Protein and Polysaccharide Conjugate Vaccines to Enteric Diseases

Surface polysaccharides of Gram-negative pathogens, capsules or lipopolysaccharides, are essential virulence factors and protective antigens. Lipopolysaccharides need to be detoxified and their O-specific polysaccharides (O-SP) isolated. Immunogenicity of polysaccharides can be improved by binding to carrier proteins. Bacterial toxins or toxoids and viral capsid proteins may be protective antigens and can also serve as carrier proteins.Salmonella typhi. (Co-principal Investigator, Feng-Ying Lin). The Vi capsular polysaccharide of S. typhi is a licensed typhoid vaccine.<P> To improve its immunogenicity in young children, Vi was conjugated to a recombinant exoprotein A of Pseudomonas aeruginosa (rEPA). A phase 3 trial of the Vi-rEPA in 11,600 Vietnamese 2-5-year olds showed an efficacy of 89% at 47 months. A study of various dosages for 2-to-5-year olds showed a higher dosage was more immunogenic. A phase 2 trial in 301 infants started in 2006. Vi-rEPA is injected concurrently with DPT at 2, 4, 6 and 12 months. Controls receive Hib-TT +DTP or DTP. IgG anti-Vi levels will be compared with those elicited in the phase 3 trial.A 10 years follow up of adults injected once with Vi-rEPA in the phase 1 study, showed a G.M. IgG anti-Vi level20 fold higher than the proposed protective level (P<0.0001). <P> The duration of serum anti-Vi IgG in children immunized with Vi-rEPA 8 years ago is under analysis and will be compared with levels 4 years ago and of those of unimmunized controls in another village.<P> Salmonella paratyphi A (SPA) is the second most common cause of enteric fever in developing countries. In our phase 1 and 2 studies, S. paratyphi A O-SP conjugated to TT was safe and immunogenic in adults, teenagers, and toddlers. A mutant S. paratyphi A was constructed by replacing the LPS elongation chain length regulator wzz gene with that of E. coli K12. A conjugate vaccine of this elongated O-SP was prepared and its immunogenicity will be evaluated in mice.E. coli O157 is a major cause of hemolytic uremic syndrome, especially in young children. In our phase 2 trial of O-SP-rEPA conjugate in 2-5 year-olds (at Carolina Medical Center), the vaccine was shown to be safe, immunogenic and elicited bactericidal antibodies; 98% had 4 fold rise of IgG anti-LPS at 6 months. A major virulence factor of E. coli O157 is the Shiga toxin II. A mutant toxoid constructed by Alison OBrien conjugated with O-SP for enhanced protection is under study. <P> Enterotoxigenic E. coli (ETEC): ETEC is the most common cause of diarrhea in developing countries. ETEC secretes two exotoxins: heat-labile toxin (LT) and heat stable toxin (ST), a polypeptide of 19 amino acids. LT is immunogenic in humans. However its efficacy against infection has not been demonstrated. ST is small, non-immunogenic and difficult to purify.A non-toxic mutant, rLT, from John Clements was formalin-treated and in mice it induced high levels of IgG anti-LT. There was a dose dependent swelling at the injection site. In collaboration with Donald Robertson, the ST was purified, to be suitable for conjugation with LT or other proteins. <P> Vibrio cholerae O1 remains a major health problem in the Indian subcontinent and in Africa. Field studies showed that the vibriocidal activity of antisera is directed towards the LPS. In our Phase 1 trial the O-SP conjugates elicited high levels of IgG anti-LPS with vibriocidal activity. Chemical synthesis of O1 V. cholera O-SP is underway.Campylobacter jejuni infection is common and may cause serious complications such as Guillain- Barre syndrome; possibly due to the structural similarity between gangliosides and lipooligosaccharides (LOS) of C. jejuni. Protein conjugates of type 2 LOS or of the de-acylated LOS elicited anti-LOS IgG and bactericidal antibodies. 28 isolates of C. jejuni from pediatric patients in Israel were analyzed for serotype distribution; a diverse serotype distribution was found. Approximately 40% of the 16 isolates contain sialic acid in their LOS and bind to human ganglioside antisera. <P> Rotavirus infection is the most common cause of infantile diarrhea worldwide. Current vaccines are reassortant whole-cell oral vaccines. One such vaccine, Rotashield, was withdrawn from the market after a suspected increase in intussusception in its recipients. We designed parenteral vaccines based on capsid proteins VP8 and VP7. Mice injected with conjugate capsid proteins had neutralizing antibodies against rotavirus of homologous (P4) and heterologous (P8) serotypes.