Distinct viral reservoirs and immune signatures in individuals on long-term antiretroviral therapy with perinatally acquired HIV-1

Abstract

Early initiation of antiretroviral therapy (ART) following HIV-1 infection restricts the size of the latent reservoir, following both horizontal and vertical infections. Here, we comprehensively profile the reservoirs and immunological milieus of nine young adults who acquired HIV-1 perinatally and remained on suppressive long-term ART (median: 20 years) since infancy (LeukoHIV cohort). Genome-intact reservoirs are markedly smaller compared to a cohort of adults on suppressive ART started in adulthood, with some LeukoHIV individuals characterized by an absence or near absence of intact proviruses in up to a billion peripheral blood mononuclear cells (PBMCs). Higher frequencies of functional CD56^bright natural killer (NK) cells with increased cytotoxic activity are detectable in the LeukoHIV cohort compared to an adult reference cohort, while one LeukoHIV participant displayed a potent HIV-1-specific CD8⁺ T cell response. Collectively, our data suggest that long-term ART initiated in early life following perinatal transmission may facilitate an immune environment better equipped to restrict the HIV-1 reservoir.

Keywords: HIV; MIP-seq; children; deep latency; leukophoresis; long-term ART; natural killer cells; perinatal infection; provirus; reservoir.

Graphical abstract

Figure 1

HIV-1 proviral reservoir dynamics of the LeukoHIV cohort compared with a cohort of elite controllers and individuals on moderate-term (8 years) suppressive ART (A–C) Frequency of total HIV-1 copies (A), genome-intact HIV-1 copies (B), and defective HIV-1 copies (C), per million PBMCs assayed, as detected by FLIP/MIP-seq. Open circle data points for cohorts in (B) indicate that the measurement is below the limit of detection. (D) Proportion of genome-intact HIV-1 copies within the total sequences identified by FLIP/MIP-seq. (E) Proportion of different sequence types of total HIV-1 sequences detected by FLIP/MIP-seq, including genome-intact, sequences with Psi defects, sequences with large deletions, sequences with premature stop codons (PSCs), hypermutated sequences, and sequences with internal inversions. (F) Parts of a whole analysis demonstrating proportions of intact sequences that are clonally identical to other sequences, and intact sequences that were detected only once, for each cohort. (G) Proportions of clonally intact proviruses within the total pool of genome-intact proviruses. (H) Frequency of replication-competent HIV-1 per million CD4⁺ cells assayed by quantitative viral outgrowth assay (QVOA). Open circle data points indicate that the measurement is below the limit of detection. (I) Maximum-likelihood phylogenetic tree of all genome-intact HIV-1 proviral sequences across the LeukoHIV cohort. Each symbol corresponds to an intact provirus detected for each member of the LeukoHIV cohort, with the symbol color relating to the respective study participant, virus clades annotated in key. As no intact sequences were detected for LeukoHIV01, no sequences are shown for that individual. Tree is rooted to a HXB2 (clade B) reference sequence. (J) The number of base pair variations within individual unique genome-intact proviruses significantly associated with autologous class I HLA alleles, as determined in a prior study. Each symbol represents one provirus. Clonal sequences were counted only once. Box and whisker plots present median, IQRs, and minimum/maximum values. (K) Proportion of wild-type clade B CTL epitopes restricted by autologous class I HLA alleles from individual unique genome-intact proviruses. Each symbol represents one provirus. Clonal sequences were counted only once. Box and whisker plots present median, IQRs, and minimum/maximum values. (L) Linear regression of the timing of ART initiation (months) against HIV-1 copies per million PBMCs (left) or log₁₀ transformed genome-intact HIV-1 copies per million PBMCs (right). Regression R² and p value presented. Spearman’s correlation rho and p value presented. Open circle data points indicate that the measurement is below the limit of detection and gray area represents the confidence interval. (A, B, C, D, G, H, J, and K) Kruskal-Wallis tests performed with false discovery rate (FDR) adjusted p values (presented as q values). (E) Fisher’s exact test performed on contingency table data, these data presented here in pie chart format, p values presented.

Figure 2

Phylogeny and chromosomal positioning of HIV-1 intact proviruses in participants LeukoHIV02, LeukoHIV04, LeukoHIV06, and LeukoHIV07 (A) Maximum-likelihood phylogenetic trees for HIV-1 intact proviruses detected by near full-length sequencing technologies for LeukoHIV02, LeukoHIV04, LeukoHIV06, and LeukoHIV07. All trees are rooted to an HXB2 reference sequence. Each symbol corresponds to a single intact sequence with the symbol shapes pertaining to the sequencing technology used to detect the respective provirus (see key). Clonal sequences are emphasized through the symbols being encircled, with clonal group numbers annotated. Sequences that differ by 1–2 base pairs from adjacent clonal sequences are labeled with an (∗). Specific proviral integration site coordinates generated through MIP-seq are documented on the trees, and the gene name is indicated where applicable. Cells assayed by FLIP/MIP-seq and QVOA are displayed with their respective tree.

Figure 3

Transcriptional behaviors of HIV-1 proviruses isolated by PRIP-seq in study participant LeukoHIV06 (A) Maximum-likelihood phylogenetic tree for individual proviruses obtained by PRIP-seq. Symbols indicate the fitness of the corresponding provirus, with the symbol color reflecting the degree of transcriptional activity observed. Chromosomal integration site coordinates and their respective gene locations are annotated on the trees. PSC, premature stop codon. (B) Circos plot representations of the locations, within the human genome, of HIV-1 transcriptionally expressed (RNA+: red) and inactive (RNA−: blue) proviruses for genic and non-genic DNA.

Figure 4

HIV-specific immune responses of the LeukoHIV cohort (A) IFN-ɣ/IL-2 FluoroSpot T assay on a representative LeukoHIV subject. (B) Scatterplots with bar (mean ± SEM) depict IL-2 (Cy3/red) or IFN-ɣ (FITC, green) spot-forming cells (SFCs) per 10⁶ peripheral blood mononuclear cells (PBMCs) after 24-h in vitro stimulation of thawed PBMCs, isolated from the LeukoHIV cohort, with HIV Gag or Env peptide pools, keyhole limpet hemocyanin (KLH, negative control), or staphylococcal enterotoxin B (SEB, positive control) on anti-IFN-ɣ and IL-2-coated plate. (C) HIV-specific CD8⁺ T cell responses in participants of focus. ELISPOT plates and the subsequent spot counts reflect the IFN-ɣ secretion in response to HLA-optimal HIV peptides or control treatments. CFSE dilution was used to measure CD8⁺ T cell proliferation (%CFSE-low), highlighting observed responses among 53 total peptides screened. LeukoHIV09 responses were recorded in response to HLA-B∗14-restricted Gag epitope DA9 (DRFYKTLRA), HLA-B∗14-restricted Env epitope (ERYLKDQQL), and HLA-B∗51-restricted Pol epitope (LPPVVAKEI); these are outlined in red, the proliferative response annotated. (D) Western blot (WB) score tabulated findings for the LeukoHIV cohort. Plasma samples collected in 2017 (left) and 2022 (right) were tested for antibodies against 9 different HIV-1 viral proteins (gp160, gp120, p66, p55/p51, gp41, p39, p31, p24, and p17). The band intensity for each viral antigen was calculated using the ImageJ software. A WB score was assigned to each participant by adding up the number of positive (1, black) and weak (0.5, gray) responses (from 0 to 9). (E) Scatterplots for WB score (2022) against Env-specific peripheral T follicular helper (pTfh) cell frequency (%) (left), Gag-specific pTfh cell frequency (%) (middle), as well as of Env-specific CD4⁺ T cell frequency (%) against the time needed to reach viral suppression in months (right), with linear regression and correlation analyses performed. Linear regression R² and p values presented. Spearman’s correlation rho and p values presented. Gray area represents the confidence interval.

Figure 5

Correlation analysis between virological data and NK cells reveals possible associations decades after virological controls (A) The differential analysis of six distinct NK cell subsets, identified according to the expression of CD56 and CD16 (gating strategy in upper left). Volcano plot in upper right shows the only 5 significantly different NK cell subsets when compared to a cohort of adult participants living with HIV. Differences are also presented as violin plots with Mann-Whitney U tests performed, p values presented. Correlation plots for the association between HIV-1 viral reservoir and (B) NK cell phenotype or (C) ADCC analysis upon stimulation with heat-inactivated autologous HIV-1-infected plasma (pHIVauto). Regression R2 and p value presented. Spearman’s correlation rho and p value presented.