAGGUGAUCAACAUGUGGCAGGAAGUGGGCAAGG CCAUGUAUGCCCCUCCCAUCAGGGGUCAGAUUAGGUGCAGCAGCAAUAUUACCGG CCUGCUACUGACCCGCGACGGCGGUAACAGCAACGAGACCAACACCACCGAGAUC UUCAGGCCUGGGGGCGGCAACAUGAAGGACAAUUGGAGGAGCGAGUUAUACAAA UAUAAGGUGGUGAGGAUUGAGCCUCUGGGUAUCGCCCCCACCAGGGCCAAGAGG AGGGUGGUGCAG SEQ ID NO:10 HIV-1 gp120-C (Strain 93MW965.26) Amino acid sequence LWVTVYYGVPVWKEAKTTLFCASEAKAYEKEVHNVWATHACVPTDPNPQEMVLENV TENFNMWKNDMVNQMHEDIISLWDQSLKPCVKLTPLCVTLNCTNANGTNNNGTVNVN DTMYGEIKNCSFNMTTELRDKKKQVYALFYKLDIVSLNENSNNSSEYRLINCNTSVITQA 35 WAS:164427.1CPKVTFDPIPIHYCAPAGYAILKCNNKTFTGIGPCKNVSTVQCTHGIKPVVSTQLLLNGSL AEEEIIVRSENLTDNVKTIIVHLNESVEIVCTRPNNNTRKSVRIGPGQTFYATGAIIGDIRQ AHCNISTIKWNKTLQGVEKKLKEHFPNKTIEFKPSSGGDLEITTHSFNCRGEFFCCNTSNL FTSNLFTDNLTNTTNITLPCRIKQIINMWQGVGRAMYAPPIAGNITCKSNITGLLLTRDGG ENNRTETFRPGGGDMKDNWRSELYKYKVVEIK PLGVAPTGAKRRVVE SEQ ID NO:11 HIV-1 gp120-C (Strain 93MW965.26) Codon optimized DNA sequence CTGTGGGTGACCGTGTACTACGGCGTGCCCGTGTGGAAGGAGGCCAAGACCACCCT GTTCTGCGCCAGCGAGGCCAAGGCCTACGAGAAGGAGGTGCACAACGTGTGGGCCA CCCACGCCTGCGTGCCCACCGACCCCAACCCCCAGGAGATGGTGCTGGAGAACGTG ACCGAGAACTTCAACATGTGGAAGAACGACATGGTGAACCAGATGCACGAGGACAT CATCAGCCTGTGGGACCAGAGCCTGAAGCCCTGCGTGAAGCTGACCCCCCTGTGCGT GACCCTGAACTGCACCAACGCCAACGGCACCAACAATAACGGCACCGTGAACGTGA ACGACACCATGTACGGCGAGATCAAGAACTGCAGCTTCAACATGACCACCGAGCTG CGGGACAAGAAGAAGCAGGTGTACGCCCTGTTCTACAAGCTGGACATCGTGAGCCT GAACGAGAACAGCAACAACAGCAGCGAGTACCGGCTGATCAACTGCAACACCAGC GTGATCACCCAGGCCTGCCCCAAGGTGACCTTCGACCCCATCCCCATCCACTACTGC GCCCCTGCCGGCTACGCCATCCTGAAGTGCAACAACAAGACCTTCACCGGCATCGG CCCCTGCAAGAACGTGAGCACCGTGCAGTGCACCCACGGCATCAAGCCCGTGGTGA GCACCCAGCTGCTGCTGAACGGCAGCCTGGCCGAGGAGGAGATCATCGTGCGGAGC GAGAACCTGACCGACAACGTGAAAACCATCATCGTGCACCTGAATGAGAGCGTGGA GATCGTGTGCACCAGGCCCAACAACAACACCCGGAAGAGCGTGCGGATCGGCCCTG GCCAGACCTTCTACGCCACCGGCGCCATCATCGGCGACATCCGGCAGGCCCACTGC AACATCAGCACCATCAAGTGGAACAAGACCCTGCAGGGCGTGGAGAAGAAGCTGA AGGAGCACTTCCCCAACAAGACCATCGAGTTCAAGCCCAGCAGCGGCGGAGACCTG GAGATCACCACCCACAGCTTCAACTGCAGGGGCGAGTTCTTCTGCTGCAACACCTCC AACCTGTTCACCAGCAATCTGTTCACCGACAACCTGACCAACACCACCAACATCACC CTGCCCTGCCGGATCAAGCAGATCATCAACATGTGGCAGGGCGTGGGCAGGGCCAT GTACGCCCCTCCCATCGCCGGCAACATCACCTGCAAGAGCAACATCACCGGCCTGCT GCTGACCCGGGACGGCGGCGAGAACAACCGGACCGAGACCTTCAGGCCCGGAGGC GGCGACATGAAGGACAACTGGCGGAGCGAGCTGTACAAGTACAAGGTGGTGGAGA TCAAG CCCCTGGGCGTGGCCCCCACCGGCGCCAAGCGCCGCGTGGTGGAG SEQ ID NO:12 HIV-1 gp120-C (Strain 93MW965.26) Codon optimized mRNA sequence CUGUGGGUGACCGUGUACUACGGCGUGCCCGUGUGGAAGGAGGCCAAGACCACCC UGUUCUGCGCCAGCGAGGCCAAGGCCUACGAGAAGGAGGUGCACAACGUGUGGGC CACCCACGCCUGCGUGCCCACCGACCCCAACCCCCAGGAGAUGGUGCUGGAGAAC GUGACCGAGAACUUCAACAUGUGGAAGAACGACAUGGUGAACCAGAUGCACGAG GACAUCAUCAGCCUGUGGGACCAGAGCCUGAAGCCCUGCGUGAAGCUGACCCCCC UGUGCGUGACCCUGAACUGCACCAACGCCAACGGCACCAACAAUAACGGCACCGU GAACGUGAACGACACCAUGUACGGCGAGAUCAAGAACUGCAGCUUCAACAUGACC ACCGAGCUGCGGGACAAGAAGAAGCAGGUGUACGCCCUGUUCUACAAGCUGGACA 36 WAS:164427.1UCGUGAGCCUGAACGAGAACAGCAACAACAGCAGCGAGUACCGGCUGAUCAACUG CAACACCAGCGUGAUCACCCAGGCCUGCCCCAAGGUGACCUUCGACCCCAUCCCC AUCCACUACUGCGCCCCUGCCGGCUACGCCAUCCUGAAGUGCAACAACAAGACCU UCACCGGCAUCGGCCCCUGCAAGAACGUGAGCACCGUGCAGUGCACCCACGGCAU CAAGCCCGUGGUGAGCACCCAGCUGCUGCUGAACGGCAGCCUGGCCGAGGAGGAG AUCAUCGUGCGGAGCGAGAACCUGACCGACAACGUGAAAACCAUCAUCGUGCACC UGAAUGAGAGCGUGGAGAUCGUGUGCACCAGGCCCAACAACAACACCCGGAAGAG CGUGCGGAUCGGCCCUGGCCAGACCUUCUACGCCACCGGCGCCAUCAUCGGCGAC AUCCGGCAGGCCCACUGCAACAUCAGCACCAUCAAGUGGAACAAGACCCUGCAGG GCGUGGAGAAGAAGCUGAAGGAGCACUUCCCCAACAAGACCAUCGAGUUCAAGCC CAGCAGCGGCGGAGACCUGGAGAUCACCACCCACAGCUUCAACUGCAGGGGCGAG UUCUUCUGCUGCAACACCUCCAACCUGUUCACCAGCAAUCUGUUCACCGACAACC UGACCAACACCACCAACAUCACCCUGCCCUGCCGGAUCAAGCAGAUCAUCAACAU GUGGCAGGGCGUGGGCAGGGCCAUGUACGCCCCUCCCAUCGCCGGCAACAUCACC UGCAAGAGCAACAUCACCGGCCUGCUGCUGACCCGGGACGGCGGCGAGAACAACC GGACCGAGACCUUCAGGCCCGGAGGCGGCGACAUGAAGGACAACUGGCGGAGCGA GCUGUACAAGUACAAGGUGGUGGAGAUCAAGCCCCUGGGCGUGGCCCCCACCGGC GCCAAGCGCCGCGUGGUGGAG SEQ ID. NO:13 HIV-1 gp120-D (Strain 92UG021.16) Amino acid sequence LWVTVYYGVPVWKEATTTLFCASDAKSYEAEAHNIWATHACVPTDPNPQEIVLENVTE NFNIWKNNMVEQMHDDIISLWDQSIKPCVKLTPLCVTLNCTEWKNATTNATNEGIGMK NCSFTEVRDKKKQAYALFYKLDVVQMNDDNSTNTSYRLINCNASTITQACPKISFEPIPI HYCAPAGFAILKCNDKKFNGTGPCKNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSK NLTNNAKIIIVHLNESVPINCTRPYDKVSYRTPIGVGRASYTTRIKGDIRQAHCNISGEKW NKTLQQVAVKLRDLLNQTAIIFKPSSGGDPEITTHSFNCGGEFFYCNTSGLFNNSVWTSN STIGANGTITLPCRIKQIINMWQGVGKAMYAPPIEGQINCSSTITGLLLTRDGGVKNNSQN ETFRPGGGDMRDNWRNELYKYKVVRIEPLGLA PTKAKRRVVE SEQ ID. NO:14 HIV-1 gp120-D (Strain 92UG021.16) Codon optimized DNA sequence CTGTGGGTGACCGTGTACTACGGCGTGCCCGTGTGGAAGGAGGCCACCACCACCCT GTTCTGCGCCAGCGACGCCAAGAGCTACGAGGCCGAGGCCCACAACATCTGGGCCA CCCACGCCTGCGTGCCCACCGACCCCAACCCCCAGGAGATCGTGCTGGAGAACGTG ACCGAGAACTTCAACATCTGGAAGAACAACATGGTGGAGCAGATGCACGACGACAT CATCAGCCTGTGGGACCAGAGCATCAAGCCCTGCGTGAAGCTGACCCCCCTGTGCGT GACCCTGAACTGCACCGAGTGGAAGAACGCCACCACAAACGCCACCAACGAGGGC ATCGGCATGAAGAACTGCAGCTTCACCGAGGTGCGGGACAAGAAGAAGCAGGCCTA CGCCCTGTTCTACAAGCTGGACGTGGTGCAGATGAACGACGATAACAGCACCAACA CCAGCTACCGGCTGATCAACTGCAACGCCAGCACCATCACCCAGGCCTGCCCCAAG ATCAGCTTCGAGCCCATCCCCATCCACTACTGCGCCCCTGCCGGCTTCGCCATCCTG AAGTGCAACGACAAGAAGTTCAACGGCACCGGCCCCTGCAAGAACGTGAGCACCGT GCAGTGCACCCACGGCATCAAGCCCGTGGTGAGCACCCAGCTGCTGCTGAACGGCA 37 WAS:164427.1GCCTGGCCGAGGAGGAGATCATCATCCGGAGCAAGAACCTGACCAACAACGCCAAG ATCATCATCGTGCACCTGAACGAGAGCGTGCCCATCAACTGCACCCGGCCCTACGAC AAGGTGAGCTACCGGACCCCCATCGGCGTGGGCAGGGCCAGCTACACCACCCGGAT CAAGGGCGACATCCGGCAGGCCCACTGCAACATCAGCGGCGAGAAGTGGAACAAG ACCCTGCAGCAGGTGGCCGTGAAGCTGCGGGACCTGCTGAACCAGACCGCCATCAT CTTCAAGCCCAGCAGCGGCGGAGACCCCGAGATCACCACCCACAGCTTCAACTGTG GCGGCGAGTTCTTCTACTGCAACACCAGCGGCCTGTTCAACAACAGCGTGTGGACCA GCAACAGCACCATCGGCGCCAACGGCACCATCACCCTGCCCTGCAGGATCAAGCAG ATCATCAACATGTGGCAGGGCGTGGGCAAGGCCATGTACGCCCCTCCCATCGAGGG CCAGATCAACTGCAGCTCCACCATCACCGGCCTGCTGCTGACCCGGGACGGCGGCG TGAAGAACAACAGCCAGAACGAGACCTTCAGGCCCGGAGGCGGCGACATGCGGGA CAACTGGCGGAACGAGCTGTACAAGTACAAGGTGGTGCGGATCGAGCCCCTGGGCC TGGCC CCCACCAAGGCCAAGCGCCGCGTGGTGGAG SEQ ID. NO: 15 HIV-1 gp120-D (Strain 92UG021.16) Codon optimized mRNA sequence CUGUGGGUGACCGUGUACUACGGCGUGCCCGUGUGGAAGGAGGCCACCACCACCC UGUUCUGCGCCAGCGACGCCAAGAGCUACGAGGCCGAGGCCCACAACAUCUGGGC CACCCACGCCUGCGUGCCCACCGACCCCAACCCCCAGGAGAUCGUGCUGGAGAAC GUGACCGAGAACUUCAACAUCUGGAAGAACAACAUGGUGGAGCAGAUGCACGAC GACAUCAUCAGCCUGUGGGACCAGAGCAUCAAGCCCUGCGUGAAGCUGACC CCCCUGUGCGUGACCCUGAACUGCACCGAGUGGAAGAACGCCACCACAAACGCCA CCAACGAGGGCAUCGGCAUGAAGAACUGCAGCUUCACCGAGGUGCGGGACAAGAA GAAGCAGGCCUACGCCCUGUUCUACAAGCUGGACGUGGUGCAGAUGAACGACGAU AACAGCACCAACACCAGCUACCGGCUGAUCAACUGCAACGCCAGCACCAUCACCC AGGCCUGCCCCAAGAUCAGCUUCGAGCCCAUCCCCAUCCACUACUGCGCCCCUGCC GGCUUCGCCAUCCUGAAGUGCAACGACAAGAAGUUCAACGGCACCGGCCCCUGCA AGAACGUGAGCACCGUGCAGUGCACCCACGGCAUCAAGCCCGUGGUGAGCACCCA GCUGCUGCUGAACGGCAGCCUGGCCGAGGAGGAGAUCAUCAUCCGGAGCAAGAAC CUGACCAACAACGCCAAGAUCAUCAUCGUGCACCUGAACGAGAGCGUGCCCAUCA ACUGCACCCGGCCCUACGACAAGGUGAGCUACCGGACCCCCAUCGGCGUGGGCAG GGCCAGCUACACCACCCGGAUCAAGGGCGACAUCCGGCAGGCCCACUGCAACAUC AGCGGCGAGAAGUGGAACAAGACCCUGCAGCAGGUGGCCGUGAAGCUGCGGGACC UGCUGAACCAGACCGCCAUCAUCUUCAAGCCCAGCAGCGGCGGAGACCCCGAGAU CACCACCCACAGCUUCAACUGUGGCGGCGAGUUCUUCUACUGCAACACCAGCGGC CUGUUCAACAACAGCGUGUGGACCAGCAACAGCACCAUCGGCGCCAACGGCACCA UCACCCUGCCCUGCAGGAUCAAGCAGAUCAUCAACAUGUGGCAGGGCGUGGGCAA GGCCAUGUACGCCCCUCCCAUCGAGGGCCAGAUCAACUGCAGCUCCACCAUCACC GGCCUGCUGCUGACCCGGGACGGCGGCGUGAAGAACAACAGCCAGAACGAGACC UUCAGGCCCGGAGGCGGCGACAUGCGGGACAACUGGCGGAACGAGCUGUACAAGU ACAAGGUGGUGCGGAUCGAGCCCCUGGGCCUGGCCCCCACCAAGGCCAAGCGCCG CGUGGUGGAG SEQ ID. NO:16 HIV-1 gp120-A / E (Consensus) 38 WAS:164427.1Amino acid sequence LWVTVYYGVPVWRDADTTLFCASDAKAHETEVHNVWATHACVPTDPNPQEIHLENVT ENFNMWKNNMVEQMQEDVISLWDQSLKPCVKLTPLCVTLNCTNANLTNNNINGSNIIG NITDEVRNCSFNMTTELRDKKQKVHALFYKLDIVQIEDNSNSSEYRLINCNTSVIKQACP KISFDPIPIHYCTPAGYAILKCNDKNFNGTGPCKNVSSVQCTHGIKPVVSTQLLLNGSLAE EEIIIRSENLTNNAKTIIVHLNKSVEINCTRPSNNTRTSITIGPGQVFYRTGDIIGDIRKAYCE INGTKWNEVLKQVTGKLKEHFNNKTIIFQPPSGGDLEITMHHFNCRGEFFYCNTTKLFNN TCIGNETMEGCNGTIILPCKIKQIINMWQGVGQAMYAPPISGRINCVSNITGILLTRDGGA NNTANETFRPGGGNIKDNWRSELYKYKVVQIEPLGIAPTRAKRRVVE SEQ. ID. NO:17 HIV-1 gp120-A / E (Consensus) Codon optimized DNA sequence CTGTGGGTCACCGTGTACTACGGCGTGCCCGTGTGGCGGGACGCCGATACCACCCTG TTCTGTGCCAGCGACGCCAAGGCCCACGAGACAGAGGTGCACAACGTGTGGGCCAC CCACGCCTGCGTGCCCACCGACCCCAACCCCCAGGAAATCCACCTGGAAAACGTGA CCGAGAACTTCAACATGTGGAAGAACAACATGGTCGAGCAGATGCAGGAAGATGTC ATCAGCCTCTGGGACCAGAGCCTGAAGCCCTGCGTGAAGCTGACCCCCCTGTGCGTG ACCCTGAACTGCACCAACGCCAACCTGACCAACAACAACATCAACGGCAGCAACAT CATCGGCAACATCACCGACGAAGTGCGGAACTGCTCCTTCAACATGACCACCGAGC TGCGGGACAAGAAACAGAAGGTGCACGCCCTGTTCTACAAGCTGGACATCGTGCAG ATCGAGGACAACAGCAACAGCAGCGAGTACCGGCTGATCAACTGCAACACCAGCGT GATCAAGCAGGCCTGCCCCAAGATCAGCTTCGACCCCATCCCCATCCACTACTGCAC CCCTGCCGGCTACGCCATCCTGAAGTGCAACGACAAGAACTTCAATGGCACCGGCC CCTGCAAGAACGTGTCCAGCGTGCAGTGCACCCACGGCATCAAGCCCGTGGTGTCC ACCCAGCTGCTGCTGAATGGCAGCCTGGCCGAGGAAGAGATCATCATCAGAAGCGA GAACCTCACCAACAATGCCAAGACCATCATCGTGCACCTGAACAAGAGCGTGGAAA TCAACTGCACCCGGCCCAGCAACAACACCCGGACCAGCATCACCATCGGCCCTGGC CAGGTGTTCTACCGGACCGGCGATATCATCGGCGATATCCGGAAGGCCTACTGCGA GATCAACGGCACCAAGTGGAACGAGGTGCTGAAGCAGGTCACAGGCAAGCTGAAA GAGCACTTCAACAACAAGACAATCATCTTCCAGCCCCCCTCTGGCGGCGACCTGGA AATCACCATGCACCACTTCAACTGTCGGGGCGAGTTCTTCTACTGCAATACCACCAA GCTGTTCAACAATACCTGCATCGGCAACGAGACAATGGAAGGCTGCAATGGCACCA TCATCCTGCCCTGCAAGATCAAGCAGATCATCAATATGTGGCAGGGCGTGGGCCAG GCTATGTACGCCCCTCCCATCAGCGGCCGGATCAACTGCGTGTCCAATATCACCGGC ATCCTGCTGACCCGGGACGGCGGAGCCAACAACACCGCCAACGAGACATTCAGACC CGGCGGAGGCAACATCAAGGACAACTGGCGGAGCGAGCTGTACAAGTACAAGGTG GTGCAGATTGAGCCCCTGGGAATCGCCCCCACCCGGGCCAAGCGGAGAGTGGTGGA A SEQ ID. NO:18 HIV-1 gp120-A / E (Consensus) Codon optimized mRNA sequence CUGUGGGUCACCGUGUACUACGGCGUGCCCGUGUGGCGGGACGCCGAUACCACCC UGUUCUGUGCCAGCGACGCCAAGGCCCACGAGACAGAGGUGCACAACGUGUGGGC CACCCACGCCUGCGUGCCCACCGACCCCAACCCCCAGGAAAUCCACCUGGAAAACG 39 WAS:164427.1UGACCGAGAACUUCAACAUGUGGAAGAACAACAUGGUCGAGCAGAUGCAGGAAG AUGUCAUCAGCCUCUGGGACCAGAGCCUGAAGCCCUGCGUGAAGCUGACCCCCCU GUGCGUGACCCUGAACUGCACCAACGCCAACCUGACCAACAACAACAUCAACGGC AGCAACAUCAUCGGCAACAUCACCGACGAAGUGCGGAACUGCUCCUUCAACAUGA CCACCGAGCUGCGGGACAAGAAACAGAAGGUGCACGCCCUGUUCUACAAGCUGGA CAUCGUGCAGAUCGAGGACAACAGCAACAGCAGCGAGUACCGGCUGAUCAACUGC AACACCAGCGUGAUCAAGCAGGCCUGCCCCAAGAUCAGCUUCGACCCCAUCCCCA UCCACUACUGCACCCCUGCCGGCUACGCCAUCCUGAAGUGCAACGACAAGAACUU CAAUGGCACCGGCCCCUGCAAGAACGUGUCCAGCGUGCAGUGCACCCACGGCAUC AAGCCCGUGGUGUCCACCCAGCUGCUGCUGAAUGGCAGCCUGGCCGAGGAAGAGA UCAUCAUCAGAAGCGAGAACCUCACCAACAAUGCCAAGACCAUCAUCGUGCACCU GAACAAGAGCGUGGAAAUCAACUGCACCCGGCCCAGCAACAACACCCGGACCAGC AUCACCAUCGGCCCUGGCCAGGUGUUCUACCGGACCGGCGAUAUCAUCGGCGAUA UCCGGAAGGCCUACUGCGAGAUCAACGGCACCAAGUGGAACGAGGUGCUGAAGCA GGUCACAGGCAAGCUGAAAGAGCACUUCAACAACAAGACAAUCAUCUUCCAGCCC CCCUCUGGCGGCGACCUGGAAAUCACCAUGCACCACUUCAACUGUCGGGGCGAGU UCUUCUACUGCAAUACCACCAAGCUGUUCAACAAUACCUGCAUCGGCAACGAGAC AAUGGAAGGCUGCAAUGGCACCAUCAUCCUGCCCUGCAAGAUCAAGCAGAUCAUC AAUAUGUGGCAGGGCGUGGGCCAGGCUAUGUACGCCCCUCCCAUCAGCGGCCGGA UCAACUGCGUGUCCAAUAUCACCGGCAUCCUGCUGACCCGGGACGGCGGAGCCAA CAACACCGCCAACGAGACAUUCAGACCCGGCGGAGGCAACAUCAAGGACAACUGG CGGAGCGAGCUGUACAAGUACAAGGUGGUGCAGAUUGAGCCCCUGGGAAUCGCCC CCACCCGGGCCAAGCGGAGAGUGGUGGAA SEQ ID. NO:19 tPA leader Amino acid sequence MDAMKRGLCCVLLLCGAVFVS SEQ ID. NO:20 tPA leader DNA sequence ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTC GTTTCG SEQ ID. NO:21 tPA leader mRNA sequence AUGGAUGCAAUGAAGAGAGGGCUCUGCUGUGUGCUGCUGCUGUGUGGAGCAGUC UUCGUUUCG SEQ ID. NO.22 Venezuelan equine encephalitis virus (VEEV) sub genomic promoter 26S GGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACAT SEQ ID. NO:23 40 WAS:164427.1Non-Structural Protein 1 (nsP1) Venezuelan equine encephalitis virus (VEEV) Amino acid sequence MEKVHVDIEE DSPFLRALQR SFPQFEVEAK QVTDNDHANA RAFSHLASKL IETEVDPSDT ILDIGSAPAR RMYSKHKYHC ICPMRCAEDP DRLYKYATKL KKNCKEITDK ELDKKMKELA AVMSDPDLET ETMCLHDDES CRYEGQVAVY QDVYAVDGPT SLYHQANKGV RVAYWIGFDT TPFMFKNLAG AYPSYSTNWA DETVLTARNI GLCSSDVMER SRRGMSILRK KYLKPSNNVL FSVGSTIYHE KRDLLRSWHL PSVFHLRGKQ NYTCRCETIV SCDGYVVKRI AISPGLYGKP SGYAATMHRE GFLCCKVTDT LNGERVSFPV CTYVPATLCD QMTGILATDV SADDAQKLLV GLNQRIVVNG RTQRNTNTMK NYLLPVVAQA FARWAKEYKE DQEDERPLGL RDRQLVMGCC WAFRRHKITS IYKRPDTQTI IKVNSDFHSF VLPRIGSNTL EIGLRTRIRK MLEEHKEPSP LITAEDVQEA KCAADEAKEV REAEELRAAL PPLAADVEEP TLEADVDLML QEAGA SEQ ID. NO:24 Non-Structural Protein 1 (nsP1) Venezuelan equine encephalitis virus (VEEV) DNA sequence ATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCA GCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATG CTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACC CATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGC ACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATA AGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAAGGAATTGGAC AAGAAAATGAAGGAGCTGGCCGCCGTCATGAGCGACCCTGACCTGGAAACTGAGAC TATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGTTTAC CAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGG AGTTAGAGTCGCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTT GGCTGGAGCATATCCATCATACTCTACCAACTGGGCCGACGAAACCGTGTTAACGG CTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGAGCGGTCACGTAGAGGGATGT CCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCTGTTGGCTC GACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATT TCACTTACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGA CGGGTACGTCGTTAAAAGAATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAG GCTATGCTGCTACGATGCACCGCGAGGGATTCTTGTGCTGCAAAGTGACAGACACAT TGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAGCTACATTGTGTG ACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTG CTGGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAA TACCATGAAAAATTACCTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAA GGAATATAAGGAAGATCAAGAAGATGAAAGGCCACTAGGACTACGAGATAGACAG TTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAACATCTATTTATAAG CGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTG CCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAAT GTTAGAGGAGCACAAGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAG CTAAGTGCGCAGCCGATGAGGCTAAGGAGGTGCGTGAAGCCGAGGAGTTGCGCGCA 41 WAS:164427.1GCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTGGAAGCCGATGTCGAC TTGATGTTACAAGAGGCTGGGGCC SEQ ID. NO:25 Non-Structural Protein 1 (nsP1) Venezuelan equine encephalitis virus (VEEV) mRNA sequence AUGGAGAAAGUUCACGUUGACAUCGAGGAAGACAGCCCAUUCCUCAGAGCUUUG CAGCGGAGCUUCCCGCAGUUUGAGGUAGAAGCCAAGCAGGUCACUGAUAAUGACC AUGCUAAUGCCAGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGG UGGACCCAUCCGACACGAUCCUUGACAUUGGAAGUGCGCCCGCCCGCAGAAUGUA UUCUAAGCACAAGUAUCAUUGUAUCUGUCCGAUGAGAUGUGCGGAAGAUCCG GACAGAUUGUAUAAGUAUGCAACUAAGCUGAAGAAAAACUGUAAGGAAAUAACU GAUAAGGAAUUGGACAAGAAAAUGAAGGAGCUGGCCGCCGUCAUGAGCGACCCU GACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUCGUGUCGCUACGAAG GGCAAGUCGCUGUUUACCAGGAUGUAUACGCGGUUGACGGACCGACAAGUCUCU AUCACCAAGCCAAUAAGGGAGUUAGAGUCGCCUACUGGAUAGGCUUUGACACC ACCCCUUUUAUGUUUAAGAACUUGGCUGGAGCAUAUCCAUCAUACUCUACCAACU GGGCCGACGAAACCGUGUUAACGGCUCGUAACAUAGGCCUAUGCAGCUCUGACGU UAUGGAGCGGUCACGUAGAGGGAUGUCCAUUCUUAGAAAGAAGUAUUUGAAACC AUCCAACAAUGUUCUAUUCUCUGUUGGCUCGACCAUCUACCACGAGAAGAGGGAC UUACUGAGGAGCUGGCACCUGCCGUCUGUAUUUCACUUACGUGGCAAGCAA AAUUACACAUGUCGGUGUGAGACUAUAGUUAGUUGCGACGGGUACGUCGUUAAA AGAAUAGCUAUCAGUCCAGGCCUGUAUGGGAAGCCUUCAGGCUAUGCUGCUACG AUGCACCGCGAGGGAUUCUUGUGCUGCAAAGUGACAGACACAUUGAACGGGGAG AGGGUCUCUUUUCCCGUGUGCACGUAUGUGCCAGCUACAUUGUGUGACCAAAUG ACUGGCAUACUGGCAACAGAUGUCAGUGCGGACGACGCGCAAAAACUGCUGGUU GGGCUCAACCAGCGUAUAGUCGUCAACGGUCGCACCCAGAGAAACACCAAUACCA UGAAAAAUUACCUUUUGCCCGUAGUGGCCCAGGCAUUUGCUAGGUGGGCAAAGG AAUAUAAGGAAGAUCAAGAAGAUGAAAGGCCACUAGGACUACGAGAUAGACAGU UAGUCAUGGGGUGUUGUUGGGCUUUUAGAAGGCACAAGAUAACAUCUAUUUAUA AGCGCCCGGAUACCCAAACCAUCAUCAAAGUGAACAGCGAUUUCCACUCAUUC GUGCUGCCCAGGAUAGGCAGUAACACAUUGGAGAUCGGGCUGAGAACAAGAAUC AGGAAAAUGUUAGAGGAGCACAAGGAGCCGUCACCUCUCAUUACCGCCGAGGACG UACAAGAAGCUAAGUGCGCAGCCGAUGAGGCUAAGGAGGUGCGUGAAGCCGAGG AGUUGCGCGCAGCUCUACCACCUUUGGCAGCUGAUGUUGAGGAGCCCACUCUGGA AGCCGAUGUCGACUUGAUGUUACAAGAGGCUGGGGCC SEQ ID. NO:26 Non-Structural Protein 2 (nsP2) Venezuelan equine encephalitis virus (VEEV) Amino acid sequence GSVETPRGLI KVTSYDGEDK IGSYAVLSPQ AVLKSEKLSC IHPLAEQVIV ITHSGRKGRY AVEPYHGKVV VPEGHAIPVQ DFQALSESAT IVYNEREFVN RYLHHIATHG GALNTDEEYY KTVKPSEHDG EYLYDIDRKQ CVKKELVTGL 42 WAS:164427.1GLTGELVDPP FHEFAYESLR TRPAAPYQVP TIGVYGVPGS GKSGIIKSAV TKKDLVVSAK KENCAEIIRD VKKMKGLDVN ARTVDSVLLN GCKHPVETLY IDEAFACHAG TLRALIAIIR PKKAVLCGDP KQCGFFNMMC LKVHFNHEIC TQVFHKSISR RCTKSVTSVV STLFYDKKMR TTNPKETKIV IDTTGSTKPK QDDLILTCFR GWVKQLQIDY KGNEIMTAAA SQGLTRKGVY AVRYKVNENP LYAPTSEHVN VLLTRTEDRI VWKTLAGDPW IKTLTAKYPG NFTATIEEWQ AEHDAIMRHI LERPDPTDVF QNKANVCWAK ALVPVLKTAG IDMTTEQWNT VDYFETDKAH SAEIVLNQLC VRFFGLDLDS GLFSAPTVPL SIRNNHWDNS PSPNMYGLNK EVVRQLSRRY PQLPRAVATG RVYDMNTGTL RNYDPRINLV PVNRRLPHAL VLHHNEHPQS DFSSFVSKLK GRTVLVVGEK LSVPGKMVDW LSDRPEATFR ARLDLGIPGD VPKYDIIFVN VRTPYKYHHY QQCEDHAIKL SMLTKKACLH LNPGGTCVSI GYGYADRASE SIIGAIARQF KFSRVCKPKS SLEETEVLFV FIGYDRKART HNSYKLSSTL TNIYTGSRLH EAGC SEQ ID. NO:27 Non-Structural Protein 2 (nsP2) Venezuelan equine encephalitis virus (VEEV) DNA sequence GGCTCAGTGGAGACACCTCGTGGCTTGATAAAGGTTACCAGCTACGATGGCGAGGA CAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAAGAGTGAAAAATT ATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAA AGGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATG CAATACCCGTCCAGGACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACG AACGTGAGTTCGTAAACAGGTACCTGCACCATATTGCCACACATGGAGGAGCGCTG AACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAGCACGACGGCGAATA CCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTA GGGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTG AGAACACGACCAGCCGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCC AGGATCAGGCAAGTCTGGCATCATTAAAAGCGCAGTCACCAAAAAAGATCTAGTGG TGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACGTCAAGAAAATGAA AGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAAC ACCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCA GAGCGCTCATAGCCATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAA CAGTGCGGTTTTTTTAACATGATGTGCCTGAAAGTGCATTTTAACCACGAGATTTGC ACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAAATCTGTGACTTCGGTC GTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACTAA GATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCAC TTGTTTCAGAGGGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAA TGACGGCAGCTGCCTCTCAAGGGCTGACCCGTAAAGGTGTGTATGCCGTTCGGTACA AGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAACATGTGAACGTCCTACTGA CCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGATAAAA ACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAA GCAGAGCATGATGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGT CTTCCAGAATAAGGCAAACGTGTGTTGGGCCAAGGCTTTAGTGCCGGTGCTGAAGA CCGCTGGCATAGACATGACCACTGAACAATGGAACACTGTGGATTATTTTGAAACG GACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTTTGGA 43 WAS:164427.1CTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATA ATCACTGGGATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCC GTCAGCTCTCTCGCAGGTACCCACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCT ATGACATGAACACTGGTACACTGCGCAATTATGATCCGCGCATAAACCTAGTA CCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACACCCACAG AGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGG GAAAAGTTGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCT ACCTTCAGAGCTCGGCTGGATTTAGGCATCCCAGGTGATGTGCCCAAATATGACATA ATATTTGTTAATGTGAGGACCCCATATAAATACCATCACTATCAGCAGTGTGAAGAC CATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATCTGAATCCCGGC GGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCAT TGGTGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACT TGAAGAGACGGAAGTTCTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCA CAATTCTTACAAGCTTTCATCAACCTTGACCAACATTTATACAGGTTCCAGACTCCAC GAAGCCGGATGT SEQ ID. NO:28 Non-Structural Protein 2 (nsP2) Venezuelan equine encephalitis virus (VEEV) mRNA sequence GGCUCAGUGGAGACACCUCGUGGCUUGAUAAAGGUUACCAGCUACGAUGGCGAG GACAAGAUCGGCUCUUACGCUGUGCUUUCUCCGCAGGCUGUACUCAAGAGUGAAA AAUUAUCUUGCAUCCACCCUCUCGCUGAACAAGUCAUAGUGAUAACACACUCUGG CCGAAAAGGGCGUUAUGCCGUGGAACCAUACCAUGGUAAAGUAGUGGUGCCAGA GGGACAUGCAAUACCCGUCCAGGACUUUCAAGCUCUGAGUGAAAGUGCCACCAUU GUGUACAACGAACGUGAGUUCGUAAACAGGUACCUGCACCAUAUUGCCACACAUG GAGGAGCGCUGAACACUGAUGAAGAAUAUUACAAAACUGUCAAGCCCAGCGAGC ACGACGGCGAAUACCUGUACGACAUCGACAGGAAACAGUGCGUCAAGAAAGAAC UAGUCACUGGGCUAGGGCUCACAGGCGAGCUGGUGGAUCCUCCCUUCCAUGAAUU CGCCUACGAGAGUCUGAGAACACGACCAGCCGCUCCUUACCAAGUACCA ACCAUAGGGGUGUAUGGCGUGCCAGGAUCAGGCAAGUCUGGCAUCAUUAAAAGC GCAGUCACCAAAAAAGAUCUAGUGGUGAGCGCCAAGAAAGAAAACUGUGCAGAA AUUAUAAGGGACGUCAAGAAAAUGAAAGGGCUGGACGUCAAUGCCAGAACUGUG GACUCAGUGCUCUUGAAUGGAUGCAAACACCCCGUAGAGACCCUGUAUAUUGACG AAGCUUUUGCUUGUCAUGCAGGUACUCUCAGAGCGCUCAUAGCCAUUAUAAGA CCUAAAAAGGCAGUGCUCUGCGGGGAUCCCAAACAGUGCGGUUUUUUUAACAUG AUGUGCCUGAAAGUGCAUUUUAACCACGAGAUUUGCACACAAGUCUUCCACAAA AGCAUCUCUCGCCGUUGCACUAAAUCUGUGACUUCGGUCGUCUCAACCUUGUUUU ACGACAAAAAAAUGAGAACGACGAAUCCGAAAGAGACUAAGAUUGUGAUUGACA CUACCGGCAGUACCAAACCUAAGCAGGACGAUCUCAUUCUCACUUGUUUCAGA GGGUGGGUGAAGCAGUUGCAAAUAGAUUACAAAGGCAACGAAAUAAUGACGGCA GCUGCCUCUCAAGGGCUGACCCGUAAAGGUGUGUAUGCCGUUCGGUACAAGGUG AAUGAAAAUCCUCUGUACGCACCCACCUCAGAACAUGUGAACGUCCUACUGACCC GCACGGAGGACCGCAUCGUGUGGAAAACACUAGCCGGCGACCCAUGGAUAAAAAC ACUGACUGCCAAGUACCCUGGGAAUUUCACUGCCACGAUAGAGGAGUGGCAA 44 WAS:164427.1GCAGAGCAUGAUGCCAUCAUGAGGCACAUCUUGGAGAGACCGGACCCUACCGACG UCUUCCAGAAUAAGGCAAACGUGUGUUGGGCCAAGGCUUUAGUGCCGGUGCUGA AGACCGCUGGCAUAGACAUGACCACUGAACAAUGGAACACUGUGGAUUAUUUUG AAACGGACAAAGCUCACUCAGCAGAGAUAGUAUUGAACCAACUAUGCGUGAGGU UCUUUGGACUCGAUCUGGACUCCGGUCUAUUUUCUGCACCCACUGUUCCGUUA UCCAUUAGGAAUAAUCACUGGGAUAACUCCCCGUCGCCUAACAUGUACGGGCUGA AUAAAGAAGUGGUCCGUCAGCUCUCUCGCAGGUACCCACAACUGCCUCGGGCAGU UGCCACUGGAAGAGUCUAUGACAUGAACACUGGUACACUGCGCAAUUAUGAUCC GCGCAUAAACCUAGUACCUGUAAACAGAAGACUGCCUCAUGCUUUAGUCCUCCAC CAUAAUGAACACCCACAGAGUGACUUUUCUUCAUUCGUCAGCAAAUUGAAG GGCAGAACUGUCCUGGUGGUCGGGGAAAAGUUGUCCGUCCCAGGCAAAAUGGUU GACUGGUUGUCAGACCGGCCUGAGGCUACCUUCAGAGCUCGGCUGGAUUUAGGCA UCCCAGGUGAUGUGCCCAAAUAUGACAUAAUAUUUGUUAAUGUGAGGACCCCAU AUAAAUACCAUCACUAUCAGCAGUGUGAAGACCAUGCCAUUAAGCUUAGCAUGU UGACCAAGAAAGCUUGUCUGCAUCUGAAUCCCGGCGGAACCUGUGUCAGCAUA GGUUAUGGUUACGCUGACAGGGCCAGCGAAAGCAUCAUUGGUGCUAUAGCGCGG CAGUUCAAGUUUUCCCGGGUAUGCAAACCGAAAUCCUCACUUGAAGAGACGGAA GUUCUGUUUGUAUUCAUUGGGUACGAUCGCAAGGCCCGUACGCACAAUUCUUAC AAGCUUUCAUCAACCUUGACCAACAUUUAUACAGGUUCCAGACUCCACGAAGCCG GAUGU SEQ ID. NO:29 Non-Structural Protein 3 (nsP3) Venezuelan equine encephalitis virus (VEEV) Amino acid sequence APSYHVVRGD IATATEGVII NAANSKGQPG GGVCGALYKK FPESFDLQPI EVGKARLVKG AAKHIIHAVG PNFNKVSEVE GDKQLAEAYE SIAKIVNDNN YKSVAIPLLS TGIFSGNKDR LTQSLNHLLT ALDTTDADVA IYCRDKKWEM TLKEAVARRE AVEEICISDD SSVTEPDAEL VRVHPKSSLA GRKGYSTSDG KTFSYLEGTK FHQAAKDIAE INAMWPVATE ANEQVCMYIL GESMSSIRSK CPVEESEAST PPSTLPCLCI HAMTPERVQR LKASRPEQIT VCSSFPLPKY RITGVQKIQC SQPILFSPKV PAYIHPRKYL VETPPVDETP EPSAENQSTE GTPEQPPLIT EDETRTRTPE PIIIEEEEED SISLLSDGPT HQVLQVEADI HGPPSVSSSS WSIPHASDFD VDSLSILDTL EGASVTSGAT SAETNSYFAK SMEFLARPVP APRTVFRNPP HPAPRTRTPS LAPSRACSRT SLVSTPPGVN RVITREELEA LTPSRTPSRS VSRTSLVSNP PGVNRVITRE EFEAFVAQQQ RFDAGA SEQ ID. NO:30 Non-Structural Protein 3 (nsP3) Venezuelan equine encephalitis virus (VEEV) DNA sequence GCACCCTCATATCATGTGGTGCGAGGGGATATTGCCACGGCCACCGAAGGAGTGAT TATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTGTGCGGAGCGCTGT ATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGA CTGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAA AGTTTCGGAGGTTGAAGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTA 45 WAS:164427.1AGATTGTCAACGATAACAATTACAAGTCAGTAGCGATTCCACTGTTGTCCACCGGCA TCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACCATTTGCTGACAGCTT TAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAATG ACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGA CGACTCTTCAGTGACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTT CTTTGGCTGGAAGGAAGGGCTACAGCACAAGCGATGGCAAAACTTTCTCATATTTGG AAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGAAATTAATGCCATGTGG CCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGCAT GAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTA GCACGCTGCCTTGCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAA AAGCCTCACGTCCAGAACAAATTACTGTGTGCTCATCCTTTCCATTGCCGAAGTAT AGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCTATATTGTTCTCACCGAAA GTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAGACGA GACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCAC CACTTATAACCGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAA GAGGAAGAAGAGGATAGCATAAGTTTGCTGTCAGATGGCCCGACCCACCAGGTGCT GCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTATCTAGCTCATCCTGGTCCAT TCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCTGGAGGG AGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAG AGTATGGAGTTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCT CCACATCCCGCTCCGCGCACAAGAACACCGTCACTTGCACCCAGCAGGGCCTGCTCG AGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAATAGGGTGATCACTAGAGAGGA GCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGAACCAGCCT GGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGT TCGTAGCACAACAACAATGACGGTTTGATGCGGGTGCA SEQ ID. NO:31 Non-Structural Protein 3 (nsP3) Venezuelan equine encephalitis virus (VEEV) mRNA sequence GCACCCUCAUAUCAUGUGGUGCGAGGGGAUAUUGCCACGGCCACCGAAGGAGUGA UUAUAAAUGCUGCUAACAGCAAAGGACAACCUGGCGGAGGGGUGUGCGGAGCGC UGUAUAAGAAAUUCCCGGAAAGCUUCGAUUUACAGCCGAUCGAAGUAGGAAAAG CGCGACUGGUCAAAGGUGCAGCUAAACAUAUCAUUCAUGCCGUAGGACCAAACUU CAACAAAGUUUCGGAGGUUGAAGGUGACAAACAGUUGGCAGAGGCUUAUGAG UCCAUCGCUAAGAUUGUCAACGAUAACAAUUACAAGUCAGUAGCGAUUCCACUG UUGUCCACCGGCAUCUUUUCCGGGAACAAAGAUCGACUAACCCAAUCAUUGAACC AUUUGCUGACAGCUUUAGACACCACUGAUGCAGAUGUAGCCAUAUACUGCAGGG ACAAGAAAUGGGAAAUGACUCUCAAGGAAGCAGUGGCUAGGAGAGAAGCAGUGG AGGAGAUAUGCAUAUCCGACGACUCUUCAGUGACAGAACCUGAUGCAGAGCUG GUGAGGGUGCAUCCGAAGAGUUCUUUGGCUGGAAGGAAGGGCUACAGCACAAGC GAUGGCAAAACUUUCUCAUAUUUGGAAGGGACCAAGUUUCACCAGGCGGCCAAG GAUAUAGCAGAAAUUAAUGCCAUGUGGCCCGUUGCAACGGAGGCCAAUGAGCAG GUAUGCAUGUAUAUCCUCGGAGAAAGCAUGAGCAGUAUUAGGUCGAAAUGCCCC GUCGAAGAGUCGGAAGCCUCCACACCACCUAGCACGCUGCCUUGCUUGUGCAUC CAUGCCAUGACUCCAGAAAGAGUACAGCGCCUAAAAGCCUCACGUCCAGAACAAA 46 WAS:164427.1UUACUGUGUGCUCAUCCUUUCCAUUGCCGAAGUAUAGAAUCACUGGUGUGCAGA AGAUCCAAUGCUCCCAGCCUAUAUUGUUCUCACCGAAAGUGCCUGCGUAUAUUCA UCCAAGGAAGUAUCUCGUGGAAACACCACCGGUAGACGAGACUCCGGAGCCAUCG GCAGAGAACCAAUCCACAGAGGGGACACCUGAACAACCACCACUUAUAACC GAGGAUGAGACCAGGACUAGAACGCCUGAGCCGAUCAUCAUCGAAGAGGAAGAA GAGGAUAGCAUAAGUUUGCUGUCAGAUGGCCCGACCCACCAGGUGCUGCAAGUCG AGGCAGACAUUCACGGGCCGCCCUCUGUAUCUAGCUCAUCCUGGUCCAUUCCUCA UGCAUCCGACUUUGAUGUGGACAGUUUAUCCAUACUUGACACCCUGGAGGGAGC UAGCGUGACCAGCGGGGCAACGUCAGCCGAGACUAACUCUUACUUCGCAAAG AGUAUGGAGUUUCUGGCGCGACCGGUGCCUGCGCCUCGAACAGUAUUCAGGAACC CUCCACAUCCCGCUCCGCGCACAAGAACACCGUCACUUGCACCCAGCAGGGCCUG CUCGAGAACCAGCCUAGUUUCCACCCCGCCAGGCGUGAAUAGGGUGAUCACUAGA GAGGAGCUCGAGGCGCUUACCCCGUCACGCACUCCUAGCAGGUCGGUCUCGAGAA CCAGCCUGGUCUCCAACCCGCCAGGCGUAAAUAGGGUGAUUACAAGAGAG GAGUUUGAGGCGUUCGUAGCACAACAACAAUGACGGUUUGAUGCGGGUGCA SEQ. ID. NO:32 Non-Structural Protein 4 (nsP4) Venezuelan equine encephalitis virus (VEEV) Amino acid sequence YIFSSDTGQG HLQQKSVRQT VLSEVVLERT ELEISYAPRL DQEKEELLRK KLQLNPTPAN RSRYQSRKVE NMKAITARRI LQGLGHYLKA EGKVECYRTL HPVPLYSSSV NRAFSSPKVA VEACNAMLKE NFPTVASYCI IPEYDAYLDM VDGASCCLDT ASFCPAKLRS FPKKHSYLEP TIRSAVPSAI QNTLQNVLAA ATKRNCNVTQ MRELPVLDSA AFNVECFKKY ACNNEYWETF KENPIRLTEE NVVNYITKLK GPKAAALFAK THNLNMLQDI PMDRFVMDLK RDVKVTPGTK HTEERPKVQV IQAADPLATA YLCGIHRELV RRLNAVLLPN IHTLFDMSAE DFDAIIAEHF QPGDCVLETD IASFDKSEDD AMALTALMIL EDLGVDAELL TLIEAAFGEI SSIHLPTKTK FKFGAMMKSG MFLTLFVNTV INIVIASRVL RERLTGSPCA AFIGDDNIVK GVKSDKLMAD RCATWLNMEV KIIDAVVGEK APYFCGGFIL CDSVTGTACR VADPLKRLFK LGKPLAADDE HDDDRRRALH EESTRWNRVG ILSELCKAVE SRYETVGTSI IVMAMTTLAS SVKSFSYLRG APITLYG SEQ ID. NO:33 Non-Structural Protein 4 (nsP4) Venezuelan equine encephalitis virus (VEEV) DNA sequence TACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACAACAAAAATCAGTAAGGCAA ACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTATGCCCCG CGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCAC ACCTGCTAACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAA CAGCTAGACGTATTCTGCAAGGCCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTG GAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTATTCATCTAGTGTGAACCGTGCCT TTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAGAGAACTTTC CGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATG GTTGACGGAGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGC 47 WAS:164427.1AGCTTTCCAAAGAAACACTCCTATTTGGAACCCACAATACGATCGGCAGTGCCTTCA GCGATCCAGAACACGCTCCAGAACGTCCTGGCAGCTGCCACAAAAAGAAATTGCAA TGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAATGTGGAATG CTTCAAGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCA TCAGGCTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGGACCAAAA GCTGCTGCTCTTTTTGCGAAGACACATAATTTGAATATGTTGCAGGACATACCAATG GACAGGTTTGTAATGGACTTAAAGAGAGACGTGAAAGTGACTCCAGGAACAAAA CATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCGCTAGCAAC AGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCT TCCGAACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGC CGAGCACTTCCAGCCTGGGGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAA AAGTGAGGACGACGCCATGGCTCTGACCGCGTTAATGATTCTGGAAGACTTAGGTGT GGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAATTTCATCAATACA TTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCT CACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTG AGAGAACGGCTAACCGGATCACCATGTGCAGCATTCATTGGAGATGACAATATCGT GAAAGGAGTCAAATCGGACAAATTAATGGCAGACAGGTGCGCCACCTGGTTGAATA TGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTATTTCTGTGGAG GGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAA AAAGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGAC AGGAGAAGGGCATTGCATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTC AGAGCTGTGCAAGGCAGTAGAATCAAGGTATGAAACCGTAGGAACTTCCATCATAG TTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGCTACCTGAGAGGG GCCCCTATAACTCTCTACGGC SEQ ID. NO:34 Non-Structural Protein 4 (nsP4) Venezuelan equine encephalitis virus (VEEV) mRNA sequence UACAUCUUUUCCUCCGACACCGGUCAAGGGCAUUUACAACAAAAAUCAGUAAGGC AAACGGUGCUAUCCGAAGUGGUGUUGGAGAGGACCGAAUUGGAGAUUUCGUAUG CCCCGCGCCUCGACCAAGAAAAAGAAGAAUUACUACGCAAGAAAUUACAGUUAAA UCCCACACCUGCUAACAGAAGCAGAUACCAGUCCAGGAAGGUGGAGAACAUGAAA GCCAUAACAGCUAGACGUAUUCUGCAAGGCCUAGGGCAUUAUUUGAAGGCA GAAGGAAAAGUGGAGUGCUACCGAACCCUGCAUCCUGUUCCUUUGUAUUCAUCU AGUGUGAACCGUGCCUUUUCAAGCCCCAAGGUCGCAGUGGAAGCCUGUAACGCCA UGUUGAAAGAGAACUUUCCGACUGUGGCUUCUUACUGUAUUAUUCCAGAGUACG AUGCCUAUUUGGACAUGGUUGACGGAGCUUCAUGCUGCUUAGACACUGCCAGUU UUUGCCCUGCAAAGCUGCGCAGCUUUCCAAAGAAACACUCCUAUUUGGAACCC ACAAUACGAUCGGCAGUGCCUUCAGCGAUCCAGAACACGCUCCAGAACGUCCUGG CAGCUGCCACAAAAAGAAAUUGCAAUGUCACGCAAAUGAGAGAAUUGCCCGUAU UGGAUUCGGCGGCCUUUAAUGUGGAAUGCUUCAAGAAAUAUGCGUGUAAUAAUG AAUAUUGGGAAACGUUUAAAGAAAACCCCAUCAGGCUUACUGAAGAAAACGUGG UAAAUUACAUUACCAAAUUAAAAGGACCAAAAGCUGCUGCUCUUUUUGCGAAG ACACAUAAUUUGAAUAUGUUGCAGGACAUACCAAUGGACAGGUUUGUAAUGGAC UUAAAGAGAGACGUGAAAGUGACUCCAGGAACAAAACAUACUGAAGAACGGCCC 48 WAS:164427.1AAGGUACAGGUGAUCCAGGCUGCCGAUCCGCUAGCAACAGCGUAUCUGUGCGGAA UCCACCGAGAGCUGGUUAGGAGAUUAAAUGCGGUCCUGCUUCCGAACAUUCAUAC ACUGUUUGAUAUGUCGGCUGAAGACUUUGACGCUAUUAUAGCCGAGCACUUC CAGCCUGGGGAUUGUGUUCUGGAAACUGACAUCGCGUCGUUUGAUAAAAGUGAG GACGACGCCAUGGCUCUGACCGCGUUAAUGAUUCUGGAAGACUUAGGUGUGGAC GCAGAGCUGUUGACGCUGAUUGAGGCGGCUUUCGGCGAAAUUUCAUCAAUACAU UUGCCCACUAAAACUAAAUUUAAAUUCGGAGCCAUGAUGAAAUCUGGAAUGUUC CUCACACUGUUUGUGAACACAGUCAUUAACAUUGUAAUCGCAAGCAGAGUGUUG AGAGAACGGCUAACCGGAUCACCAUGUGCAGCAUUCAUUGGAGAUGACAAUAUC GUGAAAGGAGUCAAAUCGGACAAAUUAAUGGCAGACAGGUGCGCCACCUGGUUG AAUAUGGAAGUCAAGAUUAUAGAUGCUGUGGUGGGCGAGAAAGCGCCUUAUUUC UGUGGAGGGUUUAUUUUGUGUGACUCCGUGACCGGCACAGCGUGCCGUGUGGCA GACCCCCUAAAAAGGCUGUUUAAGCUUGGCAAACCUCUGGCAGCAGACGAUGAA CAUGAUGAUGACAGGAGAAGGGCAUUGCAUGAAGAGUCAACACGCUGGAACCGA GUGGGUAUUCUUUCAGAGCUGUGCAAGGCAGUAGAAUCAAGGUAUGAAACCGUA GGAACUUCCAUCAUAGUUAUGGCCAUGACUACUCUAGCUAGCAGUGUUAAAUCA UUCAGCUACCUGAGAGGG GCCCCUAUAACUCUCUACGGC SEQ. ID. NO:35 Semliki Forest virus (SFV) sub genomic promoter 26S TTGGTGCGTTAATACACAGAATTCTGATTATAGCGCACTATTATAGCACC SEQ. ID. NO:36 Non-Structural Protein 1 (nsP1) Semliki Forest virus (SFV) Amino acid sequence MAAKVHVDIEADSPFIKSLQKAFPSFEVESLQVTPNDHANARAFSHLATKLIEQETDKDT LILDIGSAPSRRMMSTHKYHCVCPMRSAEDPERLVCYAKKLAAASGKVLDREIAGKITD LQTVMATPDAESPTFCLHTDVTCRTAAEVAVYQDVYAVHAPTSLYHQAMKGVRTAY WIGFDTTPFMFDALAGAYPTYATNWADEQVLQARNIGLCAASLTEGRLGKLSILRKKQL KPCDTVMFSVGSTLYTESRKLLRSWHLPSVFHLKGKQSFTCRCDTIVSCEGYVVKKITM CPGLYGKTVGYAVTYHAEGFLVCKTTDTVKGERVSFPVCTYVPSTICDQMTGILATDVT PEDAQKLLVGLNQRIVVNGRTQRNTNTMKNYLLPIVAVAFSKWAREYKADLDDEKPLG VRERSLTCCCLWAFKTRKMHTMYKKPDTQTIVKVPSEFNSFVIPSLWSTGLAIPVRSRIK MLLAKKTKRELIPVLDASSARDAEQEEKERLEAELTREALPPLVPIAPAETGVVDVDVEE LEYHAGA SEQ. ID. NO:37 Non-Structural Protein 1 (nsP1) Semliki Forest virus (SFV) DNA sequence ATGGCCGCCAAAGTGCATGTTGATATTGAGGCTGACAGCCCATTCATCAAGTCTTTG CAGAAGGCATTTCCGTCGTTCGAGGTGGAGTCATTGCAGGTCACACCAAATGACCAT GCAAATGCCAGAGCATTTTCGCACCTGGCTACCAAATTGATCGAGCAGGAGACTGA CAAAGACACACTCATCTTGGATATCGGCAGTGCGCCTTCCAGGAGAATGATGTCTAC GCACAAATACCACTGCGTATGCCCTATGCGCAGCGCAGAAGACCCCGAAAGGCTCG 49 WAS:164427.1TATGCTACGCAAAGAAACTGGCAGCGGCCTCCGGGAAGGTGCTGGATAGAGAGATC GCAGGAAAAATCACCGACCTGCAGACCGTCATGGCTACGCCAGACGCTGAATCTCC TACCTTTTGCCTGCATACAGACGTCACGTGTCGTACGGCAGCCGAAGTGGCCGTA TACCAGGACGTGTATGCTGTACATGCACCAACATCGCTGTACCATCAGGCGATGAAA GGTGTCAGAACGGCGTATTGGATTGGGTTTGACACCACCCCGTTTATGTTTGACGCG CTAGCAGGCGCGTATCCAACCTACGCCACAAACTGGGCCGACGAGCAGGTGTTACA GGCCAGGAACATAGGACTGTGTGCAGCATCCTTGACTGAGGGAAGACTCGGCAAAC TGTCCATTCTCCGCAAGAAGCAATTGAAACCTTGCGACACAGTCATGTTCTCGGTAG GATCTACATTGTACACTGAGAGCAGAAAGCTACTGAGGAGCTGGCACTTACCCTCCG TATTCCACCTGAAAGGTAAACAATCCTTTACCTGTAGGTGCGATACCATCGTATCAT GTGAAGGGTACGTAGTTAAGAAAATCACTATGTGCCCCGGCCTGTACGGTAAA ACGGTAGGGTACGCCGTGACGTATCACGCGGAGGGATTCCTAGTGTGCAAGACCAC AGACACTGTCAAAGGAGAAAGAGTCTCATTCCCTGTATGCACCTACGTCCCCTCAAC CATCTGTGATCAAATGACTGGCATACTAGCGACCGACGTCACACCGGAGGACGCAC AGAAGTTGTTAGTGGGATTGAATCAGAGGATAGTTGTGAACGGAAGAACACAGCGA AACACTAACACGATGAAGAACTATCTGCTTCCGATTGTGGCCGTCGCATTTAGCAAG TGGGCGAGGGAATACAAGGCAGACCTTGATGATGAAAAACCTCTGGGTGTCCGAGA GAGGTCACTTACTTGCTGCTGCTTGTGGGCATTTAAAACGAGGAAGATGCACACCAT GTACAAGAAACCAGACACCCAGACAATAGTGAAGGTGCCTTCAGAGTTTAACTCG TTCGTCATCCCGAGCCTATGGTCTACAGGCCTCGCAATCCCAGTCAGATCACGCATT AAGATGCTTTTGGCCAAGAAGACCAAGCGAGAGTTAATACCTGTTCTCGACGCGTCG TCAGCCAGGGATGCTGAACAAGAGGAGAAGGAGAGGTTGGAGGCCGAGCTGACTA GAGAAGCCTTACCACCCCTCGTCCCCATCGCGCCGGCGGAGACGGGAGTCGTCGAC GTCGACGTTGAAGAACTAGAGTATCACGCAGGTGCA SEQ. ID. NO:38 Non-Structural Protein 1 (nsP1) Semliki Forest virus (SFV) mRNA sequence AUGGCCGCCAAAGUGCAUGUUGAUAUUGAGGCUGACAGCCCAUUCAUCAAGUCU UUGCAGAAGGCAUUUCCGUCGUUCGAGGUGGAGUCAUUGCAGGUCACACCAAAU GACCAUGCAAAUGCCAGAGCAUUUUCGCACCUGGCUACCAAAUUGAUCGAGCAGG AGACUGACAAAGACACACUCAUCUUGGAUAUCGGCAGUGCGCCUUCCAGGAGAAU GAUGUCUACGCACAAAUACCACUGCGUAUGCCCUAUGCGCAGCGCAGAAGAC CCCGAAAGGCUCGUAUGCUACGCAAAGAAACUGGCAGCGGCCUCCGGGAAGGUGC UGGAUAGAGAGAUCGCAGGAAAAAUCACCGACCUGCAGACCGUCAUGGCUACGCC AGACGCUGAAUCUCCUACCUUUUGCCUGCAUACAGACGUCACGUGUCGUACGGCA GCCGAAGUGGCCGUAUACCAGGACGUGUAUGCUGUACAUGCACCAACAUCGCUGU ACCAUCAGGCGAUGAAAGGUGUCAGAACGGCGUAUUGGAUUGGGUUUGAC ACCACCCCGUUUAUGUUUGACGCGCUAGCAGGCGCGUAUCCAACCUACGCCACAA ACUGGGCCGACGAGCAGGUGUUACAGGCCAGGAACAUAGGACUGUGUGCAGCAU CCUUGACUGAGGGAAGACUCGGCAAACUGUCCAUUCUCCGCAAGAAGCAAUUGAA ACCUUGCGACACAGUCAUGUUCUCGGUAGGAUCUACAUUGUACACUGAGAGCAG AAAGCUACUGAGGAGCUGGCACUUACCCUCCGUAUUCCACCUGAAAGGUAAA CAAUCCUUUACCUGUAGGUGCGAUACCAUCGUAUCAUGUGAAGGGUACGUAGUU AAGAAAAUCACUAUGUGCCCCGGCCUGUACGGUAAAACGGUAGGGUACGCCGUG 50 WAS:164427.1ACGUAUCACGCGGAGGGAUUCCUAGUGUGCAAGACCACAGACACUGUCAAAGGA GAAAGAGUCUCAUUCCCUGUAUGCACCUACGUCCCCUCAACCAUCUGUGAUCAAA UGACUGGCAUACUAGCGACCGACGUCACACCGGAGGACGCACAGAAGUUGUUA GUGGGAUUGAAUCAGAGGAUAGUUGUGAACGGAAGAACACAGCGAAACACUAAC ACGAUGAAGAACUAUCUGCUUCCGAUUGUGGCCGUCGCAUUUAGCAAGUGGGCG AGGGAAUACAAGGCAGACCUUGAUGAUGAAAAACCUCUGGGUGUCCGAGAGAGG UCACUUACUUGCUGCUGCUUGUGGGCAUUUAAAACGAGGAAGAUGCACACCAUG UACAAGAAACCAGACACCCAGACAAUAGUGAAGGUGCCUUCAGAGUUUAACUCG UUCGUCAUCCCGAGCCUAUGGUCUACAGGCCUCGCAAUCCCAGUCAGAUCACGCA UUAAGAUGCUUUUGGCCAAGAAGACCAAGCGAGAGUUAAUACCUGUUCUCGACG CGUCGUCAGCCAGGGAUGCUGAACAAGAGGAGAAGGAGAGGUUGGAGGCCGAGC UGACUAGAGAAGCCUUACCACCCCUCGUCCCCAUCGCGCCGGCGGAGACGGGAGU CGUCGACGUCGACGUUGAAGAACUAGAGUAUCACGCAGGUGCA SEQ ID NO:39 Non-Structural Protein 2 (nsP2) Semliki Forest virus (SFV) Amino acid sequence GVVETPRSALKVTAQPNDVLLGNYVVLSPQTVLKSSKLAPVHPLAEQVKIITHNGRAGR YQVDGYDGRVLLPCGSAIPVPEFQALSESATMVYNEREFVNRKLYHIAVHGPSLNTDEE NYEKVRAERTDAEYVFDVDKKCCVKREEASGLVLVGELTNPPFHEFAYEGLKIRPSAPY KTTVVGVFGVPGSGKSAIIKSLVTKHDLVTSGKKENCQEIVNDVKKHRGLDIQAKTVDSI LLNGCRRAVDILYVDEAFACHSGTLLALIALVKPRSKVVLCGDPKQCGFFNMMQLKVN FNHNICTEVCHKSISRRCTRPVTAIVSTLHYGGKMRTTNPCNKPIIIDTTGQTKPKPGDIVL TCFRGWVKQLQLDYRGHEVMTAAASQGLTRKGVYAVRQKVNENPLYAPASEHVNVL LTRTEDRLVWKTLAGDPWIKVLSNIPQGNFTATLEEWQEEHDKIMKVIEGPAAPVDAFQ NKANVCWAKSLVPVLDTAGIRLTAEEWSTIITAFKEDRAYSPVVALNEICTKYYGVDLD SGLFSAPKVSLYYENNHWDNRPGGRMYGFNAATAARLEARHTFLKGQWHTGKQAVIA ERKIQPLSVLDNVIPINRRLPHALVAEYKTVKGSRVEWLVNKVRGYHVLLVSEYNLALP RRRVTWLSPLNVTGADRCYDLSLGLPADAGRFDLVFVNIHTEFRIHHYQQCVDHAMKL QMLGGDALRLLKPGGSLLMRAYGYADKISEAVVSSLSRKFSSARVLRPDCVTSNTEVFL LFSNFDNGKRPSTLHQMNTKLSAVYAGEAMHTAGC SEQ ID NO:40 Non-Structural Protein 2 (nsP2) Semliki Forest virus (SFV) DNA sequence GGGGTCGTGGAAACACCTCGCAGCGCGTTGAAAGTCACCGCACAGCCGAACGACGT ACTACTAGGAAATTACGTAGTTCTGTCCCCGCAGACCGTGCTCAAGAGCTCCAAGTT GGCCCCCGTGCACCCTCTAGCAGAGCAGGTGAAAATAATAACACATAACGGGAGGG CCGGCCGTTACCAGGTCGACGGATATGACGGCAGGGTCCTACTACCATGTGGATCG GCCATTCCGGTCCCTGAGTTTCAAGCTTTGAGCGAGAGCGCCACTATGGTGTACAAC GAAAGGGAGTTCGTCAACAGGAAACTATACCATATTGCCGTTCACGGACCGTCGCT GAACACCGACGAGGAGAACTACGAGAAAGTCAGAGCTGAAAGAACTGACGCCGAG TACGTGTTCGACGTAGATAAAAAATGCTGCGTCAAGAGAGAGGAAGCGTCGGGTTT G 51 WAS:164427.1GTGTTGGTGGGAGAGCTAACCAACCCCCCGTTCCATGAATTCGCCTACGAAGGGCTG AAGATCAGGCCGTCGGCACCATATAAGACTACAGTAGTAGGAGTCTTTGGGGTTCC GGGATCAGGCAAGTCTGCTATTATTAAGAGCCTCGTGACCAAACACGATCTGGTCAC CAGCGGCAAGAAGGAGAACTGCCAGGAAATAGTCAACGACGTGAAGAAGCACCGC GGACTGGACATCCAGGCAAAAACAGTGGACTCCATCCTGCTAAACGGGTGTCGTCG TGCCGTGGACATCCTATATGTGGACGAGGCTTTCGCTTGCCATTCCGGTACTCTGCTA GCCCTAATTGCTCTTGTTAAACCTCGGAGCAAAGTGGTGTTATGCGGAGACCCCAAG CAATGCGGATTCTTCAATATGATGCAGCTTAAGGTGAACTTCAACCACAACATC TGCACTGAAGTATGTCATAAAAGTATATCCAGACGTTGCACGCGTCCAGTCACGGCC ATCGTGTCTACGTTGCACTACGGAGGCAAGATGCGCACGACCAACCCGTGCAACAA ACCCATAATCATAGACACCACAGGACAGACCAAGCCCAAGCCAGGAGACATCGTGT TAACATGCTTCCGAGGCTGGGTAAAGCAGCTGCAGTTGGACTACCGTGGACACGAA GTCATGACAGCAGCAGCATCTCAGGGCCTCACCCGCAAAGGGGTATACGCCGTAAG GCAGAAGGTGAATGAAAATCCCTTGTATGCCCCTGCGTCGGAGCACGTGAATGTACT GCTGACGCGCACTGAGGATAGGCTGGTGTGGAAAACGCTGGCCGGCGATCCCTGGA TTAAGGTCCTATCAAACATTCCACAGGGTAACTTTACGGCCACATTGGAAGAATGG CAAGAAGAACACGACAAAATAATGAAGGTGATTGAAGGACCGGCTGCGCCTGTGGA CGCGTTCCAGAACAAAGCGAACGTGTGTTGGGCGAAAAGCCTGGTGCCTGTCCTGG ACACTGCCGGAATCAGATTGACAGCAGAGGAGTGGAGCACCATAATTACAGCATTT AAGGAGGACAGAGCTTACTCTCCAGTGGTGGCCTTGAATGAAATTTGCACCAAGTA CTATGGAGTTGACCTGGACAGTGGCCTGTTTTCTGCCCCGAAGGTGTCCCTGTATTA CGAGAACAACCACTGGGATAACAGACCTGGTGGAAGGATGTATGGATTCAATGCCG CAACAGCTGCCAGGCTGGAAGCTAGACATACCTTCCTGAAGGGGCAGTGGCATACG GGCAAGCAGGCAGTTATCGCAGAAAGAAAAATCCAACCGCTTTCTGTGCTGGACAA TGTAATTCCTATCAACCGCAGGCTGCCGCACGCCCTGGTGGCTGAGTACAAGACGGT TAAAGGCAGTAGGGTTGAGTGGCTGGTCAATAAAGTAAGAGGGTACCACGTCCTGC TGGTGAGTGAGTACAACCTGGCTTTGCCTCGACGCAGGGTCACTTGGTTGTCACCGC TGAATGTCACAGGCGCCGATAGGTGCTACGACCTAAGTTTAGGACTGCCGGCTGAC GCCGGCAGGTTCGACTTGGTCTTTGTGAACATTCACACGGAATTCAGAATCCACCAC TACCAGCAGTGTGTCGACCACGCCATGAAGCTGCAGATGCTTGGGGGAGATGCGCT ACGACTGCTAAAACCCGGCGGCAGCCTCTTGATGAGAGCTTACGGATACGCCGATA AAATCAGCGAAGCCGTTGTTTCCTCCTTAAGCAGAAAGTTCTCGTCTGCAAGAGTG TTGCGCCCGGATTGTGTCACCAGCAATACAGAAGTGTTCTTGCTGTTCTCCAACTTTG ACAACGGAAAGAGACCCTCTACGCTACACCAGATGAATACCAAGCTGAGTGCCGTG TATGCCGGAGAAGCCATGCACACGGCCGGGTGT SEQ ID NO:41 Non-Structural Protein 2 (nsP2) Semliki Forest virus (SFV) mRNA sequence GGGGUCGUGGAAACACCUCGCAGCGCGUUGAAAGUCACCGCACAGCCGAACGACG UACUACUAGGAAAUUACGUAGUUCUGUCCCCGCAGACCGUGCUCAAGAGCUCCAA GUUGGCCCCCGUGCACCCUCUAGCAGAGCAGGUGAAAAUAAUAACACAUAACGGG AGGGCCGGCCGUUACCAGGUCGACGGAUAUGACGGCAGGGUCCUACUACCAUGUG GAUCGGCCAUUCCGGUCCCUGAGUUUCAAGCUUUGAGCGAGAGCGCCACUAUGGU GUACAACGAAAGGGAGUUCGUCAACAGGAAACUAUACCAUAUUGCCGUUCACGG 52 WAS:164427.1ACCGUCGCUGAACACCGACGAGGAGAACUACGAGAAAGUCAGAGCUGAAAGAAC UGACGCCGAGUACGUGUUCGACGUAGAUAAAAAAUGCUGCGUCAAGAGAGAGGA AGCGUCGGGUUUGGUGUUGGUGGGAGAGCUAACCAACCCCCCGUUCCAUGAAUUC GCCUACGAAGGGCUGAAGAUCAGGCCGUCGGCACCAUAUAAGACUACAGUAGUA GGAGUCUUUGGGGUUCCGGGAUCAGGCAAGUCUGCUAUUAUUAAGAGCCUCGUG ACCAAACACGAUCUGGUCACCAGCGGCAAGAAGGAGAACUGCCAGGAAAUAGUCA ACGACGUGAAGAAGCACCGCGGACUGGACAUCCAGGCAAAAACAGUGGACUCCAU CCUGCUAAACGGGUGUCGUCGUGCCGUGGACAUCCUAUAUGUGGACGAGGCUUUC GCUUGCCAUUCCGGUACUCUGCUAGCCCUAAUUGCUCUUGUUAAACCUCGGAGCA AAGUGGUGUUAUGCGGAGACCCCAAGCAAUGCGGAUUCUUCAAUAUGAUGCAGC UUAAGGUGAACUUCAACCACAACAUCUGCACUGAAGUAUGUCAUAAAAGUAUAU CCAGACGUUGCACGCGUCCAGUCACGGCCAUCGUGUCUACGUUGCACUACGGAGG CAAGAUGCGCACGACCAACCCGUGCAACAAACCCAUAAUCAUAGACACCACAGGA CAGACCAAGCCCAAGCCAGGAGACAUCGUGUUAACAUGCUUCCGAGGCUGGGUAA AGCAGCUGCAGUUGGACUACCGUGGACACGAAGUCAUGACAGCAGCAGCAUCUCA GGGCCUCACCCGCAAAGGGGUAUACGCCGUAAGGCAGAAGGUGAAUGAAAAUCCC UUGUAUGCCCCUGCGUCGGAGCACGUGAAUGUACUGCUGACGCGCACUGAGGAUA GGCUGGUGUGGAAAACGCUGGCCGGCGAUCCCUGGAUUAAGGUCCUAUCAAACA UUCCACAGGGUAACUUUACGGCCACAUUGGAAGAAUGGCAAGAAGAACACGACA AAAUAAUGAAGGUGAUUGAAGGACCGGCUGCGCCUGUGGACGCGUUCCAGAACA AAGCGAACGUGUGUUGGGCGAAAAGCCUGGUGCCUGUCCUGGACACUGCCGGAA UCAGAUUGACAGCAGAGGAGUGGAGCACCAUAAUUACAGCAUUUAAGGAGGACA GAGCUUACUCUCCAGUGGUGGCCUUGAAUGAAAUUUGCACCAAGUACUAUGGAG UUGACCUGGACAGUGGCCUGUUUUCUGCCCCGAAGGUGUCCCUGUAUUACGAGAA CAACCACUGGGAUAACAGACCUGGUGGAAGGAUGUAUGGAUUCAAUGCCGCAAC AGCUGCCAGGCUGGAAGCUAGACAUACCUUCCUGAAGGGGCAGUGGCAUACGGGC AAGCAGGCAGUUAUCGCAGAAAGAAAAAUCCAACCGCUUUCUGUGCUGGACAAU GUAAUUCCUAUCAACCGCAGGCUGCCGCACGCCCUGGUGGCUGAGUACAAGACGG UUAAAGGCAGUAGGGUUGAGUGGCUGGUCAAUAAAGUAAGAGGGUACCACGUCC UGCUGGUGAGUGAGUACAACCUGGCUUUGCCUCGACGCAGGGUCACUUGGUUGU CACCGCUGAAUGUCACAGGCGCCGAUAGGUGCUACGACCUAAGUUUAGGACUGCC GGCUGACGCCGGCAGGUUCGACUUGGUCUUUGUGAACAUUCACACGGAAUUC AGAAUCCACCACUACCAGCAGUGUGUCGACCACGCCAUGAAGCUGCAGAUGCUUG GGGGAGAUGCGCUACGACUGCUAAAACCCGGCGGCAGCCUCUUGAUGAGAGCUUA CGGAUACGCCGAUAAAAUCAGCGAAGCCGUUGUUUCCUCCUUAAGCAGAAAGUUC UCGUCUGCAAGAGUGUUGCGCCCGGAUUGUGUCACCAGCAAUACAGAAGUGUUC UUGCUGUUCUCCAACUUUGACAACGGAAAGAGACCCUCUACGCUACACCAGAUGA AUACCAAGCUGAGUGCCGUGUAUGCCGGAGAAGCCAUGCACACGGCCGGGUGU SEQ ID NO:42 Non-Structural Protein 3 (nsP3) Semliki Forest virus (SFV) Amino acid sequence APSYRVKRADIATCTEAAVVNAANARGTVGDGVCRAVAKKWPSAFKGEATPVGTIKT VMCGSYPVIHAVAPNFSATTEAEGDRELAAVYRAVAAEVNRLSLSSVAIPLLSTGVFSG GRDRLQQSLNHLFTAMDATDADVTIYCRDKSWEKKIQEAIDMRTAVELLNDDVELTTD 53 WAS:164427.1LVRVHP DSSLVGRKGYSTTDGSLYSYFEGTKFNQAAIDMAEILTLWPRLQEANEQICLYALGETM DNIRSKCPVNDSDSSTPPRTVPCLCRYAMTAERIARLRSHQVKSMVVCSSFPLPKYHVD GVQKVKCEKVLLFDPTVPSVVSPRKYAASTTDHSDRSLRGFDLDWTTDSSSTASDTMSL PSLQSCDIDSIYEPMAPIVVTADVHPEPAGIADLAADVHPEPADHVDLENPIPPPRPKRAA YLASRAAERPVPAPRKPTPAPRTAFRNKLPLTFGDFDEHEVDALASGITFGDFDDVLRLG RAGA SEQ ID NO:43 Non-Structural Protein 3 (nsP3) Semliki Forest virus (SFV) DNA sequence GCACCATCCTACAGAGTTAAGAGAGCAGACATAGCCACGTGCACAGAAGCGGCTGT GGTTAACGCAGCTAACGCCCGTGGAACTGTAGGGGATGGCGTATGCAGGGCCGTGG CGAAGAAATGGCCGTCAGCCTTTAAGGGAGAAGCAACACCAGTGGGCACAATTAAA ACAGTCATGTGCGGCTCGTACCCCGTCATCCACGCTGTAGCGCCTAATTTCTCTGCC ACGACTGAAGCGGAAGGGGACCGCGAATTGGCCGCTGTCTACCGGGCAGTGGCCGC CGAAGTAAACAGACTGTCACTGAGCAGCGTAGCCATCCCGCTGCTGTCCACAGGAG TGTTCAGCGGCGGAAGAGATAGGCTGCAGCAATCCCTCAACCATCTATTCACAGCA ATGGACGCCACGGACGCTGACGTGACCATCTACTGCAGAGACAAAAGTTGGGAGAA GAAAATCCAGGAAGCCATAGACATGAGGACGGCTGTGGAGTTGCTCAATGATGACG TGGAGCTGACCACAGACTTGGTGAGAGTGCACCCGGACAGCAGCCTGGTGGGTCGT AAGGGCTACAGTACCACTGACGGGTCGCTGTACTCGTACTTTGAAGGTACGAAATTC AACCAGGCTGCTATTGATATGGCAGAGATACTGACGTTGTGGCCCAGACTGCAAGA GGCAAACGAACAGATATGCCTATACGCGCTGGGCGAAACAATGGACAACATCAGAT CCAAATGTCCGGTGAACGATTCCGATTCATCAACACCTCCCAGGACAGTGCCCTGCC TGTGCCGCTACGCAATGACAGCAGAACGGATCGCCCGCCTTAGGTCACACCAAGTT AAAAGCATGGTGGTTTGCTCATCTTTTCCCCTCCCGAAATACCATGTAGATGGGGTG CAGAAGGTAAAGTGCGAGAAGGTTCTCCTGTTCGACCCGACGGTACCTTCAGTGGTT AGTCCGCGGAAGTATGCCGCATCTACGACGGACCACTCAGATCGGTCGTTACGAGG GTTTGACTTGGACTGGACCACCGACTCGTCTTCCACTGCCAGCGATACCATGTCGCT ACCCAGTTTGCAGTCGTGTGACATCGACTCGATCTACGAGCCAATGGCTCCCATAGT AGTGACGGCTGACGTACACCCTGAACCCGCAGGCATCGCGGACCTGGCGGCAGATG TGCATCCTGAACCCGCAGACCATGTGGACCTCGAGAACCCGATTCCTCCACCGCGCC CGAAGAGAGCTGCATACCTTGCCTCCCGCGCGGCGGAGCGACCGGTGCCGGCGCCG AGAAAGCCGACGCCTGCCCCAAGGACTGCGTTTAGGAACAAGCTGCCTTTGACG TTCGGCGACTTTGACGAGCACGAGGTCGATGCGTTGGCCTCCGGGATTACTTTCGGA GACTTCGACGACGTCCTGCGACTAGGCCGCGCG GGTGCA SEQ ID NO:44 Non-Structural Protein 3 (nsP3) Semliki Forest virus (SFV) mRNA sequence GCACCAUCCUACAGAGUUAAGAGAGCAGACAUAGCCACGUGCACAGAAGCGGCUG UGGUUAACGCAGCUAACGCCCGUGGAACUGUAGGGGAUGGCGUAUGCAGGGCCG UGGCGAAGAAAUGGCCGUCAGCCUUUAAGGGAGAAGCAACACCAGUGGGCACAA 54 WAS:164427.1UUAAAACAGUCAUGUGCGGCUCGUACCCCGUCAUCCACGCUGUAGCGCCUAAUUU CUCUGCCACGACUGAAGCGGAAGGGGACCGCGAAUUGGCCGCUGUCUACCGG GCAGUGGCCGCCGAAGUAAACAGACUGUCACUGAGCAGCGUAGCCAUCCCGCUGC UGUCCACAGGAGUGUUCAGCGGCGGAAGAGAUAGGCUGCAGCAAUCCCUCAACCA UCUAUUCACAGCAAUGGACGCCACGGACGCUGACGUGACCAUCUACUGCAGAGAC AAAAGUUGGGAGAAGAAAAUCCAGGAAGCCAUAGACAUGAGGACGGCUGUGGAG UUGCUCAAUGAUGACGUGGAGCUGACCACAGACUUGGUGAGAGUGCACCCG GACAGCAGCCUGGUGGGUCGUAAGGGCUACAGUACCACUGACGGGUCGCUGUACU CGUACUUUGAAGGUACGAAAUUCAACCAGGCUGCUAUUGAUAUGGCAGAGAUAC UGACGUUGUGGCCCAGACUGCAAGAGGCAAACGAACAGAUAUGCCUAUACGCGCU GGGCGAAACAAUGGACAACAUCAGAUCCAAAUGUCCGGUGAACGAUUCCGAUUC AUCAACACCUCCCAGGACAGUGCCCUGCCUGUGCCGCUACGCAAUGACAGCA GAACGGAUCGCCCGCCUUAGGUCACACCAAGUUAAAAGCAUGGUGGUUUGCUCAU CUUUUCCCCUCCCGAAAUACCAUGUAGAUGGGGUGCAGAAGGUAAAGUGCGAGA AGGUUCUCCUGUUCGACCCGACGGUACCUUCAGUGGUUAGUCCGCGGAAGUAUGC CGCAUCUACGACGGACCACUCAGAUCGGUCGUUACGAGGGUUUGACUUGGACUGG ACCACCGACUCGUCUUCCACUGCCAGCGAUACCAUGUCGCUACCCAGUUUGCAGU CGUGUGACAUCGACUCGAUCUACGAGCCAAUGGCUCCCAUAGUAGUGACGGCUGA CGUACACCCUGAACCCGCAGGCAUCGCGGACCUGGCGGCAGAUGUGCAUCCUGAA CCCGCAGACCAUGUGGACCUCGAGAACCCGAUUCCUCCACCGCGCCCGAAGAGAG CUGCAUACCUUGCCUCCCGCGCGGCGGAGCGACCGGUGCCGGCGCCGAGAAAGCC GACGCCUGCCCCAAGGACUGCGUUUAGGAACAAGCUGCCUUUGACGUUCGGCGAC UUUGACGAGCACGAGGUCGAUGCGUUGGCCUCCGGGAUUACUUUCGGAGACUUC GACGACGUCCUGCGACUAGGCCGCGCG GGUGCA SEQ ID. NO:45 Non-Structural Protein 4 (nsP4) Semliki Forest virus (SFV) Amino acid sequence YIFSSDTGSGHLQQKSVRQHNLQCAQLDAVEEEKMYPPKLDTEREKLLLLKMQMHPSE ANKSRYQSRKVENMKATVVDRLTSGARLYTGADVGRIPTYAVRYPRPVYSPTVIERFSS PDVAIAACNEYLSRNYPTVASYQITDEYDAYLDMVDGSDSCLDRATFCPAKLRCYPKH HAYHQPTVRSAVPSPFQNTLQNVLAAATKRNCNVTQMRELPTMDSAVFNVECFKRYA CSGEYWEEYAKQPIRITTENITTYVTKLKGPKAAALFAKTHNLVPLQEVPMDRFTVDMK RDVKVTPGTKHTEERPKVQVIQAAEPLATAYLCGIHRELVRRLNAVLRPNVHTLFDMSA EDFDAIIASHFHPGDPVLETDIASFDKSQDDSLALTGLMILEDLGVDQYLLDLIEAAFGEIS SCHLPTGTRFKFGAMMKSGMFLTLFINTVLNITIASRVLEQRLTDSACAAFIGDDNIVHG VISDKLMAERCASWVNMEVKIIDAVMGEKPPYFCGGFIVFDSVTQTACRVSDPLKRLFK LGKPLTAEDKQDEDRRRALSDEVSKWFRTGLGAELEVALTSRYEVEGCKSILIAMATLA RDIKAFKKLRGPVIHLYGGPRLVR SEQ ID. NO:46 Non-Structural Protein 4 (nsP4) Semliki Forest virus (SFV) DNA sequence 55 WAS:164427.1TATATTTTCTCCTCGGACACTGGCAGCGGACATTTACAACAAAAATCCGTTAGGCAG CACAATCTCCAGTGCGCACAACTGGATGCGGTCGAGGAGGAGAAAATGTACCCGCC AAAATTGGATACTGAGAGGGAGAAGCTGTTGCTGCTGAAAATGCAGATGCACCCAT CGGAGGCTAATAAGAGTCGATACCAGTCTCGCAAAGTGGAGAACATGAAAGCCACG GTGGTGGACAGGCTCACATCGGGGGCCAGATTGTACACGGGAGCGGACGTAGGCCG CATACCAACATACGCGGTTCGGTACCCCCGCCCCGTGTACTCCCCTACCGTGATCGA AAGATTCTCAAGCCCCGATGTAGCAATCGCAGCGTGCAACGAATACCTATCCAGAA ATTACCCAACAGTGGCGTCGTACCAGATAACAGATGAATACGACGCATACTTGGAC ATGGTTGACGGGTCGGATAGTTGCTTGGACAGAGCGACATTCTGCCCGGCGAAGCTC CGGTGCTACCCGAAACATCATGCGTACCACCAGCCGACTGTACGCAGTGCCGTCCCG TCACCCTTTCAGAACACACTACAGAACGTGCTAGCGGCCGCCACCAAGAGAAACTG CAACGTCACGCAAATGCGAGAACTACCCACCATGGACTCGGCAGTGTTCAACGTGG AGTGCTTCAAGCGCTATGCCTGCTCCGGAGAATATTGGGAAGAATATGCTAAACAA CCTATCCGGATAACCACTGAGAACATCACTACCTATGTGACCAAATTGAAAGGCCC GAAAGCTGCTGCCTTGTTCGCTAAGACCCACAACTTGGTTCCGCTGCAGGAGGTTCC CATGGACAGATTCACGGTCGACATGAAACGAGATGTCAAAGTCACTCCAGGGACG AAACACACAGAGGAAAGACCCAAAGTCCAGGTAATTCAAGCAGCGGAGCCATTGG CGACCGCTTACCTGTGCGGCATCCACAGGGAATTAGTAAGGAGACTAAATGCTGTGT TACGCCCTAACGTGCACACATTGTTTGATATGTCGGCCGAAGACTTTGACGCGATCA TCGCCTCTCACTTCCACCCAGGAGACCCGGTTCTAGAGACGGACATTGCATCATTCG ACAAAAGCCAGGACGACTCCTTGGCTCTTACAGGTTTAATGATCCTCGAAGATCTAG GGGTGGATCAGTACCTGCTGGACTTGATCGAGGCAGCCTTTGGGGAAATATCCAGCT GTCACCTACCAACTGGCACGCGCTTCAAGTTCGGAGCTATGATGAAATCGGGCATGT TTCTGACTTTGTTTATTAACACTGTTTTGAACATCACCATAGCAAGCAGGGTACTGG AGCAGAGACTCACTGACTCCGCCTGTGCGGCCTTCATCGGCGACGACAACATCGTTC ACGGAGTGATCTCCGACAAGCTGATGGCGGAGAGGTGCGCGTCGTGGGTCAACATG GAGGTGAAGATCATTGACGCTGTCATGGGCGAAAAACCCCCATATTTTTGTGGGGG ATTCATAGTTTTTGACAGCGTCACACAGACCGCCTGCCGTGTTTCAGACCCACTTAA GCGCCTGTTCAAGTTGGGTAAGCCGCTAACAGCTGAAGACAAGCAGGACGAAGACA GGCGACGAGCACTGAGTGACGAGGTTAGCAAGTGGTTCCGGACAGGCTTGGGGGCC GAACTGGAGGTGGCACTAACATCTAGGTATGAGGTAGAGGGCTGCAAAAGTATCCT CATAGCCATGGCCACCTTGGCGAGGGACATTAAGGCGTTTAAGAAATTGAGAGGAC CTGTTATACACCTCTACGGCGGTCCTAGATTGGTGCGT SEQ ID. NO:47 Non-Structural Protein 4 (nsP4) Semliki Forest virus (SFV) mRNA sequence UAUAUUUUCUCCUCGGACACUGGCAGCGGACAUUUACAACAAAAAUCCGUUAGGC AGCACAAUCUCCAGUGCGCACAACUGGAUGCGGUCGAGGAGGAGAAAAUGUACCC GCCAAAAUUGGAUACUGAGAGGGAGAAGCUGUUGCUGCUGAAAAUGCAGAUGCA CCCAUCGGAGGCUAAUAAGAGUCGAUACCAGUCUCGCAAAGUGGAGAACAUGAA AGCCACGGUGGUGGACAGGCUCACAUCGGGGGCCAGAUUGUACACGGGAGCG GACGUAGGCCGCAUACCAACAUACGCGGUUCGGUACCCCCGCCCCGUGUACUCCC CUACCGUGAUCGAAAGAUUCUCAAGCCCCGAUGUAGCAAUCGCAGCGUGCAACGA AUACCUAUCCAGAAAUUACCCAACAGUGGCGUCGUACCAGAUAACAGAUGAAUAC 56 WAS:164427.1GACGCAUACUUGGACAUGGUUGACGGGUCGGAUAGUUGCUUGGACAGAGCGACA UUCUGCCCGGCGAAGCUCCGGUGCUACCCGAAACAUCAUGCGUACCACCAGCCGA CUGUACGCAGUGCCGUCCCGUCACCCUUUCAGAACACACUACAGAACGUGCUAGC GGCCGCCACCAAGAGAAACUGCAACGUCACGCAAAUGCGAGAACUACCCACCAUG GACUCGGCAGUGUUCAACGUGGAGUGCUUCAAGCGCUAUGCCUGCUCCGGAGAAU AUUGGGAAGAAUAUGCUAAACAACCUAUCCGGAUAACCACUGAGAACAUCACUA CCUAUGUGACCAAAUUGAAAGGCCCGAAAGCUGCUGCCUUGUUCGCUAAGACCCA CAACUUGGUUCCGCUGCAGGAGGUUCCCAUGGACAGAUUCACGGUCGACAUGAAA CGAGAUGUCAAAGUCACUCCAGGGACGAAACACACAGAGGAAAGACCCAAAGUCC AGGUAAUUCAAGCAGCGGAGCCAUUGGCGACCGCUUACCUGUGCGGCAUCCACAG GGAAUUAGUAAGGAGACUAAAUGCUGUGUUACGCCCUAACGUGCACACAUUGUU UGAUAUGUCGGCCGAAGACUUUGACGCGAUCAUCGCCUCUCACUUCCACCCAGGA GACCCGGUUCUAGAGACGGACAUUGCAUCAUUCGACAAAAGCCAGGACGACUCCU UGGCUCUUACAGGUUUAAUGAUCCUCGAAGAUCUAGGGGUGGAUCAGUACCUGC UGGACUUGAUCGAGGCAGCCUUUGGGGAAAUAUCCAGCUGUCACCUACCAACUGG CACGCGCUUCAAGUUCGGAGCUAUGAUGAAAUCGGGCAUGUUUCUGACUUUGUU UAUUAACACUGUUUUGAACAUCACCAUAGCAAGCAGGGUACUGGAGCAGAGACU CACUGACUCCGCCUGUGCGGCCUUCAUCGGCGACGACAACAUCGUUCACGGAGUG AUCUCCGACAAGCUGAUGGCGGAGAGGUGCGCGUCGUGGGUCAACAUGGAGGUG AAGAUCAUUGACGCUGUCAUGGGCGAAAAACCCCCAUAUUUUUGUGGGGGAUUC AUAGUUUUUGACAGCGUCACACAGACCGCCUGCCGUGUUUCAGACCCACUUAAGC GCCUGUUCAAGUUGGGUAAGCCGCUAACAGCUGAAGACAAGCAGGACGAAGACA GGCGACGAGCACUGAGUGACGAGGUUAGCAAGUGGUUCCGGACAGGCUUGGGGG CCGAACUGGAGGUGGCACUAACAUCUAGGUAUGAGGUAGAGGGCUGCAAAAGUA UCCUCAUAGCCAUGGCCACCUUGGCGAGGGACAUUAAGGCGUUUAAGAAAUUGA GA GGACCUGUUAUACACCUCUACGGCGGUCCUAGAUUGGUGCGU 57 WAS:164427.1

Claims

WHAT IS CLAIMED IS:

1. A method for immunizing a subject preventing infection by a pathogen, comprising the steps of: (1) administering to the subject an effective amount of a prime vaccine, wherein the prime vaccine comprises one or more mRNA constructs encoding one or more prime immunogens, wherein the one or more prime immunogens are selected from the group consisting of immunogens from the pathogen and modified immunogens from the pathogen; and (2) administering to the subject an effective amount of a boost vaccine, wherein the booster vaccine comprises one or more booster immunogens, wherein at least one of the booster immunogens has an amino acid sequence identity of 75% or greater to at least one of the prime immunogens, wherein the boost vaccine is in a non-mRNA vaccine modality, and wherein the boost vaccine is administered after the administration of the prime vaccine.

2. The method of claim 1, wherein at least one of the booster immunogens has an amino acid sequence identity of 85% or greater to at least one of the prime immunogens.

3. The method of claim 1, wherein at least one of the booster immunogens has an amino acid sequence identity of 90% or greater to at least one of the prime immunogens.

4. The method of claim 1, wherein at least one of the booster immunogens has an amino acid sequence identity of 95% or greater to at least one of the prime immunogens.

5. The method of any one of claims 1 to 4, wherein boost vaccine comprises a recombinant protein.

6. The method of any one of claims 1 to 5, wherein the pathogen is HIV-1, wherein the prime vaccine comprises one or more mRNA constructs encoding one or more HIV-1 envelop proteins and / or modified HIV-1 envelope proteins, and wherein the boost vaccine comprises the one or more HIV-1 envelope proteins and / or modified HIV-1 envelope proteins. 58 WAS:164427.

17. The method of claim 6, wherein the one or more HIV-1 envelop proteins are selected from the group consisting of gp120, gp140 and envelope proteins with optimized structural designs.

8. A method for introducing an immune response against HIV-1 in a subject, comprising the steps of: (1) administering to the subject an effective amount of a prime vaccine, wherein the prime vaccine comprises one or more mRNA constructs encoding one or more prime immunogens, wherein the one or more prime immunogens are selected from the group consisting of HIV-1 env immunogens and modified HIV-1 env immunogens; and (2) administering to the subject an effective amount of a boost vaccine, wherein the booster vaccine comprises one or more booster immunogens, wherein at least one of the booster immunogens has an amino acid sequence identity of 75% or greater to at least one of the prime immunogens, wherein the boost vaccine is in a non-mRNA vaccine modality, and wherein the boost vaccine is administered after the administration of the prime vaccine.

9. The method of claim 8, wherein at least one of the booster immunogens has an amino acid sequence identity of 85% or greater to at least one of the prime immunogens.

10. The method of claim 8, wherein at least one of the booster immunogens has an amino acid sequence identity of 90% or greater to at least one of the prime immunogens.

11. The method of claim 8, wherein at least one of the booster immunogens has an amino acid sequence identity of 95% or greater to at least one of the prime immunogens.

12. The method of any one of claims 8 to 11, wherein the one or more HIV-1 envelop proteins are selected from the group consisting of gp120, gp140 and HIV-1 envelope proteins with optimized structural designs.

13. The method of any one of claims 8 to 12, wherein the prime vaccine comprises one or more mRNA constructs encoding one or more HIV-1 gp120 and / or modified HIV-1 gp120, and 59 WAS:164427.1wherein the boost vaccine comprises the one or more HIV-1 gp120 protein and / or modified HIV- 1 gp120 protein.

14. An mRNA-primed vaccine formulation, comprising: (1) a mRNA component comprising one or more mRNA constructs encoding one or more prime immunogens from a pathogen; and (2) a non-mRNA component comprising one or more booster immunogens, wherein at least one of the booster immunogens has an amino acid sequence identity of 75% or greater to at least one of the prime immunogens; wherein (1) and (2) are formulated for separate administration.

15. The mRNA-primed vaccine formulation of claim 14, wherein the pathogen is HIV-1.

16. The mRNA-primed vaccine formulation of claim 14 or 15, wherein the one or more prime immunogens are selected from the group consisting of HIV-1 env immunogens and modified HIV-1 env immunogens.

17. The mRNA-primed vaccine formulation of claim 16, wherein the HIV-1 env immunogens and modified HIV-1 env immunogens are selected from the group consisting of gp120, gp140 and HIV-1 envelope proteins with optimized structural designs.

18. The mRNA-primed vaccine formulation of claim 16 or claim 17, wherein the prime vaccine comprises one or more mRNA constructs encoding one or more HIV-1 gp120 and / or modified HIV-1 gp120, and wherein the boost vaccine comprises the one or more HIV-1 gp120 protein and / or modified HIV-1 gp120 protein.

19. The mRNA-primed vaccine formulation of any one of claims 14 to 18, wherein at least one of the booster immunogens has an amino acid sequence identity of 90% or greater to at least one of the prime immunogens. 60 WAS:164427.

120. The mRNA-primed vaccine formulation of any one of claims 14 to 18, wherein at least one of the booster immunogens has an amino acid sequence identity of 95% or greater to at least one of the prime immunogens. 61 WAS:164427.1

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