Lymphatic filariasis (LF) is a profoundly disfiguring human disease caused by filarial parasite and transmitted through infected mosquitoe bite. Currently, there are no licensed vaccine to control LF. The rBmHAXT vaccine protein prepared in our laboratory is expressed as a His-tagged protein. FDA doesn’t allow tags in any recombinant vaccine developed for human use. In this study we determined if tag removal has any effect on the protein structure and vaccine efficacy. We expressed rBmHAXT protein with a TEV cleavage site in a bacterial expression system. Using TEV protease, cleaved the His-tag from the protein. Western-blot analysis using anti-His HRP antibodies and anti-rBmHAXT antibodies demonstrated that we were able to generate tag-free rBmHAXT. Circular-Dichroism spectroscopy analysis showed that the structure and folding of tag free rBmHAXT protein is like His-tagged rBmHAXT. We immunized three groups of 10 mice each with AL019 or His-tag rBmHAXT or tag-free protein. Mice were immunized four times at 2 weeks interval. The tag-free and His-tag rBmHAXT immunized mice generated comparable and significant titers of antigen-specific IgG antibodies. There were no significant differences in the levels of anti-rBmHAXT IgG isotype antibodies in the sera of tag-free or His-tag rBmHAXT immunized mice. Antibody dependent cell mediated cytotoxicity assay showed that the protective antibodies in the sera from tag-free rBmHAXT and His-tag rBmHAXT vaccinated mice killed 100% and 97.52 % of B. malayi L3 respectively. The findings from this study showed that removal of His-tag did not alter the protein structure, immunogenicity and vaccine potential of rBmHAXT.
Divya Sree Nagalati Sudhakar– Graduate Research Assistant, University of Illinois College of Medicine at Rockford
Vishal Khatri– Postdoctroal Research Associate, University of Illinois College of Medicine at Rockford
Nikhil Chauhan– Postdoctroal Research Associate, University of Illinois College of Medicine at Rockford
Ramaswamy Kalyanasundaram– Professor & Head, University of Illinois College of Medicine Rockford-Department of Biomedical Sciences