SARS-CoV-2 is the causative agent of COVID-19 and is a severe threat to global health.Patients infected with SARS-CoV-2 show a wide range of symptoms and disease severity, while limited data is available on its immunogenicity.Here, the kinetics of the development of SARS-CoV-2-specific antibody responses in relation to clinical features and dynamics of specific B-cell populations are reported. Immunophenotyping of B cells was performed by flow cytometry with longitudinally collectedPBMCs. In parallel, serum samples were analyzed for the presence of SARS-CoV-2-specific IgA, IgG, and IgM antibodies using whole proteome peptide microarrays. Soon after disease onset in a mild case, we observed an increased frequency of plasmablasts concomitantly with a strong SARS-CoV-2-specific IgA response. In contrast, a case with more severe progression showed a delayed, but eventually very strong and broad SARS-CoV-2-specific IgA response.This case study shows that determining SARS-CoV-2-specific antibody epitopes can be valuable to monitor the specificity and magnitude of the early B-cell response, which could guide the development of vaccine candidates. Follow-up studies are required to evaluate whether the kinetics and strength of the SARS-CoV-2-specific IgA response could be potential prognostic markers of viral control.

Single glycan-protein interactions are often weak, such that glycan binding partnerscommonly utilizemultiple, spatially defined binding sites to enhance binding avidity and specificity.C urrent array technologiesu sually neglect defined multivalent display.L aser-based array synthesis technology allows for flexible and rapid on-surface synthesis of different peptides. By combining this technique with click chemistry,n eo-glycopeptides werep roduced directly on a functionalized glass slide in the microarray format. Density and spatial distribution of carbohydrates can be tuned, resulting in well-defined glycan structures for multivalent display.T he two lectins concanavalin Aa nd langerin were probedw ith different glycanso nm ultivalent scaffolds, revealing strong spacing-, density-, and ligand-dependent binding. In addition, we could also measuret he surfaced issociation constant.T his approach allows for ar apid generation, screening, and optimization of am ultitudeo fm ultivalent scaffoldsfor glycan binding.

Clostridioides difficile is the causative bacterium in 15–20% of all antibiotic associated diarrheas. The symptoms associated with C. difficile infection (CDI) are primarily induced by the two large exotoxins TcdA and TcdB. Both toxins enter target cells by receptor-mediated endocytosis. Although different toxin receptors have been identified, it is no valid therapeutic option to prevent receptor endocytosis. Therapeutics, such as neutralizing antibodies, directly targeting both toxins are in development. Interestingly, only the anti-TcdB antibody bezlotoxumab but not the anti-TcdA antibody actoxumab prevented recurrence of CDI in clinical trials. In this work, 31 human antibody fragments against TcdB were selected by antibody phage display from the human naive antibody gene libraries HAL9/10. These antibody fragments were further characterized by in vitro neutralization assays. The epitopes of the neutralizing and non-neutralizing antibody fragments were analyzed by domain mapping, TcdB fragment phage display, and peptide arrays, to identify neutralizing and non-neutralizing epitopes. A new neutralizing epitope within the glucosyltransferase domain of TcdB was identified, providing new insights into the relevance of different toxin regions in respect of neutralization and toxicity.

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.

A low‐cost laser‐based printing setup is presented, which allows for the spot‐wise patterning of surfaces with defined polymer nanolayers. These nanolayer spots serve as a “solid solvent,” embedding different chemicals, chemical building blocks, materials, or precursors and can be stacked on top of each other. By melting the spot pattern, the polymer‐embedded molecules are released for chemical reaction. This enables researchers to quickly pattern a surface with different molecules and materials, mixing them directly on the surface for high‐throughput chemical synthesis to generate and screen diverse microarray libraries. In contrast to expensive ink‐jet or contact printing, this approach does not require premixing of inks, which enables in situ combinatorial mixing. Easy access and versatility of this patterning approach are shown by generating microarrays of various biomolecules, such as glycans for the first time, to screen interactions of antibodies and lectins. In addition, a layer‐by‐layer solid‐phase synthesis of peptides directly on the microarray is presented. Amino acid–containing nanolayers are repeatedly laser‐transferred and reacted with the functionalized acceptor surface in defined patterns. This simple system enables a reproducible array production, down to spot‐to‐spot distances of 100 µm, and offers a flexible and cheap alternative to expensive spotting robot technology.

Emergence and re-emergence of pathogens bearing the risk of becoming a pandemic threat are on the rise. Increased travel and trade, growing population density, changes in urbanization, and climate have a critical impact on infectious disease spread. Currently, the world is confronted with the emergence of a novel coronavirus SARS-CoV-2, responsible for yet more than 800 000 deaths globally. Outbreaks caused by viruses, such as SARS-CoV-2, HIV, Ebola, influenza, and Zika, have increased over the past decade, underlining the need for a rapid development of diagnostics and vaccines. Hence, the rational identification of biomarkers for diagnostic measures on the one hand, and antigenic targets for vaccine development on the other, are of utmost importance. Peptide microarrays can display large numbers of putative target proteins translated into overlapping linear (and cyclic) peptides for a multiplexed, high-throughput antibody analysis. This enabled for example the identification of discriminant/diagnostic epitopes in Zika or influenza and mapping epitope evolution in natural infections versus vaccinations. In this review, we highlight synthesis platforms that facilitate fast and flexible generation of high-density peptide microarrays. We further outline the multifaceted applications of these peptide array platforms for the development of serological tests and vaccines to quickly encounter pandemic threats.

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The Ebola virus (EBOV) can cause severe infections in humans, leading to a fatal outcome in a high percentage of cases. Neutralizing antibodies against the EBOV surface glycoprotein (GP) can prevent infections, demonstrating a straightforward way for an efficient vaccination strategy. Meanwhile, many different anti‐EBOV antibodies have been identified, whereas the exact binding epitopes are often unknown. Here, the analysis of serum samples from an EBOV vaccine trial with the recombinant vesicular stomatitis virus‐Zaire ebolavirus (rVSV‐ZEBOV) and an Ebola virus disease survivor, using high‐density peptide arrays, is presented. In this proof‐of‐principle study, distinct IgG and IgM antibodies binding to different epitopes of EBOV GP is detected: By mapping the whole GP as overlapping peptide fragments, new epitopes and confirmed epitopes from the literature are found. Furthermore, the highly selective binding epitope of a neutralizing monoclonal anti‐EBOV GP antibody could be validated. This shows that peptide arrays can be a valuable tool to study the humoral immune response to vaccines in patients and to support Ebola vaccine development.