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ZusammenfassungDie Erfassung von Daten zu SARS-CoV-2-Testungen in Deutschland sind für die Einschätzung des Infektionsgeschehens im Rahmen der COVID-19-Pandemie von zentraler Bedeutung. Am Robert Koch-Institut (RKI) werden dazu die Daten aus verschiedenen Systemen zur Erfassung von Labortestungen zusammengeführt. In diesem Beitrag werden zunächst bedeutsame Aspekte der Testverfahren erläutert. Nachfolgend werden die unterschiedlichen Systeme zur Erfassung von Labortestungen erläutert und Testzahlen aus der RKI-Testlaborabfrage und der laborbasierten Surveillance SARS-CoV‑2 sowie die Abrechnungsdaten der kassenärztlichen Vereinigungen zu SARS-CoV-2-Labortestungen dargestellt.Mit der RKI-Testlaborabfrage und der laborbasierten Surveillance SARS-CoV‑2 stand früh in der Pandemie eine Surveillance zur Verfügung, mit der unter den teilnehmenden Laboren Daten zu durchgeführten Testungen und Testkapazitäten ausgewertet werden können. Durch die Erfassung von positiven und negativen Testergebnissen sind Aussagen zur Gesamtzahl der durchgeführten Testungen sowie dem Anteil der positiven Testergebnisse möglich. Während die aggregierten Testzahlen bundesweit weitestgehend repräsentativ sind, ist die Repräsentativität auf Bundesland- und Landkreisebene aber nicht immer gegeben. Die Abrechnungsdaten der Kassenärztlichen Vereinigungen können die Labordaten im Nachhinein ergänzen. Sie erlauben eine retrospektive Einschätzung der Gesamtzahl von SARS-CoV-2-Tests in Deutschland, da die Leistungen der vertragsärztlichen Versorgung aller gesetzlich Krankenversicherten (ca. 85 % der Bevölkerung) enthalten sind.Die verschiedenen Systeme zur Erfassung von Labortestungen ergänzen sich gegenseitig. Die Auswertungen fließen in die Maßnahmenempfehlungen zur Pandemiebewältigung ein.

Currently, airborne transmission is seen as the most important transmission path for SARS-CoV-2. In this investigation, a classic dose-response model is used on the one hand to find out retrospectively the probable viral load of the infectious source patient at the time of transmission in 25 documented outbreaks. We showed that an infection due to airborne transmission at a distance from the infectious person was probably only possible in the 25 outbreaks examined, with attack rates of 4-100%, if the viral load had been higher than 1E+08 viral copies/ml. This demonstrates that the viral load estimated from the swab might overestimate a person’s infectivity via aerosol, because a person is generally considered infectious, independent of the transmission way, when the viral load from the swab is 1E+06 viral copies/ml.On the other hand, a possible approach is presented to predict the probable situational Attack Rate (PARs) of a group of persons in a room through aerosol particles emitted by an infectious source patient. Four main categories of influence on the risk of infection are formed: First the emitted viruses, depending on the viral load and the amount of respiratory particles, and necessary number of reproducible viruses for infection, second the room-specific data and duration of stay of the group of people, third the activity of the exposed persons, and fourth the effect of personal protection (e.g. wearing masks from infectious and/or susceptible person).Furthermore, a simplified method is presented to calculate either the maximum possible number of persons in a room, so that probably a maximum of one person becomes infected when an infectious person is in the room, or the PARs,simplefor a given number of persons, ventilation rate and time of occupancy. We additionally show, taking into account organizational preventive measures, which person-related virus-free supply air flow rates are necessary to keep the number of newly infected persons to less than 1. The simple approach makes it easy to derive preventive organizational and ventilation measures. Our results show that the volume flow rate or a person-related flow rate is a much more effective parameter to evaluate ventilation for infection prevention than the air change rate. We suggest to monitor the CO2concentration as an easy to implement and valid measurement system for indoor spaces.Finally, we show that of the three measures, besides of wearing masks and increasing ventilation, testing contributes the most to the joint protective effect. This corresponds to the classic approach to implement protection concepts: preventing the source from entering the room and emitting viruses at all. In summary, a layered approach of different measures is recommended to mutually compensate for possible failures of any one measure (e.g. incorrect execution of tests, incorrect fit of masks or irregular window opening), to increase the degree of protection and thus reduce the risk of transmission of SARS-CoV-2.

Since January 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread to become a global pandemic [1]. Active case finding, early detection and isolation of cases and their contacts are essential for breaking transmission chains. A modelling study showed that 70% of contacts should be traced in order to control the outbreak, assuming a baseline reproduction rate of 2.5 [2]. Early warning systems for the serious cross-border spread of infectious pathogens include the International Health Regulations (IHR) 2005 and the Early Warning and Response System (EWRS) for the European Union/European Economic Area (EU/EEA) countries [3,4]. Within Germany, communication channels have been established in accordance with the German Infection Protection Act (Infektionsschutzgesetz; IfSG). Cross-border contact tracing at the national level is operated by the Robert Koch Institute (RKI), the federal public health institute in Germany. The first cases of coronavirus disease 2019 (COVID-19) in Germany occurred in Bavaria at the end of January 2020 [5]. The first SARS-CoV-2 cluster also led to cross-border contacts and exposures on flights since close contacts and suspected cases travelled to Austria and Spain after exposure. This required intensive international communication to identify and share the information on contacts with the responsible health authorities. An international communication and contact tracing team (RKI IC-Team) was rapidly created in the RKI COVID-19 Emergency Operations Centre (EOC) including members of all units of the department for infectious disease epidemiology and other departments at the RKI. The core task of the team was to collect and communicate information on confirmed COVID-19 cases and their contacts to other countries in the event of cross-border relevance. In addition, incoming information on German citizens exposed abroad was communicated through the federal state health authorities to the responsible local health authorities in Germany. The spread of SARS-CoV-2 in Germany triggered the introduction of various measures: (i) mass gatherings with more than 1,000 participants were banned after calendar week 10, (ii) schools and public places were closed in several federal states, (iii) physical distancing measures of at least 1.5 m to another person were recommended, (iv) it was recommended to cancel non-essential travel and (v) quarantine measures for travellers from high risk areas entering Germany were introduced in calendar week 15. Because of the federal structure in Germany, the measures and their implementation varied between the states. This work aimed to describe the extent and course of activities resulting from information on COVID-19 exposure events with a cross-border context. Further, we discuss the challenges experienced and possible workflow improvements.

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.

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.

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 100pg/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.

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 MV vac2 genome, and the respective viruses were rescued (MV vac2 -CoV-S and MV vac2 -CoV-solS). These recombinant MVs were amplified and characterized at passages 3 and 10. The replication of MV vac2 -CoV-S in Vero cells turned out to be comparable to that of the control virus MV vac2 -GFP (encoding green fluorescent protein), while titers of MV vac2 -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 MV vac2 -CoV-S(H) or MV vac2 -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.

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.

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.

Following the publication of this article (Journal of Translational Medicine, 8:72), it was noted that a co-author had been omitted from the authors list. The submitting authors would like to apologise to Hannes Uchtenhagen for this error. The Competing interests and Authors' contributions sections have now been updated to reflect this amendment.