Impaired humoral immunity to BQ.1.1 in convalescent and vaccinated patients (preprint)

Determining SARS-CoV-2 immunity is critical to assess COVID-19 risk and the need for prevention and mitigation strategies. We measured SARS-CoV-2 Spike/Nucleocapsid seroprevalence and serum neutralizing activity against Wu01, BA.4/5 and BQ.1.1 in 1,411 individuals who received medical treatment in five emergency departments in North Rhine-Westphalia, Germany. We detected Spike-IgG in 95.6%, Nucleocapsid-IgG in 24.0% and neutralization against Wu01, BA.4/5 and BQ.1.1 in 94.4%, 85.0%, and 73.8% of participants, respectively. Neutralization against BA.4/5 and BQ.1.1 was reduced 5.6- and 23.4-fold compared to Wu01. Accuracy of S-IgG detection for determination of neutralizing activity against BQ.1.1 was reduced substantially. Furthermore, we explored previous vaccinations and infections as most important correlates of improved BQ.1.1 neutralization using multivariable and Bayesian network analyses. Given an adherence to COVID-19 vaccination recommendations of only 67.7% of all participants, we highlight the need for improvement of vaccine-uptake to reduce the COVID-19 risk in upcoming infection-waves with immune evasive variants.

The unique ORF8 protein from SARS-CoV-2 binds to human dendritic cells and induces a hyper-inflammatory cytokine storm (preprint)

The novel coronavirus pandemic, whose first outbreak was reported in December 2019 in Wuhan, China (COVID-19), is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Tissue damage caused by the virus leads to a strong immune response and activation of antigen-presenting cells, which can elicit acute respiratory distress syndrome (ARDS) characterized by the rapid onset of widespread inflammation, the so-called cytokine storm. In many viral infections the recruitment of monocytes into the lung and their differentiation to dendritic cells (DCs) are seen as a response to the viral infection. DCs are critical players in the development of the acute lung inflammation that causes ARDS. Here we focus on the interaction of the ORF8 protein, a specific SARS-CoV-2 open reading frame protein, with dendritic cells (DCs). We show that ORF8 binds to dendritic cells, causes a pre-maturation of differentiating DCs, and induces the secretion of multiple pro-inflammatory cytokines by these cells. In addition, we identified dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) as a possible interaction partner of ORF8 on dendritic cells. Blockade of ORF8 signaling leads to reduced production of IL-1{beta}, IL-6, IL-12p70, TNF-, MCP-1 (CCL2), and IL-10 by dendritic cells. Analysis of patient sera with high anti-ORF8 antibody titers showed that there was nearly no neutralization of the ORF8 protein and its function. Therefore, a neutralizing antibody that has the capacity of blocking the cytokine and chemokine response mediated by ORF8 protein might be an essential and novel additional step in the therapy of severe SARS-CoV-2 cases.

Effective high-throughput RT-qPCR screening for SARS-CoV-2 infections in children (preprint)

Systematic SARS-CoV-2 testing is a valuable tool for infection control and surveillance. However, broad application of high sensitive RT-qPCR testing in children is often hampered due to unpleasant sample collection, limited RT-qPCR capacities, and high costs. Here, we developed a high-throughput approach (Lolli-Method) for sensitive SARS-CoV-2 detection in children, combining non-invasive sample collection with an RT-qPCR-pool testing strategy. SARS-CoV-2 infections were diagnosed with sensitivities of 100% and 93.9% when viral loads were >10E6 copies/ml and >10E3 copies/ml in corresponding Naso-/Oropharyngeal-swabs, respectively. For effective application of the Lolli-Method in schools and daycare facilities, SIR-modeling indicated a preferred frequency of two tests per week. The developed test strategy was implemented in 3,700 schools and 698 daycare facilities in Germany, screening over 800,000 individuals twice per week. In a period of 3 months, 6,364 pool-RT-qPCRs tested positive (0.64%) ranging from 0.05% to 2.61% per week. Notably, infections correlated with local SARS-CoV-2 incidences as well as with a school social deprivation index. Moreover, in comparison with the alpha variant, statistical modeling revealed a 31% increase for multiple (>1 child) infections per class following infections with the delta variant. We conclude that the Lolli-Method is a powerful tool for SARS-CoV-2 surveillance and infection control in schools and daycare facilities.

No substantial pre-existing B cell immunity against SARS-CoV-2 in healthy adults (preprint)

Pre-existing immunity against SARS-CoV-2 may have critical implications for our understanding of COVID-19 susceptibility and severity. Various studies recently provided evidence of pre-existing T cell immunity against SARS-CoV-2 in unexposed individuals. In contrast, the presence and clinical relevance of a pre-existing B cell immunity remains to be fully elucidated. Here, we provide a detailed analysis of the B cell response to SARS-CoV-2 in unexposed individuals. To this end, we extensively investigated the memory B cell response to SARS-CoV-2 in 150 adults sampled pre-pandemically. Comprehensive screening of donor plasma and purified IgG samples for binding and neutralization in various functional assays revealed no substantial activity against SARS-CoV-2 but broad reactivity to endemic betacoronaviruses. Moreover, we analyzed antibody sequences of 8,174 putatively SARS-CoV-2-reactive B cells on a single cell level and generated and tested 158 monoclonal antibodies. None of the isolated antibodies displayed relevant binding or neutralizing activity against SARS-CoV-2. Taken together, our results show no evidence of relevant pre-existing antibody and B cell immunity against SARS-CoV-2 in unexposed adults.

Kinetics and Correlates of the Neutralizing Antibody Response to SARS-CoV-2 (preprint)

A detailed understanding of antibody-based SARS-CoV-2 immunity has critical implications for overcoming the COVID-19 pandemic and informing vaccination strategies. We evaluated SARS-CoV-2 antibody response dynamics in a cohort of 963 individuals over 10 months. Investigating 2,146 samples, we initially detected SARS-CoV-2 antibodies in 94.4% individuals, with 82% and 79% exhibiting serum and IgG neutralization, respectively. Approximately 3% of patients demonstrated exceptional SARS-CoV-2-neutralization, with these ‘elite neutralizers’ also possessing cross-neutralizing IgG to SARS-CoV-1. Multivariate statistical modeling revealed sero-reactivity, age and fever as key factors predicting SARS-CoV-2 neutralizing activity. A loss of anti-spike reactivity in 13% individuals was detected 10 months after infection. Neutralizing activity had half-lives of 14.7 weeks in serum versus 31.4 weeks in purified IgG, indicating a stable long-term memory IgG B-cell repertoire. Our results demonstrate a broad spectrum in the initial SARS-CoV-2-neutralizing antibody response, with sustained antibodies in majority of individuals for 10 months after mild COVID-19.Funding: This work was funded by grants to Florian Kleinfrom the German Center for Infection Research (DZIF), the German Research Foundation (DFG) CRC1279 and CRC1310, European Research Council (ERC) ERC-stG639961 and COVIM: „NaFoUniMedCovid19“ (FKZ: 01KX2021).Ethical Approval: Blood samples were collected from donors who gave their written consent under the protocols 20-1187 and 16-054, approved by the Institutional Review Board (IRB) of the University Hospital Cologne.

Evaluation of a new spike (S) protein based commercial immunoassay for the detection of anti-SARS-CoV-2 IgG (preprint)

BackgroundThe investigation of antibody response to SARS-CoV-2 represents a key aspect in facing the COVID-19 pandemic. In the present study, we compared one new and four widely used commercial serological assays for the detection of antibodies targeting S (spike) and NC (nucleocapsid) protein. MethodsSerum samples from a group of apparently non-responders, from an unbiased group of convalescent patients and from a negative control group were sim-ultaneously analyzed by the LIAISON(R) SARS-CoV-2 S1/S2 IgG test, Euroimmun anti-SARS-CoV-2 S1 IgG ELISA and IDK(R) anti-SARS-CoV-2 S1 IgG assays. IgG binding NC were detected by the Abbott SARS-CoV-2 IgG assay and by the panimmunoglobulin immunoassay Elecsys(R) Anti-SARS-CoV-2. Additionally, samples were also tested by live virus and pseudovirus neutralization tests. ResultsOverall, about 50% of convalescent patients with undetectable IgG antibodies using the commercial kit by Euroimmun were identified as IgG positive by Immundiagnostik and Roche. While both assays achieved similarly high sensitivities, Immundiagnostik correlated better with serum neutralizing activity than Roche. ConclusionsAlthough the proportion of IgG seropositive individuals appears to be higher using more sensitive immunoassays, the protective ability and the potential to serve as indirect markers of other beneficial immune responses warrants for further research.

Kinetics and correlates of the neutralizing antibody response to SARS-CoV-2 (preprint)

A detailed understanding of antibody-based SARS-CoV-2 immunity has critical implications for overcoming the COVID-19 pandemic and for informing on vaccination strategies. In this study, we evaluated the dynamics of the SARS-CoV-2 antibody response in a cohort of 963 recovered individuals over a period of 10 months. Investigating a total of 2,146 samples, we detected an initial SARS-CoV-2 antibody response in 94.4% of individuals, with 82% and 79% exhibiting serum and IgG neutralization, respectively. Approximately 3% of recovered patients demonstrated exceptional SARS-CoV-2 neutralizing activity, defining them as ‘elite neutralizers’. These individuals also possessed effective cross-neutralizing IgG antibodies to SARS-CoV-1 without any known prior exposure to this virus. By applying multivariate statistical modeling, we found that sero-reactivity, age, time since disease onset, and fever are key factors predicting SARS-CoV-2 neutralizing activity in mild courses of COVID-19. Investigating longevity of the antibody response, we detected loss of anti-spike reactivity in 13% of individuals 10 months after infection. Moreover, neutralizing activity had an initial half-life of 6.7 weeks in serum versus 30.8 weeks in purified IgG samples indicating the presence of a more stable and long-term memory IgG B cell repertoire in the majority of individuals recovered from COVID-19. Our results demonstrate a broad spectrum of the initial SARS-CoV-2 neutralizing antibody response depending on clinical characteristics, with antibodies being maintained in the majority of individuals for the first 10 months after mild course of COVID-19.

Rapid SARS-CoV-2 testing in primary material based on a novel multiplex LAMP assay (preprint)

Background: Rapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and decision-making in times of a pandemic outbreak. However, point-of-care (POC) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. The need for thermal cycling and nucleic acid isolation hampers the use of standard PCR-based methods for this purpose. Methods: To avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including loop-mediated isothermal amplification (LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK). Results: Whilst specificity of standard LAMP assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qPCR) assays yet. We describe a novel multiplexed LAMP approach and validate its sensitivity on primary samples. This approach allows for fast and reliable identification of infected individuals. Primer optimization and multiplexing helps to increase sensitivity significantly. In addition, we directly compare and combine our novel LAMP assays with SHERLOCK. Conclusion: In summary, this approach reveals one-step multiplexed LAMP assays as a prime-option for the development of easy and cheap POC test kits.

Sensitivity of commercial Anti-SARS-CoV-2 serological assays in a high-prevalence setting (preprint)

We analysed SARS-CoV-2 specific antibody responses in 42 social and working contacts of a super-spreader from the Heinsberg area in Germany. Consistent with a high-prevalence setting 26 individuals had SARS-CoV-2 antibodies determined by in-house neutralisation testing. These results were compared with four commercial assays, suggesting limited sensitivity of the assays in such a high-prevalence setting. Although SARS-CoV-2 nucleocapsid-restricted tests showed a better sensitivity, spike-based assays had a stronger correlation with neutralisation capacity.

Longitudinal isolation of potent near-germline SARS-CoV-2-neutralizing antibodies from COVID-19 patients (preprint)

The SARS-CoV-2 pandemic has unprecedented implications for public health, social life, and world economy. Since approved drugs and vaccines are not available, new options for COVID-19 treatment and prevention are highly demanded. To identify SARS-CoV-2 neutralizing antibodies, we analysed the antibody response of 12 COVID-19 patients from 8 to 69 days post diagnosis. By screening 4,313 SARS-CoV-2-reactive B cells, we isolated 255 antibodies from different time points as early as 8 days post diagnosis. Among these, 28 potently neutralized authentic SARS-CoV-2 (IC100 as low as 0.04 g/ml), showing a broad spectrum of V genes and low levels of somatic mutations. Interestingly, potential precursors were identified in naive B cell repertoires from 48 healthy individuals that were sampled before the COVID-19 pandemic. Our results demonstrate that SARS-CoV-2 neutralizing antibodies are readily generated from a diverse pool of precursors, fostering the hope of rapid induction of a protective immune response upon vaccination.