Reduction of free polysaccharide contamination in the production of a 15-valent pneumococcal conjugate vaccine

Hyeseon Yoon,Yoon Hee Whang,Inhwan Lee,Chankyu Lee,Seuk Keun Choi,Yeong Ok Baik,Anirudh K Singh,Soo Kyung Kim

doi:10.1371/journal.pone.0243909

Abstract

Glycoconjugate vaccines are vaccines in which a bacterial polysaccharide antigen is conjugated to a carrier protein to enhance immunogenicity by promoting T cell-dependent immune response. However, the free (unreacted) polysaccharides remaining after the conjugation process can inhibit the immunogenicity of a conjugate vaccine. Thus, we aimed to reduce the unbound free polysaccharides in the polysaccharide-protein conjugation process for the development of a new 15-valent pneumococcal conjugate vaccine (PCV15) by varying some factors that may affect the conjugation results such as polysaccharide/protein ratio, polysaccharide size, and concentration of a coupling agent in a conjugation reaction mixture. Concentrations of a coupling agent, carbodiimide (EDAC), and a carrier protein (CRM197) used in PCV15 production, during the conjugation process, had little effect on the content of free polysaccharides. However, the size of the polysaccharide was identified as the critical factor to control the free polysaccharide content, with an inverse relationship observed between the molecular weight of the polysaccharide and the residual free polysaccharide content after conjugation. Based on these results, a new PCV15 with low free polysaccharide contamination was produced and tested for immunogenicity using a rabbit model to show that it induces similar level of immune responses in rabbits compared to a comparator vaccine Prevnar13®.

Highlights

Pneumococci are gram-positive, aerobic diplococci bacteria that are encapsulated, and cause a range of clinical infections from mild diseases such as sinusitis and otitis media to severe diseases such as pneumonia, meningitis, and bacteremia [1]
To identify the factors affecting the content of free polysaccharides, two different sizes of type 5 capsular polysaccharide (CPS) and different concentrations of EDAC and CRM197 for the conjugation reaction were tested for serotype 5
We carried out an optimization of a new pneumococcal conjugate vaccine that contains 15 serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14,18C, 19A, 19F, 22F, and 23F) of polysaccharides by adjusting the molecular sizes of the polysaccharides used in the conjugation processes to reduce the free polysaccharide contamination

Summary

Introduction

Pneumococci are gram-positive, aerobic diplococci bacteria that are encapsulated, and cause a range of clinical infections from mild diseases such as sinusitis and otitis media to severe diseases such as pneumonia, meningitis, and bacteremia [1]. The pneumococcal capsular polysaccharide is the major virulence factor of these bacteria and is highly diverse with more than 90 different serotypes identified to date [1,3,4]. Disease development and severity are diverse with bacterial serotypes, which vary according to age, geographic area, and time [5,6]. Several pneumococcal vaccines have been developed with various serotypes, which can prevent bacterial infections caused by pneumococcal streptococci, including ear infections, sinusitis, pneumonia, bloodstream infections, and meningitis [1,7].

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