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ABSTRACTWe describe the first community-based evaluation of Shigella sonneistrain 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 × 103CFU, 2 × 104CFU, and 4 × 105CFU) were tested with cohorts of 15 adult volunteers. The vaccine was generally well tolerated at the 103- and 104-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 105-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 106peripheral blood mononuclear cells (PBMC) for the 103-, 104-, and 105-CFU doses, respectively. The IgG means were 40, 46, and 135 ASC per 106PBMC, respectively. The 104-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 104-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.

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.

ABSTRACTShigellaspp. 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 Shigellain Nha Trang, Vietnam, real-time PCR was used to detect ShigellaDNA. Randomly selected rectal swab specimens from 60 Shigellaculture-positive patients and 500 Shigellaculture-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 Shigellaspecies and enteroinvasive Escherichia coli. Shigellaspp. could be isolated from the rectal swabs of 547 of 19,206 (3%) patients with diarrhea. IpaHwas detected in 55 of 60 (93%) Shigellaculture-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 Shigellaamong patients with diarrhea may underestimate the prevalence of Shigellainfections. The clinical presentation of shigellosis may be directly related to the bacterial load.

ABSTRACTWe report the first community-based evaluation of Shigella flexneri2a strain SC602, a live, oral vaccine strain attenuated by deletion of the icsA(virG) plasmid virulence gene, given at 104CFU. 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. flexnerilipopolysaccharide. 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 103and nine received 104CFU. All volunteers who received 103CFU 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 104CFU excreted SC602. One vaccinee had a transient fever and two met the definition of diarrhea. Six volunteers that received 104CFU had an antibody-secreting cell response, and four had a serum IgA response. SC602 has now been tested at 104CFU 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.

ABSTRACTWRSd1 is a Shigella dysenteriae1 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. dysenteriae1 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 (Strr) mutant of 1617 by a filter mating procedures using a virG(icsA)deletion derivative, pΔvirG2. 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) stxABStrrmutant 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. dysenteriae1 O antigen. Vaccination with WRSd1 conferred protection against challenge with each of three virulent S. dysenteriae1 strains. Since a vaccine protecting against multiple Shigellaspecies is required for most areas where Shigellais endemic, protection studies using a combination vaccine of Shigella sonneivaccine strain WRSS1, Shigella flexneri2a 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.

ABSTRACTWe conducted a phase I trial with healthy adults to evaluate WRSS1, a live, oral ΔvirG Shigella sonneivaccine 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 × 103CFU (group 1), 3 × 104CFU (group 2), 3 × 105CFU (group 3), or 3 × 106CFU (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 Shigellaantigens occurred in some volunteers. We conclude that WRSS1 vaccine is remarkably immunogenic in doses ranging from 103to 106CFU but elicits clinical reactions that must be assessed in further volunteer trials.

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.

ABSTRACTWe studied the safety and immunogenicity of a Shigella flexneri2a vaccine comprising native S. flexneri2a lipopolysaccharide (LPS) complexed to meningococcal outer membrane proteins—proteosomes—in normal, healthy adults. A two-dose series of immunizations was given by intranasal spray, and doses of 0.1, 0.4, 1.0, and 1.5 mg (based on protein) were studied in a dose-escalating design. The vaccine was generally well tolerated. The most common reactions included rhinorrhea and nasal stuffiness, which were clearly dose related (P≤ 0.05). These reactions were self-limited and generally mild. The vaccine elicitedS. flexneri2a LPS-specific immunoglobulin A (IgA), IgG, and IgM antibody-secreting cells (ASCs) in a dose-responsive manner. At doses of 1.0 or 1.5 mg, highly significant (P< 0.001) increases in ASCs of all antibody isotypes occurred and 95% of subjects had an ASC response in at least one antibody isotype. Dose-related serum antibody responses were observed, with geometric mean two- to fivefold rises in specific serum IgA and IgG titers and two- to threefold rises in IgM in the 1.0- and 1.5-mg-dose groups (P< 0.0001 for each isotype). Elevated serum antibody levels persisted through day 70. Increases in fecal IgG and IgA and also in urinary IgA specific for S. flexneri2a LPS were demonstrated. These were most consistent and approached statistical significance (P= 0.02 to 0.12 for various measures) on day 70 after the first dose. The magnitude of immune responses to intranasally administered proteosome-S. flexneri2a LPS vaccine is similar to those reported for live vaccine candidates associated with protective efficacy in human challenge models, and further evaluation of this product is warranted.

ABSTRACTThe Shigella flexneri2a SC602 vaccine candidate carries deletions of the plasmid-borne virulence gene icsA(mediating intra- and intercellular spread) and the chromosomal locusiuc(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 × 108or 2 × 106CFU are ingested. In contrast, a dose of 104CFU was associated with transient fever or mild diarrhea in 2 of 15 volunteers. All volunteers receiving single doses of ≥104CFU excreted S. flexneri2a, and this colonization induced significant antibody-secreting cell and enzyme-linked immunosorbent assay responses against S. flexneri2a lipopolysaccharide in two-thirds of the vaccinees. Seven volunteers who had been vaccinated 8 weeks earlier with a single dose of 104CFU and 7 control subjects were challenged with 2 × 103CFU of virulent S. flexneri2a 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. flexneri2a candidate vaccine that provides protection against shigellosis in a stringent, human challenge model.

The process of Henle 407 embryonic intestinal epithelial cell infection by Shigella flexneri2a M42-43 was studied in an in vitro model system. The role of the Henle cell was assessed. It was established that entry of S. flexneriinto cells was suppressed by reagents which inhibit uptake of particles by phagocytic cells. The compounds tested included cytochalasin B, dibutyryl-cyclic adenosine monophosphate, choleragen (Vibrio choleraenterotoxin), iodoacetate, and dinitrophenol. Cytochalasin B inhibited infection at concentrations of 1.0 μg/ml or greater. Dibutyryl-cyclic adenosine monophosphate at concentrations of 1 mM and choleragen at 0.1 μg/ml caused significant suppression of infection. Iodoacetate or dinitrophenol, at 0.1 mM concentrations, inhibited internalization of virulent shigellae, and a combination of these compounds inhibited infection at 0.01 mM concentrations. Preincubation of Henle cell monolayers with the combination of iodoacetate and dinitrophenol (0.05 mM) also inhibited infection markedly. The data suggest that infection of epithelial cells by S. flexneriin vitro is accomplished by an endocytic process induced by virulent bacteria. The process appears to be similar to uptake of particles by phagocytic cells. Ultrastructural analysis by transmission electron microscopy provided corroborative evidence of phagocytosis of shigellae by Henle cells in that intracellular bacteria were often observed within membrane-limiting vacuoles resembling phagosomes.