BACKGROUND: Leishmaniasis is one of the major emerging health problems worldwide and Leishmania tropica (L. tropica) is most prevalent in the Middle East due to conflict and environmental factors, and there is no effective prevention strategy available until now. An effective vaccine has not been developed to date. DNA vaccines are considered a promising approach to protect against this infection. In this study, since vacuolar (H+)-ATPase (V-ATPase) enzyme has an essential role in the life cycle of eukaryotes, V-ATPase subunit F gene has been chosen to design DNA vaccine and evaluate its immunogenicity in BALB\c mice. METHODS: Genomic DNA was isolated from promastigote culture, synthesized complementary DNA (cDNA) after standardization of Polymerase Chain Reaction (PCR) conditions. The V-ATPase subunit F gene was placed into plasmid PCI. Then, recombinant plasmids were transformed into competent cells. Cloning was confirmed by PCR, restriction enzyme assays, and finally, DNA sequence analysis, after making miniprep from positive colonies and finally the gene was sequenced. BALB/c mice were immunized subcutaneously three times at an interval of two weeks with designed vaccine. BALB\c mice were challenged with 106 promastigotes of L. tropica 7 days post-immunization. IL-12, IFN-γ and IL-4 were quantified by RT-qPCR. RESULTS: The present study proved the existence of subunit F gene in Syrian strain of L. tropica (LCED Syrian 01) promastigotes genome. Its expression was also proved in these parasites and the gene length was 414 bp. CONCLUSION: This study showed that vaccination of BALB\c mice with this gene induced partial protection against Leishmania by reduction of lesion size by 41.9% and parasite burden reduction by 3-log in the dLNs when compared with control group. IFN-γ\IL-4 was 1.6 after challenge test, so the immune response consisted of both Th1 and Th2. Copyright
BACKGROUND: Leishmaniasis is one of the major emerging health problems worldwide and Leishmania tropica (L. tropica) is most prevalent in the Middle East due to conflict and environmental factors, and there is no effective prevention strategy available until now. An effective vaccine has not been developed to date. DNA vaccines are considered a promising approach to protect against this infection. In this study, since vacuolar (H+)-ATPase (V-ATPase) enzyme has an essential role in the life cycle of eukaryotes, V-ATPase subunit F gene has been chosen to design DNA vaccine and evaluate its immunogenicity in BALB\c mice. METHODS: Genomic DNA was isolated from promastigote culture, synthesized complementary DNA (cDNA) after standardization of Polymerase Chain Reaction (PCR) conditions. The V-ATPase subunit F gene was placed into plasmid PCI. Then, recombinant plasmids were transformed into competent cells. Cloning was confirmed by PCR, restriction enzyme assays, and finally, DNA sequence analysis, after making miniprep from positive colonies and finally the gene was sequenced. BALB/c mice were immunized subcutaneously three times at an interval of two weeks with designed vaccine. BALB\c mice were challenged with 106 promastigotes of L. tropica 7 days post-immunization. IL-12, IFN-γ and IL-4 were quantified by RT-qPCR. RESULTS: The present study proved the existence of subunit F gene in Syrian strain of L. tropica (LCED Syrian 01) promastigotes genome. Its expression was also proved in these parasites and the gene length was 414 bp. CONCLUSION: This study showed that vaccination of BALB\c mice with this gene induced partial protection against Leishmania by reduction of lesion size by 41.9% and parasite burden reduction by 3-log in the dLNs when compared with control group. IFN-γ\IL-4 was 1.6 after challenge test, so the immune response consisted of both Th1 and Th2. Copyright