CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

Methods: Matched PBMC and testis samples were collected from 3 ART- treated adults and 7 uninfected donors undergoing sex reassignment surgery. The expression of PD-1/TIGIT/LAG-3 in CD4 and CD8 T cells and PD-L1/CD155/ HLA-DR/CD80/CD86 in M ɸ /monocytes from testis and PBMC was assessed by flow cytometry. HIV genetic compartmentalization was assessed in 3 donors by applying the Wright’s measure of population subdivision (Fst) test to HIV proviral Nef sequences characterized via single-genome amplification. Results: Among CD45+ immune cells, the frequency of CD4 and CD8 T cells expressing PD-1 or TIGIT was remarkably higher in the testis than in PBMC (93.6%±6.2 vs. 31.6 ±9.1, p<0.0001 and 98.3%±6.4 vs. 36.4 ±11.8 p<0.0001), (28.2 ±4.5, vs. 13.7 ±9.3, p=0.0004 and 44.2 ±8.3 vs. 16.1 ±12.1, p<0.0001), respectively. LAG-3 expression was not different between testis and PBMC. Among CD68+CD163+ testicular M ɸ , a proportion of 73.7% (±23.9) expressed PD-L1 and 42.9% (±35.2) expressed CD155, and 92.8% (±39.1) were CD86+ while 10.1% (±9.6) were CD80+. A distinct subset of CD68+CD163- testicular M ɸ with HLA-DR-/lo expression was identified. Both M ɸ and myeloid DC displayed high levels of HLA-DR in testis compared to PBMC (p=0.004). No significant evidence for genetic compartmentalization between testis and PBMC was observed in any studied donor. In one case, multiple identical sequences were isolated from right, left testis and PBMC, consistent with migration of clonally-expanded cell populations between these sites. Conclusion: For the first time, we identified PD-1/PD-L1 and TIGIT/CD155 pathways as contributors of the human testicular immune privilege. Our results suggest that the testis does not represent a distinct viral compartment in ART- treated persons. 372 RECONSTRUCTING INTEGRATION DATES OF LATENT HIV SEQUENCES WITHIN-HOST Bradley R. Jones 1 , Natalie Kinloch 2 , Joshua J. Horacsek 2 , Bruce Ganase 1 , Marianne Harris 1 , P. Richard Harrigan 1 , R. Brad Jones 3 , Mark Brockman 2 , Jeffrey Joy 1 , Art Poon 4 , Zabrina Brumme 2 1 British Columbia Centre for Excellence in HIV/AIDS, Vancouver, BC, Canada, 2 Simon Fraser University, Burnaby, BC, Canada, 3 The George Washington University, Washington, DC, USA, 4 University of Western Ontario, London, ON, Canada Background: Given the continuous nature of within-host HIV evolution and reservoir establishment, and the long-lived nature of latently-infected cells, the reservoir in chronic infection should comprise a genetically heterogenous archive of within-host HIV evolution. Heterogeneity in reservoir age and genetic makeup could complicate immune-based HIV elimination strategies but our understanding of these parameters remains limited, in part due to a lack of methods to infer latent HIV sequence ages. We developed a phylogenetic method to reconstruct HIV integration dates within-host, and applied it to date putative latent HIV sequences in persons with long-term viremia suppression on cART. Methods: The method involves inference and optimal rooting of a maximum- likelihood phylogeny from longitudinal within-host plasma HIV RNA and putative latent sequences, followed by calibration of a linear model relating root-to-tip distances of plasma HIV RNA sequences to their sampling dates. The model is then used to convert root-to-tip distances of putative latent lineages to their establishment (integration) dates. After validating the method on simulated and published HIV sequences, we used it to reconstruct integration dates of putative reservoir sequences in two individuals with long-term viremia suppression sampled in-depth over a ~20 year period. Dated sequences included HIV isolated directly from PBMC after >10 years on suppressive cART and from low-level viremia blips on cART (presumably representing in vivo HIV release from the reservoir). All sequences were characterized by single-genome amplification. Results: For both individuals, putative reservoir sequences interspersed throughout within-host phylogenies and exhibited comparable overall diversity to pre-cART plasma RNA sequences sampled over a 10-year period. Historic within-host genetic bottleneck events were also recorded in the reservoir. Inferred proviral integration dates were consistent with the reservoir harboring both ancestral and more recent lineages, with the oldest sequence dating to 20 years prior to sampling. Sensitivity analyses confirmed that linear models can be reliably calibrated from as few as two timepoints, and that the method is robust to rooting method, thus broadening its applicability. Conclusion: Our method for reservoir dating provides a novel and potentially powerful addition to the HIV persistence research toolkit and reveals a

genetically heterogeneous reservoir that recapitulates HIV’s within-host evolutionary history.

Poster Abstracts

373 IDENTIFICATION OF MACROPHAGE RESERVOIRS THROUGH TROPISM OF HIV-1 ENVELOPES Viviane Machado 1 , Thaissa Alvarado 1 , Mario Stevenson 1 , Labelle Barrios 1 , Aubrey Morales 1 , Mark Sharkey 1 , Marco Salemi 2 , Carla Mavian 2 , Timothy J. Henrich 3 1 University of Miami, Miami, FL, USA, 2 University of Florida, Gainesville, FL, USA, 3 University of California San Francisco, San Francisco, CA, USA Background: Despite advances in antiretroviral treatment (ART), eradication of HIV-1 is still not possible due to viral persistence in cell reservoirs. Macrophages express significantly low levels of the CD4 receptor, yet they are still infected. HIV-1 replicates in tissues that are protected from the effects of ART, including resident tissue macrophages and microglia cells in the CNS, facilitating the presence of persistent viral reservoirs. Since these reservoirs are not eliminated during ART, we hypothesize that macrophages may be a source for HIV-1 reservoir in rebound viremia in individuals undergoing analytical treatment interruption (ATI). Methods: 71, 97 and 122 HIV-1 full-length envelopes were isolated by single genome amplification from three individuals at rebound plasma viremia followed ATI. To generate infectious recombinant viruses, env sequences were cloned into an infectious HIV-1 backbone, followed by transfection of HEK 293T. Monocyte-derived macrophages were infected with Env-recombinant viruses, and fusogenicity was assessed by a FRET-mediated assay. Replication capacity was monitored for 14 days by reverse transcriptase activity. Phylogenetic analysis was performed to evaluate evolutionary relationships existing among these envelopes. Results: We found that a small population of Env-recombinant viruses was able to fuse efficiently with macrophages. Of the viruses that fused with macrophages, we identified Env-recombinant viruses that were replication competent, some of which were comparable to the level of the macrophage tropic strains ADA and YU2. Phylogenetic analysis showed the presence of several distinct HIV-1 subpopulations. The relatively low diversity within each clade suggests recent diversification from the common ancestor of each clade. This suggests that several HIV-1 subpopulations persisted in the patient in distinct viral reservoirs that were re-activated during rebound.

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