Sensitive quantitation of low level free polysaccharide in conjugate vaccines by size exclusion chromatography-reverse phase liquid chromatography with UV detection.

The level of free polysaccharide is a critical quality attribute of polysaccharide-protein conjugate vaccines. The work presented describes a simple and sensitive method for the determination of low level free polysaccharides in multiple polysaccharide-protein conjugates. The method utilizes a reverse phase (RP) column to perform a size exclusion chromatography (SEC) separation of free polysaccharide and a reverse phase liquid chromatography (RPLC) separation of free protein and protein-polysaccharide conjugate. The use of phosphate buffer in the mobile phase enables the universal and sensitive detection of low level free polysaccharides at UV 200 nm. The method has been validated to monitor low level free polysaccharide (<1 %) in multiple polysaccharide-protein conjugates. The limit of quantitation is 2 µg/ml or 0.3 % free polysaccharide in 0.6 mg/ml polysaccharide-protein conjugate. The accuracy is in the range of 94.1.0-108.5 %.

Progress toward the global control of Neisseria meningitidis: 21st century vaccines, current guidelines, and challenges for future vaccine development.

The control of meningitis, meningococcemia and other infections caused by Neisseria meningitidis is a significant global health challenge. Substantial progress has occurred in the last twenty years in meningococcal vaccine development and global implementation. Meningococcal protein-polysaccharide conjugate vaccines to serogroups A, C, W, and Y (modeled after the Haemophilus influenzae b conjugate vaccines) provide better duration of protection and immunologic memory, and overcome weak immune responses in infants and young children and hypo-responsive to repeated vaccine doses seen with polysaccharide vaccines. ACWY conjugate vaccines also interfere with transmission and reduce nasopharyngeal colonization, thus resulting in significant herd protection. Advances in serogroup B vaccine development have also occurred using conserved outer membrane proteins with or without OMV as vaccine targets. Challenges for meningococcal vaccine research remain including developing combination vaccines containing ACYW(X) and B, determining the ideal booster schedules for the conjugate and MenB vaccines, and addressing issues of waning effectiveness.

Evaluation of the immunogenicity and efficacy of a Hib polysaccharide-protein conjugate vaccine by using PsaA as carrier protein / 中华微生物学和免疫学杂志

Objective To prepare a conjugate vaccine by linking Haemophilus influenzae type b (Hib)polysaccharide to PsaA protein carrier and evaluate the immunogenicity and efficacy of the conjugate vaccine. Methods A recombinant protein rPsaA,expressed by using the genetic engineering technology, was used as a protein carrier to prepare conjugate vaccine together with Hib polysaccharide. Ten mice at age of 3 weeks were immunized with the conjugate vaccine,while another 10 age-matched mice were immunized with Hib-tetanus toxoid(Hib-TT)vaccine which was produced formerly as a control. The mice treated with equal volume of PBS were set up as the negative control. The IgG antibodies in serum samples against PsaA and Hib polysaccharide were detected in two weeks after the final immunization. A suspension of Pneumococ-cus was injected into the middle ears of mice from experiment and control group. Histopathological analysis was performed to measure the clearance of bacteria in the middle ears and the severity of infection on days 3 and 7 after bacterial challenge. Results The rPsaA protein was prepared by the genetic engineering tech-nology and purified successfully with anion-exchange column. The Hib polysaccharide-PsaA protein conju-gate vaccine was prepared through a series of amide condensation reactions. The detection of IgG antibodies against PsaA protein and Hib polysaccharide in the immunized mice demonstrated that there was no signifi-cant difference with the titer of IgG against Hib polysaccharide between the mice immunized with the Hib-PsaA conjugate vaccine and those immunized with the Hib-TT vaccine. Less Pneumococcus strains were de-tected in the middle ears of mice immunized with the conjugate vaccine than those mice immunized with the Hib-TT vaccine three days after challenge. The mice from control group showed severe inflammation in the middle ears than those from experiment group. The Hib polysaccharide-PsaA protein conjugate vaccine im-proved protection against Pneumococcus infections as compared with the Hib-TT vaccine. Conclusion The rPsaA protein could be produced by genetic engineering technology and purified by anion-exchange column. The Hib polysaccharide was successfully conjugated with the rPsaA protein through amide condensation reac-tion. Both anti-PsaA and anti-Hib immune responses were induced in young mice by the injection of Hib pol-ysaccharide-PsaA protein conjugate vaccine. Apart from providing protection against Hib infection,the con-jugate vaccine might also be used for the prevention of acute otitis media caused by Pneumococcus infection.

The effect of meningococcal serogroup A polysaccharide-protein conjugate vaccine prepared with TT and rCTB on mucosal immune responses / 中华微生物学和免疫学杂志

Objective To analyze the feasibility of the recombinant cholera toxin B subunit (rCTB) as a carrier protein candidate for the preparing of polysaccharide-protein conjugate, and to discuss the immune effects of tetanus toxoid (TT) as the carrier protein in mucosal delivery vaccine. Methods The refolded pentrumer protein, rCTB was obtained by genetic engineering methods. Then conjugated the refold-ed protein with group A meningococcal polysaccharide (GAMP) using the chemical method(ADH) ,the pol-ysaccharide-protein conjugates(GAMP-rCTB) were prepared. BALB/c mice were immunized either intraper-itoneally ( i. p. ) or intranasally ( i. n. ) with GAMP-rCTB. Moreover, GAMP-TT vaccine that TT as carrier proteins was i.n. immunized to the mice. The evaluation of immunology is performed. Results The conju-gates of polysaccharide-potein with the rCTB and TT as protein carrier both are able to elicit high level of GAMP specific IgG antibody in serum after i.n. immunization, and the conjugates can also elicit specific IgA antibody in lung lavage and intestinal mucosa. Conclusion rCTB and TT can both as the protein carri-er for polysaccharide-protein conjugate as mucosal vaccine. The route of intranasal may be more ways for im-mune function than i.p. immunization when rCTB is used as the carrier of the polysaccharide-protein conju-gates.