Abstract

Background: Leishmaniasis continues to be a major health problem worldwide because the parasite has developed several strategies to resist the immune response, making the development of vaccines and treatment challenging. Methods: In the present work, we have used the CRISPR-Cas9 gene-editing system to explore the mechanisms of immune evasion used by Leishmania, and to identify new vaccine targets. Important immune-regulation genes, such as LACK, GP63, and CPB, have been successfully deleted in Leishmania major and Leishmania donovani using PCR and Sanger sequencing. Results: The immunological profile of gene-deleted BALB/c mice was assessed following infection. The modified parasites caused a greater infiltration of CD8+ IFN-γ+ T cells than did wild-type parasites. Cytokine protein levels showed elevated IFN-γ and TNF-α, and decreased IL-4, indicating a highly polarized Th1-type immune response. Immunization with genetically engineered antigens was used to assess vaccine efficacy. Immunization with antigens from the deleted parasites resulted in a significant rise in IgG antibody levels, the IgG1/IgG2a ratio, lymphocyte proliferation, and cytotoxic T cell activity. More importantly, survival rates of 80% in vaccinated mice were significantly higher after challenge with wild-type Leishmania than in 10% of mice immunized with wild-type antigens. Conclusions: The results demonstrate that host-protective immunity can be boosted by inactivating genes that contribute to immune evasion using the CRISPR-Cas9 technique. These approaches yield promising antigen sources for vaccines. The genetic modification of Leishmania species described in this research is a promising vaccine platform for developing vaccines for future generations.