Discovery of Potent STT3A/B Inhibitors and Assessment of Their Multipathogen Antiviral Potential and Safety.

In the aftermath of the COVID-19 pandemic, opportunities to modulate biological pathways common to the lifecycles of viruses need to be carefully considered. N-linked glycosylation in humans is mediated exclusively by the oligosaccharyltransferase complex and is frequently hijacked by viruses to facilitate infection. As such, STT3A/B, the catalytic domain of the OST complex, became an intriguing drug target with broad-spectrum antiviral potential. However, due to the critical role N-linked glycosylation plays in a number of fundamental human processes, the toxicological ramifications of STT3A/B inhibition required attention commensurate to that given to antiviral efficacy. Herein, we describe how known STT3A/B inhibitor NGI-1 inspired the discovery of superior tool compounds which were evaluated in in vitro efficacy and translational safety (e.g., CNS, cardiovascular, liver) studies. The described learnings will appeal to those interested in the therapeutic utility of modulating N-linked glycosylation as well as the broader scientific community.

Adaption of a conventional ELISA to a 96-well ELISA-Array for measuring the antibody responses to influenza virus proteins and vaccines.

We describe an adaptation of conventional ELISA methods to an ELISA-Array format using non-contact Piezo printing of up to 30 spots of purified recombinant viral fusion proteins and vaccine on 96 well high-protein binding plates. Antigens were printed in 1 nanoliter volumes of protein stabilizing buffer using as little as 0.25 nanograms of protein, 2000-fold less than conventional ELISA. The performance of the ELISA-Array was demonstrated by serially diluting n = 9 human post-flu vaccination plasma samples starting at a 1/1000 dilution and measuring binding to the array of Influenza antigens. Plasma polyclonal antibody levels were detected using a cocktail of biotinylated anti-human kappa and lambda light chain antibodies, followed by a Streptavidin-horseradish peroxidase conjugate and the dose-dependent signal was developed with a precipitable TMB substrate. Intra- and inter-assay precision of absorbance units among the eight donor samples showed mean CVs of 4.8% and 10.8%, respectively. The plasma could be differentiated by donor and antigen with titer sensitivities ranging from 1 × 103 to 4 × 106, IC50 values from 1 × 104 to 9 × 106, and monoclonal antibody sensitivities in the ng/mL range. Equivalent sensitivities of ELISA versus ELISA-Array, compared using plasma and an H1N1 HA trimer, were achieved on the ELISA-Array printed at 0.25 ng per 200um spot and 1000 ng per ELISA 96-well. Vacuum-sealed array plates were shown to be stable when stored for at least 2 days at ambient temperature and up to 1 month at 4-8 °C. By the use of any set of printed antigens and analyte matrices the methods of this multiplexed ELISA-Array format can be broadly applied in translational research.

Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics.

Eliciting broadly neutralizing antibodies (bNAbs) against the four dengue virus serotypes (DENV1-4) that are spreading into new territories is an important goal of vaccine design. To define bNAb targets, we characterized 28 antibodies belonging to expanded and hypermutated clonal families identified by transcriptomic analysis of single plasmablasts from DENV-infected individuals. Among these, we identified J9 and J8, two somatically related bNAbs that potently neutralized DENV1-4. Mutagenesis studies showed that the major recognition determinants of these bNAbs are in E protein domain I, distinct from the only known class of human bNAbs against DENV with a well-defined epitope. B cell repertoire analysis from acute-phase peripheral blood suggested that J9 and J8 followed divergent somatic hypermutation pathways, and that a limited number of mutations was sufficient for neutralizing activity. Our study suggests multiple B cell evolutionary pathways leading to DENV bNAbs targeting a new epitope that can be exploited for vaccine design.