Malabi M. Venkatesan

Complete Genome Sequence and Comparative Genomics of Shigella flexneri Serotype 2a Strain 2457T

ABSTRACT We determined the complete genome sequence of Shigella flexneri serotype 2a strain 2457T (4,599,354 bp). Shigella species cause >1 million deaths per year from dysentery and diarrhea and have a lifestyle that is markedly different from those of closely related bacteria, including Escherichia coli. The genome exhibits the backbone and island mosaic structure of E. coli pathogens, albeit with much less horizontally transferred DNA and lacking 357 genes present in E. coli. The strain is distinctive in its large complement of insertion sequences, with several genomic rearrangements mediated by insertion sequences, 12 cryptic prophages, 372 pseudogenes, and 195 S. flexneri-specific genes. The 2457T genome was also compared with that of a recently sequenced S. flexneri 2a strain, 301. Our data are consistent with Shigella being phylogenetically indistinguishable from E. coli. The S. flexneri-specific regions contain many genes that could encode proteins with roles in virulence. Analysis of these will reveal the genetic basis for aspects of this pathogenic organisms distinctive lifestyle that have yet to be explained.

Complete DNA Sequence and Analysis of the Large Virulence Plasmid of Shigella flexneri

ABSTRACT The complete sequence analysis of the 210-kb Shigella flexneri 5a virulence plasmid was determined.Shigella spp. cause dysentery and diarrhea by invasion and spread through the colonic mucosa. Most of the knownShigella virulence determinants are encoded on a large plasmid that is unique to virulent strains of Shigella and enteroinvasive Escherichia coli; these known genes account for approximately 30 to 35% of the virulence plasmid. In the complete sequence of the virulence plasmid, 286 open reading frames (ORFs) were identified. An astonishing 153 (53%) of these were related to known and putative insertion sequence (IS) elements; no known bacterial plasmid has previously been described with such a high proportion of IS elements. Four new IS elements were identified. Fifty putative proteins show no significant homology to proteins of known function; of these, 18 have a G+C content of less than 40%, typical of known virulence genes on the plasmid. These 18 constitute potentially unknown virulence genes. Two alleles of shet2 and five alleles ofipaH were also identified on the plasmid. Thus, the plasmid sequence suggests a remarkable history of IS-mediated acquisition of DNA across bacterial species. The complete sequence will permit targeted characterization of potential new Shigellavirulence determinants.

Shigella flexneri IpaH7.8 Facilitates Escape of Virulent Bacteria from the Endocytic Vacuoles of Mouse and Human Macrophages

ABSTRACT 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 ipaH7.8 deletion mutant ofS. 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 thatipaH7.8 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 expressingipaH7.8 restored the intracellular distribution of bacteria to that seen with the wild-type strain. Strains with deletions in ipaH4.5 oripaH9.8, 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 ipaH7.8 gene product is to facilitate the escape of the virulent bacteria from the phagocytic vacuole of monocytes and macrophages.

Phase I Evaluation of ΔvirG Shigella sonnei Live, Attenuated, Oral Vaccine Strain WRSS1 in Healthy Adults

ABSTRACT We conducted a phase I trial with healthy adults to evaluate WRSS1, a live, oral ΔvirG 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 × 103 CFU (group 1), 3 × 104 CFU (group 2), 3 × 105 CFU (group 3), or 3 × 106 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 103 to 106 CFU but elicits clinical reactions that must be assessed in further volunteer trials.

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

ABSTRACT 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 104 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 103 and nine received 104 CFU. All volunteers who received 103 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 104 CFU excreted SC602. One vaccinee had a transient fever and two met the definition of diarrhea. Six volunteers that received 104 CFU had an antibody-secreting cell response, and four had a serum IgA response. SC602 has now been tested at 104 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.

Live-attenuated Shigella vaccines

Several live-attenuated Shigella vaccines, with well-defined mutations in specific genes, have shown great promise in eliciting significant immune responses when given orally to volunteers. These responses have been measured by evaluating antibody-secreting cells, serum antibody levels and fecal immunoglobulin A to bacterial lipopolysaccharide and to individual bacterial invasion plasmid antigens. In this review, data collected from volunteer trials with live Shigella vaccines from three different research groups are described. The attenuating features of the bacterial strains, as well as the immune response following the use of different dosing regimens, are also described. The responses obtained with each vaccine strain are compared with data obtained from challenge trials using wild-type Shigella strains. Although the exact correlates of protection have not been found, some consensus may be derived as to what may constitute a protective immune response. Future directions in the field of live Shigella vaccines are also discussed.

Construction, Characterization, and Animal Testing of WRSd1, a Shigella dysenteriae 1 Vaccine

ABSTRACT 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 (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) stxAB Strr 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.

Community-Based Safety, Immunogenicity, and Transmissibility Study of the Shigella sonnei WRSS1 Vaccine in Israeli Volunteers

ABSTRACT 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 × 103 CFU, 2 × 104 CFU, and 4 × 105 CFU) 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 106 peripheral blood mononuclear cells (PBMC) for the 103-, 104-, and 105-CFU doses, respectively. The IgG means were 40, 46, and 135 ASC per 106 PBMC, 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.

Virulent Shigella flexneri Causes Damage to Mitochondria and Triggers Necrosis in Infected Human Monocyte-Derived Macrophages

ABSTRACT Shigella flexneri is a gram-negative bacterium that causes bacillary dysentery in humans that is characterized by an acute inflammatory response of the colon. The fate of phagocytes that are infected in vitro with virulent Shigella has been the subject of some investigation and debate. In this study we found that virulent Shigella caused a rapid increase in the cell membrane permeability of infected human monocyte-derived macrophages (HMDM) but not in the cell membrane permeability of monocytes, as demonstrated by the uptake of fluorescent vital dyes. Within 2 h of infection, 59% ± 6% of the HMDM and ≤4% of the monocytes were stained with propidium iodide. Treatment of the cells with the inhibitors of caspases YVAD and zVAD, the antioxidants N-acetyl-l-cysteine and butylated hydroxyanisole, or an inhibitor of NADPH oxidase, diphenyleniodonium, did not alter the infection outcome. Importantly, we found that virulent Shigella caused a rapid drop in the ATP level to about 50% in infected HMDM. Furthermore, using a combination of fluorescent vital dyes and mitochondrial membrane potential-sensitive dyes, we observed that cells that exhibited a permeable cell membrane were not stained by the mitochondrion-specific dyes, indicating that the mitochondrial membrane potential was lost in these cells. We also observed infected cells that were not stained with either type of dye, indicating that the loss of the mitochondrial membrane potential preceded the increase in cell membrane permeability. Taken together, our studies showed that virulent Shigella flexneri targets the host cell mitochondria for destruction. This activity may account for the necrotic cell death precipitated by these pathogens.

Construction and Characterization of Bivalent Shigella flexneri 2a Vaccine Strains SC608(pCFAI) and SC608(pCFAI/LTB) That Express Antigens from Enterotoxigenic Escherichia coli

ABSTRACT An invasive strain of Shigella flexneri 2a (SC608) has been developed as a vector for the expression and delivery of heterologous antigens. SC608 is an aspartate semialdehyde dehydrogenase (asd) derivative of SC602 (icsA iuc), a well-characterized live attenuated vaccine strain which has undergone several clinical trials in human volunteers. When administered orally at a single 104 (CFU) dose, SC602 is both immunogenic and efficacious against shigellosis. Using asd-based plasmid vectors, we designed SC608 to express the enterotoxigenic Escherichia coli (ETEC) fimbrial subunit CfaB (CFA/I structural subunit) alone or in combination with the E. coli B subunit of heat-labile enterotoxin (LTB). The expression of each heterologous protein in SC608 was verified by immunoblot analysis. Each strain was comparable to the parent strain, SC602, in a HeLa cell invasion assay. After intranasal immunizations of guinea pigs, serum and mucosal immune responses were detected against both Shigella lipopolysaccharide and heterologous ETEC antigens by enzyme-linked immunosorbent assay and ELISPOT analysis. All immunized animals were subsequently protected against a challenge with wild-type S. flexneri 2a in a keratoconjunctivitis Sereny test. Serum antibodies generated against LTB and CfaB demonstrated antitoxin and agglutination activities, respectively. These results suggest that CfaB and LTB expressed in SC608 retain important conformational epitopes that are required for the generation of antibodies that have functional activities. These initial experiments demonstrate that a fully invasive Shigella vaccine strain can be engineered to deliver antigens from other diarrheal pathogens.