PQBP1 Is a Proximal Sensor of the cGAS-Dependent Innate Response to HIV-1.

Dendritic cells (DCs) play a critical role in the immune response to viral infection through the facilitation of cell-intrinsic antiviral activity and the activation of adaptive immunity. HIV-1 infection of DCs triggers an IRF3-dependent innate immune response, which requires the activity of cyclic GAMP synthase (cGAS). We report the results of a targeted RNAi screen utilizing primary human monocyte-derived DCs (MDDCs) to identify immune regulators that directly interface with HIV-1-encoded features to initiate this innate response. Polyglutamine binding protein 1 (PQBP1) emerged as a strong candidate through this analysis. We found that PQBP1 directly binds to reverse-transcribed HIV-1 DNA and interacts with cGAS to initiate an IRF3-dependent innate response. MDDCs derived from Renpenning syndrome patients, who harbor mutations in the PQBP1 locus, possess a severely attenuated innate immune response to HIV-1 challenge, underscoring the role of PQBP1 as a proximal innate sensor of a HIV-1 infection.

HIV, STI and renal function testing frequency and STI history among current users of self-funded HIV pre-exposure prophylaxis, a cross-sectional study, Germany, 2018 and 2019.

IntroductionUsers of pre-exposure prophylaxis (PrEP) require periodic testing for HIV, sexually transmitted infections (STI) and renal function. Before PrEP was made free of charge through statutory health insurance in late 2019, PrEP users in Germany had to pay for testing themselves.AimWe investigated self-reported HIV, STI and renal function testing frequencies among self-funded PrEP users in Germany, factors associated with infrequent testing, and STI diagnoses.MethodsA cross-sectional anonymous online survey in 2018 and 2019 recruited current PrEP users via dating apps for men who have sex with men (MSM), a PrEP community website, anonymous testing sites and friends. We used descriptive methods and logistic regression for analysis.ResultsWe recruited 4,848 current PrEP users. Median age was 37 years (interquartile range (IQR): 30-45), 88.7% identified as male, and respectively 26.3%, 20.9% and 29.2% were tested less frequently for HIV, STI and renal function than recommended. Participants with lower STI testing frequency were significantly less likely to report STI diagnoses during PrEP use, especially among those with many partners and inconsistent condom use. Factors most strongly associated with infrequent testing included not getting tested before starting PrEP, using PrEP from informal sources and on-demand/intermittent PrEP use.DiscussionIn a setting of self-funded PrEP, many users obtained medical tests less frequently than recommended, which can lead to missed diagnoses. Barriers to testing should be addressed to enable proper medical supervision. The suitability of testing frequencies to PrEP users with less frequent risk exposures needs to be evaluated.

COVID-19: cross-border contact tracing in Germany, February to April 2020.

IntroductionThe Robert Koch Institute (RKI) managed the exchange of cross-border contact tracing data between public health authorities (PHA) in Germany and abroad during the early COVID-19 pandemic.AimWe describe the extent of cross-border contact tracing and its challenges.MethodsWe analysed cross-border COVID-19 contact tracing events from 3 February to 5 April 2020 using information exchanged through the European Early Warning Response System and communication with International Health Regulation national focal points. We described events by PHA, number of contacts and exposure context.ResultsThe RKI processed 467 events, initiating contact to PHA 1,099 times (median = 1; interquartile range (IQR): 1-2) and sharing data on 5,099 contact persons. Of 327 (70%) events with known exposure context, the most commonly reported exposures were aircraft (n = 64; 20%), cruise ships (n = 24; 7%) and non-transport contexts (n = 210; 64%). Cruise ship and aircraft exposures generated more contacts with authorities (median = 10; IQR: 2-16, median = 4; IQR: 2-11) and more contact persons (median = 60; IQR: 9-269, median = 2; IQR: 1-3) than non-transport exposures (median = 1; IQR: 1-6 and median = 1; IQR: 1-2). The median time spent on contact tracing was highest for cruise ships: 5 days (IQR: 3-9).ConclusionIn the COVID-19 pandemic, cross-border contact tracing is considered a critical component of the outbreak response. While only a minority of international contact tracing activities were related to exposure events in transport, they contributed substantially to the workload. The numerous communications highlight the need for fast and efficient global outbreak communication channels between PHA.

[Recording of SARS-CoV-2 laboratory tests in Germany]. / Erfassung der Labortestungen auf SARS-CoV-2 in Deutschland.

The collection of data on SARS-CoV­2 tests is central to the assessment of the infection rate in the context of the COVID-19 pandemic. At the Robert Koch Institute (RKI), data collected from various laboratory data recording systems are consolidated. First, this article aims to exemplify significant aspects regarding test procedures. Subsequently the different systems for recording laboratory tests are described and test numbers from the RKI test laboratory query and the laboratory-based SARS-CoV­2 surveillance as well as accounting data from the Association of Statutory Health Insurance Physicians for SARS-CoV­2 laboratory tests are shown.Early in the pandemic, the RKI test laboratory query and the laboratory-based SARS-CoV­2 surveillance became available and able to evaluate data on performed tests and test capacities. By recording the positive and negative test results, statements about the total number of tests and the proportion of positive test rates can be made. While the aggregate test numbers are largely representative nationwide, they are not always representative at the state and district level. The billing data of the Association of Statutory Health Insurance Physicians can complement the laboratory data afterwards. In addition, it can provide a retrospective assessment of the total number of SARS-CoV­2 numbers in Germany, because the services provided by statutory health insurers (around 85% of the population in Germany) are included. The various laboratory data recording systems complement one another and the evaluations flow into the recommended measures for the pandemic response.

Designed Ankyrin Repeat Protein (DARPin) to target chimeric antigen receptor (CAR)-redirected T cells towards CD4+ T cells to reduce the latent HIV+ cell reservoir.

Chimeric Antigen Receptor (CAR)-redirected T cells show great efficacy in the patient-specific therapy of hematologic malignancies. Here, we demonstrate that a DARPin with specificity for CD4 specifically redirects and triggers the activation of CAR engineered T cells resulting in the depletion of CD4+ target cells aiming for elimination of the human immunodeficiency virus (HIV) reservoir.

Exclusive Transduction of Human CD4+ T Cells upon Systemic Delivery of CD4-Targeted Lentiviral Vectors.

Playing a central role in both innate and adaptive immunity, CD4(+) T cells are a key target for genetic modifications in basic research and immunotherapy. In this article, we describe novel lentiviral vectors (CD4-LV) that have been rendered selective for human or simian CD4(+) cells by surface engineering. When applied to PBMCs, CD4-LV transduced CD4(+) but not CD4(-) cells. Notably, also unstimulated T cells were stably genetically modified. Upon systemic or intrasplenic administration into mice reconstituted with human PBMCs or hematopoietic stem cells, reporter gene expression was predominantly detected in lymphoid organs. Evaluation of GFP expression in organ-derived cells and blood by flow cytometry demonstrated exclusive gene transfer into CD4(+) human lymphocytes. In bone marrow and spleen, memory T cells were preferentially hit. Toward therapeutic applications, we also show that CD4-LV can be used for HIV gene therapy, as well as for tumor therapy, by delivering chimeric Ag receptors. The potential for in vivo delivery of the FOXP3 gene was also demonstrated, making CD4-LV a powerful tool for inducible regulatory T cell generation. In summary, our work demonstrates the exclusive gene transfer into a T cell subset upon systemic vector administration opening an avenue toward novel strategies in immunotherapy.

High secretion of interferons by human plasmacytoid dendritic cells upon recognition of Middle East respiratory syndrome coronavirus.

UNLABELLED: The Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012 as the causative agent of a severe respiratory disease with a fatality rate of approximately 30%. The high virulence and mortality rate prompted us to analyze aspects of MERS-CoV pathogenesis, especially its interaction with innate immune cells such as antigen-presenting cells (APCs). Particularly, we analyzed secretion of type I and type III interferons (IFNs) by APCs, i.e., B cells, macrophages, monocyte-derived/myeloid dendritic cells (MDDCs/mDCs), and by plasmacytoid dendritic cells (pDCs) of human and murine origin after inoculation with MERS-CoV. Production of large amounts of type I and III IFNs was induced exclusively in human pDCs, which were significantly higher than IFN induction by severe acute respiratory syndrome (SARS)-CoV. Of note, IFNs were secreted in the absence of productive replication. However, receptor binding, endosomal uptake, and probably signaling via Toll-like receptor 7 (TLR7) were critical for sensing of MERS-CoV by pDCs. Furthermore, active transcription of MERS-CoV N RNA and subsequent N protein expression were evident in infected pDCs, indicating abortive infection. Taken together, our results point toward dipeptidyl peptidase 4 (DPP4)-dependent endosomal uptake and subsequent infection of human pDCs by MERS-CoV. However, the replication cycle is stopped after early gene expression. In parallel, human pDCs are potent IFN-producing cells upon MERS-CoV infection. Knowledge of such IFN responses supports our understanding of MERS-CoV pathogenesis and is critical for the choice of treatment options. IMPORTANCE: MERS-CoV causes a severe respiratory disease with high fatality rates in human patients. Recently, confirmed human cases have increased dramatically in both number and geographic distribution. Understanding the pathogenesis of this highly pathogenic CoV is crucial for developing successful treatment strategies. This study elucidates the interaction of MERS-CoV with APCs and pDCs, particularly the induction of type I and III IFN secretion. Human pDCs are the immune cell population sensing MERS-CoV but secrete significantly larger amounts of IFNs, especially IFN-α, than in response to SARS-CoV. A model for molecular virus-host interactions is presented outlining IFN induction in pDCs. The massive IFN secretion upon contact suggests a critical role of this mechanism for the high degree of immune activation observed during MERS-CoV infection.

A Highly Immunogenic and Protective Middle East Respiratory Syndrome Coronavirus Vaccine Based on a Recombinant Measles Virus Vaccine Platform.

UNLABELLED: In 2012, the first cases of infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) were identified. Since then, more than 1,000 cases of MERS-CoV infection have been confirmed; infection is typically associated with considerable morbidity and, in approximately 30% of cases, mortality. Currently, there is no protective vaccine available. Replication-competent recombinant measles virus (MV) expressing foreign antigens constitutes a promising tool to induce protective immunity against corresponding pathogens. Therefore, we generated MVs expressing the spike glycoprotein of MERS-CoV in its full-length (MERS-S) or a truncated, soluble variant of MERS-S (MERS-solS). The genes encoding MERS-S and MERS-solS were cloned into the vaccine strain MVvac2 genome, and the respective viruses were rescued (MVvac2-CoV-S and MVvac2-CoV-solS). These recombinant MVs were amplified and characterized at passages 3 and 10. The replication of MVvac2-CoV-S in Vero cells turned out to be comparable to that of the control virus MVvac2-GFP (encoding green fluorescent protein), while titers of MVvac2-CoV-solS were impaired approximately 3-fold. The genomic stability and expression of the inserted antigens were confirmed via sequencing of viral cDNA and immunoblot analysis. In vivo, immunization of type I interferon receptor-deficient (IFNAR(-/-))-CD46Ge mice with 2 × 10(5) 50% tissue culture infective doses of MVvac2-CoV-S(H) or MVvac2-CoV-solS(H) in a prime-boost regimen induced robust levels of both MV- and MERS-CoV-neutralizing antibodies. Additionally, induction of specific T cells was demonstrated by T cell proliferation, antigen-specific T cell cytotoxicity, and gamma interferon secretion after stimulation of splenocytes with MERS-CoV-S presented by murine dendritic cells. MERS-CoV challenge experiments indicated the protective capacity of these immune responses in vaccinated mice. IMPORTANCE: Although MERS-CoV has not yet acquired extensive distribution, being mainly confined to the Arabic and Korean peninsulas, it could adapt to spread more readily among humans and thereby become pandemic. Therefore, the development of a vaccine is mandatory. The integration of antigen-coding genes into recombinant MV resulting in coexpression of MV and foreign antigens can efficiently be achieved. Thus, in combination with the excellent safety profile of the MV vaccine, recombinant MV seems to constitute an ideal vaccine platform. The present study shows that a recombinant MV expressing MERS-S is genetically stable and induces strong humoral and cellular immunity against MERS-CoV in vaccinated mice. Subsequent challenge experiments indicated protection of vaccinated animals, illustrating the potential of MV as a vaccine platform with the potential to target emerging infections, such as MERS-CoV.

Rational design of HIV vaccines and microbicides: report of the EUROPRISE network annual conference 2010.

Novel, exciting intervention strategies to prevent infection with HIV have been tested in the past year, and the field is rapidly evolving. EUROPRISE is a network of excellence sponsored by the European Commission and concerned with a wide range of activities including integrated developmental research on HIV vaccines and microbicides from discovery to early clinical trials. A central and timely theme of the network is the development of the unique concept of co-usage of vaccines and microbicides. This review, prepared by the PhD students of the network captures much of the research ongoing between the partners. The network is in its 5th year and involves over 50 institutions from 13 European countries together with 3 industrial partners; GSK, Novartis and Sanofi-Pasteur. EUROPRISE is involved in 31 separate world-wide trials of Vaccines and Microbicides including 6 in African countries (Tanzania, Mozambique, South Africa, Kenya, Malawi, Rwanda), and is directly supporting clinical trials including MABGEL, a gp140-hsp70 conjugate trial and HIVIS, vaccine trials in Europe and Africa.

SARS-CoV-2 Aerosol Transmission Indoors: A Closer Look at Viral Load, Infectivity, the Effectiveness of Preventive Measures and a Simple Approach for Practical Recommendations.

There is uncertainty about the viral loads of infectious individuals required to transmit COVID-19 via aerosol. In addition, there is a lack of both quantification of the influencing parameters on airborne transmission and simple-to-use models for assessing the risk of infection in practice, which furthermore quantify the influence of non-medical preventive measures. In this study, a dose-response model was adopted to analyze 25 documented outbreaks at infection rates of 4-100%. We show that infection was only possible if the viral load was higher than 108 viral copies/mL. Based on mathematical simplifications of our approach to predict the probable situational attack rate (PARs) of a group of persons in a room, and valid assumptions, we provide simplified equations to calculate, among others, the maximum possible number of persons and the person-related virus-free air supply flow necessary to keep the number of newly infected persons to less than one. A comparison of different preventive measures revealed that testing contributes the most to the joint protective effect, besides wearing masks and increasing ventilation. In addition, we conclude that absolute volume flow rate or person-related volume flow rate are more intuitive parameters for evaluating ventilation for infection prevention than air exchange rate.

Rational design of HIV vaccine and microbicides: report of the EUROPRISE annual conference.

EUROPRISE is a Network of Excellence sponsored from 2007 to 2011 by the European Commission within the 6th Framework Program. The Network encompasses a wide portfolio of activities ranging from an integrated research program in the field of HIV vaccines and microbicides to training, dissemination and advocacy. The research program covers the whole pipeline of vaccine and microbicide development from discovery to early clinical trials. The Network is composed of 58 partners representing more than 65 institutions from 13 European countries; it also includes three major pharmaceutical companies (GlaxoSmithKline, Novartis and Sanofi-Pasteur) involved in HIV microbicide and vaccine research. The Network displays a dedicated and informative web page: http://www.europrise.org. Finally, a distinguishing trait of EUROPRISE is its PhD School of students from across Europe, a unique example in the world of science aimed at spreading excellence through training. EUROPRISE held its second annual conference in Budapest in November, 2009. The conference had 143 participants and their presentations covered aspects of vaccine and microbicide research, development and discovery. Since training is a major task of the Network, the students of the EUROPRISE PhD program summarized certain presentations and their view of the conference in this paper.

Insights into Innate Sensing of Prototype Foamy Viruses in Myeloid Cells.

Foamy viruses (FVs) belong to the Spumaretrovirinae subfamily of retroviruses and are characterized by unique features in their replication strategy. This includes a reverse transcription (RTr) step of the packaged RNA genome late in replication, resulting in the release of particles with a fraction of them already containing an infectious viral DNA (vDNA) genome. Little is known about the immune responses against FVs in their hosts, which control infection and may be responsible for their apparent apathogenic nature. We studied the interaction of FVs with the innate immune system in myeloid cells, and characterized the viral pathogen-associated molecular patterns (PAMPs) and the cellular pattern recognition receptors and sensing pathways involved. Upon cytoplasmic access, full-length but not minimal vector genome containing FVs with active reverse transcriptase, induced an efficient innate immune response in various myeloid cells. It was dependent on cellular cGAS and STING and largely unaffected by RTr inhibition during viral entry. This suggests that RTr products, which are generated during FV morphogenesis in infected cells, and are therefore already present in FV particles taken up by immune cells, are the main PAMPs of FVs with full-length genomes sensed in a cGAS and STING-dependent manner by the innate immune system in host cells of the myeloid lineage.

Dephosphorylation of the HIV-1 restriction factor SAMHD1 is mediated by PP2A-B55α holoenzymes during mitotic exit.

SAMHD1 is a critical restriction factor for HIV-1 in non-cycling cells and its antiviral activity is regulated by T592 phosphorylation. Here, we show that SAMHD1 dephosphorylation at T592 is controlled during the cell cycle, occurring during M/G1 transition in proliferating cells. Using several complementary proteomics and biochemical approaches, we identify the phosphatase PP2A-B55α responsible for rendering SAMHD1 antivirally active. SAMHD1 is specifically targeted by PP2A-B55α holoenzymes during mitotic exit, in line with observations that PP2A-B55α is a key mitotic exit phosphatase in mammalian cells. Strikingly, as HeLa or activated primary CD4+ T cells enter the G1 phase, pronounced reduction of RT products is observed upon HIV-1 infection dependent on the presence of dephosphorylated SAMHD1. Moreover, PP2A controls SAMHD1 pT592 level in non-cycling monocyte-derived macrophages (MDMs). Thus, the PP2A-B55α holoenzyme is a key regulator to switch on the antiviral activity of SAMHD1.

Intrahepatic application of suicide gene-armed measles virotherapeutics: a safety study in transgenic mice and rhesus macaques.

Abstract Oncolytic viruses such as measles virus (MV) represent a new class of therapeutic agents that might help to overcome current limitations in cancer therapy. Although MV-based virotherapeutics already have entered clinical testing for various tumor entities, the preclinical safety of MV virotherapeutics so far has not been elucidated for particular regimens with high medical need, such as (1) direct injection into hepatic tumor sites, (2) employing high doses ibidem, and (3) concurrent usage of arming with cytotoxic genes required to further enhance oncolytic efficiency. Here, we assessed the safety and pharmacokinetics of suicide gene-armed vector MV-SCD when administered intrahepatically in two animal models, IFNAR(tm)-CD46(Ge) (interferon-α receptor deficient and CD46 MV receptor knock-in) transgenic mice and rhesus macaques (Macaca mulatta). Clinically, singular direct intrahepatic applications of MV-SCD were found to be well tolerated. Quantitative RT-PCR demonstrated the transient presence of viral RNA in various organs, whereas no shedding of infectious virus particles was observed at any time point. Histological analyses of organs did not exhibit adverse effects attributable to the test article. Blood parameters including liver enzymes revealed no deviations from normal. In both species an antiviral humoral immune response was mounted shortly after virus administration. Surprisingly, daily repeated systemic applications of MV-SCD under concomitant prodrug administration resulted in side effects in IFNAR(tm)-CD46(Ge) mice, but were less pronounced than in a 5-fluorouracil standard therapy control cohort. Taken together, these data indicate that "single shot" direct intrahepatic injections of MV-SCD in conjunction with systemic prodrug administration are safe and could be used in future virotherapeutic treatments of liver cancers.

Characterization of a monoclonal anti-capsid antibody that cross-reacts with three major primate lentivirus lineages.

Mouse monoclonal antibodies with varying specificities against the Gag capsid of simian and human immunodeficiency virus (SIV/HIV) were generated by immunizing mice with whole inactivated SIVagmTYO-1. Monoclonal antibody AG3.0 showed the broadest reactivity recognizing the Gag capsid protein (p24-27) and Gag precursors p38, p55, and p150 of HIV-1, HIV-2, SIVmac, and SIVagm. Using overlapping peptides, the AG3.0 epitope was mapped in capsid to a sequence (SPRTLNA) conserved among HIV-1, HIV-2, SIVrcm, SIVsm/mac, and SIVagm related viruses. Because of its broad cross-reactivity, AG3.0 was used to develop an antigen capture assay with a lower detection limit of 100 pg/ml HIV-1 Gag p24. Interestingly, AG3.0 was found to have a faster binding on/off rate for SIVagmVer and SIVmac Gag than for SIVagmSab Gag, possibly due to differences outside the SPRTLNA motif. In addition, the ribonucleic acid (RNA) coding for AG3.0 was sequenced to facilitate the development of humanized monoclonal antibodies.