Metal-based nanoparticles (NPs) have been shown to promote innate immunity, but they have never been shown to help in the creation of particular cellular immune responses. As a result, researchers wanted to determine if iron oxide-based NPs might be used as an adjuvant in the generation of cellular Th1, Th17, and TCD8 (Tc1) immune responses. As a subunit vaccination, a fusion protein (CMX) constituted of Mycobacterium tuberculosis antigens was employed. Co-precipitation was used to make citrate-coated MnFe2O4 NPs, which were then examined using transmission electron microscopy. Dynamic light scattering and field-emission scanning electron microscopy were used to detect the development of the protein corona in the vaccine, which was made by homogenizing NPs with the recombinant protein. Subcutaneous and intranasal methods were used to test the vaccine for the optimum vaccination route and strategy, with 21-day intervals between immunizations. 

The vaccination elicited specific (CD4+IFN-+ (Th1) and CD8+IFN-+) responses when given subcutaneously. Specific Th1, Th17 (CD4+IL-17+), and Tc1 responses were produced by intranasal immunization, primarily in the lungs. Finally, a combined vaccination approach (two subcutaneous injections followed by one intranasal immunization) elicited a Th1 (in the spleen and lungs) and splenic Tc1 response in the lungs but not a Th17 response. It showed that a subunit vaccination using iron oxide-based NPs as an adjuvant elicited cellular immunological responses (Th1, Th17, and TCD8), indicating that it has strong adjuvant properties. Furthermore, the immunological response elicited by the vaccine’s subcutaneous delivery reduced the bacterial load in Mtb-infected animals, indicating that the vaccine has room for improvement as an Mtb vaccine.