Biomedical subjects

Malabi M Venkatesan

Publications and source records attributed to Malabi M Venkatesan.

11 recordsLinked to original sources

PubMed2006

Immunogenicity and characterization of WRSF2G11: a second generation live attenuated Shigella flexneri 2a vaccine strain.

Recent clinical trials involving live attenuated Shigella vaccine strains SC602 and WRSS1 have revealed that deletion of the virG(icsA) gene dramatically reduces virulence in human volunteers. These strains can be given at low oral doses and induce a strong, and in some cases, protective immune responses. However, residual vaccine associated reactogenicity suggests that further attenuation is required. A recent clinical trial indicated that the set and sen enterotoxin genes contribute to the symptoms of fever and diarrhea observed with live Shigella vaccine strains. Based on these findings, a Shigella flexneri 2a vaccine candidate, WRSf2G11, with deletions in the virG(icsA), set and sen genes has been constructed using the lambda red recombinase system. The immunogenicity and protective efficacy of WRSf2G11 compares favorably with SC602 following either intranasal (IN) or ocular (OC) immunization of guinea pigs. Taken together, these data indicate that second generation virG-based Shigella vaccine strains which lack enterotoxin genes, such as WRSf2G11, will likely show lower levels of reactogenicity without hampering the robust immune responses achieved with previous live vaccines.

Administration, Intranasal↗

PubMed2006

An AFLP-based database of Shigella flexneri and Shigella sonnei isolates and its use for the identification of untypable Shigella strains.

Amplified fragment length polymorphism (AFLP) can be used to assess the genetic diversity of closely related microbial genomes. In this study, the first of its kind for identification of Shigella, the high discriminatory power of AFLP has been used to determine the genetic relatedness of 230 isolates of Shigella flexneri and Shigella sonnei strains. An AFLP database was generated to demonstrate its utility in the discrimination of closely related strains. Based on AFLP, S. flexneri strains could be grouped into separate clusters according to their serotypes. Within each serotype, strains demonstrated 80-100% similarity indicating that identical strains and closely related strains could be distinguished by this technique. S. flexneri 6 formed a distinct cluster with 55% similarity to the rest of the S. flexneri strains showing significant divergence from the rest of the S. flexneri strains. Significantly, S. sonnei isolates formed a distinct group and showed approximately the same level of genetic linkage to S. flexneri as Escherichia coli strains. Untypable isolates that showed conflicting agglutination reactions with conventional typing sera were identifiable by AFLP. Thus AFLP can be used for genetic fingerprinting of Shigella strains and aid in the identification of variant untypable isolates.

Bacterial Typing Techniques↗

PubMed2006

Subtractive hybridization and optical mapping of the enterotoxigenic Escherichia coli H10407 chromosome: isolation of unique sequences and demonstration of significant similarity to the chromosome of E. coli K-12.

Enterotoxigenic Escherichia coli (ETEC) is a primary cause of diarrhoea in infants in developing countries and in travellers to endemic regions. While several virulence genes have been identified on ETEC plasmids, little is known about the ETEC chromosome, although it is expected to share significant homology in backbone sequences with E. coli K-12. In the absence of genomic sequence information, the subtractive hybridization method and the more recently described optical mapping technique were carried out to determine the degree of genomic variation between virulent ETEC strain H10407 and the non-pathogenic E. coli K-12 strain MG1655. In one round of PCR-based suppression subtractive hybridization, 153 fragments representing sequences unique to strain H10407 were identified. blast searches indicated that few unique sequences showed homology to known pathogenicity island genes identified in related E. coli pathogens. A total of 65 fragments contained sequences that were either linked to hypothetical proteins or showed no homology to any known sequence in the database. The remaining sequences were either phage or prophage related or displayed homology to classifiable genes that function in various aspects of bacterial metabolism. The 153 unique sequences showed variable distribution across different ETEC strains including ETEC strain B7A, which is attenuated in virulence and lacked several H10407-specific sequences. Restriction-enzyme-based optical maps of strain H10407 were compared to in silico restriction maps of strain MG1655 and related E. coli pathogens. The 5.1 Mb ETEC chromosome was approximately 500 kb greater in length than the chromosome of E. coli K-12, collinear with it and indicated several discrete regions where insertions and/or deletions had occurred relative to the chromosome of strain MG1655. No major inversions, transpositions or gross rearrangements were observed on the ETEC chromosome. Based on comparisons with known genomic sequences and related optical-map-based restriction site similarity, the sequence of the H10407 chromosome is expected to demonstrate approximately 96 % identity with that of E. coli K-12.

Bacteriophages↗

PubMed2006

Developing live Shigella vaccines using lambda Red recombineering.

Live attenuated Shigella vaccines have shown promise in inducing protective immune responses in human clinical trials and as carriers of heterologous antigens from other mucosal pathogens. In the past, construction of Shigella vaccine strains relied on classical allelic exchange systems to genetically engineer the bacterial genome. These systems require extensive in vitro engineering of long homologous sequences to create recombinant replication-defective plasmids or phage. Alternatively, the lambda red recombination system from bacteriophage facilitates recombination with as little as 40 bp of homologous DNA. The process, referred to as recombineering, typically uses an inducible lambda red operon on a temperature-sensitive plasmid and optimal transformation conditions to integrate linear antibiotic resistance cassettes flanked by homologous sequences into a bacterial genome. Recent advances in recombineering have enabled modification of genomic DNA from bacterial pathogens including Salmonella, Yersinia, enteropathogenic Escherichia coli, or enterohemorrhagic E. coli and Shigella. These advances in recombineering have been used to systematically delete virulence-associated genes from Shigella, creating a number of isogenic strains from multiple Shigella serotypes. These strains have been characterized for attenuation using both in vivo and in vitro assays. Based on this data, prototypic Shigella vaccine strains containing multiple deletions in virulence-associated genes have been generated.

Bacteriophage lambda↗

PubMed2006

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.

Adolescent↗

PubMed2005

Shigella and Salmonella: death as a means of survival.

Shigella and Salmonella kill host cells and trigger inflammatory responses by mechanisms that are not fully understood. The goal of this review is to reevaluate key observations reported over the past 15 years and, whenever possible, to provide a chronological perspective as to how our understanding of the pathways by which Shigella and Salmonella kill host cells has evolved.

Animals↗

PubMed2005

Construction and characterization of bivalent Shigella flexneri 2a vaccine strains SC608(pCFAI) and SC608(pCFAI/LTB) that express antigens from enterotoxigenic Escherichia coli.

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 10(4) (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.

Animals↗

PubMed2005

Virulent Shigella flexneri causes damage to mitochondria and triggers necrosis in infected human monocyte-derived macrophages.

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.

Adenosine Triphosphate↗

PubMed2005

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.

Administration, Oral↗

PubMed2004

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.

Adolescent↗

PubMed2002

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.

Animals↗