CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

Methods: Serial PBMC samples were collected from five infants with neonatal HIV-1 infection who started ART within 72 hours after birth (n=4) or within 31 days after birth (n=1), and were followed for 84-96 weeks (w). Genomic DNA was subjected to near full-length amplification of single-genome templates of HIV-1. Resulting products were individually sequenced with Illumina MiSeq. Results: Intact full-genome proviral sequences represented an average of 41% of all detected sequences at baseline after delivery, compared to 21% of detected sequences after 84/96w of ART. This corresponded to an average frequency of 76 and 3 intact sequences per million PBMCs at baseline and after 84/96w of treatment, respectively, and is consistent with a half-life of 18 weeks for intact proviral sequences during the first two years of life. Viral sequence defects most frequently detected included large deletions, premature stop codons, and hypermutations. Clonally-expanded proviral sequences, defined as identical viral sequences detected more than once within the same patient, were detected at baseline, 4w, 72w and 84/96w for intact sequences, and at all time points except baseline for defective sequences. At 84/96w, 20% of all intact and 21% of all defective sequences belonged to clusters of clonally- expanded sequences. Two distinct clusters of intact proviral sequences with profound phylogenetic distance, consistent with dual transmission from a likely superinfected mother, were detected in one of the study infants at baseline and 4w; notably, only one of these proviral sequence clusters remained detectable at subsequent time points. Conclusion: ART initiated very early during neonatal HIV-1 infection leads to profound decline of intact proviral sequences in infected infants, and results in remarkably low frequencies of intact proviruses after 84/96w of treatment. Clonal expansion of viral sequences is most visible during the second year of therapy.

Methods: The phenotype and localization of GC cell subsets were defined using flow cytometry and immunohistochemistry (IHC) respectively. RNAscope; an in-situ RNA hybridization assay was used in combination with IHC to visualize the cellular localization of HIV RNA in LN sections. HIV RNA was further quantified ex-vivo in GC Tfh subsets using digital droplet PCR (ddPCR). Results: From IHC results, there was a positive correlation between the magnitude of GCs and HIV plasma viral load (r=0.6, p=0.07) in untreated individuals. Interestingly, the excessive GC T follicular helper (GCTfh) cells’ expansion observed in chronic HIV was significantly attenuated by early treatment (p=0.01). HIV Gag p24 antigen was detected almost exclusively in the GCs even after one year of cART mediated viral suppression. Specifically, follicular dendritic cells and GC Tfh cells harbored most of the detectable Gag p24. Furthermore, RNAscope for Gag-Pol multiplexed with BCL-6 confirmed GC localization of HIV RNA during early cART. Conclusion: Taken together, our results demonstrate the persistence of low level viral replication in the lymph nodes of early treated HIV infected individuals. This study highlights the need for future interventions directed at eliminating residual virus replication in tissue sanctuaries during cART. 342 THE HIV RESERVOIR DURING EARLY ANTIRETROVIRAL THERAPY AND MARAVIROC INTENSIFICATION Antoine Chaillon , Sara Gianella, Steven Lada, Maile Karris, Douglas D. Richman, Sanjay R. Mehta, Susan J. Little, Joel O. Wertheim, Davey M. Smith University of California San Diego, La Jolla, CA, USA Background: Residual viremia is common during antiretroviral therapy (ART), and could be caused by ongoing low-level virus replication or by release of viral particles from infected cells. Intensification of ART should impact ongoing viral propagation but not virion release. Here, we sought to identify HIV evolution in the context of a randomized controlled trial (RCT) of ART intensification with maraviroc (MVC) in individuals who initiated ART shortly after infection. Methods: Eighteen acutely infected men were enrolled in a RCT, and followed for a median of 107 weeks. Participants started ART with (n=9) or without (n=9) intensification with MVC within 90 days of infection. Levels of HIV DNA and cell free RNA were quantified by droplet digital PCR. Deep sequencing of C2-V3 env, gag and pol (454-Roche) was performed on longitudinally collected plasma and PBMC samples while on ART. Sequence data were analyzed for evidence of evolution by: 1) molecular diversity analysis, 2) non-parametric test for panmixia and 3) tip-date randomization technique within Bayesian framework (Figure). To validate the proposed approach, we performed the analyses mentioned above in a group of 9 ART naïve HIV infected individuals with serially collected blood plasma samples. Results: There was a longitudinal decay of HIV DNA after initiation of ART with no difference between MVC intensification groups (-0.08±0.01 log 10 copies/week in MVC+ vs -0.09±0.01 log 10 copies/week in MVC-, p=0.62). All participants had low-level residual viremia (median: 2.8 RNA copies/mL). Across participants a median of 56 (IQR:36-74), 29 (IQR:25-35) and 40 (IQR:31-54) haplotypes were generated for env, gag and pol regions, respectively. Sequence analysis and Bayesian simulations revealed no clear evidence of viral evolution during ART and no difference in viral diversity or population structure from individuals with or without MVC intensification. We confirmed in a positive control dataset the ability to reveal the temporal structure of the data using a similar framework. Conclusion: Further efforts focusing on elucidating the mechanism(s) of viral persistence in various compartments using recent sequencing technologies are still needed and potential low-level viral replication should always be considered in cure strategies.

Poster Abstracts

341 HIV RNA PERSISTS LONG-TERM IN LYMPH NODES OF INDIVIDUALS INITIATED ON ART IN FIEBIG I Omolara Baiyegunhi 1 , Funsho Ogunshola 1 , Nasreen Ismail 1 , Krista Dong 2 , Bruce D. Walker 2 , Thumbi Ndungú 1 , Zaza Ndhlovu 2 1 University of KwaZulu-Natal, Durban, South Africa, 2 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Background: One of the major barriers to achieving HIV remission through combination antiretroviral therapy (cART) is the persistence of virus in reservoirs and tissue sanctuaries. B cell follicles and germinal centres (GCs) within lymph nodes (LNs) are immune privileged sites with the potential to support low level virus replication. However, the persistence of replicating virus within this tissue compartment during cART is controversial. Furthermore, the phenotype and localization of cell subsets that support virus replication in LNs in the presence of cART is unknown. Here, we used excised LNs and paired peripheral blood samples from 17 HIV infected subjects who initiated cART in Fiebig stage I from the well-characterized FRESH cohort to investigate the persistence of virus replication and to identify cell subsets that harbor residual virus during cART. Eight chronic untreated and eight HIV negative individuals were also included in the study as controls.

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