OBJECTIVES

The importance of Leishmania infection in the Mediterranean basin is not only due to its zoonotic aspect, but also, it is important in the Veterinary Medicine because the dog is the main host. According to accepted epidemiological models, the development of a prophylactic vaccine appears to be the most effective and practical method for controlling this disease. However, currently, there is not an optimal vaccine available. The protective immunity in Leishmania infected dogs is mainly due to specific cellular immunity. Vaccination using plasmid DNA induces specific humoral and cellular immunity in a number of experimental leishmaniosis models. The aim of this preliminary study is to evaluate the safety and Leishmania-specific immune response elicited by a multi-gene DNA vaccine.

MATERIALS

 Dogs: Two Beagle dogs were used in the study. The dogs were seronegative and negative to specific cellular immunity tests.

 Vaccine: The genes gp63, LACK, TSA and kmp-11 from L. infantum were amplified through PCR and cloned independently into the pVAX expression vector.

 Vaccination protocol: Dogs were injected intramuscularly with 100 mg of each plasmid in a total volume of 1mL saline solution, four times at 3 weeks intervals.

 Blood samples were collected every 10 days.

 Evaluation of immune response: We determined Leishmania-specific total IgG and the subclasses IgG1 and IgG2 by ELISA.

 The cellular response was assessed by three different techniques: 1) Antigen-specific lymphoproliferative assay. 2) Production of IFN-gamma by PBMC using a bioassay; 3) Leishmanin skin test.

 Safety: We checked rectal temperature, local pain, and biochemistry and hematological parameters

 Statistical analysis: Student's t test for paired samples

RESULTS

The dogs did not show any adverse effects throughout all the study. The dogs did not produced antibodies against Leishmania. However, 10 days after the first booster, the lymphocytes from these dogs proliferated to Leishmania antigen and produced IFN-gamma. This response was maintained 4 weeks after the last booster. Both dogs showed a positive leishmanin skin test reaction 2 months after last vaccination.

CONCLUSION

This preliminary study shows that this multi-gene DNA vaccine is able to elicit a cellular immune response against the Leishmania parasite in the dog, which is the one supposed to be protective in this disease. The DNA vaccine did not cause any adverse effects suggesting that it is safe and can be used in large-scale studies.