To enhance the stability and immunogenicity of protective antigen (PA) of Bacillus anthracis, Multi-walled Carbon Nanotubes (MWCNTs) as a nanocarrier, was subjected to develop a new PA domain 4 (PAD4) based nanovaccine against anthrax. PAD4 protein was recombinant expressed in Escherichia coli and purified by Nickel–Nitrilotriacetic Acid (Ni–NTA) column. Purified protein was immobilized/absorbed on MWCNTs by covalently and non-covalently methods. SDS-PAGE, Bradford, TEM, SEM, AFM, FTIR, EDX and Zeta potential analyses confirmed MTWCNTs/rPAD4 conjugates. Immunogenicity examinations were performed in four booster dose for six groups of subcutaneous in mice. Determination of antibody response after second and forth booster indicated high levels of anti-rPAD4 IgG titer in serum compared with the control groups. After second booster anti-rPAD4 IgG level in MWCNTs-rPAD4 groups was significantly higher compared with rPAD4 and rPAD4 with Freund's adjuvant groups. In this booster the highest levels of anti-rPAD4 IgG titer was detected with endpoint of 20480 in covalent rPAD4-MWCNTs conjugate. While after fourth booster there was not significant difference between MWCNTs-rPAD4 and rPAD4 with Freund's adjuvant groups. Finally, the results indicated that MWCNTs with enhancing the rPAD4 stability and having the adjuvants effects can be used as an effective delivery vehicle to PA based vaccine formulation against anthrax.
