Distinct viral reservoirs in individuals with spontaneous control of HIV-1

Abstract

Sustained, drug-free control of HIV-1 replication is naturally achieved in less than 0.5% of infected individuals (here termed 'elite controllers'), despite the presence of a replication-competent viral reservoir¹. Inducing such an ability to spontaneously maintain undetectable plasma viraemia is a major objective of HIV-1 cure research, but the characteristics of proviral reservoirs in elite controllers remain to be determined. Here, using next-generation sequencing of near-full-length single HIV-1 genomes and corresponding chromosomal integration sites, we show that the proviral reservoirs of elite controllers frequently consist of oligoclonal to near-monoclonal clusters of intact proviral sequences. In contrast to individuals treated with long-term antiretroviral therapy, intact proviral sequences from elite controllers were integrated at highly distinct sites in the human genome and were preferentially located in centromeric satellite DNA or in Krüppel-associated box domain-containing zinc finger genes on chromosome 19, both of which are associated with heterochromatin features. Moreover, the integration sites of intact proviral sequences from elite controllers showed an increased distance to transcriptional start sites and accessible chromatin of the host genome and were enriched in repressive chromatin marks. These data suggest that a distinct configuration of the proviral reservoir represents a structural correlate of natural viral control, and that the quality, rather than the quantity, of viral reservoirs can be an important distinguishing feature for a functional cure of HIV-1 infection. Moreover, in one elite controller, we were unable to detect intact proviral sequences despite analysing more than 1.5 billion peripheral blood mononuclear cells, which raises the possibility that a sterilizing cure of HIV-1 infection, which has previously been observed only following allogeneic haematopoietic stem cell transplantation^2,3, may be feasible in rare instances.

Extended Data Figure 1:. Viral sequence analysis of intact HIV-1 proviruses from ECs.

(a): Genetic distance (expressed as average number of base pair substitutions) among all intact near full-length proviral sequences obtained from each study participant. Clonal sequences were considered as individual sequences; participants with at least two intact proviruses are included (n=175 intact proviral sequences from 24 ECs and n=147 intact proviral sequences from 26 ART-treated individuals). (b): Frequencies of proviral species (copies per million resting CD4⁺ T-cells) detected by IPDA from EC2. (c): Proportion of optimal CTL epitopes (restricted by autologous HLA class I isotypes) with wild-type sequences. Each dot represents one intact proviral sequence. N=182 and N=133 HIV-1 clade B intact sequences from 47 ECs and 34 ART-treated individuals are included, respectively. Optimal CTL epitopes matching the clade B consensus sequences were considered as wild-type sequences. Clonal sequences were considered as individual sequences. (d-e): Average frequencies of autologous HLA-restricted optimal CTL epitopes with wild-type sequences calculated from intact proviruses in each study participant. Clonally-expanded sequences were counted either once (d) or individually (e). Each dot represents one study participant. (f): Proportion of CTL escape variants (restricted by HLA-A01/A02 supertypes, HLA-A03 supertype, or HLA-B*27/B*57). Each dot represents one intact proviral sequence. Clonal sequences were counted individually. (g-h): Proportion of clonal intact proviruses among all intact proviruses within each study participant (g) or within all intact proviruses from ECs and ART-treated individuals(h). Study participants in whom at least two intact proviruses were detected are included in (g) and (h). (Two-tailed Mann Whitney U tests were used for panels a, c-g; two-sided Fisher’s exact test was used for panel h).

Extended Data Figure 2:. Longitudinal evolution of CD4⁺ T-cell counts and HIV-1 viral loads in EC1-EC13.

The recorded diagnosis date of HIV-1 infection for each study participant is shown as the first date on x-axis. PBMC sampling time points are indicated by red arrows.

Extended Data Figure 3:. Diagrams reflecting the structural composition of proviral reservoirs in ECs.

Virograms reflect the genetic coverage of individual sequences of proviral genomes analyzed in EC3-EC13. Numbers of total near full-length proviral sequences obtained from each individual are shown on the vertical axis; numbers of independent sequences are indicated in brackets. Open boxes indicate clonal clusters.

Extended Data Figure 4:. Highlighter plot reflecting variations in HIV-1 DNA sequences in 5’ LTR regions from intact proviruses isolated from indicated ECs, relative to HXB2.

Numbers of 5’ LTR sequences of intact proviruses obtained from each individual are shown on the vertical axis. Open boxes indicate clonal clusters.

Extended Data Figure 5:. Chromosomal integration site features of intact proviruses from ECs after counting clonal sequences individually.

(a): Heatmap indicating the relative proportion of proviral integration sites of intact proviruses in each chromosome in ECs, relative to corresponding data from long-term ART treated individuals. Proviral integration site data from prior publications are shown for comparative purposes (Veenhuis et al., Maldarelli et al, Wagner et al.); integration sites from intact and defective proviruses were not distinguished in these studies. Contributions of each chromosome to total number of genes (first row) and to total size of human genome (second row) are included as references. (b-c): Proportion of near full-length intact proviruses located in indicated genomic regions. Data from near full-length intact proviral sequences in long-term ART-treated individuals (ART) are shown for reference purpose; chromosomal integration sites from unselected (intact and defective) proviral sequences in ECs (Veenhuis et al.) and in ART-treated individuals (Maldarelli et al, Wagner et al.) are also shown for comparison. (d): SPICE diagrams demonstrating proportion of intact proviruses with indicated chromosomal integration site features in ECs and ART-treated individuals. (e-f): Chromosomal distance between integration sites of intact proviruses and the most proximal transcriptional start sites (TSS, determined by RNA-Seq) (e) or to the most proximal ATAC-Seq peak (f) in autologous total, central-memory and effector-memory CD4⁺ T-cells and in GB. Horizontal lines reflect the geometric mean. (g): Proportions of proviral sequences located in structural compartments A and B, as determined based on Hi-C-Seq data published by Rao et al. Chromosomal integration regions not covered in the study by Rao et al. were excluded from analysis. (f-g): Sequences in genomic regions included in the blacklist for functional genomics analysis identified by the ENCODE and modENCODE consortia were excluded due to absence of reliable ATAC-Seq and Hi-C-Seq reads in such repetitive regions. (a-g): All members of clonal clusters were included as individual sequences. (****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, FDR-adjusted two-sided Fisher’s exact tests were used for panels b and c; two-sided Fisher’s exact tests were used for panel d and g, FDR-adjusted two-tailed Mann Whitney U tests were used for panels e and f; all comparisons were made between ECs and reference groups).

Extended Data Figure 6:. Epigenetic features of chromosomal integration sites of intact proviruses from ECs.

(a-d): Numbers of DNA sequencing reads associated with activating (H3K27ac) or repressive (H3K27me3) histone protein modifications in proximity to integration sites from ECs and long-term ART-treated individuals; median and confidence intervals (defined by one standard deviation) of ChIP-Seq data from primary memory CD4⁺ T-cells included in the ROADMAP repository are shown. Negative distances indicate genomic regions upstream of the HIV 5’ LTR host-viral junction, while positive distances indicate regions downstream of the 3’ LTR viral-host junction. DNA sequencing reads associated with H3K36me3, an activating chromatin mark that is atypically enriched in KRAB-ZNF genes on Chromosome 19, are also shown. (e-f): Proportions of intact proviral sequences located in structural compartments A and B (and associated sub-compartments) by counting clonal sequences once (e) or by counting clonal sequences individually (f), as determined based on alignment of chromosomal integration sites of intact proviruses to Hi-C-Seq data from Jurkat cells. Chromosomal integration regions not covered in the Jurkat cell study were excluded from the analysis. Compartment B4 was not assessed in the source data for this analysis. Two-sided Fisher’s exact tests were used for statistical comparisons, nominal p-values are reported. (a-f): Sequences in genomic regions included in the blacklist for functional genomics analysis identified by the ENCODE and modENCODE consortia were excluded due to absence of reliable ChIP-Seq and Hi-C-Seq reads in such repetitive regions.

Extended Data Figure 7:. Accessory chromosomal integration site features of intact proviral sequences from ECs.

(a): Expression of host genes harboring intact proviral sequences in ECs and long-term ART-treated individuals, as determined by autologous RNA-Seq data in total, central-memory (CM) and effector-memory (EM) CD4⁺ T-cells. Gene expression percentiles are indicated. (b-c): Orientation of intact proviruses relative to host genes in ECs and long-term ART-treated individuals. All data for genic integration sites with exclusive orientation towards host genes are included. Integration site data from prior studies involving ECs (Veenhuis et al.) and ART-treated individuals (Maldarelli et al., Wagner et al.) are shown for comparative purposes. (d-e): Proportion of intact proviruses from ECs and long-term ART-treated individuals in lamina-associated domains (LAD), determined using Lamin B1-DNA adenine methyltransferase Identification (DamID) by Robson et al. for resting Jurkat cells. Integration site data from prior studies involving ECs (Veenhuis et al.) and ART-treated individuals (Maldarelli et al., Wagner et al.) are shown for comparative purposes. (b, d): Clonal proviruses were counted once; (c, e): clonal proviruses were counted as individual sequences (FDR-adjusted two-sided Fisher’s exact tests). (f): Expression of LEDGF/p75 and CPSF6 mRNA in autologous total CD4⁺ T-cells from ECs and long-term ART-treated individuals, as determined by RNA-Seq. Gene expression percentiles are indicated. (a, f): Horizontal lines reflect the geometric mean. All comparisons were made between ECs and reference groups.

Extended Data Figure 8:. Chromosomal integration site features of in-vitro infected CD4⁺ T-cells from ECs and HIV-1 negative study participants.

(a): Heatmap indicating the relative proportion of proviral integration sites in sorted GFP⁺/GFP⁻ in-vitro infected CD4⁺ T-cells (determined by LM-PCR) from ECs and HIV-1 negative study participants (HIVNs), relative to proviral integration sites of intact proviruses in each chromosome in ECs; integration sites from intact and defective proviruses were not distinguished in in-vitro infection studies. Data from GFP⁺ (n=74,055) and GFP⁻ (n=15,105) CD4⁺ T-cell populations from ECs and from GFP⁺ (n=31,682) and GFP⁻ (n=4,229) CD4⁺ T-cell populations from HIVNs were included. Contributions of each chromosome to total number of genes (first row) and to total size of human genome (second row) are included as references. (b-c): Proportion of proviral integration sites located in indicated genomic regions (b) or defined genes (c). Data from near full-length intact proviral sequences in ECs are indicated for reference. (****p<0.0001, ***p<0.001, *p<0.05, FDR-adjusted two-sided Fisher’s exact tests or two-tailed Chi-square tests were used as appropriate; p-values indicating comparisons made between ECs and each in-vitro infection group are shown in corresponding colors).

Figure 1:. Proviral reservoir landscape in HIV-1 ECs.

(a-b): Relative frequencies of total (a) and near full-length intact (b) HIV-1 DNA sequences in ECs and ART-treated individuals (ART). Grey symbols: Limit of detection (expressed as 1 copy/total number of analyzed cells without target identification). Circles: Proviral sequences obtained from unfractionated PBMC; triangles: proviral sequences retrieved from isolated CD4⁺ T-cells and normalized to PBMC. (c): Proportions of proviral sequences that are genome-intact or display defined structural defects among all proviral genomes. (d): Proportion of IPs among all proviral genomes from each study participant. Only individuals with at least one detected IP are shown. (e): Average genetic distance between distinct IPs obtained from each study participant. Participants with at least two detectable IPs are included. (f): Proportion of optimal CTL epitopes (restricted by autologous HLA class I isotypes) with wild-type clade B consensus sequences. Each dot represents one IP. Clonal sequences are counted once. (g): Diagrams reflecting all proviral HIV-1 sequences isolated from EC1 and EC2. Left vertical axis: Dates of sample collection; right vertical axis: Numbers of cells analyzed. (h): Circular maximum-likelihood phylogenetic trees for all IPs from ECs and ART-treated individuals. Dots with the same colors indicate IPs detected in the same individuals. Clonal sequences, defined by complete sequence identity, are indicated by grey arches. Bootstrap analysis with 1000 replicates was performed to assign confidence to tree nodes; bootstrap support values >70% are shown in the trees. Two-tailed Mann Whitney U tests were used for panels a-b, d-f; False Discovery Rate (FDR)-adjusted two-tailed Fisher’s exact tests were used for panel c.

Figure 2:. Increased frequency of IPs integrated in centromeric satellite DNA in ECs.

(a-e): Data indicate linear maximum-likelihood phylogenetic trees for IPs from five ECs. Coordinates and relative positioning of IS are depicted; genes harboring IS are italicized. Clonal IPs, defined by identical proviral sequences and identical corresponding IS, are highlighted in curved black boxes. Red boxes reflect multi-hit IS that cannot be definitively mapped to one particular genomic location due to positioning in repetitive centromeric satellite DNA present in multiple regions of the human genome. LAD, lamina associated domain.

Figure 3:. Preferential location of IPs from ECs in genes encoding for KRAB-ZNF proteins.

(a-f): Linear maximum-likelihood phylogenetic trees demonstrate IPs from indicated study participants. Coordinates and relative positioning of IS are depicted. Other pertinent information is as defined in the legend to Figure 2.

Figure 4:. Distinct genomic and epigenetic features of IS of IPs from ECs.

(a): Relative proportion of proviral IS of IPs in each chromosome. Contributions of each chromosome to total number of genes (first row) and to total size of human genome (second row) are included as references. (b-c): Proportion of IPs located in indicated genomic regions. (a-c): Data from IPs in ART-treated individuals (ART) and from unselected (intact and defective) proviral sequences in ECs and in ART-treated individuals^, are shown as references. (d): SPICE diagrams demonstrating proportions of IPs with indicated IS features in ECs and ART-treated individuals. (e-f): Chromosomal distance between IS of IPs and the most proximal TSS in autologous total, EM or CM CD4⁺ T-cells or from Genome Browser (GB) (e), or to the most proximal ATAC-Seq peaks (f) in autologous total, EM and CM CD4⁺ T-cells. Horizontal lines reflect the geometric mean. (g): Numbers of DNA sequencing reads associated with activating (H3K4me1) or repressive (H3K9me3) histone protein modifications in proximity to IS from ECs and long-term ART-treated individuals; median and confidence intervals (one standard deviation) of ChIP-Seq data from primary memory CD4⁺ T-cells included in the ROADMAP repository are shown. (h): Proportions of IPs located in structural compartment A and B (and associated sub-compartments), as determined by Hi-C-Seq data. IS in regions not covered in ref. were excluded. (i): Numbers of cytosine residues with indicated levels of methylation (derived from CD4⁺ T-cells in the iMethyl database) in proximity (500 or 1000 bp upstream of the 5’ LTR host-viral junction) to IS from ECs and ART-treated individuals. (j): Frequencies of HIV-1 RNA transcripts in PBMC from ECs and ART-treated individuals, normalized to the corresponding number of IPs determined by FLIP-Seq. (a-i): Clonal sequences were only counted once. (f-i): Sequences in genomic regions included in the ENCODE blacklist were excluded. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05; (b/c/d/h): two-sided Fisher’s exact tests; (e/f/j): two-sided Mann Whitney U tests; (i): two-tailed Chi-square test; (b/c/e/f/i): FDR-adjusted p-values; (d/h/j): nominal p-values. All comparisons were made between ECs and reference groups.