Cooke, G. S. | bioRxiv Science

bioRxiv2025

Comparative phenotypic, genomic and transcriptomic characterisation of two Salmonella Typhimurium strains for a first-in-human challenge model

BackgroundInvasive non-typhoidal Salmonella (iNTS) disease remains a major public health challenge in sub-Saharan Africa. Salmonella Typhimurium is responsible for the majority of cases, with specific lineages being associated with increased risk of bloodstream infection. We have recently developed a Salmonella Typhimurium controlled human infection model (CHIM) to better understand disease pathogenesis and to provide a platform to test candidate vaccines. Selecting appropriate challenge strainsis a central design consideration in developing challenge model protocols. We describe the rationale, manufacture, and detailed characterisation of the two Salmonella Typhimurium strains used in the first-in-human NTS CHIM. MethodsTwo Salmonella Typhimurium strains, 4/74 (ST19) and D23580 (ST313), were selected from the UK Health Security Agency National Collection of Type Cultures and manufactured under Good Manufacturing Practice (GMP) conditions. Challenge agent stocks underwent microbial limits testing, viability and stability assessments, and phenotypic characterisation including growth kinetics, motility, acid sensitivity, and antibiotic susceptibility. Whole-genome sequencing was performed to confirm genetic stability post-manufacture. The effect of pre-challenge handling conditions was assessed in saline and sodium bicarbonate buffers, and bacterial survival was evaluated under simulated gastric and intestinal conditions using a modified Rossett-Rice model. Transcriptomic profiling was undertaken to determine whether sodium bicarbonate exposure altered expression of key virulence genes. ResultsBoth strains retained their expected phenotypic characteristics, including reduced motility and melibiose utilisation in D23580. GMP stocks remained pure, viable, and stable post-manufacture, with no unexpected genomic mutations detected. Both strains were susceptible to clinically relevant antibiotics used in the study. Survival was maintained in neutral and mildly acidic conditions, with significant reduction below pH 3.5. Stability was preserved for up to one hour in saline buffer and during simulated gastric transit in the Rossett-Rice model. Transcriptomic analysis showed no significant changes in Salmonella pathogenicity island-1 or 2, or flagellar gene expression following sodium bicarbonate exposure ConclusionsThese findings confirm that the Salmonella Typhimurium 4/74 and D23580 strains retained genomic and phenotypic integrity post-manufacture. The resulting challenge stocks provide the foundation for an ongoing NTS CHIM that aims to advance understanding of NTS and iNTS pathogenesis and support candidate vaccine testing.

microbiology↗

bioRxiv2020

Impact of virus subtype and host IFNL4 genotype on large-scale RNA structure formation in the genome of hepatitis C virus

Mechanisms underlying the ability of hepatitis C virus (HCV) to establish persistent infections and induce progressive liver disease remain poorly understood. HCV is one of several positive-stranded RNA viruses capable of establishing persistence in their immunocompetent vertebrate hosts, an attribute associated with formation of large scale RNA structure in their genomic RNA. We developed novel methods to analyse and visualise genome-scale ordered RNA structure (GORS) predicted from the increasingly large datasets of complete genome sequences of HCV. Structurally conserved RNA secondary structure in coding regions of HCV localised exclusively to polyprotein ends (core, NS5B). Coding regions elsewhere were also intensely structured based on elevated minimum folding energy difference (MFED) values, but the actual stem-loop elements involved in genome folding were structurally entirely distinct, even between subtypes 1a and 1b. Dynamic remodelling was further evident from comparison of HCV strains in different host genetic background. Significantly higher MFED values, greater suppression of UpA dinucleotide frequencies and restricted diversification were found in subjects with the TT genotype of the rs12979860 SNP in the IFNL4 gene compared to the CC (non-expressing) allele. These structural and compositional associations with expression of interferon-{lambda}4 were recapitulated on a larger scale by higher MFED values and greater UpA suppression of genotype 1 compared to genotype 3a, associated with previously reported HCV genotype-associated differences in hepatic interferon-stimulated gene induction. Associations between innate cellular responses with HCV structure and further evolutionary constraints represents an important new element in RNA virus evolution and the adaptive interplay between virus and host.

genomics↗