CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

Conclusion: The gut mucosal epigenome appears to be altered in TGW compared to CW and MSM Thai volunteers. This occurred at several gene loci including CCR5 that are known to affect HIV susceptibility. Further investigations of biological HIV risk factors specific to TGW are needed to inform additional HIV prevention strategies 254 DECREASED EXPRESSION OF MUCOSAL TYPE I IFN RESPONSE IN HPV- INFECTED MSM PATIENTS Letizia Santinelli 1 , Alessandra Pierangeli 1 , Eugenio Nelson Cavallari 1 , Paolo Gozzo 1 , Giancarlo Ceccarelli 1 , Claudio M. Mastroianni 1 , Raphael Viscidi 2 , Guido Antonelli 1 , Gabriella d'Ettorre 1 , Carolina Scagnolari 1 1 Sapienza University of Rome, Rome, Italy, 2 Johns Hopkins University, Baltimore, MD, USA Background: Innate immunity pathways, especially those related to type I interferon (IFN-I) are involved in Human Papillomavirus (HPV) recognition and clearance. Among HIV-1 positive men who have sex with men (MSM), the extremely high incidence of HPV infection is strongly associated with an increased risk of squamous cell carcinoma of the anal canal. We hypothesized that HPV, through evasion strategies adopted to overcome the host immune defense and establish persistent infection, might target different IFN-I genes in HIV-1 MSM patients. Methods: Anal brushings were collected from 86 Caucasian MSM HIV-1 infected patients, with a median age of 46 ±11 years, on long-term antiretroviral therapy (ART), attending Policlinic Umberto I Hospital in Rome. Detection of HPV DNA and genotyping were performed by PCR and sequencing. The mRNA levels of IFN alfa, IFN beta, IFN epsilon, an emerging component of innate immune defence at mucosal sites, IFN alpha receptor (subunits R1 and R2) in anal brushings, were measured by TaqMan RT–PCR. Results: Anal HPV DNA was detected in 71 MSM patients (83%), with 43% of the cases having a high-risk (HR) HPV genotype, mainly HPV16. Out of 86 patients, 54% showed HSIL/LSIL. A decreased mucosal expression of IFN beta, IFN epsilon, IFNAR1 and IFNAR2 was recorded in HR compared to low-risk (LR) HPV positive and HPV negative patients (Mann-Whitney U test p <0.05 for all genes). No differences were found on levels of IFN-I components according to the presence or absence of SIL. By contrast, the expression of IFN beta, IFN epsilon, IFNAR1 and IFNAR2 was reduced in patients with a persistent HPV infection (18%) compared to those who spontaneously cleared the infection (11%) (Mann-Whitney U test p <0.01 for all genes). Conclusion: HPV persistent infection may dysregulate IFN-I response and contribute to the establishment of an immunosuppressive microenvironment in mucosal epithelia, which is essential for precancerous anal lesions progression. 255 HIGH-RISK HUMAN PAPILLOMAVIRUS ONCOPROTEINS DYSREGULATE INTERLEUKIN-1 SIGNALING Hee W. Kim 1 , Lk Metthew Lam 1 , Pavithra Rajagopalan 1 , Devraj Basu 1 , Elizabeth A. White 1 1 University of Pennsylvania, Philadelphia, PA, USA Background: High-risk human papillomavirus (HPV) infection causes cervical, anogenital, and oropharyngeal cancers that account for ~5% of cancer cases worldwide. The incidence of these AIDS-defining (cervical) or non-AIDS-defining (anal, oropharyngeal) malignancies is increasing among HIV-infected individuals. A mutation in any one of several components of the interleukin-1 (IL-1) signaling pathway predisposes individuals to develop HPV-associated malignancies, suggesting that IL-1 signals restrict either HPV infection or development/progression of HPV-positive neoplasia. IL-1ß is a pro- inflammatory cytokine and the IL-1 pathway is subject to complex regulation. The purpose of the study was to define the effects of high-risk HPV E6 and E7 oncoproteins on IL-1-related gene expression and signaling. Methods: We used several models to assess HPV oncoprotein-dependent changes in the IL-1 pathway. First, we compared human keratinocytes engineered to express high-risk HPV E6/E7 to negative controls. Complementary experiments used HPV-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC) cell lines. Finally, we employed a panel of HPV-positive and HPV-negative patient-derived xenografts (PDX). In each model we measured the levels of IL-1 signaling-related transcripts, the response of cells to IL-1 treatment, and the production of IL-1 upon exposure to the inflammasome- activating agent nigericin. Results: In both cell lines and PDXs, the presence of HPV oncogenes was associated with decreased expression of IL1B and increased expression of

SIGIRR, a negative regulator of IL-1 signaling. HPV16 E6/E7-expressing cells were less able to produce IL-1ß upon treatment with nigericin. The HPV16 E6/ E7-positive cells were less responsive to stimulation with IL-1ß as assessed by the transcription of IL8 and TNFA. Conclusion: High-risk HPV E6/E7 oncoproteins induced several changes in the IL-1 pathway consistent with reduced pathway activity. These changes were recapitulated in a PDX panel consisting of 8 HPV-negative and 8 HPV-positive models. HPV oncoprotein-expressing cells upregulated fewer genes related to inflammation upon treatment with IL-1ß and were impaired in the production of IL-1ß following nigericin treatment. Future studies will aim to assess the role of IL-1 dysregulation in HPV-mediated carcinogenesis. 256 25-HYDROXYCHOLESTEROL INHIBITS HERPESVIRUSES BY ACTIVATING INFLAMMATORY PATHWAYS Anna Serquiña 1 , Takanobu Tagawa 1 , Joseph Ziegelbauer 1 1 National Cancer Institute, Bethesda, MD, USA Background: Kaposi’s Sarcoma Herpesvirus (KSHV/HHV-8) expresses several viral products during latency and lytic replication cycle that block innate immune responses. It is therefore of interest to study antiviral approaches that can tip the balance and help the host mount an effective immune response. Recently, we have described how 25-hydroxycholesterol (25HC), a derivative of cholesterol, can block KSHV de novo infection of primary endothelial cells (HUVEC) at a post-entry step and decreases expression of viral genes. We wanted to determine whether 25HC inhibits other gammaherpesviruses. More importantly, we aimed to study how 25HC exerts its antiviral effect. Methods: To test the antiviral effect of 25HC against Epstein-Barr Virus (EBV), another oncogenic gammaherpesvirus (often co-infecting certain cancers with KSHV, e.g. PEL ), we performed de novo infection of primary B cells with 25HC treatment and measured apoptosis using flow cytometry. We also quantitated EBV viral transcript levels using RT-qPCR. To characterize the gene regulatory pathways triggered by 25HC, we performed RNA sequencing (RNA-Seq) of HUVEC treated with 25HC and de novo infected with KSHV. Validation was performed by RT-qPCR. Single and combinatorial siRNA knockdown of candidate target genes screened from RNA-Seq analysis were performed to identify which genes were required for the antiviral activity of 25HC. Results: We found that 25HC increased apoptosis in EBV-infected cells, decreasing the number of EBV-transformed lymphoblastoid cell lines (LCLs). 25HC downregulated an RNA Pol III-transcribed EBV transcript, but not an RNA Pol II EBV transcript. RNA-Seq showed global suppression of KSHV viral gene expression with treatment of 25HC. On the other hand, 25HC increased Type I interferon-stimulated genes (ISGs), including inflammatory cytokines and chemokines. Using single and combinatorial siRNA-mediated knockdown, we found that depletion of certain candidate genes resulted in recovery of viral gene expression, validating their contribution towards the antiviral effect of 25HC in KSHV. Conclusion: 25HC rendered EBV-infected B cells unable to form LCLs. RNA-Seq data showed induction of inflammatory cytokines due to 25HC treatment. Loss-of-function experiments confirmed their role in the antiviral activity of 25HC. Our studies aim to elucidate how we can augment these intrinsic antiviral responses to pave the way for developing therapeutic strategies for multiple viral infections. 257 ANAL HPV INFECTIONS DO NOT ASSOCIATE WITH RECTAL HIV-RNA SHEDDING IN HIV+ PATIENTS Alberto Borghetti 1 , Matteo B. Whalen 2 , Paola Cattani 2 , Veronica De Simone 3 , Simona Marchetti 4 , Marta Goglia 2 , Francesca Lombardi 2 , Davide Moschese 2 , Gianmaria Baldin 5 , Antonella Cingolani 2 , Carlo Ratto 2 , Maurizio Zazzi 6 , Simona Di Giambenedetto 2 1 Catholic University of Sacred Heart, Rome, Italy, 2 Catholic University of the Sacred Heart, Rome, Italy, 3 Fondazione Policlinico Universitario A. Gemelli IRCCS UOC di Proctologia, Rome, Italy, 4 Fondazione Policlinico Universitario A. Gemelli IRCCS UOC di Microbiologia, Rome, Italy, 5 Università Cattolica del Sacro Cuore, Rome, Italy, 6 Siena University Hospital, Siena, Italy Background: Rectal HIV-RNA shedding occurs in a not negligible proportion of HIV+ pts with undetectable plasma HIV-RNA. Since multiple HPV infections are often detected in anal swabs from HIV+ pts, a causal effect of HPV in triggering rectal HIV replication could be hypothesized. Methods: A cross-sectional, monocentric study was conducted, in which consecutively-enrolled, HIV+, virologically-suppressed pts undergoing

Poster Abstracts

87

CROI 2020