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.[unreadable] The effect of these children!|s immune sera and IgG isolated from them upon Shigella invasion into epithelial intestinal cells was studied in vitro using Caco-2 and HeLa cells. The sera inhibited shigella invasion in a type specific manner. Pretreatment of the sera or of Caco-2 cells with O-SP abrogated these effects in a type specific and dose dependent manner.[unreadable] [unreadable] Modification of O-SP by glycosylation or acetylation is important for its antigenic specificity and immunogenicity. The degree of O-acetylation and localization of the O-acetyl groups and glucose substitution of S. flexneri 2a O-SP were investigated. The structure of the repeat unit was assigned.[unreadable] [unreadable] CROSS REACTING POLYSACCHARIDES: H. influenzae types b (Hib)and a (Hia), B. pumilus: Hia, sharing a D-1,5-ribitol phosphate in its repeat unit with Hib, was a (distant) second most common H. influenzae isolate from patients, causing up to 10% of disease in some populations. Its importance has risen with the successful control of Hib disease. To provide a potential vaccine for the prevention of Hia disease, its CPS was isolated and conjugated to a recombinant S. aureus enterotoxin C1 (rSEC). Injected sc as a saline solution into 5-6 week old mice, the conjugate induced CPS antibodies with bactericidal activity.[unreadable] The cell wall polysaccharide (PS) of B. pumilus Sh 18 was reported to cross react with Hib CPS, presumably due to its D-ribitol phosphate, a common component of bacilli and staphylococcal cell wall PS. Anti Hib CPS was also shown to precipitate with the cell wall PS of L. plantarium and S. aureus. The cell wall PS of Bacillus pumilus Sh18, 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 (poly Rib-P), polyribitolphosphate substituted at C2 with N-acetyl glucosamine and polyglycerolphosphate (Gly-P). We failed to separate these components.. In addition to anti Hib this preparation bound to anti S. epidermidis, known to contain poly (gly-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; an octamer and a dodecamer with terminal keto groups were conjugated to aminooxylated BSA or tetanus toxoid. MALDI-TOF showed 10-18 saccharide chains incorporated per mole of BSA.[unreadable] The immunogenicity of these conjugates is being studied.[unreadable] [unreadable] GROUP B N. MENINGITIDIS AND ESCHERICHIA COLI K1: Group B meningococcus (GBM) and Escherichia coli k1 meningitis 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 bound to proteins as a conjugate 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. A follow up of ~50,000 person years of individuals with a past history of meningococcal infection, in Denmark and in Iceland, found no increased association with autoimmune disease in patients with GBM disease compared to those with group C meningococcal disease. We propose that clinical trials of PSA conjugate vaccines, shown to be immunogenic and protective in animals, be considered.[unreadable] [unreadable] 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) There is 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. The cholesteryl palmitoyl !Vgalactopyranoside was synthesized with an aldehydo group at the palmitoyl end which was used to bind to aminooxylated BSA. An average of 18 glycolipid groups were incorporated per BSA molecule. The immunogenicity of these conjugates is being studied in mice.[unreadable] [unreadable] BORDETELLAE: Pertussis, whooping cough in infants, is caused by B. pertussis. 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. The Lipid A and core regions of the three Bordetella species are similar but only B. parapertussis and B. bronchiseptica posses an O-SP, the structure of which was reported but the linkages to the core PS were not identified. We analyzed the carbohydrate structures of the LPS from several strains of Bordetella bronchiseptica and Bordetella parapertussis, using different chemical degradation methods, NMR spectroscopy and mass spectrometry, and identified a novel pentasaccharide that links the O-chain to the core in all LPS studied. Additionally, whereas the O-chain of these bacteria was reported to be composed of a homopolymer of 1,4-linked 2,3-diacetamido-2,3-dideoxy-??-galacturonic acid, we discovered that it contains amidated uronic acids, the number of which varies among strains.[unreadable] [unreadable] HAEMOPHILUS DUCREYI: is the cause of chancroid, a sexually transmitted disease characterized by painful genital ulceration. Chancroid also enhances the spread of HIV infection. A major virulence factor and a potentially protective antigen of H. ducreyi is its lipooligosaccharide (LOS).