EBioMedicine 57 (2020) 102830 Contents lists available at ScienceDirect EBioMedicine journal homepage: www.elsevier.com/locate/ebiom Research paper Extremely low viral reservoir in treated chronically HIV-1-infected individuals lveza,b, Victor Urreaa, Judith Dalmaua, Montse Jimeneza,b, Bonaventura Cloteta,b,c,d, Cristina Ga Valerie Monceauxe, Nicolas Huote, Lorna Lealf, Victoria Gonz alez-Solerg,h,i, Maria Gonza lez-Cao , Michaela Mu j € ller-Trutwin , Asier S e ne, Felipe Garcíae, aez-Cirio  Blancoa,d, Javier Martinez-Picadoa,d,k,*, Maria Salgadoa,** Julia a IrsiCaixa AIDS Research Institute and Institute for Health Science Research Germans Trias i Pujol (IGTP), Badalona, Spain b Autonomous University of Barcelona, Cerdanyola del Valles, Spain c ‘Lluita contra la SIDA’ Foundation, Badalona, Spain d University of Vic-Central University of Catalonia (UVic-UCC), Vic, Spain e Institut Pasteur, Unite HIV Inflammation et Persistance, Paris, France f Infectious Diseases Department, Hospital Clínic-IDIBAPS, University of Barcelona, Barcelona, Spain g Centre for Epidemiological Studies on Sexually Transmitted Diseases and HIV/AIDS of Catalonia (CEEISCAT), Health Department, Generalitat de Catalunya, Spain h Microbiology Department. Laboratori Clínic Metropolitana Nord. University Hospital “Germans Trias i Pujol”, Badalona, Spain. Department of Genetics and Microbiology. Autonomous University of Barcelona, Spain i CIBER Epidemiologia y Salud Publica (CIBERESP), Spain j Translational Cancer Research Unit, Instituto Oncolo gico Dr Rosell, Dexeus University Hospital, Barcelona, Spain k Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain A R T I C L E I N F O A B S T R A C T Article History: Background: Small viral reservoirs are found predominantly in HIV-1 controllers and individuals treated dur- Received 4 March 2020 ing acute/early HIV-1 infection. However, other HIV+ individuals could naturally also harbour low viral Revised 20 May 2020 reservoirs. Accepted 26 May 2020 Methods: We screened 451 HIV-1-infected treated-individuals with suppressed plasma viremia for at least Available online xxx 3 years and stored cryopreserved peripheral blood mononuclear cells (PBMCs). Total HIV-DNA was analysed in PBMCs with ddPCR. Individuals with <50 HIV-DNA copies/106 PBMCs constitute the ‘Low Viral Reservoir Keywords: Treated’ cohort (LoViReT). Longitudinal samples were obtained from 12 chronically treated LoViReT and HIV reservoir HIV latency compared to 13 controls (>50 HIV-DNA copies/106 PBMCs) to analyse total HIV-DNA, T-cell and NK-cell pop- total HIV-DNA ulations, HIV-1 specific antibodies, and plasma inflammation markers. immunophenotyping Findings: We found that 9.3% of the individuals screened had <50 HIV-DNA copies/106 PBMCs. At least 66% HIV-specific antibodies initiated cART during the chronic phase of HIV-1 infection (cp-LoViReT). Cp-LoViReT harboured lower levels of HIV-DNA before cART and after treatment introduction the decays were greater compared to controls. They displayed a marked decline in quantity and avidity in HIV-specific antibodies after initiation of cART. Cp-LoViReT had fewer CD8+ TTM and TEMRA in the absence of cART, and higher CD8+ TN after 18 months on therapy. Interpretation: Treated chronically HIV-1-infected LoViReT represent a new phenotype of individuals charac- terized by an intrinsically reduced viral reservoir, less impaired CD8+ T-cell compartment before cART, and low circulating HIV-1 antigens despite being treated in the chronic phase of infection. The identification of this unique group of individuals is of great interest for the design of future eradication studies. Funding: MSD Spain © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/) * Corresponding author at: IrsiCaixa AIDS Research Institute and Institute for Health Science Research Germans Trias i Pujol (IGTP), Badalona, Spain. 1. Introduction ** Corresponding author at: IrsiCaixa AIDS Research Institute and Institute for Health Science Research Germans Trias i Pujol (IGTP), Badalona, Spain E-mail addresses: jmpicado@irsicaixa.es (J. Martinez-Picado), The implementation of combination antiretroviral therapy msalgado@irsicaixa.es (M. Salgado). (cART) to treat HIV-1 infection has substantially improved the life https://doi.org/10.1016/j.ebiom.2020.102830 2352-3964/© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/) 2 lvez et al. / EBioMedicine 57 (2020) 102830 C. Ga HIV-1 infection are made at the chronic stage, when the reservoirs PANEL 1: RESEARCH IN CONTEXT are more stable [17]. Eradication strategies need to be effective in the vast majority of treated chronically HIV-1-infected individuals. Sev- Evidence before this study eral studies have described treated chronically infected individuals We searched PubMed using the terms “HIV reservoir”, “HIV with low or even undetectable levels of total HIV-1 DNA [18 20]. DNA”, or “HIV latency”, and “low”, with no restrictions by date However, no retrospective data have been reported on the joint pro- or language, for studies that assess HIV-1-infected individuals portion of individuals who achieve a low reservoir after initiation of with a very low reservoir. We found evidence of low viral reser- treatment in both the acute and the chronic phases. Furthermore, the voirs, mostly in individuals treated during acute/early infection, factors involved in achieving these low latency levels have not been elite controllers and post-treatment controllers. However, we investigated in depth. also found 4 studies that described treated chronically infected In this study, we screened the total HIV-1 DNA reservoir in 451 individuals with a low HIV reservoir and correlated low viral treated HIV-1-infected individuals with suppressed plasma viremia reservoirs with clinical parameters and/or viral rebound if ther- for at least 3 years and stored cryopreserved peripheral blood mono- apy is interrupted. nuclear cells (PBMCs) to establish the Low Viral Reservoir Treated cohort (LoViReT). We aimed to study the kinetics of these decreased Added value of this study reservoirs and to analyse associated clinical and immunological fac- tors. To do so, we focused on a subset of LoViReT individuals who ini- We demonstrate that 9% of HIV-1-infected individuals can har- tiated treatment in the chronic phase of the infection (cp-LoViReT) in bour low levels of HIV-DNA (LoViReT individuals), with 66% of order to identify strategies that could be applied in the vast majority them treated during the chronic phase of infection. Ours is the of treated HIV-1-infected individuals. first study to assess the kinetics of reservoirs in chronically HIV-1-infected individuals harbouring low levels of total HIV-1 2. Methods DNA. We found a combination of intrinsically low reservoir with enhanced decay of latency after initiation of treatment. In 2.1. Study participants addition, the LoViReT individuals had a less impaired CD8+ T compartment before initiation of treatment and lower amounts We retrospectively screened 451 HIV-1-infected subjects under- of circulating antigens after 5 years on treatment. going regular follow-up at Hospital Germans Trias i Pujol (n=319) and Hospital Clinic (n=132) in Barcelona. We included individuals Implications of all the available evidence under suppressive cART with undetectable viremia (HIV-RNA <50 Our results reveal a novel phenotype of chronically treated copies/ml) for at least 3 years and with available cryopreserved individuals characterized by a naturally reduced and more PBMCs. Demographic and clinical data were collected from the clini- ART-sensitive viral reservoir. Therefore, LoViReT individuals are cal database. The characteristics of the study participants were simi- of particular interest when designing approaches to eradicate lar to those of previously reported cohorts of individuals with low HIV, since their smaller reservoir and other characteristics HIV reservoirs in terms of sex, age, and time with infection [18 20]. might represent an advantage over individuals with a larger Treated chronically infected participants were defined as individuals reservoir. Further studies on the host and viral parameters of with more than 6 months between acquisition of HIV-1 and initiation these individuals are warranted to identify factors that are of treatment. All subjects provided their signed informed consent to potentially involved in these phenomena. Such studies should participate in the study. The study was approved by the Ethics Com- contribute to the development of more rational HIV remission mittee at both recruiting hospitals (reference #: PI-014-083). strategies based on novel concepts. 2.2. Quantification of proviral HIV-1 DNA The size of the proviral reservoir was measured in PBMCs for screening and in purified CD4+ peripheral T cells for the longitudinal expectancy of HIV-1-infected individuals. However, cART does not analysis (Miltenyi Biotech) by droplet digital polymerase chain reac- completely eliminate HIV-1, which persists as a latent infection tion (ddPCR), as previously reported, without modifications [21]. mainly in resting CD4+ T cells, thus leading to rapid relapse of viremia Cells were resuspended in lysis buffer at a concentration of 5 £ 104 if therapy is interrupted [1]. Consequently, there is emerging interest cells/ml, which consisted of UltraPureÒ DNAse-RNAse free water in developing safe and affordable curative strategies to eliminate the (Gibco, Invitrogen) containing 10 mM Tris-HCl (pH=90), 01% Triton need for lifelong therapy while improving the health of people living x-100 (Sigma), and 400 mg/ml Proteinase K (Ambion). Two different with HIV and reducing the risk of viral transmission to uninfected primer sets per subject (50 LTR and Gag [21]) were used to circumvent individuals [2 4]. potential mismatches in the viral sequences that could prevent effi- A wide variety of strategies currently focus on eradicating HIV-1 cient amplification. In addition, the RPP30 [21] housekeeping gene with the aim of reducing the latent viral reservoir to undetectable was measured in parallel to normalize sample input. PBMCs from levels [5]. Therefore, factors related to the size, distribution, and sta- HIV-negative donors were used as negative controls and assayed in bility of the viral reservoir are continuously being investigated. It has each plate to set the positive/negative threshold. The number of been postulated that the amount of HIV-1 DNA is a predictor of dis- those negative control wells was the same as that of the replicates for ease progression in primary infection [6] and during the natural each sample. Raw ddPCR data were analysed using the QX100TM course of HIV-1 infection [7]. Lower levels of HIV-1 DNA have been Droplet Reader and the software application QuantaSoft v.1.6 (Bio- observed, mostly in elite controllers, who spontaneously control viral Rad, Hercules, CA, USA). replication [8], as well as in post-treatment controllers [9], and allo- geneic stem cell transplant recipients [10 13]. 2.3. Viral tropism Various studies suggest that early initiation of cART is an impor- tant factor in reducing the size of the viral reservoir [14,15], espe- Viral coreceptor tropism was determined on proviral DNA by cially if initiated at Fiebig stage I [16]. Unfortunately, individuals are sequencing of HIV-1 gp120 hypervariable region 3 (V3). Tropism was rarely treated during the acute phase, since most new diagnoses of predicted using the Geno2Pheno algorithm (GENAFOR, Bonn, lvez et al. / EBioMedicine 57 (2020) 102830 C. Ga 3 Germany), with a false-positive rate (FPR) of 10% (non R5-tropism: after infection for co-cultures with CD8+ T-cells using an ELISA p24 FPR 10%) [22]. assay (HIV-1 p24 ELISA kit, XpressBio). 2.6. Quantification of inflammation biomarkers 2.4. Immunophenotyping and activation markers in peripheral CD4+, CD8+, and NK cells Concentrations of the pro-inflammatory or homeostatic cytokines IL-2, IL-6, IL-7, IL-10, IL-27, and IP10, as well as the coagulation bio- For T-cell immunophenotyping, cells were incubated with Fixable marker D-dimer, were quantified in plasma using a bead-based mul- Viability Stain 780 (APC H7), CD3 (SK7), CD4 (RPA-T4), and CD8 (SK1) tiplex immunoassay (ProcartaPlex, eBioscience) according to the monoclonal antibodies. T-cell maturation was based on the expres- manufacturer’s recommendations. Measurements were performed sion of CD45RA (HI100), and CD197/CCR7 (G043H7) was analysed in using a Luminex 100 instrument (Luminex Corp., Austin, TX, USA) cryopreserved PBMCs in order to define naïve (CD45RA+CCR7+, TN), and analysed using a standard curve for each cytokine. central memory (CD45RA CCR7+, TCM), effector (CD45RA CCR7 , TEF), and effector memory RA+ cells (CD45RA+CCR7 , TEMRA). CD27 2.7. HIV antibody quantification (O323) was used to differentiate effector cells into effector memory cells (CD27 CD4+ or CD27+CD8+, TEM), and transitional memory cells Specific HIV-1 antibodies were measured in plasma samples using (CD27+CD4+ or CD27 CD8+, TTM). CD4 and CD8 T cells were also ana- the HIV-1/2 VITROS assay (Ortho Clinical Diagnostics, Rochester, NY, lysed for expression of HLA-DR (L243) and CD38 (HIT2) to define acti- USA), a low sensitive (LS) version of the VITROS anti-HIV-1 assay, and vated cells (HLA-DR+CD38+); expression of CD279/PD-1 (EG12.2H7) a limiting antigen avidity assay (Lag-Avidity) (Sedia, Portland, OR, was analysed to measure exhausted cells (CD279+). TIM-3 (F38-2E2), USA), as previously described [25]. LAG-3 (11C3C65), CD32 (FUN-2), and CD2 (RPA-2.10) markers were also measured in CD4+ T cells as potential surrogate markers of the 2.8. Statistical analysis viral reservoir through analysis of CD4+TIM-3+ CD4+LAG3+, CD4+CD32+, CD4+CD32bright (to avoid potential B-cell contamination), The clinical characteristics of the study population were presented and CD4+CD2bright cells. The gating strategy is shown in Suppl. Fig. 2. as percentages for categorical variables and as median and interquar- For NK immunophenotyping, cells were incubated with the tile range (IQR) for continuous variables. The association between appropriate monoclonal antibodies and Zombie AquaTM for 30 min at clinical parameters with low levels of cell-associated total HIV-1 DNA 4°C, washed, and fixed with 4% PFA. The following monoclonal anti- were analysed using univariate and multivariate logistic regression bodies were used for surface staining: CD3 (UCHT1), CD14 (M5E2), models; the odds ratio (OR), p-value, and C-index are reported. Varia- and CD19 (SJ25C1) for the lineage negative cells and CD45 (HI30), bles associated with the LoViReT status (p<01) were considered CD16 (3G8), CD56 (NCAM16.2), CD69 (FN50), NKp46/CD335 (9E2), candidates for the final model. The multivariate model was built NKP30/CD337 (AF29-4D12), NKG2A/CD159a (REA110), NKG2C/ using a stepwise procedure and by selecting the variables associated CD159c (134591), NKB1 (DX9), KIR2D/CD158a (NKVFS1), KIR3DL1/ with LoViReT. The parameters assessed include gender, age at diag- DL2/CD158e/k (REA970), CD161(DX12), and CXCR5/CD185 (MU5U- nosis, mode of infection, AIDS events, maximum viral load reported, BEE) for NK cell phenotyping as previously reported [23]. CD4 nadir, time since HIV-1 diagnosis, viral blips (<500 copies/ml), Samples were acquired in a BD LSRFortessa or LSRII flow cytome- detectable plasma viral load (pVL; any value above limit of detection), ter (BD Bioscience) and analysed using FlowJo software (Tree Star). T time with detectable pVL, tropism, and the accumulated pVL score cells were analysed with automated detection of marker cutoffs calculated as the area of the positive pVL plot over time and normal- based on fluorescence-minus-one controls (OurFlow platform using ized (divided) by the follow-up time. The model was validated inter- R packages). nally using a 10 fold cross validation, and predictive ability was assessed using the C-index (equivalent to the AUC under the ROC 2.5. Viral inhibition by CD8+ T and NK cells curve). In the longitudinal analysis, clinical differences between groups PBMCs were thawed and cultured overnight in RPMI 1640 con- were assessed using the Mann-Whitney test in the case of continuous taining GlutaMAX, 20% FCS, penicillin (10 IU/ml), and streptomycin variables and the Fisher exact test in the case of categorical variables. (10 mg/ml). Analyses were performed as previously described [24]. Clinical variables were matched between LoViReT subjects and con- Briefly, CD4+ T cells were purified by positive selection with anti- trols using the random forest algorithm in order to avoid confound- body-coated magnetic beads (EasySep Human CD4 Positive Selection, ing factors associated with low HIV-1 DNA levels. Random forest [26] StemCell Technologies). The elution containing CD4-depleted PBMCs is a popular machine learning algorithm based on ensembles of Clas- was split and used for purification of CD8+ T-cells and NK cells by sification and Regression Trees (CART) algorithm (regression and negative selection (EasySep Human CD8+ Cell Enrichment, EasySep classification trees), which provide good predictive performance and Human NK Enrichment, StemCell Tecnhologies). Cells were separated low overfitting. In addition to the prediction model, the algorithm using a RoboSep instrument (Stemcell Technology). provides a proximity measure matrix between observations that can CD4+ T cells were activated for 3 days in the presence of 4 mg/ml be used in matching [27]. In order to produce balanced treatment PHA-L (Roche) and 100 IU/ml IL-2 (Human IL-2 IS, premium grade, and control groups in terms of all relevant clinical factors, we selected Miltenyi Biotech). During this period, NK cells were cultured in the a set of controls based on the RF proximity measure. Differences presence of IL-15 at 01 ng/ml and CD8+ T cells in the absence of cyto- within groups were assessed using the Wilcoxon signed-rank test. kines. After 3 days of culture, living activated CD4+ T cells were The analyses were performed with R (v3.4) and GraphPad (v5.01). seeded in a 96-U-well plate (106 cells/ml in triplicate) alone or in the presence of CD8+ T cells or NK cells (1:1 ratio) and then exposed to 3. Results HIV-1 BaL (CCR5 tropic strain) (10 ng p24/ml). After spinoculation (1200 x g for 1 h at room temperature), cells were cultured for 1 h at 3.1. Characteristics of the screening population 37°C and washed before culture in the presence of IL-2 (100 IU/ml). Culture supernatants were removed, and the media culture was PW Taged e analysed samples from 451 individuals with more than 3 years replenished at day 3. Levels of p24 in culture supernatants were ana- under suppressive cART (Suppl. Fig. 1). Their characteristics are sum- lysed at day 3 after infection for co-culture with NK cells and at day 7 marized in Table 1. Log10 proviral HIV-1 DNA in PBMCs was normally 4 lvez et al. / EBioMedicine 57 (2020) 102830 C. Ga Table 1 all included in the “LoViReT” (Low Viral Reservoir Treated Individu- Clinical characteristics of the subjects included in the study als) Cohort. The distribution of total HIV-1 DNA and proportion of Characteristic N (%) Median [IQR] LoViReT individuals were similar between the 2 recruiting centres (Fig. 1b). The clinical histories of LoViReT subjects confirmed that 28 Male sex 377 (836) individuals were treated in the chronic phase of the infection (>6 Region of origin Spain 236 (81) months since acquisition) and were defined as chronic phase LoViReT Europe 17 (6) subjects (cp-LoViReT). Out of the remaining LoViReT subjects, 6 were America 34 (12) treated in the acute stage, and 8 lacked proven information of their Africa 4 (1) time with infection at initiation of cART (Suppl. Fig. 1). Age at diagnosis 31 [27-37] Mode of infection MSM 252 (58) 3.2. Factors related to LoViReT status Heterosexual 87 (20) Injecting drug use 55 (13) Clinical, immunological, and virological data were collected to Other 40 (9) search for factors predicting whether the subject would belong to the AIDS events Yes 46 (10) LoViReT cohort (Table 2). No 405 (90) In the univariate analysis, being in the LoViReT cohort was associ- Zenith viral load (log10 copies/ml plasma) 49 [43-53] ated with a lower maximal reported pVL (p=00065), higher CD4+ T- CD4 nadir (cells/ml) 273 [159-358] cell nadir (p=0002), lower number of viral blips (p=008), fewer Time since HIV diagnosis (years) 13 [7-19] Viral blips 0 [0-1] detectable pVLs defined as any pVL >50 copies/ml (p=00021), and Virological failures 0 [0-2] lower time with detectable pVL (p=00078) (Table 2). In the multivar- Tropism iate regression model, only a lower maximal reported pVL, higher R5-tropic 329 (73) CD4+ nadir, and fewer detectable pVLs remained significantly associ- Non-R5 or dual 93 (21) ated with LoViReT status (Table 2). Using the c-index method to eval- Viral subtype B 403 (96) uate the predictive capacity of the multivariate model, we observed a Non B 19(4) goodness of fit of 072 after cross-validation. This is considered a Total HIV-1 DNA (copies/106 PBMCs) 1585 [787-3134] moderate predictive capacity. At the time of HIV-1 DNA measurements Thus, LoViReT individuals seemed to have lower peaks of pVL, a Age 46 [41-51] Time suppressed (years) 56 [4-8] higher CD4+ T-cell nadir, and fewer detectable pVLs during their clini- CD4 T cells (cells/ml) 676 [495-891] cal follow-up. However, this multivariate model had limited predic- CD8 T cells (cells/ml) 766 [567-1022] tive capacity. CD4/CD8 ratio 0.9 [06-12] Longitudinal analysis of HIV-1 DNA reservoirs: For further analy- ses, we focused on the cp-LoViReT population, since this cohort is less well characterized (Suppl. Fig.1). Thus, we aimed to unravel whether the cp-LoViReT reservoir decreased as a consequence of distributed (Fig. 1a). Median HIV-1 DNA was 1585 copies/106 PBMCs cART or whether these subjects had lower reservoirs before initia- (IQR, 79-313). tion of cART. According to sample availability, we selected a sub- A total of 42 individuals were under the 10th percentile (<50 HIV- group of 12 cp-LoViReT and compared them with 13 treated 1 DNA copies/106 PBMC) (93%), including 4 (09%) who had levels chronically infected matched controls (>50 HIV-1 DNA copies/106 below the limit of detection for the 2 sets of primers used. They were PBMCs) to perform a retrospective longitudinal reservoir analysis in Fig. 1. Total HIV-1 DNA. (a) Total HIV-1 DNA of 451 individuals after screening with ddPCR. Subjects with <50 copies/106 PBMCs are shown in light blue. (b) Comparative distribution of total HIV-1 DNA between the 2 recruiting centres. Subjects with <50 copies/106 PBMCs are shown in blue and red respectively to the each site. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.). lvez et al. / EBioMedicine 57 (2020) 102830 C. Ga 5 Table 2 Clinical associations with low levels of total HIV-1 DNA (LoViReT status) Clinical variables Univariate Multivariate OR [95% CI] p-value& OR [95% CI] p-value& Gender (Male) 0981 Age at diagnosis 0642 Mode of infection 0782 AIDS events 0749 Maximum viral load reported log10 067 [051-089] 0007 066 [049-089] 00069 CD4 nadir (multiples of 100) 147 [120-181] <0001 146 [119-181] 00003 Time since HIV diagnosis 0177 Viral blips (<500 copies/ml) 064 [034-105] 0083 Detectable pVL (>50 copies/ml) 072 [055-090] 0002 079 [060-098] 00286 Time with detectable pVL (years) 076 [058-094] 0008 Tropism 0394 Accumulated viral load score 078 [062-095] 0.013 At the time of HIV-1 DNA measurements Age 0531 Time suppressed (years) 0902 CD4 T cells (multiples of 100) 0648 CD8 T cells (multiples of 100) 0246 % CD4 T cells 0344 % CD8 T cells 0262 CD4/CD8 ratio 0217 & p-values based on likelihood ratio test purified CD4+ T cells. We previously ensured that there were no dif- Analysis of maturation subsets in CD8+ T cells revealed the ferences in clinical parameters between cp-LoViReT and controls expected significant increase in the frequency of TN cells after initia- (Suppl. Table 1). tion of therapy in both groups, with similar rates of increase; how- Total HIV-1 DNA was measured before cART and in several sam- ever, the frequency was higher at month 18 in cp-LoViReT than in ples after initiation of cART (median: 4 [IQR 4-5] samples per individ- controls (p=0043) (Fig. 3a,b). Although we did not observe signifi- ual) (Fig. 2a). All participants had detectable HIV-1 DNA before cant differences between the groups in the frequency of TCM (Fig. 3c), therapy, thus minimizing technical primer-mismatch hybridization we did detect a lower frequency of TTM and TEMRA cells before initia- issues. Fig. 2a shows the dynamics of total HIV-1 DNA in all the sam- tion of cART in cp-LoViReT than in controls (p=0011 for TTM and ples analysed. We observed a phase of faster decay during the first 18 p=0016 for TEMRA). This association disappeared when cART was months on cART, followed by a slower decay. introduced (Fig. 3d f). The correlation between HIV-DNA and these When we analysed the time points separately, we observed that cellular markers was assayed at each time point, although we did not before initiation of cART, cp-LoViReT already harboured significantly find statistically significant correlations (data not shown). lower levels of HIV-1 DNA (p=0002) (Fig. 2b). The differences Activated (CD38+HLA-DR+) and PD-1+ CD8+ T cells decreased sig- observed before cART were independent from pVL, since no statisti- nificantly over time in both groups (Fig. 3g,h). However, no signifi- cally significant differences were observed between the groups cant differences between groups were observed. Values for the other (Suppl. Table 1). exhaustion markers analysed, TIM-3 and LAG-3, were similar in both After 18 months of treatment, a pronounced decline in HIV-1 DNA groups (Fig. 3i,j). was observed in all those studied, although median values were In CD4+ T cells, we did not observe any significant difference lower in cp-LoViReT than in controls (p<0001). Finally, after 5 years between cp-LoViReT and controls over time in any T-cell maturation on cART, samples from both groups showed the greatest differences subset (Suppl. Fig. 3a-f). Activation, defined as co-expression of within the study (p<0001). cp-LoViReT reached a median of 54 HIV- CD38+HLA-DR+, and PD-1 expression levels decreased overtime in both 1 DNA copies/106 CD4+ T cells, which is comparable to the values we groups with no intergroup differences (Suppl. Fig. 3g,h). Similarly, we observed in the initial PBMC screening. did not find significant intergroup differences in the reservoir surrogate When we compared the decay in the HIV reservoir after 5 years markers TIM-3+, CD2bright, CD32, and LAG-3+ (Suppl. Fig. 3i l). on cART with before cART, we observed a significantly greater Additionally, immunophenotyping of NK cells was performed in decrease in cp-LoViReT subjects than in controls (16- vs. 5-fold 11 samples before initiation of cART and after 5 years on cART. In this respectively, p<0001) (Fig. 2c). In addition, a significant positive cor- case, we analysed the frequencies of NK cells and their different acti- relation was observed between these 2 time points (rho=076, vator/inhibitor receptors with a putative role in HIV control. No dif- p<0001) (Fig. 2d). ferences were found between cp-LoViReT and controls in the We observed that cp-LoViReT individuals had low viral reservoirs frequency of NK cells (Suppl. Fig. 4a) or in the expression of the C- before initiation of cART, as well as enhanced decay during type lectin-like receptors (NKG2) (Suppl. Fig. 4b-e), killer immuno- treatment. globulin-like receptors (KIR) (Suppl. Fig. 4f-g), activator natural cyto- Characterization of T-cell subsets, activation, exhaustion, and res- toxicity receptors (NCR) (Suppl. Fig. 4h-i), and CD161 (Fig Suppl. 4j). ervoir surrogate markers: Immunophenotyping of T cells was per- However, a statistically significant decrease in CD69 and CXCR5 was formed in the samples used for the longitudinal reservoir analysis. As observed for cp-LoViReT once they started cART, with similar trends expected, we found a significant increase in the frequency of CD4+ T observed in the controls (Suppl. Fig. 4k-l). cells and a significant decrease in the frequency of CD8+ T cells after In summary, we observed changes in CD8+ T-cell maturation in initiation of cART, thus leading to a significant recovery of the CD4/ cp-LoViReT with higher levels of TN in combination with lower TTM CD8 T-cell ratio. No differences were observed between cp-LoViReT and TEMRA CD8+ T cells before initiation of cART. This observation was and the control groups over time (data not shown). not associated with changes in activation or exhaustion of CD8+ T cells in this group. 6 lvez et al. / EBioMedicine 57 (2020) 102830 C. Ga Fig. 2. Longitudinal measure of total HIV-1 DNA in CD4+ T cells by ddPCR. (a) Decay in total HIV-1 DNA before and after initiation of cART. The light grey box indicates the period under cART. (b) Box plot of total HIV-1 DNA at 3 different time points: pre-cART, 18 months after initiation of cART, and 5 years after initiation of cART. (c) Fold change decay in the sample before initiation of treatment and after 5 years of cART. (d) Spearman correlation for total HIV-1 DNA pre-cART and after 5 years of cART. 3.3. CD4+ susceptibility and viral inhibition by CD8+ and NK cells there were no significant differences between groups before initia- tion of cART or after 5 years on cART. To evaluate the functionality of the T cells and NK cells in both Autologous CD8+ T cells were also tested to analyse the suppres- groups of individuals, we analysed the susceptibility of the target sion of viral replication (Fig. 4b); no significant differences between cells to HIV-1 infection and viral inhibition by CD8 and NK cells in the cp-LoViReT and controls were observed. High variability in the inhi- same samples as those used for NK immunophenotyping. Thus, we bition percentage was recorded in all the samples assayed; this could selected 11 cp-LoViReT and 11 controls with available samples before be explained by the limitation arising from the use of frozen cells in initiation of cART and after 5 years on cART. We pulsed autologous this assay. CD4+ T cells from each individual with a laboratory-adapted R5- viral Similarly, we did not find significant differences in the percentage strain for 7 days (Fig. 4a). We observed that CD4+ T cells from cp- of inhibition by autologous NK cells between groups before initiation LoViReT and controls were susceptible to HIV-1 infection and that of treatment or after 5 years on cART (Fig. 4c). lvez et al. / EBioMedicine 57 (2020) 102830 C. Ga 7 Fig. 3. Analysis of maturation subsets, activation, exhaustion, and surrogate markers in the reservoir in CD8+ T cells. (a) Median CD8+ T-cell values for the frequency of the maturation subsets (naïve, central memory, effector memory, transitional memory, and effector memory RA+) in controls and cp-LoViReT at 3 different time points: pre-cART, 18 months after initiation of cART, and 5 years after initiation of cART. Maturation stages were defined based on the 8 lvez et al. / EBioMedicine 57 (2020) 102830 C. Ga a CD4+ T cell susceptibility to HIV infection Control LoViReT 5 years on cART p24 in culture supernatants 1000 1000 1000 100 100 100 (ng/ml) 10 10 10 1 1 1 Pre-cART cART Pre-cART cART Control LoViReT b Viral inhibition by CD8+ T cells Control LoViReT 5 years on cART 100 % inhibition CD4:CD8 (1:1) 100 100 75 75 75 50 50 50 25 25 25 0 0 0 Pre-cART cART Pre-cART cART Control LoViReT c Viral inhibition by NK cells Control LoViReT 5 years on cART % inhibition CD4:NK (1:1) 100 100 100 75 75 75 50 50 50 25 25 25 0 0 0 Pre-cART cART Pre-cART cART Control LoViReT + + Fig. 4. CD4 T cell susceptibility to HIV and viral inhibition by CD8 T and NK cells. PBMC samples before initiation of cART and after 5 years on cART were used to analyse the susceptibility of CD4+ T cells to HIV BaL (CCR5 tropic strain) (10 ng p24/ml) (a). We also measured the ex vivo ability of CD8+ T cells (b) and NK cells (c) to inhibit superinfected autologous CD4+ T cells at a 1:1 ratio. The cp-LoViReT group is depicted in blue and the control group in grey. CD4+ T cells from cp-LoViReT were perfectly susceptible to HIV 3.5. HIV-1 specific antibodies infection, with no signs of distinct CD8 and NK cytotoxic activities compared with control individuals. To determine whether low reservoirs could be associated with humoral responses to HIV-1, we analysed HIV-1-specific antibodies 3.4. Inflammatory marker levels in cp-LoViReT in plasma samples before initiation of cART and after 5 years on cART in 8 cp-LoViReT and 9 controls for whom plasma samples were avail- We measured soluble plasma pro-inflammatory or homeostatic able. We observed a decrease in HIV-1-specific antibody levels in cytokines (IL-2, IL-6, IL-7, IL-10, IL-27, IP10) and the coagulation fac- both groups after initiation of cART (Fig. 5a). This decay was greater tor D-dimer in plasma samples from 8 cp-LoViReT and 9 controls in cp-LoViReT than in controls (Fig. 5a). before cART and after 5 years on cART (selected according to sample We also measured the avidity of antibodies using a limiting anti- availability). We did not find statistically significant differences either gen avidity assay. We observed trend toward a more pronounced over time or between groups (Suppl. Table 2). reduction in antibody avidity in cp-LoViReT when cART was initiated, combination of CD45RA, CD197 (CCR7), and CD27. (b) CD45RA+CD197+ (Naive), (c) CD45RA CD197+ (central memory), (d) CD45RA CD197 CD27 (transitional memory), (e) CD45RA CD197 CD27+ (effector memory), and (f) CD45RA+CD197 (effector memory RA+). (g) CD8 activation levels (HLA-DR+CD38+) in both study groups over time. (h-j) CD8+ T- cell exhaustion markers including PD-1 (CD279), TIM-3, and LAG-3. The cp-LoViReT group is depicted in blue and the control group in grey. Intragroup statistically significant differ- ences are depicted with a light blue or grey line for LoViReT and controls respectively; statistically significant differences between groups are depicted with a red line. lvez et al. / EBioMedicine 57 (2020) 102830 C. Ga 9 Fig. 5. Measurement of HIV-1-specific antibodies. Plasma samples before initiation of cART and after 5 years of cART were tested for HIV-1-specific antibody levels using a detuned version of the HIV-1 VITROS assay (a) and a limiting antigen avidity assay (b). The dotted line represents the HIV-1 antibody assay diagnostic cut-off level used to classify individuals as HIV-1-positive or -negative. The p-val- ues between groups were assessed using the Mann-Whitney test. with most individual levels falling below the cut off seen in acute Various clinical parameters were associated with LoViReT status, HIV-1-infected subjects (Fig. 5b). including a higher CD4+ nadir, lower maximum pVL, and fewer Therefore, HIV-1 antibody quantity and quality were more detectable pVLs. Nevertheless, the model is not strong enough to markedly diminished upon initiation of cART in cp-LoViReT. identify LoViReT individuals, suggesting that other viral or immuno- logical factors might be involved. The same 3 factors have been found to be closely associated with harbouring low levels of HIV-1 reservoir 4. Discussion [18,19,30,31], probably because a higher CD4+ T-cell nadir could pre- vent repopulation of the immune system with expanded latently Latent infection of CD4+ T cells is established very early after infec- infected lymphocytes. Moreover, a lower pVL peak might have pre- tion and is the major obstacle to curing HIV-1 infection [28]. A low vented massive seeding of CD4+ T cells by HIV-1. Similarly, fewer HIV-1 DNA reservoir has been associated with early treatment [14] detectable pVLs during the course of the infection might have pre- and better clinical outcome [29]. Nevertheless, the size of the HIV-1 vented replenishment of the reservoir. DNA reservoir is not necessarily associated with time to viral rebound Previous studies have reported the association between early in subjects treated early after infection and who discontinued their treatment and low viral reservoirs [14,32,33]. However, only individ- cART [16,29]. The global frequency of HIV-1-infected individuals har- uals treated in Fiebig I-IV (between 1 week and 1 month after infec- bouring low levels of HIV-1 DNA and the determinants of these levels tion) were capable of achieving low levels of total HIV-1 DNA after remain unknown. more than 3 years on treatment [14,15]. Surprisingly, if early treat- In our study, we found that 93% of the individuals screened had ment is excluded as the main cause of a remarkably low reservoir, HIV-1 DNA levels below 50 copies/106 PBMCs (LoViReT subjects). The 66% of the LoViReT subjects were treated more than 6 months after proportion of LoViReT individuals was similar in the 2 recruiting acquisition of HIV-1 (cp-LoViReT). Since eradication strategies need centres, indicating that our data might be extrapolated to other clini- to be effective in the vast majority of treated chronically infected cal sites with similar subject populations and viral subtypes. Previous individuals, it would be of considerable interest to know the mecha- studies in treated chronically infected individuals found that 28% and nism responsible for the low reservoir detected in this group of indi- 19% of individuals had HIV-1 DNA levels below 150 copies/106 PBMCs viduals. and 66 copies/106 PBMCs, respectively [18,19]. While both studies Data on the kinetics of HIV-1 reservoirs have been reported in revealed greater amounts of individuals with low HIV-1 DNA levels, treated chronically infected individuals [34 36] harbouring standard the differences observed with our results could be explained by their levels of total HIV-1 DNA or treated during primary infection [15,37]. higher cut-off for defining low HIV-1 DNA. Reanalysis of our dataset However, to our knowledge, this is the first study to address the after applying their cut-offs revealed that 33% and 19% of individuals, kinetics of reservoirs in chronically HIV-1-infected individuals har- respectively, had a “low reservoir”, thus confirming this hypothesis. bouring low levels of total HIV-1 DNA, including during the pre-cART We maintained our cut-off of 50 copies/106 PBMCs because it repre- period. Our results provide the first evidence that cp-LoViReT have sents the 10th percentile of the screened population. intrinsically lower levels of total HIV-1 DNA before starting 10 lvez et al. / EBioMedicine 57 (2020) 102830 C. Ga treatment, despite having the same levels of plasma viremia as con- tissues. However, as this was a retrospective analysis, it was not pos- trols. Besides, once cART is introduced, their reservoir seems to be sible to have access to those samples. The full size of the HIV-1 reser- more ART-sensitive, since decay of total HIV-1 DNA is faster in cp- voir was only evaluated in total HIV-1 DNA, thus enabling all forms of LoViReT. We also proved that their CD4+ T cells were susceptible to cellular HIV-1 DNA (unintegrated linear, 1-LTR and 2-LTR circular infection; therefore, the low reservoir observed is probably due to forms, and integrated forms) to be quantified. Other methods of factors that do not severely impact the viral replication cycle in the studying the HIV reservoir, such as assessment of replication-compe- cells. Thus, we cannot exclude the possibility that cp-LoViReT might tent or -defective virions and quantification of the inducible reservoir have more linear unintegrated DNA, which decays faster than inte- at the RNA level, could prove useful. However, the quantification of grated DNA in the presence of cART [38,39]. These parameters will be replicative or intact virus in individuals with very low viral reservoirs analysed in future prospective studies. Additionally, and consistent might be challenging, since they represent less than 10% of total HIV- with other authors, we found a positive correlation between pre- DNA [49]. treatment levels of total HIV-1 DNA and levels after 5 years on cART, In addition, we were unable to identify a molecular mechanism thus demonstrating the importance of certain features before initia- that could explain the limited reservoir in LoViReT individuals. Future tion of cART, such as host factors and/or the immune system, in research will be needed to unravel whether this cohort shares a com- determining subsequent reservoir size [17,40]. mon molecular mechanism that decreases the ability of the virus to A naturally low level of cell-associated HIV-1 DNA could result in a establish latency. more preserved immune system. cp-LoViReT had fewer CD8+ TTM and In conclusion, we found that some HIV-1-infected individuals TEMRA in the absence of cART, with frequencies similar to those of have low levels of total HIV-1 DNA (LoViReT subjects) and that most HIV-1-negative individuals, suggesting a less impaired CD8+ T-cell were treated in the chronic phase of the infection. Moreover, they compartment. HIV-1 infection disrupts T-cell subset homeostasis, have a less compromised CD8+ T-cell compartment and lower HIV-1- with a dramatic decrease in the frequency of CD8+ TN cells and mas- specific antibody levels, probably as a result of lower amounts of cir- sive expansion of CD8+ TTM cells in infected individuals treated during culating antigens. Among those who started treatment in the chronic both the acute phase and the chronic phase [41]. Furthermore, after phase, these are perfect candidates for the study of combined 18 months on therapy, the differences in CD8+ TN cells between cp- approaches to finding a cure for HIV-1, since we proved that cp- LoViReT and controls become statistically significant, with the result LoViReT are characterized by a viral reservoir that is intrinsically that CD8+ TN cells were more numerous than in controls. The higher reduced before initiation of cART and enhanced decay after initiation proportion of CD8+ TN, also observed in elite controllers, may suggest of treatment. shared preservation of thymic function [42]. However, in LoViReT individuals, the preserved CD8+ T-cell compartment does not seem to Declaration of Competing Interest be associated with an enhanced cytotoxic capacity of their CD8+ T cells or NK cells, in contrast to HIV-1 controllers for CD8+ T cells [43] B.C. declares that outside the submitted work has received grants and post-treatment controllers for NK cells [44]. Further exploration from Gilead, ViiV Healthcare and MSD; received consultancy fees with fresh cells, more participants, and assessment of the frequency from MSD; and a shareholder of AlbaJuna Therapeutics and AELIX of HIV-specific cells will be needed to fully elucidate this point. therapeutics. M.G-C. declares educational/consultancy fees from Finally, consistent with the kinetics of total HIV-1 DNA, the BMS, Pierre Fabre, Roche, Takeda, and AstraZeneca outside the sub- marked decline in the quantity and avidity of HIV-1-specific antibod- mitted work. A.S-C has received research grants from MSDAVENIR ies in cp-LoViReT during cART indicates a lower amount of circulating and personal fees from MSD, ViiV healthcare and Janssen, outside the HIV-1 antigen. Our findings are in line with results in treated chroni- submitted work. J.B. is the founder and CEO and a shareholder of cally infected individuals from Keating et al [45], who reported an AlbaJuna Therapeutics and receives grants from MSD and Grifols out- association between declining antibody levels during cART and lower side the submitted work. J.M-P. holds an institutional grant and has levels of antigen production, better viral control, and lower systemic received educational/consultancy fees from Merck; outside the sub- viral burdens. cp-LoViReT represent a new phenotype of individuals mitted work, he has received fees from AbiVax, AstraZeneca, Gilead characterized by low intrinsic total HIV-1 DNA, better immune pres- Sciences, Grifols, Janssen, and ViiV Healthcare. All the other authors ervation, and low circulating HIV-1 antigens despite being treated in declare no competing interests. the chronic phase of the infection. Levels of proviral DNA while on cART in some individuals of the well-characterized post-treatment Acknowledgments and elite controller cohorts were as low as those of LoViReT individu- als, with median levels of 1.7 and 1.5 log10 copies per million PBMCs, We thank the IGTP Cytometry Core Facility and staff (MA Fernan- respectively [46]; however, we can rule out an overlap between dez and G Requena) for their contribution to this publication. cohorts. LoViReT had high viral loads during chronic infection before initiation of treatment. This finding differs from the main factors Founding sources associated with elite and post-treatment controller cohorts. In addi- tion, various studies suggest that having low amounts of HIV-1 DNA This research was sponsored in part by Grifols and by Merck Sharp does not prevent early and consistent viral rebound if therapy is & Dohme Espan ~ a, S.A. (IISP 54925). The funding organizations had no interrupted in infected individuals receiving long-term treatment input in the design of the study; in the collection, analyses, or inter- [20,47,48]. Therefore, a very low reservoir is not necessarily associ- pretation of the data; writing of the manuscript; or in the decision to ated with spontaneous control of viral replication, as might be the submit the study for publication. CG is supported by the PhD fellow- case in elite controllers or post-treatment controllers. However, ship of the Spanish Ministry of Education, Culture and Sport (FPU15/ when attempting to identify combined approaches toward finding a 03698). NH received a post-doctoral grant from the Jaqueline Beytout cure for HIV-1 infection, cp-LoViReT appear to be excellent research Foundation. FG received the support of “Jose  María Segovia de Arana” participants, since they have better preserved immune cell popula- contracts (2019) and MMT from the NIH (R01AI143457). tions and start from a smaller reservoir, which might be an advantage over individuals with a larger reservoir. Authors’ Contributions Our study was subject to a series of limitations. HIV-1 DNA was only measured in peripheral blood, whereas larger HIV-1 reservoirs J.M-P. and M.S. conceived and designed the study; C.G., M.J., V.G- are known to be found in lymph nodes and gut-associated lymphoid S., V.M., N.H., and M.S. performed the experiments; C.G., V.U., M.J., V. lvez et al. / EBioMedicine 57 (2020) 102830 C. Ga 11 G-S., V.M., N.H., and M.S. analysed the data; C.G., J.D., M.J., V.G-S., V.M., [20] Calin R, Hamimi C, Lambert-Niclot S, Carcelain G, Bellet J, Assoumou L, et al. Treat- N.H., M.M-T., A.S-C,. 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