Reduction of HIV-1 Reservoir Size and Diversity After 1 Year of cART Among Brazilian Individuals Starting Treatment During Early Stages of Acute Infection

Abstract

The aim of early combined antiretroviral therapy (cART) of HIV is to limit the seeding of the viral reservoir during the initial phase of infection and, consequently, decrease intrahost viral diversity. Here, we assessed the effect of early cART on size and complexity of the proviral reservoir. Peripheral blood mononuclear cell (PBMC) and plasma samples were obtained from ten HIV-infected Brazilian individuals diagnosed at the acute phase of infection, before (PRE_ART) and 12 months (M12_ART) after suppressive cART. HIV proviral reservoir size was determined by quantitative real-time PCR; intrahost viral diversity of the env C2-V3 region was assessed by single genome amplification or next-generation sequencing in PBMC and plasma, respectively. Mean nucleotide diversity (π) and normalized Shannon entropy (H_SN) were used to infer the complexity of the viral population. Compared to PRE_ART, M12_ART saw an immunological recovery with a gain of ∼200 CD4⁺ T cells (P = 0.008) and a normalization of the CD4/CD8 ratio [1.0 (IQR: 0.88-1.18), P = 0.016], as well as a significant decrease in HIV-1 RNA (∼4 log, P = 0.004) and DNA (∼1 log, P = 0.002) levels. The median time to achieve viral suppression was 3 months (IQR: 2.8-5.8 months). The high intermixing between sequences from both visits suggests that the HIV-1 DNA reservoir remained remarkably stable under cART. After 1 year of cART, there was a minor reduction in proviral π (Pre_ART = 0.20 vs. M12_ART = 0.10; P = 0.156) but a significant decrease in H_SN (Pre_ART = 0.41 vs. M12_ART = 0.25; P = 0.019). We found no correlation between π or H_SN at Pre_ART and the rate of HIV DNA decay, T CD4⁺ counts, or CD4/CD8 ratio at M12_ART. Based on a small cohort of Brazilian infected individuals under early cART and analyses of the env region, 1 year of follow-up suggested a reservoir size reduction, allowed a significant decrease of HIV-1 complexity, and achieved immunological restoration regardless of the initial HIV-1 plasma viral load, CD4⁺ T cell counts, or HIV-1 subtype. However, further studies in the Brazilian setting aiming a longer follow-up and larger cohort are required in this field.

Keywords: HIV-1; acute infection; diversity; early cART; reservoir.

FIGURE 1

Immunological and virological measurements before and after cART initiation. CD4⁺ T cell counts (A), CD4⁺/CD8⁺ ratios (B), HIV-1 proviral load in PBMCs (C), and HIV-1 viral load in plasma (D) were measured at Pre_ART and M12_ART visits (pink circles and blue squares, respectively). P-values <0.05 were considered statistically significant.

FIGURE 2

Clinical follow-up of the ten early-treated individuals. T cell counts (cells/μL; CD4⁺, blue circles; CD8⁺, red squares) and plasma RNA viral loads (log copies/mL, green triangles) values over time after cART onset (months) are shown on the left and right Y-axis, respectively. The individuals’ identifications are indicated at the top of each graph.

FIGURE 3

Impact of early cART initiation on intrahost HIV-1 population diversity. (A) ML phylogenetic tree of the env sequences from Pre_ART and M12_ART visits. Tips’ shapes represent the viral compartment (proviral DNA, circles; plasma RNA, triangles) and are color-coded according to the visit (Pre_ART, pink; M12_ART, purple). The branches’ colors agree with the subtype assignment as indicated in the legend. Clusters from each individual are indicated by shaded gray boxes. Branch supports (aLRT-SH) are indicated at key nodes. Tips shapes marked with an “H” indicate the presence of APOBEC3G-mediated G to A hypermutations and X4 labels highlight X4-tropic sequences. Horizontal branch lengths are proportional to the bar at the bottom indicating nucleotide substitutions per site. (B) Mean nucleotide diversity (π) and normalized Shannon entropy (H_SN) indices were calculated from the proviral env sequences obtained at Pre_ART and M12_ART visits. Thick and thin lines represent the median and interquartile ranges, respectively. P-values <0.05 were considered statistically significant.

FIGURE 4

Correlations between proviral HIV-1 diversity indices and immunological and virological measurements. Mean nucleotide diversity (π) of the proviral population at Pre_ART was compared with HIV decay in PBMCs (A), T CD4⁺ cell change (B), and CD4⁺/CD8⁺ ratio in M12_ART (C). Normalized Shannon entropy (H_SN) of the proviral population at Pre_ART was compared with HIV decay in PBMCs (D), T CD4⁺ cell change (E), and CD4⁺/CD8⁺ ratio in M12_ART (F). P-values <0.05 were considered statistically significant.