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Summary The SARS-CoV-2 pandemic has unprecedented implications for public health, social life, and the world economy. Because approved drugs and vaccines are limited or not available, new options for COVID-19 treatment and prevention are in high demand. To identify SARS-CoV-2-neutralizing antibodies, we analyzed the antibody response of 12 COVID-19 patients from 8 to 69 days after diagnosis. By screening 4,313 SARS-CoV-2-reactive B cells, we isolated 255 antibodies from different time points as early as 8 days after diagnosis. Of these, 28 potently neutralized authentic SARS-CoV-2 with IC100 as low as 0.04 μg/mL, showing a broad spectrum of variable (V) genes and low levels of somatic mutations. Interestingly, potential precursor sequences were identified in naive B cell repertoires from 48 healthy individuals who 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 hope for rapid induction of a protective immune response upon vaccination., Graphical Abstract, Highlights • Isolation of highly potent SARS-CoV-2-neutralizing antibodies • Longitudinal sampling reveals early class-switched neutralizing response • SARS-CoV-2 S-protein-reactive antibodies show little somatic mutation over time • Potential antibody precursor sequences identified in SARS-CoV-2-naive individuals, In a longitudinal analysis of SARS-CoV-2-infected people, Kreer et al. find highly potent neutralizing antibodies that use a broad spectrum of variable (V) genes and show low levels of somatic mutations. They also identify potential precursor sequences of these SARS-CoV-2-neutralizing antibodies from virus-naive individuals, sampled before the COVID-19 pandemic. This could indicate that neutralizing antibodies can be readily generated from existing germline antibody sequences found in the general population.

SummaryPre-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.

Understanding antibody-based SARS-CoV-2 immunity is critical for overcoming the COVID-19 pandemic and informing vaccination strategies. We evaluated SARS-CoV-2 antibody dynamics over 10 months in 963 individuals who predominantly experienced mild COVID-19. Investigating 2,146 samples, we initially detected SARS-CoV-2 antibodies in 94.4% of individuals, with 82% and 79% exhibiting serum and IgG neutralization, respectively. Approximately 3% of individuals demonstrated exceptional SARS-CoV-2 neutralization, with these “elite neutralizers” also possessing SARS-CoV-1 cross-neutralizing IgG. Multivariate statistical modeling revealed age, symptomatic infection, disease severity, and gender as key factors predicting SARS-CoV-2-neutralizing activity. A loss of reactivity to the virus spike protein was observed in 13% of individuals 10 months after infection. Neutralizing activity had half-lives of 14.7 weeks in serum versus 31.4 weeks in purified IgG, indicating a rather long-term IgG antibody response. Our results demonstrate a broad spectrum in the initial SARS-CoV-2-neutralizing antibody response, with sustained antibodies in most individuals for 10 months after mild COVID-19., Graphical abstract, Vanshylla et al. report longitudinal antibody kinetics in a mainly mild COVID-19 convalescent cohort of 963 individuals. There is broad variation in the initial response with older age and disease severity predicting higher SARS-CoV-2 neutralizing activity. Neutralizing IgG antibodies are detectable for up to 10 months in the majority of individuals.

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® SARS-CoV-2 S1/S2 IgG test, Euroimmun anti-SARS-CoV-2 S1 IgG ELISA and IDK® 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® 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.

The current Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) pandemic is a public health emergency of international concern. Sensitive and precise diagnostic tools are urgently needed. In this study, we developed a SARS-CoV-2 spike (S1) protein enzyme-linked immunosorbent assay (ELISA) to detect SARS-CoV-2-specific antibodies. The SARS-CoV-2 S1 ELISA was found to be specific [97.8% (95% CI, 96.7% - 98.5%)], reproducible and precise (intra-assay coefficient of variability (CV) 5.3%, inter-assay CV 7.9%). A standard curve and the interpolation of arbitrary ELISA units per milliliter served to reduce the variability between different tests and operators. Cross-reactivity to other human coronaviruses was addressed by using sera positive for MERS-CoV- and hCoV HKU1-specific antibodies. Monitoring antibody development in various samples of twenty-three and single samples of twenty-nine coronavirus disease 2019 (COVID−19) patients revealed seroconversion and neutralizing antibodies against authentic SARS-CoV-2 in all cases. The comparison of the SARS-CoV-2 (S1) ELISA with a commercially available assay showed a better sensitivity for the in-house ELISA. The results demonstrate a high reproducibility, specificity and sensitivity of the newly developed ELISA, which is suitable for the detection of SARS-CoV-2 S1 protein-specific antibody responses., Highlights • A highly sensitive and specific SARS-CoV-2 S1 ELISA was developed. • A standard curve is included to reduce variability between assays and operators. • Excellent intra-assay coefficient of variability (CV) 5.3%, inter-assay CV 7.9%. • Good correlation to the virus neutralization test VNT100.

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.

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.

As exemplified by the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there is a strong demand for rapid high-throughput isolation pipelines to identify potent neutralizing antibodies for prevention and therapy of infectious diseases. However, despite substantial progress and extensive efforts, the identification and production of antigen-specific antibodies remains labor- and cost-intensive. We have advanced existing concepts to develop a highly efficient high-throughput protocol with proven application for the isolation of potent antigen-specific antibodies against human immunodeficiency virus 1, hepatitis C virus, human cytomegalovirus, Middle East respiratory syndrome coronavirus, SARS-CoV-2 and Ebola virus. It is based on computationally optimized multiplex primer sets (openPrimeR), which guarantee high coverage of even highly mutated immunoglobulin gene segments as well as on optimized antibody cloning and production strategies. Here, we provide the detailed protocol, which covers all critical steps from sample collection to antibody production within 12-14 d.

SUMMARYThe 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 (IC100as low as 0.04 μg/ml), showing a broad spectrum of V genes and low levels of somatic mutations. Interestingly, potential precursors were identified in naïve 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.

Human cytomegalovirus (HCMV) causes serious complications to immune compromised hosts. Dendritic cells (iDCgB) expressing granulocyte-macrophage colony-stimulating factor, interferon-alpha and HCMV-gB were developed to promote de novo antiviral adaptive responses. Mice reconstituted with a human immune system (HIS) were immunized with iDCgB and challenged with HCMV, resulting into 93% protection. Immunization stimulated the expansion of functional effector memory CD8+ and CD4+ T cells recognizing gB. Machine learning analyses confirmed bone marrow T/CD4+, liver B/IgA+ and spleen B/IgG+ cells as predictive biomarkers of immunization (≈87% accuracy). CD8+ and CD4+ T cell responses against gB were validated. Splenic gB-binding IgM-/IgG+ B cells were sorted and analyzed at a single cell level. iDCgB immunizations elicited human-like IgG responses with a broad usage of various IgG heavy chain V gene segments harboring variable levels of somatic hypermutation. From this search, two gB-binding human monoclonal IgGs were generated that neutralized HCMV infection in vitro. Passive immunization with these antibodies provided proof-of-concept evidence of protection against HCMV infection. This HIS/HCMV in vivo model system supported the validation of novel active and passive immune therapies for future clinical translation., Author summary Human cytomegalovirus (HCMV) is a ubiquitous pathogen. As long as the immune system is functional, T and B cells can control HCMV. Yet, for patients who have debilitated immune functions, HCMV infections and reactivations cause major complications. Vaccines or antibodies to prevent or treat HCMV are not yet approved. Novel animal models for testing new immunization approaches are emerging and are important tools to identify biomedical products with a reasonable chance to work in patients. Here, we used a model based on mice transplanted with human immune cells and infected with a traceable HCMV. We tested a cell vaccine (iDCgB) carrying gB, a potent HCMV antigen. The model showed that iDCgB halted the HCMV infection in more than 90% of the mice. We found that antibodies were key players mediating protection. Using state-of-the-art methods, we were able to use the sequences of the human antibodies generated in the mice to construct and produce monoclonal antibodies in the laboratory. Proof-of-concept experiments indicated that administration of these monoclonal antibodies into mice protected them against HCMV infection. In summary, this humanized mouse model was useful to test a vaccine and to generate and test novel antibodies that can be further developed for human use.

Recombinant vesicular stomatitis virus–Zaire Ebola virus (rVSV-ZEBOV) is the most advanced Ebola virus vaccine candidate and is currently being used to combat the outbreak of Ebola virus disease (EVD) in the Democratic Republic of the Congo (DRC). Here we examine the humoral immune response in a subset of human volunteers enrolled in a phase 1 rVSV-ZEBOV vaccination trial by performing comprehensive single B cell and electron microscopy structure analyses. Four studied vaccinees show polyclonal, yet reproducible and convergent B cell responses with shared sequence characteristics. EBOV-targeting antibodies cross-react with other Ebolavirus species, and detailed epitope mapping revealed overlapping target epitopes with antibodies isolated from EVD survivors. Moreover, in all vaccinees, we detected highly potent EBOV-neutralizing antibodies with activities comparable or superior to the monoclonal antibodies currently used in clinical trials. These include antibodies combining the IGHV3–15/IGLV1–40 immunoglobulin gene segments that were identified in all investigated individuals. Our findings will help to evaluate and direct current and future vaccination strategies and offer opportunities for novel EVD therapies. Analysis of monoclonal antibody responses to the Ebola virus vaccine rVSV-ZEBOV in human volunteers.

Background: The investigation of the antibody response to SARS-CoV-2 represents a key aspect in facing the COVID-19 pandemic. In the present study, we compared the new Immundiagnostik IDK® anti-SARS-CoV-2 S1 IgG assay with four widely-used commercial serological assays for the detection of antibodies targeting S (spike) and NC (nucleocapsid) proteins. Methods: Serum samples were taken from an unbiased group of convalescent patients and from a negative control group. Sample were simultaneously analyzed by the new Immundiagnostik IDK® anti-SARS-CoV-2 S1 IgG assay, by the DiaSorin LIAISON® SARS-CoV-2 S1/S2 IgG assay, and by the Euroimmun anti-SARS-CoV-2 S1 IgG ELISA. Antibodies binding NC were detected by the Abbott SARS-CoV-2 IgG assay and by the pan-immunoglobulin immunoassay Roche Elecsys® anti-SARS-CoV-2. Moreover, we investigated samples of a group of COVID-19 convalescent subjects that were primarily tested S1 IgG non-reactive. Samples were also tested by live virus and pseudovirus neutralization tests. Results: Overall, the IDK® anti-SARS-CoV-2 S1 IgG assay showed the highest sensitivity among the evaluated spike (S) protein-based assays. Additionally, the Immundiagnostik assay correlated well with serum-neutralizing activity. Conclusions: The novel IDK® anti-SARS-CoV-2 S1 IgG assay showed high sensitivity and specificity, representing a valid option for use in the routine diagnostic.