No abstract

The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/281576 doi: bioRxiv preprint first posted online Mar. 13, 2018; 3 that becomes most prominent around the time of bacterial clearance. Together these data

Background: Streptococcus pneumoniae frequently colonises the human nasopharynx but causes over 500,000 deaths each year from Pneumonia, Sepsis and Meningitis. Nasopharyngeal carriage is required for transmission and is a pre-requisite for disease. The conjugate polysaccharide vaccines have proven effective in decreasing disease. However, replacement of vaccine serotypes with non-vaccine types in carriage threatens the future of the vaccines' efficiency.Using an Experimental Human Pneumococcal Challenge model (EHPC) and epithelial cell culture models, we have previously shown that pneumococcal colonisation involves both direct epithelial association and micro-invasion, inducing innate immunity and clearance without overt disease. Repeated challenge in the EHPC with the same strain decreases subsequent carriage efficiency and diminishes transmission potential and/or progression to disease, suggesting active mucosal immunity in the nasopharynx.Methods and materials: We have generated live attenuated strains of 6B S. pneumoniae (AS1 and AS2) that have double virulence deletions and cannot revert to cause disease. Here, we have explored the hypothesis that despite their attenuation in a mouse model of disease, these attenuated strains retain their ability to invade the epithelium and induce epithelial-derived innate immunity in humans.Colonisation were measured by confocal microscopy and microbiology density by CFU counts. Epithelial activation was measured by flow cytometry, ELISA and RNAseq.Results: We found that both mutants colonised the human nasopharynx and formed epithelial associations with microinvasion in the EHPC model. In vitro, both mutants adhered, invaded and transmigrated across the epithelium 4-fold less than wild type. However, both mutants still resulted in secretion of IL-8, IL-6 and ICAM-1 secretion and barrier integrity was maintained. PCA analyses revealed that epithelial transcriptomic responses between wild type and the mutants generally overlapped, indicating overall similar stimulation of signaling pathways following exposure. Conclusion:The results reveal that attenuation of these pneumococcal strains has not led to loss of their ability to elicit a mucosal immune/inflammatory response. This approach provides an exciting new pipeline for the development and testing of novel vaccines. The application of these attenuated strains in the EHPC also has the potential to provide important new knowledge on the mechanisms behind bacterial clearance, transmission and disease progression during colonisation.