ABSTRACT
Recent changes in the epidemiology of meningococcal have been reported and meningococcal group W (MenW) has become the third most prevalent group isolated in Brazil in the last 10 years. In this study we have developed a conjugate vaccine for MenW using a modified reductive amination conjugation method through a covalent linkage between periodate-oxidized MenW non-O-acetylated polysaccharide and hydrazide-activated monomeric tetanus toxoid. Process control of bulks was done by physicochemical analysis including polysaccharide and protein quantification, high performance liquid chromatography - size exclusion chromatography, capillary electrophoresis, and hydrogen nuclear magnetic resonance. Conjugate bulks were best produced with concentration of polysaccharide twice as high as protein, at room temperature, and pH approximately 6.0. A scaled-up bulk (100 mg scale) was formulated and inoculated intramuscularly in mice in a dose-response study (0.1, 0.5, 1.0 and 10.0 µg of polysaccharide/dose). The immunogenicity of conjugate bulks was determined by serum bactericidal assay and ELISA assays of serum from immunized mice. ELISA and SBA titers revealed high titers of IgG and demonstrated the functionality of the antibodies produced in all doses studied 15 days after the third dose. However, significant differences were observed among them by ELISA. In conclusion, this study established the best conditions to produce MenW conjugate bulks and showed the efficacy of the obtained conjugate bulk in induce a good immune response in mice. Further experiments will need to be done to scale up the conjugation reaction and then allow the use of this conjugate in clinical trials.
Subject(s)
Meningococcal Infections/epidemiology , Meningococcal Infections/prevention & control , Meningococcal Vaccines/immunology , Neisseria meningitidis/classification , Animals , Antibodies, Bacterial , Blood Bactericidal Activity , Brazil/epidemiology , Female , Glycoconjugates , Humans , Male , Mice , Pilot Projects , Tetanus Toxoid/immunology , Vaccines, Conjugate/immunologyABSTRACT
Streptococcus pneumoniae (pneumococcus) is among the most significant causes of bacterial disease in humans. Capsular polysaccharide (CPS) production is essential for pneumococcal virulence. Pneumococcal CPS has been widely used as vaccine antigen. This study is focused on the influence of culture conditions of Streptococcus pneumoniae serotype 14 as for developing an industrial method for polysaccharide production. The pH proved to be a highly important variable in batchwise culture. Using the pH control all glucose added was consumed resulting in a four-fold increase in polysaccharide productivity relative to cultivation without pH control. S. pneumoniae is a lactic acid bacterium, so named for its primary metabolic byproduct (lactate), which has an inhibitory effect on cell growth in concentrations ranging from 4 to 5 g/L. An increase of 30 percent in polysaccharide productivity was observed using glucose pulses with 5.5 hrs of growth, resulting in a maximum polysaccharide concentration of 185.2 mg/L. Our data suggest the possibility of using a medium of non-animal origin and employing pH control for the cultivation of pneumococcus to produce a polysaccharide vaccine.