Chromatographic monoliths for high-throughput immunoaffinity isolation of transferrin from human plasma

Changes in protein glycosylation are related to different diseases and have a potential as diagnostic and prognostic disease biomarkers. Transferrin (Tf) glycosylation changes are common marker for congenital disorders of glycosylation. However, biological interindividual variability of Tf N-glycosylation and genes involved in glycosylation regulation are not known. Therefore, high-throughput Tf isolation method and large scale glycosylation studies are needed in order to address these questions. Due to their unique chromatographic properties, the use of chromatographic monoliths enables very fast analysis cycle, thus significantly increasing sample preparation throughput. Here, we are describing characterization of novel immunoaffinity-based monolithic columns in a 96-well plate format for specific high-throughput purification of human Tf from blood plasma. We optimized the isolation and glycan preparation procedure for subsequent ultra performance liquid chromatography (UPLC) analysis of Tf N-glycosylation and managed to increase the sensitivity for approximately three times compared to initial experimental conditions, with very good reproducibility. This work is licensed under a Creative Commons Attribution 4.0 International License

Collection of Monoclonal Antibodies Targeting SARS-CoV-2 Proteins

In early 2020, the COVID-19 pandemic sparked a global crisis that continues to pose a serious threat to human health and the economy. Further advancement in research is necessary and requires the availability of quality molecular tools, including monoclonal antibodies. Here, we present the development and characterization of a collection of over 40 new monoclonal antibodies directed against different SARS-CoV-2 proteins. Recombinant SARS-CoV-2 proteins were expressed, purified, and used as immunogens. Upon development of specific hybridomas, the obtained monoclonal antibody (mAb) clones were tested for binding to recombinant proteins and infected cells. We generated mAbs against structural proteins, the Spike and Nucleocapsid protein, several non-structural proteins (nsp1, nsp7, nsp8, nsp9, nsp10, nsp16) and accessory factors (ORF3a, ORF9b) applicable in flow cytometry, immunofluorescence, or Western blot. Our collection of mAbs provides a set of novel, highly specific tools that will allow a comprehensive analysis of the viral proteome, which will allow further understanding of SARS-CoV-2 pathogenesis and the design of therapeutic strategies

ChAdOx1‐S adenoviral vector vaccine applied intranasally elicits superior mucosal immunity compared to the intramuscular route of vaccination

COVID‐19 vaccines prevent severe forms of the disease, but do not warrant complete protection against breakthrough infections. This could be due to suboptimal mucosal immunity at the site of virus entry, given that all currently approved vaccines are administered via the intramuscular route. In this study, we assessed humoral and cellular immune responses in BALB/c mice after intranasal and intramuscular immunization with adenoviral vector ChAdOx1‐S expressing full‐length Spike protein of SARS‐CoV‐2. We showed that both routes of vaccination induced a potent IgG antibody response, as well as robust neutralizing capacity, but intranasal vaccination elicited a superior IgA antibody titer in the sera and in the respiratory mucosa. Bronchoalveolar lavage from intranasally immunized mice efficiently neutralized SARS‐CoV‐2, which has not been the case in intramuscularly immunized group. Moreover, substantially higher percentages of epitope‐specific CD8 T cells exhibiting a tissue resident phenotype were found in the lungs of intranasally immunized animals. Finally, both intranasal and intramuscular vaccination with ChAdOx1‐S efficiently protected the mice after the challenge with recombinant herpesvirus expressing the Spike protein. Our results demonstrate that intranasal application of adenoviral vector ChAdOx1‐S induces superior mucosal immunity and therefore could be a promising strategy for putting the COVID‐19 pandemic under control

SARS-CoV-2 Spike and Nucleocapsid Antibody Response in Vaccinated Croatian Healthcare Workers and Infected Hospitalized Patients: A Single Center Cohort Study

Studies assessing the dynamics and duration of antibody responses following SARS-CoV-2 infection or vaccination are an invaluable tool for vaccination schedule planning, assessment of risk groups and management of pandemics. In this study, we developed and employed ELISA assays to analyze the humoral responses to Nucleocapsid and Spike proteins in vaccinated health-care workers (HCW) and critically ill COVID-19 patients. Sera of more than 1000 HCWs and critically ill patients from the Clinical Hospital Center Rijeka were tested across a one-year period, encompassing the spread of major SARS-CoV-2 variants of concern (VOCs). We observed 97% of seroconversion in HCW cohort as well as sustained anti-Spike antibody response in vaccinees for more than 6 months. In contrast, the infection-induced anti-Nucleocapsid response was waning significantly in a six-month period. Furthermore, a substantial decrease in vaccinees’ anti-Spike antibodies binding to Spike protein of Omicron VOC was also observed. Critically ill COVID-19 patients had higher levels of anti-Spike and anti-Nucleocapsid antibodies compared to HCWs. No significant differences in anti-Spike and anti-Nucleocapsid antibody levels between the critically ill COVID-19 patients that were on non-invasive oxygen supplementation and those on invasive ventilation support were observed. However, stronger anti-Spike, but not anti-Nucleocapsid, antibody response correlated with a better disease outcome in the cohort of patients on invasive ventilation support. Altogether, our results contribute to the growing pool of data on humoral responses to SARS-CoV-2 infection and vaccination

Binding of the Fap2 Protein of Fusobacterium nucleatum to Human Inhibitory Receptor TIGIT Protects Tumors from Immune Cell Attack

Bacteria, such as Fusobacterium nucleatum, are present in the tumor microenvironment. However, the immunological consequences of intra-tumoral bacteria remain unclear. Here, we have shown that natural killer (NK) cell killing of various tumors is inhibited in the presence of various F. nucleatum strains. Our data support that this F. nucleatum- mediated inhibition is mediated by human, but not by mouse TIGIT, an inhibitory receptor present on all human NK cells and on various T cells. Using a library of F. nucleatum mutants, we found that the Fap2 protein of F. nucleatum directly interacted with TIGIT, leading to the inhibition of NK cell cytotoxicity. We have further demonstrated that tumor- infiltrating lymphocytes expressed TIGIT and that T cell activities were also inhibited by F. nucleatum via Fap2. Our results identify a bacterium- dependent, tumorimmune evasion mechanism in which tumors exploit the Fap2 protein of F. nucleatum to inhibit immune cell activity via TIGIT

Epitope Recognition of a Monoclonal Antibody Raised against a Synthetic Glycerol Phosphate Based Teichoic Acid

Glycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both native and synthetic structures, but no details have yet been reported on the structural basis of their interaction with antibodies. This work represents the first case study in which a monoclonal antibody, generated against a synthetic TA, was developed and employed for molecular-level binding analysis using TA microarrays, ELISA, SPR-analyses, and STD-NMR spectroscopy. Our findings show that the number and the chirality of the GroP residues are crucial for interaction and that the sugar appendage contributes to the presentation of the backbone to the binding site of the antibody

Elucidating the Structural and Minimal Protective Epitope of the Serogroup X Meningococcal Capsular Polysaccharide

Despite the considerable progress toward the eradication of meningococcal disease with the introduction of glycoconjugate vaccines, previously unremarkable serogroup X has emerged in recent years, recording several outbreaks throughout the African continent. Different serogroup X polysaccharide-based vaccines have been tested in preclinical trials, establishing the principles for further improvement. To elucidate the antigenic determinants of the MenX capsular polysaccharide, we generated a monoclonal antibody, and its bactericidal nature was confirmed using the rabbit serum bactericidal assay. The antibody was tested by the inhibition enzyme-linked immunosorbent assay and surface plasmon resonance against a set of oligosaccharide fragments of different lengths. The epitope was shown to be contained within five to six α-(1–4) phosphodiester mannosamine repeating units. The molecular interactions between the protective monoclonal antibody and the MenX capsular polysaccharide fragment were further detailed at the atomic level by saturation transfer difference nuclear magnetic resonance (NMR) spectroscopy. The NMR results were used for validation of the docking analysis between the X-ray crystal structure of the antibody (Fab fragment) and the modeled hexamer oligosaccharide. The antibody recognizes the MenX fragment by binding all six repeating units of the oligosaccharide hydrogen bonding, salt bridges, and hydrophobic interactions. studies demonstrated that conjugates containing five to six repeating units can produce high functional antibody levels. These results provide an insight into the molecular basis of MenX vaccine-induced protection and highlight the requirements for the epitope-based vaccine design

Comprehensive analysis of varicella-zoster virus proteins using a new monoclonal antibody collection

Varicella-zoster virus (VZV) is the etiological agent of chickenpox and shingles. Due to the virus's restricted host and cell typetropism and the lack of tools for VZV proteomics, it is one of the least-characterized human herpesviruses. We generated 251monoclonal antibodies (MAbs) against 59 of the 71 (83%) currently known unique VZV proteins to characterize VZV proteinexpression in vitro and in situ. Using this new set of MAbs, 44 viral proteins were detected by Western blotting (WB) and indirectimmunofluorescence (IF); 13 were detected by WB only, and 2 were detected by IF only. A large proportion of viral proteinswas analyzed for the first time in the context of virus infection. Our study revealed the subcellular localization of 46 proteins, 14of which were analyzed in detail by confocal microscopy. Seven viral proteins were analyzed in time course experiments andshowed a cascade-like temporal gene expression pattern similar to those of other herpesviruses. Furthermore, selected MAbstested positive on human skin lesions by using immunohistochemistry, demonstrating the wide applicability of the MAb collection.Finally, a significant portion of the VZV-specific antibodies reacted with orthologs of simian varicella virus (SVV), thusenabling the systematic analysis of varicella in a nonhuman primate model system. In summary, this study provides insight intothe potential function of numerous VZV proteins and novel tools to systematically study VZV and SVV pathogenesis