Antibody responses elicited by mRNA vaccination in firefighters persist six months and correlate inversely with age and directly with BMI.

Knowledge regarding the sustainability of immune responses after COVID-19 vaccination is important, e.g., to decide whom and when to booster. Thus, we analyzed antibody titers in firefighters six months after vaccination with the mRNA-based vaccine Comirnaty. SARS-CoV-2 spike-binding antibodies (bAb) were quantified and compared to peak responses determined in healthcare workers (HCW). For the firefighters, neutralizing antibodies (nAb) were also analyzed. Six months after the second vaccine dose, all analyzed firefighters had detectable bAb, and 91% exhibited nAb titers above 1:16. However, actual titers six months after vaccination were over 12-fold lower than in the HCW control group four weeks after vaccination. bAb and nAb responses showed a significant correlation, and age correlated inversely with antibody responses. Unexpectedly, participants with a body mass index over 25 had higher neutralization titers after six months. All participants with very low neutralization titers were offered booster vaccination. The booster vaccination improved the extent and sustainability of antibody responses.

Establishment and clinical validation of an in-cell-ELISA-based assay for the rapid quantification of Rabies lyssavirus neutralizing antibodies.

Neutralizing antibodies (nAbs) prevent the entry of viruses into permissive cells. Since nAbs represent correlates of protection against the Rabies lyssavirus, the presence of sufficient nAbs indicates effective vaccination. Accordingly, Rabies lyssavirus-specific nAb titers need to be determined in routine diagnostics to identify individuals being at risk of Rabies lyssavirus infections due to insufficient immunity. The current gold standard for the quantification of Rabies lyssavirus-specific nAbs is the rapid fluorescent focus inhibition test (RFFIT). However, RFFITs are expensive and labor-intensive since multiple microplate wells must be evaluated one-by-one by trained personnel through microscopic inspection, which limits the number of samples that can be processed. To overcome this disadvantage, we established a novel assay for Rabies lyssavirus-specific nAbs relying on an in-cell-ELISA (icELISA)-based neutralization test (icNT). The icNT differs from the RFFIT in the readout phase, and can be automatically quantified in minutes using broadly available microplate readers. During the establishment, icNT parameters such as antibody concentrations, permeabilization procedures, blocking reagents, infectious doses, and the duration of infection were optimized. Afterwards, a dose-dependent detection of Rabies lyssavirus neutralization was demonstrated using the WHO Standard Rabies Immunoglobulin reference. A panel of 200 sera with known RFFIT titers revealed very good sensitivity and specificity of the icNT. Furthermore, the icNT showed very good intra- and inter-assay precision. By recognizing Rabies lyssavirus-specific antigens, the assay can be applied immediately to automatically quantify the concentration of Rabies lyssavirus nAbs in routine diagnostics or for various basic research questions such as screening for antiviral compounds.

Discrepancy between frequent occurrence of COVID-19-like symptoms and low seroconversion rates among healthcare workers.

During the first wave of the pandemic, we compared the occurrence of subjectively experienced COVID-19-like symptoms and true severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroconversion rates among medical personnel in general practices. This cross-sectional study determined the SARS-CoV-2-specific immunoglobulin G (IgG) antibody status of medical staff from 100 outpatient practices in Germany. Study cohort characteristics and COVID-19-like symptoms were obtained by questionnaires. The initial screening for SARS-CoV-2-recognizing antibodies was performed using a commercial chemiluminescence microparticle immunoassay. Positive results were controlled with another approved test. Samples with discrepant results were subjected to a third IgG-binding assay and a neutralization test. A total of 861 participants were included, 1.7% (n = 15) of whom tested positive for SARS-CoV-specific IgG in the initial screening test. In 46.6% (n = 7) of positive cases, test results were confirmed by an independent test. In the eight samples with discrepant results, neither spike-specific antibodies nor in vitro neutralizing capacity were detectable, resulting in a genuine seroprevalence rate of 0.8%. 794 participants completed the questionnaire. Intriguingly, a total of 53.7% (n = 426) of them stated episodes of COVID-19-like symptoms. Except for smell and taste dysfunction, there were no significant differences between the groups with and without laboratory-confirmed SARS-CoV-2 seroconversion. Our results demonstrated that only 0.8% of participants acquired SARS-CoV-2 even though 53.7% of participants reportedly experienced COVID-19-like symptoms. Thus, even among medical staff, self-diagnosis based on subjectively experienced symptoms does not have a relevant predictive value.

A rapid test recognizing mucosal SARS-CoV-2-specific antibodies distinguishes prodromal from convalescent COVID-19.

The COVID-19 pandemic poses enormous challenges to global healthcare sectors. To prevent the overburden of medical systems, it is crucial to distinguish individuals approaching the most infectious early phase from those in the declining non-infectious phase. However, a large fraction of transmission events occur during pre- or asymptomatic phases. Especially in the absence of symptoms, it is difficult to distinguish prodromal from late phases of infection just by RT-PCR since both phases are characterized by low viral loads and corresponding high Ct values (>30). We evaluated a new rapid test detecting IgG antibodies recognizing SARS-CoV-2 nucleocapsid protein using two commercial antibody assays and an in-house neutralization test before determining suitability for testing clinical swab material. Our analyses revealed the combination of the well-known RT-PCR and the new rapid antibody test using one single clinical nasopharyngeal swab specimen as a fast, cost-effective, and reliable way to discriminate prodromal from subsiding phases of COVID-19.

A Novel In-Cell ELISA Assay Allows Rapid and Automated Quantification of SARS-CoV-2 to Analyze Neutralizing Antibodies and Antiviral Compounds.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most pressing medical and socioeconomic challenge. Constituting important correlates of protection, the determination of virus-neutralizing antibodies (NAbs) is indispensable for convalescent plasma selection, vaccine candidate evaluation, and immunity certificates. In contrast to standard serological ELISAs, plaque reduction neutralization tests (PRNTs) are laborious, time-consuming, expensive, and restricted to specialized laboratories. To replace microscopic counting-based SARS-CoV-2 PRNTs by a novel assay exempt from genetically modified viruses, which are inapplicable in most diagnostics departments, we established a simple, rapid, and automated SARS-CoV-2 neutralization assay employing an in-cell ELISA (icELISA) approach. After optimization of various parameters such as virus-specific antibodies, cell lines, virus doses, and duration of infection, SARS-CoV-2-infected cells became amenable as direct antigen source for quantitative icELISA. Antiviral agents such as human sera containing NAbs or antiviral interferons dose dependently reduced the SARS-CoV-2-specific signal. Applying increased infectious doses, the icELISA-based neutralization test (icNT) was superior to PRNT in discriminating convalescent sera with high from those with intermediate neutralizing capacities. In addition, the icNT was found to be specific, discriminating between SARS-CoV-2-specific NAbs and those raised against other coronaviruses. Altogether, the SARS-CoV-2 icELISA test allows rapid (<48 h in total, read-out in seconds) and automated quantification of virus infection in cell culture to evaluate the efficacy of NAbs and antiviral drugs using reagents and equipment present in most routine diagnostics departments.

The Human Cytomegalovirus pUL145 Isoforms Act as Viral DDB1-Cullin-Associated Factors to Instruct Host Protein Degradation to Impede Innate Immunity.

Human cytomegalovirus (HCMV) causes diseases in individuals with immature or compromised immunity. To evade immune control, HCMV evolved numerous antagonists targeting the interferon system at multiple levels. By comparative analysis of naturally arising variants of the most widely studied HCMV strain, AD169, and a panel of targeted mutants, we uncover the UL145 gene as indispensable for STAT2 downregulation. Ribosome profiling confirms the translation of the canonical pUL145 protein (pUL145-Long) and newly identifies a shorter isoform (pUL145-Short). Both isoforms recruit DDB1-containing ubiquitin ligases to induce proteasomal degradation of STAT2. An alanine-scanning mutagenesis discloses the DDB1 interaction motif of pUL145 that resembles the DDB1-binding interface of cellular substrate receptors of DDB1-containing ubiquitin ligases. Thus, pUL145 constitutes a viral DDB1-cullin-associated factor (vDCAF), which mimics cellular DCAFs to exploit the ubiquitin-proteasome system to impede antiviral immunity. Notably, the viral exploitation of the cullins can be targeted to restore the efficacy of the host immune response.