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Induction of effective memory T cells is likely to be critical to the level and duration of protection elicited by novel live oral typhoid vaccines. Using cells from volunteers who ingested Salmonella Typhi vaccine strain CVD 909, we characterized the induction of interferon (IFN)-γ-secreting central (TCM, CD45RO+CD62L+) and effector (TEM, CD45RO+CD62L−) memory T populations, and their gut-homing potential based on integrin α4/β7 expression. Both CD4+TEMand TCMpopulations secreted IFN-γ. However, although CD4+TEMexpressed, or not, integrin α4/β7, CD4+TCMcells were predominantly integrin α4/β7+. In contrast, IFN-γ-secreting CD8+cells were predominantly classical TEMand CD45RA+TEM(TEMRA, CD45RO−CD62L−) subsets. However, although CD8+TEMexpressed, or not, integrin α4/β7, CD8+TEMRAwere predominantly integrin α4/β7+. This is the first demonstration that oral immunization of humans with S. Typhi elicits diverse IFN-γ-secreting CD4+and CD8+TCMand TEMsubsets able to migrate to the gut and other lymphoid tissues.Mucosal Immunology (2008) 1, 389–398; doi:10.1038/mi.2008.30; published online 2 July 2008

A multivalent live oral vaccine against both Shigella spp. and enterotoxigenic Escherichia coli (ETEC) is being developed based on the hypothesis that protection can be achieved if attenuated shigellae express ETEC fimbrial colonization factors and genetically detoxified heat-labile toxin from a human ETEC isolate (LTh). Two detoxified derivatives of LTh, LThK63 and LThR72, were engineered by substitution-serine to lysine at residue 63, or lysine to arginine at residue 72. The genes encoding these two derivatives were cloned separately on expression plasmids downstream from the CFA/I operon. Following electroporation into S. flexneri 2a vaccine strain CVD 1204, coexpression of CFA/I and LThK63 or LThR72 was demonstrated by Western blot analysis, GM(1) binding assays, and agglutination with anti-CFA/I antiserum. Hemagglutination and electron microscopy confirmed surface expression of CFA/I. Guinea pigs immunized intranasally on days 0 and 15 with CVD 1204 expressing CFA/I and LThK63 or LThR72 exhibited high titers of both serum immunoglobulin G (IgG) and mucosal secretory IgA anti-CFA/I; 40% of the animals produced antibodies directed against LTh. All immunized guinea pigs also produced mucosal IgA (in tears) and serum IgG anti-S. flexneri 2a O antibodies. Furthermore, all immunized animals were protected from challenge with wild-type S. flexneri 2a. This prototype Shigella-ETEC hybrid vaccine demonstrates the feasibility of expressing multiple ETEC antigens on a single plasmid in an attenuated Shigella vaccine strain and engendering immune responses against both the heterologous antigens and vector strain.

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 attenuated Salmonella enterica serovar Typhi oral vaccines (e.g., CVD 908-htrA and 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 of tviA in the viaB locus. To investigate if Vi antibodies can be stimulated if Vi expression is rendered constitutive, we replaced P(tviA) in serovar Typhi vaccine CVD 908-htrA with the constitutive promoter P(tac), 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-htrA in 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).

Enteropathogenic Escherichia coli (EPEC), a leading cause of diarrhea among infants in developing countries, induces dramatic alterations in host cell architecture that depend on a type III secretion system. EspB, one of the proteins secreted and translocated to the host cytoplasm via this system, is required for numerous alterations in host cell structure and function. To determine the role of EspB in virulence, we conducted a randomized, double-blind trial comparing the ability of wild-type EPEC and an isogenic DeltaespB mutant strain to cause diarrhea in adult volunteers. Diarrhea developed in 9 of 10 volunteers who ingested the wild-type strain but in only 1 of 10 volunteers who ingested the DeltaespB mutant strain. Marked destruction of the microvillous brush border adjacent to adherent organisms was observed in a jejunal biopsy from a volunteer who ingested the wild-type strain but not from two volunteers who ingested the DeltaespB mutant strain. Humoral and cell-mediated immune responses to EPEC antigens were stronger among recipients of the wild-type strain. In addition, four of the volunteers who ingested the wild-type strain had lymphoproliferative responses to EspB. These results demonstrate that EspB is a critical virulence determinant of EPEC infections and suggest that EspB contributes to an immune response.

Salmonella enterica serovar Typhi strain CVD 908-htrA is a live attenuated strain which may be useful as an improved oral typhoid vaccine and as a vector for cloned genes of other pathogens. We conducted a phase 2 trial in which 80 healthy adults received one of two dosage levels of CVD 908-htrA in a double-blind, placebo-controlled, crossover study. There were no differences in the rates of side effects among volunteers who received high-dose vaccine (4.5 x 10(8) CFU), lower-dose vaccine (5 x 10(7) CFU), or placebo in the 21 days after vaccination, although recipients of high-dose vaccine (8%) had more frequent diarrhea than placebo recipients (0%) in the first 7 days. Seventy-seven percent and 46% of recipients of high- and lower-dose vaccines, respectively, briefly excreted vaccine organisms in their stools. All blood cultures were negative. Antibody-secreting cells producing antilipopolysaccharide (LPS) immunoglobulin A (IgA) were detected in 100 and 92% of recipients of high- and lower-dose vaccines, respectively. Almost half the volunteers developed serum anti-LPS IgG. Lymphocyte proliferation and gamma interferon production against serovar Typhi antigens occurred in a significant proportion of vaccinees. This phase 2 study supports the further development of CVD 908-htrA as a single-dose vaccine against typhoid fever and as a possible live vector for oral delivery of other vaccine antigens.

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 (DeltavirG), does not produce enterotoxin (Deltasen and Deltaset), and has limited proliferation in vivo (DeltaguaBA). 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

Attenuated Salmonella enterica serovar Typhi live vector vaccine strains are highly immunogenic in mice following intranasal but not orogastric inoculation. To elucidate the relationship between organs within which vaccine organisms are found and the induction of specific serum immunoglobulin G (IgG) antibodies, we examined the in vivo distribution of serovar Typhi vaccine strain CVD 908-htrA following intranasal administration. Vaccine organisms were cultured from the nasal lymphoid tissue (NALT), lungs, and Peyer's patches 2 min after intranasal inoculation. Vaccine organisms persisted longer in NALT than in other organs. By decreasing the volume of intranasal inoculum containing 10(9) CFU (from a single 30- or 10-microl dose to four 2.5-microl doses given over the course of 1 h), we were able to significantly reduce the number of vaccine organisms isolated from the lungs (P < 0.05) without reducing the number of vaccine organisms in NALT. Reducing the number of vaccine organisms in the lungs resulted in a significant decrease in the serum tetanus antitoxin response elicited by CVD 908-htrA expressing tetanus toxin fragment C under the control of the redox-responsive nir15 promoter. In contrast, a similar construct expressing tetanus toxin fragment C under control of the constitutive lpp promoter stimulated a strong serum IgG tetanus antitoxin response with both inoculation regimens. The data suggest that following intranasal inoculation, NALT is a sufficient inductive site for elicitation of an immune response against both the live vector and heterologous antigen and, as occurs following oral inoculation of humans, attenuated serovar Typhi vaccine organisms elicit serum IgG 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 (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.

The broad objective of the research presented here is to develop a noncatalytic plasmid maintenance system for the stabilization of multicopy expression plasmids encoding foreign antigens in a Salmonella typhi live-vector vaccine strain such as CVD 908-htrA. We have enhanced the maintenance of expression plasmids at two independent levels. First, we removed dependence upon balanced-lethal maintenance systems that involve catalytic enzymes expressed from multicopy plasmids; we accomplished this through incorporation into expression plasmids of a postsegregational killing system based on the noncatalytic hok-sok plasmid addiction system from the antibiotic resistance factor pR1. We also included at least one naturally occurring plasmid partition function in our expression plasmids, which eliminates random segregation of these plasmids, thereby enhancing their inheritance and stability; to accomplish this, we incorporated either the par locus from pSC101, the parA locus from pR1, or both. We monitored the stability of optimized expression plasmids within CVD 908-htrA by quantitating expression of a variant of green fluorescent protein (GFPuv) by using flow cytometry. In this report, we demonstrate the utility of this novel plasmid maintenance system in enhancing the stability of our expression plasmids and go on to show that as the copy number of stabilized plasmids increases, the toxicity of GFPuv synthesis also increases. The implications of these observations for the rational design of immunogenic and protective bacterial live vector vaccines are discussed.

CVD 103-HgR is a live oral cholera vaccine strain constructed by deleting 94% of the gene for the enzymatically active A subunit of cholera toxin from classical Inaba Vibrio cholerae O1 569B; the strain also contains a mercury resistance gene as an identifying marker. This vaccine was well tolerated and immunogenic in double-blind, controlled studies and was protective in open-label studies of volunteers challenged with V. cholerae O1. A randomized, double-blind, placebo-controlled, multicenter study of vaccine efficacy was designed to test longer-term protection of CVD 103-HgR against moderate and severe El Tor cholera in U.S. volunteers. A total of 85 volunteers (50 at the University of Maryland and 35 at Children's Hospital Medical Center/University of Cincinnati) were recruited for vaccination and challenge with wild-type V. cholerae El Tor Inaba. Volunteers were randomized in a double-blind manner to receive, with buffer, a single oral dose of either CVD 103-HgR (2 x 10(8) to 8 x 10(8) CFU) or placebo (killed E. coli K-12). About 3 months after immunization, 51 of these volunteers were orally challenged with 10(5) CFU of virulent V. cholerae O1 El Tor Inaba strain N16961, prepared from a standardized frozen inoculum. Ninety-one percent of the vaccinees had a >/=4-fold rise in serum vibriocidal antibodies after vaccination. After challenge, 9 (39%) of the 23 placebo recipients and 1 (4%) of the 28 vaccinees had moderate or severe diarrhea (>/=3-liter diarrheal stool) (P < 0.01; protective efficacy, 91%). A total of 21 (91%) of 23 placebo recipients and 5 (18%) of 28 vaccinees had any diarrhea (P < 0.001; protective efficacy, 80%). Peak stool V. cholerae excretion among placebo recipients was 1.1 x 10(7) CFU/g and among vaccinees was 4.9 x 10(2) CFU/g (P < 0.001). This vaccine could therefore be a safe and effective tool to prevent cholera in travelers.

Mutant diphtheria toxin molecule CRM(197) and fragments thereof were expressed in attenuated Salmonella typhi CVD 908-htrA, and the constructs were tested for their ability to induce serum antitoxin. Initially, expressed proteins were insoluble, and the constructs failed to induce neutralizing antitoxin. Soluble CRM(197) was expressed at low levels by utilizing the hemolysin A secretion system from Escherichia coli.

To provide optimum protection against classical and El Tor biotypes of Vibrio cholerae O1, a single-dose, oral cholera vaccine was developed by combining two live, attenuated vaccine strains, CVD 103-HgR (classical, Inaba) and CVD 111 (El Tor, Ogawa). The vaccines were formulated in a double-chamber sachet; one chamber contained lyophilized bacteria, and the other contained buffer. A total of 170 partially-immune American soldiers stationed in Panama received one of the following five formulations: (a) CVD 103-HgR at 10(8) CFU plus CVD 111 at 10(7) CFU, (b) CVD 103-HgR at 10(8) CFU plus CVD 111 at 10(6) CFU, (c) CVD 103-HgR alone at 10(8) CFU, (d) CVD 111 alone at 10(7) CFU, or (e) inactivated Escherichia coli placebo. Among those who received CVD 111 at the high or low dose either alone or in combination with CVD 103-HgR, 8 of 103 had diarrhea, defined as three or more liquid stools. None of the 32 volunteers who received CVD 103-HgR alone or the 35 placebo recipients had diarrhea. CVD 111 was detected in the stools of 46% of the 103 volunteers who received it. About 65% of all persons who received CVD 103-HgR either alone or in combination had a fourfold rise in Inaba vibriocidal titers. The postvaccination geometric mean titers were comparable among groups, ranging from 450 to 550. Ogawa vibriocidal titers were about twice as high in persons who received CVD 111 as in those who received CVD 103-HgR alone (600 versus 300). The addition of CVD 111 improved the overall seroconversion rate and doubled the serum Ogawa vibriocidal titers, suggesting that the combination of an El Tor and a classical cholera strain is desirable. While CVD 111 was previously found to be well tolerated in semiimmune Peruvians, the adverse effects observed in this study indicate that this strain requires further attenuation before it can be safely used in nonimmune populations.

Vibrio choleraeO1 can enter a state in which they remain viable but are non-culturable. Presumably, such bacteria can be pathogenic if they retain the capacity to proliferate in the human intestine following ingestion. Two groups of volunteeers were given inocula containing viable but non-culturable V. choleraeO1 of the attenuated vaccine strain CVD 101 (viable CVD 101 organisms readily colonize the human intestine). Volunteers in one of the two groups excreted viable CVD 101, demonstrating that, in the environment of the human intestine, previously non-culturable vibrios can regain the capacity to multiply. These observations support the proposition that viable but non-culturable bacterial enteropathogens may pose a potential threat to health.

To determine the role of Escherichia coli heat-stable enterotoxin (ST) as a virulence factor in human diarrhea, a strain that elaborates only ST (E. coli 214-4) was fed to free-living volunteers in doses of 10(6), 10(8), and 10(10) organisms. Short-lived (1 day) mild illness consisting of abdominal cramps with vomiting or diarrhea occurred in three of five individuals fed 10(8). Typical travelers' diarrhea (loose stools, abdominal cramps, and low-grade fever for 2 to 3 days) was seen in four of five volunteers given 10(10); two had brief cholera-like purging of rice-water stools. Despite fever, there was no evidence of mucosal invasion. E. coli 214-4 became the predominant coliform in stools; coproculture isolates were uniformly negative for heat-labile enterotoxin (LT), whereas most produced ST. Ten of 13 individuals developed rises in antibody to somatic E. coli antigen, and none had rises in LT antitoxin. E. coli that elaborate only ST can cause diarrheal disease in adults.

In an attempt to evaluate the immunogenicity, infectivity, transmissibility and safety of rhesus rotavirus vaccine (RRV) MMU 18006, 27 infants ages 5 to 20 months participated in two randomized, double-blind placebo controlled trials, one in a day care setting to allow for child to child contact and close surveillance and the other on an outpatient basis. Fourteen infants (mean age, 8.3 months) received 105plaqueforming units of RRV and 13 (mean age, 11.1 months) received placebo. In the eight infants who participated in the vaccine trial in the day care setting, there was no evidence of transmissibility of RRV, by either stool excretion or seroconversion. The data from both trials showed RRV to be 100 infective and immunogenic in the vaccinees. There were no gastrointestinal side effects although there was an association between vaccine administration and fever occurring on Days 3 and 4. Based on these encouraging preliminary results, further work is proceeding to evaluate this vaccine at lower doses in this age group of infants.

In this study, adult volunteers were fed tcpA and mshA deletion mutants of V. cholerae O139 strain CVD 112 to determine the role of toxin-coregulated pili (TCP) and mannose-sensitive hemagglutinin (MSHA) in intestinal colonization. Eight of 10 volunteers who received CVD 112 or CVD 112 delta mshA shed the vaccine strains in their stools; the geometric mean peak excretion for both groups was 1.4 x 10(5) CFU/g of stool. In contrast, only one of nine recipients of CVD 112 delta tcpA shed vibrios in his stool (P < 0.01); during the first 24 h after inoculation, 3 x 10(2) CFU/g was recovered from this volunteer. All recipients of CVD 112 and 8 (80%) of the recipients of CVD 112 delta mshA developed at least a fourfold rise in vibriocidal titer after immunization. In contrast, only one (11%) of the nine recipients of CVD 112 delta tcpA developed a fourfold rise in vibriocidal titer (P < 0.01). We conclude that TCP are an important colonization factor of V. cholerae O139 and probably of El Tor V. cholerae O1. In contrast, MSHA does not appear to promote intestinal colonization in humans.

Two studies of adult volunteers were performed to determine whether prior enteropathogenic Escherichia coli (EPEC) infection confers protective immunity against rechallenge. In the first study, a naive control group and volunteers who had previously ingested an O55:H6 strain were fed an O127:H6 strain. In the second study, a control group and volunteers who had previously ingested either the O127:H6 strain or an isogenic eae deletion mutant of that strain were challenged with the homologous wild-type strain. There was no significant effect of prior infection on the incidence of diarrhea in either study. However, in the homologous-rechallenge study, disease was significantly milder in the group previously challenged with the wild-type strain. Disease severity was inversely correlated with the level of prechallenge serum immunoglobulin G against the O127 lipopolysaccharide. These studies indicate that prior EPEC infection can reduce disease severity upon homologous challenge. Further studies may require the development of new model systems.

To provide optimum protection against classical and El Tor biotypes of Vibrio cholerae O1, a single-dose, oral cholera vaccine was developed by combining two live, attenuated vaccine strains, CVD 103-HgR (classical, Inaba) and CVD 111 (El Tor, Ogawa). The vaccines were formulated in a double-chamber sachet; one chamber contained lyophilized bacteria, and the other contained buffer. In the first study, 23 U.S. adult volunteers received CVD 103-HgR at 10(8) CFU plus CVD 111 at 10(8), 10(7), or 10(6) CFU, CVD 111 alone at 10(7) CFU, or placebo. In the second study, 275 Peruvian adults were randomized to receive CVD 103-HgR at 10(9) CFU plus CVD 111 at 10(9) or 10(8) CFU, CVD 111 alone at 10(9) CFU, CVD 103-HgR alone at 10(9) CFU, or placebo. Three of 15 U.S. volunteers who received CVD 111 at 10(7) or 10(8) CFU developed mild diarrhea, compared to none of 4 who received CVD 111 at 10(6) CFU and 1 of 4 who received placebo. Twelve (63%) of 19 vaccine recipients shed the El Tor vaccine strain. All but one volunteer developed significant Ogawa and Inaba vibriocidal antibody titers. Volunteers who received CVD 111 at 10(7) CFU had geometric mean Ogawa titers four to five times higher than those of volunteers who received the lower dose. In the second study, all dosage regimens were well tolerated in Peruvians. About 20% of volunteers who received CVD 111 at the high dose excreted the El Tor organism, compared to 7% in the low-dose group. CVD 111 was detected in the stools of two placebo recipients, neither of whom had symptoms or seroconverted. In all vaccine groups, 69 to 76% developed fourfold rises in Inaba vibriocidal antibodies. Among those who received the bivalent vaccine, 53 to 75% also developed significant rises in Ogawa vibriocidal antibodies. We conclude that it is feasible to produce a single-dose, oral bivalent vaccine that is safe and immunogenic against both biotypes (El Tor and classical) and both serotypes (Inaba and Ogawa) of cholera for populations in both developed and developing parts of the world.

Attenuated Salmonella typhi organisms which express genes encoding protective antigens of other pathogens have been developed for use as experimental oral vaccines. A delta asd S. typhi strain attenuated by deletions in cya, crp, and cdt which contains hepatitis B core (HBc) and pre-S genes encoded on an Asd+ pBR-based plasmid vector was constructed. Healthy adult volunteers ingested a single dose of 5 x 10(5) to 5 x 10(8) CFU of strain chi4073 (delta cya delta crp delta cdt S. typhi Ty2), 6 x 10(7) or 1 x 10(9) CFU of strain chi4632(pYA3149), a further derivative of chi4073 deleted in asd and containing the Asd+ vector without the HBc-pre-S fusion, or 3 x 10(7) or 7 x 10(8) CFU of strain X4632(pYA3167), a derivative containing the vector with the HBc-pre-S fusion. Chi4073 was generally well tolerated by 22 volunteers. No volunteer had fever or positive blood cultures; 4 of 22 volunteers shed vaccine organisms in the stool in the first 48 h only. Two of 18 volunteers who received one of the plasmid-containing derivatives of chi4073 developed low-grade fevers on day 10 or 12 after ingestion. One of these volunteers had positive blood cultures on days 7 and 8. Seven of these 18 volunteers had vaccine organisms detected in their stools in the first 48 h only. Most volunteers developed S. typhi-specific serum responses and developed S. typhi-specific antibody-secreting cells. However, no volunteer developed serum antibody to hepatitis pre-S or pre-S-specific antibody-secreting cells. Although the parent strain chi4073 was well tolerated, induced immunoglobulin G seroconversion to S. typhi lipopolysaccharide in 80 to 100% of vaccinees and stimulated specific IgA-secreting lymphocytes in 80 to 100% of vaccinees given a single oral dose of 2 x 10(7) and 5 x 10(8) CFU, chi4073 derivatives containing the Asd+ vector with and without sequences encoding the HBc-pre-S fusion caused occasional febrile reactions at high doses and did not stimulate detectable immune responses to hepatitis B antigens.

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.

Enteroaggregative Escherichia coli (EAggEC) are associated with persistent diarrhea in young children. Some of these organisms produce a low-molecular-weight, heat-stable, plasmid-encoded enterotoxin that has been named EAggEC heat-stable enterotoxin 1 (EAST1). We have cloned a 4.4-kb DNA fragment from the virulence plasmid of prototype EAggEC strain 17-2, which expresses enterotoxic activity as measured by electrogenic response in Ussing chambers mounted with rabbit ileal tissue. DNA-sequence analysis of this fragment identified an open reading frame (ORF) encoding a cysteine-rich polypeptide of 38 amino acids (M(r), 4100). Insertional and deletional mutations in this ORF resulted in loss of enterotoxic activity. The ORF was cloned into a T7 expression vector, and postinduction culture filtrates exhibited enterotoxic activity and increased ileal tissue cGMP levels. A synthetic peptide consisting of predicted amino acid residues 8-29 also showed enterotoxic activity. These data indicate that this ORF, named astA (EAggEC heat-stable enterotoxin), represents the EAST1 structural gene. EAST1 shows significant homology with the enterotoxic domain of heat-stable enterotoxin a (STa) of enterotoxigenic E. coli and with guanylin, a mammalian analog of STa. Unlike STa, which requires six cysteines and three disulfide linkages for full biological activity, both EAST1 and guanylin contain four cysteine residues. Based on the cGMP data and the sequence homology to STa and guanylin, it is predicted that EAST1 stimulates the particulate form of guanylate cyclase through the same receptor-binding region as STa and guanylin.

Vibrio cholerae O139 Bengal, although closely related to V. cholerae O1 El Tor, produces a polysaccharide capsule and has a distinct O antigen. We have identified a chromosomal region of at least 11 kb, as defined by three TnphoA mutations, that is required for the expression of both polysaccharides. Electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis show that these TnphoA mutants have lost the abilities both to express capsule and to produce lipopolysaccharide beyond the core oligosaccharide. Reactivity with O139 typing serum and resistance to serum are also lost in the mutants. DNA probes for this region do not hybridize with O1 V. cholerae but do react with other vibrios, implying that the region was recently acquired.

In the course of studies of immunity to experimental cholera in man, 105 or 106 Vibrio cholerae were given to 66 college students and other community volunteers under quarantine in an isolation ward. HLA antigen and blood group determinations were carried out to test the hypothesis that severity of clinical cholera is dependent in part upon genetically-determined host susceptibility. Fifty-five volunteers developed diarrhoea; 38 had mild illness and 17 had severe cholera (stool volume ≥ 5·0 litres). HLA antigens were found in similar frequency in volunteers with severe, mild or no diarrhoea; antigens A1, A2, A3 and B7 were most common. Blood group O, however, was found in 64% of persons with severe cholera versus 36–38% of volunteers with mild or absent illness. Thus, while no correlation was found between HLA type and severity of cholera, these results do support the claims of other investigators that blood group O is found more frequently in patients with severe cholera than in the normal population.

Despite more than a century of study, cholera still presents challenges and surprises to us. Throughout most of the 20th century, cholera was caused by Vibrio cholerae of the O1 serogroup and the disease was largely confined to Asia and Africa. However, the last decade of the 20th century has witnessed two major developments in the history of this disease. In 1991, a massive outbreak of cholera started in South America, the one continent previously untouched by cholera in this century. In 1992, an apparently new pandemic caused by a previously unknown serogroup of V. cholerae (O139) began in India and Bangladesh. The O139 epidemic has been occurring in populations assumed to be largely immune to V. cholerae O1 and has rapidly spread to many countries including the United States. In this review, we discuss all aspects of cholera, including the clinical microbiology, epidemiology, pathogenesis, and clinical features of the disease. Special attention will be paid to the extraordinary advances that have been made in recent years in unravelling the molecular pathogenesis of this infection and in the development of new generations of vaccines to prevent it.

To prepare a field site for evaluating preventive interventions against endemic shigellosis, the authors followed prospectively a cohort of 360 children (90 each of children aged 0-11, 12-23, 24-35, and 36-47 months) in Santa Julia, a low socioeconomic area in Santiago, Chile, from November 1986 through April 1989 with twice weekly household visits for diarrheal disease; infants replaced children who reached 60 months of age. Coprocultures on 2 consecutive days from children with diarrhea and from age-matched controls within the cohort were cultured for Shigella. Bacteriologic surveillance was also maintained in the health center and children's hospital serving Santa Julia. In this community, where all households had access to potable water (68% inside) and all but 3% had access to a toilet, but where there was marked crowding, the overall incidence of diarrheal disease in the cohort was low (2.26 episodes/12 child months of observation in children aged 0-11 months and 2.09 in those aged 12-23 months), yet Shigella infections were common. Shigella accounted for 10% of diarrheal episodes in the cohort (vs. 3.2% isolation rate in controls, p less than 0.0001). The incidence of shigellosis in children aged 12-47 months was 0.16 cases per 12 child months of observation; in the first 5 years of life, a child had a 67% chance of experiencing shigellosis. Shigella sonnei, Shigella flexneri 2a, and S. flexneri 6 caused greater than 79% of the infections. Shigella occurred more often in hospitalized cases of diarrhea than in age-matched cases detected in the health center or by household surveillance (p less than 0.0001). An initial episode of Shigella diarrhea did not diminish overall the risk of subsequent shigellosis but did confer 72% protection (p = 0.05) against illness due to the homologous serotype. The high rate of both S. sonnei and S. flexneri shigellosis in a population with a low background rate of diarrhea makes Santa Julia an appropriate site for assessing the efficacy and effectiveness of measures to reduce Shigella infections.

A functional tcpA gene, encoding the major subunit of toxin-coregulated pili (TCP), is necessary for Vibrio cholerae O1 Ogawa strain 395 to colonize the human intestine and confer protective immunity to virulent challenge. The immunogenicity of TCP and other antigens in experimental and naturally acquired cholera was determined. Seroconversion to cholera toxin (CT), whole cell preparations, and to Ogawa lipopolysaccharide but not to purified native TCP or to a TcpA mimiotope was found in volunteers. Local intestinal secretory immunoglobulin A from volunteers showed conversions to cholera toxin and lipopolysaccharide but not to TCP. Cholera patients in Indonesia showed a seroconversion rate to TCP of 3 of 6 and a seroconversion to a TcpA mimiotope of 1 of 6. Volunteer and patient sera showed similar vibriocidal seroconversions when assayed against either TCP-positive and TCP-negative V. cholerae O1 strains, suggesting that TCP do not contribute demonstrably to the vibriocidal antigen. We conclude that although seroconversion to TCP can occur in naturally acquired cholera, solid long-term protection can be engendered in the absence of a detectable anti-TCP immune response.

Three strains of enteropathogenic Escherichia coli (EPEC), originally isolated from humans and previously shown to cause diarrhea in human volunteers by unknown mechanisms, and one rabbit EPEC strain were shown to attach intimately to and efface microvilli and cytoplasm from intestinal epithelial cells in both the pig and rabbit intestine. The attaching and effacing activities of these EPEC were demonstrable by light microscopic examination of routine histological sections and by transmission electron microscopy. It was suggested that intact colostrum-deprived newborn pigs and ligated intestinal loops in pigs and rabbits may be useful systems to detect EPEC that have attaching and effacing activities and for studying the pathogenesis of such infections. The lesions (attachment and effacement) produced by EPEC in these systems were multifocal, with considerable animal-to-animal variation in response to the same strain of EPEC. The EPEC strains also varied in the frequency and extent of lesion production. For example, three human EPEC strains usually caused extensive lesions in rabbit intestinal loops, whereas two other human EPEC strains usually did not produce lesions in this system.

In a previous study, a seronegative child to whom attenuated A/Alaska/77-ts-1A2 virus was administered (37 degrees C shutoff temperature for plaque formation) shed virus with an altered temperature-sensitive (ts) phenotype (40 degrees C shutoff temperature) (Murphy et al., Ann. N.Y. Acad. Sci. 354:172-182, 1980; Tolpin et al., Virology 112:505-517, 1981). This ts+ virus (FV1319) was evaluated for its level of replication in hamsters and for its virulence for humans. In hamsters, FV1319 ts+ virus replicated to the same level in the nasal turbinates as that of which the A/Alaska/77 wild-type virus replicated, but its replication in the lungs was reduced 40-fold. In contrast, the A/Alaska/77-ts-1A2 reassortant achieved a titer in hamster nasal turbinates that was significantly lower (P less than 0.005) than those achieved by the wild-type and the FV1319 viruses; the A/Alaska/77-ts-1A2 reassortant was not recoverable from the lungs. In seronegative adult volunteers, the pattern of replication of the FV1319 virus was similar to that of the A/Alaska/77 wild-type virus. The illness induced by the FV1319 ts+ virus was also similar to that caused by the wild-type virus. In contrast, the A/Alaska/77-ts-1A2 reassortant was satisfactorily attenuated in adult volunteers. These results suggest that attenuation of the A/Alaska/77-ts-1A2 reassortant virus in humans is a function of the ts phenotype: loss of this phenotype restored virulence. The ability of the A/Alaska/77-ts-1A2 reassortant to lose its ts phenotype and regain virulence during growth in a permissive host limits the usefulness of the ts-1A2 reassortants as vaccine viruses for humans.

The immune response of 114 volunteers with diarrhea after experimental challenge with four strains of Vibrio cholerae O1 was characterized in a microtiter enzyme-linked immunosorbent assay antibody detection system by using a partially purified outer membrane preparation (OMP) from these strains as an antigen. Analysis of paired sera from 29 persons with noncholera diarrhea (negative control population), demonstrated that a rise in net optical density greater than 0.10 was significant. A total of 50% of the 79 cholera volunteers challenged with El Tor biotype and 54% of the 35 volunteers challenged with classical biotype had significant rises in immunoglobulin G anti-OMP. Paired sera that showed significant rises when tested against homologous OMP all manifested significant rises when also tested against a serotype-heterologous OMP. Immunoblotting techniques showed that the antigens to which antibodies were reacting were mainly protein in nature, not lipopolysaccharide. Furthermore, absorption with lipopolysaccharide decreased the optical density by a mean of only 12% (0 to 30%), corroborating that antibody was mainly directed against OMP and not lipopolysaccharide. This study indicates that there is a human immunoglobulin G response to OMP during clinical cholera infection and that this response is constant among bio- and serotypes.

Enterotoxigenic Escherichia coli (ETEC) of serotype O6:H16, biotype A, bearing colonization factor antigen II (CFA/II) possesses two distinct coli surface antigens, CS1 and CS3, whereas CFA/II-positive ETEC of serotype O8:H9 manifests only CS3. CS1 has been shown to be fimbrial in nature, but heretofore the morphology of CS3 has not been described. Accordingly, by immune electron microscopy we investigated the morphological characteristics of CS3 on bacterial cells and after purification. CS3 was found to consist of thin (2-nm), flexible, wiry, "fibrillar" fimbriae, visible both on bacteria (O6:H16, biotype A, and O8:H9 strains) and in the pure state. In contrast, CS1 exists as wider (6-nm), rigid fimbriae on the surface of O6:H16, biotype A, strains. By the use of antisera to CS1 and CS3 in immune electron microscopy, immunodiffusion in gel, and immunoblotting techniques, CS1 and CS3 were found to be immunologically as well as morphologically distinct. Six of nine volunteers who developed diarrhea after challenge with an O139:H28 ETEC strain bearing CS1 and CS3 had significant serological rises to purified CS1 and CS3 antigens, suggesting that both antigens are elaborated in vivo, play a role in pathogenesis, and stimulate an immune response.

Although serum vibriocidal activity is used extensively as a marker of immunity to O1 Vibrio cholerae, there are limitations in this assay to detect instances of reexposure. We define the conditions operative in producing secondary vibriocidal responses in North American volunteers primed with either wild-type V. cholerae 1, 4, or 6 months later. Secondary serum vibriocidal responses occurred under two distinct secondary challenge conditions. The first occurred when secondary challenge produced a breakthrough in clinical protection. Following secondary exposure, 14 of 22 (64%) and 1 of 29 (3%) subjects with and without vibrio stool excretion, respectively, had secondary responses (P < 0.001); 5 of 6 (83%) and 10 of 45 (22%) subjects with or without diarrhea, respectively, mounted a secondary response (P = 0.006). The second condition occurred in the presence of full clinical protection but was dependent on the time interval between exposure. No subject (0 to 17) vaccinated with CVD 103-HgR and given homologous wild-type challenge within 4 months mounted a secondary vibriocidal response (P = 0.0009). The majority of the serum vibriocidal activity was of the immunoglobulin M (IgM) isotype, seen in 96 and 73% of subjects following primary and secondary exposure, respectively. Vibriocidal activity in the IgG fraction following primary and secondary exposures occurred with < or = 50% of volunteers; lipopolysaccharide (LPS)-specific IgG1 and IgG3 subclass responses supported the vibriocidal isotype data. However, following primary exposure, IgG4 LPS responses predominated, occurring in 81% of responding volunteers. These data suggest that, under certain conditions of secondary exposure to V. cholerae O1 antigens, when there is sufficient active local immunity present, there is a block of vibrio antigen resampling at the M cell level. We discuss the implications of and possible explanations for these findings.

The safety and immunogenicity of the live oral attenuated vaccine strains vibrio cholerae CVD 103-HgR and Salmonella typhi Ty21a were evaluated alone or in a combined bivalent formulation in four groups composed of 185 healthy European adults. All presentations were well tolerated. The serum anti-S. typhi lipopolysaccharide immunoglobulin G and immunoglobulin A antibody responses were comparable for all groups (66 to 72% seroconversion). The serum vibriocidal antibody seroconversion rate ranged from 78 to 92.5% (P > 0.05) among the groups. However, the peak and geometric mean vibriocidal antibody titers were significantly higher (P < 0.005) in the groups which received the bivalent formulation along with two doses of Ty21a than in the group which received CVD 103-HgR followed by two doses of killed Escherichia coli K-12 placebo. The ingestion of a placebo shortly after CVD 103-HgR may have suppressed the magnitude of the immune response. These findings demonstrate the feasibility of producing multivalent live oral attenuated vaccines.

The emergence of a new agent of cholera, Vibrio cholerae O139, has prompted a reevaluation of the vibriocidal antibody assay. This assay, primarily directed to lipopolysaccharide, is an important correlate of O1 immunity. V. cholerae O139 strains are encapsulated, rendering them relatively resistant to killing by serum. Recent reports suggest that there is strain-to-strain variability in the sensitivity of the vibriocidal assay to fully encapsulated O139 strains. We have assessed a modified vibriocidal assay for fully encapsulated O139 strain AI-1837 and its unencapsulated mutant 2L in sera from 53 volunteers given wild-type AI-1837 or its attenuated derivative CVD 112 and from 48 controls challenged with V. cholerae O1 or strains of the family Enterobacteriaceae. Vibriocidal responses to the AI-1837 and 2L strains were seen in 67 and 89% of volunteers, respectively, following a single exposure to the wild-type strain. However, >50% of all controls had low-level vibriocidal responses to both strains. These nonspecific responses were transient and of the immunoglobulin G isotype. No binding activity against purified O139 lipopolysaccharide (LPS) by immunoblotting was seen in control sera. In contrast, vibriocidal assay and strain 2L LPS responses by immunoblotting were detectable in 91% of tested volunteers following a single exposure to O139. The presence of vibriocidal antibody to AI-1837 or 2L was not associated with protection in rechallenge studies with O139 strain AI-1837. The vibriocidal assay with unencapsulated strain 2L may be used to detect exposure to O139 strain AI-1837 in controlled research trials. However, its lack of specificity does not make it useful for determining exposure to V. cholerae O139 in the field.

The potential for reacquisition of ctxA genes by attenuated Vibrio cholerae O1 vaccine strain CVD 103-HgR was examined by performing a series of mating experiments under a variety of in vivo and in vitro conditions. We found no evidence that CVD 103-HgR could reacquire ctxA genes from wild-type V. cholerae O1 strains. However, if the donor V. cholerae O1 strains were genetically manipulated to add genes that allow chromosomal gene transfer, then ctxA sequences could be acquired by CVD 103-HgR. The minimal excretion of CVD 103-HgR by vaccinees and the refractoriness to reacquisition of ctxA sequences suggest that this well-tolerated, highly immunogenic live oral cholera vaccine will have a minimal environmental impact.

Peripheral blood immunoglobulin A antibody-secreting-cell (ASC) responses are thought to reflect the mucosal immune response to locally presented antigens. We evaluated the ASC response to cholera toxin (CT) and Inaba lipopolysaccharide (LPS) in 26 North American volunteers following immunization with a single oral dose of live attenuated Vibrio cholerae O1 vaccine strain CVD 103-HgR and again upon homologous wild-type challenge with V. cholerae classical Inaba 569B. Challenge occurred at either 7, 30, or 180 days after vaccination. The CT and LPS ASC responses of volunteers following vaccination (83 and 55%, respectively) were similar in magnitude and frequency to those of unvaccinated controls following wild-type challenge (80 and 60%, respectively [0.1 < or = P < or = 0.9]). The responses were primarily immunoglobulin A. Vaccinated volunteers challenged within 30 days of vaccination had reduced or nondetectable CT and LPS ASC responses. Challenge at 6 months resulted in a heightened ASC response to LPS, confirming the existence of mucosal memory. ASC responses to CT upon challenge at 6 months were detectable but not different from that seen following primary immunization, suggesting that secondary ASC responses to different antigens from a single vaccine operate independently. In spite of these variable ASC responses, the vaccine efficacy was 100% following challenge for all vaccinees. V. cholerae-specific ASC responses following antigenic reexposure gave information on the presence of mucosal B memory cells but did not correlate with protective immunity. As such, these ASC assays will have limited usefulness for evaluating vaccine responders in vaccine field trials in cholera-endemic areas where prior V. cholerae O1 exposure is unknown.

Two strains of enteroaggregative Escherichia coli of human origin fed to gnotobiotic piglets caused diarrhea or death in the majority of them. Histological examination revealed moderate hyperemia of the distal small intestine and cecum, swelling of small intestinal villi, and layers of aggregated bacteria stacked together in a mucus gel-like matrix overlying intact epithelium. These findings confirm that enteroaggregative E. coli strains produce distinctive intestinal lesions different from those caused by other major categories of diarrheagenic E. coli.

Three attenuated Salmonella typhi strains have been constructed by introducing deletions in aroC and aroD or deletions in cya and crp into one of two wild-type parent strains, Ty2 or ISP1820. These mutant strains were designated CVD 906 (ISP1820 delta aroC delta aroD), CVD 908 (Ty2 delta aroC delta aroD), and chi 3927 (Ty2 delta cya delta crp). Two studies were conducted with 36 healthy adult inpatient volunteers to determine in a double-blind fashion the safety and immunogenicity of approximately 5 x 10(4) and 5 x 10(5) CFU of each of these three vaccine candidates given as a single dose. No statistically significant difference in the incidence of reactions among vaccinees was observed. Fever (oral temperature greater than or equal to 38.2 degrees C) occurred in 2 of 12 volunteers who received CVD 906, in 0 of 12 who received CVD 908, and in 1 of 12 who received chi 3927. Vaccine bacteremia without symptoms occurred in 1 of 12 vaccinees who received CVD 906, in 0 of 12 who received CVD 908, and in 2 of 12 who received chi 3927. Overall, 19 (53%) of 36 vaccinees developed immunoglobulin G antibody to S. typhi lipopolysaccharide after vaccination, with no statistically significant differences in the rate of seroconversion among volunteers in the three groups. We conclude that defined mutations in the aromatic biosynthetic pathway and in the cyclic AMP global regulatory system attenuate S. typhi. Mutant strains CVD 906, CVD 908, and chi 3927 are highly (and approximately equally) immunogenic but possibly differ in their propensity to induce fever. Further studies are needed to document the apparent relative safety of CVD 908 as a typhoid vaccine and as a vaccine carrier of foreign antigens.

A live oral vaccine consisting of attenuated Salmonella typhi Ty21a expressing Vibrio cholerae O1 Inaba lipopolysaccharide (LPS) O antigen was constructed and tested in volunteers for safety, immunogenicity, and efficacy. Fourteen adults ingested three doses of 10(10) viable organisms with buffer. One month later, 8 vaccinees and 13 unimmunized controls were challenged with 10(6) pathogenic V. cholerae O1 E1 T or Inaba organisms. No significant adverse reactions to vaccination were observed. All volunteers had significant rises in serum immunoglobulin G (IgG) antibody to S. typhi LPS. Only 2 (14%) of 14 had significant rises in serum IgA or IgG antibody to Inaba LPS, and 5 (36%) of 14 had fourfold rises in vibriocidal antibody. In the challenge study, diarrhea occurred in 13 of 13 controls and 6 of 8 vaccinees (vaccine efficacy, 25%; P = 0.13). The vaccine significantly reduced the severity of the clinical illness (P less than 0.05) and caused decreased excretion of challenge vibrios (P less than 0.05). Although the typhoid-cholera hybrid vaccine did not provide significant protection overall against experimental cholera, this study demonstrates the importance of antibody to V. cholerae O antigen in ameliorating clinical illness and illustrates the use of an S. typhi carrier vaccine strain expressing a foreign antigen.

To address potential concerns over the release of genetically engineered live bacterial vaccines, we constructed a recombination-deficient derivative of the Vibrio cholerae O1 vaccine strain CVD103 (CVD103RM). Oral immunization of adult volunteers with CVD103RM showed that the recA mutation significantly diminished colonization ability and immunogenicity of the vaccine strain.

The serum immunoglobulin G (IgG) and mucosal secretory IgA (SIgA) response of human volunteers challenged with Vibrio cholerae O1 was analyzed for reactivity to V. cholerae O1 antigens by the immunoblot technique. Components of both in vitro- and in vivo (rabbit ligated ileal loop)-grown V. cholerae O1 were separated by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. Postchallenge serum IgG reacted uniquely with 15 antigens and with greater intensity than did prechallenge serum with at least 16 antigens. Serum IgG and SIgA reacted with antigens present in preparations from the homologous challenge strain of V. cholerae as well as antigens from strains of heterologous biotype or serotype. These heterologous antigens may represent antigens responsible for protection to rechallenge with a heterologous strain of V. cholerae. All the antigens detected by postchallenge jejunal fluid SIgA had an apparent molecular size of less than 25 kilodaltons. Serum IgG and jejunal fluid SIgA also reacted with antigens unique to in vivo-grown cells and several antigens in outer membrane preparations, suggesting that studies of protective immunity and V. cholerae O1 pathogenesis should include examination of both in vitro- and in vivo-grown V. cholerae O1 cellular antigens.

PCF8775 is a putative colonization factor complex present on the surface of 10 to 20% of enterotoxigenic Escherichia coli strains and has been reported to be composed of antigen CS6 (morphology undefined) expressed alone or together with either of the rigid fimbrial antigens CS4 and CS5. To better define the individual components of this complex and the determinants of their expression, we prepared antiserum to the PCF8775 complex as it was expressed on prototype strain E8775 and then used the antiserum to identify the subunit structure of the antigens, to study their morphology, and to detect expression of individual components of the complex after transfer of plasmids into laboratory strain HB101. CS4 was purified from strain E8775, confirmed to be fimbrial by electron microscopy, and found to be composed of a 22-kilodalton protein subunit whose N-terminal amino acid sequence (1 to 20) was similar to that of colonization factor antigen I. Transconjugants that express CS6 but not CS4 were obtained by mating prototype strain E8775 with HB101. CS6 expression was mediated by a 61-megadalton plasmid. Expression of CS6 in the transconjugants correlates with expression of a 16-kilodalton cell surface protein. The CS6 antigen was confirmed to be present on the cell surface by immunogold labeling, but its morphology was beyond the limits of resolution by electron microscopy.

Vibrio cholerae O1 A-B- vaccine strain JBK 70 and A-B+ CVD 101 prepared by recombinant DNA techniques from pathogenic EI Tor Inaba N16961 and classical Ogawa 395, respectively, were fed to 38 volunteers in single doses of 10(4) to 10(10). Although severe diarrhea did not occur in any vaccine, more than one-half developed mild diarrhea. These attenuated strains colonized well and elicited prominent vibriocidal and antitoxic (CVD 101) antibody responses. Recipients of a single dose of JBK 70 were significantly protected when challenged with 10(6) wild-type N16961. Diarrhea occurred in 7 of 8 controls but in only 1 of 10 vaccines (P less than 0.003, 89% vaccine efficacy), demonstrating the potency of immune mechanisms that do not involve cholera antitoxin. Further derivatives were prepared to explore the pathogenesis of the residual diarrhea, considering that either intestinal colonization by the vaccine itself or accessory toxins might be responsible. CVD 102, an auxotrophic mutant of CVD 101, did not cause diarrhea but colonized poorly and elicited feeble immune responses. Derivatives of JBK 70 and CVD 101 (CVD 104 and 105) deleted of genes encoding the EI Tor hemolysin still caused mild diarrhea. Genetically engineered strains can be colonizing, highly immunogenic, and protective single-dose oral vaccines, but they must be further attenuated before they can be considered for use as public health tools.

Enterohemorrhagic Escherichia coli (EHEC) of serotype O157:H7 has two putative virulence factors: (i) a fimbrial adhesin, specified by a 60-megadalton (MDa) plasmid, and (ii) bacteriophage-specified cytotoxin(s), known as Shiga-like toxin (SLT) or verotoxin. The contribution of these factors to the pathogenesis of EHEC-induced disease in gnotobiotic piglets was examined. The bacterial strains included the following: two EHEC strains and their corresponding plasmid-cured derivatives; another EHEC isolate and its derivative which had spontaneously lost the ability to produce SLT; one E. coli K-12 transconjugatant containing a 60-MDa plasmid from an EHEC strain; two K-12 strains into which an SLT-producing phage had been transduced (one of these strains also carried a 60-MDa EHEC-derived plasmid); and the parent K-12 strain. Each strain was fed to four piglets, which were observed for diarrhea and examined for development of characteristic mucosal lesions 3 or 5 days after inoculation. All 24 piglets inoculated with the three EHEC strains and their respective derivatives (two plasmid cured and one SLT negative) showed the typical mucosal lesions of bacterial attachment: effacement of microvillous border and cell membrane dissolution culminating in destruction of surface and glandular epithelium in the cecum and colon. No such lesions were observed in 12 piglets inoculated with three strains of E. coli K-12, including the strain which carried both the 60-MDa plasmid and a phage which specified production of SLT. Moderate to severe diarrhea was observed in 16 piglets inoculated with two EHEC strains and their derivatives (one plasmid cured and one SLT negative). The third EHEC strain and its plasmid-cured derivative produced fewer typical mucosal lesions and no diarrhea. The reason for the reduced virulence of this strain was not clear. These results demonstrate that neither the 60-MDa plasmid nor the capacity to produce SLT is essential for expression of virulence by E. coli O157:H7 in gnotobiotic piglets.

Ammonium sulfate-precipitated supernatants of classical enteropathogenic Escherichia coli strains were negative when investigated for enterotoxin production in rabbit ligated ileal loops, rabbit skin vascular permeability factor tests, suckling mice, and Y-1 adrenal cells. They also failed to stimulate guanylate cyclase activity in homogenates of rabbit, rat, and infant mouse intestines. Furthermore, DNA from enteropathogenic E. coli lacked sequences that encode heat-labile and heat-stable enterotoxins. These studies fail to show conventional enterotoxin synthesis by classical enteropathogenic E. coli.

The influenza A/Ann Arbor/6/60 (H2N2) cold-adapted (ca) virus was evaluated as a donor of attenuating genes to new variants of influenza A virus. This ca donor virus was mated with the A/Alaska/6/77 (H3N2) wild-type virus, and three A/Alaska/6/77 (H3N2) ca recombinant viruses were produced. The parental origin of the genes in the three ca recombinants had been determined previously (2), and their virulence for adult seronegative volunteers was assessed in the present study to identify the genes present in the ca donor virus that confer attenuation. Each of the recombinants received the hemagglutinin and neuraminidase genes from the A/Alaska/6/77 (H3N2) wild-type parent. One ca recombinant (CR-29) received all six transferable genes from the ca parent and was found to be satisfactorily attenuated in the volunteers. The two other ca recombinants received five of the six transferable genes with a wild-type gene at the M or NS locus. The pattern of infection in humans with these latter two ca recombinants was similar to the CR-29 ca recombinant. These findings demonstrate that inheritance of a gene in ca recombinants at the M or NS locus segregates independently of attenuation and suggest that the M and NS genes present in the ca donor virus are not the major determinants of attenuation conferred by this virus.