SARS-CoV-2-derived peptides define heterologous and COVID-19-induced T cell recognition.

T cell immunity is central for the control of viral infections. To characterize T cell immunity, but also for the development of vaccines, identification of exact viral T cell epitopes is fundamental. Here we identify and characterize multiple dominant and subdominant SARS-CoV-2 HLA class I and HLA-DR peptides as potential T cell epitopes in COVID-19 convalescent and unexposed individuals. SARS-CoV-2-specific peptides enabled detection of post-infectious T cell immunity, even in seronegative convalescent individuals. Cross-reactive SARS-CoV-2 peptides revealed pre-existing T cell responses in 81% of unexposed individuals and validated similarity with common cold coronaviruses, providing a functional basis for heterologous immunity in SARS-CoV-2 infection. Diversity of SARS-CoV-2 T cell responses was associated with mild symptoms of COVID-19, providing evidence that immunity requires recognition of multiple epitopes. Together, the proposed SARS-CoV-2 T cell epitopes enable identification of heterologous and post-infectious T cell immunity and facilitate development of diagnostic, preventive and therapeutic measures for COVID-19.

Biparatopic nanobodies protect mice from lethal challenge with SARS-CoV-2 variants of concern.

The ongoing COVID-19 pandemic and the emergence of new SARS-CoV-2 variants of concern (VOCs) requires continued development of effective therapeutics. Recently, we identified high-affinity neutralizing nanobodies (Nbs) specific for the receptor-binding domain (RBD) of SARS-CoV-2. Taking advantage of detailed epitope mapping, we generate two biparatopic Nbs (bipNbs) targeting a conserved epitope outside and two different epitopes inside the RBD:ACE2 interface. Both bipNbs bind all currently circulating VOCs with high affinities and are capable to neutralize cellular infection with VOC B.1.351 (Beta) and B.1.617.2 (Delta) in vitro. To assess if the bipNbs NM1267 and NM1268 confer protection against SARS-CoV-2 infection in vivo, human ACE2 transgenic mice are treated intranasally before infection with a lethal dose of SARS-CoV-2 B.1, B.1.351 (Beta) or B.1.617.2 (Delta). Nb-treated mice show significantly reduced disease progression and increased survival rates. Histopathological analyses further reveal a drastically reduced viral load and inflammatory response in lungs. These data suggest that both bipNbs are broadly active against a variety of emerging SARS-CoV-2 VOCs and represent easily applicable drug candidates.

Antibody Binding and Angiotensin-Converting Enzyme 2 Binding Inhibition Is Significantly Reduced for Both the BA.1 and BA.2 Omicron Variants.

BACKGROUND: The rapid emergence of the Omicron variant and its large number of mutations led to its classification as a variant of concern (VOC) by the World Health Organization. Subsequently, Omicron evolved into distinct sublineages (eg, BA.1 and BA.2), which currently represent the majority of global infections. Initial studies of the neutralizing response toward BA.1 in convalescent and vaccinated individuals showed a substantial reduction. METHODS: We assessed antibody (immunoglobulin G [IgG]) binding, ACE2 (angiotensin-converting enzyme 2) binding inhibition, and IgG binding dynamics for the Omicron BA.1 and BA.2 variants compared to a panel of VOCs/variants of interest, in a large cohort (N = 352) of convalescent, vaccinated, and infected and subsequently vaccinated individuals. RESULTS: While Omicron was capable of efficiently binding to ACE2, antibodies elicited by infection or immunization showed reduced binding capacities and ACE2 binding inhibition compared to wild type. Whereas BA.1 exhibited less IgG binding compared to BA.2, BA.2 showed reduced inhibition of ACE2 binding. Among vaccinated samples, antibody binding to Omicron only improved after administration of a third dose. CONCLUSIONS: Omicron BA.1 and BA.2 can still efficiently bind to ACE2, while vaccine/infection-derived antibodies can bind to Omicron. The extent of the mutations within both variants prevents a strong inhibitory binding response. As a result, both Omicron variants are able to evade control by preexisting antibodies.

NeutrobodyPlex-monitoring SARS-CoV-2 neutralizing immune responses using nanobodies.

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.

Diminishing Immune Responses against Variants of Concern in Dialysis Patients 4 Months after SARS-CoV-2 mRNA Vaccination.

Patients undergoing chronic hemodialysis were among the first to receive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccinations because of their increased risk for severe coronavirus disease and high case-fatality rates. By using a previously reported cohort from Germany of at-risk hemodialysis patients and healthy donors, where antibody responses were examined 3 weeks after the second vaccination, we assessed systemic cellular and humoral immune responses in serum and saliva 4 months after vaccination with the Pfizer-BioNTech BNT162b2 vaccine using an interferon-γ release assay and multiplex-based IgG measurements. We further compared neutralization capacity of vaccination-induced IgG against 4 SARS-CoV-2 variants of concern (Alpha, Beta, Gamma, and Delta) by angiotensin-converting enzyme 2 receptor-binding domain competition assay. Sixteen weeks after second vaccination, compared with 3 weeks after, cellular and humoral responses against the original SARS-CoV-2 isolate and variants of concern were substantially reduced. Some dialysis patients even had no detectable B- or T-cell responses.

A Novel PNGase Rc for Improved Protein N-Deglycosylation in Bioanalytics and Hydrogen-Deuterium Exchange Coupled With Mass Spectrometry Epitope Mapping under Challenging Conditions.

N-linked glycosylation is a ubiquitous posttranslational modification of proteins. While it plays an important role in the biological function of proteins, it often poses a major challenge for their analytical characterization. Currently available peptide N-glycanases (PNGases) are often inefficient at deglycosylating proteins due to sterically inaccessible N-glycosylation sites. This usually leads to poor sequence coverage in bottom-up analysis using liquid chromatography with tandem mass spectrometry and makes it impossible to obtain an intact mass signal in top-down MS analysis. In addition, most PNGases operate optimally only in the neutral to slightly acidic pH range and are severely compromised in the presence of reducing and denaturing substances, which limits their use for advanced bioanalysis based on hydrogen-deuterium exchange in combination with mass spectrometry (HDX-MS). Here, we present a novel peptide N-glycanase from Rudaea cellulosilytica (PNGase Rc) for which we demonstrate broad substrate specificity for N-glycan hydrolysis from multiply occupied and natively folded proteins. Our results show that PNGase Rc is functional even under challenging, HDX quenching conditions (pH 2.5, 0 °C) and in the presence of 0.4 M tris(2-carboxyethyl)phosphine, 4 M urea, and 1 M guanidinium chloride. Most importantly, we successfully applied the PNGase Rc in an HDX-MS workflow to determine the epitope of a nanobody targeting the extracellular domain of human signal-regulating protein alpha (SIRPα).

Chromobodies to Quantify Changes of Endogenous Protein Concentration in Living Cells.

Understanding cellular processes requires the determination of dynamic changes in the concentration of genetically nonmodified, endogenous proteins, which, to date, is commonly accomplished by end-point assays in vitro Molecular probes such as fluorescently labeled nanobodies (chromobodies, CBs) are powerful tools to visualize the dynamic subcellular localization of endogenous proteins in living cells. Here, we employed the dependence of intracellular levels of chromobodies on the amount of their endogenous antigens, a phenomenon, which we termed antigen-mediated CB stabilization (AMCBS), for simultaneous monitoring of time-resolved changes in the concentration and localization of native proteins. To improve the dynamic range of AMCBS we generated turnover-accelerated CBs and demonstrated their application in visualization and quantification of fast reversible changes in antigen concentration upon compound treatment by quantitative live-cell imaging. We expect that this broadly applicable strategy will enable unprecedented insights into the dynamic regulation of proteins, e.g. during cellular signaling, cell differentiation, or upon drug action.

Okadaic acid activates Wnt/ß-catenin-signaling in human HepaRG cells.

The lipophilic phycotoxin okadaic acid (OA) occurs in the fatty tissue and hepatopancreas of filter-feeding shellfish. The compound provokes the diarrhetic shellfish poisoning (DSP) syndrome after intake of seafood contaminated with high levels of the DSP toxin. In animal experiments, long-term exposure to OA is associated with an elevated risk for tumor formation in different organs including the liver. Although OA is a known inhibitor of the serine/threonine protein phosphatase 2A, the mechanisms behind OA-induced carcinogenesis are not fully understood. Here, we investigated the influence of OA on the ß-catenin-dependent Wnt-signaling pathway, addressing a major oncogenic pathway relevant for tumor development. We analyzed OA-mediated effects on ß-catenin and its biological function, cellular localization, post-translational modifications, and target gene expression in human HepaRG hepatocarcinoma cells treated with non-cytotoxic concentrations up to 50 nM. We detected concentration- and time-dependent effects of OA on the phosphorylation state, cellular redistribution as well as on the amount of transcriptionally active ß-catenin. These findings were confirmed by quantitative live-cell imaging of U2OS cells stably expressing a green fluorescent chromobody which specifically recognize hypophosphorylated ß-catenin. Finally, we demonstrated that nuclear translocation of ß-catenin mediated by non-cytotoxic OA concentrations results in an upregulation of Wnt-target genes. In conclusion, our results show a significant induction of the canonical Wnt/ß-catenin-signaling pathway by OA in human liver cells. Our data contribute to a better understanding of the molecular mechanisms underlying OA-induced carcinogenesis.

Monitoring interactions and dynamics of endogenous beta-catenin with intracellular nanobodies in living cells.

ß-catenin is the key component of the canonical Wnt pathway and plays a crucial role in a multitude of developmental and homeostatic processes. The different tasks of ß-catenin are orchestrated by its subcellular localization and participation in multiprotein complexes. To gain a better understanding of ß-catenin's role in living cells we have generated a new set of single domain antibodies, referred to as nanobodies, derived from heavy chain antibodies of camelids. We selected nanobodies recognizing the N-terminal, core or C-terminal domain of ß-catenin and applied these new high-affinity binders as capture molecules in sandwich immunoassays and co-immunoprecipitations of endogenous ß-catenin complexes. In addition, we engineered intracellularly functional anti-ß-catenin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins. For the first time, we were able to visualize the subcellular localization and nuclear translocation of endogenous ß-catenin in living cells using these chromobodies. Moreover, the chromobody signal allowed us to trace the accumulation of diffusible, hypo-phosphorylated ß-catenin in response to compound treatment in real time using High Content Imaging. The anti-ß-catenin nanobodies and chromobodies characterized in this study are versatile tools that enable a novel and unique approach to monitor the dynamics of subcellular ß-catenin in biochemical and cell biological assays.

Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells.

In biomedical research there is an ongoing demand for new technologies, which help to elucidate disease mechanisms and provide the basis to develop novel therapeutics. In this context a comprehensive understanding of cellular processes and their pathophysiology based on reliable information on abundance, localization, posttranslational modifications and dynamic interactions of cellular components is indispensable. Besides their significant impact as therapeutic molecules, antibodies are arguably the most powerful research tools to study endogenous proteins and other cellular components. However, for cellular diagnostics their use is restricted to endpoint assays using fixed and permeabilized cells. Alternatively, live cell imaging using fluorescent protein-tagged reporters is widely used to study protein localization and dynamics in living cells. However, only artificially introduced chimeric proteins are visualized, whereas the endogenous proteins, their posttranslational modifications as well as non-protein components of the cell remain invisible and cannot be analyzed. To overcome these limitations, traceable intracellular binding molecules provide new opportunities to perform cellular diagnostics in real time. In this review we summarize recent progress in the generation of intracellular and cell penetrating antibodies and their application to target and trace cellular components in living cells. We highlight recent advances in the structural formulation of recombinant antibody formats, reliable screening protocols and sophisticated cellular targeting technologies and propose that such intrabodies will become versatile research tools for real time cell-based diagnostics including target validation and live cell imaging. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells.

Signal-regulatory protein α (SIRPα) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRPα and the "don't eat me" ligand CD47 and as a marker to monitor macrophage infiltration into tumor lesions. To address both approaches, we developed a set of novel human SIRPα (hSIRPα)-specific nanobodies (Nbs). We identified high-affinity Nbs targeting the hSIRPα/hCD47 interface, thereby enhancing antibody-dependent cellular phagocytosis. For non-invasive in vivo imaging, we chose S36 Nb as a non-modulating binder. By quantitative positron emission tomography in novel hSIRPα/hCD47 knock-in mice, we demonstrated the applicability of 64Cu-hSIRPα-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRPα-Nbs presented in this study have potential as versatile theranostic probes, including novel myeloid-specific checkpoint inhibitors for combinatorial treatment approaches and for in vivo stratification and monitoring of individual responses during cancer immunotherapies.

A Nanobody-Based Toolset to Monitor and Modify the Mitochondrial GTPase Miro1.

The mitochondrial outer membrane (MOM)-anchored GTPase Miro1, is a central player in mitochondrial transport and homeostasis. The dysregulation of Miro1 in amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) suggests that Miro1 may be a potential biomarker or drug target in neuronal disorders. However, the molecular functionality of Miro1 under (patho-) physiological conditions is poorly known. For a more comprehensive understanding of the molecular functions of Miro1, we have developed Miro1-specific nanobodies (Nbs) as novel research tools. We identified seven Nbs that bind either the N- or C-terminal GTPase domain of Miro1 and demonstrate their application as research tools for proteomic and imaging approaches. To visualize the dynamics of Miro1 in real time, we selected intracellularly functional Nbs, which we reformatted into chromobodies (Cbs) for time-lapse imaging of Miro1. By genetic fusion to an Fbox domain, these Nbs were further converted into Miro1-specific degrons and applied for targeted degradation of Miro1 in live cells. In summary, this study presents a collection of novel Nbs that serve as a toolkit for advanced biochemical and intracellular studies and modulations of Miro1, thereby contributing to the understanding of the functional role of Miro1 in disease-derived model systems.

COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants.

As global vaccination campaigns against SARS-CoV-2 proceed, there is particular interest in the longevity of immune protection, especially with regard to increasingly infectious virus variants. Neutralizing antibodies (Nabs) targeting the receptor binding domain (RBD) of SARS-CoV-2 are promising correlates of protective immunity and have been successfully used for prevention and therapy. As SARS-CoV-2 variants of concern (VOCs) are known to affect binding to the ACE2 receptor and by extension neutralizing activity, we developed a bead-based multiplex ACE2-RBD inhibition assay (RBDCoV-ACE2) as a highly scalable, time-, cost-, and material-saving alternative to infectious live-virus neutralization tests. By mimicking the interaction between ACE2 and the RBD, this serological multiplex assay allows the simultaneous analysis of ACE2 binding inhibition to the RBDs of all SARS-CoV-2 VOCs and variants of interest (VOIs) in a single well. Following validation against a classical virus neutralization test and comparison of performance against a commercially available assay, we analyzed 266 serum samples from 168 COVID-19 patients of varying severity. ACE2 binding inhibition was reduced for ten out of eleven variants examined compared to wild-type, especially for those displaying the E484K mutation such as VOCs beta and gamma. ACE2 binding inhibition, while highly individualistic, positively correlated with IgG levels. ACE2 binding inhibition also correlated with disease severity up to WHO grade 7, after which it reduced.

Longitudinal cellular and humoral immune responses after triple BNT162b2 and fourth full-dose mRNA-1273 vaccination in haemodialysis patients.

Haemodialysis patients respond poorly to vaccination and continue to be at-risk for severe COVID-19. Therefore, dialysis patients were among the first for which a fourth COVID-19 vaccination was recommended. However, targeted information on how to best maintain immune protection after SARS-CoV-2 vaccinations in at-risk groups for severe COVID-19 remains limited. We provide, to the best of our knowledge, for the first time longitudinal vaccination response data in dialysis patients and controls after a triple BNT162b2 vaccination and in the latter after a subsequent fourth full-dose of mRNA-1273. We analysed systemic and mucosal humoral IgG responses against the receptor-binding domain (RBD) and ACE2-binding inhibition towards variants of concern including Omicron and Delta with multiplex-based immunoassays. In addition, we assessed Spike S1-specific T-cell responses by interferon γ release assay. After triple BNT162b2 vaccination, anti-RBD B.1 IgG and ACE2 binding inhibition reached peak levels in dialysis patients, but remained inferior compared to controls. Whilst we detected B.1-specific ACE2 binding inhibition in 84% of dialysis patients after three BNT162b2 doses, binding inhibition towards the Omicron variant was only detectable in 38% of samples and declining to 16% before the fourth vaccination. By using mRNA-1273 as fourth dose, humoral immunity against all SARS-CoV-2 variants tested was strongly augmented with 80% of dialysis patients having Omicron-specific ACE2 binding inhibition. Modest declines in T-cell responses in dialysis patients and controls after the second vaccination were restored by the third BNT162b2 dose and significantly increased by the fourth vaccination. Our data support current advice for a four-dose COVID-19 immunisation scheme for at-risk individuals such as haemodialysis patients. We conclude that administration of a fourth full-dose of mRNA-1273 as part of a mixed mRNA vaccination scheme to boost immunity and to prevent severe COVID-19 could also be beneficial in other immune impaired individuals. Additionally, strategic application of such mixed vaccine regimens may be an immediate response against SARS-CoV-2 variants with increased immune evasion potential.

Comparative Magnitude and Persistence of Humoral SARS-CoV-2 Vaccination Responses in the Adult Population in Germany.

Recent increases in SARS-CoV-2 infections have led to questions about duration and quality of vaccine-induced immune protection. While numerous studies have been published on immune responses triggered by vaccination, these often focus on studying the impact of one or two immunisation schemes within subpopulations such as immunocompromised individuals or healthcare workers. To provide information on the duration and quality of vaccine-induced immune responses against SARS-CoV-2, we analyzed antibody titres against various SARS-CoV-2 antigens and ACE2 binding inhibition against SARS-CoV-2 wild-type and variants of concern in samples from a large German population-based seroprevalence study (MuSPAD) who had received all currently available immunisation schemes. We found that homologous mRNA-based or heterologous prime-boost vaccination produced significantly higher antibody responses than vector-based homologous vaccination. Ad26.CoV2S.2 performance was particularly concerning with reduced titres and 91.7% of samples classified as non-responsive for ACE2 binding inhibition, suggesting that recipients require a booster mRNA vaccination. While mRNA vaccination induced a higher ratio of RBD- and S1-targeting antibodies, vector-based vaccines resulted in an increased proportion of S2-targeting antibodies. Given the role of RBD- and S1-specific antibodies in neutralizing SARS-CoV-2, their relative over-representation after mRNA vaccination may explain why these vaccines have increased efficacy compared to vector-based formulations. Previously infected individuals had a robust immune response once vaccinated, regardless of which vaccine they received, which could aid future dose allocation should shortages arise for certain manufacturers. Overall, both titres and ACE2 binding inhibition peaked approximately 28 days post-second vaccination and then decreased.

Robust and durable serological response following pediatric SARS-CoV-2 infection.

The quality and persistence of children's humoral immune response following SARS-CoV-2 infection remains largely unknown but will be crucial to guide pediatric SARS-CoV-2 vaccination programs. Here, we examine 548 children and 717 adults within 328 households with at least one member with a previous laboratory-confirmed SARS-CoV-2 infection. We assess serological response at 3-4 months and 11-12 months after infection using a bead-based multiplex immunoassay for 23 human coronavirus antigens including SARS-CoV-2 and its Variants of Concern (VOC) and endemic human coronaviruses (HCoVs), and additionally by three commercial SARS-CoV-2 antibody assays. Neutralization against wild type SARS-CoV-2 and the Delta VOC are analysed in a pseudotyped virus assay. Children, compared to adults, are five times more likely to be asymptomatic, and have higher specific antibody levels which persist longer (96.2% versus 82.9% still seropositive 11-12 months post infection). Of note, symptomatic and asymptomatic infections induce similar humoral responses in all age groups. SARS-CoV-2 infection occurs independent of HCoV serostatus. Neutralization responses of children and adults are similar, although neutralization is reduced for both against the Delta VOC. Overall, the long-term humoral immune response to SARS-CoV-2 infection in children is of longer duration than in adults even after asymptomatic infection.

Improved targeting of human CD4+ T cells by nanobody-modified AAV2 gene therapy vectors.

Adeno-associated viruses (AAV) are considered non-pathogenic in humans, and thus have been developed into powerful vector platforms for in vivo gene therapy. Although the various AAV serotypes display broad tropism, frequently infecting multiple tissues and cell types, vectors for specific and efficient targeting of human CD4+ T lymphocytes are largely missing. In fact, a substantial translational bottleneck exists in the field of therapeutic gene transfer that would require in vivo delivery into peripheral disease-related lymphocytes for subsequent genome editing. To solve this issue, capsid modification for retargeting AAV tropism, and in turn improving vector potency, is considered a promising strategy. Here, we genetically modified the minor AAV2 capsid proteins, VP1 and VP2, with a set of novel nanobodies with high-affinity for the human CD4 receptor. These novel vector variants demonstrated improved targeting of human CD4+ cells, including primary human peripheral blood mononuclear cells (PBMC) and purified human CD4+ T lymphocytes. Thus, the technical approach presented here provides a promising strategy for developing specific gene therapy vectors, particularly targeting disease-related peripheral blood CD4+ leukocytes.

Cellular and humoral immunogenicity of a SARS-CoV-2 mRNA vaccine in patients on haemodialysis.

BACKGROUND: Patients with chronic renal insufficiency on maintenance haemodialysis face an increased risk of COVID-19 induced mortality and impaired vaccine responses. To date, only a few studies have addressed SARS-CoV-2 vaccine elicited immunity in this immunocompromised population. METHODS: We assessed immunogenicity of the mRNA vaccine BNT162b2 in at-risk dialysis patients and characterised systemic cellular and humoral immune responses in serum and saliva using interferon γ release assay and multiplex-based cytokine and immunoglobulin measurements. We further compared binding capacity and neutralization efficacy of vaccination-induced immunoglobulins against emerging SARS-CoV-2 variants Alpha, Beta, Epsilon and Cluster 5 by ACE2-RBD competition assay. FINDINGS: Patients on maintenance haemodialysis exhibit detectable but variable cellular and humoral immune responses against SARS-CoV-2 and variants of concern after a two-dose regimen of BNT162b2. Although vaccination-induced immunoglobulins were detectable in saliva and plasma, both anti-SARS-CoV-2 IgG and neutralization efficacy was reduced compared to a vaccinated non-dialysed control population. Similarly, T-cell mediated interferon γ release after stimulation with SARS-CoV-2 spike peptides was significantly diminished. INTERPRETATION: Quantifiable humoral and cellular immune responses after BNT162b2 vaccination in individuals on maintenance haemodialysis are encouraging, but urge for longitudinal follow-up to assess longevity of immunity. Diminished virus neutralization and interferon γ responses in the face of emerging variants of concern may favour this at-risk population for re-vaccination using modified vaccines at the earliest opportunity. FUNDING: Initiative and Networking Fund of the Helmholtz Association of German Research Centres, EU Horizon 2020 research and innovation program, State Ministry of Baden-Württemberg for Economic Affairs, Labour and Tourism.

Immune response to SARS-CoV-2 variants of concern in vaccinated individuals.

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.

Multiplexed Serum Antibody Screening Platform Using Virus Extracts from Endemic Coronaviridae and SARS-CoV-2.

The presence of antibodies against endemic coronaviruses has been linked to disease severity after SARS-CoV-2 infection. Assays capable of concomitantly detecting antibodies against endemic coronaviridae such as OC43, 229E, NL63, and SARS-CoV-2 may help to elucidate this question. We developed a serum screening platform using a bead-based Western blot system called DigiWest, capable of running hundreds of assays using microgram amounts of protein prepared directly from different viruses. Characterization of the immunoassay for detection of SARS-CoV-2 specific antibodies revealed a sensitivity of 90.3% and a diagnostic specificity of 98.1%. Concordance analysis with the SARS-CoV-2 immunoassays available by Roche, Siemens, and Euroimmun indicates comparable assay performances (Cohen's κ ranging from 0.8874 to 0.9508). Analogous assays for OC43, 229E, and NL63 were established and combined into one multiplex with the SARS-CoV-2 assay. Seroreactivity for different coronaviruses was detected with high incidence, and the multiplex assay was adapted for serum screening.