RIG-I-like receptor activation by dengue virus drives follicular T helper cell formation and antibody production.

Follicular T helper cells (TFH) are fundamental in orchestrating effective antibody-mediated responses critical for immunity against viral infections and effective vaccines. However, it is unclear how virus infection leads to TFH induction. We here show that dengue virus (DENV) infection of human dendritic cells (DCs) drives TFH formation via crosstalk of RIG-I-like receptor (RLR) RIG-I and MDA5 with type I Interferon (IFN) signaling. DENV infection leads to RLR-dependent IKKε activation, which phosphorylates IFNα/ß receptor-induced STAT1 to drive IL-27 production via the transcriptional complex ISGF3. Inhibiting RLR activation as well as neutralizing antibodies against IL-27 prevented TFH formation. DENV-induced CXCR5+PD-1+Bcl-6+ TFH cells secreted IL-21 and activated B cells to produce IgM and IgG. Notably, RLR activation by synthetic ligands also induced IL-27 secretion and TFH polarization. These results identify an innate mechanism by which antibodies develop during viral disease and identify RLR ligands as potent adjuvants for TFH-promoting vaccination strategies.

RIG-I-like Receptor Triggering by Dengue Virus Drives Dendritic Cell Immune Activation and TH1 Differentiation.

Dengue virus (DENV) causes 400 million infections annually and is one of several viruses that can cause viral hemorrhagic fever, which is characterized by uncontrolled immune activation resulting in high fever and internal bleeding. Although the underlying mechanisms are unknown, massive cytokine secretion is thought to be involved. Dendritic cells (DCs) are the main target cells of DENV, and we investigated their role in DENV-induced cytokine production and adaptive immune responses. DENV infection induced DC maturation and secretion of IL-1ß, IL-6, and TNF. Inhibition of DENV RNA replication abrogated these responses. Notably, silencing of RNA sensors RIG-I or MDA5 abrogated DC maturation, as well as cytokine responses by DENV-infected DCs. DC maturation was induced by type I IFN responses because inhibition of IFN-α/ß receptor signaling abrogated DENV-induced DC maturation. Moreover, DENV infection of DCs resulted in CCL2, CCL3, and CCL4 expression, which was abrogated after RIG-I and MDA5 silencing. DCs play an essential role in TH cell differentiation, and we show that RIG-I and MDA5 triggering by DENV leads to TH1 polarization, which is characterized by high levels of IFN-γ. Notably, cytokines IL-6, TNF, and IFN-γ and chemokines CCL2, CCL3, and CCL4 have been associated with disease severity, endothelial dysfunction, and vasodilation. Therefore, we identified RIG-I and MDA5 as critical players in innate and adaptive immune responses against DENV, and targeting these receptors has the potential to decrease hemorrhagic fever in patients.

Investigational drugs for HIV: trends, opportunities and key players.

INTRODUCTION: Since the first antiretroviral drug was described, the field of HIV treatment and prevention has undergone two drug-based revolutions: the first one, enabled by the virtually concomitant discovery of non-nucleoside reverse transcriptase and protease inhibitors, was the inception of combined antiretroviral therapy. The second followed the creation of integrase strand-transfer inhibitors with improved safety, potency, and resistance profiles. Long-acting antiretroviral drugs, including broadly neutralizing antibodies, now offer the opportunity for a third transformational change in HIV management. AREAS COVERED: Our review focused on HIV treatment and prevention with investigational drugs that offer the potential for infrequent dosing, including drugs not yet approved for clinical use. We also discussed approved drugs for which administration modalities or formulations are being optimized. We performed a literature search in published manuscripts, conference communications, and registered clinical trials. EXPERT OPINION: While the field focuses on extending dosing intervals, we identify drug tissue penetration as an understudied opportunity to improve HIV care. We repeat that self-administration remains an essential milestone to reach the full potential of long-acting drugs. Treatments and prevention strategies based on broadly neutralizing antibodies require a deeper understanding of their antiretroviral properties.

Genotypic and Phenotypic Characterization of Replication-Competent HIV-2 Isolated from Controllers and Progressors.

Although some individuals with HIV-2 develop severe immunodeficiency and AIDS-related complications, most may never progress to AIDS. Replication-competent HIV-2 isolated from asymptomatic long-term non-progressors (controllers) have lower replication rates than viruses from individuals who progress to AIDS (progressors). To investigate potential retroviral factors that correlate with disease progression in HIV-2, we sequenced the near full-length genomes of replication-competent viruses previously outgrown from controllers and progressors and used phylogeny to seek genotypic correlates of disease progression. We validated the integrity of all open reading frames and used cell-based assays to study the retroviral transcriptional activity of the long terminal repeats (LTRs) and Tat proteins of HIV-2 from controllers and progressors. Overall, we did not identify genotypic defects that may contribute to HIV-2 non-progression. Tat-induced, LTR-mediated transcription was comparable between viruses from controllers and progressors. Our results were obtained from a small number of participants and should be interpreted accordingly. Overall, they suggest that progression may be determined before or during integration of HIV-2.

The BAF complex inhibitor pyrimethamine reverses HIV-1 latency in people with HIV-1 on antiretroviral therapy.

Reactivation of the latent HIV-1 reservoir is a first step toward triggering reservoir decay. Here, we investigated the impact of the BAF complex inhibitor pyrimethamine on the reservoir of people living with HIV-1 (PLWH). Twenty-eight PLWH on suppressive antiretroviral therapy were randomized (1:1:1:1 ratio) to receive pyrimethamine, valproic acid, both, or no intervention for 14 days. The primary end point was change in cell-associated unspliced (CA US) HIV-1 RNA at days 0 and 14. We observed a rapid, modest, and significant increase in (CA US) HIV-1 RNA in response to pyrimethamine exposure, which persisted throughout treatment and follow-up. Valproic acid treatment alone did not increase (CA US) HIV-1 RNA or augment the effect of pyrimethamine. Pyrimethamine treatment did not result in a reduction in the size of the inducible reservoir. These data demonstrate that the licensed drug pyrimethamine can be repurposed as a BAF complex inhibitor to reverse HIV-1 latency in vivo in PLWH, substantiating its potential advancement in clinical studies.

HIV-1 resistance against dolutegravir fluctuates rapidly alongside erratic treatment adherence: a case report.

OBJECTIVES: We report a case of incomplete HIV-1 suppression on a dolutegravir, lamivudine, and abacavir single-tablet regimen with the emergence of the H51Y and G118R integrase resistance mutations. METHODS: Integrase sequencing was performed retrospectively by Sanger and next-generation sequencing. Rates of emergence and decline of resistance mutations were calculated using next-generation sequencing data. Dolutegravir plasma concentrations were measured by ultra-performance liquid chromatography-tandem mass spectrometry. The effects of H51Y and G118R on infectivity, fitness, and susceptibility to dolutegravir were quantified using cell-based assays. RESULTS: During periods of non-adherence to treatment, mutations were retrospectively documented only by next-generation sequencing. Misdiagnosis by Sanger sequencing was caused by the rapid decline of mutant strains within the retroviral population. This observation was also true for a M184V lamivudine-resistant reverse transcriptase mutation found in association with integrase mutations on single HIV genomes. Resistance rebound upon treatment re-initiation was swift (>8000 copies per day). Next-generation sequencing indicated cumulative adherence to treatment. Compared to WT HIV-1, relative infectivity was 73%, 38%, and 43%; relative fitness was 100%, 35%, and 10% for H51Y, G118R, and H51Y+G118R viruses, respectively. H51Y did not change the susceptibility to dolutegravir, but G188R and H51Y+G118R conferred 7- and 28-fold resistance, respectively. CONCLUSION: This case illustrates how poorly-fit drug-resistant viruses wax and wane alongside erratic treatment adherence and are easily misdiagnosed by Sanger sequencing. We recommend next-generation sequencing to improve the clinical management of incomplete virological suppression with dolutegravir.

Inter-Laboratory Reproducibility of Inducible HIV-1 Reservoir Quantification by TILDA.

Substantial efforts to eliminate or reduce latent HIV-1 reservoirs are underway in clinical trials and have created a critical demand for sensitive, accurate, and reproducible tools to evaluate the efficacy of these strategies. Alternative reservoir quantification assays have been developed to circumvent limitations of the quantitative viral outgrowth assay. One such assay is tat/rev induced limiting dilution assay (TILDA), which measures the frequency of CD4+ T cells harboring inducible latent HIV-1 provirus. We modified pre-amplification reagents and conditions (TILDA v2.0) to improve assay execution and first internally validated assay performance using CD4+ T cells obtained from cART-suppressed HIV-1-infected individuals. Detection of tat/rev multiply spliced RNA was not altered by modifying pre-amplification conditions, confirming the robustness of the assay, and supporting the technique's amenability to limited modifications to ensure better implementation for routine use in clinical studies of latent HIV-1 reservoirs. Furthermore, we cross-validated results of TILDA v2.0 and the original assay performed in two separate laboratories using samples from 15 HIV-1-infected individuals. TILDA and TILDA v2.0 showed a strong correlation (Lin's Concordance Correlation Coefficient = 0.86). The low inter-laboratory variability between TILDAs performed at different institutes further supports use of TILDA for reservoir quantitation in multi-center interventional HIV-1 Cure trials.

Rapid SARS-CoV-2 whole-genome sequencing and analysis for informed public health decision-making in the Netherlands.

In late December 2019, a cluster of cases of pneumonia of unknown etiology were reported linked to a market in Wuhan, China1. The causative agent was identified as the species Severe acute respiratory syndrome-related coronavirus and was named SARS-CoV-2 (ref. 2). By 16 April the virus had spread to 185 different countries, infected over 2,000,000 people and resulted in over 130,000 deaths3. In the Netherlands, the first case of SARS-CoV-2 was notified on 27 February. The outbreak started with several different introductory events from Italy, Austria, Germany and France followed by local amplification in, and later also outside, the south of the Netherlands. The combination of near to real-time whole-genome sequence analysis and epidemiology resulted in reliable assessments of the extent of SARS-CoV-2 transmission in the community, facilitating early decision-making to control local transmission of SARS-CoV-2 in the Netherlands. We demonstrate how these data were generated and analyzed, and how SARS-CoV-2 whole-genome sequencing, in combination with epidemiological data, was used to inform public health decision-making in the Netherlands.

Author Correction: Rapid SARS-CoV-2 whole-genome sequencing and analysis for informed public health decision-making in the Netherlands.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

COVID-19 in health-care workers in three hospitals in the south of the Netherlands: a cross-sectional study.

BACKGROUND: 10 days after the first reported case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the Netherlands (on Feb 27, 2020), 55 (4%) of 1497 health-care workers in nine hospitals located in the south of the Netherlands had tested positive for SARS-CoV-2 RNA. We aimed to gain insight in possible sources of infection in health-care workers. METHODS: We did a cross-sectional study at three of the nine hospitals located in the south of the Netherlands. We screened health-care workers at the participating hospitals for SARS-CoV-2 infection, based on clinical symptoms (fever or mild respiratory symptoms) in the 10 days before screening. We obtained epidemiological data through structured interviews with health-care workers and combined this information with data from whole-genome sequencing of SARS-CoV-2 in clinical samples taken from health-care workers and patients. We did an in-depth analysis of sources and modes of transmission of SARS-CoV-2 in health-care workers and patients. FINDINGS: Between March 2 and March 12, 2020, 1796 (15%) of 12 022 health-care workers were screened, of whom 96 (5%) tested positive for SARS-CoV-2. We obtained complete and near-complete genome sequences from 50 health-care workers and ten patients. Most sequences were grouped in three clusters, with two clusters showing local circulation within the region. The noted patterns were consistent with multiple introductions into the hospitals through community-acquired infections and local amplification in the community. INTERPRETATION: Although direct transmission in the hospitals cannot be ruled out, our data do not support widespread nosocomial transmission as the source of infection in patients or health-care workers. FUNDING: EU Horizon 2020 (RECoVer, VEO, and the European Joint Programme One Health METASTAVA), and the National Institute of Allergy and Infectious Diseases, National Institutes of Health.