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Low-Level HIV Viremias Originate in Part From Infected Proliferating Cells
Marta E. Bull1, Sherry McLaughlin2, Hannah Huang2, Sheila Styrchak2, Jaime Soria3, Eduardo Ticona3, Alberto La Rosa4, Caroline Mitchell5, James Mullins1, Lisa Frenkel1
1 Pediatrics, University of Washington, Seattle, WA, United States. 2 Seattle Children's Research Institute, Seattle, WA, United States. 3 Hospital Nacional dos de Mayo, Lima, Peru. 4 Investigaciones Médicas en Salud (INMENSA), Lima, Peru. 5 Brigham and Women's Hospital, Harvard Medical School, Boston, MA, United States.
Background: Understanding mechanisms that allow HIV infection to persist despite suppressive antiretroviral therapy (ART) may help develop cure strategies. We observed that cells with HIV integrated into genes associated with cell proliferation or cancer form persistent clones, defined as multiple cells with identical proviruses (HIV env C2-V5) and with identical integration sites (IS). We hypothesized that these cell clones can produce virions, evident as low-level viremias (LLV).
Methods: A subset of HIV-infected ART-naïve subjects, enrolled into a 2-year observational study just before the initiation of nevirapine-based ART, were selected for this study based on detection of LLV (40-500 copies/mL) after one-year of suppressive (<40 copies/mL) ART. Samples were collected every 3 months over the 2 year study. HIV env (C2-V5) sequences were derived from peripheral blood mononuclear cells (PBMC) and plasma prior to starting ART and at a single LLV visit by single-genome-analysis (SGA). Multiple chromosomal IS and associated HIV env sequences were generated using an integration site looping assay (ISLA) (PMID25011556) from PBMC at the LLV visit that was diluted to a median single infected cell. IS were aligned using Bowtie and the location in the human genome determined using BLAT. HIV env sequences from LLV and PBMC were aligned with MUSCLE, and phylogenetic trees generated using DIVEIN. The proportion of LLV with env sequences identical to those in infected proliferating PBMC were evaluated by Fisher's exact test and distances from most recent common ancestor (MRCA) were evaluated by Wilcoxon signed rank test.
Results: Eight participants had LLV on 21/64 study visits. A median of 21 env sequences (range 19-28) were generated from a single LLV, and 90% were identical within a subject. A total of 287 IS sequences were generated from PBMC samples at the LLV visit (median 42 IS/subject, range 27-47). Proliferation of infected cells, evident from multiple cells with identical IS, was observed in 6/8 subjects. Two of these six subjects had proliferating clones with env sequences identical to those in LLV. Three of the six subjects with LLV sequences without linkage to proliferating PBMC had evidence of ongoing viral evolution, and hence continuing viral replication.
Conclusions: Clones of HIV-infected PBMC that persist during suppressive ART are capable of producing virions. LLV appear to originate from virion expression from proliferating infected cell clones or from low–level replication.