Antibody response to various domain of protective antigen in cutaneous anthrax cases in India

Background: Anthrax, a disease of bioterrorism and public health importance is caused by the Gram positive, spore-forming bacterium, Bacillus anthracis. Anthrax toxin, a tripartite toxin is composed of protective antigen (PA), lethal factor (LF) or edema factor (EF). PA is the major protein which facilitates the entry of toxin component either of lethal factor or edema factor. Recombinant PA has been a suitable target for anthrax vaccine worldwide. However, instead of full PA, its domains are reported to provide protection. LF also contributes to immuno-protection against anthrax. Therefore, in this study, a chimeric protein consisting of both, PA and LF was developed as candidate vaccine for anthrax. Methods & Materials: A chimera was made by fusion of immunodominant portion of PA (Domains 2-4) and LF (Domain 1) genes. . The construct was cloned in pET32a+ vector and expressed in E. coli host. The recombinant chimeric protein was purified by immobilized metal affinity chromatography. The 4-6 week old Balb/c mice were injected intraperitonealy with three doses of chimeric protein (20 g each mouse) at two week interval. The first dose was given with Freund’s complete adjuvant and the subesequent doses were given with incomplete Freund’s adjuvant. The serum IgG and its subtypes were determined by plate ELISA. Results: The chimeric protein (PA-LF) was purified up to homogeneity and the production yield was 15 mg/l of the shake flask culture. The chimera elicited good immune response against both the toxins i.e. PA as well as LF. The end point titre of chimeric protein was 1:1024000 by plate ELISA. An antibody titre of 1:512000 was observed in mice serum for PA protein. The same serum exhibited the titre of 1:256000 against LF protein. The end point titres of IgG1, IgG2a, IgG2b and IgG3 were 1: 512000, 1:128000, 1:256000 and 1:32000, respectively. Thus, IgG1 was predominant among all subtypes indicating that PA-LF chimera induced Th2-type immune response Conclusion: The chimera consisting of partial sequences of PA and LF can be better vaccine candidate than individual PA or LF proteins. In the present study, the recombinant protein elicited very good immune response in mice and showed Th2 type of immune response.