Antigenicity and Immunogenicity of a Synthetic Oligosaccharide-Protein Conjugate Vaccine against Haemophilus influenzae Type b

Polysaccharide-protein conjugates as vaccines have proven to be very effective in preventing Haemophilus influenzae type b infections in industrialized countries. However, cost-effective technologies need to be developed for increasing the availability of anti-H. influenzae type b vaccines in countries from the developing world. Consequently, vaccine production with partially synthetic antigens is a desirable goal for many reasons. They may be rigidly controlled for purity and effectiveness while at the same time being cheap enough that they may be made universally available. We describe here the antigenicity and immunogenicity of several H. influenzae type b synthetic oligosaccharide-protein conjugates in laboratory animals. The serum of H. influenzae type b-immunized animals recognized our synthetic H. influenzae type b antigens to the same extent as the native bacterial capsular polysaccharide. Compared to the anti-H. influenzae type b vaccine employed, these synthetic versions induced similar antibody response patterns in terms of titer, specificity, and functional capacity. The further development of synthetic vaccines will meet urgent needs in the less prosperous parts of the world and remains our major goal(AU)

Conjugados proteína-polisacárido como vacunas han demostrado ser muy eficaz en la prevención de Haemophilus influenzae tipo b infecciones en los países industrializados. Sin embargo, tecnologías rentables necesitan ser desarrolladas para incrementar la disponibilidad de anti-H. influenzae tipo b, las vacunas en los países del mundo en desarrollo. En consecuencia, la producción de vacunas con antígenos sintéticos en parte es un objetivo deseable por muchas razones. Pueden ser rígidamente controlada por la pureza y la eficacia al mismo tiempo ser lo suficientemente baratas para que sean universalmente disponibles. Se describe aquí la antigenicidad e inmunogenicidad de varias H. influenzae tipo b-oligosacáridos sintéticos conjugados proteína en animales de laboratorio. El suero de H. influenzae tipo b-reconocido nuestros animales inmunizados sintéticas H. influenzae tipo b antígenos en la misma medida que los nativos bacteriana polisacárido capsular. En comparación con el anti-H. influenzae tipo b vacuna empleada, estas versiones sintéticas similares respuesta de anticuerpos inducida por los patrones en términos de títulos, la especificidad y la capacidad funcional. El ulterior desarrollo de vacunas sintéticas satisfacer las necesidades urgentes en las regiones menos prósperas del mundo y sigue siendo nuestro principal objetivo

Antigenicity and immunogenicity of a synthetic oligosaccharide-protein conjugate vaccine against Haemophilus influenzae type b.

Polysaccharide-protein conjugates as vaccines have proven to be very effective in preventing Haemophilus influenzae type b infections in industrialized countries. However, cost-effective technologies need to be developed for increasing the availability of anti-H. influenzae type b vaccines in countries from the developing world. Consequently, vaccine production with partially synthetic antigens is a desirable goal for many reasons. They may be rigidly controlled for purity and effectiveness while at the same time being cheap enough that they may be made universally available. We describe here the antigenicity and immunogenicity of several H. influenzae type b synthetic oligosaccharide-protein conjugates in laboratory animals. The serum of H. influenzae type b-immunized animals recognized our synthetic H. influenzae type b antigens to the same extent as the native bacterial capsular polysaccharide. Compared to the anti-H. influenzae type b vaccine employed, these synthetic versions induced similar antibody response patterns in terms of titer, specificity, and functional capacity. The further development of synthetic vaccines will meet urgent needs in the less prosperous parts of the world and remains our major goal.

A synthetic conjugate polysaccharide vaccine against Haemophilus influenzae type b.

Glycoconjugate vaccines provide effective prophylaxis against bacterial infections. To date, however, no commercial vaccine has been available in which the key carbohydrate antigens are produced synthetically. We describe the large-scale synthesis, pharmaceutical development, and clinical evaluation of a conjugate vaccine composed of a synthetic capsular polysaccharide antigen of Haemophilus influenzae type b (Hib). The vaccine was evaluated in clinical trials in Cuba and showed long-term protective antibody titers that compared favorably to licensed products prepared with the Hib polysaccharide extracted from bacteria. This demonstrates that access to synthetic complex carbohydrate-based vaccines is feasible and provides a basis for further development of similar approaches for other human pathogens.

A synthetic conjugate polysaccharide vaccine against Haemophilus influenzae type b

Glycoconjugate vaccines provide effective prophylaxis against bacterial infections. To date, however, no commercial vaccine has been available in which the key carbohydrate antigens are produced synthetically. We describe the large-scale synthesis, pharmaceutical development, and clinical evaluation of a conjugate vaccine composed of a synthetic capsular polysaccharide antigen of Haemophilus influenzae type b (Hib). The vaccine was evaluated in clinical trials in Cuba and showed long-term protective antibody titers that compared favorably to licensed products prepared with the Hib polysaccharide extracted from bacteria. This demonstrates that access to synthetic complex carbohydrate-based vaccines is feasible and provides a basis for further development of similar approaches for other human pathogens(AU)

Solution conformation and dynamics of the trisaccharide fragments of the O-antigen of Vibrio cholerae O1, serotypes Inaba and Ogawa.

The conformational behavior of the trisaccharide fragments of the Ogawa and Inaba Vibrio cholera serotypes has been studied using NMR and molecular dynamics (MD). The obtained results indicate that there are no significant differences in the major conformation and in the extent of motion of the glycosidic torsions of these molecules. The differences in biological activity are probably not due to conformational effects but to van der Waals and/or hydrogen bonding interactions between the antigens and the biological receptor.

Synthesis of a sialyl-alpha-(2-->6)-lactosamine trisaccharide with a 5-amino-3-oxapentyl spacer group at C-1I.

As part of a continuing study aimed to achieve improved monoclonal antibodies against carcinoembryonic antigen (CEA) carbohydrate fragments, the synthesis of a sialyl-(2-->6)-lactosamine trisaccharide with a 5-amino-3-oxapentyl spacer group at C-1I has been developed. Two different routes to access this target are described. For this purpose 5-azido-3-oxapentyl 6-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranoside (4) was selectively beta-galactosylated in 81% yield using the crystalline 2,3-di-O-acetyl-4,6-O-benzylidene-alpha-D-galactopyranosyl trichloroacetimidate as the donor, taking advantage of the bulky phthalimido group at C-2 of 4. On the other hand, galactosylation of the suitable protected acceptor 5-azido-3-oxapentyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-beta-D-glucopyranoside with the crystalline 2,3-di-O-acetyl-4,6-O-benzylidene-alpha-D-galactosyl bromide renders the corresponding disaccharide in a moderate 58% yield. Despite the fact that the first strategy, unlike the second one, requires a hydrazinolysis-acetylation reaction at the disaccharide stage, it was found to be more convenient to access the disaccharide acceptor. Sialylation was performed using a thiophenyl donor under an NIS-TfOH activation procedure in acetonitrile to give a mixture of alpha and beta trisaccharides in 49 and 16% yields, respectively.

Synthesis of terminal disaccharide elements corresponding to the Ogawa and Inaba antigenic determinant from Vibrio cholerae O1.

Vibrio cholerae O1 LPS terminal mono- and disaccharide elements were synthesized by reduction of the azido group in several 4-amino-4,6-dideoxy-D-mannose mono- and disaccharide derivatives, followed by coupling with 2, 4-di-O-acetyl-3-deoxy-L-glycero-tetronic acid in the presence of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline. This compound represents a useful model in order to elucidate the size of the epitopes which define Ogawa and Inaba serotypes from Vibrio cholerae O1.

Synthesis and NMR analysis of 13C-labeled oligosaccharides corresponding to the major glycolipid from Mycobacterium leprae.

An improved synthesis of propyl 4-O-(3,6-di-O-methyl- beta-D-glucopyranosyl)-2,3-di-O-methyl-alpha-L-rhamnopyranoside, a disaccharide corresponding to the phenolic glycolipid of Mycobacterium leprae using a trichloroacetimidate as a glycosyl donor is described. The synthetic strategy is also applied to the preparation of three corresponding disaccharide analogues containing 13C-labeled methyl groups. The preparation of the trisaccharide, propyl 2-O-[4-O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-2,3-di-O-methyl-alpha-L - rhamnopyranosyl]-3-O-methyl-alpha-L-rhamnopyranoside is also reported. The di- and tri-saccharides were characterized by 1H and 13C NMR spectroscopy.

Conjugation of 5-azido-3-oxapentyl glycosides with thiolated proteins through the use of thiophilic derivatives.

5-Azido-3-oxa-pentyl beta-D-galactopyranoside was prepared from diethylene glycol monochlorohydrin and used as a model of oligosaccharide hapten. After deprotection, a series of amides bearing thiophilic groups had been obtained through the terminal amino function and essayed in coupling reactions with thiolated BSA. Also several Lewis human blood group oligosaccharides had been conjugated with thiolated BSA demonstrating the usefulness of the methodology.

Galactosylation with beta-galactosidase from bovine testes employing modified acceptor substrates.

In this study beta1-3 linked analogues of the T-antigen determinant were synthesized in preparative scale by transgalactosylation using beta-galactosidase from bovine testes to give synthetic antigens. Acceptors with modifications of the sugar residue such as alpha-glycosylated spacers, as well as GlcNAc-alphaOR- and 2dGal-alphaOR-substrates opened further possibilities for galactosylation.

An n.m.r. and conformational analysis of the terminal trisaccharide from the serologically active glycolipid of Mycobacterium leprae in different solvents.

The 1H- and 13C-n.m.r. spectra of allyl 2-O-[4-O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-2,3-di-O-methyl-alpha-L -rhamnopyranosyl]-3-O-methyl-alpha-L-rhamnopyranoside (3), a glycoside of the terminal trisaccharide found in the phenolic glycolipid I from Mycobacterium leprae, and those of the two component disaccharides, allyl 4-O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-2,3-di-O- methyl-alpha-L-rhamnopyranoside (1) and allyl 2-O-(2,3-di-O-methyl-alpha-L-rhamnopyranosyl)-3-O-methyl-alpha-L- rhamnopyranoside (2) have been assigned completely by 1D and 2D techniques. The preferred conformations, determined by chemical shift and n.O.e. studies, were different in D2O, CD3OD, and CDCl3. The preferred conformation of 3 accorded with the results of hard-sphere exo-anomeric (HSEA) calculations.

[Serologic demonstration of the activity of a Mycobacterium leprae antigen obtained by chemical synthesis]. / Comprobación serólogica de la actividad de un antígeno del Mycobacterium leprae obtenido por síntesis química.

Following the chemical isolation and characterization of the glycolipid phenolic I by Hunter and Brennan in 1981, derived from infected armadillo liver, studies were continued to achieve the chemical synthesis of this trisaccharide, which is part of the glycolipid and, as it has been demonstrated, was the major antigenic determinant of this substance. The synthetic antigen obtained by Fujiwara in 1984 and Gigg in 1985, was conjugated with bovine albumin. Immunodominance of the terminal residue 3,6-Di-O-methyl-glucose was confirmed by the use of ELISA, monoclonal and polyclonal antibodies. In Cuba, Mariño and Verez, based on this knowledge obtained the antigen by another way of synthesis conjugated with acrylamide against positive and negative (71%) control sera, as well as its specificity in the reaction with sera from tuberculous patients and children vaccinated with BCG (89%).

Chemical synthesis of an artificial antigen containing the trisaccharide hapten of Mycobacterium leprae.

The trisaccharide allyl O-(3,4-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl-al pha-L- rhamnopyranosyl)-(1----2)-3-O-methyl-alpha-L-rhamnopyranoside was synthesized from partially methylated monosaccharide derivatives. Condensation of 1,4-di-O-acetyl-2,3-di-O-methyl-alpha-L-rhamnopyranose promoted by boron trifluoride etherate with the appropriate alcohol proceeded stereoselectively and with very high yields. Selective deacetylation and glycosylation with 2,4-di-O-acetyl-3,6-di-O-methyl-alpha-D-glucopyranosyl bromide led to a trisaccharide. The acrylamide copolymers of mono-, di-, and tri-saccharide were compared in their ability to specifically bind antibodies from leprosy patients.

Bencilación selectiva del metil 4, 6-0-benciliden-Ó-D-manopiranósido / Selective Benzylation of Methyl 4, 6-0-benzylidene-Ó-d-mannopyranoside

Se intentó llevar a cabo la monobencilación del compuesto (II) siguiendo un método informado en la literatura y se obtuvieron resultados diferentes a los descritos. La estructura de los productos se estableció por hidrólisis suavemente ácida y oxidación peryódica. Además se corroboró ésta por espectrometría de masa, por r. m. n. y por la síntesis de uno de los productos utilizando otro método informado en la literatura. Se ensayaron varios métodos para lograr la obtención selectiva de uno de los dos isómeros monobencilados. Los mejores resultados se obtuvieron utilizando el método de laquilación interfásica. Este compuesto brinda gran interés para la síntesis de oligosacáridos(AU)