Surface polysaccharides of pathogenic bacteria, including capsular polysaccharides (CPS) or lipopolysaccharides (LPS), serve as both essential virulence factors and protective antigens. The age-related and T cell–independent immunogenicity of CPS limit their use as vaccines, especially in infants and young children. LPS are too toxic to be administered. Accordingly, their O-specific polysaccharide (O-SP), which shares the virulence-promoting and protectiveness properties of CPS, must be purified. However, O-SPS are too small to be immunogenic (haptens). Covalent binding of CPS or of O-SPS to medically useful proteins to form conjugates both increases their immunogenicity and confers T-cell dependence on these saccharides. Part of the work of this laboratory is directed at making synthetic vaccines to improve their immunogenicity.

Conjugate Vaccines against Shigellosis
The O-SPS of Shigella sonnei and of Shigella flexneri 2a were bound to bacterial toxoids. In adult army recruits and then in four- to seven-year-old children, both conjugates were safe and induced statistically significant and long-lived rises in IgG antibody levels against the homologous LPS. Similar though lesser increases in IgM and IgA anti–LPS levels were also induced. Re-injection of S. flexneri 2a conjugate induced a booster response in the recruits and the four- to seven-year-old children. A Phase III trial showed that one injection of S. sonnei O-SP, bound to a nontoxic recombinant Pseudomonas aeruginosa exoprotein A (rEPA), protected army recruits against outbreaks with this pathogen. Importantly, there was a statistically significant correlation between the levels of serum IgG anti–LPS and the efficacy of the conjugate. Two methods were developed that increased the immunogenicity of the Shigella conjugates in mice: another carrier protein, a genetically inactivated Corynebacterium diphtheriae toxin (CRM9), was found to be a superior carrier for S. sonnei O-SP, and treatment of rEPA with succinic anhydride, a nontoxic mild akylating agent that converts amino groups of proteins to carboxyls, increased the immunogenicity of S. flexneri 2a O-SP. A Phase I study in adults of these Shigella conjugates confirmed their safety and immunogenicity; the improved immunogenicity was less marked than in mice. A Phase II study in one- to four-year-old children showed an improved immunogenicity of the new S. flexneri 2a conjugate but lesser immunogenicity of the S. sonnei conjugate. A Phase III study of the modified S. flexneri 2a and the original S. sonnei conjugates are in preparation. In collaboration with the Lanzhou Vaccine Institute and Provincial Medical Center in Henan, China, a clinical trial of these two conjugates is in the planning stage.

Use of Cross-Reacting Antigens to Boost Immungenicity
To determine if concurrent administration of a cross-reacting and a homologous capsular polysaccharide (CPS) has an advantage over the use of the homologous CPS alone, we used conventional methods to isolate the cell wall polysaccharide (PS) of Bacillus pumilus, SH18, which has been reported to cross-react with the CPS of Haemophilus influenzae type b (HIb), and then investigated its structure by using gel chromatography–mass spectrometry. It was shown to contain glycerol, ribitol, and 2-acetamido-2-deoxyglucose, in a molar ratio of 0.2:1.0:0.2, and 17 percent phosphate. The CPS cross-reacted with anti Staphilococcus epidermidis as well as with the anti-HIb. Methods to prepare a conjugate of this PS are under investigation.

Other Vaccines
Neisseria meningitidis group A causes endemic and epidemic meningitis, notably in the meningitis belt of Africa. An effective and available CPS vaccine is underused. To improve the vaccine’s immunogenicity further, as was done for other CPS, we are investigating methods of binding it to a carrier protein. We developed a double mutant of Bordetella pertussis, producing a genetically inactivated toxin and deficient in FHA synthesis. Effort is currently directed toward increasing production of this B. pertussis strain as a more easily purified pertussis toxin for a monocomponent vaccine and as a carrier protein for pneumococcal type 14 CPS. Clostridium difficile is a major cause of hospital-acquired diarrhea following antibiotic usage: the diarrhea caused by the pathogen is mediated by two exotoxins, A and B. Toxin A, considered to be the major toxin, can, in extreme cases, cause pseudomembranous colitis. A genetically derived toxin mutant (rARU) induces both antitoxin and protects animals from infection with C. difficile. The succinylation of rARU improved its solubility and did not detectably affect its antigenicity. Techniques to prepare a mutant toxin A for clinical use have been worked out. Three polysaccharides of varying composition, from pneumococcus type 14, Escherichia coli K1, and S. flexneri 2a, were conjugated to succinylated rARU. The resultant conjugates induced high levels of both antipolysaccharide and antitoxin. Preparation of toxin A conjugates for clinical evaluation is under way. Borrelia burgdorferi, a spirochete transmitted though the bite of infected Ixodes ticks, is the etiologic agent of Lyme disease. A protein vaccine against it is available but is not effective under the age of 12 years. Lipopolysaccharide (LPS has been described in other spirochetes, but its presence in B. burgdorferi has been debated. So far, we have not been able to confirm its presence. The search for LPS revealed a unique glycolipid consisting of glycerol and galactose as the carbohydrate moiety. Evidence suggests that this glycolipid is surface exposed. Injected in complete Freund's adjuvant, it induced specific antibodies.

Synthetic Vaccines against Shigella dysenteriae Type 1 and Shigella sonnei
Ekborg, Sampath_Kumar, Nelson, Liptak, Pozsgay, Robbins, Schneerson
Shigella dysenteriae type 1 and Shigella sonnei are Gram-negative human pathogens that cause endemic and epidemic dysentery throughout the world. Despite their discovery many years ago, there are still no licensed vaccines against these pathogens, which have developed resistance to most available antibiotics. Our approach to vaccine development against these bacteria is based on the demonstration that serum antibodies against the O-specific polysaccharides (O-SPs) of Gram-negative bacteria are important for host protection. While O-SPs are nonimmunogenic, presumably because of their low molecular weight, covalent conjugates of an immunogenic protein and the O-SP of S. dysenteriae type 1 and S. sonnei have been shown to elicit significant anti–O-SP antibody levels of the IgG isotype, which may be boosted by repeated injections. We surmised that an improved vaccine might be constructed from chemically defined oligosaccharide fragments of O-SPs, which are devoid of biological contamination, carry a uniform molecular weight, and can be characterized by physicochemical methods.

We have chemically synthesized oligosaccharide fragments of the native O-SP of S. dysenteriae type 1 up to a tetracosasaccharide consisting of 24 monosaccharide residues. These constructs are attached to a spacer that makes bioconjugation possible through the termini of the spacer moieties (i.e., at a single site). We have been developing a new technique for chemical synthesis of extended oligosaccharides that circumvents the difficulties associated with the conventional, liquid-phase coupling of oligosaccharide building blocks and avoids the problems inherent in the solid-phase approaches. Briefly, our method employs lipophilic protecting groups in one of the blocks while using conventional protecting groups in the others. The presence of the lipophilic groups in the targeted intermediate but not in the side-products allows the isolation of the products by the solid-phase extraction technique that uses recyclable chromatographic materials and environmentally friendly alcoholic solvents. To improve the efficiency of oligosaccharide synthesis by facilitating product isolation, we have designed and synthesized several new lipophilic protecting groups, including 4-dodecyloxybenzyl, 4-dodecylbenzyl, and 4-octadecylbenzyl moieties. The utility of these groups has been documented. We are currently using our new technology for the synthesis of the oligosaccharides related to the O-SP of S. dysenteriae type 1 under clean laboratory conditions for Phase I clinical trials. So far, we have demonstrated that the immunogenicity of the synthetic saccharide-human serum albumin conjugates depends on the saccharide chain length and the saccharide density on the protein, the latter in a non-linear fashion.

Current work is directed at determining the role, if any, of the monosaccharide terminating the oligosaccharide chain. To this end, we have synthesized oligosaccharide fragments of the O-SP that differ in the terminal unit, i.e., in the repeating unit frame. We designed experiments toward making a polysaccharide mimic that might be more immunogenic when conjugated to proteins than the saccharides synthesized so far. Briefly, we designed linearly arranged "clusters" of the synthetic oligosaccharides that are interconnected by spacers through predefined sites. We demonstrated the feasibility of this approach by interconnecting six monosaccharide units. Next, we extended this approach to a synthetic octasaccharide. So far, we have succeeded in interconnecting three such units, leading to a polymer containing 24 monosaccharide residues. At present, we are trying to synthesize even longer constructs as protein conjugates for immunological experiments.

A new technique was developed for reagentless attachment of oligosaccharides to proteins. In short, the technique is based on the Diels-Alder cycloaddition between an activated double bond and a conjugated diene. The activated double bond component was introduced in the protein by using the commercial reagent 3-sulfosuccinimidyl 4-maleimidobutyrate. The carbohydrate sector was equipped with various linkers containing a 1,3 conjugated diene system. When solutions of the functionalized components were combined, conjugation took place at room temperature. pH has a marked effect on the efficiency of the coupling: lower pH (e.g., pH 5.7) favors conjugation compared with higher pH (e.g., 9.5). The conjugation was as effective in water as in pH 5.7 buffer. On the other hand, the structure of the diene system has little effect on the conjugation reaction. An advantage of this method over existing ones is that the unconjugated saccharide can be recovered with its linker for reuse in a subsequent conjugation experiment.

Synthetic Vaccines against Group A Neisseria meningitidis
Berkin , Coxon, Yergey,Pozsgay, Robbins, Schneerson
Group A Neisseria meningitidis causes endemic and epidemic meningococcal meningitis worldwide. Although a vaccine containing the purified capsular polysaccharide (CPS) of this bacterium has been available for years, it is not sufficiently immunogenic in infants, who are at the highest risk. Our program to develop a more efficient vaccine is based on the assumption that protein conjugates of relatively short fragments of the CPS (i.e., which can be made available by controlled chemical synthesis) of Group A N. meningitidis will be more immunogenic in infants than the currently available vaccine. The CPS of Group A N. meningitidis consists of alpha-linked N-acetyl-mannosaminyl residues that are interconnected by an anomerically located phosphodiester linkage. The native polysaccharide is nonstoichiometrically O-acetylated at the O-3 position. In our first approach to studying the requirements of synthetic oligosaccharides related to this polymer, we targeted the synthesis of non–O-acetylated congeners. We have designed a route to the mannosaminyl H-phosphonate moiety. Iterative condensation of this unit allowed the synthesis of a dimer and trimer of the polysaccharide's repeating unit in bioconjugatable form. We also synthesized the mannosaminyl spacer and its phosphate ester. Covalent attachment of the synthetic subunits to human serum albumin using our conjugation technology afforded glycoconjugates containing up to 30 saccharide chains per molecule of HSA. The antigenicity of these conjugates was demonstrated by the precipitin formation with the serum of a horse that was immunized with formalin-killed Group A N. meningitidis. The experiments showed that the serum recognizes as small a component of the polysaccharide as a monosaccharide. They also showed that formation of precipitation indicates that the presence of O-acetyl groups in the polymer is not essential for antigenicity. Current work is directed toward the synthesis of higher oligomers with and without O-acetyl groups for evaluation of their immunogenicies.