Modest de novo Reactivation of Single HIV-1 Proviruses in Peripheral CD4+ T Cells by Romidepsin

A cure for human immunodeficiency virus (HIV-1) is restricted by the continued presence of a latent reservoir of memory CD4+ T cells with proviruses integrated into their DNA despite suppressive antiretroviral therapy (ART). A predominant strategy currently pursued in HIV-1 cure-related research is the “kick and kill” approach, where latency reversal agents (LRAs) are used to reactivate transcription from integrated proviruses. The premise of this approach is that “kicking” latent virus out of hiding allows the host immune system to recognize and kill infected cells. Clinical trials investigating the efficacy of LRAs, such as romidepsin, have shown that these interventions do induce transient spikes in viral RNA in HIV-1-infected individuals. However, since these trials failed to significantly reduce viral reservoir size or significantly delay time to viral rebound during analytical treatment interruptions, it is questioned how much each individual latent provirus is actually “kicked” to produce viral transcripts and/or proteins by the LRA. Here, we developed sensitive and specific digital droplet PCR-based assays with single-provirus level resolution. Combining these assays allowed us to interrogate the level of viral RNA transcripts from single proviruses in individuals on suppressive ART with or without concomitant romidepsin treatment. Small numbers of proviruses in peripheral blood memory CD4+ T cells were triggered to become marginally transcriptionally active upon romidepsin treatment. These novel assays can be applied retrospectively and prospectively in HIV-1 cure-related clinical trials to gain crucial insights into LRA efficacy at the single provirus level

Administration of broadly neutralizing anti-HIV-1 antibodies at ART initiation maintains long-term CD8+ T cell immunity

In simian-human immunodeficiency virus (SHIV)-infected non-human primates, broadly neutralizing antibodies (bNAbs) against the virus appear to stimulate T cell immunity. To determine whether this phenomenon also occurs in humans we measured HIV-1-specific cellular immunity longitudinally in individuals with HIV-1 starting antiviral therapy (ART) with or without adjunctive bNAb 3BNC117 treatment. Using the activation-induced marker (AIM) assay and interferon-γ release, we observe that frequencies of Pol- and Gag-specific CD8+ T cells, as well as Gag-induced interferon-γ responses, are significantly higher among individuals that received adjunctive 3BNC117 compared to ART-alone at 3 and 12 months after starting ART. The observed changes in cellular immunity were directly correlated to pre-treatment 3BNC117-sensitivity. Notably, increased HIV-1-specific immunity is associated with partial or complete ART-free virologic control during treatment interruption for up to 4 years. Our findings suggest that bNAb treatment at the time of ART initiation maintains HIV-1-specific CD8+ T cell responses that are associated with ART-free virologic control

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 mu L. 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/m(2) 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. Copyright (C) 2022 The Author(s). Published by Elsevier Ltd

Impact of a TLR9 agonist and broadly neutralizing antibodies on HIV-1 persistence: the randomized phase 2a TITAN trial

Inducing antiretroviral therapy (ART)-free virological control is a critical step toward a human immunodeficiency virus type 1 (HIV-1) cure. In this phase 2a, placebo-controlled, double-blinded trial, 43 people (85% males) with HIV-1 on ART were randomized to (1) placebo/placebo, (2) lefitolimod (TLR9 agonist)/placebo, (3) placebo/broadly neutralizing anti-HIV-1 antibodies (bNAbs) or (4) lefitolimod/bNAb. ART interruption (ATI) started at week 3. Lefitolimod was administered once weekly for the first 8 weeks, and bNAbs were administered twice, 1 d before and 3 weeks after ATI. The primary endpoint was time to loss of virologic control after ATI. The median delay in time to loss of virologic control compared to the placebo/placebo group was 0.5 weeks (P = 0.49), 12.5 weeks (P = 0.003) and 9.5 weeks (P = 0.004) in the lefitolimod/placebo, placebo/bNAb and lefitolimod/bNAb groups, respectively. Among secondary endpoints, viral doubling time was slower for bNAb groups compared to non-bNAb groups, and the interventions were overall safe. We observed no added benefit of lefitolimod. Despite subtherapeutic plasma bNAb levels, 36% (4/11) in the placebo/bNAb group compared to 0% (0/10) in the placebo/placebo group maintained virologic control after the 25-week ATI. Although immunotherapy with lefitolimod did not lead to ART-free HIV-1 control, bNAbs may be important components in future HIV-1 curative strategies. ClinicalTrials.gov identifier: NCT03837756

Impact of a TLR9 agonist and broadly neutralizing antibodies on HIV-1 persistence:the randomized phase 2a TITAN trial

Inducing antiretroviral therapy (ART)-free virological control is a critical step toward a human immunodeficiency virus type 1 (HIV-1) cure. In this phase 2a, placebo-controlled, double-blinded trial, 43 people (85% males) with HIV-1 on ART were randomized to (1) placebo/placebo, (2) lefitolimod (TLR9 agonist)/placebo, (3) placebo/broadly neutralizing anti-HIV-1 antibodies (bNAbs) or (4) lefitolimod/bNAb. ART interruption (ATI) started at week 3. Lefitolimod was administered once weekly for the first 8 weeks, and bNAbs were administered twice, 1 d before and 3 weeks after ATI. The primary endpoint was time to loss of virologic control after ATI. The median delay in time to loss of virologic control compared to the placebo/placebo group was 0.5 weeks (P = 0.49), 12.5 weeks (P = 0.003) and 9.5 weeks (P = 0.004) in the lefitolimod/placebo, placebo/bNAb and lefitolimod/bNAb groups, respectively. Among secondary endpoints, viral doubling time was slower for bNAb groups compared to non-bNAb groups, and the interventions were overall safe. We observed no added benefit of lefitolimod. Despite subtherapeutic plasma bNAb levels, 36% (4/11) in the placebo/bNAb group compared to 0% (0/10) in the placebo/placebo group maintained virologic control after the 25-week ATI. Although immunotherapy with lefitolimod did not lead to ART-free HIV-1 control, bNAbs may be important components in future HIV-1 curative strategies. ClinicalTrials.gov identifier: NCT03837756 .</p

Redirector of Vaccine-induced Effector Responses (RoVER) for specific killing of cellular targetsResearch in context

Summary: Background: In individuals with malignancy or HIV-1 infection, antigen-specific cytotoxic T lymphocytes (CTLs) often display an exhausted phenotype with impaired capacity to eliminate the disease. Existing cell-based immunotherapy strategies are often limited by the requirement for adoptive transfer of CTLs. We have developed an immunotherapy technology in which potent CTL responses are generated in vivo by vaccination and redirected to eliminate target cells using a bispecific Redirector of Vaccine-induced Effector Responses (RoVER). Methods: Following Yellow fever (YF) 17D vaccination of 51 healthy volunteers (NCT04083430), single-epitope YF-specific CTL responses were quantified by tetramer staining and multi-parameter flow cytometry. RoVER-mediated redirection of YF-specific CTLs to kill antigen-expressing Raji-Env cells, autologous CD19+ B cells or CD4+ T cells infected in vitro with a full-length HIV-1-eGFP was assessed in cell killing assays. Moreover, secreted IFN-γ, granzyme B, and TNF-α were analyzed by mesoscale multiplex assays. Findings: YF-17D vaccination induced strong epitope-specific CTL responses in the study participants. In cell killing assays, RoVER-mediated redirection of YF-specific CTLs to autologous CD19+ B cells or HIV-1-infected CD4+ cells resulted in 58% and 53% killing at effector to target ratio 1:1, respectively. Interpretation: We have developed an immunotherapy technology in which epitope-specific CTLs induced by vaccination can be redirected to kill antigen-expressing target cells by RoVER linking. The RoVER technology is highly specific and can be adapted to recognize various cell surface antigens. Importantly, this technology obviates the need for adoptive transfer of CTLs. Funding: This work was funded by the Novo Nordisk Foundation (Hallas Møller NNF10OC0054577)

SARS-CoV-2 elicits robust adaptive immune responses regardless of disease severity

Background: The SARS-CoV-2 pandemic currently prevails worldwide. To understand the immunological signature of SARS-CoV-2 infections and aid the search and evaluation of new treatment modalities and vaccines, comprehensive characterization of adaptive immune responses towards SARS-CoV-2 is needed. Methods: We included 203 recovered SARS-CoV-2 infected patients in Denmark between April 3rd and July 9th 2020, at least 14 days after COVID-19 symptom recovery. The participants had experienced a range of disease severities from asymptomatic to severe. We collected plasma, serum and PBMC's for analysis of SARS-CoV-2 specific antibody response by Meso Scale analysis including other coronavirus strains, ACE2 competition, IgA ELISA, pseudovirus neutralization capacity, and dextramer flow cytometry analysis of CD8+ T cells. The immunological outcomes were compared amongst severity groups within the cohort, and 10 pre-pandemic SARS-CoV-2 negative controls. Findings: We report broad serological profiles within the cohort, detecting antibody binding to other human coronaviruses. 202(>99%) participants had SARS-CoV-2 specific antibodies, with SARS-CoV-2 neutralization and spike-ACE2 receptor interaction blocking observed in 193(95%) individuals. A significant positive correlation (r=0.7804) between spike-ACE2 blocking antibody titers and neutralization potency was observed. Further, SARS-CoV-2 specific CD8+ T-cell responses were clear and quantifiable in 95 of 106(90%) HLA-A2+ individuals. Interpretation: The viral surface spike protein was identified as the dominant target for both neutralizing antibodies and CD8+ T-cell responses. Overall, the majority of patients had robust adaptive immune responses, regardless of their disease severity. Funding: This study was supported by the Danish Ministry for Research and Education (grant# 0238-00001B) and The Danish Innovation Fund (grant# 0208-00018B

Single cell analysis reveals a subset of cytotoxic-like plasmacytoid dendritic cells in people with HIV-1

Summary: Human plasmacytoid dendritic cells (pDCs) play a central role in initiating and activating host immune responses during infection. To understand how the transcriptome of pDCs is impacted by HIV-1 infection and exogenous stimulation, we isolated pDCs from healthy controls, people with HIV-1 (PWH) before and during toll-like receptor 9 (TLR9) agonist treatment and performed single-cell (sc)-RNA sequencing. Our cluster analysis revealed four pDC clusters: pDC1, pDC2, cytotoxic-like pDC and an exhausted pDC cluster. The inducible cytotoxic-like pDC cluster is characterized by high expression of both antiviral and cytotoxic genes. Further analyses confirmed that cytotoxic-like pDCs are distinct from NK and T cells. Cell-cell communication analysis also demonstrated that cytotoxic-like pDCs exhibit similar incoming and outgoing cellular communicating signals as other pDCs. Thus, our study presents a detailed transcriptomic atlas of pDCs and provides new perspectives on the mechanisms of regulation and function of cytotoxic-like pDCs

Familial multiple discoid fibromas is linked to a locus on chromosome 5 including the FNIP1 gene

Previously, we reported a series of families presenting with trichodiscomas, inherited in an autosomal dominant pattern. The phenotype was named familial multiple discoid fibromas (FMDF). The genetic cause of FMDF remained unknown so far. Trichodiscomas are skin lesions previously reported to be part of the same spectrum as the fibrofolliculoma observed in Birt-Hogg-Dubé syndrome (BHD), an inherited disease caused by pathogenic variants in the FLCN gene. Given the clinical and histological differences with BHD and the exclusion of linkage with the FLCN locus, the phenotype was concluded to be distinct from BHD. We performed extensive clinical evaluations and genetic testing in ten families with FMDF. We identified a FNIP1 frameshift variant in nine families and genealogical studies showed common ancestry for eight families. Using whole exome sequencing, we identified six additional rare variants in the haplotype surrounding FNIP1, including a missense variant in the PDGFRB gene that was found to be present in all tested patients with FMDF. Genome-wide linkage analysis showed that the locus on chromosome 5 including FNIP1 was the only region reaching the maximal possible LOD score. We concluded that FMDF is linked to a haplotype on chromosome 5. Additional evaluations in families with FMDF are required to unravel the exact genetic cause underlying the phenotype. When evaluating patients with multiple trichodisomas without a pathogenic variant in the FLCN gene, further genetic testing is warranted and can include analysis of the haplotype on chromosome 5

Early intervention with 3BNC117 and romidepsin at antiretroviral treatment initiation in people with HIV-1: a phase 1b/2a, randomized trial

Attempts to reduce the human immunodeficiency virus type 1 (HIV-1) reservoir and induce antiretroviral therapy (ART)-free virologic control have largely been unsuccessful. In this phase 1b/2a, open-label, randomized controlled trial using a four-group factorial design, we investigated whether early intervention in newly diagnosed people with HIV-1 with a monoclonal anti-HIV-1 antibody with a CD4-binding site, 3BNC117, followed by a histone deacetylase inhibitor, romidepsin, shortly after ART initiation altered the course of HIV-1 infection (NCT03041012). The trial was undertaken in five hospitals in Denmark and two hospitals in the United Kingdom. The coprimary endpoints were analysis of initial virus decay kinetics and changes in the frequency of CD4+ T cells containing intact HIV-1 provirus from baseline to day 365. Secondary endpoints included changes in the frequency of infected CD4+ T cells and virus-specific CD8+ T cell immunity from baseline to day 365, pre-ART plasma HIV-1 3BNC117 sensitivity, safety and tolerability, and time to loss of virologic control during a 12-week analytical ART interruption that started at day 400. In 55 newly diagnosed people (5 females and 50 males) with HIV-1 who received random allocation treatment, we found that early 3BNC117 treatment with or without romidepsin enhanced plasma HIV-1 RNA decay rates compared to ART only. Furthermore, 3BNC117 treatment accelerated clearance of infected cells compared to ART only. All groups had significant reductions in the frequency of CD4+ T cells containing intact HIV-1 provirus. At day 365, early 3BNC117 + romidepsin was associated with enhanced HIV-1 Gag-specific CD8+ T cell immunity compared to ART only. The observed virological and immunological effects of 3BNC117 were most pronounced in individuals whose pre-ART plasma HIV-1 envelope sequences were antibody sensitive. The results were not disaggregated by sex. Adverse events were mild to moderate and similar between the groups. During a 12-week analytical ART interruption among 20 participants, 3BNC117-treated individuals harboring sensitive viruses were significantly more likely to maintain ART-free virologic control than other participants. We conclude that 3BNC117 at ART initiation enhanced elimination of plasma viruses and infected cells, enhanced HIV-1-specific CD8+ immunity and was associated with sustained ART-free virologic control among persons with 3BNC117-sensitive virus. These findings strongly support interventions administered at the time of ART initiation as a strategy to limit long-term HIV-1 persistence