Within our continued effort aimed at developing conjugate vaccines for infectious diseases from synthetic fragments of bacterial carbohydrates we have three ongoing projects. Two are concerned with a vaccine for cholera and one with a vaccine for anthrax. Existing vaccines for these diseases are based on cellular material and, in addition to having undesirable side effects, do not provide long-term immunity. Development of the two vaccines is important from both the point of view of public health and of national interest. Development of a potent vaccine for cholera is important because of the involvement of our military in protecting US interests in developing third world countries. While anthrax does not constitute a major health problem in the civilized world, new concerns regarding anthrax have emerged because of potential use of some form of Bacillus anthracis, the etiological cause of anthrax, as a biological weapon. Our work towards a potent conjugate vaccine for cholera involves synthesis of oligosaccharides that mimic the structure of O-specific polysaccharide (O-PS) of Vibrio cholerae in the form suitable for conjugation, conjugation of these antigens to suitable carriers, and serological evaluation of the immunogenicity of the resulting neoglycoconjugates. The approach towards a vaccine for anthrax is based on preparation of a neoglycoconjugate from a suitable carrier and the tetrasaccharide side chain of the major glycoprotein of Bacillus anthracis exosporium. While the work towards serological evaluation of conjugates from the Bacillus anthracis exosporium tetrasaccharide are in progress, we have focused on improving diagnostic tools for the detection of presence of anthrax spores. The immunological detection of Bacillus anthracis in various environmental samples and the discrimination from other closely related pathogens is not yet well established. To generate specific discriminating antibodies we immunized rabbits, mice and chicken with inactivated spores and, additionally, rabbits and mice with the exosporium tetrasaccharide, which is a constituent of the exosporium glycoprotein BclA and contains the newly discovered sugar anthrose. The BclA protein, is a major component of the exosporium of B. anthracis spores, and is decorated by the above tetrasaccharide. The anthrose-containing tetrasaccharide chain seems to be highly specific for B. anthracis, which makes it to a key biomarker for the detection of these spores. Different immunizations lead to anthrose-reactive polyclonal and monoclonal antibodies, which were analyzed by various methods to characterize their ability to discriminate between B. anthracis and other pathogens. Multiple applications like ELISA, indirect immunofluorescence assay and electron microscopy revealed specificity for the polyclonal and monoclonal antibodies generated for B. anthracis spore detection. All polyclonal antibodies were able to identify the tested B. anthracis strains correctly and only showed minimal cross-reactivities with other Bacillus strains. Moreover, the generated antibodies proved functional in a new capture assay for B. anthracis spores and could, therefore, be useful for the detection of spores in complex samples. In the cholera project, we have previously established that antibodies resulting from immunization of neonatal mice with the conjugate made from the hexasaccharide fragment of the O-PS of V. cholerae (serotype Ogawa) were protective. In order to determine whether transcutaneous immunization (TCI) with a V. cholerae neoglycoconjugate comprised of a synthetic terminal hexasaccharide of the O-specific polysaccharide of V. cholerae O1 (Ogawa) and BSA (CHO-BSA) could induce vibriocidal anti-V. cholerae LPS responses, we applied CHO-BSA transcutaneously in the presence or absence of immunoadjuvantative cholera toxin (CT) to mice. Transcutaneously applied neoglycoconjugate elicited prominent anti-V. cholerae LPS IgG responses in the presence of CT, but no IgM or IgA responses. CT applied on the skin induced strong IgG and IgA serum responses. TCI with neoglycoconjugate did not elicit vibriocidal responses protection in a mouse challenge assay, or stool anti-V. cholerae IgA responses, irrespective of presence or absence of CT. Our results suggest that transcutaneously applied synthetic V. cholerae neoglycoconjugate is safe and immunogenic, but predominantly induces systemic anti-LPS responses. We have recently started work on developing therapies for Fabry and Gaucher diseases, which are caused by insufficient activity of biodegrading enzymes resulting in accumulation of toxic levels of globosylceramide (Gb3). Patients suffering from Fabry disease experience, among other symptoms, myocardial infarctions, strokes and progressive kidney failure. Developing therapies for kidney diseases is one of the missions of NIDDK. Enzyme replacement therapy is somewhat successful treatment with some patients with Gaucher disease disorder, but this approach has been largely unsuccessful with Fabry disease. Even when on ERP, Fabry disease patients still experience, among other symptoms, myocardial infarctions, strokes and progressive kidney failure. An alternative therapeutic strategy for these diseases is substrate reduction therapy, which is aimed at reducing the enzymatic formation of Gb3. In collaboration with top authorities in this area we are designing a suitable substrate for the enzyme that is involved in the synthesis of Gb3. One of such compounds is likely to be 4′-deoxy-lactose. A few mg of this compound were previously synthesized in a very poor overall yield. We have considerably improved the original chemical synthesis of the compound, and its effectiveness to alleviate the condition is currently tested in a suitable enzyme system that catalyzes the formation of Gb3 from UDP-galactose and lactosylceramide.
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