EP1771571A2

EP1771571A2 - Recombinant aav based vaccine methods

Info

Publication number: EP1771571A2
Application number: EP05856895A
Authority: EP
Inventors: Anthony M. Stepan; Richard Peluso; Susan Arnold; Jason Wustner; Philip R. Johnson; Alan M. Schultz
Original assignee: Childrens Hospital Inc; Targeted Genetics Corp
Current assignee: Childrens Hospital Inc; Ampliphi Biosciences Corp
Priority date: 2004-07-30
Filing date: 2005-07-29
Publication date: 2007-04-11
Also published as: WO2006073496A3; WO2006073496A2

Abstract

The present invention relates to recombinant adeno-associated virus (rAAV) based materials and methods for eliciting an immune response to a pathogen. The present invention provides vector sets comprising a priming vector and a boosting vector, wherein at least one of the vectors in the vector set is a rAAV vector, and methods for eliciting a prophylactic immune response in a mammalian subject susceptible to infection by a pathogen, such as for example, an RNA virus, including HIV-1, and methods for eliciting a therapeutic immune response in a mammalian subject infected by a pathogen that has progressed to a disease state associated with the pathogen, such as for example, an RNA virus, including HIV-1. In particular, the present invention provides methods for eliciting an enhanced boost immune response in a mammalian subject susceptible to infection by or infected by a pathogen.

Description

RECOMBINANT AAV BASED VACCINE METHODS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. provisional application serial no.

60/592,889, filed July 30, 2004, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This application was made with Government support under Grant No. GPH-G-00-

01-00004-00 awarded by the USAID. The Government has certain rights in this invention.

BACKGROUND

[0003] Since Jenner first experimented with live cowpox vaccines in milkmaids in the late eighteenth century, a considerable focus of medicine has been the development of vaccines to prevent major diseases and resulting deaths. A number of significant vaccines have been developed to diseases such as smallpox, tuberculosis, diphtheria, rubella, and whooping cough. There remains, however, a significant number of diseases such as AIDS, Hepatitis C, malaria and herpes for which vaccine candidates remain in development.

[0004] To date successful vaccine generation has relied heavily on empirical testing and simple trial and error philosophy. Vaccines have been developed by utilizing live attenuated whole viruses (Sabin polio vaccine, measles vaccine, and chickenpox vaccine), inactivated or killed vaccines (SaIk polio vaccine and Hepatitis A), recombinant proteins (Hepatitis B) and toxoids (diphtheria). However, for pathogens such as HIV, malaria or HCV such strategies have proven to be impractical from both a safety and efficacy standpoint. Historically, for many of the successful vaccine candidates, the generation of a potent sterilizing antibody response was the correlate of vaccine efficacy and protection. For many of the pathogens for which no successful vaccine has been developed ;to date no sterilizing antibody-based immunity has been developed, that is capable of blocking entry or preventing spread of infection, leading researchers to postulate that an antibody response alone is insufficient to block entry, prevent the spread of infection and ultimately halt or slow the progression of those pathogens.

[0005] Attempts to develop a safe and effective vaccine for pathogens such as HIV, malaria, and hepatitis C have been hampered by the difficulty in defining the specific immune response necessary to either prevent disease or limit disease progression. In fact, one of the major advances in vaccine development has been the realization that a successful vaccine candidate may not necessarily prevent infection of the host with the pathogen but may reduce or limit the pathogen's spread and thereby prevent or slow the disease progression. In addition, it is also believed that an effective vaccine candidate needs to generate an effective cellular immune response in addition to antibody response in order to eliminate infected cellular reservoirs of virus. The belief that both a humoral and cellular response to pathogens such as HIV, HCV, herpes, and malaria needs to be generated has driven the vaccine field to adopt novel approaches for vaccine design. [0006] Two novel vaccine design approaches utilize vector systems that deliver the antigens of the pathogen intracellularly via viral or plasmid vector systems. These viral and plasmid based systems are designed to deliver a polynucleotide sequence to the host cell which then synthesizes the protein intracellularly thus making the antigen more available to the cellular immune system. Viral vectors utilized include avian and mammalian poxviruses, rhabdo viruses, alphaviruses, adeno- associated viruses, picornaviruses, adenoviruses, and herpes viruses.

[0007] Recently experimentation has begun to evaluate strategies of combining the various vector systems with each other or with more traditional protein based strategies as prime-boost systems. For example, a DNA-based prime vaccination is followed with an adenoviral or protein boost. These strategies are being employed in order to try and enhance the immune response of the single vector strategy alone. One of the limitations of a number of the vector strategies employed to date has been the fact that strong immune responses are developed against the vector system itself thus limiting the ability to deliver and express the antigen of the pathogen in order to boost or increase the immune response to the pathogen.

[0008] HIV is a non-oncogenic retrovirus, specifically a lentivirus, that causes Acquired

Immunodeficiency Syndrome (AIDS) in infected individuals. As assessed by the World Health Organization, more than 40 million people are currently infected with HIV and 20 million people have already perished from AIDS. Thus, HIV infection is considered a worldwide pandemic. [0009] There are two currently recognized strains of HIV₅ HIV-I and HIV-2. HIV-I is the principal causes of AIDS around the world. HIV-I has been classified based on genomic sequence variation into clades. For example, Clade B is the most predominant in North America, Europe, parts of South America and India; Clade C is most predominant in Sub-Saharan Africa; and Clade E is most predominant in southeastern Asia. HIV-I infection occurs primarily through sexual transmission, transmission from mother to child or exposure to contaminated blood or blood products.

[0010] HIV-I consists of a lipid envelope surrounding viral structural proteins and an inner core of enzymes and proteins required for viral replication and a genome of two identical linear RNAs. In the lipid envelope, viral glycoprotein 41 (gp 41)anchors another viral envelope glycoprotein 120 (gp 120) that extends from the virus surface and interacts with receptors on the surface of susceptible cells. The HIV-I genome is approximately 10,000 nucleotides in size and comprises nine genes. It includes three genes common to all retroviruses, the gag, pol and env genes. The gag gene encodes the core structural proteins, the env gene encodes the gpl20 and gp41 envelope proteins, and the pol gene encodes the viral enzymes reverse transcriptase (RT), integrase and protease (pro). The genome comprises two other genes essential for viral replication, the tat gene encoding a viral promoter transactivator and the rev gene which also facilitates gene transcription. Finally, the nef, vpu, vpr, and vif genes are unique to lentiviruses and encode polypeptides the function of which is described in Trono, Cell, 82; 189-192 (1995).

[0011] The process by which HIV-I infects human cells involves interaction of gpl20 on the surface of the virus with proteins on the surface of the cells. The common understanding is that the first step in HIV infection is the binding of HIV-I glycoprotein (gp) 120 to cellular CD4 protein. This interaction causes the viral gpl20 to undergo a conformational change and bind to other cell surface proteins, such as CCR5 or CXCR4 proteins, allowing subsequent fusion of the virus with the cell. CD4 has thus been described as the primary receptor for HIV-I while the other cell surface proteins are described as co-receptors for HIV-I. [0012] HIV-I infection is characterized by an asymptomatic period between infection with the virus and the development of AIDS. The rate of progression to AIDS varies among infected individuals. AIDS develops as CD4-positive cells, such as helper T cells and monocytes/macrophages, are infected and depleted. AIDS is manifested as opportunistic infections, increased risk of malignancies and other conditions typical of defects in cell-mediated immunity. The Centers for Disease Control and Prevention clinical categories of pediatric, adolescent and adult disease are set out in Table I of Sleasman and Goodenow, J Allergy Clin. Immunol, 111(2): S582- S592 (2003). _:

[0013] Predicting the likelihood of progression to AIDS involves monitoring viral loads

(viral replication) and measuring CD4-positive T cells in infected individuals. The higher the viral loads, the more likely a person is to develop AIDS. The lower the CD4-positive T cell count, the more likely a person a person is to develop AIDS.

[0014] At present, anti-retroviral drug therapy (ART) is the only means of treating HIV infection or preventing HIV-I transmission from one person to another. At best, even with ART, HIV-I infection; is a chronic condition that requires lifelong drug therapy and there can still be a slow progression to disease. ART does not eradicate HIV-I because the virus can persist in latent reservoirs. Moreover, treatment regimens can be toxic and multiple drugs must be used daily. There thus is an urgent need to develop effective vaccines for HIV- 1.

[0015] There are a number of HIV vaccines presently in clinical trials. Briefly, they include a gp 120 subunit vaccine by VaxGen, a combination attenuated canarypox virus (carrying HIV-I DNA) and gp 120 subunit vaccine by Aventis-Pasteur, a combination plasmid HIV-I DNA and recombinant adenovirus (carrying HIV-I DNA) vaccine by Merck, a combination plasmid HIV-I DNA and modified vaccinia Ankara (carrying HIV-I DNA) vaccine by IAVI Partners, a combination Tat-Nef subunit and gp 120 subunit vaccine by GlaxoSmithKline, an HIV-I subunit vaccine in lipopeptides by ANRS and a combination plasmid HIV-I DNA and recombinant adenovirus (carrying HIV-I DNA) vaccine by Vaccine Research Center of NIH. There are also other vaccine candidates in varying stages of pre-clinical development (concept stage to late preclinical testing). Some of these are based on viruses. [0016] One virus studied by researchers in the general vaccine field is adeno-associated virus. Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs). The nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al, J. Virol., 45: 555-564 (1983) as corrected by Ruffing et al, J. Gen. Virol, 75: 3385-3392 (1994). Cis- acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the ITRs. Three AAV promoters, p5, pi 9, and p40 (named for their relative map locations), drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and pi 9), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40)from the rep gene. Rep proteins possess multiple enzymatic properties which are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VPl, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

[0017] When wild type AAV infects a human cell, the viral genome can integrate into chromosomes resulting in latent infection of the cell. Production of infectious virus does not occur unless the cell is infected with a helper virus (for example, adenovirus or herpesvirus). In the case of adenovirus, genes El A, ElB, E2A, E4 and VA provide helper functions. Upon infection with a helper virus, the AAV provirus is rescued and amplified, and both AAV and adenovirus are produced. \

[0018] AAV possesses unique features that make it attractive as a vector for expressing immunogenic peptides/polypeptides. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. AAV infects slowly dividing _;and non-dividing cells and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element) and that integrated copies of vector in organs such as liver or muscle are very rare.

[0019] The AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication, genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA such as a gene cassette containing a promoter, a DNA of interest and a polyadenylation signal. The rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65⁰C for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0020] See for example: TR Flotte, et al., Proc Natl Acad Sci USA 93:10163-10617, 1993;

SA Afione, et al., J Virol 70:3235-3241, 1996; MG Kaplitt, et al., Nature Genetics 8:148-154, 1994; X Xiao, et al., J Virol 70:8098-8108, 1996; PD Kessler, et al., Proc Natl Acad Sci USA. 93:14082- 14087,1996; DD Koerberl, et al., Proc Natl Acad Sci USA 94:1426-1431, 1997; S Ponnazhagan, et al., Gene 190:203-210, 1997; Z Zadori, et al., Developmental Cell 1:291-302, 2001; KJ Fisher, et al.,Nature Medicine 3:306-312, 1997; RW Herzog, et al., Proc Natl Acad Sci USA 94:5804-5809, 1997; RO Snyder, et al., Human Gene Therapy 8:1891-1900, 1997; Nature Genetics 19:13-14, 1998; D Duan, k al., J.Virol. 8568-8577, 1998; Duan D, et al., J Virol. 73:161-169, 1999; N Vincent-Lacazei et al., J Virol. 73:1949-1955, 1999; C McKeon, et al., NIDDK Workshop on AAV Vectors: Gene transfer into quiescent cells. Human Gene Therapy 7:1615-1619, 1996; H Nakai, et al., J Virol 75:6969-6976, 2001; BC Schnepp, et al., J. Virol. 2003 77: 3495-350. [0021] There remains a need in the art to develop a vector system capable of delivering an effective amount of an antigen to a host in order to generate an immune response to a variety of pathogens including HIV, HCV, herpes, and malaria.

[0022] All references, patents, patent publications and patent applications disclosed herein are hereby incorporated by reference in their entirety. BRIEF SUMMARY

[0023] The present invention relates to methods for eliciting a boost immune response in an mammalian subject susceptible to infection by a pathogen or infected by a pathogen. Accordingly, the present invention provides methods for eliciting a boost immune response in a mammalian subject susceptible to infection by or infected by a pathogen comprising administering to the subject an effective amount of a boost dose of an antigen of the pathogen by a recombinant vector, wherein said mammalian subject has been administered an effective amount of a prime dose of an antigen of the pathogen by a recombinant vector; wherein at least one of the prime dose or the boost dose is administered by a recombinant adeno-associated virus (rAAV) vector in a single administration, and wherein the boost dose elicits an equal or greater measurable immune response as compared to the response elicited by a single administration of the prime dose. In some embodiments, the mammalian subject is not a mouse. In some examples, wherein the prime dose and boost dose are both administered by a rAAV vector the boost dose elicits an equal or greater measurable immune response as compared to the response elicited by a single administration of the prime dose when the prime dose and boost dose of antigen are in the same formulation. In some examples, the boost dose elicits a greater measurable immune response as compared to the response elicited by a single administration of the prime dose. In some examples, the pathogen is a viral pathogen. Viral pathogens encompassed within the present invention include, for example, human and simian immunodeficiency virus (SIV and HIV including HIV-I and HI V -2); hepatitis virus, including A, B and C; influenza virus; polio virus; measles virus; mumps virus; rubella virus; rabies virus, cytomegalovirus; SARs virus; human T-lymphotrophic virus types I and II (HTLV-I and HTLV-II); rotavirus, hantavirus, feline leukemia virus, and equine infectious encephalitis virus. In some examples, the viral pathogen is an RNA viral pathogen, such as for example, SIV and HIV; hepatitis virus; SARs virus; and rabies virus. In some examples, the pathogen is a viral pathogen, including for example an RNA viral pathogen. In other examples, the RNA viral pathogen is selected from the group consisting of HIV-I, HIV-2, SIV, hepatitis virus, SARS virus, rabies virus. In yet other examples, the RNA viral pathogen is HIV-I. In additional examples, the pathogen is HIV-I and the antigen¹ is selected from the group consisting of gag, pol, env, vif, vpr, vpx, vpu, tat, rev and nef. In yet other examples, the antigen is the gag protein. In some examples, the prime dose is administered by a rAAV and in other examples, the boost dose is administered by a rAAV. In additional examples, the prime dose and the boost dose are both administered by a rAAV. In some examples, the prime dose is administered by a rAAV comprising a capsid protein that is heterologous to a capsid protein of the rAAV that administers the boost dose. In additional examples, the prime dose is administered by a rAAV that comprises the same capsid proteins as the capsid proteins of the rAAV that administers the boost dose. In further examples, at least one of the prime dose or the boost dose is administered by a pseudotype rAAV. In further examples, the prime dose is administered by a rAAV-2 and the boost dose is administered by a pseudotype rAAV comprising AAV-I capsid proteins. In some examples, at least one of the prime dose and boost dose is administered intramuscularly. In some examples, wherein only the prime dose is administered by a rAAV, the boost dose is administered by a non rAAV vector including vectors selected from the group consisting of naked DNA, plasmid DNA, adenovirus, canarypox virus, pox virus, vaccinia virus, modified vaccinia Ankara (MVA), alphavirus, phabdovirus, picornavirus, and attenuated HIV. In some examples, wherein only the boost dose is administered by a rAAV, the prime dose is administered by a non rAAV vector including vectors selected from the group consisting of naked DNA, plasmid DNA, adenovirus, canarypox virus, pox virus, vaccinia virus, modified vaccinia Ankara (MVA), alphavirus, phabdovirus, picornavirus, and attenuated HIV. In examples wherein the prime dose or boost dose is administered by a non-rAAV vector, it maybe administered in 2 or more consecutive administrations.

[0024] The present invention also provides methods for eliciting a boost immune response in a mammalian subject susceptible to infection by or infected by HIV-I, comprising administering to the subject an effective amount of a boost dose of an antigen of HIV-I by a recombinant vector, wherein said mammalian subject has been administered an effective amount of a prime dose of an antigen of HIV-I by a recombinant vector; wherein at least one of the prime dose or the boost dose is administered by a recombinant adeno-associated virus (rAAV) vector in a single administration, and wherein the boost dose elicits an equal or greater measurable immune response as compared to the response elicited by a single administration of the prime dose. In some examples, the prime dose and boost dose are both administered by a rAAV vector and the boost dose elicits an equal or greater measurable immune response as compared to the response elicited by a single administration of the prime dose when the prime dose and boost dose of antigen are in the same formulation. In additional examples, the boost dose elicits a greater measurable immune response as compared to the response elicited by a single administration of the prime dose. In some examples, the prime dose is administered by a rAAV and in other examples, the boost dose is administered by a rAAV. In further examples, the prime dose and the boost dose are both administered by a rAAV. In some examples, the prime dose is administered by a rAAV comprising a capsid protein that is heterologous to a capsid protein of the rAAV that administers the boost dose. In other examples, the prime dose is administered by a rAAV that comprises the same capsid proteins as the capsid proteins of the rAAV that administers the boost dose. In yet other examples, at least one of the prime dose or the boost dose is administered by a pseudotype rAAV. In examples, the prime dose is administered by a rAAV-2 and the boost dose is administered by a pseudotype rAAV comprising AAV-I capsid proteins. In other examples, at least one of the prime dose or boost dose is administered intramuscularly. In some examples, the boost dose is administered by a non rAAV vector, including vectors selected from the group consisting of naked DNA, plasmid DNA, adenovirus, canarypox virus, pox virus, vaccinia virus, modified vaccinia Ankara (MVA), alphavirus, phabdovirus, picornavirus, and attenuated HIV. In other examples, the prime dose is administered by a non rAAV vector, including vectors selected from the group consisting of naked DNA, plasmid DNA, adenovirus, canarypox virus, pox virus, vaccinia virus, modified vaccinia Ankara (MVA), alphavirus, phabdovirus, picornavirus, and attenuated HIV. In yet other examples the prime dose is administered in 2 or more consecutive administrations of an individual non-rAAV vector. The present invention also provides kits that comprise a vector set of the present invention, in particular for eliciting an immune response to an HIV antigen. The present invention also relates to methods and packaging cell lines for the production of rAAV and provides methods as described herein in the examples.

[0025] The present invention also provides use of a first vector for the manufacture of a medicament foriadministration of a prime dose of an antigen of a pathogen and a second vector for the manufacture of a medicament for administration of a boost dose of an antigen of a pathogen, characterized in that at least one of the prime dose or the boost dose is administered by a recombinant adeno-associated virus (rAAV) vector in a single administration, and wherein the boost dose elicits an equal or greater measurable immune response as compared to the response elicited by administration of the prime dose, with the proviso that when both the prime dose and the boost dose are administered by rAAV, they are not both administered by AAV-2. In some examples, the boost dose is an rAAV vector in a single administration, and the prime dose is a non-rAAV vector. In some examples, the prime dose and boost dose are both administered by a rAAV and a single administration of the boost dose elicits a greater measurable immune response as compared to the response elicited by a single administration of the prime dose, wherein the measurement is performed under conditions where the prime dose and boost dose of antigen are equivalent, administered by the same route and in the same formulation. In some examples, the pathogen is a viral pathogen. In some examples, the viral pathogen is an RNA viral pathogen. In some examples, the viral pathogen is selected from group consisting of human immunodeficiency virus 1 and 2 (HIV-I and HIV-2); simian immunodeficiency virus (SIV); hepatitis virus A, B and C; influenza virus; polio virus; measles virus; mumps virus; rubella virus; rabies virus, cytomegalovirus; SARs virus; human T-lymphotrophic virus types I and II (HTLV-I and HTLV-II); rotavirus and hantavirus. In some examples, the RNA viral pathogen is HIV-I and the antigen is selected from the group consisting of gag, pol, env, vif, vpr, vpx, vpu, tat, rev and nef. In some examples, the antigen is a HIV-I gag protein. In some examples, the rAAV is selected from the group consisting of naturally occurring AAV serotypes, pseudotyped AAV, and chimeric AAV. In some examples, the boost dose is administered intramuscularly (IM) by a rAAV comprising an AAV-I capsid. In some examples,: the prime dose and the boost dose are administered by rAAVs comprising heterologous capsid proteins. In some examples, the prime dose and the boost dose are administered by rAAVs comprising homologous capsid proteins. In some examples, the prime dose is administered IM by rAAV-2 and the boost dose is administered IM in a single administration by a rAAV comprising an AAV-I capsid and wherein the boost immune response is greater as compared to the boost immune response elicited when the boost dose is administered IM by rAAV-2 in the same dose and formulation. In some examples, the prime dose is administered IM by rAAV-2 and the boost dose is administered IM in a single administration by a rAAV comprising an AAV-I capsid and the boost dose elicits a higher ratio of B-cell immune response to T-cell immune response as compared to the boost immune response elicited when the boost dose is administered IM by rAAV-2 in the same dose and formulation. In some examples, the boost dose is administered by at least one ήon-rAAV vector selected from the group consisting of naked DNA, plasmid DNA, adenovirus, canarypox virus, pox virus, vaccinia virus, modified vaccinia Ankara (MVA),

: io alphavirus, phabdovirus, picornavirus, and attenuated HIV. In some examples, the prime dose is administered by at least one non-rAAV vector selected from the group consisting of naked DNA, plasmid DNA₅ adenovirus, canarypox virus, pox virus, vaccinia virus, modified vaccinia Ankara (MVA), alphavirus, phabdovirus, picornavirus, and attenuated HIV. In some examples, the non- rAAV vector is administered in 2 or more consecutive administrations of a single vector. [0026] The invention also provides a method for generating a recombinant adenoviral vector comprising a desired polynucleotide sequence, comprising the steps of: co-transforming Escherichia CoIi bacteria with: a) a linear DNA molecule and; b) a supercoiled adenoviral vector comprising an adenoviral genome with one or more deletions; wherein the linear DNA molecule comprises a first segment of DNA comprising a desired polynucleotide sequence and a second and a third segment of adenoviral genomic DNA, each of said second and third segments being at least 500 bp and being sufficient to mediate homologous recombination with the supercoiled adenoviral vector, wherein the second and third segments flank the first segment; wherein the supercoiled adenoviral vector comprises a deletion of adenovirus transcription unit E3, but comprises a sequence encoding a functional E3 adenoviral death protein and/or a sequence encoding a functional L4 polyadenylation sequence; and wherein the supercoiled adenoviral vector comprises a bacterial origin of replication flanked on each side by segments of DNA identical to the second and third segments whereby subsequent to the step of co-transforming, the supercoiled adenoviral vector and the linear DNA molecule recombine to form a recombinant adenoviral vector comprising the desired polynucleotide sequence.

[0027] The invention also provides a method for generating a recombinant adenoviral hybrid vector comprising a desired polynucleotide sequence and an inverted terminal repeat of an adeno- associated virus^(AAV ITR), comprising the steps of: co-transforming Escherichia CoIi bacteria with: a) a linearlDNA molecule and; b) a supercoiled adenoviral vector comprising an adenoviral genome with one or more deletions; wherein the linear DNA molecule comprises a first segment of DNA comprising a desired polynucleotide sequence and an AAV ITR flanking the desired polynucleotide sequence, and a second and a third segment of adenoviral genomic DNA, each of said second andithird segments being at least 500 bp and being sufficient to mediate homologous recombination with the supercoiled adenoviral vector, wherein the second and third segments flank the first segment; wherein the supercoiled adenoviral vector comprises a deletion of adenovirus transcription unit E3, but comprises a sequence encoding a functional E3 adenoviral death protein and/or a sequence encoding a functional L4 polyadenylation sequence; and wherein the supercoiled adenoviral vector comprises a bacterial origin of replication flanked on each side by segments of DNA identical to the second and third segments whereby subsequent to the step of co-transforming, the supercoiled adenoviral vector and the linear DNA molecule recombine to form a recombinant adenoviral vector comprising the desired polynucleotide sequence and the AAV ITR. [0028] The invention also provides a method for generating a recombinant adenoviral particle comprising a desired polynucleotide sequence, comprising the steps of: a) a linear DNA molecule and; b) a supercoiled adenoviral vector comprising an adenoviral genome with one or more deletions; wherein the linear DNA molecule comprises a first segment of DNA comprising a desired polynucleotide sequence and a second and a third segment of adenoviral genomic DNA, each of said second and third segments being at least 500 bp and being sufficient to mediate homologous recombination with the supercoiled adenoviral vector, wherein the second and third segments flank the first segment; wherein the supercoiled adenoviral vector comprises a deletion of adenovirus transcription unit E3, but comprises a sequence encoding a functional E3 adenoviral death protein and/or a sequence encoding a functional L4 polyadenylation sequence; and wherein the supercoiled adenoviral vector comprises a bacterial origin of replication flanked on each side by segments of DNA identical to the second and third segments whereby subsequent to the step of co- transforming, the supercoiled adenoviral vector and the linear DNA molecule recombine to form a recombinant adenoviral vector comprising the desired polynucleotide sequence; linearizing the recombinant adenoviral vector to form a linear vector comprising termini which comprise adenoviral terminal repeats flanking the desired polynucleotide sequence ; and transfecting a mammalian cell with the linearized vector, whereby the mammalian cell produces the recombinant adenoviral particle which comprises the desired polynucleotide sequence.

[0029] The invention also provides a method for generating a recombinant adenoviral hybrid particle comprising a desired polynucleotide sequence and an inverted terminal repeat of an adeno- associated virus (AAV ITR), comprising the steps of: co-transforming Escherichia CoIi bacteria with: a) a linear DNA molecule and; b) a supercoiled adenoviral vector comprising an adenoviral genome with one or more deletions; wherein the linear DNA molecule comprises a first segment of DNA comprising a desired polynucleotide sequence and an AAV-ITR flanking the desired polynucleotide sequence, and a second and a third segment of adenoviral genomic DNA, each of said second and third segments being at least 500 bp and being sufficient to mediate homologous recombination with the supercoiled adenoviral vector, wherein the second and third segments flank the first segment; wherein the supercoiled adenoviral vector comprises a deletion of adenovirus transcription unit E3, but comprises a sequence encoding a functional E3 adenoviral death protein and/or a sequence encoding a functional L4 polyadenylation sequence; and wherein the supercoiled adenoviral vector comprises a bacterial origin of replication flanked on each side by segments of DNA identical to the second and third segments whereby subsequent to the step of co-transforming, the supercoiled adenoviral vector and the linear DNA molecule recombine to form a recombinant adenoviral vector comprising the desired polynucleotide sequence and the AAV ITR; linearizing the recombinant adenoviral vector to form a linear vector comprising termini which comprise adenoviral terminal repeats flanking the desired polynucleotide sequence and the AAT ITR; and transfecting a mammalian cell with the linearized vector, whereby the mammalian cell produces the recombinant adenoviral particle which comprises the desired polynucleotide sequence and the AAV ITR.

[0030] In some examples, the supercoiled adenoviral vector comprises a sequence encoding a functional E3 adenoviral death protein. In some examples, the supercoiled adenoviral vector comprises a sequence encoding a functional L4 polyadenylation sequence. In some examples, wherein the supercoiled adenoviral vector comprises a sequence encoding a functional E3 adenoviral death protein and a sequence encoding a functional L4 polyadenylation sequence. In some examples, the sequence encoding the functional E3 adenoviral death protein comprises about nucleotide 29,397 to about nucleotide 29,783, wherein the nucleotide numbering is based on wild type adenovirus 5 genome. In some examples, the sequence encoding the functional L4 polyadenylation sequence comprises nucleotide 28,164 to nucleotide 28,169, wherein the nucleotide numbering is based on wild type adenovirus 5 genome. Although nucleotide numbering is generally referenced to wild type adenovirus 5 genome, one skilled in the art can use other corresponding sequences from other type of adenovirus.

[0031] In some examples, the supercoiled adenoviral vector comprises a deletion of adenovirus transcription unit El. For example, the deletion of adenovirus transcription unit El is from about nucleotide 480 to about nucleotide 3533, wherein the nucleotide numbering is based on wild type adenovirus 5 genome. In some examples, when the supercoiled adenoviral vector comprises a deletion of adenovirus transcription unit El, the mammalian cell used for generating the recombinant adenoviral particle or recombinant adenoviral hybrid vector particle expresses adenovirus transcription unit El, which may be expressed transiently or stably.

[0032] In some examples, the deletion of adenovirus transcription unit E3 is from about nucleotide 28593 to about nucleotide 30470, wherein the nucleotide numbering is based on wild type adenovirus^:5 genome.

[0033] In some examples, the first segment comprises an inverted terminal repeat of an adenoviral genome.

[0034] In some examples, the supercoiled adenoviral vector comprises an inverted terminal repeat of an adenoviral genome.

[0035] In some examples, the desired polynucleotide sequence comprises a sequence encoding a multiple cloning site.

[0036] In some examples, the second segment comprises about nucleotide 34931 to about nucleotide 35935, and the third segment comprises about nucleotide 3534 to about nucleotide 5790, wherein the nucleotide numbering is based on wild type adenovirus 5 genome.

[0037] In some examples, the supercoiled adenoviral vector comprises adenovirus 5 genome from nucleotide, 3534 to nucleotide 35935 with a deletion of adenovirus transcription unit E3 except

E3 adenoviral death protein, wherein the deletion of adenovirus transcription unit E3 is from nucleotide 30470 to nucleotide 28593 and the sequence encoding the E3 adenoviral death protein is from nucleotide; 29397 to 29783.

[0038] Ih some examples, the desired polynucleotide sequence comprises a gene encoding a therapeutic product. Examples of therapeutic products include a vaccine, an antigen of a pathogen, an antigen of a viral pathogen, for example, vaccines and antigens described herein. For example, the viral antigen can be an HIV antigen (e.g., gag antigen).

[0039] The invention also provides a recombinant adenoviral vector comprising adenoviral inverted terminal repeats flanking an adenoviral genome and a desired polynucleotide sequence, wherein the adenoviral genome comprises one or more deletions, wherein the deletions comprise a deletion of adenovirus toanscription unit El and E3, wherein the adenoviral genome comprises a sequence encoding a functional E3 adenoviral death protein and/or sequence encoding a functional L4 polyadenylation site.

[0040] The invention also provides an adenoviral hybrid vector comprising adenoviral inverted terminal repeats flanking an adenoviral genome and a desired polynucleotide sequence flanked by an AAT ITR, wherein the adenoviral genome comprises one or more deletions, wherein the deletions comprise a deletion of adenovirus transcription unit El and E3, wherein the adenoviral genome comprises a sequence encoding a functional E3 adenoviral death protein and/or sequence encoding a functional L4 polyadenylation site.

[0041] In some examples, the adenoviral genome comprises a sequence encoding a functional E3 adenoviral death protein. In some examples, the adenoviral genome comprises a sequence encoding a functional L4 polyadenylation sequence. In some examples, the adenoviral genome comprises a sequence encoding a functional E3 adenoviral death protein and a sequence encoding a functional L4 polyadenylation sequence. In some examples, the sequence encoding the functional adenoviral death protein comprises about nucleotide 29,739 to about nucleotide 29,783, wherein the nucleotide numbering is based on wild type adenovirus 5. In some example, the sequence encoding the functional L4 polyadenylation sequence comprises nucleotide 28,164 to nucleotide 28,169, wherein the nucleotide numbering is based on wild type adenovirus 5. In some examples, the deletion of adenovirus transcription unit E3 is from about nucleotide 28593 to about nucleotide 3047:1, wherein the nucleotide numbering is based on wild type adenovirus 5. In some examples, the deletion of adenovirus transcription unit El is from about nucleotide 480 to about nucleotide 3533, wherein the nucleotide numbering is based on wild type adenovirus 5. In some examples, the desired polynucleotide sequence comprises a gene encoding a therapeutic product. Examples of therapeutic product includes a vaccine, an antigen of a pathogen, and an antigen of a viral pathogen, for example, vaccines and antigens described herein. For example, the viral antigen can be an HIV antigen (e.g., gag antigen).

[0042] The invention also provides a method of generating a stock of adenoviral hybrid vectors comprising infecting a mammalian cell with any of the adenoviral hybrid vector described herein.

[0043] The invention also provides a kit comprising two plasmids, wherein the first plasmid comprises (a) a bacterial origin of replication; (b) a first segment of DNA comprising a restriction enzyme site for insertion of a desired polynucleotide sequence; and (c) a second and a third segment of adenoviral genomic DNA, each of said second and third segments being at least 500 bp and being sufficient to mediate homologous recombination with an adenoviral vector, wherein the second and third segments flank the first segment; wherein the second plasmid comprises (a) an adenoviral genome comprising one or more deletions, wherein the deletions comprises a deletion of adenoviral transcription unit E3, wherein the adenoviral genome comprises a sequence encoding a functional E3 adenoviral death protein and/or a sequence encoding a functional L4 polyadenylation site; and (b) a bacterial origin of replication flanked on either side by DNA segments identical to the second and third segments in the first plasmid; wherein upon linearization of the first plasmid and co- transfection of the second plasmid of Escherichia coli bacterial cells, the second plasmid and the linearized first plasmid recombine to form a recombinant adenoviral vector comprising the restriction enzyme site. In some examples, the first segment further comprises an inverted terminal repeat of adenovirus.

[0044] The invention also provides a kit comprising two plasmids, wherein the first plasmid comprises (a) a bacterial origin of replication; (b) a first segment of DNA comprising a restriction enzyme site for insertion of a desired polynucleotide sequence and an AAV ITR flanking the restriction enzyme site; and (c) a second and a third segment of adenoviral genomic DNA, each of said second and third segments being at least 500 bp and being sufficient to mediate homologous recombination with an adenoviral vector, wherein the second and third segments flank the first segment; wherein the second plasmid comprises (a) an adenoviral genome comprising one or more deletions, wherein the deletions comprises a deletion of adenoviral transcription unit E3, wherein the adenoviral genome comprises a sequence encoding a functional E3 adenoviral death protein and/or a sequence encoding a functional L4 polyadenylation site; and (b) a bacterial origin of replication flanked on either side by DNA segments identical to the second and third segments in the first plasmid; wherein upon linearization of the first plasmid and co-transfection of the second plasmid of Escherichia coli bacterial cells, the second plasmid and the linearized first plasmid recombine to form a recombinant adenoviral vector comprising the restriction enzyme site flanked by the AAV ITR. In some examples, the first segment further comprises an inverted terminal repeat of adenovirus. ^: [0045] The kits described herein may used for generating recombinant adenoviral vectors oτ adenoviral hybrid vectors. In some examples, the adenoviral genome comprises a sequence encoding a functional E3 adenoviral death protein. In some examples, the adenoviral genome comprises a sequence encoding a functional L4 polyadenylation sequence. In some examples, the adenoviral genome comprises a sequence encoding a functional E3 adenoviral death protein and a sequence encoding a functional L4 polyadenylation sequence. In some examples, the sequence encoding the functional adenoviral death protein comprises about nucleotide 29,739 to about nucleotide 29,783, wherein the nucleotide numbering is based on wild type adenovirus 5. In some examples, the sequence encoding the functional L4 polyadenylation sequence comprises nucleotide 28,164 to nucleotide 28,169, wherein the nucleotide numbering is based on wild type adenovirus 5. In some examples, the deletion of adenovirus transcription unit E3 is from about nucleotide 28593 to about nucleotide 30471, wherein the nucleotide numbering is based on wild type adenovirus 5. In some examples, the adenoviral genome further comprises a deletion of adenovirus transcription unit El. In some examples, the deletion of adenovirus transcription unit El is from about nucleotide 480 to about nucleotide 3533, wherein the nucleotide numbering is based on wild type adenovirus 5.

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIGS. IA- 1C provide the nucleotide sequence of the rAAV-2 genome, designated herein the AAV-2/HIV gag genome (SEQ ID NO:1), as described in Example 1. [0047] FIG. 2 is a bar graph depicting antibody responses to rAAV-2/HIVgag administration in Rhesus Macaques as described in the Examples. For each set of 4 bar graphs per week, the graphs represent from left to right 3.3e9 (2/6); 3.3elO (6/6); 3.3el 1 (6/6); 3.3el2 (6/6). [0048] FIG. 3 depicts antigen-specific T cell responses to rAAV-2/HIVgag administration in

Rhesus Macaques as described in the Examples. For each set of 4 bar graphs per week, the graphs represent from left to right 3.3e9; 3.3elO; 3.3el l; 3.3el2.

[0049] FIG. 4 is a bar graph depicting ELIspot Responses after a boost dose with pseudotyped rAAV-1 (AAV-2 ITR vaccine gag-pro-ΔRT cassette within an AAV-I capsid), or rAAV-2 (AAV-2 ITR vaccine gag-pro-ΔRT cassette within an AAV-2 capsid) at 80 weeks (shown by arrow) folio-wing rAAV-2 prime dose in Rhesus Macaques at week 0. ELIspot (IFN-γ) responses are against a single peptide pool of HIV-I Clade C Gag.

[0050] FIG. 5 provides a schematic diagram of the structure of a rAAV-2 vector containing a single stranded DNA of 3171 nucleotides encoding gag, protease and part of the reverse transcriptase proteins from HIV Clade C strain (tgAAC09). The HIV coding sequence is referred to as gag-PR-ΔRT.

[0051] FIG. 6 is a bar graph depicting ELISA titers for individual animals at various weeks after a serotype rAAV-2 prime dose (AA V-2 ITR vaccine gag-pro-RT cassette within an AAV-2 capsid) followed by boost dose with rAAV-1 (AAV-2 ITR vaccine gag-pro-RT cassette within an

AAV-I capsid) at week 80 in rhesus macaques.

[0052] FIG. 7 is a plasmid map for pAdM3.1 as described herein in the examples.

[0053] FIG.8 depicts the IFN-γ ELISpot response in Macaques primed with rAAV-2

(tgAAC09) and boosted with AAV-I pseudotype.

DETAILED DESCRIPTION

[0054] The present invention relates to recombinant adeno-associated virus (rAAV) based materials and methods for eliciting an immune response to a pathogen. The present invention provides vector sets comprising a priming vector and a boosting vector, wherein at least one of the vectors in the vector set is a rAAV vector, and methods for eliciting a prophylactic immune response in a mammalian subject susceptible to infection by a pathogen, such as for example, an RNA virus, including HIV-I, and methods for eliciting a therapeutic immune response in a mammalian subject infected by a pathogen that has progressed to a disease state associated with the pathogen, such as for example, an RNA virus, including HIV-I. In particular, the present invention provides methods for eliciting a boost immune response, including an enhanced boost immune response, in a mammalian subject susceptible to infection by or infected by a pathogen comprising administering to the subject an effective amount of a prime dose of an antigen of the pathogen by a recombinant vector; and subsequently administering to said subject an effective amount of a boost dose of an antigen of the pathogen by a recombinant vector, wherein at least one of the prime dose or the boost dose is administered by a recombinant adeno-associated virus (rAAV) vector in a single administration, and wherein the boost dose elicits an equal or greater measurable immune response as compared to the response elicited by a single administration of the prime dose. In some examples, the boost dose elicits a greater measurable immune response as compared to the response elicited by a single administration of the prime dose. The present invention also provides methods for eliciting a boost immune response, including an enhanced boost immune response, in a mammalian subject susceptible to infection by or infected by a pathogen comprising administering to the subject an effective amount of a boost dose of an antigen of the pathogen by a recombinant vector, wherein said mammalian subject has been administered an effective amount of a prime dose of an antigen of the pathogen by a recombinant vector; wherein at least one of the prime dose or the boost dose is administered by a recombinant adeno-associated virus (rAAV) vector in a single administration, and wherein the boost dose elicits an equal or greater measurable immune response as compared to the response elicited by a single administration of the prime dose. In some examples, the boost dose elicits a greater measurable immune response as compared to the response elicited by a single administration of the prime dose. In some examples, the prime dose and the boost dose are both administered by a rAAV vector and the boost dose elicits a greater measurable immune response as compared to the response elicited by a single administration of the prime dose, when the prime dose and boost dose of antigen are in the same formulation. By "same formulation" is meant that pharmaceutical excipients in the formulation are immunologically equivalent. In some examples, the prime dose and the boost dose are both administered by a rAAV vector and the boost dose elicits a greater measurable immune response as compared to the response elicited by a single administration of the prime dose, when the prime dose and boost dose of antigen are equivalent and administered by the same route and in the same formulation. In examples wherein the prime dose and boost dose are administered by AAV vectors, they are not both AAV-2 serotype. [0055] The present invention is based, in part, upon the finding that in the Rhesus Macaques animal model described herein, T-cell and B-cell responses in all animals vaccinated with, that is administered, r AAV-2 expressing the gag protein of HIV-I had waned by week 80 and strongly rebounded after :a single IM administration of a boost dose of the gag protein of HIV-I with either pseudotype AAV-I, comprising AAV-I capsid proteins, or AAV-2, even in animals that received low initial rAAV-2 prime doses and made weak primary responses. Accordingly, the present invention is also based, in part, upon the finding that an AAV based HIV vaccine induces robust B- cell and T-cell responses that are persistent after a single IM administration. The present invention is also based, in part, upon the finding that in the Rhesus Macaques animal model described herein, intramuscular (IM) administration of a pseudotype r A AV-I elicited an equivalent immune response as compared to IM administration of a rAAV-2 expressing the same gag protein of HIV-I at a log₁₀ lower dose after a single administration. IM administration of the boost dose of the HIV-I gag protein with the pseudotype AAV-I elicited a greater boost immune response as compared to the boost immune response elicited by IM administration of the boost dose of the HIV-I gag protein with rAAV-2, and unexpectedly, the pseudotyped AAV-I elicited a higher ratio of B-cell response to T-cell response as compared to the boost immune response elicited by rAAV-2.

General Techniques

[0056] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), immunology, cell biology, and biochemistry which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987 and annual updates); Fundamental Virology, second edition (ed. Fields et al. Raven press) and Current Protocols in Immunology (John Wiley & Sons, Inc., NY).

[0057] As used herein, the term "prime dose" or "prime dosage" refers to the amount of antigen of the pathogen which elicits a measurable immune response in a mammalian subject as compared to the immune response in the mammalian subject in the absence of administration of the antigen. An "effective amount" of a prime dose is that amount of antigen that is capable of eliciting an immune response in the subject. An effective amount of a prime dose may be administered by a rAAV vector or a non-rAAV vector. When the prime dose is administered by a rAAV vector, a single antigen of the pathogen may be administered with a single rAAV that encodes the antigen; two or multiple antigens of the pathogen may be administered with a single rAAV vector that encodes the two or multiple antigens; or two or multiple antigens may be administered with two or multiple rAAV vectors the encode the antigens. When rAAV is used for prime, it is given as a single administration.

[0058] A "single administration" of a prime dose as used herein encompasses administration of one, two or multiple rAAV vectors encoding one, two or multiple antigens, wherein the administration of the two or multiple rAAV vectors may be simultaneous or consecutive over a matter of minutes, or hours, not to exceed about 24 hours, and in examples where rAAV is used for only one of the prime or boost administration, each individual rAAV vector encoding an antigen(s) is only administered once. When the prime dose is administered by a non-rAAV vector, it may be administered by. a single administration or multiple administrations of the non-rAAV priming vector, which may encode one or more antigens and may be administered using one or more non- AAV vectors, which may be delivered simultaneously or consecutively over a period of days, or weeks, and each individual non-rAAV vector encoding an antigen(s) is administered one or more times until a desirable immune response is achieved. Typically, if multiple administrations of a prime dose with one or more non-rAAV vectors are necessary to stimulate a subject's immune response, an effective amount of each prime dose is administered over a period of days up to and including about a week until an effective amount is achieved. By way of example, administration of a priming dose with DNA priming, that is, administering a priming dose of an antigen with a plasmid DNA vector or an adenoviral vector encoding the antigen is generally administered as about three consecutive priming doses over a period of days to weeks in order to generate an effective immune response. A prime dose may be administered to a mammalian subject susceptible to infection by a pathogen or infected by a pathogen. The subject may be a "naive" subject, that is, one not previously infected by the pathogen, or the subject may have been infected by the pathogen previously and not currently exhibiting symptoms or the subject may be currently infected by the pathogen. A desirable priming dose or dosage of antigen can be determined by routine methods known by one of skill in the art based on the vector system chosen and typically range between about for examplelθe8 to about 10el3 PFU (particle forming units) of MVA vectors, about 10e6 to about 10e9 VP (virus particles) of Adenoviral vectors and about 0.5 to about 10 mg of a DNA based vector. The present invention encompasses the use of rAAV based vector constructs comprising genetic elements from any naturally occurring AAV serotype for administration of a prime dose of an antigen as well as the use of genetically engineered rAAV, such as a "pseudotype" rAAV and "chimeric rAAV" for administration of a prime dose of an antigen. A prime dose of an antigen can be administered by any route known in the art, including but not limited to, intravenously; intramuscularly; intramucosally; including for example, nasally, vaginally, rectally; orally; intraperioneally; intradermally; subcutaneously; and interthecally, or can be introduced ex vivo, into cells which have been removed from the host. In the latter case, the transformed cells are reintroduced into the subject where an immune response can be mounted against the antigen encoded by the nucleic acid molecule. The site administration of prime dose is selected based upon the identity and condition of the subject being vaccinated, as well as the type of vector being used. [0059] A "priming vector" as used herein refers to the vector comprising nucleic acid encoding the antigen that is used to administer the prime dose. A "priming vector", "priming vectors", "priming immunization", "prime vaccine" or "prime" as used herein refer to a vector or plurality of vectors that encode(s) an antigen or antigens of the pathogen to which an immune response is to be generated. Priming vectors or vaccines of the invention are administered to the subject or host in an amount effective to elicit an immune response to a pathogen. A priming vector, as known in the art, is the first vector or plurality of vectors that is initially administered to a mammalian subject to elicit an immune response to a pathogen.

[0060] A "boost dose" or "boost dosage" as used herein refers to the amount of an antigen of a pathogen which elicits an immune response upon administration to a mammalian subject which has previously been administered a prime dose of an antigen of the pathogen. In some embodiments of the invention, an "effective amount" of boost dose, measured in DNase Resistant Particles (DRP) in the case of an rAAV vector boost dose administration, is the amount of antigen that elicits an equal or greater measurable immune response as compared to the response elicited by a single administration of the prime dose. When the prime dose and boost dose are both administered by a rAAV vector, an effective amount of the boost dose, is the amount of antigen that elicits an equal or greater measurable immune response as compared to the response elicited by a single administration of the prime dose, when the prime dose and boost dose of antigen are equivalent and administered by the rAAV by the same route and in the same formulation. An effective amount of a boost dose may be administered by a rAAV vector or a non-rAAV vector and may include a single administration of the boost dose or multiple administrations of the boost dose using one or more rAAV and/or non-AAV vectors, which may be delivered simultaneously or consecutively over a period of minutes, hours, days, or weeks. When the boost dose is administered by a rAAV vector, a single antigen of the pathogen may be administered with a single rAAV that encodes the antigen; two or multiple antigens of the pathogen may be administered with a single rAAV vector that encodes the two: or multiple antigens; or two or multiple antigens may be administered with two or multiple rAAV vectors the encode the antigens. When rAAV is used for boost, it is given as a single administration. A "single administration" of a boost dose as used herein encompasses administration of one, two or multiple rAAV vectors encoding one, two or multiple antigens, wherein the administration of the two or multiple rAAV vectors may by simultaneous or consecutive over a matter of minutes, or hours, not to exceed about 24 hours and in examples where rAAV is used for only the boost dose, each individual rAAV vector encoding an antigen(s) is only administered onpe. When the boost dose is administered by a non-rAAV vector, it may be administered by a single administration or multiple administrations of the non-rAAV priming vector, which may encode one or more antigens and may be administered using one or more non- AAV vectors, which may be delivered simultaneously or consecutively over a period of minutes, hours, days or weeks, and each individual non-rAAV vector encoding an antigen(s) can be administered one or more times until a desirable immune response is achieved. The boost dose is administered to the mammalian subject subsequent to the prime dose and in some examples, is administered to the mammalian subject after the immune response to the prime dose has waned as measured by methods known in the art, and in other examples, the boost dose is administered between from about 4 weeks, and up to about 6 weeks, or up to about 3 months, or up to about 6 months, or up to about 9 months, or up to about 12 months, or up to about 15 months, or up to about 18 months, or up to about 21 months, or up to about 24 months or longer subsequent to administration of the prime dose. In some aspects, the mammalian subject is not a mouse. In some aspects wherein the mammalian subject is a mouse, the boost dose is not administered prior to 4 months. A boost dose may be administered to a mammalian subject susceptible to infection by a pathogen or infected by a pathogen. The subject may be a "naϊve" subject, that is one not previously infected by the pathogen, or the subject may have been infected by the pathogen previously and not currently exhibiting symptoms or the subject may be currently infected by the pathogen. A boost dose may be administered to an individual who has become infected with the pathogen after the prime dose was administered but before the boost dose is administered. The present invention encompasses the use of rAAV based vector constructs comprising genetic elements from any naturally occurring AAV serotype for administration of a boost dose of an antigen as well as the use of genetically engineered rAAV, such as a "pseudotype" rAAV and "chimeric rAAV" for administration of a boost dose. A boost dose of an antigen can be administered by any route known in the art, including but not limited to, intravenously; intramuscularly; intramucosally; including for example, nasally, vaginally, rectally; orally; intraperioneally; intradermally; subcutaneously; and interthecally, or can be introduced ex vivo, into cells which have been removed from the host. In the latter case, the transformed cells are reintroduced into the subject where an immune response can be mounted against the antigen encoded by the nucleic acid molecule. The site administration of boost dose is selected based upon the identity and condition of the subject being vaccinated, as well as the type of vector being used. An "enhanced boost" as used herein refers to an immune response to an antigen of the pathogen generated subsequent to administration of a boost dose which is quantitatively or qualitatively greater in magnitude in at least one measured parameter in its immune response than a single administration of the priming dose. As used herein, a boost dose elicits an immune response that is between about 1.5 fold, about 2 fold, about 5 fold, about 10 fold, about 20 fold, and up to about 100 fold or greater in magnitude in at least one measurable parameter of an immune response as compared to the immune response elicited to a single administration of a prime dose. Measurements of immune responses are known in the art and are disclosed herein. For example, as exemplified herein in one illustrative embodiment, the antibody titer may increase from 1/25,600 at a time point post priming to 1/204,800 at a second measured time point post boosting. Alternatively, the number of antigen specific CTL, CD4 or CD8 T cells elicited by administration of a boost dose as measured by ELIspot assay, precursor frequency analysis, or flow cytometry, may increase between about 1.5 fold, about 2 fold, about 10 fold, about 20 fold, and up to about 100 fold or greater as compared to the immune response elicited by administration of a prime dose. [0061] A "boosting vector" as used herein refers to the vector comprising nucleic acid encoding the antigen that is used to administer the boost dose. A "boost vector", "boosting vector", "booster vaccination", "booster immunization", "boost vaccine" or "boost" as used herein refers to a vector of the invention that encodes an antigen or antigens of the pathogen to which an immune response is generated which is delivered, that is, administered, subsequent to a priming vector. A boost vector as used herein encodes an antigen of the pathogen that is related but not necessarily identical to the priming vector. For example, the priming vector may encode an immunogenic protein of the pathogen, or fragment thereof, while the boost vector may encode a different fragment of the protein administered with the priming vector. Alternatively, if the priming vector encodes a plurality of antigens the boosting vector may encode the same or different plurality of the antigens or a single antigen from among the plurality of antigens encoded by the priming vector. As used herein a boosting vector is administered between from about 4 weeks, and up to about 6 weeks, up to about 3 months, or up to about 6 months, or up to about 9 months, or up to about 12 months, or up to about 15 months, or up to about 18 months, or up to about 21 months, or up to about 24 months or longer subsequent to administration of the prime dose A boosting dose as administered by a boosting vector is administered in an amount effective to generate an immune response to the antigen of the pathogen that is equal to or greater than the immune response elicited to the priming dose administered by a priming vector. In some examples, the boost immune response elicited to a boosting dose administered by a boosting vector is greater than the immune response elicited to a single administration of the priming dose administered by a priming vector. Boosting vectors may be of the same vector type as the priming vector, that is both are rAAV or alternatively a boosting vector may be of a different vector construct than the priming vector however, at least one of the vectors of the set is a rAAV vector. As used herein when the priming vector and boosting vector are of the same vector type, that is, when both are rAAV, they may be of the same AAV serotype or alternatively, they may be heterologous, that is, one of the rAAV vectors is an alternate serotype rAAV, pseudotype rAAV, or chimeric rAAV vector. When the priming vector and boosting vector are both rAAV and of the same serotype, they may both be any serotype AAV, including but not limited to AAV-I, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7 or AAV-8. In some examples when the priming vector and boosting vector are both rAAV, a priming vector and boosting vector are not both AAV-2 serotype.

[0062] An "rAAV priming vector", "rAAV priming vectors", or "rAAV prime" as used herein refers to a rAAV vector or plurality of rAAV vectors of the invention that encode an antigen or antigens of the pathogen to which a immune response is to be generated. An rAAV vector of the invention may comprise polynucleotides that encode a single antigen or a plurality of antigens of the pathogen to which an immune response is to be generated. It is within the scope of the invention that a prime dose is administered with a rAAV priming vector or rAAV priming vector composition that may contain a plurality of rAAV vectors that encode multiple antigens of the pathogen. In some examples wherein a prime dose is administered by a plurality of rAAV vectors, the plurality of rAAV vectors maybe heterologous to one another (that is heterologous rAAV serotypes, or a serotype rAAV and pseudotype or chimeric rAAV) and encode the same antigen of the pathogen. [0063] An "rAAV boosting vector" "rAAV boosting vectors" or "rAAV boost" "rAAV boost vaccine" as used herein refers to an rAAV vector or plurality of rAAV vectors of the invention that encode an antigen or antigens of the pathogen to which a immune response is to be generated which is delivered subsequent to a priming vector dose of the pathogen. As used herein the rAAV boosting vector or vectors are administered as a single administration, unlike non-rAAV vectors wherein a boost dose of an antigen(s) is administered by a single vector or plurality of vectors, which can be administered consecutively or in a series of administrations over a period of minutes, hours, days, or weeks until a desirable immune response is achieved. An rAAV vector of the invention may comprise polynucleotides that encode a single antigen or a plurality of antigens of the pathogen to which an immune response is to be generated. It is within the scope of the invention that a rAAV boosting vector or rAAV boosting vector composition may contain a plurality of rAAV vectors that encode multiple antigens of the pathogen. In some examples wherein a boost; dose is administered by a plurality of rAAV vectors, the plurality of rAAV vectors maybe heterologous to one another (that is heterologous rAAV serotypes, or a serotype rAAV and pseudotype or chimeric rAAV) and encode the same antigen of the pathogen. [0064] A "vector set" or "vaccine set" as used herein comprises a priming vector or priming vaccine and a boosting vector or boosting vaccine wherein each encode at least one of a shared immunogenic determinant, a cross priming immunogenic determinant, a shared antigen, immunogenic protein or peptide, or fragment thereof.

[0065] An "antigen" refers to a molecule containing one or more epitopes (either linear, conformational or both) or immunogenic determinants that will stimulate a host's immune-system, such as a mammal's immune system, to make a humoral and/or cellular antigen-specific response. The term is used interchangeably with the term "immunogen." An antigen may be a whole protein, a truncated protein, a fragment of a protein or a peptide. Antigens may be naturally occurring, genetically engineered variants of the protein, or may be codon optimized for expression in a particular mammalian subject or host. Generally, a B-cell epitope will include at least about 5 amino acids but, can be as small as 3-4 amino acids. A T-cell epitope, such as a CTL epitope, will include at least about 7-9 amino acids, and a helper T-cell epitope at least about 12-20 amino acids. Normally, an epitope will include between about 7 and 15 amino acids, such as, 9, 10, 12 or 15 amino acids. The term "antigen" denotes both subunit antigens, (i.e., antigens which are separate and discrete from a whole organism with which the antigen is associated in nature). Antibodies such as anti-idiotype antibodies, or fragments thereof, and synthetic peptide mimotopes, that is synthetic peptides which can mimic an antigen or antigenic determinant, are also captured under the definition of antigen as used herein. For purposes of the present invention, antigens can be obtained from any of several known pathogenic viruses, bacteria, parasites and fungi. In some examples, the antigen is obtained from a viral pathogen, such as for example an RNA viral pathogen, including, but not limited to HIV, including HIV-I and HIV-II; SIV; hepatitis, including hepatitis A, B and C; SARS; and rabies. Furthermore, for purposes of the present invention, an "antigen" refers to a protein which includes modifications, such as deletions, additions and substitutions, generally conservative in nature, to the naturally occurring sequence, so long as the protein maintains the ability to elicit an immunological response, as defined herein. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the antigens. Antigens of the present invention may also be codon optimized by methods kno!wn in the art to improve their expression or immunogenicity in the host. As used herein, a "cross priming" immunogenic determinant refers to a determinant, eptitope, or antigen which is capable of eliciting an immune response to related but not identical antigenic determinants, for example a cross priming determinant for HIV would be an antigen capable of eliciting an immune response to two or multiple or all members of the HIV antigen across clades. [0066] An "immunological response" or "immune response" to an antigen, or vector or vaccine or composition comprising the antigen, is the development in a mammalian subject of a humoral and/or a cellular immune response to an antigen or antigens present in a vector set. For purposes of the present invention, a "humoral immune response" refers to an immune response mediated by antibody molecules or immunoglobulins. Antibody molecules of the present invention include the classes of IgG ( as well as subtypes IgGl, IgG 2a, and IgG2b) , IgM, IgA, IgD, and IgE. Antibodies functionally include antibodies of primary immune response as well as memory antibody responses or serum neutralizing antibodies . The antibodies of the present invention also include cross- reactive, cross protective or cross clade antibody responses. Antibodies of the present invention may serve to, but are not required to, neutralize or reduce infectivity and/or mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to the antigen of the pathogen. ;

[0067] A "cellular immune response" is one mediated by T-lymphocytes and/or other white blood cells, including without limitation NK cells and macrophages. T lymphocytes of the present invention include T cells expressing alpha beta T cell receptor subunits or gamma delta receptor expressing T cells and may be either effector or suppressor T cells. "T lymphocytes" or "T cells" are non-antibody producing lymphocytes that constitute a part of the cell-mediated arm of the immune system. T cells arise from immature lymphocytes that migrate from the bone marrow to the thymus, where they undergo a maturation process under the direction of thymic hormones. Maturing T cells become immunocompetent based on their ability to recognize and bind a specific antigen. Activation of immunocompetent T cells is triggered when an antigen binds to the lymphocyte's surface receptors It is known that in order to generate T cells responses, antigen must be synthesized within or introduced into cells, subsequently processed into small peptides by the proteasome complex, and translocated into the endoplasmic reticulum/Golgi complex secretory pathway for eventual association with major histocompatibility complex (MHC) class I proteins. Functionally cellular immunity includes antigen specific cytotoxic T cells (CTL). Antigen specific T cells, CTL, or cytotoxic T cells as used herein refers to cells which have specificity for peptide antigens presented in association with proteins encoded by the major histocompatability complex (MHC) or human leukocyte antigens (HLA) as the proteins are referred to in humans. CTLs of the present invention include activated CTL which have become triggered by specific antigen in the context of MHC; and memory CTL or recall CTL to refer to T cells that have become reactivated as a result of re-exposure to antigen as well as cross-reactive or cross clade CTL. CTLs of the present invention include CD4+ and CD8+ T cells. Activated antigen specific CTLs of the present invention promote the destruction and/or lysis of cells of the subject infected with the pathogen to which the CTL are specific, blocking pathogen entry via secretion of chemokines and cytokines including without limitation macrophage inflammatory protein 1 a (MIP-Ia), MIP-IB, and RANTES; and secretion of soluable factors that suppress infections. Cellular immunity of the present invention also refers to antigen specific response produced by the T helper subset of T cells. Helper T cells act to help stimulate the function, and focus the activity of nonspecific effector cells against cells displaying peptide in association with MHC molecules on their surface. A cellular immune response also refers to the production of cytokines, chemokines and other such molecules produced by activated T cells and/ or other white blood cells including those derived from CD4 and CD8 T cells and NK cells. A prime dose or boost dose, or a composition or vaccine comprising a prime dose or a boost dose, that elicits a cellular immune response may serve to sensitize a mammalian subject by the presentation of antigen in association with MHC molecules at the cell surface. The cell-mediated immune response is directed at, or near, cells presenting antigen at their surface. In addition, antigen-specific T-lymphocytes can be generated to allow for the future protection of an-immunized host. The ability of a particular antigen to stimulate a cell-mediated immunological response may be determined by a number of assays known in the art, such as by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, or by assaying for T-lymphocytes specific for the antigen in a sensitized subject. Such assays are well known in the art. See, e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al, Eur. J. Immunol. (1994) 24:2369-2376. Methods of measuring cell-mediated immune response include measurement of intracellular cytokines or cytokine secretion by T-cell populations, or by measurement of epitope specific T-cells (e.g., by the tetramer technique) (reviewed by McMichael, A. J., and O'Callaghan, C. A., J. Exp. Med. 187(9)1367-1371, 1998; Mcheyzer- Williams, M. G., et al, Immunol. Rev. 150:5-21, 1996; Lalvani, A., et al, J. Exp. Med. 186:859-865, 1997). An immunological response, or immune response, as used herein encompasses one which stimulates the production of CTLs, and/or the production or activation of helper T-cells and/or an antibody-mediated immune response. [0068] An "immunological response" or "immune response" as used herein encompasses at least one or more of the following effects: the production of antibodies by B-cells; and/or the activation of suppressor T-cells and/or T-cells directed specifically to an antigen or antigens present in the vectors, composition or vaccine of interest. As used herein, an "enhanced boost immune response" refers to administration of boost dose by a vector that elicits a greater measurable immune response as compared to the response elicited by a single administration of the prime dose. When the prime dose and boost dose are both administered with a rAAV, the comparison of immune response is made under conditions where the prime dose and boost dose of antigen are in the same formulation. By "same formulation" is meant that pharmaceutical excipients in the formulation are immunologically equivalent. When the prime dose and boost dose are both administered with a rAAV, the comparison of immune response is made under conditions where the prime dose and boost dose of antigen are equivalent and the prime and boost doses are administered by rAAV by the same route and in the same formulation. In some examples herein, methods are provided wherein the boost immune response to an antigen of a pathogen, in particular an antigen of HIV-I, is enhanced and. may serve to neutralize infectivity (but the methods of the present invention do not require that infectivity is neutralized), and/or mediate antibody-complement, and/or antibody dependent cell cytotoxicity (ADCC) and may provide protection to an immunized host or subject. Such responses can be determined using standard immunoassays and neutralization assays, well known in the art. The methods of the present invention which, in some examples, provide for an enhanced boost response to an antigen of a pathogen do not require that protection from the pathogen is provided to an immunized host or mammalian subject. A boost dose administered with a vector and under conditions which provide for an enhanced boost immune response may be administered to a naive individual, an individual who has been infected with a pathogen and has no symptoms or to an individual who has been infected with a pathogen and exhibits symptoms. An enhanced boost immune response may serve to reverse or slow the progression of the disease as compared to subjects who do not receive administration of a boost dose, or slow symptoms or the duration of symptoms of the disease as compared to subjects who do not receive administration of a boost dose.

[0069] An "immunogenic composition" or "vaccine compostion" is a composition that comprises an antigen or antigenic molecule or molecules wherein administration of the composition to a subject results in the development in the subject of a humoral and/or a cellular immune response to the antigen.

[0070] A "prophylactic vaccine" as used herein refers to a vaccine composition administered to a mammalian subject or host that is " immunologically naive" or has not been previously exposed to the antigen of the pathogen or one that has not generated an effective immune response to the pathogen to prevent infection or re-infection (however the present invention does not require that the infection or ire-infection is completely prevented). Prophylactic vaccines of the present invention do not necessarily generate sterilizing immunity in the host or subject to which they have been administered. [0071] A "therapeutic vaccine" as used herein refers to a vaccine composition administered to a subject or host that has been previously infected with the pathogen and has progressed to a disease state associated with the pathogen to which the vaccine is directed. [0072] As used herein the term "immunization" "immunize" or "immunized", refers to the process of administering a vaccine to a live mammalian subject or host in an effective amount to induce an immune response to the antigen of the pathogen. An "rAAV vaccine" as used herein refers to an AAy vector comprising a polynucleotide sequence not of AAV origin herein referred to also as the transgene (i.e., a polynucleotide heterologous to AAV), that encodes a peptide, polypeptide, protein or antigen of a pathogen that is capable of eliciting an immune response in a mammalian subject or host contacted with the vector. Expression of the polynucleotide may result in generation of a neutralizing antibody response and/or a cell mediated response, e.g., a cytotoxic T cell response.

[0073] T lymphocytes" or "T cells" are non-antibody producing lymphocytes that constitute a part of the cell-mediated arm of the immune system. T cells arise from immature lymphocytes that migrate from the bone marrow to the thymus, where they undergo a maturation process under the direction of thymic hormones. Here, the mature lymphocytes rapidly divide increasing to very large numbers. The maturing T cells become immunocompetent based on their ability to recognize and bind a specific aintigen. Activation of immunocompetent T cells is triggered when an antigen binds to the lymphocyte's surface receptors.

[0074] "AAV" is adeno-associated virus, and as used herein refers to the naturally occurring wild-type virus itself or derivatives thereof. The abbreviation "rAAV" refers to recombinant adeno- associated virus. The term AAV encompasses all subtypes, serotypes and pseudotypes, as well as naturally occurring and recombinant forms. A variety of AAV serotypes and strains are known in the art and are publically available from sources, such as the ATCC, and academic or commercial sources. Alternatively, sequences from AAV serotypes and strains which are published and/or available from a variety of databases may be synthesized using known techniques. As used herein, the term "serotype" refers to an AAV which is identified by and distinguished from other AAVs based on capsid protein reactivity with defined antisera. There are at least eight known serotypes of human AAV, including AAV-I through AAV-8. For example, AAV-2 serotype is used to refer to an AAV which contains capsid proteins encoded from the cap gene of AAV-2 and a genome containing 5' and 3' ITR sequences from the same AAV-2 serotype. A "pseudotyped" AAV refers to an AAV that contains capsid proteins from one serotype and a viral genome including 5 'and 3' ITRs of a different or heterologous serotype. A pseudotyped rAAV would be expected to have cell surface binding properties of the capsid serotype and genetic properties consistent with the ITR serotype. A pseudotype rAAV may comprise rAAV capsid proteins, including VPl, VP2, and VP3 capsid proteins, and ITRs from any serotype AAV, including any primate AAV serotype from AAV-I through AAV- 8, as long as the capsid protein is of a serotype heterologous to the serotype(s) of the ITRs. In a pseudotype rAAV, the 5' and 3' ITRs may be identical or heterologous. Pseudotyped rAAV are produced using standard techniques described in the art. A "chimeric" rAAV vector encompasses an AAV vector comprising heterologous capsid proteins; that is, a rAAV vector may be chimeric with respect to its capsid proteins VPl, VP2 and VP3, such that VPl, VP2 and VP3 are not all of the same serotype AAV. A chimeric AAV as used herein encompasses AAV wherein the capsid proteins VPl, VP2 and VP3 differ in serotypes, including for example but not limited to capsid proteins from AAV-I and AAV-2; are mixtures of other parvo virus capsid proteins or comprise other virus proteins or other proteins, such as for example, proteins that target delivery of the AAV to desired cells or tissues. A chimeric rAAV as used herein also encompasses a rAAV comprising chimeric 5' and 3' ITRs. The present invention encompasses chimeric rAAV vectors that comprise ITRs from different AAV serotypes, for example AAVl and AA V2, or a chimeric rAAV may comprise synthetic sequences. "rAAV vaccine" as used herein refers to an rAAV vector comprising a polynucleotide sequence heterologous to AAV, which may be a transgene or nucleic acid sequence that encodes a peptide, polypeptide, or protein capable of eliciting an immune response in a host or mammalian subject contacted with the vector. Expression of the polynucleotide may, but does not necessarily require, generation of a neutralizing antibody response and/or a cell mediated response, e.g., a cytotoxic T cell response. [0075] Gene delivery expression "vectors" as used herein include, but are not limited to, vectors derived from non-viral vectors, such as for example, bacterial nucleic acid vectors, including plasmjd vectors, and viral vectors, such as for example, adenovirus vectors, alphaviruses, pox viruses andivaccinia viruses. When used for immunization, such gene delivery expression vectors may be referred to as vaccines or vaccine vectors. As used herein, a "non-AAV" vector encompasses, but is not limited to vectors selected from the group consisting of plasmid DNA, adenovirus, canarypox virus, pox virus, vaccinia virus, modified vaccinia Ankara (MVA)₅ alphavirus, phabdovirus, and attenuated HIV.

[0076] A "nucleic acid" molecule can include, but is not limited to, procaryotic sequences, eucaryotic mRNA, cDNA from eucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. The term also captures sequences that include any of the known base analogs of DNA and RNA.

[0077] "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence. [0078] Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature. "Recombinant host cells," "host cells," "cells," "cell lines," "cell cultures," and other such terms denoting procaryotic microorganisms or eucaryotic cell lines cultured as unicellular entities, are used interchangeably, and refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell which are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition, and are covered by the above terms.

[0079] As used herein "mammalian subject" or "host" is meant any member of the subphylum chordata, including, without limitation, humans and other primates, including non- human.primates: such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered. [0080] By "pharmaceutically acceptable" or "pharmacologically acceptable" is meant a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual in a formulation or composition without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0081] A vector set comprising a priming vector and boosting vector may be administered

"prophylactically" to a mammalian subject susceptible to infection by a pathogen, that is administered prior to infection or post infection but prior to onset of the disease state associated with the pathogen, or "therapeutically", that is following induction of the disease state associated with the pathogen. Prophylactic administration means that a prime vector, boost vector or vector set is being administered to a mammalian subject susceptible to infection by a pathogen, that is administered prior to infection or post infection but prior to onset of the disease state associated with the pathogen. Therapeutic administration means that a prime vector, boost vector or vector set is being administered to a mammalian subject that has the disease state associated with the pathogen. A vector set comprising a priming vector and boosting vector may be administered to prevent infection or re-infection of a pathogen, such as by eliciting production of neutralizing antibodies or CTL; however, the present invention does not require that neutralizing antibodies are produced and does not require that protection is afforded the subject to whom the vector set is administered. A vector set comprising a priming vector and boosting vector may be administered to reduce, minimize or eliminate the symptoms of, or reduce the duration of the symptoms of infection by a pathogen or to slow the progression from initial infection to disease state. [0082] As used herein, "AIDS" refers to the symptomatic phase of HIV infection, and includes both Acquired Immune Deficiency Syndrome (commonly known as AIDS) and "ARC," or AIDS-Related Complex, as described by Adler, Brit. Med. J. 294: 1145 (1987). The immunological and clinical manifestations of AIDS are well known in the art and include, for example, opportunistic infections and cancers resulting from immune deficiency. A vector set comprising a priming vector and boosting vector may be administered "prophylactically" to a mammalian subject susceptible to AIDS or ARC, that is administered prior to infection with HIV, or post infection with HIV but prior to the appearance of symptoms or AIDS or ARC in a subject, for preventing initial infection of a subject exposed to HIV or at risk for exposure to HIV, (although the methods of the present invention do not require that HIV infection is prevented); for reducing viral burden in a subject infected with HIV; for prolonging the asymptomatic phase of HIV infection in a subject; for increasing overall health or quality of life in a subject with AIDS; and for prolonging life expectency of a subject with AIDS. An appropriate clinician can compare the effect of administration of a vector set encompassed within the present invention to a subject's condition prior to treatment, or with the expected condition of an untreated subject, to determine the outcome. [0083] Nucleic acid expression vector" or "Expression cassette" refers to an assembly which is capable of directing the expression of a sequence or gene of interest. The nucleic acid expression vector includes a promoter which is operably linked to the sequences or gene(s) of interest. Other control elements may be present as well. Expression cassettes described herein may be contained within a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include a bacterial origin of replication, one or more selectable markers, a signal which allows the plasmid construct to exist as single-stranded DNA (e.g., a M13 origin of replication), a multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).

[0084] As used herein, the term "kit" refers to components packaged or marked for use together. For example, a kit can contain a vector set of the present invention that provides for an enhanced boost immune response to an HIV-I antigen. A kit may contain the priming vector and boosting vector in separate containers and optionally, a kit further contains instructions for combining the components so as to formulate an immunogenic composition suitable for administration to a mammal.

[0085] As used herein, the singular forms "a", "an" and "the" include plural references unless explicitly stated otherwise.

Methods of eliciting an immune response to antigens of viral pathogens

[0086] The present invention encompasses methods for eliciting an immune response, and in particular encompasses methods for eliciting a boost immune response, including an enhanced boost immune response, to antigens obtainable from or derived from known viral pathogens responsible for diseases including for example, but not limited to, AIDS; hepatitis, including hepatitis A, B, and C; rabies; SARS; poliomyelitis; measles; mumps; rubella; and influenza as well as feline leukemia virus, equine infectious encephalitis, distemper, and heartworms. Viral pathogens encompassed within the present invention include, for example, human and simian immunodeficiency virus (SIV and HIV including HIV-I and HIV-2); hepatitis virus, including A, B and C; influenza virus; polio virus; measles virus; mumps virus; rubella virus; rabies virus, cytomegalovirus; SARs virus; human T-lymphotrophic virus types I and II (HTLV-I and HTLV-II); rotavirus and hantavirus. In some examples, the viral pathogen is an RNA viral pathogen, such as for example, SIV and HIV; hepatitis virus; SARs virus; and rabies virus. In some examples, the viral pathogen is HIV-I . See, e.g. Virology, 3rd Edition (W. K. Joklik ed.^" 1988); Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Rnipe, eds. 1991; Virology, 3rd Edition (Fields, B N, D M Knipe, P M Howley, Editors, 1996, Lippincott-Raven, Philadelphia, Pa.) for a description of these and other viruses. [0087] Human immunodeficiency virus is a retrovirus; two broad classes of the HIV virus have been identified, HIV- 1 and HIV-2. Three classes of HIV-I have developed across the globe: M (major), 0 (outlying) and N (new). Among the M group, which accounts for greater than 90% of reported HIV/ AIDS cases, viral envelopes have diversified so greatly that this group has been sub classified into major clades including A-D, F-H, J and K, as well as several circulating recombinant forms. Viral diversity appears to radiate out of sub-Saharan Africa, where over 28 million of the total 40 million infected persons live. HIV-2, has not spread much beyond West Africa, where it is presently endemic. Some sporadic cases have been observed elsewhere in Africa but the virus appears to be significantly less pathogenic than HIV-I. One particular subtype of HIV-I appears to have achieved phylogenetic dominance. Subtype C viruses now account for over 50% of new HIV-I infections in the world. In particular, this clade has ravaged much of sub-Saharan Africa, and is now encroaching into Indochina (Beyrer, C. et al, (2000) AIDS, 14(1): 75-83; Yu, X.F. et al, (2001) AIDS 15(4): 523-5; Piyasirisilp, S. et al, (2000) AIDS 74(23): 1128695). As used herein, the term "HIV-I" refers to all forms, subtypes and variations of the HIV-I virus, and is synonymous with the older terms HTLVIII and LAV. Various cell lines permanently infected with the HIV virus have been developed and deposited with the ATCC, including those having accession numbers CCL 214, TIB 161, CRL 1552 and CRL 8543, all of which are described in U.S. Pat. No. 4,725,669 and Gallo, Scientific American, 256:46 (1987). Details on the HlV genome may also be found in the NCBI GenBank database (i.e. Accession Nos. AF033819).

[0088] The HIV-I retroviral genome comprises genes called gag, pol and env, which code for virion proteins and enzymes. These genes are flanked at both ends by regions called long terminal repeats: (LTRs). The LTRs are responsible for pro viral integration, and transcription. They also serve as enhancer-promoter sequences. The LTRs can control the expression of the viral genes. Encapsidation of the retroviral RNAs occurs by virtue of a sequence located at the 5' end of the viral genome. With regard to the structural genes gag, pol and env themselves; gag encodes the internal structural protein of the virus. Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid). The pol gene encodes the reverse transcriptase (RT), which contains DNA polymerase, associated RNase H and integrase (IN), which mediate replication of the genome. The env gene encodes the surface (SU) glycoprotein and the transmembrane (TM) protein of the virion, which form a complex that interacts specifically with cellular receptor proteins. This interaction leads ultimately to infection by fusion of the viral membrane with the cell membrane. Co-expression of gag, pol, and env result in formation of infectious virion particles. For the purposes of immunogenic compositions and vaccine production, formation of infectious particles would result in a dangerous and unacceptably risky situation. In some examples, the present invention provides vector sets comprising priming and boosting vectors for administration of antigens of HIV-I that elicit an immune response but do not assemble into infectious or noninfectious particles. HIV also contains additional genes that code for proteins other than gag, pol and env. Additional genes in HIV are vif, vpr, vpx, vpu, tat, rev and nef. Proteins encoded by additional genes serve various functions, some of which may be duplicative of a function provided by a cellular protein. In HIV, tat acts as a transcriptional activator of the viral LTR. It binds to a stable, stein-loop RNA secondary structure referred to as TAR. Rev regulates and co-ordinates the expression of viral genes through rev-response elements (RRE).

[0089] HIV antigens as used herein include, but are not limited to gag (p55, p39, p24, pi 7 and pl5), the pol (p66/p51 and p31-34), the transmembrane glycoprotein gp41 and envelope (gpl60), including, but not limited to, for example, native gpl60, oligomeric gpl40, monomeric gpl20 as well as modified sequences of these polypeptides.. These gene products may be used alone or in combination with other HIV antigens. [0090] The present invention comprises methods for enhancing a boost immune response to

HIV-I antigens as described above and includes HIV-I antigens from a variety of families, subtypes and strains and including but not limited to isolates, HIVπib, HIVSF2, HIV-1SFI62, HIV-1_SFI7₀, HIVLAV₅ HIVLAI, HIVMN, HIV-1CM235_> HIV-1US4, other HIV-I strains. See, e.g., Myers, et al., Los Alamos Database, Los Alamos National Laboratory, Los Alamos, New Mexico; Myers, et al., Human Retroviruses and Aids, 1990, Los Alamos, New Mexico: Los Alamos National Laboratory. [0091] The HIV codon usage reflects a high content of the nucleotides A or T of the codon- triplet. The effect of the HIV-I codon usage is a high AT content in the DNA sequence that results in a decreased translation ability and instability of the mRNA. In comparison, highly expressed human codons prefer the nucleotides G or C. In some examples, the HIV-I antigen codon usage pattern is modified as described in for example, U.S. Pat. No. 6,602,705, specifically incorporated herein in its entirety by reference, so that the resulting nucleic acid coding sequence is comparable to codon usage found in highly expressed human genes.

[0092] In illustrative embodiments disclosed herein, the HIV-I antigen is a clade C gag/pro sequence comprising a deletion of nucleotides from reverse transcriptase, referred to herein as HIV- 1 Clade C gag/pro-Δ, the sequence of which was codon optimized.

[0093] Accordingly, the present invention provides vector sets that comprise a priming vector, that is, a: vector for administration of a prime dose, and a boosting vector, that is, a vector for administration of a boost dose, and methods for administering a prime dose (via the priming vector) and a boost dose (via a boosting vector) of an antigen of a pathogen to an individual susceptible to infection with a pathogen or infected with a pathogen, such as for example, HIV-I, wherein at least one vector of the vector set is a rAAV vector, and wherein the boost dose elicits an equal or greater measurable immune response as compared to the immune response elicited by a single administration of the prime dose. In examples wherein both the prime dose and boost dose are administered with a rAAV vector, the boost dose elicits an equal or greater measurable immune response as compared to the immune response elicited by a single administration of the prime dose, when the prime ;dose and boost dose of antigen are in the same formulation. In examples wherein both the prime dose and boost dose are administered with a rAAV vector, the boost dose elicits an equal or greater measurable immune response as compared to the immune response elicited by a single administration of the prime dose, when the prime dose and boost dose of antigen are equivalent and administered by the same route and in the same formulation. In some examples, the boost dose elicits a greater measurable immune response as compared to the immune response elicited by a single administration of the prime dose. In some examples, the priming vector and the boosting vector of the vector sets will comprise nucleic acid encoding at least one shared immunogenic determinant, cross-priming immunogenic determinant, antigen, immunogenic protein, peptide or fragment thereof.

[0094] In some examples, only the prime dose of the antigen of the pathogen is administered by a rAAV in a single administration, and in other examples, only the boost dose of the antigen of the pathogen is administered by a rAAV in a single administration. In yet other examples, the prime dose and boost dose are both administered by a rAAV, with each of the prime dose and boost dose administered in a single administration. In some examples, an AAV vector of the vector set encompasses any primate AAV serotype, including, for example, primate AAV serotypes 1 through 8, as well as avian AAVs known in the art. A rAAV vector may be a pseudotyped AAV. A rAAV vector may be a chimeric rAAV vector.

[0095] In some examples, wherein the priming vector is a rAAV vector, the prime dose is administered by a single administration of an amount of antigen effective to elicit an immune response, and the priming rAAV vector may be a naturally occurring AAV serotype from any species, a pseudotyped rAAV vector, or a chimeric rAAV vector. The prime dose for an antigen of a pathogen is at least about 10⁵, at least about 10⁶, at least about 10⁷, at least about 10⁸, at least about 10⁹' at least about 10¹⁰, at least about 10¹¹, at least about 10¹², at least about 10¹³ or at least about 10^14> DNAse Resistant Particles (DRPs) of rAAV. In some examples, the rAAV priming vector is a single rAAV vector comprising nucleic acid encoding a single antigen of a pathogen, such as for example a single antigen of HIV-I, or multiple antigens of the pathogen, such as for example, multiple antigens of HIV-I. In other examples, a plurality of rAAV priming vectors comprising nucleic acids encoding the same antigen (wherein the plurality of rAAV vectors are heterologous with respect to one another) or multiple antigens of the pathogen may be administered as a single administration, which include sequential administration, over a period of minutes or hours, not to exceed about 24 hours, as long as an individual rAAV vector is only administered once. In some examples, a single injection of the priming rAAV vector(s) is administered as a simple, mixed formulation or multiple injections of the vector(s) is administered sequentially within a duration of minutes, such as for example, within about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, or about 60 minutes. In some examples wherein the priming dose is administered by a plurality of rAAV vectors, it is within ordinary skill in the art to determine the ratio at which the vectors would be administered to achieve the desired immune response. [0096] Ih other examples, the priming vector is a non-rAAV vector, including plasmid

DNA, adenovirus, canarypox virus, pox virus, vaccinia virus, modified vaccinia Ankara (MVA), alphavirus, rhabdovirus, and attenuated HIV. In some examples, non-rAAV priming vectors are administered as a single dose or as a multiple dose, over a period of days or weeks using dosing strategies known in the art to be effective to achieve an appropriate prime immune response in a mammalian subject. Without being bound by theory, a single non-rAAV vector encoding an antigen may need to be administered more than once in order to achieve a desired immune response. [0097] In some examples, wherein the boosting vector is a rAAV vector, the boost dose is administered by a single administration of an amount of antigen effective to elicit an equal or greater, that is enhanced, measurable immune response, as compared to the response elicited by a single administration of the prime dose and the boosting rAAV vector may be a naturally occurring AAV serotype from any species, a pseudotyped rAAV vector, or a chimeric rAAV vector. The boost dose for an antigen of a pathogen is at least about 10⁵,.at least about 10⁶, at least about 10⁷, at least about 10⁸, at least about 10⁹' at least about 10¹⁰, at least about 10^u, at least about 10¹², or at least about 10¹³,^: at least about 10¹⁴Dnase Resistant Particles (DRPs) of rAAV. In some examples, the rAAV boosting vector is a single rAAV vector comprising nucleic acid encoding a single antigen of a pathogen, such as for example a single antigen of HIV-I, or multiple antigens of the pathogen, such as for example, multiple antigens of HIV-I. In other examples, a plurality of rAAV vectors comprising nucleic acid encoding the same or multiple antigens of the pathogen may be administered in a single administration or administered sequentially over a period of minutes, or hours, not to exceed about 24 hours. In some examples, a single injection of the boosting rAAV vector(s) is administered. In some examples wherein the boosting dose is administered by a plurality of vectors, it is within ordinary skill in the art to determine the ratio at which the vectors would be administered to achieve the desired boost immune response.

[0098] In other examples, wherein the priming vector is a rAAV vector, the boosting vector is a non-rAAV vector, including plasmid DNA, adenovirus, canarypox virus, pox virus, vaccinia virus, modified vaccinia Ankara (MVA), alphavirus, rhabdovirus, and attenuated HIV. Additional administration scenarios are provided in Table I.

TABLE 1

...W en e priming vec or an oos ing vec or are o r , a priming vec or an oos ing vec or are no o - serotype.

[0099] Ih some examples, the present invention relates to methods useful for eliciting an immune response in individuals susceptible to infection or infected but not progressed to AIDS or ARC disease by HIV-I or for eliciting therapeutic immune responses in individuals who have progressed to AIDS or ARC. In an illustrative example disclosed herein, in the Rhesus Macaques model described in the examples, T-cell and B-cell responses in all animals vaccinated with, that is administered, rAAV-2 expressing the gag protein of HIV-I had waned by week 80 and strongly rebounded after a single IM administration of a boost dose of the gag protein of HIV-I with either pseudotype AAV-I, comprising AAV-I capsid proteins, or AAV-2, even in animals that received low initial rAAV-2 prime doses and made weak primary responses. Accordingly, the present invention is also based, in part, upon the finding that an AAV based HIV vaccine induces robust B- cell and T-cell responses that are persistent after a single IM administration. The present invention is also based, inpart, upon the finding that in the Rhesus Macaques animal model described herein, intramuscular (IM) administration of a pseudotype rAAV-1 elicited an equivalent immune response as compared to IM administration of a rAAV-2 expressing the same gag protein of HIV-I at a logjo lower dose after a single administration. IM administration of the boost dose of the HIV-I gag protein with the pseudotype AAV-I elicited a greater boost immune response as compared to the boost immune response elicited by IM administration of the boost dose of the HIV-I gag protein with rAAV-2, and unexpectedly, the pseudotyped AAV-I elicited a higher ratio of B-cell response to T-cell response as compared to the boost immune response elicited by rAAV-2. In other examples, the HIV-I antigen administered in each of the priming vector and boosting vector comprises a shared immunogenic determinant, a cross priming immunogenic determinant, a shared antigen, immunogenic protein or peptide, or fragment thereof. By way of example, a priming vector for administration of a prime dose of an antigen of HIV-I may encode a rev, gag, and env antigen, or fragments thereof, and the boosting vector for administration of a boost dose subsequent to the prime dose may; encode, for example, the rev, gag, env, RT, vif, and/or tat antigen, or fragment thereof. In other examples, an antigen of HIV-I is codon optimized for expression in the particular mammalian subject. In some examples, the mammalian host is a non-human primate and in other examples is a human primate.

Methods of measuring immune response

[0100] A variety of in vitro and in vivo assays are known in the art for measuring an immune response, including measuring humoral and cellular immune responses, which include but are not limited to standard immunoassays, such as RIA, ELISA assays; intracellular staining; T cell assays including for example, lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, or by assaying for T-lymphocytes specific for the antigen in a sensitized subject. Such assays are well known in the art. See, e.g., Erickson et al., J. Immunol. (1993) 151 :4189-4199; Doe et al., Eur. J. Immunol. (1994) 24:2369-2376. Recent methods of measuring cell-mediated immune response include measurement of intracellular cytokines or cytokine secretion by T-cell populations, or by measurement of epitope specific T-cells (e.g., by the tetramer technique) (reviewed by McMichael, AJ., and O'Callaghan, C.A., J. Exp. Med. 187(9)1367-1371, 1998; Mcheyzer- Williams, M.G., et al., Immunol. Rev. 150:5-21, 1996; Lalvani, A., et al., J. Exp. Med. 186:859- 865, 1997). In illustrative embodiments disclosed herein, the enzyme-linked immunospot (ELISPOT) assay is used to detect and analyze individual cells that secrete interferon-γ (IFN- γ). ELISPOT IFN-γ assays and reagents are provided by BD Biosciences 2350 Qume Drive San Jose, CA, 95131. The ELISPOT assay is capable of detecting cytokine producing cells from both activated naϊve and memory T-cell populations and derives its specificity and sensitivity by employing highiaffinity capture and detection antibodies and enzyme-amplification. Additional information regarding the use of ELISPOT assay is provided in J. Immunol. Methods. 2001, 254(1- 2):59. Animal models, e.g. non-human primates, are known in the art and include the Rhesus Macaques model disclosed herein. See Ho et al., 2002, Cell 110:135-138. Alternatively, rodent models, such as the mouse animal model described in the examples, can be used to determine in vivo immune responses. The immune responses elicited in non-human primates, in particular in the Rhesus Macaque disclosed herein, are predicted to simulate immune responses in human primates.

AAV

[0101] Adeno-associated virus (AAV) is a non-pathogenic parvovirus, the single-stranded

DNA genome of which is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs). There are at least eight recognized serotypes of human AAV, designated as AAV-I, AAV- 2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7 and AAV-8. Although 85% of the human population is seropositive for AAV-2, the virus has never been associated with disease in humans (Berns et al., Adv. Virus Res.-, 32:243-306 (1987)). Recombinant AAV (rAAV) virions are of interest as vectors for vaccine preparations and gene therapy because of their broad host range, excellent safety profile, and duration of transgene expression in infected hosts.

[0102] The nucleotide sequences for various AAV are provided in GenBank as shown below. The complete genome of AAV 1 is provided in GenBank accession #NC_002077; the complete genome of AAV-2 is provided in GenBank accession #NC_001401 and Srivastava et al., J. Virol., 45: 555-564 (1983); the complete genome of AAV-3 is provided in GenBank accession #NC_001729; the complete genome of AAV 4 is provided in GenBank accession #NC__001829; AAV-5 genome: is provided in GenBank accession #AF085716; the complete genome of AAV-6 is provided in GenBank accession #NC_001862; and at least portions of AAV-7 and AAV-8 genomes are provided in GenBank accession #s AX753246 and AX753249, respectively. [0103] In AAV, cis-acting sequences directing viral DNA replication, encapsidation/packaging, and host cell chromosome integration are contained within the ITRs. Three AAV promoters, p5, pi 9, and p40 (named for their relative map locations), drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and pi 9), coupled with the differential splicing of the single AAV intron result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties which are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VPl, VP2, and VP3. Alternative and non-consensus translational start sites are responsible for the production of the three related capsid proteins. There is a single consensus polyadenylation site located in the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992). When AAV infects a human cell, the viral genome may integrate into the chromosome resulting in latent infection of the cell or may persist as a stable episome. Production of infectious virus does not occur unless the cell is infected with a helper virus (for example, adenovirus or herpesvirus). In the case of adenovirus, genes El A, ElB, E2A, E4 and VA provide helper functions. Upon infection with a helper virus, the AAV provirus is rescued and amplified, and both AAV and adenovirus are produced. Methods for purifying rAAV vectors from helper virus are known in the art and include methods disclosed in for example, U.S. Pat. No. 6,566,118 and PCT publication WO 98/09657.

[0104] rAAV vectors comprising an antigen of a pathogen can be produced by recombinant methods known to those of skill in the art. Where transcription of the heterologous polynucleotide encoding the antigen of the pathogen is desired in the intended target cell, it can be operably linked to its own or to a heterologous promoter, depending for example on the desired level and/or specificity of transcription within the target cell, as is known in the art. Various types of promoters and enhancers are suitable for use in this context. Constitutive promoters provide an ongoing level of gene transcription, and are preferred when it is desired that the therapeutic polynucleotide be expressed on an ongoing basis. Inducible promoters generally exhibit low activity in the absence of the inducer, and; are up-regulated in the presence of the inducer. They may be preferred when expression is desired only at certain times or at certain locations, or when it is desirable to titrate the level of expression using an inducing agent. Promoters and enhancers may also be tissue-specific: that is, they exhibit their activity only in certain cell types, presumably due to gene regulatory elements found uniquely in those cells. Illustrative examples of promoters are the SV40 late promoter from simian virus 40, the Baculovirus polyhedron enhancer/promoter element, Herpes Simplex Virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV) and various retroviral promoters including LTR elements. Inducible promoters include heavy metal ion inducible promoters (such as the mouse mammary tumor virus (mMTV) promoter or various growth hormone promoters), and the promoters from T7 phage which are active in the presence of T7 RNA polymerase. By way of illustration, examples of tissue-specific promoters include various surfactin promoters (for expression in the lung), myosin promoters (for expression in muscle), and albumin promoters (for expression in the liver). A large variety of other promoters are known and generally available in the art, and the sequences for many such promoters are available in sequence databases such as the GenBank database.

[0105] The heterologous polynucleotide may also comprise control elements that facilitate translation (such as a ribosome binding site or ¹¹RBS" and a polyadenylation signal). Accordingly, the heterologous polynucleotide will generally comprise at least one coding region operatively linked to a suitable promoter, and may also comprise, for example, an operatively linked enhancer, ribosome binding site and poly- A signal. The heterologous polynucleotide may comprise one antigen encoding region, or more than one antigen encoding region under the control of the same or different promoters. The entire unit, containing a combination of control elements and encoding region, is often referred to as an expression cassette.

[0106] The heterologous polynucleotide is integrated by recombinant techniques into or preferably in place of the AAV genomic coding region (i.e. in place of the AAV rep and cap genes), but is generally flanked on either side by AAV inverted terminal repeat (ITR) regions. This means that an ITR appears both upstream and downstream from the coding sequence, either in direct juxtaposition, preferably (although not necessarily) without any intervening sequence of AAV origin in order to reduce the likelihood of recombination that might regenerate a replication- competent AAV genome. Recent evidence suggests that a single ITR can be sufficient to carry out the functions normally associated with configurations comprising two ITRs (WO 94/13788), and vector constructs with only one ITR can thus be employed in conjunction with the packaging and production methods of the present invention.

[0107] The rAAV rep gene can also be operably linked to a heterologous promoter, whether rep is provided as part of the vector construct, or separately. Any heterologous promoter that is not strongly down-regulated by rep gene expression is suitable; but inducible promoters are preferred because constitutive expression of the rep gene can have a negative impact on the host cell. A large variety of inducible promoters are known in the art; including, by way of illustration, heavy metal ion inducible promoters (such as metallothionein promoters); steroid hormone inducible promoters (such as the MMTV promoter or growth hormone promoters); and promoters such as those from T7 phage which are active in the presence of T7 RNA polymerase.

[0108] Given the relative encapsidation size limits of various AAV genomes, insertion of a large heterologous polynucleotide into the genome necessitates removal of a portion of the AAV sequence. Removal of one or more AAV genes is in any case desirable, to reduce the likelihood of generating replication-competent AAV ("RCA"). Accordingly, encoding or promoter sequences for rep, cap, or both, may be removed, since the functions provided by these genes can be provided in trans. The resultant vector is referred to as being "defective" in these functions. In order to replicate and package the vector, the missing functions are complemented with a packaging gene, or a plurality thereof, which together encode the necessary functions for the various missing rep and/or cap gene products. The packaging genes or gene cassettes are preferably not flanked by AAV ITRs and preferably do not share any substantial homology with the rAAV genome. Thus, in order to minimize homologous recombination during replication between the vector sequence and separately provided packaging genes, it is desirable to avoid overlap of the two polynucleotide sequences. Alternatively, when insertion of a large antigen encoding polynucleotide is desired, the methods disclosed in PCT publication WO 99/60146, that describes a plurality of DNA segments, each in an individual rAAV vector, may be delivered so as to result in a single DNA molecule comprising a plurality of the DNA segments, and WO 01/25465 may be used.

[0109] AAV genomes have been introduced into bacterial plasmids by procedures such as

GC tailing (Samulski et al., 1982, Proc. Natl. Acad. Sci. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) or by direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). Transfection of such AAV recombinant plasmids into mammalian cells with an appropriate helper virus results in rescue and excision of the AAV genome free of any plasmid sequence, replication of the rescued genome and generation of progeny infectious AAV particles. Recombinant AAV vectors comprising a heterologous polynucleotide encoding an antigen of a pathogen may be constructed by substituting portions of the AAV coding sequence in bacterial plasmids with the heterologous polynucleotide. General principles of rAAV vector construction are also reviewed in for example, Carter, 1992, Current Opinions in Biotechnology, 3:533-539; and Muzyczka, 1992, Curr. Topics in Microbiol, and Immunol., 158:97-129). The AAV ITRs are generally retained, since packaging of the vector requires that they be present in cis. Other elements of the AAV genome, in particular, one or more of the packaging genes, may be omitted. The vector plasmid can be packaged into an AAV particle by supplying the omitted packaging genes in trans via an alternative source. In one approach, the sequence flanked by AAV ITRs (the rAAV vector sequence), and the AAV packaging genes to be provided in trans, are introduced into the host cell in separate bacterial plasmids. Examples of this approach are described in Ratschin et al., MoI. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81 :6466 (1984); Tratschin et al., MoI. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 MoI. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol, 63:3822-3828) have described a packaging plasmid called pAAV/Ad, which consists of Rep and Cap encoding regions enclosed by ITRs from adenovirus. A second approach is to provide either the vector sequence, or the AAV packaging genes, in the form of an episomal plasmid in a mammalian cell used for AAV replication. For example, U:S. Pat. No. 5,173,414 describes a cell line in which the vector sequence is present as a high-copy episomal plasmid. The cell lines can be transduced with the trans-complementing AAV functions rep and cap to generate preparations of AAV vector. A third approach is to provide either the vector sequence, or the AAV packaging genes, or both, stably integrated into the genome of the mammalian cell: used for replication. One exemplary technique is outlined in international patent application WO.95/13365 (Targeted Genetics Corporation and Johns Hopkins University) and corresponding U.S. Pat. No. 5,658.776 (by Flotte et al.). This example uses a mammalian cell with at least one intact copy of a stably integrated rAAV vector, wherein the vector comprises an AAV ITR and a transcription promoter operably linked to a target polynucleotide, but wherein the expression of rep is limiting. In a preferred embodiment, an AAV packaging plasmid comprising the rep gene operably linked to a heterologous AAV is introduced into the cell, and then the cell is incubated under conditions that allow replication and packaging of the AAV vector sequence into particles. A second exemplary technique is outlined in patent application WO 95/13392 (Trempe et al.). This example uses a stable mammalian cell line with an AAV rep gene operably linked to a heterologous promoter so as to be capable of expressing functional Rep protein. In various preferred embodiments, the AAV cap gene can be provided stably as well or can be introduced transiently (e.g. on a plasmid). A recombinant AAV vector can also be introduced stably or transiently. Another exemplary technique is outlined in patent application WO 96/17947 (by Targeted Genetics Corporation, J. Allen). This example uses a mammalian cell which comprises a stably integrated AAV cap gene, and a stably integrated AAV rep gene operably linked to a heterologous promoter and inducible by helper virus. In various preferred embodiments, a plasmid comprising the vector sequence is also introduced into the cells (either stably or transiently). The rescue of AAV vector particles is then initiated by introduction of the helper virus. Other methods for generating high- titer preparations of recombinant AAV vectors have been described. International Patent Application No.; PCT/US98/18600 describes culturing a cell line which can produce rAAV vector upon infection with a helper virus; infecting the cells with a helper virus, such as adenovirus; and Iy sing the cells. AAV and other viral production methods and systems are also described in, for example, WO 97/09441 (PCT/US96/14423); WO 97/08298 (PCT/US96/13872); WO 97/21825 (PCTIUS96/20777); WO 97/06243 (PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3:1124-1132.

[0110] Non-rAAV vectors, such as for example, naked plasmid DNA, adenoviruses, canarypox viruses, pox viruses, vaccinia virus, modified vaccinia Ankara (MVA), alphaviruses, and rhabdoviruses, can also be produced by methods deemed routine to the skilled artisan. Plasmid DNA can be generated by methods known in the art. A number of viral based systems have been developed for gene transfer into mammalian cells. For example, a number of adenovirus vectors and expression systems have been described. Adenoviruses persist extrachromosomally; see for example Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993) 67:5911- 5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933- 940; Barr et al., Gene Therapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988) 6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476). Another vector system useful for delivering the antigen encoding polynucleotides of the present invention is the enterically administered recombinant poxvirus vaccines described by Small, Jr., P. A., et al. (U.S. Pat. No. 5,676,950, issued Oct. 14, 1997). Additional viral vectors which will find use for delivering the nucleic acid molecules encoding the antigens of interest include those derived from the pox family of viruses, including vaccinia virus and avian poxvirus. By way of example, vaccinia virus recombinants expressing the genes can be constructed as follows. The nucleic acid encoding the particular antigen coding sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia prompter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the coding sequences of interest into the viral genome. The resulting TK-recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto; Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the genes. Recombinant avipox viruses, expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with, respect to the production of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545. A vaccinia based mfection/transfection system can be conveniently, used to provide for inducible, transient expression of the antigen coding sequences in a host cell. Ih this system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into protein by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA= (1986) 83:8122-8126. Production of AAV and non-AAV vectors

[0111] AAV vectors and virons can be produced using standard methods known to one of skill in the art. In some examples, methods for producing rAAV vectors and virions generally involve the steps of (1) introducing an AAV vector into a host cell, wherein the AAV may have certain regions/functions necessary for viral replication deleted; (2) introducing an AAV helper construct into the host cell, where the helper construct includes any necessary AAV coding regions capable of being expressed in the host cell to complement AAV viral regions/function missing from the AAV vector; (3) introducing one or more helper viruses and/or accessory function vectors as necessary into the host cell, wherein the helper virus and/or accessory function vectors provide accessory functions capable of supporting efficient recombinant AAV "rAAV") virion production in the host cell; and (4) culturing the host cell to produce rAAV virions. The AAV vector, AAV helper construct and the helper virus or accessory function vector(s) can be introduced into the host cell either simultaneously or serially, using standard transfection or transduction techniques. Additional rAAV production strategies are known in the art and are described in for example U.S. Pat. No. 5,786,211; U.S. Pat. No. 5,871,982, which discloses the use of hybrid adenovirus/AAV vectors; and U.S. Pat. No. 6,258,595, which discloses methods for helper free production of rAAV. Methods of purifying rAAV from helper virus are known in the art and disclosed in for example, U.S. Pat. No. 6,566,118 and PCT publication WO 98/09657. Production of pseudotyped rAAV is disclosed in for example PCT publication WO 01/83692. The disclosures of the foregoing patent publications are specifically incorporated herein by reference in their entirety. The rAAV vectors of the present invention are not limited in scope to any particular production or purification methods. Other methods for producing and purifying rAAV are known in the art and are encompassed within the present invention. Methods for producing non-rAAV vectors are also known in the art and are encompassed within the present invention.

Uses, Pharmaceutical compositions, and Kits

[0112] The present invention relates to rAAV based materials and methods for eliciting an immune response in a mammalian subject susceptible to infection by a pathogen or infected by a pathogen. In some examples, the present invention provides methods for eliciting an enhanced boost immune response that utilizes a vector set comprising a priming vector and a boosting vector for administration of an antigen of a pathogen, wherein at least one of the priming vector and boosting vector is a rAAV vector. In some examples, both priming vector and boosting vector are rAAV vectors. In some examples where both priming vector and boosting vector are rAAV vectors, both are not serotype rAAV-2. In some examples the pathogen is an RNA virus, such as for example, HIV-I/HIV-1, SIV, HCV, SARs virus or rabies virus. In other examples, the pathogen is an RNA virus and in yet other examples, the pathogen is HIV-I . In some examples, the HIV-I antigen administered in the prime dose by the priming vector is the same as the HIV-I antigen administered in the boost dose by the boosting vector and in other examples, the priming vector and boosting vector administer a shared immunogenic determinant, and/or a cross priming immunogenic determinant. Illustrative embodiments disclosed herein demonstrate that an AAV based HIV vaccine schedule that comprises administration of rAAV vector induces robust B-cell and T-cell responses that are persistent after a single IM administration. In the Rhesus Macaques animal model described herein, intramuscular (IM) administration of a pseudotype rAAV-1 elicited an equivalent immune response as compared to IM administration of a rAAV-2 expressing the same gag protein of HIV-I at a log₁₀ lower dose after a single administration. IM administration of the boost dose of the HIV-I gag protein with the pseudotype AAV-I elicited a greater boost immune response as compared to the boost immune response elicited by IM administration of the boost dose of the HIV-I gag protein with rAAV-2, and unexpectedly, the pseudotyped AAV-I elicited a higher ratio of B-cell response to T-cell response as compared to the boost immune response elicited by rAAV-2. A vector set comprising a priming vector and boosting vector may be administered "prophylactically" to a mammalian subject susceptible to AIDS or ARC, that is administered prior to infection, or prior to the appearance of symptoms or AIDS or ARC in a subject, for preventing initial infection of a subject exposed to HIV or at risk for exposure to HIV, although the methods of the present invention do not require that HIV infection is prevented; for reducing viral burden in a subject infected with HIV; for prolonging the asymptomatic phase of HIV infection in a subject; for increasing overall health or quality of life in a subject with AIDS; and for prolonging life expectency of a subject with AIDS. An appropriate clinician can compare the effect of administration df a vector set encompassed within the present invention to a subject's condition prior to treatment, or with the expected condition of an untreated subject, to determine whether the outcome. A vector set comprising a priming vector and boosting vector may be administered "therapeutically'' to a mammalian subject infected with HIV that has progressed to AIDS or ARC, that is administered after infection, for example to increase overall health or quality of life in a subject with AIDS; which may serve to prolong life expectency of a subject with AIDS. [0113] Compositions comprising vectors of the present invention, that is comprising a priming vector or boosting vector, may further comprise various excipients, adjuvants, carriers, auxiliary substances, modulating agents, and the like. An effective amount of a priming vector or boosting vector can be determined by one of skill in the art. Such an amount will fall in a range that can be determined through routine trials and are disclosed herein. A carrier, which is optionally present, is a molecule that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macroniolecules such as proteins, polysaccharides, polylactic acids, polyglycollic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co- glycolides), known as PLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee J P, et al., J Microencapsul. 14(2):197-210, 1997; O'Hagan D T₅ et al., Vaccine 11(2): 149-54, 1993. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents ("adjuvants"). Furthermore, the antigen may be conjugated to a bacterial toxoid, such as toxoid from diphtheria, tetanus, cholera, etc., as well as toxins derived from E. coli. Such adjuvants include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in- water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (International Publication No. WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model HOY microfiuidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi.TM. adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detoxu); (3) saponin adjuvants, such as Stimulon.TM. (Cambridge Bioscience, Worcester, Mass.) may be used or particle generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins (IL-I, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), beta chemokines (MIP, 1 -alpha, 1-beta Rantes, etc.); (6) detoxified mutants of a bacterial ADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT), particularly LT-K63 (where lysine is substituted for the wild-type amino acid at position 63) LT-R72 (where arginine is substituted for the wild-type amino acid at position 72), CT-S 109 (where serine is substituted for the wild-type amino acid at position 109), and PT-K9/G129 (where lysine is substituted for the wild-type amino acid at position 9 and glycine substituted at position 129) (see, e.g., International Publication Nos. WO93/13202 and WO92/19265); and (7) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.

[0114] The dosage regimen will also, at least in part, be determined by the potency of the modality, the vaccine delivery employed, the need of the subject and be dependent on the judgment of the practitioner.

[0115] The present invention also provides kits that comprise a vector set of the present invention and may include components necessary to administer a vector of the vector set. Thus, in some examples, the invention further provides kits for administration of a priming vector and boosting vector for inducing an immune response, in particular an immune response in an individual susceptible to infection with or infected by HIV-I, and suitable buffers and instructions for performing the methods of the present invention. In some examples, the kits comprise vector sets that comprise a priming vector and boosting vector, wherein at least one of the priming vector and boosting vector is a rAAV vector and wherein the vectors encode an antigen of HIV-I . In some examples, the kits comprise vector sets that comprise a rAAV priming vector and rAAV boosting vector, wherein the vectors encode an antigen(s) of HIV-I. Kits of the present invention may comprise any of the priming vectors (or plurality of vectors) or any of the boosting vectors (or plurality of boosting vectors) as disclosed herein. Procedures using these kits can be performed by clinical laboratories, experimental laboratories, medical practitioners, or private individuals. [0116] It is to be understood that this invention is not limited to particular examples disclosed herein, as such may vary. It is also to be understood that the examples are not intended to be limiting since the scope of the present invention is delineated by the appended claims. The examples described below may be performed and the data on the ability to generate an immune response described herein may be evaluated for any rAAV HIV antigen Viral Vector described in Example 1. Production of the vector may be by any of the methods described in Example 2. Any rAAV vector encoding an HIV antigen such as for example, a chimeric rAAV, a serotype rAAV, or a pseudotyped rAAV vector as described herein may be employed either as a single vector or a plurality of vectors as described herein.

Example 1. HIV AA V2 ITR plasmid nucleic acid expression constructs encoding HIV antigens for rAAV vector generation a. Construction of HIV nucleic acid constructs encoding HIV antigens

[0117] The HIV-I Clade C gag/pro-ΔRT nucleic acid construct (DU422 from South Africa, provided by Dr. Carolyn Williamson), was constructed as described below and codon-optimized to relieve dependency on the HIV rev-RRE machinery

[0118] The nucleic acid is a synthetic cDNA for HIVl gαg-protease (Clade C₅ strain DU422

(van Harmelen, et al, 2001)). This includes the gag and protease coding regions and an additional 306 nucleotides of HIV genome beyond the protease sequence, followed by a stop codon. This encodes the 101 amino acid N-terminal fragment of the reverse-transcriptase (RT) gene, which does not contain either of the RT active sites (Freed and Martin, 2001). This fragment was included based on reports that efficient protease activation was not confined to the gag-protease region alone (Quillent, et al, 1996). The antigen coding sequence is named gag-PR-ΔRT and was obtained from Dr. Carolyn Williamson at the University of Cape Town, South Africa. As this sequence was derived from the primary HIV isolate, it differs in three positions from the strain DU422 entry in the Los Alamos HIV database (Genbank accession # AY043175), which was obtained after propagation of the virus in culture. Because HIV uses amino acid codons that are rare in human genes, higher protein expression levels can be obtained in mammalian cells by replacing these with common human codons (Haas, et al, 1996). A synthetic cDNA optimized for human codon usage was designed by QiagenOperon (Alameda, CA) using codon usage tables from the Kazusa codon usage database known in the art and described in U.S. Pat. No. 6,602,705. This was synthesized in vitro by Retrogen (San Diego, CA) using PCR amplification methods. The coding region was 2039 nucleotides with a 13 nucleotide 5' flanking sequence which included a cloning site (Pad TTAATTAA) followed by a Kozak box (CCACC). The 11 nucleotide 3' end contained a stop codon (TGA) followed by a Pac I site. The codon optimized gag-PR-ΔRT gene sequence was inserted into the pCR-Blunt vector (InVitrogen, Carlsbad, CA) to create plasmid 2666-2716-1, which was confirmed by sequencing. Figure 1, SEQ ID NO:1, is the rAAV serotype 2 construct. [0119] A rAAV construct is prepared that contains the AAV-2 5' and 3 ' ITR vaccine cassette containing the CMV_1E promoter, intron sequences (SV40 splice donor/acceptor), nucleotides for the Clade C HIV-I gag coding region, nucleotides for the HIV-I protease coding region, nucleotides for the RT coding region, and nucleotides for S V40 polyadenylation signal (as described above) within an AAV-I capsid.

[0120] A second HIV nucleic acid construct encoding HIV antigens was constructed and codon optimized essentially as described above using sequences encoding a partial HIV RNaseH Integrase , Nef, Tat Clade A, Vif Clade C, and the bovine growth hormone polyadenylation signal (BGHpA) designated HIV-I RNaseH-INT-NEF-TatA-VifC-TatC ( also designated HIVRINTaVcTc). This construct was synthesized by Retrogen (San Diego, CA) using PCR amplification methods known in the art. The fragment was released from the Retrogen vector as a Bspl201/Sall fragment. The HIV nucleic acid construct encoding the HIV antigens was cloned into the existing plasmid containing the pCMV/HIVRTN/BGHpA plasmid containing the (CMV) IE promoter / SV40 poly A expression cassette (Boshart, et al., 1985) and the bovine growth hormone polyadenylation signal (BGHpA) by standard molecular biology techniques know in the art (SEQ ID NO:2). The clones were tested for expression by transfection into HeLa cells and assayed by Western blot with INT and VIF antibodies from the NIH AIDS Research Reference reagent Program (Vif antibody #2221, INT antibody #756). SEQ ID NO:2₅ the rAAV2 HIVRINTaVcTc nucleic acid sequence, is cloned in the Ad hybrid shuttle vector plasmid pSh420 as described in Example 2. :

[0121] A third HIV nucleic acid construct encoding HIV antigens was constructed and codon optimized essentially as described above using sequences encoding Clade C GAG/PRO/RT/BGHpA and prepared by synthesis of a HIV-I Clade C reverse transcriptase (RT) gene by Retrogen (San Diego, CA) using PCR amplification methods known in the art. The fragment was released from the Retrogen vector as a DRa/Sall fragment. The fragment was cloned into the existing pCMV-CladeCgag-pro-ΔRT described above digested with the same enzymes which removed the S V40pA sequence from the pCMV-CladeCgag-pro-ΔRT. The resulting clones encoded Gag, Pro, and the full length RT with the BGHpA sequence (SEQ ID NO:3). The clones were tested for expression by introducing them into HeLa cells and assayed by Western blot using, antibodies from the NIH AIDS Research Reference Reagent Program (gag #4121). The RT gene was sequenced using the primers PRO1, 5'-CAAATTCTGATTGAAATCTGC-3'; RTl 5' GCAAGTACACCGCCTTCACC-3'; RT2, 5' CAGCAAAGGACCTGATCGC-3' to confirm the presence of the RT open reading frame. SEQ ID NO:3, the rAAV2 Clade C GAG/PRO/RT/BGHpA nucleic acid sequence, is cloned in the Ad hybrid shuttle vector plasmid pSh420 as described in Example 2.

[0122] A fourth HIV nucleic acid construct encoding HIV antigens was constructed and codon optimized (human codon optimized) essentially as described above using sequences encoding HIV-I Clade C DU179 env gene (gpl60). This construct was synthesized by Retrogen (San Diego, CA) using PCR amplification methods. The env gene Aiery sequence was truncated from the construct in order to develop an epitope tag to distinguish the vaccine antigen from the HIV virus. The resulting plasmid was subcloned as a Notl/Sall restriction fragment into a pCMVB from Clontech (GenBank Accession #U024521) (SEQ ID NO:4). The resulting subclones were tested for expression by transfection into HeLa cells and assayed by Western blot. SEQ ID NO;4, the rAAV2 HIV-I Clade C DUl 79 env gene (gpl60), is cloned in the Ad hybrid shuttle vector plasmid pSh420 as described in Example 2.

[0123] A fifth HIV nucleic acid construct encoding HIV antigens was constructed and codon optimized essentially as described above using sequences encoding HIV-I Clade A env gene ( AF407162) along with the BGHpA sequence. This construct was prepared by Retrogen(San Diego, CA) using PCR amplification methods in conjunction with their proprietary protocols. The env gene Avery; sequence was truncated form the construct in order to develop an epitope tag to distinguish the vaccine antigen from the HIV virus. The resulting plasmid was subcloned as a Notl/Sall restriction fragment into a pCMVB from Clontech (GenBank Accession #U024521) (SEQ ID NO:5). The resulting subclones were tested for expression by transfection into HeLa cells and assayed by Western blot using an antibody against HIV-I gpl20 (YVS- 1961, Accurate Chemical & Scientific Corp., Westbury, N. Y.) SEQ ID NO:5, the rAAV2 HIV-I Clade A env gene (GenBank accession #AF407162) nucleic acid sequence, is cloned in the Ad hybrid shuttle vector plasmid pSh420 as described in Example 2. b. Construction of AAV ITR serotype 2 HIV antigen expression plasmids [0124] The expression promoter used for the constructs is the CMVI/E promoter described in (Boshart, et al, 1985) which contains a chimeric SV40 intron and the bovine growth hormone poly A (BGHpA).

[0125] The codoή optimised HIV nucleic acid constructs described in Example IA were cloned into the cytomegalovirus (CMV) IE promoter expression cassette at Columbus Children's Research Institute (CCRI, Columbus OH). The CMVI/E HIV nucleic acid constructs were each blunt ligated into Not I digested pCMVβ (Clontech, Palo Alta, CA), replacing the β-gal gene with CMVI/E HIV nucleic acid constructs to create the expression plasmid. Expression of the trans gene was verified by Western blot analysis of a transient transfection in HeLa cells. [0126] To flank the HIV antigen nucleic acid expression cassettes described above by

AA V2 ITRs, an existing plasmid as described in WO 00/73481 was used. This plasmid contained two AA V2 ITRs flanking the TNFnFc transgene. The TNFnFc transgene was excised and replaced by the HIV expression cassettes by standard molecular biology techniques know in the art. The vaccine DNA does not contain the ampicillin gene.

[0127] Example 2. Encapsidated rAAV: Viral Vector Production and Purification Methods

[0128] rAAV viral vectors are made by any of a number of methods known in the art including transient transfection strategies as described in U. S. Pat. No. 6,001,650 and 6,258,595; stable cell line strategies as fully described in WO95/34670; or shuttle vector strategies including Adenoviral hybrid vectors as described in WO96/13598 using a rep-cap cell line as described in WO99/15685 for the adenoviral-AAV hybrid vector system (Ad hybrid system). rAAV vector production requires three common elements; 1) a permissive host cell for replication which includes standard host cells known in the art including 293-A, 293-S (obtained from BioReliance), VERO, and HeLa cell lines which are applicable for the three vector production systems described herein; 2) helper virus function which as utilized herein is a wild type adenovirus type 5 virus when utilized in stable cell line manufacture and Ad hybrid vector systems or a plasmid pAd Helper 4.1 expressing the E2a, E4-orf6 and VA genes of adenovirus type 5 (Ad5) when utilized in transfection production systems; and 3) a transpackaging rep-cap construct.

[0129] The rAAV HIV plasmid constructs utilized to produce the rAAV vectors of the present invention are more fully described in Example 1. a. Transpackaging rep-cap cell lines

[0130] For the stable cell line and rep-cap transpackaging cell lines utilized for the Ad hybrid production process described below, the AAV-2 serotype vector and the rep-cap construct is described in WO95/34670. For the AAV-I capsid, AAV2 5' and 3' ITR pseudotyped constructs, a new transpackaging rep-cap plasmid is constructed containing an AAV-2 rep gene and the cap sequences of AAV 1 as depicted in SEQ ID NO:6. The plasmid encoding the rep-cap (AAV- 2/AAV-l, respectively) pseudotyped transpackaging construct under the control of the AA V2 p5 promoter as depicted in SEQ ID NO: 6 is utilized for generation of a stable pseudotyped rep-cap cell line. For the transfection system, the p5 promoter of the rep-cap (AAV2/1 respectively) pseudotyped transpackaging construct is replaced with a minimal heat shock promoter consisting of essentially a TATA box by standard molecular techniques know in the art. a. Transfection Production

[0131] Recombinant AAV vectors as produced utilizing transient transfection are produced by a standard calcium phosphate transfection methods in adherent human 293 cells, using the Ad helper, trans-packaging and AAV vector plasmids. The AAV ITR serotype 2 HIV antigen expression plasmids produced by this method are linearized within the ampicillin gene prior to transfection into the packaging cell line by digestion with Pvu I and are transfected into the C12 packaging cell line by electroporation (for AAV2 serotype vectors). C12 is an AAV-2 HeLa AA V2 packaging cell line containing the AA V2 rep and cap genes driven by the AAV p5 promoter as described more fully in Johnson WO95/34670. Cells are seeded in 24 x 96 well plates in culture medium containing 0.4 μg/mL puromycin and 0.5 mg/niL G418. After colonies appeared, these plates are replica plated to provide one set for screening and another for cell propagation. All screening assays required infection with adenovirus 5 (Ad5, provides helper function) to induce production. Briefly, cells are seeded in 225 -cm² T-flasks and grown to sub-confluency in complete DMEM (BioWhittaker™; Cambrex, Walkersville, MD). Plasmids pAd helper 4.1 SEQ ID NO:7 and AAV vector plasmid are added (2:2:1 molar ratio) to a 300 mM CaCl₂ solution. To form a calcium phosphate precipitate, the plasmids mix is added to a 2X HBS buffer (280 mM NaCl, 1.5 mM Na₂PO₄, 5 mM HEPES [pH 7.1]) and incubated for 30 sec, and then added directly onto the 293 cell monolayer. The cells are incubated for 6-8 h at 37⁰C, after which the medium was aspirated and replenished with fresh complete but serum-free DMEM. Three days post-transfection, the cells are lysed by the addition of Deoxycholate (Fisher, Houston, TX) to a final concentration of 0.5%, releasing all cellular and nuclear contents into solution. The cellular DNA is digested by the addition of Benzonase (EM Sciences, Gibbstown, NJ) at 10 U/ml for 1 hour at 37⁰C. Tween 20 (Fisher, Houston, TX) is added to a final concentration of 1% with a further incubation of 60 min at 37⁰C, followed by the addition of NaCl to a final concentration of IM₅ before clarifying through depth filters (Millipore, Bedford, MA). Clarified lysates are concentrated 15 to 20-fold by Tangential Flow Filtration (TFF) using Pellicon 2 minicassettes (110 IeDa MW cut off; Fischer, Houston, TX) and are exchanged against formulation buffer (20 mM Tris [pH 8.0], 0.2 M NaCl, 2 mM MgCl₂ and;2% glycerol). The harvested lysates are purified by ion-exchange chromatography essentially as described in U.S. Pat. No. 6,566,118. Vector-containing elutes are formulated in a buffer containing 20 mM Tris (pH 8.0), 0.2 M NaCl, 2 mM MgCl₂ and 2% glycerol, and aseptically filtered. Final vector preparations are greater than 95% pure, with low endotoxin levels (<0.5 endotoxin units (EU]/ml). The purified vectors are non-aggregated, as determined by dynamic laser light scattering analysis, using a Protein Solutions DynaPro-99 instrument (High Wycombe, UK). Vector titers are; determined by real-time PCR, using a Perkin-Elmer Applied Biosystems Prism 7900 sequence detector (Foster City, CA), and are between 5 and 20 X 10¹² DNase-resistant particles (DRP)/ml. Vector infectivity is assessed in a TCID50 assay using the HeLa-based B50 cell line as described in U.S. Pat. No. 6,475,769.

[0132] Alternatively, transfection production is performed as described below.

[0133] Cells are grown by seeding 201 flasks at a cell density of 1.5 x 106 cells per flask.

The 293 cells are placed into 5OmL of DMEM with 4mM L-glutamine and 10% FBS. The flasks are incubated for 96 hours at 37⁰C with 5% CO₂. After 96 hours of incubation, the cells are transfected using calcium phosphate. The flasks are typically 80 - 90% confluent on the day of transfection. The flasks are transfected in sets of 25. 750μg of AAV Helper plasmid containing the appropriate capsid, 1250μg of Ad Helper plasmid and 375μg of the cis plasmid are added to 104mL of 30OmM Calcium Chloride. The plasmid containing calcium chloride is slowly poured into 104mL of 2X HBS buffer and allowed to mix for 30 seconds. 8mL of the precipitate is immediately added to each flask (in a set of 25). This procedure is repeated until 200 flasks have been transfected. The flasks are placed at 37⁰C with 5% CO₂ for 6 to 8 hours. The remaining flask is trypsinized and cells counted to be used to calculate productivity.

[0134] After 6 - 8 hours, the flasks are removed from the incubator and the media is removed from each of the flasks by aspiration and replaced with 5OmL of DMEM media with 4mM L-glutamine. The flasks are incubated for 72 hours at 37⁰C with 5% CO2. [0135] After 72 hours, the flasks are removed from the incubator and tapped to release the cells. The contents of each of the flasks are emptied into 6 - 2L roller bottles. Based on the volume of each container, the amount of 10OmM MgCl₂ and 10% DOC are calculated to achieve a final concentration of 1.8mM and 0.5%, respectively. The roller bottles are placed in a 37⁰C waterbath for 10 - 20 minutes. Benzonase is added to each roller bottle to achieve a final concentration of 10 units per mL. The roller bottles are placed in a 37⁰C waterbath for 60 minutes, inverting every 15 minutes. Polysorbate 20 (T ween) is added to each roller bottle to achieve a final concentration of 1%. The roller bottles are placed in a 37°C waterbath for 60 minutes, inverting every 15 minutes. 5M NaCl is added to each roller bottle to achieve a final salt concentration of IM. [0136] The lysed material is filtered through two filters, Polygard CR Optivap XL 10 filter

(0.3 μm nominal) and Milligard Opticap Capsule (0.5μm nominal). Once filtered, the material is concentrated using tangential flow filtration (TFF). Three TFF membranes are used with a lOOKda nominal molecular weight cut off (NMWCO). The filtered material is concentrated to a volume of 50OmL. Once concentrated, the material is diafiltered with 10 diavolumes. The diafiltered material is filtered through a Suporcap 50 filter (0.45μm) and placed at -7O⁰C until purification. a. Stable Cell Line Production

[0137] Stable cell lines for rAAV viral vector production are generated by transfecting cell lines as described above and screening for stable cell lines which could be repeatedly propagated and which contained the rep-cap packaging construct as well as the AAV ITR serotype 2 HIV antigen expression plasmids stably integrated. rAAV vectors are produced and purified by methods know in the art essentially described in WO99/11764 and WO00/14205. For serotype 2 vectors containing serotype 2 capsid proteins the Cl 2 cell line described above is utilized as the packaging cell line. a. Ad hybrid production

[0138] Ad hybrid production of the rAAV HIV vaccine Vectors is performed essentially as described in WO96/13598 using a rep-cap cell line as described in WO99/15685. Briefly the system originally developed by T.C. He et al and disclosed in US Pat. No. 5,922,576 and available as AdEasy ™ kit from Qbiogene and Stratagene, was modified to produce an improved system that is capable of more efficient and higher yield generation of recombinant Adenovirus/ AAV hybrids (Ad/AAV hybrids). This approach utilizes two plasmid vector systems (a transfer or shuttle vector and a Adenovirus genome containing vector) that undergo bacterial recombination in competent E.coli yielding a recombinant Ad/AAV hybrid plasmid which was utilized to derive Ad/ AAV hybrid viral stocks as described herein. The shuttle vector described in U.S. Pat. No. 5,922,576 contains the left ITR and encapsidation sequence of adenovirus, a multiple cloning site into which the AA V2 HIV nucleic acid antigen-transgene expression cassette is inserted, and map units 9.8- 16.0 and 97.2-100 of the wild type Adenovirus type 5 genome (Ad5wt). The shuttle plasmid known in the art (U.S. issued Pat. No. 5,922,576 ) was determined by sequencing to contain a truncated left ITR and encapsidation sequence. Specifically the left ITR of the Adenovirus type 5 ITR is 385 base pairs and the shuttle vector described in U.S. Pat. No. 5,922,576 contained only 341 base pairs, a truncation of 44 base pairs. Accordingly the left adenovirus shuttle vector sequence was excised (nucleotides 1-353) by restriction enzyme digest and replaced with a PCR generated amplicon containing nucleotides 1-420 of Ad5wt. This improved shuttle vector is designated pSh420 SEQ ID NO:8. Virus produced using only this modification of the shuttle vector resulted in a log higher titer (2.42x10e8 compared to 2.3Ox 10e7 for an AAV luciferase vector) compared to the shuttle vector previously described in the art. In order to further improve the process the adenoviral vector genome plasmid of U.S. Pat. No. 5,922,576 was analyzed by comparison of the sequence to Ad5wt sequences. The resulting analysis revealed five deletion, nine insertions, and nine mis-sense mutations in addition, one of the E3 deletions (2682 bp) caused a deletion of the L4 polyadenylation signal. We replaced map units 75-100 of Ad5wt in the Adenoviral genome vector in a two step cloning process. First, pSh420 was electroporated into competent Ecoli B J5183 cells along with Ad5wt DNA resulting in homologous recombination between overlapping Ad regions as depicted in Figure 7 yielding a new E-I deleted Adenoviral genome vector designated pAdNSE-1. The pAdNSE-1 and Adenoviral genome vector described in U.S. issued Pat. No. 5,922,576 were both digested with Pacl and Spel to remove the Ad5wt map units 75-100. The Spel-Pacl fragment of the pAdNSE-1 was inserted into the Pacl -Spel digested backbone of the adenoviral vector described in the art. The new improved Adenoviral genome backbone vector was designated pAd- Ml. In order to allow for larger AAV expression cassettes the pAd-Ml was digested with Xbal and a 1878 base pair fragment of the E3 adenoviral gene was removed. This deletion allowed for insertion of a full length AAV expression cassette while retaining the L4 polyadenylation site which was deleted in the adenoviral genome vector known in the art. The kinetics of this new virus demonstrated improved titers (viral production equal to Ad5wt virus) but the kinetics of growth _. were retarded. In order to improve growth of the recombinant plaques a PCR generated sequence encoding the 11.6 kDa protein known as the adenovirus death protein was cloned into the Xbal site in the E3 region of the E3 deleted plasmid previously described. The resulting adenoviral genome plasmid designated pAdM3.1 is approximately 4587 base pairs smaller than Ad5wt and therefore has room for a full length AAV cassette without exceeding the packaging capacity of adenovirus. SEQ ID NO: 9 contains the nucleotide sequence of the pAdM3.1. The resulting improved Ad hybrid production system utilizing the improved pSh420 shuttle plasmid and pAdM3.1 adenoviral genome plasmid yielded production of infectious Ad hybrid viral particles at levels at least two logs higher than the vector system known in the art and approximating Adwt5 virus production (1.10xl0e9, 2.30xl0e7, and 2x10e9, respectively utilizing a Luciferase vector).

[0139] The Ad/ AAV hybrid viruses used to produce the rAAV vaccines of the present invention described in Example 1 are generated by cloning AAV ITR serotype 2 HIV antigen expression plasmids of Example Ib into the pSh420 shuttle plasmid by standard restriction digest and cloning techniques known in the art. Briefly, the pSh420 vector is digested with BgI 11 to linearize the plasmid within the multiple cloning site. The AAV ITR serotype 2 HIV antigen expression plasmids are excised from their expression cassettes by BgI 11 digestion, the fragments are ligated and competent recA E.coli strains known in the art including DHlOB₅ PH5α or STBL2 are transformed by electroporation using methods known in the art. The transformed bacteria are grown on Luria Broth agar plates containing 50 ug/ml kanamycin and recombinants are selected by screening colonies for appropriately sized fragments utilizing BgI 11 or Sphl restriction digests. [0140] Competent Ecoli strain BJ5183 is transformed by electroporation with supercoiled pAdM3.1, selected, expanded, and the integrity of the plasmϊd is confirmed. The psH- AAV ITR serotype 2 HIV antigen expression plasmids generated above are digested with Pmel and used to transform the pAdM3.1 competent cells by electroporation. The doubly transformed cells are grown overnight in Luria broth cultures with 50 ug/ml kanamycin and screened for recombinant Adenoviral- A AV plasmids (ad hybrid). The correct recombinant Ad hybrids are propagated by transforming STBL2 competent cells, plating and screening. In order to generate infectious Ad- AAV hybrid virus the Ad-AAV ITR serotype 2 HIV antigen expression plasmids are digested with Pac 1 and 293 cells transfected by CaC12 precipitation as know in the art. Viral plaques were selected and propagated (stock of the Ad-AAV ITR serotype 2 HIV antigen viral vectors). [0141] The Ad-AAV ITR serotype 2 HIV antigen viral vectors are used to infect a stable packaging cell line expressing rep and cap along with Ad5 wt virus as a helper virus as described in WO96/13598 using a rep-cap cell line as described in WO99/15685 for the production of the rAAV vaccine vectors of the present invention.

[0142] rAAV is purified as follows. The clarified producer cell lysate is prepared by resuspending producer cells (5x10⁶ cells/ml) in 0.5% deoxycholic acid and benzonase nuclease (35 units/ml) as described previously in Clark et ah, supra. Equilibrated POROS HE-20 resin (12.5 mM Tris, pH 8.0; 0.5 mM MgC12; 100 mM NaCl) is added to the clarified lysate (2 ml/liter) and the mixture rotated for 16 hr at 4° C to allow sufficient time for particle binding. The HE-20 resin is pelleted at 2,500 xg for 30 minutes and resuspended in 12 ml of equilibration buffer per ml of resin used. The resin as washed three times using equilibration buffer by gentle inversion and pelleted at 2,000 rpm for 10 min between each wash. After the final wash, rAAV-2 is eluted by 10 minute resuspension of the pellet in 20 ml of elution buffer (20 mM Tris, pH 8.0; 1 mM MgC12; 600 mM NaCl). Vector elution is repeated two more times (3 times total) for maximal recovery. The eluted virus is filtered through a 0.45 um membrane filter to remove resin fines prior to PI column chromatography. The virus eluate is diluted 6-fold with water to reduce the final salt concentration to ~ 100 mM. A Biocad Sprint HPLC system (PerSeptive Biosystems) is used in conjunction with a POROS PI-50 column (50 μm bead size) to generate the final purified product. A 1.7 ml column is equilibrated with 10 column volumes of 20 mM Tris, pH 7.0; 100 mM NaCl prior to application of the batched vector at a flow rate of 5 ml/min. After sample loading, the column is washed with 10 ml equilibration buffer. Bound material is eluted by application of a NaCl step gradient (0.6 M) at a flow rate of 3 ml/min, and 1 ml gradient fractions are collected. Following column purification, a small aliquot (20 μl ) of peak protein containing fractions are analyzed by SDS-PAGE and SYPRO- Orange (Molecular Probes Inc.) staining to visualize the eluted proteins. Peak virus containing fractions are pooled, dialyzed against multiple changes of 20 mM Tris, pH 8.0, 1 mM MgC12, 200 mM NaCl, and stored in aliquots at -80° C in 10% glycerol. The total protein content in vector preparations is determined using the NanoOrange Protein Quantitation Kit according to the manufacturer's instructions (Molecular Probes, Inc.).

[0143] Alternatively rAAV vector may be purified as described in WO 99/11764 and WO

00/14205 or as provided by any other method known in the art.

[0144] DRP titers are determined for purified rAAV by real time PCR methodology utilizing a Prism 7700 Taqman sequence detector system (PE Applied Biosystems) as detailed in Clark et ah, supra.

[0145] Example 3: Immunogenicity in Immunocompetent Mice

[0146] Prior to testing of rAAV-2/HIVgag in Rhesus Macaques, immunogenicity of a clinical grade production lot (lot EFX002, manufactured by Targeted Genetics Corporation, Seattle, WA) was tested in mice.

[0147] Mice were injected once in the quadriceps muscle with rAAV-2/HIVgag (1x1011

DRP) and immune responses were analyzed for twelve weeks by standard techniques using Clade B gag. In addition, muscle at the site of injection was harvested and tested for the expression of HIV- 1 gag. Gag wasi detected by a p24 antigen-capture ELISA (ZeptoMetrix Corp., Buffalo, NY). [0148] For rAAV-2 serotype 2 construct of Example 1, Figure 1, antibody titers rose over the first two months, reaching maximum at 8 weeks and remaining at maximum at 12 weeks. Antigen-specific T cell responses were also observed using an IFN-γ ELIspot kit (U-CyTech B.V., Utrecht, Netherlands) on 88 SFC/10⁶ splenocytes from one animal and 93 SFC/10⁶ splenocytes from another animal using a H2-K^d restricted HIV-I gag peptide as described in Doe and Walker, Aids, 10: 793-794 (1996). Corresponding with these immune responses is the expression of gag in the injected muscle. Gag levels peaked at three weeks after administration and gag polypeptide was still detectable at 12 weeks.

Example 4: Immune Response to rAAV vaccine in Rhesus Macaques [0149] The Rhesus Macaque model is the leading animal model for AIDS vaccine development. See, for example., Ho et al., Cell, 110:135-138 (2002). The Rhesus Macaque model is utilized to analyze the immune response to rAAV-2/HIV gag vector constructed as described in Example 1. The following methods are used for measuring the immune response data, including the ELISA data and ELISpot data.

Methods

[0150] Serum: Blood for serum is collected in glass vacutainer tubes (red top). Blood is centrifuged and aliquoted into, labeled cryovials, stored at -80°C and logged into a computer inventory.

[0151] ELISA. Purified HIV gag protein is diluted with carbonate buffer(7.5 mM Na₂CO₃-

17.4 mM NaHCO₃, pH 9.6) and then added to wells (200 ng/well) and is incubated overnight. The plates are washed with phosphate-buffered saline (PBS) and are incubated for 1 h with blocking buffer (3% bovine serum albumin plus 1% normal rabbit sera diluted in PBS). The wells are washed with PBS, diluted macaque plasma was added to the wells, and the plate was incubated for 1 hour at room temperature. The wells are washed, and rabbit anti-monkey immunoglobulin G- conjugated horseradish peroxidase antibodies (Sigma, St. Louis, Mo.) are added and incubated for 1 hour. The wells are washed, and color development is performed by using OPD FAST Tablets (Sigma) per the manufacturer's protocol. The plates are read at an optical density of 450 nm (OD₄₅o). Titers are reported as the reciprocal of the highest dilution that yields an OD reading of 0.200. ^'

[0152] T-cells: At each timepoint, 4.5 - 6 ml blood/kg body weight is drawn into CPT vacutainer blood collection tubes and PBMC are purified by centrifugation. After washing, 10 million PBMC/ml in Nunc vials are cryopreserved in freezing medium (90% fetal bovine serum & 10% DMSO) using a controlled rate freezer. Samples are transferred into a liquid nitrogen (vapor phase) freezer and logged into a computer inventory for storage. [0153] ELISpot. Frozen PBMC are shipped in a liquid nitrogen shipper to BD Biosciences

Pharmingen, where they are received and accessioned according to laboratory procedure SOP. On the day of testing, PBMC are thawed, cell count and viability determined on each sample, and the concentration adjusted as necessary.

[0154] On the same day, ELISPOT analysis for secreted IFN-γ is performed after overnight stimulation under the following conditions: 1. Using a positive control (Staphylococcus enterotoxin B (SEB)) at 0.5 μg/ml- of reaction mixture; 2. Unstimulated; 3. Using the HIV-I clade Cl Peptide Pool Lot 080702-C-GAG-l (119 15mer peptides, overlapping by 11) at a concentration of 1.5 micro-g/mL of reaction mixture. Each condition is assayed in triplicate at 2.0 x 10⁵ cells per well. [0155] The animals are sedated with ketamine (10 mg/kg) for inoculation and blood draws;

Two animals are sacrificed at week 20. Macaques are painlessly euthanized with greater than 50 mg/kg body weight of sodium pentobarbital. This method is consistent with the recommendations of the American Veterinary Medical Association's Panel on Euthanasia.

Tissue Collection and Analysis Tissue Collection

[0156] The entire injected macaque muscle (left and right quadricepts) is harvested. Each quadriceps is subdivided into 20 aliquots representing the extent of the muscle. DNA and protein lysates are then separately prepared from each aliquot. The muscle cell DNA is tested for presence of vaccine DNA using quantitative PCR as described below. Quantitative DNA Assay

[0157] One microgram of DNA from each tissue sample to be tested is subjected to TaqMan

PCR using a vaccine DNA-specific (CMV) primer and probe set in triplicate. To verify that the DNA samples are amplifiable by TaqMan PCR, 15 copies of a CMV-containing plasmid is spiked into one of the triplicate wells. If plasmid DNA- is detected in the spiked sample, then the DNA sample is considered as amplifiable, and the non-spiked duplicate wells are analyzed for the presence of DNA vaccine to determine copy number. If the plasmid spike well does not show amplification, the DNA is further purified by phenol/chloroform extraction and the TaqMan PCR is repeated. For administration of rAAV-2 gag vaccine DNA is detected in multiple aliquots of muscle from each Rhesus Macaque sacrificed at week 20. W

[0158] Immunological Analysis for rAA V-2-single administration Four groups of macaques received a single intramuscular immunization with rAAV-2 as described in Example Ia. at four dose levels ands were followed for antibody and antigen-specific T-cell responses for 79 and 71 weeks and -respectively. Antibodies to HIV-I gag rose slowly over time and were sustained. All 18 macaques in the three highest dose groups responded. The only non-responders were in the lowest dose group (3x10⁸ DRP) in which four of six monkeys did not develop measurable anti-gag antibodies in the first six months. However, two more animals in this group seroconverted by week 40. Half of the animals in this low dose group and all in the higher dose groups maintained measurable anti-gag antibody titers through 18 months, and the magnitude of the response was clearly affected by dose. Both of the highest dose groups had significantly higher titers when compared to the lower dose groups (p<0.03). However, owing to the variability in individual monkeys, the observed differences in titer between the highest close groups (3xlOⁿ vs. 3X10¹² DRP) were not statistically significant.

[0159] A similar dose response was observed in antigen-specific T cell responses as measured by ELISpot. Overall, responses gradually rose through Week 16 and persisted through six months. Differences among the three highest dose groups were not significant, again owing to the variability in individual monkeys. By six months, six of 10 animals in the two highest dose groups (two were sacrificed at 20 weeks) had persistent ELISpot responses. At one year, four animals in the study had a measurable ELISpot response.

[0160] At week 80, one low responding and one high-responding Macaque from the 3xlO⁹,

3x10¹⁰ and 3x10¹¹ DRP dose groups and one high-responding animal from the highest dose group received an intramuscular boost dose of 1x10¹² DRP tgAAC09. During eight weeks of follow-up, anamnesis was observed in both B- and T-cell responses.

[0161] One animal (AC80) in the lowest dose group had not responded with antibody to the initial immunization. After the boosting dose, it appeared to exhibit a modest primary immune response. However, all other animals made strong anamnestic antibody responses, most of which exceed in titer the response to the initial immunization. After boosting, T-cell responses behaved similarly. Animals whose T-cell response to the initial immunization was low (AC80, AC77, AC27) made stronger IFN-γ responses peaking at 2-4 weeks after the boost. Since primary responses peak at 12-16 weeks, these boosted cellular responses likely represent immunological memory. The other four animals which had made stronger initial responses that had waned by week 71, also gave boosted responses.

[0162] Example 5: Comparison of the Immune Response to rAAV vaccines in Rhesus

Macaques

Studies. ^'

Study 1 : AAV-2 single inoculation

[0163] Method: A dosing study of an AAV-2 based HIV-I vaccine in Rhesus Macaques, using the AAV-2 construct as described in Example Ia., utilizing a single intramuscular administration of 3.3xlO⁹ to 3.3xlO¹² DNase Resistant Particles (DRP) was undertaken. [0164] rAAV-2/HIVgag was immunogenic following a single dose. Both antigen-specific

T-cells responses and antibody production persisted beyond 3 months. In the macaques, rAAV- 2/HIVgag was fully immunogenic at all doses above 3x10⁹ DRP and was well-tolerated by all animals without any untoward effects. Table 2 provides the dose and formulation for the rAAV-2 vector.

TABLE 2

[0165] Results: Seroconversion to the gag protein and T-cell ELIspot responses to pooled overlapping subtype C gag 15mer peptides were induced in a dose-dependent manner.

[0166] Study 2: Single inoculation with AAV-I pseudotype rAAV expressing HIV gag. [0167] Methods: A dosing study of an AAV-I pseudotype HIV-I vaccine in Rhesus

Macaques with the AAV-I pseudotype construct described herein utilizing a single intramuscular administration of 3.3xlO⁹ to 3.3xlO¹² DNase Resistant Particles (DRP) was undertaken. [0168] Results: The data in the Rhesus Macaques model indicated that the AAV-I pseudotyped rAAV-1/HIV gag generated an equivalent immune response to the AAV-2/HIV gag as described above, at a log 10 lower dose after a single intramuscular administration. [0169] Study 3: The Rhesus Macaques from Study 1 above were re-randomized and boosted at week 80 with either the identical AAV-2/HIVgag construct, or the AAV-I pseudotyped construct in Study 2.

[0170] Results: T- and B- cell responses in all vaccinated animals had waned by week 80 but strongly rebounded after a single boost with AAV-I pseudotype rAAV/HIV gag, or AAV-2. Even animals that had received low initial AAV-2 doses and made a very weak primary response were significantly boosted after the 80 week rest. The antibody response was boosted most strongly by AAV-I pseudotype HIV gag.

[0171] See Table 3 for Individual animal ELISA data (prime), Table 4 for ELISA data from

Macaques boosted with rAAV-2, and Table 5 for Individual animal IFN-gamma ELISPOT data for administration of prime dose and boost dose of HIV-I gag with rAAV-2. See Table 6 for ELISA data from individual animals and Figure 8 and Table 7 for Individual animal IFN-gamma ELISPOT data for administration of prime dose of HIV-I gag with rAAV 2 and boost dose of HIV-I gag with pseudotyped rAAV-1.

[0172] Example 6: HIV Multi-antigenic prime/boost experiment rAAV nucleic acid compositions

[0173] The sequence of the Ad/hybrid HIV-I RNaseH-INT-NEF-TatA-VifC-TatC ( also designated HIVRINTaVcTc) shuttle plasmid is contained in SEQ ID NO:2.

[0174] The sequence of the Clade C GAG/PRO/RT/BGHpA Ad/hybrid Vector shuttle plasmid is contained in SEQ ID NO:3.

[0175] The sequence of the Ad/hybrid Vector HIV shuttle plasmid is contained in SEQ ID

NO:4. [0176] A fourth construct encoding the HIV-I Clade A env gene ( AF407162) along with the BGHpA The sequence of the Ad/hybrid Vector HIV shuttle plasmid vector is contained in SEQ ID N0:5.

Mult-antigenic rAAV vaccine production

[0177] Production and purification of the multi-antigenic HIV vaccine vectors described above is performed as described in U.S. Pat. Nos. 5, 871,982 and 6,566,118. The vaccine is formulated in buffer as described above at an equal DRP ratios of the four components for the rAAV multi-antigenic priming vector. The rAAV boosting vector utilized in the subsequent experimental methods comprises a multi-antigenic vaccine formulation or an individual vector formulated separately or any combination of the vectors.

[0178] The methods and studies previously described are performed dosing animals at doses ranging from 1-4 xlθe9 to 1-4 xl Oe 14. Individual animal data is analyzed as previously described.

Example 7: Regulated Expression of HIV antigens in adenoviral- A AV hybrid vector system (Ad hybrid system)

[0179] Due to toxicity associated with the expression of the HIV EnvA and VINNT antigens, generation of an Ad hybrid system containing these genes was problematic. To address this issue an adenoviral- AAV hybrid vector system was generated containing a tetracycline- regulated expression system (Invitrogen) to control the expression of the HIV antigens. The tetracycline regulated system consists of an expression plasmid under the control of the CMV promoter with two copies of the tetracycline operator sequence at the very 3' end of the promoter. This tet operon plasmid is used in conjunction with the Tetracycline-Regulated Expression 293 cell line (T-REx-293, Invitrogen,R710-07) which is a Human embryonic kidney cell line that stably expresses the tetracycline repressor. Gene expression is induced by the addition of tetracycline to the cultured cells.

Example 8: Generation of Ad/AAV-gpl40 env A-R

[0180] To construct the regulated Ad-AAV gpl40 envA vector, the CMV promoter with two copies of the tetracycline operator sequence was liberated from the pcDNA4/TO plasmid (Invitrogen; V1020-20). This CMV/2xTetO fragment was inserted into ρSh420AAV/Δ5'ITR/ BGHpA shuttle plasmid. The gρl40 env A (ΔAVERY) was liberated from the pSh420/AAVgpl40A plasmid and inserted into the pSh420/AAV/ (Δ5'ITR) /CMV2xTet plasmid via Hind3/Xbal sites. This resulting shuttle plasmid, the pSh420/AAV(Δ5'ITR) - gpl40 Env A-R, was subsequently used in conjunction with the T-REx-293 cells to generate an Ad-AAV hybrid vector as described in Example 2.

Example 9: Generation of Ad/AAV- VINNT-R

[0181] To construct the regulated Ad-AAV-VINNT vector the VINNT gene fragment was first liberated from the pCMV-HIVRINTaVcTc expression plasmid via Xhol/Accl digest. The pSh420/AAV(Δ5TTR) gpl40 Env A-R shuttle plasmid was digested with Sall/Clal to liberate the backbone from the Env A gene. The VINNT gene fragment was then ligated into the pSh420/AAV/CMV-tetO vector to yield pSh420/AAV(Δ5'ITR) -VINNT-R. This resulting shuttle plasmid was subsequently used in conjunction with the T-REx-293 cells to generate an Ad-AAV hybrid vector as described in Example 2.

[0182] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the descriptions and examples should not be construed as limiting the scope of the invention.

Individual Animal ELISA data

nd no data a sacrificed at week 20, no sample b inadvertent death (not vaccine-related), no sample

Table 3

ELISA data from Macaques Boosted with tgAAC09

Table 4

Individual Animal INF-γ ELISpot data

[Spot-forming cells / million PBMC]

a no data b inadvertent death (not vaccine-related), no sample

Underlined numbers represent averages of triplicate wells, normalized to 1 million PBMC, that are two standard deviations above the average of negative control (no peptide) wells for that animal at that timepoint. Other values are not significantly above negative control.

Table 5 [Spot-forming cells / million PBMC]

a no data

Underlined numbers represent averages of triplicate wells, with background (no peptide) subtracted and normalized to 1 million PBMC, that exceed the background by at least four-fold. Other values are not significantly above negative control.

W 2

Claims

CLAIMSThe claimed invention is:

1. A method for eliciting a boost immune response to a pathogen in a mammalian subject susceptible to infection by or infected by the pathogen comprising administering to the subject an effective amount of a boost dose of an antigen of the pathogen by a recombinant vector, wherein said mammalian subject has been administered an effective amount of a prime dose of an antigen of said pathogen by a recombinant vector; wherein at least one of the prime dose or the boost dose is administered by a recombinant adeno-associated virus (rAAV) vector in a single administration, and wherein the boost dose elicits an equal or greater measurable immune response as compared to the response elicited by administration of the prime dose, with the proviso that when both the prime dose and the boost dose are administered by rAAV, they are not both administered by AAV-2.

2. The method of claim 1 wherein the boost dose is an rAAV vector in a single administration, and wherein the prime dose is a non-rAAV vector.

3. The method of claim 1 wherein the prime dose and boost dose are both administered by a rAAV and a single administration of the boost dose elicits a greater measurable immune response as compared to the response elicited by a single administration of the prime dose, wherein the measurement is performed under conditions where the prime dose and boost dose of antigen are equivalent, administered by the same route and in the same formulation.

4. The method of claim 1 wherein the pathogen is a viral pathogen.

5. The method of claim 4 wherein the viral pathogen is an RNA viral pathogen.

6. The method of claim 4 wherein the viral pathogen is selected from group consisting of human immunodeficiency virus 1 and 2 (HIV-I and HIV-2); simian immunodeficiency virus (SIV); hepatitis Virus A, B and C; influenza virus; polio virus; measles virus; mumps virus; rubella virus; rabies virus, cytomegalovirus; SARs virus; human T-lymphotrophic virus types I and II (HTLV-I and HTLV-II); rotavirus and hantavirus.

7. The method of claim 5 wherein the RNA viral pathogen is HIV-I and the antigen is selected from the group consisting of gag, pol, env, vif, vpr, vpx, vpu, tat, rev and nef.

8. The method of claim 7 wherein the antigen is a HIV-I gag protein.

9. The method of claim 1 wherein the rAAV is selected from the group consisting of naturally occurring AAV serotypes, pseudotyped AAV, and chimeric AAV.

10. The method of claim 1 wherein the boost dose is administered intramuscularly (IM) by a rAAV comprising an AAV-I capsid.

11. The method of claim 3 wherein the prime dose and the boost dose are administered by rAAVs comprising heterologous capsid proteins.

12. The method of claim 3 wherein the prime dose and the boost dose are administered by rAAVs comprising homologous capsid proteins.

13. The method of claim 3 wherein the prime dose is administered IM by rAAV-2 and the boost dose is administered IM in a single administration by a rAAV comprising an AAV-I capsid and wherein the boost immune response is greater as compared to the boost immune response elicited when the boost dose is administered IM by rAAV-2 in the same dose and formulation.

14. The method of claim 3 wherein the prime dose is administered IM by rAAV-2 and the boost dose is administered IM in a single administration by a rAAV comprising an AAV-I capsid and wherein the boost dose elicits a higher ratio of B-cell immune response to T-cell immune response as compared to the boost immune response elicited when the boost dose is administered IM by rAAV-2 in the same dose and formulation.

15. The method of claim 1 wherein the boost dose is administered by at least one non- rAAV vector selected from the group consisting of naked DNA, plasmid DNA, adenovirus, canarypox virus, pox virus, vaccinia virus, modified vaccinia Ankara (MVA), alphavirus, phabdovirus, picornavirus, and attenuated HIV.

16. The method of claim 2 wherein the prime dose is administered by at least one non- rAAV vector selected from the group consisting of naked DNA, plasmid DNA, adenovirus, canarypox virus, pox virus, vaccinia virus, modified vaccinia Ankara (MVA), alphavirus, phabdovirus, picornavirus, and attenuated HIV.

17. The method of claim 1 or claim 2 wherein the non-rAAV vector is administered in 2 or more consecutive administrations of a single vector.

18. Use of a first vector for the manufacture of a medicament for administration of a prime dose of ari antigen of a pathogen and a second vector for the manufacture of a medicament for administration of a boost dose of an antigen of a pathogen, characterized in that at least one of the prime dose or the boost dose is administered by a recombinant adeno-associated virus (rAAV) vector in a single administration, and wherein the boost dose elicits an equal or greater measurable immune response as compared to the response elicited by administration of the prime dose, with the proviso that when both the prime dose and the boost dose are administered by rAAV, they are not both administered by AAV-2.

19. Use of a first vector and a second vector according to claim 18, wherein the boost dose is an rAAV vector in a single administration, and wherein the prime dose is a non-rAAV vector.

20. Use of a first vector and a second vector according to claim 18 wherein the prime dose and boost dose are both administered by a rAAV and a single administration of the boost dose elicits a greater measurable immune response as compared to the response elicited by a single administration of the prime dose, wherein the measurement is performed under conditions where the prime dose and boost dose of antigen are equivalent, administered by the same route and in the same formulation.

21. Use of a first vector and a second vector according to claim 18 wherein the pathogen is a viral pathogen.

22. Use of a first vector and a second vector according to claim 21 , wherein the viral pathogen is an RNA viral pathogen.

23. Use of a first vector and a second vector according to claim 21 , wherein the viral pathogen is selected from group consisting of human immunodeficiency virus 1 and 2 (HIV-I and HIV-2); simian immunodeficiency virus (SIV); hepatitis virus A, B and C; influenza virus; polio virus; measles virus; mumps virus; rubella virus; rabies virus, cytomegalovirus; SARs virus; human T-lymphotrophic virus types I and II (HTLV-I and HTLV-II); rotavirus and hantavirus.

24. Use of a first vector and a second vector according to claim 22, wherein the RNA viral pathogen is HIV-I and the antigen is selected from the group consisting of gag, pol, env, vif, vpr, vpx, vpu, tat, rev and nef.

25. Use of a first vector and a second vector according to claim 24, wherein the antigen is a HIV-I gag protein.

26. Use of a first vector and a second vector according to claim 18, wherein the rAAV is selected from the group consisting of naturally occurring AAV serotypes, pseudotyped AAV, and chimeric AAV.

27. Use of a first vector and a second vector according to claim 18, wherein the boost dose is administered intramuscularly (IM) by a rAAV comprising an AAV-I capsid.

28. Use of a first vector and a second vector according to claim 18, wherein the prime dose and the boost dose are administered by rAAVs comprising heterologous capsid proteins.

29. Use of a first vector and a second vector according to claim 18, wherein the prime dose and the boost dose are administered by rAAVs comprising homologous capsid proteins.

30. Use of a first vector and a second vector according to claim 18, wherein the prime dose is administered IM by rAAV-2 and the boost dose is administered IM in a single administration by a rAAV comprising an AAV-I capsid and wherein the boost immune response is greater as compared to the boost immune response elicited when the boost dose is administered IM by rAAV-2 in the same dose and formulation.

31. Use of a first vector and a second vector according to claim 18, wherein the prime dose is administered IM by rAAV-2 and the boost dose is administered IM in a single administration by a rAAV comprising an AAV-I capsid and wherein the boost dose elicits a higher ratio of B-cell immune response to T-cell immune response as compared to the boost immune response elicited when the boost dose is administered IM by rAAV-2 in the same dose and formulation.

32. Use of a first vector and a second vector according to claim 18, wherein the boost dose is administered by at least one non-rAAV vector selected from the group consisting of naked DNA, plasmid DNA, adenovirus, canarypox virus, pox virus, vaccinia virus, modified vaccinia Ankara (MVA)₅ alphavirus, phabdovirus, picornavirus, and attenuated HIV.

33. Use of a first vector and a second vector according to claim 18, wherein the prime dose is administered by at least one non-rAAV vector selected from the group consisting of naked DNA, plasmid DNA, adenovirus, canarypox virus, pox virus, vaccinia virus, modified vaccinia Ankara (MVA), alphavirus, phabdovirus, picornavirus, and attenuated HIV.

34. Use of a first vector and a second vector according to claim 32 or 33, wherein the non-rAAV vector is administered in 2 or more consecutive administrations of a single vector.

35. A method for generating a recombinant adenoviral vector comprising a desired polynucleotide sequence, comprising the steps of: co-transforming Escherichia CoIi bacteria with: a. a linear DNA molecule and; b. a supercoiled adenoviral vector comprising an adenoviral genome with one or more deletions; wherein the linear DNA molecule comprises a first segment of DNA comprising a desired polynucleotide sequence and a second and a third segment of adenoviral genomic DNA, each of said second and third segments being at least 500 bp and being sufficient to mediate homologous recombination with the supercoiled adenoviral vector, wherein the second and third segments flank the first segment; wherein the supercoiled adenoviral vector comprises a deletion of adenovirus transcription unit E3, but comprises a sequence encoding a functional E3 adenoviral death protein and/or a sequence encoding a functional L4 polyadenylation sequence; and wherein the supercoiled adenoviral vector comprises a bacterial origin of replication flanked on each side by segments of DNA identical to the second and third segments whereby subsequent to the step of co-transforming, the supercoiled adenoviral vector and the linear DNA molecule recombine to form a recombinant adenoviral vector comprising the desired polynucleotide sequence.

36. The method of claim 35, wherein the supercoiled adenoviral vector comprises a sequence encoding a functional E3 adenoviral death protein.

37. The method of claim 35, wherein the supercoiled adenoviral vector comprises a sequence encoding a functional L4 polyadenylation sequence.

38. The method of claim 35, wherein the supercoiled adenoviral vector comprises a sequence encoding a functional E3 adenoviral death protein and a sequence encoding a functional L4 polyadenylation sequence.

39. The method of claim 35, wherein the supercoiled adenoviral vector comprises a deletion of adenovirus transcription unit El .

40. The method of claim 35, wherein the deletion of adenovirus transcription unit E3 is from about nucleotide 28593 to about nucleotide 30470, wherein the nucleotide numbering is based on wild type adenovirus 5 genome.

41. The method of claim 35, wherein the sequence encoding the functional adenoviral death protein comprises nucleotide 29,397 to nucleotide 29,783, wherein the nucleotide numbering is based on wild type adenovirus 5 genome.

42. The method of claim 35, wherein the sequence encoding the functional L4 polyadenylation sequence comprises nucleotide 28,164 to nucleotide 28,169, wherein the nucleotide numbering is based on wild type adenovirus 5 genome.

43. The method of claim 35, wherein the first segment comprises an inverted terminal repeat of an adenoviral genome.

44. The method of claim 35, wherein the supercoiled adenoviral vector comprises an inverted terminal repeat of an adenoviral genome.

45. The method of claim 35, wherein the desired polynucleotide sequence comprises a sequence encoding a multiple cloning site.

46. The method of claim 35, wherein the second segment comprises about nucleotide 34931 to about nucleotide 35935, and the third segment comprises about nucleotide 3534 to about nucleotide 5790, wherein the nucleotide numbering is based on wild type adenovirus 5 genome.

47. The method of claim 35, wherein the supercoiled adenoviral vector comprises adenovirus 5 genome from nucleotide 3534 to nucleotide 35935 with a deletion of adenovirus transcription unit E3 except E3 adenoviral death protein, wherein the deletion of adenovirus transcription unit E3 is from nucleotide 30470 to nucleotide 28593 and the sequence encoding the E3 adenoviral death protein is from nucleotide 29397 to 29783.

48. The method of claim 35, wherein the desired polynucleotide sequence comprises a gene encoding a therapeutic product.

49. The method of claim 48, wherein the therapeutic product is a vaccine.

50. The method of claim 48, wherein the therapeutic product is an antigen of a pathogen.

51. The method of claim 48, wherein the therapeutic product is an antigen of a viral pathogen.

52. The method of claim 51 , wherein the antigen is an HIV antigen.

53. the method of claim 52, wherein the HIV antigen is an HIV gag antigen.

54. A method for generating a recombinant adenoviral hybrid vector comprising a desired polynucleotide sequence and an inverted terminal repeat of an adeno-associated virus (AAV ITR), comprising the steps of: co-transforming Escherichia CoIi bacteria with: a. a linear DNA molecule and; b. a supercoiled adenoviral vector comprising an adenoviral genome with one or more deletions; wherein the linear DNA molecule comprises a first segment of DNA comprising a desired polynucleotide sequence and an AAV ITR flanking the desired polynucleotide sequence, and a second and a third segment of adenoviral genomic DNA, each of said second and third segments being at least 500 bp and being sufficient to mediate homologous recombination with the supercoiled adenoviral vector, wherein the second and third segments flank the first segment; wherein the supercoiled adenoviral vector comprises a deletion of adenovirus transcription unit E3, but corriprises a sequence encoding a functional E3 adenoviral death protein and/or a sequence encoding a functional L4 polyadenylation sequence; and wherein the supercoiled adenoviral vector comprises a bacterial origin of replication flanked on each side by segments of DNA identical to the second and third segments whereby subsequent to the step of co-transforming, the supercoiled adenoviral vector and the linear DNA molecule recombine to form a recombinant adenoviral vector comprising the desired polynucleotide sequence and the AAV ITR.

55. The method of claim 54, wherein the supercoiled adenoviral vector comprises a sequence encoding a functional E3 adenoviral death protein.

56. The method of claim 54, wherein the supercoiled adenoviral vector comprises a sequence encoding a functional L4 polyadenylation sequence.

57. The method of claim 54, wherein the supercoiled adenoviral vector comprises a sequence encoding a functional E3 adenoviral death protein and a sequence encoding a functional L4 polyadenylation sequence.

58. The method of claim 54, wherein the supercoiled adenoviral vector further comprises a deletion of adenovirus transcription unit El .

59. The method of claim 54, wherein the deletion of adenovirus transcription unit E3 is from about nucleotide 28593 to about nucleotide 30471, wherein the nucleotide numbering is based on wild type adenovirus 5 genome.

60. The method of claim 54, wherein the sequence encoding the functional adenoviral death protein comprises nucleotide 29,739 to nucleotide 29,783, wherein the nucleotide numbering is based on wild type adenovirus 5 genome.

61. The method of claim 54, wherein the sequence encoding the functional L4 polyadenylation sequence comprises nucleotide 28,164 to nucleotide 28,169, wherein the nucleotide numbering is based on wild type adenovirus 5 genome.

62. The method of claim 54, wherein the first segment comprises an inverted terminal repeat of an adenoviral genome.

63. The method of claim 54, wherein the supercoiled adenoviral vector comprises an inverted terminal repeat of an adenoviral genome.

64. The method of claim 54, wherein the desired polynucleotide sequence comprises a sequence encoding a multiple cloning site.

65. The method of claim 54, wherein the second segment comprises about nucleotide 34931 to about nucleotide 35935, and the third segment comprises about nucleotide 3534 to about nucleotide 5790, wherein the nucleotide numbering is based on wild type adenovirus 5 genome.

66. The method of claim 54, wherein the supercoiled adenoviral vector comprises adenovirus 5 genome from nucleotide 3534 to nucleotide 35935 with a deletion of adenovirus transcription unit E3 except E3 adenoviral death protein, wherein the deletion of adenovirus transcription unit E3 is from nucleotide 30470 to nucleotide 28593 and the sequence encoding the E3 adenoviral death protein is from nucleotide 29397 to 29783.

67. The method of claim 54, wherein the desired polynucleotide sequence comprises a gene encoding a therapeutic product.

68. The method of claim 67, wherein the therapeutic product is a vaccine.

69. The method of claim 67, wherein the therapeutic product is an antigen of a pathogen.

70. The method of claim 67, wherein the therapeutic product is an antigen of a pathogen, an antigen of a viral pathogen.

71. The method of claim 70, wherein the antigen is an HIV antigen.

72. The method of claim 71 , wherein the HIV antigen is an HIV gag antigen.

73. A method for generating a recombinant adenoviral particle comprising a desired polynucleotide sequence, comprising the steps of: a. a linear DNA molecule and; b. a supercoiled adenoviral vector comprising an adenoviral genome with one or more deletions; wherein the linear DNA molecule comprises a first segment of DNA comprising a desired polynucleotide sequence and a second and a third segment of adenoviral genomic DNA, each of said second and third segments being at least 500 bp and being sufficient to mediate homologous recombination with the supercoiled adenoviral vector, wherein the second and third segments flank the first segment; wherein the supercoiled adenoviral vector comprises a deletion of adenovirus transcription unit E3, but comprises a sequence encoding a functional E3 adenoviral death protein and/or a sequence encoding a functional L4 polyadenylation sequence; and wherein the supercoiled adenoviral vector comprises a bacterial origin of replication flanked on each side by segments of DNA identical to the second and third segments whereby subsequent to the step of co-transforming, the supercoiled adenoviral vector and the linear DNA molecule recombine to form a recombinant adenoviral vector comprising the desired polynucleotide sequence; linearizing the recombinant adenoviral vector to form a linear vector comprising termini which comprise adenoviral terminal repeats flanking the desired polynucleotide sequence ; and transfecting a mammalian cell with the linearized vector, whereby the mammalian cell produces the recombinant adenoviral particle which comprises the desired polynucleotide sequence.

74. The method of claim 73, wherein the supercoiled adenoviral vector comprises a sequence encoding a functional E3 adenoviral death protein.

75. The method of claim 73, wherein the supercoiled adenoviral vector comprises a sequence encoding a functional L4 polyadenylation sequence.

76. The method of claim 73, wherein the supercoiled adenoviral vector comprises a sequence encoding a functional E3 adenoviral death protein and a sequence encoding a functional L4 polyadenylation sequence.

77. The method of claim 73, wherein the supercoiled adenoviral vector further comprises a deletion of adenovirus transcription unit El, and the mammalian cell expresses the adenovirus transcription unit El .

78. The method of claim 73, wherein the supercoiled adenoviral vector further comprises a deletion of adenovirus transcription unit El, and the mammalian cell stably expresses the adenovirus transcription unit El.

79. The method of claim 73, wherein the deletion of adenovirus transcription unit E3 is from about nucleotide 28593 to about nucleotide 30470 of wild type adenovirus, wherein the nucleotide numbering is based on wild type adenovirus 5 genome.

80. The method of claim 73, wherein the polynucleotide encoding the functional adenoviral death protein comprises nucleotide 29,397 to nucleotide 29,783, wherein the nucleotide numbering is based on wild type adenovirus 5 genome.

81. The method of claim 73, wherein the polynucleotide encoding the functional L4 polyadenylation sequence comprises nucleotide 28,164 to nucleotide 28,169, wherein the nucleotide numbering is based on wild type adenovirus 5 genome.

82. The method of claim 73, wherein the first segment comprises an inverted terminal repeat of an adenoviral genome.

83. The method of claim 73, wherein the supercoiled adenoviral vector comprises an inverted terminal repeat of an adenoviral genome.

84. The method of claim 73, wherein the desired polynucleotide sequence comprises a gene encoding a therapeutic product.

85. The method of claim 84, wherein the therapeutic product is a vaccine.

86. the method of claim 84, wherein the therapeutic product is an antigen of a pathogen.

87. The method of claim 84, wherein the therapeutic product is an antigen of a pathogen, an antigen of a viral pathogen.

88. The method of claim 87, wherein the antigen is an HIV antigen.

89. The method of claim 88, wherein the HIV antigen is an HIV gag antigen.

90. A method for generating a recombinant adenoviral hybrid particle comprising a desired polynucleotide sequence and an inverted terminal repeat of an adeno-associated virus (AAV ITR), comprising the steps of: co-transforming Escherichia CoIi bacteria with: a. a linear DNA molecule and; b. a supercoiled adenoviral vector comprising an adenoviral genome with one or more deletions; wherein the linear DNA molecule comprises a first segment of DNA comprising a desired polynucleotide sequence and an AAV-ITR flanking the desired polynucleotide sequence, and a second and a third segment of adenoviral genomic DNA, each of said second and third segments being at least 500 bp and being sufficient to mediate homologous recombination with the supercoiled adenoviral vector, wherein the second and third segments flank the first segment; wherein the supercoiled adenoviral vector comprises a deletion of adenovirus transcription unit E3, but comprises a sequence encoding a functional E3 adenoviral death protein and/or a sequence encoding a functional L4 polyadenylation sequence; and wherein the supercoiled adenoviral vector comprises a bacterial origin of replication flanked on each side by segments of DNA identical to the second and third segments whereby subsequent to the step of co-transforming, the supercoiled adenoviral vector and the linear DNA molecule recombine to form a recombinant adenoviral vector comprising the desired polynucleotide sequence and the AAV ITR; linearizing the recombinant adenoviral vector to form a linear vector comprising termini which comprise adenoviral terminal repeats flanking the desired polynucleotide sequence and the AAT ITR; and transfecting a mammalian cell with the linearized vector, whereby the mammalian cell produces the recombinant adenoviral particle which comprises the desired polynucleotide sequence and the AAV ITR.

91. The method of claim 90, wherein the supercoiled adenoviral vector comprises a sequence encoding a functional E3 adenoviral death protein.

92. The method of claim 90, wherein the supercoiled adenoviral vector comprises a sequence encoding a functional L4 polyadenylation sequence.

93. The method of claim 90, wherein the supercoiled adenoviral vector comprises a sequence encoding a functional E3 adenoviral death protein and a sequence encoding a functional L4 polyadenylation sequence.

94. The method of claim 90, wherein the supercoiled adenoviral vector further comprises a deletion of adenovirus transcription unit El, and the mammalian cell expresses adenovirus transcription unit El .

95. The method of claim 90, wherein the supercoiled adenoviral vector further comprises a deletion of adenovirus transcription unit El, and the mammalian cell stably expresses adenovirus transcription unit El .

96. The method of claim 94, wherein the deletion of adenovirus transcription unit E3 deletion is from about nucleotide 28593 to about nucleotide 30470, wherein the nucleotide numbering is based on wild type adenovirus 5.

97. The method of claim 94, wherein the sequence encoding the functional adenoviral death protein comprises nucleotide 29,739 to nucleotide 29,783, wherein the nucleotide numbering is based on wild type adenovirus 5.

98. The method of claim 94, wherein the sequence encoding the functional L4 polyadenylation sequence comprises nucleotide 28,164 to nucleotide 28,169, wherein the nucleotide numbering is based on wild type adenovirus 5.

99. The method of claim 94, wherein the first segment comprises an inverted terminal repeat of an adenoviral genome.

100. The method of claim 94, wherein the supercoiled adenoviral vector comprises an inverted terminal repeat of an adenoviral genome.

101. The method of claim 94, wherein the desired polynucleotide sequence comprises a gene encoding a therapeutic product.

102. The method of claim 101, wherein the therapeutic product is a vaccine.

103. The method of claim 101, wherein the therapeutic product is an antigen of a pathogen.

104. The method of claim 101, wherein the therapeutic product is an antigen of a pathogen, an antigen of a viral pathogen.

105. The method of claim 104, wherein the antigen is an HIV antigen.

106. The method of claim 105, wherein the HIV antigen is an HIV gag antigen.

107. A recombinant adenoviral vector comprising adenoviral inverted terminal repeats flanking an adenoviral genome and a desired polynucleotide sequence, wherein the adenoviral genome comprises one or more deletions, wherein the deletions comprise a deletion of adenovirus transcription unit El and E3, wherein the adenoviral genome comprises a sequence encoding a functional E3 adenoviral death protein and/or sequence encoding a functional L4 polyadenylation site.

108. An adenoviral hybrid vector comprising adenoviral inverted terminal repeats flanking an adenoviral genome and a desired polynucleotide sequence flanked by an AAT ITR, wherein the adenoviral genome comprises one or more deletions, wherein the deletions comprise a deletion of adenovirus transcription unit El and E3, wherein the adenoviral genome comprises a sequence encoding a functional E3 adenoviral death protein and/or sequence encoding a functional L4 polyadenylation site.

109. The adenoviral hybrid vector of claim 108, wherein the adenoviral genome comprises a sequence encoding a functional E3 adenoviral death protein.

110. The adenoviral hybrid vector of claim 108, wherein the adenoviral genome comprises a sequence encoding a functional L4 polyadenylation sequence.

111. The adenoviral hybrid vector of claim 108, wherein the adenoviral genome comprises a sequence encoding a functional E3 adenoviral death protein and a sequence encoding a functional L4 polyadenylation sequence.

112. The adenoviral hybrid vector of claim 108, wherein the deletion of adenovirus transcription unit E3 is from nucleotide 28593 to nucleotide 30471, wherein the nucleotide numbering is based on wild type adenovirus 5.

113. The adenoviral hybrid vector of claim 108, wherein the sequence encoding the functional adenoviral death protein comprises nucleotide 29,739 to nucleotide 29,783, wherein the nucleotide numbering is based on wild type adenovirus 5.

114. The adenoviral hybrid vector of claim 108, wherein the sequence encoding the functional L4 polyadenylation sequence comprises nucleotide 28,164 to nucleotide 28,169, wherein the nucleotide numbering is based on wild type adenovirus 5.

115. The adenoviral hybrid vector of claim 108, wherein the desired polynucleotide sequence comprises a gene encoding a therapeutic product.

116. The adenoviral hybrid vector of claim 115, wherein the therapeutic product is a vaccine.

117. The adenoviral hybrid vector of claim 115, wherein the therapeutic product is an antigen of a pathogen.

118. The adenoviral hybrid vector of claim 115, wherein the therapeutic product is an antigen of a pathogen, an antigen of a viral pathogen.

119. The adenoviral hybrid vector of claim 118, wherein the antigen is an HIV antigen.

120. The adenoviral hybrid vector of claim 119, wherein the HIV antigen is an HIV gag antigen.

121. A method of generating a stock of adenoviral hybrid vectors comprising infecting a mammalian cell with the adenoviral hybrid vector of any one of claims 108-121.

122. A kit comprising two plasmids, wherein the first plasmid comprises (a) a bacterial origin of replication; (b) a first segment of DNA comprising a restriction enzyme site for insertion of a desired polynucleotide sequence; and (c) a second and a third segment of adenoviral genomic DNA, each of said second and third segments being at least 500 bp and being sufficient to mediate homologous recombination with an adenoviral vector, wherein the second and third segments flank the first segment; wherein the second plasmid comprises (a) an adenoviral genome comprising one or more deletions, wherein the deletions comprises a deletion of adenoviral transcription unit E3, wherein the adenoviral genome comprises a sequence encoding a functional E3 adenoviral death protein and/or a sequence encoding a functional L4 polyadenylation site; and (b) a bacterial origin of replication flanked on either side by DNA segments identical to the second and third segments in the first plasmid; wherein upon linearization of the first plasmid and co-transfection of the second plasmid of Escherichia coli bacterial cells, the second plasmid and the linearized first plasmid recombine to form a recombinant adenoviral vector comprising the restriction enzyme site.

123. The kit of claim 122, wherein the first segment further comprises an inverted terminal repeat of adenovirus.

124. A kit comprising two plasmids, wherein the first plasmid comprises (a) a bacterial origin of replication; (b) a first segment of DNA comprising a restriction enzyme site for insertion of a desired polynucleotide sequence and an AAV ITR flanking the restriction enzyme sit; and (c) a second and a third segment of adenoviral genomic DNA, each of said second and third segments being at least 500 bp and being sufficient to mediate homologous recombination with an adenoviral vector, wherein the second and third segments flank the first segment; wherein the second plasmid comprises (a) an adenoviral genome comprising one or more deletions, wherein the deletions comprises a deletion of adenoviral transcription unit E3, wherein the adenoviral genome comprises a sequence encoding a functional E3 adenoviral death protein and/or a sequence encoding a functional L4 polyadenylation site; and (b) a bacterial origin of replication flanked on either side by DNA segments identical to the second and third segments in the first plasmid; wherein upon linearization of the first plasmid and co-transfection of the second plasmid of Escherichia coli bacterial cells, the second plasmid and the linearized first plasmid recombine to form a recombinant adenoviral vector comprising the restriction enzyme site flanked by the AAV ITR.

125. The kit of claim 124, wherein the first segment further comprises an inverted terminal repeat of adenovirus.