Strategy for cross-protection among Shigella flexneri serotypes.

Based upon the lipopolysaccharide (LPS) structure and antigenicity of Shigella group B, a strategy for broad cross-protection against 14 Shigella flexneri serotypes was designed. This strategy involves the use of two S. flexneri serotypes (2a and 3a), which together bear the all of the major antigenic group factors of this group. The novel attenuated strains used in these studies were S. flexneri 2a strain CVD 1207 (DeltaguaB-A DeltavirG Deltaset1 Deltasen) and S. flexneri 3a strain CVD 1211 (DeltaguaB-A DeltavirG Deltasen). Guinea pigs were immunized with an equal mixture of these strains and later challenged (Sereny test) with a wild-type S. flexneri serotype 1a, 1b, 2b, 4b, 5b, Y, or 6 strain of demonstrated virulence in the same model. Guinea pigs that were immunized with these two vaccine strains produced serum and mucosal antibodies that cross-reacted with all the S. flexneri serotypes tested (except of S. flexneri serotype 6) as assessed by enzyme-linked immunosorbent assay, immunoblotting, and slide agglutination. Furthermore, the combination vaccine conferred significant protection against challenge with S. flexneri serotypes 1b, 2b, 5b, and Y but not with serotypes 1a, 4b, or (as predicted) 6.

Further characterization of delta aroA delta virG Shigella flexneri 2a strain CVD 1203 as a mucosal Shigella vaccine and as a live-vector vaccine for delivering antigens of enterotoxigenic Escherichia coli.

The use of attenuated delta aroA delta virG Shigella flexneri 2a strain CVD 1203 as a live vector for enterotoxigenic Escherichia coli (ETEC) antigens is reported. CVD 1203 alone or expressing colonization factor antigen fimbriae and CS3 fibrillae of ETEC was given to guinea pigs and mice, orogastrically (o.g.) or intranasally (i.n.). CVD 1203 given i.n. elicited high titers of antilipopolysaccharide (anti-LPS) immunoglobulin A (IgA) and was protective in guinea pigs against a homologous conjunctival challenge. Whereas a strong IgA response against colonization factor antigen CS3, and Shigella LPS was detected in tears and serum of guinea pigs after o.g. or i.n. immunization, the i.n. route elicited significantly higher antibody titers. A strong serum IgG response was also observed against the ETEC antigens, although no serum anti-LPS IgG response was detected. The immune response in mice followed a pattern similar to that in guinea pigs, and the difference between the responses after o.g. and i.n. administration was even more remarkable.

Prevalence of Shigella enterotoxin 1 among Shigella clinical isolates of diverse serotypes.

Shigella enterotoxin 1 (ShET1) is a novel, iron-dependent, toxin encoded by chromosomal genes (set1). To determine the prevalence of this enterotoxin, 172 Shigella clinical isolates (and 10 enteroinvasive Escherichia coli [EIEC]) from distant areas worldwide, representing all 4 groups and 45 serotypes of Shigella, were screened for set1 by DNA colony hybridization and polymerase chain reaction amplification. set1 was present in all 22 Shigella flexneri 2a strains tested but was rare in isolates of other Shigella serotypes (3.3%, 5/150) and not found in EIEC (0/10). That ShET1 is found almost exclusively in S. flexneri 2a may help explain the epidemiologic predominance of this serotype in the developing world.

Evaluation of the safety, immunogenicity, and efficacy in healthy adults of four doses of live oral hybrid Escherichia coli-Shigella flexneri 2a vaccine strain EcSf2a-2.

In previous trials, live invasive Escherichia coli-Shigella flexneri 2a hybrid vaccine candidate EcSf2a-2, administered to adult volunteers as 3 doses of ca. 2 x 10(9) colony forming units (c.f.u.) spaced over one week, induced fever and/or diarrhea in 11% of subjects and provided only limited protection (36% efficacy) against illness following challenge with virulent S. flexneri 2a. We sought to improve the clinical safety of this vaccine by administering a lower inoculum, and to enhance protective immunity by administering additional booster doses at 2 weeks. Twenty-one healthy adults were immunized with ca. 7 x 10(8) c.f.u. of EcSf2a-2 on days 0, 3, 14, and 17. The vaccine consistently colonized the intestine without causing serious adverse reactions; mild diarrhea developed in one subject and low grade fever in another. Vaccination elicited an antibody secreting cell (ASC) response to lipopolysaccharide (LPS) in all subjects, which was highest on day 7 and notably diminished thereafter on days 10, 16, 21, and 24, suggesting that active mucosal immunity developed rapidly. The magnitude of the response was modest (geometric mean peak = 16 IgA ASC/10(6) peripheral blood mononuclear cells) and an IgG serological response to LPS was detected in only 19% of subjects. Following experimental challenge with virulent S. flexneri 2a administered with bicarbonate buffer, shigellosis (diarrhea, dysentery, or fever) developed in 10 of 16 vaccine recipients (63%) and in 12 of 14 unvaccinated controls (86%), resulting in a vaccine efficacy of 27% (95% confidence limits -197, 82, p = 0.15, 1-tailed).(ABSTRACT TRUNCATED AT 250 WORDS)

Outpatient studies of the safety and immunogenicity of an auxotrophic Escherichia coli K-12-Shigella flexneri 2a hybrid vaccine candidate, EcSf2a-2.

A phase II study was conducted in 244 volunteers at Fort Ord, CA, to determine the safety and immunogenicity of EcSf2a-2, a live, oral Shigella vaccine constructed by transfer of genes from Shigella flexneri to Escherichia coli K-12. In this placebo-controlled study, four doses of vaccine ranging from 2.3 to 9.0 x 10(8) colony-forming units were given on days 0, 3, 14 and 17. Vaccine shedding occurred from 1 to 3 days after each dose. The vaccine was well tolerated at every dose tested. Significant levels of IgA, IgG or IgM antibody-secreting cells (ASC) recognizing S. flexneri 2a lipopolysaccharide (LPS) were found in 94% of a volunteer subset tested 7 days after the first dose of EcSf2a-2. Seven days after the third dose, ASC were detected less often (57%), and were mainly IgA. Significant rises in serum antibody to LPS were detected in 37% of vaccine recipients.

Safety, immunogenicity, and efficacy in monkeys and humans of invasive Escherichia coli K-12 hybrid vaccine candidates expressing Shigella flexneri 2a somatic antigen.

A live, oral Shigella vaccine, constructed by transfer of the 140-MDa invasiveness plasmid from Shigella flexneri 5 and the chromosomal genes encoding the group- and type-specific O antigen of S. flexneri 2a to Escherichia coli K-12, was tested in humans. Designated EcSf2a-1, this vaccine produced adverse reactions (fever, diarrhea, or dysentery) in 4 (31%) of 13 subjects who ingested a single dose of 1.0 x 10(9) CFU, while at better-tolerated doses (5.0 x 10(6) to 5.0 x 10(7) CFU), it provided no significant protection against challenge with S. flexneri 2a. A further-attenuated aroD mutant derivative, EcSf2a-2, was then tested. Rhesus monkeys that received EcSf2a-2 in three oral doses of ca. 1.5 x 10(11) CFU experienced no increase in gastrointestinal symptoms compared with a control group that received an E. coli K-12 placebo. Compared with controls, the vaccinated monkeys were protected against shigellosis after challenge with S. flexneri 2a (60% efficacy; P = 0.001). In humans, EcSf2a-2 was well tolerated at inocula ranging from 5.0 x 10(6) to 2.1 x 10(9) CFU. However, after a single dose of 2.5 x 10(9) CFU, 4 (17%) of 23 subjects experienced adverse reactions, including fever (3 subjects) and diarrhea (209 ml) (1 subject), and after a single dose of 1.8 x 10(10) CFU, 2 of 4 subjects developed dysentery. Recipients of three doses of 1.2 to 2.5 x 10(9) CFU had significant rises in serum antibody to lipopolysaccharide (61%) and invasiveness plasmid antigens (44%) and in gut-derived immunoglobulin A antibody-secreting cells specific for lipopolysaccharide (100%) and invasiveness plasmid antigens (60%). Despite its immunogenicity, the vaccine conferred only 36% protection against illness (fever, diarrhea, or dysentery) induced by experimental challenge (P = 0.17). These findings illustrate the use of an epithelial cell-invasive E. coli strain as a carrier for Shigella antigens. Future studies must explore dosing regimens that might optimize the protective effects of the vaccine while eliminating adverse clinical reactions.

Genotypic and phenotypic characterization of an aroD deletion-attenuated Escherichia coli K12-Shigella flexneri hybrid vaccine expressing S. flexneri 2a somatic antigen.

The construction and characterization of EcSf2a-2, an aroD-deleted Escherichia coli-Shigella hybrid vaccine carrying chromosomal and plasmid genes from Shigella flexneri and expressing S. flexneri 2a somatic antigen in association with E. coli K12 core are described. Expression of hybrid lipopolysaccharide and deletion of aroD resulted in the attenuation of phenotypic characteristics associated with pathogenicity. The addition of an aroD deletion results in a requirement for an aromatic precursor of para-aminobenzoic acid (PABA), an essential bacterial metabolite not present in mammalian tissues. The biosynthesis of hybrid somatic antigen prevents expression of a Sereny-positive reaction by invasive bacteria capable of expressing a plaque-positive phenotype. A functional kcpA gene is required for expression of the plaque-positive phenotype. The presence of an aroD deletion does not interfere with expression of an invasive phenotype; however, in bacteria containing a functional kcpA gene, replication and spread by invading bacteria are limited, preventing development of the plaque-positive phenotype.

Effect of prior infection with virulent Shigella flexneri 2a on the resistance of monkeys to subsequent infection with Shigella sonnei.

All virulent shigellae have large plasmids. Plasmid-associated genes encode the expression of membrane-associated proteins (MAP), some of which correlate with the ability to invade susceptible epithelial cells. These MAP are serologically related in all of the shigella serotypes and evoke an antibody response after infection. To determine whether the MAP have a significant role in protection, 24 monkeys were infected with virulent Shigella flexneri 2a. After recovery, one group (with controls) was rechallenged with S. flexneri 2a; another group (with controls) was fed Shigella sonnei. The animals that were rechallenged with S. flexneri 2a were protected, while those that were fed S. sonnei experienced the same incidence of disease as controls. No differences in serum immune response to MAP after primary infection with S. flexneri were detected in immunoblots using lysates of S. flexneri or S. sonnei or in ELISA using water extracts of these strains.

Virulence phenotype and genetic characteristics of the T32-ISTRATI Shigella flexneri 2a vaccine strain.

The T32-ISTRATI strain, which has been used as an oral attenuated Shigella flexneri 2a vaccine, has lost the invasive phenotype due to a spontaneous deletion in the shigella virulence plasmid. This deletion has eliminated three plasmid loci (ipaBCDA, invA and virG) that are necessary for production of a positive Sereny test by Shigella species. Virulence in the Sereny test was reconstituted in the T32-ISTRATI strain by the conjugal transfer of an intact 140 M Da virulence plasmid from S. flexneri 5. The T32-ISTRATI vaccine is safe when given orally in multiple doses of 50-100 x 10(9) organisms, and both homologous and heterologous protection has been reported in large Romanian and Chinese field trials. Although the protective antigen(s) in this vaccine have not been identified, the potential use of non-invasive plasmid deletion mutants as living shigella vaccines is illustrated by the T32-ISTRATI vaccine.

An O antigen can interfere with the function of the Yersinia pseudotuberculosis invasin protein.

Escherichia coli strains harbouring the Yersinia pseudotuberculosis inv gene are able to enter cultured mammalial cells. We show here that this property is not shared by all enteric bacteria, since Shigella flexneri 2a cured of its virulence-associated plasmid and harbouring the inv gene is unable to enter mammalian cells efficiently. Mapping studies showed that the region of the chromosome responsible for this phenotype includes rfaB, a locus involved in the production of O antigen. S. flexneri 2a strains that express O antigen were unable to enter mammalian cells, even though invasin was efficiently expressed and localized, showing that this structure interferes with invasin activity. The O antigen either masks invasin or sterically hinders the ability of the mammalian cell receptor to bind this protein.

Studies in volunteers to evaluate candidate Shigella vaccines: further experience with a bivalent Salmonella typhi-Shigella sonnei vaccine and protection conferred by previous Shigella sonnei disease.

A bivalent vaccine consisting of Salmonella typhi strain Ty21a containing the 120 MDa plasmid of Shigella sonnei and expressing both S. typhi and S. sonnei lipopolysaccharides (LPS) on its surface was previously shown to protect significantly against S. sonnei disease in experimental challenge studies. However, protective efficacy could not be reconfirmed in volunteers with five subsequent lots of vaccine. One vaccine lot which resembled the initial protective lots of vaccine in biochemical and serological tests, and by electron microscopy, was administered to 16 volunteers who ingested three doses of 10(9) organisms each. Antibody secreting cells (ASC) specific for S. sonnei LPS were detected in the blood of 100% of vaccines, but no protection of these vaccines was demonstrated during a S. sonnei challenge study. To assess the ability of the volunteer model to detect infection-derived immunity, six volunteers who had had clinical shigellosis due to S. sonnei two months earlier were rechallenged with wild-type S. sonnei, together with 12 controls. Prior infection provided 100% protection against febrile illness (p = 0.05) and diarrhea (p = 0.04), thereby validating the volunteer model for assessing Shigella vaccines.

Specific immunoglobulin A-secreting cells in peripheral blood of humans following oral immunization with a bivalent Salmonella typhi-Shigella sonnei vaccine or infection by pathogenic S. sonnei.

The ability of bivalent Salmonella typhi-Shigella sonnei vaccine strain 5076-1C to stimulate an intestinal immunoglobulin A response in humans was evaluated by detecting gut-derived, trafficking antibody-secreting cells (ASC) in peripheral blood. Following vaccination, an immunoglobulin A-ASC response to O antigens of S. typhi and S. sonnei was observed in 10 of 13 and 13 of 13 vaccine recipients, respectively. Experimental challenge with pathogenic S. sonnei stimulated an ASC response to the S. sonnei O antigen in all subjects who developed clinical illness. The magnitude of the ASC response to challenge was significantly greater than that resulting from vaccination. Furthermore, compared with the response of the unimmunized controls, individuals previously immunized with 5076-1C demonstrated a significantly greater ASC response following challenge with S. sonnei.

Expression in Escherichia coli K-12 of the 76,000-dalton iron-regulated outer membrane protein of Shigella flexneri confers sensitivity to cloacin DF13 in the absence of Shigella O antigen.

One of the chromosomal segments associated with virulence in Shigella flexneri encodes the production of aerobactin and the synthesis of an iron-regulated 76-kilodalton outer membrane protein believed to be the ferric-aerobactin receptor. However, S. flexneri expressing this putative aerobactin receptor, which is slightly larger than that encoded by pColV, is insensitive to the killing action of cloacin DF13, a bacteriocin which binds to other aerobactin receptor proteins and kills the cells. In this paper we show that the conjugal transfer of DNA encoding the iron-regulated 76-kilodalton protein from S. flexneri to Escherichia coli K-12 conferred cloacin DF13 sensitivity on the recipients. However, E. coli K-12 which had also inherited genes specifying Shigella O-antigen biosynthesis remained cloacin insensitive. The data suggest that it is unwise to use cloacin DF13 sensitivity alone to screen transconjugants or clinical isolates for the expression of aerobactin receptor proteins.

Shigella vaccines.

Shigellosis remains a major public health problem in developing countries. In these nations, the disease affects young children for the most part. The infecting organism causes illness by invading the colonic mucosa. It is closely related to nonpathogenic Escherichia coli, and genetic material can be transferred from one organism to the other, a process increasing the pathogenic potential of the E. coli or reducing the virulence of the strain of Shigella. Knowledge of the genetics of virulence of shigellae enables the construction of living, attenuated oral vaccines that may offer a practical means of controlling the disease.

Electrolyte transport in rabbit cecum. I. Effect of RDEC-1 infection.

To investigate the characteristics of intestinal ion and fluid secretion induced by the adherent, effacing enteropathogenic Escherichia coli strain RDEC-1, we infected weanling rabbits with 10(7)-10(8) RDEC-1 organisms and then studied cecal ion transport under short-circuit conditions in Ussing chambers. Results in tissues with confluent adherent organisms were compared with those in uninfected ceca and in ceca stimulated with dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP). The short-circuited cecum normally absorbed Na and Cl, secreted bicarbonate (as represented by the residual ion flux), and displayed a high rate of nondiffusional Na and Cl transport. RDEC-1 infection did not alter the short-circuit current (Isc), but it increased the conductance (Gt), decreased the potential difference (PD), abolished net Na absorption, and reversed Cl absorption to secretion. The changes in Na and Cl net fluxes may be explained by inhibition of a Na-Cl linked absorptive process. In contrast, DBcAMP significantly increased the Isc, PD, and Gt, decreased net Na flux, and abolished net Cl absorption by stimulating electrogenic Cl secretion. These results suggest that RDEC-1-induced changes in cecal ion transport are not mediated by cAMP. The reduction in Na-Cl linked absorption is consistent with anatomic changes in the apical surfaces of absorptive epithelial characteristic of effacing enteroadherence, whereas the increased conductance is consistent with tight junction disruption seen with RDEC-1 infection.

Characterization of virulence marker antigen of Shigella spp. and enteroinvasive Escherichia coli.

Antisera produced in rabbits immunized with an enteroinvasive O143 strain of Escherichia coli were absorbed with an avirulent derivative of the same strain. The resulting sera have been previously shown to recognize enteroinvasive pathogens when used in an enzyme-linked immunosorbent assay. In the current study, Western blots (immunoblots) showed that such an absorbed rabbit antiserum recognized two proteins (IpaB and IpaC) which are encoded by a large, virulence-associated plasmid. These proteins are the apparent constituents of the virulence marker antigen which is expressed by shigellae and enteroinvasive E. coli.

Intracellular spread of Shigella flexneri associated with the kcpA locus and a 140-kilodalton protein.

Escherichia coli K-12 hybrids carrying both the 220-kilobase plasmid and the purE-linked kcpA locus from Shigella flexneri expressed a 140-kilodalton (kDa) protein which was recognized by convalescent sera from monkeys infected with S. flexneri. These hybrids were tested for the ability to produce plaques in HeLa cell monolayers. Hybrid strains which carried both the 220-kilobase plasmid and the kcpA locus had a plaque-forming efficiency of at least 10(-4) PFU/CFU, whereas the plaque-forming efficiency of hybrids that carried only the shigella invasion plasmid ranged from undetectable to 10(-6). Variants were purified from the rare plaques formed by E. coli hybrids that carried only the shigella invasion plasmid. These plaque-purified strains also expressed the 140-kDa protein, and they had a plaque-forming efficiency of at least 10(-4). Transduction of the purE locus from a plaque-purified hybrid into a non-plaque-forming E. coli K-12 strain did not alter the phenotype of the recipient, but conjugation of the shigella invasion plasmid into this transductant reconstituted both expression of the 140-kDa protein and the plaque-forming phenotype. Invasive E. coli K-12 hybrids carrying only the shigella invasion plasmid remained localized within discrete areas of the HeLa cell cytoplasm, whereas hybrids that also carried the S. flexneri kcpA locus grew in a dispersed pattern throughout the host cell cytoplasm. The dispersal of these organisms was inhibited by cytochalasin D, which suggested that host cell microfilaments may play a role in the intracellular spread of enteroinvasive pathogens.