Protein and Polysaccharide Conjugate Vaccines to enteric diseases

革兰氏阴性病原菌的表面多糖，即囊性脂多糖（LPS），是必不可少的毒力因子和保护性抗原。多糖的免疫原性通过与载体蛋白的共价结合而增强。为了作为疫苗，必须将LPS解毒，并将其o -特异性多糖（O-SP）分离并结合到蛋白质上。细菌毒素或类毒素和病毒衣壳蛋白可能是保护性抗原并作为载体蛋白。

Surface polysaccharides of Gram-negative pathogens, capsules or lipopolysaccharides (LPS), are essential virulence factors and protective antigens. The immunogenicity of polysaccharides is enhanced by covalent binding to carrier proteins. To serve as vaccines, LPS must be detoxified and their O-specific polysaccharides (O-SP) isolated and conjugated to proteins. Bacterial toxins or toxoids and viral capsid proteins may be protective antigens and serve as carrier proteins. The Vi capsular polysaccharide of Salmonella typhi (Vi) is a licensed vaccine with limited efficacy in children less than 5 years old. To provide a vaccine for younger children Vi was conjugated to recombinant exoprotein A of Pseudomonas aeruginosa (rEPA). A phase 3 trial of Vi-rEPA in 11,600 Vietnamese 2-to-5-year olds showed an efficacy of 89% at 47 months. In a phase 2 trial, 301 infants were injected with Vi-rEPA concurrently with DPT at 2, 4, 6 and 12 months. Controls received Hib-TT +DTP or DTP. There were no serious adverse reactions. IgG anti-Vi levels will be assayed and compared with those elicited in 2-to-5 year old children. Antibody persistence after vaccination with Vi-rEPA was evaluated in adults and in children. The G.M. IgG anti-Vi level of 18 adults 10 years after a single injection was 20 fold higher than the proposed protective level (3.52 EU). The G.M. antibody level of 97 children injected twice at 2-5 years of age 9 years later was only 2.2 fold higher than the proposed protective level (7.62 EU vs 3.52 EU; P<0.01). There was no significant difference between Vi antibody levels in this group and in those that had only one injection (7.04 EU) or no injection (8.14 EU; 8.14 vs 7.62 or 7.04, P=NS), reflecting waning of anti-Vi in young children on one hand and the development of natural antibodies on the other. Four years after a single injection into 5-to-8-year olds the G.M antibody level was 15.9 EU, significantly higher than of age-matched controls. Salmonella paratyphi A (SPA) is the second most common cause of enteric fever in developing countries. Our phase 1 and 2 studies showed that SPA O-SP conjugated to TT was safe and immunogenic in adults, teenagers, and toddlers. To improve conjugate immunogenicity, SPA LPS chain length elongation regulator wzz gene was replaced with that of E. coli K12 (from D. Kopecko). Several TT conjugates of this elongated O-SP elicited significantly higher levels of anti-LPS than those prepared with O-SP from the wild type. Enterotoxigenic E. coli (ETEC) are the most common cause of diarrhea in developing countries and in travelers to these areas. ETEC disease is mediated by two exotoxins: heat-labile toxin (LT) and heat stable toxin (ST). LT is immunogenic in humans. ST is a polypeptide of 19 amino acids; not immunogenic due to its small size. Their efficacy against infection has not been demonstrated in humans. A mutant of reduced toxicity, LT192 (from J. Clements) elicited high levels of IgG anti-LT in mice. However, there was a dosage dependent swelling at the injection site even after formalin detoxification. This adverse reaction was reduced when a double mutant LT (mutation at amino acids 192 and 211) was used as an immunogen. The wild type LT and the double mutant elicited similar IgG anti-LT levels in mice. Clinical studies are planed. Due to the high number of disulfide bonds (3 pairs), several attempts of chemical synthesis of ST have failed. In collaboration with Donald Robertson, ST was purified from E. coli. A conjugate of ST linked to bovine serum albumin was prepared and its immunogenicity will be evaluated in mice. Vibrio cholerae O1 remains a major health problem in the Indian subcontinent and in Africa. Field studies showed that serum vibriocidal activity is directed toward its LPS. In a phase 1 trial, O-SP conjugates elicited IgG anti-LPS with vibriocidal activity. To enhance its immunogenicity, a new scheme of conjugation was devised by thiolating both the carrier protein and the O-SP with heterobifunctional reagents. Antibody levels induced in mice by these conjugates were significantly higher than of those induced by the conjugate used in our phase 1 study. We also synthesized a hexamer of V. cholerae O-SP. Conjugated to TT, the hexamer was antigenic. The immunogenicity of the new bacterial O-SP conjugates and of the synthetic saccharide conjugates will be evaluated. E. coli O157 is a major cause of hemolytic uremic syndrome, especially in young children. An O157 O-SP-rEPA conjugate was injected into 55 2-to-5 years old children once or twice. The vaccine was safe and immunogenic; 6 months after injection, 98% of the children had a greater than 4 fold increase of IgG anti-LPS over their levels before immunization. The second injection did not induce a booster response. A proposal to immunize children between 2-to-10 years old in states having the highest incidence rates of E. coli O157 infection was planed in collaboration with Patricia Griffin of the Division of Enteric Disease Epidemiology, CDC and Michael Osterholm of the Center for Infectious Diseases, University of Minnesota. Rotavirus is the most common cause of infantile diarrhea worldwide. Two rotavirus vaccines are licensed, a live attenuated and a reassortant, both orally administered. We are designing a parenteral vaccine based on capsid proteins VP8 and VP7. VP8 was purified from bacular-virus infected insect cells (from T. Hoshino) and mice injected produced neutralizing antibodies against rotavirus of both homologous (P4) and heterologous (P8) serotypes. To increase the yield of VP8, its cDNA was expressed in E. coli. The recombinant VP8 elicited neutralizing antibodies to both P4 and P8 rotavirus.