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

Introduction: Users 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. Aim: We 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. Methods: A 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. Results: We 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. Discussion: In 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.Peer Reviewe

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

Einsatz von Antigentests in Einrichtungen in Deutschland - Ergebnisse einer RKI-Umfrage

Peer Reviewe

Erfassung der SARS-CoV-2-Testzahlen in Deutschland (Stand 26.8.2020)

Das Robert Koch-Institut erfasst wöchentlich die Anzahl der in Deutschland durchgeführten SARS-CoV-2-Tests, sowie einige Begleitinformationen. Hierfür werden deutschlandweit Daten von Universitätskliniken, Forschungseinrichtungen sowie klinischen und in der ambulanten Versorgung tätigen Laboren zusammengeführt. Der Artikel im Epidemiologischen Bulletin 35/2020 geht u. a. auf die Sensitivität und Spezifität der diagnostischen Tests und die Rolle falsch-positiver Testergebnisse für die Bewertung der Lage in Deutschland sowie auf die Testkapazitäten ei

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

Mechanismen der Herunterregulierung der Immunaktivierung und B-Zell Antworten in natürlichen Wirten von simianen Immundefizienzviren

Abbreviations vii Zusammenfassung xi Summary xv 1 Introduction 1 1.1 The global AIDS epidemic and its emergence 1 1.2 Phylogeny of primate lentiviruses 1 1.3 Structure and morphology of HIV and SIV 3 1.4 The replication cycle of HIV and SIV 5 1.5 Pathogenesis of HIV/SIV infection in the heterologous host 7 1.6 Immune responses in the heterologous host 8 1.6.1 Cellular immune reactions against HIV/SIV 8 1.6.2 Humoural immune response to HIV/SIV 9 1.6.3 Immune activation 11 1.7 Long-term non-progressors, highly exposed persistent seronegatives and elite controllers 12 1.8 Antiretroviral therapy 13 1.9 Non- pathogenic SIV infection of natural hosts 14 1.9.1 Levels of virus replication 14 1.9.2 Levels of mucosal CD4+ T-cells 15 1.9.3 Target cells for SIV replication and cytopathicity 16 1.10 Immune responses in natural hosts of SIV 16 1.10.1 Cellular responses 16 1.10.2 Humoural responses 17 1.10.3 Efforts to break the apparent tolerance to Gag 18 1.10.4 Immune activation in the natural hosts 19 1.13 Aim of the thesis 20 2 Materials and Methods 23 2.1 Sequencing of the AG3.0 heavy and light chain variable regions 23 2.2 Animals 23 2.3 Immunisations 24 2.4 Specimen collection 24 2.5 Determination of p27 concentration in AGM and rhesus plasma 25 2.6 Quantification of cytokines and chemokines in plasma 25 2.7 Isolation of PBMCs from AGM and rhesus blood samples 25 2.8 Sequencing of AGM and rhesus biomarker mRNAs 26 2.9 Generation of AGM biomarker cDNA for amplification and sequencing 26 2.10 Amplification of AGM biomarker cDNAs 26 2.11 Cloning of AGM and rhesus biomarker genes 31 2.12 Detection of positive bacterial clones 31 2.13 Plasmid isolation 32 2.14 Quantification of RNA and DNA 32 2.15 Sequencing of AGM and rhesus biomarker cDNAs 32 2.16 Primer design for biomarker real-time PCRs 33 2.17 Generation of positive control DNA templates for realtime-PCR 37 2.18 Testing of realtime- PCR assay efficiency with AGM and rhesus cDNAs 37 2.19 Determination of biomarker expression levels in PBMCs of chronically SIV-infected AGMs and rhesus macaques using realtime RT-PCR 38 2.20 Quantification of pDCs in PBMCs with FACS 39 2.21 Quantification of CD20+ B-cells in PBMCs with FACS 39 2.22 Interferon alpha ELISPOT 40 2.23 Total IgG ELISA 40 3 Results 43 3.1 Sequencing of the AG 3.0 antigen binding site. 43 3.2 Plasma virus load in acutely SIV-infected AGMs and rhesus macaques of the immunisation study 45 3.3 Cytokine and chemokine levels during acute SIV infection 47 3.3.1 Differences between species 47 3.3.2 Post-peak plasma cytokine elevations 58 3.4 Development of biomarker realtime PCRs 60 3.4.1 Sequencing of AGM and rhesus biomarker cDNAs 60 3.4.2 Establishing and optimising assays with AGM and rhesus cDNAs 60 3.5 Biomarker expression levels in PBMCs from chronically SIV- infected AGMs and rhesus macaques 64 3.6 Interferon regulatory factor 7 sequence 70 3.7 Interferon alpha ELISPOT 72 3.8 Quantification of antibodies and B-cells 74 3.8.1 Total IgG ELISA 74 3.8.2 Quantification of CD20+ B-cells in AGMs and rhesus macaque PBMCs 77 4 Discussion 79 4.1 The AG3.0 antibody 79 4.1.1 T-cell dependency of secondary B-cell responses to Gag 80 4.1.2 The absence of Gag-specific antibodies in SIVagm-infected AGMs in not absolute 81 4.2 Plasma cytokine profiles in acutely SIV-infected AGMs and rhesus macaques 81 4.2.1 Differences between species 81 4.2.2 Differences between immunisation groups 85 4.3 Development of realtime-PCR assays for the detection of biomarker expression levels 87 4.4 Biomarker expression levels in chronically SIV-infected AGMs and rhesus macaques 89 4.5 Plasmacytoid dendritic cells and IFN alpha production in natural hosts for SIV 95 4.5.1 Interferon alpha responses of AGM and rhesus pDCs upon TLR7/9 stimulation 95 4.5.2 Interferon regulatory factor 7 96 4.5.3 Polymorphisms in the AGM IRF-7 mRNA sequence 99 4.6 Innate activation of memory B-cells 100 4.6.1 Total IgG levels in SIV- infected AGMs and rhesus macaques 100 5 Conclusion and Perspective 102 6 Literature 105 Appendix 117 AGM biomarker sequences 117 Functions of biomarkers investigated 126 Publications 129 Acknowledgements 130The natural hosts of simian immunodeficiency virus (SIV), which include African green monkeys (AGMs), do not develop AIDS despite high viral loads. SIVagm-infected AGMs lack antibodies against the viral Gag protein, which is immunodominant in pathogenic systems, such as rhesus macaques. They do, however, develop strong humoural responses to the viral Env protein. It has been hypothesised that this lack of Gag-specific antibodies contributes to the lack of disease in AGMs, as there is a lack of immune complex deposition in the lymph nodes of natural hosts, which is in contrast to heterologous host systems where the fine structure of the lymph nodes is destroyed. AGMs may therefore have evolved a tolerance to Gag to avoid these immunopathological events. Previous experiments to break the apparent tolerance to Gag were performed by immunising AGMs with SIVagm Gag protein or SIVagm gag DNA, both approaches unexpectedly leading to the induction of Gag-specific antibodies. Upon challenge, however, the titres of such antibodies dropped to undetectable levels, thereby apparently falsifying the hypothesis that AGMs have evolved a state of immunological “tolerance” to Gag. Instead, the results suggested an active suppression of the protein-specific response in AGMs. To bypass this phenomenon, it was initially intended to generate an adeno-associated virus vector expressing the gene for a recombinant anti-Gag antibody. Upon 'gene therapy' with this construct, anti-Gag antibodies produced artificially in muscle cells of AGMs would enter the circulatory system independent of the immune system. However, evidence appeared that due to the trimeric form of the viral spike, the humoural response to Env is T-cell independent, whereas the secondary antibody response to Gag strongly depends on T-cell help. Regulatory mechanisms that suppress general T-helper cell activity would therefore have no effect on the induction of anti-Env antibodies in AGMs but would abrogate the anti-Gag response. It was therefore decided to obtain a systematic overview of immunoregulatory biomarker levels during the acute and chronic phase of infection to identify possible down-regulating mechanisms that result in the absence of anti-Gag antibodies in the natural hosts of SIV. Fortunately, sequential samples taken before and during acute and chronic infection of both macaques and AGMs were available from a previous immunisation study. Luminex multiplex assays were performed to determine the levels of 17 different pro- and anti-inflammatory markers in plasma. Distinct differences in the immune activation profiles of AGMs and rhesus macaques during the acute phase of SIV infection were identified. Rhesus macaques showed distinctly higher levels of immune activation upon SIV-infection than AGMs and there was a clear difference in the timing of peak cytokine elevations, with levels in the AGMs not only being lower, but also occurring later than in rhesus macaques. The different timing in elevations might be a critical important factor for the induction of 'tolerance' to SIV in AGMs. Real-time PCR assays were established for the detection of 34 different biomarker expression levels in the PBMCs of AGM and rhesus macaques, which now provide useful tools for characterising immune activation profiles at the mRNA level. To achieve this, it was necessary to sequence the mRNAs coding for the AGM biomarkers. Polymorphisms in the gene coding for IRF-7, a molecule involved in Toll-like receptor 7 (TLR7) signalling have been hypothesised to be responsible for lower interferon (IFN) alpha responses (and hence reduced immune activation) in plasmacytoid dendritic cells (pDCs) of sooty mangabeys (SM), another natural host species for SIV. The AGM IRF-7 sequence was determined and compared with the SM, human and rhesus sequence. One polymorphism was found to be shared in AGMs and SMs, but different to humans and rhesus. In addition, it was shown that AGM pDCs, like those of SMs, have a reduced ability to secrete Type 1 IFN following stimulation with SIV compared to those of rhesus macaques. Finally, a decrease in total IgG in AGMs and a trend towards an increase of total IgG in rhesus macaques over the course of SIV infection were demonstrated. Ultimately, two strong candidates for the dampening of T helper cell activation and hence B-cell responses to T-cell dependent antigens (such as Gag) in AGMs have been identified: The PD1/PD-L1 induced anergy of T helper cells and the severely reduced capacity for innate memory B-cell activation by TLR7 activation, due to the diminished IFN alpha responses of AGM pDCs to SIV. It is not yet known whether the phenomena of abrogated anti-Gag antibody production and reduced total IgG levels upon SIV- infection in AGMs are mere bystander effects of the infection or whether they indeed have implications for pathogenesis (such as avoiding the trapping of immune complexes in lymph nodes). Investigating these and their correlations with the candidates mentioned above should be investigated more fully in the future.Natürliche Wirte für das simiane Immundefizienzvirus (SIV), z.B. Afrikanische Grüne Meerkatzen (AGMs) entwickeln trotz hoher Viruslasten kein AIDS. Ebenfalls weisen sie keine detektierbaren Antikörpertiter gegen das virale Gag-Protein auf, das im pathogenen Wirtssytem, z.B. in Rhesusmakaken, immunodominant ist; allerdings entwickeln sie hohe Antikörpertiter gegen das virale Env-Protein. Da SIV-infizierte AGMs im Gegensatz zu Rhesusmakaken keine Ablagerungen von Immunkomplexen in Lymphknoten aufweisen und diese nicht zerstört werden, wurde bisher vermutet, dass die Abwesenheit der anti-Gag- Antikörper für den Schutz gegen AIDS in AGMs eine Rolle spielt, indem sie eine Toleranz gegenüber dem Gag-Protein entwickelt haben, um immunpathologische Auswirkungen der Infektion zu vermeiden. In zwei vorausgegangenen Studien wurde versucht, die vermutete Toleranz des Gag-Proteins zu durchbrechen. Hierfür wurden AGMs mit SIVagmGag-Protein bzw. –DNA immunisiert, was überraschenderweise in beiden Fällen Gag-spezifische Antikörper induzierte und die Hypothese der Toleranz des Gag-Proteins in AGMs widerlegte. Allerdings sanken die anti-Gag-Antikörpertiter nach SIVagm-Infektion auf undetektierbare Niveaus, was eine aktive Unterdrückung der Protein-spezifischen Antikörperantwort in AGMs suggerierte. Um dieses Phänomen zu umgehen war ursprünglich geplant, einen adeno-assoziierten viralen Vektor zu entwickeln, der einen rekombinanten anti-Gag-Antikörper exprimiert. Die mit diesem Vektor „gentherapierten“ AGMs sollten so die gewünschten Antikörper in ihren Muskelzellen produzieren, von wo aus sie unabhängig vom Immunsystem in den Blutkreislauf verteilt würden. Es wurde jedoch bekannt, dass die humorale Immunantwort gegen Env aufgrund der trimeren Form des Proteins auf der Virusoberfläche T-Zell unabhängig ist, wogegen die humorale Immunantwort gegen Gag-Proteine stark auf T-Zell Hilfe angewiesen ist. Regulatorische Mechanismen, die die Aktivität von T-helfer Zellen unterdrücken, würden daher die Ausbildung einer humoralen anti-Gag- Antwort verhindern, aber keinen Einfluss auf die Induktion von anti-Env-Antikörpern in AGMs haben. Daher wurde entschieden, einen systematischen Überblick über die Niveaus von immunregulatorischen Biomarkern während der akuten und chronischen Phase der SIV-Infektion zu schaffen, um mögliche herunterregulierende Mechanismen zu identifizieren, die in einer Abwesenheit von anti-Gag-Antikörpern in natürlichen Wirten für SIV resultieren könnten. Fortlaufende Plasma- und Zell- Proben, die während der akuten und chronischen Infektionsphase von AGMs und Rhesusmakaken gewonnen wurden, waren aus einer der vorangegangenen Immunisierungsstudien verfügbar. Luminex Multiplex Tests wurden durchgeführt, um die Niveaus von 17 pro- und anti-inflammatorischen Markern in den Plasmaproben zu bestimmen. Hier konnten starke Unterschiede in der Immunaktivierung von AGMs und Rhesusmakaken während der akuten Infektionsphase festgestellt werden, wobei letztere eine wesentlich höhere Immunaktivierung aufwiesen als die AGMs. Zusätzlich wurde ein klarer Unterschied im zeitlichen Auftreten der gemessenen Höchstwerte der Marker festgestellt, denn die Niveaus in den AGMs waren nicht nur niedriger, sondern erhöhten sich zusätzlich später als in den Rhesusmakaken. Dieser zeitliche Unterschied könnte einen wichtigen Faktor für die Entwicklung einer Immuntoleranz gegenüber SIV in AGMs darstellen. Des Weiteren wurden Real-time PCR Tests für die Detektion von Expressionsniveaus von 32 verschiedenen Biomarkern in PBMCs von AGMs und Rhesusmakaken entwickelt, die nun nützliche Werkzeuge für die Charakterisierung von Immunaktivierungsprofilen auf mRNA Niveau bereitstellen. Um diese Tests zu generieren, mussten zunächst die für die Biomarker kodierenden mRNAs der AGMs sequenziert werden. Es wurde vermutet, dass Polymorphismen im IRF-7 Gen, das für ein in den Toll-like-Rezeptor 7 (TLR7) Signalweg involviertes Molekül kodiert, für niedrigere Interferon (IFN) alpha- Ausschüttung (und daher eine niedrigere Immunaktivierung) in plasmazytoiden dendritischen Zellen (pDCs) von Rauchmangaben (einer anderen natürlichen Wirtsspezies für SIV) im Vergleich zu Rhesusmakaken verantwortlich sind. Die erhaltene AGM IRF-7 Sequenz wurde mit der von Rauchmangaben, Menschen und Rhesusmakaken verglichen und ein Polymorphismus wurde identifiziert, der nur in den natürlichen Wirten für SIV, AGMs und Rauchmangaben, aber nicht in den heterologen Wirten für SIV, Rhesusmakaken und Menschen, vorhanden ist. Zusätzlich wurde gezeigt, dass AGM pDCs nach Stimulation mit HIV oder SIV wie Rauchmangaben ebenfalls geringere Mengen IFN alpha als pDCs von Rhesusmakaken sezernieren. Des Weiteren wurde gezeigt, dass AGMs nach der SIV-infektion einen erniedrigten gesamt-Antikörpertiter im Plasma aufweisen, während dieser in Rhesusmakaken zu einer Zunahme tendiert. Schlussendlich konnten zwei Kandidaten, die für die Abschwächung der T-Helfer Zell-Aktivierung in SIV- infizierten AGMs eine Rolle spielen könnten, identifiziert werden: Eine PD1-PDL1-induzierte Anergie von T-Helfer Zellen sowie die stark verringerte Kapazität der nativen Aktivierung von Gedächtnis-B-Zellen durch eine Aktivierung von TLR7, die aus den reduzierten IFN alpha Ausschüttungen der AGM pDCs nach SIV-Stimulation resultieren. Es ist bisher nicht bekannt, ob das Phänomen der abwesenden anti-Gag-Antikörper und die Reduktion der gesamt- Antikörpertiter nach SIV-Infektion in AGMs reine Nebeneffekte der Infektion sind oder ob diese tatsächlich Auswirkungen auf die Pathogenese haben, wie z.B. ein Verhindern der Ablagerung von Immunkomplexen in lymphatischen Geweben. Gegenstand zukünftiger Studien sollte sein, die Korrelation dieser Phänomene mit den neu identifizierten Kandidaten zu untersuchen

Erfassung der SARS-CoV-2-Testzahlen in Deutschland

Im Rahmen der COVID-19-Pandemie spielt die Labordiagnostik zu SARS-CoV-2 eine entscheidende Rolle. Die Bedeutung liegt nicht nur in der diagnostischen Abklärung, sondern hat eine herausragende Stellung für die Beurteilung der epidemiologischen Entwicklung und hinsichtlich Strategien zur Verlangsamung des aktuellen Geschehens in Deutschland. Daten zur Anzahl der durchgeführten Testungen zu SARS-CoV-2 sowie zur Anzahl der Personen mit den jeweiligen Testergebnissen und den Testkapazitäten werden aktuell über verschiedene Netzwerke bzw. Umfragen erhoben. Dazu gibt der Artikel einen Überblick

Erfassung der SARS-CoV-2-Testzahlen in Deutschland

Im Rahmen der COVID-19-Pandemie spielt die Labordiagnostik zu SARS-CoV-2 eine entscheidende Rolle. Die Bedeutung liegt nicht nur in der diagnostischen Abklärung, sondern hat eine herausragende Stellung für die Beurteilung der epidemiologischen Entwicklung und hinsichtlich Strategien zur Verlangsamung des aktuellen Geschehens in Deutschland. Daten zur Anzahl der durchgeführten Testungen zu SARS-CoV-2 sowie zur Anzahl der Personen mit den jeweiligen Testergebnissen und den Testkapazitäten werden aktuell über verschiedene Netzwerke bzw. Umfragen erhoben. Dazu gibt der Artikel einen Überblick

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

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