Phd in EXPERIMENTAL MEDICINE

Thesis title: Microbial co-infections and innate immune responses in human papillomavirus mucosal infections: insights from in vivo and in vitro models

Introduction: Human papillomavirus (HPV) is the most widespread sexually transmitted infection worldwide. While most infections are cleared by the immune system within 18 months, high-risk HPV (HR-HPV) can persist and lead to invasive cancers, particularly in the anogenital tract. Comorbidities such as bacterial vaginosis (BV) and common or emerging microbial co-infections may promote HPV persistence and cancer progression by compromising mucosal integrity and immunity. Upon infection, HPV is recognized by pattern recognition receptors (PRRs), in particular toll-like receptors (TLRs), which stimulate the production of type I and type III interferons (IFN-I and IFN-III), followed by a downstream cascade of interferon-stimulated genes (ISGs) with antiviral, immunomodulatory and antiproliferative functions. However, the impact of co-infections on the innate immune response to HPV is poorly understood, in particular in the first phases on the infection. While in vitro studies provide several insights, in vivo studies have been hindered by the lack of suitable small animal models. The discovery of mouse papillomavirus (MmuPV1), which closely mimics human mucosal HPV infection, represents a milestone. Studies of MmuPV1 in immunodeficient mice have highlighted the critical role of IFN responses in controlling infection and lesion progression. In light of the aforementioned considerations, this PhD project combines microbiological/virological and viro-immunological approaches to study the prevalence of HPV infections and the effects of microbial co-infections on IFN pathways in human mucosa. It also aims to establish a MmuPV1 infection model in mouse keratinocytes and to selectively identify ISGs involved in the regulation of MmuPV1 gene expression. Matherial and Methods: Cohorts of women and men from the Cervicovaginal Pathology Unit and Proctological Clinic at Policlinico Umberto I, Sapienza University Hospital, Rome, were enrolled in this study based on strict inclusion criteria. HPV detection, genotyping, and molecular diagnosis of bacterial vaginosis (BV) in women, as well as STIs (HIV, MCPyV, JCPyV, BKPyV) in men, were performed on cervical and anal specimens. Expression levels of genes encoding TLR9, IFN-III (IFN-λ1, IFN-λ2, IFN-λ3), their specific receptor subunit IFNLR1 and ISG15, were measured using TaqMan-based RT Real-Time PCR assays. Simultaneously, mouse keratinocytes (MPEK-BL6) were infected with MmuPV1 under both proliferative and differentiation conditions. Viral gene expression was monitored over 7 days using digitalPCR (dPCR) assays targeting early E1^E4 and late L1 transcripts. ISG expression (ISG15, HERC6, PLSCR1, IFI35, IFI27, IRF7, SP100, OAS3) was measured by RT Real-Time PCR at 1-, 2-, 5-, and 7-days post-infection. Results: Among the 120 women enrolled, HPV DNA was detected in 51 (42.5%) cervical samples, with 42.9% of HPV-positive women aged 18-27 harboring HR genotypes, mainly HPV16, HPV53, and HPV18. Over 50% had bacterial vaginosis (BV) and at least one STI, with a high co-occurrence of these conditions. Notably BV-associated bacteria, G. vaginalis and A. vaginae, were more common in HPV-positive women than HPV-negative ones. To assess anal HPV prevalence, anal swabs were collected from 691 participants (n= 191 women; n= 500 men). Men had a higher HPV positivity rate than women (75.4% vs. 44.5%). Remarkably, HIV-positive men had the highest rate (83.5%), whereas HIV-negative men were more likely to be HPV-positive than women (68.3% vs. 43.6%). Around 51% of cases were positive for the quadrivalent vaccine genotypes (HPV6, 11, 16, 18), and a further 16% for the nonavalent vaccine genotypes (HPV31, 33, 45, 52, 58). Moreover, among people living with HIV (PLWH) with HPV infection, MCPyV co-infection (37.5%) significantly exceeded BKPyV (3.1%) and JCPyV (0%), with higher MCPyV viral loads observed in MCPyV/HPV/HIV co-infections. Immunological analysis revealed distinct patterns between cervical and anal mucosa. Higher levels of TLR9 and IFN-λ1 were observed in women with LR-HPV compared to HR-HPV. BV and STIs further increased IFN-λ2 and IFNLR1 in HPV-positive women. In men, anal HPV infection was associated with lower levels of TLR9, IFN-λ1 and IFN-λ3. Co-infection with HIV and HPyVs further suppressed IFN gene expression. However, consistent with cervical mucosa, IFN-λ2 expression was higher in HPV-positive men with HPyVs and HIV. In the MmuPV1 in vitro model, only early viral transcripts (E1^E4) showed moderate expression under proliferative conditions, with expression increasing over time during differentiation, where late transcripts (L1) were also detected at later time points post-infection. Analysis of antiviral ISGs revealed a significant decrease in their transcriptional levels over time, which, together with increased viral transcripts, may contribute to persistent infection. Conclusion: In conclusion, this PhD project highlights the value of a multidisciplinary approach combining epidemiological, in vivo and in vitro immunological studies to improve our understanding of the mucosal IFN response to HPV. Raising awareness of the risks of both cervical and anal HPV infection and the benefits of vaccination remains a public health priority. Molecular analyses have revealed a high prevalence of HPV, BV and other STIs in women of reproductive age, highlighting the need for integrated screening and prevention. The high prevalence of oncogenic MCPyV in the anal region, especially in PLWH co-infected with HPV, warrants further investigation. Experimental models such as MmuPV1 can better elucidate the impaired innate immune response observed in HPV infection, which is further modulated by comorbidities such as dysbiosis and microbial co-infections. Therefore, restoring innate immunity and promoting a healthy microbiome may help prevent HPV persistence and cancer progression.