Large number of rebounding/founder HIV variants emerge from multifocal infection in lymphatic tissues after treatment interruption

Abstract

Antiretroviral therapy (ART) suppresses HIV replication in most individuals but cannot eradicate latently infected cells established before ART was initiated. Thus, infection rebounds when treatment is interrupted by reactivation of virus production from this reservoir. Currently, one or a few latently infected resting memory CD4 T cells are thought be the principal source of recrudescent infection, but this estimate is based on peripheral blood rather than lymphoid tissues (LTs), the principal sites of virus production and persistence before initiating ART. We, therefore, examined lymph node (LN) and gut-associated lymphoid tissue (GALT) biopsies from fully suppressed subjects, interrupted therapy, monitored plasma viral load (pVL), and repeated biopsies on 12 individuals as soon as pVL became detectable. Isolated HIV RNA-positive (vRNA+) cells were detected by in situ hybridization in LTs obtained before interruption in several patients. After interruption, multiple foci of vRNA+ cells were detected in 6 of 12 individuals as soon as pVL was measureable and in some subjects, in more than one anatomic site. Minimal estimates of the number of rebounding/founder (R/F) variants were determined by single-gene amplification and sequencing of viral RNA or DNA from peripheral blood mononuclear cells and plasma obtained at or just before viral recrudescence. Sequence analysis revealed a large number of R/F viruses representing recrudescent viremia from multiple sources. Together, these findings are consistent with the origins of recrudescent infection by reactivation from many latently infected cells at multiple sites. The inferred large pool of cells and sites to rekindle recrudescent infection highlights the challenges in eradicating HIV.

Keywords: HIV/AIDS; founder population; treatment interruption; viral recrudescence.

Fig. 1.

Time to detectable plasma viremia and peak before restarting ART. The pVL from study entry (before treatment interruption) to restarting ART is plotted against time for each subject. The difference in time between first detection and peak represents the time required for scheduling procedures and completing the colonoscopy preparation.

Fig. 2.

Change in PB CD4 T-cell count during and after the interruption. PB CD4 T-cell count is plotted for each subject at baseline (while fully suppressed), at the time when viremia is first detected, at the peak (the day of biopsy), and again after the subject is fully suppressed. The black line indicates the mean for the group at each time point. There is no statistically significant change in CD4 T-cell count during the interruption or after pVL is again suppressed (the P value for a linear CD4 trend over time was 0.18).

Fig. 3.

HIV recrudescence from multiple anatomic sites on treatment interruption detected by ISH. A and B are of LN and ileum, respectively, obtained from subject 1,853, a 47-y-old male infected for 23 y and on ART for 15 y. pVL at the time of recrudescence was 857 copies/mL. When viewed in epipolarized light, the in situ hybridization signal for HIV RNA is a collection of green silver grains over cells or the FDCn (viral RNA in virions bound to the FDCn). (A) Arrow points to a dense cloud of virus particles attached to the FDCn. C and D are of ileum and rectum, respectively, from subject 1,064, a 61-y-old male infected for 25 y and on ART for 17 y. pVL at the time of the biopsy was 6,663 copies/mL. E and F are from LN and rectum, respectively, from subject 1,906, a 52-y-old male infected for 25 y and on ART for 7 y. pVL at the time of biopsy was 871 copies/mL.

Fig. 4.

Phylogenetic analysis of PBMCs and plasma viruses at recrudescence. Intrapatient phylogenetic analysis of eight patients revealed multiple variants initiating the reemergence of plasma viremia (blue). Additionally, PBMC-derived vRNA (orange) and vDNA (red) sequences revealed a highly divergent population likely contributing to recrudescent viremia. Patient identification and scale bar (nucleotide substitutions per site) are provided with each maximum likelihood tree along with the percentage of bootstrap support for each node with values greater than 80%.

Fig. 5.

Model of the origins of recrudescent infection from one or many cells and sites. The model is based on events in early infection after sexual exposure (18) and the main types of infected cells in LT (8, 29, 30). When infected cells are detected after STI in LT biopsies, they theoretically could represent systemic infection seeded from a single focus or multiple foci, with levels of virus in the blood below the limits of detection. However, in the multifocal model, there is a greater likelihood of sampling a site from which infection initiated. In this case, there will be many infected cells in the focus and FDC deposits of virus, reflecting the greater duration of infection. The most important distinction, however, is virus genetic diversity, which will be greater in recrudescent infection from multiple cells and sites vs. monophyletic viruses from a single reactivating focus.