Non-neutralizing Antibodies Alter the Course of HIV-1 Infection In Vivo.

Non-neutralizing antibodies (nnAbs) to HIV-1 show little measurable activity in prevention or therapy in animal models yet were the only correlate of protection in the RV144 vaccine trial. To investigate the role of nnAbs on HIV-1 infection in vivo, we devised a replication-competent HIV-1 reporter virus that expresses a heterologous HA-tag on the surface of infected cells and virions. Anti-HA antibodies bind to, but do not neutralize, the reporter virus in vitro. However, anti-HA protects against infection in humanized mice and strongly selects for nnAb-resistant viruses in an entirely Fc-dependent manner. Similar results were also obtained with tier 2 HIV-1 viruses using a human anti-gp41 nnAb, 246D. While nnAbs are demonstrably less effective than broadly neutralizing antibodies (bNAbs) against HIV-1 in vitro and in vivo, the data show that nnAbs can protect against and alter the course of HIV-1 infection in vivo. PAPERCLIP.

Bispecific IgG neutralizes SARS-CoV-2 variants and prevents escape in mice.

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-191,2. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.

Evolution of antibody immunity to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with the development of variable levels of antibodies with neutralizing activity, which can protect against infection in animal models1,2. Antibody levels decrease with time, but, to our knowledge, the nature and quality of the memory B cells that would be required to produce antibodies upon reinfection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection with SARS-CoV-2. We find that titres of IgM and IgG antibodies against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 decrease significantly over this time period, with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by fivefold in pseudotype virus assays. By contrast, the number of RBD-specific memory B cells remains unchanged at 6.2 months after infection. Memory B cells display clonal turnover after 6.2 months, and the antibodies that they express have greater somatic hypermutation, resistance to RBD mutations and increased potency, indicative of continued evolution of the humoral response. Immunofluorescence and PCR analyses of intestinal biopsies obtained from asymptomatic individuals at 4 months after the onset of coronavirus disease 2019 (COVID-19) revealed the persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 individuals. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.

Convergent antibody responses to SARS-CoV-2 in convalescent individuals.

During the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has led to the infection of millions of people and has claimed hundreds of thousands of lives. The entry of the virus into cells depends on the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. Although there is currently no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19-convalescent individuals. Plasma samples collected an average of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titres; titres were less than 50 in 33% of samples, below 1,000 in 79% of samples and only 1% of samples had titres above 5,000. Antibody sequencing revealed the expansion of clones of RBD-specific memory B cells that expressed closely related antibodies in different individuals. Despite low plasma titres, antibodies to three distinct epitopes on the RBD neutralized the virus with half-maximal inhibitory concentrations (IC50 values) as low as 2 ng ml-1. In conclusion, most convalescent plasma samples obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

Longitudinal clonal dynamics of HIV-1 latent reservoirs measured by combination quadruplex polymerase chain reaction and sequencing.

HIV-1 infection produces a long-lived reservoir of latently infected CD4+ T cells that represents the major barrier to HIV-1 cure. The reservoir contains both intact and defective proviruses, but only the proviruses that are intact can reinitiate infection upon cessation of antiretroviral therapy (ART). Here we combine four-color quantitative PCR and next-generation sequencing (Q4PCR) to distinguish intact and defective proviruses and measure reservoir content longitudinally in 12 infected individuals. Q4PCR differs from other PCR-based methods in that the amplified proviruses are sequence verified as intact or defective. Samples were collected systematically over the course of up to 10 y beginning shortly after the initiation of ART. The size of the defective reservoir was relatively stable with minimal decay during the 10-y observation period. In contrast, the intact proviral reservoir decayed with an estimated half-life of 4.9 y. Nevertheless, both intact and defective proviral reservoirs are dynamic. As a result, the fraction of intact proviruses found in expanded clones of CD4+ T cells increases over time with a concomitant decrease in overall reservoir complexity. Thus, reservoir decay measurements by Q4PCR are quantitatively similar to viral outgrowth assay (VOA) and intact proviral DNA PCR assay (IPDA) with the addition of sequence information that distinguishes intact and defective proviruses and informs reservoir dynamics. The data are consistent with the notion that intact and defective proviruses are under distinct selective pressure, and that the intact proviral reservoir is progressively enriched in expanded clones of CD4+ T cells resulting in diminishing complexity over time.

Combination therapy with anti-HIV-1 antibodies maintains viral suppression.

Individuals infected with HIV-1 require lifelong antiretroviral therapy, because interruption of treatment leads to rapid rebound viraemia. Here we report on a phase 1b clinical trial in which a combination of 3BNC117 and 10-1074, two potent monoclonal anti-HIV-1 broadly neutralizing antibodies that target independent sites on the HIV-1 envelope spike, was administered during analytical treatment interruption. Participants received three infusions of 30 mg kg-1 of each antibody at 0, 3 and 6 weeks. Infusions of the two antibodies were generally well-tolerated. The nine enrolled individuals with antibody-sensitive latent viral reservoirs maintained suppression for between 15 and more than 30 weeks (median of 21 weeks), and none developed viruses that were resistant to both antibodies. We conclude that the combination of the anti-HIV-1 monoclonal antibodies 3BNC117 and 10-1074 can maintain long-term suppression in the absence of antiretroviral therapy in individuals with antibody-sensitive viral reservoirs.

Early treatment with a combination of two potent neutralizing antibodies improves clinical outcomes and reduces virus replication and lung inflammation in SARS-CoV-2 infected macaques.

There is an urgent need for effective therapeutic interventions against SARS-CoV-2, including new variants that continue to arise. Neutralizing monoclonal antibodies have shown promise in clinical studies. We investigated the therapeutic efficacy of a combination of two potent monoclonal antibodies, C135-LS and C144-LS that carry half-life extension mutations, in the rhesus macaque model of COVID-19. Twelve young adult macaques (three groups of four animals) were inoculated intranasally and intra-tracheally with a high dose of SARS-CoV-2 and 24 hours later, treated intravenously with a high (40 mg/kg) or low (12 mg/kg) dose of the C135-LS and C144-LS antibody combination, or a control monoclonal antibody. Animals were monitored for 7 days. Compared to the control animals, animals treated with either dose of the anti-SARS-CoV-2 antibodies showed similarly improved clinical scores, lower levels of virus replication in upper and lower respiratory tract, and significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. In conclusion, this study provides proof-of-concept in support of further clinical development of these monoclonal antibodies against COVID-19 during early infection.

Sequence Evaluation and Comparative Analysis of Novel Assays for Intact Proviral HIV-1 DNA.

The HIV proviral reservoir is the major barrier to cure. The predominantly replication-defective proviral landscape makes the measurement of virus that is likely to cause rebound upon antiretroviral therapy (ART)-cessation challenging. To address this issue, novel assays to measure intact HIV proviruses have been developed. The intact proviral DNA assay (IPDA) is a high-throughput assay that uses two probes to exclude the majority of defective proviruses and determine the frequency of intact proviruses, albeit without sequence confirmation. Quadruplex PCR with four probes (Q4PCR) is a lower-throughput assay that uses limiting dilution long-distance PCR amplification followed by quantitative PCR (qPCR) and near-full-length genome sequencing (nFGS) to estimate the frequency of sequence-confirmed intact proviruses and provide insight into their clonal composition. To explore the advantages and limitations of these assays, we compared IPDA and Q4PCR measurements from 39 ART-suppressed people living with HIV. We found that IPDA and Q4PCR measurements correlated with one another, but frequencies of intact proviral DNA differed by approximately 19-fold. This difference may be in part due to inefficiencies in long-distance PCR amplification of proviruses in Q4PCR, leading to underestimates of intact proviral frequencies. In addition, nFGS analysis within Q4PCR explained that some of this difference is explained by proviruses that are classified as intact by IPDA but carry defects elsewhere in the genome. Taken together, this head-to-head comparison of novel intact proviral DNA assays provides important context for their interpretation in studies to deplete the HIV reservoir and shows that together the assays bracket true reservoir size.IMPORTANCE The intact proviral DNA assay (IPDA) and quadruplex PCR (Q4PCR) represent major advances in accurately quantifying and characterizing the replication-competent HIV reservoir. This study compares the two novel approaches for measuring intact HIV proviral DNA in samples from 39 antiretroviral therapy (ART)-suppressed people living with HIV, thereby informing ongoing efforts to deplete the HIV reservoir in cure-related trials.

Neutralizing Activity of Broadly Neutralizing anti-HIV-1 Antibodies against Primary African Isolates.

Novel therapeutic and preventive strategies are needed to contain the HIV-1 epidemic. Broadly neutralizing human antibodies (bNAbs) with exceptional activity against HIV-1 are currently being tested in HIV-1 prevention trials. The selection of anti-HIV-1 bNAbs for clinical development was primarily guided by their in vitro neutralizing activity against HIV-1 Env pseudotyped viruses. Here we report on the neutralizing activity of 9 anti-HIV-1 bNAbs now in clinical development against 126 Clade A, C, D PBMC-derived primary African isolates. The neutralizing potency and breadth of the bNAbs tested was significantly reduced compared to pseudotyped viruses panels. The difference in sensitivity between pseudotyped viruses and primary isolates varied from 3- to nearly 100-fold depending on the bNAb and the HIV-1 clade. Thus, the neutralizing activity of bNAbs against primary African isolates differs from their activity against pseudovirus panels. The data have significant implications for interpreting the results of ongoing HIV-1 prevention trials.IMPORTANCE HIV remains a major public health problem worldwide, and new therapies and preventive strategies are necessary for controlling the epidemic. Broadly neutralizing antibodies (bNAbs) have been developed in the past decade to fill this gap. The neutralizing activity of these antibodies against diverse HIV strains has mostly been measured using Env-pseudotyped viruses, which overestimate bNAb coverage and potency. In this study we measured the neutralizing activity of nine bNAbs against clade A, C, and D HIV isolates derived from cells of African patients living with HIV and produced in peripheral blood mononuclear cells. We found that the coverage and potency of bNAbs were often significantly lower than what was predicted by Env-psuedotyped viruses, and that this decrease was related to the bNAb biding site class. This data is important for the planning and analysis of clinical trials that seek to evaluate bNAbs for the treatment and prevention of HIV infection in Africa.

HIV-1 antibody 3BNC117 suppresses viral rebound in humans during treatment interruption.

Interruption of combination antiretroviral therapy in HIV-1-infected individuals leads to rapid viral rebound. Here we report the results of a phase IIa open label clinical trial evaluating 3BNC117,a broad and potent neutralizing antibody against the CD4 binding site of the HIV-1 Env protein, during analytical treatment interruption in 13 HIV-1-infected individuals. Participants with 3BNC117-sensitive virus outgrowth cultures were enrolled. Results show that two or four 30 mg kg(-1) 3BNC117 infusions,separated by 3 or 2 weeks, respectively, are generally well tolerated.Infusions are associated with a delay in viral rebound of 5-9 weeks after two infusions, and up to 19 weeks after four infusions, or an average of 6.7 and 9.9 weeks, respectively, compared with 2.6 weeks for historical controls (P < 0.00001). Rebound viruses arise predominantly from a single provirus. In most individuals,emerging viruses show increased resistance, indicating escape.However, 30% of participants remained suppressed until antibody concentrations waned below 20 µg ml(-1), and the viruses emerging in all but one of these individuals showed no apparent resistance to 3BCN117, suggesting failure to escape over a period of 9-19 weeks.We conclude that the administration of 3BNC117 exerts strong selective pressure on HIV-1 emerging from latent reservoirs during analytical treatment interruption in humans.

Longitudinal Serological Analysis and Neutralizing Antibody Levels in Coronavirus Disease 2019 Convalescent Patients.

BACKGROUND: Understanding the longitudinal trajectory of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies is crucial for diagnosis of prior infection and predicting future immunity. METHODS: We conducted a longitudinal analysis of coronavirus disease 2019 convalescent patients, with neutralizing antibody assays and SARS-CoV-2 serological assay platforms using SARS-CoV-2 spike (S) or nucleocapsid (N) antigens. RESULTS: Sensitivities of serological assays in diagnosing prior SARS-CoV-2 infection changed with time. One widely used commercial platform that had an initial sensitivity of >95% declined to 71% at 81-100 days after diagnosis. The trajectories of median binding antibody titers measured over approximately 3-4 months were not dependent on the use of SARS-CoV-2 N or S proteins as antigen. The median neutralization titer decreased by approximately 45% per month. Each serological assay gave quantitative antibody titers that were correlated with SARS-CoV-2 neutralization titers, but S-based serological assay measurements better predicted neutralization potency. Correlation between S-binding and neutralization titers deteriorated with time, and decreases in neutralization titers were not predicted by changes in S-binding antibody titers. CONCLUSIONS: Different SARS-CoV-2 serological assays are more or less well suited for surveillance versus prediction of serum neutralization potency. Extended follow-up should facilitate the establishment of appropriate serological correlates of protection against SARS-CoV-2 reinfection.

Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117.

HIV-1 immunotherapy with a combination of first generation monoclonal antibodies was largely ineffective in pre-clinical and clinical settings and was therefore abandoned. However, recently developed single-cell-based antibody cloning methods have uncovered a new generation of far more potent broadly neutralizing antibodies to HIV-1 (refs 4, 5). These antibodies can prevent infection and suppress viraemia in humanized mice and nonhuman primates, but their potential for human HIV-1 immunotherapy has not been evaluated. Here we report the results of a first-in-man dose escalation phase 1 clinical trial of 3BNC117, a potent human CD4 binding site antibody, in uninfected and HIV-1-infected individuals. 3BNC117 infusion was well tolerated and demonstrated favourable pharmacokinetics. A single 30 mg kg(-1) infusion of 3BNC117 reduced the viral load in HIV-1-infected individuals by 0.8-2.5 log10 and viraemia remained significantly reduced for 28 days. Emergence of resistant viral strains was variable, with some individuals remaining sensitive to 3BNC117 for a period of 28 days. We conclude that, as a single agent, 3BNC117 is safe and effective in reducing HIV-1 viraemia, and that immunotherapy should be explored as a new modality for HIV-1 prevention, therapy and cure.

Relationship between intact HIV-1 proviruses in circulating CD4+ T cells and rebound viruses emerging during treatment interruption.

Combination antiretroviral therapy controls but does not cure HIV-1 infection because a small fraction of cells harbor latent viruses that can produce rebound viremia when therapy is interrupted. The circulating latent virus reservoir has been documented by a variety of methods, most prominently by viral outgrowth assays (VOAs) in which CD4+ T cells are activated to produce virus in vitro, or more recently by amplifying proviral near full-length (NFL) sequences from DNA. Analysis of samples obtained in clinical studies in which individuals underwent analytical treatment interruption (ATI), showed little if any overlap between circulating latent viruses obtained from outgrowth cultures and rebound viruses from plasma. To determine whether intact proviruses amplified from DNA are more closely related to rebound viruses than those obtained from VOAs, we assayed 12 individuals who underwent ATI after infusion of a combination of two monoclonal anti-HIV-1 antibodies. A total of 435 intact proviruses obtained by NFL sequencing were compared with 650 latent viruses from VOAs and 246 plasma rebound viruses. Although, intact NFL and outgrowth culture sequences showed similar levels of stability and diversity with 39% overlap, the size of the reservoir estimated from NFL sequencing was larger than and did not correlate with VOAs. Finally, intact proviruses documented by NFL sequencing showed no sequence overlap with rebound viruses; however, they appear to contribute to recombinant viruses found in plasma during rebound.

Characterization of Intact Proviruses in Blood and Lymph Node from HIV-Infected Individuals Undergoing Analytical Treatment Interruption.

The role of lymphoid tissue as a potential source of HIV-1 rebound following interruption of antiretroviral therapy (ART) is uncertain. To address this issue, we compared the latent viruses obtained from CD4+ T cells in peripheral blood and lymph nodes to viruses emerging during treatment interruption. Latent viruses were characterized by sequencing near-full-length (NFL) proviral DNA and env from viral outgrowth assays (VOAs). Five HIV-1-infected individuals on ART were studied, four of whom participated in a clinical trial of a TLR9 agonist that included an analytical treatment interruption. We found that 98% of intact or replication-competent clonal sequences overlapped between blood and lymph node. In contrast, there was no overlap between 205 latent reservoir and 125 rebound sequences in the four individuals who underwent treatment interruption. However, rebound viruses could be accounted for by recombination. The data suggest that CD4+ T cells carrying latent viruses circulate between blood and lymphoid tissues in individuals on ART and support the idea that recombination may play a role in the emergence of rebound viremia.IMPORTANCE HIV-1 persists as a latent infection in CD4+ T cells that can be found in lymphoid tissues in infected individuals during ART. However, the importance of this tissue reservoir and its contribution to viral rebound upon ART interruption are not clear. In this study, we sought to compare latent HIV-1 from blood and lymph node CD4+ T cells from five HIV-1-infected individuals. Further, we analyzed the contribution of lymph node viruses to viral rebound. We observed that the frequencies of intact proviruses were the same in blood and lymph node. Moreover, expanded clones of T cells bearing identical proviruses were found in blood and lymph node. These latent reservoir sequences did not appear to be the direct origin of rebound virus. Instead, latent proviruses were found to contribute to the rebound compartment by recombination.

Neutralizing Activity of Broadly Neutralizing Anti-HIV-1 Antibodies against Clade B Clinical Isolates Produced in Peripheral Blood Mononuclear Cells.

Recently discovered broadly neutralizing antibodies (bNAbs) against HIV-1 demonstrate extensive breadth and potency against diverse HIV-1 strains and represent a promising approach for the treatment and prevention of HIV-1 infection. The breadth and potency of these antibodies have primarily been evaluated by using panels of HIV-1 Env-pseudotyped viruses produced in 293T cells expressing molecularly cloned Env proteins. Here we report on the ability of five bNAbs currently in clinical development to neutralize circulating primary HIV-1 isolates derived from peripheral blood mononuclear cells (PBMCs) and compare the results to those obtained with the pseudovirus panels used to characterize the bNAbs. The five bNAbs demonstrated significantly less breadth and potency against clinical isolates produced in PBMCs than against Env-pseudotyped viruses. The magnitude of this difference in neutralizing activity varied, depending on the antibody epitope. Glycan-targeting antibodies showed differences of only 3- to 4-fold, while antibody 10E8, which targets the membrane-proximal external region, showed a nearly 100-fold decrease in activity between published Env-pseudotyped virus panels and PBMC-derived primary isolates. Utilizing clonal PBMC-derived primary isolates and molecular clones, we determined that the observed discrepancy in bNAb performance is due to the increased sensitivity to neutralization exhibited by 293T-produced Env-pseudotyped viruses. We also found that while full-length molecularly cloned viruses produced in 293T cells exhibit greater sensitivity to neutralization than PBMC-derived viruses do, Env-pseudotyped viruses produced in 293T cells generally exhibit even greater sensitivity to neutralization. As the clinical development of bNAbs progresses, it will be critical to determine the relevance of each of these in vitro neutralization assays to in vivo antibody performance.IMPORTANCE Novel therapeutic and preventive strategies are needed to contain the HIV-1 epidemic. Antibodies with exceptional neutralizing activity against HIV-1 may provide several advantages to traditional HIV drugs, including an improved side-effect profile, a reduced dosing frequency, and immune enhancement. The activity of these antibodies has been established in vitro by utilizing HIV-1 Env-pseudotyped viruses derived from circulating viruses but produced in 293T cells by pairing Env proteins with a backbone vector. We tested PBMC-produced circulating viruses against five anti-HIV-1 antibodies currently in clinical development. We found that the activity of these antibodies against PBMC isolates is significantly less than that against 293T Env-pseudotyped viruses. This decline varied among the antibodies tested, with some demonstrating moderate reductions in activity and others showing an almost 100-fold reduction. As the development of these antibodies progresses, it will be critical to determine how the results of different in vitro tests correspond to performance in the clinic.

Paired quantitative and qualitative assessment of the replication-competent HIV-1 reservoir and comparison with integrated proviral DNA.

HIV-1-infected individuals harbor a latent reservoir of infected CD4+ T cells that is not eradicated by antiretroviral therapy (ART). This reservoir presents the greatest barrier to an HIV-1 cure and has remained difficult to characterize, in part, because the vast majority of integrated sequences are defective and incapable of reactivation. To characterize the replication-competent reservoir, we have combined two techniques, quantitative viral outgrowth and qualitative sequence analysis of clonal outgrowth viruses. Leukapheresis samples from four fully ART-suppressed, chronically infected individuals were assayed at two time points separated by a 4- to 6-mo interval. Overall, 54% of the viruses emerging from the latent reservoir showed gp160 env sequences that were identical to at least one other virus. Moreover, 43% of the env sequences from viruses emerging from the reservoir were part of identical groups at the two time points. Groups of identical expanded sequences made up 54% of proviral DNA, and, as might be expected, the sequences of replication-competent viruses in the active reservoir showed limited overlap with integrated proviral DNA, most of which is known to represent defective viruses. Finally, there was an inverse correlation between proviral DNA clone size and the probability of reactivation, suggesting that replication-competent viruses are less likely to be found among highly expanded provirus-containing cell clones.

Mitochondrial genome instability in colorectal adenoma and adenocarcinoma.

Mitochondrial dysfunction is regarded as a hallmark of cancer progression. In the current study, we evaluated mitochondrial genome instability and copy number in colorectal cancer using Next Generation Sequencing approach and qPCR, respectively. The results revealed higher levels of heteroplasmy and depletion of the relative mtDNA copy number in colorectal adenocarcinoma. Adenocarcinoma samples also presented an increased number of mutations in nuclear genes encoding proteins which functions are related with mitochondria fusion, fission and localization. Moreover, we found a set of mitochondrial and nuclear genes, which cooperate in the same mitochondrial function simultaneously mutated in adenocarcinoma. In summary, these results support an important role for mitochondrial function and genomic instability in colorectal tumorigenesis.

Effect of 3BNC117 and romidepsin on the HIV-1 reservoir in people taking suppressive antiretroviral therapy (ROADMAP): a randomised, open-label, phase 2A trial.

BACKGROUND: The administration of broadly neutralising anti-HIV-1 antibodies before latency reversal could facilitate elimination of HIV-1-infected CD4 T cells. We tested this concept by combining the broadly neutralising antibody 3BNC117 in combination with the latency-reversing agent romidepsin in people with HIV-1 who were taking suppressive antiretroviral therapy (ART). METHODS: We did a randomised, open-label, phase 2A trial at three university hospital centres in Denmark, Germany, and the USA. Eligible participants were virologically suppressed adults aged 18-65 years who were infected with HIV-1 and on ART for at least 18 months, with plasma HIV-1 RNA concentrations of less than 50 copies per mL for at least 12 months, and a CD4 T-cell count of greater than 500 cells per µL. Participants were randomly assigned (1:1) to receive 3BNC117 plus romidepsin or romidepsin alone in two cycles. All participants received intravenous infusions of romidepsin (5 mg/m2 given over 120 min) at weeks 0, 1, and 2 (treatment cycle 1) and weeks 8, 9, and 10 (treatment cycle 2). Those in the 3BNC117 plus romidepsin group received an intravenous infusion of 3BNC117 (30 mg/kg given over 60 min) 2 days before each treatment cycle. An analytic treatment interruption (ATI) of ART was done at week 24 in both groups. Our primary endpoint was time to viral rebound during analytic treatment interruption, which was assessed in all participants who completed both treatment cycles and ATI. We used a log-rank test to compare time to viral rebound during analytic treatment interruption between the two groups. This trial is registered with ClinicalTrials.gov, NCT02850016. It is closed to new participants, and all follow-up is complete. FINDINGS: Between March 20, 2017, and Aug 14, 2018, 22 people were enrolled and randomly assigned, 11 to the 3BNC117 plus romidepsin group and 11 to the romidepsin group. 19 participants completed both treatment cycles and the ATI: 11 in the 3BNC117 plus romidepsin group and 8 in the romidepsin group. The median time to viral rebound during ATI was 18 days (IQR 14-28) in the 3BNC117 plus romidepsin group and 28 days (21-35) in the romidepsin group B (p=0·0016). Although this difference was significant, prolongation of time to viral rebound was not clinically meaningful in either group. All participants in both groups reported adverse events, but overall the combination of 3BNC117 and romidepsin was safe. Two severe adverse events were observed in the romidepsin group during 48 weeks of follow-up, one of which-increased direct bilirubin-was judged to be related to treatment. INTERPRETATION: The combination of 3BNC117 and romidepsin was safe but did not delay viral rebound during analytic treatment interruptions in individuals on long-term ART. The results of our trial could serve as a benchmark for further optimisation of HIV-1 curative strategies among people with HIV-1 who are taking suppressive ART. FUNDING: amfAR, German Center for Infection Research.