MXPA00001299A - Immune responses against hpv antigens elicited by compositions comprising an hpv antigen and a stress protein or an expression vector capable of expression of these proteins - Google Patents

Abstract

The present invention relates to compositions for inducing an immune response, preferably a cellular, in particular a cell-mediated, cytolytic immune response, to human papillomavirus (HPV) protein antigens displayed by HPV or exhibited by infected cells including cells from cervical and other tumors. In one embodiment, compositions comprise an HPV protein antigen joined to a stress protein (or heat shock protein (Hsp)). The HPV protein antigen may be joined to the stress protein by chemical conjugation or noncovalently using linking moieties, or the HPV protein antigen and the stress protein may be joined in a fusion protein containing both HPV protein antigen and stress protein sequences. In another embodiment, compositions comprise an expression vector including, in expressible form, sequences encoding the HPV protein antigen and sequences encoding the stress protein. The expression vector can be introduced into cells of a subject, or it can be used to transduce cells of the subject ex vivo, resulting in the expression of an HPV protein antigen-stress protein fusion protein that will stimulate the subject's immune response to the HPV protein antigen. The present invention also relates to compositions comprising a stress protein linked to an HPV antigen and another pharmacologically acceptable component, to stress protein-HPV protein antigen fusions and conjugates and to expression vectors encoding and capable of directing the expression in a subject's cells of a fusion protein comprising a stress protein and an HPV protein antigen sequence. The present invention also relates to uses of these compositions to induce immune responses against HPV and HPV protein antigen-exhibiting cells including HPV-associated tumors.

Description

ANSWERS I N MU N IS ANTIGENS OF H PV PRODUCI DAS BY COMPOSITION IS UNDERSTANDING AN ANTIGEN OF H PV AND U NA TENSION PROTEIN OR A VECTOR OF EXPRESSION CAPABLE OF EXPRESSION OF THESE PROTEINS CAM PO TÉCN ICO The present invention relates generally to methods and compositions involving bound voltage proteins and human papillomavirus protein antigens to induce an immune response against human papillomavirus protein antigens.

BACKGROUND OF THE INVENTION Infection with human papillomaviruses (HPV) is common, and viruses can be transmitted sexually. It is estimated that between 20 and 80% of sexually active adults are infected. Although most infections are asymptomatic, the infection can lead to the development of genital warts and cancer of the anogenital tract. Genital warts have a frequency of 1 -5% among adults. Approximately one percent of women around the world are afflicted with cervical cancer, which is the most common cause of death in women under the age of 50. Cervical cancer is strongly associated with HPV. Frazer, Genitourin Med 72: 398-403 (1996). At present, no effective therapeutic composition or prophylactic composition, ie, vaccines, is available against H PV, and consequently, there is a need to develop effective compositions. Prospects for a conventional killed or live attenuated vaccine appear to be poor. According to Frazer, HPV has not yet been propagated in cell culture, and the tumor-promoting effects of HPV infection, as well as the specific species specificity of HPV represent additional difficulties that can not easily be overcome (Frazer, Genitourin Med 72: 398-403 (1996)). It has been proposed that the observation that the larger capsid protein, when expressed in eukaryotic cells, forms virus-like particles, which are immunogenic without helper, can provide a basis for the development of a vaccine (Christensen et al., J Gen Virol 75: 2271 -6 (1 994), see also PCT / EP95 / 03974 and PCT / US95 / 1 2914). H PV belongs to genus A of the papovaviridae family, which also includes SV40 and polyomavirus. More than 68 different types of H PV have been characterized which are highly structurally related, but less than 50% identical to the DNA sequence level. All known types are epitheliotropic viruses that infect specific types of epithelium and frequently produce epithelial proliferations. Several types were identified in common warts. It is known that twenty-three types infect the anogenital tracts female and male. The anogenital diseases caused by these types of HPV vary from Condylomata acuminata to invasive squamous cell carcinoma. HPV DNA can be identified in over 80% of women with cervical intraepithelial neoplasia or squamous intraepithelial lesions confirmed by biopsy. U how many particular types, including H PV 1 6, 1 8 and 31, are strongly associated with ^ ^ ^ ^^^^^^^^^^^^^^^ high-grade squamous intraepithelial lesions and invasive cancer of the cervix, vulva, penis and anus (Loríncz et al., Obstet Gynecol 79: 328-37 (1992)). According to Frazer, cervical cancer is 90-95% associated with HPV. Frazer, Genitourin Med 72: 398-403 (1996). HPV is not only associated with cancer of the anogenital system, but is also present in pharyngeal, laryngeal and bladder carcinomas (Brachman et al., Cancer Res 52: 4832-6 (1992): Rotóla et al., Int J Cancer 52 : 359-65 (1992)). A recent study reported that HPV DNA was also present in 30% of the lung carcinomas tested. The identified types included HPV 6, 11, 16, 18, 31 and 33 (Soini et al., Thorax 51: 887-893 (1996)). Therefore, HPV types very often associated with cancer are 6, 11, 16, 18, 31 and 33, of which HPV 16 and 18, which are detected in more than 90% of cervical carcinomas (van Driel et al., Ann Med 28: 471-477 (1996)), have been investigated very deeply. Papillomaviruses are DNA viruses that have a circular, double-stranded DNA genome of 7800 to 7900 base pairs, an unwrapped virion and an icosahedral capsid made of 72 capsomeres. The genome contains three major regions, one coding for late genes, one coding for early genes and one non-coding region (Park et al., Cancer 76: 1902-1913 (1995)). The non-coding region is also referred to as an upstream regulatory region. This region is approximately 400 base pairs long and contains an array of binding sites for the various transcription factors that control the expression of early and late genes. The region of genes ,. Finally, it has two separate open reading frames that encode viral capsid proteins L1 and L2. The L 1 protein is the major capsid protein that is highly conserved among different HPV species. The early gene region includes six open reading frames, designated E 1, E2, E4, E5, E6 and E7. The E6 and E7 proteins are critical oncoproteins for viral replication, as well as for the immortalization and transformation of host cells. The E1, E2 and E4 proteins also play an important role in the replication of viruses. In addition, E4 works in the maturation of the virus. The role of E5 is less known. Malignant tumor cells share two important growth characteristics. They are immortalized, that is, they do not age, and they are capable of growth independent of anchoring. The introduction of HPV 1 6 or HPV 1 8 DNA into immortalized rodent cells results in their transformation, that is, they acquire the ability to grow in the absence of substrate binding and the ability to form tumors when injected into mice (Crook). et al., Proc. Nati, Acad. Sci USA 85: 8820-24 (1988)). A different result is obtained when the HPV DNAs are introduced into non-immortalized, early-passage cells: the cells become immortalized, but are not transformed (Woodworth et al., Cancer Res. 48: 4620-28 (1988)). ). Thus, a pathway through which tumors develop involves a change that results in the immortalization of cells, followed by the expression of H PV genes, resulting in their transformation. H PV genes involved in the transformation of cells in vitro are those that - > "% & £ -. 4 &'.? acSC * - & - ^ - ^ \ - ^ - > afc. & tSm encode E6 and / or E7 (Bedell et al., J. Virol 61: 3635-40 (1987).) Mechanisms have been proposed by which proteins E6 and E7 can induce cell transformation (Park et al., Cancer 76. 1 902-1 91 3 (1 995), and references cited in the same.) E6 is a small polypeptide (approximately 1, 5,000 molecular weight) containing Zn binding domains.A clue for its transforming function was provided by the observation that the protein binds p53.P53 protein is a protein well-known tumor suppressor that regulates in a negative way the progression of the cell cycle and, consequently, the growth and cell division The binding of E6 to p53 results in the ubiquination and eventual deg radation of the last protein, whose process involves another cellular protein called "protein associated with E6." Consequently, the cells that express E6 will have a level of reduced salt of p53.P53 levels are elevated in response to DNA damage. Such increased levels result in enhanced expression of p21, an inhibitor of cyclin-dependent kinases, whose protein mediates cell cycle arrest. This mechanism provides cells with a window of time within which damaged DNA can be repaired before replication, which would result in the establishment of damage / manipulation. E6-mediated P6-mediated disturbance of p53 can prevent the mechanism from operating. Recently, it was also found that E6 not only affects the regulation of the cell cycle by virtue of accelerating the degradation of p53, but also, more directly, by blocking p53 from the interaction with DNA (Thomas et al., Oncogene 10: 261 -8 (1995)).

? Effffs-_fe The E7 protein is a small Zn-binding phosphoprotein (approximately 10,000 molecular weight) capable of binding the retinoblastoma gene product Rb. Rb is a tumor suppressor that binds to and inactivates the transcription factor E2F. The last factor controls the transcription of a number of genes related to growth, including those that encode thymidine kinase, c-myc, dihydrofolate reductase and alpha DNA polymerase. The formation of the Rb-E2F complex prevents the expression of the last genes in the GO and G 1 phases, restricting its expression to the S phase, where the Rb-E2F complexes are programmed to dissociate, releasing the active transcription factor E2F. The formation of Rb-E7 complexes prevents the formation of Rb-E2F complexes with the result of the shortening of the pre-S phases, that is, accelerating the progression through the cell cycle. Correlative evidence for the importance of these mechanisms is provided by observations that E6 proteins of highly oncogenic H PV types (eg, H PV 16 &; 1 8) have greater affinities for p53 than corresponding proteins of non-oncogenic types, and that E7 proteins of highly oncogenic types have higher affinities for Rb than corresponding proteins of non-oncogenic types. In a majority of cervical cancers and precursor lesions, the HPV DNA is integrated into the host cell genome (Cullen et al., J. Virol. 65: 606-1 1 2 (1 991)). It seems that in most cases, the integration involves the breakdown of genomic DNA of H PV in the E1 / E2 region, leaving the E6 / E7 region intact. A consequence of the break in the region E 1 / E2 is an interruption of the reading frame -f ^ W open that encodes two different E2 proteins, the smallest of which functions as a transcriptional repressor of the expression of early genes. This leads to an over-regulation of the expression of E6 and E7.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to compositions for inducing an immune response against a H PV protein antigen in a subject to which they are administered. The immune response may be a humoral or cellular response, in particular, a cytolytic, cell-mediated response to an H PV protein antigen. The compositions can be used prophylactically or therapeutically. In a prophylactic application, the induction of an immune response refers to the production of immune reactions on a very low background of inherent immunity. In a therapeutic application, the induction of an immune response in a subject refers to the generation of responses that exceed, either in magnitude or quality, the responses previously produced by contact with H PV protein antigens exhibited either by the virus or by infected or transformed cells of the subject. In particular embodiments, the compositions are used to generate immune responses to tumor cells that express and exhibit an H PV protein antigen. In these embodiments, the preferred HPV protein antigens targeted by the compositions are the early viral proteins E6 and E7, which are known to be consistently expressed in tumors associated with H PV. In one embodiment, the compositions comprise an HPV protein antigen linked to a strain protein (or heat shock protein (Hsp)). The HV protein antigen can bind to the stress protein by chemical conjugation, or antigen and strain protein can bind at the 5 nucleotide level, allowing the expression and isolation of a fusion protein containing both antigen and protein sequences. tensile . The compositions can be introduced into a subject or used ex vivo to stimulate and / or cause the expansion of target immune cells of the subject or target mediating HPV or cells that include tumor cells that exhibit an HPV protein antigen. The compositions are effective to stimulate an immune response when admistered as non-particulate proteinaceous solutions (eg, not as part of a virus or virus-like particle) in the absence of adjuvant. In another embodiment, the compositions comprise an expression vector including nucleic acid sequences encoding an HPV protein antigen and a strain protein. The expression vector further comprises sequence elements that direct the transcription and translation of the coding sequences and also may include elements that facilitate the delivery to, and persistence or amplification of, nucleic acids in cells of a subject. The expression vector can be introduced into cells of a subject, or it can be used to transduce a subject's cells ex vivo, resulting in the expression of an H protein protein antigen fusion protein PV-protein. tension, which will induce an immune response against the H PV protein antigen. The present invention also relates to compositions comprising a protein antigen bound to a strain protein, in combination with another pharmacologically acceptable component. In one embodiment, the composition comprises a conjugate that comprises a tension protein or an HPV protein antigen. In another modality, the composition comprises a fusion protein (eg, proteins expressed from pET65HE6 and pET65H E7), in which a strain protein is fused to an HPV protein antigen. The conjugates and fusion proteins of these compounds are also claimed as being expression vectors that encode, and are capable of directing the expression of, a fusion protein comprising a voltage protein and an HV protein antigen sequence in some cells of subject. The present invention also relates to uses of the compositions for enhancing immune responses against H PV protein antigens and, in particular embodiments, against tumors that exhibit an HPV protein antigen. The articles of the scientific literature and patent applications cited herein, especially those relating to the preparation and use of compositions of the invention, are incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic representation of the PET65H construct. Figure 2 is a schematic representation of the PET65H E6 construct. Fig. 3 is a schematic representation of the construct PET65H E7. FIG. 4 is a graph of the percentage of tumor incidence versus time after the TC-1 tumor challenge, demonstrating successful immunization of mice with Hsp65-E6 and -E7 fusion proteins against the tumor. Figure 5 is a graph of the specific percentage of cell lysis of TC-1 tumor cells versus the proportion of effector for target cells, demonstrating a cell-mediated cytolytic response against target tumor cells expressing H (E7) antigen. PV in immobilized mice. Figure 6 is a graph of the percentage of lysis of different target cells versus the proportion of effector for target cells, demonstrating the specificity of the cytolytic immune response mediated by cells against target tumor cells expressing E7 of H PV in immunized mice. Figure 7 is a graph of tumor incidence percentage in mice administered with 1.3 × 0.05 of TC-1 tumor cells and subsequently injected with saline, fusion protein.

Hsp65-E7 in saline or E7 peptide in saline / I FA versus days after administration of tumor cells. ___________________ * Figure 8 is a graph of tumor incidence percentage in mice administered with 2 × 10 5 TC-1 tumor cells and subsequently injected with saline, Hsp65-E7 fusion protein in saline or E7 peptide in saline / IFA versus days after the administration of tumor cells. Figure 9 is a schematic representation of construct pET / E7 (H). Figure 10 is a collection of graphs showing the incorporation of thymidine by cultured lymph node cells obtained from mice immunized with the fusion protein Hsp65-E7 or E7 protein. Figure 11 is a graph showing the effect of treatment with the Hsp-E7 or E7 fusion protein on tumor size in mice bearing TC-1 tumor cells. Figure 12 is a schematic representation of construct pET65C. Figure 13 is a schematic representation of the construct pET65C-E7-1N.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to compositions that induce an immune response in a human papillomavirus subject of subject cells by displaying a protein antigen of an HPV. In one embodiment, the compositions comprise an HPV protein antigen and a strain protein. In another embodiment, the compositions comprise a . «, .. expression vector capable of directing the expression of an HPV-protein protein antigen fusion protein. The compositions of the present invention can be used prophylactically to elevate immunity against an HPV protein antigen, preventing the establishment and proliferation of HPV or cells from a subject by expressing and displaying the H PV protein antigen or presenting portions of the same. The compositions can also be used therapeutically in a subject previously infected with an HVP to prevent further viral proliferation, or to eliminate subject cells that proliferate as a consequence of H PV infection, including tumors that express and exhibit an H antigen. PV or present a portion of the antigen. When an HPV protein antigen is referred to herein as an objective of an immune response induced by a composition of the present invention, it is understood that the HPV protein antigen includes a whole HPV protein or a portion of polypeptide (molecular weight greater than 10 kDa) of the HPV protein displayed on the surface of HPV, or an infected cell of a subject, as well as peptide displayed by an infected cell, as a result of processing and presentation of the H PV protein, for example, through the normal MHC class I or I I routes. The genomic sequences of many different types of HPV were cloned and characterized by DNA sequence analysis. Bacterial vectors containing complete or partial H PV genomes are available from several sources including, for example, American Tissue Culture Collection (ATCC). Additional types of HPV ..- caabas. useful for the practice of the present invention, they can be isolated and typed by methods previously established for this purpose, said methods are well known in the art. HPV expresses six or seven non-structural and two structural proteins, and each of these proteins could, in principle, serve as a target for immunoprophylactic or immunotherapeutic approaches aimed at eliminating H PV and / or infected cells. The viral capsid proteins L 1 and L 2 are the structural proteins of HPV, which are encoded by late genes. L1 is the capsid protein greater that is highly conserved among different species of H PV. The seven non-structural proteins are products of the early viral genes. The E1, E2 and #r proteins play an important role in the replication of viruses. The E4 protein functions additionally in the maturation of the virus. The role of E5 is less known. Proteins E6 and E7 are critical oncoproteins for viral replication, as well as for immortalization and transformation of host cells. These proteins, which can be incorporated in the compositions of the present invention, are referred to as HPV protein antigens. Of particular importance in the application of the present invention for the prophylactic and therapeutic treatment of cancers associated with HPV is the observation that HPV E6 and E7 proteins are consistently expressed in cervical cancers (Zur Hausen, Appl. Pathol 5: 1-9- 24 (1987); Pater and Pater, Virology 145: 31 3-8 (1 985)). Konoshita et al. , Br. J. Cancer 71: 344-9 (1,995)) have shown that the E6 and E7 genes are also expressed in lung carcinoma. Moreover, some The natural (humoral) immune response to E7 was noted in cervical cancer patients (Jochmus-Kudielka et al., J. Nat'l Cancer Inst. 81: 1 698-704 (1989)). Finally, model studies show that immunization with a modified E7 protein protects mice against a challenge with lung cells transformed with an activated c-Ha-ras gene and H6 E6 / E7 genes (Lin et al., Cancer Res. 56:21 -26 (1 996)). From the viewpoints that these proteins are normally expressed in cancers that arise as a consequence of H PV infection, that the same proteins are also the oncogenes, which most likely played a major role in the development and maintenance of the cancers, and that an immune response can be directed against these proteins, E6 and E7 are preferred targets for inm an intervention or prophylaxis, and, therefore, are preferred H-protein antigens of compounds of the present invention. to be used to prevent or treat cancer associated with H PV. It has been shown in several animal models that peptides from cytotoxic T cells (CTL) can induce protective immunity against certain viruses (Kast and Melief, Immunol.Lett.30: 229 (1 991)). It has been observed that immunosuppressed individuals more frequently develop cervical carcinoma than immunocompetent individuals (Schneider et al., Acta Cytologica 27: 220-4 (1983)). This strongly suggests that the cellular arm of the immune system, particularly the T cell system, is of greater importance in the immunological defense against malignancy associated with HPV. Evidence of support for the importance of a CTL response to produce immunity ^^ a ^ i ^^^^^ protective against cells transformed with E6 and E7, came from an experiment in which mice vaccinated with a CTL epitope relevant to HPV 16 E7 were protected against transplantable HPV 16 induced tumors (Feltkamp et al., Eur. J. Immunol., 23: 2242 (1993)). The present invention is based on our observation that the binding of a tension protein to an HPV protein antigen results in a composition that strongly stimulates cellular responses, in particular, cell-mediated cytolytic responses, against the linked HPV protein antigen, said responses can kill cells exhibiting the HPV antigen. An HPV protein antigen of the present invention can be any polypeptide encoded with HPV. In addition, it may be a portion of an HPV protein, provided that the portion, when bound with a tension protein, retains the ability to induce an immune response against the HPV protein antigen displayed by infected cells, or shown by HPV. . Compositions of the invention comprising several portions of an HPV antigen instead of a complete HPV protein antigen can be produced by routine methods, such as those described below or in textbooks of biochemistry and molecular biology. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (1989); Deutxcher, M., Guide to Protein Purification Methods Enzymology (Guide for Protein Purification Methods, Enzymology) vol. 182, Academic Press, Inc., San Diego, CA (1990). Each composition that has a particular portion of an HPV protein antigen can be "Tested for the degree and quality of the immune response against the HPV protein antigen in experiments, such as those described in the examples described below. Minimally, a H PV protein antigen in a composition of the present invention will contain at least one B or T cell epitope (e.g., a CTL or an auxiliary cell epitope T) of an HPV protein. When referring to compositions of the present invention, the term "H PV protein antigen" includes portions of an HPV protein antigen, provided that such portions retain the ability to elicit an immune response against the protein antigen of HPV. H PV exhibited by cells infected or shown by HPV. E6 and particularly E7 are transforming proteins. In compositions that include as the HPV protein antigen, an E6 or E7 protein sequence of HVP, or a portion sufficiently complete to retain the transformation capacity, the transforming nature of the antigen may or may not represent a substantial risk, depending of the method by which the H PV antigen is manufactured. For example, in cases in which a H PV protein antigen or a composition that includes such an antigen is prepared by recombinant techniques that carry a risk of DNA contamination, it may be prudent to attempt steps to eliminate the transforming ability of the antigen. . When compositions are used that include an expression vector that directs the expression in a subject of a fusion protein including an E6 or E7 complete protein sequence of H PV or a sufficiently complete portion to retain the transforming capacity, it may be necessary to eliminate sequences that make the transformation of protein product. The non-transforming variants of E6 and E7 were obtained by fusing sequences of E6 and E7 (PCT / AU95 / 00868). Consequently, it is possible that certain fusion proteins, including E6 or E7 sequences and voltage protein sequences, may no longer be transformative. Alternatively, the sequences can be selectively deleted from E6 or E7 using techniques that are well known in the art, and which have also been described in PCT / AU95 / 00868. Deletions can be made in expression vectors, which express E6 or E7 sequences alone or in vectors expressing fusion proteins of E6- or E7-tension proteins. The results of such manipulations can be assessed by testing the transforming capacity of suppression proteins in a transfection experiment. For example, cells of N I H3T3 can be transfected with an expression vector including a gene for a deletion protein, and transforming capacity can be estimated by colony formation assay on soft agar. This particular test is also described in PCT / AU95 / 00868. In one embodiment of the present invention, the compositions are comprised of two portions: a tension protein and an HPV protein antigen against which a cellular immune response is desired. The two portions can be connected by conjugation, that is, through a covalent link between the tension protein and the HPV protein antigen. Hermanson, G .T. , Bioconjugate Techniques, Academic Press, I nc. , San Diego, CA (1996); Lussow, A. R. et al. , Eur. J. Immun. . j «WI < .aatelA-21: 2297-2302 (1 991); Barrios, C. et al. , Eur. J. Immun. 22: 1 365-1372 (1992)). Alternatively, recombinant techniques can be used to connect and express the two portions, said techniques result in a recombinant fusion protein, which includes the tension protein and the H PV protein antigen in a single molecule. This makes it possible to produce and purify a simple recombinant molecule in the production process. The two portions can also be attached non-covalently. Any of several known high affinity interactions can be adapted to connect the two portions non-covalently. For example, a group of biotin can be added to an HVP protein antigen, and the strain protein to be linked can be expressed as an avidin-strain protein fusion protein. The avid ina-strain protein fusion protein will bind strongly to the biotinylated H PV protein antigen. Analogously, portions of the H PV protein antigens may go to a full voltage protein or portions of the strain protein, and portions of a strain protein may bind to a complete HPV protein antigen, or portions of the HPV protein antigen, provided that the respective portions are sufficient to induce an immune response against the H PV protein antigen in a subject to whom it is admired. In another embodiment, the compositions comprise an expression vector capable of directing the expression of a fusion protein of HV protein antigen-tension protein. -s ^ Swl ^. i-e, - Any suitable tension protein (heat shock protein (hsp)) can be used in the compositions of the present invention. For example, as described in the examples, Hsp60 and / or Hsp70 can be used. Returning to stress proteins, in general, the 5 cells respond to a tensioner (usually heat shock treatment) by increasing the expression of a group of genes commonly referred to as tension or heat shock genes. Heat shock treatment involves exposure of cells or organisms at temperatures that are one to several degrees Celsius above the temperature to which the cells adapt. In coordination with the induction of such genes, the corresponding voltage protein levels increase in stressed cells. As used herein, a "tension protein", also known as a "heat shock protein" or "Hsp", is a protein that is encoded by a voltage gene, and consequently it is normally produced in significantly greater amounts over the contact or exposure of the tensioner for the organism. A "voltage gene", also known as a "heat shock gene" is used herein as a gene that is activated or over-regulated detectably otherwise. to contact or exposure of an organism (containing the gene) to a tensioner, such as heat shock, hypoxia, glucose deprivation, heavy metal salts, inhibitors of energy metabolism and electron transport, and protein denaturants , or certain benzoquinone ansamycins. Nover, L., Heat Shock Response, CRC Press, Inc., Boca Raton, FL (1 991). The "voltage gene" also includes bs & a ^ t. homologous genes within families of known voltage genes, such as, certain genes within the families of tension genes Hsp70 and Hsp90, even when such homologous genes are not induced by themselves by a tensioner. Each of the tense voltage and tension protein gene terms, as used in the present specification, may be included in the other, unless the context indicates otherwise. In particular embodiments, for example, in cases involving chemical conjugates between a strain protein and an HPV protein antigen, the strain proteins used in the present invention are isolated strain proteins, which means that the proteins of Stress have been selected and separated from the host cell in which they were produced. Such isolation can be performed as described herein and using routine protein isolation methods known in the art. Maniatis et al. , Molecular Cloning, A Laboratory Manual (Molecular Cloning, a laboratory manual), Cold Springer Harbor Laboratory, Cold Spring Harbor, N.Y. (1 982); Sambrook et al. , Molecular Cloning: A Laboratory Manual (Molecular Cloning: A Laboratory Manual), 2nd ed. , Cold Spring Harbor Laboratory Press (1,989); Deutscher, M. , Guide to Protein Purification Methos Enzymology, vol. 1 82, Academic Pres, I nc. , San Diego, CA (1990). In bacteria, the predominant stress proteins are proteins with molecular sizes of approximately 70 to 60 kDa, respectively, which are commonly referred to as Hsp70 and Hsp60, respectively. These and other specific tension proteins and the genes encoding them are discussed further below. In bacteria, Hsp70 and Hsp60 normally represent approximately 1-3% of the cellular protein based on the staining pattern using sodium dodecyl sulfate polyacrylamide gel electrophoresis and the Coomassie blue stain, but accumulates at levels as high as 25% under tension conditions. Tension proteins seem to participate in important cellular processes, such as protein synthesis, intracellular transport and assembly and disassembly of protein complexes. It appears that the increased amounts of stress proteins synthesized during stress serve mainly to minimize the consequences of protein cleavage. Actually, the pre-exposure of the cells to gently tensioning conditions that induce the synthesis of tension proteins, provides protection to the cells from the damaging effects of a subsequent, more extreme tension. The main tension proteins appear to be expressed in each organism and tissue type examined so far. In addition, it appears that strain proteins represent the most highly conserved group of proteins identified to date. For example, when comparing stress proteins in widely differing organisms, Hsp90 and Hsp70 exh iben 50% or greater identity at the level of amino acids and share many simulations in non-identical positions. It is noted that similar or greater levels of homology exist between different members of a particular strain protein family within the species.

The genes that encode stress proteins can be present in a single copy or in multiple copies, not identical in the genome of a cell or organism. For example, it has been shown that the human genome contains at least one copy of a hsp l OO gene, at least two different hsp90 genes, up to ten hsp70 genes, of which at least several are non-identical copies, several T-complex genes (Tcp genes) and at least one gene encoding the mitochondrial related HspdO protein, as well as at least three copies of small hsp genes encoding Hsps in the 20-30 kDa molecular size range. In most families of tension genes, there is at least one gene whose level of expression is relatively high and is either completely constitutive or only inducible with mild heat shock. Additionably, various families of tension genes include members that are not over-regulated by heat but by other signals, such as, increased calcium levels, etc. Tension proteins, particularly Hsp70, Hsp60, Hsp20-30 and Hsp1 0, are among the main determinants recognized by the host immune system in the immune response for infection by Mycobacterium tuberculosis and Mycobacterium leprae. Young, R.A. and Elliott, T.J. , Streess Proteins, Infection and Immune Surveillance (Proteins of tension, infection and immune survival), Cell 50: 5-8 (1 989). Additionally, some rat arthritogenic T cells recognize epitopes of hsp60. Van Eden, W. et al. , Nature 331: 1 71-1,373 (1,988). However, individuals, including healthy individuals, with no history of mycobacterial infection or autoimmune disease also carry T cells that recognize both bacterial and human Hsp60 epitopes; a considerable fraction of T cells in healthy individuals that are characterized by expression of the gamma-delta T cell receptor, recognize both their own and foreign tension proteins. O'Brien, R. et al. , cell 57: 664-674 (1989). Thus, individuals, even healthy individuals, possess populations of T cells that recognize both epitopes of their own and extraneous stress proteins. This system, which recognizes epitopes of tension proteins, presumably constitutes an "early defense system" against invading organisms. M urray, P.J. and Young, R.A. , J. Bacteriol 1 74: 41 93-6 (1992). The system can be maintained by frequent stimulation by bacteria and viruses. As discussed earlier, healthy individuals have T cell populations that recognize their own tension proteins. Thus, the presence of self-reactive T cells is compatible with normal health and does not cause autoimmune diseases; this demonstrates the safety of tension proteins within an individual. The safety of tension proteins is further demonstrated by the success and relative safety of BCG vaccinations (Bacille Calmette Guerin, a strain of Mycobacterium bovis), which induce an immune response against tension proteins, which is also protective against Mycobacterium tuberculosis. . The families of the genes and tension proteins for use in the present invention are those well known in the art and include, for example, Hsp 1 00-200, Hspl OO, Hsp90, Lon, Hsp70, Hsp60, TF55, Hsp40, FKBPs , cyclophilins, Hsp20-30, CIpP, GrpE, Hspl O, ubiquitin, calnexin and disulfide protein isomerases. Macario, A.J. L., Cold Spring Harbor Laboratory Res. 25: 59-70, 1995; Parsell, D.A. & Lindquist, S. Ann. Rev. Genet. 27: 437-496 (1992); US patent no. 5.232, 833 (Sanders et al.). A particular group of tension proteins includes 5 Hsp90, Hsp70, Hsp60, Hsp20-30, in addition preferably Hsp70 and Hsp60. Examples of Hsp1 00-200 include Grp1 70 (for protein regulated with glucose). Grp1 70 resides in the ER membrane, in the pre-golgi compartment, and can play a role in the folding and assembly of immunoglobulins. 10 Examples of Hsp1 00 include Hsp1 1 0 of mammal, Yeast Hsp1 04, CIpA, CIpB, CIpC, CIpX and CIpY. Hsp 1 04 of yeast and CIpA of E. coli, form hexameric and teramética particles of CI pB of E. coli, whose assembly seems to require the nucleotide binding of adenine. Clp protease provides a 750 kDa heteroligomer composed of dpP 15 (a proteolytic subunit) and of CIpA. CIpB-Y are structurally related to CIpA, although unlike CIpA they do not appear to be complex with dpP. Examples of Hsp90 include HtpG in E. coli, Hsp83 and Hsc83 in yeast and Hsp90alpha, Hsp? Obeta and Grp94 in humans. Hsp90 binds protein groups 20, such proteins are usually cellular regulatory molecules, such as, steroid hormone receptors (e.g., glucocorticoid, estrogen, progesterone and testosterone receptors), transcription factors and protein kinases that play a role in the Signal transduction mechanisms. The Hsp90 proteins also iVda áSíßt m '*. .. > i- »a * ¿aii? .f. they participate in the formation of large and abundant protein complexes that include other tension proteins. Lon is a tetrameric protein that functions as an ATP-dependent protease that tastes unnatural proteins in E. coli. Examples of Hsp70 include Hsp72 and Hsc73 from mammalian cells, DnaK of bacteria, particularly mycobacteria such as Mycobacterium leprae, Mycobacterium tuberculosis and Mycobacterium bovis (such as, Bacille-Calmette Guerin: referred to herein as Hsp71), DnaK of Escherichia coli, yeast and other prokaryotes, and Bi P and Grp78 . Hsp70 is capable of specifically binding ATP, as well as split polypeptides and peptides, thereby participating in the folding and unfolding of proteins, as well as in the assembly and disassembly of protein complexes. Examples of Hsp60 include Hsp65 from mycobacteria. Bacterial Hsp60 is also commonly known as GroEL, such as, the GroEL from E. coli. Hsp60 forms large homo-oligomeric complexes and seems to play a key role in protein folding. Homologs of Hsp60 are present in chloroplasts and eukaryotic mitochondria. Examples of TF55 include Tcpl, TriC and thermosome. Proteins normally occur in the cytoplasm of eukaryotes and some archaebacteria, and form rings of multiple members, promoting the folding of proteins. They are also weakly homologous to Hsp60. Examples of hsp40 include DnaJ of prokaryotes, such as, £. coli and mycobacteria and HSJ 1, HDJ 1 and Hsp40. Hsp40 plays a role as a molecular chaperone in the doubling of proteins, thermotolerance and DNA replication, among other cellular activities. Examples of FKBPs include FKBP 1 2, FKBP 1 3, FKBP25 and FKBP59, Fprl and Nepl. The proteins normally have peptidyl-propyl isomerase activity and interact with imunsuppressants, such as, FK506 and rapamycin. The proteins are normally found in the cytoplasm and the endoplasmic reticulum. Examples of cyclophilins include cyclophilins A, B, and C. the proteins have peptidyl-propyl isomerase activity and interact with the immunosuppressant cyclosporin A. The cyclosporin A protein binds to calcineurin (a protein phosphatase). Hsp20-30 is also referred to as a small Hsp. Hsp20-30 is normally found in large homo-oligomeric complexes, or possibly also hetero-oligomeric complexes, where an organism or cell type expresses several different types of small Hsps. Hsp20-30 interacts with cytoskeletal structures, and a regulatory role can be played in the polymepzation / depolymerization of actin. Hsp20-30 is rapidly phosphorylated on stress or exposure of resting cells to growth factors. Homologues of Hsp20-30 include alpha-crystalline. CIpP is an E. coli protease involved in the degradation of abnormal proteins. Homologs of CIpP are found in chloroplasts. CIpP forms a hetero-oligomeric complex with CIpA. GrpE is an E. coli protein of approximately 20 kDa, which is involved in both the rescue of proteins damaged by stress, as well as in the degradation of damaged proteins. GrpE plays a role in the regulation of strain gene expression in E. coli. Examples of Hspl O include GroES and Cpn 1 0. Hspl O is usually found in £. coli and in mitochondria and chloroplasts of eukaryotic cells. Hsp l O forms a seven-membered ring that is associated with Hsp60 oligomers. Hspl O is also involved in protein folding. Ubiq uitin has been found to bind proteins in coordination with protein proteolytic removal by ATP-dependent cytosolic proteases. In particular embodiments, the tension proteins of the present invention are obtained from enterobacteria, mycobacteria (particularly, M. leprae, M. tuberculosis, M. vaccae, M. smegmatis and M. bovis), E. coli, yeast, Drosophila, vertebrates, birds, chickens, mammals, rats, mice, primates or humans. The tension proteins can be in the form of acidic or basic salts, or in neutral form. In addition, individual amino acid residues can be modified by oxidation or reduction. Additionally, various substitutions, deletions or additions can be made to the amino acid or nucleic acid sequences, the net effect of which is to further retain or intensify the increased biological activity of the stress protein. Due to the degeneracy of the code, for example, there may be considerable variation in nucleotide sequences encoding the same amino acid sequence. The present invention is also suitable for use with protein portions of tension proteins or peptides derived from tension proteins, provided that such portions or peptides include the epitopes involved with the enhancement of the immune response to the chosen HPV protein antigen. Portions of strain proteins can be obtained by fragmentation using proteinases, or by recombinant methods, such as the expression of only part of a nucleotide sequence encoding a tension protein (either alone or fused with another acid sequence). n ucleic encoding protein). The peptides can also be produced by such methods, or by chemical synthesis. Stress proteins may include mutations introduced in particular places by a variety of known techniques. See, for example, Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Laboratory Press (1,989); Drinkwater and Klinedinst Proc. Nati Acad. Sci. USA 83: 3402-3406 (1986); Liao and Wise, Gene 88: 1 07- 1 1 1 (1 990); Horwitz et al. , Genome 3: 1 1 2-1 1 7 (1 989). The term "tension protein", as used herein, is intended to include such portions and peptides of a tension protein. Methods for identifying a gene or protein under consideration as a gene or protein tension are well known in the art. For example, the conservation of the genes and proteins of a particular tension protein family allows the comparison of the nucleotide or amino acid sequence of the gene / protein under consideration with well-known stress genes, such as, DnaK, GroEL or DnaJ, for example, by nucleic acid hybridization or -t-faith-a ". ^. Mv & Ji &tík? Á & sequencing of nucleic acids or amino acids, followed by com pation analysis by computer. Voellmy, R., et al. , Proc. Nat'l Acad. Sci. USA 82: 4949-4953 (1985). Alternatively, an assay can be used to identify and / or discriminate between essential structural features and / or functional properties of a selected tension protein. For example, an expression library can be classified using anti-Hsp antibodies. Hsp90 is well known to bind benzoquinone geldanamycin ansamycin with high affinity. Therefore, an expression library could be classified with geldanamycin to discover putative homologs of Hsp90 as proteins that bind the ansamycin of benzoquinone. The nature of the protein encoded by the isolated nucleic acid could be further confirmed by other assays, including antibody-based assays. Antibodies: A Laboratory Manual (Antibodies: A Laboratory Manual), Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press (1 988). In addition, the biological activity of a given protein group can be exploited. Guidon, P.T. , and Hightower, L. E., Biochem. 25: 3231-3239 (1986). For example, Hsp70 is capable of specifically binding ATP, as well as split polypeptides and peptides in the assembly of complexes of proteins. Thus, mixing a protein under consideration with a sample comprising polypeptides, peptides, or appropriate ATP, followed by the determination of the presence or absence of the production of protein-protein or protein-nucleotide complexes, indicates the apparent presence or absence of a protein. Hsp70 protein or gene, said presence or absence may É & & to, . iki ^ mí ^ m ^ be confirmed using other assays, such as antibody-based assays. The tension protein, tension protein portion, tension protein homologue and the H PV protein antigen to which the tension protein is conjugated or bound non-covalently, present in the composition, can be produced or obtained using known techniques. For example, the tension protein and / or the antigen can be obtained (isolated) from a source, in which it is known to occur, can be produced and harvested from cell cultures or, in the case of the antigen, can be obtained from cells infected, it can be produced by cloning, if necessary, and expressing a gene encoding the voltage protein or the desired antigen, or it can be synthesized chemically. Additionally, a nucleic acid sequence encoding the tension protein or the desired antigen can be chemically synthesized. A fusion protein including a strain protein and a H PV protein antigen can be produced by recombinant means. For example, a nucleic acid encoding the strain protein can be attached to either end of a nucleic acid sequence encoding the H PV protein antigen, such that the two protein coding sequences are sharing a reading frame. translational common and can be expressed as a fusion protein including the H PV protein antigen and the tension protein. The combined sequence is inserted into a suitable vector, chosen based on the desired expression characteristics and the nature of the host cell. In the examples provided below, the nucleic acid sequences are assembled into a suitable vector for protein expression in the E coli bacterium. Following expression in the chosen host cell, the fusion protein can be purified by routine biochemical separation techniques or by immunoaffinity methods, using an antibody for one or another part of the fusion protein. Alternatively, the selected vector can add a tag to the fusion protein sequence, for example, an oligohistidine tag, as described in the examples presented below, allowing the expression of a labeled fusion protein that can be purified by affinity methods, using an antibody or other material having an appropriately high affinity for the tag. Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (1 989); Deutscher, M. , Guide to Protein Purification Methods Enzymology, vol. 182, Academic Press, I nc. , San Diego, CA (1990). If a suitable vector is used for expression in mammalian cells, for example, one of the vectors discussed below, the fusion protein can be expressed and purified from mammalian cells. Alternatively, the mammalian expression vector (including fusion protein coding sequences) can be administered to a subject for direct expression of the fusion protein in the cells of the subject. A nucleic acid encoding a fusion protein including a strain protein and a protein H PV antigen can also be chemically produced and then inserted into a suitable vector for the production and purification of the protein. , ^ Á & , 8 * ^ > j ^ w? g fusion protein, or administration to a subject. Finally, the fusion protein can also be prepared chemically. Compositions comprising a strain protein and a H PV antigen described herein, can be used to enhance an immune response, particularly, a cell-mediated cytolytic response, against a H PV, or transformed or H PV-infected cell, which expresses an HPV antigen. Preferably, the compositions will contain protein antigen sequences from the particular HVP type against which proteins the immune response will be produced. Compositions comprising a strain protein and a H PV antigen described herein, can be administered to a subject in a variety of ways. Routes of administration include intradermal, transdermal (e.g., slow release polymers), intramuscular, intraperitoneal, intravenous, subcutaneous, oral, epidural, and intranasal routes. Any other convenient route of administration may be used, eg, infusion or bolus injection, or absorption through the epithelial or mucocutaneous coatings. In addition, the compositions described herein may contain and be administered together with other pharmacologically acceptable components, such as, biologically active agents (e.g., auxiliaries, such as, alum), surfactants (e.g., glycerides), excipients (e.g., lactose), carriers, diluents, and vehicles. Additionally, the compositions can be used ex vivo as a means to stimulate white blood cells obtained from a subject to produce, expand and propagate antigen-specific immune cells of • SSV protein in vitro, which are subsequently re-introduced into the subject. In addition, a tension protein-HPV protein antigen fusion protein can be administered by live m expression of a nucleic acid encoding such protein sequences in a human subject. The expression of such nucleic acid can also be achieved ex vivo, as a means to stimulate white blood cells obtained from a subject, to produce, expand and propagate HPV antigen-specific immune cells in vitro, which are subsequently re-introduced into the subject. Suitable expression vectors for directing the expression of HPV-protein protein antigen fusion proteins can be selected from a wide variety of vectors currently used in the field. Preferred vectors will be those that are capable of producing high levels of expression, as well as they are effective to transduce a gene of interest. For example, the recombinant adenovirus vector pJM17 (All et al., Gene Therapy 1: 367-84 81994); Berkner KL, Biotechniques 6,616-24, 1988), second-generation adenovirus vectors DE1 / DE4 (Wang and Finer, Nature Medicine 2: 714-6 (1996)), or adeno-associated viral vector AAV / Neo (Wall-20). Cacho et al., J. Immunotherapy 11: 231-7 (1992)). In addition, recombinant retroviral vectors MFG (Jaffee et al., Cancer Res. 53: 2221-6 (1993)) or LN, LNSX, LNCX, LXSN (Miller and Rosman, Biotechniques 7: 980-9 (1989)) can be used. Vectors based on Herpes simplex virus, such as, pHSV1 (Geller et al., Proc. Nat'l Acad. Sci 87: 8950-4 (1990) or viral vaccinia vectors, such as MVA (Sutter and Moss, Proc. Nat'l Acad. Sci. 89: 10847-51 (1992)) can serve as alternatives. Frequently used specific expression units including promoter and 3 'sequences are those found in the plasmid CDNA3 (Invitrogen), plasmid AH5, pRC / CMV (Invitrogen), pCMU II (Paabo et al., EMBO J. 5.1921-1927 (1986 )), pZip-Neo SV (Cepko et al., Cell 37: 1053-1062 (1984)) and pSRa (DNAX, Palo Alto, CA). The introduction of genes into units and / or expression vectors can be achieved using genetic engineering techniques, as described in manuals such as Molecular Cloning and Current Protocols in Molecular Biology (Sambrook, J et al., Molecular Cloning, Cold Spring Harbor Press (1989); Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience (1989)). A resulting expressible nucleic acid can be introduced into cells of a human subject by any method capable of placing the nucleic acid in cells in an expressible form, for example, as part of a viral vector, as described above, a naked plasmid or other DNA, or encapsidated in focused liposomes or erythrocyte ghosts (Friedman, T., Science 244: 1275-1281 (1989); Rabinovich, NR et al., Science, 265: 1401-1404 (1994)). Transduction methods include direct injection into tissues and tumors, liposomal transfection (Fraley et al., Nature 370: 111-117 (1980)), receptor-mediated endocytosis (Zatloukal et al., Ann. NY Acad. Sci. 660: 136-153 (1992)), and * - # **. - - ^ x *, ^ gene transfer mediated by particle bombardment (Eisenbraun et al., DNA &Cell Biol. 2: 791-797 (1992)). The amount of HPV protein strain and antigen protein (fused, conjugated or non-covalently bound as discussed above) in the compositions of the present invention is an amount that produces an effective immunostimulatory response in a subject. An effective amount is such an amount that when administered results in an induction of an immediate response. In addition, the amount of protein tension and antigen of H PV protein administered to the subject will vary depending on a variety of factors, including the H PV protein antigen and strain protein employed, size, age, body weight, general health , sex and diet of the subject, as well as his general immunological sensitivity. The adjustment and manipulation of established dose ranges are within the skill of those skilled in the art. For example, the amount of tension protein and antigen can be formed from about 1 microgram to about 1 gram, preferably from about 100 microgram to about 1 gram, and from about 1 milligram to about 1 gram. An effective amount of a composition comprising an expression vector is such an amount that when admistered, it induces an immune response against the HPV protein antigen, which it encodes. Additionally, the amount of expression vector administered to the subject will vary depending on a variety of factors, including the HPV protein antigen and tension protein expressed, the size, age, body weight, general health, sex and diet of the subject, as well as as of its general immunological sensitivity. Additional factors that need to be considered are the application route and the type of vector used. For example, when a prophylactic or therapeutic treatment is performed without a viral vector containing a nucleic acid encoding an HPV-protein protein antigen fusion protein, the effective amount will be in the range of 10 4 to 10 12 of defective virus in replication, free of auxiliary, per kg of body weight, preferably in the range of 10 5 to 10 11 viruses per kg of body weight, and most preferably in the range of 10 6 to 10 10 viruses per kg of body weight. The present invention is illustrated by the following examples, which are not intended to be limiting in any way.

EXAMPLES EXAMPLE 1: ISOLATION OF RECOMBINANT TENSION PROTEINS Recombinant mycobacterial Hsp71 Plasmid Y3111 contains an hsp71 gene of M. tuberculosis functionally inserted between expression control sequences (Mehlert, A. and Young, D.B., Mol.Microbiol.3: 125-130 (1989)). E. coli strain CG2027 (obtained from C. Georgopoulos, University of Geneva, Switzerland), containing a truncated dnaK gene, was transformed with the plasmid Y3111 by standard procedures. (Maniatis et al., Molecular Cloning, ' JE te ^ i * A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1 982)). Bacteria were grown containing the plasmid Y31 1 1 overnight in 2xYT medium (20 g of Tryptone, 1 0 g of yeast extract, 10 g of NaCl per liter) containing 10 micrograms / ml of ampicillin at 37 ° C, with stirring 8250 rpm). A 10% glycerol stock solution was prepared from this stock and stored at -70 ° C. Several scrapes of the frozen glycerol stock solution were used to inoculate a large culture, which was incubated as before for 48 h. When the optical density at 590 nm reached 2.5 to 3.5, the cells were harvested by centrifugation. The following steps were performed at 4 ° C. The cell pellet was resuspended in 3 ml of lysis buffer per gram of pelleted cells. The lysis buffer composition was 1 mM Tris-HCl., 2 mM ethylenediamine tetraacetate (EDTA), 5 mM beta-mercaptoethanol, 1 microgram / ml aprotinin, 1 microgram / ml leupeptin, and 1 microgram / ml pepstatin. Lysozyme was added to the cell suspension to a final concentration of 0.14 mg / ml. The suspension was then frozen at -70 ° C. The cell suspension was thawed, and the cells were disrupted by infection. The sonicate was subjected to centrifugation at 1, 000 rpm for 30 min (JA-1 7, Beckmann rotor). Solid (N H) 2 SO 4 was added to the supernatant solution until the solution was 65% saturated with (NH) 2 SO. After 30 min of incubation, the mixture was centrifuged as before. The pellet dissolved in shock absorber A of Q SEPHAROSE. To this solution were added 10 micrograms / ml of aproten, 10 micrograms / ml of leupeptin and 1 microgram / ml of pepstatin, and the solution was dialysed overnight against 65 volumes of buffer A of Q SEPHAROSE. Q SEPHAROSE buffer A contained 30 mM Tris-HCl (pH 7.5), 1 mM EDTA, 5 mM beta-mercaptoethanol. The dialyzed solution was clarified by centrifugation as described above. The dialyzed solution was applied to a column of Q SEPHAROSE (Pharmacia) balanced in buffer A of Q SEPHAROSE. The column was washed with 2 volumes of the same buffer. The levigation was performed with a gradient from 0 to 600 mM NaCl. Fractions were tested by SDSPAGE and stained with Coomassie Blue for the presence of a larger 71 kDA polypeptide (ie, the recombinant M. tuberculosis Hsp71 protein). The fractions containing the polypeptide were deposited, and the deposit was brought to 65% saturation by the addition of solid (N H 4) 2 SO 4. The mixture was centrifuged as described above, the pellet was dissolved in ATP Start buffer (50 mM Tps-HCl, (pH 8.0), 20 mM NaCl, 5 mM MgCl2, 5 mM beta-mercaptoethanol, 0.1 mM EDTA) , and the resulting protein solution was dialyzed overnight against 65 volumes of the same buffer and clarified by centrifugation. The dialyzed protein solution was then applied to a column of ATP-agarose (Fluka) equilibrated in ATP Start buffer. The column was washed with 1 volume of ATP Start buffer with 1 M NaCl. The levigation was achieved with ATP Start buffer supplemented with 1 mM ATP. The levigado was brought to 65% saturation with (NH4) 2SO4, and the precipitated protein was collected as described above. The centrifugation pellet was dissolved in, and dialyzed against, 200 volumes of Blue SEPHAROSE buffer (30 mM Tris-HCl (pH 7.5), 25 mM MgCl, 5 mM beta-mercaptoethanol). The dialyzed protein solution from the last step was applied to a Blue SEPHAROSE column (Pharmacia) equilibrated with Blue SEPHAROSE buffer. The column was washed with 1.5 column volumes of the same buffer. The fractions of flow through and wash (containing Hsp71) were collected as a simple deposit. The purity of the final preparation was assessed by SDS-PAGE and Coomasie Blue spotting, by western blot analysis (Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Springer Harbor, NY (1982)); (See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, NY (1989)), using mouse monoclonal antibodies specific for mycobacterial Hsp71 and DnaK of £. coli, respectively, and by assays of ATPase activity. The preparations are usually more than 90% pure, based on the staining pattern of the preparation on gels stained with Coomassie blue, and preferably more than 95% pure, and contain less than 1% GroEL of £. coli and DnaK for £. coli not detectable.

Recombinant mycobacterial HSP65 Plasmid RIB 1300 contains an hsp65 gene of M. bovis BCG functionally inserted between expression control sequences (Thole, JER et al., J. Exp. Med. 178: 343-8 (1993). E. coli strain M1546 with plasmid RIB1300 (Thole, JER, supra) using standard procedures, Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). containing plasmid RIB 1300 was grown to saturation in NCZYM medium (10 g of NZ Amin A, 5 g of Bacto yeast extract, 1 g of Casamino acids, 5 g of NaCl, 2 g of (NH4) 2 SO4-7H2O per liter) containing 200 micrograms / ml of ampicillin at 28 ° c and under agitation (250 rpm) This culture was used to inoculate a larger culture, which was grown under the same conditions as the inoculum culture, until the density optics at 590 nm of the crop was between 0.3 and 0.6. The recombinant protein was initiated by rapidly raising the culture temperature to 42 ° C by incubation in a hot water bath. The culture was maintained at this temperature for 3 h with agitation. The bacteria were then harvested by centrifugation and resuspended in 6 volumes by weight of bacterial pellet lysis buffer. The lysis buffer contained 10 mM Tris-HCl (pH 8.0), 10 mM ethylenediamine tetraacetate (EDTA), 0.1 mM PMSF and 0.1% BA RIVM (0.104 g of 4-amino-benzamidine-HCl, 0.066 g of epsilon acid). caproic amino by 50 ml). Lysozyme was added at a concentration of 0.1 mg / ml, and the suspension was frozen at -70 ° C. The bacterial suspension was thawed and placed at 4 ° C. The following operations were performed at this temperature. The complete lysis of bacteria was achieved by sonication. The sonicate was centrifuged at 17,000 rpm for 30 min in a JA-17 (Beckman) rotor. Saturated (NH) 2SO was added to the supernatant solution until 20% saturation was achieved. The precipitates were removed by centrifugation (see above) and discarded. The supernatant solution was brought to 55% saturation by the addition of saturated (N H) 2 SO 4. The pellet resulting from the subsequent centrifugation was dissolved in TE buffer (10 mM Tris-HCl (pH 8.0), 5 mM beta-mercaptoethanol, 1 mM EDTA). The protein solution in TE was then dialyzed against 50 volumes of TE buffer. After centrifugation (as before) to remove the precipitated material, the dialyzed protein solution was applied to a DEAE SEPHAROSE column (Pharmacia). After washing with TE buffer, the proteins were levigated with a gradient of 0-300 μM of NaCl in TE buffer. Fractions containing Hsp65 from M. bovis BCG were identified by SDS-PAGE and stained with Coomassie blue and deposited. 1 μg / ml of aprotinin, 1 μg / ml of leupeptin and 1 μg / ml of pepstatin were added to the tank, which was then concentrated in a Am icon cell using a YM30 membrane. The concentrated deposit was applied to a column of S-200 SEPHACRYL (Pharmacia) equilibrated with buffer S200 (Na2H PO4 1 0 mM (pH 6.8), NaCl 1 50 mM and beta-mercaptoethanol 1 5 m M). The levigation was carried out with the same shock absorber. The fractions were tested for the presence of mycobacterial Hsp65 as before, and the positive fractions containing highly purified portein were deposited and dialysed overnight against HAP buffer (10 mM Na2HPO (pH 6.8), 15 mM beta-mercaptoethanol). The dialyzed deposit was applied to a hydroxyapatite column (Bio-Rad, Bio-Gel HTP Gel) balanced in HAP buffer. The column was washed with 3 column volumes of 1 mM MgCl 2 and 15 mM beta-mercaptoethanol, and then with 1 mM Na 2 HPO (pH 6.8) and 15 mM beta-mercaptoethanol. The protein was levigated with a 10-60 mM phosphate gradient. The fractions were tested as before, and the positive fractions were deposited, concentrated and exchanged in 0.85% NaCl by means of gel filtration through PD10 (Pharmacia). The purity of mycobacterial Hsp65 was assessed by SDS-PAGE and stained with Coomassie blue, as well as by western blot analysis using antibodies specific for DnaK and GroEL of £. coli The preparations were normally more than 90% pure, and contained no more than 0.5% GroEL of £. coli and 0.1-0.2% of DnaK of £. coli, respectively. The hsp preparations can be depyrogenated, either by affinity chromatography on DetoxyGel resin (Pierce), addition of polymyxin B or by extraction with detergents, such as, Triton X-114 or X-100. The reduction in the lipopolysaccharide content can be followed by the limulus amylocyte assay (LAL; Biowhittaker, QCL 1000). The Hsp preparations can be stored in buffer at -70 ° C, or can be maintained, preferably at -70 ° C, as dry pellets after lyophilization.

EXAMPLE 2: "Ü * K" • J * lfá ^ S tmm- PREPARATION OF PROTEIN ANTENGINE CONJUGATES - TENSION PROTEIN This example is provided as an illustration of the techniques that can be used to prepare conjugates between a voltage protein and a protein antigen. protein, in this example, a peptide derived from influenza virus nucleoprotein (NP).

Synthesis of tension protein (Hsp71) and antigen (NP.B) Hsp71 of M. tuberculosis was prepared as described in Example 1. The peptide NP (referred to herein as NP.B; Motal, UMA et al., Eur. J. Immunol 25.11214 (1995) and references therein) with the amino acid sequence [CjVQLASNENMETM (SEQ ID NO: 1, the peptide contains an extra amino-terminal cysteine residue) corresponding to residues 363-374 in the complete NP and containing a known CTL epitope (H-2b-restricted) was produced synthetically (0.25 mM scale) on a peptide synthesizer from Applied Biosystems model 431A using Fmoc (9-fluorenylmethyloxycarbonyl) as the alpha- amino protector and HMP resin (Wang) as the solid support. All synthesis chemicals and amino acids were purchased from Applied Biosystems. NP.B was cut from the support and the side chain protective groups were removed by incubating under continuous stirring of NP B-resin for 3 hours in 2 ml of a mixture prepared by combining 10 ml of trifluoroacetic acid, 0.5 ml of water, 0.75 g of crystalline phenol, 0.2 ml of ethanedithiol and 0.5 ml of thioanisole. The cut mixture was filtered in 40 ml of ice-cold diethyl ether. The insoluble material was collected by centrifugation at 5000 x g for 8 min. The ether was decanted and the pellet was washed three times by resuspension in cold diethyl ether, followed by centrifugation. After the last wash, the pellet was air dried, captured in distilled water and lyophilized.

Chemical conjugation of peptide N P. B to Hsp71 and diphtheria toxoid The conjugations were carried out both with Hsp71 and with the commonly used carrier protein of toxoid de difteria (abbreviated DT; DT was obtained from Wako Chemical), to provide a standard of efficiency comparisons of the specific stimulation of CTL activity. Activated carrier protein solutions: Nine mg of Hsp71 was dissolved in 4.5 ml of 0.1 M sodium borate buffer, pH 8.0. Sulfo-MBS (m-maleim idobenzoyl-N-hydroxy-sulfosuccinimide ester) (2.3 mg in 1000 ul of dimethyl sulfoamine) was added to the protein, and the reaction mixture was incubated for 1 hour at room temperature. The pH was then adjusted to 6.0 and the reaction mixture was dialyzed overnight at 4 ° C against 1 liter of 20 mM sodium phosphate and 50 mM NaCl, pH 5.6. DT was treated similarly. Reduced peptide solutions: For each conjugation reaction, 3 mg of peptide was dissolved in 1000 ul of 0.1 M beta-mercaptoethanol. After 1 hour of incubation to allow the reduction of the peptide, the reducing agent was removed by drying the reaction mixture in a SpeedVac centrifuge. The peptide was redissolved in 0.5 ml of distilled water, to which aliquots of 5 ul of 1 N NaOH were added, until the peptide was completely dissolved. For conjugation experiments with DT, 6 mg of peptide was reduced and then redissolved in 1 ml of water. The pH of the solutions of activated carrier proteins was adjusted to 6.8 using 0.1 N NaOH. The solution containing 3 mg of activated carrier protein was reacted with 0.5 ml of reduced peptide solution (or 1 ml of reduced peptide solution for the preparation). of conjugates with DT) for 3 hours at room temperature with continuous mixing. To remove the unreacted peptide, the solution containing the resulting conjugate was dialyzed overnight at 4 ° C against 1 liter of 20 mM sodium phosphate and 150 mM NaCl, pH 7. The protein concentration was determined by BCA assay. The efficiency of conjugation achieved by this procedure has been determined in previous pilot experiments using radiolabeled NP.B peptide. The peptide: protein ratio was found to be 17.5 for conjugate of NP.B-Hsp71 (71. NP) and 10.1 for NP.B-DT (DT.NP).

EXAMPLE 3: PREPARATION OF FUSION GENES HSP-E6 AND HSP-E7 Preparation of bacterial expression vector pET65H Plasmid RIB1300 contains an hsp65 gene from BCG of Mycobacterium bovis (Thole, J.E.R. et al., J. Exp. Med. 178: 343-8 (1993)). A pair of primers was synthesized for the amplification of the hsp65 gene in an automated oligonucleotide synthesizer and purified using - -. Ax. * S £ S * iá routine procedures. The forward primer included an Ndel restriction site and had the nucleotide sequence 5 'AAT CAC TTC CAT ATG GCC AAG ACÁ ATT. The reverse primer included EcoRI and Nhel sites flanking a stop codon and had the 5 'nucleotide sequence CGC TCG GAC GAA TTC TCA GCT AGC GAA ATC CAT GCC. The polymerase chain reaction (PCR) was performed using the above primer pair and pRI B1 300 as the DNA template. The PCR fragments were double digested with restriction endonucleases Ndel and EcoRI and ligated to dEltA double-digested pET28a (I nvitrogen) using routine subcloning procedures (Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, NY (1989). The competent cells transform £. Coli strain DHdalfa were transformed with the ligation mixture and platinized on agar containing 1 00 ug / ml of ampicillin. Colonies were isolated from the transformed cells, and plasmid DNA was prepared and analyzed for the presence of hsp65 gene and vector sequences by restriction mapping and nucleotide sequencing. The correct constructs (called pET65H) including the mycobacterial hsp65 gene were identified and transformed into £. coli strain BL21 (DE3; Novagen) for expression analysis of the hsp65 gene. A schematic map of construct pET65H is shown in Fig. 1 . To test the expression of Hsp65, transformed bacteria of strain BL21 were grown, induced and harvested using the manufacturer's instructions (Novagen). The bacteria were lysed and soluble material, as well as material solubilized from inclusion bodies by hydrochloride . «Afigásagttasg. guanidinium (Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, NY (1989)) were subjected to electrophoresis on SDS-PAGE and subjected to anti-Hsp65 'immunoblot' using an antibody specific monoclonal for mycobacterial tension protein.

Preparation of a construct for the expression of an HPV16 Hsp65-E6 fusion protein in bacteria A complete HPV16 E6 coding region was inserted into the carboxy-terminal end of the hsp65 gene in pET65H. Plasmid pHPV16 contains a complete HPV16 genome in the Bluescript SK vector (ATCC 45113). For the nucleotide sequence of the HPV16 genome, see Seedorf et al. Virology 145: 181-5 (1985). A pair of primers was synthesized for the amplification of the E6 gene in an automated oligonucleotide synthesizer and purified using routine procedures. The forward primer included a Nhel restriction site and had the nucleotide sequence 5 'AAA AGC AGA GCT AGC ATG CAC CAA AAG. The reverse primer included EcoRI and a stop codon and had the nucleotide sequence 5 'CTC CAT GAA TTC TTA CAG CTG GGT. The polymerase chain reaction (PCR) was performed using the above primer pair and pHPV16 as the DNA template. The PCR fragments were double digested with restriction endonucleases Nhel and EcoRI and ligated to pET65H double digested with Nhel / EcoRI using routine subcloning procedures (Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Lab. Cold Spring Harbor, NY (1 989)). Competent cells were transformed into £ transformation. coli strain DHdalfa with the ligation mixture and platinized on agar containing 1 00 ug / ml of ampicillin. Colonies of transformed cells were isolated and plasmid DNA was prepared and analyzed for the presence of hsp65-E6 fusion gene and vector sequences by restriction mapping and nucleic acid sequencing. The correct constructs (called pET65HE6) including the fusion gene hsp65-E6 were identified and transformed into £. coli strain BL21 (DE3; Novagen) for expression analysis of the fusion gene. A skeletal map of construct pET65HE6 is shown in Fig. 2. To test the expression of Hsp65-E6, BL2 1 strain bacteria transformed with pET65H E6 were grown, induced and harvested using the manufacturer's instructions (Novagen). Bacteria were used and the soluble material, as well as insoluble material inclusion bodies by guanidinium hydrochloride (Maniatis et al Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, NY (1989)) subjected to electrophoresis in SDS-PAGE. As a standard, purified Hsp65 was run in parallel. The expression of Hsp65-E6 was assessed by the appearance of a band of staining strongly with Coomassie blue that migrates slightly slower (apparent molecular weight (MOO) of approximately 73 kDa) than authentic Hsp65 (apparent molecular weight of approximately 56 kDa) in m samples of bacteria transformed with pET65HE6 that was not present in corresponding untransformed bacteria.

Preparation of a construct for the expression of a fusion protein of Hsp65-H P 1 6 E7 in bacteria A complete ET coding region of H PV1 6 was inserted into the carboxy-terminal end of the hsp65 gene in pET65H. A pair of primers was synthesized for the amplification of the E7 gene in an automated oligonucleotide synthesizer and purified using routine procedures. The forward primer included a Nhel restriction site and had the nucleotide sequence 5 'AAC CCA CCT GCT AGC ATG CAT GGA GAT. The reverse primer included EcoRI and a stop codon and had the sequence of n ucleotide 5 'AGC CAT GAA TTC TTA TGG TTT CTG. The polymerase chain reaction (PCR) was performed using the above pair of ini- tiators and pH PV16 as DNA template. The PCR fragments were digestated twice with restriction endonucleases Nhel and EcoRI and ligated to pET65H digested twice with Nhel / EcoRI using routine subcloning procedures. The competent cells in transformation of £. Coli strain DHdalfa were transformed with the ligation mixture and platinized on agar containing 1 00 ug / ml of ampicillin. Colonies of transformed cells were isolated, and plasmid DNA was prepared and analyzed for the presence of the hsp65-E7 fusion gene and vector sequences by restriction mapping and nucleic acid sequencing. The correct constructs (called pET65H E7) including the hsp65-E7 fusion gene were identified and transformed into £. coli strain BL21 (DE3; I nvitrogen) for analysis of expression of the fusion gene. A schematic map of the pET65HE6 construct is shown in Fig. 3 To test the expression of Hsp65-E7, BL21 strain bacteria transformed with pET65HE7 were grown, induced and harvested using routine procedures. Bacteria were lysed and soluble material as well as material solubilized from inclusion bodies by guanidinium hydrochloride, subjected to electrophoresis on SDS-PAGE and subjected to anti-E7 blot using a monoclonal antibody specific for HPV16 E7 (Zymed Laboratory Inc ., catalog number 28-0006). EXAMPLE 4: EXPRESSION AND PURIFICATION OF FUSION PROTEINS Expression and Purification of Hsp65-E6 Fusion Protein: Procedure 1 The construct pET65HE6 was transformed into £. coli strain BL21 (DE3; Novagen), and transformed cells were grown in 6 liters of 2xYT medium cultures (20 g of tryptone, 10 g of yeast extract, 10 g of NaCl per liter) containing 30 ug / ml of kanamycin at 30 ° C. For each culture, when the density reached 0.5 (OD590), fusion protein expression was induced by 0.5 mM isopropylthio-galactopyranoside, and the culture was incubated for an additional three hours at 37 ° C. The cells were harvested by centrifugation, suspended in 90 ml of lysis buffer (10 mM Tris-HCl, 0.5 mM beta-mercaptoethanol, pH 7.5) containing 200 micrograms / ml of lysozyme, and frozen at -70 ° C. 25 One day later, the frozen cell suspension was thawed in a , - * t-, * «-. ^^ t ^ * -. Fe-mary bath at 37 ° C, and supplemented with 2 micrograms / ml aprotinin, 2 micrograms / ml leupeptin, 2 micrograms / ml pepstatin and 2 mM PMSF. All subsequent steps were performed at 0-4 ° C. The lysis of the cells was by sonication, and the insoluble material was collected by centrifugation at 1, 7,000 rpm for 1 5 min (JA-1 7 rotor, Beckmann). The pelleted material was washed twice with lysis buffer and then solubilized, aided by sonication, in 90 ml of buffer A (50 mM Tris-HCl, pH 7.5, 6 M guanidinium hydrochloride). The insoluble material was removed by centrifugation as before. The solubilized material was then applied to a column containing 50 ml of nickel-laden metal chelating resin (Chelating Sepharose Fast Flow; Pharmacia) which had been equilibrated with buffer A. The bound fusion protein was slowly doubled back into the resin with a gradient of NaCl 0-1 M. The resin was washed with five volumes of buffer B (1 M NaCl) to remove the residual guanidinium hydrochloride, and with five buffer volumes C (50 mM im idazole, pH 7.5, 0.5 m beta-mercaptoethanol, 50 mM NaCl) to remove contaminating proteins. The fusion protein was levigated with six volumes of a 50-500 μm linear imidazole gradient in C-buffer. The deposited protein was dial-up overnight against approximately 40 volumes of Dulbecco's phosphate-buffered saline (KH2PO4 2.7 mM, 4.3 mM Na2HPO4, 2.7 mM KCI, 0.1 37 M NaCl), was concentrated by ultrafiltration (Amicon, 30 kDa molecular weight cutoff) and passed through an Detoxigel equil ibrada resin in phosphate buffered solution of Dulbecco for endotoxin removal.

Expression and Purification of Hsp65-E6 Fusion Protein: Procedure 2 The pET65H E6 construct was transformed into £. coli strain BL21 (DE3; Novagen), and the transformed cells were grown in 12 liters of 2xYT medium cultures (20 g of Tryptone, 10 g of yeast extract, 1.0 g of NaCl per liter) containing 30 ug / ml of kanamycin at 30 ° C. For each culture, when the density reached 0.5 (OD590), the expression of fusion protein was induced by 0.5 mM isopropylthio-galactopyranoside, and the culture was incubated for an additional three hours at 37 ° C. The cells were harvested by centrifugation, suspended in 1 80 ml of lysis buffer (10 mM Tris-HCl, 0.5 mM beta-mercaptoethanol, pH 7.5) containing 200 ug / ml of lysozyme, and frozen at -70 °. C. One day later, the frozen cell suspension was thawed in a water bath at 37 ° C and supplemented with 2 micrograms / ml of aprotinin, 2 micrograms / ml of leupeptin, 2 micrograms / ml of pepstatin and 2 mM PMSF. All subsequent steps were performed at 0-4 ° C. Cell lysis was by sonication, and the insoluble material was collected by centrifugation at 1 7,000 rpm for 1 5 min (JA-1 7 rotor, Beckmann). The pelleted material was washed twice with lysis buffer and then solubilized, assisted by sonication, in 1 80 ml of buffer A (50 mM Tris-HCl, pH 7.5, 6 M guanid hydrochloride). The insoluble material was removed by centrifugation as before. The solubilized material was then applied to a column containing 50 ml of nickel-laden metal chelating resin (Chelating Sepharose Fast Flow; Pharmacia) which had been equilibrated with buffer A. The bound protein was washed with buffer D (buffer A 5). % Triton X1 00) and then slowly doubled back into the resin with a gradient of 0-1 M NaCl. The resin was washed with five volumes of buffer E (1M NaCl, 1% Triton X1 00) and five volumes of buffer B (1M NaCl) to remove the residual guanidinium hydrochloride, and with five volumes of buffer F ( 50 mM imidazole, pH 7.5, 0.5 mM beta-mercaptoethanol, 0.5 M NaCl, 1 5% g licerol) to remove contaminating proteins. The fusion protein was levigated with six volumes of a 50-500 mM linear imidazole gradient in F buffer. The deposited levigated protein was dialyzed overnight against 40 volumes of G buffer (30 mM Tris-HCl, pH 6.5, 2 mM EDTA, 5 mM beta-mercaptoethanol, 15% or glycerol) and applied to a 50 ml SP-Sepharose column equilibrated in the same buffer. The fusion protein (approximately 42 mg, representing approximately 50% of the total fusion protein present in the unfractionated extract) was recovered in the flow through fraction, dialyzed overnight against 40 volumes of phosphate buffered saline. of Dulbecco (KH2PO4 2.7 mM, Na2H PO4 4.3 mM, KCI 2.7 mM, NaCl 0. 1 37 M), and concentrated by ultrafiltration (Amicon, molecular weight cut-off of 30 kDa).

The hsp65-E7 fusion protein was expressed and purified by the same procedures. The purity of the fusion proteins was estimated by SDS-PAGE and stained with Coomassie blue of gels. The proteins were normally 70-90% pure after procedure 1, and approximately 95% pure after the preferred procedure 2. The -tsi? / • »? ÍMÍ * ¿-. . ? - Mf, endotoxin levels after purification using procedure 2 were below 20 EU / mg protein.

EXEM PLO 5. I N M U N IZAC I N N E N HSP65-E6 AND HSP65-E7 Female C57 / BL / 6 mice, six to eight weeks old, were obtained from Charles River Laboratory (St. Constant, Quebec, Canada). Groups of six to eight mice per group were subcutaneously immunized at the nape with equal amounts of fusion proteins Hsp65-E6 and Hsp65-E7 in Dulbecco's phosphate-buffered saline, as described under Example 4. Total adm merges were 20 micrograms or 200 micrograms, respectively. The negative control immunizations were with incomplete Freund's assistant in saline (I FA), and positive control immunizations with 1000 micrograms of an E7 peptide from H PV1 6, including residues 44 to 62 in I FA and solution saline. In relation to the doses of fusion proteins applied, this amount of peptide E7 administered represented an excess of 20 or 200 times, respectively. Immunizations were repeated 14 days later. Twelve days after the second immunization, the mice were challenged by subcutaneous injection in a shaved area of the back of the mice, with 1.3 x 1 05 of tumor cells expressing E7 of the TC-1 line. Tumor incidence was recorded as the presence or absence of tumor based on visual observation and palpation every two days for fifteen ; 3-1 »MSPgi.a¡ days. The TC-1 tumor cell line expressing the HPV16 E7 protein was derived from primary lung cells of C57 / BI / 6 mice by immortalization and transformation with HPV16 E6 and E7 genes and a human c-Ha-ras gene, as described by Lin et al (Cancer Res. 56: 21-26 (1996)). For tumor inoculation, TC-1 cells, supplied by Dr. T.-C. Wu (The Johns Hopkins Medical Institutions, Baltimore, MD), were grown at 60-70% confluency in RPMI1640 medium supplemented with 105 fetal calf serum (Hyclon, Logan, UT), non-essential amino acids, glutamine, pyruvate, gentamicin, beta-mercaptoethanol, 0.5 mg / ml of Geneticin® (Life Technologies, Grand Island, NY) and 0.2 mg / ml of hygromycin B at 37 ° C. Cells were harvested by trypsinization and resuspended in Hank's buffer at 6.5x105 cells / ml. The results of such an experiment are shown in Fig. 4. Shortly after the challenge, the incidence of tumor rose in all treatment groups (between 5 and 15 days after the challenge). While the incidence remained high for the IFA group, it fell dramatically to 0% for groups treated with 200 micrograms of fusion protein mix or with E7 peptide in IFA. An intermediate result was obtained for the group treated with 20 micrograms of fusion proteins. Thus, immunization with mixtures of fusion proteins Hsp65-E6 and Hsp65-E7, in the absence of any auxiliary, effectively protected the mice from a lethal challenge with tumor cells expressing E7. A summary of the results is shown in Table 1. __. * - * - "& * a *». "tofáfe i ^^ iS TABLE 1 RESPONSE TO A SECOND CHALLENGE WITH TUMOR TC-1 Immunization Group Percentage of tumor incidence * After the 1st challenge After the 2nd challenge (Day 54) (Day 79) IFA or nothing ** 83 (5/6) 100 (4/4) Micrograms of 25 (2/8) 25 (1/4) fusion proteins Micrograms of 0 (0/8) 0 ( 0/4) Peptide E7 fusion proteins in IFA 0 (0/8) 0 (0/4) * In parentheses, the number of animals with tumor / total number of animals per group is given. In the right column, the animals were monitored for the presence or absence of tumor for an additional 25 days (after the second challenge). ** As a control for the second tumor challenge, a group of non-immunized animals was included for comparison.

EXAMPLE 6: EXPOSURE OF IMMUNIZED ANIMALS TO A SECOND CHALLENGE WITH TUMOR CELLS To assess the longevity of the immune response for antigens of HPV, the groups of four surviving animals from the previous experiment (at day 54), both from groups treated with fusion proteins and from the E7 / IFA peptide group, were challenged a second time with 1.3 x 105 TC-tumor cells. 1 live per animal. As a control, a group of mice without affectation was included, exposed to the same tumor challenge. The tumor incidence was assessed 25 days later. The results are shown in Table 1. The animals previously immunized with fusion proteins or with E7 / IFA peptide were completely or almost completely protected from the second challenge, while the non-immunized animals showed a tumor incidence of 100%.

EXAMPLE 7: CYTOLYTIC ACTIVITY OF SPLINOCYTES OF ANIMALS IMMUNIZED AND NOT IMMUNIZED Groups of two mice from animals immunized with fusion protein, peptide or non-immunized were euthanized by cerivcal dislocation, and their spleens were removed. Single-cell suspensions of extracted spleens were prepared and washed once in Hank's buffer solution, supplemented with 5% fetal calf serum. The lymphoid cells were re-stimulated by culturing 20 x 106 viable cells with 2 x 106 TC-1 cells treated with mitomycin C for five days in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 microM 2-mercaptoethanol and 50 micrograms / ml gentamicin sulfate at 37 ° C and 5% CO2 Splenocytes (effector cells) were then harvested and used in the described CTL activity assay ahead. TC-1 and equal cell lines of type HLA EL4 and MC57G that do not express an E7 epitope were used., as target cells The cells were incubated for 90 min with 1 50 uCi of Na251CrO4 and, in the case of EL4 cells, also with 1 0 ug of peptide366-37 of influenza virus nucleoprotein per 1 06 cells. Following extensive washing to remove excess radiolabel, 5x1 03 target cells were co-cultured with re-stimulated effector cells to various proportions of target cell, effector. After 4-5 hours of incubation, the culture plates were centrifuged for 5 min at 200 x g, and 1 000 ul aliquots of supernatant solutions containing radiolabel released from cells were collected in Beckman Ready Caps. The radioactivity was measured by liquid scintillation counting. To determine the spontaneously released radioactivity and total releasable radioactivity, solutions of culture supernatants containing target cells alone or of target cells lysed by the addition of Triton X1 00 were collected, and the radioactivity was determined as before. The results were expressed as% corrected lysis, calculated based on the following formula: % corrected lysis = 1 00 x (cpmpru eba - cpmespon) / (cpmtota? - cpmespon), where cpmprue a is the radioactivity released from a particular co-cupon, cpm8Spon Is the radioactivity released spontaneously from a culture of target cells and cpmfotai is the radioactivity released by Triton lysis X1 00 of target cells. The CTL assays were performed in triplicate, and averaged values were provided. The results of an experiment using TC-1 cells as target cells are shown in Fig. 5. 40% and 25% lysis of the TC-1 cells were mediated by effector cells (used as a 100-fold excess on target cells) obtained from animals immunized with 200 micrograms and 20 micrograms of fusion proteins, respectively. In a second experiment, the specificity of CTL activity was tested using splenocytes from animals immunized with 20 micrograms of fusion proteins. The results shown in Fig. 6 demonstrate the effective lysis of TC-1 cells transformed with E6 / E7 of HPV16. Lysis of two different cell types (EL4 and MC57G) that do not express an HPV antigen occurred with much reduced efficiency, demonstrating the specificity of the CTL response produced by immunization with fusion proteins Hsp65-E6 and -E7 .

EXAMPLE 8: TUMOR REGRESSION TC-1 IN MICE AFTER TREATMENT WITH FUSION PROTEIN HSP65-E7 Three groups of eight C57 / BIJ6 mice were used in this experiment. Each animal was inoculated with 1.3x105 cells subcutaneously in a shaved area of the back. Two days later, a first group was given saline (negative control), a second group 100 micrograms / animal of fusion protein Hsp65-E7 in saline and a third group 100 micrograms / animal of peptide E7 in saline and IFA (positive control). All injections (0.2 ml) were given subcutaneously at the nape of the neck). Fourteen days later (16 days after the tumor was inoculated), the injections were repeated. Beginning one day after tumor inoculation and every second day thereafter, mice were examined for the presence or absence of tumor by visual inspection and palpation. Fig. 7 revealed that 9 days after tumor inoculation all treatment groups showed maximum tumor incidence 5 (expressed as a percentage of animals showing tumors), ranging from 85 to 100%. However, by day 17, the group treated with the fusion protein Hsp65-E7, as well as the given group with peptide E7 in I FA showed a highly significant decrease in the incidence of tumor that leveled out at approximately 1 5% . In contrast, the treated group with saline continued to have almost 1 00% tumor incidence throughout the rest of the observation period. These results demonstrate that the Hsp65-E7 fusion protein, administered in the absence of helper, induces the drastic regression of a tumor induced with H PV 15 Results of a similar experiment are shown in Fig 8. In this experiment, the animals were inoculated with a higher tumor dose (2x1 05 / animal) than in the previous experiment. The results obtained were closely similar to those of the previous experiment. EXAMPLE 9: COMPARISON OF THE CAPACITY OF PROTEIN E7 AND PROTEIN OF FUSION HSP65-E7 FOR INDUC I R I NMUN ITS CELLULAR RESPONSES The Hsp65-E7 fusion protein was produced and purified as described in Example 4. The full-length HPV E7 protein was obtained by the following procedure. The E7 gene was amplified from HPV1 6 genomic DNA (pSK / H PV1 6 obtained from American Tissue Culture Collection) using AmpliTaq DNA polymerase (Perkin Elmer). The forward primer (5'-AAC CCA GCT GCT AGC ATG CAT GGA GAT-3 ') contained a Nhel site immediately upstream of the ATG start codon, while the reverse primer (5'-AGC CAT GAA TTC TTA) TGG TTT CTG-3 ') contained an EcoRI site immediately downstream of the stop codon TAA of the E7 coding sequence. The PCR product was digested with Nhel and EcoRI, was purified from an agarose gel, and ligated to pET28a that had been digested with the same restriction enzymes. Transformation of bacteria, isolation of colonies containing recombinants, and preparation of plasmid DNA from expanded colonies were performed by standard procedures. See, for example, Ausubel et al. (eds.), Short Protocols in Molecular Biology, (Short Protocols in Molecular Biology), 3rd edition (John Wiley & amp; amp;; Sons, I nc. nineteen ninety five) . The identity of the resulting plasmid construct, pET / E7 (H) was confirmed by diagnostic restriction digestion and DNA sequence analysis. A schematic map of pET / ET (H) is presented in Figure 9. Twelve liters of 2xYT medium containing 30 μg / ml kanamycin were inoculated with a culture of £. coli BL21 (DE3) containing pET / E7 (H) and incubated overnight at 30 ° C with aeration. When an optical density of 0.5 was reached, the culture was induced with 0.5 mM I PTG for three hours. The cells were then harvested by centrifugation, resuspended in 1 80 ml of lysis buffer (10 mM Tris-HCl, pH 7.5, 0.5 mM 2-mercaptoethanol) supplemented with 200 μg / ml lysozyme and frozen at -70. ° C during the night. The cell suspension was frozen in a water bath at 37 ° C in the presence of 2 μg / ml of aprotinin, 2 μg / μl of leupeptin and 2 μg / ml of pepstatin. Following the addition of 2 mM PMSF, the cell suspension was subjected to vigorous sonication and insoluble proteins were collected by centrifugation. Protein pellets were resuspended twice in lysis buffer, rescreened and harvested by centrifugation. Protein pellets were resuspended twice in lysis buffer, resuspended and harvested by centrifugation. The protein pellets were then solubilized by sonication in buffer A (50 mM Tris-HCl, pH 7.5, 6 mM idone hydrochloride, 1 mM 2-mercaptoethanol) and the insoluble material was removed by centrifugation. The solubilized proteins were applied to a 50 ml Ni-Ni chelating column (2.6 cm x 1 2 cm; Pharmacia), which had been pre-equilibrated in buffer A. The bound protein was washed with 5 volumes of buffer bed. E (buffer A with 2% Triton X-1 00) and refolded with a gradient of sodium hydrochloride hydrochloride (0.1 M NaCl / 6.0 M guanidine hydrochloride, 5 bed volumes) in the presence of 1 5 of Triton X-1 00. The re-folded protein was washed with 5 volumes of buffer bed F (30 mM Tris-HCl, pH 7.5, 1 M NaCl, 1 5% glycerol, 2% of Triton X-1 00, 2-mercaptoethanol 1 mM) and subsequently with 5 volumes of cushion bed G (buffer F without Triton X-1 00) to remove Triton X-1 00. The column was further washed with 5 volumes of buffer H (50 mM imidazole, pH 7.5, NaCl 0.5 M, 15% glycerol, 1 mM 2-mercaptoethanol) to remove weakly bound proteins. The E7 protein was levigated with a gradient of linear im idazol (50 μM to 1 M imidazole in H buffer). The E7 protein was concentrated and dialyzed against Dulbecco's phosphate buffered saline, supplemented with 25% glycerol. The soluble protein was stored at -70 ° C. It was found that the preparation of E7 is essentially homogeneous by SDS-PAGE and protein staining. The endotoxin concentrations of the protein preparations were assessed by the lipid ammocyte assay and found to be no greater than 50 endotoxin units per milligram of protein. Groups of five C57BL / 6 mice were immunized by tail-base injection with saline, or 0.055, 0.55 or 1.8 nanomoles of E7 protein or Hsp65-E7 fusion protein in ina salt solution. The injection volumes were 0.2 ml. Ten days later, the inguinal lymph nodes (LN) of each animal were removed aseptically, and LN was extracted from each of the five animals in a group. Cell suspensions were prepared by a standard procedure. For each deposit of LN cells (2 x 106 cells / ml), flat-bottom 96-well plates with 4 x 1 05 cells per well were seeded. Cells were probed in triplicate for proliferative responses for the addition of either medium, 1 0 or 50 μg / ml of E7 protein, 1 0 or 1 00 μg / ml of Hsp65 * E7 fusion protein "or 1 or 10 μg / ml. ml of peptide E7 ("pep"); residues 44-57). Following the additions, the cells were incubated for four days at 37 ° C and 5% OO2. Tritiated thymidine (1 μCi) was added to each culture. After 1.5 hours of additional incubation, the cells were harvested and prepared by scintillation counting. The data are presented in Figure 1 0 as average cpm of incorporated radioactivity +/- standard deviation. The different panels show assays with LN cells from animals immunized with the different amounts of fusion protein Hsp65-E7 or protein E7 declared above. The results show that immunization with Hsp65-E7 fusion protein induces cellular immunity to the fusion protein by itself (upper and left central), as well as the E7 protein (central left). Recognition of the E7 protein is further demonstrated by the observed induction of proliferation by E7 peptide, said peptide is known to represent an epitope of T helper cells (left central). In contrast, no proliferative responses were observed with immune-mediated LN cells immunized with different amounts of E7 protein (right central and bottom) or immunized simulated with saline. Cells prepared from animals immunized with E7 protein were viable, as evidenced by their ability to proliferate in response to T cell mitogen ConA. In summary, when compared to immunization with E7, immunization with fusion protein Hsp65-E7 produces a cellular immune response higher than E7, as assessed by the LN cells of immunized animals.

EJ £ t SO 10: TREATMENT WITH FUSION PROTEIN HSP65-E7 CAUSES REGRESSION OF ESTABLISHED TUMORS CONSIDERABLE To test the efficacy of the Hsp65-E7 fusion protein in tumor therapy, C57 / BL / 6 mice were inoculated with 1.3 x 105 TC-1 tumor cells by subcutaneous injection in a shaved area of the back. Seven days later, when all the animals had developed palpable / measurable tumors, the animals were arbitrarily assigned to three treatment groups. Each group included 12-14 animals. All treatments were by subcutaneous injection of a volume of 0.2 ml at the nape of the neck. The first group was injected with 100 μg of fusion protein Hsp65-E7, the second group with 20 μg of E7 protein (corresponding to a similar molar quantity as 100 μg of fusion protein) and the third group with saline. Beginning one day after the tumor inoculation, the mice were visually inspected and palpated for the presence of tumor. Tumor volumes were determined using calibrators in two orthogonal directions. Volumes were obtained from these measurements using the conversion formula described by Naito et al., Cancer Research 464109 (1986). The results are presented in Figure 11 as the average tumor volume in each group +/- standard error.

The results show that the treatment with the fusion protein Hsp65-E7 resulted in complete regression of established, considerable tumors. The effect was manifest in each of the treated animals. In contrast, neither sham treatment nor treatment with E7 protein caused more than a transient regression of the tumors. A statistical evaluation of tumor measurements, made at three late moments in the experiment, is presented in Table 2. As is evident from the calculated p-values, the effects on the tumor size of the treatment with Hsp65- fusion protein E7, are statistically different from the effects of the other treatments.

TABLE 2 COMPARISON STATISTIC SITUATION OF GRU POS OF TRATAM I ENTO Note that in the previous Examples, the Hsp65-E7 protein was produced as a histidine-labeled protein. To clearly demonstrate that the observed therapeutic effects were in no way related to the presence in the fusion protein of an oligo-isthidine sequence, Hsp65-E7 fusion protein was prepared and used lacking the histidine tag in this Example. The fusion protein was obtained using the following procedure. For the construction of plasmid ET65C (Figure 1 2), the 5 hsp65 gene from Bacil lus Calmette Guerin (BCG) was amplified from plasmid RI B 1 300 (Van Edén et al., Nature 331: 1 71, 1 988 ) using AmpliTaq DNA polymerase (Perkin Elmer). The forward forward primer used (5'-TTC GCC ATG GCC AAG ATT GCG-3 ') contained an Ndel site that included the ATG start codon of the hsp65 gene, and the reverse primer (5'-CGC TCG GAC GCT AGC TCA CAT ATG GAA ATC CAT GCC-3 ') contained a site Ndel immediately downstream of the stop codon TGA of the coding sequence of Hsp65 and an N hel site immediately downstream. The PCR product was digested with Ncol and Nhel and ligated to pET28a digested in a similar manner. The ligation mixture was transformed in DH5a of £. coli, and resistant colonies were isolated and amplified to the antibiotic for the preparation of the plasmid DNA. Plasmid ET65C was identified by diagnostic restriction digests and DNA sequence analysis. To prepare the plasmid Et65C / E7-1 N containing a gene from Hsp65-Eu fusion protein without label, the E7 gene was amplified from genomic DNA of H PV 1 6 (pSK / H PV1 6 obtained from American Tissue Culture Collection) using Am pliTaq DNA polymerase (Perkin Elmer). The forward primer (5'-CCA GCT GTA CAT ATG CAT GGA GAT-3 ') contained an Ndel site that included the start codon of ATG, while the reverse primer (5'-AGC CAT GAA TTC TTA TGG TTT CTG-3 ') contained '& & * - * T- ^ .j & an EcoRI site immediately downstream of the stop codon TAA of the E7 coding sequence. The PCR product was digested with Ndel and EcoRI, purified from an agarose gel, and labeled to pET65C which had been digested with the same restriction enzymes. Transformation of 5 bacteria, isolation of colonies containing recombinants and preparation of plasmid DNA from expanded colonies were performed by standard procedures. See, for example, Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3rd edition (John Wiley &Sons, I nc 1 995). The identity of the resulting plasmid construct, pET65C / E7-1 N was confirmed by diagnostic restriction digestion and DNA sequence analysis. A schematic map of pET65C / E7-1 N is presented in Figure 1 3. Twelve liters of 2xYT medium containing 30 μg / ml kanamycin was inoculated with a culture of £. coli BL21 (DE3) containing pET65C / E7-15 N and incubated overnight at 30 ° C with aeration. When an optical density of 0.5 was reached, the culture was induced with 0.5 mM I PTG for three hours. The cells were then harvested by centrifugation, resuspended in 1 80 μl of 1 μm 8Tris-HCl lysis buffer, pH 7.5, 0.5 μM 2-mercaptoethanol), supplemented with 200 μg / ml lysozyme and frozen at -70 ° C overnight. The cell suspensions were thawed in a water bath at 37 ° C in the presence of 2 μg / ml of aprotinin, 2 μg / ml of leupeptin, and 2 μg / ml of pepstatin. Following the addition of 2 mM PMSF, the cell suspensions were subjected to vigorous sonication, and the insoluble proteins were removed by centrifugation. Most protein Hsp65-E7 without Marking was found in the soluble protein fraction. To remove the endotoxin, 1% Triton X-114 was added to the soluble protein fraction, and the mixture was chilled on ice (for 5 minutes or more) and mixed thoroughly. The mixture was then heated for 10 minutes in a waterbath at 30 ° C and then subjected to centrifugation at 24 ° C. The clear supernatant fraction (upper layer) was transferred to a clean tube. The centrifugation was repeated 3-6 times to remove residual Triton X-114. The supernatant fraction was then subjected to fractionation of ammonium sulfate. The fusion protein was recovered in the fraction of 0-15% ammonium sulfate (w / v). The precipitated protein was dissolved in buffer B (30 mM Tris-HCl, pH 7.5, 3 M urea, 1 mM EDTA, 1 mM 2-mercaptoethanol) and applied to a 170 ml Source Q column (3.5 cm x 20 cm; Pharmacia) pre-equilibrated with buffer B. The column was washed with 3 bed volumes of buffer C (buffer B minus urea) and then with 3 bed volumes of buffer D (buffer C supplemented with 250 mM NaCl). The fusion protein was levigated with a linear salt gradient (250 mM -1 M NaCl in buffer C) (tank A) and then with 6 M guanidinium hydrochloride (buffer A) (tank B). Deposit B was applied to a chelating column with Ni of 50 ml (2.6 cm x 12 cm, Pharmacia) pre-equilibrated with buffer A. The bound protein was washed with 5 volumes of buffer bed E (buffer A with 2% Triton X -100) and doubled again with a gradient of guanidinium hydrochloride-sodium chloride (0.1 M naCl / 6.0M guanidinium hydrochloride, 5 bed volumes) in the presence of 15 Triton X-100. The re-folded protein c * 4 ¿8 * Eia¡ > »Was washed with 5 volumes of buffer bed F (30 mM Tris-HCl, pH 7.5, 1 M NaCl, 15% glycerol, 2% Triton X-1 00, 1 mM 2-mercaptoethanol) and subsequently with 5 volumes of cushion bed G (shock absorber F without Triton X-1 00) to remove Triton X-1 00. The column was further washed with 5 volumes of buffer bed H (imidazole 50 mM, pH 7.5, NaCl 0.5 M, 1 5 % glycerol, 1 mM 2-mercaptoethanol)) to remove weakly bound proteins. The fusion protein was levigated with a linear imidazole gradient (50 μM to 1 μM imidazole in H buffer). The unlabeled Hsp65-E7 protein was concentrated and dialyzed against Dulbecco's phosphate buffered saline, supplemented with 25% glycerol. The soluble protein was stored at -70 ° C. Analysis by SDS-PAGE and protein staining revealed that the preparation was approximately 90% pure. As an additional demonstration of the minor importance of the histadine tag, the efficiencies of Hsp65-E7 labeled with histidine and unlabeled Hsp65-E7 were compared directly in an experiment substantially identical to that described above. It was found that the two fusion proteins retrodegrade tumors with similar efficacy.

EQUIVALENTS Those skilled in the art will know, or will be able to inquire, using nothing more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is intended that these and other equivalents be encompassed by the following claims.

Claims (44)

1. REIVI NDICATIONS 1 . A composition for inducing an immune response against an H PV protein antigen in a subject, the composition comprising an H PV protein antigen linked to a strain protein.
2. 2. The composition of claim 1, wherein the immune response is a cellular immune response.
3. 3. The composition of claim 1, wherein the tension protein is a mycobacterial strain protein.
4. 4. The composition of claim 1, wherein the H PV protein antigen is the E6 or E7 protein.
5. 5. The composition of claim 1, wherein the tension protein is of the Hsp60 or Hsp70 family.
6. 6. A composition for inducing an immune response against an HVP protein antigen in a subject, the composition comprising an HPV protein antigen conjugated to a strain protein. The composition of claim 6, wherein the immune response is a cellular immune response. 8. The composition of claim 6, wherein the tension protein is a mycobacterial strain protein. The composition of claim 6, wherein the H PV protein antigen is the E6 or E7 protein. The composition of claim 6, wherein the tension protein is of the HspdO or Hsp70 family. eleven . A composition for inducing an immune response against an HPV protein antigen in a subject, the composition comprising a fusion protein, comprising an HPV protein antigen fused to a strain protein. The composition of claim 1, wherein the immune response is a cellular immune response. The composition of claim 1, wherein the tension protein is a mycobacterial strain protein. 14. The composition of the 1 1 re-excitation, where the H PV protein antigen is the E6 or E7 protein. The composition of claim 1, wherein the tension protein is of the HspdO or Hsp70 family. 1 6. A composition comprising an HPV protein antigen linked to a tension protein, in combination with another pharmacologically acceptable component.
7. 7. The composition of claim 16, wherein the tension protein is a mycobacterial strain protein. The composition of claim 1 6, wherein the H PV protein antigen is the E6 or E7 protein 19. The composition of claim 16, wherein the strain protein is of the Hsp60 or Hsp70 family. 20. A conjugate comprising an HVP protein antigen conjugated to a tension protein. twenty-one . The conjugate of claim 20, wherein the tension protein is a mycobacterial strain protein. 22. The conjugate of claim 20, wherein the H PV protein antigen is the E6 or E7 protein. 23. The conjugate of claim 20, wherein the tension protein is of the Hsp60 or Hsp70 family. 24. A fusion protein comprising an H PV protein antigen fused to a strain protein. 25. The fusion protein of claim 24, wherein the tension protein is a mycobacterial strain protein. 26. The fusion protein of claim 24, wherein the H PV protein antigen is the E6 or E7 protein. 27. The fusion protein of claim 24, wherein the tension protein is of the Hsp60 or Hsp70 family. 28. The fusion protein of claim 24, wherein the H PV protein antigen is E6 or E7 and the strain protein is of the Hsp60 or Hsp70 family. 29. A composition for inducing an immune response against an HPV protein antigen in a subject, the composition comprising an expression vector that encodes and is capable of directing the expression of the fusion protein of claim 24 in cells of the subject. 30. The composition of claim 29, wherein the immune response is a cellular immune response. 31 A composition for inducing an immune response against an HPV protein antigen in a subject, the composition comprising an expression vector that encodes and is capable of directing the expression of the fusion protein of claim 28 in cells of the subject. 32. The composition of claim 31, wherein the immune response is a cellular immune response. 33. An expression vector that encodes and is capable of directing the expression of the fusion protein of claim 24 in cells of a subject. 34. An expression vector that encodes and is capable of directing the expression of the fusion protein of claim 28 in cells of a subject. 35. A method for inducing an immune response against an HPV protein antigen, comprising administering to a subject an effective amount of the expression vector of claim 33. 36. A method for inducing an immune response against a protein antigen. of HPV, comprising administering to a subject an effective amount of the expression vector of claim 34. 37. A method for inducing an immune response against an HPV protein antigen, comprising administering to a subject an effective amount of the composition of the invention. claim 1 6. 38. A method for inducing an immune response against an HPV protein antigen, comprising administering to a subject an effective amount of the conjugate of claim 20. 39. A method for inducing an immune response against a protein antigen. of HPV, comprising administering to a subject an effective amount of the fusion protein of claim 24. A method for treating a tumor exhibiting a H PV protein antigen, comprising administering to a subject an effective amount of the expression vector of claim 33. 41. A method for treating a tumor exhibiting a H PV protein antigen, comprising admixing to a subject an effective amount of the expression vector of claim 34. 42. A method for treating a tumor expressing an HPV protein antigen. , comprising administering to a subject an effective amount of the composition of claim 1. 43. A method for treating a tumor expressing a H PV protein antigen, comprising administering to a subject an effective amount of the composition of the Claim 20. 44. A method for treating a tumor expressing an HPV protein antigen, comprising administering to a subject an effective amount of the composition of claim 24. SUMMARY The present invention relates to compositions for inducing an immune response, preferably a cellular immune response, in particular a cytolytic, cell-mediated immune response, to human papillomavirus (HPV) protein antigens shown by HPV or exhibited by infected cells that they include cervical tumor cells and other tumors. In one embodiment, the compositions comprise an H PV protein antigen linked to a strain protein (or heat shock protein (Hsp)). The HPV protein antigen can be bound to the tension protein by chemical conjugation or non-covalently using linker portions, or the H PV protein antigen and the strain protein can be bound in a fusion protein containing both sequences. HPV protein antigen and tension protein. In another embodiment, the compositions comprise an expression vector that includes, in an expressible form, sequences encoding the H PV protein antigen and sequences encoding the strain protein. The expression vector can be introduced into cells from a subject, or it can be used to transduce cells from the subject ex vivo, resulting in the expression of an HPV-protein protein antigen fusion protein, which will stimulate the immune response of the subject. subject to the HPV protein antigen. The present invention also relates to compositions comprising a voltage protein linked to a H PV antigen and another pharmacologically acceptable component, to conjugates and fusions of HPV protein tension-antigen protein and to expression vectors which encode and are capable of directing expression in subject cells of a fusion protein, comprising a strain protein sequence and HPV protein antigen. The present invention also relates to uses of these compositions to induce immune responses against H PV and cells. that exhibit H PV protein antigen, including tumors associated with H PV