The first workshop dedicated to Lassa virus–specific correlates of protection (CoP) was held in 2024 and was convened by the Coalition for Epidemic Preparedness Innovations (CEPI). Experts from multiple disciplines reviewed existing knowledge and identified gaps in understanding Lassa virus- and vaccine-induced immune responses. Discussions covered key areas including epidemiology, immunogenicity, preclinical and clinical research, data science, and regulatory considerations, with the goal of pinpointing opportunities to discover CoP.

MVA-MERS-S, a vaccine candidate against Middle East respiratory syndrome (MERS), was recently evaluated in a randomized, placebo-controlled, double-blind phase 1b clinical trial to assess its safety, immunogenicity, and optimal dosing in healthy adults in Hamburg and Rotterdam. A three-dose regimen was safe and elicited robust spike-specific antibody responses. We extended this trial to assess the two-year durability of MERS-CoV-specific antibody and T cell responses in 48 study participants of the Hamburg cohort. Our findings show that immune responses remain detectable for at least 24 months after the third vaccination. Antibodies persisted at levels comparable to the peak response observed after the second vaccination and were able to cross-neutralize MERS-CoV spike mutants. Although the immune correlates of protection against MERS remain unknown, the observed durability of humoral and cellular immune responses supports the potential of MVA-MERS-S as a promising MERS vaccine candidate and highlights the importance of a booster dose in sustaining long-term immunity.

BACKGROUND:Invasive mould infections (IMI) cause substantial morbidity and mortality in populations at risk. Novel treatment approaches are urgently needed. Targeting immune checkpoints may reverse hyporesponsiveness of the innate and adaptive immune systems. METHODS:In this prospective, observational study, we investigated immune checkpoint expression levels on immune cells in patients with invasive aspergillosis (IA; n = 25) and mucormycosis (MU; n = 7). Healthy controls (HC; n = 5) and patients with matched haematological diseases but without IMI served as control populations (CP; n = 10). Multicolour flow cytometry analysis was used to compare immune cell subsets and the expression of immune-regulatory molecules in peripheral blood mononuclear cells (PBMCs). RESULTS:Lymphocyte subsets and immune phenotypes in PBMCs were similar between patients with IMI and haematological CP, except for regulatory T cells, which were increased in PBMCs of patients with IA and MU compared to HCs. In IA and MU, PBMCs showed increased expression of immune checkpoint molecules compared to healthy controls and matched haematological CP, with this effect being more pronounced in IA than in MU. We found heterogeneous, disease-, molecule-, and patient-specific expression patterns of immune checkpoint molecules. For example, PD-1 expression was highest in MU PBMCs, followed by IA PBMCs, while HC PBMCs showed lower expression levels. Overall mortality in our patient population was 44.0% (IPA) and 80.0% (MU). CONCLUSIONS:We report an immune phenotype consistent with T-cell exhaustion in IMI, indicating potential contributions from haematological treatment, underlying disease, and infection. However, the primary underlying cause remains unclear and requires further investigation. A marker that was notably higher in IMI patients was PD-1, and treatment approaches specifically targeting this molecule may be promising.

Background MERS-CoV is a respiratory pathogen with a case-fatality rate of 36%, and for which no vaccines currently licensed. MVA-MERS-S is a candidate vaccine based on recombinant modified vaccinia virus Ankara (MVA). In this study, the safety, immunogenicity, and optimal dose schedule of MVA-MERS-S was assessed in individuals with previous exposure to SARS-CoV-2 infections and vaccines. Methods We conducted a multicentre, double-blind, randomised controlled phase 1b clinical trial at two university medical centres in Germany and the Netherlands. Healthy volunteers aged 18-55 years were assigned by computer randomisation to receive three intramuscular injections of 107 or 10$ plaque-forming units (PFU) of MVA-MERS-S, with two treatment groups each of either 28-day or 56-day intervals between the initial two doses, and one control arm that received only placebo, at a ratio of 2:2:2:2:1. The third dose was given after 224 days. The sponsor, clinical laboratory staff, and participants were masked to both vaccine dose and dosing interval. The primary outcome safety, assessed in the all participants who had received at least one injection; daily solicited vaccine reactions were recorded after each dose for 7 days, unsolicited adverse events for 28 days, and serious adverse events throughout study. The secondary outcome was humoral immunogenicity, measured with vaccine-induced geometric mean antibody concentrations and seroconversion rates, analysed in all participants who received at least three allocated treatments. This study is registered at ClinicalTrials.gov (NCT04119440) and is completed. Findings Between 26 July, 2021, and 3 March, 2022, 244 volunteers were screened, 177 of whom were eligible 140 were randomly assigned either to the 28-day 107 PFU group (n=32), 56-day 107 PFU group (n=31), 28-day 10$ PFU group (n=31), 56-day 10$ PFU group (n=30), or placebo group (n=16). In total, 178 doses were administered of 107 PFU of MVA-MERS-S, 174 of 10$ PFU, and 164 doses of placebo, and 139 participants received least one injection. 73 (53%) were female and 66 (48%) were male. No serious vaccine-related adverse events occurred. Solicited local reactions were mild in 288 (93%, 95% CI 90-96) of 309 reports and consisted primarily of pain tenderness. Pain or tenderness (of any severity) occurred after 69 (39%, 32-46) of 178 107 PFU injections, 138 (79%; 73-85) of 174 10$ PFU injections, and 18 (11%; 7-11) of 164 placebo injections. Of 595 reported solicited systemic reactions, 479 (81%, 77-83) were graded as mild. Systemic reactions of any grade occurred after 77 (43%; 36-51) 107 PFU injections, 102 (59%; 51-66) 10$ PFU injections, and 67 (41%; 34-49) of 164 placebo injections. At 28 days after the second dose, MERS-CoV neutralising antibodies were highest for participants assigned to 56-day 10$ PFU, with geometric mean ratios of 7.2 (95% CI 3.9-13.3) for the 56-day 10$ PFU group versus the 28-day 10$ PFU group (p<00001), 3.9 (2.1-7.2) for the 56-day 10$ PFU group versus the 56-day 107 PFU group (p=0.0031), and 5.4 (2.9-10.0) for the 56-day 10$ PFU group versus the 28-day 107 PFU group (p=0.0003). Interpretation MVA-MERS-S was safe and immunogenic in individuals with previous and concurrent SARS-CoV-2 exposure. The second vaccination with the 10$ PFU dose of MVA-MERS-S elicited a stronger humoral immune response when administered 56 days after the first dose than a 28-day interval. Further studies are needed to verify these findings in groups at risk for MERS-CoV exposure, and at risk of severe disease, including older individuals and those with relevant comorbidities. Funding Coalition for Epidemic Preparedness Innovations, the German Centre for Infection Research, and the German Research Foundation.Copyright (c) 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.

In response to the COVID-19 pandemic, multiple vaccines were developed using platforms such as viral vectors and mRNA technology. Here, we report humoral and cellular immunogenicity data from human phase 1 clinical trials investigating two recombinant Modified Vaccinia virus Ankara vaccine candidates, MVA-SARS-2-S and MVA-SARS-2-ST, encoding the native and the prefusion-stabilized SARS-CoV-2 spike protein, respectively. MVA-SARS-2-ST was more immunogenic than MVA-SARS-2-S, but both were less immunogenic compared to licensed mRNA- and ChAd-based vaccines in SARS-CoV-2 naïve individuals. In heterologous vaccination, previous MVA-SARS-2-S vaccination enhanced T cell functionality and MVA-SARS-2-ST boosted the frequency of T cells and S1-specific IgG levels when used as a third vaccination. While the vaccine candidate containing the prefusion-stabilized spike elicited predominantly S1-specific responses, immunity to the candidate with the native spike was skewed towards S2-specific responses. These data demonstrate how the spike antigen conformation, using the same viral vector, directly affects vaccine immunogenicity in humans.

Objective Preventing severe COVID-19 remains a priority globally, particularly in the immunocompromised population. As shown in healthy individuals, immunity against SARS-CoV-2 can be yielded by previous infection, vaccination, or both (hybrid immunity). The objective of this observation study was to investigate hybrid immunity in patients with chronic lymphocytic leukemia (CLL). Methods/Results Blood samples of six patients with CLL were collected 55 days after fourth COVID-19 vaccination. All patients had a SARS-CoV-2 infection within 12 months before the second booster (fourth vaccination). SARS-CoV-2 spike receptor binding domain (RBD)-specific IgG antibodies were detectable in 6/6 (100.0%) CLL patients after four compared to 4/6 (66.7%) after three vaccinations. The median number of SARS-CoV-2 spike-specific T cells after repeated booster vaccination plus infection was 166 spot-forming cells (SFC) per million peripheral blood mononuclear cells. Overall, 5/5 (100%) studied patients showed a detectable increase in T cell activity. Conclusion Our data reveal an increase of cellular and humoral immune response in CLL patients after fourth COVID-19 vaccination combined with SARS-CoV-2 infection, even in those undergoing B cell-depleting treatment. Patients with prior vaccination failure now show a specific IgG response. Future research should explore the duration and effectiveness of hybrid immunity considering various factors like past infection and vaccination rates, types and numbers of doses, and emerging variants.

IntroductionVaccine platforms such as viral vectors and mRNA can accelerate vaccine development in response to newly emerging pathogens, as demonstrated during the COVID-19 pandemic. However, the differential effects of platform and antigen insert on vaccine immunogenicity remain incompletely understood. Innate immune responses induced by viral vector vaccines are suggested to have an adjuvant effect for subsequent adaptive immunity. Integrating data on both innate and adaptive immunity, systems vaccinology approaches can improve the understanding of vaccine-induced immune mechanisms.MethodsTwo vaccine candidates against SARS-CoV-2, both based on the viral vector Modified Vaccinia virus Ankara (MVA) and encoding the native (MVA-SARS-2-S) or prefusion-stabilized spike protein (MVA-SARS-2-ST), were evaluated in phase 1 clinical trials (ClinicalTrials.gov: NCT04569383, NCT04895449). Longitudinal dynamics of innate and early adaptive immune responses induced by vaccination in SARS-CoV-2-naïve individuals were analyzed based on transcriptome and flow cytometry data, in comparison to the licensed ChAd and mRNA vaccines.ResultsCompared to MVA-SARS-2-S, MVA-SARS-2-ST (encoding the prefusion-stabilized spike protein) induced a stronger transcriptional activation early after vaccination, as well as higher virus neutralizing antibodies. Positive correlations were observed between innate and adaptive immune responses induced by a second MVA-SARS-2-ST vaccination. MVA-, ChAd- and mRNA-based vaccines induced distinct immune signatures, with the overall strongest transcriptional activation as well as monocyte and circulating T follicular helper (cTFH) cell responses induced by ChAd.DiscussionOur findings suggest a potential impact of the spike protein conformation not only on adaptive but also on innate immune responses. As indicated by positive correlations between several immune parameters induced by MVA-SARS-2-ST, the distinct transcriptional activation early after vaccination may be linked to the induction of classical monocytes and activation of cTFH1 cells, which may in turn result in the superior adaptive immunogenicity of MVA-SARS-2-ST, compared to MVA-SARS-2-S. Overall, our data demonstrate that both the vaccine platform and antigen insert can affect innate immune responses and subsequent vaccine immunogenicity in humans.

Licensed vaccines against the Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging pathogen of concern, are lacking. The modified vaccinia virus Ankara vector-based vaccine MVA-MERS-S, expressing the MERS-CoV-spike glycoprotein (MERS-S), is one of 3 candidate vaccines in clinical development and elicits robust humoral and cellular immunity. Here, we identified for the first time a MERS-S-specific CD8(+) T-cell epitope in an HLA-A*03:01/HLA-B*35:01-positive vaccinee using a screening assay, intracellular cytokine staining, and in silico epitope prediction. As evidence from MERS-CoV infection suggests a protective role of long-lasting CD8(+) T-cell responses, the identification of epitopes will facilitate longitudinal analyses of vaccine-induced T-cell immunity.

Besides neutralizing antibodies, which are considered an important measure for vaccine immunogenicity, Fc-mediated antibody functions can contribute to antibody-mediated protection. They are strongly influenced by structural antibody properties such as subclass and Fc glycan composition. We here applied a systems serology approach to dissect humoral immune responses induced by MVA-MERS-S, an MVA-vectored vaccine against the Middle East respiratory syndrome coronavirus (MERS-CoV). Building on preceding studies reporting the safety and immunogenicity of MVA-MERS-S, our study highlights the potential of a late boost, administered one year after prime, to enhance both neutralizing and Fc-mediated antibody functionality compared to the primary vaccination series. Distinct characteristics were observed for antibodies specific to the MERS-CoV spike protein S1 and S2 subunits, regarding subclass and glycan compositions as well as Fc functionality. These findings highlight the benefit of a late homologous booster vaccination with MVA-MERS-S and may be of interest for the design of future coronavirus vaccines.

Abstract Background Recombinant Modified Vaccinia virus Ankara (MVA) is a replication-deficient viral vector platform with a favorable safety profile that is currently developed for multiple indications, including vaccines against emerging infections. We assessed the safety and immunogenicity of two MVA-based vaccine candidates against COVID-19 encoding the native or pre-fusion stabilized SARS-CoV-2 spike antigen, namely MVA-SARS-2-S and MVA-SARS-2-ST, in two clinical Phase I trials. Methods We conducted a first-in-human trial of the vaccine candidate MVA-SARS-2-S (NCT04569383) and a Phase Ib trial of the successor vaccine MVA-SARS-2-ST (NCT05226390) in healthy, SARS-CoV-2 immune-naïve adults aged between 18-55 (MVA-SARS-2-S) and 18-64 (MVA-SARS-2-ST) years. MVA-SARS-2-S expresses the native, full-length spike glycoprotein of SARS-CoV-2 (S), while MVA-SARS-2-ST delivers a pre-fusion stabilized spike antigen (ST). Both trials were designed as open-label dose-escalation trials and vaccinations were administered as a prime-boost regimen on days 0 and 28. Detailed safety and immunogenicity assessments were conducted at multiple timepoints throughout the study until day 168. Results In NCT04569383, 30 individuals received either 1 x 107 ± 0.5 log IU (IU=International Units, n=15, lower dose) or 1 x 108 ± 0.5 log IU (n=15, higher dose) of MVA-SARS-2-S. In NCT05226390, 8 participants received a lower (≥ 1 x 107 IU) and 7 participants a higher (≥ 5 x 107 IU) dose of MVA-SARS-2-ST. Both vaccines were well-tolerated and no vaccine-related severe adverse events (SAEs) occurred in either trial. However, seroconversion rates, measured as binding anti-SARS-CoV-2 S1 antibodies, could only be observed in 3/15 participants in the high dose cohort and none in the low dose cohort after vaccination with MVA-SARS-2-S. In contrast, all participants who received MVA-SARS-2-ST (n=15) achieved seroconversion independent of administered dose. Conclusion Both vaccine candidates were safe and well-tolerated. While MVA-SARS-2-S did not induce adequate immunogenicity, vaccination with the pre-fusion stabilized successor vaccine MVA-SARS-2-ST induced seroconversion in all vaccinees, demonstrating the significance of SARS-CoV-2 spike antigen conformation for vaccine immunogenicity. Disclosures Anahita Fathi, MD, Biotest: Honoraria Sibylle C. Mellinghoff, MD, Gilead: Grant|Octapharma: Advisor/Consultant

Abstract Modified vaccinia virus Ankara (MVA) is used as a vaccine against monkeypox virus and as a viral vaccine vector. MVA-MERS-S is a vaccine candidate against Middle East respiratory syndrome (MERS)–associated coronavirus. Here, we report that cross-reactive monkeypox virus neutralizing antibodies were detectable in only a single study participant after the first dose of MVA-MERS-S vaccine, in 3 of 10 after the second dose, and in 10 of 10 after the third dose.

Abstract Background The clinical course of COVID-19 patients ranges from asymptomatic infection, via mild and moderate illness, to severe disease and even fatal outcome. Biomarkers which enable an early prediction of the severity of COVID-19 progression, would be enormously beneficial to guide patient care and early intervention prior to hospitalization. Methods Here we describe the identification of plasma protein biomarkers using an antibody microarray-based approach in order to predict a severe cause of a COVID-19 disease already in an early phase of SARS-CoV-2 infection. To this end, plasma samples from two independent cohorts were analyzed by antibody microarrays targeting up to 998 different proteins. Results In total, we identified 11 promising protein biomarker candidates to predict disease severity during an early phase of COVID-19 infection coherently in both analyzed cohorts. A set of four (S100A8/A9, TSP1, FINC, IFNL1), and two sets of three proteins (S100A8/A9, TSP1, ERBB2 and S100A8/A9, TSP1, IFNL1) were selected using machine learning as multimarker panels with sufficient accuracy for the implementation in a prognostic test. Conclusions Using these biomarkers, patients at high risk of developing a severe or critical disease may be selected for treatment with specialized therapeutic options such as neutralizing antibodies or antivirals. Early therapy through early stratification may not only have a positive impact on the outcome of individual COVID-19 patients but could additionally prevent hospitals from being overwhelmed in potential future pandemic situations.

The Middle East respiratory syndrome (MERS) is a respiratory disease caused by MERS coronavirus (MERS-CoV). In follow up to a phase 1 trial, we perform a longitudinal analysis of immune responses following immunization with the modified vaccinia virus Ankara (MVA)-based vaccine MVA-MERS-S encoding the MERS-CoV-spike protein. Three homologous immunizations were administered on days 0 and 28 with a late booster vaccination at 12 ± 4 months. Antibody isotypes, subclasses, and neutralization capacity as well as T and B cell responses were monitored over a period of 3 years using standard and bead-based enzyme-linked immunosorbent assay (ELISA), 50% plaque-reduction neutralization test (PRNT50), enzyme-linked immunospot (ELISpot), and flow cytometry. The late booster immunization significantly increases the frequency and persistence of spike-specific B cells, binding immunoglobulin G1 (IgG1) and neutralizing antibodies but not T cell responses. Our data highlight the potential of a late boost to enhance long-term antibody and B cell immunity against MERS-CoV. Our findings on the MVA-MERS-S vaccine may be of relevance for coronavirus 2019 (COVID-19) vaccination strategies.

The SARS-CoV-2 spike (S) glycoprotein is synthesized as a large precursor protein and must be activated by proteolytic cleavage into S1 and S2. A recombinant modified vaccinia virus Ankara (MVA) expressing native, full-length S protein (MVA-SARS-2-S) is currently under investigation as a candidate vaccine in phase I clinical studies. Initial results from immunogenicity monitoring revealed induction of S-specific antibodies binding to S2, but low-level antibody responses to the S1 domain. Follow-up investigations of native S antigen synthesis in MVA-SARS-2-S–infected cells revealed limited levels of S1 protein on the cell surface. In contrast, we found superior S1 cell surface presentation upon infection with a recombinant MVA expressing a stabilized version of SARS-CoV-2 S protein with an inactivated S1/S2 cleavage site and K986P and V987P mutations (MVA-SARS-2-ST). When comparing immunogenicity of MVA vector vaccines, mice vaccinated with MVA-SARS-2-ST mounted substantial levels of broadly reactive anti-S antibodies that effectively neutralized different SARS-CoV-2 variants. Importantly, intramuscular MVA-SARS-2-ST immunization of hamsters and mice resulted in potent immune responses upon challenge infection and protected from disease and severe lung pathology. Our results suggest that MVA-SARS-2-ST represents an improved clinical candidate vaccine and that the presence of plasma membrane–bound S1 is highly beneficial to induce protective antibody levels.

Vaccine development is essential for pandemic preparedness. We previously conducted a Phase 1 clinical trial of the vector vaccine candidate MVA-MERS-S against the Middle East respiratory syndrome coronavirus (MERS-CoV), expressing its full spike glycoprotein (MERS-CoV-S), as a homologous two-dose regimen (Days 0 and 28). Here, we evaluate the safety (primary objective) and immunogenicity (secondary and exploratory objectives: magnitude and characterization of vaccine-induced humoral responses) of a third vaccination with MVA-MERS-S in a subgroup of trial participants one year after primary immunization. MVA-MERS-S booster vaccination is safe and well-tolerated. Both binding and neutralizing anti-MERS-CoV antibody titers increase substantially in all participants and exceed maximum titers observed after primary immunization more than 10-fold. We identify four immunogenic IgG epitopes, located in the receptor-binding domain (RBD, n = 1) and the S2 subunit ( n = 3) of MERS-CoV-S. The level of baseline anti-human coronavirus antibody titers does not impact the generation of anti-MERS-CoV antibody responses. Our data support the rationale of a booster vaccination with MVA-MERS-S and encourage further investigation in larger trials. Trial registration: Clinicaltrials.gov NCT03615911.

The coronavirus SARS-CoV-2 is the causative agent for the disease COVID-19. To capture the IgA, IgG, and IgM antibody response of patients infected with SARS-CoV-2 at individual epitope resolution, we constructed planar microarrays of 648 overlapping peptides that cover the four major structural proteins S(pike), N(ucleocapsid), M(embrane), and E(nvelope). The arrays were incubated with sera of 67 SARS-CoV-2 positive and 22 negative control samples. Specific responses to SARS-CoV-2 were detectable, and nine peptides were associated with a more severe course of the disease. A random forest model disclosed that antibody binding to 21 peptides, mostly localized in the S protein, was associated with higher neutralization values in cellular anti-SARS-CoV-2 assays. For antibodies addressing the N-terminus of M, or peptides close to the fusion region of S, protective effects were proven by antibody depletion and neutralization assays. The study pinpoints unusual viral binding epitopes that might be suited as vaccine candidates.

Memory B cells (MBCs), part of the immune response elicited by infection or vaccination, can persist in lymphoid organs and peripheral blood and are capable of rapid reactivation upon secondary antigen exposure. Here, we describe a flow cytometric assay to identify antigen-specific MBCs from peripheral blood mononuclear cells and characterize their isotypes and activation status. We detail steps to use fluorescently labeled antigen probes derived from the SARS-CoV-2 spike protein. These can be adapted to detect MBCs against other antigens.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). A better understanding of its immunogenicity can be important for the development of improved diagnostics, therapeutics, and vaccines. Here, we report the longitudinal analysis of three COVID-19 patients with moderate (#1) and mild disease (#2 and #3). Antibody serum responses were analyzed using spike glycoprotein enzyme linked immunosorbent assay (ELISA), full-proteome peptide, and glycan microarrays. ELISA immunoglobulin A, G, and M (IgA, IgG, and IgM) signals increased over time for individuals #1 and #2, whereas #3 only showed no clear positive IgG and IgM result. In contrast, peptide microarrays showed increasing IgA/G signal intensity and epitope spread only in the moderate patient #1 over time, whereas early but transient IgA and stable IgG responses were observed in the two mild cases #2 and #3. Glycan arrays showed an interaction of antibodies to fragments of high-mannose and core N-glycans, present on the viral shield. In contrast to protein ELISA, microarrays allow for a deeper understanding of IgA, IgG, and IgM antibody responses to specific epitopes of the whole proteome and glycans of SARS-CoV-2 in parallel. In the future, this may help to better understand and to monitor vaccination programs and monoclonal antibodies as therapeutics.

Vaccination with the adenoviral-vector-based AstraZeneca ChAdOx1 nCov-19 (Vaxzevria) vaccine is efficient and safe. However, in rare cases vaccinated individuals developed life-threatening thrombotic complications, including thrombosis in cerebral sinus and splanchnic veins. Monitoring of the applied vector in vivo represents an important precondition to study the molecular mechanisms underlying vaccine-driven adverse effects now referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT). We previously have shown that digital PCR (dPCR) is an excellent tool to quantify transgene copies in vivo. Here, we present a highly sensitive dPCR for in situ quantification of ChAdOx1 nCoV-19 copies. Using this method, we quantified vector copies in human plasma 24, 72, and 168 h post vaccination and in a variety of murine tissues in an experimental vaccination model 30 min post injection. We describe a method for high-sensitivity quantitative detection of ChAdOx1 nCoV-19 with possible implications to elucidate the mechanisms of severe ChAdOx1 nCov-19 vaccine complications.

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.