WO2009110950A1 - Vaccins à souches atténuées de listeria et d'adénovirus, et méthodes d'utilisation - Google Patents

Vaccins à souches atténuées de listeria et d'adénovirus, et méthodes d'utilisation Download PDF

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WO2009110950A1
WO2009110950A1 PCT/US2008/088543 US2008088543W WO2009110950A1 WO 2009110950 A1 WO2009110950 A1 WO 2009110950A1 US 2008088543 W US2008088543 W US 2008088543W WO 2009110950 A1 WO2009110950 A1 WO 2009110950A1
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antigen
another embodiment
listeria
present
subject
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Fred R. Frankel
Zhongxia Li
Ruth Ruprecht
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The Trustees Of The University Of Pennsylvania
Dana-Farber Cancer Institute, Inc.
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
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    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
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    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
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    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16271—Demonstrated in vivo effect
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Definitions

  • This invention provides a combination of attenuated Listeria strains and attenuated adenovirus strains for use as vaccines and methods of using and generating the same.
  • Attenuated or inactivated bacterial or viral vaccines often only induce immunization for a short period of time and immunity is limited to a humoral response. Further, traditional attenuated or inactivated bacterial or viral vaccines vaccines do not elicit the cytotoxic T- lymphocyte (CTL) immune response necessary for the lysis of tumor cells and cells infected with intracellular pathogens.
  • CTL cytotoxic T- lymphocyte
  • bacterial vaccine vectors such as Listeria monocytogenes (LM), a beta hemolytic gram positive facultative intracellular microbe.
  • LM Listeria monocytogenes
  • Many vaccine strategies depend on a prime/boost protocol, which leads to a selective increase of memory T cells specific for the shared antigen of the prime and boost vectors, while minimizing the undesired T cells induced by the individual vectors.
  • DNA vaccines have been used for the primary immunization, having the virtue of introducing few irrelevant antigens to the system.
  • responses to DNA vaccines tend to be weak unless supplemented with molecules that induce additional inflammatory reactions that mimic responses to a pathogen.
  • the virtue of more complex vectors is that they prime innate and adaptive immune responses through their interaction with pattern-recognition receptors on antigen-presenting cells (APCs), initiating inflammatory reactions that are a prerequisite for a good response.
  • APCs antigen-presenting cells
  • Prime and boost vaccines that can be administered mucosally and effectively induce strong mucosal immunity is desirable for preventing and treating disease associated with agents that infect the mucosa.
  • the present invention provides, in one embodiment, a method of inducing an antigen- specific cell- mediated immune response in a subject, comprising the steps of: administering to said subject a recombinant strain of Listeria comprising said antigen; and administering to said subject an attenuated recombinant adenovirus comprising said antigen; thereby inducing an antigen- specific cell-mediated immune response.
  • the present invention provides a vaccine comprising an effective amount of a recombinant strain of Listeria comprising an antigen; and an attenuated recombinant adenovirus comprising said antigen.
  • the present invention provides a method of inducing an antigen- specific cell-mediated immune response in a subject, comprising the steps of: mucosally administering to said subject a recombinant strain of Listeria comprising said antigen; and mucosally administering to said subject an attenuated recombinant adenovirus comprising said antigen; thereby inducing an antigen- specific cell-mediated immune response.
  • FIG. 1 Immune response after one or more oral L. monocytogenes primes followed by a systemic (i.m.) boost by adenovirus type5. Mice were infected orally by gastric intubation with 1.5 x 10 10 attenuated Lm-gag one or more times as indicated (four weeks between infections) followed four weeks later with an i.m. infection with 5 x 10 9 particles of replication-defective Ad5-gag. Control samples were taken before Ad5-gag infection (3xLm (memory)) and at seven days after a single Ad5 infection (IxAd (effector)).
  • lymphocytes were isolated from the indicated tissues (A-C) and assessed by FACS analysis for activated (CDl Ia+), Gag-specific CD8 + T cells, or for intracellular cytokine expression (D). The numbers indicate the percent of CD8 + T cells within the gated boxes.
  • CD8 + T cells from the latter were analyzed 9 days later for the percentage of Gag-tetramer + CDl Ia + cells that expressed IFN- ⁇ in response to stimulation with HIV- Gagi 97 _ 205 peptide.
  • the left peak in this histogram resulted from stimulating the cells with a control nonrelevant peptide, lymphocytic choriomeningitis virus-nucleoprotein 118-126 (LCMV-NPn8-i26)-
  • Figure 2 In vivo cytolytic activity in immune mice after two different prime/boost protocols.
  • mice were immunized by a single oral infection with rLm-g ⁇ g (1.5 X 10 10 CFU) followed 4 wk later by im boost with ⁇ Ad5-gag (5 X 10 9 vps). After the boost, spleens were isolated 8 days (effector) or 5 wk (memory) later and assayed for cytolytic activity either 2 h or 16 h after i.v. transfusion of mice with a mixture of control target cells (left peak, low CFSE in each diagram) or Gag-peptide-labeled target cells (right peak, high CFSE in diagrams where detectable). For each analysis, splenocytes from two mice were combined.
  • the numbers indicate the percentage of cytolysis of the Gag-labeled targets relative to that of naive controls.
  • the 16-h CTL activity for rLm-g ⁇ g-only memory mice was 8% and for rAd5- gag-on ⁇ y effector mice, it was 45% (not shown).
  • B and C In vivo cytolytic activity in vagina, iliac lymph nodes, and spleen after ivag rhm-gag/rAd5-gag prime-boost vaccination (B) and the effect of CD8 T cell depletion on this activity (C).
  • mice were immunized with a single ivag infection with rLm-gag (5 X 10 9 CFU) followed 4 weeks later with a single ivag boost with ⁇ Ad5-gag (1 X 10 10 vps).
  • Tissues were isolated 8 days (effector) or 5 wk (memory) after the boost for assay of cytolytic activity at either 2 or 16 h (B) or 3 h (C) after i.v. transfusion of mice with a mixture of control target cells (left peak, low CFSE in each diagram) and Gag-peptide-labeled target cells (right peak, high CFSE in each diagram).
  • the numbers indicate the percentage of cytolysis of the Gag-labeled targets relative to appropriate naive controls and are given as mean values. In all cases, the SEM was ⁇ 4% of the mean values shown.
  • the tissues from five mice were combined with the exception of the naive vagina, from which the tissues from seven mice were combined. For analysis of other tissues, 2-3 mice were combined.
  • T cell depletion of effector mice was achieved with two administrations (0.25 mg each; i.p.) of anti-CD8 (clone 2.43) or anti-CD4 (clone GK 1.5) rat anti-mouse mAbs or control rat Ig.
  • Figure 3 The recall response of immune mice and protection against vaccinia virus challenge.
  • Groups of mice were immunized by a single oral infection with attenuated Lm-gag followed 30 days later by intramuscular boost with Ad5-gag (A, B) or by intra-vaginal infections with both Lm-gag and, 30 days later, Ad5-gag (C, D).
  • Ad5-gag Ad5-gag
  • C, D Ad5-gag
  • mice were challenged i.p. with 1 x 10 7 pfu Vac-gag (A, B) or ivag with 8 x 10 7 Vac-gag (C, D).
  • Spleen (A) was examined for Gag-specific CD8 + T cell memory (Lm (ig)/Ad (im)) and its recall after vaccinia challenge (Lm (ig)/Ad (im) + Vac(ip)). The same animals were assessed for their protection against replication of the Vac-gag virus in ovaries (B).
  • vagina (C) was examined for Gag-specific CD8 + T cell memory (Lm (ivg)/Ad (ivg)) and its recall after challenge (Lm(ivg)/Ad(ivg) + Vac(ivg)). The animals were assessed for their protection against replication of the Vac-gag virus in ovaries and oviduct (D). The threshold for virus detection is shown by dotted lines.
  • FIG. 4 Bacterial translocation across the vaginal epithelium and immunity generated after ivag infection by L. monocytogenes .
  • DLNs refers to the combined inguinal and iliac draining lymph nodes of vagina.
  • mice were immunized with attenuated Lm-gag by the oral (ig, 1.5 x 10 10 cfu), intra-rectal (ir, 5 x 10 9 ) or intra- vaginal (ivg, 5 x 10 9 ) routes of infection, followed 35 days later either by (C) an intramuscular boost (im) (5 x 10 9 vps) or (D) an intra-vaginal boost (ivg, 10 10 vps) with Ad5-gag.
  • the numbers shown are the percent Gag-specific CD8 + T cells in spleen and vagina at six days after the boost.
  • the percent Gag-specific CD8 + T cells in all naive samples was ⁇ 0.6%; in Lm- gag-only memory mice in spleen and vagina by all routes was 1% or less; in Ad5-gag-only effector mice, in spleen was ⁇ 0.5% (ivg) or ⁇ 4% (im), and in vagina, ⁇ 13% (ivag) or ⁇ 8% (im).
  • This invention provides, in one embodiment, a method of inducing an antigen- specific immune response in a subject, comprising the steps of: administering to said subject a recombinant strain of Listeria comprising an antigen; and administering to said subject a recombinant adenovirus comprising said antigen; thereby inducing an antigen- specific immune response in said subject.
  • said immune response is cell-mediated.
  • said cell-mediated immune response is a CD8 + T cell immune response.
  • said recombinant adenovirus is attenuated.
  • said recombinant Listeria is attenuated.
  • This invention provides, in one embodiment, a method of inducing an antigen- specific cell- mediated immune response in a subject, comprising the steps of: administering to said subject a recombinant strain of Listeria comprising an antigen; and administering to said subject an attenuated recombinant adenovirus comprising said antigen; thereby inducing an antigen- specific cell-mediated immune response in said subject.
  • said antigen- specific cell-mediated immune response is induced in a vaginal tissue of said subject.
  • this invention provides a method of inducing an antigen- specific CD8 + T cell immune response in a subject, comprising the steps of: administering to said subject a recombinant strain of Listeria comprising an antigen; and administering to said subject an attenuated recombinant adenovirus comprising said antigen; thereby inducing an antigen- specific CD8 + T cell immune response in said subject.
  • said antigen- specific CD8 + T cell immune response is induced in a vaginal tissue of said subject.
  • This invention provides, in one embodiment, a method of inducing an antigen- specific cell- mediated immune response in a subject, comprising the steps of: mucosally administering to said subject a recombinant strain of Listeria comprising an antigen; and mucosally administering to said subject an attenuated recombinant adenovirus comprising said antigen; thereby inducing an antigen- specific cell- mediated immune response in said subject.
  • said antigen- specific cell-mediated immune response is induced in a vaginal tissue of said subject.
  • said antigen- specific cell-mediated immune response is induced systemically in said subject.
  • this invention provides a method of inducing an antigen- specific CD8 + T cell immune response in a subject, comprising the steps of: mucosally administering to a subject a recombinant strain of Listeria comprising an antigen; and mucosally administering to a subject an attenuated recombinant adenovirus comprising an antigen; thereby inducing an antigen- specific CD8 + T cell immune response in said subject.
  • said antigen- specific CD8 + T cell immune response is induced in a vaginal tissue of said subject.
  • said antigen- specific CD8 + T cell immune response is induced systemically in said subject.
  • this invention provides a method of inducing an antigen- specific cell- mediated immune response in a subject, comprising the steps of: administering to said subject a recombinant strain of an intracellular bacterium comprising an antigen; and administering to said subject an attenuated recombinant virus comprising said antigen; thereby inducing an antigen- specific cell- mediated immune response in said subject.
  • said intracellular bacterium is a Listeria.
  • said virus is an adenovirus.
  • said antigen- specific cell-mediated immune response is induced in a vaginal tissue of said subject.
  • this invention provides a method of inducing an antigen- specific CD8 + T cell immune response in a subject comprising the steps of: administering to said subject a recombinant strain of an intracellular bacterium comprising an antigen; and administering to said subject an attenuated recombinant virus comprising said antigen; thereby inducing an antigen- specific cell-mediated immune response in said subject.
  • said intracellular bacterium is a Listeria.
  • said virus is an adenovirus.
  • said antigen- specific cell-mediated immune response is induced in a vaginal tissue of said subject.
  • said intracellular bacterium is Mycobacterium, Listeria, and Salmonella, Chlamydia, Rickettsia Lawsonia, or Brucella.
  • said intracellular bacterium is Salmonella typhimurium, while in one embodiment, said intracellular bacterium is Mycobacterium bovis, which in one embodiment, is the BCG strain of Mycobacterium bovis.
  • the recombinant Listeria strain is Listeria seeligeri.
  • the recombinant Listeria strain is Listeria grayi.
  • the recombinant Listeria strain is Listeria ivanovii.
  • the recombinant Listeria strain is Listeria welshimeri. In another embodiment, the recombinant Listeria strain is Listeria innocua. In another embodiment, the recombinant Listeria strain is Listeria monocytogenes, which in one embodiment, is serotypes 1, 2, 3, 4a, 4b, 4d, 4e, or a combination thereof, and in another embodiment, EGD-e strain. In one embodiment, the Listeria monocytogenes is serovars l/2a, l/2b, or 4b. In another embodiment, the recombinant Listeria strain is a recombinant strain of any other Listeria species known in the art. Each possibility represents a separate embodiment of the present invention.
  • the recombinant virus is a poxvirus, retrovirus, herpes virus, polio virus, adeno-associated virus or an adenovirus.
  • said poxvirus is vaccinia. Methods of attenuating such viruses as well as inserting heterologous genes are known in the art.
  • a recombinant Listeria strain of the present invention has been passaged through an animal host.
  • the passaging maximizes efficacy of the strain as a vaccine vector.
  • the passaging stabilizes the immunogenicity of the Listeria strain.
  • the passaging stabilizes the virulence of the Listeria strain.
  • the passaging increases the immunogenicity of the Listeria strain.
  • the passaging increases the virulence of the Listeria strain.
  • the passaging removes unstable sub-strains of the Listeria strain.
  • the passaging reduces the prevalence of unstable sub-strains of the Listeria strain.
  • the recombinant Listeria comprises the antigen, while in another embodiment, the recombinant Listeria expresses the antigen, while in another embodiment, the recombinant Listeria secretes the antigen.
  • the recombinant adenovirus comprises the antigen, while in another embodiment, the recombinant adenovirus expresses the antigen.
  • the antigen is endogenous to Listeria.
  • the antigen is endogenous to adenovirus.
  • the antigen is heterologous to Listeria, heterologous to adenovirus, or heterologous to both Listeria and adenovirus.
  • the Listeria strain contains a genomic insertion of a gene encoding an antigen-containing recombinant peptide, which in one embodiment is via homologous recombination, and in another embodiment, via transposon insertion. These and other methods are well known in the art.
  • the Listeria strain comprises a plasmid vector comprising a gene encoding an antigen-containing recombinant peptide.
  • an antigen-containing recombinant peptide is a heterologous antigen.
  • the Listeria strain comprises a gene encoding an antigen of interest.
  • the antigen of interest is a heterologous antigen.
  • the present invention provides that a recombinant strain of Listeria is an attenuated recombinant strain of Listeria.
  • the compositions and methods of the present invention provide attenuated strains of Listeria, wherein the bacteria have been attenuated by the introduction of auxotrophic mutations in the Listeria genomic DNA.
  • the present invention provides that these strains are herein referred to as attenuated auxotrophic strains or "AA strains" of Listeria.
  • Attenuated Listeria strains for use in the compositions and methods of the present invention comprise defects, which in one embodiment, are mutations, deletions, or insertions which disrupt gene product structure or function, in one or more of the Listeria monocytogenes virulence genes, which in one embodiment, is prfa, acta, mpl, plcB, plcAB, MyA, or inlAB gene.
  • the prfA gene is overexpressed and the actA gene is deleted.
  • Listeria attenuation is via deletion of the Lm actA gene (either alone or with the plcB or inlB genes), which in one embodiment, stops both intracellular movement and cell-to-cell spreading of bacteria while maintaining immunogenicity (see, for example, Angelakopoulos et al. Infect Immun. 2002;70(7):3592-601; Goossens et aL ⁇ tf Immunol. 1995;7(5):797-805; Brockstedtet al. Proc Natl Acad Sci USA. 2004;101(38):13832- 7).
  • the uvrA and uvrB genes of Lm are deleted, which, in one embodiment, encode nucleotide excision repair genes (Brockstedt et al., Nat Med. 2005;l l(8):853-60).
  • the term "attenuation" as used herein refers to a diminution in the ability of the bacterium to cause disease in an animal.
  • the present invention provides that the pathogenic characteristics of the attenuated Listeria strain have been decreased compared with wild- type Listeria.
  • the attenuated Listeria is capable of growth and maintenance in culture.
  • the present invention provides that an attenuated strain of Listeria is thus one which does not kill an animal to which it is administered, or is one which is lethal to an animal only when the number of bacteria administered is vastly greater, in one embodiment, 10 times greater, in another embodiment, 100 times greater, and in another embodiment, 200 times greater than the number of wild-type, non- attenuated bacteria required to kill such an animal.
  • the present invention provides that an attenuated bacterium should also be construed to mean one which is incapable of replication in a typical or the general environment because the nutrient required for its growth is not present therein.
  • the present invention provides that the bacterium is limited to replication in a controlled environment wherein the required nutrient is provided.
  • the present invention provides that the attenuated strains of the present invention are therefore environmentally safe in that they are incapable of uncontrolled replication.
  • the present invention provides that any Listeria species capable of infectious disease may be genetically attenuated according to the methods of the present invention to yield a useful and safe bacterial vaccine provided the attenuated Listeria species exhibits an LD 50 in a host organism that is significantly greater than that of the non-attenuated wild- type species.
  • the present invention provides that strains of Listeria other than L. monocytogenes may be used for the generation of attenuated mutants for use as vaccines.
  • the present invention provides that the Listeria strain useful for the generation of attenuated vaccines is L. monocytogenes .
  • the present invention provides that the auxotrophic attenuated strains of Listeria that are auxotrophic for D-alanine are contemplated as part of this invention.
  • the present invention provides that the term "auxotrophic for D-alanine", as used herein, describes an auxotrophic attenuated strain of Listeria unable to synthesize D-alanine, or, in another embodiment, Listeria that cannot grow in the absence of D-alanine and therefore requires exogenously added D-alanine for growth.
  • the present invention provides that the D-alanine is required for the synthesis of the peptidoglycan component of the cell wall of virtually all bacteria, and is found almost exclusively in the microbial world.
  • the present invention provides that the wild- type Listeria species synthesize D-alanine and thus do not require exogenously added D-alanine for g &rowth.
  • the present invention provides that the D-alanine auxotrophic mutants useful as vaccine vectors may be generated in a number of ways.
  • disruption of both alanine racemase gene (da ⁇ ) or the D-amino acid aminotransferase gene (dat), each of which is involved in D-alanine synthesis generates an auxotrophic attenuated strain of Listeria which required exogenously added D-alanine for growth.
  • the present invention provides that the generation of auxotrophic attenuated strains of Listeria deficient in D-alanine synthesis may be accomplished in a number of ways that are well known to those of skill in the art, including deletion mutagenesis, insertion mutagenesis, and mutagenesis which results in the generation of frame shift mutations, mutations which effect premature termination of a protein, or mutation of regulatory sequences which affect gene expression.
  • the present invention provides that mutagenesis can be accomplished using recombinant DNA techniques or using traditional mutagenesis technology, which in one embodiment, entails using mutagenic chemicals or radiation and subsequent selection of mutants.
  • the present invention provides deletion mutants.
  • the present invention provides that the mutants of D-alanine which are generated according to the protocols of the present invention may be tested for the ability to grow in the absence of D-alanine in a simple laboratory culture assay. In another embodiment, the present invention provides that those mutants which are unable to grow in the absence of this compound are selected for further study. In another embodiment, any other Listeria auxotroph known to one of skill in the art is used by the vaccines and methods of the present invention.
  • the present invention provides that other genes involved in D-alanine synthesis are used as targets for mutagenesis of Listeria.
  • the present invention provides that genes which are involved in the synthesis of other metabolic components in a bacterial cell are also useful targets for the generation of attenuated auxotrophic mutants of Listeria, which mutants may also be capable of serving as bacterial vaccine vectors for use in the methods of the present invention.
  • the present invention provides that the generation and characterization of such other auxotrophic attenuated strains of Listeria may be accomplished in a manner known to one of skill in the art.
  • the present invention provides that additional potential useful targets for the generation of additional auxotrophic strains of Listeria include the genes involved in the synthesis of the cell wall component D-glutamic acid.
  • D-glutamic acid auxotrophic mutants it is necessary to inactivate the dot gene, which is involved in the conversion of D- glu+pyr to alpha-ketoglutarate+D-ala and the reverse reaction.
  • the present invention provides that other potential useful targets for the generation of additional auxotrophic strains of Listeria are the genes involved in the synthesis of diamimopimelic acid.
  • auxotrophic strains of Listeria for use in the compositions and methods of the instant invention are Listeria with mutations in a gene involved in the synthesis of phenylalanine, which in one embodiment, is prephenate dehydratase (Alexander et al, Infection and Immunity, 1993, 63:2245- 2248, which is incorporated herein by reference).
  • auxotrophic mutants and methods for making auxotrophic mutants are known in the art (Marquis et al., Infection and Immunity, 1993, 61:3756-3760; Camilli et aU. Bacteriol. 1990 JuI: 172(7):3738-44; Rouquette et al. FEMS Microbiol Lett, 1995, 133.1-2: 77-83; Sleator et al. Appl Environ Microbiol. 2001 Jun;67(6):2571-7, which are incorporated herein by reference in their entirety).
  • the present invention provides that the introduction of DNA encoding a heterologous antigen into a strain of Listeria may be accomplished, for example, by the creation of a recombinant Listeria in which DNA encoding the heterologous antigen is harbored on a vector, such as a plasmid for example, which plasmid is maintained and expressed in the Listeria species.
  • a vector such as a plasmid for example, which plasmid is maintained and expressed in the Listeria species.
  • the present invention provides that DNA encoding the heterologous antigen may be stably integrated into the Listeria chromosome by employing, for example, transposon mutagenesis or by homologous recombination.
  • the present invention provides for producing recombinant Listeria having a gene encoding a heterologous antigen integrated into the chromosome thereof, via the induction of homologous recombination between a temperature sensitive plasmid comprising DNA encoding the antigen and Listeria chromosomal DNA.
  • a heterologous antigen in Listeria monocytogenes include plasmid-based expression systems and chromosome expression systems.
  • chromosomal based method is described in Frankel et al. (1995, J. Immunol. 155:4775- 4782) and Mata et al. (2001, Vaccine 19:1435-1445), which are incorporated herein by reference in their entirety. Briefly, a gene encoding the antigen of interest is placed, along with a suitable promoter and signal sequence, between two regions of DNA homologous to a region of the Listeria chromosome.
  • This homologous recombination allows specific integration of the antigen in the Listeria chromosome.
  • the cassette comprising the antigen and the homologous DNA is ligated into a temperature- sensitive plasmid incapable of replication at temperatures above 40° C.
  • the plasmid further comprises drug-resistance markers for selection and plasmid maintenance purposes.
  • the manipulation and replication of this plasmid usually takes place in E. coli, because of its rapid replication and ease of transformation compared to Listeria. Because Listeria is a gram positive organism and E.
  • the drug resistance genes can be specific to each category of organism, or there may be two copies of the same drug resistance gene effective in both types of organism, but under the control of separate gram positive and gram negative promoters.
  • the plasmid is transformed into Listeria monocytogenes (LM) by direct conjugation with the E. coli comprising the plasmid, or by lysis and isolation of the plasmid from the E. coli, followed by electroporation of competent LM.
  • LM Listeria monocytogenes
  • a plasmid may be integrated into a desired region of the Listeria chromosome using the two-step allelic exchange method of Camilli et al. (1992, MoI. Microbiol. 8:143- 157; incorporated herein by reference). Briefly, the Listeria is passaged at greater than 40° C to prevent plasmid replication. Integration of the plasmid into the Listeria chromosome is selected by growth at 40° C in the presence of a selecting drug, e.g. chloramphenicol. After selection of transformants, bacteria are passaged at 30° C and selected for drug sensitivity to screen for Listeria in which excision of extraneous vector sequences has occurred.
  • a selecting drug e.g. chloramphenicol
  • a second chromosomal method of producing Listeria strains comprising a heterologous antigen is described by Lauer et al. (2002, J. Bacteriol. 184:4177-4186; incorporated herein by reference) may be utilized. This method does not require allelic exchange, but instead requires two phage-based integration vectors. This method utilizes one or two drug resistance genes, resulting in a Listeria organism comprising resistance to one or more drugs.
  • a third method of expressing foreign antigen in Listeria is to express the antigen episomally from a plasmid. This method is described in Ikonomidis et al. (1994 J. Exp. Med. 180: 2209 - 2218) and Gunn et al. (2001, J Immunol 167: 6471-6479), which are incorporated herein by reference. This method has the advantage that the gene does not have to be integrated into the chromosome and can be expressed in multiple copies, which may enhance immunogenicity.
  • the present invention provides that stable transformants of Listeria which express the desired antigen may be isolated and characterized.
  • the present invention provides that method of homologous recombination is advantageous in that site-directed insertion of DNA encoding the heterologous antigen is effected, thereby minimizing the possibility of disruption of other areas of the Listeria chromosome which may be essential for growth of this organism.
  • the present invention provides transposon insertion for introducing a gene encoding a heterologous antigen integrated into the chromosome of a recombinant Listeria for the compositions and methods of the present invention.
  • the present invention provides that several approaches are employed to express a heterologous antigen in Listeria species as will be understood by one skilled in the art once armed with the present disclosure.
  • the present invention provides that genes encoding heterologous antigens are preferably designed to either facilitate secretion of the heterologous antigen from the bacterium or to facilitate expression of the heterologous antigen on the Listeria cell surface.
  • the present invention provides that expression of a fusion protein of the present invention is directed by a L. monocytogenes promoter, regulatory sequence, signal sequence, or a combination thereof.
  • sequences derived from the Listeria My gene which encodes LLO, the Listeria p60 gene (Bouwer et al., 1996, Infect. Immun. 64:2515-2522) and in one embodiment, the Listeria actA gene which encodes a surface protein necessary for L. monocytogenes actin assembly may be used.
  • the present invention provides that other promoter sequences which might be useful in some circumstances include the plcA gene which encodes PI-PLC, the Listeria mpl gene, which encodes a metalloprotease, the Listeria /?/c5 gene encoding a phospholipase C, and the Listeria inlA gene which encodes internalin, a Listeria membrane protein.
  • the present invention provides that the data presented herein indicate that certain auxotrophic attenuated strains of Listeria may undergo osmotic lysis following infection of a host cell.
  • the present invention provides that if the Listeria which is introduced into the host cell comprises a vector, the vector is released into the cytoplasm of the host cell.
  • the present invention provides that the vector may comprise DNA encoding a heterologous antigen.
  • the present invention provides that uptake into the nucleus of the vector DNA enables transcription of the DNA encoding the heterologous antigen and subsequent expression of the antigen in and/or secretion of the same from the infected host cell.
  • the present invention provides that the vector is a plasmid that is capable of replication in Listeria.
  • the present invention provides that the vector encodes a heterologous antigen, wherein expression of the antigen is under the control of eukaryotic promoter/regulatory sequences.
  • typical plasmids having suitable promoters that might be employed include, but are not limited to, pCMVbeta comprising the immediate early promoter/enhancer region of human cytomegalovirus, and those which include the S V40 early promoter region or the mouse mammary tumor virus LTR promoter region.
  • a promoter for use in the compositions and methods of the present invention is a CMV early enhancer/chicken ⁇ actin (CAG) promoter.
  • CAG CMV early enhancer/chicken ⁇ actin
  • the present invention provides that the use of a suicide plasmid to conditionally suppress the attenuation of the Listeria auxotrophic attenuated strain by temporarily supplying the missing enzyme or enzymes to the bacterium for synthesis of the essential nutrient.
  • a suitable suicide plasmid includes pKSW.
  • this plasmid contains a gram positive (for use in Listeria), temperature- sensitive replication system such that growth at 37-4O 0 C inhibits plasmid replication in Listeria.
  • this plasmid also contains an E. coli replication system which is not temperature-sensitive.
  • the present invention provides that the plasmid, or even more temperature-sensitive derivatives thereof, may be further modified by inserting an alanine racemase gene into the plasmid, which modified plasmid is then inserted into an auxotrophic attenuated strain of Listeria.
  • the present invention provides that the Listeria cells having the plasmid inserted therein, are replicated at 3O 0 C for a short period of time in order that some molecules of racemase are accumulated in the cytoplasm.
  • the present invention provides that the attenuated recombinant adenoviruses and Listeria cells, so replicated are then injected into an animal or a human, wherein plasmid replication then ceases because of the temperature sensitive nature of the replication system at 37 0 C.
  • the nutrient media utilized for growing a culture of a Listeria strain is LB. In another embodiment, the nutrient media is TB. In another embodiment, the nutrient media is a modified, animal-product free Terrific Broth. In another embodiment, the nutrient media is a defined media. In another embodiment, the nutrient media is a defined media of the present invention. In another embodiment, the nutrient media is any other type of nutrient media known in the art. Each possibility represents a separate embodiment of the present invention. [0049] In another embodiment, in the methods, vaccines, and compositions of the present invention, the step of growing is performed with a shake flask. In another embodiment, the flask is a baffled shake flask.
  • the growing is performed with a batch fermenter. In another embodiment, the growing is performed with a stirred tank or flask. In another embodiment, the growing is performed with an airflit fermenter. In another embodiment, the growing is performed with a fed batch. In another embodiment, the growing is performed with a continuous cell reactor. In another embodiment, the growing is performed with an immobilized cell reactor. In another embodiment, the growing is performed with any other means of growing bacteria that is known in the art. Each possibility represents a separate embodiment of the present invention.
  • a constant pH is maintained during growth of the culture (e.g. in a batch fermenter).
  • the pH is maintained at about 7.0.
  • the pH is about 6.
  • the pH is about 6.5.
  • the pH is about 7.5.
  • the pH is about 8.
  • the pH is 6.5-7.5.
  • the pH is 6-8.
  • the pH is 6-7.
  • the pH is 7-8.
  • a constant temperature is maintained during growth of the culture.
  • the temperature is maintained at about 37 0 C.
  • the temperature is 37 0 C.
  • the temperature is 25 0 C.
  • the temperature is 27 0 C.
  • the temperature is 28 0 C.
  • the temperature is 3OC.
  • the temperature is 32 0 C.
  • the temperature is 34 0 C.
  • the temperature is 35 0 C.
  • the temperature is 36 0 C.
  • the temperature is 38 0 C.
  • the temperature is 39 0 C.
  • a constant dissolved oxygen concentration is maintained during growth of the culture.
  • the dissolved oxygen concentration is maintained at 20% of saturation.
  • the concentration is 15% of saturation.
  • the concentration is 16% of saturation.
  • the concentration is 18% of saturation.
  • the concentration is 22% of saturation.
  • the concentration is 25% of saturation.
  • the concentration is 30% of saturation.
  • the concentration is 35% of saturation.
  • the concentration is 40% of saturation.
  • the concentration is 45% of saturation.
  • the concentration is 50% of saturation.
  • the concentration is 55% of saturation.
  • the concentration is 60% of saturation. In another embodiment, the concentration is 65% of saturation.
  • the concentration is 70% of saturation. In another embodiment, the concentration is 75% of saturation. In another embodiment, the concentration is 80% of saturation. In another embodiment, the concentration is 85% of saturation. In another embodiment, the concentration is 90% of saturation. In another embodiment, the concentration is 95% of saturation. In another embodiment, the concentration is 100% of saturation. In another embodiment, the concentration is near 100% of saturation.
  • the Listeria culture is flash-frozen in liquid nitrogen, followed by storage at the final freezing temperature.
  • the culture is frozen in a more gradual manner; e.g. by placing in a vial of the culture in the final storage temperature.
  • the culture is frozen by any other method known in the art for freezing a bacterial culture. Each possibility represents a separate embodiment of the present invention.
  • the storage temperature of the culture is between -20 and -80 degrees Celsius ( 0 C). In another embodiment, the temperature is significantly below -2O 0 C. In another embodiment, the temperature is not warmer than - 7O 0 C. In another embodiment, the temperature is -7O 0 C. In another embodiment, the temperature is about - 7O 0 C. In another embodiment, the temperature is -2O 0 C. In another embodiment, the temperature is about - 2O 0 C. In another embodiment, the temperature is -3O 0 C. In another embodiment, the temperature is -4O 0 C. In another embodiment, the temperature is -5O 0 C. In another embodiment, the temperature is -6O 0 C.
  • the temperature is -8O 0 C. In another embodiment, the temperature is -30 - -7O 0 C. In another embodiment, the temperature is -40 - -7O 0 C. In another embodiment, the temperature is -50 - - 7O 0 C. In another embodiment, the temperature is -60 - -7O 0 C. In another embodiment, the temperature is - 30 - -8O 0 C. In another embodiment, the temperature is -40 - -8O 0 C. In another embodiment, the temperature is -50 - -8O 0 C. In another embodiment, the temperature is -60 - -8O 0 C. In another embodiment, the temperature is -70 - -8O 0 C. In another embodiment, the temperature is colder than -7O 0 C. In another embodiment, the temperature is colder than -8O 0 C. Each possibility represents a separate embodiment of the present invention.
  • the recombinant Listeria strain utilized in methods of the present invention has been stored in a frozen cell bank.
  • the recombinant Listeria strain has been stored in a lyophilized cell bank.
  • the cell bank of methods, vaccines, and compositions of the present invention is a master cell bank.
  • the cell bank is a working cell bank.
  • the cell bank is Good Manufacturing Practice (GMP) cell bank.
  • the cell bank is intended for production of clinical-grade material.
  • the cell bank conforms to regulatory practices for human use.
  • the cell bank is any other type of cell bank known in the art. Each possibility represents a separate embodiment of the present invention.
  • Good Manufacturing Practices are defined, in another embodiment, by (21 CFR 210-211) of the United States Code of Federal Regulations. In another embodiment, “Good Manufacturing Practices” are defined by other standards for production of clinical-grade material or for human consumption; e.g. standards of a country other than the United States. Each possibility represents a separate embodiment of the present invention.
  • a recombinant Listeria strain utilized in methods of the present invention is from a batch of vaccine doses.
  • the present invention provides that using the intravenous inoculation of BALB/c mice with an attenuated Listeria, the lethal dose at which 50% of inoculated animals survive (LD 50 ) is preferably increased above the LD 50 of wild- type Listeria by at least about 10-fold. In another embodiment, the present invention provides that using the intravenous inoculation of BALB/c mice with an attenuated Listeria, the LD 50 is preferably increased above the LD 50 of wild-type Listeria by at least about 50-fold.
  • the present invention provides that using the intravenous inoculation of B ALB/c mice with an attenuated Listeria, the LD 50 is preferably increased above the LD 50 of wild- type Listeria by at least about 100-fold. In another embodiment, the present invention provides that using the intravenous inoculation of BALB/c mice with an attenuated Listeria, the LD 5O is preferably increased above the LD 50 of wild-type Listeria by at least about 500-fold. In another embodiment, the present invention provides that using the intravenous inoculation of BALB/c mice with an attenuated Listeria, the LD 50 is preferably increased above the LD 50 of wild- type Listeria by at least about 1, 000-fold.
  • the present invention provides that using the intravenous inoculation of BALB/c mice with an attenuated Listeria, the LD 50 is preferably increased above the LD 50 of wild-type Listeria by at least about 10,000-fold. In another embodiment, the present invention provides that using the intravenous inoculation of BALB/c mice with an attenuated Listeria, the LD 50 is preferably increased above the LD 50 of wild- type Listeria by at least about 100,000-fold.
  • the present invention provides that the attenuated recombinant adenovirus comprises a chimeric coat protein. In another embodiment, the present invention provides that a chimeric coat protein reduces the immunogenicity of an attenuated recombinant adenovirus. In another embodiment, the present invention provides that a chimeric coat protein reduces the immunogenicity of an adenovirus of the invention thus the LD 50 of such an adenovirus strain is increased by about 10 fold compared to an adenovirus of the serotype of the present invention that does not comprise a chimeric coat protein.
  • the present invention provides that a chimeric coat protein reduces the immunogenicity of an adenovirus of the invention thus the LD 50 of such an adenovirus strain is increased by about 50 fold compared to an adenovirus of the serotype of the present invention that does not comprise a chimeric coat protein.
  • the present invention provides that a chimeric coat protein reduces the immunogenicity of an adenovirus of the invention thus the LD 50 of such an adenovirus strain is increased by about 100 fold compared to an adenovirus of the serotype of the present invention that does not comprise a chimeric coat protein.
  • the present invention provides that a chimeric coat protein reduces the immunogenicity of an adenovirus of the invention thus the LD 50 of such an adenovirus strain is increased by about 1000 fold compared to an adenovirus of the serotype of the present invention that does not comprise a chimeric coat protein.
  • the present invention provides that a chimeric coat protein reduces the immunogenicity of an adenovirus of the invention thus the LD 50 of such an adenovirus strain is increased by about 10000 fold compared to an adenovirus of the serotype of the present invention that does not comprise a chimeric coat protein.
  • the present invention provides that a chimeric coat protein reduces the immunogenicity of an adenovirus of the invention thus the LD 50 of such an adenovirus strain is increased by about 100,000 fold compared to an adenovirus of the serotype of the present invention that does not comprise a chimeric coat protein.
  • the present invention provides that a chimeric coat protein reduces the immunogenicity of an adenovirus of the invention thus the LD 50 of such an adenovirus strain is increased by about 1x10 7 fold compared to an adenovirus of the serotype of the present invention that does not comprise a chimeric coat protein.
  • the present invention provides that the attenuated recombinant adenovirus comprises a coat protein derived from serotype 5, serotype 26, serotype 48, or a combination thereof. In another embodiment, the present invention provides that the attenuated recombinant adenovirus comprises a coat protein derived from serotype 5 and serotype 26. In another embodiment, the present invention provides that the attenuated recombinant adenovirus comprises a coat protein derived from serotype 26 and serotype 48. In another embodiment, the present invention provides that the attenuated recombinant adenovirus comprises a coat protein derived from serotype 5 and serotype 48. In another embodiment, the present invention provides that the attenuated recombinant adenovirus is an attenuated recombinant adenovirus type 5.
  • an attenuated recombinant adenovirus in the compositions and methods of the present invention is derived from adenovirus type 1 , adenovirus type 2, adenovirus type 5, adenovirus type 6, adenovirus type 8, adenovirus type 19, adenovirus type 37, adenovirus type 4, adenovirus type 7, adenovirus type 40, adenovirus type 41, adenovirus type 7, adenovirus type 26, adenovirus type 48, or a combination thereof.
  • recombinant Listeria infects mucosal tissue.
  • attenuated recombinant adenovirus infects mucosal tissue.
  • recombinant adenovirus of the present invention has been passaged through an animal host.
  • passaging decreases the virulence of the recombinant adenovirus.
  • the present invention provides that the recombinant adenovirus of the invention is used as both gene delivery and gene therapy tools.
  • the present invention provides that the recombinant adenovirus or attenuated adenovirus of the invention comprises deletions or inactivating mutations in the El, E3 or both El and E3 regions.
  • the present invention provides that the El deletion prevents the recombinant adenovirus from replicating and therefore no cell lysis occurs.
  • the present invention provides that the recombinant adenovirus of the invention is packaged into a complementing cell line, i.e. a cell line that provides the El products in trans (e.g. QBIHEK 293A cells), making viral replication possible.
  • a recombinant micro-organism of the invention is replication-competent. In another embodiment, a recombinant micro-organism of the invention is replication-defective. In another embodiment, a recombinant micro-organism of the invention is live-attenuated. In one embodiment, a recombinant adenovirus of the invention is replication-competent. In another embodiment, a recombinant adenovirus of the invention is replication-defective. In another embodiment, a recombinant adenovirus of the invention is live-attenuated.
  • the present invention provides that the E3 region, not essential for viral growth, is also deleted. In another embodiment, the present invention provides that these two deletions allow the introduction of the transgene of interest into the virus. In another embodiment, the present invention provides that the transgene of interest is the antigen of the present invention.
  • the E2 and/or E4 region of adenovirus early genes are deleted or rendered non-functional by a mutation.
  • a helper-dependent adenovirus or gutless adenovirus in which all viral coding sequences are deleted (for e.g., Parks et al, Proc. Natl. Acad. Sci. USA 93: 13565-13570, 1996), and only comprises the adenovirus inverted terminal repeats and packaging sequence ( ⁇ ), is used in the compositions and methods of the present invention.
  • helper-dependent adenovirus or gutless adenovirus in which all viral coding sequences are deleted (for e.g., Parks et al, Proc. Natl. Acad. Sci. USA 93: 13565-13570, 1996), and only comprises the adenovirus inverted terminal repeats and packaging sequence ( ⁇ ), is used in the compositions and methods of the present invention.
  • the present invention provides that the recombinant adenovirus is used in vaccination by expressing a gene product that triggers an immune response.
  • the present invention provides that recombinant adenovirus comprising the antigen of the present invention is used to boost an immune response primed by an attenuated Listeria comprising the antigen of the present invention.
  • the present invention provides that the recombinant adenovirus is a human virus and infects human cells.
  • the present invention provides that human proteins expressed by a recombinant adenovirus vector have identical post-translational modifications as native proteins.
  • the present invention provides that the recombinant adenovirus is well tolerated, with post-infection viability of the host cells being at least 40%. In another embodiment, the present invention provides that the recombinant adenovirus is well tolerated, with post- infection viability of the host cells being at least 50%. In another embodiment, the present invention provides that the recombinant adenovirus is well tolerated, with post-infection viability of the host cells being at least 60%. In another embodiment, the present invention provides that the recombinant adenovirus is well tolerated, with post-infection viability of the host cells being at least 70%.
  • the present invention provides that the recombinant adenovirus is well tolerated, with post- infection viability of the host cells being at least 80%. In another embodiment, the present invention provides that the recombinant adenovirus is well tolerated, with post-infection viability of the host cells being at least 90%. In another embodiment, the present invention provides that the recombinant adenovirus is well tolerated, with post- infection viability of the host cells being 100%.
  • the present invention provides that the recombinant adenovirus remains epichromosomal, i.e. does not integrate into the host chromosome, which in one embodiment, prevents the recombinant adenovirus from inactivating genes or from activating oncogenes.
  • the vaccines of the invention stimulate a CTL immune response against an infectious agent or a tumor cell
  • the auxotrophic attenuated Listeria and the adenovirus strain each comprise a vector encoding a heterologous antigen that may be expressed using a eukaryotic expression system.
  • the present invention provides that a vector is propagated in the auxotrophic attenuated strain of Listeria concomitant with the propagation of the auxotrophic attenuated strain itself.
  • the present invention provides that the vector may be, for example, a plasmid that is capable of replication in the auxotrophic attenuated strain or the vector may be a lysogenic phage.
  • the present invention provides that a cytotoxic T-cell response in a mammal is defined as the generation of cytotoxic T cells capable of detectably lysing cells presenting an antigen against which the T-cell response is directed.
  • the present invention provides that the T-cell response is directed against a heterologous antigen expressed delivered to a cell via the vaccine of the present invention.
  • the present invention provides that assays for a cytotoxic T-cell response are well known in the art and include, for example, a chromium release assay (Frankel et al., 1995, J. Immunol. 155:4775-4782).
  • an assay for released lactic acid dehydrogenase may be performed using a Cytotox 96 kit obtained from Promega Biotech, WI.
  • the present invention provides that the antigen-specific CD8 + T cell immune response is in a mucosal tissue a subject.
  • a mucosal tissue comprises vaginal tissue, rectal tissue, intestinal tissue, nasal tissue, urethral tissue, sublingual tissue, buccal tissue, oral tissue, or a combination thereof.
  • a vaccine or other composition of the present invention is given mucosally.
  • a virus or bacteria of the present invention replicates in the mucosa.
  • a virus or bacteria of the present invention generates an immune response in the mucosa.
  • the present invention provides that vaginal tissue comprises muscle tissue. In another embodiment, the present invention provides that vaginal tissue comprises the cervix. In another embodiment, the present invention provides that vaginal tissue comprises the vulva. In another embodiment, the present invention provides that vaginal tissue comprises the uterus. In another embodiment, the present invention provides that vaginal tissue comprises the Mons Veneris. In another embodiment, the present invention provides that vaginal tissue comprises Bartholin's glands. In another embodiment, the present invention provides that vaginal tissue comprises the hymen. In another embodiment, the present invention provides that vaginal tissue comprises any combination of the tissues described hereinabove.
  • the present invention provides that rectal tissue comprises muscle tissue. In another embodiment, the present invention provides that the rectum comprises the anus. In another embodiment, the present invention provides that the rectum comprises the rectal ampulla. In another embodiment, the present invention provides that the rectum comprises any combination of the tissues described hereinabove.
  • the present invention provides that intestinal tissue comprises muscle tissue. In another embodiment, the present invention provides that intestinal tissue comprises a duodenum. In another embodiment, the present invention provides that intestinal tissue comprises a jejunum. In another embodiment, the present invention provides that intestinal tissue comprises an ileum. In another embodiment, the present invention provides that intestinal tissue comprises the pyloric sphincter of the stomach. In another embodiment, the present invention provides that intestinal tissue comprises the colon. In another embodiment, the present invention provides that intestinal tissue comprises the anal canal. In another embodiment, the present invention provides that intestinal tissue comprises any combination of the tissues described hereinabove.
  • the present invention provides that the heterologous antigen is used alone (i.e., in the absence of fused Listeria sequences). In another embodiment, the present invention provides that it may be advantageous to fuse thereto signal sequences for cell surface expression and/or secretion of the heterologous antigen. In another embodiment, the present invention provides that the procedures for accomplishing this are well know in the art of bacteriology and molecular biology.
  • the antigen is a eukaryotic antigen. In another embodiment, the antigen is a bacterial antigen. In another embodiment, the antigen is a viral antigen. In another embodiment, the antigen is derived from a pathogen. In another embodiment, the antigen comprises an immunogenic fragment derived from a pathogen. In another embodiment, the antigen is an HIV antigen. In another embodiment, HIV antigen is an HIV- 1 antigen. In another embodiment, HIV antigen is an HIV-2 antigen. In another embodiment, the antigen is a tumor antigen. In another embodiment, the antigen is a cancer- related antigen.
  • the present invention provides that a heterologous antigen encoded by the auxotrophic attenuated strain of Listeria and/or attenuated recombinant adenovirus is one which when expressed by Listeria and/or attenuated recombinant adenovirus is capable of providing protection in an animal against challenge by the infectious agent from which the heterologous antigen was derived, or which is capable of affecting tumor growth and/or metastasis in a manner which is of benefit to a host
  • administration of the compositions of the present invention has therapeutic or prophylactic effects, wherein the object is to prevent or lessen the targeted infection, pathologic condition or disorder.
  • the antigen within compositions and in the methods of the present invention prevents, inhibits, or suppresses tumor growth and/or metastasis.
  • compositions of the present invention may suppress, inhibit, prevent, reduce the severity of, delay the onset of, reduce symptoms associated with the disease, disorder or condition, delay progression, expedite remission, induce remission, augment remission, speed recovery, increase efficacy of or decrease resistance to alternative therapeutics, delay the onset of symptoms, prevent relapse to a disease, decrease the number or frequency of relapse episodes, increase latency between symptomatic episodes, reduce the severity of symptoms, reduce the severity of an acute episode, reduce the number of symptoms, reduce the incidence of disease-related symptoms, reduce the latency of symptoms, ameliorate symptoms, reduce secondary symptoms, reduce secondary infections, prolong patient survival, or a combination thereof.
  • symptoms are primary, while in another embodiment, symptoms are secondary.
  • the present invention provides that a heterologous antigen which may be introduced into an auxotrophic attenuated strain of Listeria and/or attenuated recombinant adenovirus by way of DNA encoding the same thus includes any antigen which when expressed by Listeria serves to elicit a cellular immune response which is of benefit to the host in which the response is induced.
  • heterologous antigens include those produced by infectious agents, wherein an immune response directed against the antigen serves to prevent or treat disease caused by the agent.
  • Such heterologous antigens include, but are not limited to, viral, bacterial, fungal or parasite surface proteins and any other proteins, glycoproteins, lipoprotein, glycolipids, and the like.
  • the present invention provides that heterologous antigens also include those which provide benefit to a host organism which is at risk for acquiring or which is diagnosed as having a tumor.
  • the present invention provides that the host organism is preferably a mammal and most preferably, is a human.
  • the host organism is a vertebrate.
  • the host is murine, canine, feline, bovine, ovine, or porcine.
  • the present invention provides that the term "heterologous antigen," as used herein, is meant a protein or peptide, a glycoprotein or glycopeptide, a lipoprotein or lipopeptide, or any other macromolecule which is not normally expressed in Listeria and/or attenuated recombinant adenovirus, which substantially corresponds to the same antigen in an infectious agent, a tumor cell or a tumor-related protein.
  • the present invention provides that the heterologous antigen is expressed by an auxotrophic attenuated strain of Listeria and/or attenuated recombinant adenovirus, and is processed and presented to cytotoxic T-cells upon infection of mammalian cells by the auxotrophic attenuated strain and/or attenuated recombinant adenovirus.
  • the present invention provides that the heterologous antigen expressed by Listeria species and/or attenuated recombinant adenovirus need not precisely match the corresponding unmodified antigen or protein in the tumor cell or infectious agent so long as it results in a T-cell response that recognizes the unmodified antigen or protein which is naturally expressed in the mammal.
  • "antigen" is a substance, usually a protein or polypeptide, that elicits a detectable immune response when introduced into a subject to which it is heterologous.
  • the immune response comprises the formation of antibodies that react with the antigen.
  • an "antigen” refers to a polypeptide used to elicit an immune response when introduced into a subject to which it is heterologous.
  • a reporter protein or polypeptide that is not specifically being used to elicit an immune response is not an antigen.
  • the present invention provides that the term "tumor-related antigen," as used herein, refers to an antigen which affects tumor growth or metastasis in a host organism.
  • the present invention provides that the tumor-related antigen may be an antigen expressed by a tumor cell, or it may be an antigen which is expressed by a non-tumor cell, but which when so expressed, promotes the growth or metastasis of tumor cells.
  • the present invention provides that the types of tumor antigens and tumor- related antigens which may be introduced into Listeria and/or attenuated recombinant adenovirus by way of incorporating DNA encoding the same include any known or heretofore unknown tumor antigen.
  • the present invention provides that the antigen is an HIV antigen, a malaria antigen, an influenza antigen, a Hepatitis A antigen, a Hepatitis B antigen, a Hepatitis C antigen, a Hepatitis E antigen, or a tuberculosis antigen.
  • the present invention provides that the HF/ antigen is an HIV-Gag antigen, an HIV- Vif antigen, an HIV-Tat antigen, an HIV-Nef antigen, HIV-Env antigen, or a SIV-Gag antigen.
  • the present invention provides that the HIV antigen is an HIV-PoI, HIV-Rev, HIV- Vpr, HIV- Vpu, or HIV-Tev antigen.
  • the malaria antigen is Apical membrane antigen 1 (AMA-I); Acidic basic repeat antigen (ABRA or p 101); Gametocyte antigen 11.1; Circumsporozoite protein 1 (CSP-I); Erythrocyte binding proteins; P.
  • PfEMP-I falciparum erythrocyte membrane protein 1
  • Glutamate-rich protein Glutamate-rich protein
  • Heat shock proteins Histidine-rich protein 2 (HRP-2); Knob-associated histidine-rich protein (KAHRP); Mature- parasite-infected erythrocyte membrane antigen (MESA/PfEMP-2); Merozoite surface protein 1 (MSP-I); Merozoite surface protein 2 (MSP-2); Ring-infected erythrocyte surface antigen (RESA/Pf 155); Assorted references about rhoptry proteins; S antigen; Pf 332 protein.
  • influenza antigen is haemagglutinin (HA), nucleoprotein (NP); Envelope protein matrix (M); Neuraminidase surface enzyme protein; PA.
  • hepatitis B antigen is HBeAb, HBe, HBeAg, the hepatitis B surface antigen (HBsAg) or the hepatitis B core antigen (HBcAg).
  • the tuberculosis antigen is ESAT-6; CFP-IO; 38kD Antigen.
  • the present invention provides that the heterologous antigen useful in vaccine development may be selected using knowledge available to the skilled artisan, and many antigenic proteins which are expressed by tumor cells or which affect tumor growth or metastasis or which are expressed by infectious agents are known in the art.
  • the present invention provides that viral antigens which may be considered as useful as heterologous antigens include but are not limited to the nucleoprotein (NP) of influenza virus and the Gag protein of HIV.
  • the viral antigen is the p24 protein of the gag gene, which in one embodiment, makes up the viral capsid.
  • the viral antigen is the p6, p7, or pl7 protein of the gag gene, which in one embodiment, comprise the nucleocapsid, and in another embodiment, provide a protective matrix.
  • heterologous antigens include, but are not limited to, HIV Env protein (in one embodiment, gpl60), which in one embodiment, is cleaved into gpl20 and gp41 by a host cell protease, which in one embodiment, is Furin; HIV Negative Factor (Nef) protein, HIV Pol protein, HIV Trans-Activator of Transcription (Tat), or HIV Viral infectivity factor (Vif) protein.
  • the viral antigen is the reverse transcriptase, integrase or protease proteins of the HIV pol gene.
  • a heterologous antigen of the present invention is HIV Regulator of Virion (Rev), HIV Viral Protein R (Vpr), HIV Viral Protein U (Vpu), or HIV Tev, which in one embodiment, is present in only a few HIV- 1 isolates and comprises a fusion of portions of the tat, env, and rev genes, and codes for a protein with some of the properties of Tat, but little or none of the properties of Rev.
  • a viral antigen for use in the compositions and methods of the present invention are targets for cytotoxic T lymphocytes (CTL).
  • CTL cytotoxic T lymphocytes
  • a viral antigen for use in the compositions and methods of the present invention are expressed on the host cell surface.
  • a viral antigen of the present invention elicits a detectable immune response within the compositions of the present invention, which in one embodiment, is humoral immunity, and in another embodiment, is cellular immunity, and in another embodiment, both humoral and cellular immunity.
  • the present invention provides that other viral antigens such as herpesvirus proteins may be useful.
  • the heterologous antigens need not be limited to being of viral origin.
  • the present invention provides that parasitic antigens, such as, for example, malarial antigens, are included, as are fungal antigens, bacterial antigens and tumor antigens.
  • the antigen of interest is an immunogenic peptide derived from a tumor or, in another embodiment, the antigen of interest is an antigen that is cancer-related.
  • a cancer-related antigen is a tumor antigen, which in one embodiment, is expressed by a solid tumor.
  • a cancer-related antigen is expressed by cancer cells that are not part of a solid tumor.
  • the antigen of interest is an immunogenic peptide derived from metastasis.
  • the antigen of interest is an immunogenic peptide derived from cancerous cells.
  • the antigen of interest is a pro-angiogenesis immunogenic peptide.
  • the antigen of interest is Human Papilloma Virus-E7 (HPV-E7) antigen. In another embodiment, the antigen of interest is HPV-E6. In another embodiment, the antigen of interest is a Her/2- neu antigen. In another embodiment, the antigen of interest is Prostate Specific Antigen (PSA). In another embodiment, the antigen of interest is Prostate Stem Cell Antigen (PSCA). In another embodiment, the antigen of interest is Stratum Corneum Chymotryptic Enzyme (SCCE) antigen. In another embodiment, the antigen of interest is Wilms tumor antigen 1 (WT-I). In another embodiment, the antigen of interest is a B-cell receptor (BCR) antigen.
  • PSA Prostate Specific Antigen
  • PSCA Prostate Stem Cell Antigen
  • SCCE Stratum Corneum Chymotryptic Enzyme
  • WT-I Wilms tumor antigen 1
  • the antigen of interest is a B-cell receptor (BCR) antigen.
  • the antigen of interest is Telomerase. In another embodiment, the antigen of interest is Proteinase 3. In another embodiment, the antigen of interest is Tyrosinase Related Protein 2 (TRP2). In another embodiment, the antigen of interest is High Molecular Weight Melanoma Associated Antigen (HMW-MAA). In another embodiment, the antigen of interest is Testisin. In another embodiment, the antigen of interest is NY ESO 1 antigen. In another embodiment, the antigen of interest is MAGEb. In another embodiment, the antigen of interest is any other antigen of interest known in the art. Each possibility represents a separate embodiment of the present invention.
  • the antigen of interest is MAGE 1, MAGE 2, MAGE 3, MAGE 4, p97 melanoma antigen, Ras peptide, p53 peptide, HPV El, HPV E2, muc-1, KLH, CEA (carcinoembryonic antigen), gplOO, MARTl, human telomerase reverse transcriptase (hTERT), proteinase 3, WT-I, HPV 16/18 antigen, TRP-2, gp-100, tyrosinase, HSP-70, beta-HCG, synovial sarcoma, X (SSX)-2, carcinoembryonic antigen (CEA), MAGE-A, interleukin-13 Receptor alpha (IL13-R alpha), Carbonic anhydrase IX (CAIX), survivin, or a combination thereof.
  • heterologous antigens include, but are not limited to, the bcr/abl antigen in leukemia, HPVE6 and E7 antigens of the oncogenic virus associated with cervical cancer, the MAGEl and MZ2-E antigens in or associated with melanoma, and the MVC-I and HER-2 anti 'g6e l ns in or associated with breast cancer.
  • the present invention provides that the HIV- 1 antigens or proteins that are used to generate a vaccine in accordance with this invention are the HIV Env protein or its component parts, gpl20 and gp 41, HIV Gag, HIV Nef, HIV Vif and HIV Pol or its component parts, reverse transcriptase and protease.
  • the HIV- 1 antigen is an HIV-Rev, HIV- Vpr, HIV- Vpu, or HIV-Tev antigen.
  • a heterologous antigen may be any of the antigens mentioned herein.
  • the compositions for use in the present invention may comprise antigens that are commonly used in the art for vaccinations.
  • the antigens are described herein.
  • such antigens are viral coat proteins.
  • the compositions of the present invention and their methods of use of the present invention may be used to deliver DNA vaccines.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating Human Immunodeficiency Virus (HIV) in a subject comprising administering a vaccine of the present invention.
  • HIV Human Immunodeficiency Virus
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating Acquired Immunodeficiency Syndrome (AIDS) in a subject comprising administering a vaccine of the present invention.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating cervical cancer or head and neck cancer in a subject comprising administering a vaccine of the present invention, thereby treating, inhibiting, abrogating, or ameliorating cervical cancer or head and neck cancer in a subject.
  • AIDS Acquired Immunodeficiency Syndrome
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating breast cancer or ovarian cancer in a subject comprising administering a vaccine of the present invention, thereby treating, inhibiting, abrogating, or ameliorating cervical cancer or head and neck cancer in a subject.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating melanoma, ovarian cancer, or lung cancer in a subject comprising administering a vaccine of the present invention, thereby treating, inhibiting, abrogating, or ameliorating melanoma, ovarian cancer, or lung cancer in a subject.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating leukemia in a subject comprising administering a vaccine of the present invention, thereby treating, inhibiting, abrogating, or ameliorating leukemia in a subject.
  • the present invention provides a method of inhibiting or abrogating, a tumor in a subject comprising administering a vaccine of the present invention, thereby inhibiting or abrogating a tumor in a subject.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating prostate cancer in a subject comprising administering a vaccine of the present invention, thereby treating, inhibiting, abrogating, or ameliorating prostate cancer in a subject.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating lymphoma in a subject comprising administering a vaccine of the present invention, thereby treating, inhibiting, abrogating, or ameliorating lymphoma in a subject.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating renal cancer in a subject comprising administering a vaccine of the present invention, thereby treating, inhibiting, abrogating, or ameliorating renal cancer in a subject.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating skin cancer in a subject comprising administering a vaccine of the present invention, thereby treating, inhibiting, abrogating, or ameliorating skin cancer in a subject.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating mucosal cancer, which in one embodiment, is esophageal cancer, gastric mucosal cancer, stomach cancer, or cancer of the esophagogastric junction.
  • the antigen of interest is an immunogenic peptide derived from an influenza antigen.
  • the antigen of interest is an immunogenic peptide derived from a Hepatitis A antigen.
  • the antigen of interest is an immunogenic peptide derived from a Hepatitis B antigen.
  • the antigen of interest is an immunogenic peptide derived from a Hepatitis C antigen.
  • the antigen of interest is an immunogenic peptide derived from a Hepatitis E antigen.
  • the antigen of interest is an immunogenic peptide derived from or a tuberculosis antigen.
  • the antigen of interest is an immunogenic peptide derived from an infectious organism.
  • the antigen of interest is an immunogenic peptide which activates the immune system during the course of an infectious disease in a subject.
  • the present invention provides that antigens are processed via both the MHC class I and class II pathways to induce strong CTL responses, which in one embodiment are CD4 + T cell responses, and in another embodiment, CD8 + T cell responses, and in another embodiment, both CD4 + and CD8 + T cell responses.
  • the present invention provides that antigens are processed via the MHC class I pathway.
  • the present invention provides that antigens are processed via the MHC class II pathway.
  • the present invention provides an attenuated strain of L. monocytogenes expressing HIV-gag (attenuated Lm-gag).
  • the present invention provides that immunizing with an attenuated strain of L.
  • the present invention provides that immunizing with an attenuated strain of L. monocytogenes expressing HIV-gag results in Gag-specific CD8 + T cell levels in spleen ranging from 5 to 15% of total CD8 + T cells. In another embodiment, the present invention provides that immunizing with an attenuated strain of L. monocytogenes expressing HIV-gag results in Gag-specific CD8 + T cell levels in spleen ranging from 10 to 15% of total CD8 + T cells.
  • the present invention provides that immunizing with an attenuated strain of L. monocytogenes expressing HIV-gag results in Gag-specific CD8 + T cell levels in spleen ranging from 7 to 12% of total CD8 + T cells. In another embodiment, the present invention provides that immunizing with an attenuated strain of L. monocytogenes expressing HIV-gag results in Gag-specific CD8 + T cell levels in spleen ranging from 5 to 8% of total CD8 + T cells. In another embodiment, the present invention provides that immunizing with an attenuated strain of L.
  • the present invention provides that immunizing with an attenuated strain of L. monocytogenes expressing HIV-gag results in Gag-specific CD8 + T cell levels in spleen ranging from 15 to 30% of total CD8 + T cells. In another embodiment, the present invention provides that immunizing with an attenuated strain of L. monocytogenes expressing HIV-gag results in Gag-specific CD8 + T cell levels in spleen ranging from 25 to 40% of total CD8 + T cells .
  • the present invention provides that immunizing with an attenuated strain of L. monocytogenes expressing HIV-gag results in Gag-specific CD8 + T cell levels in spleen ranging from 30 to 50% of total CD8 + T cells.
  • an antigen used by the methods of the present is derived from or associated with the following organisms and/or diseases: Acanthamoeba, acquired immunodeficiency syndrome, adenovirus, Aedes albopictus, Aedes japonicus mosquito, African sleeping sickness, AHD, AIDS, alveolar hydatid disease, amebiasis, American trypanosomiasis, amnesic shellfish, Ancylostoma, Angiostrongylus, angiostrongyliasis, animal-borne diseases, Anisakis, anisakiasis, anthrax, antibiotic resistance, antimicrobial resistance, arboviral encephalitis, arboviral encephalitides, arenavirus infections, ascariasis, ascarids, Ascaris lumbricoides, aseptic (viral) meningitis, Asian mosquito, Aspergillus, aspergillosis, astrovirus infection, B.
  • Acanthamoeba acquired immunodeficiency
  • cepacia cepacia
  • Babesia babesiosis
  • Bacillus anthracis Bacterial and Mycotic Diseases
  • bacterial meningitis balantidiasis
  • Balantidium Bartonella henselae
  • Baylisascaris Bayou virus
  • bilharzia Black Creek Canal virus
  • Blastocystis hominis blastomycosis
  • body lice Bordetella pertussis
  • Borrelia burgdorferi botulism
  • bovine spongiform encephalopathy Brainerd diarrhea, broad (fish) tapeworm
  • Brucella brucellosis
  • Brugia malayi infection Brugia timori infection
  • BSE Burkholderia cepacia
  • Burkholderia pseudomallei calicivirus infection
  • Campylobacter campylobacteriosis
  • Candida candidiasis
  • Capillaria capillariasis
  • Cat scratch disease cat
  • an antigen used by the methods of the present is derived from or associated with the following organisms and/or diseases: dengue fever, dengue hemorrhagic fever, dengue hemorrhagic fever/dengue fever, dengue virus infection, diarrhea, diarrheagenic Escherichia coli, Dientamoeba fragilis infection, diphtheria, Diphyllobothrium infection, diphyllobothriasis, Dipylidium infection, disparities, dog flea tapeworm infection, dogs, dracunculiasis, drinking water safety, drug resistance Drug Service, CDC, ear infection, East African trypanosomiasis, Eastern equine encephalitis, Ebola hemorrhagic fever, Ebola virus infection, EBV, echinococcosis, echovirus infection, E.
  • coli infection Ehrlichia infection, ehrlichiosis, elephantiasis, emerging infectious diseases (listing, sites and publications about), encephalitis, encephalitis, arboviral, encephalitis, Eastern equine, encephalitis, Japanese, encephalitis, La Crosse, encephalitis, St.
  • an antigen used by the methods of the present is derived from or associated with the following organisms and/or diseases: Gambian sleeping sickness, GAS infection, gastroenteritis, viral, GBS infection, genital candidiasis, gerbils, German measles, Giardia infection, giardiasis, Global Migration and Quarantine, Division of, Gnathostoma infection, gnathostomiasis, gonorrhea, group A streptococcal infection, group B streptococcal infection, guinea pigs, Guinea worm disease, Haemophilus ducreyi infection, Haemophilus influenzae serotype b infection, hamsters, pet (diseases people can get from them), hand, foot, and mouth disease, hand hygiene in healthcare settings, Hansen's disease, hantavirus pulmonary, syndrome, head lice infestation, Helicobacter pylori infection, hematologic diseases, hemophilia,
  • pylori infection human ehrlichiosis, human immunodeficiency virus infection, human parainfluenzavirus infection, human parvovirus B 19 infection, hymenolepiasis, Hymenolepis infection, iguanas, infectious mononucleosis, influenza, insects and their relatives (listing, disease information by type), intestinal roundworm infection, Iodamoeba buetschlii infection, Isospora infection,
  • an antigen used by the methods of the present is derived from or associated with the following organisms and/or diseases: Japanese encephalitis, kala-azar, Kawasaki syndrome, Laboratory Network, Measles, La Crosse encephalitis, Lassa fever, LCMV, Legionella pneumophila infection, Legionnaires' disease, legionellosis, Leishmania infection, leishmaniasis, leprosy, Leptospira infection, leptospirosis, lice infestation, Listeria monocytogenes infection, listeriosis, Loa loa infection, Lockjaw, Lyme disease, lymphatic filariasis, lymphedema, lymphocytic choriomeningitis, MAC infection, mad cow disease, malaria, Marburg hemorrhagic fever, Marburg virus infection, marine toxins, measles, melioidosis, meningococcal disease, meningitis, Methicillin Resistant Staphylococcus aureus (M
  • an antigen used by the methods of the present is derived from or associated with the following organisms and/or diseases: Naegleria infection, necrotizing fasciitis, Neisseria gonorrhoeae infection, neurocysticercosis, neurotoxic shellfish poisoning, new variant Creutzfeldt-Jakob disease, New York-1 virus infection, Nipah virus infection, Nocardia infection, nocardiosis, nonpathogenic intestinal amebae infection, non-polio enterovirus infection, Norovirus infection, Norwalk and Norwalk- like virus infection, nosocomial infections, nvCJD, ocular larva migrans, Onchocerca volvulus infection, onchocerciasis, OPC, opisthorchiasis, Opisthorchis infection, orf virus infection, oropharyngeal candidiasis, otitis media, paragonimiasis, Paragonimus infection, paralytic shellfish poisoning, parasit
  • an antigen used by the methods of the present is derived from or associated with the following organisms and/or diseases: Q fever, rabies, rabies virus infection, raccoon roundworm infection, rat bite fever, rats, respiratory syncytial virus infection, rhinitis, Rickettsia rickettsii infection, Rickettsial diseases, Rift Valley fever, Rift Valley fever virus infection, ringworm, river blindness, RMSF, Rocky Mountain spotted fever, rotavirus, rotavirus infection, roundworm infection, intestinal, roundworm infection (parasitic), RSV infection, rubella, rubeola, runny nose, RVF infection, Salmonella infection, salmonellosis, Salmonella enteritidis infection, Salmonella typhi infection, Sarcoptes scabei infestation, SARS, scabies, scarlet fever, Schistosoma infection, schistosomiasis, Scientific Resources Program, scombrotoxic fish poisoning
  • an antigen used by the methods of the present is derived from or associated with the following organisms and/or diseases: Taenia infection, taeniasis, Taenia solium infection, tapeworm (broad or fish) infection, tapeworm infection, TB, tetanus, three-day measles, thrush, tick-borne diseases (partial list), tick-borne relapsing fever, tick typhus, toxic shock syndrome, Toxocara canis, Toxocara cati, Toxocara infection, toxocariasis, Toxoplasma infection, toxoplasmosis, Treponema pallidum infection, Trichinella infection, trichinellosis, trichinosis, Trichomonas infection, trichomoniasis, trichuriasis, Trichuris infection, Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense, Trypanosoma cruzi infection,
  • the present invention provides methods, compositions, and vaccines for treating, inhibiting, or abrogating HIV/ AIDS.
  • the present invention provides that route of vaccination can result in a selective distribution of tissue lymphocytes and that mucosal vaccination can provide effective local protection at the site of antigen encounter.
  • the present invention provides an approach designed to enhance protection against vaginal HIV transmission, using a unique prime/boost protocol that employs two live mucosal vectors, Listeria monocytogenes (Lm) and adenovirus type 5 (Ad5), to elevate the abundance of memory CD8 + T cells in the vaginal lamina limbal.
  • Lm Listeria monocytogenes
  • Ad5 adenovirus type 5
  • the present invention provides an L. monocytogenes/adenovinis5 prime- boost protocol.
  • the present invention provides use of two attenuated live agents that express a common HIV antigen but signal through different innate response receptors as a means to elicit a stronger and more diverse response.
  • the present invention provides an enhancement of oral vaccination with an attenuated strain of L. monocytogenes expressing HIV-gag by boosting with an attenuated adenovirus type 5 HIV-gag recombinant (Ad5-gag).
  • the Listeria and adenovirus-containing compositions of methods, vaccines, and compositions of the present invention are each, in another embodiment, an immunogenic composition.
  • the composition is inherently immunogenic by virtue of its comprising a Listeria strain and/or an adenovirus strain of the present invention.
  • the composition further comprises an adjuvant. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides that the DNA encoding the heterologous antigen which is expressed in the vaccine comprising the Listeria and adenovirus strains of the invention must be preceded by a suitable promoter to facilitate such expression.
  • the present invention provides that the appropriate promoter/regulatory and signal sequences to be used will be readily apparent to those skilled in the art of molecular biology.
  • the present invention provides that a method of inducing an antigen- specific cell-mediated immune response in a subject, comprising the steps of: administering to a subject a recombinant strain of Listeria expressing an antigen; and administering to a subject an attenuated recombinant adenovirus comprising an antigen, results in an induction of an antigen- specific cell-mediated immune response in a mucosal tissue.
  • the present invention provides that a method of inducing an antigen- specific cell-mediated immune response in a subject, comprising the steps of: administering to a subject a recombinant strain of Listeria expressing an antigen; and administering to a subject an attenuated recombinant adenovirus comprising an antigen, results in an induction of an antigen- specific cell-mediated immune response in a vaginal tissue.
  • the present invention provides that a method of inducing an antigen- specific cell-mediated immune response in a subject, comprising the steps of: administering to a subject a recombinant strain of Listeria expressing an antigen; and administering to a subject an attenuated recombinant adenovirus comprising an antigen, results in an induction of an antigen- specific cell-mediated immune response in a peritoneal tissue.
  • the present invention provides that a method of inducing an antigen- specific cell-mediated immune response in a subject, comprising the steps of: administering to a subject a recombinant strain of Listeria expressing an antigen; and administering to a subject an attenuated recombinant adenovirus comprising an antigen, results in an induction of an antigen- specific cell-mediated immune response in a rectal tissue.
  • the present invention provides that the administration of an auxotrophic attenuated strain of Listeria to a mammal results in the development of a host cytotoxic T cell (CTL) response directed against Listeria following survival of the auxotrophic attenuated strain in the mammal for a time sufficient for the development of the CTL response.
  • CTL cytotoxic T cell
  • the present invention provides that an auxotrophic attenuated strain comprises a heterologous gene, wherein the gene is expressed by the auxotrophic attenuated strain.
  • the present invention provides that auxotrophic attenuated strains encoding additional heterologous genes are useful as bacterial vector vaccines for the prevention and/or treatment of infection caused by any number of infectious agents.
  • the present invention provides that auxotrophic attenuated strains encoding additional heterologous genes are useful as bacterial vector vaccines for the prevention and/or treatment of infection caused by any number of viruses. In another embodiment, the present invention provides that auxotrophic attenuated strains encoding additional heterologous genes are useful as bacterial vector vaccines for the prevention and/or treatment of tumors in mammals. In another embodiment, the present invention provides that auxotrophic attenuated strains encoding additional heterologous genes are useful as bacterial vector vaccines for the prevention and/or treatment of cancers in mammals. In another embodiment, the present invention provides that auxotrophic attenuated strains encoding additional heterologous genes are useful as bacterial vector vaccines for the prevention and/or treatment of metastasis in mammals.
  • the present invention provides a vaccine comprising an effective amount of a recombinant Listeria comprising an antigen; and an effective amount of an attenuated recombinant adenovirus comprising said antigen.
  • the vaccine comprising recombinant Listeria and recombinant adenovirus is a synergistic vaccine, which in one embodiment, refers to a vaccine in which the two vaccine components, when combined, work together so the total effect is greater than the sum of the individual two components.
  • the present invention provides a vaccine composition comprising an effective amount of a recombinant strain of Listeria comprising an antigen; and an attenuated recombinant adenovirus comprising an antigen.
  • the present invention provides a vaccine composition comprising an effective amount of a recombinant strain of Listeria comprising a first antigen; and an attenuated recombinant adenovirus comprising a second antigen.
  • both first and second antigens are distinct antigens from the same infectious agent or tumor.
  • the present invention provides a vaccine composition comprising an effective amount of a recombinant strain of Listeria and an attenuated recombinant adenovirus comprising the same antigen.
  • the present invention provides that the term "vaccine,” as used herein, is meant a combined population of bacteria and adenovirus which, when inoculated into a mammal, has the effect of stimulating a cellular immune response comprising a T cell response.
  • the present invention provides that the T cell response may be a cytotoxic T cell response directed against macromolecules produced by the bacteria.
  • the T cell response may be a cytotoxic T cell response directed against macromolecules produced by the virus.
  • the T cell response may be a cytotoxic T cell response directed against the antigen expressed by the bacteria or virus.
  • the present invention provides that the induction of a T cell response comprising other types of T cells by the vaccine of the invention is also contemplated.
  • the present invention provides that vaccination with the recombinant Listeria vector induces both CD4 + T cells and CD8 + T cells.
  • a vaccine of the present invention elicits an immune response to the antigen.
  • a vaccine of the present invention has therapeutic or prophylactic effects, wherein the object is to prevent or lessen the targeted infection, pathologic condition or disorder.
  • the present invention provides that the induced CD4 + T cells are responsible for the synthesis of cytokines, such as interferon-. gamma., IL-2 and TNF-alpha.
  • the present invention provides that the CD8 + T cells are cytotoxic T cells and also secrete cytokines such as interferon-gamma. and TNF-alpha.
  • the present invention provides that these cells and the molecules synthesized therein play a role in the infection and subsequent protection of the host against the heterologous genes expressed by the Listeria.
  • the present invention provides that the cytokines produced by these cells activate additional T cells and also macrophages and recruit polymorphonuclear leukocytes to the site of infection.
  • the present invention provides that either prophylactic or therapeutic vaccines, or both, are contemplated as being within the scope of the present invention, that is, vaccines which are administered either prior to or subsequent to the onset of disease or exposure to an infectious are included in the invention.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating HIV infection in a subject comprising administering a vaccine comprising an effective amount of a recombinant Listeria comprising an antigen; and an effective amount of an attenuated recombinant adenovirus comprising said antigen.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating cervical cancer in a subject comprising administering a vaccine comprising an effective amount of a recombinant Listeria comprising an antigen; and an effective amount of an attenuated recombinant adenovirus comprising said antigen.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating head and neck cancer in a subject comprising administering a vaccine comprising an effective amount of a recombinant Listeria comprising an antigen; and an effective amount of an attenuated recombinant adenovirus comprising said antigen.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating breast cancer in a subject comprising administering a vaccine comprising an effective amount of a recombinant Listeria comprising an antigen; and an effective amount of an attenuated recombinant adenovirus comprising said antigen.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating ovarian cancer in a subject comprising administering a vaccine comprising an effective amount of a recombinant Listeria comprising an antigen; and an effective amount of an attenuated recombinant adenovirus comprising said antigen.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating melanoma in a subject comprising administering a vaccine comprising an effective amount of a recombinant Listeria comprising an antigen; and an effective amount of an attenuated recombinant adenovirus comprising said antigen.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating lung cancer in a subject comprising administering a vaccine comprising an effective amount of a recombinant Listeria comprising an antigen; and an effective amount of an attenuated recombinant adenovirus comprising said antigen.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating a tumor in a subject comprising administering a vaccine comprising an effective amount of a recombinant Listeria comprising an antigen; and an effective amount of an attenuated recombinant adenovirus comprising said antigen.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating prostate cancer in a subject comprising administering a vaccine comprising an effective amount of a recombinant Listeria comprising an antigen; and an effective amount of an attenuated recombinant adenovirus comprising said antigen.
  • the present invention provides a method of treating, inhibiting, abrogating, or ameliorating lung cancer in a subject comprising administering a vaccine comprising an effective amount of a recombinant Listeria comprising an antigen; and an effective amount of an attenuated recombinant adenovirus comprising said antigen.
  • an effective amount is determined using routine experimentation, as is known to those of skill in the art. In one embodiment, an effective amount is determined by the weight of the subject, by the immuno-compromised status of the subject, and by other criteria known in the art.
  • An effective dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models to achieve an induction of an immune response using techniques that are well known in the art.
  • Dosage amount and interval may be adjusted individually.
  • the vaccine formulations of the invention may be administered in about 1 to 3 doses for a 1-36 week period.
  • 1 or 2 doses are administered, at intervals of about 3 weeks to about 4 months.
  • booster vaccinations may be given periodically thereafter.
  • Alternative protocols may be appropriate for individual subjects.
  • a suitable dose is an amount of the vaccine formulation that, when administered as described above, is capable of raising an immune response in an immunized subject sufficient to protect the animal from an infection or from the development of a tumor for at least 4 to 12 months.
  • a suitable dose is an amount of the vaccine formulation that, when administered as described above, is capable of raising an immune response in an immunized subject sufficient to treat an animal with an infection or a tumor so that improvement can be measured using standard techniques.
  • the amount of the antigen present in a dose ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 ⁇ g. Suitable dose range will vary with the route of injection and the size of the patient, but will typically range from about 0.1 mL to about 5 mL.
  • the solution used for freezing contains another colligative additive or additive with anti-freeze properties, in place of glycerol.
  • the solution used for freezing contains another colligative additive or additive with anti-freeze properties, in addition to glycerol.
  • the additive is mannitol.
  • the additive is DMSO.
  • the additive is sucrose.
  • the additive is any other colligative additive or additive with anti-freeze properties that is known in the art. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides mucosal vaccination by the intravaginal route. In another embodiment, the present invention provides mucosal vaccination by the intranasal route. In another embodiment, the present invention provides mucosal vaccination by the intrarectal route. In another embodiment, the present invention provides mucosal vaccination by the intraintestinal route. In another embodiment, the present invention provides mucosal vaccination by the intraperitoneal route. In another embodiment, the present invention provides mucosal vaccination by the intramuscular route.
  • the present invention provides that L. monocytogenes and attenuated recombinant adenovirus are able to directly infect and initiate an immune response at the vaginal mucosa.
  • the present invention provides mucosal vaccination by the intravaginal route which results in induction of T cell immunity in the vagina.
  • the present invention provides mucosal vaccination by the intrarectal route which results in induction of T cell immunity in the vagina.
  • the present invention provides mucosal vaccination by the intrarectal route which results in induction of T cell immunity in the rectum.
  • the present invention provides mucosal vaccination by the intravaginal route which results in the induction of systemic T cell immunity.
  • the present invention provides a systemic route of vaccination which results in systemic T cell immunity. In another embodiment, the present invention provides a systemic route of vaccination which results in induction of T cell immunity in the vagina. [00133] In another embodiment, the present invention provides that L. monocytogenes and/or attenuated recombinant adenovirus are able to cross the mucosal barrier of the vagina, inducing CD8 + T cells in spleen, liver, lungs and vagina.
  • the present invention provides that the vaccines of the present invention are administered to a host vertebrate animal. In another embodiment, the present invention provides that the vaccines of the present invention are administered to a mammal. In another embodiment, the present invention provides that the vaccines of the present invention are administered to a human. In another embodiment, the present invention provides that the vaccines of the present invention are administered in combination with a pharmaceutically acceptable carrier.
  • the present invention provides that the vaccines of the present invention are administered in an amount effective to induce an immune response to the heterologous antigen which the Listeria and adenovirus species have been modified to express.
  • the present invention provides that the amount of vaccine to be administered is routinely determined by one of skill in the art when in possession of the present disclosure.
  • a pharmaceutically acceptable carrier may include, but is not limited to, sterile distilled water, saline, phosphate buffered solutions or bicarbonate buffered solutions.
  • a pharmaceutically acceptable carrier selected and the amount of carrier to be used will depend upon several factors including the mode of administration and the age and disease state of the patient.
  • the present invention provides that administration of the vaccine may be by an oral route, or it may be parenteral, intranasal, intramuscular, intravascular, intrarectal, intraperitoneal, intravaginal, intramucosal, subcutaneous, intradermal, intra-tumoral, or any one of a variety of routes of administration known in the art.
  • the present invention provides that the route of administration is selected in accordance with the type of infectious agent or tumor to be treated.
  • the recombinant Listeria or adenovirus of the compositions and methods of the present invention is administered orally.
  • the recombinant Listeria or adenovirus of the compositions and methods of the present invention is administered intravaginally or intrarectally. In one embodiment, the recombinant Listeria or adenovirus of the compositions and methods of the present invention is administered intramuscularly.
  • the recombinant Listeria and adenovirus of the compositions and methods of the present invention are administered via the same route of administration. In another embodiment, the recombinant Listeria and adenovirus of the compositions and methods of the present invention are administered via distinct routes of administration. In one embodiment, the recombinant Listeria and adenovirus of the compositions and methods of the present invention are administered concurrently, while in another embodiment, the recombinant Listeria and adenovirus of the compositions and methods of the present invention are administered at separate time points. In one embodiment, several priming vaccinations are provided before one or more boosting vaccinations. In another embodiment, several boosting vaccinations are provided after one or more priming vaccinations. In one embodiment, the priming or boosting vaccination, or both, may be administered one, two, three, four, or five times or more.
  • the present invention provides that the vaccines of the present invention may be administered in the form of elixirs, capsules or suspensions for oral administration or in sterile liquids for parenteral or intravascular administration.
  • the present invention provides that the vaccine may also be administered in conjunction with a suitable adjuvant, which adjuvant will be readily apparent to the skilled artisan.
  • the present invention provides that the immunogenicity of the auxotrophic attenuated strain of the invention is enhanced by a booster comprising an attenuated recombinant adenovirus.
  • the adjuvant of methods, vaccines, and compositions of the present invention is Montanide ISA 51.
  • Montanide ISA 51 contains a natural metabolizable oil and a refined emulsifier.
  • the adjuvant is GM-CSF.
  • the adjuvant is KLH.
  • Recombinant GM-CSF is a human protein grown, in another embodiment, in a yeast (S. cerevisiae) vector. GM-CSF promotes clonal expansion and differentiation of hematopoietic progenitor cells, APC, and dendritic cells and T cells.
  • the adjuvant is a cytokine. In another embodiment, the adjuvant is a growth factor. In another embodiment, the adjuvant is a cell population. In another embodiment, the adjuvant is QS21. In another embodiment, the adjuvant is Freund's incomplete adjuvant. In another embodiment, the adjuvant is aluminum phosphate. In another embodiment, the adjuvant is aluminum hydroxide. In another embodiment, the adjuvant is BCG. In another embodiment, the adjuvant is alum. In another embodiment, the adjuvant is an interleukin. In another embodiment, the adjuvant is an unmethylated CpG oligonucleotide. In another embodiment, the adjuvant is quill glycosides. In another embodiment, the adjuvant is monophosphoryl lipid A.
  • the adjuvant is liposomes. In another embodiment, the adjuvant is a bacterial mitogen. In another embodiment, the adjuvant is a bacterial toxin. In another embodiment, the adjuvant is a chemokine. In another embodiment, the adjuvant is any other type of adjuvant known in the art.
  • the vaccine of methods, vaccines, and compositions of the present invention comprises two of the above adjuvants. In another embodiment, the vaccine comprises more than two of the above adjuvants. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides that for treatment of cancer, the vaccine of the invention may be used to protect people at high risk for cancer.
  • the vaccine may be used as an immuno therapeutic agent for the treatment of cancer following debulking of tumor growth by surgery, conventional chemotherapy, or radiation treatment.
  • the present invention provides that the patients receiving such treatment may be administered a vaccine which expresses a desired tumor antigen for the purpose of generating a CTL response against any residual tumor cells in the individual.
  • the present invention provides that the vaccine of the present invention may also be used to inhibit the growth of any previously established tumors in a human by either eliciting a CTL response directed against the tumor cells per se, or by eliciting a CTL response against cells which synthesize tumor promoting factors, wherein such a CTL response serves to kill those cells thereby diminishing or ablating the growth of the tumor.
  • the present invention provides vaccines for subjects that have a high risk for developing a specific cancer, which in one embodiment, comprises patients with specific genetic profiles known in the art to indicate an increased susceptibility to developing a cancer (Monhandas 2001 Current Science 81(5): 482-488, incorporated herein by reference).
  • mutation of the BRCAl and BRC A2 may indicate higher susceptibility to breast cancer, ovarian cancer, or both.
  • the present invention provides the administration of a vaccine to a human for the purpose of preventing, alleviating, or ablating an HIV infection.
  • the protocol which is described herein for the administration of a vaccine for the purpose of treating HIV infection is provided as an example of how to administer an attenuated auxotrophic Listeria strain as a vaccine to a human.
  • this protocol should not be construed as being the only protocol which can be used, but rather, should be construed merely as an example of the same.
  • the present invention provides that other protocols will become apparent to those skilled in the art when in possession of the present invention.
  • the present invention provides a vaccine for preventing infectious diseases. In another embodiment, the present invention provides a vaccine for managing infectious diseases. In another embodiment, the present invention provides a vaccine for treating infectious diseases. In another embodiment, the present invention provides a vaccine for inhibiting infectious diseases. In another embodiment, the present invention provides a vaccine for ameliorating infectious diseases.
  • the present invention provides vaccines that depend for efficacy on the establishment of potent, long-lived memory T cells, ready to expand rapidly to express killing functions on reexposure to antigen.
  • the present invention provides that effector cells generated by the prime/boost protocol are capable of generating a population of long-lived memory cells, which in one embodiment, are mucosal memory cells.
  • the culture e.g. the culture of a Listeria vaccine strain and/or attenuated recombinant adenovirus vaccine strain that are used to produce a batch of Listeria and/or attenuated recombinant adenovirus vaccine doses
  • the culture is inoculated from a cell bank.
  • the culture is inoculated from a frozen stock.
  • the culture is inoculated from a starter culture.
  • the culture is inoculated from a colony.
  • the culture is inoculated at mid-log growth phase.
  • the culture is inoculated at approximately mid-log growth phase.
  • the culture is inoculated at another growth phase.
  • the present invention provides that the vaccine of the invention may be maintained in storage until use. Storage may comprise freezing the vaccine, or maintaining the vaccine at 4 0 C, room temperature, or the vaccine may first be lyophilized and then stored.
  • the present invention provides that the attenuated recombinant adenovirus administered to a subject with the attenuated recombinant Listeria comprises the same heterologous antigen as the Listeria. In another embodiment, the present invention provides that the attenuated recombinant adenovirus is administered to a subject after the attenuated recombinant Listeria is administered to a subject. In another embodiment, the present invention provides that the attenuated recombinant adenovirus is administered to a subject before the attenuated recombinant Listeria is administered to a subject.
  • the present invention provides that the attenuated recombinant adenovirus is administered to a subject a day after the attenuated recombinant Listeria is administered to a subject. In another embodiment, the present invention provides that the attenuated recombinant adenovirus is administered to a subject 1-3 days after the attenuated recombinant Listeria is administered to a subject. In another embodiment, the present invention provides that the attenuated recombinant adenovirus is administered to a subject 2-4 days after the attenuated recombinant Listeria is administered to a subject.
  • the present invention provides that the attenuated recombinant adenovirus is administered to a subject 3-6 days after the attenuated recombinant Listeria is administered to a subject. In another embodiment, the present invention provides that the attenuated recombinant adenovirus is administered to a subject 4-10 days after the attenuated recombinant Listeria is administered to a subject. In another embodiment, the present invention provides that the attenuated recombinant adenovirus is administered to a subject 8-15 days after the attenuated recombinant Listeria is administered to a subject.
  • the present invention provides that the attenuated recombinant adenovirus is administered to a subject 10-15 days after the attenuated recombinant Listeria is administered to a subject. In another embodiment, the present invention provides that the attenuated recombinant adenovirus is administered to a subject 15-25 days after the attenuated recombinant Listeria is administered to a subject. In another embodiment, the present invention provides that the attenuated recombinant adenovirus is administered to a subject 20-30 days after the attenuated recombinant Listeria is administered to a subject.
  • the present invention provides that the attenuated recombinant adenovirus is administered to a subject 25-40 days after the attenuated recombinant Listeria is administered to a subject. In another embodiment, the present invention provides that the attenuated recombinat adenovirus is administered to a subject 6 to 18 weeks after the attenuated recombinant Listeria is administered to a subject.
  • the present invention provides that the attenuated recombinant adenovirus and the attenuated recombinant Listeria are administered in 1-10 cycles. In another embodiment, the present invention provides that the attenuated recombinant adenovirus and the attenuated recombinant Listeria are administered in 2-4 cycles. In another embodiment, the present invention provides that the attenuated recombinant adenovirus and the attenuated recombinant Listeria are administered in 5-10 cycles. In another embodiment, the present invention provides that a person of skill in the art will determine the protocol of administering the attenuated recombinant adenovirus and the attenuated recombinant Listeria of the present invention.
  • the present invention provides that the attenuated recombinant adenovirus is administered to a subject a day before the attenuated recombinant Listeria is administered to a subject. In another embodiment, the present invention provides that the attenuated recombinant adenovirus is administered to a subject 1-3 days before the attenuated recombinant Listeria is administered to a subject. In another embodiment, the present invention provides that the attenuated recombinant adenovirus is administered to a subject 2-4 days before the attenuated recombinant Listeria is administered to a subject.
  • the present invention provides that the attenuated recombinant adenovirus is administered to a subject 3-6 days before the attenuated recombinant Listeria is administered to a subject. In another embodiment, the present invention provides that the attenuated recombinant adenovirus is administered to a subject 4-10 days before the attenuated recombinant Listeria is administered to a subject. In another embodiment, the present invention provides that the attenuated recombinant adenovirus is administered to a subject 8-15 days before the attenuated recombinant Listeria is administered to a subject.
  • the present invention provides that the attenuated recombinant adenovirus is administered to a subject 10-15 days before the attenuated recombinant Listeria is administered to a subject. In another embodiment, the present invention provides that the attenuated recombinant adenovirus is administered to a subject 15-25 days before the attenuated recombinant Listeria is administered to a subject. In another embodiment, the present invention provides that the attenuated recombinant adenovirus is administered to a subject 20-30 days before the attenuated recombinant Listeria is administered to a subject.
  • the present invention provides that the attenuated recombinant adenovirus is administered to a subject 25-40 days before the attenuated recombinant Listeria is administered to a subject. In another embodiment, the present invention provides that the attenuated recombinant adenovirus is administered to a subject 6 to 18 weeks before the attenuated recombinant Listeria is administered to a subject.
  • adenovirus is used as the prime and Listeria as the boost
  • Listeria is used as the prime
  • adenovirus is used as the boost.
  • the prime is administered one time, two times, three times, four times, five times, or more
  • the boost is administered one time, two times, three times, four times, five times, or more.
  • the present invention provides Listeria as an oral, intravaginal, or intrarectal vaccine along with a systemic boost by adenovirus.
  • the present invention provides that boost increases the number of antigen-specific CD8 + T cells after priming with an attenuated strain of L. monocytogenes by 10- 10,000% ( Figure 1). In another embodiment, the present invention provides that boost increases the number of antigen- specific CD8 + T cells after priming with an attenuated strain of L. monocytogenes by 10- 100% . In another embodiment, the present invention provides that boost increases the number of antigen- specific CD8 + T cells after priming with attenuated strain of L. monocytogenes by 100-500%. In another embodiment, the present invention provides that boost increases the number of antigen- specific CD8 + T cells after priming with attenuated strain of L. monocytogenes by 500- 1000% .
  • the present invention provides that boost increases the number of antigen-specific CD8 + T cells after priming with attenuated strain of L. monocytogenes by 1000-1500%. In another embodiment, the present invention provides that boost increases the number of antigen-specific CD8 + T cells after priming with attenuated strain of L. monocytogenes by 1500-5000%. In another embodiment, the present invention provides that boost increases the number of antigen- specific CD8 + T cells after priming with attenuated strain of L. monocytogenes by 3000-6000%. In another embodiment, the present invention provides that boost increases the number of antigen- specific CD8 + T cells after priming with attenuated strain of L. monocytogenes by 5000-8000%. In another embodiment, the present invention provides that boost increases the number of antigen- specific CD8 + T cells after priming with attenuated strain of L. monocytogenes by 8000-10000%.
  • the present invention provides mucosal vaccination by the intravaginal route. These data supported the efficacy of the prime/boost protocol.
  • the present invention provides that L. monocytogenes is able to directly infect and initiate an immune response at the vaginal mucosa.
  • the present invention provides that adenovirus infect the vagina.
  • the present invention provides a prime/boost protocols initiated by a single dose of attenuated Lm-gag deposited either on the vaginal or rectal mucosa or administered orally.
  • the present invention provides that infection with Ad5-gag by either a systemic route or mucosally into the vaginal canal takes place at 1-10 weeks after Lm-gag administration. In another embodiment, the present invention provides that infection with Ad5-gag by either a systemic route or mucosally into the vaginal canal takes place at 2-4 weeks weeks after Lm-gag administration. In another embodiment, the present invention provides that infection with Ad5-gag by either a systemic route or mucosally into the vaginal canal takes place at 4-8 weeks after Lm-gag administration. In another embodiment, the present invention provides that infection with Ad5-gag by either a systemic route or mucosally into the vaginal canal takes place at 7-10 weeks after Lm-gag administration.
  • the present invention provides that the mucosal Lm-gag infection followed with a systemic boost by Ad5-gag results in high levels of antigen- specific T cells elicited in both the spleen and vagina.
  • the present invention provides that infection with Ad5-gag by either a systemic route or mucosally into the vaginal canal at 1-10 weeks after Lm-gag administration.
  • the present invention provides that the prime/boost protocol generates a strong and more diverse immune response than possible with either agent alone, or as is commonly practiced, using a DNA vaccine to prime either agent.
  • the present invention provides that the prime/boost protocol generates a synergistic immune response compared with vaccination with either a " 1 gOe"-nt alone.
  • the present invention provides that attenuated forms of two live vectors, L. monocytogenes and adenovirus type 5, in a heterologous prime/boost protocol generates high levels of vaginal immunity.
  • the present invention provides that these two agents together yield far and unexpectedly superior responses than either alone.
  • the present invention provides that mucosal and systemic infections by a variety of pathogens leads to wide dissemination of effector and memory CD4 + and CD8 + T cells to all peripheral nonlymphoid tissue.
  • the present invention provides that regional priming of T cells leads to the imprinting of an "area code" that allows the selective recruitment of T cell subsets to the priming site.
  • the present invention provides that skin infection by vaccinia virus at the base of a mouse tail leads to CD8 + T cell activation in the draining inguinal lymph nodes, upregulation of T cell skin-homing molecules and their preferential infiltration into tail skin after infection.
  • the present invention provides that the immune system targets effector T cells to the site of pathogen encounter to arrest an infection in situ, but protects distant tissues against dissemination of the agent by also superimposing a more promiscuous T cell migration behavior.
  • the present invention provides enhancement of the number of protective T cells in vaginal lamina intestinal by vaccinating at that site with two strong mucosal agents.
  • the present invention provides that L. monocytogenes is able to cross the mucosal barrier of vagina, inducing CD8 + T cells in spleen, liver, lungs and vagina.
  • the present invention provides that attenuated recombinant adenovirus infects the vagina and induce a CD8 + T cell response.
  • a recombinant Listeria strain of the present invention is not antibiotic- resistant. In another embodiment, the Listeria strain of the present invention does not contain an antibiotic- resistance gene. In another embodiment, a recombinant Listeria strain of the present invention does not contain an antibiotic-resistance gene at the SepA location in the chromosome.
  • any of the compositions of this invention will comprise an attenuated Listeria and an attenuated Adenovirus, in any form or embodiment as described herein. In some embodiments, any of the compositions of this invention will consist of an attenuated Listeria and an attenuated Adenovirus, in any form or embodiment as described herein. In some embodiments, of the compositions of this invention will consist essentially of an attenuated Listeria and an attenuated Adenovirus, in any form or embodiment as described herein.
  • the term “comprise” refers to the inclusion of an attenuated Listeria and an attenuated Adenovirus, as well as inclusion of other active agents, such as adjuvants and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc., as are known in the pharmaceutical and vaccine industries.
  • the term “consisting essentially of” refers to a composition, whose only active ingredient is the indicated active ingredient, however, other compounds may be included which are for stabilizing, preserving, etc. the formulation, but are not involved directly in the therapeutic effect of the indicated active ingredient.
  • the term “consisting essentially of may refer to components which facilitate the release of the active ingredient.
  • the term “consisting” refers to a composition, which contains the active ingredient and a pharmaceutically acceptable carrier or excipient.
  • the bacteria were administered by gavage at a dose of 1.5 x 10 10 .
  • Intrarectal infections were delivered by a 2-cm catheter at the same dose.
  • 5 x 10 9 bacteria were deposited into the vaginal canal.
  • Intravaginal infections by all vectors (Lm-gag, Ad5-gag and vaccinia-gag) were preceded at -5 days with subcutaneous injection of 2 mg medroxyprogesterone acetate (Depo-Provera®, Pfizer) to thin the vaginal mucosa.
  • the animals were anesthetized by i.p.
  • ketamine 150 mg/kg body weight
  • xylazine 10 mg/kg
  • vaginal mucus was removed by use of a fine calcium alginate swab and the vector was deposited and left in the vagina during a 60-min period of anesthesia.
  • Replication-defective El E3-deleted adenovirus type 5-HIV-gag (Ad5-gag; G. Pavlakis and H. Ertl) was administered i.m. at 5 x 10 9 virus particles or at 1 x 10 10 virus particles intravaginally. Isolation of lymphocytes
  • Splenocyte suspensions were obtained by pressing the tissue through a nylon mesh screen, followed by lysis of red blood cells using ACK lysis buffer.
  • Lymphocytes from PPs were prepared by digestion with collagenase D and DNase I at 37 0 C for 30 minutes and then incubated in the presence of 5mM EDTA for 5 min. The digested tissues were teased into suspension and filtered through nylon mesh to remove debris.
  • Liver and lung lymphocytes were obtained from mice perfused with PBS plus heparin and purified between 44% and 67.5% percoll layers (Pharmacia), followed by ACK lysis of red blood cells.
  • Vaginal tissue was minced, incubated with 0.5 mg/ml Dispase II (Roche Diagnostics) in PBS at 37 0 C for 15 min, then further incubated in 5 ml PBS containing 0.22 ⁇ g of collagenase D (Roche Diagnostics), 75 ⁇ g of DNase (Roche Diagnostics), and 500 U of hyaluronidase (Sigma) at 37 0 C for 45 min with shaking, and then pressed through 70- ⁇ m mesh. Cells were washed 2 times with PBS, and one time in 2.5% BSA-2 mM EDTA in PBS.
  • the cells were then washed, fixed in 2% (wt/vol) paraformaldehyde/PBS, and analyzed on a FACSCalibur flow cytometer.
  • vaginal lymphocytes cells were blocked with anti-CD26/32 at 4 0 C for 10 min, and then stained in 2.5% BSA- 2mM EDTA in PBS for 1 hr at 4 0 C, adding 7- AAD to identify dead cells during the last 15 min, washed with 2 mM EDTA in PBS, and analyzed.
  • Flowjo software (Tree Star, Inc.) was used to interpret the data.
  • lymphocytes were cultured for 5h with Golgistop (BD PharMinegen, San Diego, CA), with 5 ⁇ g/ml peptide. Cells were then stained for surface molecules, fixed, and cell membranes were permeablized in cytofix/cytoperm solution and stained with FITC-anti-IFN- ⁇ (clone XMG 1.2; BD) and APC-anti-TNF- ⁇ (clone MP6-XT22; eBioscience, San Diego, CA) in Perm/Wash solution. Cells were washed and fluorescence intensity was measured using a FACSCalibur flow cytometer.
  • Golgistop BD PharMinegen, San Diego, CA
  • target cells were pooled splenocytes of naive mice, pooled and labeled at 2xlO 7 cells/ml with 6 ⁇ M CFSE or 0.3 ⁇ M CFSE (Molecular Probes, Eugene, OR) for 10 min in the dark, quenched with an equal volume of 100% FCS at RT for 1 min, followed by two washes with RPMI 1640 medium, 10% FCS. The CFSEhigh cells were further incubated with 10 "6 M HIV-I Gag 197-205 peptide. After washing, 10 7 cells of each population were mixed and injected i.v. into groups (5 or more/group) of recipient mice.
  • 6 ⁇ M CFSE or 0.3 ⁇ M CFSE Molecular Probes, Eugene, OR
  • Vvkl vaccinia-gag virus
  • EXAMPLE 1 AN L. MONOCYTOGENES! ADEHO VIRUS PRIME-BOOST PROTOCOL
  • Figure ID demonstrates that the splenic T cells were functional. All of the tetramer-positive cells of spleen expressed both IFN- ⁇ and TNF- ⁇ . The immunized animals were tested for in vivo cytolytic activity directed against Gag-presenting target cells.
  • Figure 2A shows that the immune mice lysed all Gag-targets at 16-h after their injection, and that this level of activity was seen as early as two hours after injecting the target cells.
  • the individual vectors resulted in significantly lower activities.
  • Memory cells retained the cytolytic activity, since after 42 days the mice continued to show high activity, even in the stringent 2-hours assay.
  • Figure 3A To assess the ability of these memory T cells, shown in Figure 3A, to mount a recall response, three weeks after the boost mice were challenged i.p. with vaccinia-gag virus. A large expansion of the antigen- specific CD8 + T cells resulted (Figure 3A), and the mice were able to block most replication of the virus ( Figure 3B).
  • Listeria prime-adenovirus boost is an efficacious means to inducing cellular immune responses.
  • Figure 4C shows that when any of the mucosal Lm-gag infections was followed with a systemic boost by Ad5-gag, high levels of antigen- specific T cells were elicited in both the spleen (18-21%) and vagina (14-26%). Conversely, when the mucosal Lm-gag primes were followed by a vaginal (mucosal) boost by Ad5-gag ( Figure 4D), a redistribution of CD8 T cells to the lamina intestinal of the vagina was observed.
  • the resulting level of Gag-specific CD8 + T cells in spleen was surprisingly low (2-6%), while exaggerated numbers of antigen- specific CD8 + T cells accumulated in vaginal tissue (21-62%). This was particularly evident after intravaginal administration of both vectors.
  • This ivag/ivag regimen also elicited strong Gag-specific responses in other peripheral tissues (30% and 40% in lung and liver, respectively, not shown), but vagina showed the highest relative abundance of antigen- specific T cells.
  • ivag/ivag-immunized mice were depleted of either CD4 or CD8 T cells by the injection of depleting rat anti-mouse mAbs (Fig. 2C). Whereas sham-immunized mice showed no cytolytic activity in their spleens or in the draining iliac lymph nodes, immunized mice injected with control rat Ig showed 84-91% cytolysis of Gag peptide-labeled targets in these tissues at 3 h after target cell injection.
  • T EM effector memory T cell
  • T CM central memory T cell
  • Table 1 Retention of Gag-specific memory CD8 T cells in vagina and other tissues after intravaginal L. monocytogenes -gag prime and adenovirus type 5-gag boost*
  • mice were infected intravaginally with 5 x 10 attenuated Lm-gag. At 30 days, the animals were boosted with 10 10 particles of Ad5-gag. Eight days (effector) or 35 days (memory) later, lymphocytes were stained to detect Gag-specific and CD62L-positive CD8 + T cells . Data are from one experiment and reflect results of two additional independent experiments. Vaginal tissues were combined before cell isolation. t r The numbers represent the percent of activated Gag-specific cells relative to total CD8 + T cells. *This row shows the fraction of tetramer-negative CD8 + T cells negative or positive for CD62L. ⁇ These rows show the fraction of tetramer-positive CD8 + T cells negative or positive for CD62L.
  • FIG. 3C To examine characteristics of the vaginal memory T cells (seen in Figure 3C), their in vivo cytolytic activity and their ability to be reactivated and expand upon re-exposure to antigen was measured.
  • Figure 2B shows that in the vagina and its draining lymph node in the immune memory mice, the sensitive assay at 2 hr after target cell transfer showed significant cytolysis (69%).
  • the recall response of the memory T cells was examined by infection of the memory animals with an unrelated virus, recombinant vaccinia-gag. As shown in Figure 3C, despite the existing high level of memory cells in the vagina, the new challenge elicited a further increase.
  • the number of Gag-specific CD8 T cells in vagina of these mice increased from less than 5 in a naive animal to ⁇ 1 x 10 4 in an immune mouse. Further, spleens of the ivag/ivag mice contained as many as 5 x 10 5 Gag-specific CD8 T cells.
  • using heterologous live vectors to both prime and boost a response may be useful because different pathogens vary in their intracellular habitat, their survival, the induction of inflammation and the nature of T cell priming and memory generation.
  • mixed live agents different TLRs and sensing receptors on dendritic cell subsets are triggered and brought into play. These in turn lead to activation of varied accessory cells and chemokine responses. These multiple inputs can lead to immune diversity and immune synergy.

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Abstract

La présente invention concerne des vaccins comprenant des souches recombinantes atténuées de Listeria et des souches recombinantes atténuées d'adénovirus, qui expriment un antigène hétérologue. L'invention concerne également des méthodes de production et d'utilisation de ces vaccins.
PCT/US2008/088543 2007-12-31 2008-12-30 Vaccins à souches atténuées de listeria et d'adénovirus, et méthodes d'utilisation WO2009110950A1 (fr)

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US11446369B2 (en) 2007-05-10 2022-09-20 Advaxis, Inc. Compositions and methods comprising KLK3 or FOLH1 antigen
US9644212B2 (en) 2008-05-19 2017-05-09 Advaxis, Inc. Dual delivery system for heterologous antigens
US9650639B2 (en) 2008-05-19 2017-05-16 Advaxis, Inc. Dual delivery system for heterologous antigens
US10016617B2 (en) 2009-11-11 2018-07-10 The Trustees Of The University Of Pennsylvania Combination immuno therapy and radiotherapy for the treatment of Her-2-positive cancers
EP2621527A4 (fr) * 2010-10-01 2015-12-09 Univ Pennsylvania Utilisation de vecteurs de vaccin de listeria pour renverser l'insensibilité au vaccin chez des individus infectés par des parasites
US9226958B2 (en) 2010-10-01 2016-01-05 University Of Georgia Research Foundation, Inc. Use of Listeria vaccine vectors to reverse vaccine unresponsiveness in parasitically infected individuals
US9943590B2 (en) 2010-10-01 2018-04-17 The Trustees Of The University Of Pennsylvania Use of Listeria vaccine vectors to reverse vaccine unresponsiveness in parasitically infected individuals
US9463227B2 (en) 2011-03-11 2016-10-11 Advaxis, Inc. Listeria-based adjuvants
US10064898B2 (en) 2011-03-11 2018-09-04 Advaxis, Inc. Listeria-based adjuvants
US10058599B2 (en) 2012-03-12 2018-08-28 Advaxis, Inc. Suppressor cell function inhibition following Listeria vaccine treatment

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