Design, Construction and Immunogenicity Assessment of pEGFP-N1-KMP11-GP96 (Fusion) as a DNA Vaccine Candidate against Leishmania major Infection in BALB/c Mice
Background: KMP-11 (Kinetoplastid membrane protein-Π) exists in all species of kinetoplastid family. It is fully conserved and the protein produced by this gene can induce a very high cellular immune response. We aimed to design a suitable construction for a Leishmania major DNA vaccine and evaluate the protective efficacy of it as a candidate for DNA vaccine against cutaneous leishmaniasis in BALB/c mice.
Methods: This experimental study was conducted in Tehran City, Iran, between April 20, 2015 and May 30, 2016. KMP-11 gene of L. major (MRHO/IR/75/ER, Iranian strain) and NT-GP96 of Xenopus GP96 DNA from a pBluescript-GP96 plasmid were amplified by PCR and the purified PCR products were cloned into the pJET1.2/blunt plasmid vector, then, subcloned into pEGFP-N1 plasmid as an expression vector. Finally, the KMP-11 gene was fused with GP96 and afterward the combination cloned in pEGFP-N1. All the cloned genes confirmed by enzyme digestions. Then, four groups of mice were immunized with PBS, pEGFP-N1, pEGFP-N1-KMP, and pEGFP-N1-fusion. Four weeks after immunization, all animals were challenged with L. major virulent promastigotes.
Results: The constructed fusion potentially showed an ability to elicit Th1 responses that led to cutaneous lesion healing. Interestingly, the group received KMP11-GP96 –GFP showed the highest ratio of IFN- γ /IL-4 and IgG2a/IgG1 compare to other groups. No side effect was observed after using the fusion in the mice.
Conclusion: The constructed fusion could well stimulate both the cellular and humoral immune systems that led to cutaneous lesion healing in mice.
2. Connell ND, Medina-Acosta E, McMaster WR et al. Effective immunization against cutaneous leish-maniasis with recombinant bacilli Calmette Guerin expressing the Leishmania surface protein gp63. Proc Natl Acad Sci. 1993; :11473-7.
3. Skeiky YA, Benson DR, Guderian JA et al. Im-mune responses of leishmaniasis patients to heat shock proteins of Leishmania species and humans. Infect Immun.1995; 63(10): 4105-14.
4. Webb JR, Campos-Neto A, Ovendale PJ et al. Human and murine immune responses to a novel L. major recombinant protein encoded by members of a multicopy gene family. Infect Immunol. 1998; 66:3279.
5. Sjolander A, Baldwin TM, Curtis J, Handman E. Induction of a Th1 immune response and simulta-neous lack of activation of a Th2 response are re-quired for generation of immunity to leishmaniasis. J Immunol. 1998;160: 3949.
6. Solioz N, Blum-Tirouvanziam U, Jacqute R et al. The protective capacities of histon H1 against ex-perimental murine cutaneous leishmaniasis. Vac-cine. 1999; 18: 850-859.
7. Ziaee Hezarjaribi H, Ghaffarifar F, Dalimi A et al. Effect of IL-22 on DNA vaccine encoding LACK gene of L. major in BALB/c mice. Exp Parasitol. 2013;134 :341–348.
8. Jorjani O, Ghaffarifar F, Sharifi Z et al. LACK Gene’s immune response induced by cocktail DNA vaccine with IL-12 gene against cutaneous leishmaniasis in BALB/c mice. Avicenna J Med Biotechnol. 2018; 10, 3:134-140.
9. Ghaffarifar F, Jorjani O, Sharifi Z et al. Enhance-ment of immune response induced by DNA vac-cine cocktail expressing complete LACK and TSA genes against Leishmania major. APMIS. 2013;121(4):290-8.
10. Maspi N, Ghaffarifar F, Sharifi Z. Dalimi A. Co-delivery of DNA vaccination encoding LeIF gene and IL-12 increases protection against Leishmania major infection in BALB/c mice. Parasite Immunol. 2016; 38:228–235
11. Maspi N, Ghaffarifar F, Sharifi Z et al. Immuno-genicity and efficacy of a bivalent DNA vaccine containing LeIF and TSA genes against murine cu-taneous leishmaniasis. APMIS. 2017a; 125: 249–258.
12. Maspi N, Ghaffarifar F, Sharifi Z et al. Comparative assessment of induced immune responses follow-ing intramuscular immunization with fusion and cocktail of LeIF, LACK and TSA genes against cu-taneous leishmaniasis in BALB/c mice. Arch Im-munol Therap Exp. 2018 ,66(1):55-64.
13. Maspi N, Ghaffarifar F, Sharifi Z et al. DNA vac-cination with a plasmid encoding LACK-TSA fu-sion against Leishmania major infection in BALB/c mice. Malaysian J Pathol. 2017c; 39(3):267–275.
14. King DL, Chang YD, Turco SJ. Cell surface lipo-phosphoglycan of Leishmania donovani. Mol Biochem Parasitol. 1987; 24: 47-53.
15. Russo DM, Turco SJ, Burns JM Jr, Reed SG. Stimulation of human T lymphocytes by Leishmania lipophosphoglycan-associated proteins. J Immunol. 1992; 148: 202-207.
16. Todoli F, Solano-Gallego L, de Juan R et al. Hu-moral and in vivo cellular immunity against the raw insect-derived recombinant Leishmania infantum anti-gens KMPII, TRYP, LACK, and papLe22 in dogs from an endemic area. Am J Trop Med Hyg. 2010; 83 (6):1287–94.
17. Yang Y, Li Z. Roles of heat shock protein GP96 in the ER quality control: redundant or unique func-tion? Mol Cells. 2005; 20 (2):173-182.
18. Binder RJ, Vatner R, Srivastava P. The heat-shock protein receptors: some answers and more ques-tions. Tissue Antigens. 2004; 64:442–451
19. Li HT, Yan JB, Li J et al. Enhancement of humoral immune responses to HBsAg by heat shock pro-tein GP96 and its N-terminal fragment in mice. World J Gastroenterol. 2005; 11:2858–2863.
20. Nasiri V, Dalimi A, Ghaffarifar F, et al. Immuno-genicity and efficacy of live L. arentolae expressing KMP11-NTGP96 -GFP fusion as a vaccine candi-date against experimental viscer-al leishmaniasis caused by L. Infantum. Iran J Parasi-tol. 2016; 11(2): 144–158.
21. Sundar S, More DK, Singh MK et al. Failure of pentavalent antimony in visceral leishmaniasis in In-dia: report from the center of the Indian epidemic. Clin Infect Dis. 2000; 31(4):1104-7.
22. Garmory HS, Brown KA, Titball RW. DNA vac-cines: improving expression of antigens. Genet Vaccines Ther. 2003; 1(1):2.
23. WHO. Control of the leishmaniases: report of a meeting of the WHO Expert Commitee on the Control of Leishmaniases, Geneva, 22-26 March 2010, no. 949. p. 186.
24. Nagill R, Kaur S. Vaccine candidates for leishmani-asis: a review. Int Immunopharmacol. 2011; 11(10):1464–88.
25. Borja-Cabrera GP, Santos FN, Bauer FS et al. Im-munogenicity assay of the Leishmune vaccine against canine visceral leishmaniasis in Brazil. Vac-cine. 2008; 26(39): 4991–7.
26. Trujillo C, Ramirez R, Velez ID, Berberich C. The humoral immune response to the kinetoplastid membrane protein-11 in patients with American leishmaniasis and Chagas disease prevalence of IgG subclasses and mapping of epitopes. Immunol Lett.1999; 70:203-209.
27. Ramirez JR, Gilchrist K, Robledo S et al. Attenuat-ed Toxoplasma gondii ts-4 mutants engineered to express the Leishmania antigen KMP-11 elicit a spe-cific immune response in BALB/c mice. Vaccine. 2001; 20:455-461.
28. Uzonna JE, Wei G, Yurkowski D, Bretscher P. Immune elimination of Leishmania major in mice: Implications for immune memory, vaccination, and reactivation disease. J Immunol. 2001; 167, 6967–6974.
29. Yamakami K, Akao S, Sato M et al. A single intra-dermal administration of soluble leishmanial antigen and plasmid expressing interleukin-12 protects BALB/c mice from Leishmania major infection. Par-asitol Int. 2001; 50, 81–91.
30. Jaafari MR, Ghafarian A, Farrokh-Gisour A et al. Immune response and protection assay of recom-binant major surface glycoprotein of Leishmania (rgp63) reconstituted with liposomes in BALB/c mice. Vaccine. 2006; 24, 5708–5717.
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.