Strain Selection for Generation of O-Antigen-Based Glycoconjugate Vaccines against Invasive Nontyphoidal <i>Salmonella</i> Disease

<div><p>Nontyphoidal <i>Salmonellae</i>, principally <i>S</i>. Typhimurium and <i>S</i>. Enteritidis, are a major cause of invasive bloodstream infections in sub-Saharan Africa with no vaccine currently available. Conjugation of lipopolysaccharide O-antigen to a carrier protein constitutes a promising vaccination strategy. Here we describe a rational process to select the most appropriate isolates of <i>Salmonella</i> as source of O-antigen for developing a bivalent glycoconjugate vaccine. We screened a library of 30 <i>S</i>. Typhimurium and 21 <i>S</i>. Enteritidis in order to identify the most suitable strains for large scale O-antigen production and generation of conjugate vaccines. Initial screening was based on growth characteristics, safety profile of the isolates, O-antigen production, and O-antigen characteristics in terms of molecular size, O-acetylation and glucosylation level and position, as determined by phenol sulfuric assay, NMR, HPLC-SEC and HPAEC-PAD. Three animal isolates for each serovar were identified and used to synthesize candidate glycoconjugate vaccines, using CRM₁₉₇ as carrier protein. The immunogenicity of these conjugates and the functional activity of the induced antibodies was investigated by ELISA, serum bactericidal assay and flow cytometry. <i>S</i>. Typhimurium O-antigen showed high structural diversity, including O-acetylation of rhamnose in a Malawian invasive strain generating a specific immunodominant epitope. <i>S</i>. Typhimurium conjugates provoked an anti-O-antigen response primarily against the O:5 determinant. O-antigen from <i>S</i>. Enteritidis was structurally more homogeneous than from <i>S</i>. Typhimurium, and no idiosyncratic antibody responses were detected for the <i>S</i>. Enteritidis conjugates. Of the three initially selected isolates, two <i>S</i>. Typhimurium (1418 and 2189) and two <i>S</i>. Enteritidis (502 and 618) strains generated glycoconjugates able to induce high specific antibody levels with high breadth of serovar-specific strain coverage, and were selected for use in vaccine production. The strain selection approach described is potentially applicable to the development of glycoconjugate vaccines against other bacterial pathogens.</p></div