Within our continued effort aimed at developing conjugate vaccines for infectious diseases from synthetic fragments of bacterial polysaccharides 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, as such, have undesirable properties and do not provide long-term immunity.  Development of such 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.  In the cholera project, we are still trying to simplify the synthesis of the hexasaccharide fragment of the O-specific polysaccharide of Vibrio cholerae O1 using glycosyl donors and glycosyl acceptors having the N-(3-deoxy-L-glycero-tetronamido) side chain already in place.  In addition, we investigate the role of details in the architecture of neoglycoconjugates upon immunogenicity.  We have previously evaluated serological responses of conjugates from the hexasaccharide that mimics the O-PS of Vibrio cholerae O:1.  Those conjugates were found potent immunogens, which also elicited in mice bactericidal and protective antibodies for Vibrio cholerae O:1.  To approximate a clinically relevant vaccine, we prepared conjugates composed of synthetic mono-, di- or hexasaccharide fragments of the O-specific polysaccharide of V. cholerae O1, serotype Inaba and Ogawa as the B cell epitopes and recombinant Pseudomonas aeruginosa exotoxin[unreadable] A (rEPA).  Compared to EPA (Pseudomonas aeruginosa exotoxin A), rEPA has one of the glutamic acids in EPA replaced with aspartic acid.  rEPA has been used previously as a carrier in conjugate vaccines.  Monosaccharide conjugates boosted the humoral responses to the hexasaccharide conjugates.  Prior exposure to purified Ogawa lipopolysaccharide (LPS) enabled contra-serotype hexasaccharide conjugates to boost the vibriocidal response, but Inaba LPS did not prime for an enhanced vibriocidal response by a contra-serotype conjugate.  Prior exposure to the carrier, and priming B cells with the LPS of either serotype, resulted in enhanced vibriocidal titers if the Ogawa hexasaccharides were used, but a diminished response to the Inaba LPS.  These studies demonstrate that the functional B cell epitopes on the LPS differ from those of the neoglycoconjugates and that the order of immunization and the serotype of the boosting conjugate can influence the epitope specificity and function of the antisera.  The novel finding that Ogawa neoglycoconjugates can boost Inaba-primed mice while Inaba neoglycoconjugates can not boost Ogawa primed mice highlights the issue of immunodominant LPS epitopes that we propose, based on experimental evidence, differ between Ogawa and Inaba LPS.  Thus, neoglycoconjugates can be used as boosters for cholera vaccine responses that have waned.  The neoglycoconjugates are an attractive alternative because they can be delivered parenterally[unreadable] without the attending inflammation of LPS, and they have a carrier component to enhance B cell memory and antibody-isotype switching. [ unreadable] Regarding our work towards a vaccine for cholera caused by the strain Vibrio cholerae O:139, we have prepared the fully protected form of the spacer-equipped, phosphorylated di-, tri- and tetrasaccharide fragments of the O-specific polysaccharide. [ unreadable] Within the anthrax project, our work toward the vaccine is complicated by the fact that the mode of linkage of the tetrasaccharide side chain in the exosporium glycoprotein is not known.  We can expect optimum immune response from the neoglycoconjugate only when all epitopes in the antigenic component of the neoglycoconjugate are identical to those in the anthrax spores, including the anomeric configuration at the site of attachment of the tetrasaccharide in the glycoprotein.  With that information unknown we will have to synthesize two linker-equipped tetrasaccharide, one with a- and one with b-linked spacer.  We have previously reported the synthesis of the b-linked tetrasaccharide, and we have now synthesized the a-tetrasaccharide in the fully protected form. [ unreadable] We also continue our international collaboration with Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovak Republic, on mass spectroscopy of carbohydrates that are involved as tools in the life sciences.  In this line of work we have investigated mass spectroscopic fragmentation of synthetic fragments of the O-PS of Vibrio cholerae O:1 and some analogs thereof.