HK1164896B

HK1164896B - Antiviral vaccines with improved cellular immunogenicity

Info

Publication number: HK1164896B
Application number: HK12105453.5A
Authority: HK
Inventors: Dan H. Barouch; Bette T. Korber; William M. Fischer
Original assignee: Beth Israel Deaconess Medical Center; Los Alamos National Security, Llc
Priority date: 2008-11-18
Filing date: 2009-11-18
Publication date: 2016-06-10

Description

Antiviral vaccines with improved cellular immunogenicity

Statement of federally sponsored research

This study was partially sponsored by NIH Foundation Nos. U19-AI066305 and U19-AI 078526. The government has certain rights in the invention.

Technical Field

The present invention provides compositions, methods and kits for treating or preventing viral infections. The multivalent (e.g., bivalent) vaccines described herein incorporate computationally optimized viral polypeptides that can increase the diversity or breadth and depth of the cellular immune response of the vaccinated subject.

Background

Vaccines that elicit a cellular immune response against the virus must reflect the global viral diversity in order to effectively treat or prevent viral infections. For example, the initiation of a strong and diverse HIV-1 specific T cell response may be critical for an effective HIV-1 vaccine. Cytotoxic T Lymphocyte (CTL) responses are associated with a slow disease progression in humans, and the importance of CTL responses in non-human primate vaccination models is well established. Since the highly variable envelope (Env) is the primary target for neutralizing antibodies against HIV, and vaccine antigens also need to be tailored to elicit these antibody responses, T cell vaccine components can target more conserved proteins to elicit responses that are more likely to be cross-reactive. But even the most conserved HIV-1 proteins are so diverse that mutation will be a problem. Artificial central sequence vaccination methods, such as consensus and progenitor HIV-1 sequences, can essentially "split" the inter-strain differences and stimulate responses with enhanced cross-reactivity compared to the native strain vaccines. The consensus antigen represents the synthetic antigen sequence, which is the only best "average level" among all circulating strains. Although these antigens are capable of eliciting direct cellular immune responses, the breadth and magnitude of these responses has not been greatly improved in previous vaccine strategies.

The development of next generation vaccines for the treatment or prevention of viral infections must lead to an increase in the breadth of cellular immunity in order for the vaccination results to be successful. The need for such vaccines is particularly acute when treating or preventing HIV-1.

Summary of The Invention

In a first aspect, the invention features a vaccine for treating or reducing the risk of viral infection in a mammal, such as a human, that includes at least two different optimized viral polypeptides (e.g., 2, 3, 4, 5 or more different optimized viral polypeptides), wherein the optimized viral polypeptides correspond to the same viral gene product. In one embodiment, the viral infection is caused by a retrovirus, reovirus, picornavirus, togavirus, orthomyxovirus, paramyxovirus, calicivirus, arenavirus, flavivirus, filovirus, bunyavirus, coronavirus, astrovirus, adenovirus, papilloma virus, parvovirus, herpesvirus, hepadnavirus, poxvirus, or polyomavirus. In other embodiments, the retrovirus is human immunodeficiency virus type 1 (HIV-1), and the viral gene product comprises Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr, or Vpu. In another embodiment, the vaccine comprises only two (no more than two) optimized viral polypeptides corresponding to one of Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr, or Vpu viral gene products. In another embodiment, the vaccine does not include optimized viral polypeptides corresponding to Gag and Nef. In yet another embodiment, the vaccine comprises at least two different optimized viral polypeptides (e.g., 2, 3, 4, 5 or more different optimized viral polypeptides) for a first viral gene product selected from Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr and Vpu, and one or more different optimized viral polypeptides (e.g., 2, 3, 4, 5 or more different optimized viral polypeptides) for a second viral gene product different from the first viral gene product selected from Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr and Vpu.

In a second aspect, the invention features a vaccine for treating or reducing the risk of infection by human immunodeficiency virus type 1 (HIV-1) in a mammal, such as a human, that includes an optimized viral polypeptide having at least 7 contiguous amino acids (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 100, 150, 175, 200, 250, 300, 350, 400, 450, 500 or more contiguous amino acids in length) that is complementary to SEQ id no: 1-29 has at least 85% amino acid sequence identity (identity). In one embodiment, the optimized viral polypeptide has at least 7 contiguous amino acids (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 100, 150, 175, 200, 250, 300, 350, 400, 450, 500 or more contiguous amino acids in length) that are identical to SEQ ID NOS: 1-29, have amino acid sequence identity. In another embodiment, the optimized viral polypeptide has the amino acid sequence of SEQ ID NOS: 1-29, or a pharmaceutically acceptable salt thereof. In yet another embodiment, the vaccine comprises at least two optimized viral polypeptides selected from any one or more of groups a) -k): a) SEQ ID NOS: 1 and 2; b) SEQ ID NOS: 3. 4 and 5; c) SEQ ID NOS: 6 and 7; d) SEQ ID NOS: 8-12; e) SEQ ID NOS: 13. 14 and 15; f) SEQ ID NOS: 16. 17 and 18; g) SEQ ID NOS: 19 and 20; h) SEQ ID NOS: 21. 22 and 23; i) SEQ ID NOS: 24 and 25; j) SEQ ID NOS: 26 and 27; k) SEQ ID NOS: 21-22. In another embodiment, the vaccine may comprise a pair of optimized viral polypeptides selected from any one of the above groups a) -k), and one or more different optimized viral polypeptides from the same or different groups a) -k). In other embodiments, the vaccine may comprise at least three or four or more optimized viral polypeptides from one or more of groups a) -k).

In a third aspect, the invention features a vaccine for treating or reducing the risk of viral infection in a mammal, such as a human, comprising at least two different pairs of optimized viral polypeptides, wherein each pair of optimized viral polypeptides corresponds to the same viral gene product, and wherein only two (nomore than two) optimized viral polypeptides that have been incorporated into the vaccine correspond to the same viral gene product. In one embodiment, the vaccine comprises at least three pairs of different optimized viral polypeptides. In another embodiment, the vaccine comprises at least four pairs of different optimized viral polypeptides. In one embodiment, the viral infection is caused by a retrovirus, reovirus, picornavirus, togavirus, orthomyxovirus, paramyxovirus, calicivirus, arenavirus, flavivirus, filovirus, bunyavirus, coronavirus, astrovirus, adenovirus, papilloma virus, parvovirus, herpesvirus, hepadnavirus, poxvirus, or polyomavirus. In other embodiments, the retrovirus is human immunodeficiency virus type 1 (HIV-1), and the viral gene product comprises Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr, or Vpu. In yet another embodiment, the vaccine comprises only two optimized viral polypeptides corresponding to one of the viral gene products Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr or Vpu. In another embodiment, the vaccine does not include optimized viral polypeptides corresponding to Gag and Nef. In yet another embodiment, the vaccine comprises at least three pairs of different optimized viral polypeptides corresponding to any three of the viral gene products Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr or Vpu. In another embodiment, the vaccine comprises at least four pairs of different optimized viral polypeptides corresponding to any four of the viral gene products Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr or Vpu.

In an embodiment of any of the first three aspects of the invention, the vaccine elicits a cellular immune response against the viral gene product. In another embodiment, the vaccine elicits a cellular immune response against HIV-1. In yet another embodiment, the nucleotide sequence of at least one different optimized viral polypeptide is encoded by a nucleic acid or vector. In one embodiment, the vector is a recombinant adenovirus, such as a serotype 26 adenovirus (Ad26), a serotype 34 adenovirus (Ad34), a serotype 35 adenovirus (Ad35), a serotype 48 adenovirus (Ad48), or a serotype 5HVR48 adenovirus (Ad5HVR 48). In yet another embodiment, the vaccine is combined with a pharmaceutically acceptable carrier, excipient, or diluent.

In a fourth aspect, the invention features a nucleotide comprising a nucleotide sequence of an optimized viral polypeptide having at least 7 contiguous amino acids (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 100, 150, 175, 200, 250, 300, 350, 400, 450, 500 or more contiguous amino acids in length) that is substantially identical to the nucleotide sequence of SEQ ID NOS: 1-29 has at least 85% amino acid sequence identity. In one embodiment, the optimized viral polypeptide has at least 7 contiguous amino acids (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 100, 150, 175, 200, 250, 300, 350, 400, 450, 500 or more contiguous amino acids in length) that is substantially identical to the sequence of SEQ id no: 1-29, has sequence identity. In another embodiment, the optimized viral polypeptide has the amino acid sequence of SEQ ID NOS: 1-29, or a pharmaceutically acceptable salt thereof. In yet another embodiment, the nucleic acid comprises a vector. In one embodiment, the vector is a recombinant adenovirus, such as a serotype 26 adenovirus (Ad26), a serotype 34 adenovirus (Ad34), a serotype 35 adenovirus (Ad35), a serotype 48 adenovirus (Ad48), or a serotype 5HVR48 adenovirus (Ad5HVR 48).

In a fifth aspect, the invention features an optimized viral polypeptide having at least 7 contiguous amino acids (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 100, 150, 175, 200, 250, 300, 350, 400, 450, 500 or more contiguous amino acids in length) that hybridizes to SEQ ID NOS: 1-29 has at least 85% amino acid sequence identity. In one embodiment, the optimized viral polypeptide has at least 7 contiguous amino acids (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 100, 150, 175, 200, 250, 300, 350, 400, 450, 500 or more contiguous amino acids in length) that is substantially identical to the sequence of SEQ ID NOS: 1-29, and any one of the amino acid sequences described herein is identical. In another embodiment, the optimized viral polypeptide has the amino acid sequence of seq id NOS: 1-29, or a pharmaceutically acceptable salt thereof.

In a sixth aspect, the invention features a method of treating or reducing the risk of a viral infection in a mammal, such as a human, by administering a vaccine or nucleic acid of the invention. In one embodiment, the viral infection is caused by a retrovirus, reovirus, picornavirus, togavirus, orthomyxovirus, paramyxovirus, calicivirus, arenavirus, flavivirus, filovirus, bunyavirus, coronavirus, astrovirus, adenovirus, papilloma virus, parvovirus, herpesvirus, hepadnavirus, poxvirus, or polyomavirus. In yet another embodiment, the retrovirus is human immunodeficiency virus type 1 (HIV-1), and the viral gene product comprises Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr, or Vpu. In one embodiment, the vaccine or nucleic acid elicits a cellular immune response against the viral gene product.

In a seventh aspect, the invention features a method of making a vaccine for treating or reducing the risk of viral infection in a mammal, such as a human, by synthesizing a vaccine according to the invention.

In an eighth aspect, the invention features a method of making a vaccine for treating or reducing the risk of viral infection in a mammal, such as a human, by contacting a nucleic acid of the invention with a cell and isolating an optimized viral polypeptide.

In an embodiment of the seventh or eighth aspect of the invention, the optimized viral polypeptide elicits a cellular immune response when administered to a mammal. The cellular immune response may be directed against a viral gene product. In another embodiment, the viral infection is caused by a retrovirus, reovirus, picornavirus, togavirus, orthomyxovirus, paramyxovirus, calicivirus, arenavirus, flavivirus, filovirus, bunyavirus, coronavirus, astrovirus, adenovirus, papilloma virus, parvovirus, herpesvirus, hepadnavirus, poxvirus, or polyomavirus. In yet another embodiment, the retrovirus is human immunodeficiency virus type 1 (HIV-1), and the viral gene product comprises Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr, or Vpu.

In a ninth aspect, the invention features a kit that includes the vaccine of the invention, a pharmaceutically acceptable carrier, excipient, or diluent, and instructions for use thereof. In one embodiment, the kit further comprises an adjuvant.

In a final aspect, the invention features a kit that includes a nucleic acid of the invention, a pharmaceutically acceptable carrier, excipient, or diluent, and instructions for use thereof. In one embodiment, the kit further comprises an adjuvant.

In embodiments of all aspects of the invention, the optimized viral polypeptide is encoded by a nucleotide sequence optimized for expression in humans (e.g., any one of SEQ ID NOS: 5, 10, 11, 12, 15, 18, and 23).

Definition of

An "optimized viral polypeptide" or "computationally optimized viral polypeptide" refers to an immunogenic polypeptide that is not a naturally occurring viral peptide, polypeptide or protein. Optimization of viral polypeptide sequences is initially generated by modifying the amino acid sequence of one or more naturally occurring viral gene products (e.g., peptides, polypeptides, and proteins) to increase the breadth, strength, depth, or longevity of the anti-viral immune response (e.g., cellular or humoral immune response) generated following immunization of a mammal (e.g., a human) such as, for example, when incorporated into a vaccine of the present invention. Thus, the optimized viral polypeptide may correspond to a "parental" viral gene sequence; alternatively, the optimized viral polypeptide may not correspond to a particular "parental" viral gene sequence, but may correspond to the sequence of a variant or quasi-species-generated analog of the virus. Modifications to the viral gene sequence included in the optimized viral polypeptide include amino acid additions, substitutions and deletions. In one embodiment of the invention, the optimized viral polypeptide is a composite or fused amino acid sequence of two or more naturally occurring viral gene products (e.g., natural or clinical viral isolates), wherein each potential antigenic epitope (e.g., each contiguous or overlapping amino acid sequence of 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids in length) is analyzed and modified to increase the immunogenicity of the optimized viral polypeptide produced. Optimized viral polypeptides corresponding to different viral gene products may also be fused to facilitate their incorporation into the vaccines of the present invention. Methods of generating optimized viral polypeptides in, for example, Fisher et al, "multivalent Vaccine for Optimal Coverage of Potential T cell epitopes in Global HIV-1 Variants (temporal Coverage of Potential T-CeIlepipopes in Global HIV-I Variants)" nat. Med.13 (1): 100-106(2007) and international patent application publication WO 2007/024941, which are incorporated herein by reference. Once the optimized viral polypeptide sequences are generated, the corresponding polypeptides can be produced or managed by standard techniques (e.g., recombinant viral vectors such as the adenoviral vectors disclosed in international patent application publications WO 2006/040330 and WO2007/104792, incorporated herein by reference).

"pharmaceutically acceptable carrier" refers to a carrier that is physiologically acceptable to the mammal being treated and that retains the therapeutic properties of the compound with which it is co-administered. A typical pharmaceutically acceptable carrier is normal saline. Other physiologically acceptable carriers and formulations thereof are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (18 th., ed.a. gennaro, 1990, Mack Publishing Company, Easton, PA), herein incorporated by reference.

"vector" refers to a DNA construct comprising a promoter operably linked to a downstream gene or coding region (e.g., a cDNA or genomic DNA fragment encoding a polypeptide or polypeptide fragment). Upon introduction of the vector into a recipient cell (e.g., prokaryotic or eukaryotic cells, such as bacteria, yeast, insect cells, mammalian cells, depending on the promoter in the expression vector) or organism (including, for example, humans), the cell is capable of expressing the vector-encoded mRNA, which is then translated into the encoded optimized viral polypeptide described herein. Vectors for in vitro transcription/translation are well known in the art and are further described herein. The vector may be a genetically engineered plasmid, a virus or an artificial chromosome derived from, for example, a bacteriophage, adenovirus, retrovirus, poxvirus, or herpes virus.

"viral gene product" refers to any naturally occurring viral peptide, polypeptide, or protein, or fragment thereof. In one embodiment of the invention, the viral gene product is derived from human immunodeficiency virus type 1 (HIV-1). HIV-1 viral gene products include Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr, and Vpu polypeptides.

Drawings

FIG. 1 is a chart illustrating the expanded breadth of computationally optimized HIV-1Gag, Pol, and Env viral polypeptides for the global potential T cell epitope (PTE) peptide in rhesus monkeys. Animals immunized with the optimized viral polypeptide (blue) reacted with the memory peptide pool in the greatest number.

FIG. 2 is a graph showing that computationally modified HIV-1Gag, Pol, and Env viral polypeptides expand the breadth of epitope-specific cellular immune responses.

FIG. 3 illustrates the breadth of cellular immune responses detected in rhesus macaques immunized with HIV-1 viral gene products Gag, Pol, and Env from the computationally modified viral polypeptides of the invention, as well as in animals vaccinated with the consensus HIV-1 antigen or HIV-1 clade C isolated antigen. Animals immunized with the optimized viral polypeptide (blue) reacted with the memory peptide pool in the greatest number. Since the animals were inbred, the pools varied from animal to animal. Gag, Pol, and Env each elicit a variety of cellular immune responses and may share a pattern of response.

Fig. 4A-4C are graphs showing potential epitopes shared between different vaccines (bivalent mosaic sequence (Mos2), M consensus sequence (Mcon), and optimized clade C (optc)) tested with viral polypeptides (Pol (fig. 4A), Gag (fig. 4B), and Env (fig. 4C)). Fig. 4A-C show the relative coverage of the full-length genome of the current HIV database and PTE peptides by different vaccine candidates.

Figure 5 is a graph showing that the number of responses (each response considered to be an independent event, whether overlapping or not) of PTE peptides to a bivalent mosaic (Mos2) vaccine is greater than the number of responses to a group M consensus (Mcon) vaccine and a native virus vaccine (optimized clade C (native C (best))), which has been selected to produce optimal coverage of group M pool (OptC) vaccine antigens. Figure 5 shows the number of PTE peptide responses per animal for proteins, CD8+ T cells, and CD4+ T cells. Statistically, Mos2 > Mcon to OptC (Mcon shows a trend of more responses than OptC). Wilcoxon p value of Mos2 compared to Mcon: the value of p is 0.001058.

Figure 6 is a graph showing the number of PTE peptides that elicit a T cell response. The PTE peptide with a median of 16 (range: 12-29) in the bivalent mosaic (Mos2) vaccine elicited a response within CD8+ T cells, whereas the peptide with a median of only 6 (range: 0-7) in the Mcon and OptC peptides of only 3 (range; 0-3) elicited a response within CD8+ T cells. A PTE peptide with a median of 4 (range: 2-6) in the bivalent mosaic (Mos2) vaccine triggered a response within CD4+ T cells, while a peptide with a median of only 1 (range: 0-2) in the Mcon and OptC peptides of only 0.5 (range: 0-2) triggered a response within CD4+ T cells. Thus, the trend of response is Mos2 > Mcon > OptC.

Figure 7 is a schematic diagram summarizing the profile of all CD8+ T cell Gag PTE peptides recognized by T cells from each animal studied (see example 3 below). Animal numbers, peptide pools and peptide numbers mark the boundaries of each active peptide. Groups are symbolized by: mos 2;ConM; + -, OptC. Gag is included herein as an example. Even if the animals are outcrossingCD8 responses still tended to cluster. Mosaic vaccines have potential advantages over monovalent vaccines. Mosaic (chimera) has a better chance of stimulating a response, which interacts with the more common variants. Mosaic type also stimulates multiple responses to different forms present in the mixture. Thus, the mosaic type has the potential to block the general escape pathway. In our studies, mosaic vaccines tend to stimulate T cell responses, recognizing more overlapping peptides. There are many hot spots where active peptides are localized. The PTE peptides were designed to maximize coverage of potential epitopes (or 9 amino acid stretches of nonamers) of the HIV-1M group in peptide reagents used to evaluate vaccines. Inevitably, there is much overlap in the PTE peptides, but due to the algorithms, the overlap is usually that of certain variations.Fig. 7 discloses SEQ ID NO: 42.

FIG. 8 is a schematic diagram summarizing the map (mapping) of the CD4+ T cell Gag PTE peptide recognized by the T cells of each animal studied.Fig. 8 discloses SEQ ID NO: 43.

figure 9 is a graph depicting a typical pattern of PTE responses against either the ConM vaccine or the best natural vaccine, aligning the peptides that elicit the response with those of the relevant region of the vaccine. A good match of stable peptide fragments with identity between the vaccine and the target PTE peptide is essential to achieve a response to these vaccines.FIG. 9 discloses SEQ ID NOS 44-57 in order of appearance, respectively.

Figure 10 is a graph illustrating that a mosaic vaccine generates multiple responses, identifying multiple variant overlapping peptides with insignificant antigenic competition and with a broad local response. In particular, four labile (variable) PTE peptides are recognized. In addition, in the overlap region, both mosaic forms and a combination of both are identified. Finally, the new form (S) is identified.FIG. 10 discloses in order of occurrence respectively SEQ ID NOS 58-63。

FIG. 11 is a graph illustrating a typical pattern of CD8+ PTE peptide response in mosaic vaccinated animals (361-07). 22 PTE peptides were tested and 8 CD8 response regions were identified; 5 areas are included in one or moreAnother variable peptide with amino acid matching in mosaic sequence (mosaic). 5 CD4 response regions were identified. Thus, more variable peptides are seen in a given region in the T cell response to mosaic sequences. This seems to be especially true for the CD8T cell response. This may be the result of eliciting multiple T cell clones to recognize epitope variants, and this may block appropriate escape routes. Not only are there more responses, but they are deeper and cover more variants.FIG. 11 discloses in sequence of occurrence the sequences as depicted in SEQ ID NOS 64-101, respectively CD8 response. Separately disclosed in order of appearance CD4 as described in SEQ ID NOS 102-117 And (6) responding.

Figure 12 is a graph showing the number of overlapping labile PTE peptides that span the region targeted by vaccine-induced T cells.

Figure 13 is a graph showing that 2 mosaic antigen vaccines generate more T cell responses against regions containing one or more overlapping PTE peptides relative to Mcon and OptC vaccines. Figure 13 is similar to figure 5, showing monkeys in the same order from right to left, but with the scale changed to reflect the number of responses to a region containing one or more overlapping PTE peptides rather than a single peptide.

Figure 14 is a graph showing the number of T cell responses following administration of bivalent mosaic vaccine (Mos2), Mcon vaccine, and OptC vaccine to animals. The bivalent mosaic (Mos2) vaccine elicited a median 8 response in CD8+ T cells, whereas the Mcon and OptC vaccines elicited CD8+ T cell responses with a median of only 3 (range: 0-6) and 1.5 (range: 0-5) peptides, respectively. The bivalent mosaic (Mos2) vaccine elicited a median 3 (range: 2-5) response in CD4+ T cells, whereas the Mcon and OptC vaccines elicited CD4+ T cell responses with median only 1 (range: 0-2) and 0.5 (range: 0-2), respectively. Thus, the trend of response is Mos2 > Mcon > OptC.

Figure 15 is a graph showing that mosaic vaccines are able to elicit more responses to cross-react with clade C native proteins than do clade C (clade C) native vaccines: GAG integration peptide (fermented peptide) represents 5 proteins. Animals vaccinated with the group M consensus sequence or best coverage clade C native protein responded 0-2 to peptides from these proteins, while animals vaccinated with the mosaic vaccine were able to respond 1-5 peptide pools. Mosaic vaccines elicit more responses to each test protein than Mcon or optimal C sequence. The T cell response elicited by the mosaic vaccine also recognized a more integrated peptide group throughout the authentic Gag protein. 10-12 subpools-10 × pentadecameric peptide (except 96ZM Gag, which is 5 × icosameric peptide).

Figure 16 is a graph showing that the mosaic design is stable to changes in viral polypeptides (e.g., Gag M) over time.

Figure 17 is a graph showing that coverage optimized with nonamers is stable (shown as Gag) near (e.g., octa-dodecamer) optimal length.

FIG. 18 is a graph showing that increasing the number of mutations increases the coverage, but only with decreasing recovery (Gag) (shown).

FIGS. 19A-19B are graphs showing the breadth and magnitude of epitope-specific T lymphocyte responses to PTE peptide. FIG. 19A is a graph showing the number of epitope-specific CD4+ (top) and CD8+ (bottom) T lymphocytes responding to a single PTE peptide after a single immunization with rAd26 vector expressing mosaic sequence (blue), M consensus sequence (green), clade B + clade C (purple), or best natural clade C (red) HIV-1Gag, Pol, and Env antigens. A single monkey is depicted on the x-axis. The different shading of each color reflects the response to different antigens (Gag, Pol, Env). FIG. 19B is a graph showing the number of CD4+ (top) and CD8+ (bottom) T lymphocyte response regions.

FIGS. 20A-20C show a scheme showing localization (mapping) to HIV-1Gag (FIG. 20A)(SEQ ID NO：118)Pol (FIG. 20B)(SEQ ID NO：119)And Env (FIG. 20C)(SEQ ID NO：120)CD8+ T lymphocyte response to PTE peptide 4 weeks after immunization of the protein sequence. Color representation accepts mosaic sequence (blue), M consensus sequence (green), clade B + clade C (purple)Color) or the best natural clade C (red) of the monkeys for HIV-1Gag, Pol, and Env antigens. For each epitope, monkey numbering, antigen (G, Gag; P, Pol; E, Env), subpool numbering and individual PTE peptide numbering are shown.

FIGS. 21A-21C show a scheme showing localization to HIV-I Gag (FIG. 21A)(SEQ ID NO：121)Pol (FIG. 21B)(SEQ ID NO：122)And Env (FIG. 21C)(SEQ ID NO：123)CD4+ T lymphocyte response to PTE peptide 4 weeks after immunization of the protein sequence. Color indicates monkeys receiving mosaic sequence (blue), M consensus (green), clade B + clade C (purple), or best natural clade C (red) HIV-1Gag, Pol, and Env antigens. For each epitope, monkey numbering, antigen (G, Gag; P, Pol; E, Env), subpool numbering and individual PTE peptide numbering are shown.

Figure 22 shows a diagram showing the alignment of vaccine sequences with active PTE peptides in all monkeys after 4 weeks of immunization with rAd26 vectors expressing mosaic sequences, M consensus, clade B + clade C or best natural clade C HIV-1Gag, Pol and Env antigens. For each monkey, the vaccine sequence is shown at the top and the active PTE peptide is shown below the vaccine sequence represented by the antigen (G, Gag; P, Pol; E, Env) and PTE peptide numbering. The minimum overlap area is shown in bold. Sequence polymorphisms between two mosaic sequences or two clade B + clade C antigens are shown in blue. The difference between the vaccine sequence and the active PTE peptide is shown in red.FIG. 22 shows The sequences disclose SEQ ID NOS 124-640, respectively.

The smallest region within a peptide that may contain an epitope of the immune response is bolded in the vaccine based on the overlap between the active peptides at the time of their occurrence. If there are no overlapping peptides, we speculate that the epitope may be anywhere in the peptide, so the entire region is bold. We were unable to distinguish between different T cell response targeting epitopes with different boundaries within the peptide, or more promiscuous clonal T cell responses that can tolerate variation when variants are present; both cases are advantageous in vaccine immune responses. The number of targeted regions corresponds to the minimum number of T cell responses required to account for the data.

Amino acids at vaccine and peptide mismatches are written in red; they are in bold red if they fall into a region likely to carry an epitope. When multiple peptides overlap, amino acid differences outside the overlap region are labeled in non-bold red.

The vaccine is always on top. Letters and peptide numbers representing each protein (Gag for G, Pol for P, and Envelope for E) were used to label each active PTE peptide. Protein and HXB2 codes were attached to each peptide.

For mosaic sequences and clade B + C vaccines, each contained 2 antigens and were included in the alignment; amino acid differences in the vaccine are labeled blue, as are active peptides with variant amino acids in the second mosaic sequence. At each position where the two vaccine antigens have differences, the active peptides are also marked in bold to show that the position including the two variants may affect the vaccine immune response and make the breadth and depth larger.

For example, the first vaccine summarized is the clade B + C vaccine, and animals 287-95 are the first animals listed in response. There were 3 CD8 responses against the PTE peptide and 1 against CD 4. The two CD8 peptides E26 and E282 showed substantial overlap, and therefore both may be targets of the same CTL response; we also noted therefore only 2CD 8 response regions, and 1 CD4 response region. For each response region, we written the number of overlapping peptides in each region (e.g., CD 8: 12CD 4: 1) to assess the depth of the response; two are red, indicating that the overlap region is variable in active peptides. If the vaccine is different, such as D/E in the second active area, the label is blue. Only the overlapping areas are bold. H in E282 was not found in both vaccines and was therefore marked red; it is therefore bold in the overlap region. Each active peptide has its protein and corresponding HXB2 numbering, labeled on the right.

FIGS. 23A-23C are graphs showing the magnitude of T lymphocyte responses from lowest to highest ranking for all Gag, Pol, and Env specific CD8+ (FIGS. 23A and 23B) and CD4+ (FIG. 23C).

FIGS. 24A-C show the depth of epitope-specific T lymphocyte responses to PTE peptides. Figure 24A is a diagram showing an example of the T lymphocyte response to localization in monkeys 366 who received the best natural clade C antigen. Figure 24B is a graph showing an example of a localized T lymphocyte response in monkeys 361 that received a bivalent mosaic antigen. In FIGS. 24A and 24B, the vaccine sequences are shown at the top (OptC; Mos1, Mos2) and the active PTE peptides are shown below the vaccine sequences represented by antigen (G, Gag; P, Pol; E, Env) and PTE peptide numbering. The minimum overlap area is shown in bold. Sequence polymorphisms between the two mosaic antigens are shown in blue. The difference between the vaccine sequence and the active PTE peptide is shown in red. The complete alignment of all positive peptides consisting of the response regions is shown in figure 22. FIG. 24C is a graph showing the depth of CD4+ (top) and CD8+ (bottom) T lymphocyte responses following immunization with rAd26 vector expressing mosaic, M consensus, clade B + clade C, or optimal native clade C antigen. A single monkey is depicted on the x-axis. For each epitope region, one response variation (light shading) or > 1 response variation (dark shading) is shown.FIG. 24A discloses SEQ ID NOS in order of appearance 641-650. FIG. 24B discloses SEQ ID NOS 651-685 in order of appearance, respectively.

FIG. 25 is a graph showing the breadth of epitope-specific T lymphocyte responses to HIV-1Gag peptides from clades A, B and C. The breadth of the cellular immune response was assessed using a sub-pool of overlapping peptides from the following HIV-1Gag strains: clade C DU422, clade C ZM651, consensus C, consensus a and consensus B. The number of positive subpools was shown after a single immunization with rAd26 vectors expressing mosaic sequence (blue), M consensus sequence (green), clade B + clade C (purple) or optimized natural clade C (red) HIV-1Gag, Pol and Env antigens. A single monkey is depicted on the x-axis.

FIGS. 26A-D are graphs showing cellular and humoral immune responses following boosting. The magnitude (fig. 26A) and breadth (fig. 26B) of individual T lymphocyte responses at week 4 post-priming (left side of each panel) and week 44 post-boost (right side of each panel) are shown for each monkey. Monkeys were primed with rAd26 vector at week 0 and boosted with rAd5HVR48 vector expressing mosaic sequence, M consensus sequence, or best natural clade C HIV-I Gag, Pol, and Env antigens at week 40. A single monkey is depicted on the x-axis. In fig. 26A, red indicates CD8+ T lymphocyte responses, blue indicates CD4+ T lymphocyte responses, and the responses observed at both time points are plotted by lines and the responses observed at only one time point are plotted by dots. In fig. 26B, the different shading for each color reflects the response to different antigens (Gag, Pol, Env). Figure 26C is a graph showing Env-specific ELISA endpoint titers at 0, 4, 44 weeks. Figure 26D is a graph showing neutralizing antibody (NAb) titers at week 44 against grade 1 clade a (DJ263.8), clade B (sf162.ls) and clade C (MW965.26) viruses. NAb titers against murine leukemia virus were used as negative controls, and were < 20 in all samples.

Figure 27 is a graph showing the theoretical coverage of PTE peptides by different vaccine antigens. The percentage of 9 amino acid PTE peptides covered by mosaic sequence (blue), M consensus sequence (green), clade B + clade C (purple) or optimized natural clade C (red) HIV-1Gag, Pol and Env antigens is shown.

Detailed Description

The invention features an optimized viral polypeptide computationally derived from a naturally occurring viral gene product. Following immunization of a subject (e.g., a human) with one or more optimized viral polypeptides of the invention or a vaccine (e.g., vector) incorporating one or more optimized viral polypeptides of the invention, the optimized viral polypeptides of the invention increase the breadth and depth of virus-specific immunity (e.g., cellular immunity, such as a T cell-based immune response). The present invention provides vaccines that can be administered to a subject (e.g., a human) that has or is at risk of infection with a virus. The vaccines of the present invention incorporate at least two different optimized viral polypeptides represented by each of the corresponding viral gene products. The inventors have found that the incorporation of two different optimized viral polypeptides leads to an increased coverage and immunogenic epitope expression in the vaccine, resulting in an increased total number of virus-specific immune responses after vaccination of a subject. The invention also provides methods of administering and preparing vaccines, vectors and optimized viral polypeptides for use in subjects (e.g., humans). The compositions, methods, and kits described herein can significantly increase the diversity, breadth, and/or depth of a virus-specific cellular immune response by providing at least two different optimized viral polypeptides.

Optimized viral polypeptides of the invention

The present invention provides multivalent (e.g., bivalent) vaccines that incorporate computationally optimized (computationally-optimized) viral polypeptides corresponding to and derived from naturally-transmitted viral gene products. Multivalent mosaic proteins are assembled from native sequences by (in silico) recombination and optimization performed in silico to provide maximized coverage of potential T cell epitopes (PTEs) for a given valency. Mosaic antigens are full-length proteins designed to retain native antigen expression and processing.

The inventors have found that immunization with two different optimized viral polypeptides corresponding to and derived from a single viral gene product (i.e., a bivalent vaccine) is capable of eliciting significantly higher numbers of cellular immune responses (e.g., T cell responses) than traditional monovalent or multivalent vaccines incorporating a naturally occurring polypeptide from the same viral gene product (e.g., a sequence based on a clinical isolate), or a consensus sequence of naturally occurring polypeptides from the same viral gene product.

Thus, with respect to vaccines incorporating computationally optimized (or in silico optimized) viral polypeptides whose sequences provide maximized coverage of non-rare short segments of circulating viral sequences, the vaccine can enhance the breadth and depth of the immune response.

A genetic algorithm is used to create a set of optimized viral polypeptides as a "mosaic" mixture of fragments of any set of naturally occurring viral gene product sequences, provided as input data. This genetic algorithm strategy uses unaligned protein sequences from the general viral population as the input data set, and therefore has the advantage of being "alignment independent". The artificially optimized viral polypeptides they create are similar to viral proteins found in nature, but do not occur naturally. Genetic algorithms can be tailored to optimize viral polypeptides of different lengths depending on the intended target or desired immune response. Since most T cell epitopes are nine amino acids in length, the genetic algorithm utilized to design optimized viral polypeptides according to the present invention is based on optimizing each contiguous nonamer amino acid sequence of a given viral gene product (e.g., HIV-1 Gag). According to this approach, nonamers that do not occur or are very rare in nature (for example) can be excluded — an improvement over consensus sequence-based vaccine strategies, as the latter may contain some nonamers that are rare or not at all occurring in nature (for example). The definition of fitness for use in genetic algorithms is that the most "suitable" multivalent mixture is the combination of input viral sequences that yields the best coverage (highest score of perfect match) of all nonamers in the population, and meets the constraint of no nonamer deletions or rareness in the population. Genetic algorithms for producing optimized viral polypeptides of the invention are further described in international patent application publication WO 2007/024941, incorporated herein by reference.

In one embodiment, the invention provides multivalent (e.g., bivalent) HIV-1 vaccines that incorporate a single optimized HIV-1 polypeptide (e.g., the polypeptide described in SEQ ID NOS: 1-29). In another embodiment, the invention features a multivalent vaccine that incorporates two or more optimized HIV-1 polypeptides. In each case, the optimized HIV-1 polypeptides were based on all HIV-1 variants circulating worldwide, designated HIV-1 major (M) group. The inventors generated an optimized set of HIV-1 polypeptides (SEQ ID NOS: 1-29) that increased the breadth and depth of cellular immunity based on M sets of mosaic genes that each utilized only two variants (e.g., two polypeptide sequences, each of Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr, and Vpu). We have obtained new and surprising results in Rhesus monkeys (Rhesus macaques), using these optimized HIV-1 polypeptides in multivalent (e.g., bivalent) HIV-1 group M vaccines, elicited significantly greater breadth and depth of HIV-1 specific cellular immune responses than the other two leading vaccine antigen strategies (M consensus antigen and best natural clade C antigen).

The present invention provides fusions of optimized viral polypeptides corresponding to different viral gene products. The genetic algorithms described above can be used to generate fusion polypeptides for use in the vaccines of the present invention. For example, optimized HIV-1 polypeptide fusions of Gag/Nef (SEQ ID NOS: 19-20), Gag/Pol (SEQ ID NOS: 21-27), Gag/Pol/Nef (SEQ ID NOS: 28-29) can be incorporated into the vectors of the present invention for administration to a subject (e.g., a human) infected with HIV-1 or at risk of infection with HIV-1. The vaccines of the invention (whether in the form of polypeptides or nucleic acids) may also include one or more non "mosaic" polypeptides (or sequences encoding them, respectively), such as, for example, the best clade C sequence (SEQ ID NOS: 30-36) or a consensus sequence (SEQ ID NOS: 37-39).

The optimized viral polypeptides disclosed in the present invention can be prepared conventionally by chemical synthesis techniques, such as Merrifield, j.amer.chem.soc.85: 2149(1963) (see also, for example, Stemmer et al, 164Gene 49 (1995)). For example, the vaccine can be readily prepared using Solid Phase Peptide Synthesis (SPPS). Automated solid phase synthesis can be performed using any of a number of well known, commercially available automated synthesizers, such as the ABI433A peptide synthesizer available from Applied Biosystems. Alternatively, the optimized viral polypeptides of the invention can be recombinantly produced by transfecting or transducing a cell or organism with a nucleic acid or vector (e.g., a viral vector, such as an adenovirus) that effects intracellular expression of the optimized viral polypeptide. Nucleic acids and vectors encoding the nucleotide sequences of the optimized viral polypeptides of the invention can be synthesized by well-known recombinant DNA techniques, including those described herein.

Vaccines of the invention

The invention also features a vaccine that can be administered to a patient already infected with a virus or at risk of infection with a virus (e.g., HIV-1). The vaccine of the invention comprises at least one optimized viral polypeptide of the invention, as discussed herein. The vaccine of the invention may be a nucleic acid (e.g., a recombinant immunogenic component (e.g., a subunit) or a whole organism (e.g., a whole virus) viral vector) encoding two or more nucleotide sequences of the optimized viral polypeptides of the invention. Nucleic acids include vectors (e.g., viral vectors, such as adenoviruses) incorporating two or more nucleotide sequences of the optimized viral polypeptides of the invention. The optimized viral polypeptides of the invention, as well as vaccines, nucleic acids and vectors incorporating the optimized viral polypeptides, can be recombinantly expressed in a cell or organism, or can be administered directly to a subject (e.g., a human) that has or is at risk of having an infection with a virus.

Vectors of the invention

The invention also features vectors encoding one or more nucleotide sequences (e.g., DNA or RNA) of the optimized viral polypeptides of the invention. The vector can be a vector (e.g., a liposome), a plasmid, a cosmid, a yeast artificial chromosome, or a virus that includes a nucleotide sequence that encodes one or more of the optimized viral polypeptides of the invention. The vector can include additional nucleic acid sequences from several sources.

Vectors encoding one or more optimized viral polypeptides of the invention can be constructed using any recombinant molecular biology technique known in the art. After transfection or transduction of the target cell or organism, the vector may be extrachromosomal or may integrate into the chromosome of the host cell. The nucleic acid component of the vector may be present in single or multiple copy number per target cell, and may be linear, circular, or tandem.

The vectors of the invention may also include Internal Ribosome Entry Site (IRES) sequences to effect expression of multiple peptides or polypeptide chains in a single nucleic acid transcription. For example, a vector of the invention can encode one or more optimized viral polypeptides of the invention as well as other polypeptides (e.g., a detectable label such as Green Fluorescent Protein (GFP)).

The vectors of the invention further include gene expression elements that facilitate the expression of the optimized viral polypeptides of the invention. Gene expression elements that facilitate expression of the vectors encoding the optimized viral polypeptides described herein include, but are not limited to, (a) regulatory sequences, such as viral transcription promoters and enhancer elements thereof, such as the SV40 early promoter, the Rous (Rous) sarcoma virus LTR, and the Moloney (Moloney) murine leukemia virus LTR; (b) for example, splicing regions and polyadenylation sites from the late region of SV 40; and (c) a polyadenylation site, e.g., in SV 40. Also included are plasmids derived from replication, antibiotic resistance or selectable genes, multiple cloning sites (e.g., restriction endonuclease sites), and other viral gene sequences (e.g., sequences encoding viral structural, functional, or regulatory elements, such as HIV Long Terminal Repeat (LTR)).

The vectors of the invention may also include optimized viral polypeptides of the invention optimized for expression in humans, such as, for example, the nucleic acid sequences of SEQ ID NOS: 11, 14-18 and 23.

The vectors of the invention may be designed to include a Multiple Cloning Site (MCS) containing the following enzyme sites: Xbal-EcoRI-Kozak-start.. terminate-BamHI-NheI; and the following sequences: TCTAGA GAATTC GCCACC [ ATG gene TAA TGA ] GGATCCGCTAGC. Vectors with this MCS can be used with optimized viral polypeptides that do not have internal Xbal, EcoRI, BamHI, NheI sites nor 6 or more segments of C or G.

In vivo administration

The invention features a method for in vivo administration of one or more vaccines of the invention (e.g., encoding two or more optimized viral polypeptides of the invention) to a subject (e.g., a human) to facilitate expression of the two or more optimized viral polypeptides of the invention. Following administration of a vaccine to a subject, one or more optimized viral polypeptides of the invention will be expressed that are capable of eliciting a protective or therapeutic immune response (e.g., a cellular or humoral immune response) against a viral immunogen.

Several vectors can be used to deliver nucleotide sequences encoding one or more optimized viral polypeptides of the invention directly to a subject (e.g., a human). Vectors of the invention include viruses, naked DNA, oligonucleotides, cationic lipids (e.g., liposomes), cationic polymers (e.g., polysomes), viral particles, and dendrimers. The present invention provides for the in vitro transfection or transduction of cells (e.g., blood cells) followed by administration of these cells to a donor subject to express an optimized viral polypeptide of the present invention having immunogenic properties. Cells that can be isolated and transfected or transduced in vitro according to the methods of the present invention include, but are not limited to, blood cells, skin cells, fibroblasts, endothelial cells, skeletal muscle cells, liver cells, prostate epithelial cells, and vascular endothelial cells. Stem cells are also suitable for transfection or transduction with the vectors of the invention. Totipotent, pluripotent, multipotent, or unipotent stem cells, including bone marrow progenitor cells and Hematopoietic Stem Cells (HSCs), can be isolated and transfected or transduced with a vector encoding one or more optimized viral polypeptides of the invention and administered to a subject according to the methods of the invention.

The transfection or transduction methods used to express the optimized viral vectors of the present invention have a strong impact on the strength and longevity of protein expression in transfected or transduced cells and in subjects subsequently receiving the cells. The present invention provides vectors that have a short life span (e.g., adenoviral vectors) or a long life span (e.g., retroviral vectors) in nature. Regulatory sequences (e.g., promoters and enhancers) are known in the art and can be used to regulate protein expression. Transfected or transduced cell types also have a strong influence on the strength and longevity of protein expression. For example, cell types with high turnover rates can be expected to have shorter protein expression cycles.

In vitro transfection and transduction

The invention also features a method for transfection and transduction of cells (e.g., blood cells, such as lymphocytes) in vitro, followed by administration of the cells to a subject (e.g., a human). In one embodiment, the cells are autologous cells of the subject being treated. Cells may be transfected or transduced in vitro with one or more vectors encoding one or more nucleotide sequences for optimized viral polypeptides of the invention to enable transient or long-term expression of the optimized viral polypeptides in the treated subject. Administration of these modified cells to a subject will express one or more optimized viral vectors described herein to elicit a protective or therapeutic immune response (e.g., a cellular or humoral immune response) against the viral immunogen.

Several vectors can be used to deliver nucleotide sequences encoding one or more optimized viral polypeptides of the invention to cells (e.g., blood cells, such as lymphocytes). Vectors of the invention include viruses, naked DNA, oligonucleotides, cationic lipids (e.g., liposomes), cationic polymers (e.g., polysomes), viral particles, and dendrimers. The present invention provides for the in vitro transfection or transduction of cells (e.g., blood cells) followed by administration of these cells to a donor subject to express an optimized viral polypeptide having immunogenic properties as described herein. Cells that can be isolated and transfected or transduced in vitro according to the methods of the present invention include, but are not limited to, blood cells, skin cells, fibroblasts, endothelial cells, skeletal muscle cells, liver cells, prostate epithelial cells, and vascular endothelial cells. Stem cells are also suitable cells for transfection or transduction with the vectors of the invention. Totipotent, pluripotent, multipotent or unipotent stem cells, including bone marrow progenitor cells and Hematopoietic Stem Cells (HSCs), can be isolated and transfected or transduced with a vector encoding one or more optimized viral polypeptides of the invention and administered to a subject according to the methods described herein.

Viral vectors

Viral vectors encoding one or more nucleotide sequences of the optimized viral polypeptides of the invention may be used as vaccines of the invention. For example, one or more nucleotide sequences of the optimized viral polypeptides of the invention can be recombinantly inserted into a nucleotide sequence of a native or modified (e.g., attenuated) viral genome suitable for transduction (e.g., in vivo administration) of a subject or a cell isolated from a subject (e.g., in vitro transduction followed by administration of the cell to the subject). Additional modifications may be made to the virus to increase infectivity or tropism (e.g., pseudotyped), to reduce or eliminate replication capacity, or to reduce immunogenicity of viral components (e.g., all components not associated with an immunogenic vaccine agent). The vectors of the invention may be expressed by the transduced cell and secreted into the extracellular space or left in the expressing cell (e.g., as intracellular molecules or presented on the cell surface). Chimeric or pseudotyped viral vectors may also be used to transduce cells to achieve expression of one or more of the optimized viral polypeptides of the invention. Typical vectors are described below.

Adenoviral vectors

Recombinant adenoviruses offer several significant advantages when used as vectors for expressing one or more optimized viral polypeptides as described herein. Viruses can be prepared at high titers, can infect non-replicating cells, and can also provide high transduction of target cells in vitro upon exposure to a population of target cells. In addition, adenoviruses do not integrate their DNA into the genome of the host. Thus, its use as an expression vector reduces the risk of inducing spontaneous proliferation abnormalities. In animal models, adenovirus vectors are typically found to mediate high levels of expression for about a week. The duration of transgene expression (expression of a nucleic acid encoding an optimized viral polypeptide of the invention) can be extended by using cell or tissue specific promoters. Other improvements in the molecular engineering of the adenoviral vector itself result in more durable transgene expression and less inflammation. This is seen in the so-called "second generation" vectors with specific mutations in additional early adenovirus genes, as well as in the "lifeless" vectors (Engelhardt et al, Proc. Natl. Acad. Sci. USA 91: 6196 (1994)) in which virtually all viral genes have been deleted using the Cre-Lox strategy and Kochanek et al, Proc. Natl. Acad. Sci. USA 93: 5731(1996), each of which is incorporated herein by reference.

The rare serotypes and chimeric adenoviral vectors disclosed in International patent application publications WO 2006/040330 and WO2007/104792 (each incorporated herein by reference) are particularly useful as vectors in the present invention. For example, recombinant adenoviruses rAd26, rAd34, rAd35, rAd48, and rAd5HVR48 can encode one or more optimized viral polypeptides of the invention. One or more recombinant viral vectors encoding the optimized viral polypeptides of the invention can be administered to a subject to treat or prevent a viral infection.

Adeno-associated virus (AAV)

Adeno-associated virus (AAV), derived from non-pathogenic parvovirus, can also be used to express optimized viral polypeptides of the present invention, since these vectors elicit a cellular immune response that is nearly vector-free and results in transgene expression lasting months in most experimental systems.

Retroviruses

Retroviruses are used for expression of the optimized viral polypeptides of the invention. Unlike adenovirus, the reverse transcribed genome is RNA-based. When a retrovirus infects a cell, its RNA is introduced into the cell along with several enzymes. Viral RNA molecules from retroviruses will produce double-stranded copies of DNA through a process called reverse transcription, called a provirus. Upon transport to the nucleus, the proviral DNA integrates into the host cell chromosome, permanently altering the genome of the transduced cell and any progeny cells derived from the cell. The ability to permanently introduce genes into cells or organisms is an inevitable feature of retroviruses used for gene therapy. Retroviruses include lentiviruses, which are a family of viruses including Human Immunodeficiency Virus (HIV), which includes several accessory proteins to facilitate viral infection and proviral integration. Currently, "third generation" lentiviral vectors are characterized by complete lack of replication capacity, broad tropism, and enhanced Gene transfer capacity to mammalian cells (see, e.g., Mangeat and Trono, Human Gene Therapy 16 (8): 913(2005) and Wiznerowicz and Trono, Trends Biotechnol.23 (1): 42(2005), each of which is incorporated herein by reference).

Other viral vectors

In addition to adenoviruses and retroviruses, other viral vectors and techniques are known in the art for expressing the optimized viral polypeptides of the invention in cells (e.g., blood cells, such as lymphocytes) or subjects (e.g., humans). These include poxviruses (e.g., vaccinia virus and modified vaccinia virus ankara (MVA); see, e.g., U.S. patent nos. 4,603,112 and 5,762,938, each of which is incorporated herein by reference), herpes viruses, togaviruses (e.g., venezuelan equine encephalitis virus; see, e.g., U.S. patent No. 5,643,576, incorporated herein by reference), picornaviruses (e.g., poliovirus; see, e.g., U.S. patent No. 5,639,649, incorporated herein by reference), baculoviruses, and others described by wattapitayakul and Bauer (biomed. pharmacother, 54: 487(2000), incorporated herein by reference).

Other expression vectors: naked DNA and oligonucleotides

Naked DNA or oligonucleotides encoding one or more of the optimized viral polypeptides of the invention may also be used to express these polypeptides in cells (e.g., blood cells such as lymphocytes) or in subjects (e.g., humans). See, for example, Cohen, Science 259: 1691 + 1692 (1993); fynan et al, proc.natl.acad.sci.usa, 90: 11478 (1993); and Wolff et al, BioTechniques 11: 474485(1991), each of which is incorporated herein by reference. This is the simplest non-viral transfection method. Effective methods for delivering naked DNA are the use of, for example, electroporation and "gene gun", which inject DNA-coated gold particles into cells using high pressure gas and carrier particles (e.g., gold).

Lipid complexes and polymer complexes

To enhance delivery of nucleic acids encoding the optimized viral polypeptides of the invention into cells or subjects, lipid complexes (e.g., liposomes) and polymeric complexes can be used to protect vector DNA from undesirable degradation during transfection. Plasmid DNA may cover lipids in tissue structures like micelles or liposomes. When this tissue structure is complexed with DNA, it is called a lipoplex. There are three types of lipids, anionic (negatively charged), neutral or cationic (positively charged). Lipid complexes utilizing cationic lipids have proven useful for gene delivery. Cationic lipids, due to their positive charge, naturally form complexes with negatively charged DNA. Also due to their charge, they interact with the cell membrane, and endocytosis of the lipid complex occurs, releasing the DNA into the cytoplasm. Cationic lipids also protect DNA from degradation by cells.

Complexes of polymers with DNA are called polymeric complexes (polyplex). Most polymeric complexes consist of cationic polymers, the production of which is regulated by ionic interactions. The large difference between the mode of action of the polymer complex and the lipid complex is that the polymer complex cannot release its DNA load into the cytoplasm and therefore, must be co-transfected with an endosomolytic agent (to lyse the endosomes generated during endocytosis of the cell) such as inactivated adenovirus. However, this is not always the case; polymers such as polyethyleneimine have their own methods of endosome disruption, as do chitosan and trimethyl chitosan.

Typical cationic lipids and polymers that can be used to bind to nucleic acids encoding the optimized viral polypeptides described herein to form a lipid or polymeric complex include, but are not limited to, polyethyleneimine, lipofectin (lipofectin), lipofectamine (lipofectamine), polylysine, chitosan, trimethyl chitosan, and alginate.

Hybridization method

Several hybrid approaches to gene transfer combine two or more techniques. Viral particles, for example, bind lipid complexes (e.g., liposomes) to inactivated viruses. This approach has been shown to result in more efficient gene transfer in respiratory epithelial cells than viral or liposomal approaches alone. Other methods involve mixing other viral vectors with cationic lipids or hybrid viruses. Each of these methods can be used to facilitate transfer of a nucleic acid encoding an optimized viral polypeptide of the invention into a cell (e.g., a blood cell, such as a lymphocyte) or a subject (e.g., a human).

Dendritic polymers

Dendrimers can also be used to transfer nucleic acids encoding the optimized viral polypeptides described herein into cells (e.g., blood cells, such as lymphocytes) or subjects (e.g., humans). Dendrimers are highly branched spherical macromolecules. The surface of the microparticle can function in many ways, and many properties of the resulting construct are determined by its surface. It is especially possible to build cationic dendrimers (i.e. dendrimers with a positive surface charge). When present in genetic material such as DNA or RNA, charge complementarity results in transient binding of the nucleic acid to the cationic dendrimer. Once the target is reached, the dendrimer-nucleic acid complex is taken up into the cell by endocytosis.

In vivo administration

The invention features an in vivo method of immunizing a subject (e.g., a human) with a vaccine of the invention. In one embodiment, one or more vaccines of the present invention can be administered directly to a subject to elicit a protective or therapeutic immune response (e.g., a cellular or humoral immune response) against the virus (HIV-1). Alternatively, a vector encoding one or more optimized viral polypeptides of the invention as described above can be administered directly to a subject to prevent or treat a viral infection. Vectors (e.g., viral vectors) that effectively transfect or transduce one or more cells in vivo are capable of eliciting a broad, durable, and effective immune response in a treated subject. After transfer of the nucleic acid component of the expression vector to a host cell (e.g., blood cells, such as lymphocytes), the host cell produces and presents or secretes the vaccine of the invention, which is then used to activate components of the immune system, such as Antigen Presenting Cells (APCs), T cells, and B cells, resulting in establishment of immunity.

Pharmaceutical composition

The invention features vaccines, vectors, and optimized viral polypeptides of the invention in combination with one or more pharmaceutically acceptable excipients, diluents, buffers, or other acceptable carriers. The formulation of the vaccine, vector or optimized viral polypeptide will use or achieve the expression of an effective amount of the optimized viral polypeptide immunogen. That is, an amount of antigen will be included that results in a specific and sufficient immune response in the subject being treated (e.g., a human) to protect the subject from subsequent exposure to a virus (e.g., HIV-1) or to treat an existing viral infection. For example, the formulation of the vaccine of the present invention may achieve a certain amount of antigen expression, which results in a broad and specific cellular immune response in the subject. Subjects treated with the vaccines, vectors, or optimized viral polypeptides of the invention can also produce anti-viral antibodies (e.g., neutralizing antibodies) that can provide protective or therapeutic benefits to the subject. The vaccine, vector or optimized viral polypeptide of the present invention may be administered directly to a subject, either alone or in combination with any pharmaceutically acceptable carrier, salt or adjuvant known in the art.

Pharmaceutically acceptable salts may include non-toxic acid addition salts or metal complexes commonly used in the pharmaceutical industry. Examples of the acid addition salts include organic acids such as acetic acid, lactic acid, pamoic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, palmitic acid, suberic acid, salicylic acid, tartaric acid, methanesulfonic acid, toluenesulfonic acid, trifluoroacetic acid or the like; copolymer acids such as tannic acid, carboxymethyl cellulose or the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, or the like. The metal complex includes zinc, iron, and the like.A typical pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and formulations thereof are known to those skilled in the art and are described, for example,Remington′s Pharmaceutical Sciences，(18^thversion), ed.a.gennaro, 1990, Mack Publishing Company, Easton, PA.

The prophylactic or therapeutic amount of the vaccine, vector or optimized viral polypeptide of the present invention may be administered orally, parenterally (e.g., intramuscularly, intraperitoneally, intravenously or subcutaneously, by inhalation, intradermally by injection, by eye drop or implantation), nasally, vaginally, rectally, sublingually or topically, in admixture with a pharmaceutically acceptable carrier suitable for the route of administration. The concentration of the vaccine, vector or optimized viral polypeptide of the invention in the formulation may vary from about 0.1 to 100% by mass.

Formulations for parenteral administration of compositions containing the vaccines, vectors or optimized viral polypeptides of the invention include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of suitable carriers include propylene glycol, polyethylene glycol, vegetable oils, gelatin, hydrogenated naphthalenes, and injectable organic esters such as ethyl oleate. Such formulations may also contain additives such as preservatives, wetting agents, emulsifying agents and dispersing agents. Biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compounds. Other potentially useful parenteral delivery systems comprising the vaccine, vector or optimized viral polypeptide compositions of the present invention include ethylene vinyl acetate copolymer microparticles, osmotic pumps, implantable infusion systems and liposomes.

Liquid formulations may be sterilized by, for example, filtration through a bacterial filter, by adding a sterilizing agent to the composition, or by irradiating or heating the composition. Alternatively, it may be produced as a sterile, solid composition which is immediately ready for use after dissolution in sterile water or some other sterile injectable medium.

Compositions for rectal or vaginal administration containing the vaccines, vectors or optimized viral polypeptides of the invention are preferably suppositories, which may contain excipients other than the active substance, such as cocoa butter or a suppository wax. Compositions for nasal or sublingual administration are also prepared using standard excipients known in the art. Formulations for administration by inhalation may contain excipients such as lactose, or may be aqueous solvents containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solvents, administered in the form of nasal drops or sprays, or as a gel.

The amount of active ingredient in the compositions of the present invention may vary. One skilled in the art will appreciate that the exact individual dosage may be adjusted depending on a variety of factors, including the peptide administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the nature of the disease in the subject, and the age, weight, health, and sex of the patient. In addition, the severity of the disease being treated by the vaccine, vector or optimized viral polypeptide also has an effect on the dosage level. Typically, the dosage level is between 0.1. mu.g/kg body weight and 100mg/kg body weight, administered in a single dose or divided into multiple doses per day. Preferably, the typical dosage range is between 250. mu.g/kg body weight to 5.0mg/kg body weight per day. Due to the different efficiencies of the different routes of administration, a wide variation in the required dosage is expected. For example, oral administration is generally expected to require higher dosage levels than intravenous administration. These dosage level variations can be adjusted using standard empirical procedures for optimization as is well known in the art. In general, the exact prophylactically or therapeutically effective dose can be determined by the attending physician with reference to the factors identified above.

The amount of the vaccine, vector or optimized viral polypeptide of the present invention present in each dose administered to a patient is selected in view of considerations of age, weight, sex, general physical condition of the patient and the like. The amount of vaccine, carrier or optimized viral polypeptide required to induce an immune response (e.g., a cellular immune response) or produce an exogenous effect in a patient without significant adverse side effects varies depending on the pharmaceutical composition used and the presence of optional adjuvants. The initial dose optionally follows repeated boosts if desired. The methods may involve chronic administration of a vaccine, vector or optimized viral polypeptide as described herein. For therapeutic or prophylactic use, repeated doses of immunizing vaccines, vectors or optimized viral polypeptides are desirable, such as a yearly boost or boosts at other time intervals. The dose administered will, of course, depend on known factors such as the pharmacodynamic characteristics of the particular vaccine, vector or optimized viral polypeptide and its mode and route of administration; age, health, and weight of the recipient; the nature and extent of the symptoms, the kind of concurrent treatment, the frequency of treatment and the desired effect vary. The vaccines, vectors, or optimized viral polypeptides of the invention can be administered to subjects at risk of acute infection due to needle stick or maternal infection during long-term treatment. The dose frequency of this "acute" infection can range from daily doses to once or twice weekly intramuscular or intravenous infusions for about 6 weeks. Vaccines, vectors, or optimized viral polypeptides can also be administered to patients already infected, or patients with late stage infection with a virus (e.g., HIV-1) in long-term therapy. The frequency of chronic administration in an infected patient can range from daily doses to once or twice weekly intramuscular or intravenous injections and depends on the half-life of the immunogen present in the vaccine, vector or optimized viral polypeptide of the invention.

Adjuvant

The vaccines against viruses described herein for vaccinating a mammal (e.g., a human) in need thereof can be co-administered or sequentially administered with one or more pharmaceutically acceptable adjuvants to increase the immunogenicity of the vaccine. Adjuvants approved for use in humans include aluminum salts (alum). These adjuvants have been used in certain vaccines, including hepatitis b, diphtheria, rabies, polio and influenza vaccines. Other useful adjuvants include Complete Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), Muramyl Dipeptide (MDP), synthetic analogs of MDP, N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2- [1, 2-dipalmitoyl-s-glycero-3- (hydroxyphosphoryloxy) ] acetamide (MTP-PE) and compositions comprising a metabolizable oil and an emulsifier, wherein the oil and emulsifier are present in the form of an oil-in-water emulsion having substantially all of the oil droplets of less than one micron in diameter.

Reagent kit

The invention provides kits comprising a pharmaceutical composition comprising a therapeutically effective amount of a vaccine, vector or optimized viral polypeptide of the invention for preventing or treating a viral infection and a pharmaceutically acceptable carrier. The kit includes instructions for use to allow a clinician (e.g., a doctor or nurse) to administer the composition contained therein.

Preferably, the kit comprises a multi-pack single dose pharmaceutical composition containing an effective amount of a vaccine, vector or optimized viral polypeptide according to the invention. Alternatively, the kit may comprise the instruments or devices necessary for administration of the pharmaceutical composition. For example, the kits of the invention can provide one or more pre-filled syringes containing an effective amount of a vaccine, vector or optimized viral polypeptide of the invention. In addition, the kit may also include other components, such as instructions for use or a schedule of administration of a pharmaceutical composition containing the vaccine, vector or optimized viral polypeptide of the invention to a patient already infected with or at risk of infection with a virus.

It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, methods and kits of the present invention without departing from the spirit or scope of the invention. It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

Detailed Description

The invention is illustrated by the following examples, which are not intended to limit the invention.

Example 1

Mosaic antigen Gag, Pol, Nef and Env sequences (SEQ ID NOS: 1-8) were constructed using genetic algorithms as discussed above. These sequences were then modified to have utility for vaccine development by eliminating cleavage/fusion activity in Env (SEQ ID NOS: 9-11), eliminating catalytic activity in Pol (SEQ ID NOS: 12-14), eliminating tetradecylation sites in Nef (SEQ ID NOS: 16-18), and constructing fusion constructs including GagNef, GagPol, or GagPolNef (SEQ ID NOS: 19-29). The best natural clade C genes (SEQ ID NOS: 30-36) are also depicted in alignment.

Example 2

Expression of Gag, Pol and Env genes with 3X 10 from M consensus sequences (group 1), bivalent M mosaic sequences (group 2) or best native clade C sequences (group 3)¹⁰The vp rAd26 vector was used to immunize 20 rhesus monkeys. The M consensus sequence represents the synthetic sequence, which represents the only best "average level" of the global epidemic. Bivalent M mosaic sequences are described above. The best natural clade C sequence is the naturally occurring sequence from the true clade C HIV-1 virus that is the most "consensus-like" in nature. The breadth of cellular immunity was assessed by assessing the number of responding peptides from the global set of potential T cell epitope (PTE) peptides. The PTE peptides represent > 85% of the global HIV-1 sequence and are freely available from NIH.

The results show that the novel bivalent M-mosaic sequence is significantly superior to the other two main antigen concepts. As shown in table 1, the bivalent M mosaic antigen caused a significant increase in the breadth of Gag-specific, Env-specific, Pol-specific and total T lymphocyte responses compared to the M consensus antigen and the best natural clade C antigen. (mean represents mean # epitope for each group of monkeys; SEM represents standard error of mean).

Table 1: extended breadth of mosaic HIV-1Gag/Pol/Env antigens in rhesus monkeys against global PTE peptides

Example 3

3 × 10 expressing Gag, Pol and Env genes with M consensus sequences (group 1; n ═ 7), bivalent M mosaic sequences (group 2; n ═ 7) or optimal natural clade C sequences (group 3; n ═ 6) as described in example 2¹⁰The vp rAd26 vector was used to immunize rhesus monkeys. The breadth of cellular immunity was assessed by assessing the number of responding peptides from the global potential epitope (PTE) peptide group.

As an indication (readout ), we assessed the CD4/CD8 IFN-. gamma.enzyme linked immunosorbent assay (ELISPOT) response (amplitude) to the integrated PTE peptide. Comprehensive mapping of epitopes using pentadecylic PTE peptides to assess the number of positives (positive is defined as every 10⁶55 Spot Forming Cells (SFC) in 4 × background and PBMC). An integrated set of overlapping peptides across 5 Gag proteins was also tested to compare responses to a complete set of proteins.

The results show that the bivalent M-mosaic sequence is significantly superior to the other two main antigen concepts (Mcon and OptC).

Example 4

We used modeling to verify that the T cell response we observed increased due to the mosaic vaccine. We fit a Poisson regression model, predict the number of active peptides as a function of vaccine, polypeptide and T cell type, and then gradually eliminate the interaction. We observed that while the mosaic vaccine produced a highly significant increase in the number of positive PTE responses, it crossed all polyproteins and T cell types more or less uniformly. Thus, the number of peptides with a positive effect in an animal can be predicted by combining contributions that depend on the T cell type, the polypeptide and the vaccine that the animal receives, respectively.

These models also included random effects to account for animal-to-animal differences. This is a designed precaution to generate more reliable p-values by properly assigning the predictive power of the model.

We observed the following effects:

a) the CD8 response was much greater than the CD4 response, 4.37 fold, p < 2X 10^-16；

b) The response in gp160 was less than that in Gag or pol, 0.54, p-0.000830, and there was no significant difference between Gag and pol (even when normalized by sequence length, there was more chance to react since pol is twice the length of Gag); and

c) mosaic vaccines produce significantly more positive responses than Mcon (3.6 fold, p ═ 6.26 × 10)^-11) Whereas OptC produces less, although the difference between Mcon-OptC is not significant.

Example 5

If we only consider the minimum number of responses elicited by the vaccine and detected by the PTE peptides, then all peptides overlapping > 8 amino acids were counted only once ignoring variation, and the mosaic vaccine still produced a greater number of responses to different regions.

For CD8, each overlapping peptide group was counted only once:

statistical summary:

mos2 > Mcon OptC (Mcon shows a tendency to respond more than OptC)

Wilcoxon p value of Mos2 compared to Mcon: p value of 0.0009992

Wilcoxon p values of Mcon compared to optimal C (sequence): p value of 0.2351

The groups are summarized as follows:

vaccine	Minimum value	1^stQu.	Median number	Mean number of	3^rdQu.	Maximum value
							Mos2.cd8	7	7.5	8	9.4	11	14
Mcon.cd8	0	3	3	3.3	4	6
							OptC.cd8	0	1	1.5	2	4.25	5

For CD4, each overlapping peptide group was counted only once (there was little overlap in CD4, so this was nearly the same as the first count).

And (4) statistical summary:

mos2 > Mcon OptC (Mcon shows a tendency to respond more than OptC)

Wilcoxon p value of Mos2 compared to Mcon: p value of 0.00198

Wilcoxon p values of Mcon compared to optimal C (sequence): p value is 0.099

The groups are summarized as follows:

vaccine

Minimum value

1^stQu.

Median number

Mean number of

3^rdQu.

Maximum value

Mos2.cd4	2	2.5	3	3.4	4.5	5
							Mcon.cd4	0	1	1	1.3	2	2
OptC.cd4	0	0	0.5	0.67	1	2

Example 6: poisson regression with only one count per overlapping peptide group

Using overlapping PTE peptides, we determined that the following is roughly the same as the results discussed in example 4 above, with each positive PTE response counted separately;

a) the CD8 response was much greater than the CD4 response, 2.8 fold, p ≈ 1 × 10^-7；

b) Mosaic vaccines produce significantly more positive responses than Mcon (2.84 fold, p ≈ 4.3 × 10)^-7) Whereas OptC produces less, although the difference between Mcon-OptC is not significant; and

c) the response to Pol was greater than that to Gag, and more than to gp160, but only the difference between Pol-gp160 was significant, about 2-fold, with p < 0.001.

Example 7:

the following table is a record of the total response to Gag, Pol and Env responses for three vaccine responses in 7 animals vaccinated with 2 (valency) mosaic sequences (Mos2) or Mcon, and 6 animals vaccinated with the best natural clade c (optc):

the OptC vaccine produced a mean response in all monkeys that was slightly lower than the CD8+ T cell response per protein. Mcon vaccines show about 1 response per protein. Differences in proteins were only observed with Mos2, where Env generally had less response than Gag or Pol.

Each protein in the Mos2 vaccine elicits many responses and contributes to the overall response. After modification to inactivate pol and deletion of the cleavage and fusion domains in Env, the relative lengths of the consensus proteins are: env is 671 amino acids, Pol is 851, and Gag is 498 (1.35: 1.7: 1).

Summary of the invention

The breadth is as follows: the 2 (valency) mosaic sequence vaccine elicits a T cell response that recognizes more epitope regions than the M consensus sequence or the single best natural strain.

Depth: the diversity of PTE peptide recognition suggests that both forms in the 2 (valent) mosaic sequence can elicit different T cell responses to the variant peptide, increasing the likelihood of cross-reactivity.

Example 8:

the mosaic HIV-1 vaccine provided by the invention expands the breadth and depth of cellular immune response in rhesus monkeys. We constructed mosaic HIV-1Gag, Pol and Env antigens that optimized the coverage of PTE on HIV-1 group M sequences, including all major HIV-1 clades and recombination lines in the Los Alamos HIV-1 sequence database. The competition problem of theoretical coverage and practicality is balanced by using a bivalent mosaic strategy. The bivalent mosaic HIV-1Gag, Pol and Env antigens greatly expanded the breadth and breadth (depth) of epitope-specific CD8+ and CD4+ T lymphocytes in rhesus monkeys relative to the immune response observed in rhesus monkeys using consensus and native sequence HIV-1 antigens.

We immunized 27 outbred rhesus monkeys with a single injection of recombinant adenovirus serotype 26 (rAd26) vector expressing the following antigens: (i) a bivalent mosaic sequence (N ═ 7), (ii) an M consensus sequence (N ═ 7), (iii) a bivalent combination clade B and clade C (N ═ 7), or (iv) an optimal natural clade C (N ═ 6) HIV-1Gag, Pol, and Env antigens. The total dose is 3 × 10¹⁰The viral particles of rAd26 vector expressing these antigens were administered once by intramuscular injection to each animal. In the los alamos HIV-1 sequence database, the best clade C antigen is the native strain sequence, which is selected to provide maximized PTE coverage of the clade C sequence (discussed in materials and methods below). Fourth week after immunization with pools and subpools that included all PTE found in at least 15% of HIV-1 group M sequences,we assessed the breadth and breadth (depth) of the HIV-1 specific T lymphocyte response elicited by the vaccine by the IFN-. gamma.ELISPOT method. All individual peptide responses were resolved and a cytoablative IFN- γ ELISPOT method was performed to determine whether active peptides represent CD8+ or CD4+ T lymphocyte epitopes.

The total number of Gag, Pol and Env-specific cellular immune responses to PTE peptides elicited by mosaic antigens was 3.8 times higher than the number of responses induced by consensus or native sequence antigens (FIG. 19A; P1X 10;)^-11Mosaic sequences compared to the consensus antigen, the second highest group, based on the poisson regression model). CD8+ responded 4.4-fold more than CD4+ T lymphocytes (P < 10)^-11) The response to Env is less than that to Gag or Pol (P < 0.0007). The median of the CD8+ T lymphocyte responses was the highest mosaic vaccine followed by the consensus, combination B + C and native clade C vaccines (the median of the responses in each animal in each group was 16, 5, 3 and 2, respectively). Although overall CD4+ T lymphocyte responses were minor, the same pattern of association appeared with the highest number of CD4+ T lymphocyte responses to the mosaic vaccine, followed by the consensus, combined B + C and native clade C vaccines (median responses in each animal in each group were 4, 1 and 0.5, respectively). There was no statistical difference in the number of responses of CD8+ and CD4+ T lymphocytes elicited by the consensus, combined B + C and native clade C vaccines.

The PTE peptide comprises multiple overlapping sequences, reflecting naturally occurring HIV-1 sequence polymorphisms, and thus PTE peptide responses include recognition of a particular epitope (breadth) and cross-recognition of variants of that epitope (depth). We performed a conservative analysis of breadth by assessing the number of active epitope regions in each monkey, where all active PTE peptides overlapping by 8 or more amino acids were counted as one event. In this conservation analysis we still observed that mosaic antigens caused a 3.1-fold increase in the number of Gag, Pol and Env active epitope regions compared to either the common antigen or the native sequence antigen (fig. 19B; P ═ 1.6 x 10-7, poisson regression). Epitope regions showed clustering in animals as evidenced by regions of high epitope density (FIGS. 20A-20C and 21A-21C). A complete alignment of all positive peptides consisting of the response region is shown in figure 22.

These data indicate that mosaic antigens greatly enhance the breadth of the cellular immune response compared to M consensus sequences and natural clade C antigens. Bivalent mosaic antigens were also shown to outperform bivalent combinations of clade B and clade C antigens (fig. 19A and 19B), indicating that the increased breadth is due to the design of mosaic sequences and does not simply reflect the use of two different antigen sequences per protein. To determine whether the increased breadth induced by mosaic antigens compromised the efficacy of the response, we evaluated the magnitude of all individual CD8+ and CD4+ T lymphocyte responses. The magnitude of these responses proved to be similar in all groups (fig. 23; P-0.58 and P-0.99, two-sided Kolmogorov-Smirnov test). Thus, mosaic antigens expanded the breadth of cellular immunity without compromising the magnitude of individual epitope-specific responses, suggesting that antigenic competition and immunodominant limitations do not limit the immunogenicity of mosaic antigens in this study.

Next we describe the depth of the cellular immune response elicited by the different vaccine protocols. We define depth as the number of different PTE peptides elicited simultaneously for a particular epitope region. Inducing responses to multiple common epitope variants may increase immune coverage of infected viral sequences, block common escape pathways in vivo, or force the virus down to a third escape pathway, which incurs a high fitness cost. Consensus and native sequence antigens elicit responses characterized by a high degree of sequence identity between the vaccine sequence and the active PTE peptide, such as in monkey 366 receiving the native clade C antigen (fig. 24A; see also fig. 22). In contrast, mosaic antigens elicit responses characterized by multiple active PTE peptides in specific epitope regions. These peptides represent common variants and generally reflect polymorphisms contained in mosaic vaccine sequences, such as responses in monkeys 361 (figure 24B; see also figure 22). A summary of all epitope-specific responses in these animals shows that mosaic antigens increase the frequency of cellular immune responses to peptides with two or more targeted variants compared to consensus or native sequence antigens (FIG. 24C; P ═ 0.001, Wilcoxon rank-sum test of mosaic sequences compared to consensus antigen-second highest group).

To supplement the analysis with PTE peptides, we also evaluated the breadth of cellular immune responses in monkeys vaccinated with the traditional overlapping peptides covering 5 different Gag sequences (clade C DU422, clade C ZM651, consensus C, consensus a and consensus B). The breadth of cellular immunity was determined by assessing the response to a sub-pool of 10 overlapping peptides throughout each Gag sequence. Mosaic antigens elicited a greater breadth of T lymphocyte responses against all tested Gag sequences compared to either the consensus or native sequence antigens (FIG. 25; P ═ 1X 10^-7Two-term regression). Thus, mosaic antigens increase the breadth of cellular immunity, not only against the PTE peptide, but also against the authentic Gag peptide from clades A, B and C. The mosaic antigen was even shown to be superior to the optimal native clade C antigen in inducing a response against the clade C Gag peptide. Furthermore, mosaic antigens elicited similar responses to Gag peptides from multiple clades, while native clade C antigens showed reduced responses to clade a and clade B Gag peptides (fig. 25).

To evaluate the robustness of these observations, we used a total dose of 3X 10 at week 40¹⁰Viral particles of the heterologous vector rAd5HVR48 expressing HIV-1Gag, Pol and Env antigens matching the sequences used for the primary immunization were boosted against monkeys receiving mosaic antigens, consensus antigens and the best natural clade C antigen. The breadth of cellular immunity was determined by assessing responses to a subpool of 10 PTE peptides at week 4 (post-prime) and week 44 (post-boost). Most of the CD8+ and CD4+ T lymphocyte responses observed after priming were amplified after boosting (fig. 26A, red and blue lines), and many new responses were also detected (fig. 26A red and blue dots). At week 44, the magnitude of the single cell immune response proved similar among the groups (fig. 26A). However, after the boost, the number of subpool responses elicited by mosaic antigens (median 27 per animal response) was still much higher than the number of subpool responses elicited by consensus antigens (median 11 per animal response) or optimal native clade C antigens (median 10 per animal response) (fig. 26)B) In that respect Before and after the boost, there was more response elicited by the mosaic vaccine in each animal compared to either the consensus vaccine (consensus vaccine) or the natural clade C vaccine (P < 0.001, Wilcoxon rank-sum test for all pairwise comparisons).

After boosting, we also measured Env-specific humoral immune responses by enzyme-linked immunosorbent assay (ELISA) (fig. 26C) and fluorescent enzyme-based pseudovirus neutralization assay (fig. 26D). All groups showed ELISA titers similar to clade C gp140 and neutralizing antibody (NAb) responses similar to grade 1 clade C virus MW 965.26. Mosaic antigens elicited slightly higher Nab responses to the grade 1 clade B virus sf162.ls compared to the consensus or native clade C antigen (P ═ 0.02, Wilcoxon rank sum test), although we did not detect any Nab response to the grade 2 virus in any group.

Our data indicate that mosaicism of HIV-1Gag, Pol and Env antigens increases the breadth and depth of epitope-specific cellular immune responses in rhesus monkeys compared to consensus or native sequence antigens, well consistent with theoretical predictions (FIG. 27). The surprising results of mosaic antigens in this study may reflect the fact that rAd26 vector is particularly effective for eliciting CD8+ T lymphocyte responses, and the fact that mosaic antigens may be particularly effective for increasing the breadth of CD +8T lymphocytes (fig. 19A and 19B).

The breadth of Gag-specific cellular immune responses has been shown to be critical for SIV inhibition and human HIV-1 inhibition in rhesus monkeys. Furthermore, in stage 2B of the STEP study, rAd 5-based HIV-1 vaccine candidates expressing native clade B Gag, Pol and Nef antigens elicited only a limited breadth of HIV-1 specific cellular immune responses, and no vaccine advantage was observed. The vaccines in the STEP study developed epitope-specific T lymphocyte responses with a median of only 2-3, including epitope-specific responses to Gag with a median of only 1, and the breadth of this very narrow cellular immune response may provide insufficient immune coverage for a diverse range of infectious viruses. It has been reported that viral escape from CD8+ T lymphocytes occurs rapidly during acute HIV-1 infection, which may also prove that the cellular immune response elicited by the vaccine against common epitope variants is crucial. Taken together, these studies underscore the necessity to develop HIV-1 vaccine strategies that increase the breadth and depth of cellular immunity.

Since we evaluated the mosaic HIV-1 antigen in this study, we were unable to evaluate the protective efficacy of these vaccine regimens against SIV challenge. However, we have previously reported that the breadth of the SIV-specific cellular immune response elicited by rAd vectors in rhesus monkeys correlates with protective efficacy against SIV challenge (Liu et al, Nature 457: 87, 2009). We also show that the cellular immune response to variant epitopes (variant epitopes) in rhesus monkeys can block SIV mutational evolution (Barouch et al, nat. Immunol.6: 247, 2005), suggesting a biological relevance to extend the depth of cellular immunity. Modeling the protective efficacy of mosaic vaccines against SIV challenge in non-human primates has inherent limitations because the observed diversity of the SIV and HIV-1 group M sequences is greatly different and is influenced by different potential biological factors. For example, the selective pressure of CD8+ T lymphocytes in natural hosts such as the black and white brow monkey (sootty mangabeys) appears to be much less than in humans. Thus, further evaluation of mosaic antigens as candidates for HIV-1 vaccines can benefit from clinical trials.

In summary, we demonstrate that bivalent mosaicism of HIV-1Gag, Pol and Env antigens greatly expands the breadth and depth of cellular immunity in rhesus monkeys. These findings are of great significance for HIV-1 vaccine development, as global viral diversity and viral escape from the cellular immune response represent important obstacles to T-cell based HIV-1 vaccine development. Mixtures of bivalent mosaic antigens are also of practical and potentially feasible for clinical development. Increasing the valency of the mosaic antigen may further improve coverage. Finally, the mosaic antigen strategy is generalizable and can be used for other genetically diverse pathogens besides HIV-1.

Materials and methods

Antigen design and vector production. Bivalent mosaic HIV-1Gag, Pol and Env antigens were constructed to provide optimal coverage of HIV-1 group M sequences in the Ross alamus HIV-1 sequence database essentially as described (1, 2). The best native clade C antigen was selected as the sequence that provided the best PTE coverage of the clade C sequence in the los alamos HIV-1 sequence database (c.in. -.70177Gag, c.za.04.04zask208b1 Pol, c.sn.90.90se 364 Env). Clade B antigens were selected as close to or consensus sequences (b.cam-1 Gag, b.iiibpol, b.con Env) and used to complement the best clade C antigens in the bivalent clade B + C vaccine approach. Pol antigens contain RT and IN, are PR-free, and include point mutations to eliminate the catalytic activity (Priddy et al, Clinical infectious diseases 46: 1769, 2008). The Env gp140 antigen contains point mutations to eliminate cleavage and fusion activity. The vaccine sequences are depicted in figure 27. Recombinants expressing these antigens, incompletely replicated 26 serotype adenovirus (rAd26) and the hexon chimeric rAd5HVR48 vector were grown in PER.55K cells and purified essentially by the double CsCl gradient sedimentation (Abbink et al, J.Virol.81: 4654, 2007, and Roberts et al, Nature 441: 239, 2006).

Animal and immunization. 27 inbred rhesus monkeys that did not express the MHC class I allele Mamu-A × 01 were housed at the New England Primate Research Center (NEPRC) in south town (Southborough), Mass. Comprising 3 x 10¹⁰Immunization of viral particles expressing mosaic sequences, M consensus sequences, clade B + clade C or best natural clade C rAd26 or rAd5HVR48 vectors with HIV-1Gag, Pol and Env antigens was given as 1ml injections by intramuscular injection of bilateral quadriceps at weeks 0 and 40. All animal studies were approved by our Institutional Animal Care and Use Committee (IACUC).

IFN-gamma ELISPOT method. HIV-1 specific cellular immune responses in vaccinated monkeys were evaluated essentially by the interferon-gamma (IFN-. gamma.) ELISPOT method described (Roberts et al, Nature 441: 239, 2006, and Liu et al, Nature 457: 87, 2009). HIV-1Gag, Pol, and Env potential T cell epitope (PTE) peptides obtained from NIHAIDS study and reference reagent program, which includes all PTEs found in at least 15% of HIV-1 group M sequences and HIV-1Gag peptides from clade C DU422, clade CZM651, consensus C, consensus a and consensus B strains. Anti-human IFN-. gamma.10. mu.g/ml in endotoxin-free Dulbecco's PBS (D-PBS) (BD biosciences) were coated overnight at 100. mu.l/well on 96-well multi-well filter plates (Mllipore). The plates were then washed three times with D-PBS containing 0.25% Tween 20 (D-PBS/Tween), blocked with D-PBS containing 5% FBS for 2 hours at 37 ℃, washed three times with D-PBS/Tween, rinsed with RMPI 1640 containing 10% FBS to remove Tween 20, washed with 2. mu.g/ml of each peptide and 2X 10⁵PBMCs were incubated in triplicate in 100 μ l reaction volumes. After incubation for 18 hours at 37 ℃, the plates were washed nine times with PBS/tween and once with distilled water. The plates were then incubated with 2. mu.g/ml biotin-labeled anti-human IFN-. gamma. (BD Biosciences) for 2 hours at room temperature, washed six times with PBS/Tween, and incubated with a 1: 500 dilution of streptavidin-alkaline phosphatase (southern Biotechnology Associates) for 2 hours. After five washes with PBS/tween and one wash with PBS, plates were developed with nitroblue tetrazolium/5-bromo-4 chloro-3-indole-phosphate chromogen (Pierce), stopped by washing with tap water, air dried, and read using an ELISPOT plate reader (Cellular technology ltd). Calculate every 10⁶Spot-forming cells (SFC) of PBMC. Each 10 in the background medium⁶PBMC are typically < 15 SFC. Positive responses were defined as every 10⁶> 55 SFCs in PBMC and > 4-fold background.

Epitope mapping. Comprehensive localization of CD8+ and CD4+ T lymphocyte epitopes was performed using Gag, Pol, and Env PTE peptides obtained from the NIH AIDS study and reference reagent program. IFN-. gamma.ELISPOT was performed at week 4 after the initial immunization with the complete peptide pool and the subpool containing 10 PTE peptides. All peptide subpools with positive responses were deconvoluted and the PTE peptide whose epitope had a single 15 amino acids was confirmed. The cytoreductive IFN-. gamma.ELISPOT method was then performed to determine whether the active peptides represent CD8+ or CD4+ T lymphocyte epitopes. Partial epitope mapping using the PTE subpool was also performed 4 weeks after the boost, i.e., at week 44. All ambiguous responses were retested and considered positive if confirmed. Partial epitope mapping (mapping) was also performed using a subpool containing 10 overlapping Gag peptides to assess the breadth of HIV-1Gag from different clades.

A humoral immunoassay. Env-specific humoral immune responses were evaluated by direct ELISA and fluorescent-based pseudovirus neutralization assays using the HIV-1 clade C Env gp140 essentially as described (Montefiori, evaluation of neutralizing antibodies against HIV, SIV and SHIV in the fluorescent reporter assay (Evaluating neutralizing antibodies against HIV, SIV and SHIV in luciferase reporter assays), Current Protocols in immunology, Coligan, Kruisbeam, Margulies, Shevach, Strober and Coico, Ed. (John Wiley & Sons, 2004, pp.1-15)).

And (5) carrying out statistical analysis. All statistical analyses were performed using the R software package (Team, Foundation for statistical Computing, Vienna, Austria, 2009). To analyze the breadth of cellular immune responses to the localized PTE peptide (fig. 19A), we fitted a poisson regression model to predict the amount of active peptide based on the vaccine groups, antigens (Gag, Pol, Env) and lymphocyte subpopulations (CD4, CD 8). Our model included stochastic effects to accommodate animal-to-animal variation and was fitted with the lme4 library in the R software package (Pinheiro, Springer, New York (2000)). The data fit well to the model (discrete parameter 1.0) and there was no significant interaction between the three illustrative factors. For example, a 3.8 fold increase in the number of PTE peptides recognised by monkeys receiving mosaic antigen compared to monkeys receiving consensus or native sequence antigen (figure 19A) is equally applicable to PTE from Gag, Pol and Env and to responses generated by CD8+ and CD4+ T lymphocytes. Analysis of the number of active epitope regions (fig. 19B) also included a poisson regression model with random effects and fit well (coefficient of dispersion 0.87) without any significant interaction. Comparison of the magnitude of CD8+ and CD4+ T lymphocyte responses (fig. 23) was performed using a two-sided Kolmogorov-Smimov assay. Non-parametric tests were also performed to compare the breadth and depth of each monkey response between different vaccines (fig. 19A and 24C). We initially used the Kruskal-Wallis test to determine if there were differences between the 4 vaccine groups. This was highly significant in each case, so we subsequently evaluated all pairwise comparisons between 4 vaccine groups using the Wilcoxon rank sum (rank-sum) test. In each comparison, the mosaic vaccine elicited significantly more responses in each monkey than the other three vaccines. To analyze the breadth of response to HIV-1Gag from different clades (fig. 25), we fit the data to a two-term regression model. These models use vaccine groups as explanatory variables and include random effects to account for animal-to-animal and strain-to-strain variations. The data are somewhat discrete, but animals receiving the mosaic vaccine still elicited a significantly greater number of responses. PTE coverage was assessed using tools provided in the los alamos HIV-1 sequence database.

Sequence appendix

I. Bivalent M mosaic sequence ENV GP160, GAG, POL, NEF sequence

Mosaic sequence ENV1GP160(AA sequence)

SEQ ID NO：1

MRVTGIRKNYQHLWRWGTMLLGILMICSAAGKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDDVRNVTNNATNTNSSWGEPMEKGEIKNCSFNITTSIRNKVQKQYALFYKLDWPIDNDSNNTNYRLISCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSENFTNNAKTIMVQLNVSVEINCTRPNNNTRKSIHIGPGRAFYTAGDIIGDIRQAHCNISRANWNNTLRQIVEKLGKQFGNNKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTKLFNSTWTWNNSTWNNTKRSNDTEEHITLPCRIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGNDTSGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTTVPWNASWSNKSLDKIWNNMTWMEWEREINNYTSLIYTLIEESQNQQEKNEQELLELDKWASLWNWFDISNWLWYIKIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTRLPAPRGPDRPEGIEEEGGERDRDRSVRLVDGFLVLIWDDLQSLCLFSYHRLRDLLLIVELLGRRGWEALKYWWNLLQYWSQELKNSAISLLNATAVAVAEGTDRVIEALQRACRAILHIPRRIRQGLERLLL

Mosaic sequence ENV2GP160(AA sequence)

SEQ ID NO：2

MRVRGIQRNWPQWWIWGILGFWMIIICRVMGNLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTENFNMWKNDMVDQMHEDIIRLWDQSLKPCVKLTPLCVTLECRNVRNVSSNGTYNIIHNETYKEMKNCSFNATTVVEDRKQKVHALFYRLDIVPLDENNSSEKSSENSSEYYRLINCNTSAITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNETVNITCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNLSRDGWNKTLQGVKKKLAEHFPNKTINFTSSSGGDLEITTHSFNCRGEFFYCNTSGLFNGTYMPNGTNSNSSSNITLPCRIKQIINMWQEVGRAMYAPPIAGNITCRSNITGLLLTRDGGSNNGVPNDTETFRPGGGDMRNNWRSELYKYKVVEVKPLGVAPTEAKRRVVEREKRAVGIGAVFLGILGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQTRVLAIERYLQDQQLLGLWGCSGKLICTTAVPWNTSWSNKSQTDIWDNMTWMQWDKEIGNYTGEIYRLLEESQNQQEKNEKDLLALDSWKNLWNWFDITNWLWYIKIFIMIVGGLIGLRIILGVLSIVRRVRQGYSPLSFQTLTPNPRGLDRLGRIEEEGGEQDRDRSIRLVNGFLALAWDDLRSLCLFSYHQLRDFILIVARAVELLGRSSLRGLQRGWEALKYLGNLVQYWGLELKKGAISLLDTIAIAVAEGTDRIIELIQSICRAIRNIPRRIRQGFEASLL

Mosaic sequence GAG1(AA sequence)

SEQ ID NO：3

MGARASVLSGGELDRWEKIRLRPGGKKKYRLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALEKIEEEQNKSKKKAQQAAADTGNSSQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSNKGRPGNFLQNRPEPTAPPEESFRFGEETTTPSQKQEPIDKEMYPLASLKSLFGNDPSSQ

Mosaic sequence GAG2(AA sequence)

SEQ ID NO：4

MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFALNPGLLETSEGCKQIIKQLQPALQTGTEELRSLFNTVATLYCVHAEIEVRDTKEALDKIEEEQNKSQQKTQQAKEADGKVSQNYPIVQNLQGQMVHQPISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPVAPGQMREPRGSDIAGTTSNLQEQIAWMTSNPPIPVGDIYKRWIILGLNKIVRMYSPTSILDIKQGPKEPFRDYVDRFFKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPSHKARVLAEAMSQTNSTILMQRSNFKGSKRIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFRFEETTPAPKQEPKDREPLTSLRSLFGSDPLSQ

Embedded sequence POL1(AA sequence)

SEQ ID NO：5

FFRENLAFQQGEAREFPSEQTRANSPTSRELQVRGDNPHSEAGAERQGTLNFPQITLWQRPLVSIKVGGQIREALLDTGADDTVLEDINLPGKWKPKMIGGIGGFIKVRQYDQILIEICGKKAIGTVLVGPTPVNIIGRNMLTQLGCTLNFPISPIETVPVKLKPGMDGPRVKQWPLTEEKIKALTAICEEMEKEGKITKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEGFRKYTAFTIPSTNNETPGIRYQYNVLPQGWKGSPAIFQCSMTRILEPFRAKNPEIVIYQYMDDLYVGSDLEIGQHRAKIEELREHLLKWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYPGIKVRQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGHDQWTYQIYQEPFKNLKTGKYAKMRTAHTNDVKQLTEAVQKIAMESIVIWGKTPKFRLPIQKETWETWWTDYWQATWIPEWEFVNTPPLVKLWYQLEKDPIAGVETFYVDGAANRETKLGKAGYVTDRGRQKIVSLTETTNQKTELQAIYLALQDSGSEVNIVTDSQYALGIIQAQPDKSESELVNQIIEQLIKKERVYLSWVPAHKGIGGNEQVDKLVSSGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDQCQLKGEAMHGQVDCSPGIWQLDCTHLEGKIILVAVHVASGYIEAEVIPAETGQETAYFILKLAGRWPVKVIHTDNGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIKDYGKQMAGADCVAGRQDED

Embedded sequence POL2(AA sequence)

SEQ ID NO：6

FFRENLAFPQGKAREFSSEQTRANSPTRRELQVWGRDNNSLSEAGADRQGTVSFSFPQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIGGIGGFIKVRQYDQIPIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNFPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVKQLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMRGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETFYVDGAANRETKLGKAGYVTDRGRQKVVSLTDTTNQKTELQAIHLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSRGIRKVLFLDGIDKAQEEHEKYHSNWRAMASEFNLPPIVAKEIVASCDKCQLKGEAIHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHTDNGSNFTSATVKAACWWAGIKQEFGIPYNPQSQGVVESINKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGEYSAGERIVDIIASDIQTKELQKQITKIQNFRVYYRDSRDPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDED

Mosaic sequence NEF1(AA sequence)

SEQ ID NO：7

MGGKWSKSSVVGWPAIRERMRRAEPAADGVGAVSRDLEKHGAITSSNTAANNADCAWEAQEEEEVGFPVRPQVPLRPMTYKGALDLSHFLKEKGGLEGLIYSQKRQDILDLWVYHTQGYFPDWQNYTPGPGIRYPLTFGWCFKLVPVEPEKIEEANEGENNSLLHPMSQHGMDDPEKEVLMWKFDSRLAFHHMARELHPEYYKDC

Mosaic sequence NEF2(AA sequence)

SEQ ID NO：8

MGGKWSKSSIVGWPAVRERIRRAEPAAEGVGAASQDLDKYGALTSSNTAATNADCAWLEAQEDEEVGFPVKPQVPLRPMTYKAAFDLSFFLKEKGGLDGLIYSKKRQEILDLWVYNTQGFFPDWQNYTPGPGVRYPLTFGWCFKLVPVDPREVEEANKGENNCLLHPMNLHGMDDPEREVLVWRFDSRLAFHHMAREKHPEYYKNC

Bivalent M mosaic sequence ENV GP140 sequence (cleavage/fusion deficient)

Mosaic sequence ENV1GP140(AA sequence)

SEQ ID NO：9

MRVTGIRKNYQHLWRWGTMLLGILMICSAAGKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDDVRNVTNNATNTNSSWGEPMEKGEIKNCSFNITTSIRNKVQKQYALFYKLDVVPIDNDSNNTNYRLISCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSENFTNNAKTIMVQLNVSVEINCTRPNNNTRKSIHIGPGRAFYTAGDIIGDIRQAHCNISRANWNNTLRQIVEKLGKQFGNNKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTKLFNSTWTWNNSTWNNTKRSNDTEEHITLPCRIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGNDTSGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQSEKSAVGIGAVFLGFLGAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTTVPWNASWSNKSLDKIWNNMTWMEWEREINNYTSLIYTLIEESQNQQEKNEQELLELDKWASLWNWFDISNWLW

Mosaic sequence ENV2GP140(AA sequence)

SEQ ID NO：10

MRVRGIQRNWPQWWIWGILGFWMIIICRVMGNLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTENFNMWKNDMVDQMHEDIIRLWDQSLKPCVKLTPLCVTLECRNVRNVSSNGTYNIIHNETYKEMKNCSFNATTVVEDRKQKVHALFYRLDIVPLDENNSSEKSSENSSEYYRLINCNTSAITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNETVNITCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNLSRDGWNKTLQGVKKKLAEHFPNKTINFTSSSGGDLEITTHSFNCRGEFFYCNTSGLFNGTYMPNGTNSNSSSNITLPCRIKQIINMWQEVGRAMYAPPIAGNITCRSNITGLLLTRDGGSNNGVPNDTETFRPGGGDMRNNWRSELYKYKVVEVKPLGVAPTEAKRRVVESEKSAVGIGAVFLGILGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQTRVLAIERYLQDQQLLGLWGCSGKLICTTAVPWNTSWSNKSQTDIWDNMTWMQWDKEIGNYTGEIYRLLEESQNQQEKNEKDLLALDSWKNLWNWFDITNWLW

MOS3 ENV GP140(AA sequence)

678AA

SEQ ID NO：11

MRVKGIRKNYQHLWKWGTMLLGMLMICSAAEQLWVTVYYGVPVWRDAETTLFCASDAKAYEREVHNIWATHACVPTDPNPQEIVLENVTEEFNMWKNDMVEQMHTDIISLWDESLKPCVKLAPLCVTLNCTNANLNCTNDNCNRTVDKMREEIKNCSFNMTTELRDKKQKVYALFYKLDIVPIEKNSSEYRLINCNTSTITQACPKVTFEPIPIHYCTPAGFAILKCKDKKFNGTGPCKNVSTVQCTHGIKPVISTQLLLNGSLAEGEIIIRSENITNNAKTIIVQLNESVVINCTRPGNNTRKSVRIGPGQAFYATGEIIGDIRQAYCNISRAKWNNTLKQIVTKLKEQFKNKTIVFNQSSGGDPEITTHSFNCGGEFFYCNTTQLFNSTWNSNSTWNDTTGSVTEGNDTITLPCRIKQIVNMWQRVGQAMYAPPIEGNITCKSNITGLLLVRDGGNINRTNETFRPGGGNMKDNWRSELYKYKWEIKPLGVAPTRAKRRVVESEKSAVGLGAVFLGFLGTAGSTMGAASLTLTVQARQVLSGIVQQQSNLLKAIEAQQHLLKLTVWGIKQLQARILAVERYLRDQQLLGIWGCSGKLICTTNVP WNSSWSNKSQEEIWNNMTWMQWDREISNYTDTIYRLLEDSQNQQEKNEQDLLALDKWASLWNWFSITNWLW

Bivalent M mosaic sequence POL sequence (extensive inactivation, deletion of PR, 9A inactivating mutations to eliminate catalytic activity)

Embedded sequence POL1(AA sequence)

SEQ ID NO：12

MAPISPIETVPVKLKPGMDGPRVKQWPLTEEKIKALTAICEEMEKEGKITKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLAVGDAYFSVPLDEGFRKYTAFTIPSTNNETPGIRYQYNVLPQGWKGSPAIFQCSMTRILEPFRAKNPEIVIYQYMAALYVGSDLEIGQHRAKIEELREHLLKWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYPGIKVRQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGHDQWTYQIYQEPFKNLKTGKYAKMRTAHTNDVKQLTEAVQKIAMESIVIWGKTPKFRLPIQKETWETWWTDYWQATWIPEWEFVNTPPLVKLWYQLEKDPIAGVETFYVAGAANRETKLGKAGYVTDRGRQKIVSLTETTNQKTALQAIYLALQDSGSEVNIVTASQYALGIIQAQPDKSESELVNQIIEQLIKKERVYLSWVPAHKGIGGNEQVDKLVSSGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDQCQLKGEAMHGQVDCSPGIWQLACTHLEGKIILVAVHVASGYIEAEVIPAETGQETAYFILKLAGRWPVKVIHTANGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVASMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIKDYGKQMAGADCVAGRQDED

Embedded sequence POL2(AA sequence)

SEQ ID NO：13

MAPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLAVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDIVIYQYMAALYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVKQLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMRGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETFYVAGAANRETKLGKAGYVTDRGRQKVVSLTDTTNQKTALQAIHLALQDSGLEVNIVTASQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSRGIRKVLFLDGIDKAQEEHEKYHSNWRAMASEFNLPPIVAKEIVASCDKCQLKGEAIHGQVDCSPGIWQLACTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHTANGSNFTSATVKAACWWAGIKQEFGIPYNPQSQGVVASINKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGEYSAGERIVDIIASDIQTKELQKQITKIQNFRVYYRDSRDPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDED

MOS3 POL V3(AA sequence)

851AA

SEQ ID NO：14

MAPISPIDTVPVTLKPGMDGPKIKQWPLTEEKIKALTEICTEMEKEGKISRIGPENPYNTPVFAIKKKNSTRWRKLVDFRELNKKTQDFWEVQLGIPHPAGLKKKRSVTVLAVGDAYFSVPLDKDFRKYTAFTIPSVNNETPGVRYQYNVLPQGWKGSPAIFQCSMTKILEPFRAQNPEIVIYQYVAALYVGSDLEIEQHRTKIEELRAHLLSWGFTTPDKKHQREPPFLWMGYELHPDRWTVQPIELPEKESWTVNDIQKLVGKLNWASQIYPGIKVKQLCRLLRGAKALTEVIPLTKEAELELAENREILREPVHGVYYDPSKDLVAEIQKQGQDQWTYQIYQEPYKNLKTGKYARKRSAHTNDVRQLTEAVQKIALESIVIWGKIPKFRLPIQRETWETWWTEYWQATWIPDWEFVNTPPLVKLWYQLEKEPIAGAETFYVAGASNRETKIGKAGYVTDKGRQKVVSLTETTNQKAALQAIQLALQDSGPEVNIVTASQYVLGIIQAQPDRSESELVNQIIEELIKKEKVYLSWVPAHKGIGGNEQVDKLVSAGIRKILFLDGIDKAQEEHERYHSNWRTMASDFNLPPIVAKEIVANCDKCQLKGEAMHGQVDCSPGMWQLACTHLEGKIIIVAVHVASGYMEAEVIPAETGQETAYYILKLAGRWPVKVVHTANGSNFTSTTVKAACWWANVTQEFGIPYNPQSQGVIASMNKELKKIIGQVREQAEHLKTAVQMAVLIHNFKRRGGIGGYSAGERIVDIIATDIQTRELQKQIIKIQNFRVYFRDSRDPVWKGPAKLLWKGEGAVVIQDNSEIKVVPRRKVKIIRDYGKQMAGDDCVAGRQDEDQ

Bivalent M mosaic sequence GAG sequences

MOS3 GAG (AA sequence)

508 AA

SEQ ID NO：15

MGARASVLSGGKLDAWEKIRLRPGGKKKYKLKHIVWASRELDRFALNPGLLETAEGCQQIIEQLQPALQTGSEELKSLYNTVAVLYCVHQRIDVKDTKEALDKIEEIQNKSKQKTQQAAADTGSSSKVSQNYPIVQNAQGQMVHQALSPRTLNAWVKVVEEKGFNPEVIPMFSALAEGATPQDLNMMLNIVGGHQAAMQILKDTINEEAADWDRLHPVHAGPIPPGQMREPRGSDIAGTTSTPQEQIGWMTSNPPVPVGEIYKRWIIMGLNKIVRMYSPVSILDIKQGPKESFRDYVDRFFKVLRAEQATQEVKNWMTETLLIQNANPDCKSILRALGPGASLEEMMTACQGVGGPSHKARILAEAMSQANNTNIMMQRGNFKGQKRIKCFNCGKEGHLARNCRAPRKRGCWKCGREGHQMKDCNERQANFLGKIWPSSKGRPGNFPQSRPEPTAPLEPTAPPAEPTAPPAESFGFGEEITPSPKQEQKDREPLTSLKSLFGSDPLLQ

Bivalent M mosaic sequence NEF sequence (replacement of 2G positions by A to delete tetradecylation site)

MOS1 NEF

(206AA)

SEQ ID NO：16

MAGKWSKSSVVGWPAIRERMRRAEPAADGVGAVSRDLEKHGAITSSNTAANNADCAWLEAQEEEEVGFPVRPQVPLRPMTYKGALDLSHFLKEKGGLEGLIYSQKRQDILDLWVYHTQGYFPDWQNYTPGPGIRYPLTFGWCFKLVPVEPEKIEEANEGENNSLLHPMSQHGMDDPEKEVLMWKFDSRLAFHHMARELHPEYYKDC

MOS2 NEF

(206AA) -replacement of 2G positions with A to delete the tetradecylation site

SEQ ID NO：17

MAGKWSKSSIVGWPAVRERIRRAEPAAEGVGAASQDLDKYGALTSSNTAATNADCAWLEAQEDEEVGFPVKPQVPLRPMTYKAAFDLSFFLKEKGGLDGLIYSKKRQEILDLWVYNTQGFFPDWQNYTPGPGVRYPLTFGWCFKLVPVDPREVEEANKGENNCLLHPMNLHGMDDPEREVLVWRFDSRLAFHHMAREKHPEYYKNC

MOS3 NEF

(208AA)

SEQ ID NO：18

MAGKWSKRSVVGWPAVRERMRRTEPAAEGVGAVSQDLDKHGALTSSNTAHNNADCAWLQAQEEEEEVGFPVRPQVPVRPMTYKAAVDLSHFLKEKGGLEGLIHSQKRQEILDLWVYHTQGFFPDWHNYTPGPGTRFPLTFGWCYKLVPVDPKEVEEANEGENNCLLHPMSQHGMEDEDREVLKWKFDSSLARRHMARELHPEFYKDCL

Bivalent M mosaic sequence GAGNEF fusion sequence

Mosaic sequence GAGNEF1(AA sequence)

SEQ ID NO：19

MGARASVLSGGELDRWEKIRLRPGGKKKYRLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALEKIEEEQNKSKKKAQQAAADTGNSSQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSNKGRPGNFLQNRPEPTAPPEESFRFGEETTTPSQKQEPIDKEMYPLASLKSLFGNDPSSQAGKWSKSSVVGWPAIRERMRRAEPAADGVGAVSRDLEKHGAITSSNTAANNADCAWLEAQEEEEVGFPVRPQVPLRPMTYKGALDLSHFLKEKGGLEGLIYSQKRQDILDLWVYHTQGYFPDWQNYTPGPGIRYPLTFGWCFKLVPVEPEKIEEANEGENNSLLHPMSQHGMDDPEKEVLMWKFDSRLAFHHMARELHPEYYKDC

Mosaic sequence GAGNEF2(AA sequence)

SEQ ID NO：20

MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFALNPGLLETSEGCKQIIKQLQPALQTGTEELRSLFNTVATLYCVHAEIEVRDTKEALDKIEEEQNKSQQKTQQAKEADGKVSQNYPIVQNLQGQMVHQPISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEΞAAEWDRLHPVHAGPVAPGQMREPRGSDIAGTTSNLQEQIAWMTSNPPIPVGDIYKRWIILGLNKIVRMYSPTSILDIKQGPKEPFRDYVDRFFKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPSHKARVLAEAMSQTNSTILMQRSNFKGSKRIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFRFEETTPAPKQEPKDREPLTSLRSLFGSDPLSQAGKWSKSSIVGWPAVRERIRRAEPAAEGVGAASQDLDKYGALTSSNTAATNADCAWLEAQEDEEVGFPVKPQVPLRPMTYKAAFDLSFFLKEKGGLDGLIYSKKRQEILDLWVYNTQGFFPDWQNYTPGPGVRYPLTFGWCFKLVPVDPREVEEANKGENNCLLHPMNLHGMDDPEREVLVWRFDSRLAFHHMAREKHPEYYKNC

Bivalent M mosaic sequence GAGPOL fusion sequence (variant 3; POL extensively inactivated, deletion PR, 9A inactivating mutations to abolish catalytic activity)

Mosaic sequence GAGPOL 1V 3(AA sequence)

SEQ ID NO：21

MGARASVLSGGELDRWEKIRLRPGGKKKYRLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALEKIEEEQNKSKKKAQQAAADTGNSSQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSNKGRPGNFLQNRPEPTAPPEESFRFGEETTTPSQKQEPIDKEMYPLASLKSLFGNDPSSQMAPISPIETVPVKLKPGMDGPRVKQWPLTEEKIKALTAICEEMEKEGKITKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLAVGDAYFSVPLDEGFRKYTAFTIPSTNNETPGIRYQYNVLPQGWKGSPAIFQCSMTRILEPFRAKNPEIVIYQYMAALYVGSDLEIGQHRAKIEELREHLLKWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYPGIKVRQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGHDQWTYQIYQEPFKNLKTGKYAKMRTAHTNDVKQLTEAVQKIAMESIVIWGKTPKFRLPIQKETWETWWTDYWQATWIPEWEFVNTPPLVKLWYQLEKDPIAGVETFYVAGAANRETKLGKAGYVTDRGRQKIVSLTETTNQKTALQAIYLALQDSGSEVNIVTASQYALGIIQAQPDKSESELVNQIIEQLIKKERVYLSWVPAHKGIGGNEQVDKLVSSGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDQCQLKGEAMHGQVDCSPGIWQLACTHLEGKIILVAVHVASGYIEAEVIPAETGQETAYFILKLAGRWPVKVIHTANGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVASMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIKDYGKQMAGADCVAGRQDED

Mosaic sequence GAGPOL 2V 3(AA sequence)

SEQ ID NO：22

MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFALNPGLLETSEGCKQIIKQLQPALQTGTEELRSLFNTVATLYCVHAEIEVRDTKEALDKIEEEQNKSQQKTQQAKEADGKVSQNYPIVQNLQGQMVHQPISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPVAPGQMREPRGSDIAGTTSNLQEQIAWMTSNPPIPVGDIYKRWIILGLNKIVRMYSPTSILDIKQGPKEPFRDYVDRFFKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPSHKARVLAEAMSQTNSTILMQRSNFKGSKRIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFRFEETTPAPKQEPKDREPLTSLRSLFGSDPLSQMAPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLAVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDIVIYQYMAALYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVKQLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMRGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETFYVAGAANRETKLGKAGYVTDRGRQKVVSLTDTTNQKTALQAIHLALQDSGLEVNIVTASQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSRGIRKVLFLDGIDKAQEEHEKYHSNWRAMASEFNLPPIVAKEIVASCDKCQLKGEAIHGQVDCSPGIWQLACTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHTANGSNFTSATVKAACWWAGIKQEFGIPYNPQSQGVVASINKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGEYSAGERIVDIIASDIQTKELQKQITKIQNFRVYYRDSRDPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDED

MOS3 GAG-POL V3(AA sequence)

1359 aa-intact GAG and modified POL GAG-POL fusion

SEQ ID NO：23

MGARASVLSGGKLDAWEKIRLRPGGKKKYKLKHIVWASRELDRFALNPGLLETAEGCQQIIEQLQPALQTGSEELKSLYNTVAVLYCVHQRIDVKDTKEALDKIEEIQNKSKQKTQQAAADTGSSSKVSQNYPIVQNAQGQMVHQALSPRTLNAWVKVVEEKGFNPEVIPMFSALAEGATPQDLNMMLNIVGGHQAAMQILKDTINEEAADWDRLHPVHAGPIPPGQMREPRGSDIAGTTSTPQEQIGWMTSNPPVPVGEIYKRWIIMGLNKIVRMYSPVSILDIKQGPKESFRDYVDRFFKVLRAEQATQEVKNWMTETLLIQNANPDCKSILRALGPGASLEEMMTACQGVGGPSHKARILAEAMSQANNTNIMMQRGNFKGQKRIKCFNCGKEGHLARNCRAPRKRGCWKCGREGHQMKDCNERQANFLGKIWPSSKGRPGNFPQSRPEPTAPLEPTAPPAEPTAPPAESFGFGEEITPSPKQEQKDREPLTSLKSLFGSDPLLQMAPISPIDTVPVTLKPGMDGPKIKQWPLTEEKIKALTEICTEMEKEGKISRIGPENPYNTPVFAIKKKNSTRWRKLVDFRELNKKTQDFWEVQLGIPHPAGLKKKRSVTVLAVGDAYFSVPLDKDFRKYTAFTIPSVNNETPGVRYQYNVLPQGWKGSPAIFQCSMTKILEPFRAQNPEIVIYQYVAALYVGSDLEIEQHRTKIEELRAHLLSWGFTTPDKKHQREPPFLWMGYELHPDRWTVQPIELPEKESWTVNDIQKLVGKLNWASQIYPGIKVKQLCRLLRGAKALTEVIPLTKEAELELAENREILREPVHGVYYDPSKDLVAEIQKQGQDQWTYQIYQEPYKNLKTGKYARKRSAHTNDVRQLTEAVQKIALESIVIWGKIPKFRLPIQRETWETWWTEYWQATWIPDWEFVNTPPLVKLWYQLEKEPIAGAETFYVAGASNRETKIGKAGYVTDKGRQKVVSLTETTNQKAALQAIQLALQDSGPEVNIVTASQYVLGIIQAQPDRSESELVNQIIEELIKKEKVYLSWVPAHKGIGGNEQVDKLVSAGIRKILFLDGIDKAQEEHERYHSNWRTMASDFNLPPIVAKEIVANCDKCQLKGEAMHGQVDCSPGMWQLACTHLEGKIIIVAVHVASGYMEAEVIPAETGQETAYYILKLAGRWPVKVVHTANGSNFTSTTVKAACWWANVTQEFGIPYNPQSQGVIASMNKELKKIIGQVREQAEHLKTAVQMAVLIHNFKRRGGIGGYSAGERIVDIIATDIQTRELQKQIIKIQNFRVYFRDSRDPVWKGPAKLLWKGEGAVVIQDNSEIKVVPRRKVKIIRDYGKQMAGDDCVAGRQDEDQ

Bivalent M mosaic sequence GAGPOL fusion sequence (variant 4; POL minimal inactivation, intact PR-RT-IN)

Mosaic sequence GAGPOL 1V 4(AA sequence)

SEQ ID NO：24

MGARASVLSGGELDRWEKIRLRPGGKKKYRLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALEKIEEEQNKSKKKAQQAAADTGNSSQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSNKGRPGNFLQNRPEPTAPPEESFRFGEETTTPSQKQEPIDKEMYPLASLKSLFGNDPSSQRENLAFQQGEAREFPSEQTRANSPTSRELQVRGDNPHSEAGAERQGTLNFPQITLWQRPLVSIKVGGQIREALLATGADDTVLEDINLPGKWKPKMIGGIGGFIKVGQYDQILIEICGKKAIGTVLVGPTPVNIIGRNMLTQLGCTLNFPISPIETVPVKLKPGMDGPRVKQWPLTEEKIKALTAICEEMEKEGKITKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEGFRKYTAFTIPSTNNETPGIRYQYNVLPQGWKGSPAIFQCSMTRILEPFRAKNPEIVIYQYMDHLYVGSDLEIGQHRAKIEELREHLLKWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYPGIKVRQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGHDQWTYQIYQEPFKNLKTGKYAKMRTAHTNDVKQLTEAVQKIAMESIVIWGKTPKFRLPIQKETWETWWTDYWQATWIPEWEFVNTPPLVKLWYQLEKDPIAGVETFYVDGAANRETKLGKAGYVTDRGRQKIVSLTETTNQKTELQAIYLALQDSGSEVNIVTDSQYALGIIQAQPDKSESELVNQIIEQLIKKERVYLSWVPAHKGIGGNEQVDKLVSSGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDQCQLKGEAMHGQVDCSPGIWQLACTHLEGKIILVAVHVASGYIEAEVIPAETGQETAYFILKLAGRWPVKVIHTDNGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIKDYGKQMAGADCVAGRQDED

Mosaic sequence GAGPOL 2V 4(AA sequence)

SEQ ID NO：25

MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFALNPGLLETSEGCKQIIKQLQPALQTGTEELRSLFNTVATLYCVHAEIEVRDTKEALDKIEEEQNKSQQKTQQAKEADGKVSQNYPIVQNLQGQMVHQPISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPVAPGQMREPRGSDIAGTTSNLQEQIAWMTSNPPIPVGDIYKRWIILGLNKIVRMYSPTSILDIKQGPKEPFRDYVDRFFKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPSHKARVLAEAMSQTNSTILMQRSNFKGSKRIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFRFEETTPAPKQEPKDREPLTSLRSLFGSDPLSQRENLAFPQGKAREFSSEQTRANSPTRRELQVWGRDNNSLSEAGADRQGTVSFSFPQITLWQRPLVTIKIGGQLKEALLATGADDTVLEEMNLPGRWKPKMIGGIGGFIKVGQYDQIPIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNFPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDIVIYQYMDHLYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVKQLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMRGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETFYVDGAANRETKLGKAGYVTDRGRQKVVSLTDTTNQKTELQAIHLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSRGIRKVLFLDGIDKAQEEHEKYHSNWRAMASEFNLPPIVAKEIVASCDKCQLKGEAIHGQVDCSPGIWQLACTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHTDNGSNFTSATVKAACWWAGIKQEFGIPYNPQSQGVVESINKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGEYSAGERIVDIIASDIQTKELQKQITKIQNFRVYYRDSRDPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDED

IX. bivalent M mosaic sequence GAGPOL fusion sequence (variant 5; POL minimal inactivation, deletion PR)

Mosaic sequence GAGPOL 1V 5(AA sequence)

SEQ ID NO：26

MGARASVLSGGELDRWEKIRLRPGGKKKYRLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALEKIEEEQNKSKKKAQQAAADTGNSSQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSNKGRPGNFLQNRPEPTAPPEESFRFGEETTTPSQKQEPIDKEMYPLASLKSLFGNDPSSQMAPISPIETVPVKLKPGMDGPRVKQWPLTEEKIKALTAICEEMEKEGKITKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEGFRKYTAFTIPSTNNETPGIRYQYNVLPQGWKGSPAIFQCSMTRILEPFRAKNPEIVIYQYMDHLYVGSDLEIGQHRAKIEELREHLLKWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYPGIKVRQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGHDQWTYQIYQEPFKNLKTGKYAKMRTAHTNDVKQLTEAVQKIAMESIVIWGKTPKFRLPIQKETWETWWTDYWQATWIPEWEFVNTPPLVKLWYQLEKDPIAGVETFYVDGAANRETKLGKAGYVTDRGRQKIVSLTETTNQKTELQAIYLALQDSGSEVNIVTDSQYALGIIQAQPDKSESELVNQIIEQLIKKERVYLSWVPAHKGIGGNEQVDKLVSSGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDQCQLKGEAMHGQVDCSPGIWQLACTHLEGKIILVAVHVASGYIEAEVIPAETGQETAYFILKLAGRWPVKVIHTDNGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIKDYGKQMAGADCVAGRQDED

Mosaic sequence GAGPOL 2V 5(AA sequence)

SEQ ID NO：27

MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFALNPGLLETSEGCKQIIKQLQPALQTGTEELRSLFNTVATLYCVHAEIEVRDTKEALDKIEEEQNKSQQKTQQAKEADGKVSQNYPIVQNLQGQMVHQPISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPVAPGQMREPRGSDIAGTTSNLQEQIAWMTSNPPIPVGDIYKRWIILGLNKIVRMYSPTSILDIKQGPKEPFRDYVDRFFKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPSHKARVLAEAMSQTNSTILMQRSNFKGSKRIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFRFEETTPAPKQEPKDREPLTSLRSLFGSDPLSQMAPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDIVIYQYMDHLYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVKQLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMRGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETFYVDGAANRETKLGKAGYVTDRGRQKVVSLTDTTNQKTELQAIHLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSRGIRKVLFLDGIDKAQEEHEKYHSNWRAMASEFNLPPIVAKEIVASCDKCQLKGEAIHGQVDCSPGIWQLACTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHTDNGSNFTSATVKAACWWAGIKQEFGIPYNPQSQGVVESINKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGEYSAGERIVDIIASDIQTKELQKQITKIQNFRVYYRDSRDPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDED

X. bivalent M mosaic sequence GAGPOLNEF fusion sequence (POL extensively inactivated, deleted PR)

Mosaic sequence GAGPOLNEF1(AA sequence)

SEQ ID NO：28

MGARASVLSGGELDRWEKIRLRPGGKKKYRLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALEKIEEEQNKSKKKAQQAAADTGNSSQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSNKGRPGNFLQNRPEPTAPPEESFRFGEETTTPSQKQEPIDKEMYPLASLKSLFGNDPSSQMAPISPIETVPVKLKPGMDGPRVKQWPLTEEKIKALTAICEEMEKEGKITKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLAVGDAYFSVPLDEGFRKYTAFTIPSTNNETPGIRYQYNVLPQGWKGSPAIFQCSMTRILEPFRAKNPEIVIYQYMAALYVGSDLEIGQHRAKIEELREHLLKWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYPGIKVRQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGHDQWTYQIYQEPFKNLKTGKYAKMRTAHTNDVKQLTEAVQKIAMESIVIWGKTPKFRLPIQKETWETWWTDYWQATWIPEWEFVNTPPLVKLWYQLEKDPIAGVETFYVAGAANRETKLGKAGYVTDRGRQKIVSLTETTNQKTALQAIYLALQDSGSEVNIVTASQYALGIIQAQPDKSESELVNQIIEQLIKKERVYLSWVPAHKGIGGNEQVDKLVSSGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDQCQLKGEAMHGQVDCSPGIWQLACTHLEGKIILVAVHVASGYIEAEVIPAETGQETAYFILKLAGRWPVKVIHTANGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVASMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIKDYGKQMAGADCVAGRQDEDMAGKWSKSSVVGWPAIRERMRRAEPAADGVGAVSRDLEKHGAITSSNTAANNADCAWLEAQEEEEVGFPVRPQVPLRPMTYKGALDLSHFLKEKGGLEGLIYSQKRQDILDLWVYHTQGYFPDWQNYTPGPGIRYPLTFGWCFKLVPVEPEKIEEANEGENNSLLHPMSQHGMDDPEKEVLMWKFDSRLAFHHMARELHPEYYKDC

Mosaic sequence GAGPOLNEF2(AA sequence)

SEQ ID NO：29

MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFALNPGLLETSEGCKQIIKQLQPALQTGTEELRSLFNTVATLYCVHAEIEVRDTKEALDKIEEEQNKSQQKTQQAKEADGKVSQNYPIVQNLQGQMVHQPISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPVAPGQMREPRGSDIAGTTSNLQEQIAWMTSNPPIPVGDIYKRWIILGLNKIVRMYSPTSILDIKQGPKEPFRDYVDRFFKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPSHKARVLAEAMSQTNSTILMQRSNFKGSKRIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFRFEETTPAPKQEPKDREPLTSLRSLFGSDPLSQMAPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLAVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDIVIYQYMAALYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVKQLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMRGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETFYVAGAANRETKLGKAGYVTDRGRQKVVSLTDTTNQKTALQAIHLALQDSGLEVNIVTASQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSRGIRKVLFLDGIDKAQEEHEKYHSNWRAMASEFNLPPIVAKEIVASCDKCQLKGEAIHGQVDCSPGIWQLACTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHTANGSNFTSATVKAACWWAGIKQEFGIPYNPQSQGVVASINKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGEYSAGERIVDIIASDIQTKELQKQITKIQNFRVYYRDSRDPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDEDMAGKWSKSSIVGWPAVRERIRRAEPAAEGVGAASQDLDKYGALTSSNTAATNADCAWLEAQEDEEVGFPVKPQVPLRPMTYKAAFDLSFFLKEKGGLDGLIYSKKRQEILDLWVYNTQGFFPDWQNYTPGPGVRYPLTFGWCFKLVPVDPREVEEANKGENNCLLHPMNLHGMDDPEREVLVWRFDSRLAFHHMAREKHPEYYKNC

XI optimal clade C ENV GP160, GAG, POL, NEF sequences

Best clade C ENV GP160(SN90.90.SE364) (AA sequences)

SEQ ID NO：30

MRVTGMLRNCQPWWIWGILGFWMLLIYNVGGNLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTEYFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCRNVTTSNNATSNDNPNGEIKNCSFNITTELRDKRRNEYALFYRLDIVPLSGSKNSSNSSEYRLINCNTSAITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENLTNNAKTIIVHLNESIEIVCARPNNNTRKSMRIGPGQTFYATGDIIGDIRQAHCNISGNWNATLEKVKGKLQEHFPGKNISFEPSSGGDLEITTHSFNCRGEFFYCDTSKLFNGTTHTANSSITIQCRIKQIINMWQGVGRAIYAPPIAGNITCKSNITGLLLTRDGGTLNNDTEKFRPGGGDMRDNWRSELYKYKVVEIKPLGIAPTKAKRRVVEREKRAVGIGAVFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQTRVLAIERYLKDQQLLGIWGCSGKIICTTAVPWNTSWSNKSLEDIWDNMTWMQWDREINNYTSIIYSLLEESQNQQEKNEKDLLALDSWNNLWNWFNITKWLWYIKIFIMIVGGLIGLRIIFAVLSIVNRVRQGYSPLSFQTLIPNPRGPDRLGRIEEEGGEQDRDRSIRLVNGFLAIAWDDLRSLCLFSYRRLRDFILIVARAVELLIQRGWETLKYLGSLPQYWGLELKKSAISLLDTIAITVAEGTDRIIELVQRICRAISNIPRRIRQGFEAALQ

Optimal clade C GAG (IN.70177) (AA sequence)

SEQ ID NO：31

MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFALNPGLLETSEGCKQILKQLQPALQTGTEELRSLYNTVATLYCVHAGIEVRDTKEALDKIEEEQNKGQQKTQQAKGADGKVSQNYPIVQNLQGQMVHQAISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIAWMTNNPPVPVGDIYKRWIILGLNKIVRMYSPVSILDIKQGPKEPFRDYVDRFFKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPSHKARVLAEAMSQTGSTIMMQRSNFKGSKRIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFRFEETTPAPKQELKDREPLTSLKSLFGSDPLSQ

Best clade C POL (ZA.04.04ZASK208B1) (AA sequence)

SEQ ID NO：32

FFRENLAFQQGEAREFPSEQARANSPTSREFQVRGDNPCSEAGVKGQGTLNFPQITLWQRPLVSIKVGGQVKEALLDTGADDTVLEEINLPGKWKPKMIGGIGGFIKVRQYDQILIEICGKKAIGTVLVGPTPVNIIGRNMLTQLGCTLNFPISPIETVPVKLKPGMDGPKIKQWPLTEEKIKALMAICEEMEKEGKITKIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDESFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRAKNPEIVIYQYMDDLYVGSDLEIGQHRAKIEELREHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYSGIKVRQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGYDQWTYQIYQEPFKNLKTGKYAKMRTAHTNDVKQLTEAVQKIALESIVIWGKTPKFRLPIQKETWEIWWTDYWQATWIPEWEFVNTPPLVKLWYQLEKEPIAGAETFYVDGAANRETKIGKAGYVTDKGRQKIVTLTETTNQKTELQAIQLALQDSGSEVNIVTDSQYALGIIQAQPDKSESELVNQIIEQLINKERVYLSWVPAHKGIGGNEQVDKLVSSGIRKVLFLDGIDKAQEEHEKYHSNWRAMASEFNLPPVVAKEIVASCDKCQLKGEAIHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYMEAEVIPAETGQETAYYILKLAGRWPVKVIHTDNGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIKDYGKQMAGADCVAGRQDED

Best clade C NEF (ZA00.1170MB) (AA sequence)

SEQ ID NO：33

MGGKWSKSSIVGWPDVRERMRRTEPAAEGVGAASQDLDKYGALTSSNTTHNNADCAWLEAQEEGEVGFPVRPQVPLRPMTYKGAFDLSFFLKEKGGLDGLIYSKKRQEILDLWVYHTQGFFPDWQNYTPGPGVRYPLTFGWCFKLVPVDPREVEEANKGENNCLLHPMSLHGMEDEEREVLKWEFDSSLARRHLARELHPEYYKDC

Optimal clade C ENV GP140 sequence (cleavage/fusion deficient)

Best clade C ENV GP140(SN.90.90.SE364) (AA sequences)

SEQ ID NO：34

MRVTGMLRNCQPWWIWGILGFWMLLIYNVGGNLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTEYFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCRNVTTSNNATSNDNPNGEIKNCSFNITTELRDKRRNEYALFYRLDIVPLSGSKNSSNSSEYRLINCNTSAITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENLTNNAKTIIVHLNESIEIVCARPNNNTRKSMRIGPGQTFYATGDIIGDIRQAHCNISGNWNATLEKVKGKLQEHFPGKNISFEPSSGGDLEITTHSFNCRGEFFYCDTSKLFNGTTHTANSSITIQCRIKQIINMWQGVGRAIYAPPIAGNITCKSNITGLLLTRDGGTLNNDTEKFRPGGGDMRDNWRSELYKYKVVEIKPLGIAPTKAKRRVVESEKSAVGIGAVFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQTRVLAIERYLKDQQLLGIWGCSGKIICTTAVPWNTSWSNKSLEDIWDNMTWMQWDREINNYTSIIYSLLEESQNQQEKNEKDLLALDSWNNLWNWFNITKWLW

Optimal clade C POL sequence (extensive inactivation, deletion of PR)

Best clade C POL (ZA.04.04ZASK208B1) (AA sequence)

SEQ ID NO：35

MAPISPIETVPVKLKPGMDGPKIKQWPLTEEKIKALMAICEEMEKEGKITKIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLAVGDAYFSVPLDESFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRAKNPEIVIYQYMAALYVGSDLEIGQHRAKIEELREHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYSGIKVRQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGYDQWTYQIYQEPFKNLKTGKYAKMRTAHTNDVKQLTEAVQKIALESIVIWGKTPKFRLPIQKETWEIWWTDYWQATWIPEWEFVNTPPLVKLWYQLEKEPIAGAETFYVAGAANRETKIGKAGYVTDKGRQKIVTLTETTNQKTALQAIQLALQDSGSEVNIVTASQYALGIIQAQPDKSESELVNQIIEQLINKERVYLSWVPAHKGIGGNEQVDKLVSSGIRKVLFLDGIDKAQEEHEKYHSNWRAMASEFNLPPVVAKEIVASCDKCQLKGEAIHGQVDCSPGIWQLACTHLEGKVILVAVHVASGYMEAEVIPAETGQETAYYILKLAGRWPVKVIHTANGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVASMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIKDYGKQMAGADCVAGRQDED

XIV. best clade C GAGNEF fusion sequences

Best clade C GAGNEF (IN.70177-ZA00.1170MB) (AA sequence)

SEQ ID NO：36

MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFALNPGLLETSEGCKQILKQLQPALQTGTEELRSLYNTVATLYCVHAGIEVRDTKEALDKIEEEQNKGQQKTQQAKGADGKVSQNYPIVQNLQGQMVHQAISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIAWMTNNPPVPVGDIYKRWIILGLNKIVRMYSPVSILDIKQGPKEPFRDYVDRFFKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPSHKARVLAEAMSQTGSTIMMQRSNFKGSKRIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFRFEETTPAPKQELKDREPLTSLKSLFGSDPLSQAGKWSKSSIVGWPDVRERMRRTEPAAEGVGAASQDLDKYGALTSSNTTHNNADCAWLEAQEEGEVGFPVRPQVPLRPMTYKGAFDLSFFLKEKGGLDGLIYSKKRQEILDLWVYHTQGFFPDWQNYTPGPGVRYPLTFGWCFKLVPVDPREVEEANKGENNCLLHPMSLHGMEDEEREVLKWEFDSSLARRHLARELHPEYYKDC

XV. consensus sequence

M consensus sequence ENV

SEQ ID NO：37

MRVRGIQRNCQHLWRWGTLILGMLMICSAAENLWVTVYYGVPVWKEANTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTNVNVTNTTNNTEEKGEIKNCSFNITTEIRDKKQKVYALFYRLDVVPIDDNNNNSSNYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCNDKKFNGTGPCKNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENITNNAKTIIVQLNESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNISGTKWNKTLQQVAKKLREHFNNKTIIFKPSSGGDLEITTHSFNCRGEFFYCNTSGLFNSTWIGNGTKNNNNTNDTITLPCRIKQIINMWQGVGQAMYAPPIEGKITCKSNITGLLLTRDGGNNNTNETEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRWESEKSAVGIGAVFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTTVPWNSSWSNKSQDEIWDNMTWMEWEREINNYTDIIYSLIEESQNQQEKNEQELLALDKWASLWNWFDITNWLW

M consensus sequence GAG

SEQ ID NO：38

MGARASVLSGGKLDAWEKIRLRPGGKKKYRLKHLVWASRELERFALNPGLLETSEGCKQIIGQLQPALQTGSEELRSLYNTVATLYCVHQRIEVKDTKEALEKIEEEQNKSQQKTQQAAADKGNSSKVSQNYPIVQNLQGQMVHQAISPRTLNAWVKVIEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPIPPGQMREPRGSDIAGTTSTLQEQIAWMTSNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFFKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVLAEAMSQVTNAAIMMQRGNFKGQRRIIKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSNKGRPGNFLQSRPEPTAPPAESFGFGEEITPSPKQEPKDKEPPLTSLKSLFGNDPLSQ

M consensus sequence POL

SEQ ID NO：39

MAPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALTEICTEMEKEGKISKIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRTQNPEIVIYQYMDHLYVGSDLEIGQHRAKIEELREHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYPGIKVKQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQDQWTYQIYQEPFKNLKTGKYAKMRSAHTNDVKQLTEAVQKIATESIVIWGKTPKFRLPIQKETWETWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIAGAETFYVDGAANRETKLGKAGYVTDRGRQKVVSLTETTNQKTELQAIHLALQDSGSEVNIVTDSQYALGIIQAQPDKSESELVNQIIEQLIKKEKVYLSWVPAHKGIGGNEQVDKLVSTGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPIVAKEIVASCDKCQLKGEAMHGQVDCSPGIWQLACTHLEGKIILVAVHVASGYIEAEVIPAETGQETAYFILKLAGRWPVKVIHTDNGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQITKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVAGRQDED

Other embodiments

Although the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims

1. A vaccine for treating or reducing the risk of a viral infection in a mammal, comprising:

at least two different optimized viral HIV-1 polypeptides, or

One or more vectors encoding said at least two different optimized viral HIV-1 polypeptides,

wherein the at least two different optimized viral HIV-1 polypeptides correspond to the same viral HIV-1 gene product, wherein the viral HIV-1 gene product is selected from the group consisting of Gag, Pol, and Env, and

wherein the at least two different optimized Gag polypeptides are the amino acid sequences of SEQ ID NOs 3 and 4, respectively;

wherein the at least two different optimized Pol polypeptides are the amino acid sequences of SEQ ID NO 12 and 13, respectively; and

wherein the at least two different optimized Env polypeptides are the amino acid sequences of SEQ ID NO 9 and 10, respectively.

2. The vaccine of claim 1, wherein the at least two optimized viral HIV-1 polypeptides are Gag polypeptides of the amino acid sequences of SEQ ID NOs 3 and 4, respectively.

3. The vaccine according to claim 1, wherein said at least two optimized viral HIV-1 polypeptides are Pol polypeptides of the amino acid sequences of SEQ ID NO 12 and 13, respectively.

4. The vaccine of claim 2, further comprising at least two optimized Pol virus HIV-1 polypeptides having the amino acid sequences of SEQ ID NOs 12 and 13, respectively.

5. The vaccine of claim 4, further comprising at least two optimized Env virus HIV-1 polypeptides having the amino acid sequences of SEQ ID NO 9 and 10, respectively.

6. The vaccine of claim 1, wherein the vaccine comprises one or more of the vectors encoding at least two optimized Gag virus HIV-1 polypeptides having amino acid sequences of SEQ ID NOs 3 and 4, respectively.

7. The vaccine according to claim 6, wherein said vaccine further comprises one or more of said vectors encoding at least two optimized Pol virus HIV-1 polypeptides, the amino acid sequences of said at least two optimized Pol virus HIV-1 polypeptides being SEQ ID NO 12 and 13, respectively.

8. The vaccine of claim 7, wherein the optimized Gag and Pol polypeptides are encoded by the same vector.

9. The vaccine of claim 7, wherein said vaccine further comprises one or more of said vectors encoding at least two optimized Env virus HIV-1 polypeptides having the amino acid sequences of SEQ ID NO 9 and 10, respectively.

10. The vaccine of claim 9, wherein the optimized Gag, Pol, and Env polypeptides are encoded by the same vector.

11. The vaccine of any one of claims 1 and 6-10, wherein the vector is selected from the group consisting of adenovirus serotype 26(Ad26), adenovirus serotype 34(Ad34), adenovirus serotype 35(Ad35), adenovirus serotype 48(Ad48), or adenovirus serotype 5HVR48 (Ad5HVR48), poxvirus, modified vaccinia virus ankara (MVA).

12. The vaccine of claim 11, wherein the vector is adenovirus serotype 26(Ad 26).

13. The vaccine of claim 11, wherein the vector is a modified vaccinia virus ankara (MVA).

14. The vaccine of any one of claims 1-10, wherein the vaccine further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

15. A method of preparing a vaccine for treating or reducing the risk of HIV-1 infection in a human, the method comprising synthesizing a vaccine according to any one of claims 1 to 14.

16. A kit, comprising:

a) the vaccine of any one of claims 1 to 13;

b) a pharmaceutically acceptable carrier, excipient or diluent;

c) instructions for its use; and, optionally

d) An adjuvant.

17. A polypeptide of the amino acid sequence of SEQ ID NO. 9.

18. Use of a polypeptide according to claim 17 in the manufacture of a medicament for treating or reducing the risk of HIV-1 infection in a human, wherein the medicament comprises a pharmaceutically acceptable carrier, excipient or diluent.

19. A vector encoding the polypeptide of claim 17.

20. Use of a vector according to claim 19 in the manufacture of a medicament for treating or reducing the risk of HIV-1 infection in a human, wherein the medicament comprises a pharmaceutically acceptable carrier, excipient or diluent.