Search

We describe the first community-based evaluation of Shigella sonnei strain WRSS1, a live, oral candidate vaccine attenuated by a 212-bp deletion in the virG (or icsA ) plasmid virulence gene. Three single-dose regimens of WRSS1 (5 × 10 3 CFU, 2 × 10 4 CFU, and 4 × 10 5 CFU) were tested with cohorts of 15 adult volunteers. The vaccine was generally well tolerated at the 10 3 - and 10 4 -CFU doses. There were no fevers and there was one report of moderate diarrhea in 30 vaccinees; five additional vaccinees reported mild diarrhea. At the 10 5 -CFU dose, there were two reports of low-grade fevers and four reports of moderate diarrhea. The geometric means for immunoglobulin A (IgA) antibody-secreting cells (ASC) against lipopolysaccharide (LPS) were 30, 75, and 193 ASC per 10 6 peripheral blood mononuclear cells (PBMC) for the 10 3 -, 10 4 -, and 10 5 -CFU doses, respectively. The IgG means were 40, 46, and 135 ASC per 10 6 PBMC, respectively. The 10 4 -CFU dose of WRSS1 gave the best balance of safety and immunogenicity, since all vaccinees had a significant IgA ASC response and 73% had a response of more than 50 ASC. The anti-LPS seroconversion rate (threefold) for IgA was 60% and the IgG rate was 27% for the 10 4 -CFU cohort. Each vaccinee and a cohabitating household contact delivered daily perianal stool swabs for bacteriological culture. WRSS1 colonized vaccinees for a median of 5 days, and one individual excreted WRSS1 intermittently for 23 days. None of the 45 household contacts were colonized with WRSS1 after a cumulative 192 days of cohabitation with colonized vaccinees, suggesting that adventitious vaccine spread was not common in the community setting.

Addition of the cholera toxin B subunit to oral and intranasal proteosome-Shigella flexneri 2a lipopolysaccharide vaccines improved their immunogenicities. Enhancement of anti-O-Shigella immunoglobulin A levels was most evident in lung lavages following oral immunization and in lung and intestinal fluids when suboptimal doses were used with either immunization route.

Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of diarrhea among Israeli soldiers serving in field units. Two double-blind placebo-controlled, randomized trials were performed among 155 healthy volunteers to evaluate the safety and immunogenicity of different lots of the oral, killed ETEC vaccine consisting of two doses of whole cells plus recombinantly produced cholera toxin B subunit (rCTB). The two doses of vaccine lot E005 and the first dose of vaccine lot E003 were well tolerated by the volunteers. However, 5 (17%) vaccinees reported an episode of vomiting a few hours after the second dose of lot E003; none of the placebo recipients reported similar symptoms. Both lots of vaccine stimulated a rate of significant antibody-secreting cell (ASC) response to CTB and to colonization factor antigen I (CFA/I) after one or two doses, ranging from 85 to 100% and from 81 to 100%, respectively. The rate of ASC response to CS2, CS4, and CS5 was slightly lower than the rate of ASC response induced to CTB, CFA/I, and CS1. The second vaccine dose enhanced the response to CTB but did not increase the frequencies or magnitude of ASC responses to the other antigens. The two lots of the ETEC vaccine induced similar rates of serum antibody responses to CTB and CFA/I which were less frequent than the ASC responses to the same antigens. Based on these safety and immunogenicity data, an efficacy study of the ETEC vaccine is under way in the Israel Defense Force.

Microbiological surveys in areas where diarrheal disease is endemic implicate Shigella species as etiologic agents in at least 20% of diarrheal cases. Shigella flexneri 2a is usually the most prevalent species and serotype in these areas (8, 14, 30). Shigellae are extraordinarily adept intestinal pathogens, as evidenced by their small infectious doses (7). Shigella infection is usually transmitted by the fecal-oral route and can be manifested as uncomplicated watery diarrhea. A more definitive manifestation of shigellosis is dysentery, i.e., frequent passage of small-volume stools with gross blood, mucus, and fecal leukocytes. Constitutional symptoms (e.g., fever, rectal tenesmus, and headache) also characterize severe disease. Colonoscopy of patients infected with either Shigella dysenteriae or S. flexneri reveals diffuse erythema, focal hemorrhages, and inflammatory changes resembling ulcerative colitis. Rectal biopsies taken during the early stages of infection reveal aphthoid lesions overlying small lymphoid follicles (23). These clinical findings are consistent with experimental observations made with the rabbit ileal loop model, suggesting that Shigella initiates intestinal infections by invading the follicle-associated membranous cells (28). Experiments employing polarized epithelial cells as a model of the intestinal epithelium suggest that shigellae invade enterocytes through the basolateral membrane. Internalized bacteria subsequently spread within infected cells by organizing host cell actin into a cytoskeleton-based motor (10, 22). Genetic analysis has shown that this spreading phenotype is dependent upon a plasmid-borne virulence gene designated icsA (2) or virG (22). The 120-kDa protein expressed by this plasmid-carried gene acts as a recruiter for cytosolic nucleators of filamentous actin (10). This actin is concentrated at the distal poles of septating shigellae, and the resulting comet-like tails provide a motive force for the bacteria within the cytoplasm of infected epithelial cells (36). The mobilized bacteria impinge on the inner face of the host cell plasma membrane, and they spread into contiguous epithelial cells via membrane protrusions (10). Intragastric challenge of rhesus monkeys, a primate model of intestinal shigellosis, demonstrates that icsA-mediated intercellular spread of shigellae is a key step in pathogenesis. For example, endoscopy of asymptomatic animals challenged with an icsA mutant of S. flexneri serotype 5 reveals only scattered nodular abscesses rather than the hemorrhagic ulcerations and diffuse mucosal inflammation seen in animals challenged with the virulent parent strain (32). icsA mutants are also avirulent in the Sereny guinea pig keratoconjunctivitis model of suppurative Shigella infection (13, 26). Deletion of the iuc chromosomal locus (encoding aerobactin) partially attenuates S. flexneri in the Sereny guinea pig test and in the rabbit ileal loop model (24). Intragastric inoculation with either an icsA single mutant or an icsA iuc double mutant protects rhesus monkeys against subsequent challenge with the virulent S. flexneri 5 parent strain (32, 33). Epidemiological and clinical studies indicate that an episode of shigellosis elicits substantial immunity against subsequent disease caused by the same Shigella serotype (6, 8, 15, 19). In a rational approach to Shigella vaccine development, we and others have constructed genetically attenuated vaccines designed to establish asymptomatic infections that induce protective immune responses. However, the ideal balance of safety and efficacy in attenuated Shigella vaccines has been elusive (11, 15–18, 20, 25). Current research suggests that icsA iuc mutants could serve as attenuated Shigella vaccines, and the SC602 ΔicsA Δiuc S. flexneri 2a candidate was constructed to test this combination of attenuating mutations in volunteers. Here we describe a preliminary dose selection study, two expanded dose selection studies, and an efficacy (challenge) study of SC602 that were performed in the clinical inpatient ward of the U.S. Army Medical Research Institute for Infectious Diseases (USAMRIID), Ft. Detrick, Md. (Parts of this work were previously presented at the 96th General Meeting of the American Society for Microbiology, 19 to 23 May 1996, and at the 98th General Meeting of the American Society for Microbiology, 17 to 21 May 1998.)

The safety and immunogenicity of investigational conjugates, composed of the O-specific polysaccharides of Shigella sonnei and Shigella flexneri type 2a covalently bound to Pseudomonas aeruginosa recombinant exoprotein A (rEPA), were evaluated in 192 Israeli soldiers. None had significant local reactions or fever. Fourteen days after injection, 90% of S. sonnei-rEPA recipients and 73 to 77% of S. flexneri-rEPA recipients had a fourfold or greater increase in serum immunoglobulin G (IgG) and IgA anti-lipopolysaccharide (anti-LPS) levels; at 2 years, these remained higher than at prevaccination (P < 0.01). There was a fourfold or greater increase in IgM anti-LPS in 20% of vaccinees at 2 weeks, but levels returned to prevaccination values at 6 to 12 months. IgG was the highest and most sustained class of LPS antibodies. Reinjection at day 42 did not boost antibody levels. Eighteen of 23 (78%) who received S. sonnei-rEPA and 13 of 19 (68%) who received S. flexneri-rEPA. had significant IgA-secreting cell responses. Significant IgG antibody-secreting cell responses were detected in 19 of 23 (83%) and 11 of 19 (58%) volunteers following vaccination with S. sonnei-rEPA and S. flexneri 2a-rEPA, respectively. On the basis of these data, further evaluation of the Shigella conjugates for protective efficacy in field trials in Israel was started.