CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

250 COMPARTMENTALIZED HIV-1 IS FOUND IN THE SEMEN OF MEN WITH AND WITHOUT URETHRITIS Olivia Council 1 , Lihua Ping 1 , Chase McCann 1 , Gerald Tegha 2 , Irving Hoffman 1 , Debbie Kamwendo 2 , Mitch Matoga 2 , Myron S. Cohen 1 , Ronald Swanstrom 1 1 University of North Carolina Chapel Hill, Chapel Hill, NC, USA, 2 University of North Carolina Project–Malawi, Lilongwe, Malawi Background: HIV-1 RNA can be found in the semen of most untreated HIV- infected men and may represent local viral replication or migration of virus or infected cells into the genital tract from the periphery. Inflammation appears to mediate these processes as men with HIV-1 and a concurrent sexually- transmitted infection (STI) have a higher HIV-1 semen viral load. Inflammation likely increases shedding by recruiting CD4+ T cells into the genital tract that are either uninfected and can amplify virus in that compartment or are infected and can release virus from the periphery into the genital tract. In this study, we tested the hypothesis that inflammation due to STI-associated urethritis increases migration of HIV-infected cells into the genital tract, causing HIV-1 in the semen to be genetically similar to virus in the blood (equilibrated). Similarly, we hypothesize that in the absence of inflammation, viral lineages are more likely to replicate independently in the genital tract and become genetically distinct from virus in the periphery (compartmentalized). Methods: Paired blood and semen samples were collected from HIV-infected, ART-naïve men in Lilongwe, Malawi with and without symptomatic urethritis. HIV-1 RNA was extracted from the samples and cDNA was synthesized. Each cDNA was tagged with a unique 11 nucleotide Primer ID. This Primer ID ‘tag’ is maintained throughout subsequent amplification steps and reduces PCR and sequencing errors. Approximately 600 bases of the HIV-1 env V1-V3 region were deep sequenced using the MiSeq platform. Phylogenetic trees were then constructed using sequences from the blood and semen and examined for the presence of compartmentalization in the genital tract. Results: We sequenced samples frommen with (n=16) and without (n=8) urethritis. Overall, we observed some degree of viral compartmentalization in the sequences of 7/16 (44%) men with urethritis and in 3/8 (38%) of men without urethritis. Thus, in this cohort, urethritis did not alter the prevalence of HIV-1 compartmentalization in semen. Conclusion: We observed varying degrees of HIV-1 compartmentalization in the genital tract of approximately 40% of men analyzed, regardless of the presence or absence of urethritis. Deep sequencing with Primer ID revealed compartmentalization in the semen of a surprisingly high fraction of ART-naïve men. The existence of compartmentalized viral lineages in the genital tract has important implications for understanding the biology of transmission.

drivers of the HIV-2 benign course, prompting us to investigate here, for the first time, the ability of Tfh to support HIV-2 infection. Methods: CD4+ T cell subsets from tonsillar tissue were sort-purified based on the expression of CXCR5, PD-1 and ICOS and infected in vitro with HIV-2 and HIV-1 primary isolates with distinct co-receptor tropism. PBMCs were isolated from freshly collected peripheral blood of untreated HIV-2-infected individuals and CD4+ T cell subsets were sort-purified based on CCR7, CD45RA and CXCR5 expression. Viral DNA and mRNA were measured by real-time quantitative PCR. (CXCR5+ICOSbrightPD-1bright). Of note, CXCR4 usage was not associated with higher reservoirs in the case of HIV-2 primary isolates, in contrast with the typical pattern observed in HIV-1. Moreover, Tfh supported productive HIV-2 infection, as attested by the increase in viral DNA levels after TCR stimulation, along with evidence of infectious viruses in culture supernatants. Importantly, sort-purified Tfh from the blood of untreated HIV-2-infected individuals confirmed that this subset is also a main HIV-2 reservoir in vivo. This reservoir was effectively controlled, since these patients featured no detectable plasma viral load. Conclusion: We reveal that Tfh support productive HIV-2 infection and are a preferential target in HIV-2-infected individuals. Our data are in agreement with a link between Tfh infection and sustained titers of HIV-specific nAbs. Identifying the host factors responsible for the controlled HIV-2 infection will help clarify the mechanisms responsible for the pathogenic nature of HIV-1 and may uncover strategies to achieve a functional HIV cure. 249 NEUTROPHIL EXTRACELLULAR TRAPS PREVENT HIV INFECTION IN THE GENITAL TRACT Fiona D. Barr 1 , Christina Ochsenbauer 2 , Charles Wira 1 , Marta Rodriguez- Garcia 1 1 Dartmouth College, Hanover, NH, USA, 2 University of Alabama at Birmingham, Birmingham, AL, USA Background: Women worldwide acquire HIV mainly through sexual intercourse. However, the low transmission rate per sexual act indicates that local innate immune mechanisms contribute to HIV prevention. Neutrophils represent 10-20% of the genital immune cells in healthy women. Neutrophils participate in mucosal protection against bacterial and fungal pathogens through different mechanisms, including the release of Neutrophil Extracellular Traps (NETs). NETs are DNA fragments associated with antimicrobial granular proteins. Despite neutrophil abundance and central contributions to innate immunity in the genital tract, their role in protection against HIV acquisition is unknown. Methods: Genital tract tissues obtained from hysterectomies (ectocervix, endocervix and endometrium) were enzymatically digested to generate mixed cell suspensions and isolate neutrophils. Mixed cell suspensions (immune cells and fibroblasts) or purified neutrophils were stimulated ex-vivo with GFP-tagged HIV viral-like particles (HIV-VLP). NET release was quantified and characterized using time-lapse imaging and confocal microscopy. To measure anti-HIV activity of pre-formed NETs, NETs were induced using calcium ionophore, a known inducer of NETosis. NETs were recovered by centrifugation, and incubated with replication-competent HIV for 1h prior to addition of CD4+ T cells. HIV infection was evaluated after 7 days by flow cytometry and p24 ELISA. Results: Stimulation of genital neutrophils induced the release of NETs within minutes of viral exposure (P=0.0005). NET release resulted in immediate entrapment of HIV-VLP, which co-localized with extracellular DNA strands, as demonstrated by time-lapse imaging and confocal microscopy. Incubation of HIV with pre-formed NETs resulted in complete inhibition of HIV infection of CD4+ T cells. Viral inactivation was irreversible, given that treatment of HIV-NET complexes with DNAse, to degrade NETs and release potentially infectious virions, did not restore infection. Confocal microscopic analysis of NETs revealed the presence of HNP 1-3, LL-37 and myeloperoxidase, all of which have known anti-HIV activity. Conclusion: Genital neutrophils recognize and respond to HIV with the release of NETs, which inactivate the virus and prevent the infection of target cells. This could represent a novel mucosal protection mechanism for HIV-acquisition not previously considered. Our findings could open new avenues for research and strategies for HIV prevention. Viral infectivity was assessed using a TZM-bl reporter cell line. Results: Our data showed that HIV-2 is able to infect Tfh

Poster Abstracts

251LB WITHDRAWN

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CROI 2018