HPV Immunological Markers of Cervical Persistent Infection and Oncogenesis

The end point of this study is to analyze host factors, including virus-specific immunity and HPV-related parameters influencing the onset and progression of squamous intraepithelial lesion (SIL), in order to define risk stratification and tailoring treatment, developing widely accessible and non-invasive assays immunoscore with high predictive value.

The progression of squamous intraepithelial lesion (H SIL) at 2 years in women with abnormal pap smear will also be evaluated (primary end point) and the immune host profile after anti-HPV prophylactic and therapeutic vaccination, in women with squamous intraepithelial lesions and with persistence of infection will be analyzed (secondary end point).

The investigators will analyze the patients, enrolled prospectively with abnormal pap smear every six months up to two years after diagnosis. At each time point, during colposcopy, peripheral blood (30 mL) and cervical samples (vaginal washing, cervical biopsy and pap-smear) will be collected for the characterization of local and peripheral immune response. Viral load, HPV integration will be assessed on cytological and tissue samples obtained from the patients at time of diagnosis and/or curative surgery.

For the secondary endpoints, the investigators will enroll patients with confirmed HPV infection, undergoing therapeutic and prophylactic HPV vaccination. The vaccination will be performed immediately after HPV lesion diagnosis. Follow-up program implies colposcopy every six months for 2 years. The investigators will analyze HPV-specific immune response every six months, and viral load and HPV integration at the time of diagnosis and at the end of the follow-up.

To evaluate antigen-specific proliferative response, PBMCs will be stimulated in triplicate in 96-well round-bottom plates with antigens L1, E6, E7 proteins of HPV- 16 and -18 and human Actin peptide pools for 7 days. Culture medium was RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin solution, 10% of heat inactivated FBS, 1 mM Sodium Pyruvate, 100 µM non-essential amino acids, and 50 µM 2-Mercaptoethanol. After culture, cells will be washed, stained with Live/Dead Fixable Violet Dye ) and subsequently with CD3, CD4, CD8, CD25, CD278 (ICOS). Finally, cells will be washed and resuspended in PBS 1% paraformaldehyde.

A Cell Proliferation Index (CPI) for antigen-specific expanded T-cells will be determined by subtracting the percentage of CD25+ICOS+ CD3+CD4+ or CD3+CD8+ detected in PBMC incubated with actin peptides from the percentage of CD25+ICOS+ T-cell subsets detected in PBMC incubated with HPV peptides. A CPI <1.5% will be considered negative while a value ≥1.5% will be considered positive.

Flow cytometry analyses were performed with a FACS Canto II flow cytometer and BD DIVA software.

For the analysis of HPV-related features, molecular approaches will be used. HPV DNA will be extracted from biopsies and lesion swabs using automatized systems after specific lysis treatment for tissues.

HPV DNA will be then quantified using real time PCR specific for L1/E2 and E6 regions; in detail MY 09/11 fragment of L1 gene will be used. Results will be expressed as the number of copies/100.000 cells and normalized using a housekeeping gene.

For the differential quantification of integrated or episomal HPV DNA a specific standardized system for the amplification of ORF E2 and E6 will be used.