Investigation of in Vitro Immunostimulating Effects of FreezeThaw Antigens against Visceral Leishmaniasis Caused by L. Infantum

Abstract

Background: Visceral leishmaniasis, also known as Kala-Azar among the public, is among the notifiable infectious diseases. The fact that an effective and safe drug or vaccine formulation suitable for use on humans has not been developed causes the disease threat to continue. The aim of this study is to determine the immune system stimulating properties and cytotoxicity of the formulations obtained by combining the antigen obtained from Leishmania infantum parasites with different adjuvants. Methods: L. infantum (EP 126) antigens were obtained by the freeze-thaw method. Freund's adjuvant (Complete and Incomplete), a classical adjuvant, was used in the studies. Mouse macrophage (J774) and mouse fibroblast (L929) cell lines were used for cytotoxicity studies. Additionally, the responses at both 10ug/ml and 40ug/ml were investigated to ensure that the developed formulations were easily adaptable to in vivo. While the cytotoxicity of the formulations was determined by the MTT cell viability analysis method, the antileishmanial property was determined by the Nitric Oxide (NO) determination method. Results: It was determined that all the developed formulations did not have a toxic effect on the host cell, the immunostimulating activity increased with increasing concentration, and the highest value was obtained at a concentration of 200 µg/ml. Cell viability at the maximum concentration was found to be between 78% and 89% for both J774 and L929 cells. The highest cell viability values and NO production abilities (37% increase compared to the control group) were obtained with Incomplete Freund's adjuvant (40 ug/ml formulation). Conclusion: It has been understood that the combination of antigens obtained by the freeze-thaw method with Freunds adjuvant (incomplete) supports NO production, thus having an immunostimulating effect.