Reactions And Immunochemistry Of Carbohydrates

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

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. 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. Since making conjugates aplying squaric acid chemistry has not always been reproducible, we have focused on examining variables that affect conjugation by this method. Methyl 6-hydroxyhexanoyl glycoside of lactose was used as a model oligosaccharide. The objective was to revise the original protocol to arrive at a reproducible and a more economical protocol than the existing one. Accordingly, the foregoing lactose glycoside was treated with each of 1,2-diaminoethane or hydrazine hydrate, and the corresponding amino amide and acyl hydrazide were treated with each of squaric acid dimethyl, diethyl, dibutyl, and didecyl esters. The objective or the experiment was to establish if any of these derivatives would be the preferred reagent for conjugation. The monoesters were conjugated to bovine serum albumine (BSA) at different concentrations of hapten using 0.05 and 0.5 M pH 9 borate buffer. Maximum loading was achieved faster and the conjugation efficiency was higher when conjugation was conducted at higher concentration of both hapten and buffer. Conjugations involving haptens prepared from hydrazide were generally slower and less efficient than those with compounds made from amino amide. Maintaining pH 9 during conjugation was found to be the single most important factor to make the conjugation a fast, highly efficient and reproducible process. When the pH of the conjugation mixture fell during the reaction, resulting in decreased reaction rate or even termination of the conjugation process, normal course of the conjugation reaction could be restored by addition of buffer salts. Hydrolysis studies of monoesters formed from amino amides involving different, above mentioned alkyl groups showed that under the conjugation conditions the decyl ester was the most stable one and that the methyl compound was the one most readily hydrolyzed. Stability of monoesters prepared form hydrazide was alike and comparable to the decyl ester. No definite advantage was found to the use of any of the four dialkyl squarate reagents (methyl-, ethyl-, butyl-, and decyl-) for conversion of carbohydrate derivatives to species amenable for conjugation. Nevertheless, dimethyl squarate seemed to be the most convenient reagent since it is a crystalline, easy-to-handle, and commercially available material with very good reactivity. Using that reagent, based on other findings of the study, the original protocol for conjugation was revised, and the use of large excess of hapten is no longer required. The 20% excess of hapten normally required by the refined, more efficient protocol is about the same as in the syntheses of small organic molecules. Thus, recovery of the excess of the synthetic, labor intensive hapten becomes a non-issue. In continuation of our work on the anthrax project, we have revised our original synthesis of the anthrax spores-specific tetrasaccharide. In anticipation of need of a large amount of the antigen, we made the new synthesis suitable for large-scale preparation. We have made 3 g of the tetrasaccharide in form suitable for conjugation. Considering the current use of 2-5 micrograms of carbohydrate per dose, the amount prepared is sufficient for 600,0001.000,000 immunizations. This with the ability to prepare neoglycoconjugates efficiently and in a predictable way allows us to conclude that we have reached a stage when the synthesis of complex oligosaccharide antigens and preparation of neoglycoconjugates is no longer the bottleneck in the process of developing conjugate vaccines. In addition, we have used the synthesized, linker-equipped tetrasaccharide and the previously synthesized all theoretically possible fragments thereof to develop an important diagnostic tool for anthrax. It is a microarray platform obtained by immobilizing bacterial signature carbohydrates onto epoxide-modified glass slides. The carbohydrate microarray platform was probed with sera from non-melioidosis and melioidosis (Burkholderia pseudomallei) individuals. The platform was also probed with sera from rabbits vaccinated with Bacillus anthracis spores and Francisella tularensis bacteria. By employing this microarray platform, we were able to detect and differentiate B. pseudomallei, B. anthracis and F. tularensis antibodies in infected patients, and infected or vaccinated animals. These antibodies were absent in the sera of nave test subjects. This array is a multiplex carbohydrate microarray for the detection of all three biothreat bacterial infections including, melioidosis, anthrax and tularemia with one, multivalent device.