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Objective: Cardiovascular complications and congenital malformations are known traits in Turner syndrome (TS), which increases mortality. Women with TS have varying phenotype and cardiovascular risks. A biomarker assessing the risk for cardiovascular complications could potentially reduce mortality in high-risk TS and reduce screening in TS participants with low cardiovascular risk.Design, patients, participants and measurements: As part of a study initiated in 2002, 87 TS participants and 64 controls were invited to magnetic resonance imaging of the aorta, anthropometry, and biochemical markers. TS participants were re-examined thrice lastly in 2016. The focus of this paper is the additional measurements of transforming growth factor beta (TGFβ), matrix metalloproteinase (MMP's), tissue inhibitor of matrix metalloproteinase (TIMP), peripheral blood DNA and their associations with TS and the cardiovascular risk and congenital heart disease.Results: TS participants had lower TGFβ1 and TGFβ2 values compared to controls. snp11547635 heterozygosity was not associated with any biomarkers but was associated with increased risk of aortic regurgitation. TIMP4 and TGFβ1 were correlated with the aortic diameter at several measuring positions. During follow-up, the antihypertensive treatment decreased the descending aortic diameter and increased TGFβ1 and TGFβ2 levels in TS.Conclusion: TGFβ and TIMP's are altered in TS and may play a role in the development of coarctation and dilated aorta. snp11547635 heterozygosity was not found to impact biochemical markers. Further studies should investigate these biomarkers to further unravel the pathogenesis of the increased cardiovascular risk in TS participants. Background: The combined effectiveness of remdesivir and dexamethasone in subgroups of hospitalised patients with COVID-19 is poorly investigated. Methods: In this nationwide retrospective cohort study, we included 3826 patients with COVID-19 hospitalised between February 2020 and April 2021. The primary outcomes were use of invasive mechanical ventilation and 30-day mortality, comparing a cohort treated with remdesivir and dexamethasone with a previous cohort treated without remdesivir and dexamethasone. We used inverse probability of treatment weighting logistic regression to assess associations with progression to invasive mechanical ventilation and 30-day mortality between the two cohorts. The analyses were conducted overall and by subgroups based on patient characteristics. Results: Odds ratio for progression to invasive mechanical ventilation and 30-day mortality in individuals treated with remdesivir and dexamethasone compared to treatment with standard of care alone was 0.46 (95% confidence interval, 0.37–0.57) and 0.47 (95% confidence interval, 0.39–0.56), respectively. The reduced risk of mortality was observed in elderly patients, overweight patients and in patients requiring supplemental oxygen at admission, regardless of sex, comorbidities and symptom duration. Conclusions: Patients treated with remdesivir and dexamethasone had significantly improved outcomes compared to patients treated with standard of care alone. These effects were observed in most patient subgroups.

The complement system which is part of the innate immune response against invading pathogens, represents a powerful mechanism for killing of infected cells. Utilizing direct complement recruitment for complement-mediated elimination of HIV-1-infected cells is underexplored. We developed a novel therapeutic modality to direct complement activity to the surface of HIV-1-infected cells. This bispecific complement engager (BiCE) is comprised of a nanobody recruiting the complement-initiating protein C1q, and single-chain variable fragments of broadly neutralizing antibodies (bNAbs) targeting the HIV-1 envelope (Env) protein. Here, we show that two anti-HIV BiCEs targeting the V3 loop and the CD4 binding site, respectively, increase C3 deposition and mediate complement-dependent cytotoxicity (CDC) of HIV-1 Env expressing Raji cells. Furthermore, anti-HIV BiCEs trigger complement activation on primary CD4 T cells infected with laboratory-adapted HIV-1 strain and facilitates elimination of HIV-1-infected cells over time. In summary, we present a novel approach to direct complement deposition to the surface of HIV-1-infected cells leading to complement-mediated killing of these cells.

OBJECTIVES: To identify individual characteristics associated with serological COVID-19 vaccine responsiveness and durability of vaccine-induced antibodies.METHODS: Adults without history of SARS-CoV-2 infection from the Danish population scheduled for SARS-CoV-2 vaccination were enrolled in this parallel group, phase IV study. SARS-CoV-2 Spike IgG and Spike-ACE2-receptor-blocking antibodies were measured at days 0, 21, 90 and 180. Vaccine responsiveness was categorized according to Spike IgG and Spike-ACE2-receptor-blocking levels at day 90 post-1st vaccination. Non-durable vaccine-response was defined as day 90 responders that no longer had significant responses by day 180.RESULTS: Of 6544 participants completing two vaccine doses (median age 64, interquartile range:54-75), 3654 (55.8%) received BTN162b2, 2472 (37.8%) mRNA-1273, and 418 (6.4%) ChAdOx1 followed by a mRNA vaccine. Levels of both types of antibodies increased from baseline to day 90 and then decreased to day 180. The decrease was more pronounced for levels of Spike-ACE2-receptor-blocking antibodies than for Spike IgG. Proportions with vaccine hypo-responsiveness and lack of durable response were 5.0% and 12.1% for Spike IgG; 12.7% and 39.6% for Spike-ACE2-receptor-blocking antibody levels, respectively. Male sex, vaccine type and number of co-morbidities were associated with all four outcomes. Additionally, age >=75y was associated with hypo-responsiveness for Spike-ACE2-receptor-blocking antibodies (adjusted odds-ratio:1.59, 95% confidence interval:1.25-2.01) but not for Spike IgG.CONCLUSIONS: Comorbidity, male sex and vaccine type were risk factors for hypo-responsiveness and non-durable response to COVID-19 vaccination. The functional activity of vaccine-induced antibodies declined with increasing age and had waned to pre-2nd vaccination levels for most individuals after 6 months.

Genetic polymorphisms at the IFNL4 loci are known to influence the clinical outcome of several different infectious diseases. Best described is the association between the IFNL4 genotype and hepatitis C virus clearance. However, an influence of the IFNL4 genotype on the adaptive immune system was suggested by several studies but never investigated in humans. In this cross-sectional study, we have genotyped 201 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive participants for 3 IFNL4 polymorphisms (rs368234815, rs12979860, and rs117648444) and stratified them according to the IFNλ4 activity. Based on this stratification, we investigated the association between the IFNL4 genotype and the antibody as well as the CD8+ T cell response in the acute phase of the SARS-CoV-2 infection. We observed no differences in the genotype distribution compared with a Danish reference cohort or the 1,000 Genome Project, and we were not able to link the IFNL4 genotype to changes in either the antibody or CD8+ T cell responses of these patients.

An effective but balanced cellular and inflammatory immune response may limit the severity of coronavirus disease (COVID-19), whereas uncontrolled inflammation leads to disease progression. Older age is associated with higher risk of COVID-19 and a worse outcome, but the underlying immunological mechanisms for this age-related difference are not clear. We investigated the impact of age on viral replication, inflammation, and innate and adaptive cellular immune responses in 205 hospitalized COVID-19 patients. During the early symptomatic phase of COVID-19, we found that patients above 65 years had significantly higher viral load, higher levels of proinflammatory markers, and inadequate mobilization and activation of monocytes, dendritic cells, natural killer cells, and CD8 T cells compared to those below 65 years. Our study points toward age-related deficiencies in the innate immune cellular response to SARS-CoV-2 as a potential cause of poorly controlled viral replication and inflammation during the early symptom phase and subsequent disease progression.

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.

BackgroundThe current treatment for HIV-1 is based on blocking various stages in the viral replication cycle using combination antiretroviral therapy (ART). Even though ART effectively controls the infection, it is not curative, and patients must therefore continue treatment life-long.AimHere we review recent literature investigating the single or combined effect of toll-like receptor (TLR) agonists and broadly neutralizing antibodies (bNAbs) with the objective to evaluate the evidence for this combination as a means towards an HIV-1 cure.ResultsMultiple preclinical studies found significantly enhanced killing of HIV-1 infected cells by TLR agonist-induced innate immune activation or by Fc-mediated effector functions following bNAb administration. However, monotherapy with either agent did not lead to sustained HIV-1 remission in clinical trials among individuals on long-term ART. Notably, findings in non-human primates suggest that a combination of TLR agonists and bNAbs may be able to induce long-term remission after ART cessation and this approach is currently being further investigated in clinical trials.ConclusionPreclinical findings show beneficial effects of either TLR agonist or bNAb administration for enhancing the elimination of HIV-1 infected cells. Further, TLR agonist-mediated stimulation of innate effector functions in combination with bNAbs may enhance antibody-dependent cellular cytotoxicity and non-human primate studies have shown promising results for this combination strategy. Factors such as immune exhaustion, proviral bNAb sensitivity and time of intervention might impact the clinical success.

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)

Background TLR9 agonists are being developed as immunotherapy against malignancies and infections. TLR9 is primarily expressed in B cells and plasmacytoid dendritic cells (pDCs). TLR9 signalling may be critically important for B cell activity in lymph nodes but little is known about the in vivo impact of TLR9 agonism on human lymph node B cells. As a pre-defined sub-study within our clinical trial investigating TLR9 agonist MGN1703 (lefitolimod) treatment in the context of developing HIV cure strategies ( NCT02443935 ), we assessed TLR9 agonist-mediated effects in lymph nodes. Methods Participants received MGN1703 for 24 weeks concurrent with antiretroviral therapy. Seven participants completed the sub-study including lymph node resection at baseline and after 24 weeks of treatment. A variety of tissue-based immunologic and virologic parameters were assessed. Findings MGN1703 dosing increased B cell differentiation; activated pDCs, NK cells, and T cells; and induced a robust interferon response in lymph nodes. Expression of Activation-Induced cytidine Deaminase, an essential regulator of B cell diversification and somatic hypermutation, was highly elevated. During MGN1703 treatment IgG production increased and antibody glycosylation patterns were changed. Interpretation Our data present novel evidence that the TLR9 agonist MGN1703 modulates human lymph node B cells in vivo. These findings warrant further considerations in the development of TLR9 agonists as immunotherapy against cancers and infectious diseases. Fund This work was supported by Aarhus University Research Foundation , the Danish Council for Independent Research and the NovoNordisk Foundation . Mologen AG provided study drug free of charge.

This discussion paper addresses the safety of HIV cure studies, particularly those involving stopping antiretroviral therapy, known as an analytic treatment interruption (ATI) in the context of the SARS-CoV-2 pandemic. More than 30 studies listed on ClinicalTrials.gov include an ATI and many others were planned to begin over the next 12 months but most were halted due to the COVID-19 pandemic. We consider the ethics, risks and practical considerations to be taken into account before re-opening HIV cure clinical trials, noting the specific risks of ATI in the context of circulating SARS-CoV-2.

The coronavirus responsible for COVID-19, SARS-CoV-2, utilizes a viral membrane spike protein for host cell entry. For the virus to engage in host membrane fusion, SARS-CoV-2 utilizes the human transmembrane surface protease, TMPRSS2, to cleave and activate the spike protein. Camostat mesylate, an orally available well-known serine protease inhibitor, is a potent inhibitor of TMPRSS2 and has been hypothesized as a potential antiviral drug against COVID-19. In vitro human cell and animal studies have shown that camostat mesylate inhibits virus-cell membrane fusion and hence viral replication. In mice, camostat mesylate treatment during acute infection with influenza, also dependent on TMPRSS2, leads to a reduced viral load. The decreased viral load may be associated with an improved patient outcome. Because camostat mesylate is administered as an oral drug, it may be used in outpatients as well as inpatients at all disease stages of SARS-CoV-2 infection if it is shown to be an effective antiviral agent. Clinical trials are currently ongoing to test whether this well-known drug could be repurposed and utilized to combat the current pandemic. In the following, we will review current knowledge on camostat mesylate mode of action, potential benefits as an antiviral agent and ongoing clinical trials.

Background Although persistent symptoms after coronavirus disease 2019 (COVID-19) are emerging as a major complication to the infection, data on the diversity and duration of symptoms are needed. Methods Patients aged ≥18 years with a positive polymerase chain reaction (PCR) test for severe acute respiratory syndrome coronavirus 2 who were hospitalized at the Department of Infectious Diseases, Aarhus University Hospital, Denmark, in the period from March 11 to May 15 were offered follow-up after hospitalization. On admission, a comprehensive symptom and medical history was collected, including demographic characteristics, duration of symptoms, comorbidities, and concomitant medications. At discharge, patients were offered follow-up consultations—either by telephone or at an in-person visit—at 6 and 12 weeks at our post-COVID-19 outpatient clinic to assess whether symptoms present at admission had resolved. Results During the inclusion period, 71 patients were admitted with COVID-19. Of these, 10 patients died, 3 were transferred to another region, 4 declined to participate, and 5 were lost to follow-up before the 12-week evaluation. Thus, 49 patients were included. Overall, 96% reported 1 or more persisting symptoms at 12-week follow-up. The main symptoms were fatigue, dyspnea, cough, chemosensory dysfunction, and headache. Conclusions A wide range of persistent symptoms in patients recovering from COVID-19 were present 12 weeks after hospitalization, calling for larger descriptive studies and interdisciplinary research collaborations.

Summary: Changes in antibody glycosylation are linked to inflammation across several diseases. Alterations in bulk antibody galactosylation can predict rheumatic flares, act as a sensor for immune activation, predict gastric cancer relapse, track with biological age, shift with vaccination, change with HIV reservoir size on therapy, and decrease in HIV and HCV infections. However, whether changes in antibody Fc biology also track with reservoir rebound time remains unclear. The identification of a biomarker that could forecast viral rebound time could significantly accelerate the downselection and iterative improvement of promising HIV viral eradication strategies. Using a comprehensive antibody Fc-profiling approach, the level of HIV-specific antibody Fc N-galactosylation is significantly associated with time to rebound after treatment discontinuation across three independent cohorts. Thus virus-specific antibody glycosylation may represent a promising, simply measured marker to track reservoir reactivation.

The SARS-CoV-2 pandemic currently prevails worldwide. To understand the immunological signature of SARS-CoV-2 infections and aid the search for treatments and vaccines, comprehensive characterization of adaptive immune responses towards SARS-CoV-2 is needed. We investigated the breadth and potency of antibody-, and T-cell immune responses, in 203 recovered SARS-CoV-2 infected patients who presented with asymptomatic to severe infections. We report very broad serological profiles with cross-reactivity to other human coronaviruses. Further, >99% had SARS-CoV-2 epitope specific antibodies, with SARS-CoV-2 neutralization and spike-ACE2 receptor interaction blocking observed in 95% of individuals. A significant positive correlation between spike-ACE2 blocking antibody titers and neutralization potency was observed. SARS-CoV-2 specific CD8+T-cell responses were clear and quantifiable in 90% of HLA-A2+individuals. 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 disease severity.Author summarySARS-CoV-2 can cause severe and deadly infections. However, the immunological understanding of this viral infection is limited. Currently, several vaccines are being developed to help limit transmission and prevent the current pandemic. However, basic understanding of the adaptive immune response developed during SARS-CoV-2 infections is needed to inform further vaccine development and to understand the protective properties of the developed immune response. We investigated, the adaptive immune response developed during SARS-CoV-2 infections in recovered patients experiencing a full spectrum of disease severity, from asymptomatic infections to severe cases requiring hospitalization. We used a novel multiplex serological platform, cell-based neutralization assays and dextramer flow cytometry assays to characterize a broad and robust humoral and cellular immune response towards SARS-CoV-2. We found that the vast majority of recovered individuals have clear detectable and functional SARS-CoV-2 spike specific adaptive immune responses, despite diverse disease severities. The detection of both a humoral and cellular functional spike specific immune response in the vast majority of the individuals, irrespective of asymptomatic manifestations, supports vaccine designs currently underway, and encourages further exploration of whether primary infections provide protection to reinfection.

PURPOSE OF REVIEW: Antiretroviral therapy (ART) is extremely effective in controlling HIV-1 infection; however, ART is not curative. Here, we review broadly neutralizing anti-HIV-1 antibodies (bNAbs) combined with latency-reversing agents (LRAs) or immune modulators as strategy for achieving long-term HIV-1 remission. RECENT FINDINGS: Clinical trials testing the effect of a single intervention such as a LRA 'shock and kill', immune modulator or bNAbs among HIV-1 infected individuals on long-term suppressive ART have not lead to long-term HIV-1 remission when ART is stopped. Novel combinations of interventions designed to eliminate infected cells and enhance immune-effector functions are being investigated. Findings in nonhuman primates (NHPs) of such combinations are very promising and clinical trials are now ongoing. These trials will provide the first indication of the efficacy of combinations of bNAbs and LRA or immune modulators for achieving durable HIV-1 remission. SUMMARY: bNAbs facilitate the elimination of HIV-1 infected cells and boost immune responses. Preclinical findings show that these effects can be harnessed by simultaneous administration of LRAs or immune modulators such as Toll-like receptor agonists. The clinical success of such combination strategies may be impacted by factors such as immune exhaustion, bNAbs sensitivity as well as the pharmacodynamics of the investigational compounds.

Humanized mouse models are used extensively in research involving human pathogens and diseases. However, most of these models require preconditioning. Radio-active sources have been used routinely for this purpose but safety issues have motivated researchers to transition to chemical or X-ray based preconditioning. In this study, we directly compare 350 kV X-ray and Cs-137 low-dose precondition of NOG mice before human stem cell transplantation. Based on flow cytometry data, we found that engraftment of human cells into the mouse bone marrow was similar between radiation sources. Likewise, human engraftment in the peripheral blood was comparable between Cs-137 and three different X-ray doses with equal chimerization kinetics. In primary lymphoid organs such as the thymus and lymph nodes, and spleen, liver and lung, human-to-mouse chimerization was also comparable between irradiation sources. Development of different CD4 and CD8 T cells as well as these cells’ maturation stages, i.e. from naïve to effector and memory subsets were generally analogous. Based on our results, we conclude that there are no discernable differences between the two sources in the low-dose spectrum investigated. However, while we encourage the transition to X-ray-based sources, we recommend all research groups to consider technical specifications and dose-finding studies. Humanized mouse models are used extensively in research involving human pathogens and diseases. However, most of these models require preconditioning. Radio-active sources have been used routinely for this purpose but safety issues have motivated researchers to transition to chemical or X-ray based preconditioning. In this study, we directly compare 350 kV X-ray and Cs-137 low-dose precondition of NOG mice before human stem cell transplantation. Based on flow cytometry data, we found that engraftment of human cells into the mouse bone marrow was similar between radiation sources. Likewise, human engraftment in the peripheral blood was comparable between Cs-137 and three different X-ray doses with equal chimerization kinetics. In primary lymphoid organs such as the thymus and lymph nodes, and spleen, liver and lung, human-to-mouse chimerization was also comparable between irradiation sources. Development of different CD4 and CD8 T cells as well as these cells' maturation stages, i.e. from naïve to effector and memory subsets were generally analogous. Based on our results, we conclude that there are no discernable differences between the two sources in the low-dose spectrum investigated. However, while we encourage the transition to X-ray-based sources, we recommend all research groups to consider technical specifications and dose-finding studies.

Background: The trans-membrane protease serine 2 (TMPRSS2) is essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry and infection. Efficacy and safety of TMPRSS2 inhibitors in patients with coronavirus disease 2019 (Covid-19) have not been evaluated in randomized trials.Methods: We conducted an investigator-initiated, double-blind, randomized, placebo-controlled multicenter trial in patients hospitalized with confirmed SARS-CoV-2 infection from April 4, to December 31, 2020. Within 48 h of admission, participants were randomly assigned in a 2:1 ratio to receive the TMPRSS2 inhibitor camostat mesilate 200 mg three times daily for 5 days or placebo. The primary outcome was time to discharge or clinical improvement measured as ≥2 points improvement on a 7-point ordinal scale. Other outcomes included 30-day mortality, safety and change in oropharyngeal viral load.Findings: 137 patients were assigned to receive camostat mesilate and 68 to placebo. Median time to clinical improvement was 5 days (interquartile range [IQR], 3 to 7) in the camostat group and 5 days (IQR, 2 to 10) in the placebo group ( P = 0·31). The hazard ratio for 30-day mortality in the camostat compared with the placebo group was 0·82 (95% confidence interval [CI], 0·24 to 2·79; P = 0·75). The frequency of adverse events was similar in the two groups. Median change in viral load from baseline to day 5 in the camostat group was -0·22 log 10 copies/mL ( p Interpretation: Under this protocol, camostat mesilate treatment was not associated with increased adverse events during hospitalization for Covid-19 and did not affect time to clinical improvement, progression to ICU admission or mortality. ClinicalTrials.gov Identifier: NCT04321096. EudraCT Number: 2020-001200-42.

DESIGN: This was an exploratory, single-arm clinical trial that tested the immune enhancement effects of 24-weeks of Toll-like receptor 9 (TLR9) agonist (MGN1703; Lefitolimod; 60 mg × 2 weekly) therapy. METHODS: We enrolled HIV-1-infected individuals on suppressive combination antiretroviral therapy. Safety was assessed throughout the study. The primary outcome was reduction in total CD4 T-cell viral DNA levels. Secondary outcomes included safety, detailed immunological and virological analyses, and time to viral rebound (viral load > 5000 copies/ml) after randomization into an analytical treatment interruption (ATI). RESULTS: A total of 12 individuals completed the treatment phase and nine completed the ATI. Adverse events were limited and consistent with previous reports for MGN1703. Although the dosing regimen led to potent T-cell activation and increased HIV-1-specific T-cell responses, there were no cohort-wide changes in persistent virus (total CD4 T cells viral DNA; P = 0.34). No difference in time to rebound was observed between the ATI arms (log rank P = 0.25). One of nine ATI participants, despite harboring a large replication-competent reservoir, controlled viremia for 150 days via both HIV-1-specific cellular and antibody-mediated immune responses. CONCLUSION: A period of 24 weeks of MGN1703 treatment was safe and improved innate as well as HIV-1-specific adaptive immunity in HIV-1+ individuals. These findings support the incorporation of TLR9 agonism into combination HIV-1 cure strategies. TRIAL NAME AND REGISTRATION: TLR9 Enhancement of antiviral immunity in chronic HIV-1 infection: a phase 1B/2A trial; ClinicalTrials.gov NCT02443935.

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., 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.

Summary Analytical antiretroviral treatment interruption (ATI) is an important feature of HIV research, seeking to achieve sustained viral suppression in the absence of antiretroviral therapy (ART) when the goal is to measure effects of novel therapeutic interventions on time to viral load rebound or altered viral setpoint. Trials with ATIs also intend to determine host, virological, and immunological markers that are predictive of sustained viral control off ART. Although ATI is increasingly incorporated into proof-of-concept trials, no consensus has been reached on strategies to maximise its utility and minimise its risks. In addition, differences in ATI trial designs hinder the ability to compare efficacy and safety of interventions across trials. Therefore, we held a meeting of stakeholders from many interest groups, including scientists, clinicians, ethicists, social scientists, regulators, people living with HIV, and advocacy groups, to discuss the main challenges concerning ATI studies and to formulate recommendations with an emphasis on strategies for risk mitigation and monitoring, ART resumption criteria, and ethical considerations. In this Review, we present the major points of discussion and consensus views achieved with the goal of informing the conduct of ATIs to maximise the knowledge gained and minimise the risk to participants in clinical HIV research.

HIV reservoirs persist in infected individuals despite combination antiretroviral therapy and can be identified in secondary lymphoid tissues, in intestinal tissues, in the central nervous system as well as in blood. Clinical trials have begun to explore effects of small molecule interventions to perturb the latent viral infection, but only limited information is available regarding the impacts of HIV cure-related clinical interventions on viral reservoirs found in tissues. Of the 14 HIV cure-related clinical trials since 2012 that have evaluated the effects of small molecule interventions in vivo, four trials have examined the impacts of the interventions in peripheral blood as well as other tissues that harbor persistent HIV. The additional tissues examined include cerebral spinal fluid, intestines and lymph nodes. We provide a comparison contrast analyses of the data across anatomical compartments tested in these studies to reveal where peripheral blood analyses reflect outcomes in other tissues as well as where the data reveal differences between tissue outcomes. We also summarize the current knowledge on these topics and highlight key open questions that need to be addressed experimentally to move the HIV cure research field closer to the development of an intervention strategy capable of eliciting long-term antiretroviral free remission of HIV disease.

Histone deacetylase inhibitors (HDACi) modulate the transcriptional activity of all cells, including innate and adaptive immune cells. Therefore, we aimed to evaluate immunological effects of treatment with the HDACi panobinostat in HIV-infected patients during a clinical phase IIa latency reversal trial. Using flow cytometry, we investigated changes in T cell activation (CD69, CD38, HLA-DR) and the expression of CD39 and CTLA4 on regulatory T cells (Tregs). Whole-blood stimulations were performed and cytokine responses measured using Luminex. Gene expression in purified peripheral blood mononuclear cells (PBMCs) was evaluated using an Affymetrix HTA 2.0 gene chip. We found that proportions of CD4+ and CD8+ T cells expressing CD69 increased 24 h after initial panobinostat administration (P < 0.01), followed by an increase in the proportions of CD38+ HLA-DR+-coexpressing CD4+ T cells on day 4 (P = 0.02). Concurrently, proportions of Tregs increased by 40% (P = 0.003). Treg CTLA4 median fluorescent intensity (MFI) increased by 25% (P = 0.007), and CD39 MFI on CD39+ Treg increased by 12% (P = 0.02). Lipopolysaccharide (LPS)-induced inflammatory responses (interleukin-1β [IL-1β], IL-6, IL-12p40, and tumor necrosis factor alpha [TNF-α]) in whole blood were significantly downregulated 4 days after initial dosing. Lastly, panobinostat induced significant changes in the overall gene expression pattern (fold change, >1.5; false-discovery-rate [FDR]-corrected P

Toll-like receptor 9 (TLR9) agonists are being developed for treatment of colorectal and other cancers, yet the impact of these drugs on human intestines remains unknown. This, together with the fact that there are additional potential indications for TLR9 agonist therapy (e.g., autoimmune and infectious diseases), led us to investigate the impact of MGN1703 (Lefitolimod) on intestinal homeostasis and viral persistence in HIV-positive individuals. Colonic sigmoid biopsies were collected (baseline and week four) from 11 HIV+ individuals on suppressive antiretroviral therapy, who received MGN1703 (60 mg s.c.) twice weekly for 4 weeks in a single-arm, phase 1b/2a study. Within sigmoid mucosa, global transcriptomic analyses revealed 248 modulated genes (false discovery rate

The effect of treatment with histone deacetylase inhibitors on the immune system in HIV-infected individuals is not clear. Analysis of results from a clinical trial in which 15 HIV-infected individuals received 12 doses of panobinostat identified a significant impact on both T cell activation status and regulatory T cell suppressive marker expression and a reduced level of monocytic responsiveness to inflammatory stimuli. These changes were substantiated by global gene expression analysis. Collectively, the results suggest that panobinostat has multiple effects on innate and adaptive immune responses. Importantly, all the effects were transient, and further panobinostat treatment did not cause persistent long-term changes in gene expression patterns in HIV-infected individuals., Histone deacetylase inhibitors (HDACi) modulate the transcriptional activity of all cells, including innate and adaptive immune cells. Therefore, we aimed to evaluate immunological effects of treatment with the HDACi panobinostat in HIV-infected patients during a clinical phase IIa latency reversal trial. Using flow cytometry, we investigated changes in T cell activation (CD69, CD38, HLA-DR) and the expression of CD39 and CTLA4 on regulatory T cells (Tregs). Whole-blood stimulations were performed and cytokine responses measured using Luminex. Gene expression in purified peripheral blood mononuclear cells (PBMCs) was evaluated using an Affymetrix HTA 2.0 gene chip. We found that proportions of CD4+ and CD8+ T cells expressing CD69 increased 24 h after initial panobinostat administration (P < 0.01), followed by an increase in the proportions of CD38+ HLA-DR+-coexpressing CD4+ T cells on day 4 (P = 0.02). Concurrently, proportions of Tregs increased by 40% (P = 0.003). Treg CTLA4 median fluorescent intensity (MFI) increased by 25% (P = 0.007), and CD39 MFI on CD39+ Treg increased by 12% (P = 0.02). Lipopolysaccharide (LPS)-induced inflammatory responses (interleukin-1β [IL-1β], IL-6, IL-12p40, and tumor necrosis factor alpha [TNF-α]) in whole blood were significantly downregulated 4 days after initial dosing. Lastly, panobinostat induced significant changes in the overall gene expression pattern (fold change, >1.5; false-discovery-rate [FDR]-corrected P

BACKGROUND: Immune priming before reversal of latency might be a component of a functional HIV cure. To assess this concept, we assessed if therapeutic HIV immunisation followed by latency reversal would affect measures of viral transcription, plasma viraemia, and reservoir size in patients with HIV on suppressive antiretroviral therapy.METHODS: In this single-arm, phase 1B/2A trial, we recruited adults treated at the Department of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark (aged ≥18 years) with successfully treated HIV-1 with plasma RNA loads of less than 50 copies per mL for the previous year and CD4 counts of at least 500 cells per μL. Exclusion criteria included CD4 counts of less than 200 cells per μL within the past 2 years, active hepatitis B or C infections, and clinically significant cardiac disease, including QTc prolongation. Participants received six therapeutic intradermal HIV-1 immunisations with 12 mg/mL Vacc-4x and 0·6 mg/mL rhuGM-CSF over 12 weeks (at 0 weeks, 1 week, 2 weeks, 3 weeks, 11 weeks, and 12 weeks) before receiving 5 mg/m(2) intravenous romidepsin once a week for 3 weeks. This procedure was followed by analytical treatment interruption. Coprimary outcomes were changes in copies of HIV-1 DNA (total and integrated) per million CD4 T cells and infectious units per million (IUPM) resting memory CD4 T cells established by viral outgrowth, assessed in all patients receiving at least one dose of active treatment with assessable data. We assessed total HIV-1 DNA at screening, before romidepsin treatment, and 6 weeks after romidepsin treatment. We assessed integrated viral DNA at baseline, before romidepsin treatment, and 8 weeks after romidepsin treatment. We assessed IUPM at screening, 2 weeks before romidepsin treatment, and 6 weeks after romidepsin treatment. This trial is registered at ClinicalTrials.gov, number NCT02092116.FINDINGS: Between May 19, 2014, and Oct 8, 2014, we enrolled 20 individuals, of whom 17 completed all Vacc-4x and rhuGM-CSF administrations and romidepsin infusions. 16 of 17 had assessable total HIV-1 DNA, 15 of 17 had assessable integrated HIV-1 DNA, and six of 17 had assessable IUPM at baseline and at one or more timepoints after study treatment. Total HIV-1 DNA declined from screening to 6 weeks after romidepsin treatment (mean reduction 39·7%, 95% CI -59·7 to -11·5; p=0·012). The decrease in integrated HIV-1 DNA from baseline to 8 weeks after romidepsin treatment was not significant (19·2%, -38·6 to 6·3; p=0·123). Among the six assessable participants, the mean reduction in IUPM from screening to 6 weeks after romidepsin treatment was 38·0% (95% CI -67·0 to -8·0; p=0·019). Of 141 adverse events, 134 (95%) were grade 1 and seven (5%) were grade 2-3.INTERPRETATION: This in-vivo combinatorial approach provides the first evidence for the feasibility of a combined shock and kill strategy, but also emphasises that further optimisation of this strategy is needed to achieve a sizeable effect on the latent reservoir that will translate into clinically measurable benefits for people living with HIV-1.FUNDING: Bionor Pharma, the Research Council of Norway, and SkatteFUNN.

Toll-like receptor (TLR) agonists are potent enhancers of innate antiviral immunity and may also reverse HIV-1 latency. Therefore, TLR agonists have a potential role in the context of a “shock-and-kill” approach to eradicate HIV-1. Our extensive preclinical evaluation suggests that a novel TLR9 agonist, MGN1703, may indeed perform both functions in an HIV-1 eradication trial. Peripheral blood mononuclear cells (PBMCs) from aviremic HIV-1-infected donors on antiretroviral therapy (ART) that were incubated with MGN1703 ex vivo exhibited increased secretion of interferon alpha (IFN-α) ( P = 0.005) and CXCL10 ( P = 0.0005) in culture supernatants. Within the incubated PBMC pool, there were higher proportions of CD69-positive CD56 dim CD16 + NK cells ( P = 0.001) as well as higher proportions of CD107a-positive ( P = 0.002) and IFN-γ-producing ( P = 0.038) NK cells. Incubation with MGN1703 also increased the proportions of CD69-expressing CD4 + and CD8 + T cells. Furthermore, CD4 + T cells within the pool of MGN1703-incubated PBMCs showed enhanced levels of unspliced HIV-1 RNA ( P = 0.036). Importantly, MGN1703 increased the capacity of NK cells to inhibit virus spread within a culture of autologous CD4 + T cells assessed by using an HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) ( P = 0.03). In conclusion, we show that MGN1703 induced strong antiviral innate immune responses, enhanced HIV-1 transcription, and boosted NK cell-mediated suppression of HIV-1 infection in autologous CD4 + T cells. These findings support clinical testing of MGN1703 in HIV-1 eradication trials. IMPORTANCE We demonstrate that MGN1703 (a TLR9 agonist currently undergoing phase 3 clinical testing for the treatment of metastatic colorectal cancer) induces potent antiviral responses in immune effector cells from HIV-1-infected individuals on suppressive antiretroviral therapy. The significantly improved safety and tolerability profiles of MGN1703 versus TLR9 agonists of the CpG-oligodeoxynucleotide (CpG-ODN) family are due to its novel “dumbbell-shape” structure made of covalently closed, natural DNA. In our study, we found that incubation of peripheral blood mononuclear cells with MGN1703 results in natural killer cell activation and increased natural killer cell function, which significantly inhibited the spread of HIV in a culture of autologous CD4 + T cells. Furthermore, we discovered that MGN1703-mediated activation can enhance HIV-1 transcription in CD4 + T cells, suggesting that this molecule may serve a dual purpose in HIV-1 eradication therapy: enhanced immune function and latency reversal. These findings provide a strong preclinical basis for the inclusion of MGN1703 in an HIV eradication clinical trial.

Research challenges associated with understanding HIV persistence during antiretroviral therapy can be categorized as temporal, spatial and combinatorial. Temporal research challenges relate to the timing of events during establishment and maintenance of HIV persistence. Spatial research challenges regard the anatomical locations and cell subsets that harbor persistent HIV. Combinatorial research challenges pertain to the order of administration, timing of administration and specific combinations of compounds to be administered during HIV eradication therapy. Overcoming these challenges will improve our understanding of HIV persistence and move the field closer to achieving eradication of persistent HIV. Given that humanized mice and non-human primate HIV models permit rigorous control of experimental conditions, these models have been used extensively as in vivo research platforms for directly addressing these research challenges. The aim of this manuscript is to provide a comprehensive review of these recent translational advances made in animal models of HIV persistence.

Adjunct therapy with the histone deacetylase inhibitor (HDACi) romidepsin increases plasma viremia in HIV patients on combination antiretroviral therapy (cART). However, a potential concern is that reversing HIV latency with an HDACi may reactivate the virus in anatomical compartments with suboptimal cART concentrations, leading to de novo infection of susceptible cells in these sites. We tested physiologically relevant romidepsin concentrations known to reactivate latent HIV in order to definitively address this concern. We found that romidepsin significantly inhibited HIV infection in peripheral blood mononuclear cells and CD4 + T cells but not in monocyte-derived macrophages. In addition, romidepsin impaired HIV spreading in CD4 + T cell cultures. When we evaluated the impact of romidepsin on quantitative viral outgrowth assays with primary resting CD4 + T cells, we found that resting CD4 + T cells exposed to romidepsin exhibited reduced proliferation and viability. This significantly lowered assay sensitivity when measuring the efficacy of romidepsin as an HIV latency reversal agent. Altogether, our data indicate that romidepsin-based HIV eradication strategies are unlikely to reseed a latent T cell reservoir, even under suboptimal cART conditions, because romidepsin profoundly restricts de novo HIV infections.