ORIGINAL RESEARCH published: 06 May 2020 doi: 10.3389/fimmu.2020.00823 HIVconsv Vaccines and Romidepsin in Early-Treated HIV-1-Infected Individuals: Safety, Immunogenicity Edited by: and Effect on the Viral Reservoir Carolina Garrido, University of North Carolina at (Study BCN02) Chapel Hill, United States Reviewed by: Beatriz Mothe 1,2,3*† , Miriam Rosás-Umbert 1,4† , Pep Coll 1 , Christian Manzardo 5 , Paul W. Denton, Maria C. Puertas 1 , Sara Morón-López 1 , Anuska Llano 1 , Cristina Miranda 2 , University of Nebraska Omaha, Samandhy Cedeño 1 , Miriam López 2 , Yovaninna Alarcón-Soto 6 , Guadalupe Gómez Melis 6 , United States Klaus Langohr 6 , Ana M. Barriocanal 4,7 , Jessica Toro 2 , Irene Ruiz 5 , Cristina Rovira 5 , Jonathan Li, Antonio Carrillo 7 , Michael Meulbroek 8 , Alison Crook 9 , Edmund G. Wee 9 , Jose M. Miró 5 , Brigham and Women’s Hospital, Bonaventura Clotet 1,2,3,4 , Marta Valle 4,10 , Javier Martinez-Picado 1,3,11 , Tomáš Hanke 9,12 , United States Christian Brander 1,3,11† , José Moltó 2,7† and the BCN02 Study Investigators *Correspondence: 1 Beatriz Mothe IrsiCaixa AIDS Research Institute-HIVACAT, Badalona, Spain, 2 Fundació Lluita contra la Sida, Infectious Diseases bmothe@irsicaixa.es Department, Hospital Universitari Germans Trias i Pujol, Badalona, Spain, 3 Faculty of Medicine, Universitat de Vic-Central de Catalunya (UVic-UCC), Vic, Spain, 4 Department of Cellular Biology, Physiology and Immunology, Universitat Autonoma de † These authors have contributed Barcelona (UAB), Barcelona, Spain, 5 Hospital Clinic- IDIBAPS, University of Barcelona, Barcelona, Spain, 6 Departament equally to this work d’Estadística i Investigació Operativa, Universitat Politècnica de Catalunya/BARCELONATECH, Barcelona, Spain, 7 Department of Infectious Diseases, Germans Trias i Pujol Research Institute, Badalona, Spain, 8 Projecte dels Specialty section: NOMS-Hispanosida, BCN Checkpoint, Barcelona, Spain, 9 The Jenner Institute, University of Oxford, Oxford, This article was submitted to United Kingdom, 10 Pharmacokinetic/Pharmacodynamic Modeling and Simultation, Institut de Recerca de l’Hospital de la Vaccines and Molecular Therapeutics, Santa Creu i Sant Pau-IIB Sant Pau, Barcelona, Spain, 11 ICREA, Barcelona, Spain, 12 Joint Research Center for Human a section of the journal Retrovirus Infection, Kumamoto University, Kumamoto, Japan Frontiers in Immunology Received: 13 February 2020 Kick&kill strategies combining drugs aiming to reactivate the viral reservoir with Accepted: 09 April 2020 Published: 06 May 2020 therapeutic vaccines to induce effective cytotoxic immune responses hold potential Citation: to achieve a functional cure for HIV-1 infection. Here, we report on an open-label, Mothe B, Rosás-Umbert M, Coll P, single-arm, phase I clinical trial, enrolling 15 early-treated HIV-1-infected individuals, Manzardo C, Puertas MC, testing the combination of the histone deacetylase inhibitor romidepsin as a Morón-López S, Llano A, Miranda C, Cedeño S, López M, Alarcón-Soto Y, latency-reversing agent and the MVA.HIVconsv vaccine. Romidepsin treatment resulted Melis GG, Langohr K, Barriocanal AM, in increased histone acetylation, cell-associated HIV-1 RNA, and T-cell activation, which Toro J, Ruiz I, Rovira C, Carrillo A, Meulbroek M, Crook A, Wee EG, were associated with a marginally significant reduction of the viral reservoir. Vaccinations Miró JM, Clotet B, Valle M, boosted robust and broad HIVconsv-specific T cells, which were strongly refocused Martinez-Picado J, Hanke T, toward conserved regions of the HIV-1 proteome. During a monitored ART interruption Brander C, Moltó J and the BCN02 Study Investigators (2020) HIVconsv phase using plasma viral load over 2,000 copies/ml as a criterium for ART resumption, Vaccines and Romidepsin in 23% of individuals showed sustained suppression of viremia up to 32 weeks without Early-Treated HIV-1-Infected Individuals: Safety, Immunogenicity evidence for reseeding the viral reservoir. Results from this pilot study show that the and Effect on the Viral Reservoir combined kick&kill intervention was safe and suggest a role for this strategy in achieving (Study BCN02). an immune-driven durable viremic control. Front. Immunol. 11:823. doi: 10.3389/fimmu.2020.00823 Keywords: romidepsin, HDAC inhibitor, kick&kill strategy, HIVconsv, early-treatment Frontiers in Immunology | www.frontiersin.org 1 May 2020 | Volume 11 | Article 823 Mothe et al. Control After Kick&Kill and Early-cART INTRODUCTION RNA inversely correlated with time to rebound, supporting that, in the absence of an enhanced HIV-1-specific CTL response, Current antiretroviral therapy (ART) effectively suppresses HIV- viral reactivation might facilitate viral rebound once ART is 1 replication, thus preventing disease progression. However, the interrupted (35). infection remains chronic given that a latent HIV-1 reservoir, Here, in this single-arm, open-label, phase I, proof-of-concept established early after infection, persists despite suppressive study, referred to as BCN02 trial (NCT02616874), we assessed ART (1). Upon ART discontinuation, integrated replication- the safety, tolerability, immunogenicity and effect on the viral competent proviruses in the reservoir drive a rapid viral rebound reservoir of a kick&kill strategy consisting of the combination (2). Therapeutic vaccination has been proposed as a possible of HIVconsv vaccines with RMD in suppressed early-treated approach to induce an effective immune control able to contain HIV-1-infected individuals. Participants were rolled-over from rebounding virus (3). the therapeutic vaccine trial BCN01 (NCT01712425), in which Most therapeutic vaccines tested in the past expressed one or individuals who started ART during acute/recent HIV-1 infection several HIV-1 proteins, which expanded HIV-1-specific CD8+ had received a prime/boost regimen of the ChAdV63.HIVconsv cytotoxic T lymphocyte (CTL) responses to varying levels. and MVA.HIVconsv vaccines (CM) (20). Three years after, However, the responses were ineffective in controlling viremia BCN01 participants who had shown sustained viral suppression after ART interruption, likely because of their suboptimal and who accepted to participate in BCN02 study were immunized magnitude, breadth, width, specificity, and/or polyfunctionality with two doses of MVA.HIVconsv, before and after three weekly- (4–8), raising the need for novel immunogens and delivery doses of RMD, followed by a monitored antiretroviral pause methods to tackle HIV-1 diversity and the virus’ ability to escape. (MAP) for a period of 32 weeks to assess the ability of the New vaccines strategies are being developed to maximize the intervention to control viral rebound. vaccine coverage of circulating viruses using multivalent mosaic immunogens designed in silico (9, 10). Alternatively, vaccines designs are tested that aim to focus the CTL responses toward MATERIALS AND METHODS more conserved and protective regions of the virus, which are Study Design and Interventions less likely to mutate and escape the T-cell response (11–15). The BCN02 clinical trial was an investigator initiated phase Among the latter, the HIVconsv immunogen is one of the most I, open-label, single-arm, multicenter, single-country study advanced vaccine candidates in clinical development. HIVconsv to assess the safety, tolerability and efficacy of a combined immunogen consists of a chimeric protein assembled from kick&kill strategy in suppressed HIV-1-infected patients that 14 highly conserved domains derived from HIV-1 genes Gag, had initiated ART during acute/recent HIV-infection. Individuals Pol, Vif, and Env alternating, for each domain, the consensus were rolled over from vaccine trial BCN01 (20) and invited sequence of the four major HIV-1 clades A, B, C, and D (12). to participate after 3 years on suppressive ART. A complete Upon delivery to both HIV-1-negative and positive individuals list of inclusion/exclusion criteria is available in the Study by heterologous prime/boost regimens as DNA or in simian Protocol (Appendix). The study took place between February adenovirus of chimpanzee (ChAdV) and poxvirus MVA vectors, 2016 and October 2017 at two HIV-1 units from universitary HIVconsv vaccines were safe and induced CD8+ T cells with hospitals (Hospital Universitari Germans Trias i Pujol -HUGTIP, broad inhibitory capacity of HIV-1 in vitro, but showed no effect Badalona and Hospital Clínic, Barcelona) and a community on the viral reservoir (16–23). center (BCN-Checkpoint, Barcelona). Before inclusion in the To overcome the limitations of therapeutic vaccines - study, all participants signed an informed consent previously administered alone- in targeting the viral reservoir, vaccines discussed, reviewed and approved by the Community Advisory are combined with latency reversing agents (LRA) in the Board of the Barcelona-based vaccine program (HIVACAT). so called kick&kill strategies (24). This approach intends to The study was approved by the institutional ethical review activate transcription of HIV-1 using small molecules able to board of the participating institutions (Reference Nr AC-15- disrupt the viral latency and facilitate effective sensing and 108-R) and by the Spanish Regulatory Authorities; and was clearance of infected cells by vaccine-elicited HIV-1-specific conducted in accordance to the principles of the Helsinki CTL (25, 26). Histone deacetylase inhibitors (HDACi) have Declaration and local personal data protection law (LOPD been proposed as potential HIV-1 LRA (27–31). Romidepsin 15/1999). The MVA.HIVconsv vaccine was GMP manufactured (RMD; Istodax R , Celgene Ltd.) is a potent HDACi approved for at IDT Biologika GmbH, Germany, and supplied for the the treatment of cutaneous and peripheral T-cell lymphomas, study under an investigator initiated clinical trial contract which has been shown to induce HIV-1 transcription both agreement. Risk of Genetically Modified Organism release to in vitro and in vivo (32, 33). The REDUC trial combined the environment was evaluated by the Spanish Ministry of RMD with Vacc-4x and rhuGM-CSF in chronically suppressed Environment (B/ES/12/09). RMD was supplied for the study HIV-1-positive individuals, resulting in a mean reduction of by Celgene Ltd. (Couvet, Switzerland) under an investigator 39.7% in total HIV-1 DNA (34). However, this intervention initiated clinical trial contract agreement. failed to delay viral rebound after ART interruption, suggesting The BCN02 trial design is summarized in Figure 1A. that the reservoir-purge effect was not sufficient and/or the After their inclusion in the study (week 0), all participants vaccine-induced response was unable to eliminate cells actively received a first dose of 2 × 108 plaque-forming units (pfu) of replicating HIV-1. In fact, the increase in cell-associated HIV-1 MVA.HIVconsv (MVA1 ) administered intramuscularly, followed Frontiers in Immunology | www.frontiersin.org 2 May 2020 | Volume 11 | Article 823 Mothe et al. Control After Kick&Kill and Early-cART FIGURE 1 | Trial design. (A) Schematic study design. (B) Consolidated Standards of Reporting Trials (CONSORT) flow diagram for the trial. *Viral rebound during MAP was defined as pVL >20 copies/ml and # criteria for ART resumption included pVL over 2,000 copies/ml in two consecutive determinations, CD4+ cell counts decrease over 50% and/or below 500 cells/mm3 and/or development of clinical symptoms suggestive of an acute retroviral syndrome. MVA, MVA.HIVconsv vaccine; RMD, romidepsin; MAP, monitored antiretroviral pause; ART, antiretroviral therapy; pVL, plasma HIV-1 viral load. by three weekly doses of RMD of 5 mg/m2 BSA infused to compensate for any potential impairment in the previous intravenously over 4 hours (RMD1−2−3 ), and by a second dose vaccine-induced response caused by RMD. Following RMD of 2 × 108 pfu of MVA.HIVconsv (MVA2 ) 4 weeks after RMD3 prescription information, participants received prophylactic Frontiers in Immunology | www.frontiersin.org 3 May 2020 | Volume 11 | Article 823 Mothe et al. Control After Kick&Kill and Early-cART antiemetic treatment with ondasetron before and during 3 days were solicited prospectively for a minimum of 7 days following after each RMD dose. each immunization and RMD administration. Both local and Eight weeks after MVA2 (week 17), eligible participants systemic AE were graded according to the Division of AIDS initiated a MAP for a maximum of 32 weeks (MAP0−32 ) or (DAIDS) Table for Grading the Severity of Adult and Pediatric until any ART resumption criteria were met. To be eligible for Adverse Events, version 2.0, November 2014, accessible online the MAP, participants had to maintain undetectable HIV-1 pVL at https://rsc.niaid.nih.gov/sites/default/files/daids-ae-grading- and meet the immune futility criteria, defined as showing a table-v2-nov2014.pdf. AE were specified as unrelated, unlikely, net increase in HIVconsv-specific immune response with MVA2 probably or definitely related to the investigational products by boost measured in an ex vivo IFN-γ ELISPOT assay. During the investigator. MAP, participants were allowed to choose either the hospital units or BCN-Checkpoint community center for the follow up visits. Symptoms suggestive of acute retroviral syndrome Determination of RMD Pharmacokinetics and sexually transmitted diseases were solicited and viral load The concentration of RMD in plasma was determined, for was tested using the finger-tip Xpert HIV-1 Qual kit (Cepheid, RMD1 , at the end of the infusion (4 h) and 4.5, 5, 6, 8, 12, Sunnyvale, CA, US) in all visits. When a positive result was and 24 h after and, for RMD2 and RMD3 , at the end of the obtained in the Xpert HIV-1 Qual, participants were called infusion and 12 h after. RMD concentrations were measured by in for a confirmatory quantitative pVL within the next 24 h. liquid chromatography-mass spectrometry/mass spectrometry If pVL was confirmed to be over 20 copies/ml, a visit was (LC-MS/MS), according to a validated method. A population scheduled 3 days after to closely monitor viral rebound and be pharmacokinetic model for RMD was developed using non- able to offer prompt ART resumption if required. Details on linear mixed-effects modeling with the computer program viral load management during MAP are described in the Study NONMEM version 7.3 (Icon Development Solution, Ellicot City, Protocol (Appendix). After ART ressumption, participants were MD) (36). Bayesian estimates of the individual parameters of followed at 4, 12, and 24 weeks to assure that they re-attained RMD were used to simulate individual drug concentrations, viral suppression. and RMD area under the concentration-time curve (AUC0- inf) was calculated for each individual on each occasion using Study Population a non-compartmental approach (Winnonlin software; Phoenix, BCN02 participants were adult (≥18 years) HIV-1-infected version 7.0). individuals, who had initiated ART >6 months after estimated date of HIV-1 acquisition and who had received a prime/boost heterologous vaccination regimen using ChAdV63.HIVconsv- MVA.HIVconsv in the parental BCN01 study (20). To be Flow Cytometry Determination of acH3 and eligible for BCN02, participants had to maintain optimal HIV- Activation of T Cells 1 suppression during at least 3 years and CD4+ cell counts The levels of histone H3 acetylation in lymphocytes (based ≥500 cells/mm3 at BCN02 baseline visit. Main exclusion criteria on FSC/SCC scatter) were determined by flow cytometry from included active hepatitis B or C, history of AIDS-defining samples taken before (0 h) and at the end of each RMD infusion disease, treatment for cancer or lymphoproliferative disease (RMD1−2−3 ) (4 h), at 8 and 24 h (+1 day) RMD1 , and at 72 h (+3 within 1 year before study entry or use of immunosuppressants days) and 7 days after (RMD1−2−3 ). Cryopreserverd PBMC were within the 3 months prior to the screening visit. Concomitant thawed 4 h before use, and 500,000 cells were blocked with 600 ul treatment with strong CYP3A4 inhibitors was not permitted, but of PBS/10% FBS for 20 min and stained with polyclonal rabbit switching ART to a non-boosted integrase-inhibitor raltegravir- anti-acetyl histoneH3 (10 µg/ml, MerckMillipore #06–599) or or dolutegravir-based regimen at least 4 weeks before baseline normal rabbit serum (control stain, LifeTechnologies #10510) visit was allowed for those patients receiving ART containing for 30 min. Cells were washed and subsequently incubated ritonavir or cobicistat at screening. with donkey anti-rabbit IgG(H+L) (6 µg/mL, LifeTechnologies #A21206) for 30 min at room temperature in the dark. Cells Study Endpoints were washed, re-suspended in 150 µl PBS and analyzed. ∼50,000 The primary endpoint of this study was to assess the safety, events were acquired per sample. The median fluorescence tolerability and the effect on the viral reservoir size of the intensity (MFI) for each sample was calculated by substracting combined treatment with HIVconsv vaccines and RMD given the background MFI (isotype control stain) from the anti-acetyl as a latency reversing agent. Secondary endpoints included the histoneH3 stain. extent and specificity of the CTL response and the effect of the Activation of T cells was determined based upon HLA-DR intervention in controlling viral rebound after ART interruption. expression on CD3+ T cells. Cryopreserved PBMC were thawed, Other secondary endpoints included RMD pharmacokinetics and 1,000,000 cells were stained with CD3 APC-Cy7, CD4 FITC, and the effects of RMD on histone acetylation in lymphocytes, CD8 BV510 and HLA-DR PECy7 (BioLegend #344818, 300538, induction of viral transcription, changes in T-cell activation 301048, and 307616, respectively). Cells were collected on an surface markers, and quantification of plasma viremia. LSRII instrument (BD), and data analyzed according to the Safety and tolerability were evaluated by the development of gating criteria shown in Supplementary Figure 1 using FlowJo grade ≥3 and serious adverse events (AE). Local and systemic AE 10 software. Frontiers in Immunology | www.frontiersin.org 4 May 2020 | Volume 11 | Article 823 Mothe et al. Control After Kick&Kill and Early-cART Quantification of Cell-Associated (CA) IL-2 (39). Before their use in ELISPOT assays, the expanded cells HIV-1 RNA in CD4+ T Cells were washed twice with R10 and incubated overnight at 37 ◦ C in Cell-associated HIV-1 RNA was quantified in peripheral CD4+ the absence of IL-2 and individual OLP were added at 5 µg/ml. T cells by ddPCR (One-Step RT-ddPCR Advanced Kit for 100,000 cells were used per well as in the direct ex vivo assay. Probes, BioRad) from samples taken before (0 h) and at the Spots were counted using an automated Cellular Technology end of each RMD infusion (4 h), at 8 and 24 h (+1 day) after Limited (C.T.L., OH, USA) ELISPOT Reader Unit. The threshold RMD1 , and 72 h (+3 days) and 7 days after RMD1−2−3 . CA for positive responses was set at ≥50 SFC/106 PBMC (5 spots per HIV-1 RNA was quantified using two different primers/probe well), > the mean number of SFC in negative control wells plus 3 sets annealing to the 5′ LTR and GAG conserved regions of SD of the negative control wells, or > 3× the mean of negative HIV-1, to circumvent potential primer mismatch in individuals’ control wells, whichever was higher. To avoid overestimating viral sequence as previously described (37). HIV-1 transcription the breadth of responses, positive responses to two consecutive levels were normalized to the housekeeping gene TATA-binding 15-mer overlapping peptide were counted as one response. The protein (TBP) and shown as relative to levels before RMD1 . highest magnitude of the sequential responses was taken as the magnitude for each identified response. Ultra-Sensitive Determination of Residual Viremia Quantification of HIV-1 Reservoir To evaluate HIV-1 RNA below 20 copies/ml, 4–8 ml of plasma To quantify the size of the peripheral blood proviral reservoir, samples taken before (0 h) and at the end of each RMD infusion lysed extracts from CD4+ T cells were used to measure total (4 h), at 8 and 24 h (+1 day) after RMD1 , and 72 h (+3 days) and CA HIV-1 DNA by ddPCR. Primers and probes for the RPP30 7 days after RMD1−2−3 were ultracentrifugated at 170,000 g at cellular gene were used for input normalization. 4◦ C for 30 min and viral RNA was extracted automatically using the m2000sp Abbot device. HIV-1 RNA copies were quantified Statistical Analysis using the Abbott Real-Time HIV-1 assay (Abbott Molecular Inc.) Qualitative variables were represented as mean absolute and in-house calibration curve sets as described (38). The limit and relative frequencies, whereas quantitative variables were of detection (2 HIV-1 RNA copies/mL) was calculated relative to represented as mean or median and range. Safety endpoints the plasma volume. are summarized by the number and percentage of participants reporting local and systemic AE and their grading. The Wilcoxon Vaccine Immunogenicity signed rank test was used to test whether the viral reservoir and Total HIV-1 and HIVconsv-specific T cells were assessed ex vivo the immune parameter changed as an effect of the intervention, using cryopreserved PBMC obtained the day of vaccination and without correction for multiple comparisons. The maximum 1 week afterwards, 3 weeks after MVA1 , and 4 weeks after MVA2 breadth of the T-cell response per individual was estimated using an IFN-γ-detecting enzyme-linked immunoabsorbent as the number of P1-P6 pools eliciting a positive response spot assay (ELISPOT IFN-γ Mabtech kit). 15-mer peptides throughout the study and the number of individual OLP eliciting overlapping by 11 amino acid were combined into 6 pools of a response at peak immunogenicity time point from the mapping 32–33 peptides per pool corresponding to the HIVconsv vaccine assay. Reservoir size and immunogenicity were analyzed using insert (P1-P6, total n = 166 peptides, IN pools) (Figure 3A) GraphPad Prism (v5.01) for Mac OS X (San Diego, CA). and 12 pools of 39–67 peptides per pool spanning the rest To evaluate the effect of the intervention on viral rebound, of the HIV-1 viral protein sequences (OUT pools for “outside a positive pre-defined efficacy signal was established if at the immunogen,” obtained through the NIH AIDS Reagent least over 20% of patients remained with pVL below 2,000 Program). All peptides pools were tested in duplicates. The copies/ml at week 12 of MAP, considering previous data final concentration of individual peptides in the ELISPOT assay suggesting that early treatment initiation could favor delayed was 1.57 µg/ml. Medium only was used as no-peptide negative viral rebound/spontaneous viral control in up to 15% of control in quadruplicate wells, and PHA (50 µg/ml) and a individuals (40). However, BCN02 was an exploratory pilot trial CEF peptide pool (2 µg/ml) consisting of 23 previously defined and, due to the absence of a control arm and its final small sample human CD8+ T-cell epitopes from cytomegalovirus, Epstein- size, the nature of this study only allowed to detect trends in Barr virus and influenza virus (C.T.L. OH, USA) were added as virological effects, which collectively, could be useful to design positive controls. future studies. To detect possible factors associated to the viremic To address the breadth of the vaccine-induced response at control observed during the MAP phase, univariate log-binomial the peak immunogenicity time point, an IFN-γ ELISPOT assay regression models were used (41). This model uses the logarithm with in vitro expanded T cells was performed on stored samples as a link function, and is a generalized lineal model for a binary from week 10 and 13 to test individual overlapping peptides outcome where the error terms follows a binomial distribution. covering the HIVconsv immunogen sequence (n = 166 OLP). The effect size measure of the model is the relative risk. Because Briefly, cryopreserved PBMC were thawed and incubated for 3 h of the low number of MAP-C (n = 3), multivariate log-binomial at 37◦ C in R10 before stimulation with an anti-CD3 monoclonal regression models were not fitted. The significance threshold antibody during 2–4 weeks in RPMI 1640 supplemented with for all univariate analyses was set at a two-sided α = 0.05. The FBS and penicillin/streptomycin with 50 U/ml of recombinant analyses were performed with R Core Team (42) (v3.0.2). Frontiers in Immunology | www.frontiersin.org 5 May 2020 | Volume 11 | Article 823 Mothe et al. Control After Kick&Kill and Early-cART RESULTS symptoms started within 4 h after RMD3 and therefore, the SAE was considered as possibly related to RMD. Participants Enrolled in the Study No laboratory abnormalities related to MVA1−2 were Between February 29th and September 15th 2016, 15 out of reported. All laboratory abnormalities related to RMD were grade the 22 eligible BCN01 participants were enrolled in BCN02. 1-2 (n = 22), the most frequent being hypophosphatemia (8 Seven declined to participate due to their inability to attend all events) and thrombocytopenia (5 events), except from one case the scheduled visits. Baseline characteristics of trial participants of grade 4 creatinine kinase elevation with normal eGFR which are summarized in Table 1. All 15 participants received two resolved within 7 days without sequelae. Noteworthy, CD4+ T- doses MVA.HIVconsv (MVA1−2 ) and three doses of romidepsin cell counts showed a transient decrease by a median of 248 (RMD1−2−3 ) as shown in the study chronogram (Figure 1A), cells/mm3 3 days after each RMD administration which was not and were included in the safety, immunogenicity and reservoir fully recovered by day 7 after RMD3 (Supplementary Figure 2). analyses. One participant was not eligible for MAP due Overall, both MVA.HIVconsv and RMD at the regimen to immune futility pre-defined criteria and 14 participants and dose administered in this study were well-tolerated underwent a MAP for a maximum of 32 weeks. Retrospective and safety profiles were consistent with data previously analyses of stored plasma samples obtained during MAP revealed reported (20, 34). the presence of antiretroviral drugs in some samples from one During the MAP, 12 (86%) participants reported a total of 58 participant, whose MAP data were censored for the viral rebound AE, which were all grade 1-2 and not suggestive of acute retroviral kinetics analyses (Figure 1B). syndrome (not shown). Grade 1 anxiety was observed in one participant who repeatedly declined psychological support (same Safety of MVA.HIVconsv and RMD participant with protocol violation during the MAP). Administrations All participants reported adverse events (AE) related to both study investigational medicinal products. A total of 333 AE were RMD Pharmacokinetics and recorded during the study intervention phase, 129 after MVA1−2 Pharmacodynamics and 204 after RMD1−2−3 , which were mostly mild or moderate Pharmacokinetics of RMD was comparable to previously (grade 1-2) (n = 318, 95%). The most frequent AE related described data (43). A population pharmacokinetic model to MVA.HIVconsv, summarized in Table 2, were local pain at adequately describing RMD concentrations in plasma was the injection site and a flu-like syndrome consisting of fatigue, developed (36). According to the individual profiles simulated headache, myalgia, and/or low-grade temperature (<38◦ C). using the model, each infusion was followed by a rapid and Regarding AE related to RMD (Table 3), the most common polyexponential decline in RMD concentrations in plasma, grade 1–2 events were headache, fatigue, and gastro-intestinal reaching nearly undetectable levels by 24 h after dosing symptoms. Despite prophylactic ondansetron treatment, 4 (27%) (Figure 2A). individuals vomited the days of RMD administration. One Regarding the direct effect of RMD on chromatin and participant experienced a grade 4 AE consisting in a sepsis induction of viral transcription, histone H3 acetylation (acH3) by Shigella sonnei that required hospital admission for 24 h, thus fulfilling the criteria of serious adverse event (SAE). The TABLE 2 | Summary of adverse events related to MVA.HIVconsv vaccinations (n = 15). TABLE 1 | Demographic, clinical, and treatment characteristics of study patients Grade 1 Grade 2 Grade 3 Grade 4 Total at study entry (n = 15). n n n n n (%) Age (years) 43 (33–51) Injection site reaction Gender (M/F), n 14/1 Local pain 7 4 2 0 13 (87) MSM/HTS, n 14/1 Redness 1 0 0 0 1 (7) Time since HIV-1 acquisition to ART (days) 92 (28–164) Induration 0 0 0 0 0 (0) Pre ART log10 HIV-1 RNA (copies/ml) 4.9 (3.2–5.8) Systemic adverse events Time on ART (years) 3.23 (3.03–3.77) Fatigue 7 4 2 0 13 (87) ART regimen, n (%) Headache 5 3 1 0 9 (60) TDF/FTC/RAL 11 (73) Myalgia 4 3 2 0 9 (60) ABC/3TC/RAL 2 (13) Fever 5 0 0 0 5 (33) ABC/3TC/DTG 2 (13) Anorexia 3 0 1 0 4 (27) CD4+ T-cell counts (cells/mm3 ) 728 (416–1,408) Sweating 2 2 0 0 4 (27) Ratio CD4/CD8 1.37 (0.97–1.93) Nausea 2 0 1 0 3 (20) Median (range) is shown unless otherwise described. M, male; F, female; MSM, Abdominal pain 0 1 1 0 2 (13) men who have sex with men; HTS, heterosexual; ART, antiretroviral therapy; TDF, Flatulence 1 0 0 0 1 (7) Tenofovir Disoproxil Fumarate; FTC, Emtricitabine; RAL, Raltegravir; ABC, Abacavir; 3TC, Lamivudine; DTG, Dolutegravir. Somnolence 1 0 0 0 1 (7) Frontiers in Immunology | www.frontiersin.org 6 May 2020 | Volume 11 | Article 823 Mothe et al. Control After Kick&Kill and Early-cART TABLE 3 | Summary of adverse events related to RMD1−2−3 treatment (n = 15). (ELISPOT) assay using 6 peptide pools covering the HIVconsv immunogen sequence (P1-P6) on week 0 (day of MVA1 ), 1, 3 (day Grade 1 Grade 2 Grade 3 Grade 4 Total n n n n n (%) of RMD1 ), 9 (day of MVA2 ), 10, and 13. A total of 90 samples were obtained, of which 3 (3%) were censored due to low positive Headache 9 5 0 0 14 (93) controls and/or high background. All 15 participants showed Fatigue 9 5 0 0 14 (93) an absolute increase in HIVconsv-specific IFN-γ-producing T Nausea 4 7 0 0 11 (73) cells during the study, either after MVA1 (Wilcoxon signed-rank, Anorexia 8 1 0 0 9 (60) p = 0.0007) or after MVA2 (Wilcoxon signed-rank, p = 0.0017) Abdominal pain 5 2 0 0 7 (47) (Figure 3B). Median (range) total frequencies of HIVconsv- Metallic taste 5 1 0 0 6 (40) specific T cells reached 1,965 (530-6,901) spot-forming cells Constipation 4 2 0 0 6 (40) (SFC)/106 PBMC at the peak immunogenicity time point, Abdominal 4 1 0 0 5 (33) which represented an absolute median increase of 1,600 (300– distension 6,621) SFC/106 PBMC from baseline (Wilcoxon signed-rank, Vomits 4 0 0 0 4 (27) p <0.0001). Sweating 2 2 0 0 4 (27) Over the intervention phase, participants responded to median Palpitations 3 0 0 0 3 (20) (range) of 5 (2–6) peptide pools (Supplementary Figure 5). Myalgia 1 1 0 0 2 (13) To map the maximum vaccine-induced breadth at peak Rash 0 2 0 0 2 (13) immunogenicity time point (weeks 10–13), in vitro expanded Dry mouth 1 0 0 0 1 (7) T cells responding toward individual OLPs covering the ECG: ST-elevation 1 0 0 0 1 (7) HIVconsv immunogen were assessed. Median (range) of 8 Anxiety 0 1 0 0 1 (7) (3–16) IFN-γ-producing responses to individual OLPs were Libido decrease 0 1 0 0 1 (7) found, with a dominance in Pol-specific T cells, consistent with Somnolence 1 0 0 0 1 (7) the immunogen composition (Figure 3C). Sepsis by Shigella 0 0 0 1 1 (7) The dominance of HIVconsv-specific responses was calculated sonnei (SAE) at each time point as the percentage of HIVconsv-specific T- Hypotension 0 1 0 0 1 (7) cell frequencies divided by the total HIV-1 proteome-specific T-cell frequencies. At the moment of HIV diagnoses, HIVconsv responses were subdominant (<10% being HIVconsv-specfic) and peaked after the CM vaccination reaching a median increased rapidly during each RMD infusion, remained high (range) of 58% (7%−100%) of the total anti-HIV-1 T-cell during 4 h, and returned to baseline values 3 days after each responses (BCN01 parental study) (20). In BCN02, 2 years from dose (Figure 2B), which is consistent with previous reports (33, the last HIVconsv vaccination, the increase in the frequency 34). HIV-1 transcription transiently increased in parallel, with of HIVconsv-specific T-cell responses after MVA1 or MVA2 changes more pronouned after RMD2 and RMD3 (Figure 2C). further shifted the patterns of T-cell immuno-dominance toward These changes were more evident without normalization HIVconsv with median (range) of 85% (54%–100%) of the total for house-keeping genes (Supplementary Figure 3) possibly anti-HIV-1 T-cell responses at peak immunogenicity time point reflecting the general increase in histone acetylation levels (44) being HIVconsv specific (Figure 3D). induced by RMD. Increases in T-cell activation, measured by the proportion of CD3+ /HLA-DR+ cells, were observed 3 days after Effects on the HIV-1 Reservoir each RMD dose. Over the course of the three RMD doses, T-cell All participants had detectable viral reservoirs, as measured by activation increased in a progressive manner and was maintained total CD4+ T cell-associated HIV-1 DNA, throughout the study. up to 1 week after RMD3 (Figure 2D), suggesting a cumulative Results from 2 samples out of a total of 60 were considered effect of RMD. invalid and were censored. At BCN02 study entry, median To evaluate changes in levels of quantifiable plasma HIV- (range) reservoir size was of 140 (17–752) HIV-1 DNA copies/106 1 RNA, an ultrasensitive single copy assay was used. Kinetics CD4+ T-cells (Figure 4). Proviral DNA showed a tendency to of plasma HIV-1 RNA levels did not follow a clear pattern further decrease from baseline to week 17 (Wilcoxon signed- (Figure 2E), despite the increasing percentage of participants rank, p = 0.0599, Figure 4) to median (range) levels of 120 with detectable low-level viremia at the end of each RMD dose (11-680) copies/106 CD4+ Tcells. (Supplementary Figure 4). Collectively, we reproduced effects on acH3, HIV-1 transcription and T cell activation previously Monitored Antiretroviral Pause (MAP) reported in chronically infected individuals (34), suggesting that Participants undergoing MAP were monitored weekly for the a lower viral reservoir level achieved by early-treatment initiation first 12 weeks and every 2 weeks thereafter for a maximum does not preclude the reactivation potential of RMD. of 32 weeks (MAP0−32 ). Criteria for ART resumption included pVL over 2,000 copies/ml in two consecutive determinations, MVA.HIVconsv Immunogenicity CD4+ cell counts decrease over 50% and/or below 500 cells/mm3 Total HIV-1 and HIVconsv-specific T cells were assessed ex vivo and/or development of clinical symptoms suggestive of an acute by an IFN-γ-detecting enzyme-linked immunoabsorbent spot retroviral syndrome. Frontiers in Immunology | www.frontiersin.org 7 May 2020 | Volume 11 | Article 823 Mothe et al. Control After Kick&Kill and Early-cART FIGURE 2 | Pharmacokinetic and pharmacodynamic effects of RMD. (A) Mean individual predictions of RMD plasma concentrations. (B) Levels of histone H3 acetylation in peripheral lymphocytes. (C) Viral transcription levels expressed as changes from pre-RMD1 levels of cell-associated HIV-1 RNA in peripheral CD4+ T-cells. (D) Levels of T-cell activation (CD3+ /HLA-DR+ cells). (E) individual determinations of pVL. Median of frequencies and IQR (error bars) are represented. Wilcoxon signed-rank p-values compare each represented time point with the corresponding values preceding each RMD administration. *p < 0.05. **p < 0.01. ***p < 0.001 and ****p < 0.0001. The p value resulting from the comparison between the value at day 0 of RMD1 and 7 days after RMD3 is shown in red. Frontiers in Immunology | www.frontiersin.org 8 May 2020 | Volume 11 | Article 823 Mothe et al. Control After Kick&Kill and Early-cART FIGURE 3 | Vaccine immunogenicity. (A) Schematic representation of the selected regions in the HIV-1 proteome from different clades included in the HIVconsv immunogen and distribution of 6 peptide pools (P1-P6) and individual overlapping 15-mer peptides (OLP) used in the IFN-γ ELISPOT assays. (B) Magnitude (sum of SFU/106 PBMC to pools P1-P6) of vaccine-induced responses over the BCN02 study. Horizontal and error bars represent median and IQR, respectively, and p-values correspond to comparisons between the indicated time points using the Wilcoxon signed-rank test. (C) Breadth of vaccine-elicited responses toward individual OLP included in the indicated HIVconsv regions. Horizontal and error bars represent median and IQR, respectively. (D) Average distribution of total HIV-1 T-cells according to their specificity at the indicated time points. HIVconsv-specific responses are shown in blue. Pie charts are scaled according to the total frequencies of responses. IN are peptide pools corresponding to the HIVconsv vaccine insert and OUT peptide pools spanning the rest of HIV-1 proteome “outside the immunogen”. All participants rebounded (detectable pVL over 20 copies/ml) 32 weeks (MAP-C, from MAP-Controllers). Highest peak pVL during MAP (Figure 5A). Median (range) time to first detectable determination in the MAP-C was of 3,110 copies/ml at week pVL was 13 (7–35) days with median (range) of first detectable 12 of MAP in the absence of symptoms followed by 2,460 and pVL of 122 (28–3,410) copies/ml. Ten participants resumed ART 1,100 three and six days later (participant decision to stay off). before MAP12 (MAP-NC for MAP-Non-controllers) with median At week 32, two out of the three MAP-C accepted to stay off (range) time to resume ART of 28 (16–59) days. All MAP-NC cART out of the protocol and were followed under standard of resumed ART due to the viral load criteria, with median (range) care. One MAP-C showed a late rebound after 48 weeks off cART pVL of 19,250 (2,900–179,000) copies/ml at the moment of ART and the other one, voluntary resumed cART after 1.5 years off resumption (Figure 5B). None of the participants resumed ART cART despite sustained low-level viremia. All participants who due to immune or clinical criteria. A “late-rebounder” presented restarted ART reached viral re-suppression within 6 months. No the first detectable pVL 5 weeks after ART interruption (MAP5 , evidence of emergence of drug resistance was detected. pVL of 59 copies/ml) and was able to maintain viral load below To assess re-seeding of the viral reservoir during the MAP, 2,000 for 3 more weeks, resuming ART at MAP8 . In addition total HIV-1 DNA was measured at MAP0 (n = 13), on the day to the “late-rebounder,” 3 (23%) other participants remained of ART resumption (n = 10), and 6 months after for the 10 MAP- off ART with sustained pVL <2,000 copies/ml for a total of NC (n = 8 available), and at MAP32 for the three MAP-C. We did Frontiers in Immunology | www.frontiersin.org 9 May 2020 | Volume 11 | Article 823 Mothe et al. Control After Kick&Kill and Early-cART FIGURE 4 | Viral reservoir. (A) Total HIV-1 DNA copies/106 CD4+ T cells for each participant are shown at study entry (week 0), week 3 (day of RMD1 ), and week 6 (1 week after RMD3 ) and at week 17 (8 weeks after MVA2 ). Horizontal and error bars represent median and IQR, respectively, and p-values correspond to comparisons between the indicated time points using the Wilcoxon signed-rank test. (B) Changes in proviral DNA throughout the study with respect to baseline (week 0), which is considered to be 1. not observe any significant change in total HIV-1 DNA during treated during acute/recent HIV-1 infection, we show that MAP in participants with early ART resumption (Wilcoxon combination of the HIVconsv vaccines with RMD as a latency signed-rank, p = 0.5759 for MAP-NC) and, noteworthy, nor reversing agent was safe, highly immunogenic, and induced in the three participants with sustained low level viremia for bursts of viral transcription. The combined intervention resulted 32 weeks. Moreover, one of the three MAP-C showed a 2-fold in a tendency toward a reduction in proviral DNA levels and was reduction in the HIV-1 DNA (from 34 at MAP0 to 16 copies/106 followed by a prolonged viremic control in 23% of participants CD4+ cells at MAP32 , Figure 5C). This was the only participant after ART interruption without evidence of reservoir re-seeding. undergoing MAP carrying HLA alleles associated with natural Early-treated individuals typically show less immune HIV control (HLA-B∗ 27:01/HLA-B∗ 51:01) (45, 46). exhaustion (47) and reduced frequency of immune-escaped viral variants compared to individuals initiating ART at Factors Influencing Viral Rebound Kinetics later stages of HIV-1 infection (48), offering a potentially Supplementary Table 1 shows the summary of variables favorable setting to induce a protective immune response explored to explain the binary outcome defined as MAP-NC upon therapeutic vaccinations. In the parental BCN01 trial, vs. MAP-C. The estimated relative risks obtained from the a prime/boost vaccine regimen with ChAdV63.HIVconsv log-binomial models for different covariates analyzed are shown and MVA.HIVconsv induced high frequencies of T cells with in Figure 5D. high in vitro suppressive capacity that markedly shifted the Univariate log-binomial regression models used to detect focus of the CD8+ CTL response toward HIVconsv sequences factors associated with virologic control during MAP revealed that are subdominant during natural infection (20). In the that pVL before ART initiation (pre-ART pVL) was the only BCN02 trial, after 3 years of viral suppression and 2 years factor statistically significantly associated with control of viral since the last vaccination in BCN01, booster MVA.HIVconsv rebound after ART interruption. For each log increase on the vaccinations were still immunogenic and further increased pre-ART pVL, the probability of becoming a MAP-C decreased breadth, magnitude and immunodominance of CTL responses by 66% (RR 0.34; 95% CI 0.14, 0.79). Interestingly, albeit not toward HIVconsv sequences. statistically significant in the univariate models, the 3 MAP-C Along with a strong vaccine-elicited CTL activity (“kill”), the had among the lowest reservoir levels at treatment interruption ability to simultaneously induce reactivation of the viral reservoir time point (16, 54 and 122 copies/106 CD4+ T cells) –consistent (“kick”) is a critical feature for the success of the kick&kill with lower pre-ART pVL– and showed the highest shift in CTL strategy (24). A 3-dose regimen of weekly RMD at 5 mg/m2 immunodominance pattern after vaccination (>700 HIVconsv- BSA was selected based on results from previous trials (33, 34). specific SFC/106 PBMCs and >75% of HIVconsv dominance at Consistently, we observed a direct in vivo effect of RMD in peak immunogenicity time point, Supplementary Figure 6). histone 3 acetylation upon each RMD dose, which was followed by changes in cell-associated HIV-1 RNA levels. Conversely, a DISCUSSION placebo-controlled dose-escalating trial (ACTG 5315) testing 3 RMD doses (5 mg/m2 BSA) administered every two weeks in In this single-arm, open-label, phase I, proof-of-concept chronically suppressed individuals, did not show changes in viral trial performed in HIV-1-infected ART-suppressed individuals transcription (49). The weekly administration regimen and the Frontiers in Immunology | www.frontiersin.org 10 May 2020 | Volume 11 | Article 823 Mothe et al. Control After Kick&Kill and Early-cART FIGURE 5 | Monitored antiretroviral pause. (A) Individual pVL during MAP is shown for each participant in different colors (n = 13). Lines are interrupted on day of ART resumption. Dotted lines represent detection limit and threshold for ART resumption (20 and 2,000 copies/ml, respectively). (B) Proportion of individuals remaining off ART during MAP. (C) Total HIV-1 DNA copies/106 CD4+ T cells are shown for each participant at MAP initiation, at day of ART ressumption, and 24 weeks after ART. P-value corresponds to comparison between MAP initiation and the day of ART resumption for each individual using the Wilcoxon signed-rank test. Individuals with sustained low-level viremia over 32 weeks are shown in red. (D) Estimated relative risks and 95% confidence intervals for the durable control of pVL during MAP obtained from univariate log-binomial regression models. intensive sampling after each RMD dose in our study allowed of the current study, elimination of reactivated cells by vaccine- us to detect increases in CA HIV-1 RNA above 2-fold in 80% of elicited T cells may have additionally blunted quantification of individuals at any time point after RMD administrations. These plasma viremia. changes were followed by consistent increases in T cell activation A critical objective of the use of LRA in a kick&kill markers, which remarkably, were maintained one week after strategy is to mobilize and ultimately reduce the viral reservoir. RMD3 , likely reflecting a direct and cumulative effect of weekly Our findings showed that, despite robust immunogenicity of RMD dosing and an effective induction of viral transcription. HIVconsv vaccines and at least partial reactivation of the viral Noteworthy, changes above 2.1-fold in CA-RNA have been reservoir induced by RMD, the net effect on the proviral DNA estimated to occur in <5% of repeated measurements in an levels was modest. All participants had detectable levels of individual (50). HIV-1 DNA at the time of treatment interruption although a Despite the induction of viral transcription, the kinetics of tendency toward a decrease by 19.3% from baseline to week 17 plasma HIV-1 RNA followed an unclear pattern, similar to (Wilcoxon signed-rank, p = 0.0599) was detected. Conversely, previous studies showing variable changes in plasma viremia a mean 39.7% decrease in reservoir size was observed in following LRA administration (34, 51, 52). This variability might the REDUC trial (34). This discrepancy between REDUC and reflect suboptimal potency of the agents tested so far and/or the BCN02 results may be explained by the inclusion of early- reactivation of predominantly defective proviruses. In the context treated individuals in our study, in which already baseline Frontiers in Immunology | www.frontiersin.org 11 May 2020 | Volume 11 | Article 823 Mothe et al. Control After Kick&Kill and Early-cART levels of proviral DNA were substantially lower, challenging the The safety and tolerability profiles of MVA.HIVconsv and quantification of the effects of the intervention on HIV-1 DNA RMD were similar to those reported in previous studies (19, levels. We acknowledge that the translation of HIV-1 protein 20, 34). However, there was a SAE in one participant. This case expression into antigen presentation—even in case of defective highlights the need for planning intensive monitoring in this proviruses (1)—upon LRA reactivation is poorly understood. kind of pilot trials, even if not powered to detect low-frequency Likewise, the ability of LRA-induced HIV-1 protein expression AE, and points toward the need for a trade-off between the to effectively induce recognition and killing by CD8+ T cells number of participants potentially put at risk in well-powered remains to be fully elucidated (53) and therefore, the potential controlled trials and for caution with the use of uncompletely effects of further RMD administrations on the viral reservoir characterized agents in large numbers of individuals. Given that are to be determined. A potential significant toxicity—suggested natural PTC rates are considered to be up to 13% in early- in in vitro assays (54)—on vaccine-induced T cells might also treated individuals, powering trials to show viral control efficacy have limited our capability to observe a further reduction in after an ATI becomes challenging (60). Despite frequent clinical the reservoir size in our study. This hypothesis is consistent monitoring for pVL and access to psychological support, protocol with the fact that HIVconsv vaccines induced higher levels violation during MAP occurred in one individual, probably due of activated T cells compared to Vacc-4x vaccination in the to anxiety secondary to the antiretroviral interruption. This case REDUC trial. Nevertheless, the potential toxicity of RMD in vivo, warrants close psychological management in longer term ATIs. its relationship with RMD exposure and, ultimately, whether We fully acknowledge the limitations of the small sample vaccine-induced T cells were able to sensor and remove infected size and lack of a control arm in the present study. Therefore, cells in response to HIV-1 reactivation remains to be determined. we interpret these results with caution and regard this study To attain a functional cure, a persistent immune-mediated only as hypothesis-generating trial for future interventions. control of residual HIV-1 might be as relevant as achieving an BCN02 eligibility was restricted to vaccinated participants in the absolute reduction on the proviral DNA levels. In this regard, parental open-label BCN01 trial. This intrinsic restriction limited the three BCN02 MAP-C, were among the subjects with both the sample size to a small number of previously vaccinated lower viral reservoir levels at MAP and higher vaccine-induced individuals and precluded the inclusion of a control arm. responses. In our study, having lower pre-ART pVL was the only At the time of trial design, interventional trials including an outstanding marker associated with viral control during MAP, ATI were typically small and included very conservative ART which correlates with the size of the viral reservoir after ART resumption criteria (58, 61). Furthermore, ATI acceptability suppression. These relationships are consistent with previous by participants, risks of HIV-1 transmission to others in the studies suggesting a role of a low viral reservoir on analytical absence of available PrEP, and potential viral re-seeding upon treatment interruptions (ATI) outcomes (55, 56). Furthermore, treatment interruption were of special concern in early-treated after ART discontinuation, MAP-C did not show an initial burst individuals, who had limited viral reservoirs both in size and in pVL followed by a fast post-peak control as described in diversity (48). several post treatment controllers (PTC) (8, 57). Collectively, Altogether, the results from this pilot study suggest a the findings from this and other studies suggest that a small potential role for kick&kill strategies in inducing durable reservoir size, resulting from early ART or another intervention, immune-mediated HIV-1 control in a proportion of early- may be essential to achieve sustained post-intervention control treated individuals. In view of these results, future controlled (55) but also, that a potent vaccine-induced immune pressure studies to identify the mechanisms underlying sustained HIV-1 might contribute to prevent a peak burst of viremia and maintain suppression are warranted. suppressed viremia for a substantial period of time. This control, mediated by immune pressure, is supported by the absence of re- seeding of the viral reservoir in the BCN02 MAP-C, in contrast DATA AVAILABILITY STATEMENT to reports from previous ATI trials (58, 59). All datasets generated for this study are included in the The interpretation of the outcome of kick&kill studies may article/Supplementary Material. be confounded by individuals controlling HIV rebound after treatment interruption without the need for a prior therapeutic intervention. The prevalence and mechanisms driving such PTC ETHICS STATEMENT in natural HIV infection are not well-understood. A recent metanalysis (CHAMP study) including 14 interruption trials The studies involving human participants were reviewed and estimated a 13% rate of PTC among early-treated individuals approved by Hospital Germans Trias i Pujol and Hospital Clínic (57). Importantly, and in contrast to the three BCN02 MAP-C (AC-15-108-R). The patients/participants provided their written who did not show a transient high burst of viremia, 32% of the 61 informed consent to participate in this study. PTCs analyzed in the CHAMP metanalysis had peaks of viremia ranging from 1,000 to over 10,000 copies/ml within the first AUTHOR CONTRIBUTIONS 24 weeks after treatment interruption. Thus, in addition to the different behavior of the 3 MAP-C (23%) with respect to the PTC, BM, JM, BC, and CB conceived and designed the study. CMa, according to this metanalysis, the BCN02 trial may have missed TH, JMM, JM-P, AB, PC, MM, and MV contributed to the study additional MAP-C due to the conservative ART resumption design. CMi, CMa, IR, JT, ACa, MR-U, and MP contributed criteria used (two consecutive pVL over 2,000 copies/ml). contributed to data management. BM, MR-U, MP, ML, SM-L, AL, Frontiers in Immunology | www.frontiersin.org 12 May 2020 | Volume 11 | Article 823 Mothe et al. Control After Kick&Kill and Early-cART JM, PC, ACa, CMi, SC, AB, and CMa performed the experiments. FUNDING JM, BM, MP, MV, YA-S, GM, and KL undertook the statistical analysis. ACr and TH contributed with reagents, materials, BCN02 study was an investigator-initiated study funded by analysis tools. BM, JM, and CB drafted the manuscript. TH, MR- the ISCIII PI15/01188 grant, the HIVACAT Catalan research U, CMa, MP, SM-L, JM-P, JMM, MV, and BC participated in program for an HIV vaccine and the Fundació Gloria Soler. study analyses and revised the manuscript critically for important Some sub-analyses were partly funded by the European Union’s intellectual content. All authors reviewed and approved the final Horizon 2020 research and innovation program under grant version of the manuscript. agreement 681137-EAVI2020 and by NIH grant P01-AI131568. The GMP manufacture of the MVA.HIVconsv vaccines was INVESTIGATORS MEMBERS OF THE jointly funded by the Medical Research Council (MRC) UK and the UK Department for International Development (DFID) BCN02 under the MRC/DFID Concordat agreements (G0701669) and kindly provided through an IIS agreement. Romidepsin was IrsiCaixa AIDS Research Institute-HIVACAT, Hospital kindly provided by Celgene Ltd through an IIS agreement. BM Universitari Germans Trias i Pujol, Badalona, Spain: Susana held a post-doctoral fellowship grant from ISCIII (JR 13/00024) Benet, Christian Brander, Samandhy Cedeño, Bonaventura from 2014–16. CMa held a post-doctoral research grant (PERIS- Clotet, Pep Coll, Anuska Llano, Javier Martinez-Picado, 2016/2020) from the Departament de Salut de la Generalitat de Marta Marszalek, Sara Morón-López, Beatriz Mothe, Roger Catalunya, Barcelona, Spain during the conduct of the study. Paredes, Maria C. Puertas, Miriam Rosás-Umbert, Marta SM-L held a Ph.D. grant from DGR (2013FI_B 00275) from Ruiz-Riol. Fundació Lluita contra la Sida, Infectious Diseases 2013–16. JM received a 80:20 research grant from IDIBAPS, Department, Hospital Universitari Germans Trias i Pujol, Barcelona, Spain, during 2017–21. GM, KL, and YS have been Badalona, Spain: Roser Escrig, Silvia Gel, Miriam López, partially supported by Grant MTM2015-64465-C2-1-R from Cristina Miranda, José Moltó, Jose Muñoz, Nuria Perez-Alvarez, Ministerio de Economía y Competitividad. Jordi Puig, Boris Revollo, Jessica Toro. Germans Trias i Pujol Research Institute, Badalona, Spain: Ana María Barriocanal, Cristina Perez-Reche. Clinical Pharmacology Unit, Hospital ACKNOWLEDGMENTS Universitari Germans Trias i Pujol, Badalona, Spain: Magí Farré. Pharmacokinetic/pharmacodynamic modeling and simultation, We would like to thank i2e3 for providing medical writing Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau-IIB support. We especially thank Agueda Fernandez, Rafaela Ayen, Sant Pau, Barcelona, Spain: Marta Valle. Hospital Clinic- Lucía Gómez and Carmen Ligero for their technical assistance. HIVACAT, IDIBAPS, University of Barcelona, Barcelona, Spain: Special thanks to all the volunteers participating in this study for Christian Manzardo, Juan Ambrosioni, Irene Ruiz, Cristina their perseverance and dedication. Rovira, Carmen Ligero, Emma Fernández, and Jose M. Miró. Projecte dels NOMS-Hispanosida, BCN Checkpoint, Barcelona, SUPPLEMENTARY MATERIAL Spain: Antonio Carrillo, Michael Meulbroek, Ferran Pujol and Jorge Saz. The Jenner Institute, The Nuffield Department of The Supplementary Material for this article can be found Medicine, University of Oxford, UK: Nicola Borthwick, Alison online at: https://www.frontiersin.org/articles/10.3389/fimmu. Crook, Edmund G. Wee and Tomáš Hanke. 2020.00823/full#supplementary-material REFERENCES adenovirus type 5 vaccine expressing the HIV-1 core protein. J Infect Dis. (2010) 202:705–16. doi: 10.1086/655468 1. Bruner KM, Murray AJ, Pollack RA, Soliman MG, Laskey SB, Capoferri AA, 6. 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(2014) 10:e1004287. doi: 10.1371/journal.ppat.1004287 Copyright © 2020 Mothe, Rosás-Umbert, Coll, Manzardo, Puertas, Morón-López, 55. Li JZ, Etemad B, Ahmed H, Aga E, Bosch RJ, Mellors JW, Llano, Miranda, Cedeño, López, Alarcón-Soto, Melis, Langohr, Barriocanal, Toro, et al. The size of the expressed HIV reservoir predicts timing Ruiz, Rovira, Carrillo, Meulbroek, Crook, Wee, Miró, Clotet, Valle, Martinez-Picado, of viral rebound after treatment interruption. AIDS. (2016) Hanke, Brander, Moltó and the BCN02 Study Investigators. This is an open-access 30:343–53. doi: 10.1097/01.aids.0000499516.66930.89 article distributed under the terms of the Creative Commons Attribution License (CC 56. Williams JP, Hurst J, Stöhr W, Robinson N, Brown H, Fisher M, et al. HIV- BY). The use, distribution or reproduction in other forums is permitted, provided 1 DNA predicts disease progression and post-treatment virological control. the original author(s) and the copyright owner(s) are credited and that the original Elife. (2014) 3:e03821. doi: 10.7554/eLife.03821 publication in this journal is cited, in accordance with accepted academic practice. 57. Namazi G, Fajnzylber JM, Aga E, Bosch RJ, Acosta EP, Sharaf R, et al. No use, distribution or reproduction is permitted which does not comply with these The control of HIV after antiretroviral medication pause (CHAMP) study: terms. Frontiers in Immunology | www.frontiersin.org 15 May 2020 | Volume 11 | Article 823