Original Article

Immunogenicity and Efficacy of Live L. tarentolae Expressing KMP11-NTGP96-GFP Fusion as a Vaccine Candidate against Experimental Visceral Leishmaniasis Caused by L. infantum


Background: The aim of present study was to evaluate the protective efficacy of live recombinant L. tarentolae expressing KMP11-NTGP96-GFP fusion as candidates for live engineered recombinant vaccine against visceral leishmaniasis in BALB/c mice.

Methods: KMP-11 and NT-GP96 genes cloned into the pJET1.2/blunt cloning vector and then into pEGFP-N1 expression vector. The KMP-11, NT-GP96 and GFP fused in pEGFP-N1 and subcloned into Leishmanian pLEXSY-neo vector. Finally this construct was transferred to L. tarentolae by electroporation. Tranfection was confirmed by SDS-PAGE, WESTERN blot, flowcytometry and RT-PCR. Protective efficacy of this construct was evaluated as a vaccine candidate against visceral leishmaniasis. Parasite burden, humoral and cellular immune responses were assessed before and at 4 weeks after challenge.

Results: KMP- NT-Gp96-GFP Fusion was cloned successfully into pLEXSY -neo vector and this construct successfully transferred to L. tarentolae. Finding indicated that immunization with L. tarentolae tarentolae-KMP11-NTGP96-GFP provides significant protection against visceral leishmaniasis and was able to induce an increased expression of IFN-γ and IgG2a. Following challenge, a reduced parasite load in the spleen of the KMP11-NTGP96-GFP immunized group was detected.

Conclusion: The present study is the first to use a combination of a Leishmania antigen with an immunologic antigen in live recombinant L. tarentolae and results suggest that L. tarentolae-KMP11-NTGP96-GFP could be considered as a potential tool in vaccination against visceral leishmaniasis and this vaccination strategy could provide a potent rout for future vaccine development. 

Dumonteil E, Maria Jesus R-S, Javier E-O, Maria del Rosario G-M. DNA vaccines induce partial protection against L. mexicana. Vaccine. 2003; 21(17):2161–8.

Joshi S, Rawat K, Yadav NK, Kumar V, Siddiqi MI, Dube A. Visceral Leishmaniasis: Advancements in Vaccine Development via Classical and Molecular Approaches. Front Immunol. 2014; 5:380.

Edrissian Gh H, Nadim A, Alborzi A V, Ardehali S. Visceral leishmaniasis: the Iranian experiences. Arch Iran Med. 1998; 1(1):22–6.

Edrissian Gh H. Visceral leishmaniasis in Iran and the role of serological tests in diagnosis and epidemiological studies. Parasitol 21st Century ICOPA VIII, Izmir, Turkey CAB Int. 1996;63–78.

Mohebali M, Edrissian Gh H, Nadim A, Hajjaran H, Akhoundi B, Hooshmand B, et al. Application of direct agglutination test (DAT) for the diagnosis and seroepide-miological studies of visceral leishmaniasis in Iran. Iran J Parasitol. 2006; 1(1):15–25.

Mohebali M. Visceral leishmaniasis in Iran: Review of the Epidemiological and Clinical Features. Iran J Parasitol; 2013;8(3):348–58.

Mohebali M, Hajjaran H, Hamzavi Y, Mobedi I, Arshi S, Zarei Z, et al. Epidemiological aspects of canine visceral leishmaniosis in the Islamic Republic of Iran. Vet Parasitol. 2005; 129(3):243–51.

Croft SL. Recent developments in the chemotherapy of leishmaniasis. Trends Pharmacol Sci. 1988; 9(10):376–81.

Murray HW, Berman JD, Wright SD. Immunochemotherapy for intracellular L. donovani infection: γ interferon plus pentavalent antimony. J Infect Dis. 1988; 157(5): 973–8.

Lonso CAA, Berberich C, Requena JM, Alonso C. Cloning of Genes and Expression and Antigenicity Analysis of the L. infantum KMP-11 Protein. Exp Parasitol. 1997; 85(1):105–8.

Kurtzhals JAL, Hey AS, Jardim A, Kemp M, SChaefer K, Odera EO, et al. Dichotomy of the human T cell response to Leishmania antigens. II. Absent or Th2‐like response to gp63 and Thl-like response to lipophosphoglycan-associated protein in cells from cured visceral leishmaniasis patients. Clin Exp Immunol; 1994; 96(3):416–21.

Todolí F, Galindo I, Gómez-Sebastián S, Pérez-Filgueira M, Escribano JM, Alberola J, et al. Dynamics and predictive potential of antibodies against insect-derived recombinant L. infantum proteins during chemotherapy of naturally infected dogs. Am J Trop Med Hyg 2010; 82(5):795–800.

Tolson DL, Jardim A, Schnur LF, Stebeck C, Tuckey C, Beecroft RP, et al. The kinetoplastid membrane protein 11 of L. donovani and African trypanosomes is a potent stimulator of T-lymphocyte proliferation. Infect Immun. 1994; 62(11):4893–9.

Fuertes MA, Pérez JM, Soto M, López MC, Alonso C. Calcium-induced conformational changes in L. infantum kinetoplastid membrane protein-11. JBIC J Biol Inorg Chem. 2001; 6(1):107–17.

Srivastava P. Interaction of heat shock proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses. Annu Rev Immunol. Annual Reviews 4139 El Camino Way, USA; 2002; 20(1):395–425.

Nicchitta CV. Re-evaluating the role of heat-shock protein–peptide interactions in tumour immunity. Nat Rev Immunol. 2003; 3(5):427–32.

Li H-T, Yan J-B, Li J, Zhou M-H, Zhu X-D, Zhang Y-X, et al. Enhancement of humoral immune responses to HBsAg by heat shock protein gp96 and its N-terminal fragment in mice. World J Gastroenterol. 2005; 11(19):2858–63.

Yan J, Liu X, Wang Y, Jiang X, Liu H, Wang M, et al. Enhancing the potency of HBV DNA vaccines using fusion genes of HBV‐specific antigens and the N‐terminal fragment of gp96. J Gene Med. 2007; 9(2):107–21.

Zahedifard F, Gholami E, Taheri T, Taslimi Y, Doustdari F, Seyed N, et al. Enhanced Protective Efficacy of Nonpathogenic Recombinant L. tarentolae Expressing Cysteine Proteinases Combined with a Sand Fly Salivary Antigen. PLoS Negl Trop Dis. 2014; 8(3):e2751.

Zhang WW, Charest H, Matlashewski G. The expression of biologically active human p53 in L. cells: a novel eukaryotic system to produce recombinant proteins. Nucleic Acids Res. 1995; 23(20):4073–80.

Hughes AL, Piontkivska H. Phylogeny of Trypanosomatidae and Bodonidae (Kinetoplastida) based on 18S rRNA: evidence for paraphyly of Trypanosoma and six other genera. Mol Biol Evol. 2003; 20(4):644–52.

Clayton C, Häusler T, Blattner J. Protein trafficking in kinetoplastid protozoa. Microbiol Rev. 1995; 59(3):325–44.

Breitling R, Klingner S, Callewaert N, Pietrucha R, Contreras R, Geyer A, et al. Non-pathogenic trypanosomatid protozoa as a platform for protein research and production. Protein Expr Purif. 2002; 25(2):209–18.

Hemayatkar M, Mahboudi F, Majidzadeh-A K, Davami F, Vaziri B, Barkhordari F, et al. Increased expression of recombinant human tissue plasminogen activator in L. tarentolae. Biotechnol J. 2010; 5(11):1198–206.

Papadopoulou B, Roy G, Ouellette M. A novel antifolate resistance gene on the amplified H circle of L.. EMBO J. 1992; 11(10):3601–8.

Coligan JE, Dunn BM, Speicher DW, Winfeild PT. Short protocols in protein science: a compendium of methods from Current protocols in protein science. John Wiley & Sons Inc; 2003.

da Costa AV, Huerre M, Delacre M, Auriault C, Costa JMC, Verwaerde C, et al. IL-10 leads to a higher parasite persistence in a resistant mouse model of L. major infection. Parasitol Int. 2002; 51(4):367–79.

Buffet PA, Sulahian A, Garin YJ, Nassar N, Derouin F. Culture microtitration : a sensitive method for quantifying L. infantum in tissues of infected mice. Culture Microtitration : a Sensitive Method for Quantifying L. infantum in Tissues of Infected Mice. Antimicrob Agents Chemother. Am Soc Microbiol; 1995; 39(9):2167–9.

Mutiso JM, Macharia JC, Kiio MN, Ichagichu JM, Rikoi H, Gicheru MM. Development of Leishmania vaccines: predicting the future from past and present experience. J Biomed Res. 2013;27(2):85–102.

Sundar S, More DK, Singh MK, Singh VP, Sharma S, Makharia A, et al. Failure of pentavalent antimony in visceral leishmaniasis in India: report from the center of the Indian epidemic. Clin Infect Dis. 2000; 31(4):1104–7.

Khamesipour A, Rafati S, Davoudi N, Maboudi F, Modabber F. Leishmaniasis vaccine candidates for development: a global overview. Indian J Med Res. 2006; 123(3):423.

Myler PJ, Fasel N. Leishmania: after the genome. Horizon Scientific Press; 2008.

Gradoni L. Canine Leishmania vaccines: still a long way to go. Vet Parasitol. 2015; 208(1-2):94-100.

Mohebali M, Fallah E, Jamshidi Sh HH. Vaccine trial against canine visceral leishmaniasis in the Islamic Republic of Iran. East Mediterr Heal J. 1998; 4(2):234–8.

Mohebali M, Khamesipour A, Mobedi I, Zarei Z, Hashemi-Fesharki R. Double-blind randomized efficacy field trial of alum precipitated autoclaved L. major vaccine mixed with BCG against canine visceral leishmaniasis in Meshkin-Shahr district, IR Iran. Vaccine. 2004; 22(29):4097–100.

Barati M, Mohebali M, Alimohammadian MH, Khmesipour A, Keshavarz H, Akhoundi B, et al. Double-Blind Randomized Efficacy Field Trial of Alum Precipitated Autoclaved L. major (Alum-ALM) Vaccine Mixed With BCG Plus Imiquimod Vs. Placebo Control Group. Iran J Parasitol.; 2015;10(3):351.

Stebeck CE, Beecroft RP, Singh BN, Jardim A, Olafson RW, Tuckey C, et al. Kinetoplastid membrane protein-11 (KMP-11) is differentially expressed during the life cycle of African trypanosomes and is found in a wide variety of kinetoplastid parasites. Mol Biochem Parasitol. 1995;71(1):1–13.

Ramírez JR, Berberich C, Jaramillo A, Alonso C, Vélez ID. Molecular and antigenic characterization of the L. (Viannia) panamensis kinetoplastid membrane protein-11. Mem Inst Oswaldo Cruz. 1998; 93(2):247–54.

Jensen AT, Gasim S, Ismail A, Gaafar A, Kurtzhals JA, Kemp M, El Hassan AM, Kharazmi A TT. Humoral and cellular immune responses to synthetic peptides of the L. donovani kinetoplastid membrane protein‐11. Scand J Immunol.; 1998; 48(1):103–9.

Trujillo C, Ramı́rez R, Vélez 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(3):203–9.

Marañón C, Thomas MC, Planelles L, López MC. The immunization of A2/K b transgenic mice with the KMP11-HSP70 fusion protein induces CTL response against human cells expressing the T. cruzi KMP11 antigen: identification of A2-restricted epitopes. Mol Immunol. 2001; 38(4):279–87.

Flechas ID, Cuellar A, Cucunubá ZM, Rosas F, Velasco V, Steindel M, et al. Characterising the KMP-11 and HSP-70 recombinant antigens’ humoral immune response profile in chagasic patients. BMC Infect Dis. 2009; 9:186.

Ramírez JR, Gilchrist K, Robledo S, Sepúlveda JC, Moll H, Soldati D, et al. Attenuated Toxoplasma gondii ts-4 mutants engineered to express the L. antigen KMP-11 elicit a specific immune response in BALB/c mice. Vaccine. 2001; 20(3-4):455–61.

Chen C-H, Wang T-L, Hung C-F, Yang Y, Young RA, Pardoll DM, et al. Enhancement of DNA vaccine potency by linkage of antigen gene to an HSP70 gene. Cancer Res. 2000; 60(4):1035–42.

Baker-LePain JC, Sarzotti M, Fields TA, Li C-Y, Nicchitta C V. GRP94 (gp96) and GRP94 N-terminal geldanamycin binding domain elicit tissue nonrestricted tumor suppression. J Exp Med. 2002; 196(11):1447–59.

Li H, Zhou M, Han J, Zhu X, Dong T, Gao GF, et al. Generation of murine CTL by a hepatitis B virus-specific peptide and evaluation of the adjuvant effect of heat shock protein glycoprotein 96 and its terminal fragments. J Immunol. J Immunol. 2005; 174(1):195–204.

Cargnelutti DE, Salomón MC, Celedon V, García Bustos MF, Morea G, Cuello-Carrión FD, et al. Immunization with antigenic extracts of Leishmania associated with Montanide ISA 763 adjuvant induces partial protection in BALB/c mice against L.(L.) amazonensis infection. J Microbiol Immunol Infect. 2016; 49(1):24-32.

Fiuza JA, Gannavaram S, Santiago HDC, Selvapandiyan A, Souza DM, Passos LSA, et al. Vaccination using live attenuated L. donovani centrin deleted parasites induces protection in dogs against Leishmania infantum. Vaccine. 2015; 33(2):280–8.

Soleimani M, Mahboudi F, Davoudi N, Amanzadeh A, Azizi M, Adeli A, et al. Expression of human tissue plasminogen activator in the trypanosomatid protozoan L. tarentolae. Biotechnol Appl Biochem. 2007; 48(1):55–61.

Phan H-P, Sugino M, Niimi T. The production of recombinant human laminin-332 in a L. tarentolae expression system. Protein Expr Purif.; 2009; 68(1):79–84.

Kushnir S, Gase K, Breitling R, Alexandrov K. Development of an inducible protein expression system based on the protozoan host L. tarentolae . Protein Expr Purif. 2005;42(1):37–46.

Fritsche C, Sitz M, Weiland N, Breitling R, Pohl H-D. Characterization of the growth behavior of L. tarentolae: a new expression system for recombinant proteins. J Basic Microbiol. 2007; 47(5):384–93.

McCall LI, Zhang WW, Ranasinghe S, Matlashewski G. Leishmanization revisited: Immunization with a naturally attenuated cutaneous L. donovani isolate from Sri Lanka protects against visceral leishmaniasis. Vaccine. 2013; 31(10):1420-5.

Soto M, Requena JM, Quijada L, Alonso C. Multicomponent chimeric antigen for serodiagnosis of canine visceral leishmaniasis. J Clin Microbiol. 1998; 36(1):58–63.

Molano I, Alonso MG, Miron C, Redondo E, Requena JM, Soto M, et al. A L. infantum multi-component antigenic protein mixed with live BCG confers protection to dogs experimentally infected with L. infantum. Vet Immunol Immunopathol. 2003; 92(1):1–13.

Carcelen J, Iniesta V, Fernandez-Cotrina J, Serrano F, Parejo JC, Corraliza I, et al. The chimerical multi-component Q protein from L. in the absence of adjuvant protects dogs against an experimental L. infantum infection. Vaccine. 2009; 27(43):5964–73.

IssueVol 11 No 2 (2016) QRcode
SectionOriginal Article(s)
KMP-11 NT-GP96 L. tarentolae L. infantum Visceral leishmaniasis Vaccine

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How to Cite
NASIRI V, DALIMI A, GHAFFARIFAR F, BOLHASSANI A. Immunogenicity and Efficacy of Live L. tarentolae Expressing KMP11-NTGP96-GFP Fusion as a Vaccine Candidate against Experimental Visceral Leishmaniasis Caused by L. infantum. Iran J Parasitol. 2016;11(2):144-158.