An inducible HIV reservoir in resting CD4 T cells (CD3+CD4+CD25-CD69-HLA-DR-) is well characterized and is the focus of most reservoir studies and assays. However, all CD4 T cells have the potential to harbor latent HIV. We compared inducible and infectious HIV from total and resting CD4 T cells.
Total and resting CD4 T cells were isolated by negative selection from blood donors on long-term ART, stimulated with PMA/ionomycin, and virus production was measured by qPCR for HIV RNA copies. Quantitative viral outgrowth assays (QVOA, 21-day endpoint) were performed in parallel with total and resting CD4 T cells. Near-full length single genome sequencing (SGS) was performed on p24-positive viral outgrowth cultures. Cell associated HIV DNA (CA-DNA) and unspliced HIV RNA (CA-RNA) were quantified by qPCR normalized to CCR5 DNA.
11 donors on suppressive ART for a median of 9 years (range: 3-13) were studied. There was no significant difference in mean CA-DNA (727+/-578 vs. 695+/-492 copies/million cells) or CA-RNA (257+/-211 vs. 342+/-309 copies/per million cells) between total and resting CD4 cells. Virus release was infrequent from unstimulated total and resting CD4 cells. Inducible virus production from total and resting CD4 cells varied by donor (Figure), but was higher from total than resting CD4 cells (mean 2.1-fold higher; p=0.024, signed rank test). The ratio of inducible virus production from total to resting CD4 cells was positively correlated with the percent of activated (CD25+/CD69+/HLA-DR+) total CD4 cells (rho=0.74, p=0.046). Infectious virion release (IUPM) was detected in only 6 of 11 donors, but was higher from total CD4 cells in 5 of the 6 positive donors (mean 1.4-fold higher [range 1.5-16]; p=0.11, signed rank test). SGS analysis of outgrowth viruses revealed different viral clones recovered from total CD4 vs. resting CD4 cells. Inducible virus production correlated strongly with IUPM (Figure) for both total and resting CD4 cell populations (rho=0.87, p=0.0005; rho=0.68, p=0.022), suggesting that inducible virus production is a good marker of the latent infectious reservoir in both cell types.
Latent HIV exists in both resting and non-resting CD4 T cells. Different clonal virus populations can be recovered from the two cell types. Inducible proviruses in all CD4 T cells should be considered when evaluating strategies to reduce the size of the HIV reservoir. Focusing only on resting CD4 T cells is likely to underestimate the latent HIV reservoir.