Characterization of <i>Shigella</i> virulence factor intracellular spread A (IcsA, or VirG) functional epitopes against <i>S. flexneri</i> 2a and <i>S. sonnei</i> invasion and adherence

There are no licensed vaccines against <i>Shigella</i>, a leading cause of children's diarrhea and travelers' diarrhea. To develop a cross-protective vaccine against heterogeneous <i>Shigella</i> species and serotypes, we attempted to apply an epitope- and structure-based vaccinology platform, multiepitope fusion antigen, to construct an optimal polyvalent chimeric immunogen with functional epitopes from the key <i>Shigella</i> virulence determinants. With <i>Shigella</i> invasion plasmid antigens B and D functional epitopes identified in recent studies, in this study, we focused on <i>Shigella</i> intracellular spread protein A (IcsA; also known as virulence gene G, VirG), a multifunctional virulence factor that plays roles in bacterial adherence, invasion, and particularly intracellular and intercellular spread. We <i>in silico</i> predicted continuous B-cell immunodominant epitopes from the IcsA functional passenger domain, and we fused each epitope to a non-homologous carrier protein, <i>Escherichia coli</i> adhesin CS4 subunit CsaB, for epitope fusions. We then immunized mice with each epitope fusion and examined antibody functions against <i>Shigella</i> bacterial invasion and adherence. Eleven IcsA B-cell epitopes were identified and fused to CsaB. Mice intramuscularly immunized with each epitope fusion developed IgG responses. Mouse serum antibodies significantly reduced <i>Shigella sonnei</i> or <i>Shigella flexneri</i> 2a bacterial invasion or adherence <i>in vitro</i>, particularly from the fusion with epitope #2 (₁₂₂GDNNDGNSCGGNG₁₃₄), #3 (₁₅₆GGSGADHNGDGGE₁₆₈), #7 (₃₀₉YIISGKEDDGTQNV₃₂₅), #9 (₄₈₅WNDTDGDSHG₄₉₄), or #10 (₅₀₇TILADNLSHHNIN₅₂₃). These IcsA epitopes are more effective in inducing functional antibodies, suggesting their antigenic utility in constructing an optimal polyvalent <i>Shigella</i> immunogen and developing a cross-protective vaccine, and perhaps in better understanding their role in IcsA biogenesis.IMPORTANCEAn effective <i>Shigella</i> vaccine is urgently needed to reduce <i>Shigella</i>-associated diarrhea and dysentery. <i>Shigella</i> IcsA (or VirG) plays a crucial role in <i>Shigella</i> bacterial pathogenesis and is conserved across <i>Shigella</i> species and serotypes, thus making IcsA an excellent antigen target in <i>Shigella</i> vaccine development. Identification of the functional epitopes of the IcsA passenger domain from this study enables us to construct an optimal epitope- and structure-based polyvalent immunogen for a cross-protective multivalent <i>Shigella</i> vaccine. Unlike the lipopolysaccharide-based whole-cell or subunit vaccine candidates that are serotype-specific, a multivalent vaccine carrying functional epitopes of conserved virulence determinants would be cross-protective against the heterogeneous <i>Shigella</i> species and serotypes, effectively preventing shigellosis and dysentery. Data from this study may also provide insightful information for a better understanding of IcsA biogenesis.