Mycobacterium tuberculosis (M.tb.) is an obligatory aerobic, intracellular bacillus that causes the death of over 3 million people every year. It resides within macrophages of the host, and completely evades the immune surveillance by secreting proteins that are immunogenic and provoke protective immunity. The study aims at developing nanoparticles carrying two secretory proteins of M. tb - CFP-10 and CFP-21 and evaluating their potential to invoke an immune response coupled with the oxidative stress when encapsulated in chitosan nanoparticles. Chitosan nanoparticles were prepared in the size range of ~250 to ~300nm having a zeta potential +41mV, and an entrapment efficiency for CFP-10 ~16% and for CFP-21 ~18%. The FTIR studies indicated that the terminal phosphate group of TPP gets bound with amine (NH2) group of chitosan by ionic bond. The characteristic peak of C-N (1250-1375 cm-1) was present in FTIR of Chitosan along with other peaks. But in chitosan nanoparticles there was a distinct shift of the peak to due to the wagging of NH2 bond. The cytokine levels of IFN-γ, TNF-α, IL-12, IL-17, IL-2, IL-10 and IL-4 were observed to be significantly increased for CHNP CFP-10 and CHNP CFP-21. CFP-10 and CFP-21 per se primed cells demonstrated a Th1 biased T cell response in an ex vivo assay. To further analyze the potential of the nanoparticles to cause oxidative stress, various biochemical assays such as Glutathione S-transferases (GST), reduced Glutathione (GSH), Glutathione Peroxidase, Glutathione Reductase and Nitric oxide (NO) were determined in the mice treated with CFP-10, CFP21, void CHNP, CHNP CFP-10 and CHNP CFP-21 in liver, lung and spleen post 7 days and 21 days of injection. The GST levels were lowered indicating oxidative stress in all the organs on Day 7. But post Day 21 of injections, enhanced GST levels indicated reduced or no oxidative stress in the tissues. The enhanced levels of IFN-γ and IL-12 clearly indicate a Th1 response coupled with low levels of GSH. Therefore, an interplay of immune response, ROS and RNS created by secretory proteins of M.tb. encapsulated in nanoparticles indicated interesting results which warrant detailed evaluation on the signaling pathways to ascertain the extent of interdependence.
Key words: Mycobacterium tuberculosis, Protective immunity, Chitosan nanoparticles, Immune response, Oxidative stress