WO2005002512A2 - Immunotherapeutic vaccine strategy - Google Patents

Abstract

The present invention relates to a fusion protein which comprises an epitope tag, a protein translocation domain, a ubiquitin domain, and a pathogen derived immunogen. The epitope tag, the protein translocation domain, the ubiquitin domain, and the pathogen derived immunogen are directly or indirectly coupled together. Alternatively, the present invention is directed to a nucleic acid construct encoding a fusion protein where the nucleic acid construct comprises a nucleic acid molecule encoding an epitope tag, a nucleic acid molecule encoding a protein translocation domain, a nucleic acid molecule encoding a ubiquitin domain, and a nucleic acid molecule encoding a pathogen derived immunogen. The nucleic acid molecules encoding the epitope tag, the protein translocation domain, the ubiquitin domain, and the pathogen derived immunogen are directly or indirectly coupled together. Methods of vaccinating a subject against a pathogen by administering the fusion protein or the nucleic acid construct to the subject under conditions effective to vaccinate the subject against the pathogen are also disclosed.

Description

IMMUNOTHERAPEUTIC VACCINE STRATEGY

[0001] This application claims the benefit of U.S. Provisional Patent

Application Serial No. 60/479,815, filed June 19, 2003.

FIELD OF THE INVENTION

[0002] The present invention is directed to an immunotherapeutic vaccine strategy.

BACKGROUND OF THE INVENTION

[0003] Certain human papillomaviruses (HPNs) infect mucosal epithelial tissues and cause a variety of hyperproliferative epithelial disorders ranging from benign oral/anogenital warts and laryngeal papillomatosis to malignancy and its precursor lesions. It is known, for example, that infection with certain HPN genotypes is a necessary prerequisite for the development of invasive uterine cervical carcinoma. This disease is the most common female malignancy in resource-poor regions. Thus, there is an urgent need for effective methods of controlling HPN disease. Cellular immune mechanisms are known to play a critical role in controlling HPN-associated disease, and there is evidence to suggest the potential efficacy of immunotherapeutic strategies designed to target certain well-defined viral molecular targets for the purpose of induction or enhancement of cytotoxic T lymphocyte (CTL) responses against them. HPN recombinant virus-like particles (NLPs) are promising candidates for HPN immunoprophylaxis. Importantly, NLPs are able to activate dendritic cells directly, and, as exogenously administered immunogens, can enter directly the Major Histocompatibility Complex (MHC) Class I antigen-processing pathway for CTL induction. Thus, NLPs can also be used to target heterologous immunogens into the Class I pathway, and to deliver plasmid DΝA vectors to mediate de novo expression of heterologous antigens. SUMMARY OF THE INVENTION

[0004] A first embodiment of the present invention relates to a fusion protein which comprises an epitope tag, a protein translocation domain, a ubiquitin domain, and a pathogen derived immunogen. The epitope tag, the protein translocation domain, the ubiquitin domain, and the pathogen derived immunogen are directly or indirectly coupled together.

[0005] -Another embodiment of the present invention is directed to a nucleic acid construct encoding the fusion protein. The nucleic acid construct comprises a nucleic acid molecule encoding an epitope tag, a nucleic acid molecule encoding a protein translocation domain, a nucleic acid molecule encoding a ubiquitin domain, and a nucleic acid molecule encoding a pathogen derived immunogen. The nucleic acid molecules encoding the epitope tag, the protein translocation domain, the ubiquitin domain, and the pathogen derived immunogen are directly or indirectly coupled together.

[0006] A method of vaccinating a subject against a pathogen by administering the fusion protein to the subject under conditions effective to vaccinate the subject against the pathogen is also disclosed.

[0007] Another aspect of the present invention relates to method of vaccinating a subject against a pathogen by administering the nucleic acid construct to the subject under conditions effective to vaccinate the subject against the pathogen [0008] Also disclosed is a viral vector comprising the nucleic acid construct.

[0009] Using the present invention, proteins can be targeted into the Major

Histocompatibility Complex (MHC) Class I pathway via proteasomal degradation mediated by assembly of a polyubiquitin chain attached to an accessible lysine or arginine residue in the target protein (Sijts, et al., "The Role of the Ubiquitin- Proteasome Pathway in MHC Class I Antigen Processing: Implications for Vaccine Design," Curr. Mol. Med. 1 :665 (2001), which is hereby incorporated by reference in its entirety). A major influence on the rate at which polyubiquitination occurs is the identity of the N-terminal residue of the target protein (Bachmair et al., "In Vivo Half- Life of a Protein is a Function of its Amino-Terminal Residue," Science 234:179 (1986), which is hereby incorporated by reference in its entirety). The "N end rule" relates the half-life of a protein to the identity of its N-terminal residue in that certain non-methionine residues, notably Arg or Lys, appearing at the amino-terminus will target a peptide for rapid degradation by the 26S proteasome (Narshavsky, "The Ν- End Rule: Functions, Mysteries, Uses," Proc. Νafl. Acad. Sci. USA 93:12142 (1996), which is hereby incorporated by reference in its entirety). This phenomenon has been utilized to enhance antigen-specific induction of CTL. For example, by fusing a peptide antigen of interest (i.e., "target peptide") at the C-terminus of ubiquitin in such a way as to place an arginine residue at the amino-terminus of the target peptide mediates enhanced degradation of the target peptide, which facilitates presentation of target peptide-derived peptides in association with MHC Class I. Thus, CTL responses against specific pathogens can be enhanced by fusion of proteins derived from those pathogens to ubiquitin.

[00010] It has also been shown in mice that intraperitoneal injection of a foreign protein fused to the protein transduction domain (PTD) from the human immunodeficiency virus (HIN) TAT protein results in delivery of the biologically active fusion protein to all tissues (Schwarze et al., "In Vivo Protein Transduction: Delivery of a Biologically Active Protein into the Mouse," Science 285:1569 (1999), which is hereby incorporated by reference in its entirety) and Ho et al., "Synthetic Protein Transduction Domains: Enhanced Transduction Potential In Vitro and In Nivo," Cancer Res. 61 :474 (2001), which are hereby incorporated by reference in its entirety). Thus, direct delivery of proteins into patients for immunotherapy is feasible. [00011] Here, a method of immunization is described involving administration of a purified recombinant fusion protein containing an epitope tag (e.g., six histidine residues, also known as a "6-His Tag") fused with a PTD sequence (e.g., HIN tat PTD; YGRKKRRQRRR) (SEQ. ID NO. 1), in turn fused with the coding sequence of human ubiquitin, in turn fused with a pathogen-derived immunogen. The method involves placement of an arginine or tyrosine codon at the amino-terminus of the protein target of interest.

[00012] Expression of this fusion protein in a recombinant expression system is followed by purification via utilization of the epitope tag, which results in the production and purification of a protein product that can be administered by a variety of routes, with or without an adjuvant. Fusion of the target peptide with a PTD sequence is designed to promote rapid dissemination and uptake into a variety of host cells, and once taken up, the fused ubiquitin moiety facilitates efficient proteasomal degradation, which in turn enhances presentation of pathogen-derived peptides in association with host MHC Class I. Enhanced systemic presentation of pathogen- derived peptides thus facilitates efficient immune activation and enhancement of antigen-specific CD4+ and CD8+ cellular immune.

[00013] Human Papillomavirus (HPN) Virus-like Particles (VLPs) are promising vaccine candidates for prophylaxis against anogenital HPV disease (Rose et al., "Human Papillomavirus Infections," in G. J. Galasso et al. (ed.), Antiviral Agents and Human Viral Diseases, pp. 343-368 (4 ed. 1997); Schiller et al., "Papillomavirus-Like Particle Vaccines," J. Νatl. Cancer Inst. Monogr. 28:50-4 (2001); and Schiller, "Papillomavirus-Like Particle Vaccines for Cervical Cancer," Molec. Med. Today 5:209-215 (1999), which are hereby incorporated by reference in their entirety). Applicant was among the first to show that VLPs form readily following recombinant expression of papillomavirus LI protein (Hagensee et al., "Self- Assembly of Human Papillomavirus Type 1 Capsids by Expression of the LI Protein Alone or by Coexpression of the LI and L2 Capsid Proteins," J. Virol. 67:315-322 (1993); Kirnbauer et al., "Papillomavirus LI Major Capsid Protein Self- Assembles into Virus-Like Particles That are Highly Immunogenic," Proc. Νat'l. Acad. Sci. USA 89:12180-84 (1992); Rose et al., "Expression of Human Papillomavirus Type 11 LI Protein in Insect Cells: In Vivo and In Vitro Assembly of Viruslike Particles," J. Virol. 67:1936-1944 (1993), which are hereby incorporated by reference in their entirety), and that they closely approximate the antigenic properties of native anogenital HPV virions (Rose et al., "Serological Differentiation of Human Papillomavirus Types 11, 16 and 18 Using Recombinant Virus-Like Particles," Gen. Virol. 75:2445-2449 (1994) and Rose et al., "Human Papillomavirus (HPV) Type 11 Recombinant Virus-like Particles Induce the Formation of Neutralizing Antibodies and Detect HPV-specific Antibodies in Human Sera," J. Gen. Virol. 75:2075-79 (1994), which are hereby incorporated by reference in their entirety). Applicant has also shown that VLP immunization elicits high-titer virion-neutralizing antibody responses (Rose et al., "Human Papillomavirus (HPV) Type 11 Recombinant Virus-like Particles Induce the Formation of Neutralizing Antibodies and Detect HPV-specific Antibodies in Human Sera," J. Gen. Virol. 75:2075-79 (1994) and White et al., "In Vitro Infection and Type-Restricted Antibody-Mediated Neutralization of Authentic Human Papillomavirus Type 16," J. Virol. 72:959-64 (1998), which are hereby incorporated by reference in their entirety), that have been correlated with protection from infectious challenge in relevant experimental animal models (Breitburd et al., "Immunization With Viruslike Particles From Cottontail Rabbit Papillomavirus (CRPV) Can Protect Against Experimental CRPV Infection," J. Virol. 69:3959-63 (1995); Kirnbauer et al., "Virus-Like Particles of Bovine Papillomavirus Type 4 in Prophylactic and Therapeutic Immunization," Virol. 219:37-44 (1996) and Suzich et al. "Systemic Immunization With Papillomavirus LI Protein Completely Prevents the Development of Viral Mucosal Papillomas," Proc. NatT. Acad. Sci. USA 92:11553-57 (1995), which are hereby incorporated by reference in their entirety). VLPs are now being evaluated in clinical studies as a parenteral vaccine modality and have been found to be safe, well-tolerated, and highly immunogenic in human subjects (Evans et al., "A Phase 1 Study of a Recombinant Viruslike Particle Vaccine Against Human Papillomavirus Type 11 in Healthy Adult Volunteers," J. Infect. Pis. 183:1485-93 (2001) and Harro et al., "Safety and Immunogenicity Trial in Adult Volunteers of a Human Papillomavirus 16 LI Virus- Like Particle Vaccine." J. NatT. Cancer Inst. 93:284-92 (2001), which are hereby incorporated by reference in their entirety). VLP mucosal immunogenicity has also been demonstrated (Balmelli et al., "Nasal Immunization of Mice With Human Papillomavirus Type 16 Virus-Like Particles Elicits Neutralizing Antibodies in Mucosal Secretions," J. Virol. 72:8220-29 (1998); Dupuy et al., "Nasal Immunization of Mice With Human Papillomavirus Type 16 (HPV-16) Virus-Like Particles or With the HPV-16 LI Gene Elicits Specific Cytotoxic T Lymphocytes in Vaginal Draining Lymph Nodes," J. Virol. 73:9063-71 (1999); and Rose et al., "Oral Vaccination of Mice With Human Papillomavirus Virus-Like Particles Induces Systemic Virus- Neutralizing Antibodies," Vaccine 17:2129-35 (1999), which are hereby incorporated by reference in their entirety). Applicant has shown, for example, that VLPs are immunogenic when administered orally and induce strong, durable responses in serum and genital mucosal secretions (Gerber et al., "Human Papillomavirus Virus- Like Particles are Efficient Oral Immunogens When Co-Administered With Escherichia Coli Heat-Labile Enterotoxin Mutant R192G or CpG DNA," J. Virol. 75:4752-60 (2001), which is hereby incorporated by reference in its entirety). [00014] VLPs immunogenicity may derive from an inherent ability to activate antigen-presenting cells. For example, addition of intact HPV-16 VLPs, but not intact polyomavirus VLPs or disordered HPV-16 capsomeres, was found to invoke acute maturation of bone marrow-derived dendritic cells, as judged by upregulation of proinflammatory cytokines IL-6 and TNF-alpha (Lenz et al., "Papillomavirus-Like Particles Induce Acute Activation of Dendritic Cell," J. Immunol. 166:5346-55 (2001), which is hereby incorporated by reference in its entirety). This ability has been confirmed in other studies in which HPV-16 L1/L2-E7 chimeric VLPs (cVLPs) were found to bind to human peripheral blood-derived dendritic cells, and to significantly upregulate CD80 and CD83, and to enhance secretion of IL-12 (Rudolf et al., "Human Dendritic Cells are Activated by Chimeric Human Papillomavirus Type-16 Virus-Like Particles and Induce Epitope-Specific Human T Cell Responses In Vitro," J. Immunol. 166:5917-24 (2001), which is hereby incorporated by reference in its entirety). Importantly, in that study human dendritic cells loaded with L1/L2-E7 cVLPs were found to induce an HLA-*0201-restricted human T cell response in vitro that was specific for E7-derived peptides (Rudolf et al., "Human Dendritic Cells are Activated by Chimeric Human Papillomavirus Type-16 Virus-Like Particles and Induce Epitope-Specific Human T Cell Responses In Vitro," J. Immunol. 166:5917- 24 (2001), which is hereby incorporated by reference in its entirety). Thus, immature human dendritic cells can be activated by VLPs, and can induce epitope-specific T cell responses.

[00015] VLPs can enter directly the Major Histocompatibility Complex Class I

(MHC Class I) antigen processing pathway (Bachmann et al., "Dendritic Cells Process Exogenous Viral Proteins and Virus-Like Particles for Class I Presentation to CD8+ Cytotoxic T Lymphocytes," Eυr. J. Immunol. 26:2595-600 (1996) and Gissmann et al., "Therapeutic Vaccines for Human Papillomaviruses," Intervirol. 44:167-75 (2001), which are hereby incorporated by reference in their entirety), and thus can be used to deliver heterologous antigens for induction of antigen-specific CTL (Kaufmann et al., "HPV16 L1E7 Chimeric Virus-Like Particles Induce Specific HLA-Restricted T Cells in Humans After In Vitro Vaccination," Int. J. Cancer 92:285-93 (2001) and Schafer et al., "Immune Response to Human Papillomavirus 16 L1E7 Chimeric Virus-Like Particles: Induction of Cytotoxic T Cells and Specific Tumor Protection," Int. J. Cancer 81 :881-88 (1999), which are hereby incorporated by reference in their entirety). HPV-16 E7 has been fused, for example, with carboxy- terminally truncated LI to form cVLPs that, when administered to mice, were found to induce E7-specific CTL (Kaufmann et al., "HP VI 6 L1E7 Chimeric Virus-Like Particles Induce Specific HLA-Restricted T Cells in Humans After In Vitro Vaccination," Int. J. Cancer 92:285-93 (2001); Muller et al., "Chimeric Papillomavirus-Like Particles," Virol. 234:93-111 (1997); and Schafer et al., "Immune Response to Human Papillomavirus 16 L1E7 Chimeric Virus-Like Particles: Induction of Cytotoxic T Cells and Specific Tumor Protection," Int. J. Cancer 81:881-88 (1999), which are hereby incorporated by reference in their entirety). Immunization with L1/E7 cVLPs was also found to inhibit the outgrowth of E7-expressing tumors in mice, and to induce HLA-restricted T cells in humans following vaccination in vitro (Kaufmann et al., "HPV16 L1E7 Chimeric Virus-Like Particles Induce Specific HLA-Restricted T Cells in Humans After In Vitro Vaccination," Int. J. Cancer 92:285-93 (2001); Muller et al., "Chimeric Papillomavirus-Like Particles," Virol. 234:93-111 (1997); and Schafer et al., "Immune Response to Human Papillomavirus 16 L1E7 Chimeric Virus-Like Particles: Induction of Cytotoxic T Cells and Specific Tumor Protection," Int. J. Cancer 81:881-88 (1999), which are hereby incorporated by reference in their entirety). Alternatively, as discussed above, cVLPs can be formed by co-expression of non-fused LI with HPV L2 fused in-frame with an antigen of interest (Greenstone et al., "Chimeric Papillomavirus Virus-Like Particles Elicit Antitumor Immunity Against the E7 Oncoprotein in an HPV16 Tumor Model," Proc. NatT. Acad. Sci. USA 95:1800-05 (1998), which is hereby incorporated by reference in its entirety). Co-expression of HPV-16 LI with HPV-16 L2/E7 fusion protein mediated formation of L1/L2-E7 cVLPs that were immunogenic in mice, and protected immunized mice from challenge with a tumor cell line constitutively expressing E7 (Greenstone et al., "Chimeric Papillomavirus Virus-Like Particles Elicit Antitumor Immunity Against the E7 Oncoprotein in an HP VI 6 Tumor Model," Proc. NatT. Acad. Sci. USA 95:1800-05 (1998), which is hereby incorporated by reference in its entirety). This effect was observed in MHC class II knockout mice, but not in β2-microglobulin or perform knockout mice, suggesting the induction of E7-specific MHC Class I- restricted CTL ((Greenstone et al., "Chimeric Papillomavirus Virus-Like Particles Elicit Antitumor Immunity Against the E7 Oncoprotein in an HPV16 Tumor Model," Proc. NatT. Acad. Sci. USA 95: 1800-05 (1998), which is hereby incorporated by reference in its entirety). Thus, cVLPs are potentially useful for therapy of HPV disease, and this is now being investigated (Schiller et al., "Papillomavirus-Like Particle Vaccines," J. NatT. Cancer Inst. Monogr. 28:50-54 (2001), which is hereby incorporated by reference in its entirety).

[00016] Targeting proteins into the Major Histocompatibility Complex (MHC)

Class I pathway via proteasomal degradation involves assembly of a polyubiquitin chain attached to an accessible lysine or arginine residue in the target protein (Sijts et al., "The Role of the Ubiquitin-Proteasome Pathway in MHC Class I Antigen Processing: Implications for Vaccine Design," Curr. Mol. Med. 1:665-76 (2001), which is hereby incorporated by reference in its entirety). A major influence on the rate at which polyubiquitination occurs is the identity of the N-terminal residue of the target protein (Bachmair et al., "In Vivo Half-Life of a Protein is a Function of its Amino-Terminal Residue," Science 234:179-86 (1986), which is hereby incorporated by reference in its entirety). The "N end rule" relates the half-life of a protein to the identity of its N-terminal residue in that certain non-mMethionine residues, notably Arg or Lys, appearing at the amino-terminus will target a peptide for rapid degradation by the 26S proteasome (Varshavsky, "The N-End Rule: Functions, Mysteries, Uses," Proc. NatT. Acad. Sci. USA 93:12142-49 (1996), which is hereby incorporated by reference in its entirety). This phenomenon has been utilized to enhance antigen-specific induction of CTL. For example, it was shown that HIV Nef fused with Ub and expressed in a vaccinia vector induced Nef-specific CTL responses, and mice immunized in this way were protected from lethal challenge with syngeneic tumor cells expressing Nef (Tobery et al., "Cutting Edge: Induction of Enhanced CTL-Dependent Protective Immunity In Vivo by N-End Rule Targeting of a Model Tumor Antigen," J. Immunol. 162:639-42 (1999), which is hereby incorporated by reference in its entirety). In that study, protection from challenge was found to correlate with the magnitude of the CTL response (Tobery et al., "Cutting Edge: Induction of Enhanced CTL-Dependent Protective Immunity In Vivo by N-End Rule Targeting of a Model Tumor Antigen," J. Immunol. 162:639-42 (1999), which is hereby incorporated by reference in its entirety). In another study, cotranslational ubiquitination combined with N end rule targeting was found to significantly reduce the half-life of Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1), which resulted in increased CTL activity against that antigen (Tellam et al., "Targeting of EBNA1 for Rapid Intracellular Degradation Overrides the Inhibitory Effects of the Gly-Ala Repeat Domain and Restores CD8+ T Cell Recognition," J. Biol. Chem. 276:33353-60 (2001), which is hereby incorporated by reference in its entirety). In addition, N end rule targeting was found to restore endogenous processing of HLA class I-restricted CTL epitopes within EBNA1 for immune recognition by human EBV-specific CTLs (Tellam et al., "Targeting of EBNA1 for Rapid Intracellular Degradation Overrides the Inhibitory Effects of the Gly-Ala Repeat Domain and Restores CD8+ T Cell Recognition," J. Biol. Chem. 276:33353-60 (2001), which is hereby incorporated by reference in its entirety). Other investigators have used N end rule targeting to generate DNA immunization vectors encoding Ub fused with antigens derived from M. tuberculosis and found in mice that such immunization was , associated with reduced growth of tubercle bacilli in lung and spleen following aerosol challenge (Delogu et al., "DNA Vaccine Combinations Expressing Either Tissue Plasminogen Activator Signal Sequence Fusion Proteins or Ubiquitin- Conjugated Antigens Induce Sustained Protective Immunity in a Mouse Model of Pulmonary Tuberculosis," Infect. Immun. 70:292-302 (2002), which is hereby incorporated by reference in its entirety). Thus, N end rule targeting can improve CTL responses in vivo, which suggests the potential utility of this strategy for enhancement of HPV-specific CTL activity.

DETAILED DESCRIPTION OF THE INVENTION

[00017] A first embodiment of the present invention relates to a fusion protein which comprises an epitope tag, a protein translocation domain, a ubiquitin domain, and a pathogen derived immunogen. The epitope tag, the protein translocation domain, the ubiquitin domain, and the pathogen derived immunogen are directly or indirectly coupled together. [00018] The epitope tag is one or more histidine tags.

[00019] The protein translocation domain can be HIV tat, interleukin-lβ, acid and basic fibroblast growth factors, angiogenin, homeoprotein Antennapedia,

Schwannoma derived growth factor, or Herpes Simplex Virus VP22 protein.

[00020] The ubiquitin domain is human ubiquitin.

[00021] The pathogen derived immunogen can be an HPV protein, an HIV protein, gplOO, MART-1, tyrosinase, MAGE-1, MAGE-2, MAGE-3, MAGE-3b,

MAGE-4, MAGE-4a, MAGE-4b, MAGE-5a, MAGE-5b, MAGE-6, MAGE-8,

MAGE-9, MAGE-10, MAGE-11, MAGE-41, MAGE-Xp, BAGE, N- acetylglucosaminyltransferase-N Intron, MUM-1, MUM- lb, MUM-lc, ErbB-2 (Her-

2/neu), CDK4, or prostate specific antigen.

[00022] In one embodiment, the pathogen derived immunogen is an HPN protein, including LI, L2, El, E2, E4, E5, E6, or E7.

[00023] In another embodiment, the pathogen derived immunogen is an HIV protein, such as Tat, Rev, Νef, Gag, Pol, Env, Vif, Vpr, and Vpu.

[00024] The fusion protein can further include intervening amino acids located between the protein translocation domain and the ubiquitin domain. The intervening amino acids may be arginine or tyrosine.

[00025] Another embodiment of the present invention is directed to a nucleic acid construct encoding a fusion protein. The nucleic acid construct comprises a nucleic acid molecule encoding an epitope tag, a nucleic acid molecule encoding a protein translocation domain, a nucleic acid molecule encoding a ubiquitin domain, and a nucleic acid molecule encoding a pathogen derived immunogen. The nucleic acid molecules encoding the epitope tag, the protein translocation domain, the ubiquitin domain, and the pathogen derived immunogen are directly or indirectly coupled together.

[00026] The nucleic acid construct can be used to prepare the fusion protein of the present invention by recombinant protein production techniques which are well known to those skilled in the art. Typically, this involves inserting the nucleic acid construct into any of the many available expression vectors and cell systems using reagents that are well known in the art. Suitable vectors include, but are not limited to, the following viral vectors such as baculo virus lambda vector system gtl 1, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/- (see "Sfratagene Cloning Systems" Catalog (1993) from Sfratagene, La Jolla, CA, which is hereby incorporated by reference in its entirety), pQE, pIH821, pGEX, pET series (see F.W. Studier et. al, "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes," Gene Expression Technology vol. 185 (1990), which is hereby incorporated by reference in its entirety), and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual. Second Edition, Cold Spring Harbor Press, NY (1989), and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., which are hereby incorporated by reference in their entirety. [00027] U.S. Patent No. 4,237,224 issued to Cohen and Boyer, which is hereby incorporated by reference in its entirety, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eukaryotic cells grown in tissue culture. [00028] Certain "control elements" or "regulatory sequences" are also incorporated into the vector-construct. These include non-translated regions of the vector, promoters, and 5' and 3' untranslated regions which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used.

[00029] A constitutive promoter is a promoter that directs expression of a gene throughout the development and life of an organism. Examples of some constitutive promoters that are widely used for inducing expression of transgenes include the nopaline synthase (NOS) gene promoter, from Agrobacterium tumefaciens (U.S. Patent No. 5,034,322 issued to Rogers et al., which is hereby incorporated by reference in its entirety), the cauliflower mosaic virus (CaMV) 35S and 19S promoters (U.S. Patent No. 5,352,605 issued to Fraley et al., which is hereby incorporated by reference in its entirety), those derived from any of the several actin genes, which are known to be expressed in most cells types (U.S. Patent No. 6,002,068 issued to Privalle et al., which is hereby incorporated by reference in its entirety), and the ubiquitin promoter, which is a gene product known to accumulate in many cell types.

[00030] An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer, the DNA sequences or genes will not be transcribed. The inducer can be a chemical agent, such as a metabolite, growth regulator, herbicide, or phenolic compound, or a physiological stress directly imposed upon the plant such as cold, heat, salt, toxins, or through the action of a pathogen or disease agent such as a virus or fungus.

[00031] The DNA construct of the present invention also includes an operable

3' regulatory region, selected from among those which are capable of providing correct transcription termination and polyadenylation of mRNA for expression in the host cell of choice, operably linked to a modified trait DNA molecule of the present invention. A number of 3 ' regulatory regions are known to be operable in plants. Exemplary 3' regulatory regions include, without limitation, the nopaline synthase ("nos") 3' regulatory region (Fraley, et al., "Expression of Bacterial Genes in Plant Cells," Proc. Nat'l Acad. Sci. USA 80:4803-07 (1983), which is hereby incorporated by reference in its entirety) and the cauliflower mosaic virus ("CaMV") 3' regulatory region (Odell, et al., "Identification of DNA Sequences Required for Activity of the Cauliflower Mosaic Virus 35S Promoter." Nature 313(6005) :810-12 (1985), which is hereby incorporated by reference in its entirety).

[00032] The different components described above can be ligated together to produce the expression systems which contain the DNA constructs of the present invention, using well known molecular cloning techniques as described in Sambrook et al., Molecular Cloning: A Laboratory Manual Second Edition, Cold Spring Harbor Press, NY (1989), and Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., which are hereby incorporated by reference in their entirety.

[00033] The DNA construct of the present invention is configured to encode

RNA molecules which are translatable. As a result, that RNA molecule will be translated at the ribosomes to produce the protein encoded by the DNA construct. Production of proteins in this manner can be increased by joining the cloned gene encoding the DNA construct of interest with synthetic double-stranded oligonucleotides which represent a viral regulatory sequence (i.e., a 5' untranslated sequence) (U.S. Patent No. 4,820,639 to Gehrke, and U.S. Patent No. 5,849,527 to Wilson, which are hereby incorporated by reference in their entirety). [00034] Once the DNA construct of the present invention has been prepared, it is ready to be incorporated into a host cell. Accordingly, another aspect of the present invention relates to a recombinant host cell containing one or more of the DNA constructs of the present invention. Basically, this method is carried out by transforming a host cell with a DNA construct of the present invention under conditions effective to yield transcription of the DNA molecule in the host cell, using standard cloning procedures known in the art, such as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Springs Laboratory, Cold Springs Harbor, New York (1989), which is hereby incorporated by reference in its entirety. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.

[00035] Preferably, transformed cells are first identified using a selection marker simultaneously introduced into the host cells along with the nucleic acid construct of the present invention. Suitable selection markers include, without limitation, markers encoding for antibiotic resistance, such as the nptll gene which confers kanamycin resistance (Fraley, et al., Proc. Natl. Acad. Sci. USA 80:4803-07 (1983), which is hereby incorporated by reference in its entirety), and the genes which confer resistance to gentamycin, G418, hygromycin, streptomycin, spectinomycin, tetracycline, chloramphenicol, and the like. Cells or tissues are grown on a selection medium containing the appropriate antibiotic, whereby generally only those transformants expressing the antibiotic resistance marker continue to grow. Other types of markers are also suitable for inclusion in the expression cassette of the present invention. For example, a gene encoding for herbicide tolerance, such as tolerance to sulfonylurea is useful, or the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J. 2:1099-1104 (1983), which is hereby incorporated by reference in its entirety). Similarly, "reporter genes," which encode for enzymes providing for production of an identifiable compound are suitable. The most widely used reporter gene for gene fusion experiments has been uidA, a gene from Escherichia coli that encodes the β-glucuronidase protein, also known as GUS. Jefferson et al., "GUS Fusions: β Glucuronidase as a Sensitive and Versatile Gene Fusion Marker in Higher Plants," EMBO J. 6:3901-07 (1987), which is hereby incorporated by reference in its entirety. Similarly, enzymes providing for production of a compound identifiable by luminescence, such as luciferase, are useful. The selection marker employed will depend on the target species; for certain target species, different antibiotics, herbicide, or biosynthesis selection markers are preferred.

[00036] Cells selected by means of an inhibitory agent or other selection marker are then tested for the acquisition of the viral gene by Southern blot hybridization analysis, using a probe specific to the viral genes contained in the given cassette used for transformation (Sambrook et al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor, New York: Cold Spring Harbor Press (1989), which is hereby incorporated by reference in its entirety).

[00037] An immunotherapeutic vaccine can be formulated from either the fusion protein or the nucleic acid construct. The vaccine further includes a carrier. [00038] -Another aspect of the present invention is directed to a method of vaccinating a subject against a pathogen by administering the fusion protein to the subject under conditions effective to vaccinate the subject against the pathogen is also disclosed.

[00039] Preferably, the pathogen being vaccinated against is either HPV or fflV.

[00040] Effective amounts of the fusion protein will depend upon the mode of administration, frequency of administration, nature of the treatment, age, and condition of the individual to be treated, and the type of pharmaceutical composition used to deliver the compound into a living system. Effective levels of the fusion protein may range from 50 nM to 5 μM (given to experimental animals as 20-30 mg/kg twice daily for ten days), depending upon the compound, system, experimental and clinical endpoints, and toxicity thresholds. While individual doses vary, optimal ranges of effective amounts may be determined by one of ordinary skill in the art. For fusion proteins that are involved in clinical trials for other indications, the safe and effective dosages identified in such trials can be considered when selecting dosages for treatments according to the present invention. [00041] The fusion protein used according to the methods of the present invention can be administered alone or as a pharmaceutical composition, which includes the compound(s) and a pharmaceutically-acceptable carrier. The fusion protein is typically provided as a pharmaceutical composition. The pharmaceutical composition can also include suitable excipients, or stabilizers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions. Typically, the composition will contain from about 0.01 to 99 percent, preferably from about 5 to 95 percent of active compound(s), together with the carrier. [00042] The fusion protein, when combined with pharmaceutically or physiologically acceptable carriers, excipients, or stabilizers, whether in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions, can be administered orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, mtraarterially, intralesionally, transdermally, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes (i.e., inhalation).

[00043] For most therapeutic purposes, the fusion protein can be administered orally as a solid or as a solution or suspension in liquid form, via injection as a solution or suspension in liquid form, or via inhalation of a nebulized solution or suspension. The solid unit dosage forms can be of the conventional type. The solid form can be a capsule, such as an ordinary gelatin type containing the compounds of the present invention and a carrier, for example, lubricants and inert fillers such as, lactose, sucrose, or cornstarch. In another embodiment, these compounds are tableted with conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, or gelatin, disintegrating agents, such as cornstarch, potato starch, or alginic acid, and a lubricant, like stearic acid or magnesium stearate.

[00044] For injectable dosages, solutions or suspensions of these materials can be prepared in a physiologically acceptable diluent with a pharmaceutical carrier.

Such carriers include sterile liquids, such as water and oils, with or without the addition of a surfactant and other pharmaceutically and physiologically acceptable carrier, including adjuvants, excipients, or stabilizers. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose, and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.

[00045] For use as aerosols, the compound in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The materials of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.

[00046] For transdermal routes, the compound is present in a carrier which forms a composition in the form of a cream, lotion, solution, and/or emulsion. The composition can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.

[00047] The present invention also relates to method of vaccinating a subject against a pathogen by administering the nucleic acid construct to the subject under conditions effective to vaccinate the subject against the pathogen.

[00048] This method is carried out by administering the nucleic acid molecule of the present invention using the previously-described modes.

[00049] Preferably, the pathogen being vaccinated against is either HPV or fflV.

[00050] The introduction of a particular foreign or native gene into a mammalian host is facilitated by first introducing the gene sequence into a suitable nucleic acid vector. "Vector" is used here to mean any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which is capable of transferring gene sequences between cells. Thus, the term includes cloning and expression vectors, as well as viral vectors. The nucleic acid molecules of the present invention maybe inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art.

[00051] Examples of viruses which have been employed as vectors for the transduction and expression of exogenous genes in mammalian cells include the SV40 virus (Innis et al., "Chromatin Structure of Simian Virus 40-pBR322 Recombinant Plasmids in COS-1 Cells," Mol. Cell Biol. 3(12):2203-10 (1983); Okaya a et al., "Bacteriophage Lambda Vector for Transducing a cDNA Clone Library into Mammalian Cells," Mol. Cell Biol. 5(5V.1136-42 (1985), which are hereby incorporated by reference in their entirety) and bovine papilloma virus (Meneguzzi et al., "Plasmidial Maintenance in Rodent Fibroblasts of a BPVl-ρBR322 Shuttle Vector Without Immediately Apparent Onco genie Transformation of the Recipient Cells," EMBOJL 3(2):365-71 (1984); DiMaio et al., "Bovine Papillomavirus Vector that Propagates as a Plasmid in Both Mouse and Bacterial Cells," Proc. NatT. Acad. Sci. USA 79(13):4030-34 (1982); Lusky et al., "Characterization of the Bovine Papilloma Virus Plasmid Maintenance Sequences," Cell 36(2) :391-401 (1984); Giri et al., "Comparative Studies of the Expression of Linked Escherichia coli gpt Gene and BPV-1 DNAs in Transfected Cells," Virol. 127(2):385-96 (1983), which are hereby incorporated by reference in their entirety), the retrovirus Moloney murine sarcoma virus (Perkins et al., "Design of a Retrovirus-Derived Vector for Expression and Transduction of Exogenous Genes in Mammalian Cells," Mol. Cell Biol. 3(6):1123- 32 (1983); Lee et al., "DNA Clone of Avian Fujinami Sarcoma Virus with Temperature-Sensitive Transforming Function in Mammalian Cells," J. Virol. 44(1):401-12 (1982); Curran et al., "FBJ Murine Osteosarcoma Virus: Identification and Molecular Cloning of Biologically Active Proviral DNA," J. Virol. 44(2):674-82 (1982); Gazit et al., "Mammalian Cell Transformation by a Murine Retrovirus Vector Containing the Avian Erythroblastosis Virus erbB Gene," J. Virol. 60(1): 19-28 (1986), which are hereby incorporated by reference in their entirety), and HIV-based viruses.

[00052] A number of adenovirus (Ad) based gene delivery systems have also been developed. Human adenoviruses are double-stranded DNA viruses which enter cells by receptor-mediated endocytosis. These viruses are particularly well suited for gene therapy, because they are easy to grow and manipulate and they exhibit a broad host range in vivo. Adenovirus is easily produced at high titers and is stable so that it can be purified and stored. Even in the replication-competent form, adenoviruses generally cause only low level morbidity and are not associated with human malignancies. Furthermore, Ad infects both dividing and non-dividing cells; a number of tissues which are targets for gene therapy comprise largely non-dividing cells (U.S. Patent No. 6,171,855 to Askari, which is hereby incorporated by reference in its entirety). For descriptions of various adenovirus-based gene delivery systems, see, e.g., Haj-Ahmad et al., "Development of a Helper-Independent Human Adenovirus Vector and Its Use in the Transfer of the Herpes Simplex Virus Thymidine Kinase Gene," J. Virol. 57(l):267-74 (1986); Bett et al., "Packaging Capacity and Stability of Human Adenovirus Type 5 Vectors," J. Virol. 67(10):5911- 21 (1993); Mittereder et al., "Evaluation of the Efficacy and Safety of in vitro, Adeno virus-Mediated Transfer of the Human Cystic Fibrosis Transmembrane Conductance Regulator cDNA." Hum. Gene Ther. 5(6):717-29 (1994); Seth et al., "Mechanism of Enhancement of DNA Expression Consequent to Cointernalization of a Replication-Deficient Adenovirus and Unmodified Plasmid DNA," J. Virol. 68(2):933-40 (1994); Barr et al., "Efficient Catheter-Mediated Gene Transfer into the Heart Using Replication-Defective Adenovirus," Gene Ther. l(l):51-8 (1994); Berkner et al., "Development of Adenovirus Vectors for the Expression of Heterologous Genes," Biotechniques 6(7):616-29 (1988); Rich et al., "Development and Analysis of Recombinant Adenoviruses for Gene Therapy of Cystic Fibrosis," Hum. Gene Ther. 4(4) :461-76 (1993), which are hereby incorporated by reference in their entirety.

[00053] Retroviral vectors, capable of integration into the cellular chromosome, have also been used for the identification of developmentally important genes via insertional mutagenesis (see, e.g., U.S. Patent No. 6,207,455 to Chang, which is hereby incorporated by reference in its entirety). Retroviral vectors are also used in therapeutic applications (e.g., gene therapy), in which a gene (or genes) is added to a cell to replace a missing or defective gene or to inactivate a pathogen such as a virus. The members of the family Retro viridae are characterized by the presence of reverse transcriptase in their virions (U.S. Patent No. 6,207,344 to Chang, which is hereby incorporated by reference in its entirety). The family is divided into three subfamilies: (1) Oncovirinae, including all the oncogenic retroviruses, and several closely related non-oncogenic viruses; (2) Lentivirinae, the "slow retroviruses," discussed in greater detail below, and (3) Spumavirinae, the "foamy" retroviruses that induce persistent infections, generally without causing any clinical disease (U.S. Patent No. 6,218,181 to Verma et al., which is hereby incorporated by reference in its entirety). Some of the retroviruses are oncogenic (i.e., tumorigenic), while others are not. The oncoviruses induce sarcomas, leukemias, lymphomas, and mammary carcinomas in susceptible species (U.S. Patent No. 6,033,905 to Wilson et al., which is hereby incorporated by reference in its entirety). Retroviruses infect a wide variety of species, and may be transmitted both horizontally and vertically. They are integrated into the host DNA, and are capable of transmitting sequences of host DNA from cell to cell. This has led to the development of retroviruses as vectors for various purposes including gene therapy. For example, the majority of the approved gene transfer trials in the United States rely on replication-defective retroviral vectors harboring a therapeutic polynucleotide sequence as part of the retroviral genome (Miller et al., "Gene Transfer by Retrovirus Vectors Occurs Only in Cells that are Actively Replicating At The Time of Infection," Mol. Cell Biol. 10(8):4239-4442 (1990); Cornetta et al., "No Retroviremia or Pathology in Long-term Follow-up of Monkeys Exposed to Amphotropic Retrovirus," Hum. Gene Ther. 2(3):215-19 (1991), which are hereby incorporated by reference in their entirety). As is known in the art, the major advantages of retroviral vectors for gene therapy are the high efficiency of gene transfer into certain types of replicating cells, the precise integration of the transferred genes into cellular DNA, and the lack of further spread of the sequences after gene transfer (U.S. Patent No. 6,033,905 to Wilson et al., which is hereby incorporated by reference in its entirety).

[00054] As used herein, the term "lentivirus" refers to a group (or genus) of retroviruses that give rise to slowly developing disease. Viruses included within this group include HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2), the etiologic agent of the human acquired immunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, the caprine arthritis-encephalitis virus, which causes immune deficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia, and encephalopathy in horses; feline immunodeficiency virus (FIN), which causes immune deficiency in cats; bovine immune deficiency virus (BIN), which causes lymphadenopathy, lymphocytosis, and possibly central nervous system infection in cattle; and simian immunodeficiency virus (SIN), which cause immune deficiency and encephalopathy in sub-human primates. Diseases caused by these viruses are characterized by a long incubation period and protracted course. Usually, the viruses latently infect monocytes and macrophages, from which they spread to other cells. HIN, FIN, and SIN also readily infect T lymphocytes (i.e., T-cells). Lentivirus virions have bar-shaped nucleoids and contain genomes that are larger than other retroviruses. Lentiviruses use tRΝA^lys as primer for negative-strand synthesis, rather than the tRNA^pro commonly used by other infectious mammalian retroviruses. The lentiviral genomes exhibit homology with each other, but not with other retroviruses (Davis et al., Microbiology, 4th ed., J.B. Lippincott Co., Philadelphia, Pa., pp. 1123-51 (1990), which is hereby incorporated by reference in its entirety). An important factor in the disease caused by these viruses is the high mutability of the viral genome, which results in the production of mutants capable of evading the host immune response. The advantage of lentiviruses is the ability for sustained transgene expression. Thus, in one embodiment of the present invention, a lentiviral vector is employed to provide long-term expression of the neurotrophic transgene in a target cell.

[00055] Adeno-associated viruses (AAV) may also be employed as a vector in the present invention. AAV is a small, single-stranded (ss) DNA virus with a simple genomic organization (4.7 kb) that makes it an ideal substrate for genetic engineering. Two open reading frames encode a series of rep and cap polypeptides. Rep polypeptides (rep78, rep68, rep62, and rep40) are involved in replication, rescue, and integration of the AAV genome. The cap proteins (VP1, VP2, and VP3) form the virion capsid. Flanking the rep and cap open reading frames at the 5' and 3' ends are 145 bp inverted terminal repeats (ITRs), the first 125 bp of which are capable of forming Y- or T-shaped duplex structures. Of importance for the development of AAV vectors, the entire rep and cap domains can be excised and replaced with a therapeutic or reporter transgene (B. J. Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155-168 (1990), which is hereby incorporated by reference in its entirety). It has been shown that the ITRs represent the minimal sequence required for replication, rescue, packaging, and integration of the AAV genome (U.S. Patent No. 5,871,982 to Wilson et al., which is hereby incorporated by reference in its entirety).

[00056] As noted above, viral vectors have been successfully employed in order to increase the efficiency of introducing a recombinant vector into suitably sensitive host cells. Therefore, viral vectors are particularly suited for use in the present invention, including any adenoviral (Ad), retroviral, lentiviral, or adeno- associated viral (AAV) vectors described above or known in the art. Current research in the field of viral vectors is producing improved viral vectors with high-titer and high-efficiency of transduction in mammalian cells (see, e.g., U.S. Patent No. 6,218,187 to Finer et al., which is hereby incorporated by reference in its entirety). Such vectors are suitable in the present invention, as is any viral vector that comprises a combination of desirable elements derived from one or more of the viral vectors described herein. It is not intended that the expression vector be limited to a particular viral vector.

[00057] Certain "control elements" or "regulatory sequences" are also incorporated into the vector-construct. The term "control elements" refers collectively to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these control elements need always be present so long as the selected coding sequence is capable of being replicated, transcribed, and translated in an appropriate host cell. Some of these control elements have been described above. [00058] Following transfection of an appropriate host with the viral vector of the present invention, the virus is propagated in the host and collected. Generally, this involves collecting the cell supernatants at periodic intervals, and purifying the viral plaques from the crude lysate, using techniques well-known in the art, for example, cesium chloride density gradient. The titer (pfu/ l) of the virus is determined, and can be adjusted up (by filtration, for example), or down (by dilution with an appropriate buffer/medium), as needed. In the present invention, typical Ad titers are in the range of 10¹⁰-10¹² pfu/ml.

[00059] "Subject" is meant herein to include any member of the class

Mammalia including, without limitation, humans and nonhuman primates, such as chimpanzees and other apes and monkey species; farm animals including cattle, sheep, pigs, goats and horses; domestic animals including cats and dogs; laboratory animals including rodents such as mice rats, and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adults and post-natal (newborn) subjects, as well as fetuses, are intended to be covered.

[00060] The recombinant viruses of the present invention may be administered to a subject, using the formulations and modes previously described. [00061] The recombinant viruses of this invention may be administered in sufficient amounts to transfect the desired cells and provide sufficient levels of integration and expression of the selected transgene to provide a therapeutic benefit without undue adverse effects or with medically acceptable physiological effects which can be determined by those skilled in the medical arts. Conventional and pharmaceutically acceptable parenteral routes of administration include direct delivery to the target organ, tissue, or site, are encompassed by the present invention. [00062] Dosages of the recombinant virus will depend primarily on factors, such as the condition being treated, the selected fusion protein, the age, weight, and health of the patient, and may thus vary among patients. A therapeutically effective human dosage of the viruses of the present invention is believed to be in the range of about 5 ml of saline solution containing concentrations of from about 2.5 X 10 pfu/ml to 2.5 X 10¹² pfu/ml virus of the present invention. The dosage will be adjusted to balance the therapeutic benefit against any side effects. The levels of expression of the selected gene can be monitored to determine the selection, adjustment, or frequency of dosage administration. EXAMPLES

Example 1 - Production of Hybrid VLPs (hVLPs).

[00063] Methods that are well established will be used to generate recombinant baculoviruses that express HPN-16 L1/L2-E6, L1/L2-E7, and L1/L2-E2 proteins for assembly of hybrid NLPs. Complete genomic clones of several HPN genotypes are available (i.e., types 6, 11, 16, 18, 31, 33, 35, 45, and 58). HPN-16 early region coding sequences, which will be the initial focus shall be fused at the C-terminus of HPN-16 L2 by PCR. Briefly, L2 will be modified by replacement of the normal stop codon with an Eco RI site, which will be used to accept HPN-16 early region gene sequences amplified by PCR using primers containing Eco RI sites. Fusion sequences will be constructed in a baculovirus transfer vector (pNL-1392/1393; kindly provided by V. Luckow and M. Summers; Texas A&M University, College Station, Tx) as previously described (Rose et al., "Expression of Human Papillomavirus Type 11 LI Protein in Insect Cells: In Vivo and In Vitro Assembly of Viruslike Particles," L Virol. 67:1936-44 (1993) and Rose et al., "Expression of the Full-Length Products of the Human Papillomavirus Type 6b (HPV-6b) and HPV-11 L2 Open Reading Frames by Recombinant Baculovirus, and -Antigenic Comparisons With HPV-11 Whole Virus Particles," J. Gen. Virol. 71:2725-29 (1990), which are hereby incorporated by reference in their entirety). Constructs will be used to generate recombinant baculoviruses by co-transfection of Spodoptera frugiperda (Sf-9) cells (ATCC) along with purified baculovirus DΝA (Baculogold™, Pharmingen, Carlsbad, CA). Following incubation at 27°C for 72-96 hours, cell culture supernatants containing recombinants will be passaged in fresh Sf-9 cells for amplification followed by plaque-purification. Purified recombinant isolates will be used to infect Trichoplusia ni (T. ni) cells (the kind gift of Dr. Robert Granados, Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, Y) to select for optimal expression by Western blot immunoassay. Optimally expressing strains will then be used in co- infection experiments along with an HPV-16 LI baculovirus, which is already available (Rose et al., "Serological Differentiation of Human Papillomavirus Types 11, 16 and 18 Using Recombinant Virus-Like Particles," J. Gen. Virol. 75:2445-49 (1994), which is hereby incorporated by reference in its entirety), to generate hybrid VLPs. For control purposes, viral early region proteins will also be expressed in bacteria as His-tagged fusion proteins (FPs). Purified His-tagged FPs will be administered to control mice to assess baseline immunogenicity against which relative immunogenicities of novel proposed immunogens will be assessed. Purified His- tagged FPs will also be used for pre/post-immune stimulation of lymphocytes in vitro.

Example 2 - Construction of Ubiquitin (Ub)/HPV Fusion Vectors.

[00064] To complement this work with hybrid VLPs, Ubiquitin (Ub) fusion constructs containing E6 (Ub/E6), E7 (Ub/E7), and E2 (Ub/E2) will be constructed for direct immunization, and for use in studies involving co-administration of E6, E7 and/or E2 constructs with hybrid VLPs (i.e., for VLP -mediated gene delivery). The full-length Ub gene will be amplified by polymerase chain reaction (PCR) from genomic DNA extracted from a spontaneously immortalized epidermal keratinocyte cell line (i.e., HaCaT cells, obtained from N. Fusenig, (Boukamp et al., "Normal Keratinization in a Spontaneously Immortalized Aneuploid Human Keratinocyte Cell Line," J. Cell Biol. 106:761-71 (1988), which is hereby incorporated by reference in its entirety). In these experiments, Ub/HPV fusion vectors will be constructed with stabilizing Methionine (M) or de-stabilizing Arginine (R) at the N-terminus of the HPV sequence, essentially as described (Tobery et al., "Targeting of HIV-1 Antigens for Rapid Intracellular Degradation Enhances Cytotoxic T Lymphocyte (CTL) Recognition and the Induction of De Novo CTL Responses In Vivo After Immunization," J. Exp. Med. 185:909-20 (1997), which is hereby incorporated by reference in its entirety) to generate a total of six constructs (i.e., UbME6, UbRE6, UbME7, UbRE7, UbME2, and UbRE2). Before administration in mice, constructs will be tested for expression in cell culture via standard transfection protocols (i.e., FuGene and Lipofectamine 2000) and by hVLP-mediated gene delivery, followed by Western blot immunoassay with HPV-specific reagents.

Example 3 - Construction of Novel Multipartite Fusion Proteins.

[00065] Ubiquitin fusion proteins will also be modified by insertion of a protein translocation domain (PTD) derived from HIV tat. His-tagged tat PTD/Ubiquitin/HPV E region constructs will be expressed in E. coli and purified via the His epitope tag.

Example 4 - Immunogenicity Testing.

[00066] For immunogenicity testing, applicant will utilize female BALB/c and

C57BL/6 mice. While much of the work will be carried out as described, these designs are provided as examples to illustrate the types of experiments and procedures envisioned to be required to achieve the stated objectives. [00067] Initial experiments will be designed to determine relative immunogenicities of HPV-L1/L2-E6, /L2-E7 and /L2-E2 hybrid VLPs when administered by parenteral injection, alone or in combination, at a standard dose level (i.e., 0.3 μg) (Gerber et al., "Human Papillomavirus Virus-Like Particles are Efficient Oral Immunogens When Co-Administered With Escherichia Coli Heat-Labile Enterotoxin Mutant R192G or CpG DNA," J. Virol. 75:4752-60 (2001), which is hereby incorporated by reference in its entirety). Groups often mice will be used throughout to ensure adequate statistical power for interpretation. Applicant previously determined that primary and booster immunizations by intramuscular injection of 0.3 μg of purified HPV-16 or HPV-18 LI VLPs, without adjuvant, was adequate for induction of high-titer serum neutralizing antibody responses in BALB/c mice (Gerber et al., "Human Papillomavirus Virus-Like Particles are Efficient Oral Immunogens When Co-Administered With Escherichia Coli Heat-Labile Enterotoxin Mutant R192G or CpG DNA," J. Virol. 75:4752-60 (2001), which is hereby incorporated by reference in its entirety). Applicant will here use ELISPOT and ICS assays to characterize the ability of L1/L2-E6 VLPs to elicit E6-specific CD4+/CD8+ responses, using purified recombinant E6 protein for immunostimulation in vitro. Peripheral blood mononuclear cells (PBMC) recovered from mice immunized with either native or hybrid (i.e., E6-containing) L1/L2 VLPs will be simulated in vitro with purified recombinant E6 protein and cytokine responses will be evaluated in ELISPOT/ICS assays.

[00068] Applicant's previous work demonstrates minimum oral dose levels of

VLPs and LT(R192G) to be 5 μg each. Previously, it was found that CpG DNA also significantly enhanced VLP oral immunogenicity (Gerber et al., "Human Papillomavirus Virus-Like Particles are Efficient Oral Immunogens When Co- Administered With Escherichia Coli Heat-Labile Enterotoxin Mutant R192G or CpG DNA," J. Virol. 75:4752-60 (2001), which is hereby incorporated by reference in its entirety). It is expected that CpG DNA will be particularly useful in the proposed studies as it has a demonstrated ability to activate innate immune mechanisms, and to enhance T helper type 1 (Thl) cytokine profiles, which are recognized as important for induction of antigen-specific CTL. Before further evaluation of this potentially useful adjuvant for oral immunization, it will be necessary to perform a dose-ranging study to determine whether it is possible to obtain a similar effect by administering a lower dose level. In this and all other experiments involving oral immunization, immunogen(s) and adjuvants will be administered by gavage, as previously described (Gerber et al., "Human Papillomavirus Virus-Like Particles are Efficient Oral Immunogens When Co- Administered With Escherichia Coli Heat-Labile Enterotoxin Mutant R192G or CpG DNA," J. Virol. 75:4752-60 (2001), which is hereby incorporated by reference in its entirety). Experimental results from 2A1 may indicate that levels of antibody responses and CTL activity are comparable when hNLPs are administered parenterally either alone or in combination; the same experimental design will be employed for evaluation of hNLP oral immunogenicity. As above, cytokine responses generated after stimulation of peripheral blood mononuclear cells will be evaluated in ELISPOT/ICS assays using purified recombinant versions of the same proteins as antigens for in vitro stimulation. [00069] The relative immunogenicities of hNLPs will be compared when administered parenterally without adjuvant vs orally with LT(R192G). Systemic and mucosal humoral and cellular responses will be evaluated as previously described (Gerber et al., "Human Papillomavirus Virus-Like Particles are Efficient Oral Immunogens When Co- Administered With Escherichia Coli Heat-Labile Enterotoxin Mutant R192G or CpG DNA," J. Virol. 75:4752-60 (2001), which is hereby incorporated by reference in its entirety).

[00070] Antigen-specific immune responses can be modulated by co- administration of specific immunomodulatory factors (Moore et al., "Effects of -Antigen and Genetic Adjuvants on Immune Responses to Human Immunodeficiency Virus DNA Vaccines in Mice," J. Virol. 76:243-50 (2002), which is hereby incorporated by reference in its entirety). This possibility will be investigated by co- administration of DNA plasmids that encode murine cytokine and/or hematopoietic growth factors (e.g., Interleukin-12 (IL-12) and/or Granulocyte-Macrophage Colony- Stimulating Factor (GM-CSF)). Co-administration of these genes has been found to alter immune responses, and antigen-specific effects have been noted (Moore et al., "Effects of Antigen and Genetic Adjuvants on Immune Responses to Human Immunodeficiency Virus DNA Vaccines in Mice," J. Virol. 76:243-50 (2002), which is hereby incorporated by reference in its entirety). For example, IL-12 is able to enhance IFN-γ and immunoglobulin G (IgG) responses to Nef, whereas GM-CSF is more active with regard to Env-specific IFN-γ and CTL responses. These immunomodulatory factors will be co-administered with VLPs containing HPV early region gene products (L2 fusion proteins and/or Ubiquitin expression constructs) and in this way assess whether it may be possible to preferentially induce a Thl response.

Example 5 - Ub Fusion Constructs. [00071] Ub DNA fusion vectors will be evaluated similarly utilizing parenteral and topical (e.g., transdermal and/or vaginal) routes of delivery. Optimization of dose level, route of administration, adjuvant and/or cytokine use will be performed as outlined above and will be guided by results from those experiments. Relative ' magnitudes of antigen-specific T cell specificities induced after immunization with hVLP or corresponding UB DNA fusion vector will be assessed. [00072] Experiments similar to those outlined above will be performed to assess potential for synergistic enhancement of specific CTL activity by co- administration of hVLP/UB DNA constructs (i.e., VLP -mediated delivery of UB constructs). Dose, route, and adjuvant/cytokine effects will also be evaluated. [00073] Potential efficacies of prime/boost strategies will be assessed that will involve primary immunizations with DNA constructs followed by boosting with hVLPs (and vice versa). [00074] Novel fusion proteins consisting of an epitope tag (i.e., 6-Histidine; for purification purposes), HIV Tat PTD (to mediate rapid uptake in vivo), human Ubiquitin, and an HPV early region sequence of interest will be expressed in E. coli. Following purification, these novel immunogens will be administered in mice by intravenous and intraperitoneal routes. Given the well-documented ability of HIV Tat PTD to mediate rapid systemic dissemination and uptake of fused proteins in a variety of tissue types, this approach is expected to mediate rapid systemic distribution, uptake and processing of PTD/Ubiquitin/HPV fusion proteins, and thus may mediate induction of robust MHC class I-restricted responses against specific HPV early region gene products. Control comparisons will be performed using the same HPV early region sequences expressed without Tat PTD and/or Ubiquitin to provide a benchmark for assessment of CTL induction.

Example 6 - Measurement of IFN-γ Production by ELISPOT.

[00075] Briefly, the ELISPOT assay for detection of gamma interferon (IFN-γ) release is performed as follows. Nitrocellulose-backed 96-well plates (Millipore) are coated overnight at 4°C with 0.1 ml/well of murine IFN-γ-specific monoclonal antibody (R4; ATCC). Plates are washed with PBS and blocked (lhr; room temperature) with medium supplemented with 10% fetal calf serum. Two dilutions (5x10⁵ and 2.5x10⁵) of freshly isolated splenocytes and 2 μg/ml specific peptide are added to wells, and plates are incubated overnight (37°C; 5% CO ). Cells incubated without peptide provide a negative control. Following this, cells are again washed with PBS, and secondary antibody biotin conjugate is added (XMG1.2; 1 μg/ml (Pharmingen)), and plates are incubated 3 hours at room temperature. Wells are again washed with PBS and Alkaline phosphatase-conjugated streptavidin (Sigma) is added (1 : 1,000 dilution; 1 hour). Wells are again washed with PBS and spots are developed by adding substrate (DAB; Sigma). Following 15 minutes of incubation, wells are washed with water, dried, and spots are counted under a dissection microscope.

Example 7 - Intracellular Cytokine Staining.

[00076] CTL activity induction will be assessed using a modified intracellular cytokine staining (ICS) assay as previously described (Jin et al., "Safety and Immunogenicity of ALVAC vCP1452 and Recombinant gpl60 in Newly Human Immunodeficiency Virus Type 1 -Infected Patients Treated With Prolonged Highly Active Antiretroviral Therapy," J. Virol. 76:2206-16 (2002), which is hereby incorporated by reference in its entirety). Briefly, aliquots of 1 x 10⁶ lymphocytes obtained from either spleen or lymph nodes of mice will be stimulated with recombinant immunogens generated for this purpose for 6 or 20 hours (dependent upon which time frame provides optimal CTL stimulation), 10 μg/ml of Brefeldin A (Golgiplug™, PharMingen, San Diego, CA) will be added during the last five hours of stimulation. Cells are then stained with anti-CD3PE, anti-CD4APC, and anti- CDδPerCP (Becton-Dickson, San Jose, CA) antibodies for 30 minutes at 4°C. After washing, cells are permeabilized with CytoFix/Cytoperm solution (PharMingen, San Diego, CA), then stained intracellularly with an anti-IFN-γ-FITC antibody (PharMingen, San Diego CA) before fluorescence-activated cell sorting (FACS) and analysis with CellQuest software (Becton-Dickinson, San Jose, CA). Results are expressed as the percentage of CD8+ T cells producing IFN-γ. In all experiments, staphylococcal enterotoxin B (SEB) will serve as a positive control.

Example 8 - Tumor Challenge Studies.

[00077] Relative abilities of the various proposed immunogens to enhance host immune capabilities will be evaluated in a murine in vivo challenge model. C57BL/6 and BALB/c mice will be immunized with the most promising immunogen formulations (to be determined in vitro; see above), and then challenged with tumor cell lines expressing target antigens. Line 1 cells (Brown et al., "Tumours Can Act as Adjuvants for Humoral Immunity," Immυn. 102:486-97 (2001), which is hereby incorporated by reference in its entirety) are H-2D, and therefore are compatible with BALB/c mice, whereas B16 cells (H-2B) are compatible with C57BL/6 mice. Both lines are well-characterized and suitable for this purpose.

[00078] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED:

1. A fusion protein comprising: an epitope tag; a protein translocation domain; a ubiquitin domain; and a pathogen derived immunogen, wherein the epitope tag, the protein translocation domain, the ubiquitin domain, and the pathogen derived immunogen are directly or indirectly coupled together.

2. The fusion protein according to claim 1, wherein the epitope tag is one or more histidine tags.

3. The fusion protein according to claim 1 , wherein the protein translocation domain is selected from the group consisting of HIV tat, interleukin-lβ, acid and basic fibroblast growth factors, angiogenin, homeoiprotem Antennapedia, Schwannoma derived growth factor, and Herpes Simplex Virus VP22 protein.

4. The fusion protein according to claim 1, wherein the ubiquitin domain is human ubiquitin.

5. The fusion protein according to claim 1, wherein the pathogen derived immunogen is selected from the group consisting of an HPV protein, an HIV protein, gplOO, MART-1, tyrosinase, MAGE-1, MAGE-2, MAGE-3, MAGE-3b, MAGE-4, MAGE-4a, MAGE-4b, MAGE-5a, MAGE-5b, MAGE-6, MAGE-8, MAGE-9, MAGE-10, MAGE-11, MAGE-41, MAGE-Xp, BAGE, N- acetylglucosaminyltransferase-V Intron, MUM-1, MUM- lb, MUM-lc, ErbB-2 (Her- 2/neu), CDK4, and prostate specific antigen.

6. The fusion protein according to claim 5, wherein the pathogen derived immunogen is an HPV protein selected from the group consisting of LI, L2, El, E2, E4, E5, E6, and E7.

7. The fusion protein according to claim 5, wherein the pathogen derived immunogen is an HIV protein selected from the group consisting of Tat, Rev, Nef, Gag, Pol, Env, Vif, Vpr, and Vpu.

8. The fusion protein according to claim 1 further comprising: one or more amino acids located between the protein translocation domain and the ubiquitin domain.

9. The fusion protein according to claim 8, wherein the amino acids are arginine or tyrosine.

10. An immunotherapeutic vaccine comprising: the fusion protein according to claim 1 and a carrier.

11. A nucleic acid construct encoding a fusion protein, wherein the nucleic acid construct comprising: a nucleic acid molecule encoding an epitope tag; a nucleic acid molecule encoding a protein translocation domain; a nucleic acid molecule encoding a ubiquitin domain; and a nucleic acid molecule encoding a pathogen derived immunogen, wherein the nucleic acid molecules encoding the epitope tag, the protein translocation domain, the ubiquitin domain, and the pathogen derived immunogen are directly or indirectly coupled together.

12. The nucleic acid construct according to claim 11, wherein the epitope tag is one or more histidine tags.

13. The nucleic acid construct according to claim 11 , wherein the protein translocation domain is selected from the group consisting of of HIV tat, interleukin- lβ, acid and basic fibroblast growth factors, angiogenin, homeopvotew. Antennapedia, Schwannoma derived growth factor, and Herpes Simplex Virus VP22 protein.

14. The nucleic acid construct according to claim 11 , wherein the ubiquitin domain is human ubiquitin.

15. The nucleic acid construct according to claim 11, wherein the pathogen derived immunogen is selected from the group consisting of an HPV protein, an HIV protein, gplOO, MART-1, tyrosinase, MAGE-1, MAGE-2, MAGE-3, MAGE-3b, MAGE-4, MAGE-4a, MAGE-4b, MAGE-5a, MAGE-5b, MAGE-6, MAGE-8, MAGE-9, MAGE-10, MAGE-11, MAGE-41, MAGE-Xp, BAGE, N- acetylglucosaminyltransferase-V Intron, MUM-1, MUM- lb, MUM-lc, ErbB-2 (Her- 2/neu), CDK4, and prostate specific antigen.

16. The nucleic acid construct according to claim 15, wherein the pathogen derived immunogen is an HIV protein selected from the group consisting of Tat, Rev, Nef, Gag, Pol, Env, Vif, Vpr, and Vpu.

17. The nucleic acid construct according to claim 15, wherein the pathogen derived immunogen is an HPV protein selected from the group consisting of LI, L2, El, E2, E4, E5, E6, and E7.

18. The nucleic acid construct according to claim 11 further comprising: an intervening nucleic acid molecule encoding one or more amino acids and located between the nucleic acid encoding the protein translocation domain and the nucleic acid molecule encoding the ubiquitin domain.

19. The nucleic acid construct according to claim 18, wherein the one or more amino acids are arginine or tyrosine.

20. An immunotherapeutic vaccine comprising: the nucleic acid construct according to claim 11 and a carrier.

21. A method of vaccinating a subj ect against a pathogen, said method comprising: administering the fusion protein according to claim 1 to the subject under conditions effective to vaccinate the subject against the pathogen.

22. The method according to claim 21 , wherein said administering is carried out by parenteral, mucosal, or transdermal routes.

23. The method according to claim 21, wherein the pathogen is selected from the group consisting of HPV and HIV.

24. A method of vaccinating a subject against a pathogen, said method comprising: administering the nucleic acid construct according to claim 11 to the subject under conditions effective to vaccinate the subject against the pathogen.

25. The method according to claim 24, wherein said administering is carried out by parenteral, mucosal, or transdermal routes.

26. The method according to claim 24, wherein the pathogen is selected from the group consisting of HPV and HIV. _;

27. The method according to claim 24, the nucleic acid construct is in a viral vector.

28. The method according to claim 27, wherein the viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an adeno-associated viral vector.

29. A viral vector comprising the nucleic acid construct according to claim 11.

30. The viral vector according to claim 29, wherein the viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an adeno-associated viral vector.