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NONSUPPRESSIBLE VIREMIA ON ART FROM LARGE CELL CLONES CARRYING INTACT PROVIRUSES
Elias K. Halvas1, Kevin Joseph1, Leah D. Brandt1, Johannes C. Botha2, Michele Sobolewski1, Jana L. Jacobs1, Brandon F. Keele3, Mary F. Kearney4, John M. Coffin5, Jason W. Rausch4, Shuang Guo6, Xiaolin Wu6, Stephen H. Hughes4, John W. Mellors1
1University of Pittsburgh, Pittsburgh, PA, USA,2Stellenbosch University, Cape Town, South Africa,3AIDS and Cancer Virus Program, Frederick, MD, USA,4National Cancer Institute, Frederick, MD, USA,5Tufts University, Boston, MA, USA,6Leidos Biomedical Research, Inc, Frederick, MD, USA
Clinically detectable viremia on ART is generally attributed to virus replication from incomplete adherence and/or drug resistance. One case of infectious viremia from a large cell clone with an intergenic intact provirus has been reported in an individual with metastatic cancer (Simonetti, PNAS 2016). We studied individuals referred for clinically detectable viremia despite receiving potent ART, adherence counseling, and in some cases, regimen switches or intensification.
Peripheral blood mononuclear cells (PBMCs) and plasma were collected at two or more time points from donors with plasma HIV RNA >20 copies/ml occurring for >6 months on combination ART. Single-genome sequencing was performed on plasma HIV RNA, cell-associated HIV DNA (CAD), and p24+ culture supernatants from quantitative viral outgrowth assays (qVOA). The clonal cellular origin of viremia was assessed by phylogenetics and integration site analysis (ISA), and confirmed by sequencing the integrated provirus and the flanking host sequences.
Across the 10 individuals referred, median plasma HIV-1 RNA was 97.5 cps/mL (range 40 to 356 cps/mL) after a median of 10 years on ART. One donor (A-04) had phylogenetic evidence of virus evolution and accumulation of resistance mutations and was not analyzed further. Each of the other 9 donors had multiple identical single-genome HIV RNA sequences in plasma that did not change over time and lacked resistance to the current ART regimen. In 6 of 9 donors, HIV sequences from plasma matched proviral sequences in PBMC. Plasma HIV RNA and proviral sequences were identical to HIV RNA in p24+ qVOA wells for 4 donors (C02, C03, R09, T13). The integration sites for the intact proviruses producing viremia were mapped to the human genome for 3 donors (4th pending). Integrations were in introns of the MATR3, ZNF268, and ABCA11P genes for C02, C03, and R09, respectively. The provirus in MATR3 and ZNF268 were in the opposite orientation to the gene, whereas the ABCA11P integrant was in the same orientation. The intact provirus comprised 4.2-15.4% of all proviruses in PBMC with amplifiable pro/pol sequences.
Large cell clones carrying intact proviruses can produce clinically relevant levels of viremia and should be considered in managing patients. The mechanisms involved in clonal expansion and persistence of cells with intact proviruses that produce viremia need to be understood to effectively target the HIV reservoir.