Several candidate vaccines against Shigella spp. are in development, but the lack of a clear correlate of protection from challenge with the induction of adequate immune responses among the youngest age groups in the developing world has hampered Shigella vaccine development over the past several decades. Bioconjugation technology, exploited here for an Shigella flexneri 2a candidate vaccine, offers a novel and potentially cost-effective way to develop and produce vaccines against a major pathogen of global health importance. Flexyn2a, a novel S. flexneri 2a bioconjugate vaccine made of the polysaccharide component of the S. flexneri 2a O-antigen, conjugated to the exotoxin protein A of Pseudomonas aeruginosa (EPA), was evaluated for safety and immunogenicity among healthy adults in a single-blind, phase I study with a staggered randomization approach. Thirty subjects (12 receiving 10 μg Flexyn2a, 12 receiving Flexyn2a with aluminum adjuvant, and 6 receiving placebo) were administered two injections 4 weeks apart and were followed for 168 days. Flexyn2a was well-tolerated, independently of the adjuvant and number of injections. The Flexyn2a vaccine elicited statistically significant S. flexneri 2a lipopolysaccharide (LPS)-specific humoral responses at all time points postimmunization in all groups that received the vaccine. Elicited serum antibodies were functional, as evidenced by bactericidal activity against S. flexneri 2a. The bioconjugate candidate vaccine Flexyn2a has a satisfactory safety profile and elicited a robust humoral response to S. flexneri 2a LPS with or without inclusion of an adjuvant. Moreover, the bioconjugate also induced functional antibodies, showing the technology's features in producing a promising candidate vaccine. (This study has been registered at ClinicalTrials.gov under registration no. NCT02388009.)

Studies were undertaken to manufacture a multivalentShigellainactivated whole-cell vaccine that is safe, effective, and inexpensive. By using several formalin concentrations, temperatures, and incubation periods, an optimized set of inactivation conditions was established forShigella flexneri2a,S. sonnei, andS. flexneri3a to produce inactivated whole cells expressing a full repertoire of Ipa proteins and lipopolysaccharide (LPS). The inactivation conditions selected were treatment with 0.2% formalin (S. flexneri2a and 3a) or 0.6% formalin (S. sonnei) for 48 h at 25°C. Vaccine formulations prepared under different inactivation conditions, in different doses (10E5, 10E7, and 10E9 cells), and with or without the inclusion of double-mutant heat-labile toxin (dmLT) were evaluated in mice. Two intranasal immunizations with ≥10E7 inactivated whole cells resulted in high levels of anti-Invaplex and moderate levels of LPS-specific IgG and IgA in serum and in lung and intestinal wash samples. Addition of dmLT to the vaccine formulations did not significantly enhance humoral immunogenicity. Minimal humoral responses for IpaB, IpaC, or IpaD were detected after immunization with inactivated wholeShigellacells regardless of the vaccine inactivation conditions. In guinea pigs, monovalent formulations ofS. flexneri2a of 3a orS. sonneiconsisting of 10E8, 10E9, or 10E10 cells were protective in a keratoconjunctivitis assay. A trivalent formulation provided protection against all three serotypes (S. flexneri2a,P= 0.018;S. flexneri3a,P= 0.04;S. sonnei,P< 0.0001). The inactivatedShigellawhole-cell vaccine approach incorporates an uncomplicated manufacturing process that is compatible with multivalency and the future development of a broadly protectiveShigellavaccine.

Diarrheal diseases are a leading cause of global morbidity and mortality affecting all ages, but especially children under the age of five in resource-limited settings. Shigella is a leading contributor to diarrheal diseases caused by bacterial pathogens and is considered a significant antimicrobial resistance threat. While improvements in hygiene, and access to clean water help as control measures, vaccination remains one of the most viable options for significantly reducing morbidity and mortality. Flexyn2a is a bioconjugate vaccine manufactured using novel conjugation methodologies enzymatically linking the O-polysaccharide of S. flexneri 2a to exotoxin A of Pseudomonas aeruginosa. The protective capacity of Flexyn2a was assessed in a controlled human infection model after two intramuscular immunizations. Immune responses pre- and post-immunization and/or infection were investigated and are described here. Flexyn2a induced lipopolysaccharide (LPS)-specific serum IgG responses post-immunization which were associated with protection against shigellosis. Additionally, several other immune parameters, including memory B cell responses, bactericidal antibodies and serum IgA, were also elevated in vaccinees protected against shigellosis. Immunization with Flexyn2a also induced gut-homing, LPS-specific IgG and IgA secreting B cells, indicating the vaccine induced immune effectors functioning at the site of intestinal infection. Collectively, the results of these immunological investigations provide insights into protective immune mechanisms post-immunization with Flexyn2a which can be used to further guide vaccine development and may have applicability to the larger Shigella vaccine field. Funding for this study was provided through a Wellcome Trust grant.

The ability of genetically detoxified lipopolysaccharide (LPS) to stimulate adaptive immune responses is an ongoing area of investigation with significant consequences for the development of safe and effective bacterial vaccines and adjuvants. One approach to genetic detoxification is the deletion of genes whose products modify LPS. The msbB1 and msbB2 genes, which encode late acyltransferases, were deleted in the Shigella flexneri 2a human challenge strain 2457T to evaluate the virulence, inflammatory potential, and acquired immunity induced by strains producing underacylated lipid A. Consistent with a reduced endotoxic potential, S. flexneri 2a msbB mutants were attenuated in an acute mouse pulmonary challenge model. Attenuation correlated with decreases in the production of proinflammatory cytokines and in chemokine release without significant changes in lung histopathology. The levels of specific proinflammatory cytokines (interleukin-1β [IL-1β], macrophage inflammatory protein 1α [MIP-1α], and tumor necrosis factor alpha [TNF-α]) were also significantly reduced after infection of mouse macrophages with either single or double msbB mutants. Surprisingly, the msbB double mutant displayed defects in the ability to invade, replicate, and spread within epithelial cells. Complementation restored these phenotypes, but the exact nature of the defects was not determined. Acquired immunity and protective efficacy were also assayed in the mouse lung model, using a vaccination-challenge study. Both humoral and cellular responses were generally robust in msbB -immunized mice and afforded significant protection from lethal challenge. These data suggest that the loss of either msbB gene reduces the endotoxicity of Shigella LPS but does not coincide with a reduction in protective immune responses.

The global diarrheal disease burden for Shigella, enterotoxigenic Escherichia coli (ETEC), and Campylobacter is estimated to be 88M, 75M, and 75M cases annually, respectively. A vaccine against this target trio of enteric pathogens could address about one-third of diarrhea cases in children. All three of these pathogens contribute to growth stunting and have demonstrated increasing resistance to antimicrobial agents. Several combinations of antigens are now recognized that could be effective for inducing protective immunity against each of the three target pathogens in a single vaccine for oral administration or parenteral injection. The vaccine combinations proposed here would result in a final product consistent with the World Health Organization’s (WHO) preferred product characteristics for ETEC and Shigella vaccines, and improve the vaccine prospects for support from Gavi, the Vaccine Alliance, and widespread uptake by low- and middle-income countries’ (LMIC) public health stakeholders. Broadly protective antigens will enable multi-pathogen vaccines to be efficiently developed and cost-effective. This review describes how emerging discoveries for each pathogen component of the target trio could be used to make vaccines, which could help reduce a major cause of poor health, reduced cognitive development, lost economic productivity, and poverty in many parts of the world.