The HIV-1 reservoir landscape in persistent elite controllers and transient elite controllers

The HIV-1 reservoir landscape in persistent elite controllers and transient elite controllers Carmen Gasca-Capote, … , Xu G. Yu, Ezequiel Ruiz-Mateos J Clin Invest. 2024;134(8):e174215. https://doi.org/10.1172/JCI174215. Graphical abstract Clinical Medicine AIDS/HIV Virology Find the latest version: https://jci.me/174215/pdf The Journal of Clinical Investigation CLINICAL MEDICINE 1 Introduction Elite controllers (ECs) represent a small subset of people living with HIV-1 (PLHIV), less than 1%, who can maintain undetectable viral load in the absence of antiretroviral therapy (ART) (1–3). ECs consist of a heterogeneous and dynamic group regarding virological, immunological, and clinical factors (4–7). This has enabled us to classify ECs in 2 different phenotypes: persistent elite controllers (PCs), who maintain the virological control indefinitely over time; BACKGROUND. Persistent controllers (PCs) maintain antiretroviral-free HIV-1 control indefinitely over time, while transient controllers (TCs) eventually lose virological control. It is essential to characterize the quality of the HIV reservoir in terms of these phenotypes in order to identify the factors that lead to HIV progression and to open new avenues toward an HIV cure. METHODS. The characterization of HIV-1 reservoir from peripheral blood mononuclear cells was performed using nextgeneration sequencing techniques, such as full-length individual and matched integration site proviral sequencing (FLIP-Seq; MIP-Seq). RESULTS. PCs and TCs, before losing virological control, presented significantly lower total, intact, and defective proviruses compared with those of participants on antiretroviral therapy (ART). No differences were found in total and defective proviruses between PCs and TCs. However, intact provirus levels were lower in PCs compared with TCs; indeed the intact/ defective HIV-DNA ratio was significantly higher in TCs. Clonally expanded intact proviruses were found only in PCs and located in centromeric satellite DNA or zinc-finger genes, both associated with heterochromatin features. In contrast, sampled intact proviruses were located in permissive genic euchromatic positions in TCs. CONCLUSIONS. These results suggest the need for, and can give guidance to, the design of future research to identify a distinct proviral landscape that may be associated with the persistent control of HIV-1 without ART. FUNDING. Instituto de Salud Carlos III (FI17/00186, FI19/00083, MV20/00057, PI18/01532, PI19/01127 and PI22/01796), Gilead Fellowships (GLD22/00147). NIH grants AI155171, AI116228, AI078799, HL134539, DA047034, MH134823, amfAR ARCHE and the Bill and Melinda Gates Foundation. The HIV-1 reservoir landscape in persistent elite controllers and transient elite controllers Carmen Gasca-Capote,1 Xiaodong Lian,2,3 Ce Gao,2,3 Isabelle C. Roseto,2,3 María Reyes Jiménez-León,1 Gregory Gladkov,2,3 María Inés Camacho-Sojo,1 Alberto Pérez-Gómez,1 Isabel Gallego,1 Luis E. Lopez-Cortes,4,5,6,7 Sara Bachiller,1,8 Joana Vitalle,1 Mohamed Rafii-El-Idrissi Benhnia,1,8 Francisco J. Ostos,1,8 Antonio R. Collado-Romacho,9 Jesús Santos,10 Rosario Palacios,10 Cristina Gomez-Ayerbe,10 Leopoldo Muñoz-Medina,11 Andrés Ruiz-Sancho,11 Mario Frias,7,12 Antonio Rivero-Juarez,7,12 Cristina Roca-Oporto,1 Carmen Hidalgo-Tenorio,13 Anna Rull,7,14 Julian Olalla,15 Miguel A. Lopez-Ruz,13 Francesc Vidal,7,14 Consuelo Viladés,7,14 Andrea Mastrangelo,16 Matthias Cavassini,17 Nuria Espinosa,1 Matthieu Perreau,16 Joaquin Peraire,7,14 Antonio Rivero,7,12 Luis F. López-Cortes,1 Mathias Lichterfeld,2,3 Xu G. Yu,2,3 and Ezequiel Ruiz-Mateos1 1Institute of Biomedicine of Seville (IBiS), Virgen del Rocio University Hospital, Spanish National Research Council (CSIC), University of Seville, Clinical Unit of Infectious Diseases, Microbiology and Parasitology, Seville, Spain. 2Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts, USA. 3Infectious Disease Division, Brigham and Women’s Hospital, Boston, Massachusetts, USA. 4Clinical Unit of Infectious Diseases and Microbiology, Virgen Macarena University Hospital, Seville, Spain. 5Department of Medicine and Microbiology, School of Medicine and 6IBiS, Virgen Macarena University Hospital, CSIC, University of Seville, Seville, Spain. 7CIBERINFEC, Institute of Health Carlos III (ISCIII), Madrid, Spain. 8Department of Medical Biochemistry, Molecular Biology, and Immunology, School of Medicine, University of Seville, Seville, Spain. 9Infectious Diseases Unit, Internal Medicine Service, Torrecardenas University Hospital, Almeria, Spain. 10Infectious Diseases, Microbiology and Preventive Medicine Unit, Virgen de la Victoria University Hospital, Malaga, Spain. 11Unit of Infectious Diseases, San Cecilio University Hospital, Biohealth Research Institute, IBS-Granada, Granada, Spain. 12Service of Infectious Diseases, Reina Sofía University Hospital, Maimonides Biomedical Research Institute of Cordoba (IMIBIC), Córdoba University, Cordoba, Spain. 13Unit of Infectious Diseases, Virgen de las Nieves University Hospital, Biohealth Research Institute, IBS-Granada, Granada, Spain. 14Joan XXIII University Hospital of Tarragona, IISPV, University of Rovira i Virgili, Tarragona, Spain. 15Internal Medicine Department, Costa Del Sol Hospital, Marbella, Spain. 16Service of Immunology and Allergy, Lausanne University Hospital and 17Service of Infectious Diseases, Lausanne University Hospital, University of Lausanne, Lausanne, Switzerland. Conflict of interest: ERM and CGC are authors on the patent “Methods to determine the thymic function by droplet digital PCR system” under patent number EP23383145. Copyright: © 2024, Gasca-Capote et al. This is an open access article published under the terms of the Creative Commons Attribution 4.0 International License. Submitted: July 25, 2023; Accepted: February 13, 2024; Published: February 20, 2024. Reference information: J Clin Invest. 2024;134(8):e174215. https://doi.org/10.1172/JCI174215. The Journal of Clinical Investigation CLINICAL MEDICINE 2J Clin Invest. 2024;134(8):e174215 https://doi.org/10.1172/JCI174215 mediated selection factors that preferentially eliminate intact proviruses in accessible chromatin (18, 21). Despite all these advances, the HIV reservoir quality in PCs and TCs is unknown. It is important to elucidate the multiple mechanisms involved in the persistent natural virological control (22) since it would facilitate the future design of immunotherapies to bias the reservoir to a deep latency state compatible with permanent virological remission off ART. In this work, we comprehensively characterize the HIV reservoir and immunological footprints in TCs before losing the virological control compared with PCs who have been controlling the virus for a median of 25 years in absence of ART. Results Characteristics and clinical parameters of study participants. Twentyseven ECs, with undetectable viral load in the absence of ART for at least 1 year of follow-up, were included in the study (5). Ten ECs were classified as TCs after experiencing a loss of virological control during more than 1 year of follow up, and 17 as PCs after maintaining persistent virological control during the follow-up period (5). Characteristics and clinical parameters of PCs and TCs are detailed in Table 1. The PC group was older and presented a longer time since HIV diagnosis than the TC group (Table 1). There were no differences in the remaining variables. Fifty-three percent of PCs presented protective HLA-alleles, HLA-B27/B57, versus 20 percent of TCs. Viral loads (HIV-RNA copies/mL), CD4+ T cells (cells/mm3), and the CD4/CD8 ratio from the 10 TCs, before and after losing the virological control, are shown in Figure 1. The studied time points of the TCs preceded the loss of the virological control from 0.3 to 2 years. PCs have maintained undetectable viral load with no ART for a median of 25.5 [IQR, 22.3–31.3] years with a median of 791 [IQR, 647–1013] CD4+ T cells/mm3. Distinct HIV-1 reservoir landscape in PCs compared to TCs before losing virological control. The analysis of proviral sequences was performed using full length individual proviral sequencing (FLIP-Seq). The number of cells assayed, clades, and the total, intact and defective proviral sequences of the PCs and TCs are detailed in Supplemental Table 1 and 2; supplemental material available online with this article; https://doi.org/10.1172/ JCI174215DS1, respectively. The individual HIV-1 proviral genome analysis was not different in the total (intact plus defective) (P = 0.167) (Figure 2A) nor the defective provirus levels (P = 0.141) (Figure 2B) between PCs (n = 17) and TCs (n = 10). Interestingly, the NFL intact provirus levels were significantly increased in TCs in comparison with PCs (P = 0.006) (Figure 2C) and importantly, no genome-intact HIV-1 was detected in 70.59% of PCs (indicated as grey dots in Figure 2C). Consequently, the intact/defective HIV-DNA ratio was significantly higher in TCs compared with PCs, counting the clones (P = 0.013) or not (P = 0.005) (Supplemental Figure 1, A and B, respectively). Significant differences were found in the total (Fig- and transient elite controllers (TCs), who lose the virological control after being able to control viral replication without ART (5, 8). Previous studies have focused on distinguishing both phenotypes, PC and TC, to determine the causes of the loss of the virological control and to define the best model of persistent viral remission. It is known that PCs have different immunological (5), proteomic (9), metabolomic (10), microRNA (11), and virological profiles (5) compared with TCs before losing the virological control. TCs, in comparison with PCs, are characterized by displaying a weaker HIV-specific T cell response with more limited polyfunctionality (5), a higher expression of markers associated with inflammation (9), a dysregulation of metabolic parameters (10, 11) and a higher viral diversity in env and gag genes (5) even one year before losing virological control. Together, these results suggest that PCs can serve as a premier model for understanding immune pathways responsible for spontaneous cures of HIV and for designing new HIV cure and treatment strategies (12). Nevertheless, the mechanisms that allow PCs to maintain viral control in the absence of ART have not been fully characterized. Studies focused on this phenotype, also known as exceptional ECs (13), concluded that PCs generally present a low HIV reservoir and absence of viral diversity and evolution in HIV-1 env and gag sequences (5, 13). Next-generation sequencing (NGS) techniques have changed the paradigm in the characterization of HIV reservoirs. NGS amplifies and sequences single, near full-length (NFL) HIV-1 proviruses, distinguishing between intact and defective provirus and identifying the integration site of the provirus in the genome (14, 15). A distinct proviral reservoir landscape has been associated with the natural control of the HIV-1 infection (16–19), in which intact proviruses from a subset of ECs were preferentially integrated into centromeric satellite DNA or in Krüppel-associated box domain containing zinc finger genes, related to heterochromatin features (20) that do not support effective viral transcription (16). These findings suggest that the quality rather than the quantity of the HIV reservoir plays a crucial role in the search of a functional cure for HIV-1 infection (16). Nevertheless, the mechanisms that lead to this distinct, beneficial, intact proviral reservoir in ECs remain unclear, and may be associated with immune- Table 1. Characteristics and clinical parameters of study participants Variables PC (n = 17) TC (n = 10) P value Female, n (%) 6 (35.3) 4 (40.0) 0.810 Time before losing viral control (years) [range] n/a 1.2 [0.5-1.7] n/a Age (years) [range] 55 [47 – 57] 41 [37-51] 0.040 Time since HIV diagnosis (years) [range] 25.5 [22.3–31.3] 16.8 [13.1–19.5] 0.005 CD4+ T cell counts (cells/mm3) [range] 791 [647–1013] 805 [644–902] 0.874 CD4/CD8 ratio [range]A1 [0.5–1.3] 1 [0.6–1.6] 0.905 HLA-B57, n (%) 6 (35.3) 2 (20) 0.410 HLA-B27, n (%) 3 (17.7) 00.167 HLA-B27/B57, n (%) 9 (53.0) 2 (20) 0.099 Categorical variables are expressed as numbers and percentages (%), and continuous variables are expressed as median (interquartile ranges). χ2 and Mann-Whitney U tests were used to compare categorical and continuous variables, respectively. P < 0.05 was considered statistically significant. ACD8+ T cell count not available in TC1 and TC3. The Journal of Clinical Investigation CLINICAL MEDICINE 3 J Clin Invest. 2024;134(8):e174215 https://doi.org/10.1172/JCI174215 sequences from PCs and TCs (Figure 3B). The proportion of intact proviruses (P = 0.006) and hypermutations (P = 0.013) were higher in TCs compared with PCs. However, the large deletion (LD) genome-defective provirus levels were lower in TCs compared with PCs (P = 0.004). No differences were found in the proportion of packaging signal defect (PSI) (P = 0.518), premature stop codon (PMSC) (P > 0.999) and internal inversion (P = 0.226) (Figure 3B) between PCs and TCs. Nevertheless, the proportion of sequences ure 2A), defective (Figure 2B), and intact provirus levels (Figure 2C) of PCs and TCs compared with participants on ART. PCs with higher levels of intact proviruses (n = 3) (Figure 2C) were derived from clonally expanded HIV-1 infected cells that accounted for 100% of all intact proviruses (Figure 3A; left panel). However, clonally expanded intact proviruses were not observed in TCs (Figure 3A; right panel). Further, significant differences were found in the proportion of nonclonal intact and defective Figure 1. CD4+ T cell counts, viral load levels, and the CD4/CD8 ratio in TCs. CD4+ T cell counts and the CD4/CD8 ratio were represented in the left axes (CD4+ T cell count in orange and CD4/CD8 ratio in green) and viral load levels in the right axes (red). CD8+ T cell counts were unavailable in TC1 and TC3. The black dot represents the studied time point that preceded the loss of the virological control. The Journal of Clinical Investigation CLINICAL MEDICINE 4J Clin Invest. 2024;134(8):e174215 https://doi.org/10.1172/JCI174215 the virological control. Further, no clonal intact proviruses were detected in these participants (Figure 4; TC1 and TC2). Cell-associated HIV-1 RNA levels in PCs compared with TCs before losing virological control. We also found differences in cell-associ- ated HIV-1 RNA levels, which were higher in TCs (n = 5) compared with PCs (n = 14) (P = 0.018) (Figure 5A). Higher levels of cell-as- sociated HIV-1 RNA detected in PCs corresponded to PC1 (4,602 copies per 106 TATA-box binding protein (TBP) RNA) (Figure 5A and Figure 4; PC1) and PC2 (347 copies per 106 TBP RNA) (Figure with internal inversion, counting clonal sequences, was significantly higher in PCs compared with TCs (P = 0.005), since internal inversion accounted for 57.9% of the HIV-1 total proviruses (81.8% were clonal) in the PC10 (Supplemental Figure 2). Distinct integration sites of intact genome proviruses in PCs compared with TCs before losing the virological control. For a more in-depth analysis, together with FLIP-Seq, we assayed the corresponding intact provirus chromosomal integration site by matched integration site and proviral sequencing (MIP-Seq) and integration site loop amplification (ISLA). We analyzed the HIV-1 chromosomal integration sites of the intact proviruses of PCs and TCs (Figure 4). The intact provirus chromosomal integration sites are detailed in Supplemental Table 3. We focused on participants with intact genome proviruses and available samples to perform integration site analysis: 2 PCs (PC1 and PC2) that presented higher intact proviruses, derived from clonally identical sequences, and 2 TCs (TC1 and TC2) 0.3 and 0.6 years before losing the virological control, respectively. PC1 and PC2 have maintained ART-free HIV-1 control for 37 and 31 years, and TC1 and TC2 for 9 and 15 years until losing the virological control, respectively. PC1 presented 2 large clonal genome-intact proviral sequences located in centromeric satellite DNA and in genes that encode members of the Zinc Finger Nucleases (ZNF) protein family, ZNF26 (Figure 4; PC1). PC2 also presented 2 large clonal genome-intact proviral sequences, both located in ZNF genes, ZNF160 and ZNF607 (Figure 4; PC2) on chromosome 19. In contrast to PCs, TC1 and TC2 intact proviruses were located in genic regions before losing Figure 2. Analysis of HIV-1 proviral sequences in PCs, TCs, and participants on ART. (A) Total, (B) defective, and (C) intact provirus levels in PCs, TCs, and participants on ART. Grey dots represent values below the limit of detection (expressed as 0.05 copy/total number of analyzed cells without target identification). PCs and TCs are represented by unique identifiers (Supplemental Tables 1 and 2). Each dot represents a participant. Mann-Whitney U test was used to compare PCs, TCs, and participants on ART. P < 0.05 was considered statistically significant. **P ≤ 0.01, ****P ≤ 0.0001. Figure 3. Genome-proviral sequences in PCs and TCs. (A) Circular maximumlikelihood phylogenetic trees for all genome-intact proviral sequences from PCs and TCs. HXB2 was used as the reference HIV-1 sequence. Dots with the same colors represent genome-intact proviral sequences from the same participant. Clonal sequences are indicated by black arches. PCs and TCs are represented by unique identifiers (Supplemental Tables 1 and 2). (B) Proportions of nonclonal genome-proviral sequences. Intact and defective proviruses as packaging signal defect (PSI), large deletion (LD), premature stop codon (PMSC) hypermutations and internal inversion, were included. FDR-adjusted 2-tailed Fisher’s exact tests were used to compare PCs and TCs. P < 0.05 was considered statistically significant. *P ≤ 0.05, **P ≤ 0.01. The Journal of Clinical Investigation CLINICAL MEDICINE 5 J Clin Invest. 2024;134(8):e174215 https://doi.org/10.1172/JCI174215 no genome-intact HIV-1 proviruses were found at 2 different time points, 2010 and 2017, in a total of 9.9 million cells. No differences were found in the defective provirus levels (Figure 6; PC6). We also analyzed the viral reservoir of one TC, TC10, who maintained virological control for 29 years, at 3 different time points, 13, 3, and 2 years before losing virological control, in a total of 8.2 million cells. Interestingly, intact HIV-1 proviruses were found 2 years before losing control, not being detected 13 and 3 years preceding aborted virological control (Figure 6; TC10). High defective provirus levels, mainly with hypermutations, were found even 13 years before losing virological control. Distinct signature of immune selection in intact and defective proviral sequences of PCs and TCs. We analyzed the frequencies of amino acid variations associated with sensitivity or resistance to broadly neutralizing antibodies (bnAbs) recognizing the CD4 binding site, the V2/V3 envelope regions, or the membrane proximal external region (MPER), as previously reported (23), per intact and defective proviral sequence in PCs and TCs. Overall, TCs presented higher number of amino acid changes associated with resistance to bnAbs per intact (P = 0.021) (Figure 7A) and defective provirus (P = 0.007) (Figure 7B) than PCs. These differences were more remarkable for antibodies targeting the CD4 binding site (P = 0.011) and for antibodies recognizing the V2 envelope region (P < 0.0001) in intact provirus (Supplemental Figure 3A) and MPER regions (P = 0.080) in defective proviruses (Supplemental Figure 3B). Interestingly, intact-genome sequences with higher number of bnAb-resis- tance sites in TCs (Figure 7A) belonged to the participant who was 5A and Figure 4; PC2). Interestingly, cell-associated HIV-1 RNA was not detected in 5 PCs who corresponded with those with no intact proviruses detected (grey dots in Figure 5A). Cell-associat- ed HIV-1 RNA levels significantly correlated with total (Figure 5B) and intact proviruses (Figure 5C) but not with defective proviruses (Figure 5D). Excluding PC1, the correlation remained significant between cell-associated HIV-1 RNA and intact proviruses, finding a trend with total proviruses. Longitudinal analysis of viral reservoir landscape in PCs and TCs. Genome-intact HIV-1 proviruses were not detected in the 70.59% of the PCs (grey dots in Figure 2C). Consequently, we analyzed the reservoir of 4 of these PCs (PC3, PC4, PC5, and PC6) with available samples at different time points to evaluate possible changes in the viral reservoir over time (Figure 6). PC3 has maintained virological control for 27 years and no genome-intact HIV-1 proviruses were found in 3 different time points, 2009, 2018 and 2019, in a total of 21.6 million cells assayed. Differences in the defective provirus levels were found at different time points (Figure 6; PC3). PC4 has maintained virological control for 33 years and no genome-intact HIV-1 proviruses were found at 2 different time points, 2017 and 2019, in a total of 9.2 million cells. Differences in the defective provirus levels were found at different time points (Figure 6; PC4). PC5 has maintained virological control for 24 years and no genome-intact HIV-1 proviruses were found at 2 different time points, 2017 and 2018, in a total of 15 million cells. There were no differences in the defective provirus levels (Figure 6; PC5). PC6 has maintained virological control for 27 years and Figure 4. Simultaneous analysis of HIV-1 proviral sequences and integration sites in linear maximum-likelihood phylogenetic trees. Coordinates and relative positioning of integration sites are indicated. Clonal genome proviral sequences, defined by identical proviral sequences and identical corresponding integration sites, are highlighted in black boxes. The rest of the symbols represent different types of defective proviruses. The Journal of Clinical Investigation CLINICAL MEDICINE 6J Clin Invest. 2024;134(8):e174215 https://doi.org/10.1172/JCI174215 closer to losing virological control, 0.3 years before the loss (Figure 4; TC1). On the contrary, PCs presented higher frequencies of amino acid variations associated with sensitivity to bnAbs per intact (P = 0.065) (Figure 7C) and defective proviruses (P = 0.020) (Figure 7D). These differences were more pronounced for antibodies recognizing the V2 envelope region (P = 0.023) and MPER region (P = 0.046) (Supplemental Figure 4A) in intact provirus and the CD4 binding sites (P = 0.047) and V3 envelope region (P = 0.021) in defective proviruses (Supplemental Figure 4B). Unexpectedly, the frequencies of amino acid variations associated with sensitivity to bnAbs recognizing the V3 envelope region in intact proviruses were significantly higher in TCs (P = 0.020) (Supplemental Figure 4A) whereas the frequencies of amino acid variations associated with resistance to bnAbs recognizing the same region were lower (P = 0.022) compared with PCs (Supplemental Figure 3A). We next analyzed signs of cytotoxic T lymphocyte–driven (CTL-driven) immune pressure in the proviral sequences of PCs and TCs. No differences were found in the proportion of WT CTL or escape variant per intact (Supplemental Figure 5, A and B) and defective proviruses (Supplemental Figure 5, C and D) of PCs and TCs. Distinct Gag-specific T cell responses in PCs and TCs. Immunological differences between PCs and TCs have previously been associated with the loss of the virological control (5). We compared and associated immune parameters of 14 PCs and 5 TCs with the quality of the HIV-1 reservoir. No differences in the magnitude of the response, assayed by cytokine production (IL-2, TNFA, and IFNG) after stimulation with Gag peptides in PCs and TCs, were observed neither in CD4+ nor in CD8+T cells (for gating strategy, see Supplemental Figure 6). However, a lower frequency of polyfunctionality, defined as simultaneous production of IFNG, TNFA, IL-2, CD107a, and perforin (PRF) per T cell in response to Gag stimulation, was observed in central memory (CM) CD4+ T cells in TCs compared with PCs (P = 0.049) (Figure 8A). Significant positive correlations were found between total proviruses and the frequency of HIV-specific total memory (P = 0.037; r=0.900) and CM CD4+ T cell response (P = 0.037; r=0.900) (Figure 8B) in TCs but not in PCs (P = 0.311; r=0.292 and P = 0.383; r=0.253, respectively) (Figure 8C). Distinct HIV-specific CD8+ T cell proliferation in PCs and TCs. Given the importance of the CD8+ T cell role in the spontaneous virological control (24), we analyzed CD8+ T cell proliferation in PCs and TCs after stimulation with an HIV (Gag)-specific peptide pool. Comparing the experimental condition (Figure 9, A–D; right panel) with the negative control (Figure 9, A–D; middle panel) Figure 5. Cell-associated HIV-1-RNA in PCs and TCs. (A) Cell-associated HIV-1-RNA, expressed as copies per 106 TBP RNA. Correlation between cell-associ- ated HIV-1 RNA and (B) total, (C) intact and (D) defective genome proviruses in PCs and TCs. Each dot represents a participant. PCs and TCs are represented by unique identifiers (Supplemental Tables 1 and 2). Correlations were performed also excluding the participant in the red circle (PC1), in this case statistics are indicated in red. Mann-Whitney U test was used to compare PCs and TCs. P < 0.05 was considered statistically significant. *P ≤ 0.05. Spearman test was used for nonparametric correlations. The Journal of Clinical Investigation CLINICAL MEDICINE 7 J Clin Invest. 2024;134(8):e174215 https://doi.org/10.1172/JCI174215 we found that TC2 (Figure 4; TC2) and TC4 (Figure 9, A and B, respectively) presented a higher CD8+ T cell proliferation before losing the virological control than PC1 (Figure 4; PC1) and PC7 (Figure 9, C and D, respectively). After that, to prove the lack of proliferation in PCs, we analyzed the CD8+ T cell proliferation in 2 longitudinal samples, T0 (Figure 9C) and T1 (Figure 9E), 1 year after T0, of PC1 (Figure 4; PC1). The CD8+ T cell proliferation was not changed over time, since no differences were found between experimental (Figure 9, C and E; right panel) and negative control (Figure 9, C and E; middle panel) in any of the time points. The positive control stimulated with Staphylococcal enterotoxin B (SEB) validated the efficacy of the assay (Figure 9, A–E; left panel). Thymic function in PCs compared with TCs before losing virological control. Thymic function has been associated with HIV disease progression (25). We assayed the sj/β-TREC ratio in 11 PCs and 10 TCs by droplet digital PCR (ddPCR). We observed that sj/β-TREC ratio was significantly increased in TCs in comparison with PCs (P = 0.024) (Figure 10A). Nevertheless, these differences were colineal with age, as we observed an inverse correlation between age and sj/β-TREC ratio (r = –0.410; P = 0.065) (Figure 10B) and TCs were younger than PCs (Table 1). However, a significant positive correlation was found between sj/β-TREC ratio and the levels of intact proviruses in TCs (r = 0.709; P = 0.022) (Figure 10C) but not in PCs (data not shown). Discussion Despite the recent advances in measuring and characterizing the HIV reservoir (26, 27), the characteristics and distinctive features of PC and TC HIV-1 reservoirs remain poorly defined. Understanding the mechanisms responsible for the maintenance and loss of virological control in ECs is important to design functional or sterilizing cure strategies. The present study, which comprehensively analyzed the quality of the TC HIV-1 reservoir before losing virological control, demonstrates a distinct and dynamic viral reservoir landscape in TCs compared with PCs. Our analysis showed, as indicative factors of the viral rebound, higher intact proviruses and cell-associated HIV-1 RNA levels, and a higher viral diversity with no detectable intact provirus clones in TCs compared with PCs. Importantly, intact proviruses may be more likely to be located in permissive genic euchromatic positions in TCs, although our results are limited by sample size. These findings are crucial since they could be consistent with TCs having subclinical HIV-1 replication before losing virologic control; analysis of additional measures of active virus replication are suggested to test this hypothesis further. Despite that no differences were observed in defective provirus levels between PCs and TCs, a higher proportion of hypermutations was found in TCs and may facilitate the HIV-1 escape from the immune system by increasing the genetic diversity and the evolution of viral variants, as previously reported (28). As opposed to TCs, genome-intact proviral sequences were not detected in 70.59% of PCs after analyzing millions of cells. It is notable that no cell-associated HIV-1 RNA was detected in 5 of these PCs. The absence of genome-intact proviral sequences in a large number of analyzed cells has previously been associated with a spontaneous cure of HIV-1 infection in the Berlin (16, 29) Figure 6. Longitudinal evolution of genome proviral reservoir landscape in PCs and TCs. Total, intact, and defective proviruses levels in PC3, 4, 5, 6, and TC10 over time. Figure 7. Analysis of bnAb resistance and sensitivity signature sites in intact and defective proviral sequences of PCs and TCs. Number of bnAb resistance sites per (A) intact and (B) defective provirus in PCs and TCs. Number of bnAb sensitivity sites per (C) intact and (D) defective provirus in PCs and TCs. Each dot represents an intact or defective proviral sequence. PCs and TCs are represented by unique identifiers (Supplemental Table 1 and 2). Mann-Whitney U test was used to compare PCs and TCs. P < 0.05 was considered statistically significant. *P ≤ 0.05, **P ≤ 0.01. The Journal of Clinical Investigation CLINICAL MEDICINE 8J Clin Invest. 2024;134(8):e174215 https://doi.org/10.1172/JCI174215 Moreover, clonal-intact proviral sequences presented the same integration site, confirming their role in the reservoir persistence in PCs (37). Interestingly, both large clones in PC2 were integrated in ZNF genes (37) located in defined regions that are occupied by heterochromatin proteins of chromosome 19 (20, 34), as previously reported in a subset of ECs (16). Unlike PCs, we did not detect intact clonally expanded HIV-1 infected cells in TCs (5). These data suggest that the intact proviral reservoir of PCs, in contrast with TCs, seems mostly fueled by clonal proliferation of latently infected cells harboring early seeded intact proviruses. This fact resembled our previous findings in the overall EC population (16), probably because the more comprehensive analysis was performed in those ECs with intact proviral clones, consistent with the PC phenotype described in the present study. In addition, cell-associated HIV-1 RNA levels were significantly higher in TCs and positively correlated with total and intact proviruses. Notably, higher cell-associated HIV-1 RNA levels in PCs corresponded to PC1 and PC2, the participants that presented clonal intact provirus sequences located in centromeric satellite DNA or ZNF genes, reassuring the importance of the quality, rather than the quantity, of viral reservoir. This fact may indicate a production of viral proteins by defective proviruses in PCs (38), mostly driven by the higher proportion of LD proviruses observed in this phenotype (39) that could act as a therapeutic vaccine and could magnify the antiviral host immune activity in PCs. and Esperanza patients by the same techniques used in this study (30). Although we cannot confirm with our data that these participants, with virological control without ART for a median of 25 years, have achieved a spontaneous cure of HIV-1 infection, further studies can now be guided by our findings to study a larger number of cells from PBMCs and different anatomical compartments (31), such as lymphoid tissue, known for containing the majority of the viral reservoir (32, 33). These results suggest that the idea of a possible spontaneous cure of HIV-1 in the Esperanza patient is not anecdotic, and a higher number of ECs with this reservoir profile might be spontaneously cured. This hypothesis is supported by the longitudinal analysis of the HIV-1 reservoir in 4 of the PCs. Genome-intact HIV-1 proviruses were not detected in these participants during the follow up, some of them with samples 10 years apart. The detectable proviral DNA was completely defective in all the studied time points and, thus, unable to produce infectious virions. These data strongly suggest the absence of enough genome-intact proviruses to cause loss of control and support additional research to strengthen evidence for this hypothesis The other profile found in PCs consisted of participants who presented higher genome-intact proviral levels, derived completely from clonally expanded HIV-1 infected T cells and preferentially located in centromeric satellite DNA or ZNF genes, both associated with heterochromatin regions, generally disfavored for proviral integration and linked to deep viral latency (20, 34–36). Figure 8. HIV-1-specific T-cell response in PCs and TCs. (A) HIV-1-specific CM CD4+ T cell polyfunctionality with up to 5 functional responses to Gag stimulation per T cell in PCs and TCs. The 5 functional responses to Gag stimulation represent the simultaneous production of IFNG, TNFA, IL-2, CD107a, and PRF per T cell. IFNG, TNFA, IL-2, CD107a, and PRF are shown in arcs in the polyfunctional distribution. Pestle and Spice were used for analysis. Correlations between Gag-specific CM T cell response with total HIV DNA levels (106 peripheral blood mononuclear cells [PBMCs]) in (B) TCs and (C) PCs. Each dot represents a participant. PCs and TCs are represented by unique identifiers (Supplemental Table 1 and 2). Spearman test was used for nonparametric correlations. *P ≤ 0.05.