Glycoconjugation of Shigella flexneri type 2a O-polysaccharide with CRM197 as a potential vaccine candidate for shigellosis

• Published in: Biologicals 2021-07, Vol.72, p.1-9
• Main Authors: Narayana, P.V.S.L.S.S., Dutta, Jayati Ray
• Format: Article
• Language: English
• Subjects: Adipic acid dihydrazide; CRM197; Glycoconjugate; O-specific polysaccharide; Shigellosis
• ISSN: 1045-1056; 1095-8320
• DOI: 10.1016/j.biologicals.2021.07.001

Shigellosis, a diarrheal disorder caused by an entero-invasive bacterium Shigella, is a major concern among children often leading to mortality. As most of these strains have developed universal antibiotic resistance, the development of a vaccine is crucial in combating the infection. The O-specific polysaccharide (O-PSs) from S. flexneri type 2a is considered to be the major disease-causing antigen in shigellosis. Therefore, the O-PSs conjugated with carrier proteins, can serve as a potential high molecular weight vaccine candidate. Accordingly, in the present study, O-PS extracted from S. flexneri 2a is conjugated with Cross-Reactive Material (CRM197), a non-toxic mutant of diphtheria toxin. We derivatized CRM197 and O-PS separately with adipic acid dihydrazide (ADH) and reacted with their counterparts to probe the conjugation efficacy. Among the two strategies, the CRM197-ADH treated with O-PS has yielded a stable glycoconjugate of 311 kDa. The conjugation efficiency has been probed by estimating the free protein, free O-PS and O-PS:CRM197 ratio using slot-blot, size exclusion and high-performance anion exchange chromatography techniques. The conjugate exhibited enhanced shelf-life of three months. The cytotoxicity studies with Vero/MRC-5 cells have confirmed the non-toxicity of the conjugate, which makes the glycoconjugate a potential vaccine candidate for shigellosis. •CRM197-S. flexneri O-PS conjugate as a potential vaccine candidate for shigellosis.•ADH was used to separately activate CRM197 and O-PS for efficient conjugation.•ADH-CRM197 + O-PS conjugate yielded a high molecular weight immunogen (>300 kDa).•The glycoconjugate exhibited sustained shelf-life of over three months.