Comparative investigation of in vivo immune response induced by various L. infantum antigens, obtained that are with different techniques, for vaccine development

Leishmaniasis is one of the most important public health problem both of world and Turkey which is a tropical disease caused by the obligate intracellular parasites Leishmania species. 350 millions people are under the risk of this disease in the world. The disease is endemic in 98 countries including Turkey that is the number of infected people is 12 million. Visceral leishmaniasis (VL) which is the different clinical forms of the disease can cause death if not treated. Widely seen as the leading cause of disease against Leishmaniasis yet fully developed lack of a safe and effective vaccine formulation comes. There are three generations vaccines developed up to day. Each of three generation vaccines have distinct disadvantages. Although the current strategy for vaccination against leishmaniasis is based on the use of recombinant antigens, whole parasite vaccines are still attractive in terms of cost, safety, and stability of their biochemical composition and antigenicity. Whole soluble Leishmania antigens obtained with freeze/thawed method contain several epitopes to protect against disease and can interact with different MHC molecules play an important role in the development of vaccines. But, the biggest disadvantage of this type of vaccine formulation is using antigen alone or together with adjuvant which can not generate an adequate immune response. For increasing the immune response generated from killed parasites antigens is very important to investigate appropriate adjuvants in the present. Polyoxidonium (PO) is FDA approved and water soluble polymer that has an effective immunostimulating (immune stimulating) activity for the complex treatment of both acute and chronic infections in humans and has been recognized as an effective, safe immunomodulator. This situation can allow opportunity to use of PO in the further vaccines development against the other infectious diseases. However, there are no studies about immunogenic properties of combinations with PO and killed Leishmania antigen in the literature. Accordingly this thesis purpose of visceral leishmaniasis vaccine against development of freeze/thawed and autoclaving obtained by killed Leishmania infantum antigens PO with adjuvant vaccine formulations created and the resulting formulations immunogenic activity in vitro and in vivo, for the first time to investigate. For this purpose, firstly, Leishmania antigens were obtained which killed by freeze / thawed and using autoclave and formulations were obtained that have different consantration with PO adjuvant. Then, toxixity of formulation was examined with MTT assay on J774 macrophage and L929 fibroblast cell culture. And then, nontoxic formulations, that are determined to be, were imvestigated product potential of NO (nitric oxide) in macrophage cell culture and ability to killing parasites on infected macrophages cells. According to results of in vitro, to choose the best formulation with PO adjuvant for antigens, were obtained by both freeze/thawed and autoclaved, and the efficacy of these formulations was investigated on in vivo animal models. Both two of four immunization on Balb/C mice, was taken blood serum of mice and amount of the antibody has been identified by ELISA. Also, spleen lenfosites were isolated from mice, that killed after completion of immunizations, and IL-4, IL-10, IL-12 and TFN- α cytokines, secretion from lenfosites, are were identified by a commercial kit. According to the results, examined antigens and polymers concenrations did not show toxic effect compared to control on J774 macrophage and L929 mice fibroblast cell culture. When determined to efficacy of formulations on NO products of macrophages, PO-D/Ç Ag combination (250 µg/ml:100 µg/ml) and PO-autoclaved Ag combination (250 µg/ml:100 µg/ml), are 4 fold increase NO product, were determined to show a maximum affect of NO product of macrophage cells compared control. Thus, results of studying on in vitro, its found that, the most appropriate vaccine formulation is PO-D/Ç Ag combination (250 µg/ml:100 µg/ml) and PO-autoclaved Ag combination (250 µg/ml:100 µg/ml). Then, determined formulations were tested on Balb/c mice. Antibody levels of both PO-D/Ç Ag combination and PO-autoclaved Ag combination have been identified to increase compared to just antigens or adjuvant applied groups, on serum samples taken from mice. But the highest antibody level, it was shown applied PO-D/Ç Ag combination groups. Moreover, IL-12 and TNF- α cytokine levels were increased by PO-D/Ç Ag combination and PO-autoclaved Ag combination, on the other hand, levels of IL-4 and IL-10 were not change on obtained spleen lenfosite culture after the killing of mice. The obtained results clearly demostrate that combination of killed Leishmania antigens with PO, are obtained different way, induced both antibody level and TH1 immune response which induced by IL-12 and TNF-α cytokine in mice. Thus for the first time in this study, derived from different methods formulations prepared killed parasites with PO adjuvants, vaccine candidates have been shown to have significant potential. Killed Leishmania antigens and their formulations with PO has comprising future thought to play an important role in the development of vaccines against leishmaniasis. Thus for the first time in this thesis vaccine candidate formulations containing the whole L. infantum antigens with PO were obtained against the development of a vaccine for visceral leishmaniasis. Also for the first time in the development of vaccines against leishmaniasis macrophage cell system has been shown to be suitable for the selection of vaccine candidates. Developed new vaccine candidate may include first generation vaccine candidates are in the future. Keywords: L.infantum, PO, MTT, cell culture, nitric oxide, vaccine, cytokine