Academic literature on the topic 'C-polysaccharide'

Cette thèse issue d'une collaboration entre l'Université Lyon 1 et Sanofi Pasteur (SP) porte sur l'étude du procédé de dépolymérisation et d'activation d'un polyoside capsulaire. Cette réaction est la première étape du couplage d'un vaccin (Menactra®) antiméningococcique conjugué (polyoside du méningocoque de groupe C conjugué à l'anatoxine diphtérique). L'objectif de ce travail, réalisé dans le cadre d'un programme d'amélioration de la conformité et de la robustesse des procédés de SP, est la compréhension du mécanisme et l'optimisation des paramètres clés de cette réaction. Le procédé de couplage de ce vaccin tel qu'il est décrit par SP comporte 3 étapes clés : la dépolymérisation/activation du polyoside par le peroxyde d'hydrogène, la dérivatisation par un "linker" et le greffage à la protéine. Si les 2 dernières étapes sont des réactions chimiques bien connues, la première qui permet, à la fois de réduire la masse molaire du polyoside et de générer des groupements réducteurs, est plus obscure. Une stratégie a été élaborée afin de comprendre cette réaction. Dans un premier temps, l'étude poussée du procédé a permis d'identifier les paramètres impactant la cinétique de dépolymérisation et l'activité réductrice. Ensuite, l'analyse structurale, par diverses techniques, du polyoside dépolymérisé a confirmé l'activation. Enfin, la caractérisation de modifications chimiques de macromolécules étant relativement complexe, de plus petits modèles (monomère, tétramère) ont été utilisés et ont permis d'établir un mécanisme réactionnel de la dépolymérisation du polyoside. A partir de ces résultats, plusieurs solutions ont été proposées à l'industriel pour améliorer le rendement et/ou la robustesse du procédé<br>This PhD work, initiated by Sanofi Pasteur in collaboration with the University of Lyon 1, concerns the study of the Menactra® vaccine, a glycoconjugate vaccine produced by covalently coupling Neisseria meningitidis serogroups A, C, W135, Y capsular polysaccharides to diphtheria toxoid. The objective was to better understand the chemistry involved in the conjugation process of the vaccine, in order to improve the process robustness and the overall conjugated yields with particular emphasis on the serogroup C. The conjugation process can be divided into 3 key steps: depolymerization/activation by hydrogen peroxide, derivatization, and conjugation. While the 2 last steps of the process are well known in bioconjugation chemistry, the exact mechanism of the first step, which serves 2 purposes, first to reduce the polysaccharide molecular weight and second, to generate the reducing groups on the polysaccharide chain, is poorly understood. An overall strategy for the characterization of the serogroup C polysaccharide depolymerization process was successfully applied to understand this reaction. We first provided a process description of this step, identified and optimized the key process parameters. Then, the structural comparison of the polysaccharide before and after the depolymerization obtained with specified analytical methods gave important information on the mechanism. Finally, well defined sialic and tetrasialic acids were reacted with H2O2 to complete the elucidation of this complex mechanism. From these results, several solutions were proposed to the industrial to improve the yield and the robustness

C-reactive protein (CRP) is a part of the innate immune system, is synthesized in the liver, its blood level increases in inflammatory states, and it binds to Streptococcus pneumoniae. The conformation of CRP is altered under conditions mimicking an inflammatory milieu and this non-native CRP also binds to immobilized/aggregated/pathogenic proteins. Experiments in mice have revealed that one of the functions of CRP is to protect against pneumococcal infection. For protection, CRP must be injected into mice within two hours of administering pneumococci, thus, CRP is protective against early-stage infection but not against late-stage infection. It is unknown how CRP protects or why CRP does not protect against late-stage infection. The hypotheses are that the protection requires complement activation by CRP-pneumococci complexes and that CRP cannot protect if pneumococci have time to recruit complement inhibitor factor H on their surface to become complement attack-resistant. To test these hypotheses, we generated CRP mutants by site-directed mutagenesis: a mutant that binds to pneumococci but does not activate complement and a mutant that binds to immobilized factor H. We found that mutant CRP incapable of activating complement was not protective against infection and that mutant CRP capable of binding to factor H was protective against both early and late stage infections. Additional experiments showed that CRP enhances the effects of the antibiotic clarithromycin in reducing bacteremia in infected mice. Moreover, we observed that mutant CRP capable of binding to factor H bound to several proteins immobilized on plastic, suggesting that CRP recognizes a pattern, probably an amyloid-like structure, on immobilized proteins. Indeed, mutant CRP, after binding to amyloid b peptides, prevented the formation of pathogenic amyloid fibrils. Lastly, employing a hepatic cell line, we investigated the mechanism of CRP expression in response to pro-inflammatory cytokines. We found that the transcription factor C/EBPb and two C/EBP-binding sites on the CRP promoter were critical for inducing CRP expression. We conclude that complement activation is necessary for CRP-mediated protection against infection, that CRP functions in two structural conformations, that CRP and clarithromycin act synergistically, that CRP has anti-amyloidogenic properties, and the increased CRP expression requires C/EBPb.

The anti-pneumococcal function of native C-reactive protein (CRP) involves its binding to phosphocholine molecules present on Streptococcus pneumoniae and subsequent activation of the complement system. However, when pneumococci recruit complement inhibitory protein factor H on their surface, they escape complement attack. Non-native forms of CRP have been shown to bind immobilized factor H. Accordingly, we hypothesized that modified CRP would bind to factor H on pneumococci, masking its complement inhibitory activity, allowing native CRP to exert its anti-pneumococcal function. As reported previously, native CRP protected mice from lethal pneumococcal infection when injected 30 minutes before infection but not when injected 24 hours after infection. However, a combination of native and mutant CRP was found to protect mice even when administered 24 hours after infection. Therefore, it is concluded that while native CRP is protective only against early-stage infection, a combination of native and mutant CRP offers protection against late-stage infection.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-12-10T12:32:54Z No. of bitstreams: 1 iaralice-souza.pdf: 1338553 bytes, checksum: 59fa26df56bce23aa1b5861c460f716f (MD5)<br>Made available in DSpace on 2012-12-10T12:32:54Z (GMT). No. of bitstreams: 1 iaralice-souza.pdf: 1338553 bytes, checksum: 59fa26df56bce23aa1b5861c460f716f (MD5) Previous issue date: 2001<br>Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.<br>Neisseria meningitidisdo grupo C é uma bactéria encapsulada causadora dediversas doenças, está associada à altas taxas de mortalidade e portanto é de grande importância para a saúde pública. Bio-Manguinhos está desenvolvendo uma vacina conjugada formada pela ligação covalente do polissacarídeo capsular àanatoxina tetânica e esta vacina, atualmente, está sendo avaliada em estudos clínicosde Fase II em crianças de 1 a 9 anos. A quantificação de componentes livres como polissacarídeos e proteínas faz parte do controle de processo de vacinas conjugadas e tem o objetivo de evitar o aparecimento de reações adversas exacerbadas e/ou redução da imunogenicidade do componente vacinal. A Organização Mundial de Saúde preconiza níveis máximos de proteína livre no conjugado vacinal de 5%, mas não estabelece um limite máximo para o polissacarídeo livre para a vacina conjugada contra o grupo C. Desta forma, o objetivo deste estudo foi desenvolver e validar métodos de controle de qualidade adequados para separar e quantificar estes componentes livres presentes na vacina meningocócica C conjugada brasileira, utilizando a técnica de eletroforese capilar (EC). Para a separação da proteína livre foram comparados os modos de eletroforese capilar de zona livre (CZE) e cromatografia eletrocinética micelar (MEKC). Diferentes condições de migração da amostravariando-se parâmetros como pH, temperatura, tensão, concentração do tampão, ciclodextrinas e de surfactante dodecil sulfato de sódio (SDS) foram estudadas. Os resultados demonstraram que a melhor separação do conjugado foi obtida por MEKC utilizando tampão tetraborato de sódio (TBNa) 150 mM, 25 mM de SDS, 60°C, 30 kV e pH 9,3. Entretanto, nos modos de EC estudados não foi possível obter a separação completa dos componentes, sendo necessária a utilização de outro processo. Por outro lado, por CZE foi possível observar a separação da proteína ativada da nativa, demonstrando a necessidade de otimização da reação de ativação da proteína, a fim de aumentar orendimento da reação de conjugação. A separação completa do açúcar livre presente no conjugado foi obtida empregando CZE utilizando tampão TBNa 50 mM, 40°C, 30 kV e pH 10. Com as condições escolhidas foi possível determinar o conteúdo de polissacarídeo livre nos lotes de conjugado e validar o método proposto, que se mostrou linear na faixa de 0,047 a 0,164 mg/mL, apresentou efeito matriz, 0,0154 mg/mL de limite de detecção e 0,0454mg/mL de limite de quantificação. Após as etapas de validação, foram quantificados alguns lotes de conjugado e observou-se um alto teor de açúcar livre nos lotes com longo período de estocagem a 4°C. Desse modo, fez-se a avaliação de um lote recentemente produzido e obteve-se o valor de 19,08% de polissacarídeo livre. A fim de estimar o tempo de estocagem máximo do conjugado foram realizadas análises com 30, 60 e 90 dias de estocagem a 4°C. Os valores encontrados até 60 dias não foram significativamente diferentes dosdeterminados no tempo zero. No entanto, com 90 dias de estocagem ocorreu uma modificação do perfil do conjugado que impossibilitou a sua quantificação. A metodologia desenvolvida e validada será introduzida no controle de qualidade do lote de conjugado que será submetido aos estudos clínicos de Fase III e na rotina da vacina conjugada estudada. Além disto, o conhecimento adquirido poderá ser empregado no controle de qualidade de outras vacinas conjugadas contra bactérias encapsuladas de interesse epidemiológico no país.<br>Neisseria meningitidisgroup C is an encapsulated bacterium that causes several diseases and is associated with high mortality rates becoming a serious public health problem. Bio-Manguinhos is developing a conjugate vaccine constituted by covalent attachment of capsular polysaccharide to tetanus toxoid, which iscurrently being evaluated in Phase II clinical studies in children between 1-9 years. Free components quantification is a vaccine process control assay and intended to prevent exacerbated adverse reactions occurrence and/or vaccine immunogenicity reduction. The World Health Organization recommends 5% of free protein maximum level in the conjugate vaccine, but does not set a limit for the free polysaccharide contents. Thus, the aim of this study was to develop and validate quality control methods appropriate to separate and quantify free components present in the conjugate vaccine against N. meningitidisgroup C, using capillary electrophoresis (CE) technique. For free protein separation, free capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were compared and different sample migration conditions were studied by varying parameters such as pH, temperature, voltage, buffer concentration, cyclodextrin and surfactant sodium dodecyl sulfate (SDS). The results showed that the best separation was obtained by MEKC using sodium tetraborate buffer (TBNA) 150 mM, 25 mM SDS, 60°C, 30 kV and pH 9.3. H owever, the CE did not induce a complete separation of the components suggesting that other techniques should be necessary. On the other hand, native and activated protein separation was possible using CZE, demonstrating the necessity of optimize protein activation reaction in order to increase the conjugation reaction yield. The total free sugar conjugate was completely separated from the conjugate by CZE using 50 mM TBNA buffer, 40°C, 30 kV and pH 10. In these conditions it was possible to determine the free polysaccharide content and validate the proposed method, which was linear in 0.047 to 0.164 mg/mL range, showed a matrix effect, 0.0154 mg/mL of detection limit and 0.0454 mg/mL ofquantification limit.After the validation steps, some conjugate batches were quantified and high levels of free sugar were observed in batches storaged at 4°C for long periods. On the other hand a conjugate batch recently produced was evaluated and showed19.08% of free polysaccharide. In order to estimate the maximum storage time a conjugate batch was analyzed30, 60 and 90 days after the production steps. The values found up to 60 days were not significantly different from that one determined at the initial time. However, with 90 days of storage there was a change in the conjugate profile that impaired its quantification. The methodology developed and validated will be used to evaluate the conjugate batch that will be submitted to Phase III clinical studies and in the routine quality controlof the conjugate vaccine. Moreover, the acquiredknowledge could be used in quality control of other conjugate vaccines against encapsulated bacteria of epidemiological importancein the country.