Title:

Methods and compositions relating to HPV-associated pre-cancerous and cancerous growths, including CIN

Document Type and Number:

United States Patent 7410758

Abstract:

The present invention concerns the use of E6 and/or E7 peptides from human papilloma virus (HPV) to evaluate a cell-mediated response in a patient infected with HPV to determine the prognosis for that patient with respect to the development or recurrence of pre-cancerous or cancerous growths, including cervical intraepithelial neoplasia (CIN).

Inventors:

Sastry, Jagannadha K. (Katy, TX, US)
Tortolero-luna, Guillermo (Houston, TX, US)
Follen, Michele (Houston, TX, US)

Plaque It!

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of immunology, virology, and oncology. More particularly, it concerns diagnostic and therapeutic methods related to the development and recurrence of pre-cancerous and cancerous growths or lesions, including cervical intraepithelial neoplasia (CIN), caused by human papilloma virus (HPV).

2. Description of Related Art

Cervical cancer is the second most common malignancy in women worldwide, accounting for 15% of all cancers diagnosed in women (Parkin et al., 1993). In the United States, cervical cancer is one of the most common neoplasm of the female genital tract. Laboratory and epidemiological research has focused on the etiological role of some types of human papilloma virus (HPV) in the pathogenesis of cervical neoplasia (Brinton, 1992; Munoz et al., 1992). Overall, HPV DNA has been detected in more than 79% of specimens of women with definite cervical disease. The most prevalent HPV type is HPV 16, which is detected in high-grade squamous intraepithelial lesions and cancer (Lörincz et al., 1992). Results from epidemiological studies support an association between cervical neoplasia and HPV, which is markedly stronger with HPV type 16 (Morrison et al., 1991; Koutsky et al., 1992; and Munoz et al., 1992). Neoplasia is characterized by abnormal growth of cells, which often results in the invasion of normal tissues, e.g., primary tumors or the spread to distant organs, e.g., metastasis.

The E6 and E7 genes of HPV 16 are frequently co-expressed and are most abundant viral transcripts in biopsies from HPV 16 positive cervical carcinoma (Wettstein, 1990; Seedorf et al., 1987). There is a strong evidence that co-expression of both E6 and E7 open reading frames is necessary and sufficient for efficient malignant transformation of a variety of mammalian cells (Munger et al., 1989). Furthermore, continued expression of the E6 and E7 regions of the viral genome appears to be required to maintain the malignant phenotype (von Knebel Doeberitz et al., 1988).

While some HPV infected patients develop cervical neoplasia, others do not. Also there is a high rate of spontaneous regression observed indicating the role of host immune responses. The induction of a cytotoxic T-lymphocyte (CTL) response constitutes a significant defense mechanism against viral infections; occasionally, a virus-specific. CTL response can render full protection without a concomitant antibody response (Sastry et al., 1992; Bevan, 1989; Lukacher, 1984). Based on reports in the literature describing a relation between increased prevalence of anti-HPV antibodies, in particular those directed against the E 7 oncoprotein, with severity of the cervical disease (Cason et al., 1992; Hamsikova et al., 1994; Jha et al., 1993), it has been suggested that HPV-specific humoral response may not play a protective role against HPV-associated cervical neoplasia (Nakagawa et al., 1996). On the other hand, it has been reported that individuals with defects in CMI have an increased prevalence of HPV-associated cervical neoplasia, indicating that T cells participate in the control of HPV-associated neoplasia in humans (Nakagawa et al., 1996; Tsukui et al.; 1996; Feltkamp et al., 1993 and Clerici et al., 1997). Decreased IL-2 production and proliferative responses to mitogens such as PHA and concanavalin-A have been observed in patients with invasive cervical carcinoma (Park et al., 1992). A number of in vitro and in vivo strategies have been described to identify peptides from HPV-16 E6, E7, and L1 proteins that induce T-cell activity in mice and humans (Feltkamp et al., 1993; Strang et al., 1990; Tindel et al., 1991; Shepherd et al., 1992; Stauss et al., 1992; Kast et al., 1993). Typically, induction of virus-specific CTLs can be effected by infection with a virus or recombinant virus that expresses a viral gene product. The viral gene product is processed and presented as a peptide on the surface of infected cells in association with an MHC class I molecule for recognition by the CTL (Unanue, 1989).

Additionally, research efforts have concentrated on identifying and characterizing HIV peptides that elicit a viral-specific CTL response. Townsend et al. illustrated the concept of using T-cell epitopes in proteins as vaccine candidates when their group demonstrated the use of short synthetic peptides from influenza nucleoprotein as epitopes for CTL responses (Townsend et al., 1986). The inventors and others have reported using synthetic peptides to generate virus-specific CTLs in vivo (Kast et al, 1991; Aichele et al., 1990; Deres et al, 1989; Sastry et al., 1992; Sastry et al., 1994; Casement et al., 1995) against influenza, lymphocytic choriomeningitis, Sendai virus and HIV.

Over 90% of cervical carcinomas express human papillomavirus (HPV) E6 and E7 proteins. These unique antigens are ideal targets for the development of cytotoxic T-lymphocytes (CTL) for antitumor immunotherapy. Synthetic peptides have been identified corresponding with the E6 and E7 oncoproteins of HPV-16 that were effective in including HPV-specific CTL responses in vivo (Sarkar et al., 1995). Recently, Nakagawa et al. reported that systemic T-cell proliferative responses and CTL responses to HPV-16 peptides and proteins were detectable in many virgin as well as sexually active women without cervical lesions but not in those with active disease (Nakagawa et al., 1997). Similarly, Tsukui et al. reported that TH lymphocyte response, particularly IL-2 production, to HPV antigens was greater among cytologically normal women than in women with different degrees of progressive cervical neoplasia (Tsukui et al., 1996). Also, Clerici et al observed that production of TH1 cytokines (IL-2 and IFN-γ) which potentially enhances CMI, to be defective in women with extensive HPV infection and that progression to CIN to be associated with a shift from TH1 to TH2 cytokine production (Clerici et al., 1997). Employing a long term in vitro stimulation protocol for determining the TH activity Kadish et al. reported that lymphoproliferative responses to specific HPV peptides were associated with HPV clearance and regression to CIN (Kadish et al., 1997). On the other hand, de Gruijil et al. reported that T-cell proliferative responses to HPV16 E7 peptides correlated with persistence of HPV infection, but antigen-specific IL-2 production was associated with both virus clearance as well as progression of cervical lesions (de Gruijil et al., 1996).

A common clinical management strategy for CIN patients includes excisional or ablative treatment. However, follow-up studies indicate that a significant number of patients experience recurrence. At present no clear understanding exists regarding the development of pre-cancerous or cancerous growths, their recurrence, or disease-free status in the patients who have undergone ablative or excisional treatment for CIN. Better and improved strategies for effective diagnostics of HPV-associated pre-cancerous or cancerous growths and lesions is needed.

SUMMARY OF THE INVENTION

The present invention is based on the observation that a cell-mediated immune (CMI) response by patients infected with human papilloma virus (HPV) against E6 and/or E7 peptides of HPV is correlated with their prognosis. A cell-mediated immune response is indicative of a reduced risk for the development of pre-cancerous or cancerous growths in the genitourinary tract, particularly the cervix, than the risk for a patient who does not exhibit a cell-mediated immune response; in other words, a patient who exhibits a positive cell-mediate immune response to particular E6 and/or E7 peptides has a good prognosis with respect to the development of HPV-associated pre-cancerous or cancerous growths. Alternatively, a patient who exhibits no or a low CMI response to an E6 or E7 proteinaceous compound of HPV has a greater risk of a bad prognosis with respect to physiological effects as a result of HPV infection. Thus, the present invention encompasses compositions and methods for identifying patients at risk for HPV-related hyperproliferative conditions, including warts, CIN, and malignant growths or other pre-cancerous or cancerous growths; the invention is particularly suited to evaluating patients for recurrence of a hyperproliferative condition. As used herein, the terms “growth” and “lesion” are used interchangeably. Also, the term “pre-cancerous or cancerous growth” refers to HPV-associated growths. In addition to pre-cancerous or cancerous growths or lesions on the cervix, such growths or lesions may occur throughout the urogenitary tract and they include perineal, vulvar and penile growths or lesions. Patients for whom the methods may be applied include any mammals capable of HPV infection; in some embodiments, the patient is specifically contemplated to be a human, either male or female.

In some embodiments the present invention encompasses methods for determining the possibility of the development or recurrence of a pre-cancerous or cancerous growth in a patient infected with human papilloma virus. In some cases, the patient has been treated for the growth. The methods involve employing the following steps: obtaining a sample from the patient; incubating the sample with at least one E6 or E7 peptide of HPV; and assaying the sample for a cell-mediated immune (CMI) response against the peptide. A cell-mediated immune response against an E6 or E7 peptide, or a combination thereof indicates a reduced risk of recurrence as compared to a person who does not exhibit such a response. A pre-cancerous growth frequently observed with the development of cervical cancer is cervical intraepithelial neoplasia or CIN. In some embodiments of the invention, the method of the invention is used with respect to patients who have or had CIN of any stage (CIN 1, CIN 2, or CIN 3 or squamous intraepithelial lesions (SIL), low grade-SIL (L-SIL) and high grade SIL (H-SIL)). Furthermore, in other embodiments, the methods may be implemented with patients who have or had more severe stages of hyperproliferative growth than CIN, such as a malignancy or cancerous growth. As used in this application, the term “recurrence” refers to the appearance of a pre-cancerous or cancerous growth, or a reappearance of the first growth, or evidence thereof, after a first pre-cancerous or cancerous growth was reduced, eliminated, or treated. As used herein, the term “incubating” refers to exposing or contacting the sample with a composition that includes a peptide.

The claimed methods have applicability to human papilloma virus infections. The human papilloma virus may be a high grade or high risk type, such as HPV 16, 18, 31, 45, 56, or 58. In some embodiments, the human papilloma virus is HPV 16. In other embodiments, the human papilloma virus is a intermediate risk type, such as HPV 33, 35, 37, 51, 52, 59, 66, or 68. In still further embodiments the HPV is a low risk or low grade type associated with warts, such as type 6, 11, 26, 40, 42, 43, 44, 53, 55, 62, or 66.

The sample will include cells that give rise to a cell-mediated immune response. In some embodiments, the sample is a blood sample or serum sample, while in other embodiments, the sample is obtained by lavage, smear, or swab of the area suspected of infection or known to be infected, such as in the vaginal, cervical, or penile area. Peripheral blood mononuclear cells (PBMC) can render a cell-mediated immune response and any sample containing such cells can also be employed in methods of the invention. In some embodiments, it is contemplated that cells from a sample are incubated in media after they have been obtained but before they are assayed. It is contemplated that the sample may be incubated up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more hours and up to 1, 2, 3, 4, 5, 6, or 7 days, and up to 1, 2, 3, 4, or 5 or more weeks and up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months in media prior to assaying. It is also contemplated that the cells may be incubated in media and/or stored under conditions of sub-zero degrees centrigrade prior to assaying. The cells themselves or the cell culture supernatant (media, not intact cells) may be used for subsequent assays for a cell-mediated immune response. In some embodiments, the cells are incubated between 2 and 8 hours—in some cases for 6 hours—in media prior to performing intracellular cytokine analysis by flow cytometry. In other embodiments, the cells are incubated in media between 2 days and 20 days—in some cases 15 days—in media prior to performing chromium release assays to determine cytotoxic T lymphocyte (CTL) activity.

Methods of the present invention involve determining whether a patient exhibits a cell-mediated immune response against HPV peptides. In several embodiments, the peptides are E6 or E7 peptides meaning they have an amino acid sequence that is at least 90% identical over its length to a contiguous amino acid sequence in an E6 or E7 polypeptide. Specifically contemplated is the use of a peptide comprising 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more contiguous amino acids of SEQ ID NO:19 (E6 from HPV 16) or SEQ ID NO:20 (E7 from HPV 16). It is contemplated that in some embodiments that peptides of only one sequence are tested—for example, an E6 peptide or in another example, an E7 peptide—while in other embodiments, multiple sequences may be tested. In one embodiment, an E6 and an E7 peptide are employed in methods of the invention. In other embodiments, at least two E6 peptides (referring to at least two different E6 sequences), at least two E7 peptides (referring to at least two different E7 sequences), or both may be implemented. It is contemplated that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or more E6 or E7 peptides may be employed, as well as any combination of E6 or E7 peptides thereof. In still further embodiments the E6 peptide is K9L, E10I, C10R, Q15L, V10C, P9L, P10I, Q20P, R16R, or G10S, or a combination thereof. In specific embodiments, the following E6 peptides are employed individually or as a cocktail that includes one or more of the following peptides: K9L, E10I, C10R, Q15L, or V10C. While in other embodiments, an E7 peptide is T10Q, M9T, D9L, Q19D, R9F, R9V, L9V, G10C, or D20C, or a combination thereof. In certain embodiments, the following E7 peptides are employed individually or as a cocktail Q19D, R9F, R9V, L9V, G10C. Furthermore, a cocktail that includes at least one E6 peptide and one E7 peptide from the following is contemplated: K9L, E10I, C10R, Q15L, V10C, Q19D, R9F, R9V, L9V, or G10C. In some embodiments, it is specifically contemplated that one or more peptides in the cocktails described above be excluded. It is also specifically contemplated that compositions discussed with respect to diagnostic methods of the invention may also be applied to preventative or therapeutic methods of the invention.

Methods of the invention concern a cell-mediated immune (CMI) response against human papilloma virus. There are different ways of identifying and evaluating a cell-mediated immune response (distinguished from a serum or antibody-mediated immune response). In one embodiment of the invention, T cell proliferation is measured. T-cell proliferation may be assayed by measuring incorporation of tritiated thymidine. A proliferative response of equal to or greater than 2.0 using an SI scale to at least one E6 or E7 peptide is considered positive and is indicative a patient with a reduced risk of recurrence of a pre-cancerous or cancerous growth or lesion. A proliferative response of equal to or greater than 3.0 using an SI scale to at least one E6 or E7 peptide is indicative of a cell mediated response, and thus, identifies a patient with a improved prognosis with respect to the development of pre-cancerous or cancerous growths. Alternatively, a patient who has an SI of less than 2.0, including an SI of zero, would be considered to have a low or no cell-mediated immune response to the E6 or E7 peptide(s) and would be considered as having an increased risk for the development or recurrence of pre-cancerous or cancerous growths.

A cell mediated response can also be measured using non-radioactive means such as an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) dye or reduction assay, which is a calorimetric assay for live cells (T cell proliferation) (daCosta et al., 1999), or the alamar Blue assay, another colorimetric assay that measures IL-2-responding cells (Gloeckner et al, 2001; Kwack et al, 2000).

In another embodiment of the invention, assaying for a cell-mediated immune response involves measuring TH1 or TH2 cytokine amounts. Even if a patient does not exhibit a CMI response by a T-cell proliferation assay, an increased risk of recurrence may be associated with the production of a TH2 cytokine, such as IL-10. A reduced risk of recurrence is observed with a patient who exhibits production of a TH1 cytokine such as IFN-γ and IL-2 in response to an E6 and/or E7 peptide. In some examples, the amount of a TH1 cytokine is measured, such as IL-2, interferon (IFN) γ, tumor necrosis factor (TNF) α, or TNF-β, IL-3, IL-12, IL-15, IL-16, IL-17, or IL-18. In a specific embodiment, the amount of IL-18 is measured. In additional examples, the amount of a TH2 cytokine is measured, such as IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-13 or IL-14.

Measuring a CMI response may be accomplished by an immunoassay, such as ELISA or a radioimmunoassay, or by flow-cytometry. In embodiments of the invention, a sample may be assayed more than once either as duplicate samples or by different assays. In some embodiments, more than one sample is obtained from the patient. The multiple samples may be the same type, for example, multiple blood sample, or they may be different types, for example, a blood sample and a vaginal swab.

Patients for whom the methods of the invention have applicability include patients not yet diagnosed with HPV but suspected of having HPV, patients once infected with HPV but no longer showing signs of HPV infection, patients known to be infected with HPV, patients with a pre-cancerous or cancerous growth on or around the cervix or other genitourinary area who may or may not know she is infected with HPV, patients whose pre-cancerous or cancerous growth(s) have been treated successfully or unsuccessfully, and patients who have had at least one recurrence of a pre-cancerous or cancerous growth(s). A pre-cancerous or cancerous growth or lesion refers to a hyperproliferative cells whose growth is not controlled, and includes pre-neoplasias, such as CIN and neoplasias—benign and malignant—involving squamous epithelial cells and atypical squamous cells of uncertain significance (ASCUS). It is contemplated that a patient have more than one growth or lesion. Treatment for any growths may involve surgery—ablative or excisional—as well as conventional cancer therapy and treatment against HPV. Such treatments include chemotherapy, radiation therapy, hormonal therapy, immunotherapy, administration of foscarnet, Thiovir, thiovir analogs (BioKeys), podofilox, podophyllin, trichloracetic acid (TCA), or 5-fluorouracil (5-FU), intralesional or intransal interferon, Imiquimid cream. Ablative techniques include the use of liquid nitrogen, electrocautery or electrodissection, surgical excision, or laser technology. A successful treatment refers to treatment that completely removes any signs of a growth, while a partially successful treatment refers to a treatment that affects the growth by reducing its size or growth rate, or preventing its enlargement, or reducing the number of growths if there is more than one. Patients once infected with HPV may at later times not exhibits signs of an HPV infection. However, it is believed such patients may still experience recurrence of a pre-cancerous or cancerous growth, like patients who have signs of continued HPV infection.

In some methods of the invention, the patient is evaluated to determine whether he/she is infected with HPV. In further embodiments, a serotyping of HPV is also included or is part of the initial determination of infection. In still further embodiments, the patient is evaluated to determine whether she has a pre-cancerous or cancerous growth, and if it is cancerous, whether the growth is benign or malignant.

Methods of the invention include embodiments in which the sample is obtained from the patient at least one month after treatment for a pre-cancerous or cancerous growth. The patient may have undergone treatment for at least one pre-cancerous or cancerous growth, such as by some form of ablation.

The present invention also includes therapeutic methods that may be employed with the diagnostic methods of the invention. In some embodiments of the invention, a patient is identified as having an increased risk for the development of recurrence of pre-cancerous or cancerous growths. A course of action that was not previously considered prior to the patient being identified as having that increased risk may be undertaken. In some embodiments, a patient who would not otherwise be treated is administered preventative treatment against pre-cancerous or cancerous growths or examined more frequently, or both. Preventative treatments are treatments administered in the absence of physiological signs of pre-cancerous or cancerous growths; “therapeutic treatment” encompasses medical treatment of a physiological condition that the patient exhibits. These preventative treatments include the use of therapeutic treatments for both HPV infection and HPV-associated pre-cancerous and cancerous growths, as described above.

In some embodiments, a preventative method to protect against or reduce the risk of the development of pre-cancerous and cancerous growths involves immunotherapy with HPV E6 and E7 peptides disclosed herein. If a patient is identified as having a low or no cell-mediated immune response against a particular E6 or E7 peptide, or against a combination of such peptides, a peptide or peptides of E6 or E7 sequence may be administered to the patient to elicit a CMI response. Such peptides include any E6 or E7 peptide, specifically including all or part of the peptides of Table 3. Also, peptides from an E6 or E7 polypeptide, such as those discussed with respect to diagnostic methods of the invention, may be employed in preventative methods as well. It is contemplated that the patient may be administered a composition containing one or more peptide sequences, and in some embodiments, with an adjuvant, liposome-based compound, or both. In further embodiments, the patient is administered peptides more than one time.

In some embodiments, there is a method for preventing recurrence of a pre-cancerous or cancerous growth, such as CIN, in a patient infected with HPV and treated for the growth by identifying a patient at risk for recurrence of an HPV-associated growth using methods disclosed herein; and, treating the patient to prevent or treat any recurrence. Treatment options may involve surgery—ablative or excisional—as well as conventional cancer therapy and treatment against HPV, as described above. In some embodiments, the treatment is the immunotherapy treatment involving at least one E6 or E7 peptide from HPV described above.

Furthermore, the present invention also encompasses kits for determining the possibility of recurrence of a pre-cancerous or cancer growth in a patient once infected with HPV and treated for the growth comprising, in a suitable container means, at least one E6 or E7 peptide from HPV and an antibody that allows the detection of a cell-mediated immune response against the peptide. In some embodiments, the antibody is attached to a non-reacting structure on which a sample can be applied, such as a plate with wells. In further embodiments, the non-reacting structure has membrane, which can be affixed or attached to the structure. In some embodiments, the kit can be used in an Enzyme-Linked Immunospot (ELISPOT) Assay to detect, and in some embodiments, quantitate, cytokine secreting cells. In still further embodiments, the kit includes an antibody against a TH1 or TH2 cytokine disclosed herein. Other embodiments include a detection reagent to detect the included antibody. A detection reagent is any compound that allows the detection of another compound, including reagents that allow detection visually, such as by a colorimetric detection reagent.

The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1A-B. Proliferative responses to different E6 and E7 synthetic peptides by PBMC from women in four different study groups. Women in group 1 were normal (CIN ⁽⁻⁾/HPV ⁽⁻⁾, n=6), in group 2 were freshly diagnosed to be HPV-associated CIN (CIN ⁽⁺⁾/HPV ⁽⁺⁾, n=31), group 3 were disease-free post-treatment (Recur ⁽⁻⁾, n=22) and group 4 were with disease recurrence (Recur ⁽⁺⁾, n=10). PBMC from women in the four different groups were tested for proliferative responses to peptides from the E6 or E7 oncoproteins of HPV-16. A. The stimulation index (SI) values calculated as fold increase in ³[H] thymidine incorporation in peptide-treated samples over medium controls were shown for each patient for each of the peptides tested. B. A summary of positive responses to E6, E7, or both peptides by each group. Group numbers are indicated on the x-axis, while the percentage of positive responders in indicated on the y-axis.

FIG. 2A-D. Proliferative responses of PBMC from patients in groups 3 (Recur ⁽⁻⁾) and 4 (Recur ⁽⁺⁾) to synthetic peptides from the E6 and E7 oncoproteins of HPV-16. PBMC from women in these groups were tested for proliferative responses to a control peptide (control), and 7 and 8 peptides each from the E6 and E7 oncoproteins, respectively, of HPV-16. Representative data showing SI values from two patients each from group 3 (panels A and B) and group 4 (panels C and D) are shown.

FIG. 3. Production of TH1 cytokines by PBMC from women in group 3 in response to stimulation with selected E6 and E7 peptides. PBMC from women in groups 3 (Recur ⁽⁻⁾) and 4 (Recur ⁽⁺⁾) were cultured in the presence of the E6 peptide Q15L and E7 peptide Q19D for two days and the supernatant fluid in each case was analyzed for the presence of various TH1 (IL-2, IFN-γ, IL-12) and TH2 (IL-4, IL-10) cytokines by ELISA. The amount of each cytokine produced, after adjusting to medium controls was shown for each of the patient tested from groups 3 and 4.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Human Papilloma Virus (HPV) infection is a major risk factor for cervical cancer, and there is an association between strong HPV-specific cell mediated immunity and less severe stages of CIN. A common clinical management strategy for CIN patients includes excisional or ablative treatment but, follow up studies indicate that a significant number of patients experience recurrence. At present no clear understanding exists regarding disease recurrence or disease free status in these patients. Prognostics and treatment or preventative therapies against CIN in both infected, uninfected and persons who undergo a recurrence of the disease are critical. Many treatment therapies have been tested and implemented but they have yet to eliminate the disease or prevent the recurrence of the disease. Significant number of patients experience recurrence of CIN but there are no means available to test the possibility and probability of recurrence.

The present invention provide a methods for determining the possibility of recurrence of CIN as a prognostic or biomarker in a patient infected by HPV and treated for CIN. The method involves the assay and analysis of a cell mediated immune response against peptides of HPV oncoproteins such as E6 and E7. The method also helps in identifying an HPV-infected patient at risk for recurrence of CIN. The present invention, further, makes use of targeted delivery systems, kits and immunotherapeutic measures to prevent the recurrence of CIN and to diagnose a patient with high risk of CIN.

I. HPV

Human papillomavirus (HPV) has been identified previously as an important cofactor in the development of stages of cervical neoplasia and cancer. Infection with HPV is however insufficient to cause cervical cancer. Not all women infected woth HPV develop cervical cancer. Women are often treated for dysplastic cervical disease detected at the annual Pap Smear. Despite the existence of Pap smear screening, epidemiological investigations continue to implicate HPV as the single greatest risk factor for progression to cervical neoplasia and cancer. Cervical Intraepithelial Neoplasia (CIN) is a type of cervical cancer caused by Human Papilloma Virus (HPV). HPV is associated with development of cervical cancer, specifically HPV types 16, 18, 31, 45, 56 and 58 These comprise the High Grade Type/High Risk type of HPV. Intermediate grade/risk type include HPV 33, 35, 37, 51, 52, 59, 66, and 68. Other Low Grade Types/Low Risk Type associated with warts are types 6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 62, and 66. These low grade types are not malignant in nature. The HPV genome is presented in an episomal (nonintegrated, circular) form in CIN, whereas the genome is often integrated into host DNA in invasive cervical carcinoma. The E6 and E7 oncoproteins, expressed by high-risk types of HPV, appear critical to the malignant transformation of cervical squamous epithelium, as a consequence of their ability to bind and then inactivate two important tumor suppressor genes, p53 and retinoblastoma gene (Rb). The inactivation of these tumor suppressor proteins appears to be a critical component of the oncogenic potential of HPV.

A. Diagnosis and Treatment of Cervical Cancer

Human Papillomavirus has been identified previously to be associated with the development of cervical carcinoma, a malignant condition which appears to be preceded by several stages of cervical intraepithelial neoplasia (CIN). Despite the existence of Pap smear screening, epidemiological investigations continue to implicate HPV as the single greatest risk factor for progression to CIN, many investigations continue to search for host and/or viral markers that will help identify women infected with HPV who are at risk for CIN. Equally important is the possibility of recurrence of CIN in patients who have been treated. Follow up studies in patients who have undergone excisional or ablative treatment indicate that a significant number of patients experience recurrence. Therefore, it is very important to be able to evaluate the possibility of recurrence of CIN. A prognosis of recurrence would allow a doctor to consider preventative options or therapy options.

Since the first reports linking HPV with cervical cancer appeared in the early 1980s (zur Hausen, 1994), it has become generally accepted that high-risk HPV types contribute to the initiation and progression of preinvasive intraepithelial lesions to carcinoma. Indeed, it has been noted that HPV infection culminates in a distinctive cytopathology in Pap smears, characterized by perinuclear clearing with associated nuclear atypia (Kurman et al, 1994). These HPV changes have been combined with mild dysplasia into the designation LSIL in the revised Bethesda terminology (Kurman and Solomon, 1994). The usefulness of HPV testing is complicated by the fact that there is a need to distinguish between low-risk (L-SIL) and high-risk (H-SIL) HPV types (only the latter pose a significant risk of association with dysplasia→carcinoma progression), and the actual risk of progression. In the former case, a new hybrid capture test for HPV distinguishes high-risk HPV types (Sherman et al., 1995; Poijak et al, 1999; Clavel et al, 2000). DNA image cytometry may be employed in addition to the methods of the invention to diagnose patients at risk for cervical lesions (Lorenzato et al., 2001).

1. Pap Smear

Over the last fifty years, Papanicolaou Smear (“Pap Smear”) has become the cornerstone of efforts to reduce cervical cancer mortality. Pap Smear is effective because it identifies the earliest stages of cervical cancer. Current estimates are that 60-70 million Pap Smears are done in the U.S. each year. Pap Smear has thus become a norm in the detection of cervical cancer. In spite of its broad acceptance in the medical community, studies indicate that Pap Smear screenings will fail to detect from 50%-80% of low grade cancerous lesions, and even 15%-30% of high grade cancerous lesions.

The first step of any cytological diagnostic method is obtaining suitable Pap smear cells for review. In a conventional Pap smear test, a cytologist examines an exfoliative cell specimen, obtained by scraping some cells from the lining of the cervix, smearing the cells onto a slide and staining with Papanicolaou stain. The cytologist examines the stained smears for the presence of abnormal-looking cells that indicate the presence of a malignant condition. The term “malignant condition” refers to the presence of dysplasia including adenocarcinoma in situ (AIS), invasive carcinoma (CA), neoplastic, malignant or tumor cells or the like.

In the method of the invention an exfoliative cell specimen is obtained from a patient, who may or may not harbor a malignant condition. The specimen may be obtained by rotating a cervical sampling device, such as a swab, spatula, or cytobrush along a portion of cervix or vaginal mucosa to obtain a cell sample. A suitable specimen will contain endocervical cells with squamous and/or glandular cells.

The exfoliative cell specimen is generally smeared on the slide to provide a thin layer of the specimen on the surface of the slide. However, the manual observation of cellular abnormalities or the automated analysis of cytological material can be optimized by preparing “monolayers” of cells on the specimen slides. A “monolayer” is defined as substantially two-dimensional layer of uniformly distributed cellular material, predominantly made up of single cells and small clusters of cells.

When conducting Pap Smear screenings, a gynecologist collects exfoliated cells from the surface of the cervix and places them on slides that are sent to cytologists for further examination. Cytologists then review the cells placed on the slides and look for abnormal cells. If abnormal cells are found, the Pap Smear is considered to be positive. If no abnormal cells are found, the Pap Smear is considered to be negative. Pap Smear screening is generally recognized as a practical and economical procedure for the early detection of cervical cancer. In the present invention HPV positivity was determined by Virapap/Viratype assay (Technologies Inc., Gaithersburg, Md.).

2. Colposcopy

While the Pap Smear process is designed for initial screening, colposcopy and related procedures are generally used to confirm Pap Smear abnormalities and to grade cancerous and potential cancerous lesions. Since its introduction in 1925, colposcopy has acquired wide recognition as a follow-up clinical procedure for patients identified by Pap Smear screening as having possible cervical abnormalities. It is generally recognized that colposcopy is highly effective in evaluating patients with abnormal Pap Smears and has therefore become the standard of medical care in the Western world for this circumstance. It is estimated that approximately 4 million colposcopy examinations are currently performed in the U.S. each year.

3. Fluorescence Spectroscopy

Another method for detecting pre-cancerous and cancerous growths or lesions involves fluorescence spectroscopy, which has the capability to quickly, non-invasively and quantitatively probe the biochemical and morphological changes that occur as tissue becomes neoplastic. The altered biochemical and morphological state of the neoplastic tissue is reflected in the spectral characteristics of the measured fluorescence. U.S. Pat. Nos. 6,258,576 and 6,135,965 discuss diagnosis of cervical squamous intraepithelial (CIN) lesions and are specifically incorporated by reference.

4. Treatment of Precancerous and Cancerous Growth

A treatment is intended to effect an elimination, reduction or retardation of the growth. The cancerous growths can treated by excision or ablative procedures. In addition to the immunotherapy discussed in further detail below, the following treatments may be employed therapeutically or preventatively in methods of the invention.

i) Chemotherapy

Cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments. Combination chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing.

ii) Radiotherapy

Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are used herein to describe the process by which a therapeutic or diagnostic peptide or polynucleotide, or a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.

iii) Genes

In yet another embodiment, the secondary treatment is a gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as a chimeric polypeptide of the present invention. Delivery of a chimeric polypeptide in conduction with a second vector encoding one of the following gene products will have a combined anti-hyperproliferative effect on target tissues. Alternatively, a single vector encoding both genes may be used. A variety of proteins are encompassed within the invention, including inducers of cell proliferation such as growth factor receptors, inhibitors of cellular proliferation such as tumor suppressors, and regulators of apoptosis.

iv) Ablative Procedures

A majority of persons with any cancer will generally undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a pre-cancer or cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of pre-cancerous or cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.

v) Other Agents

It is contemplated that other agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adehesion, or agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers. Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1beta, MCP-1, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands such as Fas/Fas ligand, DR4 or DR5/TRAIL would potentiate the apoptotic inducing abililties of the present invention by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyerproliferative efficacy of the treatments. Inhibitors of cell adehesion are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy.

Hormonal therapy may also be used in conjunction with the present invention or in combination with any other cancer therapy previously described. The use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.

v) Anti-Viral Agents

A patient infected with HPV may be treated with anti-viral agents alone or in combination with anti-cancer therapies. An “anti-viral agent” refers to a composition that prevents or inhibits viral infection; prevents or inhibits the progression of a viral infection; reduces the infectivity of the virus; prevent, inhibits, or reduces the physiological symptoms of viral infection; prevents or reduces the incidence of viral activation; inhibits a cell that is a viral host; induces a host cell to undergo apoptosis; clears virus from all or part of the body; induces the virus to become inactive; or any combination of the above.

Agents used against HPV include administration of foscarnet, Thiovir, thiovir analogs (BioKeys), podofilox, podophyllin, trichloracetic acid (TCA), or 5-fluorouracil (5-FU), intralesional or intransal interferon, or Imiquimid cream. Other agents are disclosed in U.S. Pat. Nos. 6,245,568, 6,238,659, and 6,214,874.

II. Proteins and Peptides Selection, Synthesis and Use

In the present invention, peptides are employed in diagnostic and treatment methods. These peptides correspond to HPV 16 oncoproteins.

A. Proteinaceous Compositions

In certain embodiments, the present invention concerns novel compositions comprising at least one proteinaceous molecule as can be seen in peptides with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and in the polypeptides of SEQ ID NO: 20 and SEQ ID NO: 21. As used herein, a “proteinaceous molecule,” “proteinaceous composition,” “proteinaceous compound,” “proteinaceous chain” or “proteinaceous material” generally refers, but is not limited to, a protein of greater than about 200 amino acids or the full length endogenous sequence translated from a gene; a polypeptide of greater than about 100 amino acids; and/or a peptide of from about 3 to about 100 amino acids. All the “proteinaceous” terms described above may be used interchangeably herein.

In certain embodiments the size of the at least one proteinaceous molecule may comprise, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 or greater amino molecule residues, and any range derivable therein. Peptides of the invention may comprise 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or up to 100 contiguous amino acids from SEQ ID NOS: 1-21, inclusive. SEQ ID NOS: 1 to 10 are peptides from the E6 polypeptide of HPV, while SEQ ID NOS: 11-19 are peptides from the E7 polypeptide of HPV. SEQ ID NOS: 20 and 21 are polypeptide sequences for HPV oncoproteins E6 and E7 respectively The GenBank accession number for E6 in HPV 16 is AF327851 (SEQ ID NO:26), while the number for E7 in HPV 16 is U76404 (SEQ ID NO:27), which are both specifically incorporated by reference. Based on Table 3, it is understood that the peptides specifically contemplated as part of the invention include the following E6 peptides: K9L (aa 18-26 of SEQ ID NO:26), E10I (aa 23-34 of SEQ ID NO:26), C10R (aa 37-46 of SEQ ID NO:26), Q15L (aa 43-57 of SEQ ID NO:26), V10C (aa 49-58 of SEQ ID NO:26), P9L (aa 66-74 of SEQ ID NO:26), P10I (aa 102-111 of SEQ ID NO:26), Q20P (aa 97-116 of SEQ ID NO:26), R16R (aa 131-146 of SEQ ID NO:26), G10S (aa 141-150 of SEQ ID NO:26), or a combination thereof. Based on Table 3, it is further understood that the peptides specifically contemplated as part of the invention include the following E7 peptides: T10Q (aa 7-15 of SEQ ID NO:27), M9T (aa 12-20 of SEQ ID NO:27), D9L (aa 14-22 of SEQ ID NO:27), Q19D (aa 44-62 of SEQ ID NO:27), R9F (aa 49-57 of SEQ ID NO:27), R9V (aa 66-74 of SEQ ID NO:27), L9V (aa 82-90 of SEQ ID NO:27), G10C (aa 85-94 of SEQ ID NO:27), D20C (aa 75-94 of SEQ ID NO:27), or a combination thereof.

As used herein, an “amino molecule” refers to any amino acid, amino acid derivative or amino acid mimic as would be known to one of ordinary skill in the art. In certain embodiments, the residues of the proteinaceous molecule are sequential, without any non-amino molecule interrupting the sequence of amino molecule residues. In other embodiments, the sequence may comprise one or more non-amino molecule moieties. In particular embodiments, the sequence of residues of the proteinaceous molecule may be interrupted by one or more non-amino molecule moieties.

Accordingly, the term “proteinaceous composition” encompasses amino molecule sequences comprising at least one of the 20 common amino acids in naturally synthesized proteins, or at least one modified or unusual amino acid, including but not limited to those shown on Table 1 below.

TABLE 1

Modified and Unusual Amino Acids
Abbr.	Amino Acid	Abbr.	Amino Acid

Aad	2-Aminoadipic acid	EtAsn	N-Ethylasparagine
Baad	3-Aminoadipic acid	Hyl	Hydroxylysine
Bala	β-alanine, β-Amino-propionic acid	AHyl	allo-Hydroxylysine
Abu	2-Aminobutyric acid	3Hyp	3-Hydroxyproline
4Abu	4-Aminobutyric acid, piperidinic	4Hyp	4-Hydroxyproline
	acid
Acp	6-Aminocaproic acid	Ide	Isodesmosine
Ahe	2-Aminoheptanoic acid	AIle	allo-Isoleucine
Aib	2-Aminoisobutyric acid	MeGly	N-Methylglycine,
			sarcosine
Baib	3-Aminoisobutyric acid	MeIle	N-Methylisoleucine
Apm	2-Aminopimelic acid	MeLys	6-N-Methyllysine
Dbu	2,4-Diaminobutyric acid	MeVal	N-Methylvaline
Des	Desmosine	Nva	Norvaline
Dpm	2,2′-Diaminopimelic acid	Nle	Norleucine
Dpr	2,3-Diaminopropionic acid	Orn	Ornithine
EtGly	N-Ethylglycine

In certain embodiments the proteinaceous composition comprises at least one protein, polypeptide or peptide. In further embodiments the proteinaceous composition comprises a biocompatible protein, polypeptide or peptide. As used herein, the term “biocompatible” refers to a substance which produces no significant untoward effects when applied to, or administered to, a given organism according to the methods and amounts described herein. Organisms include, but are not limited to, Such untoward or undesirable effects are those such as significant toxicity or adverse immunological reactions. In preferred embodiments, biocompatible protein, polypeptide or peptide containing compositions will generally be mammalian proteins or peptides or synthetic proteins or peptides each essentially free from toxins, pathogens and harmful immunogens.

Proteinaceous compositions may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteinaceous compounds from natural sources, or the chemical synthesis of proteinaceous materials. The nucleotide and protein, polypeptide and peptide sequences for various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (http://www.ncbi.nlm.nih.gov/). The coding regions for these known genes may be amplified and/or expressed using the techniques disclosed herein or as would be know to those of ordinary skill in the art. Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art.

In certain embodiments a proteinaceous compound may be purified. Generally, “purified” will refer to a specific or protein, polypeptide, or peptide composition that has been subjected to fractionation to remove various other proteins, polypeptides, or peptides, and which composition substantially retains its activity, as may be assessed, for example, by the protein assays, as would be known to one of ordinary skill in the art for the specific or desired protein, polypeptide or peptide.

It is contemplated that virtually any protein, polypeptide or peptide containing component may be used in the compositions and methods disclosed herein. However, it is preferred that the proteinaceous material is biocompatible. In certain embodiments, it is envisioned that the formation of a more viscous composition will be advantageous in that will allow the composition to be more precisely or easily applied to the tissue and to be maintained in contact with the tissue throughout the procedure. In such cases, the use of a peptide composition, or more preferably, a polypeptide or protein composition, is contemplated. Ranges of viscosity include, but are not limited to, about 40 to about 100 poise. In certain aspects, a viscosity of about 80 to about 100 poise is preferred.

B. Peptides Selection, Synthesis and Use

Peptide sequences corresponding to E6 and E7 oncoproteins of HPV 16 are selected on the basis of the amphipathic structures and information related to known T-cell epitopes described in literature.

The peptides of the invention can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. Small synthetic peptide sequences, typically less than 100 residues in length, are conventionally prepared using stepwise solid-phase synthesis. Such solid phase synthesis makes use of an insoluble resin support for a growing oligomer. A sequence of subunits, destined to comprise a desired polymer, are reacted together in sequence on the support. A terminal amino acid is attached to the solid support in an initial reaction, either directly or through a keying agent. The terminal residue is reacted, in sequence, with a series of further residues such as amino acids or blocked amino acid moieties to yield a growing oligomer attached to the solid support through the terminal residue. At each stage in the synthetic scheme, unreacted reactant materials are washed out or otherwise removed from contact with the solid phase. The cycle is continued with a pre-selected sequence of residues until the desired polymer has been completely synthesized, but remains attached to the solid support. The polymer is then cleaved from the solid support and purified for use. The foregoing general synthetic scheme was developed, by R. B. Merrifield for use in the preparation of certain peptides (Merrifield, 1986). These peptides can be synthesized either on a modified Vega 250 automatic peptide synthesizer (Vega Biochemicals, Tucson, Ariz.) or by the “bag method” as mentioned by Houghten (Houghten, 1985). Also see, for example, Stewart and Young, (1984); Tam et al., (1983); and Barany and Merrifield (1979), each incorporated herein by reference.

Short peptide sequences, or libraries of overlapping peptides, usually from about 6 up to about 35 to 50 amino acids, which correspond to the selected regions described herein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides. Peptides with at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or up to about 100 contiguous amino acid residues of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 are contemplated by the present invention.

The compositions of the invention may include a peptide that has been modified to enhance its activity or to render it biologically protected. Biologically protected peptides have certain advantages over unprotected peptides when administered to human subjects and, as disclosed in U.S. Pat. No. 5,028,592, incorporated herein by reference, protected peptides often exhibit increased pharmacological activity.

Compositions for use in the present invention may also comprise peptides that include all L-amino acids, all D-amino acids, or a mixture thereof. The use of D-amino acids may confer additional resistance to proteases naturally found within the human body and are less immunogenic and can therefore be expected to have longer biological half lives.

III. Protein Purification

Peptides and proteins derived from HPV can be purified in many ways. Generally, “purified” will refer to a specific protein, polypeptide, or peptide composition that has been subjected to fractionation to remove various other proteins, polypeptides, or peptides, and which composition substantially retains its activity, as may be assessed, for example, by the protein assays, as described herein below, or as would be known to one of ordinary skill in the art for the desired protein, polypeptide or peptide.

Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.

Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide, such as peptides derived from E6 and E7 oncoprotein. The term “purified protein or peptide” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state. A purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.

Generally, “purified” will refer to a protein or peptide composition, that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a “-fold purification number.” The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

There is no general requirement that the protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater “-fold” purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsorption, less zone spreading and the elution volume is related in a simple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (e.g., alter pH, ionic strength, and temperature.).

A particular type of affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography. Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.

The matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability. The ligand should be coupled in such a way as to not affect its binding properties. The ligand also should provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand. One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.

IV. Nucleic Acids

A. Screening of DNA

In the present invention, screening of nucleic acids may be employed not only for screening a sample for infection but also, for detecting the possibility of recurrence of the disease. Screening procedures which rely on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided the appropriate probe is available. Oligonucleotide probes, which correspond to a part of the sequence encoding the protein in question, can be synthesized chemically. This requires that short, oligopeptide stretches of amino acid sequence must be known. The DNA sequence encoding the protein can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. It is possible to perform a mixed addition reaction when the sequence is degenerate. This includes a heterogeneous mixture of denatured double-stranded DNA. For such screening, hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA. Hybridization is particularly useful in the detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present. In other words, by using stringent hybridization conditions directed to avoid non-specific binding, it is possible, for example, to allow the autoradiographic visualization of a specific cDNA done by the hybridization of the target DNA to that single probe in the mixture which is its complete complement (Wallace et al., 1981). The use of a probe or primer of between 13 and 100 nucleotides, preferably between 17 and 100 nucleotides in length, or in some aspects of the invention up to 1-2 kilobases or more in length, allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over contiguous stretches greater than 20 bases in length are generally preferred, to increase stability and/or selectivity of the hybrid molecules obtained. One will generally prefer to design nucleic acid molecules for hybridization having one or more complementary sequences of 20 to 30 nucleotides, or even longer where desired. Such fragments may be readily prepared, for example, by directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.

Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNAs and/or RNAs or to provide primers for amplification of DNA or RNA from samples. Depending on the application envisioned, one would desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of the probe or primers for the target sequence.

For applications requiring high selectivity, one will typically desire to employ relatively high stringency conditions to form the hybrids. For example, relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50° C. to about 70° C. Such high stringency conditions tolerate little, if any, mismatch between the probe or primers and the template or target strand and would be particularly suitable for isolating specific genes or for detecting specific mRNA transcripts. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.

For certain applications, for example, site-directed mutagenesis, it is appreciated that lower stringency conditions are preferred. Under these conditions, hybridization may occur even though the sequences of the hybridizing strands are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and/or decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C. to about 55° C., while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. to about 55° C. Hybridization conditions can be readily manipulated depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl ₂, 1.0 mM dithiothreitol, at temperatures between approximately 20° C. to about 37° C. Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂, at temperatures ranging from approximately 40° C. to about 72° C.

In certain embodiments, it will be advantageous to employ nucleic acids of defined sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of being detected. In preferred embodiments, one may desire to employ a fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a detection means that is visibly or spectrophotometrically detectable, to identify specific hybridization with complementary nucleic acid containing samples.

In general, it is envisioned that the probes or primers described herein will be useful as reagents in solution hybridization, as in PCR™, for detection of expression of corresponding genes, as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to hybridization with selected probes under desired conditions. The conditions selected will depend on the particular circumstances (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Optimization of hybridization conditions for the particular application of interest is well known to those of skill in the art. After washing of the hybridized molecules to remove non-specifically bound probe molecules, hybridization is detected, and/or quantified, by determining the amount of bound label. Representative solid phase hybridization methods are disclosed in U.S. Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods of hybridization that may be used in the practice of the present invention are disclosed in U.S. Pat. Nos. 5,849,481, 5,849,486 and 5,851,772. The relevant portions of these and other references identified in this section of the Specification are incorporated herein by reference.

B. Synthesis of DNA

The synthesis of DNA sequences is frequently the method of choice when the entire sequence of amino acid residues of the desired polypeptide product is known. When the entire sequence of amino acid residues of the desired polypeptides is not known, the direct synthesis of DNA sequences is not possible and the method of choice is the synthesis of cDNA sequences. Among the standard procedures for isolating cDNA sequences of interest is the formation of plasmid- or phage-carrying cDNA libraries which are derived from reverse transcription of mRNA which is abundant in donor cells that have a high level of genetic expression. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned. In those cases where significant portions of the amino acid sequence of the polypeptide are known, the production of labeled single or double-stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single-stranded form.

1. Biochips

Methods of isolating arrays of biomolecules on various supports, referred to as biochips, have been developed and have been employed in DNA synthesis, sequencing, mutation studies, gene expression analysis and gene discovery. Biochips are useful in the present invention as it enables one to identify the markers for pathological states, in this case, HPV infection that may be of subsequent diagnostic value.

Use of a biochip involves the hybridization of a labeled molecule or pool of molecules to the targets immobilized on the biochip. The labeled molecules are normally cDNA copies of the mRNA content of a cell or tissue. In this instance the number of copies of each distinct type of cDNA reflects the number of copies of the corresponding mRNA species in the initial isolate. In general terms, the intensity of hybridization to the target immobilized on the biochip is proportional to the concentration of the cDNA and thus measurement of hybridization intensity enables the relative amount of the mRNA in the initial isolate to be deduced. A relative amount of the same mRNA in two different mRNA isolated can be determined by comparing the intensities of hybridization to the same target spot between two samples. These measurements can be used to identify markers for particular cell types or pathological states that can be of subsequent diagnostic value. Alternatively, sharp increases in the abundance of particular mRNAs in a given disease state can indicate a possible target for drug attack, thereby providing novel therapeutic targets.

C. Nucleic Acid Amplification Reaction

Nucleic acid molecules can be detected using a variety of techniques, including amplification reactions. The present invention contemplates using these amplification reactions for detecting cell mediated immune response or to identify a patient who is infected with HPV and/or have a precancerous or cancerous growth. For example, a cell-mediated immune response can be detected by RT-PCR of a TH1 or TH2 cytokine disclosed herein.

1. Polymerase Chain Reaction (PCR™)

Nucleic acid used as a template for amplification is isolated from cells contained in the biological sample, according to standard methodologies (Sambrook, 1989). The nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a cDNA.

Pairs of primers that selectively hybridize to nucleic acids corresponding to a K _ATPchannel protein or a mutant thereof are contacted with the isolated nucleic acid under conditions that permit selective hybridization. The term “primer,” as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred.

Once hybridized, the nucleic acid:primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as “cycles,” are conducted until a sufficient amount of amplification product is produced.

Next, the amplification product is detected. In certain applications, the detection may be performed by visual means. Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax technology).

A number of template dependent processes are available to amplify the marker sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, and each incorporated herein by reference in entirety.

Briefly, in PCR™, two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If the marker sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.

A reverse transcriptase PCR™ (RT-PCR™) amplification procedure may be performed in order to quantify the amount of mRNA amplified or to prepare cDNA from the desired mRNA. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al., 1989. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641, filed Dec. 21, 1990, incorporated herein by reference. Polymerase chain reaction methodologies are well known in the art.

2. Other Nucleic Acid Amplification Reactions

Another method for amplification is the ligase chain reaction (“LCR”), disclosed in EPA No. 320 308, incorporated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR™, bound ligated units dissociate from the target and then serve as “target sequences” for ligation of excess probe pairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, incorporated herein by reference, may also be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence that can then be detected.

An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention.

Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids that involves multiple rounds of strand displacement and synthesis, i.e., nick translation. A similar method, called Repair Chain Reaction (RCR), involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. Target specific sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having 3′ and 5′ sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample. Upon hybridization, the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated.

Still another amplification methods described in GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety, may be used in accordance with the present invention. In the former application, “modified” primers are used in a PCR™-like, template- and enzyme-dependent synthesis. The primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR Gingeras et al., PCT Application WO 88/10315, incorporated herein by reference. In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has target specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization. The double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNA's are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target specific sequences.

Davey et al. (EPA No. 329 822, incorporated herein by reference in its entirety) disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H(RNase H, an RNase specific for RNA in duplex with either DNA or RNA). The resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5′ to its homology to the template. This primer is then extended by DNA polymerase (exemplified by the large “Klenow” fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then reenter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.

Miller et al (PCT Application WO 89/06700, incorporated herein by reference in its entirety) disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA (“ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts. Other amplification methods include “RACE” and “one-sided PCR” (Frohman, 1990, incorporated by reference).

Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting “di-oligonucleotide”, thereby amplifying the di-oligonucleotide, may also be used in the amplification step of the present invention.

D. Detection of Nucleic Acids

Following any amplification, it may be desirable to separate the amplification product from the template and/or the excess primer. The detection of nucleic acids may be useful in identifying a cell mediated immune response, a patient who is infected with HPV and/or a patient who has a precancerous or cancerous growth.

In one embodiment, amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al., 1989). Separated amplification products may be cut out and eluted from the gel for further manipulation. Using low melting point agarose gels, the separated band may be removed by heating the gel, followed by extraction of the nucleic acid.

Separation of nucleic acids may also be effected by chromatographic techniques known in art. There are many kinds of chromatography which may be used in the practice of the present invention, including adsorption, partition, ion-exchange, hydroxylapatite, molecular sieve, reverse-phase, column, paper, thin-layer, and gas chromatography as well as HPLC.

In certain embodiments, the amplification products are visualized. A typical visualization method involves staining of a gel with ethidium bromide and visualization of bands under UV light. Alternatively, if the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the separated amplification products can be exposed to x-ray film or visualized under the appropriate excitatory spectra.

In one embodiment, following separation of amplification products, a labeled nucleic acid probe is brought into contact with the amplified marker sequence. The probe preferably is conjugated to a chromophore but may be radiolabeled. In another embodiment, the probe is conjugated to a binding partner, such as an antibody or biotin, or another binding partner carrying a detectable moiety.

In particular embodiments, detection is by Southern blotting and hybridization with a labeled probe. The techniques involved in Southern blotting are well known to those of skill in the art (see Sambrook et al., 1989). One example of the foregoing is described in U.S. Pat. No. 5,279,721, incorporated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.

HPV infection can also be detected by catalyzed signal amplified calorimetric DNA in situ hybridization (CSAC-ISH) (GenPoint system, DAKO) (Birner et al., 2001).

Other methods of nucleic acid detection that may be used in the practice of the instant invention are disclosed in U.S. Pat. Nos. 5,840,873, 5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729, 5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, 5,861,244, 5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124, 5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227, 5,932,413 and 5,935,791, each of which is incorporated herein by reference.

V. Cell Mediated Immunity (CMI):

Some methods of the claimed invention take advantage of T-cell responses by using them as a prognostic indicator of recurrence or as a preventative therapy against the development of CIN. More particularly, the methods assay for CMI responses to synthetic peptides from E6 and E7 oncoproteins of HPV 16. The E6 and E7 genes of HPV 16 are frequently co-expressed and are most abundant viral transcripts in biopsies from HPV 16 positive cervical carcinoma (Wettstein, 1990; Seedorf et al., 1987). There is a strong evidence that co-expression of both E6 and E7 open reading frames is necessary and sufficient for efficient malignant transformation of a variety of malignant transformation of a variety of mammalian cells (Munger et al., 1989). Furthermore, continued expression of the E6 and E7 regions of the viral genome appears to be required to maintain the malignant phenotype (von Knebel Doeberitz et al., 1988).

Most viral infections in immune competent mammals result in a cell-mediated immune response against the virus infected cells, the net effect being lysis of the cells. During viral infections, viral proteins are synthesized in the cell for inclusion into new viral particles. Some of those endogenous viral proteins also are degraded and transported into the class I antigen presentation pathway, where the foreign antigens associate with a class I MHC molecule. This peptide-MHC complex then is transported to the surface of the cells where the foreign peptide is presented, in the context of

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