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The humoral immune response to SARS-CoV-2 is a benchmark for immunity and detailed analysis is required to understand the manifestation and progression of COVID-19, monitor seroconversion within the general population, and support vaccine development. The majority of currently available commercial serological assays only quantify the SARS-CoV-2 antibody response against individual antigens, limiting our understanding of the immune response. To overcome this, we have developed a multiplex immunoassay (MultiCoV-Ab) including spike and nucleocapsid proteins of SARS-CoV-2 and the endemic human coronaviruses. Compared to three broadly used commercial in vitro diagnostic tests, our MultiCoV-Ab achieves a higher sensitivity and specificity when analyzing a well-characterized sample set of SARS-CoV-2 infected and uninfected individuals. We find a high response against endemic coronaviruses in our sample set, but no consistent cross-reactive IgG response patterns against SARS-CoV-2. Here we show a robust, high-content-enabled, antigen-saving multiplex assay suited to both monitoring vaccination studies and facilitating epidemiologic screenings for humoral immunity towards pandemic and endemic coronaviruses., Serology is an important way to monitor SARS-CoV-2 infection in the population and support vaccine development. Here the authors develop a multiplex immunoassay including spike and nucleocapsid proteins of SARS-CoV-2 and the endemic human coronaviruses with high specificity and sensitivity.

BACKGROUND: Until now, information on the spread of SARS-CoV-2 infections in Germany has been based mainly on data from the public health offices. It may be assumed that these data do not include many cases of asymptomatic and mild infection. METHODS: We determined seroprevalence over the course of the pandemic in a sequential, multilocal seroprevalence study (MuSPAD). Study participants were recruited at random in seven administrative districts (Kreise) in Germany from July 2020 onward; each participant was tested at two different times 3–5 months apart. Test findings on blood samples were used to determine the missed-case rate of reported infections, the infection fatality rate (IFR), and the association between seropositivity and demographic, socio-economic, and health-related factors, as well as to evaluate the self-reported results of PCR and antigenic tests. The registration number of this study is DRKS00022335. RESULTS: Among non-vaccinated persons, the seroprevalence from July to December 2020 was 1.3–2.8% and rose between February and May 2021 to 4.1–13.1%. In July 2021, 35% of tested persons in Chemnitz were not vaccinated, and the seroprevalence among these persons was 32.4% (07/2021). The surveillance detection ratio (SDR), i.e., the ratio between the true number of infections estimated from seroprevalence and the actual number or reported infections, varied among the districts included in the study from 2.2 to 5.1 up to December 2020 and from 1.3 to 2.9 up to June 2021, and subsequently declined. The IFR was in the range of 0.8% to 2.4% in all regions except Magdeburg, where a value of 0.3% was calculated for November 2020. A lower educational level was associated with a higher seropositivity rate, smoking with a lower seropositivity rate. On average, 1 person was infected for every 8.5 persons in quarantine. CONCLUSION: Seroprevalence was low after the first wave of the pandemic but rose markedly during the second and third waves. The missed-case rate trended downward over the course of the pandemic.

In light of the COVID-19 pandemic, there is an ongoing need for diagnostic tools to monitor the immune status of large patient cohorts and the effectiveness of vaccination campaigns. Here, we present 11 unique nanobodies (Nbs) specific for the SARS-CoV-2 spike receptor-binding domain (RBD), of which 8 Nbs potently inhibit the interaction of RBD with angiotensin-converting enzyme 2 (ACE2) as the major viral docking site. Following detailed epitope mapping and structural analysis, we select two inhibitory Nbs, one of which binds an epitope inside and one of which binds an epitope outside the RBD:ACE2 interface. Based on these, we generate a biparatopic nanobody (bipNb) with viral neutralization efficacy in the picomolar range. Using bipNb as a surrogate, we establish a competitive multiplex binding assay ("NeutrobodyPlex") for detailed analysis of the presence and performance of neutralizing RBD-binding antibodies in serum of convalescent or vaccinated patients. We demonstrate that NeutrobodyPlex enables high-throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, to monitor immune status or to guide vaccine design.

SARS-CoV-2 is evolving with mutations in the receptor binding domain (RBD) being of particular concern. It is important to know how much cross-protection is offered between strains following vaccination or infection. Here, we obtain serum and saliva samples from groups of vaccinated (Pfizer BNT-162b2), infected and uninfected individuals and characterize the antibody response to RBD mutant strains. Vaccinated individuals have a robust humoral response after the second dose and have high IgG antibody titers in the saliva. Antibody responses however show considerable differences in binding to RBD mutants of emerging variants of concern and substantial reduction in RBD binding and neutralization is observed against a patient-isolated South African variant. Taken together our data reinforce the importance of the second dose of Pfizer BNT-162b2 to acquire high levels of neutralizing antibodies and high antibody titers in saliva suggest that vaccinated individuals may have reduced transmission potential. Substantially reduced neutralization for the South African variant further highlights the importance of surveillance strategies to detect new variants and targeting these in future vaccines., Here, the authors characterize the antibody response from vaccinated (Pfizer BNT-162b2), infected and uninfected individuals against emerging variants of concern of SARS-CoV-2, finding reduced neutralization of a South African isolate. High IgG titers in the saliva of vaccinees suggest that transmission may be reduced.

Facing the worldwide disease progression of COVID-19 caused by the SARS-CoV-2 virus, the situation is highly critical and there is an unmet need for effective vaccination, reliable diagnosis and therapeutic intervention. Neutralizing binding molecules such as antibodies or derivatives thereof have become important tools for acute treatment of COVID-19. Additionally, such binders provide the unique possibility to monitor the emergence and presence of a neutralizing immune response in infected or vaccinated individuals. Here we describe a set of 11 unique nanobodies (Nbs), originated from an immunized alpaca which bind with high affinities to the glycosylated SARS-CoV-2 Spike receptor domain (RBD). Using a multiplex in vitro binding assay we showed that eight of the selected Nbs effectively block the interaction between RBD, S1-domain and homotrimeric Spike protein with the angiotensin converting enzyme 2 (ACE2) as the viral docking site on human cells. According to competitive binding analysis and detailed epitope mapping, we grouped all Nbs blocking the RBD:ACE2 interaction in three distinct Nb-Sets and demonstrated their neutralizing effect with IC50 values in the low nanomolar range in a cell-based SARS-CoV-2 neutralization assay. Tested Nb combinations from different sets showed substantially lower IC50 values in both functional assays indicating a profound synergistic effect of Nbs simultaneously targeting different epitopes within the RBD. Finally, we applied the most potent Nb combinations in a competitive multiplex binding assay which we termed NeutrobodyPlex and detected a neutralizing immune response in plasma samples of infected individuals. We envisage that our Nbs have a high potential for prophylactic as well as therapeutic options and provide a novel approach to screen for a neutralizing immune response in infected or vaccinated individuals thus helping to monitor the immune status or to guide vaccine design.

Given the importance of the humoral immune response to SARS-CoV-2 as a global benchmark for immunity, a detailed analysis is needed to monitor seroconversion in the general population, understand manifestation and progression of COVID-19 disease, and ultimately predict the outcome of vaccine development. In contrast to currently available serological assays, which are only able to resolve the SARS-CoV-2 antibody response on an individual antigen level, we developed a multiplex immunoassay, for which we included spike and nucleocapsid proteins of SARS-CoV-2 and the endemic human coronaviruses (NL63, OC43, 229E, HKU1) in an expanded antigen panel. Compared to three commercialin vitrodiagnostic tests, our MULTICOV-AB assay achieved the highest sensitivity and specificity when analyzing a well-characterized sample set of SARS-CoV-2 infected and uninfected individuals. Simultaneously, high IgG responses against endemic coronaviruses became apparent throughout all samples, but no consistent cross-reactive IgG response patterns could be defined. In summary, we have established and validated, a robust, high-content-enabled, and antigen-saving multiplex assay MULTICOV-AB, which is highly suited to monitor vaccination studies and will facilitate epidemiologic screenings for the humoral immunity toward pandemic as well as endemic coronaviruses.

Influenza A virus (IAV), a major airborne pathogen, is closely associated with significant morbidity and mortality. The primary target for influenza virus replication is the respiratory epithelium, which reacts to infection by mounting a multifaceted antiviral response. A part of this mucosal host defense is the generation of reactive oxygen species (ROS) by NADPH oxidases. Duox1 and Duox2 are the main ROS-producing enzymes in the airway epithelium, but their contribution to mammalian host defense is still ill defined.To gain a better understanding of Duox function in respiratory tract infections, human differentiated lung epithelial cells and an animal model were used to monitor the effect of epithelial ROS on IAV propagation. IAV infection led to coordinated up-regulation of Duox2 and Duox-mediated ROS generation. Interference with H2O2 production and ROS signaling by oxidase inhibition or H2O2 decomposition augmented IAV replication. A nuclear pool of Duox enzymes participated in the regulation of the spliceosome, which is critical for alternative splicing of viral transcripts and controls the assembly of viable virions. In vivo silencing of Duox increased the viral load on intranasal infection with 2009 pandemic H1N1 influenza virus.This is the first study conclusively linking Duox NADPH oxidases with the antiviral mammalian immune response. Further, ROS generated by Duox enzymes localized adjacent to nuclear speckles altered the splicing of viral genes.Duox-derived ROS are host protective and essential for counteracting IAV replication.

SummaryReactive oxygen species (ROS) play key roles in mucosal defense, yet how they are induced and the consequences for pathogens are unclear. We report that ROS generated by epithelial NADPH oxidases (Nox1/Duox2) during Campylobacter jejuni infection impair bacterial capsule formation and virulence by altering bacterial signal transduction. Upon C. jejuni invasion, ROS released from the intestinal mucosa inhibit the bacterial phosphotyrosine network that is regulated by the outer-membrane tyrosine kinase Cjtk (Cj1170/OMP50). ROS-mediated Cjtk inactivation results in an overall decrease in the phosphorylation of C. jejuni outer-membrane/periplasmic proteins, including UDP-GlcNAc/Glc 4-epimerase (Gne), an enzyme required for N-glycosylation and capsule formation. Cjtk positively regulates Gne by phosphorylating an active site tyrosine, while loss of Cjtk or ROS treatment inhibits Gne activity, causing altered polysaccharide synthesis. Thus, epithelial NADPH oxidases are an early antibacterial defense system in the intestinal mucosa that modifies virulence by disrupting bacterial signaling.