Multicomponent polysaccharide-protein bioconjugation in the development of antibacterial glycoconjugate vaccine candidates.

A new synthetic strategy for the development of multivalent antibacterial glycoconjugate vaccines is described. The approach comprises the utilization of an isocyanide-based multicomponent process for the conjugation of functionalized capsular polysaccharides of S. pneumoniae and S. Typhi to carrier proteins such as diphtheria and tetanus toxoids. For the first time, oxo- and carboxylic acid-functionalized polysaccharides could be either independently or simultaneously conjugated to immunogenic proteins by means of the Ugi-multicomponent reaction, thus leading to mono- or multivalent unimolecular glycoconjugates as vaccine candidates. Despite the high molecular weight of the two or three reacting biomolecules, the multicomponent bioconjugation proved highly efficient and reproducible. The Ugi-derived glycoconjugates showed notable antigenicity and elicited good titers of functional specific antibodies. To our knowledge, this is the only bioconjugation method that enables the incorporation of two different polysaccharidic antigens to a carrier protein in a single step. Applications in the field of self-adjuvanting, eventually anticancer, multicomponent vaccines are foreseeable.

Caracterización de conjugados inmunogénicos de polisacárido capsular Streptococcus pneumoniae serotipo 14 / Characterization of capsular polysaccharide immunogenic conjugates from Streptococcus pneumoniae serotype 14

Las vacunas conjugadas que consisten en polisacáridos bacterianos unidos a través de un enlace covalente a una proteína portadora, han tenido un gran impacto en los esquemas de vacunación infantil, disminuyendo de forma dramática la incidencia de infecciones bacterianas. En el caso de Streptococcus pneumoniae, a pesar de que se han descrito más de 90 serotipos basados en la estructura de las cápsulas polisacarídicas y que al menos 23 tienen una importancia clínica demostrada, solo un número limitado de siete, o más recientemente 10 y 13, están incluidos en las vacunas conjugadas licenciadas. Por otra parte, la necesidad creciente de estas vacunas en el mundo requiere la incorporación de nuevos productores que se enfrentan a una elevada complejidad tecnológica, pues en todo el procedimiento de conjugación no se pueden afectar las características estructurales por las que el polisacárido es reconocido inmunológicamente. Este trabajo implementó un procedimiento de conjugación para el polisacárido de la cápsula de Streptococcus pneumoniae serotipo 14. El procedimiento comprendió la fragmentación, oxidación peryódica y posterior conjugación del polisacárido a anatoxina tetánica o diftérica. Cada intermedio fue caracterizado por métodos físico-químicos. En todas las reacciones se obtuvieron rendimientos superiores al 50%. Los conjugados generaron altos títulos de anticuerpos específicos de tipo IgG y memoria inmunológica. Se concluyó que el procedimiento permitió la obtención de conjugados inmunogénicos de serotipo 14(AU)

Conjugate vaccines consisting of bacterial polysaccharides linked through a covalent bond to a carrier protein have a major impact on childhood immunization schemes which have dramatically decrease the incidence of bacterial infections. In the case of Streptococcus pneumoniae more than 90 serotypes have been reported, based on the structure of the polysaccharide capsules and at least 23 of them have demonstrated clinical importance. A limited number of 7 or more recently 10 and 13 are included in licensed conjugate vaccines. On the other hand, the increasing need for these vaccines worldwide requires the incorporation of new manufacturers who are facing highly complex technology since the entire conjugation process can not affect the structural features for which the polysaccharide is immunologically recognized. Our paper provides a conjugation procedure for the capsular polysaccharide of Streptococcus pneumoniae serotype 14. The process includes fragmentation, peryodic oxidation and subsequent conjugation to tetanus toxoid or diphtheria toxoid to the polysaccharide, each intermediate was characterized by physico-chemical methods. Yields higher than 50% were obtained in all reactions. The conjugates generated high titers of IgG specific antibodies and immunological memory. In conclusion, the procedure allows immunogenic conjugates of serotype 14(AU)

Relevance of O-acetyl and phosphoglycerol groups for the antigenicity of Streptococcus pneumoniae serotype 18C capsular polysaccharide.

Capsular polysaccharides are important virulence factors of Streptococcus pneumoniae. The polysaccharide has been used as a component of vaccines against pneumococcal diseases either as plain polysaccharide or better conjugated to a protein. The last one is the vaccine of choice to target child protection. The immune responses depend on several polysaccharide physicochemical properties that can be affected during either purification or modification in the case of conjugate vaccines. In serotype 18C, the repeating unit has a complex structure having a branched pentasaccharide with two apparently labile subtituents: glycerol-phosphate and O-acetyl group. The loss of these groups may potentially reduce the ability of the 18C polysaccharide to induce the desired immune response. Therefore, the relationship of both groups with the antigenicity and immunogenicity of 18C capsular polysaccharide is explored. It is shown that glycerol-phosphate must be preserved for conserving adequate antigenicity of the 18C capsular polysaccharide. At the same time, it was proved that O-acetyl groups do not play any role for the antigenicity and immunogenicity.

High-performance reverse phase chromatography with fluorescence detection assay for characterization and quantification of pneumococcal polysaccharides.

A methods using high-performance reverse phase (RP) chromatography with fluorescence detection, has been developed to determine the composition and identity of Streptococcus pneumoniae capsular polysaccharide used in formulating conjugate vaccine for prevention of pneumococcal infection. For the monosaccharide composition, the polysaccharides were subjected to hydrofluoric acid (HF) hydrolysis followed by trifluoroacetic acid (TFA). After acid hydrolysis, the released monosaccharides were re-N-acetylated and labeled with 2-aminobenzamide (2AB) by reductive amination reaction. High-performance RP chromatography was performed on C18 TSKODS 120T column. Nuclear magnetic resonance was used to confirm chemical structure and purity of pneumococcal capsular polysaccharides.

Enzyme-linked immunosorbent assay for quantitative determination of capsular polysaccharide production in Streptococcus pneumoniae clinical isolates.

A simple, specific, sensitive and reproducible ELISA has been developed to quantify the level of CPS (capsular polysaccharide) production in supernatants of Streptococcus pneumoniae cell cultures. CPSs from Strep. pneumoniae have been widely used as vaccine antigens. The quantification method is based on two type-23F serotype-specific polyclonal antibodies: IgG, purified from sera of mice immunized with a pneumococcal type-23F CPS conjugate, used in the coating step, and a serotype-specific rabbit serum as the second antibody. Solutions of purified type-23F CPS were used as standards. The relationship between A(492) and type-23F CPS concentration was linear over the range 1-310 ng/ml (r=0.989), with 1 ng/ml as the lower limit of sensitivity. The specificity of ELISA was assessed because purified type-19F CPS and cell-wall polysaccharide samples were not detected after their evaluation by the ELISA described in the present study. Repeatability and intermediate precision of the assay were good, the coefficients of variation being 3 and 10% respectively. This ELISA allowed selection of an appropriate vaccine strain, for a natural polysaccharide vaccine, among several 23F pneumococcal clinical isolates and constituted a valuable analytical tool for Strep. pneumoniae fermentation and CPS purification follow-up.

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.