Evaluation of SARS-CoV-2-Specific T-Cell Activation with a Rapid On-Chip IGRA.

Interferon-gamma release assays (IGRAs) that measure pathogen-specific T-cell response rates can provide a more reliable estimate of protection than specific antibody levels but have limited potential for widespread use due to their workflow, personnel, and instrumentation demands. The major vaccines for SARS-CoV-2 have demonstrated substantial efficacy against all of its current variants, but approaches are needed to determine how these vaccines will perform against future variants, as they arise, to inform vaccine and public health policies. Here we describe a rapid, sensitive, nanolayer polylysine-integrated microfluidic chip IGRA read by a fluorescent microscope that has a 5 h sample-to-answer time and uses ∼25 µL of a fingerstick whole blood sample. Results from this assay correlated with those of a comparable clinical IGRA when used to evaluate the T-cell response to SARS-CoV-2 peptides in a population of vaccinated and/or infected individuals. Notably, this streamlined and inexpensive assay is suitable for high-throughput analyses in resource-limited settings for other infectious diseases.

SARS-CoV-2 Epitopes following Infection and Vaccination Overlap Known Neutralizing Antibody Sites.

Identification of epitopes targeted following virus infection or vaccination can guide vaccine design and development of therapeutic interventions targeting functional sites, but can be laborious. Herein, we employed peptide microarrays to map linear peptide epitopes (LPEs) recognized following SARS-CoV-2 infection and vaccination. LPEs detected by nonhuman primate (NHP) and patient IgMs after SARS-CoV-2 infection extensively overlapped, localized to functionally important virus regions, and aligned with reported neutralizing antibody binding sites. Similar LPE overlap occurred after infection and vaccination, with LPE clusters specific to each stimulus, where strong and conserved LPEs mapping to sites known or likely to inhibit spike protein function. Vaccine-specific LPEs tended to map to sites known or likely to be affected by structural changes induced by the proline substitutions in the mRNA vaccine's S protein. Mapping LPEs to regions of known functional importance in this manner may accelerate vaccine evaluation and discovery of targets for site-specific therapeutic interventions.

Nanopore-based disease diagnosis using pathogen-derived tryptic peptides from serum.

Nanopore sensors have shown great utility in nucleic acid detection and sequencing approaches. Recent studies also indicate that current signatures produced by peptide-nanopore interactions can distinguish high purity peptide mixtures, but the utility of nanopore sensors in clinical applications still needs to be explored due to the inherent complexity of clinical specimens. To fill this gap between research and clinical nanopore applications, we describe a methodology to select peptide biomarkers suitable for use in an immunoprecipitation-coupled nanopore (IP-NP) assay, based on their pathogen specificity, antigenicity, charge, water solubility and ability to produce a characteristic nanopore interaction signature. Using tuberculosis as a proof-of-principle example in a disease that can be challenging to diagnose, we demonstrate that a peptide identified by this approach produced high-affinity antibodies and yielded a characteristic peptide signature that was detectable over a broad linear range, to detect and quantify a pathogen-derived peptide from digested human serum samples with high sensitivity and specificity. This nanopore signal distinguished serum from a TB case, non-disease controls, and from a TB-case after extended anti-TB treatment. We believe this assay approach should be readily adaptable to other infectious and chronic diseases that can be diagnosed by peptide biomarkers.