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Patent application title: Immunological Compositions for HIV
James T. Tartaglia (Nazareth, PA, US)
Sanjay Gurunathan (Nazareth, PA, US)
Jerome H. Kim (Bethesda, MD, US)
Donald Francis (S. San Francisco, CA, US)
IPC8 Class: AA61K3921FI
USPC Class:
Class name: Disclosed amino acid sequence derived from virus retroviridae (e.g., feline leukemia, etc.) immunodeficiency virus (e.g., hiv, etc.)
Publication date: 2012-12-06
Patent application number: 20120308593
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The disclosure relates to immunological compositions for vaccinating
human beings against infection by the Human Immunodeficiency Virus (HIV).
1. A method for protectively immunizing a human being against human
immunodeficiency virus (HIV) by administering to the human being at least
one dose of a first composition comprising a viral vector encoding an HIV
polypeptide or fragment or derivative thereof and subsequently
administering to the human being at least one dose of a second
composition comprising the HIV polypeptide or fragment or derivative
thereof, wherein a protective immune response directed against HIV is
2. A method for protectively immunizing a human being against human immunodeficiency virus (HIV) by administering to the human being a vaccine consisting essentially of: a first composition and a second composition, the first composition consisting essentially of a live, attenuated viral vector encoding at least one HIV gp120 or fragment or derivative thereof and, optionally, at least one additional HIV polypeptide or fragment or derivative thereof, the second composition consisting essentially of al least one HIV gp120 polypeptide or fragment or derivative thereof and, optionally, at least one additional HIV polypeptide or fragment or derivative thereof; the method comprising the steps of administering the first composition to the human being and subsequently administering to the human being at least one composition or combination of compositions selected from the group consisting of: the second composition alone: the first and second compositions, optionally separately or together as a single dose; at least one additional dose of the first composition followed by at least one dose of the second composition; the second composition followed by at least one additional dose of the first composition, optionally followed by at least one additional dose of the first and/or second composition; wherein a protective immune response against HIV is induced in the human being.
3. The method of claim 1 wherein at least one of the compositions comprises an amino acid sequence corresponding to that of a herpes simplex vims (HSV).
4. The method of claim 3 wherein the HSV amino acid sequence comprises SEQ ID NO.: 7.
5. The method of claim 1 wherein one of the compositions comprises SEQ ID NO. 7.
6. The method of claim 1, further comprising administering the vaccine is administered to a population of human beings and at least about one-third of that population is protected from infection by HIV.
7. The method of claim 1, wherein the first composition is administered repeatedly prior to at least one administration of the second composition, with the time between administrations is of sufficient length to allow for the development of an immune response within the human being.
8. The method of claim 4, wherein the second composition is co-administered with the first composition.
9. The method of claim 1, wherein administration of both the first and second compositions is via a route selected from the group consisting of mucosal, intradermal, intramuscular, subcutaneous, via skin scarification, intranodal, or intratumoral.
10. The method of claim 1, wherein administration is at separate sites in the human being.
11. The method of claim 1 wherein the amount of viral vector administered in each dose is the equivalent of about 10.sup.7 CCID50 and the total amount of polypeptide administered in each dose is about 600 μg.
12. The method of claim 1 wherein the viral vector is selected from the group consisting of retrovirus, adenovirus, adeno-associated virus (AAV), alphavirus, herpes virus, and poxvirus.
13. The method of claim 12 wherein the viral vector is a poxvirus.
14. The method of claim 13 wherein the poxvirus vector is selected from the group consisting of vaccinia, NYVAC, Modified Virus Ankara (MVA), avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.
15. The method of claim 14 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).
16. The method of claim 1 wherein the HIV polypeptide or HIV gp120 is derived from an HIV virus selected from the group consisting of HIV-1, HIV-2, and HIV-3, wherein the first and second composition contain the same or different HIV polypeptides and/or gp120.
17. The method of claim 16 wherein the HIV-1 is HIV-1 subtype A1, HIV-1 subtype A2, HIV-1 subtype A3, HIV-1 subtype A4, HIV-1 subtype B, HIV-1 subtype C, HIV-1 subtype D, HIV-1 subtype E, HIV-1 subtype F1, HIV-1 subtype F2, HIV-1 subtype G, HIV-1 subtype H, HIV-1 subtype J and HIV-1 subtype K.
18. The method of claim 16 wherein the HIV-2 is selected from the group consisting of HIV-2 subtype A, HIV-2 subtype B, HIV-2 subtype C, HIV-2 subtype D, and HIV-2 subtype E.
19. The method of claim 1 further comprising administering a composition comprising at least one additional HIV immunogen selected from the group consisting of gag, pol, nef, a variant thereof, and a derivative thereof.
20. The method of claim 1 wherein the first or second composition additionally contain at least one additional HIV immunogen selected from the group consisting of gag, the protease component encoded by pol, nef, a variant thereof, and a derivative thereof.
21. The method of claim 1 wherein the viral vector encodes at least one polypeptide selected from the group consisting of HIV gp120 MN 12-485, HIV gp120 A244 12-484, and HIV gp120 GNE8 12-477.
22. The method of claim 1 wherein the viral vector encodes at least HIV gp120 MN 12-485 and HIV gp120 GNE8 12-477, or at least HIV gp120 MN 12-485 and HIV gp120 A244 12-484.
23. The method of claim 1 wherein the viral vector is ALVAC-HIV (vCP1521).
24. The method of claim 1 wherein the viral vector comprises the nucleic acid sequence of SEQ ID NO. 5.
25. The method of claim 1 wherein the second composition is AIDSVAX® B/B or AIDSVAX® B/E.
26. The method of claim 1 wherein the viral vector is ALVAC-HIV (vCP1521) and the second composition is AIDSVAX® B/E.
[0001] This application claims priority to U.S. Ser. No. 61/243,522 filed Sep. 17, 2009.
[0002] The present invention relates generally to the field of immunology and, in particular to methods and compositions for immunizing and generating protection in a host against infection and disease with HIV.
[0003] Human immunodeficiency virus (HIV) is a human retrovirus and is the etiological agent of acquired immunodeficiency syndrome (AIDS). Despite the passage of more than 20 years since the discovery of HIV, no effective vaccine has been found to either ameliorate the disease or to prevent infection. By the end of the year 2007, more than 30 million people worldwide were infected with HIV, with more than 20 million of those people living in sub-Saharan Africa (Report on the Global AIDS Epidemic, Joint United Nations Programme on HIV/AIDS (UNAIDS), 2008).
[0004] A hallmark of resistance to future viral infection, is the generation of `neutralizing antibodies` capable of recognizing the viral pathogen. Another measure is cellular immunity against infected ceils. In typical viral infections, generation of neutralizing antibodies and cellular immunity heralds recovery from infection. In HIV-1 infection, however, neutralizing antibodies and cellular immunity appear very early during the infection and have been associated with only a transient decrease in viral burden. In spite of the generation of neutralizing antibodies and cellular immunity, viral replication in HIV-1 infection rebounds and AIDS (acquired immune deficiency syndrome) develops. Thus, in HIV-1 infection, neutralizing antibodies and cellular immunity are not accurate measures of protective immunity. Protective immunity, meaning the vaccinees are protected against new infections by HIV, is a major unaccomplished goal of those skilled in the art.
[0005] Several potential vaccines have been tested in humans but found not to be protective. For examples, polypeptide vaccines based on gp120 have been tested (e.g., AIDSVAX® B/B, AIDSVAX® B/E (Vaxgen)) as solo vaccines or together in a prime-boost format, but have not shown protection against HIV infection (McCarthy, M. Lancet. 362(9397): 1728 (2003); Nitayaphan, et al. J. Inf. Dis. 190:702-6 (2004); Pitisuttithum, P. 11th Conf. Retr. Opp. Inf. 2004. 115: Abstract 107). Many studies have also been performed using animal models (e.g., monkeys). However, while primate data are instructive they also highlight the gaps in our understanding of immunological mechanism that mediate vaccine associated protection and emphasize the need to conduct human efficacy studies to test promising candidate vaccines empirically.
[0006] ALVAC-HIV (vCP1521) vaccine is a preparation of recombinant canarypox-derived virus expressing the products of the HIV-1 env and gag genes. The genes are inserted into the C6 locus under the control of the vaccinia virus H6 and I3L promoters respectively. The gp120 env sequence is derived from the HIV-92TH023 (subtype E) strain, but the anchoring part of gp41 is derived from the HIV-LAI (subtype B) strain. ALVAC-HIV infected cells present env and gag proteins in a near-native conformation (Fang, et al. J. Infect Dis. 180 (4): 1122-32 (1999)). In addition, intracellular processing of the HIV-1 proteins via the MHC class I pathway facilitates stimulation of cytotoxic T-lymphocytes. Part of the rationale for use of Gag from a subtype B in Thailand is that portions of the gag gene are conserved among virus subtypes. Therefore, gag-specific CTL elicited by vCP1521 may cross-react with CTL epitopes on non-subtype B primary viruses. Data from an AVEG-sponsored prime-boost trial (vCP205 alone or boosted with Chiron SF2 gp120/MF59) showed that CD8.sup.+ CTL from some vaccine recipients recognized target cells infected with non-subtype B viruses, including subtype E (Ferrari, et al. Proc. Natl. Acad. Set. USA, 94;1396-401 (1997)).
[0007] In view of this data, several attempts have been made to provide protection using a prime-boost immunization format (McNeil, et al. Science. 303:961 (2004)). For example, AIDSVAX® B/E (VaxGen) and ALVAC-HIV have been used as the prime and boost compositions, respectively, and shown to induce neutralizing antibodies (Karnasuta, et al Vaccine, 23: 2522-2529 (2005). In one safety trial, neutralizing antibodies were observed m 84% to 100% of subjects, cytotoxic lymphocytes (CTL) were observed in 16-25% of subjects, and lympho-proliferation was observed in 55-93% of subjects. In another safety trial, neutralizing antibodies were observed in 31% to 71% of subjects, cytotoxic lymphocytes (CTL) were observed in about 25% of subjects, and lympho-proliferation was observed in 58-71% of subjects. However, protection against infection by HIV was not shown, leading some to question the value of such a combination vaccine (Burton, et al. Science. 303; 316 (2004); Letters to the Editor. Science. 305:177-180 (2004)).
[0008] To date, no vaccine has shown protection against HIV infection. Thus, there is a clear need in the art for compositions and methods for protectively immunizing humans against HIV. Such compositions and methods are provided by this disclosure.
[0009] FIG. 1. Comparison of the predicted amino acid sequence for A244 rgp120/HIV-1 protein (gd244) with the predicted sequence for MN rgp120/HIV-1 protein (MN.mature).
[0010] FIG. 2. A. Map of ALVAC-HIV (vCP1521) genome. B. Nucleotide sequence of the HIV insert contained within ALVAC-HIV (vCP1521).
[0011] FIG. 3. Efficacy of the AIDSVAX® B/E/ALVAC-HIV prime-boost vaccine.
[0012] The reagents and methodologies described herein may be used to protectively immunize a human being against human immunodeficiency virus, which has not been previously demonstrated In one embodiment, a two-part composition including a first composition comprising an expression vector encoding an HIV immunogen, and a second composition comprising a polypeptide derived from or representing an HIV immunogen is provided. As shown herein, administration of the first composition prior to the second composition, and then administering the first, and second compositions, protects human beings against infection by HIV. In an exemplary embodiment, the first composition includes a live, attenuated viral vector encoding at least one HIV immunogen, and the second composition includes an HIV immunogen in the form of a polypeptide. In certain embodiments, a method for protectively immunizing a human being against human immunodeficiency virus (HIV) by administering to the human being a vaccine composition. The composition consists essentially of a first composition and a second composition, the first composition consisting essentially of a live, attenuated avipox (e.g., canarypox, ALVAC) vector encoding multiple HIV immunogens, and the second composition including one or more polypeptides corresponding in amino acid sequence to at least a portion of HIV gp120. In some embodiments, at least one of the compositions comprises an amino acid sequence corresponding to that of HIV and at least one amino acid sequence corresponding to a herpes simplex virus (HSV). In some embodiments, the first composition is administered to the human being and the first and second compositions are subsequently administered to the human being. In certain embodiments, the compositions are administered via the intramuscular route. Using this method, it has been found for the first time that human beings may be protected from infection by HIV. In certain embodiments, the method involves administering the vaccine to a population of human beings and at least about one-third of that population is protected from infection by HIV.
[0013] The present invention provides compositions and methodologies useful for treating and/or preventing conditions relating to an infectious or other agent(s) such as a tumor cell by stimulating an immune response against such an agent. In general, the immune response results from expression of an immunogen derived from or related to such an agent following administration of a nucleic acid vector encoding the immunogen, for example. In certain embodiments, multiple immunogens (which may be the same or different) are utilized. In other embodiments, variants or derivatives (i.e., by substitution, deletion or addition of amino acids or nucleotides encoding the same) of an immunogen or immunogens (which may be the same or different) may be utilized.
[0014] An immunogen may be a moiety (e.g., polypeptide, peptide or nucleic acid) that induces or enhances the immune response of a host to whom or to which the immunogen is administered. An immune response may be induced or enhanced by either increasing or decreasing the frequency, amount, or half-life of a particular immune modulator (e.g, the expression of a cytokine, chemokine, co-stimulatory molecule). This may be directly observed within a host cell containing a polynucleotide of interest (e.g., following infection by a recombinant virus) or within a nearby cell or tissue (e.g., indirectly). The immune response is typically directed against a target antigen. For example, an immune response may result from expression of an immunogen in a host following administration of a nucleic acid vector encoding the immunogen to the host. The immune response may result in one or more of an effect (e.g., maturation, proliferation, direct- or cross-presentation of antigen, gene expression profile) on cells of either the innate or adaptive immune system. For example, the immune response may involve, effect, or he detected in innate immune cells such as, for example, dendritic cells, monocytes, macrophages, natural killer cells, and/or granulocytes (e.g., neutrophils, basophils or eosinophils). The immune response may also involve, effect, or be detected in adaptive immune cells including, for example, lymphocytes (e.g., T cells and/or B cells). The immune response may be observed by detecting such involvement or effects including, for example, the presence, absence, or altered (e.g., increased or decreased) expression or activity of one or more immunomodulators such as a hormone, cytokine, interleukin (e.g., any of IL-1 through IL-35), interferon (e.g., any of IFN-I (IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-τ, IFN-ζ, IFN-ω), IFN-II (e.g., IFN-γ), IFN-III (IFN-λ1, IFN-λ2, IFN-λ3)), chemokine (e.g., any CC cytokine (e.g., any of CCL1 through CCL28), any CXC chemokine (e.g., any of CXCL1 through CXCL24), Mipla), any C chemokine (e.g., XCL1, XCL2), any CX3C chemokine (e.g., CX3CL1)), tumor necrosis factor (e.g., TNF-α, TNF-β)), negative regulators (e.g., PD-1, IL-T) and/or any of the cellular components (e.g., kinases, lipases, nucleases, transcription-related factors (e.g., IRF-1,IRF-7, STAT-5, NFKB, STAT3, STAT1, IRF-10), and/or cell, surface markers suppressed or induced by such immunomodulators) involved in the expression of such immunomodulators. The presence, absence or altered expression may be detected within cells of interest or near those cells (e.g., within a ceil culture supernatant, nearby cell or tissue in vitro or in vivo, and/or in blood or plasma). Administration, of the immunogen may induce (e.g., stimulate a de novo or previously undetected response), or enhance or suppress an existing response against the immunogen by, for example, causing an increased antibody response (e.g., amount of antibody, increased affinity/avidity) or an increased cellular response (e.g., increased number of activated T cells, increased affinity/avidity of T cell receptors, cytoxicity including but not limited to antibody-dependent cellular cytotoxicity (ADCC), proliferation). In the case of HIV infections, no clear correlates of immunity have been associated with immunity (especially protective immunity), but any of the measures described herein may be helpful in. determining the usefulness of the compositions and methods described herein. Some immune responses may, in the case of a viral immunogen, lead to decreased viral load in, or lead to elimination of the virus from, a host. In certain embodiments, the immune response may be protective, meaning that the immune response may be capable of preventing initiation or continued infection of or growth within a host and/or by eliminating an agent (e.g., a causative agent, such as HIV) from the host. In some instances, elimination of an agent from the host may mean that the vaccine is therapeutic. In some embodiments, a composition comprising an immunogen may be administered to a population of hosts (e.g., human beings) and determined to provide protective immunity to only a portion of that population. The composition may therefore be considered to protect a portion of that population (e.g., about 1/10, 1/4, 1/3, 1/2, or 3/4 of the population). The proportion of the population that is protected may be calculated and thereby provide the efficacy of the composition in that population (e.g., about 10%, 25%, 33%, 50%, or 75% efficacy).
[0015] With respect to HIV, immunogens may be selected from any HIV isolate (e.g., any primary or cultured HIV-1, HIV-2, and/or HIV-3 isolate, strain, or clade). As is well-known in the art, HIV isolates are now classified into discrete genetic subtypes. HIV-1 is known to comprise at least ten subtypes (A1, A2, A3, A4, B, C, D, E, F1, F2, G, H, J and K) (Taylor et al, NEJM, 359(18): 1965-1966 (2008)). HIV-2 is known to include at least five subtypes (A, B, C, D, and E). Subtype B has been associated with the HIV epidemic in homosexual men and intravenous drug users worldwide. Most HIV-1 immunogens, laboratory adapted isolates, reagents and mapped epitopes belong to subtype B. In sub-Saharan Africa, India, and China, areas where the incidence of new HIV infections is high, HIV-1 subtype B accounts for only a small minority of infections, and subtype HIV-1 C appears to be the most common infecting subtype. Thus, in certain embodiments, it may be preferable to select immunogens from particular subtypes (e.g., HIV-1 subtypes B and/or C). It may be desirable to include immunogens from multiple HIV subtypes (e.g.,HIV-1 subtypes B and C, HIV-2 subtypes A and B, or a combination of HIV-1, HIV-2, and/or HIV-3 subtypes) in a single immunological composition. Suitable HIV immunogens include HIV envelope (env; e.g., NCBI Ref. Seq. NP--057856, or as shown in any of FIGS. 1, 2 and/or SEQ ID NOS. 1-6), gag (e.g., p6, p7, p17, p24, GenBank AAD39400.1), the protease encoded by pol (e.g., UniProt P03366), nef (e.g., GenBank CAA41585.1; Shugars, et al. J. Virol, Aug. 1993, pp. 4639-4650 (1993)), as well as variants, derivatives, and fusion proteins thereof, as described by, for example, Gomez et al. Vaccine, Vol. 25, pp. 1969-1992 (2007). Immunogens (e.g., env and pol) may be combined as desired. Immunogens from different HIV isolates (e.g., HIV-1 A1 env and HIV-1 A2 env) may also be combined (e.g., AIDSVAX B/B® and AIDSVAX® B/E). In some embodiments, at least one of the compositions comprises an amino acid sequence corresponding to that of HIV and at least one amino acid sequence corresponding to a herpes simplex virus (HSV). In some embodiments, the HSV antigen may be the glycoprotein D (gD) leader sequence shown in FIG. 1. In certain embodiments, the HSV amino acid sequence comprises KYALADASLKMADPNRFRGKDLPVLDQL (SEQ ID NO. 7), or a fragment or derivative thereof. In some embodiments, at least one of the compositions comprises SEQ ID NO. 7. In some embodiments, a suitable immunogen may be the polypeptide gd244 as shown in FIG. 1, or a fragment thereof. In others, a suitable immunogen may be the polypeptide "MN.mature" shown in FIG. 1, or a fragment thereof. Suitable strains and combinations may be selected by the skilled artisan as desired.
[0016] In some embodiments, a method for protectively immunizing a human being against human immunodeficiency virus (HIV) by administering to the human being at least one dose of a first composition comprising a viral vector encoding an HIV polypeptide or fragment or derivative thereof and subsequently administering to the human being at least one dose of a second, composition comprising the HIV polypeptide or fragment or derivative thereof wherein a protective immune response directed against HIV results, is provided. In some embodiments, a method for protectively immunizing a human being against human immunodeficiency virus (HIV) by administering to the human being a vaccine consisting essentially of a first composition and a second composition, the first composition consisting essentially of a live, attenuated viral vector encoding at least one HIV gp120 or fragment or derivative thereof and, optionally, at least one additional HIV polypeptide or fragment or derivative thereof, the second composition consisting essentially of at least, one HIV gp120 polypeptide or fragment or derivative thereof and, optionally, at least one additional HIV polypeptide or fragment or derivative thereof; the method comprising the steps of administering the first composition to the human being and subsequently administering to the human being at least one composition or combination of compositions selected from the group consisting of: the second composition alone; the first and second compositions, optionally separately or together as a single dose; at least one additional dose of the first composition followed by at least one dose of the second composition; the second composition followed by at least one additional dose of the first composition, optionally followed by at least one additional dose of the first and/or second composition; wherein a protective immune response against HIV is induced in the human being, is provided. In some embodiments, the compositions are administered together (e.g., at essentially the same time (e.g., simultaneously) to the same or different sites of a host) or separately (e.g., either in time or site of administration in the host).
[0017] In certain embodiments, at least one of the compositions comprises an amino acid sequence corresponding to that of a herpes simplex virus (HSV) (e.g., glycoprotein D (gD) leader sequence shown in FIG. 1). In certain embodiments, the HSV amino acid sequence may include, for example, KYALADASLKMADPNRFRGKDLPVLDQL (SEQ ID NO. 7), or a fragment thereof. In some embodiments, the HSV amino acid sequence may be that shown in the polypeptide "MN .mature" shown in FIG. 1 (SEQ ID NO.: 3), or a fragment thereof. In some embodiments, the HSV amino acid sequence may be that shown in the polypeptide gd244 as shown in FIG. 1 (SEQ ID NO.: 4), or a fragment thereof. In some embodiments, the gp120 amino acid, sequence may be SEQ ID NO.: 5. In some embodiments, at least one of the compositions may comprise a polypeptide of any one or more of SEQ ID NOS. 3, 4, 5, or 7. By "comprise a polypeptide" is meant both a polypeptide per se and/or one encoded by a nucleic acid contained within an expression vector. Variations and derivatives of the polypeptides referred to herein may also be suitable, as could be determined by one of skill in the art.
[0018] In some embodiments, the first composition is administered repeatedly prior to at least one administration of the second composition, where the time between administrations is of sufficient length to allow for the development of an immune response within the human being. In some embodiments, the methods described herein comprise administering the vaccine is administered to a population of human beings such that at least about one-third of that population is protected from infection by HIV. In some embodiments, the first composition is administered repeatedly prior to at least one administration of the second composition, with the time between administrations is of sufficient length to allow for the development of an immune response within the human being. In certain embodiments, administration of either or both the first and second compositions is via a route selected from the group consisting of mucosal, intradermal, intramuscular, subcutaneous, via skin scarification, intranodal, or intratumoral. The dose of the compositions may vary, but in some embodiments, the amount of viral vector administered in each dose is the equivalent of about 107 CCID50 and the total amount of polypeptide administered in each dose is about 600 μg. In some embodiments, the viral vector may be a poxviral vector such as vaccinia, NYVAC, Modified Virus Ankara (MVA), avipox, canarypox, ALVAC, ALVAC(2), fowlpox, or TROVAC. In some embodiments, the viral vector may be ALVAC-HIV (vCP1521). The viral vector may comprise the nucleic acid sequence of SEQ ID NO. 1 or 5, for example. The second composition may be AIDSVAX® B/B or AIDSVAX® B/E. In some embodiments, the viral vector may be ALVAC-HIV (vCP1521) and the second composition may be AIDSVAX® B/E.
[0019] In certain embodiments, the HIV polypeptide or HIV gp120 is derived from an HIV virus selected from the group consisting of HIV-1, HIV-2, and HIV-3, wherein the first and second composition contain the same or different HIV polypeptides and/or gp120. The HIV-1 may be, for example, HIV-1 subtype A1, HIV-1 subtype A2, HIV-1 subtype A3, HIV-1 subtype A4, HIV-1 subtype B, HIV-1 subtype C, HIV-1 subtype D, HIV-1 subtype E, HIV-1 subtype F1, HIV-1 subtype F2, HIV-1 subtype G, HIV-1 subtype H, HIV-1 subtype J and HIV-1 subtype K. The HIV-2 may be, for example, HIV-2 subtype A, HIV-2 subtype B, HIV-2 subtype C, HIV-2 subtype D, and HIV-2 subtype E. The viral vector may encode, for example, at least one polypeptide selected from the group consisting of HIV gp120 MN 12-485, HIV gp120 A244 12-484, and HIV gp120 GNE8 12-477.
[0020] Where multiple HIV immunogens are used, the at least one additional HIV immunogen may be, for example, gag, pol, nef, a variant thereof and a derivative thereof. Thus, is some embodiments, the first or second composition additionally contain at least one additional HIV immunogen selected from the group consisting of gag, the protease component encoded by pol, nef, a variant thereof and a derivative thereof.
[0021] In preferred embodiments of the present invention, vectors are used to transfer a nucleic acid sequence encoding a polypeptide to a cell. A vector is any molecule used to transfer a nucleic acid sequence to a host cell. In certain cases, an expression vector is utilized. An expression vector is a nucleic acid molecule that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control the expression of the transferred nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and splicing, if introns are present. Expression vectors typically comprise one or more flanking sequences operably linked to a heterologous nucleic acid sequence encoding a polypeptide. As used herein, the term operably linked refers to a linkage between polynucleotide elements in a functional relationship such as one in which a promoter or enhancer affects transcription of a coding sequence. Flanking sequences may be homologous (i.e., from the same species and/ or strain as the host cell), heterologous (i.e., from a species other than the host, cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), or synthetic, for example.
[0022] In certain embodiments, it is preferred that the flanking sequence is a transcriptional regulatory region that drives high-level, gene expression in the target cell. The transcriptional regulatory region may comprise, for example, a promoter, enhancer, silencer, repressor element, or combinations thereof. The transcriptional regulatory region may be either constitutive, tissue-specific, cell-type specific (i.e., the region is drives higher levels of transcription in a one type of tissue or cell as compared to another), or regulatable (i.e., responsive to interaction with a compound such as tetracycline). The source of a transcriptional regulatory region may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence functions in a cell by causing transcription of a nucleic acid within that cell. A wide variety of transcriptional regulatory regions may be utilized in practicing the present invention.
[0023] In some embodiments, derivatives of polypeptides, peptides, or polynucleotides incorporated into or expressed by the vectors described herein including, for example, fragments and/or variants thereof may be utilized. Derivatives may result from, for example, substitution, deletion, or addition of amino acids or nucleotides from or to the reference sequence (e.g., the parental sequence). A derivative of a polypeptide or protein, for example, typically refers to an amino acid sequence that is altered with respect to the referenced polypeptide or peptide. A derivative of a polypeptide typically retains at least one activity of the polypeptide. A derivative will typically share at least approximately 60%, 70%, 80%, 90%, 95%, or 99% identity to the reference sequence. With respect to polypeptides and peptides, the derivative may have "conservative" changes, wherein a substituted amino acid has similar structural, or chemical properties. A derivative may also have "nonconservative" changes. Exemplary, suitable conservative amino acid substitutions may include, for example, those shown in Table 1:
TABLE-US-00001 TABLE 1 Original Exemplary Preferred Residues Substitutions Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln Gln Asp Gln Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleucine Leu Leu Norleucine, Ile, Val, Met, Ala, Phe Ile Lys Arg, 1,4 Diamino-butyric Acid, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Norleucine Leu
[0024] Other amino acid substitutions may be considered non-conservative. Derivatives may also include amino acid or nucleotide deletions and/or additions/insertions, or some combination of these. Guidance in determining which amino acid residues or nucleotides may be substituted, inserted, or deleted without abolishing the desired activity of the derivative may be identified using any of the methods available to one of skill in the art.
[0025] Derivatives may also refer to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide may include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide may encode a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide may be one modified by glycosylation, pegylation, biotinylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
[0026] The phrases "percent identity" and "% identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any .fragment length supported by the sequences shown herein, in the tables, FIGS. or Sequence Listing, may be used to describe a length over which percentage identity may be measured. Percent identity can be measured both globally or locally. Examples of alignment algorithms known in the art for global alignments are ones which attempt to align every residue in every sequence, such as the Needleman-Wunsch algorithm. Local alignment algorithmns are useful for dissimilar sequences that contain regions of similar sequence motifs within their larger sequence, such as the Smith-Waterman algorithm.
[0027] As mentioned above, this disclosure relates to compositions comprising recombinant vectors, the vectors per se, and methods of using the same. A "vector" is any moiety (e.g., a virus or plasmid) used to carry, introduce, or transfer a polynucleotide or interest to another moiety (e.g., a host cell). In certain cases, an expression vector is utilized. An expression vector is a nucleic acid molecule containing a polynucleotide of interest encoding a polypeptide, peptide, or polynucleotide and also containing other polynucleotides that, direct and/or control the expression of the polynucleotide of interest. Expression includes, but is not limited to, processes such as transcription, translation, and/or splicing (e.g., where introns are present).
[0028] Viral vectors that may be used include, for example, retrovirus, adenovirus, adeno-associated virus (AAV), alphavirus, herpes virus, and poxvirus vectors, among others. Many such viral vectors are available in the art. The vectors described herein may be constructed using standard recombinant techniques widely available to one skilled in the art. Such techniques may be found in common molecular biology references such as Molecular Cloning: A laboratory Manual (Sambrook, et al, 1989. Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), and PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.).
[0029] Suitable retroviral vectors may include derivatives of lentivirus as well, as derivatives of murine or avian retroviruses. Exemplary, suitable retroviral vectors may include, for example, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV). A number of retroviral vectors can incorporate multiple exogenous polynucleotides. As recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided by, for example, helper cell lines encoding retrovirus structural genes. Suitable helper cell lines include Ψ2, PA317 and PA12, among others. The vector virions produced using such cell lines may then be used to infect a tissue cell line, such as NIH 3T3 cells, to produce large quantities of chimeric retroviral virions. Retroviral vectors may be administered by traditional methods (i.e., injection) or by implantation of a "producer cell line" in proximity to the target cell population (Culver, K., et al, 1994, Hum. Gene Ther., 5 (3): 343-79; Culver, K., et. al., Cold Spring Harb. Symp. Quant. Biol., 59: 685-90); Oldfield, E., 1993, Hum. Gene Ther., 4 (1): 39-69). The producer cell line is engineered to produce a viral vector and releases viral particles in the vicinity of the target cell. A portion of the released viral particles contact the target cells and infect those cells, thus delivering a nucleic acid encoding an immunogen to the target cell. Following infection of the target cell expression of the polynucleotide of interest from the vector occurs.
[0030] Adenoviral vectors have proven especially useful for gene transfer into eukaryotic cells (Rosenfeld, M., et al., 1991, Science, 252 (5004): 431-4; Crystal, R., et at, 1994, Nat. Genet, 8 (1): 42-51), the study eukaryotic gene expression (Levrero, M., et al., 1991, Gene, 101 (2); 195-202), vaccine development (Graham, F. and Prevec, L., 1992, Biotechnology, 20: 363-90), and in animal models (Stratford-Perricaudet, L., et al., 1992, Bone Marrow Transplant, 9 (Suppl. 1): 151-2; Rich, et at, 1993, Hum. Gene Ther., 4 (4): 461-76). Experimental routes for administrating recombinant Ad to different tissues in vivo have included intratracheal instillation. (Rosenfeld, M, et. al., 1992, Cell, 68 (1): 143-55) injection into muscle (Quantify B., et al., 1992, Proc. Natl. Acad. Sci. U.S.A., 89 (7): 2581-4), peripheral intravenous injection (Herz, J., and Gerard, R., 1993, Proc. Natl. Acad. Sci. U.S.A., 90 (7): 2812-6) and/or stereotactic inoculation to brain (Le Gal La Salle, G., et al., 1993, Science, 259 (5097): 988-90), among others.
[0031] Adeno-associated virus (AAV) demonstrates high-level infectivity, broad host range and specificity in integrating into the host cell genome (Hermonat, P., et at, 1984, Proc, Natl. Acad. Sci. U.S.A., 81 (20); 6466-70). And Herpes Simplex Virus type-1 (HSV-1) is yet another attractive vector system, especially for use in the nervous system because of its neurotropic property (Geller, A., et al., 1991, Trends Neurosci., 14 (10): 428-32; Glorioso, et al, 1995, Mol. Biotechnol. 4 (1): 87-99; Glorioso, et al., 1995, Annu. Rev. Microbiol., 49: 675-710).
[0032] Alphavirus may also be used to express the immunogen in a host. Suitable members of the Alphavirus genus include, among others, Sindbis virus, Semliki Forest virus (SFV), the Ross River virus and Venezuelan, Western and Eastern equine encephalitis viruses, among others. Expression systems utilizing alphavirus vectors are described in, for example, U.S. Pat. Nos. 5,091,309; 5,217,879; 5,739,026; 5,766,602; 5,843,723; 6,015,694; 6,156,558; 6,190,666; 6,242,259; and, 6,329,201; WO 92/10578; Xiong et: al. Science, Vol 243, 1989, 1188-1191; Liliestrom, et al. Bio/Technology, 9: 1356-1361, 1991. Thus, the use of alphavirus as an expression system is well known by those of skill in the art.
[0033] Poxvirus is another useful expression vector (Smith, et al, 1983, Gene, 25 (1): 21-8; Moss, et al, 1992, Biotechnology, 20: 345-62; Moss, et al, 1992, Curr. Top. Microbiol. Immunol., 158: 25-38; Moss, et al. 1991. Science, 252: 1662-1667). The most often utilized poxviral vectors include vaccinia and derivatives therefrom such as NYVAC and MVA, and members of the avipox genera such as fowlpox, canarypox, ALVAC, and ALVAC(2), among others.
[0034] An exemplary suitable vector is NYVAC (vP866) which was derived from the Copenhagen vaccine strain of vaccinia virus by deleting six nonessential regions of the genome encoding known or potential virulence factors (see, for example, U.S. Pat. Nos. 5,364,773 and 5,494,807). The deletion loci were also engineered as recipient loci for the insertion of foreign genes. The deleted regions are: thymidine kinase gene (TK: J2R); hemorrhagic region (u; B13R+B14R); A type inclusion body region (ATI; A26L); hemagglutinin gene (HA; A56R); host range gene region (C7L-K1L); and, large subunit, ribonucleotide reductase (I4L). NYVAC is a genetically engineered vaccinia virus strain that was generated by the specific deletion of eighteen open reading frames encoding gene products associated with virulence and host range. NYVAC has been show to be useful for expressing TAs (see, for example, U.S. Pat. No. 6,265,189). NYVAC (VP866), vP994, vCP205, vCP1433, placZH6H4Lreverse, pMPC6H6K3E3 and pC3H6FHVB were also deposited with the ATCC under the terms of the Budapest Treaty, accession numbers VR-2559, VR.-2558, VR-2557, VR-2556, ATCC-97913, ATCC-97912, and ATCC-97914, respectively.
[0035] Another suitable virus is the Modified Vaccinia Ankara (MVA.) virus which was generated by 516 serial passages on chicken embryo fibroblasts of the Ankara strain of vaccinia virus (CVA) (for review, see Mayr, A., et al. Infection 3, 6-14 (1975)). It was shown in a variety of animal models that the resulting MVA was significantly avirulent (Mayr, A. & Danner, K. (1978) Dev. Biol. Stand. 41: 225.34) and has been tested in clinical trials as a smallpox, vaccine (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167, 375-390 (1987), Sticl et al, Dtsch. med. Wschr. 99,2386-2392 (1974)). MVA has also been engineered for use as a viral vector far both recombinant gene expression studies and as a recombinant vaccine (Sutter, G, et al. (1994), Vaccine 12: 1032-40; Blanc-hard et al., 1998, J Gen Virol 79, 1159-1167; Carroll & Moss, 1997, Virology 238, 198-211; Altenberger, U.S. Pat, No. 5,185,144; Ambrosini et al., 1999, J Neurosci Res 55(5), 569). Modified virus Ankara (MVA) has been previously described in, for example, U.S. Pat. Nos. 5,185,146 and 6,440,422: Sutter, et al. (B. Dev. Biol. Stand. Basel, Karger 84:195-200 (1995)); Antoine, et al. (Virology 244: 365-396, 1998); Sutter et al. (Proc. Natl. Acad. Sci. USA 89: 10847-10851, 1992); Meyer et al. (J. Gen. Virol. 72: 1031-1038, 1991); Mahnel, et al. (Berlin Munch, Tierarztl, Wochenschr. 107; 253-256, 1994); Mayr et al. (Zbl. Bakt. Hyg. I, Abt. Org. B 167: 375-390 (1987); and, Stickl et al. (Dtsch. med. Wschr. 99: 2386-239:2 (1974)). An exemplary MVA is available from the ATCC under accession numbers VR-1508 and VR-1566.
[0036] ALVAC-based recombinant viruses (i.e., ALVAC-1 and ALVAC-2) are also suitable for use in practicing the present, invention (see, for example, U.S. Pat. No. 5,756,103). ALVAC(2) is identical to ALVAC(1) except that ALVAC(2) genome comprises the vaccinia E3L and K3L genes under the control of vaccinia promoters (U.S. Pat. No. 6,130,066; Beattie et al., 1995a, 1995b, 1991; Chang et al., 1992; Davies et al., 1993). Both ALVAC(1) and ALVAC(2) have been, demonstrated to be useful in expressing foreign DNA sequences, such as TAs (Tartaglia et al., 1993 a,b; U.S. Pat. No. 5,833,975). ALVAC was deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, USA, ATCC accession number VR-2547, Vaccinia virus host range genes (e.g., C18L, C17L, C7L K1L, E3L, B4R, B23R, and B24R) have also been shown to be expressible in canarypox (e.g., U.S. Pat. No. 7,473,536).
[0037] Another useful poxvirus vector is TROVAC. TROVAC refers to an attenuated fowlpox that was a plaque-cloned isolate derived from the FP-1 vaccine strain of fowlpoxvirus which is licensed for vaccination of 1 day old chicks. TROVAC was likewise deposited under the terms of the Budapest Treaty with the ATCC, accession number 2553.
[0038] "Non-viral" plasmid vectors may also be suitable for use. Plasmid DNA molecules comprising expression cassettes for expressing an immunogen may be used for "naked DNA" immunization. Preferred plasmid vectors are compatible with bacterial, insect, and/or mammalian host cells. Such vectors include, for example, PCR-II, pCR3, and pcDNA3.1 (Invitrogen, San Diego, Calif.), pBSII (Stratagene, La Jolla, Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL (BineBacII, Invitrogen), pDSR-alpha (PCT pub. No. WO 90/14363) and pFastBacDual (Gibco-BRL, Grand Island, N.Y.) as well as Bluescript® plasmid derivatives (a high copy number COLE1-based phagemid, Stratagene Cloning Systems, La Jolla, Calif.), PCR cloning plasmids designed for cloning Taq-amplified PCR products (e.g., TOPO® TA cloning® kit, PCR2.1® plasmid derivatives, Invitrogen, Carlsbad, Calif.).
[0039] Bacterial vectors may also be suitable for use. These vectors include, for example, Shigella, Salmonella (e.g., Darji, et. al. Cell, 91: 765-775 (1997); Woo, et at Vaccine, 19; 2945-2954 (2001)), Vibrio cholerae, Lactobacillus, Bacille calmette guerin (BCG), and Streptococcus (e.g., WO 88/6626, WO 90/0594, WO 91/33157, WO 92/1796, and WO 92/21376). Many other non-viral plasmid expression vectors and systems are known in the art and could be used with the current invention.
[0040] Nucleic acid delivery or transformation techniques that may be used include DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO4 precipitation, gene gun techniques, electroporation, and colloidal dispersion systems, among others. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome, which are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo. RNA, DNA and Intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, R., et al. Trends Biochem. Sci., 6: 77 (1981)). The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerois, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
[0041] Strategies for improving the efficiency of nucleic acid-based immunization may also be used including, for example, the use of self-replicating viral replicons (Caley, et al. Vaccine, 17; 3124-2135 (1999); Dubensky, et al. Mol. Med 6: 723-732 (2000); Leitner, et al. Cancer Res. 60: 51-55 (2000)), codon optimization (Liu, et al. Mol. Ther., 1: 497-500 (2000); Dubensky, supra; Huang, et al. J. Virol. 75: 4947-4951 (2001)), in vivo electroporation (Widera, et al. J. Immunol. 164: 4635-3640 (2000)), incorporation of CpG stimulatory motifs (Gurunathan, et al, Ann, Rev, Immunol. 18: 927-974 (2000); Leitner, supra), sequences for targeting of the endocytic or ubiquitin-processing pathways (Thomson, et al. J. Virol. 72: 2246-2252 (1998); Velders, et al. J. Immunol. 166: 5366-5373 (2001)), and/or prime-boost regimens (Gurunathan, supra; Sullivan, et al. Nature, 408: 605-609 (2000); Hanke, et al. Vaccine, 16: 439-445 (1998); Amara, et al. Science, 292; 69-74 (2001)), Other methods are known in the art, some of which are described below.
[0042] In other embodiments, it may be advantageous to combine or include within the compositions or recombinant vectors additional polypeptides, peptides or polynucleotides encoding one or more polypeptides or peptides that function as "co-stimulatory" component(s). Such co-stimulatory components may include, for example, cell surface proteins, cytokines or chemokines in a composition of the present invention. The co-stimulatory component may be included in the composition as a polypeptide or peptide, or as a polynucleotide encoding the polypeptide or peptide, for example. Suitable co-stimulatory molecules include, for instance, polypeptides that bind members of the CD28 family (i.e., CD28, ICOS; Hutloff et al. Nature 1999, 397: 263-265; Peach, et al. J Exp Med 1994, 180: 2049-2058) such as the CD28 binding polypeptides B7.1 (CD80: Schwartz, 1992; Chen et al, 1992; Ellis, et al. J. Immunol, 156(8): 2700-9) and B7.2 (CD86; Ellis, et al. J. Immunol., 156(8); 2700-9); polypeptides which bind members of the integrin family (i.e., LFA-1 (CD11a/CD18); Sedwick, et al J Immunol 1999, 162: 1367-1375; Wulfing, et al. Science 1998, 282: 2266-2269; Lub, et al. Immunol Today 1995, 16: 479-483) including members of the ICAM family (i.e., ICAM-1, -2 or -3); polypeptides which bind CD2 family members (i.e., CD2, signalling lymphocyte activation molecule (CDw150 or "SLAM"; Aversa, et al. J Immunol 1997, 158; 4036-4044) such as CD58 (LFA-3; CD2 ligand: Davis, et al. Immunol Today 1996, 17; 177-187) or SLAM ligands (Sayos, et al. Nature 1998, 395: 462-469); polypeptides which bind heat stable antigen (HSA or CD24; Zhou, et al. Eur J Immunol 1997, 27; 2524-2528); polypeptides which bind to members of the TNF receptor (TNFR) family (i.e., 4-IBB (CD137; Vinay, et al. Semin Immunol 1998, 10: 481-489)), OX40 (CD 134; Weinberg, et al. Semin Immunol 1998, 10:471-480; Higgins, et al. J Immunol 1999, 162: 486-493), and CD27 (Lens, et al. Semin Immunol 1998, 10: 491-499)) such as 4-IBBL (4-IBB ligand; Vinay, et al. Semin Immunol 1998, 10: 481-48; DeBenedette, et al. J Immunol 1997, 158: 551-559), TNFR associated factor-I (TRAF-1; 4-IBB ligand; Saoulli, et al. J Exp Med 1998, 187: 1849-1862, Arch, et al. Mol Cell Biol 1998, 18: 558-565), TRAF-2 (4-IBB and OX40 ligand; Saoulli, et al. J Exp Med 1998, 187: 1849-1862; Oshima, et al. Int Immunol 1998, 10: 517-526, Kawamata, et al. J Biol Chem 1998, 273: 5808-5814), TRAF-3 (4-IBB and OX40 ligand; Arch, et al. Mol Cell Biol 1998, 18: 558-565; Jang, et al. Biochem Biophys Res Commun 1998, 242: 613-620; Kawamata S, et al. J Biol Chem 1998, 273: 5808-5814), OX40L (OX40 ligand; Gramaglia, et al. J Immunol 1998, 161; 6510-6517), TRAF-5 (OX40 ligand; Arch, et al. Mol Cell Biol 1998, 18: 558-565; Kawamata, et al. J Biol Chem 1998, 273: 5808-5814), and CD70 (CD27 ligand; Couderc, et al. Cancer Gene Ther., 5(3): 163-75). CD154 (CD40 ligand or "CD40L"; Guranaraan, et al. J. Immunol, 1998, 161: 4563-4571; Sine, et al. Hum. Gene Ther., 2001, 12: 1091-1102) Other co-stimulatory molecules may also be suitable for practicing the present invention.
[0043] One or more cytokines may also be suitable co-stimulatory components or "adjuvants", either as polypeptides or being encoded by nucleic acids contained within the compositions of the present invention (Parmiani et al. Immunol Lett 2000 Sep 15; 74(1): 41-4; Berzofsky, et al. Nature Immunol. 1: 209-219). Suitable cytokines include, for example, interleukin-2 (IL-2) (Rosenberg, et al. Nature Med 4; 321-327 (1998)), IL-4, IL-7, IL-12 (reviewed by Pardoll, 1992; Harries, et al. J. Gene Med. 2000 Jul-Aug; 2(4); 243-9: Rao, et al. J. Immunol 156; 3357-3365 (1996)), IL-15 (Xin, et al. Vaccine, 17:858-866, 1999), IL-16 (Cruikshank, et al. J. Leuk Biol. 67(6): 757-66, 2000), IL-18 (J. Cancer Res. Clin. Oncol. 2001. 127(12): 718-726), GM-CSF (CSF (Disis, et al. Blood, 88: 202-210 (1996)), tumor necrosis factor-alpha (TNF-α), or interferon-gamma (INF-γ). Other cytokines may also be suitable for practicing the present invention.
[0044] Chemokines may also be utilized. For example, fusion proteins comprising CXCL10 (IP-10) and CCL7 (MCP-3) fused to a tumor self-antigen have been shown to induce anti-tumor immunity (Biragyn, et al. Nature Biotech, 1999, 17: 253-258). The chemokines CCL3 (MIP-1α) and CCL5 (RANTES) (Boyer, et al. Vaccine, 1999, 17 (Supp. 2): S53-S64) may also be of use in practicing the present invention. Other suitable chemokines are known in the art.
[0045] It is also known In the art that suppressive or negative regulatory immune mechanisms may be blocked, resulting in enhanced immune responses. For instance, treatment with anti-CTLA-4 (Shrikant, et al. Immunity, 1996, 14; 145-155; Sutmuller, et al. J. Exp. Med, 2001, 194: 823-832), anti-CD25 (Sutmuller, supra), anti-CD4 (Matsui, et al. J. Immunol., 1999, 163: 184-193), the fusion protein IL13Ra2-Fc (Terabe, et al. Nature Immunol., 2000, 1: 515-520), and combinations thereof (i.e., anti-CTLA-4 and anti-CD25, Sutmuller, supra) have been shown to upregulate anti-tumor immune responses and would be suitable in practicing the present invention.
[0046] An immunogen may also be administered in combination with one or more adjuvants to boost the immune response. Adjuvants may also be included to stimulate or enhance the immune response against the immunogen. Non-limiting examples of suitable adjuvants include those of the gel-type (i.e., aluminum hydroxide/phosphate ("alum adjuvants"), calcium phosphate), of microbial origin (muramyl dipeptide (MDP)), bacterial exotoxins (cholera toxin (CT), native cholera toxin subunit B (CTB), E. coli labile toxin (LT), pertussis toxin (PT), CpG oligonucleotides, BCG sequences, tetanus toxoid, monophosphoryl lipid A (MPL) of, for example , E. coli, Salmonella minnesota, Salmonella typhimurium, or Shigella exseri), particulate adjuvants (biodegradable, polymer microspheres), immunostimulatory complexes (ISCOMs)), oil-emulsion and surfactant-based adjuvants (Freund's incomplete adjuvant (FIA), microfluidized emulsions (MF59, SAF), saponins (QS-21)), synthetic (muramyl peptide derivatives (murabutide, threony-MDP), nonionic block copolymers (L121), polyphosphazene (PCCP), synthetic polynucleotides (poly A:U, poly I:C), thalidomide derivatives (CC-4407/ACTIMID)), RH3-ligand, or polylactide glycolide (PLGA) microspheres, among others. Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity. Suitable mutants or variants of adjuvants are described, e.g., in WO 95/17211 (Arg-7- Lys CT mutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant). Additional LT mutants that can be used in the methods and compositions of the invention include, e.g., Ser-63-Lys, Ala-69-Gly, Glu-110-Asp, and Glu-112-Asp mutants. Other suitable adjuvants are also well-known in the art.
[0047] As an example, metallic salt adjuvants such alum adjuvants are well-known in the art as providing a safe excipient with adjuvant activity. The mechanism of action of these adjutants are thought to include the formation of an antigen depot such that antigen may stay at the site of injection for up to 3 weeks after administration, and also the formation of antigen/metallic salt complexes which are more easily taken up by antigen presenting cells. In addition to aluminium, other metallic salts have been used to adsorb antigens, including salts of zinc, calcium, cerium, chromium, iron, and berilium. The hydroxide and phosphate salts of aluminium are tire most common. Formulations or compositions containing aluminium salts, antigen, and an additional immunostimulant are known in the art. An example of an immunostimulant is 3-de-O-acylated monophosphoryl lipid A (3D-MPL).
[0048] Any of these components may be used alone or in combination with other agents. For instance, it has been shown that a combination of CD80, ICAM-1 and LFA-3 ("TRICOM") may potentiate anti-cancer immune responses (Hodge, et al. Cancer Res. 59: 5800-5807 (1999). Other effective combinations include, for example, IL-12+GM-CSF (Ahlers, et al. J. Immunol., 158; 3947-3938 (1.997); Iwasaki, et al. J. Immunol 158: 4591-4601 (1997)), IL-12+GM-CSF+TNF-α (Ahlers, et al Int. Immunol 13: 897-908 (2001)), CD80+IL-12 (Fruend, et al. Int. J. Cancer, 85: 508-517 (2000); Rao, et al. supra), and CD86+GM-CSF+IL-12 (Iwasaki, supra). One of skill in the art would be aware of additional combinations useful in carrying out the present invention. In addition, the skilled artisan would be aware of additional reagents or methods that may be used to modulate such mechanisms. These reagents and methods, as well as others known by those of skill in the art, may be utilized in practicing the present invention.
[0049] Other agents that may be utilized in conjunction with the compositions and methods provided herein include anti-HIV agents including, for example, protease inhibitor, an HIV entry inhibitor, a reverse transcriptase inhibitor, and/or or an anti-retroviral nucleoside analog. Suitable compounds include, for example, Agenerase (amprenavir), Combivir (Retrovir/Epivir), Crixivan (indinavir), Emtriva (emtricitabine), Epivir (3tc/lamivudine), Epzicom, Fortovase/Invirase (saquinavir), Fuzeon (enfuvirtide), Hivid (ddc/zalcitabine), Kaletra (lopinavir), Lexiva (Fosamprenavir), Norvir (ritonavir), Rescriptor (delavirdine), Retrovir/AZT (zidovudine), Reyatax (atazanavir, BMS-232632), Sustiva (efavirenz), Trizivir (abacavir/zidovudine/lamivudine), Truvada (Emtricitabine/Tenofovir DF), Videx (ddl/didanosine), Videx EC (ddl, didanosine), Viracept (nevirapine), Viread (tenofovir disoproxil fumarate), Zerit (d4T/stavudine), and Ziagen (abacavir). Other suitable agents are known to those of skill in the art. Such agents may either be used prior to, during, or after administration of the compositions and/or use of the methods described herein.
[0050] Administration of a composition of the present invention to a host may be accomplished using any of a variety of techniques known to those of skill in the art. The composition(s) may be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals (i.e., a "pharmaceutical composition"). The pharmaceutical composition is preferably made in the form of a dosage unit containing a given amount of DNA, viral vector particles, polypeptide, peptide, or other drug candidate, for example. A suitable daily dose for a human or other mammal may vary widely depending on the condition of the patient, and other factors, but, once again, can be determined using routine methods. The compositions are administered to a patient in a form and amount sufficient to elicit a therapeutic effect. Amounts effective for this use will depend on various factors, including for example, the particular composition, of the vaccine regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician. The dosage regimen for immunizing a host or otherwise treating a disorder or a disease with a composition of this invention is based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods.
[0051] In general, recombinant viruses maybe administered in compositions in a dosage amount of about 104 to about 109 pfu per inoculation; often about 104 pfu to about 106 pfu, or as shown in the Examples, 107 to 103 pfu. Higher dosages such as about 104 pfu to about 1010 pfu, e.g., about 105 pfu to about 109 pfu, or about 106 pfu to about 108 pfu, or about 107 pfu can also be employed. Another measure commonly used is cell culture infective dose (CCID50); suitable CCID50 ranges for administration include about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 1010 CCID50. Ordinarily, suitable dosage amounts of plasmid or naked DNA are about 1 μg to about 100 mg, about 1 mg, about 2 mg, but lower levels such as 0.1 to 1 mg or 1-10 μg may be employed. For polypeptide compositions (e.g., AIDSVAX compositions), a suitable amount may be 1-1000 μg. Without limiting the possible sub-ranges within that dosage range, particular embodiments may employ 5, 10,20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, and 1000 μg. A typical exemplary dosage of polypeptide may be, for example, about 50-250 μg, about 250-500 μg, 500-750 μg, or about 1000 μg of polypeptide. Low dose administration may typically utilize a dose of about 100 μg or less. High dose administration may typically utilize a dose of 300 μg or more. In referring to the amount of polypeptide in a dose, it is to be understood that the amount may refer to the amount of a single polypeptide or, where multiple polypeptides are administered, to the total amount of all polypeptides (e.g., 300 μg each of two polypeptides for a total administration of 600 μg). The AIDSVAX® compositions described herein are typically but not necessarily administered in a total dosage of 200 μg or 600 μg (e.g., recombinant MN and GNE8 gp120, or recombinant MN and A244 gp120). "Dosage" may refer to that administered in a single or multiple doses, including the total of all doses administered. Actual dosages of such compositions can be readily determined by one of ordinary skill in the field of vaccine technology.
[0052] The pharmaceutical composition may be administered orally, parentally, by inhalation spray, rectally, intranodally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of a nucleic acid, polypeptide, or peptide as a pharmaceutical composition. A "pharmaceutical composition" is a composition comprising a therapeutically effective amount of a nucleic acid or polypeptide. The terms "effective amount" and "therapeutically effective amount" each refer to the amount of a nucleic acid or polypeptide used to observe the desired therapeutic effect (e.g., induce or enhance and immune response).
[0053] Injectable preparations, such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Suitable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution, among others. For instance, a viral vector such as a poxvirus may be prepared in 0.4% NaCl or a Tris-HCl buffer, with or without a suitable stabilizer such as lactoglutamate, and with or without freeze drying medium. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic, mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
[0054] Pharmaceutical compositions may take any of several forms and may be administered by any of several routes. The compositions are administered via a parenteral route (e.g., intradermal, intramuscular, subcutaneous, skin, scarification) to induce an immune response in the host. Alternatively, the composition may be administered directly into a tissue or organ such, as a lymph node (e.g., intranodal) or tumor mass (e.g., intratumoral). Preferred embodiments of administratable compositions include, for example, nucleic acids, viral particles, or polypeptides in liquid preparations such as suspensions, syrups, or elixirs. Preferred injectable preparations include, for example, nucleic acids or polypeptides suitable for parental, subcutaneous, intradermal, intramuscular or intravenous administration such as sterile suspensions or emulsions. For example, a naked DNA molecule and/or recombinant poxvirus may separately or together be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like. The composition may also be provided in lyophilized form for reconstituting, for instance, in isotonic aqueous, saline buffer. In addition, the compositions can be co-administered or sequentially administered with one another, other antiviral compounds, other anti-cancer compounds and/or compounds that reduce or alleviate ill effects of such agents.
[0055] As previously mentioned, while the compositions described herein may be administered as the sole active agent, they can also be used in combination with one or more other compositions or agents (i.e., other immunogens, co-stimulatory molecules, adjuvants). When administered as a combination, the individual components can be formulated as separate compositions administered at the same time or different times, or the components can be combined as a single composition. In one embodiment, a method of administering to a host a first form of an immunogen and subsequently administering a second form of the immunogen, wherein tie first and second forms are different, and wherein administration of the first form prior to administration of the second form enhances the immune response resulting from administration of the second form relative to administration of the second form alone, is provided. Also provided are compositions for administration to the host. For example, a two-part immunological composition where the first part of the composition comprises a first form of an immunogen and the second part comprises a second form of the immunogen, wherein the first and second parts are administered together or separately from one another such that administration of the first form enhances the immune response against the second form relative to administration of the second form alone, is provided. The immunogens, which may be the same or different, are preferably derived from the infectious agent or other source of immunogens. The multiple immunogens may be administered together or separately, as a single or multiple compositions, or in single or multiple recombinant vectors. For instance, a viral vector encoding an immunogen may be initially administered and followed by one or more subsequent administrations with, a second form of the immunogen (e.g., a polypeptide). The different forms may differ in either or both of the form of delivery (e.g., viral vector, polypeptide) or in the immunogens represented by each form. It is preferred that the forms, however, induce or enhance the immune response against a particular target (e.g., HIV-1). For instance, as shown herein, a viral vector encoding a viral antigen (e.g., HIV gp120) may be administered to a human being. This may then be followed by administration of the viral vector along with a polypeptide representing the same or a similar viral antigen (e.g., HIV gp120). For prime-boost applications (e.g., ALVAC-HIV and AIDSVAX® ), ALVAC-HIV is typically administered in a 107 CCID50 dosage (the "priming" dose), and then subsequently re-administered at the same or different dosage along with a suitable dosage (e.g., 600 μg total, the "boosting" dose) of polypeptide (e.g., AIDSVAX® B/B or B/E). ALVAC-HIV is a preparation of live attenuated, recombinant canarypox virus (ALVAC(1)) expressing gene products from the HIV-1 env (clade E in vCP1521 and clade B in vCP205), gag (clade B), and protease (clade B) coding sequences (FIG. 2). Exemplary, non-limiting prime-boost combinations may include ALVAC-HIV (vCP205) and AIDSVAX® B/B or ALVAC-HIV (vCP1521) and AIDSVAX® B/E, as the HIV clades from which the gp120 immunogen is derived in those combinations are the same. Typically, both the priming and boosting doses are administered via the same route (e.g., intramuscular, intradermal) but the routes of administration may also be different. Typically, the priming and boosting doses are administered to different parts of the body, but the doses may also be administered to the same part of the body. "Along with" may mean that the two forms are administered as separate compositions, as part of a single composition, at separate sites of the body, or at the same site of the body, depending on the particular protocol. Variations of such exemplary dosing regimens may be made by those of skill in the art.
[0056] A kit comprising a composition of the present invention is also provided. The kit can include a separate container containing a suitable carrier, diluent or excipient. The kit may also include additional components for simultaneous or sequential-administration. In one embodiment such a kit may include a first form of an immunogen and a second form of the immunogen. Additionally, the kit can include instructions for mixing or combining ingredients and/or administration. A kit may provide reagents for performing screening assays, such as one or more PCR primers, hybridization probes, and/or biochips, for example.
[0057] A better understanding of the present invention and of its many advantages will be had from the following examples, given by way of illustration.
Example 1
Immunological Compositions
A. First Composition: Viral Vector
[0058] ALVAC-HIV is a preparation of live attenuated, recombinant canarypox virus (ALVAC(1)) expressing gene products from the HIV-1 env (clade E in vCP1521 and clade B in vCP205), transmembrane anchoring portion of gp41 (clade B:LAI), gag (clade B:LAI), and protease (clade B:LAI) coding sequences and cultured in chick embryo fibroblast cells. These vectors were generated by co-insertion of genes encoding HIV-1 gene products into the ALVAC(1) genome at the C6 insertion site using standard techniques (FIG. 2A). The HIV-1 sequences contained within ALVAC-HIV (vCP1521) are shown in FIG. 2B and SEQ ID NOS.: 5 and 6. These sequences include: 1) the region of the env gene encoding the extracellular envelope gp120 moiety of TH023 strain of HIV-1 linked to the sequences encoding the HIV-1 transmembrane anchor sequence of gp41 (28 amino acids), under the control of the vaccinia virus H6 promoter; and, 2) the gag gene encoding the entire Gag protein, and a portion of the pol sequences of LAI strain of HIV-1 sufficient to encode the protease function, under the control of the same vaccinia virus promoter I3L.
[0059] ALVAC-HIV was produced by inoculation of the ALVAC-HIV working seed lot in primary chick embryo fibroblasts and cultivation in roller bottles. Alter viral amplification, the infected cells were harvested and disrupted by sonication and cell debris removed by centriftigation. An equal volume of stabilizer (lactoglutamate) was blended with the supernatant and the suspension filtered through a 4.5 μm membrane. The clarified suspension was filled into vials and stored at ≦-35° C. At this step, the biological substance is the clarified harvest. End stage manufacturing of the vaccine entails blending of the clarified harvest with, the freeze drying medium under sterile conditions. This blend (final bulk product) was prepared and then filled and freeze dried.
[0060] Immunoprecipitation analyses were performed using radiolabeled lysates derived from uninfected CEF cells or ceils infected with either ALVAC(1) parental virus or ALVAC-HIV. Immunoprecipitation was performed using human serum derived from HIV-seropositive individuals (anti-HIV). Results with anti-HIV demonstrated expression of gp120, the 55 kDa precursor Gag polypeptide, and intermediate and completely processed forms of Gag including the major capsid protein, p24 in ALVAC-HIV infected CEF cells but not from cells infected with ALVAC parental vims.
[0061] Regarding ALVAC-HIV (vCP1521), FACS (Fluorescent Activated Cell Sorter) scan analyses with human anti-HIV antibody demonstrated expression of gp120 on the surface of infected HeLa, but not the parental virus. PCR amplification of the inserted sequences and those of ALVAC was performed. DMA analysis was performed by agarose gel electrophoresis followed by ethidium bromide staining to confirm the identity of the amplified fragments according to their molecular size. Restriction analysis was performed on viral DNA derived from ALVAC-HIV (vCP1521) infected cells to confirm proper insertion of the gp120TM and gag expressing cassettes. The nucleotide sequence of inserted genes was confirmed by sequencing on the Working Seed Lot (passage 6).
[0062] ALVAC-HIV (vCP1521) genetic stability was confirmed by immunoplaqne assay after several passages on CEF. Immunoplaque analysis consisted of detecting Env and Gag expression in viral plaques by monoclonal antibodies. Analysis was performed after 7 passages (i.e. at the production lot level) and after 10 passages (i.e. 3 passages beyond the production lot level).
[0063] ALVAC-HIV (vCP1521) was formulated as a lyophilized vaccine for injection and reconstituted with 1.0 mL of sterile sodium chloride solution (NaCl 0.4%) for a single dose. The composition of the vaccine after reconstitution with 1.0 mL NaCl 0.4% included ALVAC-HIV (vCP1521); ≧106.0 CCID50 and excipients (TRIS-HCl buffer 10 mM, pH 9, 0.25 mL; stabilizer (lactoglutamate), 0.25 ml; freeze-drying medium, 0.50 mL; and NaCl, 4 mg). The appearance of the lyophilisate was homogeneous, white to beige; residual moisture was ≦3%; reconstitution time was ≦3 minutes; the appearance after reconstitution was a limpid to slightly opalescent solution, colorless with possible presence of particles or filaments; pH between 7.0 and 8.0; osmolality between 350 to 700 mOsmol/kg; BSA content of ≦50 ng/dose; bacterial, endotoxins content of ≦10 IU/dose. The ALVAC-HIV (vCP1521) was stored at 2-8° C. without freezing and administered within 2 hours of reconstitution. Prior to reconstitution, the vial was allowed to come to room temperature. Each vial was reconstituted with the diluent supplied, 1.0 mL 0.4% NaCl for administration by slow injection, into the vial containing the lyophilized ALVAC-HIV (e.g., using a 25 gauge, 5/8- inch needle). The vial was allowed to sit for approximately three minutes, and then gently swirled. The vial was then inverted and the contents withdrawn into a syringe,
B. Second Composition: Polypeptide
[0064] Recombinant gp120 is an envelope glycoprotein with an apparent molecular mass of about 120,000 daltons. Approximately 50% of the molecular mass is accounted for by extensive glycosylation of the protein. AIDSVAX® vaccines are highly purified mixtures of gp120 proteins from HIV-1 produced by recombinant DNA procedures using Chinese hamsters ovary (CHO) cell expression. Molecular epidemiologic analyses of virus circulating in the US has documented polymorphisms occur at the major neutralizing epitopes of gp120. Analysis of breakthrough infections in Phase I and Phase II trials of MN rgp120/HIV-1 revealed that most contained amino acid substitutions that differed from MN rgp120/HIV-1 at epitopes important for virus neutralization (specifically at the V2, V3 and C4 domains). After examining a variety of US strains, GNE8 was selected because amino acid sequences at sites know to be target of neutralizing antibodies differed from. MM and possessed common polymorphisms that complemented MN at major neutralizing epitopes. Thus, an exemplary polypeptide composition is AIDSVAX® B/B (VaxGen), which contains a bivalent polypeptide vaccine containing the HIV-1 type B epitopes MN recombinant glycoprotein (rgp)120 (amino acids 12-485 of MN gp120) and GNE8 rpg120 (amino acids 12-477 of GNE8 gp120) at a one-to-one ratio. Another exemplary polypeptide composition for use where subtypes B and E are prevelant (e.g., Thailand) is AIDSVAX® B/E, which contains the subtype B antigen MN rgp120 (as described above) and the subtype E antigen A244 (CM244) recombinant glycoprotein 120 (ammo acids 12-484 of A244 gp120) at a one-to-one ratio. The subtype E antigen is derived from, the A244 (CM244) strain of HIV-1, which is isolated from Chiang Mai in Northern Thailand and represents about 75% of the incident infections in intravenous drug users (IVDUs) in Bangkok, A244 rgp120/HIV-1 is derived from a primary, macrophage or NSI viral type and, like GNE8 rgp120/HIV-1, requires the chemokine receptor CCR5 to bind with CD4 cells.
[0065] In both AIDSVAX® B/B and AIDSVAX® B/E, gp120 of the MN, GNE8, or A244 strains are each expressed as an amino-terminal fusion protein, with a 27 amino acids of the herpes simplex virus type I gD protein. The gD sequence facilitates gp120 expression and provides an epitope that can be used in a generic immunoaffinity purification process. The amino acid residues equivalent to the mature, native gp120 for each particular HIV-1 isolate utilized are; 12 to 485 for the MN isolate, 12 to 477 for the GNE8 isolate, and 12 to 484 for the A244 isolate.
[0066] The recombinant gp120 polypeptides are produced in genetically modified CHO cell line. The CHO cells secrete the rgp120/HIV-1 molecule into the culture medium, and the protein is purified by a generic purification process for gp120 that includes immunoaffinity chromatography. AIDSVAX® bivalent vaccines are supplied as a sterile suspension in single-use glass vials. Each vial has a nominal content of 1 mL (300 μ/mL) of each rgp120/HIV-1 protein adsorbed onto a total of 0.6 mg aluminum hydroxide gel adjuvant.
Example 2
Clinical Trial Design and Results
[0067] A prime-boost immunization protocol using as the "prime" composition ALVAC-HIV (vCP1521) and die "boost" composition AIDSVAX® B/E was tested in a formal phase III clinical trial. The trial described herein was a community-based, randomized (vaccine; placebo=1:1), multicenter, double blind, placebo-controlled clinical trial conducted in Thailand. The primary objective of the study was to determine if vaccination with ALVAC-HIV (vCP1521) and AIDSVAX® B/E could prevent HIV infection in healthy Thai adults. Thus, infection rates, as well as plasma viral load and CD4.sup.+ T cell counts in volunteers developing HIV infection during the trial, were assessed. The statistical assumptions of the study required that 16,000 persons enroll into the study. Intramuscular vaccinations (e.g., deltoid muscle) for each individual occurred during a 24-week period (0, 4, 12, 24 weeks). Following vaccination, the volunteers were tested for the presence of HIV in their plasma every 6 months tor 3 years.
[0068] The "first composition" (ALVAC-HIV), as described in Example 1A above was initially administered to patients as the priming dose at months 0, 1, 3 and 6 (weeks 0, 4, 12 and 24). The "second composition" (AIDSVAX® B/E) was later administered at months 3 and 6 (weeks 12 and 24 as for ALVAC-HIV) as the boosting dose. ALVAC-HIV doses included approximately 107 CCID50. AIDSVAX® B/E doses included approximately 600 μg of polypeptide (300 μg of each of B and E). There was a 3 year follow-up with vaccine volunteers (Rerks-Ngram, 2009, NEJM 361:2209). The trial design is summarized in Table 2, as shown below:
TABLE-US-00002 TABLE 2 RV144 Trial Design Weeks Group Number 0 4 12 24 I 8,000 ALVAC ALVAC ALVAC ALVAC Placebo Placebo Placebo + Placebo + AIDSVAX AIDSVAX Placebo Placebo II 8,000 ALVAC- ALVAC- ALVAC- ALVAC- HIV HIV HIV + HIV + AIDSVAX ® A1DSVAX ® B/E B/E
[0069] As shown in FIG. 3, the two-part composition was used in a prime-boost format to successfully vaccinate human beings with an efficacy of 31.2% (e.g., about one-third of the population). The population to whom a placebo was administered exhibited 74 HIV infections over the testing period, while those administered the ALVAC-HIV/AIDSVAX® B/E two-part composition ("vaccine") exhibited 51 HIV infections over the testing period. The difference in the number of HIV infections between the two groups was statistically significant (p=0.039). In patients that were infected by HIV after vaccination, neither the setpoint viral load nor mean CD4+ T cell counts were significantly different from placebo at 30 months post-vaccination. This is the first vaccine shown to reduce the risk of HIV infection in human beings compared to a placebo. The observed vaccine efficacy was 31.2% (p=0.0385, O'Brien-Fleming adjusted 95% CI 1.1, 52.1) using the Cox Proportional Hazard method. Thus, this is the first demonstration of a safe, efficacious vaccine that is capable of protecting human beings from infection by HIV.
[0070] It is to be understood that any reference to a particular range includes all individual values and sub-ranges within that range as if each were individually listed herein. All references cited within this application are incorporated by reference in their entirety. While the present invention has been described in terms of the preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations that come within the scope of the invention as claimed.
Burton, et al Science, 303:316 (2004)
[0071] deBruyn, et al. Vaccine. 22:704-13 (2004) Esposito, et al (eds): Arch. Virol Supp.2. NY, Springer-Verlag, pp 91-102 (1991)
Fang, et al J. Infect, Dis. 180 (4): 1122-32 (1999)
[0072] Ferrari, et al. Proc. Natl. Acad. Sci. USA 94:1396-401 (1997) Klausner, et al. Science, 300:2036-39 (2003) Letters to the Editor. Science, 305:177-180 (2004) McCarthy M. Lancet. Nov 22;362(9397):1728 (2003) McNeil, et al. Science, 303:961 (2004) Meeting Summary: AIDS Vaccine Trials, Considerations for Phase III Trial Design and Endpoints, National Institutes of Health, NIAID, Nov. 16,2001. Retrieved from the World Wide Web on Aug. 3, 2004 at http://www.niaid.nih.gov/vrc. NIAID Phase III HIV vaccine trial to determine correlates of protection will not proceed NIAID News, Feb. 25,2002. Retrieved from the World Wide Web on Aug. 9, 2004 at http:/www2.niaid.nih.gov/newsroom/releases/phase3hiv.htm. Nitayaphan, et al. J. Inf. Dis. 190:702-6 (2004) Rerks-Ngarm et al. NEJM, 361:2209 (2009) Tangcharoensathien, et al. Health Policy. 57:111-139(2001) Tartaglia, et al., (eds): AIDS Research Reviews, Vol. 3. New York, Marcel Dekker, pp 361-378(1993).
[0073] Tovanabutra S, et al. AIDS Vaccine 2003, New York, N.Y.; Abstract number 463.
Trinvuthipoong, D. Science, 303:954-5 (2004)
Sequence CWU
711478DNAHuman immunodeficiency virus 1ggtacctgtg tggaaagaag caaccaccac tctattttgt gcatcagatg ctaaagcata 60tgatacagag gcacataatg tttgggccac acatgcctgt gtacccacag accccaaccc 120acaagaagta gaattggtaa atgtgacaga aaattttaac atgtggaaaa ataacatggt 180agaacagatg catgaggata taatcagttt atgggatcaa agcctaaagc catgtgtaaa 240attaacccca ctctgtgtta ctttaaattg cactgatttg aggaatacta ctaataccaa 300taatagtact gataataaca atagtaaaag cgagggaaca ataaagggag gagaaatgaa 360aaactgctct ttcaatatca ccacaagcat aggagataag atgcagaaag aatatgcact 420tctttataaa cttgatatag aaccaataga taatgatagt accagctata ggttgataag 480ttgtaatacc tcagtcatta cacaagcttg tccaaagata tcctttgagc caattcccat 540acactattgt gccccggctg gttttgcgat tctaaagtgt aacgataaaa agttcagtgg 600aaaaggatca tgtaaaaatg tcagcacagt acaatgtaca catggaatta ggccagtagt 660atcaactcaa ctgctgttaa atggcagtct agcagaagaa gaggtagtaa ttagatctga 720ggatttcact gataatgcta aaaccatcat agtacatctg aaagaatctg tacaaattaa 780ttgtacaaga cccaactaca ataaaagaaa aaggatacat ataggaccag ggagagcatt 840ttatacaaca aaaaatataa aaggaactat aagacaagca cattgtatca ttagtagagc 900aaaatggaat gacactttaa gacagatagt tagcaagtta aaagaacaat ttaagaataa 960aacaatagtc tttaatccat cctcaggagg ggacccagaa attgtaatgc acagttttaa 1020ttgtggaggg gaatttttct actgtaatac atcaccactg tttaatagta tttggaatgg 1080taataatact tggaataata ctacagggtc aaataacaat atcacacttc aatgcaaaat 1140aaaacaaatt ataaacatgt ggcagaaagt aggaaaagca atgtatgccc ctcccattga 1200aggacaaatt agatgttcat caaatattac agggctacta ttaacaagag atggtggtga 1260ggacacggac acgaacgaca ccgagatctt cagacctgga ggaggagata tgagggacaa 1320ttggagaagt gaattatata aatataaagt agtaacaatt gaaccattag gagtagcacc 1380caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagcgatag gagctctgtt 1440ccttgggttc ttaggagcag caggaagcac tatgggcg 14782492PRTHuman immunodeficiency virus 2Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu Phe Cys Ala Ser Asp1 5 10 15Ala Lys Ala Tyr Asp Thr Glu Ala His Asn Val Trp Ala Thr His Ala 20 25 30Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Val Glu Leu Val Asn Val 35 40 45Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln Met His 50 55 60Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro Cys Val Lys65 70 75 80Leu Thr Pro Leu Cys Val Thr Leu Asn Cys Thr Asp Leu Arg Asn Thr 85 90 95Thr Asn Thr Asn Asn Ser Thr Asp Asn Asn Asn Ser Lys Ser Glu Gly 100 105 110Thr Ile Lys Gly Gly Glu Met Lys Asn Cys Ser Phe Asn Ile Thr Thr 115 120 125Ser Ile Gly Asp Lys Met Gln Lys Glu Tyr Ala Leu Leu Tyr Lys Leu 130 135 140Asp Ile Glu Pro Ile Asp Asn Asp Ser Thr Ser Tyr Arg Leu Ile Ser145 150 155 160Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Ile Ser Phe Glu 165 170 175Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys 180 185 190Cys Asn Asp Lys Lys Phe Ser Gly Lys Gly Ser Cys Lys Asn Val Ser 195 200 205Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr Gln Leu 210 215 220Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Val Ile Arg Ser Glu225 230 235 240Asp Phe Thr Asp Asn Ala Lys Thr Ile Ile Val His Leu Lys Glu Ser 245 250 255Val Gln Ile Asn Cys Thr Arg Pro Asn Tyr Asn Lys Arg Lys Arg Ile 260 265 270His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Lys Asn Ile Lys Gly 275 280 285Thr Ile Arg Gln Ala His Cys Ile Ile Ser Arg Ala Lys Trp Asn Asp 290 295 300Thr Leu Arg Gln Ile Val Ser Lys Leu Lys Glu Gln Phe Lys Asn Lys305 310 315 320Thr Ile Val Phe Asn Pro Ser Ser Gly Gly Asp Pro Glu Ile Val Met 325 330 335His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Thr Ser Pro 340 345 350Leu Phe Asn Ser Ile Trp Asn Gly Asn Asn Thr Trp Asn Asn Thr Thr 355 360 365Gly Ser Asn Asn Asn Ile Thr Leu Gln Cys Lys Ile Lys Gln Ile Ile 370 375 380Asn Met Trp Gln Lys Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Glu385 390 395 400Gly Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg 405 410 415Asp Gly Gly Glu Asp Thr Asp Thr Asn Asp Thr Glu Ile Phe Arg Pro 420 425 430Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr 435 440 445Lys Val Val Thr Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys 450 455 460Arg Arg Val Val Gln Arg Glu Lys Arg Ala Ala Ile Gly Ala Leu Phe465 470 475 480Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly 485 4903472PRTHuman immunodeficiency virus 3Val Pro Val Trp Lys Glu Ala Asp Thr Thr Leu Phe Cys Ala Ser Asp1 5 10 15Ala Lys Ala His Glu Thr Glu Val His Asn Val Trp Ala Thr His Ala 20 25 30Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Ile Asp Leu Glu Asn Val 35 40 45Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln Met Gln 50 55 60Glu Asp Val Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro Cys Val Lys65 70 75 80Leu Thr Pro Pro Cys Val Thr Leu His Cys Thr Asn Ala Asn Leu Thr 85 90 95Lys Ala Asn Leu Thr Asn Val Asn Asn Arg Thr Asn Val Ser Asn Ile 100 105 110Ile Gly Asn Ile Thr Asp Glu Val Arg Asn Cys Ser Phe Asn Met Thr 115 120 125Thr Glu Leu Arg Asp Lys Lys Gln Lys Val His Ala Leu Phe Tyr Lys 130 135 140Leu Asp Ile Val Pro Ile Glu Asp Asn Asn Asp Ser Ser Glu Tyr Arg145 150 155 160Leu Ile Asn Cys Asn Thr Ser Val Ile Lys Gln Pro Cys Pro Lys Ile 165 170 175Ser Phe Asp Pro Ile Pro Ile His Tyr Cys Thr Pro Ala Gly Tyr Ala 180 185 190Ile Leu Lys Cys Asn Asp Lys Asn Phe Asn Gly Thr Gly Pro Cys Lys 195 200 205Asn Val Ser Ser Val Gln Cys Thr His Gly Ile Lys Pro Val Val Ser 210 215 220Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Ile Ile Ile225 230 235 240Arg Ser Glu Asn Leu Thr Asn Asn Ala Lys Thr Ile Ile Val His Leu 245 250 255Asn Lys Ser Val Val Ile Asn Cys Thr Arg Pro Ser Asn Asn Thr Arg 260 265 270Thr Ser Ile Thr Ile Gly Pro Gly Gln Val Phe Tyr Arg Thr Gly Asp 275 280 285Ile Ile Gly Asp Ile Arg Lys Ala Tyr Cys Glu Ile Asn Gly Thr Glu 290 295 300Trp Asn Lys Ala Leu Lys Gln Val Thr Glu Lys Leu Lys Glu His Phe305 310 315 320Asn Asn Lys Pro Ile Ile Phe Gln Pro Pro Ser Gly Gly Asp Leu Glu 325 330 335Ile Thr Met His His Phe Asn Cys Arg Gly Glu Phe Phe Tyr Cys Asn 340 345 350Thr Thr Arg Leu Phe Asn Asn Thr Cys Ile Ala Asn Gly Thr Ile Glu 355 360 365Gly Cys Asn Gly Asn Ile Thr Leu Pro Cys Lys Ile Lys Gln Ile Ile 370 375 380Asn Met Trp Gln Gly Ala Gly Gln Ala Met Tyr Ala Pro Pro Ile Ser385 390 395 400Gly Thr Ile Asn Cys Val Ser Asn Ile Thr Gly Ile Leu Leu Thr Arg 405 410 415Asp Gly Gly Ala Thr Asn Asn Thr Asn Asn Glu Thr Phe Arg Pro Gly 420 425 430Gly Gly Asn Ile Lys Asp Asn Trp Arg Asn Glu Leu Tyr Lys Tyr Lys 435 440 445Val Val Gln Ile Glu Pro Leu Gly Val Ala Pro Thr Arg Ala Lys Arg 450 455 460Arg Val Val Glu Arg Glu Lys Arg465 4704472PRTHuman immunodeficiency virus 4Val Pro Val Trp Arg Asp Ala Asp Thr Thr Leu Phe Cys Ala Ser Asp1 5 10 15Ala Lys Ala His Glu Thr Glu Val His Asn Val Trp Ala Thr His Ala 20 25 30Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Ile Asp Leu Glu Asn Val 35 40 45Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln Met Gln 50 55 60Glu Asp Val Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro Cys Val Lys65 70 75 80Leu Thr Pro Leu Cys Val Thr Leu His Cys Thr Asn Ala Asn Leu Thr 85 90 95Lys Ala Asn Leu Thr Asn Val Asn Asn Arg Thr Asn Val Ser Asn Ile 100 105 110Ile Gly Asn Ile Thr Asp Glu Val Arg Asn Cys Ser Phe Asn Met Thr 115 120 125Thr Glu Leu Arg Asp Lys Lys Gln Lys Val His Ala Leu Phe Tyr Lys 130 135 140Leu Asp Ile Val Pro Ile Glu Asp Asn Asn Asp Asn Ser Lys Tyr Arg145 150 155 160Leu Ile Asn Cys Asn Thr Ser Val Ile Lys Gln Ala Cys Pro Lys Ile 165 170 175Ser Phe Asp Pro Ile Pro Ile His Tyr Cys Thr Pro Ala Gly Tyr Ala 180 185 190Ile Leu Lys Cys Asn Asp Lys Asn Phe Asn Gly Thr Gly Pro Cys Lys 195 200 205Asn Val Ser Ser Val Gln Cys Thr His Gly Ile Lys Pro Val Val Ser 210 215 220Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Ile Ile Ile225 230 235 240Arg Ser Glu Asp Leu Thr Asn Asn Ala Lys Thr Ile Ile Val His Leu 245 250 255Asn Lys Ser Val Val Ile Asn Cys Thr Arg Pro Ser Asn Asn Thr Arg 260 265 270Thr Ser Ile Thr Ile Gly Pro Gly Gln Val Phe Tyr Arg Thr Gly Asp 275 280 285Ile Ile Gly Asp Ile Arg Lys Ala Tyr Cys Glu Ile Asn Gly Thr Glu 290 295 300Trp Asn Lys Ala Leu Lys Gln Val Thr Glu Lys Leu Lys Glu His Phe305 310 315 320Asn Asn Lys Pro Ile Ile Phe Gln Pro Pro Ser Gly Gly Asp Leu Glu 325 330 335Ile Thr Met His His Phe Asn Cys Arg Gly Glu Phe Phe Tyr Cys Asn 340 345 350Thr Thr Arg Leu Phe Asn Asn Thr Cys Ile Ala Asn Gly Thr Ile Glu 355 360 365Gly Cys Asn Gly Asn Ile Thr Leu Pro Cys Lys Ile Lys Gln Ile Ile 370 375 380Asn Met Trp Gln Gly Ala Gly Gln Ala Met Tyr Ala Pro Pro Ile Ser385 390 395 400Gly Thr Ile Asn Cys Val Ser Asn Ile Thr Gly Ile Leu Leu Thr Arg 405 410 415Asp Gly Gly Ala Thr Asn Asn Thr Asn Asn Glu Thr Phe Arg Pro Gly 420 425 430Gly Gly Asn Ile Lys Asp Asn Trp Arg Asn Glu Leu Tyr Lys Tyr Lys 435 440 445Val Val Gln Ile Glu Pro Leu Gly Ala Ala Pro Thr Arg Ala Lys Arg 450 455 460Arg Val Val Glu Arg Glu Lys Arg465 47053796DNAArtificial SequenceHuman immunodeficiency virus 5agtacaataa aaagtattaa ataaaaatac ttacttacga aaaaatgact aattagctat 60aaaaacccgg ggttatccct gcctaactct attcactaca gagagtacag caaaaactat 120tcttaaacct accaagcctc ctactatcat tatgaataat cttttttctc tctccaccac 180tcttcttttg gcgcgggtgg gtgctattcc tagtggttca atttgtacta ctttatattt 240atataattca cttctccaat tgtcctttat atttcctcct ccaggtctga aggtctcgtt 300agtattattg agaccaccat ctcttgtcaa tagtattcct gtaatatttg atacacaatt 360aattcttcca ctgatgggag gagcatacat tgcttgtcct gctccctgcc acatgtttat 420aatttgcttt atcttgcatg gaagtatgat agtgccatta cacccctcca tggtttcatt 480ttccatgcaa gtattattaa acagtcgtgt tgtattgcaa tagaagaatt cccctctaca 540attaaaatga tgcattgtaa tttctagatc tcctcctgag ggtggttgaa agattattgt 600cttattatta aagtgctctt ttaatttttt agttaccttt tttaaaactt cattccattt 660tgttccatta atctcacaat atgcttttct tatatctcct attatgtctc ctgttctgta 720gaatacttgt cctggtccta tatttatact tgttcttgta ttgttggagg gtctggtaca 780attgatttct acagatttat taaggtgcac tattatggtt ttggcattgt ttgtgagatt 840ttcagatctg attattatct cttcttctgc tagactgcca tttaacagca attgagttga 900taccacaggc ttaattccat gtgtgcattg tactgagctg acatttttac atggccctgt 960cccattgaaa ttcttatcat tacactttaa aatcgcataa ccagctggag tacaataatg 1020tataggaatt ggatcaaagg atatctttgg acaagcctgc ttaatgactg aagtattaca 1080atttattaac ctatactcac tactactcgt gttatcttca attggtacta tatcaagctt 1140gtagaatagt gcatggacct tctgcttctt atctcttagt tctgtggtca tattaaaaga 1200acagtttctt acttcatctg ttatatttcc tattatgtta gggacattgg ttatgttctt 1260gacattggtc acattagcat tggtacaatt taaagtaacg cagagaggag ttaactttac 1320acatggtttt agactttgat cccataaact gattacatcc tcctgcatct gctctaccat 1380gttatttttc cacatgttaa aattttctgt tacattttcc aggtgtaatt cttgtgggtt 1440ggggtctgtg ggtacacagg catgtgtggc ccagacattg tgcgcttctg tctcttgtgc 1500tttggcatct gatgcacaaa atagggtggt atctgcatct ctccatacag gaaccccata 1560ataaactgta acccacaagt tgtctgaggc actacaaatt atcaccaacc caaggatcaa 1620agtcccccat ttccacaagt ttggccaagt catctgtgtc tccttcactc tcattacgat 1680acaaacttaa cggatatcgc gataatgaaa taatttatga ttatttctcg ctttcaattt 1740aacacaaccc tcaagaacct ttgtatttat tttcactttt taagtataga ataaagaagc 1800tctaattaat taagctacaa atagtttcgt tttcaccttg tctaataact aattaattaa 1860cccggatctt gagataaagt gaaaatatat atcattatat tacaaagtac aattatttag 1920gtttaatcat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgatgggaaa 1980aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa 2040gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta 2100gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat 2160tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca 2220aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaaaaaa gcacagcaag 2280cagcagctga cacaggacac agcaatcagg tcagccaaaa ttaccctata gtgcagaaca 2340tccaggggca aatggtacat caggccatat cacctagaac tttaaatgca tgggtaaaag 2400tagtagaaga gaaggctttc agcccagaag tgatacccat gttttcagca ttatcagaag 2460gagccacccc acaagattta aacaccatgc taaacacagt ggggggacat caagcagcca 2520tgcaaatgtt aaaagagacc atcaatgagg aagctgcaga atgggataga gtgcatccag 2580tgcatgcagg gcctattgca ccaggccaga tgagagaacc aaggggaagt gacatagcag 2640gaactactag tacccttcag gaacaaatag gatggatgac aaataatcca cctatcccag 2700taggagaaat ttataaaaga tggataatcc tgggattaaa taaaatagta agaatgtata 2760gccctaccag cattctggac ataagacaag gaccaaaaga accctttaga gactatgtag 2820accggttcta taaaactcta agagccgagc aagcttcaca ggaggtaaaa aattggatga 2880cagaaacctt gttggtccaa aatgcgaacc cagattgtaa gactatttta aaagcattgg 2940gaccagcggc tacactagaa gaaatgatga cagcatgtca gggagtagga ggacccggcc 3000ataaggcaag agttttggct gaagcaatga gccaagtaac aaattcagct accataatga 3060tgcagagagg caattttagg aaccaaagaa agattgttaa gtgtttcaat tgtggcaaag 3120aagggcacac agccagaaat tgcagggccc ctaggaaaaa gggctgttgg aaatgtggaa 3180aggaaggaca ccaaatgaaa gattgtactg agagacaggc taatttttta gggaagatct 3240ggccttccta caagggaagg ccagggaatt ttcttcagag cagaccagag ccaacagccc 3300caccagaaga gagcttcagg tctggggtag agacaacaac tccccctcag aagcaggagc 3360cgatagacaa ggaactgtat cctttaactt ccctcagatc actctttggc aacgacccct 3420cgtcacaata aagatagggg ggcaactaaa ggaagctcta ttagatacag gagcagatga 3480tacagtatta gaagaaatga gtttgccagg aagatggaaa ccaaaaatga tagggggaat 3540tggaggtttt atcaaagtaa gacagtatga tcagatactc atagaaatct gtggacataa 3600agctataggt acagtattag taggacctac acctgtcaac ataattggaa gaaatctgtt 3660gactcagatt ggttgcactt taaattttta acccgggctg cagctcgagg aattcttttt 3720attgattaac tagtcaaatg agtatatata attgaaaaag taaaatataa atcatataat 3780aatgaaacga aatatc 379663796DNAArtificial SequenceHuman immunodeficiency virus 6tcatgttatt tttcataatt tatttttatg aatgaatgct tttttactga ttaatcgata 60tttttgggcc ccaataggga cggattgaga taagtgatgt ctctcatgtc gtttttgata 120agaatttgga tggttcggag gatgatagta atacttatta gaaaaaagag agaggtggtg 180agaagaaaac cgcgcccacc cacgataagg atcaccaagt taaacatgat gaaatataaa 240tatattaagt gaagaggtta acaggaaata taaaggagga ggtccagact tccagagcaa 300tcataataac tctggtggta gagaacagtt atcataagga cattataaac tatgtgttaa 360ttaagaaggt gactaccctc ctcgtatgta acgaacagga cgagggacgg tgtacaaata 420ttaaacgaaa tagaacgtac cttcatacta tcacggtaat gtggggaggt accaaagtaa 480aaggtacgtt cataataatt tgtcagcaca acataacgtt atcttcttaa ggggagatgt 540taattttact acgtaacatt aaagatctag aggaggactc ccaccaactt tctaataaca 600gaataataat ttcacgagaa aattaaaaaa tcaatggaaa aaattttgaa gtaaggtaaa 660acaaggtaat tagagtgtta tacgaaaaga atatagagga taatacagag gacaagacat 720cttatgaaca ggaccaggat ataaatatga acaagaacat aacaacctcc cagaccatgt 780taactaaaga
tgtctaaata attccacgtg ataataccaa aaccgtaaca aacactctaa 840aagtctagac taataataga gaagaagacg atctgacggt aaattgtcgt taactcaact 900atggtgtccg aattaaggta cacacgtaac atgactcgac tgtaaaaatg taccgggaca 960gggtaacttt aagaatagta atgtgaaatt ttagcgtatt ggtcgacctc atgttattac 1020atatccttaa cctagtttcc tatagaaacc tgttcggacg aattactgac ttcataatgt 1080taaataattg gatatgagtg atgatgagca caatagaagt taaccatgat atagttcgaa 1140catcttatca cgtacctgga agacgaagaa tagagaatca agacaccagt ataattttct 1200tgtcaaagaa tgaagtagac aatataaagg ataatacaat ccctgtaacc aatacaagaa 1260ctgtaaccag tgtaatcgta accatgttaa atttcattgc gtctctcctc aattgaaatg 1320tgtaccaaaa tctgaaacta gggtatttga ctaatgtagg aggacgtaga cgagatggta 1380caataaaaag gtgtacaatt ttaaaagaca atgtaaaagg tccacattaa gaacacccaa 1440ccccagacac ccatgtgtcc gtacacaccg ggtctgtaac acgcgaagac agagaacacg 1500aaaccgtaga ctacgtgttt tatcccacca tagacgtaga gaggtatgtc cttggggtat 1560tatttgacat tgggtgttca acagactccg tgatgtttaa tagtggttgg gttcctagtt 1620tcagggggta aaggtgttca aaccggttca gtagacacag aggaagtgag agtaatgcta 1680tgtttgaatt gcctatagcg ctattacttt attaaatact aataaagagc gaaagttaaa 1740ttgtgttggg agttcttgga aacataaata aaagtgaaaa attcatatct tatttcttcg 1800agattaatta attcgatgtt tatcaaagca aaagtggaac agattattga ttaattaatt 1860gggcctagaa ctctatttca cttttatata tagtaatata atgtttcatg ttaataaatc 1920caaattagta cccacgctct cgcagtcata attcgccccc tcttaatcta gctacccttt 1980tttaagccaa ttccggtccc cctttctttt ttatatttaa ttttgtatat catacccgtt 2040cgtccctcga tcttgctaag cgtcaattag gaccggacaa tctttgtagt cttccgacat 2100ctgtttatga ccctgtcgat gttggtaggg aagtctgtcc tagtcttctt gaatctagta 2160atatattatg tcatcgttgg gagataacac acgtagtttc ctatctctat tttctgtggt 2220tccttcgaaa tctgttctat ctccttctcg ttttgttttc attctttttt cgtgtcgttc 2280gtcgtcgact gtgtcctgtg tcgttagtcc agtcggtttt aatgggatat cacgtcttgt 2340aggtccccgt ttaccatgta gtccggtata gtggatcttg aaatttacgt acccattttc 2400atcatcttct cttccgaaag tcgggtcttc actatgggta caaaagtcgt aatagtcttc 2460ctcggtgggg tgttctaaat ttgtggtacg atttgtgtca cccccctgta gttcgtcggt 2520acgtttacaa ttttctctgg tagttactcc ttcgacgtct taccctatct cacgtaggtc 2580acgtacgtcc cggataacgt ggtccggtct actctcttgg ttccccttca ctgtatcgtc 2640cttgatgatc atgggaagtc cttgtttatc ctacctactg tttattaggt ggatagggtc 2700atcctcttta aatattttct acctattagg accctaattt attttatcat tcttacatat 2760cgggatggtc gtaagacctg tattctgttc ctggttttct tgggaaatct ctgatacatc 2820tggccaagat attttgagat tctcggctcg ttcgaagtgt cctccatttt ttaacctact 2880gtctttggaa caaccaggtt ttacgcttgg gtctaacatt ctgataaaat tttcgtaacc 2940ctggtcgccg atgtgatctt ctttactact gtcgtacagt ccctcatcct cctgggccgg 3000tattccgttc tcaaaaccga cttcgttact cggttcattg tttaagtcga tggtattact 3060acgtctctcc gttaaaatcc ttggtttctt tctaacaatt cacaaagtta acaccgtttc 3120ttcccgtgtg tcggtcttta acgtcccggg gatccttttt cccgacaacc tttacacctt 3180tccttcctgt ggtttacttt ctaacatgac tctctgtccg attaaaaaat cccttctaga 3240ccggaaggat gttcccttcc ggtcccttaa aagaagtctc gtctggtctc ggttgtcggg 3300gtggtcttct ctcgaagtcc agaccccatc tctgttgttg agggggagtc ttcgtcctcg 3360gctatctgtt ccttgacata ggaaattgaa gggagtctag tgagaaaccg ttgctgggga 3420gcagtgttat ttctatcccc ccgttgattt ccttcgagat aatctatgtc ctcgtctact 3480atgtcataat cttctttact caaacggtcc ttctaccttt ggtttttact atccccctta 3540acctccaaaa tagtttcatt ctgtcatact agtctatgag tatctttaga cacctgtatt 3600tcgatatcca tgtcataatc atcctggatg tggacagttg tattaacctt ctttagacaa 3660ctgagtctaa ccaacgtgaa atttaaaaat tgggcccgac gtcgagctcc ttaagaaaaa 3720taactaattg atcagtttac tcatatatat taactttttc attttatatt tagtatatta 3780ttactttgct ttatag 3796728PRTherpes simplex virus 1 7Lys Tyr Ala Leu Ala Asp Ala Ser Leu Lys Met Ala Asp Pro Asn Arg1 5 10 15Phe Arg Gly Lys Asp Leu Pro Val Leu Asp Gln Leu 20 25
Patent applications by SANOFI PASTEUR, INC.
Patent applications in class Immunodeficiency virus (e.g., HIV, etc.)
Patent applications in all subclasses Immunodeficiency virus (e.g., HIV, etc.)
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