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Recombinant A-B+ Vibrio cholerae O1 strain CVD 103-HgR is a safe, highly immunogenic, single-dose live oral vaccine in adults in industrialized countries. Safety, excretion, immunogenicity, vaccine transmissibility, and environmental introduction of CVD 103-HgR were investigated among 24- to 59-month-old children in Jakarta. In 81 households, 1 child was randomly allocated a single dose of vaccine (5 x 10(9) cfu) and another, placebo. Additionally, 139 unpaired children were randomly allocated vaccine or placebo. During 9 days of follow-up, diarrhea or vomiting did not occur more often among vaccines than controls. Vaccine was minimally excreted and was isolated from no controls and from 1 (0.6%) of 177 unvaccinated family contacts. A 4-fold or higher rise in serum vibriocidal antibody was observed in 75% of vaccines (10-fold rise in geometric mean titer over baseline). Of 135 paired placebo recipients or household contacts, 5 had vibriocidal seroconversions. Moore swabs placed in sewers and latrines near 97 households failed to detect vaccine. These observations pave the way for a large-scale field trial of efficacy.

A promising live attenuated typhoid vaccine candidate strain for mucosal immunization was developed by introducing a deletion in theguaBAlocus of pathogenicSalmonella entericaserovar Typhi strain Ty2. The resultant ΔguaBAmutant, serovar Typhi CVD 915, has a gene encoding resistance to arsenite replacing the deleted sequence withinguaBA, thereby providing a marker to readily identify the vaccine strain. CVD 915 was compared in in vitro and in vivo assays with wild-type strain Ty2, licensed live oral typhoid vaccine strain Ty21a, or attenuated serovar Typhi vaccine strain CVD 908-htrA(harboring mutations inaroC, aroD, andhtrA). CVD 915 was less invasive than CVD 908-htrAin tissue culture and was more crippled in its ability to proliferate after invasion. In mice inoculated intraperitoneally with serovar Typhi and hog gastric mucin (to estimate the relative degree of attenuation), the 50% lethal dose of CVD 915 (7.7 × 107CFU) was significantly higher than that of wild-type Ty2 (1.4 × 102CFU) and was only slightly lower than that of Ty21a (1.9 × 108CFU). Strong serum O and H antibody responses were recorded in mice inoculated intranasally with CVD 915, which were higher than those elicited by Ty21a and similar to those stimulated by CVD 908-htrA. CVD 915 also elicited potent proliferative responses in splenocytes from immunized mice stimulated with serovar Typhi antigens. Used as a live vector, CVD 915(pTETlpp) elicited high titers of serum immunoglobulin G anti-fragment C. These encouraging preclinical data pave the way for phase 1 clinical trials with CVD 915.

Live oral Ty21a and parenteral Vi polysaccharide vaccines provide significant protection against typhoid fever, albeit by distinct immune mechanisms. Vi stimulates serum immunoglobulin G Vi antibodies, whereas Ty21a, which does not express Vi, elicits humoral and cell-mediated immune responses other than Vi antibodies. Protection may be enhanced if serum Vi antibody as well as cell-mediated and humoral responses can be stimulated. Disappointingly, several new attenuatedSalmonella entericaserovar Typhi oral vaccines (e.g., CVD 908-htrAand Ty800) that elicit serum O and H antibody and cell-mediated responses following a single dose do not stimulate serum Vi antibody. Vi expression is regulated in response to environmental signals such as osmolarity by controlling the transcription oftviAin theviaBlocus. To investigate if Vi antibodies can be stimulated if Vi expression is rendered constitutive, we replaced PtviAin serovar Typhi vaccine CVD 908-htrAwith the constitutive promoter Ptac, resulting in CVD 909. CVD 909 expresses Vi even under high-osmolarity conditions and is less invasive for Henle 407 cells. In mice immunized with a single intranasal dose, CVD 909 was more immunogenic than CVD 908-htrAin eliciting serum Vi antibodies (geometric mean titer of 160 versus 49,P= 0.0007), whereas O antibody responses were virtually identical (geometric mean titer of 87 versus 80). In mice challenged intraperitoneally with wild-type serovar Typhi 4 weeks after a single intranasal immunization, the mortality of those immunized with CVD 909 (3 of 8) was significantly lower than that of control mice (10 of 10,P= 0.043) or mice given CVD 908-htrA(9 of 10,P= 0.0065).

The DeltaguaBA Shigella flexneri 2a vaccine candidate, CVD 1204, was evaluated as a delivery system for the non-toxic C-terminal of tetanus toxin (fragment C), either as a polypeptide expressed in the bacteria or as a DNA vaccine. CVD 1204 was transformed with plasmid pTETnir15 which encodes the fragment C gene (tetC) under the control of the inducible prokaryotic nir15 promoter or a DNA vaccine plasmid pcDNA3tetC which encodes tetC under the eukaryotic hCMV promoter. Guinea pigs immunised intranasally (i.n.) with either recombinant strain mounted a secretory immune response against S. flexneri 2a Lipopolysaccharide (LPS) and were protected against ocular challenge with wild-type S. flexneri 2a. Both strains were effective in eliciting a serum IgG response against fragment C in guinea pigs following i.n. immunisation. Furthermore, serum from guinea pigs immunised with CVD 1204(pTETnir15) contained tetanus toxin neutralising antibodies. These results demonstrate that this S. flexneri 2a vaccine candidate can serve as a vehicle for the delivery of foreign antigens to the systemic immune system while retaining its capacity to serve as a mucosal Shigella vaccine.

A phase 1 clinical trial was conducted among 35 healthy adult volunteers to evaluate the safety, immunogenicity, and shedding of different doses of CVD 1207, a live attenuated Shigella flexneri 2a vaccine candidate with specific deletion mutations in virG , sen , set , and guaBA . CVD 1207 retains the ability to invade epithelial cells but cannot effectively spread intercellularly after invasion (Δ virG ), does not produce enterotoxin (Δ sen and Δ set ), and has limited proliferation in vivo (Δ guaBA ). In a consecutive fashion, groups of three to seven subjects ingested a single oral dose of CVD 1207 at an inoculum of either 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 CFU. CVD 1207 was remarkably well-tolerated at inocula as high as 10 8 CFU. In comparison, one of 12 subjects who received 10 9 CFU experienced mild diarrhea and another experienced a single episode of emesis. One of five subjects who received 10 10 CFU experienced watery diarrhea and emesis. All subjects who ingested doses of 10 8 to 10 10 CFU excreted the vaccine; in 23 of 25, the duration of excretion was ≤3 days. A dose-related, immunoglobulin A antibody-secreting cell (ASC) response to S. flexneri 2a O-specific lipopolysaccharide was seen, with geometric mean peak values of 6.1 to 35.2 ASCs/10 6 peripheral blood mononuclear cells (PBMC) among recipients of 10 7 to 10 10 CFU. The cytokine response to Shigella -specific antigens observed in volunteers' PBMC following vaccination suggested a Th1 pattern with stimulation of gamma interferon and absence of interleukin 4 (IL-4) or IL-5. CVD 1207 represents a Shigella live oral vaccine strain prepared from wild-type S. flexneri 2a by rational use of recombinant DNA technology that achieves a remarkable degree of attenuation compared with earlier recombinant strains, even when administered at high dosage.

Shigella flexneri 2a strain CVD 1204, which was constructed by introducing a specific, in-frame deletion mutation in the guaB-A operon, was compared with deltaaroA strain CVD 1201. CVD 1204 was less invasive for HeLa cells than CVD 1201, whereas following invasion, the abilities of the two mutants to proliferate intracellularly were similarly impaired. The reduction in invasiveness was independent of the guanine auxotrophic phenotype and fully recovered when the chromosomal deletion mutation in CVD 1204 was repaired. Following inoculation of the conjunctival sac of guinea pigs (Serény test) at high doses (10(9) CFU per eye), both strains evoked minimal, short- lived conjunctival inflammation, which was significantly milder with strain CVD 1204. Double mutant deltaguaB-A deltavirG (also called icsA) strain CVD 1205 induced, after a single intranasal dose, high mucosal immunoglobulin A antilipopolysaccharide titers, which were significantly boosted further following a second dose of vaccine given 14 days later. Upon Serény test challenge with wild-type S. flexneri 2a, CVD 1205-vaccinated animals were significantly protected against keratoconjunctivitis (zero of eight vaccinees versus five of seven controls, P = 0.03; vaccine efficacy, 100%). CVD 1205 is an attractive candidate for human clinical trials.

Attenuated Salmonella typhi vaccine strain CVD 908, which harbors deletion mutations in aroC and aroD, has been shown to be well-tolerated and highly immunogenic, eliciting impressive serum antibody, mucosal IgA and cell-mediated immune responses. A further derivative prepared by introducing a deletion in htrA (which encodes a heat-shock protein that also has activity as a serine protease in CVD 908 (Chatfield et al., unpublished data) resulted in CVD 908-htrA. In phase 1 clinical trials, CVD 908-htrA appears very attractive as a live oral vaccine candidate. Both CVD 908 and CVD 908-htrA are useful as live vector vaccines to deliver foreign antigens to the immune system. Conditions that enhance the expression and immunogenicity of foreign antigens carried by CVD 908 and CVD 908-htrA are being investigated.

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.

We have employed a molecular genetic approach to characterize the nature of enteroinvasive Escherichia coli (EIEC) enterotoxic activity, as previously observed in Ussing chambers (A. Fasano, B.A. Kay, R.G. Russell, D.R. Maneval, Jr., and M.M. Levine, Infect. Immun. 58:3717-3723, 1990). The screening of TnphoA mutants of EIEC yielded a single insertion mutant which had significantly reduced levels of enterotoxic activity in the Ussing chamber assay. DNA flanking the insertion was used as a probe to screen for EIEC cosmid clones which conferred secretogenic activity. Such screening resulted in the identification of two overlapping cosmid clones which elicited significant changes in mucosal short-circuit current (Isc). Subcloning and nucleotide sequence analysis of a DNA fragment from one of the cosmid clones led to the identification of a single open reading frame which conferred this enterotoxic activity. By DNA hybridization, this gene (designated sen for shigella enterotoxin) was found in 75% of EIEC strains and 83% of Shigella strains and was localized to the inv plasmid of Shigella flexneri 2457T. By PCR, a sen gene with 99.7% nucleotide identity was cloned and sequenced from 2457T. A deletion in the EIEC sen gene was constructed by allelic exchange, resulting in significantly lower rises in Isc than were elicited by the wild-type parent; however, significant enterotoxic activity remained in the sen deletion mutant. To purify the Sen protein, the gene was cloned into the multiple cloning site of the expression vector pKK223-3. Purification of the sen gene product yielded a protein with a molecular mass of 63 kDa which elicited rises in Isc in the Ussing chamber. We believe that the sen gene product may constitute all or part of a novel enterotoxin in EIEC and Shigella spp.

Culture filtrates of Shigella flexneri 2a strain M4243 grown in iron-depleted medium, caused significant fluid accumulation in rabbit ileal loops. Also, when tested in Ussing chambers, a greater rise in potential difference and short circuit current was seen with such filtrates compared with the medium control. Analogous filtrates from two M4243 derivatives lacking the 140-MD invasiveness plasmid (either M4243avir or BS103) retained 60-65% of the wild-type enterotoxic activity. Ultrafiltration and gel exclusion size fractionation of M4243 filtrate revealed that the activity was approximately 60 kD. SDS-PAGE performed on this fraction showed 18 bands, 5 of which reacted with human convalescent sera. Genes encoding this enterotoxin, named ShET1 for Shigella enterotoxin 1, were cloned from the S. flexneri 2a chromosome, and two separate open reading frames of 534 and 186 bp were sequenced. These observations suggest that S. flexneri 2a elaborates two distinct enterotoxins: ShET1, encoded by genes located on the chromosome, and ShET2, encoded by a gene on the 140-MD invasiveness plasmid. ShET1, which is composed of two distinct subunits and is elaborated in vivo, where it elicits an immune response, may be important in the pathogenesis of diarrheal illness due to S. flexneri 2a.

rcsp, encoding amino acids 21-398 of Plasmodium falciparum circumsporozoite protein (CSP), under control of tacP was integrated into the chromosomal delta aroC locus of attenuated delta aroC, delta aroD Salmonella typhi CVD 908. By immunoblot and ELISA, rCSP expression was greater from a multicopy plasmid than from the single chromosomal gene. CVD 908 omega (delta aroC1019::tacP-rcsp) was well tolerated by 10 volunteers who were fed two doses of 5 x 10(7) organisms 8 days apart. Seven subjects excreted the vaccine strain for 1-3 days. All subjects developed serologic responses to O and H antigens of the live vector, whereas 3 vaccinees responded to the foreign antigen: 1 developed an 80-fold rise in serum anti-sporozoite antibody, another had a 4-fold rise in antibody to a recombinant portion of CSP (residues 309-345), while a third vaccinee developed CSP-specific CD8+ cytotoxic T lymphocyte activity. This is the first report of attenuated S. typhi eliciting a human serologic or a cytotoxic T lymphocyte response to a foreign protein. Improved foreign gene expression should enhance immunogenicity.

The best correlate of protection against cholera is the level of serum vibriocidal antibodies, which are primarily directed against the O antigen of Vibrio cholerae O1 and lyse V. cholerae in the presence of complement. We established the timing of peak vibriocidal antibody response using sera from safety/immunogenicity studies of live oral cholera vaccines CVD 103-HgR, CVD 103-HgR2 and CVD 110 among immunologically naive North Americans and Colombians. The serum reciprocal vibriocidal antibody titre was consistently higher 10 days postimmunization than on either day 7 or day 14. This study suggests that recent phase 2 studies of CVD 103-HgR may have underestimated the peak vibriocidal titre by collecting serum on days 7-8 rather than on day 10; future studies of live oral cholera vaccines should take these results into account to obtain the best measurement of peak immunological responses. Because of the rapid drop in vibriocidal antibody titres about 2 weeks after immunization, care must be exercised in comparing immunogenicity of different vaccine candidates, formulations, dosage levels and immunization schedules.

We have identified and characterized a secreted protein, designated Pic, which is encoded on the chromosomes of enteroaggregative Escherichia coli (EAEC) 042 and Shigella flexneri 2457T. The product of the pic gene is synthesized as a 146.5-kDa precursor molecule which is processed at the N and C termini during secretion, allowing the release of a mature protein (109.8 kDa) into the culture supernatant. The deduced amino acid sequence of Pic shows high homology to autotransporter proteins, particularly a subgroup termed the SPATEs (serine protease autotransporters of the Enterobacteriaceae ). Present in all members of this subgroup is a motif similar to the active sites of certain serine proteases. Pic catalyzes gelatin degradation, which can be abolished by disruption of the predicted proteolytic active site. Functional analysis of the Pic protein implicates this factor in mucinase activity, serum resistance, and hemagglutination. Our data suggest that Pic may be a multifunctional protein involved in enteric pathogenesis.

Attenuated Salmonella typhi strain CVD 915, harboring a deletion in guaBA that interrupts the biosynthesis of guanine nucleotides, was evaluated as a live vector vaccine for delivering foreign antigens utilizing prokaryotic or eukaryotic expression systems. Plasmids pTETnir15 and pcDNA3tetC encoding fragment C (Frag C) of tetanus toxin under the control of prokaryotic or eukaryotic promoters, respectively, were introduced into CVD 915 and administered intranasally to mice. Purified pcDNA3tetC and Frag C were given intramuscularly. High titers of serum IgG1, IgG2a, and IgG2b antibodies against Frag C were elicited by CVD 915(pTETnir15) and CVD 915(pcDNA3tetC). These responses were significantly higher than those induced by pcDNA3tetC. Proliferative responses and IL-2 and IFN-gamma production were observed in splenocytes exposed to S. typhi antigens and Frag C. We conclude that CVD 915 is a highly efficient live vector to carry foreign genes under eukaryotic or prokaryotic control and elicit potent immune responses.

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 (Δ guaB-A ΔvirG Δset1 Δsen ) and S. flexneri 3a strain CVD 1211 (Δ guaB-A ΔvirG Δsen ). 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.

Attenuated Salmonella typhi are attractive for use as live vector vaccines to express protozoal antigens and deliver them to the human immune system. The gene encoding the mature form of Leishmania mexicana mexicana gp63 under control of tac promoter was integrated into the delta aroC locus of the chromosome of attenuated delta aroC, delta aroD S. typhi strain CVD 908. After oral immunization of BALB/c mice with two 1 x 10(9) colony forming unit doses given 21 days apart, CVD 908 omega (delta aroC::Ptac-gp63) elicited a broad T cell-mediated immune response against L. m. mexicana gp63 as demonstrated by: (1) lymphoproliferative response to fixed whole L. m. mexicana promastigotes; (2) activation of IL-2 (but not IL-4)-producing lymphocytes; (3) appearance of cytotoxic T cells against mouse mastocytoma cells expressing gp63. This T-cell mediated immune response was associated with significant protection in F1 (BALB/cXC57Bl/6) mice challenged in their footpads with a wild type strain of L. m. mexicana.

We evaluated the safety and immunogenicity of attenuated Shigella flexneri 2a vaccine candidate CVD 1203, which harbors precise deletions in the plasmid gene virG and in the chromosomal gene aroA. CVD 1203 invades epithelial cells but undergoes minimal intracellular proliferation and cell-to-cell spread. Fasting healthy volunteers, aged 18 to 40 years, were randomly allocated (double-blind design) to receive either CVD 1203 vaccine or placebo, along with sodium bicarbonate buffer, on days 0 and 14, as follows. At the time of the first inoculation, 10 subjects received placebo (group 1) and 22 subjects received either 1.5 x 10(8) (group 2; 11 subjects) or 1.5 x 10(9) (group 3; 11 subjects) CFU of CVD 1203. Fourteen days later, subjects from group 1 received 1.2 x 10(6) CFU of CVD 1203 and subjects from groups 2 and 3 received 1.2 x 10(8) vaccine organisms. Clinical tolerance was dose dependent. After a single dose of CVD 1203 at 10(6), 10(8), or 10(9) CFU, self-limited (

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.

We engineered an oral Shigella vaccine prototype that can invade intestinal epithelial cells but cannot undergo extensive intracellular replication or extend to adjacent epithelial cells. Strain CVD 1203, derived from wild-type Shigella flexneri 2a by introducing deletions in chromosomal aroA and invasion plasmid virG, was highly attenuated in the Sereny test. Two 10(9)-CFU orogastric doses (2 weeks apart) stimulated production of secretory immunoglobulin A antibodies to S. flexneri 2a and protected against conjunctival sac challenge with virulent S. flexneri 2a.

Considerable progress has been made in the last decade in developing vaccines against the enteric infections of greatest public health importance. A quadrivalent rotavirus vaccine consisting of rhesus rotavirus vaccine (which contains serotype 3 neutralization antigen) and three reassortant viruses (rhesus virus expressing neutralization antigens of serotypes 1, 2 or 4) is undergoing placebo-controlled field trials of efficacy in the USA and in two developing countries. Two new vaccines against typhoid fever (oral Ty21a and parenteral Vi polysaccharide) have been licensed in many countries. Even newer generations of typhoid vaccines are undergoing clinical testing, including new attenuated S. typhi strains and Vi polysaccharide-carrier protein conjugate vaccines. Two inactivated oral cholera vaccines, consisting of inactivated V. cholerae 01 bacteria alone or in combination with purified B subunit of cholera toxin, each conferred 50–53% protection over 3 years in a field trial in Bangladesh where subjects were immunized with a three-dose regimen. In extensive clinical trials in adults and children in less-developed countries, an engineered live oral cholera vaccine, strain CVD 103-HgR, has been shown to be well tolerated and highly immunogenic following administration of just a single oral dose; a large-scale field trial in 70000 subjects is underway to investigate the efficacy of this vaccine. Several candidate vaccines against Shigella and enterotoxigenic Escherichia coli are in clinical trials. Accumulating knowledge on pathogenesis of enteric infections and advances in mucosal and cellular immunology, coupled with the application of modern biotechnology, have resulted in a plethora of vaccine candidates. It is expected that in future years efforts will be directed to construct vaccines against other enteric pathogens.

We describe here a new tissue culture method for prolonged laboratory maintenance of tachyzoites of the highly virulent RH strain of Toxoplasma gondii. Using a rapidly proliferating murine tumor cell line (YAC-1), the method described is easy to perform and is as or more efficient (both in terms of yield and cost) than other traditional methods for maintenance of the parasite. Furthermore, upon prolonged maintenance (greater than 160 days) in YAC-1 tissue culture, the pathogenicity of the parasite, as well as its capacity to elicit an immune response, are comparable to that of organisms maintained in mice. We conclude therefore, that the method described herein is a suitable alternative to the traditional method of maintenance of virulent RH strain T. gondii tachyzoites.