Preliminary Studies Of DNA Vaccine With Amastin Gene Against Leishmania Donovani

Visceral leishmaniasis caused by Leishmania donovani is a serious disease andthe mortality is over 90 percent by estimation. From the data of WHO/TDR, 500thousands people got this disease every year and the total patients of the world isabout 2500 thousands. In the modem era, the general measures to control thisdisease is using stibine sodium gluconate for treatment of patients or catching andkilling the infected dogs to control infective agent; the second is killing sandfly tobreak the rout of transmission. However, drugs for this disease have somedisadvantage characters, such as long term of usage, obvious side effect, lack of oraladministration and expensive cost exceeding patient's economic endurance,especially for progressing countries. Today, the appearing of some strains ofanti-stibine sodium gluconate and the emergence of admixture of AIDS and visceralleishmaniasis influence the word condition of controlling this disease. Thus, findingout a safe, effective, and low cost vaccine is urgent for clinical controlling visceralleishmaniasis.Today, the appearance of the "third-generation vaccine"—nucleic acid vaccinepresents a good perspective for controlling leishmaniasis. Some researchers reportedtheir observations. Here, on the basis of many literatures, the amastin gene fromLeishmania donovani is firstly selected as candidate gene and combinated with ctxBgene encoding cholera toxin B subunit, then made into two DNA vaccines with amastin gene. Subsequently, the two DNA vaccines with amastin were injected intomice respectively, and an evaluation of immunogenicity and protective immunityand the adjuvantive effect of CTB were carried out. This stuty consists of five parts:In the first part, amastin gene from DNA of two Leishmania donovani isolateswas amplified respectively by PCR. Amastin gene then was cloned into prokaryoticexpression plasmid pET-32a(+). The prokaryotic expression recombinant plasidpET-amastin and pET-dog-amastin were constructed and sequenced, and a fusionprotein about 40kD was obtained by inducing pET-amastin in prokaryotic expressionsystem.In the second part, the fused gene ctxB/amastin based on ctxB and amastin wasobtained by SOEing (gene splicing by overlap extension) and cloned into pET-32a(+). A fusion protein about 54kD was obtained by inducing the recombinant plasmidpET-ctxB/amastin in prokaryotic expression system.In the third part, the amastin gene and fused ctxB/amastin gene were cloned intoeukaryotic expression plasmid pcDNA3.1(+) and constructing the recombinantplasmid pcDNA3.1-amastin and pcDNA3.1-ctxB/amastin. Then the two recombinantplasmids were transfected into NIH3T3 cell in vitro and their transient expressionwas detected by immunofluorescence. The transfected cells were screened by G418and RNA was extracted from the survival cells. DNA fragment about 552bp and921bp was got by RT-PCR. These data suggest that the two plasmids got stableexpression in NIH3T3 cells. In addition, the protein about 20kD and 34kDrespectively was detected by western blot.In the fourth part, the two eukaryotic expression recombinant plasmidsconstructed was used as DNA vaccine to inject into BALB/c mice and then the levelof antibody in serum, proliferative response of spleen-derived lymphocytes, level ofIFN-γ, IL-2 and IL-4 production in lymphocytes culture supemant, and cytotoxicityof spleen-derived lymphocytes was detected in different time. The results showedthat immunogenicity of group pcDNA3.1-amastin and pcDNA3.1-ctxB/amastin wasstronger than the control group's; humoral immunity and cellular immunity of grouppcDNA3.1-ctxB/amastin was stronger than group pcDNA3.1-amastin. In the fifth part, the two eukaryotic expression recombinant plasmids was used asthe DNA vaccine to inject into BALB/c mice. The mice were challenged byLeishmania donovani promastigote after the second immunization. The challengedmice were sacrificed after 6 weeks and their liver and spleen was extracted toobserve the number of amastigote by impression smear and histopathology by tissuesection. The results showed that the liver and spleen from mice injected with DNAvaccine includig amastin gene have fewer amastigote than the control group and themice injected with pcDNA3.1-ctxB/amastin kept the least. In addition, the liver andspleen from mice injected with DNA vaccine with amastin gene have little change.These data suggest that vaccination with DNA encoding amastin confers protectiveimmunity to mice infected with Leishmania donovani and the mice injected withpcDNA3.1-ctxB/amastin got stronger protective immunity.All data in this study suggest that the two DNA vaccines with amastin gene havesome degree of immunogenicity and protective immunity. When CTB adjuvant isadded, they have stronger immunogenicity and protective immunity. Among them,vaccine of ctxB/amastin has the stronger ability of inducing cellular immunity.Because leishmania is the intracellular parasite, cellular immunity can suppress itsproliferation. Thus, constructing the recombinant gene of candidate gene andadjuvant has the great significance and this study provides instruction for developingthe safe and effective DNA vaccines against leishmaniasis. So far, similary study hasnot been found.