Supplemental Material for Rhodehouse et al., 2018

Supplementary data files - figures, tables and microscope image

The latent reservoir of inducible, infectious HIV-1 does not decrease despite decades of antiretroviral therapy.

HIV-1 persists in a latent reservoir in resting CD4+ T cells despite antiretroviral therapy (ART). The reservoir decays slowly over the first 7 years of ART (t1/2 = 44 months). However, whether decay continues with long-term ART is unclear. Recent integration site studies indicate gradual selection against inducible, intact proviruses, raising speculation that decades of ART might allow treatment interruption without viral rebound. Therefore, we measured the reservoir in 42 people on long-term ART (mean 22 years) using a quantitative viral outgrowth assay. After 7 years of ART, there was no long-term decrease in the frequency of inducible, replication-competent proviruses but rather an increase with an estimated doubling time of 23 years. Another reservoir assay, the intact proviral DNA assay, confirmed that reservoir decay with t1/2 of 44 months did not continue with long-term ART. The lack of decay reflected proliferation of infected cells. Most inducible, replication-competent viruses (79.8%) had env sequences identical to those of other isolates from the same sample. Thus, although integration site analysis indicates changes in reservoir composition, the proliferation of CD4+ T cells counteracts decay, maintaining the frequency of inducible, replication-competent proviruses at roughly constant levels over the long term. These results reinforce the need for lifelong ART

Antigen-driven clonal selection shapes the persistence of HIV-1 infected CD4+ T cells in vivo

Clonal expansion of infected CD4+ T cells is a major mechanism of HIV-1 persistence and a barrier to achieving a cure. Potential causes are homeostatic proliferation, effects of HIV-1 integration, and interaction with antigens. Here, we show that it is possible to link antigen responsiveness, the full proviral sequence, the integration site, and the T cell receptor β-chain (TCRβ) sequence to examine the role of recurrent antigenic exposure in maintaining the HIV-1 reservoir. We isolated CMV- and Gag-responding CD4+ T cells from 10 treated individuals. Proviral populations in CMV-responding cells were dominated by large clones, including clones harboring replication-competent proviruses. TCRβ repertoires showed high clonality driven by converging adaptive responses. Although some proviruses were in genes linked to HIV-1 persistence (BACH2, STAT5B, MKL1), the proliferation of infected cells under antigenic stimulation occurred regardless of the site of integration. Paired TCRβ and integration site analysis showed that infection could occur early or late in the course of a clone's response to antigen and could generate infected cell populations too large to be explained solely by homeostatic proliferation. Together, these findings implicate antigen-driven clonal selection as a major factor in HIV-1 persistence, a finding that will be a difficult challenge to eradication efforts