Please use this identifier to cite or link to this item: http://prr.hec.gov.pk/jspui/handle/123456789/1139
Title: Preparation of polysaccharide-protein conjugates of Vi negative Salmonella Typhi as potential candidates for vaccines
Authors: Salman, Muhammad
Keywords: Natural Sciences
Biology
Biotechnology
Issue Date: 2014
Publisher: Pakistan Institute of Engineering and Applied Sciences Nilore Islamabad, Pakistan
Abstract: Salmonella enterica serovar Typhi (S. Typhi) is the cause of typhoid fever in humans. Although typhoid fever has almost vanished from developed countries, it is still a major cause of morbidity and mortality in the developing world. Its global annual incidence has increased from 21 million cases to 26.9 million cases during the period 2000 to 2010. Typhoid fever is curable with high dose of several antimicrobials but the treatment is time consuming and expensive. Vaccines are the eventual source of prevention from typhoid fever. At present, licensed typhoid vaccines are based on Vi polysaccharides. These vaccines are not useful for children of less than 2 years of age and are ineffective against the infections caused by Vi negative S. Typhi strains. O-Specific polysaccharides (OSP) from lipopolysaccharides (LPS) are universally present in both Vi positive and Vi negative S. Typhi and their conjugation with a potential immunogenic protein can make them useful for children below 2 years of age and to elicit humoral as well as cellular immune response. Therefore, we planned to prepare polysaccharide-protein conjugates of Vi negative S. Typhi as potential vaccine candidates. Initially, we purified OSP from the extracted LPS of a local Vi negative S. Typhi isolate and checked its antigenicity against polyclonal mice antisera. To conjugate a carrier protein with the purified OSP, we used the recombinant exoprotein A of Pseudomonas aeruginosa (rEPA) and human serum albumin (HSA). For production of recombinant rEPA, full length rEPA gene was cloned in expression vector. Protein expression was optimized using different isopropyl-β-D- thiogalactoside (IPTG) concentrations, various temperatures and post-induction time. The expressed protein was purified on Ni-NTA chromatography column from soluble fraction as well as from inclusion bodies. His-tag was removed from rEPA using tobacco etch virus (TEV) protease. Polyacrylamide gel and Western blot procedures were performed to check the purity of the protein. We synthesized four glycoconjugate vaccine candidates of Vi negative S. Typhi OSP using sodium cyanoborohydride (reductive amination) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP). Each of the conjugate was injected to mice on day 1, day 21 and day 42. The immune response (IgG) against prepared conjugates were determined using enzyme-linked immunosorbent assay (ELISA) and the results were interpreted by one way ANOVA using all conjugate groups vs. OSP control group. The conjugate 2 elicited significantly high (P = 0.0001) antibody titer, while the titers produced by conjugate 1 (P = 0.037), conjugate 3 (P = 0.302) and conjugate 4 (P = 0.416) were not significantly higher than that of the control group. We conclude that the conjugate 2 (OSP-rEPA) has the potential to be evaluated further and recommended for the clinical trials.
URI:  http://prr.hec.gov.pk/jspui/handle/123456789//1139
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