Response To Polysaccharide/Oligosaccharide/Peptide-Prote

Child Health And Human Development

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Abstract

SHIGELLAE: Surface polysaccharides of pathogenic bacteria, including capsular polysaccharides (CPS) or the O-specific polysaccharide (O-SP) of lipopolysaccharides (LPS), serve both as essential virulence factors and as protective antigens. Covalent binding of CPS or of O-SP to medically-useful proteins to form conjugates both increases their immunogenicity and confers T-cell dependence to these saccharides making them suitable vaccines for infants and children. The O-SP of Shigella sonnei bound to recombinant non toxic P. aeruginosa exoprotein A (rEPA) and of S. flexneri 2a bound to the succinylated, exoprotein A (rEPA-succ) were safe and induced IgG antibodies to the homologous LPS in 1-4 year-olds. A randomized, blinded, phase 3 study of these conjugates in 1-4 year-olds with each conjugate serving as a control for the other is in progress. CROSS REACTING POLYSACCHARIDES: H. influenzae type b (Hib), Bacillus pumilis: A study of 10 year old children injected in infancy with Hib-TT showed a GM serum level of 4.16 mcg of IgG anti-Hib/mL with no change in this level after a DT booster but with an increase of IgG anti-TT from 0.09 to 4.58 IU/mL. To investigate if concurrent administration of a cross-reacting polysaccharide along with a homologous CPS has an advantage over the use of the homologous CPS alone, the cell wall polysaccharide (PS) of Bacillus pumilis Sh18, cross reactive with Hib CPS, was isolated and its structure investigated using GC-MS, NMR, fast atom bombardment and several sugar degrading techniques. It was shown to be composed of polyribitolphosphate, polyribitolphosphate substituted at C2 with N-acetyl glucosamine and polyglycerolphosphate. In addition to the anti-Hib it reacted with anti-Staphylococcus epidermidis known to contain polyribitol phosphate (poly Rib-P) in its teichoic acid. The Sh18 PS was conjugated to carrier proteins and its immunogenicity evaluated in general purpose mice. Conjugate-induced antibodies reacted with the homologous and with several cross-reacting polysaccharides. Poly (Rib-P) was synthesized to investigate its cross reactivity with poly (Rib-P) containing polysaccharides. NEISSERIA MENINGITIDIS: N. Meningitidis group A (Men. A): causes endemic and epidemic meningitis, notably in the meningitis belt of Africa. A CPS vaccine, effective and available, is underutilized. To improve its immunogenicity, Men. A CPS was conjugated to BSA using ADH as a linker. Contrary to the CPS alone the conjugates were immunogenic in mice, with booster responses after the 2nd and 3rd injections and bactericidal activity related to IgG anti-CPS levels. Conjugates of the cross reactive polysaccharides, E. coli K93 and B. pumilus Sh17, did not induce anti-Men. A CPS. Outbreaks of meningitis due to N. meningitides group W135 were reported recently in West Africa, Saudi Arabia, Europe, and Australia. Similar to other polysaccharides W135 CPS is not immunogenic in young children. We prepared conjugates of this CPS bound to a recombinant Staphylococcus aureus enterotoxin C1 (rSEC) using ADH as a linker. Opposed to the CPS alone the conjugates induced IgG anti-CPS in mice, with booster responses upon reinjection. Neutralizing SEC antibodies were induced as well. Injection of Group A and W135 conjugates combined induced antibody levels comparable to those of each alone. Group B N. meningitidis (GBM) and Escherichia coli k1 continue to cause serious and difficult to treat diseases and there is no licensed vaccine for their prevention. The polysaccharide, alpha (2-8) N-acetylneuraminic acid (PSA), is the capsular polysaccharide (CPS) of GBM and of E. coli K1 and a component of mammalian glycopeptides. Because it is a "self antigen", vaccines designed to induce PSA immune responses have been considered as a potential cause of immunopathology. Antibodies to PSA bind to human tissues in-vitro but have not been shown to induce pathology in-vivo. The incidence, severity, and nature of systemic infection caused by GBM are similar to those caused by the closely related group C meningococci (GCM). As shown for other polysaccharides, there is an age-related acquisition of serum PSA antibodies in humans: most human neonates and adults have IgG anti-PSA. Purified PSA is not immunogenic but as a component of bacteria or conjugated to proteins this CPS induces antibodies of the 3 major isotypes with its IgM and IgG components being protective in in-vitro and in-vivo models. Despite its similarity to mammalian glycoproteins, there are no data indicating an association of IgG anti-PSA with immunopathology in experimental animals or in humans. In collaboration with Mark Miller, FIC, the Lanspitali University Hospital, Iceland, and the Statens Serum Institut, Denmark, we are conducting long term examination of survivors of groups B and C meningococcal meningitis to unearth any unusual diseases considered to be "autoimmune". We propose that clinical trials of PSA conjugate vaccines, shown to be immunogenic and protective in animals, be considered. BORRELIA BURGDORFERI, a spirochete transmitted through the bite of infected Ixodes ticks, is the etiologic agent of Lyme disease. A protein vaccine against it is not effective below the age of 12 years, and has been taken off the market recently. LPS has been described in other spirochetes but its presence in B. burgdorferi has been debated. We have not been able to confirm its presence. The search for LPS revealed 2 unique glycolipids: cholesteryl 6-O-acyl-beta-D-galactopyranoside (BbGL I) and 1,2-di-O-acyl-3-O-alpha-D-galactopyranosyl-sn-glycerol (BbGL II) We found evidence that they are surface exposed. Injected in various formulations into mice BbGL I induced specific antibodies, in order of induced levels: CFA, PBS, DMSO, and squalene. Investigation of the effect of BbGL-I and II upon human PMNs and monocytes showed increased levels of IFN-gamma, IL-4 and TNF-alpha secreted in their presence. BORDETELLAE: B. parapertussis causes pertussis in humans, though of a milder form and lower frequency than B. pertussis. B. bronchiseptica causes respiratory infection in animals, rarely in humans. B. parapertussis and B. bronchiseptica do not produce pertussis toxin. They reportedly share their LPS structure, which is different from that of B. pertussis, but variants in the LPS of B. bronchiseptica were found. The LPS of B. parapertussis and of 2 strains of B. bronchiseptica were purified and their O-SP isolated. Their structural and immunological properties are being studied. HAEMOPHILUS DUCREYI: is the cause of chancroid, a sexually transmitted disease characterized by painful genital ulceration. Importantly, chancroid also enhances the spread of HIV infection. A major virulence factor and a potentially protective antigen of H. ducreyi is its lipooligosaccharide (LOS). The LOS was isolated, its lipid A hydrolyzed and the O-Specific oligosaccharide (O-SP) bound to carrier proteins by two new methods: 1. binding the carbonyl group at the KDO of the reducing end of the O-SP to an aminooxy group of a bifunctional linker, prepared in our laboratory, bound to carrier proteins; 2. using adipic acid dihydrazide to bind to the carbonyl group of the O-SP and to benzaldehyde derivatized carrier proteins. These conjugates preserved the external, nonreducing end of the O-SP and precipitated with H. ducreyi antisera. Their immunogenicity in mice is being investigated.