Mapping the immunopeptidome of seven SARS-CoV-2 antigens across common HLA haplotypes.

Most COVID-19 vaccines elicit immunity against the SARS-CoV-2 Spike protein. However, Spike protein mutations in emerging strains and immune evasion by the SARS-CoV-2 virus demonstrates the need to develop more broadly targeting vaccines. To facilitate this, we use mass spectrometry to identify immunopeptides derived from seven relatively conserved structural and non-structural SARS-CoV-2 proteins (N, E, Nsp1/4/5/8/9). We use two different B-lymphoblastoid cell lines to map Human Leukocyte Antigen (HLA) class I and class II immunopeptidomes covering some of the prevalent HLA types across the global human population. We employ DNA plasmid transfection and direct antigen delivery approaches to sample different antigens and find 248 unique HLA class I and HLA class II bound peptides with 71 derived from N, 12 from E, 28 from Nsp1, 19 from Nsp4, 73 from Nsp8 and 45 peptides derived from Nsp9. Over half of the viral peptides are unpublished. T cell reactivity tested against 56 of the detected peptides shows CD8+ and CD4+ T cell responses against several peptides from the N, E, and Nsp9 proteins. Results from this study will aid the development of next-generation COVID vaccines targeting epitopes from across a number of SARS-CoV-2 proteins.

Escherichia coli Promoters with Consistent Expression throughout the Murine Gut.

Advanced microbial therapeutics have great potential as a novel modality to diagnose and treat a wide range of diseases. Yet, to realize this potential, robust parts for regulating gene expression and consequent therapeutic activity in situ are needed. In this study, we characterized the expression level of more than 8000 variants of the Escherichia coli sigma factor 70 (σ70) promoter in a range of different environmental conditions and growth states using fluorescence-activated cell sorting and deep sequencing. Sampled conditions include aerobic and anaerobic culture in the laboratory as well as growth in several locations of the murine gastrointestinal tract. We found that σ70 promoters in E. coli generally maintain consistent expression levels across the murine gut (R2: 0.55-0.85, p value < 1 × 10-5), suggesting a limited environmental influence but a higher variability between in vitro and in vivo expression levels, highlighting the challenges of translating in vitro promoter activity to in vivo applications. Based on these data, we design the Schantzetta library, composed of eight promoters spanning a wide expression range and displaying a high degree of robustness in both laboratory and in vivo conditions (R2 = 0.98, p = 0.000827). This study provides a systematic assessment of the σ70 promoter activity in E. coli as it transits the murine gut leading to the definition of robust expression cassettes that could be a valuable tool for reliable engineering and development of advanced microbial therapeutics.