Establishment of a Shigella sonnei human challenge model in Thailand.

In order to establish a human challenge model of Shigella related disease for vaccine testing, a dose-escalating inpatient trial was performed. Three groups of 12 healthy adult volunteers were orally challenged with 93,440 and 1680 CFU of Shigella sonnei strain 53G. Subjects were admitted to the Vaccine Trial Centre (VTC) at Mahidol University in Bangkok, Thailand. The primary purpose of this study was to identify the dose of S. sonnei 53G required to elicit clinical disease in at least 70% of Thai adult subjects. At the highest dose of 1680 CFU, the attack rate was 75%, while at the two lower doses, the attack rate was approximately 50%. This human challenge model, which is the first of its kind in an endemic region, will provide an opportunity for S. sonnei vaccine evaluation in endemic populations.

Comparative evaluation of the antibody in lymphocyte supernatant (ALS) and enzyme-linked immunospot (ELISPOT) assays for measuring mucosal immune responses to Shigella antigens.

Accurately assessing mucosal immune responses to candidate vaccines remains a technical challenge. ELISPOT is widely used as a surrogate of mucosal immune response by directly enumerating circulating antibody secreting cells (ASCs), while antibody in lymphocyte supernatant (ALS) titers the total amount of antibody secreted by ASC ex vivo using ELISA. ALS is more practical than ELISPOT because the ASC supernatant is frozen for ELISA that can be conducted at any time, with any antigen, and in any laboratory. We compared IgA and IgG responses to serotype-specific Shigella LPS using ELISPOT and ALS in subjects following vaccination or infection with Shigella. ALS results correlated well with ELISPOT results, and the ALS method was both sensitive and specific for the detection of antibody responses against Shigella LPS. Based on these observations, the ALS assay is a practical and flexible alternative to ELISPOT for measuring mucosal IgA responses to Shigella LPS antigen.

Safety, dose, immunogenicity, and transmissibility of an oral live attenuated Shigella flexneri 2a vaccine candidate (SC602) among healthy adults and school children in Matlab, Bangladesh.

In double-blind trials in Bangladesh, 88 adults, and 79 children (8-10 years) were randomized to receive either a single oral dose of 1 × 10(4), 1 × 10(5) or 1 × 10(6)CFU of SC602 (a live, attenuated Shigella flexneri 2a strain vaccine) or placebo. In the adult outpatient 1 × 10(6) CFU group, severe joint pain and body aches were reported by one and two vaccinees respectively. In the adult inpatient trial, SC602 was isolated from 3 volunteers, pre-vaccination antibody titers were high, and fourfold increases in serum IgG anti-LPS responses were observed in 2 of 5 subjects of the 1 × 10(6)CFU group. None of the volunteers developed diarrhea. Overall, SC602 was found to be associated with minimal vaccine shedding, minimal reactogenicity, no transmission risk, and low immune stimulation.

Vaccination of children in low-resource countries against Shigella is unlikely to present an undue risk of reactive arthritis.

Shigellosis is a major cause of morbidity and mortality among children in low-resource countries. Promising vaccine strategies in development include genetically attenuated Shigella, killed whole cell vaccines, subcellular vaccines, and O-polysaccharide-protein conjugates. There is a concern that Shigella vaccines could either induce reactive arthritis or could prime vaccinees for arthritis after a subsequent exposure to the pathogen because shigellosis is associated with reactive arthritis, especially in patients expressing the HLA B27 histocompatibility antigen. Our understanding of the pathogenesis of reactive arthritis is incomplete, and even surrogate biomarkers of bacterial arthritogenic activity have not yet been identified. Nonetheless, all of the Shigella vaccine strategies currently in development are designed to limit inflammation and intracellular antigen persistence that could trigger arthritogenic sequelae. The relatively low occurrence of the HLA B27 phenotype in most Shigella endemic areas, and the rarity of reported reactive arthritis in these populations, suggests that vaccination with attenuated, killed, or subcellular vaccines may not increase the background incidence of arthritic sequelae. More importantly, incidence rates of shigellosis in children living in low-resource countries suggest that, during maturation, the entire pediatric population may be infected with Shigella-possibly with devastating consequences. Therefore, clinical trials of candidate Shigella vaccines should be pursued aggressively in the developing world, beginning with a Phase 1 in HLA B27-negative volunteers, but proceeding to Phase 2 and Phase 3 in unscreened volunteers. Post-vaccination monitoring for possible reactive arthritis should be included in all clinical protocols.

Safety and immunogenicity of WRSd1, a live attenuated Shigella dysenteriae type 1 vaccine candidate.

Among Shigella serotypes Shigella dysenteriae type 1 produces the most severe disease, including cases of hemolytic-uremic syndrome and pandemic outbreaks. WRSd1 is a live S. dysenteriae 1 strain attenuated by deletion of the virG(icsA) gene, which encodes a protein that mediates intercellular spread, and stxA and stxB, which encode the Shiga toxin. In this Phase I trial five groups of eight subjects ingested escalating doses of WRSd1 ranging from 10(3) to 10(7)CFU. No subject experienced fever or shigellosis, but 20% had diarrhea. Approximately two-thirds of subjects developed an IgA-ASC response to LPS. Days of fecal shedding of the vaccine strain, but not dose ingested, correlated with stronger immune responses. These results suggest that to be effective an attenuated Shigella vaccine must colonize well.

Community-based safety, immunogenicity, and transmissibility study of the Shigella sonnei WRSS1 vaccine in Israeli volunteers.

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 x 10(3) CFU, 2 x 10(4) CFU, and 4 x 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.

Detection of Shigella by a PCR assay targeting the ipaH gene suggests increased prevalence of shigellosis in Nha Trang, Vietnam.

Shigella spp. are exquisitely fastidious gram-negative organisms which frequently escape detection by traditional culture methods. To get a more complete understanding of the disease burden caused by Shigella in Nha Trang, Vietnam, real-time PCR was used to detect Shigella DNA. Randomly selected rectal swab specimens from 60 Shigella culture-positive patients and 500 Shigella culture-negative patients detected by population-based surveillance of patients seeking care for diarrhea were processed by real-time PCR. The target of the primer pair is the invasion plasmid antigen H gene sequence (ipaH), carried by all four Shigella species and enteroinvasive Escherichia coli. Shigella spp. could be isolated from the rectal swabs of 547 of 19,206 (3%) patients with diarrhea. IpaH was detected in 55 of 60 (93%) Shigella culture-positive specimens, whereas it was detected in 87 of 245 (36%) culture-negative patients free of dysentery (P < 0.001). The number of PCR cycles required to detect a PCR product was highest for culture-negative, nonbloody diarrheal specimens (mean number of cycles to detection, 36.6) and was lowest for children with culture-positive, bloody diarrheal specimens (mean number of cycles, 25.3) (P < 0.001). The data from real-time PCR amplification indicate that the culture-proven prevalence of Shigella among patients with diarrhea may underestimate the prevalence of Shigella infections. The clinical presentation of shigellosis may be directly related to the bacterial load.

Two studies evaluating the safety and immunogenicity of a live, attenuated Shigella flexneri 2a vaccine (SC602) and excretion of vaccine organisms in North American volunteers.

We report the first community-based evaluation of Shigella flexneri 2a strain SC602, a live, oral vaccine strain attenuated by deletion of the icsA (virG) plasmid virulence gene, given at 10(4) CFU. The primary objectives of this trial were to determine the safety and immunogenicity of the vaccine and to determine the duration of colonization. Four of 34 volunteers experienced transient fevers, and three reported diarrhea during the first 3 days of the study. Half of the volunteers mounted a positive serum immunoglobulin A (IgA) response to S. flexneri lipopolysaccharide. All but one of the volunteers excreted the vaccine in their stools for 1 to 33 days, and this excretion was often intermittent. Data from the community-based study were supplemented with an inpatient trial in which three volunteers received 10(3) and nine received 10(4) CFU. All volunteers who received 10(3) CFU excreted SC602 and had an IgA antibody-secreting cell response. Two of these had a serum IgA response. Six of the nine volunteers who received 10(4) CFU excreted SC602. One vaccinee had a transient fever and two met the definition of diarrhea. Six volunteers that received 10(4) CFU had an antibody-secreting cell response, and four had a serum IgA response. SC602 has now been tested at 10(4) CFU in a total of 58 volunteers. The cumulative results of these clinical trials, reported here and previously (Coster et al., Infect. Immun. 67:3437-3443, 1999), have demonstrated that SC602 is a substantially attenuated candidate vaccine that can evoke protection against the most severe symptoms of shigellosis in a stringent human challenge model of disease.

Construction, characterization, and animal testing of WRSd1, a Shigella dysenteriae 1 vaccine.

WRSd1 is a Shigella dysenteriae 1 vaccine containing deletions of the virG(icsA) gene required for intercellular spreading and a 20-kb chromosomal region encompassing the Shiga toxin genes (stxAB). WRSd1 was constructed from S. dysenteriae 1 strain 1617 that was originally isolated during the 1968 to 1969 epidemic of Shiga dysentery in Guatemala. The virG(icsA) deletion was constructed from a streptomycin-resistant (Str(r)) mutant of 1617 by a filter mating procedures using a virG(icsA) deletion derivative, pDeltavirG2. A colony that was invasive for HeLa cells and negative for the virG(icsA) gene by Southern blotting was grown anaerobically on plates containing chlorate for selection of resistant colonies that had lost the entire Shiga toxin gene. A virG(icsA) stxAB Str(r) mutant selected from the chlorate plates was designated WRSd1. This candidate vaccine was evaluated for safety, immunogenicity, and protective efficacy using the guinea pig keratoconjunctivitis model. WRSd1 was Sereny negative, and two applications of this strain to the cornea elicited a significant protective immune response against the S. dysenteriae 1 O antigen. Vaccination with WRSd1 conferred protection against challenge with each of three virulent S. dysenteriae 1 strains. Since a vaccine protecting against multiple Shigella species is required for most areas where Shigella is endemic, protection studies using a combination vaccine of Shigella sonnei vaccine strain WRSS1, Shigella flexneri 2a vaccine strain SC602, and WRSd1 were also performed. Guinea pigs vaccinated with a mixture of equal amounts of the three vaccine strains were protected against challenge with each of the homologous virulent strains. Unlike WRSS1 and SC602, however, the level of protection afforded by WRSd1 in a combination vaccine was lower than the protection elicited by a pure culture. A current Good Manufacturing Practice product of WRSd1 given intragastrically to rhesus monkeys proved safe and immunogenic.

Phase I evaluation of delta virG Shigella sonnei live, attenuated, oral vaccine strain WRSS1 in healthy adults.

We conducted a phase I trial with healthy adults to evaluate WRSS1, a live, oral Delta 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 x 10(3) CFU (group 1), 3 x 10(4) CFU (group 2), 3 x 10(5) CFU (group 3), or 3 x 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) CFU but elicits clinical reactions that must be assessed in further volunteer trials.