Early nucleocapsid-specific T cell responses associate with control of SARS-CoV-2 in the upper airways and reduced systemic inflammation before seroconversion (preprint)

Despite intensive research since the emergence of SARS-CoV-2, it has remained unclear precisely which components of the early immune response protect against the development of severe COVID-19. To address this issue, we performed a comprehensive immunogenetic and virologic analysis of nasopharyngeal and peripheral blood samples obtained during the acute phase of infection with SARS-CoV-2. We found that soluble and transcriptional markers of systemic inflammation peaked during the first week after symptom onset and correlated directly with the upper airways viral loads (UA-VLs), whereas the contemporaneous frequencies of circulating viral nucleocapsid (NC)-specific CD4+ and CD8+ T cells correlated inversely with various inflammatory markers and UA-VLs. In addition, we observed high frequencies of activated CD4+ and CD8+ T cells in acutely infected nasopharyngeal tissue, many of which expressed genes encoding various effector molecules, such as cytotoxic proteins and IFN-γ. The presence of functionally active T cells in the infected epithelium was further linked with common patterns of gene expression among virus-susceptible target cells and better local control of SARS-CoV-2. Collectively, these results identified an immune correlate of protection against SARS-CoV-2, which could inform the development of more effective vaccines to combat the acute and chronic illnesses attributable to COVID-19.

From first to second wave: follow-up of the prospective Covid-19 cohort (KoCo19) in Munich (Germany) (preprint)

Background: In the 2nd year of the Covid-19 pandemic, knowledge about the dynamics of the infection in the general population is still limited. Such information is essential for health planners, as many of those infected show no or only mild symptoms and thus, escape the surveillance system. We therefore aimed to describe the course of the pandemic in the Munich general population living in private households from April 2020 to January 2021. Methods: The KoCo19 baseline study took place from April to June 2020 including 5313 participants (age 14 years and above). From November 2020 to January 2021, we could again measure SARS-CoV-2 antibody status in 4,433 of the baseline participants (response 83%). Participants were offered a self-sampling kit to take a capillary blood sample (dry blood spot; DBS). Blood was analysed using the Elecsys(R) Anti-SARS-CoV-2 assay (Roche). Questionnaire information on socio-demographics and potential risk factors assessed at baseline was available for all participants. In addition, follow-up information on health-risk taking behaviour and number of personal contacts outside the household (N=2768) as well as leisure time activities (N=1263) were collected in summer 2020. Results: Weighted and adjusted (for specificity and sensitivity) SARS-CoV-2 sero-prevalence at follow-up was 3.6% (95% CI 2.9-4.3%) as compared to 1.8% (95% CI 1.3-3.4%) at baseline. 91% of those tested positive at baseline were also antibody-positive at follow-up. While sero-prevalence increased from early November 2021 to January 2021, no indication of geospatial clustering across the city of Munich was found, although cases clustered within households. Taking baseline result and time to follow-up into account, men and participants in the age group 20-34 years were at the highest risk of sero-positivity. In the sensitivity analyses, differences in health-risk taking behaviour, number of personal contacts and leisure time activities partly explained these differences. Conclusion: The number of citizens in Munich with SARS-CoV-2 antibodies was still below 5% during the 2nd wave of the pandemic. Antibodies remained present in the majority of baseline participants. Besides age and sex, potentially confounded by differences in behaviour, no major risk factors could be identified. Non-pharmaceutical public health measures are thus still important.

In search for the SARS-CoV-2 protection correlate: A head-to-head comparison of two quantitative S1 assays in a group of pre-characterized oligo-/asymptomatic patients (preprint)

Background Quantitative serological assays detecting response to SARS-CoV-2 infection are urgently needed to quantify immunity. This study analyzed the performance and correlation of two independent quantitative anti-S1 assays in oligo-/asymptomatic individuals from a previously characterized population-based cohort. Methods A total of 362 samples included 108 from individuals who had viral RNA detected in pharyngeal swabs, 111 negative controls and 143 samples with positive serology but not confirmed by RT-PCR. Blood plasma was tested with quantitative assays Euroimmun Anti-SARS-CoV-2 QuantiVac ELISA (IgG) (EI-S1-IgG-quant) and Roche Elecsys ® Anti-SARS-CoV-2 CoV-2 S (Ro-RBD-Ig-quant), which were compared with each other and with confirmatory tests, including wild-type virus micro-neutralization (NT) and GenScript ® cPass™. Results were analyzed using square roots R of coefficients of determination for association among continuous variables and non-parametric tests for paired comparisons. Results Quantitative anti-S1 serology correlated well with each other (96%/97% for true-positives and true-negatives, respectively). Antibody titers decreased over time (from <30 days to >240 days after initial positive RT-PCR). Agreement with GenScript-cPass was 96%/99% for true-positives and true-negatives, respectively, for Ro-RBD-Ig-quant and 93%/97% for EI-S1-IgG-quant. Ro-RBD-Ig-quant allowed a distinct separation between positive and negative values, and less non-specific reactivity compared with EI-S1-IgG-quant. Raw values (with 95% CI) ≥28.7 U/mL (22.6–36.4) for Ro-RBD-Ig-quant and ≥49.8 U/mL (43.4–57.1) for EI-S1-IgG-quant predicted virus neutralization >1:5 in 95% of cases. Conclusions Both quantitative anti-S1 assays, Ro-RBD-Ig-quant and EI-S1-IgG-quant, may replace direct neutralization assays in quantitative measurement of immune protection against SARS-CoV-2 in certain circumstances in the future. Key points Two quantitative anti-S1 assays showed similar performance and a high level of agreement with direct virus neutralization and surrogate neutralization tests, arguing for their utility in quantifying immune protection against SARS-CoV-2.

A Serology Strategy for Epidemiological Studies Based on the Comparison of the Performance of Seven Different Test Systems - The Representative COVID-19 Cohort Munich (preprint)

BackgroundSerosurveys are essential to understand SARS-CoV-2 exposure and enable population-level surveillance, but currently available tests need further in-depth evaluation. We aimed to identify testing-strategies by comparing seven seroassays in a population-based cohort. MethodsWe analysed 6,658 samples consisting of true-positives (n=193), true-negatives (n=1,091), and specimens of unknown status (n=5,374). For primary testing, we used Euroimmun-Anti-SARS-CoV-2-ELISA-IgA/IgG and Roche-Elecsys-Anti-SARS-CoV-2; and virus-neutralisation, GeneScript(R)cPass, VIRAMED-SARS-CoV-2-ViraChip(R), and Mikrogen-recomLine-SARS-CoV-2-IgG, including common-cold CoVs, for confirmatory testing. Statistical modelling generated optimised assay cut-off-thresholds. FindingsSensitivity of Euroimmun-anti-S1-IgA was 64.8%, specificity 93.3%; for Euroimmun-anti-S1-IgG, sensitivity was 77.2/79.8% (manufacturers/optimised cut-offs), specificity 98.0/97.8%; Roche-anti-N sensitivity was 85.5/88.6%, specificity 99.8/99.7%. In true-positives, mean and median titres remained stable for at least 90-120 days after RT-PCR-positivity. Of true-positives with positive RT-PCR (<30 days), 6.7% did not mount detectable seroresponses. Virus-neutralisation was 73.8% sensitive, 100.0% specific (1:10 dilution). Neutralisation surrogate tests (GeneScript(R)cPass, Mikrogen-recomLine-RBD) were >94.9% sensitive, >98.1% specific. Seasonality had limited effects; cross-reactivity with common-cold CoVs 229E and NL63 in SARS-CoV-2 true-positives was significant. ConclusionOptimised cut-offs improved test performances of several tests. Non-reactive serology in true-positives was uncommon. For epidemiological purposes, confirmatory testing with virus-neutralisation may be replaced with GeneScript(R)cPass or recomLine-RBD. Head-to-head comparisons given here aim to contribute to the refinement of testing-strategies for individual and public health use.

Prevalence and Risk Factors of Infection in the Representative COVID-19 Cohort Munich (preprint)

Background: Population-based studies investigating the dynamics of the SARS-CoV-2 pandemic are needed. Here, we report on baseline findings from April through June 2020 of a prospective cohort study in Munich, Germany.Methods: We drew a representative sample of 2994 private households. The 5313 participating household members 14 years and older completed questionnaires and provided blood samples. SARS-CoV-2 seropositivity was defined as Roche N pan-Ig ≥ 0·4218. We adjusted the prevalence for the sampling design, sensitivity, and specificity. We investigated risk factors for SARS-CoV-2 seropositivity and geospatial transmission patterns by generalized linear mixed models and permutation tests.Findings: Seropositivity for SARS-CoV-2 specific antibodies was 1·82% (95% confidence interval (CI) 1·28-2·37%) as compared to 0·46% PCR-positive cases officially registered in Munich. Loss of the sense of smell or taste was associated with seropositivity (odds ratio (OR) 47·4; 95% CI 7·2-307·0) and infections clustered within households. Participants suffering from respiratory allergies (OR 2·7; 95% CI 0·9-8·6) and working in high-risk jobs (OR 2·0; 95% CI 0·5-6·7) showed non-significantly increased odds for SARS-CoV-2 seropositivity.Interpretation: Applying a validated assay, we demonstrate a low SARS-CoV-2 seroprevalence in the Munich population 14 years and older towards the end of the first pandemic wave. However, we noted official sub-registration at this early stage of the pandemic.Funding: Bavarian State Ministry of Science and the Arts, University Hospital of LMU Munich, Helmholtz Centre Munich, University of Bonn, and University of Bielefeld.Declaration of Interests: FF, TF, DM, LO, and VT report grants from the Bavarian State Ministry of Science and the Arts during the conduct oft he study. TF reports grants from the University Hospital of LMU Munich, Helmholtz Center Munich, University of Bonn, University of Bielefeld, and German Ministry for Education and Research during the conduct of the study. JH reports grants from the German Federal Ministry of Education and Research during the conduct of the study. MH and AW report personal fees and non-financial support, LO and MP report non-financial support from Roche Diagnostics. MH, LO, MP, and AW report non-financial support from Euroimmun, Viramed, and Mikrogen. MH, MP, and AW report grants, non-financial support, and other from German Center for Infection Research (DZIF). FF, MH, LO, MP, VT, and AW report grants and non-financial support from the Government of Bavaria. MH, LO, MP, and AW report non-financial support from BMW, Mercedes Benz, Munich Police, and Accenture. MH and AW report personal fees and non-financial support from Dr. Box Betrobox during the conduct of the study. LO and MP report non-financial support from Dr. Box Betrobox. MH and AW have a patent Sample System for Sputum Diagnostics of SARS-CoV-2 pending. DM reports to be a a sub-investigator on a Phase I SARS-CoV-2 vaccine trial and on a Phase I rabies vaccine trial, both sponsored by CureVac AG. MP and AW report non-financial support from Dr. Becker MVZ. VT reports support from CureVac AG outside the submitted work. AW reports personal fees and other from Haeraeus Sensors. AW reports non-financial support from Bruker Daltronics outside the submitted work. AW is involved in other different patents and companies not in relation with the serology of SARS-CoV-2. All other authors report nothing to disclose.Ethics Approval Statement: Prior to study initiation, this study had been approved by therespective Institutional Review Board.

Protocol of a population-based prospective COVID-19 cohort study Munich, Germany (KoCo19) (preprint)

Background: The SARS-CoV-2 pandemic is leading to the global introduction of public health interventions to prevent the spread of the virus and avoid the overload of health care systems, especially for the most severely affected patients. Scientific studies to date have focused primarily on describing the clinical course of patients, identifying treatment options and developing vaccines. In Germany, as in many other regions, current tests for SARS-CoV2 are not being conducted on a representative basis and in a longitudinal design. Furthermore, knowledge about the immune status of the population is lacking. Yet these data are needed to understand the dynamics of the pandemic and to thus appropriately design and evaluate interventions. For this purpose, we recently started a prospective population-based cohort in Munich, Germany, with the aim to better understand the state and dynamics of the pandemic. Methods: In 100, randomly selected constituencies out of 755, 3,000 Munich households are identified via random route and offered enrollment into the study. All household members are asked to complete a baseline questionnaire and subjects [≥]14 years of age are asked to provide a venous blood sample of [≤]3 ml for the determination of SARS-CoV-2 IgG/IgA status. The residual plasma and the blood pellet are preserved for later genetic and molecular biological investigations. For twelve months, each household member is asked to keep a diary of daily symptoms, whereabouts and contacts via WebApp. If symptoms suggestive for COVID-19 are reported, family members, including children <14 years, are offered a pharyngeal swab taken at the Division of Infectious Diseases and Tropical Medicine, LMU University Hospital Munich, for molecular testing for SARS-CoV-2. In case of severe symptoms, participants will be transferred to a Munich hospital. For one year, the study teams re-visits the households for blood sampling every six weeks. Discussion: With the planned study we will establish a reliable epidemiological tool to improve the understanding of the spread of SARS-CoV-2 and to better assess the effectiveness of public health measures as well as their socio-economic effects. This will support policy makers in managing the epidemic based on scientific evidence.