CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

338 A METHOD TO DETERMINE BOTH THE INTEGRATION SITES AND SEQUENCES OF HIV-1 PROVIRUSES Sean Patro 1 , Leah D. Brandt 2 , Michael J. Bale 1 , Elias K. Halvas 2 , Michele Sobolewski 2 , Jenna Hasson 1 , Aurelie Niyongabo 1 , Xiaolin Wu 3 , Shuang Guo 3 , Steven G. Deeks 4 , Stephen H. Hughes 1 , John M. Coffin 5 , Jason W. Rausch 1 , John W. Mellors 2 , Mary F. Kearney 1 1 National Cancer Institute, Frederick, MD, USA, 2 University of Pittsburgh, Pittsburgh, PA, USA, 3 Leidos Biomedical Research, Inc, Frederick, MD, USA, 4 University of California San Francisco, San Francisco, CA, USA, 5 Tufts University, Boston, MA, USA Background: Most of the HIV-1 reservoir on ART is likely contained within clonally-expanded cells carrying intact proviruses. Current methods used to characterize the reservoir include near full-length single-genome sequencing (NFL-SGS) and integration site analysis. However, new technologies are needed to link proviruses to their integration sites. We describe a method, called full- length integrated proviral single-genome sequencing (FLIP-SGS), to solve this problem. Methods: Genomic DNA from PBMC or lymph node mononuclear cell samples (LNMC) from 4 donors was diluted to much less than one provirus per well. An in-house optimized multiple-displacement amplification (MDA) method was performed on each of the wells, generating multiple copies of genomic DNA in each well. Aliquots of the MDA products were then used to obtain the integration sites and to PCR amplify and sequence the corresponding proviruses. The near full length (NFL) sequences were compared to the sequences of viruses obtained in quantitative viral outgrowth assays (qVOA) to identify clones with replication-competent proviruses. Results: FLIP-SGS was applied to evaluate identical P6-PR-RT sequences identified by standard SGS in PBMC or LNMC. We obtained the integration sites and NFL sequences from several clones that contained defective proviruses and one clone with an intact provirus that matched the NFL sequence of an infectious virus identified by qVOA. In 3 donors, identical sequences identified by P6-PR-RT SGS were confirmed to be of clonal origin by FLIP-SGS (identical integration sites) but we also found proviruses with identical P6-PR-RT sequences that had different integration sites, i.e. “false clones.” Such false clones were more common in donors with low proviral diversity or with drug resistant variants. Conclusion: We describe a newmethod that can link the sequence of a provirus with its integration site. This method can differentiate 1) identical proviral sequences that are within a cell clone from those that are not; and 2) intact from defective proviruses in cell clones. We identified a second in vivo clone that contains a replication-competent provirus, providing additional evidence that the HIV-1 reservoir is comprised, at least in part, of infectious proviruses in clonally expanded cells. In donors with low proviral diversity or other genetic bottlenecks (e.g. selection of drug resistant variants), identical proviral sequences may or may not be in clones of cells. 339 DISTINCT CHROMOSOMAL POSITIONS OF INTACT HIV-1 PROVIRUSES Kevin Einkauf 1 , Guinevere Q. Lee 1 , Ce Gao 2 , Radwa Sharaf 1 , Xiaoming Sun 2 , Stephane Hua 2 , Samantha M. Chen 2 , Chenyang Jiang 1 , Xiaodong Lian 1 , Fatema Z. Chowdhury 2 , Eric Rosenberg 3 , Tae-Wook Chun 4 , Jonathan Z. Li 1 , Xu G. Yu 2 , Mathias Lichterfeld 1 1 Brigham and Women’s Hospital, Boston, MA, USA, 2 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA, 3 Massachusetts General Hospital, Boston, MA, USA, 4 National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA Background: CD4+ T cells harboring integrated HIV-1 DNA represent a long-lasting viral reservoir that can persist for decades in infected individuals despite effective antiretroviral therapy (ART). A small minority of these proviral sequences are genetically intact yet transcriptionally silent during ART, though the mechanisms that maintain this viral latency remain unclear. Chromosomal positions of intact proviruses may critically influence viral transcriptional activity, but have been insufficiently characterized in the past, primarily due to the lack of experimental techniques enabling simultaneous analysis of proviral sequences and corresponding integration sites. Methods: Proviral HIV-1 sequences from CD4+ T cells of 3 long-term ART-treated participants were diluted to single genomes and subjected to whole-genome amplification using phi29 polymerase. Products containing 1,000-10,000 copies of an identical proviral sequence were split and separately used for near full-length viral sequencing and for integration site analysis using integration-site loop amplification or ligation-mediated PCR. Chromatin

in defective virus per million between these sites (median=789, IQR=357- 3642; median=584, IQR=299-1530; P=0.95) The ratio of intact to total virus was median=9.9%. Only a small proportion of the intact provirus population was inducible (median=5.7%, IQR=3.3-10.8%) with an even smaller subset of inducible defective provirus (median=1.3%, IQR=0.9-2.3%). Intact and inducible provirus correlated (r=0.779, P=0.0006). Conclusion: Using two novel assays to analyze different properties of the HIV-1 LR we found no quantitative differences between the peripheral blood and lymph nodes. Taken together with previously reported data showing no difference in the distribution of HIV-1 proviral sequence variants between these sites, we conclude that levels of intact and inducible HIV-1 in blood provide a reasonable approximation of frequencies in the lymph nodes. 337LB GENUINE HIV-1 RESERVOIRS FORM IN URETHRAL MACROPHAGES OF ART-SUPPRESSED PATIENTS Yonatan Ganor 1 , Fernando Real 1 , Alexis Sennepin 1 , Charles-Antoine Dutertre 1 , Eliseo Eugenin 2 , Remi Cheynier 1 , Marc Revol 1 , Sarra Cristofari 1 , Anne Hosmalin 1 , Morgane Bomsel 1 1 INSERM, Paris, France, 2 Rutgers University, Newark, NJ, USA Background: HIV-1 eradication requires the elimination/reduction of the HIV-1 reservoir pool mainly characterized until now within T-cells. Yet, residual viremia in HIV-1-infected cART-suppressed individuals originates not only from T-cells but also frommacrophages that posses all required characteristics to form an additional long-lived HIV-1 reservoir. Hence, macrophages, a initial target of HIV-1 infection in the genital mucosa, are tissues-resident cells that resist the cytopathic effects of HIV-1 infection, are long-lived, can self-renew, accumulate infectious virus in intracellular virus-containing compartments (VCC), and produce infectious virus upon stimulation in-vitro. Methods: We used whole penile tissues from HIV-1-infected cART-suppressed individuals with undetectable plasma viral loads obtained upon transgender surgery and searched by PCR, FISH and microscopy for HIV-1 DNA, RNA, p24 and intact virions. Tissue viral outgrowth was used to detect infectious reactivation- competent virus. Results: We show that urethral macrophages contain integrated HIV-1 DNA (Fig1A), RNA (Fig1C), proteins (Fig1D) and intact virions in virus- containing compartments (VCCs)(Fig1E), whereas viral components remain undetectable in urethral T-cells. Moreover, urethral cells specifically release replication-competent infectious HIV-1 following re-activation with the macrophage activator lipopolysaccharide (LPS), while the T-cell activator phytohaemagglutinin (PHA) is ineffective (Fig1B). HIV-1 urethral reservoirs localize preferentially in a newly identified subset of transitional M1/M2 urethral macrophages, highly expressing IL-1-receptor, CD206 and IL-4-receptor, but not CD163. Finally, macrophage reservoirs form longlasting conjugates with CD8+T-cells resisting killing suggestive of a mechanism of enhanced inflammation that participates in reservoir persistence. Conclusion: Altogether, by demonstrating that replication-competent HIV reservoirs form in tissue macrophages, these results challenge the dogma is that HIV reservoirs principally reside in T-cells. Systematic investigation of the presence of HIV reservoirs in other human tissues is now clearly necessary and would be crucial for shock and kill strategies aimed at reservoir elimination.

Poster Abstracts

CROI 2019 123

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