Optimization of the Process for Preparing Bivalent Polysaccharide Conjugates to Develop Multivalent Conjugate Vaccines against Streptococcus pneumoniae or Neisseria meningitidis and Comparison with the Corresponding Licensed Vaccines in Animal Models

Objective:This study aimed to describe,optimize and evaluate a method for preparing multivalent conjugate vaccines by simultaneous conjugation of two different bacterial capsular polysaccharides(CPs)with tetanus toxoid(TT)as bivalent conjugates.Methods:Different molecular weights(MWs)of polysaccharides,activating agents and capsular polysaccharide/protein(CP/Pro)ratio that may influence conjugation and immunogenicity were investigated and optimized to prepare the bivalent conjugate bulk.Using the described method and optimized parameters,a 20-valent pneumococcal conjugate vaccine and a bivalent meningococcal vaccine were developed and their effectiveness was compared to that of corresponding licensed vaccines in rabbit or mouse models.Results:The immunogenicity test revealed that polysaccharides with lower MWs were better for Pn1-TT-Pn3 and MenA-TT-MenC,while higher MWs were superior for Pn4-TT-Pn14,Pn6A-TT-Pn6B,Pn7F-TT-Pn23F and Pn8-TT-Pn11A.For activating polysaccharides,1-cyano-4-dimethylaminopyridinium tetrafluoroborate(CDAP)was superior to cyanogen bromide(CNBr),but for Pn1,Pn3 and MenC,N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride(EDAC)was the most suitable option.For Pn6A-TT-Pn6B and Pn8-TT-Pn11A,rabbits immunized with bivalent conjugates with lower CP/Pro ratios showed significantly stronger CP-specific antibody responses,while for Pn4-TT-Pn14,higher CP/Pro ratio was better.Instead of interfering with the respective immunological activity,our bivalent conjugates usually induced higher IgG titers than their monovalent counterparts.Conclusion:The result indicated that the described conjugation technique was feasible and efficacious to prepare glycoconjugate vaccines,laying a solid foundation for developing extended-valent multivalent or combined conjugate vaccines without potentially decreased immune function.

From Monovalent to Multivalent Vaccines, the Exploration for Potential Preventive Strategies Against Hand, Foot, and Mouth Disease(HFMD)

Hand,foot,and mouth disease(HFMD)recently emerged as a global public threat.The licensure of inactivated enterovirus A71(EV-A71)vaccine was the first step in using a vaccine to control HFMD.New challenges arise from changes in the pathogen spectrum while vaccines directed against other common serotypes are in the preclinical stage.The mission of a broad-spectrum prevention strategy clearly favors multivalent vaccines.The development of multivalent vaccines was attempted via the simple combination of potent monovalent vaccines or the construction of chimeric vaccines comprised of epitopes derived from different virus serotypes.The present review summarizes recent advances in HFMD vaccine development and discusses the next steps toward a safe and effective HFMD vaccine that is capable of establishing a crossprotective antibody response.

Immune responses against Schistosoma japonicum after vaccinating mice with a multivalent DNA vaccine encoding integrated membrane protein Sj23 and cytokine interleukin-12

Background The vaccination of mice with DNA encoding single candidate antigens has failed to induce significant protection against Schistosoma japonicum (S. japonicum) challenge infections In this study, we evaluated the feasibility of using a multivalent DNA vaccine which co expressed S japonicum integral membrane protein Sj23 and murine cytokine IL 12 to induce protective immune responses Methods The plasmid pVIVO2 IL12 Sj23, a eukaryotic expression vector expressing Sj23 and murine IL 12 simultaneously, was constructed, identified, and tested for expression in vitro Its ability to protect against S japonicum challenge infections was analyed according to worm reduction rate and egg reduction rate after vaccination of BALB/c mice The serum levels of specific IgG antibody were determined by enzyme linked immuno sorbent assay (ELISA) and Western blot analysis Using cultured spleen cells, IFN γ and IL 4 post stimulation were quantified by ELISA The phenotypes of splenocyte populations were analyzed by flow cytometry (FCM) Results The plasmid DNA pVIVO2 IL12 Sj23 was proven to express well in vitro by transient transfection of HEK 293 cells Immunization resulted in a worm reduction rate of 45 53% and egg reduction rate of 58 35% ELISA and Western blot analysis indicated that immunized mice generated specific IgG against Sj23 Spleen cells showed significant increases in IFN γ but decreases in IL 4 No significant differences in CD4 + and CD8 + subgroup ratios were observed after the challenges Conclusions The multivalent DNA vaccine pVIVO2 IL12 Sj23 is sufficient to elicit moderate but highly significant levels of protective immunity against challenge infections Cytokine IL 12, as a gene adjuvant, was able to enhance the Th1 responses and, hence, the protective immunity