Isolation and characterization of a Shigella flexneri invasin complex subunit vaccine.
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The invasiveness and virulence of Shigella spp. are largely due to the expression of plasmid-encoded virulence factors, among which are the invasion plasmid antigens (Ipa proteins). After infection, the host immune response is directed primarily against lipopolysaccharide (LPS) and the virulence proteins (IpaB, IpaC, and IpaD). Recent observations have indicated that the Ipa proteins (IpaB, IpaC, and possibly IpaD) form a multiprotein complex capable of inducing the phagocytic event which internalizes the bacterium. We have isolated a complex of invasins and LPS from water-extractable antigens of virulent shigellae by ion-exchange chromatography. Western blot analysis of the complex indicates that all of the major virulence antigens of Shigella, including IpaB, IpaC, and IpaD, and LPS are components of this macromolecular complex. Mice or guinea pigs immunized intranasally with purified invasin complex (invaplex), without any additional adjuvant, mounted a significant immunoglobulin G (IgG) and IgA antibody response against the Shigella virulence antigens and LPS. The virulence-specific response was very similar to that previously noted in primates infected with shigellae. Guinea pigs (keratoconjunctivitis model) or mice (lethal lung model) immunized intranasally on days 0, 14, and 28 and challenged 3 weeks later with virulent shigellae were protected from disease (P<0.01 for both animal models). (+info)
Safety and immunogenicity of improved Shigella O-specific polysaccharide-protein conjugate vaccines in adults in Israel.
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Data suggest that the O-specific polysaccharide (O-SP) domain of the lipopolysaccharide (LPS) of Shigella species is both an essential virulence factor and a protective antigen and that a critical level of serum immunoglobulin G (IgG) to this antigen will confer immunity to shigellosis. Because covalent attachment of polysaccharides to proteins increases their immunogenicity, especially in infants and in young children, the O-SP of Shigella species were bound to medically useful proteins, and the safety and immunogenicity of the resultant conjugates were confirmed in adults and 4- to 7-year-old children. Succinylation of the carrier protein improved the immunogenicity of Shigella conjugates in mice and increased their yield. Based on these results, a clinical trial of O-SP conjugates of Shigella sonnei and Shigella flexneri 2a bound to succinylated mutant Pseudomonas aeruginosa exotoxin A (rEPAsucc) or native or succinylated Corynebacterium diphtheriae toxin mutant (CRM9 or CRM9succ) was conducted in healthy adults. The conjugates were safe and immunogenic. S. sonnei-CRM9, S. sonnei-CRM9succ, and S. sonnei-rEPAsucc elicited significant rises of geometric mean (GM) IgG anti-LPS within 1 week of injection (P < 0.001). At 26 weeks, the GM anti-LPS levels elicited by these three conjugates were similar and higher than their prevaccination levels (P < 0.0001). GM IgG anti-LPS levels elicited by S. flexneri 2a-rEPAsucc were significantly higher than those elicited by S. flexneri 2a-rCRM9succ at all intervals after injection. At 26 weeks, the levels of IgG anti-LPS in vaccinees were higher than their prevaccination levels (P < 0.0001). The serum antibody responses were specific, as there was no significant rise of anti-LPS to the heterologous O-SP in any vaccinee. Both conjugates elicited statistically significant rises of serum antibodies to the injected carrier protein. At 6 months, these five Shigella conjugates elicited higher fold rises than similar conjugates (D. N. Taylor et al., Infect. Immun. 61:3678-3687, 1993). Based on these data, we chose S. sonnei-CRM9 and S. flexneri 2a-rEPAsucc for evaluation in children. (+info)
Safety and immunogenicity of a proteosome-Shigella flexneri 2a lipopolysaccharide vaccine administered intranasally to healthy adults.
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We studied the safety and immunogenicity of a Shigella flexneri 2a vaccine comprising native S. flexneri 2a 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 < or = 0.05). These reactions were self-limited and generally mild. The vaccine elicited S. flexneri 2a 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. flexneri 2a 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. flexneri 2a 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. (+info)
Phase I evaluation of delta virG Shigella sonnei live, attenuated, oral vaccine strain WRSS1 in healthy adults.
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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. (+info)
Construction, characterization, and animal testing of WRSd1, a Shigella dysenteriae 1 vaccine.
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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. (+info)
Molecular cloning and characterization of genes for Shigella sonnei form I O polysaccharide: proposed biosynthetic pathway and stable expression in a live salmonella vaccine vector.
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The gene region for biosynthesis of Shigella sonnei form I O polysaccharide (O-Ps) and flanking sequences, totaling >18 kb, was characterized by deletion analysis to define a minimal construct for development of Salmonella-based live vaccine vector strains. Lipopolysaccharide (LPS) expression and DNA sequence studies of plasmid deletion derivatives indicated form I O-Ps expression from a 12.3-kb region containing a putative promoter and 10 contiguous open reading frames (ORFs), one of which is the transposase of IS630. A detailed biosynthetic pathway, consistent with the predicted functions of eight of the nine essential ORFs and the form I O-Ps structure, is proposed. Further sequencing identified partial IS elements (i.e., IS91 and IS630) and wzz upstream of the form I coding region and a fragment of aqpZ and additional full or partial IS elements (i.e., IS629, IS91, and IS911) downstream of this region. The stability of plasmid-based form I O-Ps expression was greater from low-copy vectors than from high-copy vectors and was enhanced by deletion of the downstream IS91 from plasmid inserts. Both core-linked (i.e., LPS) and non-core-linked (i.e., capsule-like) surface expression of form I O-Ps were detected by Western blotting and silver staining of polyacrylamide gel electrophoresis-separated Shigella and Escherichia coli extracts. However, salmonellae, which have a core that is chemically dissimilar to that of shigellae, expressed only non-core-linked surface-associated form I O-Ps. Finally, attenuated Salmonella enterica serovar Typhi live vaccine vector candidates, containing minimal-sized form I operon constructs, elicited immune protection in mice against virulent S. sonnei challenge, thereby supporting the promise of live, oral vaccines for the prevention of shigellosis. (+info)
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.
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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. (+info)
Deletion in the Shigella enterotoxin genes further attenuates Shigella flexneri 2a bearing guanine auxotrophy in a phase 1 trial of CVD 1204 and CVD 1208.
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BACKGROUND: We created a live, attenuated, oral Shigella vaccine by constructing a lineage of guanine auxotrophs and conducted a double-blind, placebo-controlled trial to ascertain (1) the attenuation profile of Delta guaBA Shigella flexneri 2a, which harbors deletions in the guanine nucleotide synthesis pathway (CVD 1204); (2) additional attenuation conferred by deletions in set and sen genes encoding Shigella enterotoxins (ShETs) 1 and 2, respectively (CVD 1208); and (3) the relative immunogenicity of these constructs. METHODS: Inpatient volunteers received a single oral dose of CVD 1204, CVD 1208 (10(7), 10(8), or 10(9) cfu), or placebo. Clinical, immunologic, and microbiologic responses were evaluated. RESULTS: Reactogenicity occurred in 8 of 23 recipients of CVD 1204, characterized by diarrhea (30%), fever (22%), and/or dysentery (17%), but in only 1 (5%) of 21 recipients of CVD 1208 (brief fever) (P=.02, Fisher's exact test). Antilipopolysaccharide responses, as measured by antibody-secreting cell, serum, or fecal antibody levels, occurred in 67%, 71%, and 100% of recipients of CVD 1204 and in 86%, 43%, and 100% of recipients of CVD 1208 at doses of 10(7), 10(8), and 10(9) cfu, respectively. CONCLUSIONS: We conclude that 1 or both ShETs are virulence determinants in humans; their inactivation, in combination with Delta guaBA, leads to a well-tolerated and immunogenic Shigella vaccine candidate. (+info)