WO2015009946A1 - Méthode visant à renforcer la réponse immunitaire face aux antigènes du vih - Google Patents

Méthode visant à renforcer la réponse immunitaire face aux antigènes du vih Download PDF

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WO2015009946A1
WO2015009946A1 PCT/US2014/047056 US2014047056W WO2015009946A1 WO 2015009946 A1 WO2015009946 A1 WO 2015009946A1 US 2014047056 W US2014047056 W US 2014047056W WO 2015009946 A1 WO2015009946 A1 WO 2015009946A1
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hiv
dose
weeks
boosting
composition
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PCT/US2014/047056
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English (en)
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Rama R. AMARA
Harriet L. Robinson
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Emory University
Geovax, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to methods of immunization as well as methods for improving an immune response to an antigen. More specifically the present invention relates to administration regimes that promote improved cellular and/or humoral immune responses to an HIV antigen(s) than conventional
  • HIV human immunodeficiency virus
  • a particular approach involves priming the immune response with plasmid DNA followed by boosting with a recombinant poxvirus booster, such as a modified vaccinia virus (MVA) Ankara (Amara et al. 2001 Science 292:69-74 and
  • the prime consists of a recombinant viral vector, such as MVA.
  • a recombinant viral vector such as MVA.
  • the prime and boost may lead to a diminished immune response similar to preexisting immunity to the recombinant vector.
  • a major challenge has been raising antibody responses that are long lasting.
  • the decline of antibody correlated with lower levels of prevention of infection.
  • the HIV envelope glycoprotein in its natural form, is heavily glycosylated and is slow to elicit high avidity antibody.
  • the present invention is directed to methods of HIV immunization, as well as methods improving an immune response to an HIV antigen(s).
  • the methods of the present invention provide an improved cellular and/or humoral immune response compared to administration of HIV antigens by conventional methods.
  • the present invention is a method of immunization, comprising (i) administering a priming composition comprising one or more genes encoding an HIV antigen; (ii) administering a first dose of a boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding an HIV antigen, wherein the vector expresses one or more recombinant HIV antigens, and (iii) administering a second dose of a boosting composition between about 12 and 20 weeks after the first dose.
  • the priming composition is DNA plasmid.
  • the priming composition is a DNA plasmid
  • HIV antigen selected from a Clade A G, B or C antigen, or combinations thereof.
  • the boosting composition comprises one or more genes encoding an HIV antigen selected from a Clade A G, B or C antigen, or combinations thereof.
  • the one or more genes of the priming composition and the one or more genes of the boosting composition are the same. In a further embodiment, the boosting compositions are the same.
  • the viral vector further comprises one or more genes encoding an HIV enzyme, wherein the gene encoding the HIV enzyme have been has been modified.
  • the gene encoding the HIV enzyme has been modified to possess safety mutations.
  • the HIV enzyme is selected from reverse transcriptase or protease.
  • the second dose is administered between about 14 and about 20 weeks after the first dose, more particularly, about 16 weeks after the first dose.
  • the method may comprise one or more additional steps.
  • the method comprises one or more priming steps, boosting steps, or a combination thereof.
  • the method results in an improved immune response relative to the same method, but where the second dose is administered less than 12 weeks after the first dose, more particularly, about 8 weeks after the first dose.
  • the present invention is a method of enhancing
  • immunization comprising (i) administering a first dose of a boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding an HIV antigen, wherein the vector expresses one or more recombinant HIV antigens, and (ii) administering a second dose of a boosting composition between about 12 and 20 weeks after the first dose.
  • the boosting composition comprises one or more genes encoding an HIV antigen selected from a Clade A G, B or C antigen, or combinations thereof.
  • the boosting compositions are the same.
  • the viral vector further comprises one or more genes encoding an HIV enzyme, wherein the gene encoding the HIV enzyme have been has been modified.
  • the gene encoding the HIV enzyme has been modified to possess safety mutations.
  • the HIV enzyme is selected from reverse transcriptase or protease.
  • the second dose is administered between about 14 and about 20 weeks after the first dose, more particularly, about 16 weeks after the first dose.
  • the method may involve one or more additional steps.
  • the method may involve one or more additional steps.
  • the method comprises one or more boosting steps.
  • the methods produces an improved immune response relative to the same method in which the second dose is administered less than 12 weeks after the first dose, more particularly, about 8 weeks after the first dose.
  • the present invention is a method of enhancing B cell antibody response, comprising (i) administering a priming composition comprising one or more genes encoding an HIV antigen, wherein the vector expresses one or more recombinant HIV antigens , (ii) administering a first dose of a boosting
  • composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding an HIV antigen, wherein the vector expresses one or more recombinant HIV antigens, and (iii) administering a second dose of a boosting composition between about 12 and 20 weeks after the first dose.
  • the boosting composition comprises one or more genes encoding an HIV antigen selected from a Clade A G, B or C antigen, or combinations thereof.
  • the boosting compositions are the same.
  • the viral vector further comprises one or more genes encoding an HIV enzyme, wherein the gene encoding the HIV enzyme have been has been modified.
  • the gene encoding the HIV enzyme has been modified to possess safety mutations.
  • the HIV enzyme is selected from reverse transcriptase or protease.
  • the second dose is administered between about 14 and about 20 weeks after the first dose, more particularly, about 16 weeks after the first dose.
  • the method may involve one or more additional steps.
  • the method comprises one or more boosting steps.
  • the methods produces an improved immune response relative to the same method in which the second dose is administered less than 12 weeks after the first dose, more particularly, about 8 weeks after the first dose.
  • the present invention is a method of enhancing immunization, comprising (i) administering a first dose of a boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding an HIV antigen, wherein the vector expresses one or more recombinant HIV antigens, (ii) administering a second dose of a boosting composition; and (iii) administering a third dose of the boosting composition between about 12 and about 20 weeks after the second dose, more particularly, about 14 and about 18 weeks after the second dose, even more particularly, about 16 weeks after the second dose.
  • the boosting composition comprises one or more genes encoding an HIV antigen selected from a Clade A G, B or C antigen, or combinations thereof.
  • the boosting compositions are the same.
  • the method produces an improved immune response relative to the same method in which the third dose is administered less than 12 weeks after the second dose, more particularly, about 8 weeks after the first dose.
  • the invention is a method of enhancing immunization, comprising (i) administering a boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding an HIV antigen, wherein the vector expresses one or more recombinant HIV antigens, and (ii) administering a boosting composition comprising an adenovirus vector comprising one or more genes encoding an HIV antigen, wherein the step (i) and step (ii) may occur in either order but are separated by a period of between about 12 and 20 weeks, more particularly, about 14 to about 18 weeks, even more particularly, about 16 weeks.
  • the method produces an improved immune response relative to methods in which step (i) and (ii) are separated by a period of about less than 12 weeks.
  • the improved immune response of the method of the present invention may have one or more characteristics.
  • the immune response is improved with respect to avidity, B cell response or T cell response.
  • the immune response is improved with respect to B cell memory. In exemplary embodiments, the immune response is improved with respect to antibody titer.
  • the immune response is improved with respect to avidity.
  • the immune response is improved with respect to CD8+ T cell response.
  • the immune response is improved with respect to CD4+ T cell response.
  • the present invention is a method comprising (i) administering a priming composition comprising one or more genes encoding an HIV antigen, wherein the priming composition is a plasmid vector; (ii)
  • a first dose of a boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding an HIV antigen, wherein the vector expresses one or more recombinant HIV antigens, and (iii) administering a second dose of a boosting composition between about 12 and 20 weeks after the first dose.
  • the HIV antigens in steps (i)-(iii) are the same.
  • the method comprising one or more additional steps, such as one or more additional priming or boosting steps.
  • the method results in increased antibody titers relative to the same method, but where the second dose of the boosting composition is administered less than 12 weeks after the first dose, more particularly, about 8 weeks after the first dose.
  • the method results in increased avidity relative to the same method, but where the second dose of the boosting composition is administered less than 12 weeks after the first dose, more particularly, about 8 weeks after the first dose.
  • FIG. 1 provides a schematic of DNA/SIV239 vaccine.
  • SIV239 Gag (gag) SIV239 Gag (gag)
  • FIG. 2 provides a graph showing SIV239 envelope specific binding antibody titers in serum at 2 weeks following the second MVA boost of a DDM_M regimen. Binding antibody titers were measured using an ELISA.
  • FIG. 3 provides a graph showing SIV239 envelope specific avidity index (Al) in serum at 2 weeks following the second MVA boost of a DDM_M regimen. Avidity Index was measured using an ELISA.
  • D DNA SIV vaccine expressing SIV239 Gag, Pol, Env, tat and rev; M, MVA/SIV vaccine expressing SIV239 Gag, Pol and Env.
  • FIG. 4 provides a graph showing the net response (MFI-Blank) for two antigens Con6 gp120 and gp41 . These results show a trend for increased magnitude by adding a third MVA boost four months (16 weeks) after the second MVA boost.
  • FIG. 5 provides a graph showing increased trend in avidity index with increased delay between first and second MVA boosts. Avidity is determined for the immunodominant region of gp41 as shown by citrate wash assay. DDMM or DgDgMM uses a 2 month (8 week) delay between MVA (M) boosts while
  • FIG. 8 provides a plot demonstrating that a third spaced MVA boost inhanced durability as shown by the difference in the slopes for the T2 (DgDgMM_M) and T3(DgDgM_M) groups. The last two MVA boosts were spaced by 4 month (16 weeks).
  • the present invention relates to method for immunization, and more particularly, methods for HIV immunization.
  • the invention also extends to methods for improving an immune response to an antigen(s), and more particularly, for improving an immune response to an HIV antigen(s).
  • the methods of the present invention improves the immune response to HIV antigens relative to conventional administration regimes.
  • the present invention relates to a "prime and boost"
  • immunization regime in which the immune response induced by administration of a priming composition comprising an antigen is boosted by administration of a boosting composition, one or more times.
  • Antibody or “antibody molecule” refers to any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen, such as epitopes of an apoptosis modulator protein.
  • the term includes polyclonal, monoclonal, chimeric, and bispecific antibodies.
  • antibody or antibody molecule contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule such as those portions known in the art as Fab, Fab', F(ab').sub.2 and F(v).
  • Antigen refer to a short peptide sequence or oligosaccharide, that is specifically recognized or specifically bound by a component of the immune system.
  • Booster or “booster composition” refers to a second or later vaccine dose given after the primary dose(s) to increase the immune response to the original vaccine antigen(s).
  • the vaccine given as the booster dose may or may not be the same as the primary vaccine.
  • Gene is used broadly to refer to any nucleic acid associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. The term “gene” applies to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.
  • HIV-1 Immunodeficiency Virus.
  • HIV-2 There are many different strains of HIV-1 .
  • the strains of HIV-1 can be classified into three groups: the "major” group M, the "outlier” group 0 and the “new” group N. These three groups may represent three separate introductions of simian
  • clade A is a group of organisms, such as a species, whose members share homologous features derived from a common ancestor.
  • “Host cell” means a cell which contains a heterologous nucleic acid, such as a vector, and supports the replication and/or expression of the nucleic acid, and optionally production of one or more encoded products including a polypeptide and/or a virus.
  • Immune response signifies any reaction produced by an antigen, such as a viral antigen, in a host having a functioning immune system. Immune responses may be either humoral in nature, that is, involve production of immunoglobulins or antibodies, or cellular in nature, involving various types of B and T lymphocytes, dendritic cells, macrophages, antigen presenting cells and the like, or both.
  • Immune responses may also involve the production or elaboration of various effector molecules such as cytokines, lymphokines and the like. Immune responses may be measured both in in vitro and in various cellular or animal systems.
  • the term “improving” or “enhancing” with respect to an immune response means increasing the magnitude, breadth and/or duration of the immune response.
  • Immunity refers to natural or acquired resistance provided by the immune system to a specific disease. Immunity may be partial or complete, specific or nonspecific, long-lasting or temporary.
  • Immuno effector cells refers to cells capable of binding an antigen and which mediate an immune response. These cells include, but are not limited to, T cells, B cells, monocytes, macrophages, dendritic cells, NK cells and cytotoxic T lymphocytes (CTLs).
  • Nucleic acid refers to single-stranded or double-stranded deoxyribonucleotide or ribonucleotide polymers, or chimeras or analogues thereof.
  • Protein Protein
  • polypeptide and “peptide” are used interchangeably, unless otherwise indicated, to refer to a polymeric form of amino acids.
  • Principal or “subject” refers to a vertebrate, preferably a mammal, more preferably a human.
  • Recombinant indicates that the material (e.g., a nucleic acid or protein) has been artificially or synthetically (non-naturally) altered by human intervention. The alteration can be performed on the material within, or removed from, its natural environment or state. Specifically, when referring to a virus, e.g., an influenza virus, the virus is recombinant when it is produced by the expression of a recombinant nucleic acid.
  • Vaccine refers to a pharmaceutical composition comprising at least one immunologically active component that induces an immunological response in an animal and possibly but not necessarily one or more additional components that enhance the immunological activity of said active component. A vaccine may additionally comprise further components typical to pharmaceutical compositions.
  • the immunologically active component of a vaccine may comprise complete virus particles in either their original form or as attenuated particles in a "modified live vaccine” (MLV) or particles inactivated by appropriate methods in a “killed vaccine” (KV).
  • MMV modified live vaccine
  • KV killed vaccine
  • vaccine and “vaccine composition” are used interchangeably in the present invention.
  • Variant refers to a sequence that is naturally found in a subject or a virus.
  • human genes often contain single nucleotide polymorphisms that are present in certain individuals within a population. Viruses often acquire
  • Vector refers to the means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components.
  • Vectors include plasmids, viruses, bacteriophage, pro-viruses, phagemids, transposons, and artificial chromosomes, and the like, that replicate autonomously or can integrate into a chromosome of a host cell.
  • a vector can also be a naked RNA
  • vector encompasses vectors capable of promoting expression, as well as replication, of a nucleic acid incorporated therein.
  • the nucleic acid to be expressed is "operably linked" to a promoter and/or enhancer, and is subject to transcription regulatory control by the promoter and/or enhancer.
  • Therapeutic vaccine is a vaccine designed to boost the immune response to an antigen in a person already exposed to the antigen.
  • “Therapeutically effective amount” refers to an amount sufficient to prevent, correct and/or normalize an abnormal physiological response
  • HIV human immunodeficiency virus
  • Clade A viruses are genetically the most divergent and were the most common HIV-1 subtype in Africa early in the epidemic. With the rapid spread of HIV-1 to southern Africa during the mid to late 1990s, clade C viruses have become the dominant subtype and now account for 48% of HIV-1 infections worldwide. Clade B viruses, the most intensively studied HIV-1 subtype, remain the most prevalent isolates in Europe and North America. Gag
  • Retroviral Gag proteins are generally synthesized as polyprotein precursors; the HIV-1 Gag precursor has been named, based on its apparent molecular mass, Pr55 Gag .
  • the mRNA for Pr55 Gag is the unspliced 9.2-kb transcript that requires Rev for its expression in the cytoplasm.
  • PR viral protease
  • MA is localized immediately inside the lipid bilayer of the viral envelope
  • CA forms the outer portion of the cone-shaped core structure in the center of the particle
  • NC is present in the core in a ribonucleoprotein complex with the viral RNA genome.
  • the HIV Pr55 Gag precursor oligomerizes following its translation and is targeted to the plasma membrane, where particles of sufficient size and density to be visible by EM are assembled. Formation of virus-like particles by Pr55 Gag is a self-assembly process, with critical Gag-Gag interactions taking place between multiple domains along the Gag precursor. The assembly of virus-like particles does not require the participation of genomic RNA (although the presence of nucleic acid appears to be essential), pol-encoded enzymes, or Env
  • the pol gene Downstream of gag lies the most highly conserved region of the HIV genome, the pol gene, which encodes three enzymes: PR, RT, and IN. RT and IN are required, respectively, for reverse transcription of the viral RNA genome to a double-stranded DNA copy, and for the integration of the viral DNA into the host cell chromosome.
  • PR plays a critical role late in the life cycle by mediating the production of mature, infectious virions.
  • the pol gene products are derived by enzymatic cleavage of a 160-kd Gag-Pol fusion protein, referred to as Pr160 Gag" Po1 . This fusion protein is produced by ribosomal frame-shifting during translation of Pr55 Gag .
  • the frame-shifting mechanism for Gag-Pol expression also utilized by many other retroviruses, ensures that the pol-derived proteins are expressed at a low level, approximately 5% to 10% that of Gag.
  • the N- terminus of Pr160 Gag"Po1 is myristylated and targeted to the plasma membrane.
  • retroviral Gag proteins are initially synthesized as polyprotein precursors that are cleaved to generate smaller products. Subsequent studies demonstrated that the processing function is provided by a viral rather than a cellular enzyme, and that proteolytic digestion of the Gag and Gag-Pol precursors is essential for virus infectivity. Sequence analysis of retroviral PRs indicated that they are related to cellular "aspartic" proteases such as pepsin and renin. Like these cellular enzymes, retroviral PRs use two apposed Asp residues at the active site to coordinate a water molecule that catalyzes the hydrolysis of a peptide bond in the target protein.
  • retroviral PRs function as true dimers.
  • X-ray crystallographic data from HIV- 1 PR indicate that the two monomers are held together in part by a four-stranded antiparallel .beta. -sheet derived from both N- and C-terminal ends of each monomer.
  • the substrate-binding site is located within a cleft formed between the two monomers.
  • the HIV PR dimer contains flexible "flaps" that overhang the binding site and may stabilize the substrate within the cleft; the active-site Asp residues lie in the center of the dimer.
  • the primary sequences of retroviral PRs are highly divergent, yet their structures are remarkably similar.
  • Retroviral RTs have three enzymatic activities: (a) RNA-directed DNA polymerization (for minus-strand DNA synthesis), (b) RNaseH activity (for the degradation of the tRNA primer and genomic RNA present in DNA-RNA hybrid intermediates), and (c) DNA-directed DNA polymerization (for second- or plus-strand DNA synthesis).
  • the mature HIV-1 RT holoenzyme is a heterodimer of 66 and 51 kDa subunits.
  • the 51 -kDa subunit (p51 ) is derived from the 66-kDa (p66) subunit by proteolytic removal of the C-terminal 15-kd RNaseH domain of p66 by PR.
  • the crystal structure of HIV-1 RT reveals a highly asymmetric folding in which the
  • orientations of the p66 and p51 subunits differ substantially.
  • the p66 subunit can be visualized as a right hand, with the polymerase active site within the palm, and a deep template-binding cleft formed by the palm, fingers, and thumb subdomains.
  • the polymerase domain is linked to RNaseH by the connection subdomain.
  • the active site, located in the palm, contains three critical Asp residues (1 10, 185, and 186) in close proximity, and two coordinated Mg 2+ ions. Mutation of these Asp residues abolishes RT polymerizing activity.
  • the orientation of the three active-site Asp residues is similar to that observed in other DNA polymerases (e.g., the Klenow fragment of E. coli DNA poll).
  • the p51 subunit appears to be rigid and does not form a polymerizing cleft; Asp 1 10, 185, and 186 of this subunit are buried within the molecule. Approximately 18 base pairs of the primer-template duplex lie in the nucleic acid binding cleft, stretching from the polymerase active site to the RNaseH domain.
  • retrovirus replication is the insertion of a DNA copy of the viral genome into the host cell chromosome following reverse transcription.
  • the integrated viral DNA (the provirus) serves as the template for the synthesis of viral RNAs and is maintained as part of the host cell genome for the lifetime of the infected cell.
  • Retroviral mutants deficient in the ability to integrate generally fail to establish a productive infection.
  • Retroviral IN proteins are composed of three structurally and functionally distinct domains: an N-terminal, zinc-finger-containing domain, a core domain, and a relatively nonconserved C-terminal domain. Because of its low solubility, it has not yet been possible to crystallize the entire 288-amino-acid HIV-1 IN protein. However, the structure of all three domains has been solved independently by x- ray crystallography or NMR methods. The crystal structure of the core domain of the avian sarcoma virus IN has also been determined.
  • the N-terminal domain (residues 1 to 55), whose structure was solved by NMR spectroscopy, is composed of four helices with a zinc coordinated by amino acids His-12, His-16, Cys-40, and Cys-43.
  • the structure of the N-terminal domain is reminiscent of helical DNA binding proteins that contain a so-called helix-turn-helix motif, however, in the HIV-1 structure this motif contributes to dimer formation. Initially, poor solubility hampered efforts to solve the structure of the core domain.
  • polynucleotidyl transferases in HIV-1 IN these are Asp-64, Asp-1 16 and Glu- 152, the so-called D,D-35-E motif. Mutations at these positions block HIV IN function both in vivo and in vitro.
  • the close proximity of these three amino acids in the crystal structure of both avian sarcoma virus and HIV-1 core domains supports the hypothesis that these residues play a central role in catalysis of the polynucleotidyl transfer reaction that is at the heart of the integration process.
  • the C-terminal domain whose structure has been solved by NMR methods, adopts a five-stranded P-barrel folding topology reminiscent of a Src homology 3 (SH3) domain.
  • SH3 Src homology 3
  • the HIV Env glycoproteins play a major role in the virus life cycle. They contain the determinants that interact with the CD4 receptor and coreceptor, and they catalyze the fusion reaction between the lipid bilayer of the viral envelope and the host cell plasma membrane. In addition, the HIV Env glycoproteins contain epitopes that elicit immune responses that are important from both diagnostic and vaccine development perspectives.
  • the HIV Env glycoprotein is synthesized from the singly spliced 4.3-kb Vpu/Env bicistronic mRNA; translation occurs on ribosomes associated with the rough endoplasmic reticulum (ER).
  • the 160-kDa polyprotein precursor (gp160) is an integral membrane protein that is anchored to cell membranes by a hydrophobic stop-transfer signal in the domain destined to be the mature TM Env
  • glycoprotein gp41 .
  • the gp160 is cotranslationally glycosylated, forms disulfide bonds, and undergoes oligomerization in the ER. The predominant oligomeric form appears to be a trimer, although dimers and tetramers are also observed.
  • the gp160 is transported to the Golgi, where, like other retroviral envelope precursor proteins, it is proteolytically cleaved by cellular enzymes to the mature SU glycoprotein gp120 and TM glycoprotein gp41 .
  • the cellular enzyme responsible for cleavage of retroviral Env precursors following a highly conserved Lys/Arg-X-Lys/Arg-Arg motif is furin or a furin-like protease, although other enzymes may also catalyze gp160 processing. Cleavage of gp160 is required for Env-induced fusion activity and virus infectivity. Subsequent to gp160 cleavage, gp120 and gp41 form a noncovalent association that is critical for transport of the Env complex from the Golgi to the cell surface. The gp120-gp41 interaction is fairly weak, and a substantial amount of gp120 is shed from the surface of Env- expressing cells.
  • a primary function of viral Env glycoproteins is to promote a membrane fusion reaction between the lipid bilayers of the viral envelope and host cell
  • the methods of the present invention involves a prime-boost strategy, wherein the immune response to a priming composition comprising an antigen is boosted by the administration, one or more times, of a boosting composition.
  • vaccinia virus vectors for example, a replication-deficient strain such as modified vaccinia Ankara (MVA) or NYVAC (Tartaglia et al. 1992 Virology 1 18:217-232); an avipox vector such as fowlpox or canarypox, e.g. the strain known as ALVAC (Paoletti et al. 1994 Dev Biol Stand 82:65-69); adenovirus vectors; vesicular stomatitis virus vectors; or alphavirus vectors.
  • vaccinia virus vectors for example, a replication-deficient strain such as modified vaccinia Ankara (MVA) or NYVAC (Tartaglia et al. 1992 Virology 1 18:217-232); an avipox vector such as fowlpox or canarypox, e.g. the strain known as ALVAC (Paoletti et al. 1994 Dev
  • Vectors useful as a priming composition in the method of the present invention have been engineered to one or more antigens.
  • the antigens encoded by the vector are typically proteinaceous.
  • serial arrays of amino acid residues, linked through peptide bonds can be obtained by using recombinant techniques to express DNA (e.g., as was done for the vaccine inserts described and exemplified herein), purified from a natural source, or synthesized.
  • the priming composition comprises one HIV antigen.
  • the priming composition is a vector that encodes one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) HIV antigens selected from the group of: Gag, Pol, Env (e.g., gp160, gp120, and gp41 ), Tat, Rev, Vpu, Nef, Vif, and Vpr.
  • the priming composition is plasmid DNA engineered to express one or more HIV antigens.
  • the priming composition is a viral vector modified to encode express one or more HIV antigens, such as modified
  • MVA vaccinia virus
  • Antigens are provided as non-infective virus like particles (VLPs) which present antigens for immune system recognition in a form similar to native virus.
  • the priming composition may include nucleic acids representing one or more genes found in one or more HIV clades or any fragments or derivatives thereof that, when expressed, elicit an immune response against the virus (or viral clade) from which the nucleic acid was derived or obtained.
  • the nucleic acids may be purified from HIV or they may have been previously cloned, subcloned, or synthesized and, in any event, can be the same as or different from a naturally- occurring nucleic acid sequence.
  • the priming composition also contains genes encoding or more viral (e.g., HIV) enzymes, wherein those genes are modified to prevent enzymatic activity.
  • viral e.g., HIV
  • the gene sequence is be modified by deleting or replacing one or more nucleic acids, and those deletions or substitutions can result in a gene product that has less enzymatic activity than its wild type counterpart (e.g., less integrase activity, less reverse transcriptase (RT) activity, or less protease activity).
  • the priming composition is a plasmid DNA (plasmid vector) encoding: (a) a Gag protein in which one or both zinc fingers have been inactivated; (b) a Pol protein in which (i) the integrase activity has been inhibited by deletion of some or all of the pol sequence, (ii) the polymerase, strand transfer, and/or RNase H activity of reverse transcriptase has been inhibited by one or more point mutations within the pol sequence and (iii) the proteolytic activity of the protease has been inhibited by one or more point mutations; and (c) Env, Tat, Rev, and Vpu, with or without mutations.
  • plasmid vector encoding: (a) a Gag protein in which one or both zinc fingers have been inactivated; (b) a Pol protein in which (i) the integrase activity has been inhibited by deletion of some or all of the pol sequence, (ii) the polymerase, strand
  • plasmids encoding the antigens just described can be combined with (e.g., mixed with) other plasmids that encode antigens obtained from, or derived from, a different HIV clade (or subtype or recombinant form thereof).
  • the inserts per se are also within the scope of the disclosure.
  • the inserts may contain sequences that encode one or more conserved protein sequences and/or may contain one or more designer sequences (e.g., mosaic sequences that contain a sequence from one or more HIV clades).
  • vectors suitable for use as priming compositions in the method of the present invention include plasmids encoding (a) a Gag protein (e.g., a Gag protein in which one or both of the zinc fingers have been inactivated); (b) a Pol protein (e.g., a Pol protein in which integrase, RT, and/or protease activities have been inhibited); (iii) a Vpu protein (which may be encoded by a sequence having a mutant start codon); and (iii) Env, Tat, and/or Rev proteins (in a wild type or mutant form).
  • a Gag protein e.g., a Gag protein in which one or both of the zinc fingers have been inactivated
  • a Pol protein e.g., a Pol protein in which integrase, RT, and/or protease activities have been inhibited
  • Vpu protein which may be encoded by a sequence having a mutant start codon
  • plasmids encoding the antigens just described can be combined with (e.g., mixed with) other plasmids that encode antigens obtained from, or derived from, a different HIV clade (or subtype or recombinant form thereof).
  • the priming composition is a vector encoding: (a) a Gag protein in which one or more of the zinc fingers has been inactivated to limit the packaging of viral RNA; (b) a Pol protein in which (i) the integrase activity has been inhibited by deletion of some or all of the pol sequence and (ii) the polymerase, strand transfer and/or RNase H activity of reverse transcriptase has been inhibited by one or more point mutations within the pol sequence; and (c) Env, Tat, Rev, and Vpu, with or without mutations.
  • vectors suitable for use as the priming composition in the method of the present invention include plasmids encoding (a) a Gag protein (e.g., a Gag protein in which one or both of the zinc fingers have been inactivated); (b) a Pol protein (e.g., a Pol protein in which integrase, RT, and/or protease activities have been inhibited; a Vpu protein (which may be encoded by a sequence having a mutant start codon); and (c) Env, Tat, and/or Rev proteins (in a wild type or mutant form).
  • a Gag protein e.g., a Gag protein in which one or both of the zinc fingers have been inactivated
  • a Pol protein e.g., a Pol protein in which integrase, RT, and/or protease activities have been inhibited
  • Vpu protein which may be encoded by a sequence having a mutant start codon
  • MVA 65A/G examples of such recombinant MVA vectors possessing safety mutations include MVA 65A/G, MVA 62B and MVA 71 C described in WO2006/026667.
  • the priming composition is a vector which comprises an insert encoding one or more antigens that elicit an immune response against an HIV of a subtype or recombinant form, said insert encoding:
  • the integrase activity is inhibited by the deletion of integrase
  • the reverse transcriptase activity is inhibited by amino acid changes D185N, W266T and E478Q, and
  • the protease activity is inhibited by amino acid change selected from L90M, G48V, R25N, or D25A.
  • the HIV is clade A/G, B or C.
  • the priming composition is a vector which comprises an insert encoding one or more clade A/G HIV antigens that elicit an immune response against a clade A G HIV, where the insert encodes:
  • amino acid change selected from L90M, G48V or R25N, and
  • the priming composition is a vector which comprises an insert encoding one or more clade B HIV antigens that elicit an immune response against a clade B HIV, where the insert encodes:
  • the integrase activity is inhibited by the deletion of integrase
  • the reverse transcriptase activity is inhibited by amino acid changes D185N, W266T and E478Q, and
  • the priming composition is vector which comprises an insert encoding one or more clade C HIV antigens that elicit an immune response against a clade C HIV, where the insert encodes:
  • the plasmid vectors of the present disclosure may be referred to herein as, inter alia, expression vectors, expression constructs, plasmid vectors or, simply, as plasmids, regardless of whether or not they include a vaccine insert (i.e., a nucleic acid sequence that encodes an antigen or immunogen). Similar variations of the term “viral vector” may appear as well (e.g., a "poxvirus vector,” a "vaccinia vector,” a "modified vaccinia Ankara vector,” or an "MVA vector”). The viral vector may or may not include a vaccine insert.
  • the recombinant MVA vaccinia virus can be prepared in any suitable manner.
  • the recombinant MVA vaccine virus is prepared as follows: A DNA-construct which contains a DNA-sequence which codes for a foreign polypeptide flanked by MVA DNA sequences adjacent to a naturally occurring deletion, e.g. deletion III, or other non-essential sites, within the MVA genome, is introduced into cells infected with MVA, to allow homologous recombination. Once the DNA-construct has been introduced into the eukaryotic cell and the foreign DNA has recombined with the viral DNA, it is possible to isolate the desired recombinant vaccinia virus in a manner known per se, preferably with the aid of a marker.
  • the DNA-construct to be inserted can be linear or circular.
  • a plasmid or polymerase chain reaction product is preferred.
  • the DNA-construct contains sequences flanking the left and the right side of a naturally occurring deletion, e.g. deletion III, within the MVA genome.
  • the foreign DNA sequence is inserted between the sequences flanking the naturally occurring deletion.
  • regulatory sequences which are required for the transcription of the gene, to be present on the DNA.
  • promoters are known to those skilled in the art, and include for example those of the vaccinia 1 1 kDa gene as are described in EP-A-198,328, and those of the 7.5 kDa gene (EP-A-1 10,385).
  • the DNA- construct can be introduced into the MVA infected cells by transfection, for example by means of calcium phosphate precipitation (Graham et al. 1973 Virol 52:456-467; Wigler et al. 1979 Cell 16:777-785), by means of electroporation (Neumann et al. 1982 EMBO J. 1 :841 -845), by microinjection (Graessmann et al. 1983 Meth Enzymol 101 :482-492), by means of liposomes (Straubinger et al. 1983 Meth Enzymol 101 :512-527), by means of spheroplasts (Schaffher 1980 PNAS USA 77:2163-2167) or by other methods known to those skilled in the art.
  • transfection for example by means of calcium phosphate precipitation (Graham et al. 1973 Virol 52:456-467; Wigler et al. 1979 Cell 16:777-785), by means of electroporation (Neumann et al. 1982 EMBO J
  • regulatory sequences for expression of the encoded antigen will include a natural, modified or synthetic poxvirus promoter.
  • promoter is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA).
  • Operably linked means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
  • DNA operably linked to a promoter is "under transcriptional initiation regulation” of the promoter.
  • Other regulatory sequences including terminator fragments, polyadenylation sequences, marker genes and other sequences may be included as appropriate, in accordance with the knowledge and practice of the ordinary person skilled in the art: see, for example, Moss, B. (2001 ).
  • Poxyiridae the viruses and their replication.
  • the method of the present invention involves a prime-boost strategy in which a boosting composition is administered, one or more times, to boost the immune response to the primary immunization (i.e., a priming composition).
  • a boosting composition is administered, one or more times, to boost the immune response to the primary immunization (i.e., a priming composition).
  • a priming composition Any suitable boosting composition may be used, including but not limited to, viral vectors such as modified vaccinia virus.
  • the boosting composition comprises one or more genes encoding one or more antigens, and in a particular embodiment, one or more genes encoding antigens common to the priming compositions.
  • Vaccinia viruses have been used to engineer viral vectors for recombinant gene expression and as recombinant live vaccines (Mackett et al., Proc. Natl. Acad. Sci. U.S.A. 79:7415-7419; Smith et al., Biotech. Genet. Engin. Rev. 2:383-407, 1984).
  • DNA sequences that encode selected HIV antigens can be introduced into the genomes of vaccinia viruses. If the gene is integrated at a site in the viral DNA that is non-essential for the life cycle of the virus, it is possible for the newly produced recombinant vaccinia virus to be infectious (i.e., able to infect foreign cells) and to express the integrated DNA sequences.
  • the modified vaccinia Ankara (MVA) virus was generated by long-term serial passages of the Ankara strain of vaccinia virus on chicken embryo fibroblasts (CVA; see Mayr et al., Infection 3:6- 14, 1975).
  • the MVA virus is publicly available from the American Type Culture Collection (ATCC; No. VR-1508; Manassas, Va.). The desirable properties of the MVA strain have been demonstrated in clinical trials (Mayr et al., Gottbl.
  • the boosting composition is a recombinant MVA viral vector encoding one or more HIV antigens.
  • the antigens encoded by rMVA are typically proteinaceous.
  • serial arrays of amino acid residues, linked through peptide bonds can be obtained by using recombinant techniques to express DNA (e.g., as was done for the vaccine inserts described and exemplified herein), purified from a natural source, or synthesized.
  • the boosting composition is a recombinant MVA viral vector encoding one or more HIV antigens common to the priming composition.
  • the boosting composition is a recombination MVA vector that encodes the same HIV antigens as the priming composition, or variants thereof.
  • the vector used as the priming composition encodes one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) HIV antigens selected from the group of: Gag, Pol, Env (e.g., gp160, gp120, and gp41 ), Tat, Rev, Vpu, Nef, Vif, and Vpr.
  • Antigens are provided as non-infective virus like particles (VLPs) which present antigens for immune system recognition in a form similar to native virus.
  • VLPs virus like particles
  • the boosting composition can include nucleic acids representing one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) genes found in one or more HIV clades or any fragments or derivatives thereof that, when expressed, elicit an immune response against the virus (or viral clade) from which the nucleic acid was derived or obtained.
  • the nucleic acids may be purified from HIV or they may have been previously cloned, subcloned, or synthesized and, in any event, can be the same as or different from a naturally-occurring nucleic acid sequence.
  • clade of the inserts is designated by the last letter.
  • clade B inserts are designated for example MVA62B
  • clade AG inserts are designated for example MVA65AG
  • clade C inserts are designated for example MVA70C and MVA71 C.
  • vaccinia viruses have been used to engineer viral vectors for recombinant gene expression and for the potential use as recombinant live vaccines (Mackett, M. et al 1982 PNAS USA 79:7415-7419; Smith, G. L. et al. 1984 Biotech Genet Engin Rev 2:383-407).
  • This entails DNA sequences (genes) which code for foreign, antigens being introduced, with the aid of DNA
  • vaccinia viruses into the genome of the vaccinia viruses. If the gene is integrated at a site in the viral DNA which is non-essential for the life cycle of the virus, it is possible for the newly produced recombinant vaccinia virus to be infectious, that is to say able to infect foreign cells and thus to express the integrated DNA sequence (EP Patent Applications No. 83,286 and No. 1 10,385).
  • the recombinant vaccinia viruses prepared in this way can be used, on the one hand, as live vaccines for the prophylaxis of infectious diseases, on the other hand, for the preparation of heterologous proteins in eukaryotic cells.
  • modified vaccinia Ankara has been generated by long-term serial passages of the Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr, A. et al. 1975 Infection 3:6-14; Swiss Patent No. 568,392).
  • the MVA virus is publicly available from American Type Culture Collection as ATCC No.: VR-1508.
  • MVA is distinguished by its great attenuation, that is to say by diminished virulence and ability to replicate in primate cells while maintaining good immunogenicity.
  • the MVA virus has been analyzed to determine alterations in the genome relative to the parental CVA strain. Six major deletions of genomic DNA (deletion I, II, III, IV, V, and VI) totaling 31 ,000 base pairs have been identified (Meyer, H. et al. 1991 J Gen Virol 72:1031 -1038). The resulting MVA virus became severely host cell restricted to avian cells.
  • MVA is characterized by its extreme attenuation. When tested in a variety of animal models, MVA was proven to be avirulent even in
  • MVA replication in human cells was found to be blocked late in infection preventing the assembly to mature infectious virions. Nevertheless, MVA was able to express viral and recombinant genes at high levels even in non- permissive cells and was proposed to serve as an efficient and exceptionally safe gene expression vector (Sutter, G. and Moss, B.
  • novel vaccinia vector vaccines were established on the basis of MVA having foreign DNA sequences inserted at the site of deletion III within the MVA genome (Sutter, G. et al. 1994 Vaccine 12:1032-1040).
  • the boosting composition is a recombinant MVA viral vector encoding: (a) a Gag protein in which one or both zinc fingers have been inactivated; (b) a Pol protein in which (i) the integrase activity has been inhibited by deletion of some or all of the pol sequence, (ii) the polymerase, strand transfer, and/or RNase H activity of reverse transcriptase has been inhibited by one or more point mutations within the pol sequence and (iii) the proteolytic activity of the protease has been inhibited by one or more point mutations; and (c) Env, Tat, Rev, and Vpu, with or without mutations.
  • the MVA viral vector encoding the antigens just described can be combined with (e.g., mixed with) other vectors that encode antigens obtained from, or derived from, a different HIV clade (or subtype or recombinant form thereof).
  • the inserts per se are also within the scope of the disclosure.
  • the inserts may contain sequences that encode one or more conserved protein sequences and/or may contain one or more designer sequences (e.g., mosaic sequences that contain a sequence from one or more HIV clades).
  • the boosting composition is a recombinant MVA viral vector encoding (a) a Gag protein (e.g., a Gag protein in which one or both of the zinc fingers have been inactivated); (b) a Pol protein (e.g., a Pol protein in which integrase, RT, and/or protease activities have been inhibited); (c) a Vpu protein (which may be encoded by a sequence having a mutant start codon); (d) and Env, Tat, and/or Rev proteins (in a wild type or mutant form).
  • a Gag protein e.g., a Gag protein in which one or both of the zinc fingers have been inactivated
  • a Pol protein e.g., a Pol protein in which integrase, RT, and/or protease activities have been inhibited
  • Vpu protein which may be encoded by a sequence having a mutant start codon
  • Env, Tat, and/or Rev proteins
  • vectors encoding the antigens just described can be combined with (e.g., mixed with) other vectors that encode antigens obtained from, or derived from, a different HIV clade (or subtype or recombinant form thereof).
  • the boosting composition is a recombinant MVA viral vector encoding: (a) a Gag protein in which one or more of the zinc fingers has been inactivated to limit the packaging of viral RNA; (b) a Pol protein in which (i) the integrase activity has been inhibited by deletion of some or all of the pol sequence and (ii) the polymerase, strand transfer and/or RNase H activity of reverse transcriptase has been inhibited by one or more point mutations within the pol sequence; and (c) Env, Tat, Rev, and Vpu, with or without mutations.
  • boosting compositions suitable for use in the present invention include recombinant MVA viral vectors encoding (a) a Gag protein (e.g., a Gag protein in which one or both of the zinc fingers have been inactivated); (b) a Pol protein (e.g., a Pol protein in which integrase, RT, and/or protease activities have been inhibited; (c) a Vpu protein (which may be encoded by a sequence having a mutant start codon); and (d) Env, Tat, and/or Rev proteins (in a wild type or mutant form).
  • a Gag protein e.g., a Gag protein in which one or both of the zinc fingers have been inactivated
  • a Pol protein e.g., a Pol protein in which integrase, RT, and/or protease activities have been inhibited
  • Vpu protein which may be encoded by a sequence having a mutant start codon
  • MVA 65A/G examples of such recombinant MVA vectors possessing safety mutations include MVA 65A/G, MVA 62B and MVA 71 C described in WO2006/026667.
  • the boosting composition is an MVA viral vector which comprises an insert encoding one or more antigens that elicit an immune response against an HIV of a subtype or recombinant form, said insert encoding (a) a HIV-1 Gag protein in which both zinc fingers have been inactivated by amino acid changes C392S, C395S, C413S and C416S; and
  • the protease activity is inhibited by amino acid change selected from L90M, G48V, R25N, or D25A.
  • the HIV is clade A/G, B or C.
  • the vector comprises an insert encoding one or more clade A G HIV antigens that elicit an immune response against a clade A G HIV, where the insert encodes:
  • HIV-1 Gag protein in which both zinc fingers have been inactivated by amino acid changes C390S, C393S, C41 1 S and C414S, a HIV-1 Pol protein in which
  • the protease activity is inhibited by amino acid change selected from L90M, G48V or R25N, and clade A/G Vpu, Env, Tat, and Rev.
  • the vector comprises an insert encoding one or more clade B HIV antigens that elicit an immune response against a clade B HIV, where the insert encodes:
  • the vector comprises an insert encoding one or more clade C HIV antigens that elicit an immune response against a clade C HIV, where the insert encodes:
  • the integrase activity is inhibited by the deletion of integrase
  • the reverse transcriptase activity is inhibited by amino acid changes D185N, W266T and E478Q
  • the protease activity is inhibited by amino acid change D25A
  • the present invention provides immunization methods as well as methods of improving the immune response to an antigen(s) and more particularly, to immunization with an HIV antigen(s).
  • the present invention provides a method of immunization that employs a prime-boost strategy, in which the immune response to administration of a priming composition comprising one or more antigens (e.g., plasmid DNA, viral vector or an infectious agent) is then boosted by the administration, one or more times, of a boosting composition.
  • a priming composition comprising one or more antigens (e.g., plasmid DNA, viral vector or an infectious agent) is then boosted by the administration, one or more times, of a boosting composition.
  • the boosting composition may be the same or different than the priming composition and each boosting composition administered (if more than one) may be the same or different. Any of the priming and boosting compositions described above are suitable for use with the methods described here.
  • a recombination MVA vector is administered as a boosting composition for administration schedules that use a plasmid DNA vaccine.
  • Such protocols include priming with a DNA vaccine (D) and boosting with an MVA vector (M).
  • D DNA vaccine
  • M MVA vector
  • Such administration protocols can use multiple permutations of DNA and MVA administration to achieve appropriate immune responses.
  • Such protocols include but are not limited to DMM, DMMM, DDMM, DDMMM,
  • MVA vectors are used for both priming and boosting purposes.
  • Such protocols include but are not limited to MM, MMM, and MMMM.
  • one, two, three, four, five, six, seven, eight, nine, ten or more than ten MVA boosts are administered.
  • Vectors can be administered alone (i.e., a plasmid can be administered on one or several occasions with or without an alternative type of vaccine formulation (e.g., with or without administration of protein or another type of vector, such as a viral vector)) and, optionally, with an adjuvant or in conjunction with (e.g., prior to) an alternative booster immunization (e.g., a live-vectored vaccine such as a recombinant modified vaccinia Ankara vector (MVA)) comprising an insert that may be distinct from that of the "prime" portion of the immunization or may be a related vaccine insert(s).
  • an alternative booster immunization e.g., a live-vectored vaccine such as a recombinant modified vaccinia Ankara vector (MVA)
  • a live-vectored vaccine such as a recombinant modified vaccinia Ankara vector (MVA)
  • MVA modified vaccinia Ankara
  • the antigen(s) encoded by the respective priming and boosting compositions need not be identical, but should share at least one CD8 + T cell epitope.
  • the antigen may correspond to a complete antigen, or a fragment thereof.
  • Peptide epitopes or artificial strings of epitopes may be employed, more efficiently cutting out unnecessary protein sequence in the antigen and encoding sequence in the vector or vectors.
  • One or more additional epitopes may be included, for instance epitopes which are recognized by T helper cells, especially epitopes recognized in individuals of different HLA types.
  • the boosting composition is a vector that can contain at least some of the sequence contained with the plasmid administered as the "prime" portion of the inoculation protocol (e.g., sequences encoding one or more, and possibly all, of the same antigens).
  • a live-vectored vaccine e.g., an MVA vector
  • a plasmid-based (or "DNA") vaccine e.g., an MVA vector
  • the disclosure features compositions having only viral vectors (with, optionally, one or more (e.g., two, three, four, five, or six) of any of the inserts described here, or inserts having their features) and methods of administering them.
  • the viral-based regimens e.g., "MVA only” or “MVA-MVA” vaccine regimens
  • the MVAs in any vaccine can be in any proportion desired.
  • the immunization protocol employs only plasmid-based immunogens, only viral-carried immunogens, or a
  • the method of the present invention involves administering the compositions of the disclosure to a subject who has not yet become infected with a pathogen (thus, the terms "subject” or "patient,” as used herein
  • the method of the present invention elicits an immune response that decreases either the risk or rate of infection in a patient (e.g., by at least
  • the method of the present invention involves
  • administering the compositions described herein can be administered as therapeutic vaccines (e.g., to a subject or patient exposed to or already infected with an HIV of any clade, including those presently known as clades A-L or mutant or recombinant forms thereof).
  • the method involves administering the compositions described herein in an experimental context, for instance in investigation of mechanisms of immune responses to an antigen of interest, e.g. protection against HIV or AIDS.
  • the in which the immune response to administration of a priming composition comprising one or more HIV antigens is then boosted by the administration, one or more times, of a boosting composition comprising one or more HIV antigens.
  • the boosting composition may be the same or different than the priming composition and each boosting composition administered (if more than one) may be the same or different.
  • a recombination MVA vector comprising one or more HIV antigens is administered as a boosting composition for administration schedules that use a plasmid DNA vaccine comprising one or more HIV antigens.
  • HIV immunization protocols include but are not limited to DMM, DMMM, DDMM, DDMMM, DDDMM, and DDDMMM.
  • a recombinant MVA vectors comprising one or more HIV antigens is administered for both priming and boosting purposes.
  • Such protocols include but are not limited to MM, MMM, and MMMM.
  • the present invention is an immunization method comprising (i) administering a priming composition comprising a DNA plasmid comprising one or more genes encoding an HIV antigen; (ii) administering a first dose of a boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding an HIV antigen; and (iii) administering a second dose of a boosting composition between about 12 and 20 weeks after the first dose, more particularly between about 14 and about 18 weeks after the first dose, even more particularly, about 16 weeks after the first dose.
  • the HIV antigens are the same in step (i)-(iii).
  • the method further comprises one or more additional steps, including, for example, the administration of one or more additional doses of the priming composition or a different priming composition (i.e., a second priming
  • boosting composition and/or one or more additional doses of the boosting composition or a different boosting composition (i.e., a second boosting composition).
  • the present invention is an immunization method comprising (i) administering a priming composition comprising a DNA plasmid comprising one or more genes encoding an HIV antigen; (ii) administering a first dose of a boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding an HIV antigen; and (iii) administering a second dose of a boosting composition; and (iii) administering a third dose of the boosting composition between about 12 and 20 weeks after the second dose, more particularly between about 14 and about 18 weeks after the second dose, even more particularly, about 16 weeks after the second dose.
  • the HIV antigens are the same in step (i)-(iii).
  • the method further comprises one or more additional steps, including, for example, the administration of one or more additional doses of the priming composition or a different priming composition (i.e., a second priming
  • boosting composition and/or one or more additional doses of the boosting composition or a different boosting composition (i.e., a second boosting composition).
  • the present invention is an immunization method comprising (i) administering a priming composition comprising a DNA plasmid comprising one or more genes encoding an HIV antigen; (ii) administering a first boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding an HIV antigen; and (iii) administering a second boosting compositing comprising an adenovirus vector comprising one or more genes encoding an HIV antigen between about 12 and 20 weeks after administering the first boosting composition, more particularly between about 14 and about 18 weeks after administering the first boosting composition, even more particularly, about 16 weeks after administering the first boosting composition.
  • the HIV antigens are the same in step (i)-(iii).
  • the method further comprises one or more additional steps, including, for example, the administration of one or more additional doses of the priming composition or a different priming composition (i.e., a second priming
  • composition and/or the administration of one or more additional doses of the first and/or second boosting composition or a different boosting composition (i.e., a third boosting composition).
  • the present invention is an immunization method comprising (i) administering a priming composition comprising a DNA plasmid comprising one or more genes encoding an HIV antigen; (ii) administering a first boosting composition comprising n adenovirus vector comprising one or more genes encoding an HIV antigen; and (iii) administering a second boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding an HIV antigen between about 12 and 20 weeks after the first boosting composition, more particularly between about 14 and about 18 weeks after administering the first boosting composition, even more particularly, about 16 weeks after administering the first boosting composition.
  • the HIV antigens are the same in step (i)-(iii).
  • the method further comprises one or more additional steps, including, for example, the administration of one or more additional doses of the priming composition or a different priming composition (i.e., a second priming composition) and/or the administration of one or more additional doses of the first and/or second boosting composition or a different boosting composition (i.e., a third boosting
  • the present invention is an immunization method comprising (i) administering a priming composition comprising a viral vector (e.g., a recombinant MVA viral vector) comprising one or more genes encoding an HIV antigen; (ii) administering a first dose of a boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding an HIV antigen; and (iii) administering a second dose of a boosting composition between about 12 and 20 weeks after the first dose, more particularly between about 14 and about 18 weeks after the first dose, even more particularly, about 16 weeks after the first dose.
  • the HIV antigens are the same in step (i)-(iii).
  • the method further comprises one or more additional steps, including, for example, the administration of one or more additional doses of the priming composition or a different priming composition (i.e., a second priming composition) and/or one or more additional doses of the boosting composition or a different boosting composition (i.e., a second boosting composition).
  • additional steps including, for example, the administration of one or more additional doses of the priming composition or a different priming composition (i.e., a second priming composition) and/or one or more additional doses of the boosting composition or a different boosting composition (i.e., a second boosting composition).
  • the present invention is an immunization method comprising (i) administering a priming composition comprising a viral vector (e.g., a recombinant MVA viral vector) comprising one or more genes encoding an HIV antigen; (ii) administering a first dose of a boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding an HIV antigen; and (iii) administering a second dose of a boosting composition; and (iii) administering a third dose of the boosting composition between about 12 and 20 weeks after the first dose, more particularly between about 14 and about 18 weeks after the first dose, even more particularly, about 16 weeks after the second dose.
  • a priming composition comprising a viral vector (e.g., a recombinant MVA viral vector) comprising one or more genes encoding an HIV antigen
  • administering a first dose of a boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more
  • the HIV antigens are the same in step (i)-(iii).
  • the method further comprises one or more additional steps, including, for example, the administration of one or more additional doses of the priming composition or a different priming composition (i.e., a second priming composition) and/or one or more additional doses of the boosting composition or a different boosting composition (i.e., a second boosting composition).
  • the present invention is a method of improving B cell antibody response comprising (i) administering a priming composition comprising a viral vector (e.g., a recombinant MVA viral vector) comprising one or more genes encoding an HIV antigen; (ii) administering a first dose of a boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding an HIV antigen; and (iii) administering a second dose of a boosting composition; and (iii) administering a third dose of the boosting composition between about 12 and 20 weeks after the first dose, more
  • a priming composition comprising a viral vector (e.g., a recombinant MVA viral vector) comprising one or more genes encoding an HIV antigen
  • administering a first dose of a boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding an HIV antigen
  • a second dose of a boosting composition comprising
  • the method further comprises one or more additional steps, including, for example, the
  • a different priming composition i.e., a second priming composition
  • one or more additional doses of the boosting composition or a different boosting composition i.e., a second boosting composition
  • the present invention is an immunization method comprising (i) administering a priming composition comprising a viral vector (e.g., a recombinant MVA viral vector) comprising one or more genes encoding an HIV antigen; (ii) administering a first boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding an HIV antigen; and (iii) administering a second boosting composition comprising an adenovirus vector comprising one or more genes encoding an HIV antigen between about 12 and 20 weeks after administering the first boosting
  • a priming composition comprising a viral vector (e.g., a recombinant MVA viral vector) comprising one or more genes encoding an HIV antigen
  • a first boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding an HIV antigen
  • a second boosting composition comprising an adenovirus vector comprising one or more genes encoding
  • the method further comprises one or more additional steps, including, for example, the administration of one or more additional doses of the priming composition or a different priming composition (i.e., a second priming composition) and/or the administration of one or more additional doses of the first and/or second boosting composition or a different boosting composition (i.e., a third boosting
  • the present invention is an immunization method comprising (i) administering a priming composition comprising a viral vector (e.g., a recombinant MVA viral vector) comprising one or more genes encoding an HIV antigen; (ii) administering an first boosting composition comprising an adenovirus vector comprising one or more genes encoding an HIV antigen; and (iii) administering a second boosting composition comprising a modified vaccinia Ankara viral vector comprising one or more genes encoding an HIV antigen between about 12 and 20 weeks after the first boosting composition, more particularly between about 14 and about 18 weeks after administering the first boosting composition, even more particularly, about 16 weeks after administering the first boosting composition.
  • a priming composition comprising a viral vector (e.g., a recombinant MVA viral vector) comprising one or more genes encoding an HIV antigen
  • administering an first boosting composition comprising an adenovirus vector comprising one or more genes encoding an
  • the HIV antigens are the same in step (i)-(iii).
  • the method further comprises one or more additional steps, including, for example, the administration of one or more additional doses of the priming composition or a different priming composition (i.e., a second priming composition) and/or the administration of one or more additional doses of the first and/or second boosting composition or a different boosting composition (i.e., a third boosting composition).
  • the boost composition is administered about 1 to 12 months after administration of the prior dose of priming or boosting composition, preferably about 1 to 6 months, preferably about 1 to 4 months, preferably about 1 to 3 months.
  • the second boost i.e., the second dose of the boosting composition or administration of the second boosting composition
  • the improved immune response of the method of the present invention may have one or more characteristics.
  • the immune response is improved with respect to avidity, B cell response or T cell response.
  • the immune response is improved with respect to B cell memory.
  • the immune response is improved with respect to antibody titer.
  • the immune response is improved with respect to avidity.
  • the immune response is improved with respect to CD8+ T cell response.
  • the immune response is improved with respect tto CD4+ T cell response.
  • Administration is preferably in a "prophylactically effective amount" or a
  • therapeutically effective amount (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the subject.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, or in a veterinary context a veterinarian, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, 1980, Osol, A. (ed.).
  • the priming composition is a plasmid DNA
  • a boosting composition comprising a recombinant MVA viral vector at a dose of 10 6 to 10 9 infectious virus particles/injection, or more particularly, about 1 x10 8 pfu .
  • administration of priming composition, boosting composition, or both priming and boosting compositions is intradermal, intramuscular or mucosal immunization.
  • Administration of MVA vaccines may be achieved by using a needle to inject a suspension of the virus.
  • a needleless injection device to administer a virus suspension (using, e.g., Biojector.TM. needleless injector) or a resuspended freeze-dried powder containing the vaccine, providing for manufacturing individually prepared doses that do not need cold storage. This would be a great advantage for a vaccine that is needed in rural areas of Africa.
  • Components e.g., vectors to be administered in accordance with the present invention may be formulated in pharmaceutical compositions.
  • compositions may comprise a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material may depend on the route of administration, e.g. intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
  • Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included as required.
  • a slow-release formulation may be employed. Following production of MVA particles and optional formulation of such particles into compositions, the particles may be administered to an patient or subject, such as a human or other primate. Administration may be to another mammal, e.g. rodent such as mouse, rat or hamster, guinea pig, rabbit, sheep, goat, pig, horse, cow, donkey, dog or cat.
  • rodent such as mouse, rat or hamster, guinea pig, rabbit, sheep, goat, pig, horse, cow, donkey, dog or cat.
  • a composition may be administered alone or in combination with other
  • priming and boosting compositions may include an adjuvant, such as granulocyte macrophage-colony stimulating factor (GM-CSF) or encoding nucleic acid therefor.
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • IMMUNIZATIONS ENHANCES THE MAGNITUDE OF IMMUNOGEN-SPECIFIC ANTIBODY RESPONSE IN RHESUS MACAQUES
  • Rhesus macaques were immunized with a DNA MVA vaccine expressing SIV239 Gag, Pol, and Env. All animals received two immunizations with DNA on weeks 0 and 8, followed by two immunizations with MVA. One group received MVA immunizations on weeks 16 and 24 (8 weeks interval) and the other group received MVA immunizations on weeks 16 and 32 (16 weeks interval). SIV239 Env-specific binding antibody was measured at 2 weeks after the final MVA boost to test if increasing the rest period between two MVA immunizations from 8 weeks to 16 weeks influenced the antibody titers.
  • T cell responses Intracellular cytokine production was assessed as previously described with a few modifications. Briefly, 2 million PBMCs were stimulated in 200 ⁇ RPMI with 10% FBS in a 5-ml polypropylene tube. SIV-specific cytokine production was assessed as previously described with a few modifications. Briefly, 2 million PBMCs were stimulated in 200 ⁇ RPMI with 10% FBS in a 5-ml polypropylene tube. SIV-specific cytokine production was assessed as previously described with a few modifications. Briefly, 2 million PBMCs were stimulated in 200 ⁇ RPMI with 10% FBS in a 5-ml polypropylene tube. SIV-specific cytokine production was assessed as previously described with a few modifications. Briefly, 2 million PBMCs were stimulated in 200 ⁇ RPMI with 10% FBS in a 5-ml polypropylene tube. SIV-specific cytokine production was assessed as previously described with a few modifications. Briefly, 2 million PBMCs were stimulated
  • Staphylococcal enterotoxin B was used as a positive control at 1 g/ml. Stimulations were performed in presence of anti-CD28 and anti-CD49d Abs (1 pg/ml; BD Pharmingen, San Diego, CA). For all stimulations, cells were incubated at 37°C in the presence of 5% CO2 for 6 h. Brefeldin A (10 pg/ml) and Golgi-stop (1 ug/ml) were added for the last 4 h of incubation.
  • CD3+CD8+CD4- cells were considered CD8 T cells. These CD4 or CD8 T cells were then gated for cytokine-positive cells. Responses that were greater than 0.01 % of respective total CD4 or CD8 T cells were considered positive.
  • T cells were subjected to tetramer staining and typing for the presence of CD4 and CD8 Tcells. This was done using a mixture of the following Abs and Gag- CM9 tetramer conjugated to allophycocyanin: anti-human CD3-Alexa Fluor 700 (clone SP34-2; BD Pharmingen), anti human CD4-PerCP (clone L200, BD Pharmingen), anti-human CD8-AmCyan (clone SK1 ; BD Biosciences), anti- human CD28-PE-Cy7 (clone CD28.2; Beckman Coulter, Brea, CA), and anti- human CD95-Pacific blue (clone DX2; Invitrogen, Carlsbad, CA). The levels of CD4 T cells in intestinal biopsies are presented as a percentage of total CD3+ T cells.
  • SIV Env-specific binding Abs were measured with ELISA using tissue culture-produced SIV Env, captured on a Con A-coated plate as described previously (39). Briefly, ELISA plates (Costar;
  • the avidity index was calculated by dividing the dilution of the serum that gave an OD of 0.5 with NaSCN treatment by the dilution of the serum that gave an OD of 0.5 without NaSCN treatment and multiplying by 100.
  • Each assay included one plate with a standard serum with known avidity. Interassay variation in the avidity index for the standard serum was ⁇ 3 for an index of 27.
  • Measurements for total IgA, anti-SIV env IgA, or anti-SIV gag,pol IgA or IgG were done by ELISA using microtiter plates coated respectively with 100 ⁇ 0.5 g/ml goat anti-monkey IgA (Rockland, Gilbertsville, PA), 1 g/ml SIVmac251 rgp130 (ImmunoDiagnostics, Woburn, MA), or 1/400 SIVmac251 viral lysate (Advanced Biotechnologies, Columbia, MD), which lacks detectable envelope protein at this dilution. These ELISAs and the serum standards have been described previously (39).
  • SIV RNA plasma load Quantitation of SIV RNA plasma load.
  • the SIV copy number was determined using a quantitative real-time PCR as previously described. All specimens were extracted and amplified in duplicate, with the mean results reported.
  • total RNA was extracted from about 1 million cells obtained from gut biopsies and used for quantitative real-time PCR analyses.
  • results in FIG.2 show that increasing the rest period significantly increases the antibody titers to the SIV Env in the MVA immunogen.
  • results in FIG. 3 show that the long rest also significantly increases the avidity of anti-Env antibody.
  • IMMUNIZATIONS ENHANCES THE MAGNITUDE OF IMMUNOGEN-SPECIFIC ANTIBODY RESPONSE IN HUMANS A phase 1 placebo controlled clinical trial to evaluate the safety and
  • a heterologous prime-boost regimen consisting of two injections of GEO-D02 DNA vaccine (D) or GEO-D03 DNA vaccine (Dg) followed by two or three injections of modified vaccinia Ankara (MVA)/HIV62B (MVA62B).
  • GEO-D02 DNA vaccine a plasmid DNA expressing HIV-1 proteins Gag, PR, RT, Env, Tat, Rev, and Vpu in an insert also known as JS7.
  • the DNA vaccine has been vialed at a concentration of 3 mg/mL. Both the 0.3 mg and 3 mg injections will be administered as a 1 mL intramuscular (IM) injection into the deltoid.
  • IM intramuscular
  • GEO-D03 DNA vaccine (Dg): a 9.9 kb plasmid DNA expressing HIV-1 proteins Gag, PR, RT, Env, Tat, Rev, and Vpu, and human granulocyte-macrophage colony stimulating factor (GM-CSF).
  • the DNA vaccine has been vialed at a concentration of 3 img/mL Both the 0.3 mg and 3 mg injections will be
  • IM intramuscular
  • Formulation Buffer for dilution of GEO-D03 and GEO-D02 DNA Phosphate- buffered saline (PBS), EDTA (ethylenediamine tetraacetic acid), and ethanol.
  • MVA HIV62B (MVA62B) vaccine M: a highly attenuated vaccinia virus expressing HIV-1 gag, pol, and env genes from the same HIV-1 sequences present in the GEOD03 DNA vaccine.
  • the MVA62B vaccine has been vialed at 1 x108 50% tissue culture infective dose (TCID50)/ml_.
  • TCID50 tissue culture infective dose
  • a 1 x10 8 TCID50 dose will be administered as a 1 ml_ IM injection into the deltoid.
  • Placebo for GEO-D03 DNA Sodium Chloride for Injection USP, 0.9%.
  • the GEOD03 DNA placebo will be administered as a 1 ml_ IM injection into the deltoid.
  • Placebo for MVA62B Sodium Chloride for Injection USP, 0.9%.
  • the MVA62B placebo will be administered as a 1 ml_ IM injection into the deltoid.
  • Immunization protocols were evaluated to determine the effect of delaying MVA boost on immune response from 8 weeks to 16 weeks.
  • Another object was to determine the effect of adding a third MVA boost having a longer delay after the second boost (16 weeks) than between first and second boosts (8 weeks).
  • FIG. 4 provides a graph showing the net response (MFI-Blank) for two antigens Con6 gp120 and gp41 .
  • FIG. 5 provides a graph showing increased trend in avidity index with increased delay between first and second MVA boosts.
  • Avidity is determined for the immunodominant region of gp41 as shown by citrate wash assay.
  • the DDMM or DgDgMM regimes have a 2 month (8 week) delay between MVA (M) boosts while DgDgM_M has a 4 month (16 week) delay between MVA (M) boosts.
  • DgDgMM_M the increased avidity index with an added third MVA boost after a 4 month (16 week) delay after the second MVA boost
  • DgDgMM_M compare to DgDgMM regime
  • FIG. 8 provides a plot demonstrating that a third spaced MVA boost inhanced durability as shown by the difference in the slopes for the T2 (DgDgMM_M) and T3(DgDgM_M) groups. The last two MVA boosts were spaced by 4 month (16 weeks).
  • T2.3 M is 2 weeks after the third MVA boost (peak response)
  • T2.6mo is 6 months after the third MVA boost (contracted response)
  • T3.2M is 2 weeks after the second MVA boost
  • T3.6mo 6 months after the second MVA boost
  • HVTN HIV Vaccine Trials Network
  • GLP lab practices

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Abstract

La présente invention concerne des méthodes d'immunisation contre le VIH, ainsi que des méthodes permettant d'améliorer la réponse immunitaire face à un ou des antigènes du VIH. Les méthodes de la présente invention permettent d'obtenir, de façon avantageuse, une réponse immunitaire cellulaire et/ou humorale améliorée par rapport à ce que l'on observe en cas d'administration d'antigènes du VIH par des méthodes traditionnelles. L'invention concerne, selon un mode de réalisation, une méthode d'immunisation, comprenant les étapes consistant (i) à administrer une composition pour primo-vaccination contenant un ou plusieurs gènes codant pour un antigène du VIH; (ii) à administrer une première dose d'une composition de rappel contenant un vecteur viral modifié de la vaccine Ankara comprenant un ou plusieurs gènes codant pour un antigène du VIH, ledit vecteur exprimant un ou plusieurs antigènes recombinés du VIH, et (iii) à administrer une seconde dose d'une composition de rappel environ 12 à 20 semaines après la première.
PCT/US2014/047056 2013-07-17 2014-07-17 Méthode visant à renforcer la réponse immunitaire face aux antigènes du vih WO2015009946A1 (fr)

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