Quantitative viral outgrowth assay (QVOA) and infectious units per million (IUPM)

QVOAs were performed as previously described (Crooks et al., 2015). Briefly,~5 × 10⁷ million resting CD4+ T cells, defined as CD4⁺ CD45⁺ CD3⁺ CD69⁻ CD25⁻ CD8⁻ CD14⁻ HLA-DR⁻, were isolated via negative selection from PBMC (Keedy et al., 2009). CD4+ T cells were maximally stimulated in limiting dilutions with phytohemagglutinin (PHA) (Remel-PHA; Thermo Scientific), IL-2, and irradiated CD8-depleted PBMC from a seronegative donor, which were added twice to cultures to support virus propagation. The CD8-depleted PBMC were obtained from seronegative donors previously screened to ensure adequate CCR5 expression. Culture supernatants were collected on days 15 and 19 and assayed for p24 expression by ELISA (ABL, Rockville, MD). Only cultures that contained an equivalent or greater level of p24 antigen on day 19 compared with day 15 were scored as positive. A maximum likelihood method was used on day 15 data to estimate the frequency of latent HIV infection, reported as bias-corrected Infectious Units Per Million (IUPM) (Supplementary file 1; Trumble et al., 2017). For 10 participants, longitudinal IUPM measurements were available. IUPM values measured in the 2 years prior to initial T cell mapping were averaged (range of IUPM values: 1–6, mean number of IUPM values/participant: 2). The limit of detection was defined as ≤1 p24 positive well (range 12–36 cultures, mean 18 cultures) at the highest input of cells (2.5 million cells).