Characterisation of genital and systemic biomarkers of cervical HPV viral load in women at risk of cervical intra­epithelial neoplasia.

Abstract

Cervical cancer is the fourth most common cancer worldwide and the second most common among South African women. Over 90% of all cervical cancer cases and mortalities occur in low- and middle-income countries (LMICs). The persistence of infection with high-risk Human papillomaviruses (HPV) drives the development and progression of cervical intraepithelial neoplasia (CIN), a precursor for cervical cancer. Women living with HIV (WLWH) have a higher incidence of CIN and cervical cancer and experience higher rates of CIN recurrence after treatment when compared with HIV-negative women. Effective intervention against HPV and CIN in WLWH will require a thorough understanding of how virological parameters and host immune responses, which comprises the role of T-cell responses and the resultant cytokines profiles, impact their viral clearance and CIN regression, progression, and recurrence. Such information will also guide on the biomarkers that can be used to predict or monitor their treatment outcomes. However, there is a paucity of information on virological and immunological basis for CIN recurrence in this population. Therefore, we aimed to characterize the virological and immune biomarkers of HPV-associated abnormalities and determine their relationship with CIN treatment outcomes in 96 women living with HIV. The study participants were between the ages of 18-65 years old and were recruited from the King Edward's Hospital in Durban, South Africa, from April 2012 to April 2015. All participants presented with abnormal pap smears at enrolment, required colposcopy, and were treated for CIN by Large Loop Excision of the Transformation Zone (LLETZ). Participants were subsequently followed up at six month intervals for a year after treatment. For these analyses, they were then stratified into “No-recurrence” and “Recurrence” groups based on clinical CIN detection at either six months or one year post-treatment, with 69 participants being in the “No-recurrence” group and 27 being in the “Recurrence” group. Genital and blood samples were collected at study entry, 6 months, and 1 year after treatment. Virological assays on the number of HPV types and HPV viral loads were done on the cervicovaginal lavage (CVL) andcytobrush samples, respectively, by conducting HPV genotyping at baseline using Roche Linear array and then quantifying high-risk HPV viral load at all study visits using Hybrid Capture II (HCII) assay. Immune correlates of CIN recurrence were assessed by measuring anti-HPV IFN-γ+ CD4+ and CD8+ T cell responses in peripheral blood mononuclear cells of 28 women (16 in the “No-recurrence” group and 12 in the “Recurrence” group) using intracellular cytokine staining at baseline and 1-year post-treatment. Genital and peripheral cytokine responses were also measured in serum and CVL of 39 women (20 in the “Norecurrence” group and 19 in the “Recurrence” group) using a multiplexed Enzyme-Linked Immunosorbent Assay (ELISA). Women who experienced CIN recurrence after excisional surgical treatment had higher numbers of highrisk HPV types at enrolment (p=0.008) and higher high-risk HPV viral loads at all time points (p<0.001).Notably, the combined presence of both high-risk HPV types and high HPV viral load was the best predictor for CIN recurrence in a multivariable regression model (odds ratio 1.985, 95% CI 1.020 to 3.865, p=0.044). There were no differences in T cell responses between the two groups, suggesting minimal impact of naturally elicited systemic anti-HPV T-cell responses on treatment success. Interestingly, the frequencies of IFN-γ+ producing CD8 T cells to the E7 oncoprotein at the pre-treatment time point were significantly higher in women with CIN 3 when compared to those with CIN 2 (p=0.033) and had a trend for a positive correlation with HPV viral loads (Rho= 0.38, p=0.08), suggesting the dependence of naturally elicited pretreatment anti-HPV CD8 T-cell responses on antigen load. Our study also revealed stable kinetics of the anti-HPV16 and anti-HPV18 E6- and E7-specific T cell immunity post-treatment in blood for both “Norecurrence” and “Recurrence” groups. These data reinforce the potential role of combining HPV genotyping, HPV viral load quantification, and holistic immune response assessment, comprising both systemic and local immunity, as prognostic tools in identifying women at the highest risk of CIN recurrence after treatment, especially for women living with HIV. The study also sheds light on the potential role of viral persistence and immune modulation in CIN recurrence.