The behavior of Shigella flexneri ipaH mutants was studied in human monocyte-derived macrophages (HMDM), in 1-day-old human monocytes, and in J774 mouse macrophage cell line. In HMDM, strain pWR700, an ipaH 7.8 deletion mutant of S. flexneri 2a strain 2457T, behaved like the wild-type strain 2457T. This strain caused rapid host cell death by oncosis, and few bacterial CFU were recovered after incubation in the presence of gentamicin as previously described for 2457T-infected HMDM. However, analysis of bacterial compartmentalization within endocytic vacuoles with gentamicin and chloroquine indicated that more pWR700 than 2457T was present within the endocytic vacuoles of HMDM, suggesting that ipaH 7.8 deletion mutant transited more slowly from the vacuoles to the cytoplasm. In contrast to findings with HMDM, CFU recovered from pWR700-infected mouse J774 cells were 2 to 3 logs higher than CFU from 2457T-infected J774 cells. These values exceeded CFU recovered after infection of J774 cells with plasmid-cured avirulent strain M4243A1. Incubation with gentamicin and chloroquine clearly showed that pWR700 within J774 cells was mostly present within the endocytic vacuoles. This distribution pattern was similar to that seen with M4243A1 and contrasted with the pattern seen with 2457T. Complementation of pWR700 with a recombinant clone expressing ipaH 7.8 restored the intracellular distribution of bacteria to that seen with the wild-type strain. Strains with deletions in ipaH 4.5 or ipaH 9.8 , however, behaved like 2457T in both HMDM and J774 cells. The distribution profile of pWR700 in 1-day-old monocytes was similar to that seen in J774 cells. Like infected J774 cells, 1-day-old human monocytes demonstrated apoptosis upon infection with virulent Shigella. These results suggest that a role of the ipaH 7.8 gene product is to facilitate the escape of the virulent bacteria from the phagocytic vacuole of monocytes and macrophages.

A Xgtll expression library of TnS-tagged invasion plasmid pWR110 (from Shigellaflexneri serotype 5, strain M9OT-W) contained a set of recombinants encoding a 60-kilodalton protein (designated IpaH) recognized by rabbit antisera raised against S. flexneri invasion plasmid antigens

The invasiveness and virulence of Shigella spp. are largely due to the expression of plasmid-encoded virulence factors, among which are the invasion plasmid antigens (Ipa proteins). After infection, the host immune response is directed primarily against lipopolysaccharide (LPS) and the virulence proteins (IpaB, IpaC, and IpaD). Recent observations have indicated that the Ipa proteins (IpaB, IpaC, and possibly IpaD) form a multiprotein complex capable of inducing the phagocytic event which internalizes the bacterium. We have isolated a complex of invasins and LPS from water-extractable antigens of virulent shigellae by ion-exchange chromatography. Western blot analysis of the complex indicates that all of the major virulence antigens of Shigella, including IpaB, IpaC, and IpaD, and LPS are components of this macromolecular complex. Mice or guinea pigs immunized intranasally with purified invasin complex (invaplex), without any additional adjuvant, mounted a significant immunoglobulin G (IgG) and IgA antibody response against the Shigella virulence antigens and LPS. The virulence-specific response was very similar to that previously noted in primates infected with shigellae. Guinea pigs (keratoconjunctivitis model) or mice (lethal lung model) immunized intranasally on days 0, 14, and 28 and challenged 3 weeks later with virulent shigellae were protected from disease (P < 0.01 for both animal models).Shigellosis is a leading cause of human diarrheal disease. Each year millions of cases occur, particularly in developing countries, with over 1 million cases resulting in death (15). The constant emergence of antibiotic resistance in Shigella spp. (12), even to the newest antibiotics, underscores the need for an effective vaccine to help control Shigella disease. Vaccine strategies must consider the need for protection against four species of Shigella (S. flexneri, S. sonnei, S. dysenteriae, and S. boydii) with over 45 different serotypes, and also enteroinvasive Escherichia coli (EIEC), as cross-protection is not significant between the species. Historically, successful Shigella vaccines have emphasized presentation of lipopolysaccharide (LPS) in a manner that will elicit protection. Such vaccines include live attenuated vaccines (25, 32) and delivery of Shigella LPS or O polysaccharides with carriers such as proteosomes (28), tetanus toxoid (6), or ribosomes (16). Of these vaccine approaches, only the live attenuated vaccines utilize the native invasiveness of the shigellae to deliver the LPS and other antigens to the mucosal immune system, presumably via the follicle-associated epithelium (33). The residual pathogenicity of the attenuated vaccine strains may limit this approach unless further attenuation is achieved (7).The pathogenesis of Shigella is attributed to the organism's ability to invade, replicate intracellularly, and spread intercellularly within the colonic epithelium. The invasion of host cells by Shigella spp. is a complex multifactorial event in which many different bacterial proteins are involved. M...

The Shigella flexneri 2a SC602 vaccine candidate carries deletions of the plasmid-borne virulence gene icsA (mediating intra-and intercellular spread) and the chromosomal locus iuc (encoding aerobactin) (S. Barzu, A. Fontaine, P. J. Sansonetti, and A. Phalipon, Infect. Immun. 64:1190-1196, 1996). Dose selection studies showed that SC602 causes shigellosis in a majority of volunteers when 3 ؋ 10 8 or 2 ؋ 10 6 CFU are ingested. In contrast, a dose of 10 4 CFU was associated with transient fever or mild diarrhea in 2 of 15 volunteers. All volunteers receiving single doses of >10 4 CFU excreted S. flexneri 2a, and this colonization induced significant antibody-secreting cell and enzyme-linked immunosorbent assay responses against S. flexneri 2a lipopolysaccharide in two-thirds of the vaccinees. Seven volunteers who had been vaccinated 8 weeks earlier with a single dose of 10 4 CFU and 7 control subjects were challenged with 2 ؋ 10 3 CFU of virulent S. flexneri 2a organisms. Six of the control volunteers developed shigellosis with fever and severe diarrhea or dysentery, while none of the vaccinees had fever, dysentery, or severe symptoms (P ‫؍‬ 0.005). Three vaccinees experienced mild diarrhea, and these subjects had lower antibody titers than did the fully protected volunteers. Although the apparent window of safety is narrow, SC602 is the first example of an attenuated S. flexneri 2a candidate vaccine that provides protection against shigellosis in a stringent, human challenge model.

We conducted a phase I trial with healthy adults to evaluate WRSS1, a live, oral ⌬virG Shigella sonnei vaccine candidate. In a double-blind, randomized, dose-escalating fashion, inpatient volunteers received a single dose of either placebo (n ‫؍‬ 7) or vaccine (n ‫؍‬ 27) at 3 ؋ 10 3 CFU (group 1), 3 ؋ 10 4 CFU (group 2), 3 ؋ 10 5 CFU (group 3), or 3 ؋ 10 6 CFU (group 4). The vaccine was generally well tolerated, although a low-grade fever or mild diarrhea occurred in six (22%) of the vaccine recipients. WRSS1 was recovered from the stools of 50 to 100% of the vaccinees in each group. The geometric mean peak anti-lipopolysaccharide responses in groups 1 to 4, respectively, were 99, 39, 278, and 233 for immunoglobulin (IgA) antibody-secreting cell counts; 401, 201, 533, and 284 for serum reciprocal IgG titers; and 25, 3, 489, and 1,092 for fecal IgA reciprocal titers. Postvaccination increases in gamma interferon production in response to Shigella antigens occurred in some volunteers. We conclude that WRSS1 vaccine is remarkably immunogenic in doses ranging from 10 3 to 10 6