WO2015009946A1

WO2015009946A1 - Method of increasing immune response to hiv antigens

Info

Publication number: WO2015009946A1
Application number: PCT/US2014/047056
Authority: WO
Inventors: Rama R. AMARA; Harriet L. Robinson
Original assignee: Emory University; Geovax Inc
Current assignee: Emory University; Geovax Inc
Priority date: 2013-07-17
Filing date: 2014-07-17
Publication date: 2015-01-22
Anticipated expiration: 2016-01-17

Abstract

The present invention is directed to methods of HIV immunization, as well as methods improving an immune response to an HIV antigen(s). Advantageously, the methods of the present invention provide an improved cellular and/or humoral immune response compared to administration of HIV antigens by conventional methods. In one aspect, 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.

Description

METHOD OF INCREASING IMMUNE RESPONSE TO HIV ANTIGENS

Cross Reference to Related Applications

This application claims the benefit of U.S. Provisional Patent Application

61/847,108 filed July 17, 2013, which is incorporated in its entirety.

Field of the Invention

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

administration regimes.

Background of the Invention

Many novel candidates and approaches have been developed in the pursuit of an effective vaccine for the human immunodeficiency virus (HIV) vaccine.

One promising approach to HIV vaccination is a prime-boost approach. 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

Robinson et al. 2000 AIDS Rev. 2:105-1 10) or a VLP-protein boost plus IL- 12/GM-CSF (O'Neill et al. 2003 AIDS Res. Hum. Retrovir. 19: 883-890 and O'Neill et al. 2002 J. Med. Primatol. 31 : 217-227). The DNA phme-MVA approach has been shown to be improved by the use of multiple HIV-1 gene regions on the same vector (Amara et al. 2002 J. Virol. 76:6138-6146).

In another approach, the prime consists of a recombinant viral vector, such as MVA. However, using the same recombinant viral vector for the prime and boost may lead to a diminished immune response similar to preexisting immunity to the recombinant vector (Vogels et al 2003 J. Virol. 77: 8263-8271 ). For HIV vaccines, a major challenge has been raising antibody responses that are long lasting. In the RV144 Thai trial, the decline of antibody correlated with lower levels of prevention of infection. (Robb et al., Lancet Infect Dis 12:531 - 537.) The HIV envelope glycoprotein, in its natural form, is heavily glycosylated and is slow to elicit high avidity antibody. Many protective functions of antibody are mediated by the Fc region of bound antibody initiating antibody-dependent cell mediated killing, phagocytosis, and complement-mediated killing. These killing functions are triggered by oligomerization of Fc receptors - thus they require the binding of multiple antibodies to be triggered, which in turn means that the avidity of a response is important to the efficacy of antibody initiating Fc- mediated mechanisms. (See Robinson, H.L., Expert Opin Biol Ther 13:197-207).

There remains a need for novel approaches to providing an effective HIV vaccine and more particularly, an approach that permits improved cellular and/or humor immunity.

Summary of the Invention

The present invention is directed to methods of HIV immunization, as well as methods improving an immune response to an HIV antigen(s). Advantageously, the methods of the present invention provide an improved cellular and/or humoral immune response compared to administration of HIV antigens by conventional methods. In one aspect, 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. In one embodiment, the priming composition is DNA plasmid.

In a particular embodiment, the priming composition is a DNA plasmid

comprising one or more genes encoding an HIV antigen selected from a Clade A G, B or C antigen, or combinations thereof.

In another embodiment, 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.

In a further embodiment, 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.

In another embodiment, 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. In a particular embodiment, the gene encoding the HIV enzyme has been modified to possess safety mutations. In a particular embodiment, the HIV enzyme is selected from reverse transcriptase or protease.

In a further embodiment, 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.

Optionally, the method may comprise one or more additional steps. In a particular embodiment, the method comprises one or more priming steps, boosting steps, or a combination thereof. In exemplary embodiments, 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.

In a second aspect, 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.

In one embodiment, 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.

In a further embodiment, the boosting compositions are the same.

In another embodiment, 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. In a particular embodiment, the gene encoding the HIV enzyme has been modified to possess safety mutations. In a particular

embodiment, the HIV enzyme is selected from reverse transcriptase or protease. In a further embodiment, 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. In a particular

embodiment, the method comprises one or more boosting steps. In exemplary embodiments, 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.

In a third aspect, 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. In one embodiment, 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.

In a further embodiment, the boosting compositions are the same.

In a further embodiment, 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. In a particular

embodiment, the method comprises one or more boosting steps.

In exemplary embodiments, 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.

In a fourth aspect, 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.

In a further embodiment, the boosting compositions are the same. In exemplary embodiments, 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. In a fifth aspect, 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.

In exemplary embodiments, 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. In a particular embodiment, the immune response is improved with respect to avidity, B cell response or T cell response.

In exemplary embodiments, the immune response is improved with respect to B cell memory. In exemplary embodiments, the immune response is improved with respect to antibody titer.

In exemplary embodiments, the immune response is improved with respect to avidity.

In exemplary embodiments, the immune response is improved with respect to CD8+ T cell response.

In exemplary embodiments, the immune response is improved with respect to CD4+ T cell response. In a specific embodiment, 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)

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. In a particular embodiment, the HIV antigens in steps (i)-(iii) are the same. Optionally, the method comprising one or more additional steps, such as one or more additional priming or boosting steps.

In a particular embodiment, 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.

In a particular embodiment, 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.

Brief Description of Drawings

FIG. 1 provides a schematic of DNA/SIV239 vaccine. SIV239 Gag (gag),

Protease (PR) and Reverse Transcriptase (RT), Envelope (env), Tat and Rev are expressed by a CMV immediate early promoter (CMVIE) and intron A. x, mutations that inactivate packaging of RNA in Gag. This plasmid also contains mutations in PR and RT that inactivate their enzymatic functions. 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. D, DNA/SIV vaccine expressing SIV239 Gag, Pol, Env, tat and rev; M, MVA/SIV vaccine expressing SIV239 Gag, Pol and Env. 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

DgDgM_M is a 4 month (16 week) delay between MVA (M) boosts. Also shown is 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 (P=0.01 ) and the DgDgM_M regime (P=0.05).

FIG. 6 provides a graph showing the increased avidity index of a 4 month (16 week) MVA boost (P=0.05). These data also demonstrate the increase in avidity index with adding a third boost with a 4 month (16 week) delay after the second MVA boost. (P=0.01 ).

FIG. 7 provides a graph demonstrating the results of TZM neutralization assay (MN is HIV-MN). Also shown are the results of a ADCC assay sing BAL gp120- coated CEM NKR cells. These results show an increased magnitude of ADCC. (p=0.001 ).

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).

Detailed Description of the Invention

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). Advantageously, and unexpectedly, the methods of the present invention improves the immune response to HIV antigens relative to conventional administration regimes.

More particularly, 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.

Definitions:

As used herein:

"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. As used herein, 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.

"Challenge" refers to the deliberate exposure of a subject to a vaccine antigen.

"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" signifies what one of skill to one skilled in the art to refer to Human

Immunodeficiency Virus. There are two types of HIV: HIV-1 and 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

immunodeficiency virus into humans. Within the M-group there are at least ten subtypes or clades: e.g., clade A, B, C, D, E, F, G, H, I, J, and K. A "clade" 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.

"Immune 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," "polynucleotide," "polynucleotide sequence" and "nucleic acid sequence" refer to single-stranded or double-stranded deoxyribonucleotide or ribonucleotide polymers, or chimeras or analogues thereof.

"Protein," "polypeptide," and "peptide" are used interchangeably, unless otherwise indicated, to refer to a polymeric form of amino acids.

"Patient" 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). The terms "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. For example, human genes often contain single nucleotide polymorphisms that are present in certain individuals within a population. Viruses often acquire

spontaneous mutations in their nucleic acid after serial passage in vitro or upon replication in an infected subject. Mutations within HIV sequences may confer resistance to drug treatment or alter the rate of infection or replication of the virus in a subject. Several natural variant sequences of HIV clades are known in the art (see, for example, the Los Alamos DNA Database website). "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

polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that are not autonomously replicating. The term vector encompasses vectors capable of promoting expression, as well as replication, of a nucleic acid incorporated therein. Typically, 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

HIVs and Their Replication

The etiological agent of acquired immune deficiency syndrome (AIDS) is recognized to be a retrovirus exhibiting characteristics typical of the lentivirus genus, referred to as human immunodeficiency virus (HIV).

The earliest phylogenetic analyses of HIV-1 isolates focused on samples from Europe/North America and Africa; discrete clusters of viruses were identified from these two areas of the world.

All of the HIV-1 group M subtypes can be found in Africa. 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

The Gag proteins of HIV, like those of other retroviruses, are necessary and sufficient for the formation of noninfectious, virus-like particles. 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. As noted previously, the mRNA for Pr55^Gag is the unspliced 9.2-kb transcript that requires Rev for its expression in the cytoplasm. When the pol ORF is present, the viral protease (PR) cleaves Pr55^Gag during or shortly after budding from the cell to generate the mature Gag proteins p17 (MA), p24 (CA), p7 (NC), and p6. In the virion, 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, and 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

glycoproteins, but the production of infectious virions requires the encapsidation of the viral RNA genome and the incorporation of the Env glycoproteins and the Gag-Pol polyprotein precursor Pr160^Gag"Po1. Pol

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. Like Pr55^Gag, the N- terminus of Pr160^Gag"Po1 is myristylated and targeted to the plasma membrane.

Protease

Early pulse-chase studies performed with avian retroviruses clearly indicated that 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. Unlike the cellular aspartic proteases, which function as pseudodimers (using two folds within the same molecule to generate the active site), 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. Like their cellular homologs, 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. Interestingly, although some limited amino acid homology is observed surrounding active-site residues, the primary sequences of retroviral PRs are highly divergent, yet their structures are remarkably similar.

Reverse Transcriptase

By definition, retroviruses possess the ability to convert their single-stranded RNA genomes into double-stranded DNA during the early stages of the infection process. The enzyme that catalyzes this reaction is RT, in conjunction with its associated RNaseH activity. 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 Mg²⁺ 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.

Integrase

A distinguishing feature of 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.

The integration of viral DNA is catalyzed by integrase, a 32-kDa protein generated by PR-mediated cleavage of the C-terminal portion of the HIV-1 Gag- Pol polyprotein. 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.

However, attempts at crystallography were successful when it was observed that a Phe-to-Lys change at IN residue 185 greatly increased solubility without disrupting in vitro catalytic activity. Each monomer of the HIV-1 IN core domain (IN residues 50 to 212) is composed of a five-stranded P-sheet flanked by helices; this structure bears striking resemblance to other polynucleotidyl transferases including RNaseH and the bacteriophage MuA transposase. Three highly conserved residues are found in analogous positions in other

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. Recently, the x-ray structures of SIV and Rous sarcoma virus IN protein fragments encompassing both the core and C-terminal domains have been solved.

Env

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

membranes. This membrane fusion event enables the viral core to gain entry into the host cell cytoplasm. A number of regions in both gp120 and gp41 have been implicated, directly or indirectly, in Env-mediated membrane fusion. Studies of the HA2 hemagglutinin protein of the orthomyxoviruses and the F protein of the paramyxoviruses indicated that a highly hydrophobic domain at the N-terminus of these proteins, referred to as the fusion peptide, plays a critical role in membrane fusion. Mutational analyses demonstrated that an analogous domain was located at the N-terminus of the HIV-1 , HIV-2, and SIV TM glycoproteins.

Nonhydrophobic substitutions within this region of gp41 greatly reduced or blocked syncytium formation and resulted in the production of noninfectious progeny virions. Priming Compositions

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.

Any suitable priming composition may be used, including but not limited to a infectious agent or a vector such as a plasmid DNA or viral vector. Examples of suitable viral vectors include but are not limited to 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.

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. In any event, 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.

In one embodiment, the priming composition comprises one HIV antigen. In a particular embodiment, 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. In exemplary embodiments, the priming composition is plasmid DNA engineered to express one or more HIV antigens. In other exemplary embodiments, the priming composition is a viral vector modified to encode express one or more HIV antigens, such as modified

(recombinant) vaccinia virus (MVA). Additional discussion of MVA and vaccinia virus vectors is provided below.

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.

In exemplary embodiments, the priming composition also contains genes encoding or more viral (e.g., HIV) enzymes, wherein those genes are modified to prevent enzymatic activity. For example, 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).

In a particular embodiment, 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.

As is true for plasmids encoding other antigens, 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 (sans vector) are also within the scope of the disclosure. As described herein, 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).

Other 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). As is true for plasmids encoding other antigens, 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).

In one embodiment, 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. Other 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).

Examples of such recombinant MVA vectors possessing safety mutations include MVA 65A/G, MVA 62B and MVA 71 C described in WO2006/026667.

In one embodiment, 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:

(a) a HIV-1 Gag protein in which both zinc fingers have been inactivated by amino acid changes C392S, C395S, C413S and

C416S;

and

(b) a HIV-1 Pol protein in which

(i) the integrase activity is inhibited by the deletion of integrase, (ii) the reverse transcriptase activity is inhibited by amino acid changes D185N, W266T and E478Q, and

(iii) the protease activity is inhibited by amino acid change selected from L90M, G48V, R25N, or D25A.

In a particular embodiment, the HIV is clade A/G, B or C.

In one embodiment, 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:

(a) a HIV-1 Gag protein in which both zinc fingers have been inactivated by amino acid changes C390S, C393S, C41 1 S and C414S,

(b) a HIV-1 Pol protein in which

(i) the integrase activity is inhibited by the deletion of integrase,

(ii) the reverse transcriptase activity is inhibited by amino acid changes D185N, W266T and E478Q, and

(iii) the protease activity is inhibited by amino acid change selected from L90M, G48V or R25N, and

(c) clade A/G Vpu, Env, Tat, and Rev.

In one embodiment, 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:

(a) a HIV-1 Gag protein in which both zinc fingers have been inactivated by amino acid changes C392S, C395S, C413S and C416S,

(b) a HIV-1 Pol protein in which

(iii) the protease activity is inhibited by amino acid change D25A, and

clade B Vpu, Env, Tat, and Rev.

In one embodiment, 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:

(b) a HIV-1 Pol protein in which

(i) the integrase activity is inhibited by the deletion of integrase,

(iii) the protease activity is inhibited by amino acid change D25A, and

(c) clade B Vpu, Env, Tat, and Rev.

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. In one embodiment, 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. For the expression of a DNA sequence or gene, it is necessary for regulatory sequences, which are required for the transcription of the gene, to be present on the DNA. Such regulatory sequences (called 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.

Within the MVA vector, regulatory sequences for expression of the encoded antigen will include a natural, modified or synthetic poxvirus promoter. By

"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. In Fields Virology, D. M. Knipe, and P. M. Howley, eds. (Philadelphia, Lippincott Williams & Wilkins), pp. 2849-2883. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology (1998 Ausubel et al. eds., John Wiley & Sons). Promoters for use in aspects and embodiments of the present invention must be compatible with poxvirus expression systems and include natural, modified and synthetic sequences.

Boosting Compositions

As discussed above, 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). 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.

Several attenuated strains of vaccinia virus have been developed to avoid undesired side effects of smallpox vaccination. 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., Zentralbl.

Bakteriol. 167:375-390, 1978; Stickl et al., Dtsch. Med. Wschr. 99:2386-2392, 1974; see also, Sutter and Moss, Proc. Natl. Acad. Sci. U.S.A. 89:10847-10851 , 1992). During these studies in over 120,000 humans, including high-risk patients, no side effects were associated with the use of MVA vaccine.

In one embodiment, the boosting composition is a recombinant MVA viral vector encoding one or more HIV antigens. The antigens encoded by rMVA are typically proteinaceous. In any event, 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.

In a particular embodiment, the boosting composition is a recombinant MVA viral vector encoding one or more HIV antigens common to the priming composition. In a specific embodiment, the boosting composition is a recombination MVA vector that encodes the same HIV antigens as the priming composition, or variants thereof. In a particular embodiment, 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.

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.

Recombinant MVA vectors suitable as boosting compositions are described for example in WO2006/026667 and WO2002/072754 which are incorporated by reference in their entirety. In this work, clade of the inserts is designated by the last letter. For example, clade B inserts are designated for example MVA62B, clade AG inserts are designated for example MVA65AG, and clade C inserts are designated for example MVA70C and MVA71 C.

More recently, 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

recombination techniques, 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.

For vector applications health risks would be lessened by the use of a highly attenuated vaccinia virus strain. Several such strains of vaccinia virus were especially developed to avoid undesired side effects of smallpox vaccination. Thus, the modified vaccinia Ankara (MVA) 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.

Furthermore, MVA is characterized by its extreme attenuation. When tested in a variety of animal models, MVA was proven to be avirulent even in

immunosuppressed animals. More importantly, the excellent properties of the MVA strain have been demonstrated in extensive clinical trials (Mayr A. et al. 1978 Zentralbl Bakteriol [B] 167:375-390; Stickl et al. 1974 Dtsch Med Wschr 99:2386-2392). During these studies in over 120,000 humans, including high-risk patients, no side effects were associated with the use of MVA vaccine. 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. 1992 PNAS USA 89:10847- 10851 ). Additionally, 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 recombinant MVA vaccinia viruses can be prepared as described above. In exemplary embodiments, 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 (sans vector) are also within the scope of the disclosure. As described herein, 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).

In exemplary embodiments, 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). As is true for vectors encoding other antigens, 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).

In exemplary embodiments, 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.

Other 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).

In one embodiment, 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

(b) a HIV-1 Pol protein in which

(i) the integrase activity is inhibited by the deletion of integrase,

In a particular embodiment, the HIV is clade A/G, B or C.

In one embodiment, 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:

(a) a 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

(i) the integrase activity is inhibited by the deletion of integrase,

(iii) the protease activity is inhibited by amino acid change selected from L90M, G48V or R25N, and clade A/G Vpu, Env, Tat, and Rev.

In one embodiment, 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:

(b) a HIV-1 Pol protein in which

(i) the integrase activity is inhibited by the deletion of integrase,

(iii) the protease activity is inhibited by amino acid change D25A, and

(c) clade B Vpu, Env, Tat, and Rev.

In one embodiment, 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:

(b) a HIV-1 Pol protein in which

(i) the integrase activity is inhibited by the deletion of integrase, (ii) the reverse transcriptase activity is inhibited by amino acid changes D185N, W266T and E478Q, and (iii) the protease activity is inhibited by amino acid change D25A, and

(C) clade B Vpu, Env, Tat, and Rev.

Methods

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).

In exemplary embodiments, 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. 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.

In one embodiment, 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). 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,

DDDMM, and DDDMMM.

In another embodiment, MVA vectors are used for both priming and boosting purposes. Such protocols include but are not limited to MM, MMM, and MMMM.

In some embodiments, 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). For example, GM-CSF or other adjuvants known to those of skill in the art. The adjuvant can be a "genetic adjuvant" (i.e., a protein delivered by way of a DNA sequence).

The antigen(s) encoded by the respective priming and boosting compositions (however many boosting compositions are employed) 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.

In one embodiment, 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). Similarly, as described further below, one can immunize a patient (or elicit an immune response, which can include multi-epitope CD8⁺ T cell responses) by administering a live-vectored vaccine (e.g., an MVA vector) without administering a plasmid-based (or "DNA") vaccine. Thus, in alternative embodiments, 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) are the same as those described herein for "DNA-MVA" regimens, and the MVAs in any vaccine can be in any proportion desired. For example, in any case (whether the immunization protocol employs only plasmid-based immunogens, only viral-carried immunogens, or a

combination of both), one can include an adjuvant and administer a variety of antigens, including those obtained from any HIV clade, by way of the plurality of vectors administered. In one embodiment, 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

encompasses apparently healthy or non-HIV-infected individuals). In exemplary embodiments, 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

10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%).

In another embodiment, 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). In yet another embodiment, 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.

In a particular embodiment, the in which the immune response to administration of a priming composition comprising one or more HIV 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 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.

In one embodiment, 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. Such HIV immunization protocols include but are not limited to DMM, DMMM, DDMM, DDMMM, DDDMM, and DDDMMM.

In another embodiment, 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.

In exemplary embodiments, 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. In a particular embodiment, the HIV antigens are the same in step (i)-(iii). Optionally, 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).

In exemplary embodiments, 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. In a particular embodiment, the HIV antigens are the same in step (i)-(iii). Optionally, 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

In exemplary embodiments, 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. In a particular embodiment, the HIV antigens are the same in step (i)-(iii).

Optionally, 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).

In exemplary embodiments, 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. In a particular embodiment, the HIV antigens are the same in step (i)-(iii). Optionally, 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).

In exemplary embodiments, 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. In a particular embodiment, the HIV antigens are the same in step (i)-(iii). Optionally, 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).

In exemplary embodiments, 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. In a particular embodiment, the HIV antigens are the same in step (i)-(iii). Optionally, 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).

In exemplary embodiments, 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

particularly between about 14 and about 18 weeks after the first dose, even more particularly, about 16 weeks after the second dose. In a particular embodiment, the HIV antigens are the same in step (i)-(iii). Optionally, 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).

In exemplary embodiments, 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

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. In a particular embodiment, the HIV antigens are the same in step (i)-(iii). Optionally, 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). In exemplary embodiments, 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. In a particular embodiment, the HIV antigens are the same in step (i)-(iii). Optionally, 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). In a particular embodiment, 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. In a particular embodiment, the second boost (i.e., the second dose of the boosting composition or administration of the second boosting composition) is administered about 1 to 10 months after administration of the priming

composition, preferably about 1 to 6 months, preferably about 1 to 4 months, preferably about 1 to 3 months. The improved immune response of the method of the present invention may have one or more characteristics. In a particular embodiment, the immune response is improved with respect to avidity, B cell response or T cell response. In exemplary embodiments, the immune response is improved with respect to B cell memory.

In exemplary embodiments, the immune response is improved with respect to antibody titer.

In exemplary embodiments, the immune response is improved with respect tto CD4+ T cell response.

Dosing

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.).

In one preferred regimen, the priming composition is a plasmid DNA

administered at a dose of 250 g to 2.5 mg/injection, followed by administration of a boosting composition comprising a recombinant MVA viral vector at a dose of 10⁶ to 10⁹ infectious virus particles/injection, or more particularly, about 1 x10⁸ pfu .

Administration

Preferably, 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. An alternative is the use of 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. These

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.

For intravenous, cutaneous, subcutaneous, intramuscular or mucosal injection, or injection at the site of affliction, 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. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, 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.

A composition may be administered alone or in combination with other

treatments, either simultaneously or sequentially dependent upon the condition to be treated. Either or both of the priming and boosting compositions may include an adjuvant, such as granulocyte macrophage-colony stimulating factor (GM-CSF) or encoding nucleic acid therefor.

Further aspects and embodiments of the present invention will be apparent to those of ordinary skill in the art, in view of the above disclosure and following experimental exemplification, included by way of illustration and not limitation, and with reference to the attached figures. Examples

EXAMPLE 1 : INCREASING THE REST PERIOD BETWEEN TWO MVA

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.

Methods:

Immunizations and challenge. Young adult Indian rhesus macaques from the Yerkes breeding colony were cared for under guidelines established by the Animal Welfare Act and the National Institutes of Health (Bethesda, MD) Guide for the Care and Use of Laboratory Animals using protocols approved by the Emory University (Atlanta, GA) Institutional Animal Care and Use Committee. Macaques were typed for the Mamu-A^*01 , Mamu B^*08, and Mamu B^*17 alleles as described previously (Hammarlund et al., Nature Medicine 9:1 131 -1 137; Lai et al., Vaccine 30:1737-1745.; Kannanganant, J Virol 2014). Of the 35 macaques, eight were vaccinated with a DNA MVA SIV vaccine, 12 (six Mamu-A^*01 , six Mamu-A^*01 -negative) were vaccinated with the DNA/MVA SIV vaccine with CD40L in the DNA and 15 were unvaccinated controls. The DNA and rMVA immunizations were delivered i.m in PBS using a hypodermic needle in the outer thigh. The DNA immunogen expressed SIV239 Gag-Pol, Env, Tat, and Rev. The DNA immunogen was constructed by replacing the EcoRI— Nhel fragment of SHIV DNA construct (Patel et al., Proceedings of the National

630 Academy of Sciences 1 10:2975-2980) containing HIV-1 89.6 Tat, Rev, and Env genes with an EcoRM 19 Nhel fragment containing SIV Tat, Rev, and Env. Two MVA recombinants, one expressing SIV239 Gag-Pol (Giorgi et al, Journal of Infectious Diseases 179:859-870) and the other expressing SIV239 Env (Xiao et al., J Virol 84:7161 -7173), were premixed and used for immunizations. The DNA was delivered at 3 mg/dose, and the rMVA was delivered at 1 10⁸ PFU/dose. At 20-24 weeks after the final rMVA booster, animals were challenged with weekly doses of SIVE660 intrarectally using a pediatric feeding tube 15 to 20 cm into the rectum. Dr. Vanessa Hirsch at the National Institutes of Health provided the challenge stock.

T cell responses. Intracellular cytokine production was assessed as previously described with a few modifications. Briefly, 2 million PBMCs were stimulated in 200 μΙ RPMI with 10% FBS in a 5-ml polypropylene tube. SIV-specific

stimulations were conducted using a single pool of 125 SIV239 Gag peptides, two pools of 225 SIV239 Env peptides (National Institutes of Health AIDS

Research and Reference Reagent Program, Germantown, MD). All peptides were 15-mers overlapping by 1 1 . 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. At the end of stimulation, cells were washed once with PBS containing 2% FBS, surface stained with anti-human CD4-PerCP (clone L200; BD Pharmingen), and anti-human CD8-AmCyan (clone SK1 ; BD Biosciences, San Jose, CA), fixed with Cytofix/Cytoperm (BD Pharmingen), and permeabilized with 1 * Permwash (BD Pharmingen). Cells were then stained using a mixture of Abs containing anti human CD3-Pacific blue (clone SP34-2; BD Pharmingen), anti-human IFN-γ Alexa 700 (clone B27; BD Pharmingen), anti-human IL-2- allophycocyanin (clone MQ1 -17H12; BD Pharmingen), and anti-human TNF-a- PE-Cy7 (clone Mab1 1 ; eBioscience, San Diego, CA), washed twice with Permwash, once with 2% FBS in PBS, and resuspended in 1 % formalin in PBS. Approximately 500,000 lymphocytes were acquired on the LSRII (BD

Imnnunocytonnetry Systems, San Jose, CA) and analyzed using FlowJo software (Tree Star, Ashland, OR). Lymphocytes were identified based on their scatter pattern, and CD3+CD8-CD4+ cells were considered CD4 T cells, and

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.

Measurement of binding Ab responses. 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;

Corning Life Sciences, Lowell, MA) were coated with concanavalin A (25 pg/ml) overnight at 4°C. Plates were washed and incubated with 100 μΙ of Triton X-100 disrupted undiluted 239 virus-like particle supernatant (generated by transient transfection of 293T cells with the earlier-described SIV239 DNA vaccine expressing Gag, Pol, and Env) or with SIVE660 grown in rhesus PBMC for 1 h. Plates were washed and blocked for 1 h (PBS-Tween with 4% whey and 5% dry milk). Test sera was added to duplicate wells in serial 3-fold dilutions and incubated for 1 h. Plates were then washed, and bound Ab was detected using peroxidase-conjugated anti-monkey IgG (Accurate Chemical and Scientific, Westbury, NY) and tetramethylbenzidine substrate (KPL, Gaithersburg, MD). Reactions were stopped with 100 μΙ 2N H2SO4. Each plate included a standard curve generated using goat anti-monkey IgG and rhesus macaque IgG (both from Accurate Chemical and Scientific Corp.) as previously (39). Standard curves were fitted and sample concentrations interpolated as micrograms of Ab per milliliter of serum using SOFTmax 2.3 software (Molecular Devices, Sunnyvale, CA). The concentrations of IgG are relative to our standard curve, not absolute values. A NaSCN displacement ELISA assay modeled after that described by Vermont et al. (44) was used for determining avidity. This assay was conducted using parallel titrations of test sera in our standard ELISA assay. After the binding of the test sera, the parallel titrations were treated for 10 min at room

temperature with PBS or 1 .5 M NaSCN (prepared fresh in PBS). Then, the relative levels of bound Ab were determined using the standard ELISA procedure (see earlier). 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 μΙ 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). Plates were developed by consecutive treatments with biotinylated goat anti-monkey IgA (a Diagnostics, San Antonio, TX) or biotinylated goat anti human IgG (SouthernBiotech, Birmingham, AL), avidin-peroxidase,

tetramethylbenzidine, and 2N H₂SO₄. For rectal secretions, the concentration of anti-env or anti-gag, pol IgA was divided by the total IgA concentration to obtain specific IgA activity. Samples were considered IgA Ab-positive if the env or gag, pol specific IgA activity was greater than or equal to 0.145 or 0.224, respectively. These cutoffs represent the mean sp. act. + 3 SD previously established for rectal secretions from naive macaques. Measurement of neutralizing antibody. SIV-specific neutralization was measured as a function of reductions in luciferase reporter gene expression after a single round of infection in TZM-bl cells as described. TZM-bl cells were obtained from the National Institutes of Health AIDS Research and Reference Reagent

Program as contributed by John Kappes and Xiaoyun Wu.

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. For viral load determinations in gut, total RNA was extracted from about 1 million cells obtained from gut biopsies and used for quantitative real-time PCR analyses.

Results

Results in FIG.2 show that increasing the rest period significantly increases the antibody titers to the SIV Env in the MVA immunogen. Similarly, results in FIG. 3 show that the long rest also significantly increases the avidity of anti-Env antibody. These results are very important for the efficacy of HIV vaccines as our previous studies have shown that the avidity of anti-Env antibody contributes significantly for protection against a pathogenic SIVE660 infection in macaques (1. 2).

Lai L, Kwa S, Kozlowski PA, Montefiori DC, Ferrari G, Johnson WE, Hirsch V, Villinger F, Chennareddi L, Earl PL, Moss B, Amara RR, Robinson HL.

Prevention of infection by a granulocyte-macrophage colony-stimulating factor co-expressing DNA/modified vaccinia Ankara simian immunodeficiency virus vaccine. J Infect Dis. United States201 1 . p. 164-73.

Kwa S, Lai L, Gangadhara S, Siddiqui M, Pillai BV, LaBranche CC, Yu T, Moss B, Montefiori DC, Robinson HL, Kozlowski P, Amara RR. CD40L- adjuvanted DNA/MVA SIV239 vaccine enhances SIV-specific humoral and cellular immunity, and improves protection against a heterologous SIVE660 mucosal challenge. J Virol. 2014:in Press.

EXAMPLE 2: INCREASING THE REST PERIOD BETWEEN TWO MVA

IMMUNIZATIONS ENHANCES THE MAGNITUDE OF IMMUNOGEN-SPECIFIC ANTIBODY RESPONSE IN HUMANS A phase 1 placebo controlled clinical trial to evaluate the safety and

immunogenicity of a prime-boost vaccine regimen of GEO-D03 DNA and MVA HIV62B vaccines in healthy, HIV-1 -uninfected vaccinia na^'fve adult participants Primary objective:

To assess the safety and tolerability of 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).

Study products and routes of administration:

GEO-D02 DNA vaccine (D): 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.

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

administered as a 1 ml_ intramuscular (IM) injection into the deltoid.

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_. A 1 x10⁸ 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 . 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. 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. Also shown is 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 (P=0.01 ) and the DgDgM_M regime (P=0.05).

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 is 6 months after the second MVA boost

All assays were conducted in HIV Vaccine Trials Network (HVTN) endpoint labs under good laboratory practices (GLP) certified conditions.

Claims

1 . 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.

2. The method of claim 1 , wherein the priming composition is DNA plasmid.

3. The method of claim 1 , wherein the priming composition is a DNA plasmid comprising one or more genes encoding an HIV antigen selected from a Clade A G, B or C antigen, or combinations thereof.

4. The method of claim 1 , wherein 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.

5. The method of claims 1 -4, wherein the one or more genes of the priming composition and the one or more genes of the boosting composition are the same.

6. The method of claim 1 , wherein the boosting compositions are the same.

7. The method of claim 1 , wherein 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. In a particular embodiment, the gene encoding the HIV enzyme has been modified to possess safety mutations. In a particular embodiment, the HIV enzyme is selected from reverse transcriptase or protease.

8. The method of claim 1 , wherein 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.

9. The method of any of claims 1 -8, further comprising one or more priming steps, boosting steps, or a combination thereof.

10. The method of any of claims 1 -9, wherein 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.

1 1 . 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.

12. The method of claim 1 1 , wherein 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.

13. The method of claim 1 1 , wherein the boosting compositions are the same.

14. The method of claim 1 1 , wherein 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. In a particular embodiment, the gene encoding the HIV enzyme has been modified to possess safety mutations. In a particular embodiment, the HIV enzyme is selected from reverse transcriptase or protease.

15. The method of claim 1 1 , wherein 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.

16. The method of any of claims 1 1 -15, further comprising one or more boosting steps.

17. The method of any of claims 1 1 -16, wherein 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.

18. 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.

19. The method of claim 18, wherein 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.

20. The method of claim 18, wherein the boosting compositions are the same.

21 . The method of claim 18, wherein 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. In a particular embodiment, the gene encoding the HIV enzyme has been modified to possess safety mutations. In a particular embodiment, the HIV enzyme is selected from reverse transcriptase or protease.

22. The method of claim 18, wherein 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.

23. The method of any of claims 18-22, further comprising one or more boosting steps.

24. The method of any of claims 18-23, wherein 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.

25. The method of any of claims 1 -24, resulting in an improved immune response.

26. The method of claim 25, wherein the immune response is improved with respect to avidity, B cell response or T cell response.

27. The method of claim 25, wherein the immune response is improved with respect to B cell memory.

28. The method of claim 25, wherein the immune response is improved with respect to antibody titer.

29. The method of claim 25, wherein the immune response is improved with respect to avidity.

30. The method of claim 25, wherein the immune response is improved with respect to CD8+ T cell response.

31 . The method of claim 25, wherein the immune response is improved with respect to CD4+ T cell response.