Allelic variation in class I HLA determines CD8+ T cell repertoire shape and cross-reactive memory responses to SARS-CoV-2.

Effective presentation of antigens by human leukocyte antigen (HLA) class I molecules to CD8+ T cells is required for viral elimination and generation of long-term immunological memory. In this study, we applied a single-cell, multiomic technology to generate a unified ex vivo characterization of the CD8+ T cell response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) across four major HLA class I alleles. We found that HLA genotype conditions key features of epitope specificity, TCRα/ß sequence diversity, and the utilization of pre-existing SARS-CoV-2-reactive memory T cell pools. Single-cell transcriptomics revealed functionally diverse T cell phenotypes of SARS-CoV-2-reactive T cells, associated with both disease stage and epitope specificity. Our results show that HLA variations notably influence the CD8+ T cell repertoire shape and utilization of immune recall upon SARS-CoV-2 infection.

Profiling SARS-CoV-2 HLA-I peptidome reveals T cell epitopes from out-of-frame ORFs.

T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection, but the repertoire of naturally processed and presented viral epitopes on class I human leukocyte antigen (HLA-I) remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post infection using mass spectrometry. We found HLA-I peptides derived not only from canonical open reading frames (ORFs) but also from internal out-of-frame ORFs in spike and nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and individuals with COVID-19 that exceeded responses to canonical peptides, including some of the strongest epitopes reported to date. Whole-proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights, as well as the discovery of out-of-frame ORF epitopes, will facilitate selection of peptides for immune monitoring and vaccine development.

Controlled loading of building blocks into temporary self-assembled scaffolds for directed assembly of organic nanostructures.

Using temporary self-assembled scaffolds to preorganize building blocks is a potentially powerful method for the synthesis of organic nanostructures with programmed shapes. We examined the underlying phenomena governing the loading of hydrophobic monomers into lipid bilayer interior and demonstrated successful control of the amount and ratio of loaded monomers. When excess styrene derivatives or acrylates were added to the aqueous solution of unilamellar liposomes made from saturated phospholipids, most loading occurs within the first few hours. Dynamic light scattering and transmission electron microscopy revealed no evidence of aggregation caused by monomers. Bilayers appeared to have a certain capacity for accommodating monomers. The total volume of loaded monomers is independent of monomer structure. X-ray scattering showed the increase in bilayer thickness consistent with loading monomers into bilayer interior. Loading kinetics is inversely proportional to the hydrophobicity and size of monomers. Loading and extraction kinetic data suggest that crossing the polar heads region is the rate limiting step. Consideration of loading kinetics and multiple equilibria are important for achieving reproducible monomer loading. The total amount of monomers loaded into the bilayer can be controlled by the loading time or length of hydrophobic lipid tails. The ratio of loaded monomers can be varied by changing the ratio of monomers used for loading or by the time-controlled replacement of a preloaded monomer. Understanding and controlling the loading of monomers into bilayers contributes to the directed assembly of organic nanostructures.