HIV-1 reservoir evolution in infants infected with clade C from Mozambique

Abstract

Background: The persistence of HIV-1-infected cells during antiretroviral therapy is well documented but may be modulated by early initiation of antiretroviral therapy in infants.

Methods: Here, we longitudinally analyzed the proviral landscape in nine infants with vertical HIV-1 infection from Mozambique over a median period of 24 months, using single-genome, near full-length, next-generation proviral sequencing.

Results: We observed a rapid decline in the frequency of intact proviruses, leading to a disproportional under-representation of intact HIV-1 sequences within the total number of HIV-1 DNA sequences after 12-24 months of therapy. In addition, proviral integration site profiling in one infant demonstrated clonal expansion of infected cells harboring intact proviruses and indicated that viral rebound was associated with an integration site profile dominated by intact proviruses integrated into genic and accessible chromatin locations.

Conclusion: Together, these results permit rare insight into the evolution of the HIV-1 reservoir in infants infected with HIV-1 and suggest that the rapid decline of intact proviruses, relative to defective proviruses, may be attributed to a higher vulnerability of genome-intact proviruses to antiviral immunity. Technologies to analyze combinations of intact proviral sequences and corresponding integration sites permit a high-resolution analysis of HIV-1 reservoir cells after early antiretroviral treatment initiation in infants.

Keywords: Evolution; HIV-1; Infants; Integration sites; Reservoir.

Figure 1.

Longitudinal quantification of the proviral reservoir size in nine infants over the period of 24 months. (a) Longitudinal trends of total HIV copies per million PBMCs. Infant trends are distinguished by color. (b) Longitudinal absolute copies of genome-intact HIV per million PBMCs. (c) Longitudinal absolute copies of genome-defective HIV per million PBMCs. (d) Cumulative proportions of different proviruses detected from the nine infants at three different TPs grouped by age: TP1 = 2–3 months old, TP2 = 5–6 months old and TP3 = 19–36 months old. Statistical significance was tested using the chi-square test. Proportions of intact (Blue), large deletion (Red), large deletion with hypermut (Green), large deletion with internal inversion (Orange), hypermut (Black) and premature stop codon (Brown). (e) Maximum-likelihood phylogenetic tree of HIV-1 DNA intact genomes from eight infants. Proviral sequences generated from infants’ samples are differentiated by color coding: infant 1 (Blue), infant 2 (Red), infant 3 (Green), infant 5 (Brown), infant 6 (Black), infant 7 (Turquise), infant 8 (Tangerine/Orange), infant 9 (Sky blue). FLIP-Seq, full-length individual proviral sequencing; MIP-Seq, Matched integration site and proviral sequencing; PBMC, peripheral blood mononuclear cell; TP, timepoint.

Figure 2.

(a) Longitudinal viral load dynamics of infant 5 followed for a duration for 24 months. The gray bars indicate the duration of antiretroviral therapy. Arrows indicate the timepoint where specimens were collected: at age of 6 months old, 3.7 million PBMCs were assayed, at age of 10 months old, plasma specimen was analyzed and at age of 19 months old 900,000 PBMCs were assayed. (b) Proportions of different proviruses detected from infant 5 at two different TPs: The TP1 was at 6 months old and TP2 at 19 months old. Proportions of proviruses are differentiated by color: intact (Blue), large deletion (Red), large deletion with hypermut (Green), hypermut (Black). (c) Maximum-likelihood phylogenetic tree of HIV-1 DNA intact genomes from infant 5. Proviral sequences generated from the infant’s samples at different timepoints and using different assays are differentiated by color and shape. Red circles (FLIP-Seq) sequences from 2019, Blue circles (FLIP-Seq) sequences from 2018, Red squares (MIP-Seq) sequences from 2019 and Green Triangles represent sequences from plasma. (d) Chromosomal distance between integration sites of genome-intact proviral sequences and the most proximal transcriptional start sites, to the most proximal ATAC-seq peaks in total CD4+ T cells, numbers of DNA-sequencing reads in association with activating and repressive histone protein modifications in proximity to integration sites. CD, clusters of differentiation; FLIP-Seq, full-length individual proviral sequencing; IS, integration site; MIP-Seq, Matched integration site and proviral sequencing; PBMC, peripheral blood mononuclear cell; TP, timepoint.