Surface polysaccharides of Gram-negative enteric pathogens, capsules or lipopolysaccharides, are both essential virulence factors and protective antigens. The immunogenicity of these polysaccharides is enhanced by binding to carrier proteins. SALMONELLA TYPHI: Sequential clinical studies in adults and in children in Vietnam, an area with a high attack rate of typhoid, showed that vaccines of the capsular polysaccharide (Vi) conjugated to the recombinant Pseudomonas aeruginosa exoprotein A (rEPA) were safe and elicited high serum IgG antibody levels to the Vi. A Phase 3 trial of the conjugate vaccine in about 12,000 2-5 year old children showed an efficacy of Vi-rEPA of 89% after 48 months of surveillance. A minimal protective level of serum IgG anti-Vi was estimated to be 3.7 ELISA units. A dosage study in Northern Vietnam conducted in children 2-5 years old showed the 25 mcg dose elicited the highest response, followed by the 12.5 mcg dose. There was no statistical difference between the two dosages. However, children injected with a 5 mcg dose had significantly lower Vi antibody levels than those who received the higher dosages. A clinical trial of Vi-rEPA injected with DTP in Vietnam has been approved and will commence soon. SALMONELLA PARATYPHI A: is the second most common cause of enteric fever in Southeast Asia. A conjugate vaccine of the O-specific (O-SP) polysaccharide of S. paratyphi A and rEPA was found to be safe and immunogenic in adults, teenagers, and 2-4 year old children. The O-acetyl content of the PS was found to be related directly to the immunogenicity of the conjugate. In southeast China, cases of paratyphi A have exceeded those of typhi. A phase 3 clinical trial is planned in China with S. paratyphi A O-SP-rEPA conjugate vaccine manufactured by the Chinese under NIH guidance. ESCHERICHIA COLI O157: is an emerging pathogen causing hemorrhagic colitis and hemolytic uremic syndrome especially in young children. A Phase 1 study of E. coli O157 O-specific polysaccharide-rEPA conjugate demonstrated safety and immunogenicity in adult volunteers. In a phase 2 study of this conjugate, 55 children 2-5 years old were injected once or twice. The vaccine was found to be safe and Immunogenetic; 98% of children responded with a greater than 4 fold rise of IgG anti-LPS at 6 months after the first injection. No booster response was found following a second dose 6 weeks after the first one. Genetically detoxified Shiga toxin I and II have been purified from a mutant E. coli O157 and will be conjugated to the O-SP to make a bivalent vaccine. As the major reservoir of E. coli O157 is cattle, an LPS-protein conjugate vaccine was tested in cattle and found to be safe and immunogenic. A challenge study showed an inverse correlation between the serum IgG anti-LPS level and the degree of colonization with E. coli O157. ESCHERICHIA COLI -ENTEROTOXIGENIC ESCHERICHIA COLI: E. coli that secrete cholera-like toxin (ETEC) are a cause of diarrhea in most countries. These illnesses are commonly known as Traveler?s Diarrhea but their incidence, morbidity, and mortality is highest in developing countries. These ETEC are distributed in only few of the many O serotypes of this Genus. ETEC may secrete an exotoxin similar in structure and action to cholera toxin and known as the heat-labile toxin (LT) or a polypeptide of 19 amino acids called the stable toxin (ST). About 40% of ETEC in strains from patients secrete LT alone, 20% secrete both LT and ST, and 60% secrete ST alone. Although there is good information from animal models, scientists have not studied either the immunogenicity or protective actions of LT administered parenterally. Conclusions about the protective activity of LT antibodies have been drawn from animals injected with LT admixed with Freund?s adjuvants, from humans infected with ETEC, or from clinical trials with cross-reactive cholera toxin. The limitations of these approaches to evaluating the usefulness of LT as a vaccine are well-known. Purified LT and a recombinant mutant non-toxic protein (rLT) have been obtained from John Clements, Tulane University. The mutant LT, treated with low concentrations of formaldehyde as is done for diphtheria and pertussis toxin cross-reacting mutants, was injected into young out bred mice and evaluated for its immunogenicity. Treatment of the LT and the rLT established that the latter, treated with formaldehyde, was the most immunogenic material. The effect of adsorbing these proteins on to aluminium hydroxide is now under study. We plan to modify the disulfide bonds of a synthetic ST to reduce toxicity and improve solubility. Studies are underway to develop conjugates of ST with LT or rLT to improve immunogenicity. Our overall objective is to study the clinical effect of parenteral administration of LT and a conjugate of LT with ST for prevention of Traveler's diarrhea. VIBRIO CHOLERA O1 AND O139 are the major serotypes found in cholera infections. Conjugates synthesized with the capsular polysaccharide of O139 and the O-SP of LPS of O1 elicited vibriocidal antibodies in mice. Detoxified LPS from both O1 Inaba and Ogawa serotypes have been used. In mice the conjugates elicited higher antibody levels than the LPS alone. Synthesized O-SP of Ogawa will be prepared and conjugated to a protein carrier. The immunogenicity of cholera vaccines prepared with native or chemically synthesized O-SP will be compared in animal studies. V. cholera O139, similar to O1, secrets cholera toxin and causes sporadic outbreaks in Southeast Asia. Unlike serotype O1, type O139 contains a capsule. We found that convalescent human sera contained high levels of vibriocidal IgM antibodies to the capsule. IgM secretion is usually short-lived and it does not penetrate the gut lumen to lyse the organisms. We conjugated V. cholera O139 CPS with diphtheria mutant toxin, H21G. In mice this conjugate showed high levels of vibriocidal IgG antibodies. A phase 1 study of monovalent conjugate vaccines and a trivalent vaccine of these 3 major V. cholera strains is planned. SALMONELLA TYPHIMURIUM causes most salmonella infections in developed countries. Antibiotic resistant strains are common among disease isolates. We compared the O-specific polysaccharide structures of two of the most common and antibiotic resistant strains. The O-acetyl group on the abequose was found to be a major antigenic site and an immunodominant determinant. Strains that are O-acetyl positive reacted poorly with antibodies from O-acetyl negative strains. Selection of strains for a vaccine will therefore rely on an epidemiologic survey and bacteriologic identification. CAMPYLOBACTER JEJUNI: infection is one of the most common enteric infections around the world, and one of the most common types in the U.S. is type HS:2. Campylobacter is microaerophilic and fermentation in liquid media has been difficult. Various media and fermentation conditions were studied and requirements for growth in liquid media were established. SDS-PAGE silver staining of its purified LPS indicated it was a lipooligosaccharide (LOS). Chemical analysis of the O-SP showed keto groups and no sialic acid. Conjugates prepared with LOS or with de-acylated LOS were immunogenic and antibodies elicited by these vaccines showed bactericidal activity in the presence of complement. THE EFFECT OF LOW BIRTH WEIGHT ON THE IMMUNE RESPONSE IN LATE ADULTHOOD: was studied using Vi as a probe. Substantial evidence links small size at birth (not due to prematurity) to susceptibility in later life to chronic disease. Some components of the immune function may be programmed in early life. We investigated the association between birth size and response to vaccination with Vi-rEPA in a cohort of 257 adults (mean age: 29.4 y; 146 men) born in an urban slum in Lahore, Pakistan, during 1964?1978.
