“Gently Rough”: The Vaccine Potential of aSalmonella entericaRegulatory Lipopolysaccharide Mutant

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Abstract
Background. An alternative to multivalent vaccines could be to construct strains capable of conferring broad protection through shared antigens. Down-regulation of immunodominant major antigens has been proposed to enhance the immunogenicity of conserved antigens. Methods. The protection provided by an aroA as well as structural and regulatory lipopolysaccharide (LPS) mutants of Salmonella enterica serovar Typhimurium against homologous and heterologous challenges was assessed in the murine model of typhoid. The reactivity and cross-reactivity of the immune sera raised was tested by enzyme-linked immunospot assay and immunoblots. Conserved outer membrane proteins were identified by mass spectrometry. Results. Unlike any structural LPS mutants, the regulatory mutant lacking RfaH was finely balanced between safety and immunogenicity, and its vaccine potential was comparable to that of the well-characterized ΔaroA mutant. Loss of the transcriptional antiterminator RfaH resulted in a heterogeneous length of LPS chains, designated here as the “gently rough” phenotype. Our study also provides evidence that the rough phenotype enhances the immunogenicity of minor antigens, which may improve cross-protection against heterologous bacteria. A panel of conserved antigens shared by members of the Enterobacteriaceae family was identified as abundant porins and lipoprotein antigens. Conclusions. Fine-tuned down-regulation of immunodominant epitopes can create live vaccine strains that are not only desirably attenuated but that also exhibit an improved cross-protective potential.