Structural and immunological characterization of E. coli derived recombinant CRM197 protein used as carrier in conjugate vaccines

It is established that the immunogenicity of Polysaccharides is enhanced by coupling them to carrier proteins. Cross reacting material (CRM₁₉₇), a nontoxic variant of diphtheria toxin (DT) is widely used carrier protein for polysaccharide conjugate vaccines. Conventionally, CRM₁₉₇ is isolated by fermentation of Corynebacterium diphtheriae C7 (β₁₉₇) cultures, which often suffers from low yield. Recently, several recombinant approaches have been reported with robust processes and higher yields, which will improve the affordability of CRM₁₉₇-based vaccines. Vaccine manufacturers require detailed analytical information to ensure that the CRM₁₉₇ meets quality standards and regulatory requirements. In the present manuscript we have described detailed structural characteristics of Escherichia coli based recombinant CRM₁₉₇ (rCRM₁₉₇) carrier protein. The crystal structure of the E. coli based rCRM₁₉₇ was found to be identical with the reported crystal structure of the C7 CRM₁₉₇ produced in C. diphtheriae C7 strain (Protein Data Bank (PDB) ID: 4EA0)_. The crystal structure of rCRM₁₉₇ was determined at 2.3 Å resolution and structure was submitted to the PDB with accession number ID 5I82. This is the first report of a crystal structure of E. coli derived recombinant CRM₁₉₇ carrier protein. Furthermore, the rCRM₁₉₇ was conjugated to Vi polysaccharide to generate Typhoid conjugate vaccine (Vi-rCRM₁₉₇) and its immunogenicity was evaluated in Balb/C Mice. The Vi-rCRM₁₉₇ conjugate vaccine was found to generate strong primary α-Vi antibody response and also showed a booster response after subsequent vaccination in mice. Overall data suggest that E. coli based recombinant CRM₁₉₇ exhibits structural and immunological similarity with the C7 CRM₁₉₇ and can be used as a carrier protein in conjugate vaccine development.