More than 50 years of research has yielded numerous Shigella vaccine candidates that have exemplified both the promise of vaccine-induced prevention of shigellosis and the impediments to developing a safe and effective vaccine for widespread use, a goal that has yet to be attained. This Review discusses the most advanced strategies for Shigella vaccine development, the immune responses that are elicited following disease or vaccination, the factors that have accelerated or impeded Shigella vaccine development and our ideas for the way forward.At the end of the 19th century, as epidemics of bacillary dysentery accompanied by high mortality spread across Japan, the young microbiologist Kiyoshi Shiga examined dysenteric stools and isolated a bacterium that was agglutinated by serum from convalescent patients but not from patients with acute disease [1][2][3] (FIG. 1). That bacterium -known today as Shigella dysenteriae 1 -was the first identified member of the genus Shigella. Four Correspondence to M.L. mlevine@medicine.umaryland.edu. Competing interests statement:The authors declare no competing financial interests. Links NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptShigella species (or groups) are now recognized: S. dysenteriae (group A), which has 15 serotypes; Shigella flexneri (group B), which has 14 classical serotypes and subserotypes; Shigella boydii (group C), which has 20 serotypes; and Shigella sonnei (group D), which has single serotype 4 (TABLE 1).There has been resurgent interest in Shigella as a human pathogen, driven by the availability of more precise data on the disease burden 5-8 , emerging antibiotic resistance 9,10 and the fact that mucosally invasive Shigella, which often cause dysentery (gross blood in diarrhoeal stools), are less amenable to the salutary effects of oral rehydration than non-invasive pathogens that cause watery diarrhoea, such as Vibrio cholerae and enterotoxigenic Escherichia coli. The target populations for the use of Shigella vaccines include infants and young children in developing countries (in which the peak incidence occurs at 12-47 months of age and the S. flexneri serotypes predominate) 5-7 .S. dysenteriae 1, which produces Shiga toxin and typically carries R factors that encode resistance to multiple antibodies, waxes and wanes as a cause of epidemic severe disease in the world's least developed countries [11][12][13][14] Few bacterial pathogens have had their pathogenesis or interactions with mammalian tissues elucidated so precisely at the cellular and subcellular levels as Shigella spp. 20,21 Nevertheless, progress in attaining safe and effective Shigella vaccines has faltered. Herein, we review recent and old clinical trials that have evaluated the safety, immunogenicity and efficacy of candidate Shigella vaccines. We relate the bassis for the most popular strategies (BOX 2), the relevance of the different immune responses measured, the factors that have favoured or impeded vaccine development and, most importantly, the less...

Shigella case isolates from the Global Enteric Multicenter Study were serotyped to guide vaccine development. A quadrivalent vaccine that includes O antigens from S. sonnei, S. flexneri 2a, S. flexneri 3a, and S. flexneri 6 should provide broad protection.

The Shigella species cause millions of cases of watery or bloody diarrhea each year, mostly in children in developing countries. While many aspects of Shigella colonic cell invasion are known, crucial gaps in knowledge regarding how the bacteria survive, transit, and regulate gene expression prior to infection remain. In this study, we define mechanisms of resistance to bile salts and build on previous research highlighting induced virulence in Shigella flexneri strain 2457T following exposure to bile salts. Typical growth patterns were observed within the physiological range of bile salts; however, growth was inhibited at higher concentrations. Interestingly, extended periods of exposure to bile salts led to biofilm formation, a conserved phenotype that we observed among members of the Enterobacteriaceae. Characterization of S. flexneri 2457T biofilms determined that both bile salts and glucose were required for formation, dispersion was dependent upon bile salts depletion, and recovered bacteria displayed induced adherence to HT-29 cells. RNAsequencing analysis verified an important bile salt transcriptional profile in S. flexneri 2457T, including induced drug resistance and virulence gene expression. Finally, functional mutagenesis identified the importance of the AcrAB efflux pump and lipopolysaccharide O-antigen synthesis for bile salt resistance. Our data demonstrate that S. flexneri 2457T employs multiple mechanisms to survive exposure to bile salts, which may have important implications for multidrug resistance. Furthermore, our work confirms that bile salts are important physiological signals to activate S. flexneri 2457T virulence. This work provides insights into how exposure to bile likely regulates Shigella survival and virulence during host transit and subsequent colonic infection.

Francisella tularensis, an aerobic, non-spore-forming, gram-negative coccobacillus, is the causative agent of tularemia. We reported previously that F. tularensis live vaccine strain (LVS) elicited strong, dose-dependent NF-B reporter activity in Toll-like receptor 2 (TLR2)-expressing HEK293T cells and proinflammatory gene expression in primary murine macrophages. Herein, we report that F. tularensis LVS-induced murine macrophage proinflammatory cytokine gene and protein expression are overwhelmingly TLR2 dependent, as evidenced by the abrogated responses of TLR2 ؊/؊ macrophages. F. tularensis LVS infection also increased expression of TLR2 both in vitro, in mouse macrophages, and in vivo, in livers from F. tularensis LVS-infected mice. Colocalization of intracellular F. tularensis LVS, TLR2, and MyD88 was visualized by confocal microscopy. Signaling was abrogated if the F. tularensis LVS organisms were heat or formalin killed or treated with chloramphenicol, indicating that the TLR2 agonist activity is dependent on new bacterial protein synthesis. F. tularensis LVS replicates in macrophages; however, bacterial replication was not required for TLR2 signaling because LVS⌬guaA, an F. tularensis LVS guanine auxotroph that fails to replicate in the absence of exogenous guanine, activated NF-B in TLR2-transfected HEK293T cells and induced cytokine expression in wild-type macrophages comparably to wild-type F. tularensis LVS. Collectively, these data indicate that the primary macrophage response to F. tularensis LVS is overwhelmingly TLR2 dependent, requires de novo bacterial protein synthesis, and is independent of intracellular F. tularensis replication.

Renewed awareness of the significant morbidity and mortality that Shigella causes among young children in developing countries combined with technological innovations in vaccinology has led to the development of novel vaccine strategies in the past five years. Along with advancement of classical vaccines in clinical trials and new sophisticated measurements of immunological responses, much new data has been produced lending promise to the potential for production of safe and effective Shigella vaccines. Herein we review the recent progress in Shigella vaccine development within the framework of persistent obstacles.

The macrophage proinflammatory response to Francisella tularensis (Ft) live vaccine strain (LVS) was shown previously to be TLR2 dependent. The observation that intracellular Ft LVS colocalizes with TLR2 and MyD88 inside macrophages suggested that Ft LVS might signal from within the phagosome. Macrophages infected with LVSΔiglC, a Ft LVS mutant that fails to escape from the phagosome, displayed greatly increased expression of a subset of TLR2-dependent, proinflammatory genes (e.g., Tnf) but decreased expression of others (e.g., Ifnb1). This latter subset was similarly mitigated in IFN-β−/− macrophages indicating that while Ft LVS-induced TLR2 signaling is necessary, cytosolic sensing of Ft to induce IFN-β is required for full induction of the macrophage proinflammatory response. Although LVSΔiglC greatly increased IL-1β mRNA in wild-type macrophages, protein secretion was not observed. IL-1β secretion was also diminished in Ft LVS-infected IFN-β−/− macrophages. rIFN-β failed to restore IL-1β secretion in LVSΔiglC-infected macrophages, suggesting that signals in addition to IFN-β are required for assembly of the inflammasome and activation of caspase-1. IFN-β plays a central role in controlling the macrophage bacterial burden: bacterial recovery was greater in IFN-β−/− than in wild-type macrophages and treatment of Ft LVS-infected macrophages with rIFN-β or 5,6-dimethylxanthenone-4-acetic acid, a potent IFN-β inducer, greatly decreased the intracellular Ft LVS burden. In toto, these observations support the hypothesis that the host inflammatory response to Ft LVS is complex and requires engagement of multiple signaling pathways downstream of TLR2 including production of IFN-β via an unknown cytosolic sensor and activation of the inflammasome.

We conclude that 1 or both ShETs are virulence determinants in humans; their inactivation, in combination with Delta guaBA, leads to a well-tolerated and immunogenic Shigella vaccine candidate.

Enteropathogenic Escherichia coli (EPEC) are diarrhoeagenic E. coli, and are a significant cause of gastrointestinal illness among young children in developing countries. Typical EPEC are identified by the presence of the bundle-forming pilus encoded by a virulence plasmid, which has been linked to an increased severity of illness, while atypical EPEC lack this feature. Comparative genomics of 70 total EPEC from lethal (LI), non-lethal symptomatic (NSI) or asymptomatic (AI) cases of diarrhoeal illness in children enrolled in the Global Enteric Multicenter Study was used to investigate the genomic differences in EPEC isolates obtained from individuals with various clinical outcomes. A comparison of the genomes of isolates from different clinical outcomes identified genes that were significantly more prevalent in EPEC isolates of symptomatic and lethal outcomes than in EPEC isolates of asymptomatic outcomes. These EPEC isolates exhibited previously unappreciated phylogenomic diversity and combinations of virulence factors. These comparative results highlight the diversity of the pathogen, as well as the complexity of the EPEC virulence factor repertoire.