Intradermal Immunization of Leishmania donovani Centrin Knock-Out Parasites in Combination with Salivary Protein LJM19 from Sand Fly Vector Induces a Durable Protective Immune Response in Hamsters
Background Visceral leishmaniasis (VL) is a neglected tropical disease and is fatal if untreated. There is no vaccine available against leishmaniasis. The majority of patients with cutaneous leishmaniasis (CL) or VL develop a long-term protective immunity after cure from infection, which indicates that development of an effective vaccine against leishmaniasis is possible. Such protection may also be achieved by immunization with live attenuated parasites that do not cause disease. We have previously reported a protective response in mice, hamsters and dogs with Leishmania donovani centrin gene knock-out parasites (LdCen-/-), a live attenuated parasite with a cell division specific centrin1 gene deletion. In this study we have explored the effects of salivary protein LJM19 as an adjuvant and intradermal (ID) route of immunization on the efficacy of LdCen-/- parasites as a vaccine against virulent L. donovani. Methodology/Principal Findings To explore the potential of a combination of LdCen-/- parasites and salivary protein LJM19 as vaccine antigens, LdCen-/- ID immunization followed by ID challenge with virulent L. donovani were performed in hamsters in a 9-month follow up study. We determined parasite burden (serial dilution), antibody production (ELISA) and cytokine expression (qPCR) in these animals. Compared to controls, animals immunized with LdCen-/- + LJM19 induced a strong antibody response, a reduction in spleen and liver parasite burden and a higher expression of pro-inflammatory cytokines after immunization and one month post-challenge. Additionally, a low parasite load in lymph nodes, spleen and liver, and a non-inflamed spleen was observed in immunized animals 9 months after the challenge infection. Conclusions Our results demonstrate that an ID vaccination using LdCen-/-parasites in combination with sand fly salivary protein LJM19 has the capability to confer long lasting protection against visceral leishmaniasis that is comparable to intravenous or intracardial immunization.
References
[1]
Desjeux P. Leishmaniasis: Current situation and new perspectives. Comp Immunol Microbiol Infect Dis. 2004;27(5):305–18. pmid:15225981 doi: 10.1016/j.cimid.2004.03.004
[2]
Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS One [Internet]. 2012;7(5):e35671. Available from: doi: 10.1371/journal.pone.0035671. pmid:22693548
[3]
Croft SL, Coombs GH. Leishmaniasis—Current chemotherapy and recent advances in the search for novel drugs. Trends Parasitol. 2003;19(11):502–8. pmid:14580961 doi: 10.1016/j.pt.2003.09.008
[4]
Minodier P, Jurquet AL, Noel G, Uters M, Laporte R, Garnier JM. [Leishmaniasis treatment]. Arch Pediatr. 2010;17(6):838–9. doi: 10.1016/S0929-693X(10)70137-8. pmid:20654919
[5]
Costa CHN. How effective is dog culling in controlling zoonotic visceral leishmaniasis? A critical evaluation of the science, politics and ethics behind this public health policy. Rev Soc Bras Med Trop [Internet]. 2011;44(2):232–42. Available from: pmid:21468480 doi: 10.1590/s0037-86822011005000014
[6]
Okwor I, Uzonna J. Vaccines and vaccination strategies against human cutaneous leishmaniasis. Hum Vaccin. 2009;5(May):291–301. doi: 10.4161/hv.5.5.7607
[7]
Buxbaum LU, Denise H, Coombs GH, Alexander J, Mottram JC, Scott P. Cysteine protease B of Leishmania mexicana inhibits host Th1 responses and protective immunity. J Immunol. 2003;171(7):3711–7. pmid:14500670 doi: 10.4049/jimmunol.171.7.3711
[8]
Sp？th GF, Lye L-F, Segawa H, Sacks DL, Turco SJ, Beverley SM. Persistence without pathology in phosphoglycan-deficient Leishmania major. Science. 2003;301(2003):1241–3. doi: 10.1126/science.1087499
[9]
Stewart J, Curtis J, Spurck TP, Ilg T, Garami A, Baldwin T, et al. Characterisation of a Leishmania mexicana knockout lacking guanosine diphosphate-mannose pyrophosphorylase. Int J Parasitol [Internet]. 2005;35(8):861–73. Available from: pmid:15936761 doi: 10.1016/j.ijpara.2005.03.008
[10]
Silvestre R, Cordeiro-Da-Silva A, Santarém N, Vergnes B, Sereno D, Ouaissi A. SIR2-deficient Leishmania infantum induces a defined IFN-gamma/IL-10 pattern that correlates with protection. J Immunol [Internet]. 2007;179(5):3161–70. Available from: pmid:17709531 doi: 10.4049/jimmunol.179.5.3161
[11]
Cabral SM, Silvestre RL, Santarém NM, Tavares JC, Silva AF, Cordeiro-da-Silva A. A Leishmania infantum cytosolic tryparedoxin activates B cells to secrete interleukin-10 and specific immunoglobulin. Immunology [Internet]. 2008;123(4):555–65. Available from: pmid:18028371
[12]
Selvapandiyan A, Dey R, Duncan R, Nylén S, Nakhasi HL, Sacks DL, et al. Intracellular Replication-Deficient Leishmania donovani Induces Long Lasting Protective Immunity against Visceral Leishmaniasis. J Immunol [Internet]. 2009;183(3):1813–20. Available from: doi: 10.4049/jimmunol.0900276. pmid:19592661
[13]
Fiuza JA, Santiago HDC, Selvapandiyan A, Gannavaram S, Ricci ND, Bueno LL, et al. Induction of immunogenicity by live attenuated Leishmania donovani centrin deleted parasites in dogs. Vaccine [Internet]. 2013;31(14):1785–92. Available from: doi: 10.1016/j.vaccine.2013.01.048. pmid:23398933
[14]
Dey R, Natarajan G, Bhattacharya P, Cummings H, Dagur PK, Terrazas C, et al. Characterization of cross-protection by genetically modified live-attenuated Leishmania donovani parasites against Leishmania mexicana. J Immunol [Internet]. 2014;193(7):3513–27. Available from: pmid:25156362 doi: 10.4049/jimmunol.1303145.
[15]
Titus RG, Gueiros-Filho FJ, de Freitas L a, Beverley SM. Development of a safe live Leishmania vaccine line by gene replacement. Proc Natl Acad Sci U S A. 1995;92(October 1995):10267–71. doi: 10.1073/pnas.92.22.10267
[16]
Papadopoulou B, Roy G, Breton M, Kündig C, Dumas C, Fillion I, et al. Reduced infectivity of a Leishmania donovani biopterin transporter genetic mutant and its use as an attenuated strain for vaccination. Infect Immun [Internet]. 2002;70(1):62–8. Available from: pmid:11748164
[17]
Saravia NG, Escorcia B, Osorio Y, Valderrama L, Brooks D, Arteaga L, et al. Pathogenicity and protective immunogenicity of cysteine proteinase-deficient mutants of Leishmania mexicana in non-murine models. Vaccine [Internet]. 2006;24(19):4247–59. Available from: pmid:16216395 doi: 10.1016/j.vaccine.2005.05.045
[18]
Fiuza JA, Gannavaram S, Santiago HDC, Selvapandiyan A, Souza DM, Passos LSA, et al. Vaccination using live attenuated Leishmania donovani centrin deleted parasites induces protection in dogs against Leishmania infantum. Vaccine [Internet]. 2015;33(2):280–8. Available from: pmid:25475955 doi: 10.1016/j.vaccine.2014.11.039.
[19]
Valenzuela JG, Belkaid Y, Garfield MK, Mendez S, Kamhawi S, Rowton ED, et al. Toward a defined anti-Leishmania vaccine targeting vector antigens: characterization of a protective salivary protein. J Exp Med [Internet]. 2001;194(3):331–42. Available from: pmid:11489952
[20]
Andrade BB, De Oliveira CI, Brodskyn CI, Barral a., Barral-Netto M. Role of sand fly saliva in human and experimental leishmaniasis: Current insights. Scand J Immunol. 2007;66(2–3):122–7. pmid:17635789 doi: 10.1111/j.1365-3083.2007.01964.x
[21]
Chagas AC, Oliveira F, Debrabant A, Valenzuela JG, Ribeiro JMC, Calvo E. Lundep, a sand fly salivary endonuclease increases Leishmania parasite survival in neutrophils and inhibits XIIa contact activation in human plasma. PLoS Pathog [Internet]. 2014;10(2):e1003923. Available from: doi: 10.1371/journal.ppat.1003923. pmid:24516388
[22]
Titus RG, Ribeiro JM. Salivary gland lysates from the sand fly Lutzomyia longipalpis enhance Leishmania infectivity. Science. 1988;239(4845):1306–8. pmid:3344436 doi: 10.1126/science.3344436
[23]
Kamhawi S. The biological and immunomodulatory properties of sand fly saliva and its role in the establishment of Leishmania infections. Microbes Infect. 2000;2:1765–73. pmid:11137049 doi: 10.1016/s1286-4579(00)01331-9
[24]
Oliveira F, Lawyer PG, Kamhawi S, Valenzuela JG. Immunity to distinct sand fly salivary proteins primes the anti-Leishmania immune response towards protection or exacerbation of disease. PLoS Negl Trop Dis [Internet]. 2008;2(4):e226. Available from: doi: 10.1371/journal.pntd.0000226. pmid:18414648
[25]
Collin N, Gomes R, Teixeira C, Cheng L, Laughinghouse A, Ward JM, et al. Sand fly salivary proteins induce strong cellular immunity in a natural reservoir of visceral leishmaniasis with adverse consequences for Leishmania. PLoS Pathog [Internet]. 2009;5(5):e1000441. Available from: doi: 10.1371/journal.ppat.1000441. pmid:19461875
[26]
Gomes R, Teixeira C, Teixeira MJ, Oliveira F, Menezes MJ, Silva C, et al. Immunity to a salivary protein of a sand fly vector protects against the fatal outcome of visceral leishmaniasis in a hamster model. Proc Natl Acad Sci U S A. 2008;105(22):7845–50. doi: 10.1073/pnas.0712153105. pmid:18509051
[27]
Gomes R, Oliveira F, Teixeira C, Meneses C, Gilmore DC, Elnaiem D-E, et al. Immunity to sand fly salivary protein LJM11 modulates host response to vector-transmitted leishmania conferring ulcer-free protection. J Invest Dermatol [Internet]. 2012;132(12):2735–43. Available from: doi: 10.1038/jid.2012.205. pmid:22739793
[28]
Tavares NM, Silva R a, Costa DJ, Pitombo M a, Fukutani KF, Miranda JC, et al. Lutzomyia longipalpis saliva or salivary protein LJM19 protects against Leishmania braziliensis and the saliva of its vector, Lutzomyia intermedia. PLoS Negl Trop Dis [Internet]. 2011;5(5):e1169. Available from: doi: 10.1371/journal.pntd.0001169. pmid:21655303
[29]
Roatt BM, Aguiar-Soares RDDO, Vitoriano-Souza J, Coura-Vital W, Braga SL, Corrêa-Oliveira R, et al. Performance of LBSap vaccine after intradermal challenge with L. infantum and saliva of Lu. longipalpis: immunogenicity and parasitological evaluation. PLoS One [Internet]. 2012;7(11):e49780. Available from: doi: 10.1371/journal.pone.0049780. pmid:23189161
[30]
Zahedifard F, Gholami E, Taheri T, Taslimi Y, Doustdari F, Seyed N, et al. Enhanced Protective Efficacy of Nonpathogenic Recombinant Leishmania tarentolae Expressing Cysteine Proteinases Combined with a Sand Fly Salivary Antigen. PLoS Negl Trop Dis. 2014;8(3):e2751. doi: 10.1371/journal.pntd.0002751. pmid:24675711
[31]
MOHEBALI M. Double-blind randomized efficacy field trial of alum precipitated autoclaved Leishmania major vaccine mixed with BCG against canine visceral leishmaniasis in Meshkin-Shahr district, I.R. Iran. Vaccine [Internet]. 2004;22(29–30):4097–100. Available from: pmid:15364462
[32]
Mutiso JM, Macharia JC, Gicheru MM. Immunization with Leishmania vaccine-alum-BCG and montanide ISA 720 adjuvants induces low-grade type 2 cytokines and high levels of IgG2 subclass antibodies in the vervet monkey (Chlorocebus aethiops) model. Scand J Immunol. 2012;76(5):471–7. doi: 10.1111/j.1365-3083.2012.02764.x. pmid:22862788
[33]
Belkaid Y, Von Stebut E, Mendez S, Lira R, Caler E, Bertholet S, et al. CD8+ T cells are required for primary immunity in C57BL/6 mice following low-dose, intradermal challenge with Leishmania major. J Immunol [Internet]. 2002;168(8):3992–4000. Available from: pmid:11937556 doi: 10.4049/jimmunol.168.8.3992
[34]
Gomes RB, Brodskyn C, de Oliveira CI, Costa J, Miranda JC, Caldas A, et al. Seroconversion against Lutzomyia longipalpis saliva concurrent with the development of anti-Leishmania chagasi delayed-type hypersensitivity. J Infect Dis [Internet]. 2002;186(10):1530–4. Available from: pmid:12404176 doi: 10.1086/344733
[35]
Méndez S, Gurunathan S, Kamhawi S, Belkaid Y, Moga M a, Skeiky Y a, et al. The potency and durability of DNA- and protein-based vaccines against Leishmania major evaluated using low-dose, intradermal challenge. J Immunol [Internet]. 2001;166(8):5122–8. Available from: pmid:11290794 doi: 10.4049/jimmunol.166.8.5122
[36]
Peters a M, Fowler JC, Britton TB, Solanki CK, Ballinger JR, Ravichandran D, et al. Functional variation in lymph node arrangements within the axilla. Lymphat Res Biol [Internet]. 2009;7(3):139–44. Available from: pmid:19778201 doi: 10.1089/lrb.2008.1021.
[37]
Ribeiro-Gomes FL, Roma EH, Carneiro MBH, Doria N a., Sacks DL, Peters NC. Site-dependent recruitment of inflammatory cells determines the effective dose of Leishmania major. Infect Immun. 2014;82(7):2713–27. doi: 10.1128/IAI.01600-13. pmid:24733090
[38]
McCall L-I, Zhang W-W, Matlashewski G. Determinants for the development of visceral leishmaniasis disease. PLoS Pathog [Internet]. 2013;9(1):e1003053. Available from: doi: 10.1371/journal.ppat.1003053. pmid:23300451
[39]
Romano A, Doria NA, Mendez J, Sacks DL PN. No Title. J Immunol. 2015;195(8):3816–27. pmid:26371247 doi: 10.4049/jimmunol.1500752
[40]
Ghosh A, Zhang WW, Matlashewski G. Immunization with A2 protein results in a mixed Th1/Th2 and a humoral response which protects mice against Leishmania donovani infections. Vaccine. 2001;20(1–2):59–66. pmid:11567746 doi: 10.1016/s0264-410x(01)00322-x
[41]
Garg R, Gupta SK, Tripathi P, Hajela K, Sundar S, Naik S, et al. Leishmania donovani: identification of stimulatory soluble antigenic proteins using cured human and hamster lymphocytes for their prophylactic potential against visceral leishmaniasis. Vaccine [Internet]. 2006;24(15):2900–9. Available from: pmid:16448729 doi: 10.1016/j.vaccine.2005.12.053
[42]
Chávez-Fumagalli M a., Costa M a F, Oliveira DM, Ramírez L, Costa LE, Duarte MC, et al. Vaccination with the Leishmania infantum ribosomal proteins induces protection in BALB/c mice against Leishmania chagasi and Leishmania amazonensis challenge. Microbes Infect. 2010;12(12–13):967–77. doi: 10.1016/j.micinf.2010.06.008. pmid:20601076
[43]
Maroof A, Brown N, Smith B, Hodgkinson MR, Maxwell A, Losch FO, et al. Therapeutic vaccination with recombinant adenovirus reduces splenic parasite burden in experimental visceral leishmaniasis. J Infect Dis [Internet]. 2012;205(5):853–63. Available from: doi: 10.1093/infdis/jir842. pmid:22301630
[44]
Gerth AJ, Lin L, Peng SL. T-bet regulates T-independent IgG2a class switching. Int Immunol. 2003;15(8):937–44. pmid:12882831 doi: 10.1093/intimm/dxg093
[45]
Guha R, Gupta D, Rastogi R, Vikram R, Krishnamurthy G, Bimal S, et al. Vaccination with leishmania hemoglobin receptor-encoding DNA protects against visceral leishmaniasis. Sci Transl Med [Internet]. 2013;5(202):202ra121. Available from: pmid:24027025 doi: 10.1126/scitranslmed.3006406.
[46]
Da Silva R a a, Tavares NM, Costa D, Pitombo M, Barbosa L, Fukutani K, et al. DNA vaccination with KMP11 and Lutzomyia longipalpis salivary protein protects hamsters against visceral leishmaniasis. Acta Trop [Internet]. 2011;120(3):185–90. Available from: doi: 10.1016/j.actatropica.2011.08.007. pmid:21875567
[47]
Ghalib HW, Whittle JA, Kubin M, Hashim FA, el-Hassan AM, Grabstein KH, et al. IL-12 enhances Th1-type responses in human Leishmania donovani infections. J Immunol. 1995;154:4623–9. pmid:7722314
[48]
Kenney RT, Sacks DL, Gam a a, Murray HW, Sundar S. Splenic cytokine responses in Indian kala-azar before and after treatment. J Infect Dis. 1998;177(3):815–8. pmid:9498473 doi: 10.1086/517817
[49]
Dey R, Majumder N, Bhattacharyya Majumdar S, Bhattacharjee S, Banerjee S, Roy S, et al. Induction of host protective Th1 immune response by chemokines in Leishmania donovani-infected BALB/c mice. Scand J Immunol [Internet]. 2007;66(6):671–83. Available from: pmid:18021365 doi: 10.1111/j.1365-3083.2007.02025.x
[50]
Banerjee A, De M, Ali N. Complete cure of experimental visceral leishmaniasis with amphotericin B in stearylamine-bearing cationic liposomes involves down-regulation of IL-10 and favorable T cell responses. J Immunol. 2008;181(2):1386–98. pmid:18606693 doi: 10.4049/jimmunol.181.2.1386
[51]
Kobayashi M, Fitz L, Ryan M, Hewick RM, Clark SC, Chan S, et al. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J Exp Med. 1989;170(3):827–45. pmid:2504877 doi: 10.1084/jem.170.3.827
[52]
Trinchieri G. Interleukin-12 and its role in the generation of TH1 cells. Immunol Today. 1993;14(7):335–8. pmid:8103338 doi: 10.1016/0167-5699(93)90230-i
[53]
Trinchieri G. Interleukin-12 and interferon-gamma. Do they always go together? Am J Pathol. 1995;147(6):1534–8. pmid:7495276
