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Title
The majority of the replication-competent virus in the latent reservoir originates from viruses circulating near the time of ART initiation
Presenter
Sarah B. Joseph
Authors
S.B. Joseph1, M.-R. Abrahams2, N. Garrett3, S. Zhou4, M. Moeser4, W. Burgers2, L. Tyers2, D. Matten5, C. Anthony2, O. Council1, N. Archin6,7, D.M. Margolis6,7, S. Abdool Karim3,8, R. Swanstrom4,9, C. Williamson2,3
Institutions
1University of North Carolina at Chapel Hill, Department of Microbiology and Immunology, Chapel Hill, United States, 2University of Cape Town Faculty of Health Sciences, Division of Medical Virology, Institute of Infectious Disease and Molecular Medicine, Cape Town, South Africa, 3Centre for the AIDS Programme of Research in South Africa (CAPRISA), Durban, South Africa, 4University of North Carolina at Chapel Hill, UNC Center for AIDS Research, Chapel Hill, United States, 5University of Cape Town Faculty of Health Sciences, Division of Medical Virology, institute of Infectious Disease and Molecular Medicine, Cape Town, South Africa, 6University of North Carolina at Chapel Hill, Division of Infectious Diseases, School of Medicine, Chapel Hill, United States, 7University of North Carolina at Chapel Hill, UNC HIV Cure Center, Chapel Hill, United States, 8Columbia University, Department of Epidemiology, New York City, United States, 9University of North Carolina at Chapel Hill, Department of Biochemistry and Biophysics, Chapel Hill, United States

Background: HIV-1 persists during suppressive therapy in latently infected cells. These cells have been observed in all HIV-infected people even if treated very early, but little is known about when viruses enter the latent reservoir. Such knowledge would inform our understanding of the dynamics of this reservoir.
Methods: Plasma samples were collected longitudinally from 9 HIV-infected, ART-naive women enrolled in the CAPRISA 002 acute infection cohort who initiated therapy when CD4+ T cell count dropped below 500 cells/µl (mean = 4.5 yr p.i.). After prolonged ART (mean= 5.1 yr), resting CD4+ T cells were isolated from blood and cultured in a quantitative viral outgrowth assay (QVOA) to sample virus in the latent reservoir. vRNA from plasma collected longitudinally pre-ART was analyzed by MiSeq deep sequencing across the genome, and PacBio was used to analyze vRNA from p24+ QVOA outgrowth wells to generate near full-length sequences. Phylogenetic analyses were performed to compare these two types of sequences.
Results: Of the genetically unique outgrowth viruses sampled from each participant, 60 to 100% were related most closely to virus circulating in the year prior to ART initiation, and 0 to 22% to virus circulating during the first two years of infection. This suggests that the start of therapy is associated with the formation of the majority of the persistent reservoir. In sampling 6-44 outgrowth viruses per person, between 0 and 50% of these viruses were genetically identical indicating they were derived from clonally expanded cells, with half arising well before ART initiation and the other half proximal to the start of ART.
Conclusions: We used virus evolution off therapy as a molecular clock to date formation of the latent reservoir. Unexpectedly, the majority of the viruses that we detected entered the reservoir near the time of therapy. Our results support several models including ones in which ART-induced changes to the host immune environment promote latency in HIV-infected cells and/or increase the half-life of latently infected cells. These findings provide unique insights into the formation of the latent reservoir and suggest that interventions at the time of ART initiation may significantly reduce the size of the latent reservoir.


Representative example of the phylogenetic relationship between pre-ART plasma sequences and sequences from the replication-competent reservoir
[Representative example of the phylogenetic relationship between pre-ART plasma sequences and sequences from the replication-competent reservoir ]