DNA vaccination with a plasmid encoding LACK-TSA fusion against Leishmania major infection in BALB/c mice.

Vaccination would be the most important strategy for the prevention and elimination of leishmaniasis. The aim of the present study was to compare the immune responses induced following DNA vaccination with LACK (Leishmania analogue of the receptor kinase C), TSA (Thiol-specific-antioxidant) genes alone or LACK-TSA fusion against cutaneous leishmaniasis (CL). Cellular and humoral immune responses were evaluated before and after challenge with Leishmania major (L. major). In addition, the mean lesion size was also measured from 3th week post-infection. All immunized mice showed a partial immunity characterized by higher interferon (IFN)-γ and Immunoglobulin G (IgG2a) levels compared to control groups (p<0.05). IFN-γ/ Interleukin (IL)-4 and IgG2a/IgG1 ratios demonstrated the highest IFN-γ and IgG2a levels in the group receiving LACK-TSA fusion. Mean lesion sizes reduced significantly in all immunized mice compared with control groups at 7th week post-infection (p<0.05). In addition, there was a significant reduction in mean lesion size of LACK-TSA and TSA groups than LACK group after challenge (p<0.05). In the present study, DNA immunization promoted Th1 immune response and confirmed the previous observations on immunogenicity of LACK and TSA antigens against CL. Furthermore, this study demonstrated that a bivalent vaccine can induce stronger immune responses and protection against infectious challenge with L. major.

Codelivery of DNA vaccination encoding LeIF gene and IL-12 increases protection against Leishmania major infection in BALB/c mice.

Previous studies have shown that Leishmania elongation initiation factor (LeIF) antigen causes a partial immunity against leishmaniasis. The antigen develops type I immunity by overexpression of inflammatory cytokines such as interleukin-12 (IL-12), IFN-γ and TNF-α. Therefore, We evaluated immune responses induced by the LeIF gene against Leishmania major infection. Immunization with LeIF gene alone or with IL-12 induced Th1 response and produced higher IFN-γ and lower IL-4 levels by splenocytes than control groups (P < 0·05) and also ratios of IFN-γ/IL-4 were 11·68 to 18·53 times more in immunized groups than control groups after challenge. In addition, analysis of humoral immune response revealed that immunized mice had more IgG2a levels than both control groups (P < 0·05). On the other hand, lesion size was less for immunized animals than control groups from 4th week after challenge (P < 0·05). The percentage reduction in lesion size was 29·30% for LeIF and 51·98% for LeIF + IL-12 than PBS at 12th week post-infection. Spleen parasite burden decreased in all immunized groups in comparison with control groups (P < 0·05). The results indicated that LeIF gene induced partial immunity against L. major infection in BALB/c mice. However, LeIF plus IL-12 group showed more potent immunity with smaller lesions than other groups.

Cloning and Constructing a Plasmid Encoding Leishmania Eukaryotic Initiation Factor Gene of Leishmania major Fused with Green Fluorescent Protein Gene as a Vaccine Candidate.

OBJECTIVE: Leishmaniasis is usually treated with chemotherapy; however, toxicity, resistance and high-cost limit use of the chemical drugs. Leishmania eukaryotic initiation factor (LeIF) protein acts the same as interleukin (IL)-12 and reduces the secretion of IL-4 in lymph node cells of mice infected with Leishmania major. The aim of this study was cloning of the gene encoding LeIF antigen into eukaryotic expression plasmid pEGFP-N1. METHODS: DNA was extracted from Iranian strain of the L major (MRHO/IR/75/ER) promastigotes. The full-length sequence of LeIF was amplified with Pfu DNA polymerase using a specific primer. The amplified LeIF was cloned into a pJET1.2/blunt vector. Then this fragment was digested with HindIII and EcoRI and was subcloned into the pEGFP-N1 vector. Confirmation of the cloning was done by colony polymerase chain reaction (PCR). RESULTS: Leishmania eukaryotic initiation factor gene was successfully cloned and subcloned into pJET1.2 and pEGFP-N1 plasmids, respectively. The results of colony PCR, restriction analysis and sequencing confirmed them. CONCLUSIONS: We cloned LeIF gene which could be expressed in eukaryotic cells in vivo and could be used as a vaccine candidate against leishmaniasis in future studies.