The National Institute of Diabetes & Digestive & Kidney Diseases

Our primary focus is to further the development of conjugate vaccines from synthetic carbohydrate antigens. Ultimately, we would like to develop reliable protocols for preparation of neoglycoconjugates which could become substitutes for traditional vaccines based on antigens present in e.g. attenuated cells. Such vaccines are often pyrogenic or have other undesirable effects. As antigenic components of our immunogens, we use synthetic oligosaccharides that mimic the structure of polysaccharides present on surface of bacterial pathogens. Since there is virtually an infinite number of choices of architectonic details a synthetic neoglycoconjugate can incorporate, part of our work involves studies of the effects of variables such are size of the carbohydrate antigen, type of linker, linking chemistry, type of carrier, etc., upon immunogenicity and protctive capacity. In addition to obtaining potent immunogens, we expect our studies to result in findings of general utility in synthetic vaccine preparation. Our approach involves five stages:

1. Synthesis of fragments of antigenic polysaccharides as well as deoxy and deoxyfluoro analogs thereof.
2. Studies of binding of the above ligands with antibodies to the native antigen and identification of critical hydrogen bonding interactions.
3. Identification of the immunologically dominant oligosaccharide sequence in the antigenic polysaccharide.
4. Chemical conjugation of such fragment(s) to suitable carrier(s), to obtain neoglycoconjugates.
5. Probing the antigenicity, immunogenicity and protective capacity of neoglycoconjugate(s).

Our Section has studied the interaction of carbohydrate antigens and antibodies for many years using the above approach. As a result, we have been able to obtain a great deal of detailed information on binding on the molecular level. Following the same concept, ground work was laid for development of immunogens for Shigella dysenteria type 1. The current objective of our work is development of synthetic vaccines for cholera and anthrax.

In addition, we often engage in collaborative research with universities and other scientific institutions.

(Click image to enlarge)
Model of the monoclonal anti Vibrio cholerae O1, serotype Ogawa antibody–O-SP complex based on the X-ray structue of the crystalline complex of Fab with the synthetic disaccharide that mimics the terminus of the bacterial O-PS. Individual perosamine residues are shown in different colors; the light and heavy chains of the Ab variable domains are shown in light and dark blue, respectively. The O-SP Ag was built assuming that all glycosidic linkages display the same dihedral angles observed for the Fab-bound disaccharide, giving rise to an extended polysaccharide conformation. The upstream terminal perosamine is bound inside the Ab-binding cavity (the 2-O-methyl group at the center of the interface is shown in yellow). As revealed by the Fab–disaccharide structure, the second perosamine residue is positioned at the exterior of the binding site and makes fewer contacts with Ab residues, whereas subsequent sugar residues are not involved in the interaction. Synthesis of the disaccharide carried out at Carbohydrate Work Group, LMC; Crystallography performed at Institute Pasterur, Paris, France. Taken from Villeneuve et al., PNAS, Vol. 97 (2000) 8433–8438.

Hou SJ, Saksena R, Kovac P Preparation of glycoconjugates by dialkyl squarate chemistry revisited. Carbohydr Res(343): 196-210, 2008. [Full Text/Abstract]

Adamo, R., Kovác, P. Glycosylation under thermodynamic control. Synthesis of the di- and the hexasaccharide fragments of the O-SP of Vibrio cholerae O:1, serotype Ogawa, from fully functionalized building blocks Eur. J. Org. Chem.: 988-1000, 2007.

Saksena R, Adamo R, Kovac P Immunogens related to the synthetic tetrasaccharide side chain of the Bacillus anthracis exosporium. Bioorg Med Chem(15): 4283-310, 2007. [Full Text/Abstract]

Wang D, Carroll GT, Turro NJ, Koberstein JT, Kovac P, Saksena R, Adamo R, Herzenberg LA, Herzenberg LA, Steinman L Photogenerated glycan arrays identify immunogenic sugar moieties of Bacillus anthracis exosporium. Proteomics(7): 180-4, 2007. [Full Text/Abstract]

Wade TK, Saksena R, Shiloach J, Kovac P, Wade WF Immunogenicity of synthetic saccharide fragments of Vibrio cholerae O1 (Ogawa and Inaba) bound to Exotoxin A. FEMS Immunol Med Microbiol(48): 237-51, 2006. [Full Text/Abstract]

Saksena R, Ma X, Wade TK, Kovác P, Wade WF Length of the linker and the interval between immunizations influences the efficacy of Vibrio cholerae O1, Ogawa hexasaccharide neoglycoconjugates. FEMS Immunol Med Microbiol(47): 116-28, 2006. [Full Text/Abstract]

Ruttens B, Kovac P Synthesis of spacer-equipped phosphorylated di-, tri- and tetrasaccharide fragments of the O-specific polysaccharide of Vibrio cholerae O139. Carbohydr Res(341): 1077-80, 2006. [Full Text/Abstract]

Provenzano D, Kovac P, Wade WF The ABCs (Antibody, B cells, and Carbohydrate epitopes) of cholera immunity: considerations for an improved vaccine. Microbiol Immunol(50): 899-927, 2006. [Full Text/Abstract]

Taylor, R. K., Kirn, T. J., Bose, N., Stonehouse, E., Tripathi, S. A., Kovác, P., Wade, W. F. Progress towards development of a cholera subunit vaccine Chemistry & Biodiversity(1): 2036-2057, 2004.

Kovác, P Quantity over Quality?: An Open Letter to the Community of Chemists Chemistry and Biodiversity(1): 606-608, 2004.

Meeks M. D., Saksena, R., MA, X. , Wade T. K. , R. K. Taylor, Kovác, P., Wade W. F Synthetic Fragments of Vibrio cholerae O1 Inaba O-SP Bound to a Protein Carrier are Immunogenic in Mice but do not Induce Protective Antibodies Infection and Immunity(72): 4090-4101, 2004.

Ma, X., Saksena, R., Chernyak, A., Kovác, P. Neoglycoconjugates from synthetic tetra- and hexasaccharides that mimic the terminus of the O-PS of Vibrio cholerae O:1, serotype Inaba Org. Biomol. Chem.(1): 775-784, 2003.

Chernyak A Kondo S Wade TK Meeks MD Alzari PM Fournier JM Taylor RK Kovac P Wade WF Induction of protective immunity by synthetic Vibrio cholerae hexasaccharide derived from V. cholerae O1 Ogawa lipopolysaccharide bound to a protein carrier. J Infect Dis (185): 950-62, 2002. [Full Text/Abstract]

Chernyak A Karavanov A Ogawa Y Kovac P Conjugating oligosaccharides to proteins by squaric acid diester chemistry: rapid monitoring of the progress of conjugation, and recovery of the unused ligand. Carbohydr Res (330): 479-86, 2001. [Full Text/Abstract]