Related Experiment Video
Updated: Jun 11, 2025
Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction
Published on: May 28, 2012
Phenotypic variation in the lipopolysaccharide O-antigen of Salmonella Paratyphi A and implications for vaccine
Elli Mylona1, Joana Pereira-Dias1, Jacqueline A Keane1
1Cambridge Institute of Therapeutic Immunology & Infectious Disease (CITIID), Department of Medicine, University of Cambridge, Cambridge, UK.
Abstract:
Enteric fever remains a major public health problem in South and Southeast Asia. The recent roll-out of the typhoid conjugate vaccine protecting against S. Typhi exhibits great promise for disease reduction in high burden areas. However, some endemic regions remain vulnerable to S. Paratyphi A due to a lack of licensed vaccines and inadequate WASH. Several developmental S. Paratyphi A vaccines exploit O-antigen as the target antigen. It has been hypothesised that O-antigen is under selective and environmental pressure, with mutations in O-antigen biosynthesis genes being reported, but their phenotypic effects are unknown. Here, we aimed to evaluate O-antigen variation in S. Paratyphi A originating from Nepal, and the potential effect of this variation on antibody binding. O-antigen variation was determined by measuring LPS laddering shift following electrophoresis; this analysis was complemented with genomic characterisation of the O-antigen region. We found structural O-antigen variation in <10 % of S. Paratyphi A organisms, but a direct underlying genetic cause could not be identified. High-content imaging was performed to determine antibody binding by commercial O2 monoclonal (mAb) and polyclonal antibodies, as well as polyclonal sera from convalescent patients naturally infected with S. Paratyphi A. Commercial mAbs detected only a fraction of an apparently "clonal" bacterial population, suggesting phase variation and nonuniform O-antigen composition. Notably, and despite visible subpopulation clusters, O-antigen structural changes did not appear to affect the binding ability of polyclonal human antibody considerably, which led to no obvious differences in the functionality of antibodies targeting organisms with different O-antigen conformations. Although these results need to be confirmed in organisms from alternative endemic areas, they are encouraging the use of O-antigen as the target antigen in S. Paratyphi A vaccines.
