Quantitative NMR for the structural analysis of novel bivalent glycoconjugates as vaccine candidates.

Novel unimolecular bivalent glycoconjugates were assembled combining several functionalized capsular polysaccharides of Streptococcus pneumoniae and Neisseria meningitidis to a carrier protein by using an effective strategy based on the Ugi 4-component reaction. The development of multivalent glycoconjugates opens new opportunities in the field of vaccine design, but their high structural complexity involves new analytical challenges. Nuclear Magnetic Resonance has found wide applications in the characterization and impurity profiling of carbohydrate-based vaccines. Eight bivalent conjugates were studied by quantitative NMR analyzing the structural identity, the content of each capsular polysaccharide, the ratios between polysaccharides, the polysaccharide to protein ratios and undesirable contaminants. The qNMR technique involves experiments with several modified parameters for obtaining spectra with quantifiable signals. In addition, the achieved NMR results were combined with the results of colorimetric assay and Size Exclusion HPLC for assessing the protein content and free protein percentage, respectively. The application of quantitative NMR showed to be efficient to clear up the new structural complexities while allowing the quantitative assessment of the components.

Quantitative assessment of C-polysaccharide in capsular polysaccharides of Streptococcus pneumoniae by 31PNMR.

Capsular polysaccharides of Streptococcus pneumoniae are key components of commercially available anti-pneumococcal vaccines; meanwhile C-polysaccharide is considered an impurity. World Health Organization recommends a strict control over the presence of this biomolecule due to the possibility of introducing an undesired response. An alternative way for assessing this impurity is focused on detect the phosphocholine residues by means of quantitative 1H-NMR. This could be tricky due to the amounts of this substituent may vary generating two C-polysaccharides forms. In this work we propose an improved quantitative NMR methodology based on 31P-NMR for the quantification of C-polysaccharide on capsular polysaccharide preparations. The technique also focuses on phosphocholine but, conversely to above-mentioned methods, allows to discriminate between phosphocholine linked in different positions. The methodology was run on samples of eleven vaccine serotypes, including seven with phosphate groups. From a rational acceptance criterion of 10 wt%, the method allows to quantified from 30 µg of the impurity in 3 mg of total polysaccharide (1 wt%) with a signal/noise ratio of 16:1. Repeatability and intermediate precision evaluation showed a relative standard deviation of 3.33 % and 8.34 % respectively. Additionally, the method provides information about structural identity of phosphate contained in capsular polysaccharides and C-polysaccharide species. This constitutes a new contribution from the NMR that highlights the power of these techniques for assessing imperative parameters in carbohydrate-based vaccines.

Synthetic, Zwitterionic Sp1 Oligosaccharides Adopt a Helical Structure Crucial for Antibody Interaction.

The zwitterionic Streptococcus pneumoniae serotype 1 polysaccharide (Sp1) is an important anchor point for our immune system to act against streptococcal infections. Antibodies can recognize Sp1 saccharides, and it has been postulated that Sp1 can elicit a T-cell-dependent immune reaction as it can be presented by MHC-II molecules. To unravel the molecular mode of action of this unique polysaccharide we here describe the chemical synthesis of a set of Sp1 fragments, ranging from 3 to 12 monosaccharides in length. We outline a unique synthetic approach to overcome the major synthetic challenges associated with the complex Sp1 structure and provide a stereoselective route of synthesis for the oligosaccharide backbone as well as a strategy to introduce the carboxylic acid functions. Molecular dynamics (MD) simulations together with NMR spectroscopy studies reveal that the oligosaccharides take up helical structures with the nona- and dodecasaccharide completing a full helical turn. The 3D structure of the oligosaccharides coincides with the topology required for good interaction with anti-Sp1 antibodies, which has been mapped in detail using STD-NMR. Our study has revealed the Sp1 nona- and dodecasaccharides as promising synthetic antigens, displaying all (3D) structural elements required to mimic the natural polysaccharide and required to unravel the molecular mode of action of these unique zwitterionic polysaccharides.

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.

Influence of the Co-Administration of Heptavalent Conjugate Vaccine PCV7-TT on the Immunological Response Elicited by VA-MENGOC-BC® and Heberpenta®-L in Rabbits.

Finlay Vaccine Institute is developing a new heptavalent conjugate vaccine against Streptococcus pneumoniae. As infants are the target population, PCV7-TT will be necessarily co-administered with other vaccines, and then, the interactions represent a concern. The aim of this work is to evaluate the possible immunological interferences in rabbits as animal experimental model. Rabbits were immunized with Heberpenta®-L, VA-MENGOC-BC®, and PCV7-TT. Blood samples were taken fourteen days after final immunization for obtaining sera. Antibody responses to all antigens were evaluated by indirect ELISA. Functional responses against diphtheria and tetanus toxoid were done by in vivo seroneutralization assay. No interference was observed by PCV7-TT over the humoral response against diphtheria toxoid and meningococcal antigens (p > 0.05). A nonstatistically significant reduction (p > 0.05) was observed in the case of the humoral response against Haemophilus influenzae type b oligosaccharide. Concomitant administration of Heberpenta®-L and PCV7-TT increased twice the antibody titers as well as the protective activity against tetanus toxoid, but no statistical differences were found. The co-administration did not induce a reduction in the percent of responders against pneumococcal polysaccharides contained in PCV7-TT vaccine. Concomitant administration of PCV7-TT did not induce interferences over the evaluated antigens of Heberpenta®-L and VA-MENGOC-BC®. Also, no interference was observed on the immune response elicited by PCV7-TT. These preclinical results suggest that PCV7-TT will not result in a serious problem over the immune response elicited by the licensed vaccines Heberpenta®-L and VA-MENGOC-BC®. However, the clinical interference could be strictly studied during clinical trials in infants.

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)