Abstract:

Visceral leishmaniasis (VL) is one of the most important tropical diseases worldwide. Although chemotherapy has been widely used to treat this disease, problems related to the development of parasite resistance and side effects associated with the compounds used have been noted. Hence, alternative approaches for VL control are desirable. Some methods, such as vector control and culling of infected dogs, are insufficiently effective, with the latter not ethically recommended. The development of vaccines to prevent VL is a feasible and desirable measure for disease control; for example, some vaccines designed to protect dogs against VL have recently been brought to market. These vaccines are based on the combination of parasite fractions or recombinant proteins with adjuvants that are able to induce cellular immune responses; however, their partial efficacy and the absence of a vaccine to protect against human leishmaniasis underline the need for characterization of new vaccine candidates. This review presents recent advances in control measures for VL based on vaccine development, describing extensively studied antigens, as well as new antigenic proteins recently identified using immuno-proteomic techniques.

Keywords:
Vaccine. Visceral leishmaniasis; Recombinant proteins; Immuno-proteomic approach; Hypothetical proteins.

INTRODUCTION

Leishmaniasis is a disease complex caused by different species of protozoan parasites of the genus Leishmania¹1. Grimaldi Jr G, Tesh RB. Leishmaniases of the New World: current concepts and implications for future research. Clin Microbiol Rev 1993; 6:230-250.. The disease causes high levels of morbidity and mortality worldwide, where approximately 1-1.5 million cases of tegumentary leishmaniasis (TL) and 0.2-0.5 million cases of visceral leishmaniasis (VL) are registered annually²2. Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 2012; 7:e35671.. VL is caused by parasites of the Leishmania donovani complex, including the species L. donovani and Leishmania infantum³3. Kedzierski L, Sakthianandeswaren A, Curtis JM, Andrews PC, Junk PC, Kedzierska K. Leishmaniasis: current treatment and prospects for new drugs and vaccines. Curr Med Chem 2009; 16:599-614.. In the Americas, VL is a zoonotic disease caused by L. infantum, where dogs are considered the main domestic reservoirs of the parasites⁴4. World Health Organization. Control of the leishmaniases. Report of a Meeting of the WHO Expert Committee on the Control of Leishmaniases. Geneva: 2010. World Health Organ Tech Rep Ser 2010; 949:22-26.^{) (5}5. Roatt BM, Aguiar-Soares RDO, Coura-Vital W, Ker HG, Moreira ND, Vitoriano-Souza J. Immunotherapy and immunochemotherapy in visceral leishmaniasis: promising treatments for this neglected disease. Front Immunol 2014; 5:272.. In human VL, the outcomes of infection can vary from an asymptomatic and/or subclinical disease to a form with acute symptoms; the disease carries a high risk of mortality in the absence of an adequate treatment⁶6. Michel G, Pomares C, Ferrua B, Marty P. Importance of worldwide asymptomatic carriers of Leishmania infantum (L. chagasi) in human. Acta Trop 2011; 119:69-75.^{) (7}7. Apa H, Devrim I, Bayram N, Deveci R, Demir-Özek G, Carti ÖU. Liposomal amphotericin B versus pentavalent antimony salts for visceral Leishmania in children. Turk J Pediatr 2013; 55:378-383..

Chemotherapy based on the administration of pentavalent antimonials has been used to treat VL; however, these products present problems related to their toxicity⁸8. Thakur CP, Sinha GP, Pandey AK, Kumar N, Kumar P, Hassan SM, et al. Do the diminishing efficacy and increasing toxicity of sodium stibogluconate in the treatment of visceral leishmaniasis in Bihar, India, justify its continued use as a first-line drug? An observational study of 80 cases. Ann Trop Med Parasitol 1998; 92:561-569.^{) (9}9. 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; 31:1104-1107.^{) (10}10. Croft SL, Coombs GH. Leishmaniasis-current chemotherapy and recent advances in the search for novel drugs. Trends Parasitol 2003; 19:502-508.. Other drugs, such as pentamidine, miltefosine, and amphotericin B also present issues of toxicity and/or high cost¹¹11. Annaloro C, Olivares C, Usardi P, Onida F, Della-Volpe A, Tagliaferri E, et al. Retrospective evaluation of amphotericin B deoxycholate toxicity in a single centre series of haematopoietic stem cell transplantation recipients. J Antimicrob Chemother 2009; 63:625-626.^{) (12}12. Caldeira LR, Fernandes FR, Costa DF, Frézard F, Afonso LC, Ferreira LA. Nanoemulsions loaded with amphotericin B: a new approach for the treatment of leishmaniasis. Eur J Pharm Sci 2015; 70:125-131.^{) (13}13. Chávez-Fumagalli MA, Ribeiro TG, Castilho RO, Fernandes SOA, Cardoso VN, Coelho CSP, et al. New delivery systems for amphotericin B applied to the improvement of leishmaniasis treatment. Rev Soc Bras Med Trop 2015; 48:235-242.^{) (14}14. Martins VT, Chávez-Fumagalli MA, Costa LE, Martins AMCC, Lage PS, Lage DP, et al. Antigenicity and protective efficacy of a Leishmania amastigote-specific protein, member of the super-oxygenase family, against visceral leishmaniasis. PLoS Negl Trop Dis 2013; 7:e2148.. Early diagnosis of VL could allow for more effective treatment of the disease; however, parasitological diagnosis, based on the direct observation of amastigote forms has low sensitivity and requires invasive collection procedures¹⁵15. Srividya G, Kulshrestha A, Singh R, Salotra P. Diagnosis of visceral leishmaniasis: developments over the last decade. Parasitol Res 2012; 110:1065-1078.. The detection of Leishmania deoxyribonucleic acid (DNA) using the polymerase chain reaction (PCR) technique is highly specific; however, its sensitivity is variable¹⁶16. Singh DP, Goyal RK, Singh RK, Sundar S, Mohapatra TM. In search of an ideal test for diagnosis and prognosis of kala-azar. J Health Popul Nutr 2010; 28:281-285.^{) (17}17. Duarte MC, Pimenta DC, Menezes-Souza D, Magalhães RD, Diniz JL, Costa LE, et al. Proteins selected in Leishmania (Viannia) braziliensis by an immunoproteomic approach with potential serodiagnosis applications for tegumentary leishmaniasis. Clin Vaccine Immunol 2015; 22:1187-1196.. Serological tests based on the detection of antileishmanial antibodies in patient serum samples are also employed for the diagnosis of VL; however, these are also associated with issues related to sensitivity and/or specificity, depending on the antigens targeted¹⁸18. Peruhype-Magalhães V, Machado-de-Assis TS, Rabello A. Use of the Kala-Azar Detect(r) and IT-LEISH(r) rapid tests for the diagnosis of visceral leishmaniasis in Brazil. Mem Inst Oswaldo Cruz 2012; 107:951-952.^{) (19}19. Abass E, Bollig N, Reinhard K, Camara B, Mansour D, Visekruna A, et al. rKLO8, a novel Leishmania donovani - derived recombinant immunodominant protein for sensitive detection of visceral leishmaniasis in Sudan. PLoS Negl Trop Dis 2013; 7:e2322..

Evidence of life-long immunity to leishmaniasis has also inspired the development of prophylactic vaccination protocols against the disease, although few have progressed beyond the experimental stage¹⁴14. Martins VT, Chávez-Fumagalli MA, Costa LE, Martins AMCC, Lage PS, Lage DP, et al. Antigenicity and protective efficacy of a Leishmania amastigote-specific protein, member of the super-oxygenase family, against visceral leishmaniasis. PLoS Negl Trop Dis 2013; 7:e2148.. An ideal vaccine candidate against leishmaniasis should be safe, affordable to the population, and able to induce both cluster of differentiation 4⁺ (CD4⁺⁾ and cluster of differentiation 8⁺ (CD8⁺⁾ T cell responses and long-term immunological memory, which could be boosted by natural infections, thus reducing the number of vaccine doses required. In addition, an ideal vaccine should be effective against different Leishmania species and stable at room temperature or at 4°C, to eliminate the need for storage at -20°C or -80°C²⁰20. Reed SG, Coler RN, Mondal D, Kamhawi S, Valenzuela JG. Leishmania vaccine development: exploiting the host-vector-parasite interface. Expert Rev Vaccines 2016; 15:81-90.. However, the induction and maintenance of long-lasting immunity and protection against different Leishmania species are very difficult to achieve, since the majority of candidate vaccines are composed of antigens that only offer species-specific protection²¹21. Rachamim N, Jaffe CL. Pure protein from Leishmania donovani protects mice against both cutaneous and visceral leishmaniasis. J Immunol 1993; 150:2322-2331.^{) (22}22. Afrin F, Anam K, Ali N. Induction of partial protection against Leishmania donovani by promastigote antigens in negatively charged liposomes. J Parasitol 2000; 86:730-735.^{) (23}23. Bhowmick S, Ravindran R, Ali N. IL-4 contributes to failure, and colludes with IL-10 to exacerbate Leishmania donovani infection following administration of a subcutaneous leishmanial antigen vaccine. BMC Microbiol 2014; 14:8.^{) (24}24. Iborra S, Parody N, Abánades DR, Bonay P, Prates D, Novais FO, et al. Vaccination with the Leishmania major ribosomal proteins plus CpG oligodeoxynucleotides induces protection against experimental cutaneous leishmaniasis in mice. Microbes Infect 2008; 10:1133-1141.^{) (25}25. Kumari S, Kumar A, Samant M, Singh N, Dube A. Discovery of novel vaccine candidates and drug targets against visceral leishmaniasis using proteomics and transcriptomics. Curr Drug Targets 2008; 9:938-947.^{) (26}26. Stäger S, Smith DF, Kaye PM. Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis. J Immunol 2000; 165:7064-7071..

Protective immunity against VL is based on the development of an antigen- and parasite-specific T-helper 1 (Th1)-type cellular response, primed by the production of interferon-gamma (IFN-γ), interleukin-2 (IL-2), interleukin-12 (IL-12), granulocyte macrophage colony-stimulating factor (GM-CSF), and other cytokines²⁷27. Agallou M, Smirlis D, Soteriadou KP, Karagouni E. Vaccination with Leishmania histone H1-pulsed dendritic cells confers protection in murine visceral leishmaniasis. Vaccine2012; 30:5086-5093.^{) (28}28. Das A, Ali N. Vaccine prospects of killed but metabolically active Leishmania against visceral leishmaniasis. Expert Rev Vaccines 2012; 11:783-785.^{) (29}29. Murray HW, Cervia JS, Hariprashad J, Taylor AP, Stoeckle MY, Hockman H. Effect of granulocyte-macrophage colony-stimulating factor in experimental visceral leishmaniasis. J Clin Invest 1995; 95:1183-1192.^{) (30}30. Dumas C, Muyombwe A, Roy G, Matte C, Ouellette M, Olivier M, et al. Recombinant Leishmania major secreting biologically active granulocyte-macrophage colony-stimulating factor survives poorly in macrophages in vitro and delays disease development in mice. Infect Immun 2003; 71:6499-6509.^{) (31}31. Lage DP, Martins VT, Duarte MC, Garde E, Chávez-Fumagalli MA, Menezes-Souza D, et al. Prophylactic properties of a Leishmania-specific hypothetical protein in a murine model of visceral leishmaniasis. Parasite Immunol 2015; 37:646-656.^{) (32}32. Martins VT, Lage DP, Duarte MC, Costa LE, Garde E, Rodrigues MR, et al. A new Leishmania-specific hypothetical protein, LiHyT, used as a vaccine antigen against visceral leishmaniasis. Acta Trop 2016; 154:73-81.. The induction of CD4⁺ Th1 cell responses against parasite antigens is crucial in controlling primary infection, when cytokines such as IFN-γ induce nitric oxide production by activated phagocytic cells able to kill internalized parasites³³33. Green SJ, Mellouk S, Hoffman SL, Meltzer MS, Nacy CA. Cellular mechanisms of nonspecific immunity to intracellular infection: cytokine-induced synthesis of toxic nitrogen oxides from L-arginine by macrophages and hepatocytes. Immunol Lett 1990; 25:15-19.^{) (34}34. Blackwell JM, Fakiola M, Ibrahim ME, Jamieson SE, Jeronimo SB, Miller EN, et al. Genetic susceptibility to leishmanial infections: studies in mice and man. Parasite Immunol 2009; 31:254-266.. Concomitantly to the role of CD4⁺ T cells, CD8⁺ T cells also contribute to protection against disease, and have an important role in controlling primary infections by increasing the Th1 response through a mechanism dependent on IFN-γ production³⁵35. Kharazmi A, Kemp K, Ismail A, Gasim S, Gaafar A, Kurtzhals JA, et al. T-cell response in human leishmaniasis. Immunol Lett 1999; 65:105-108.. In contrast, cytokines such as interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-18 (IL-18), and transforming growth factor beta (TGF-β) represent disease promoting molecules which inhibit the Th1 response, contributing to the deactivation of infected macrophages and, consequently, to the development of disease³⁶36. Wilson ME, Jeronimo SM, Pearson RD. Immunopathogenesis of infection with the visceralizing Leishmania species. Microb Pathog 2005; 38:147-160.^{) (37}37. Stäger S, Alexander J, Carter KC, Brombacher F, Kaye PM. Both interleukin-4 (IL-4) and IL-4 receptor alpha signaling contribute to the development of hepatic granulomas with optimal antileishmanial activity. Infect Immun 2003; 71:4804-4807.^{) (38}38. Afonso LC, Scott P. Immune responses associated with susceptibility of C57BL/10 mice to Leishmania amazonensis. Infect Immun 1993; 61:2952-2959..

In recent years, proteomic screening studies have revealed a number of antigenic proteins specific to the Leishmania genus and frequently annotated as hypothetical proteins in genome databases. This review explores recent developments and discusses the prospects for vaccine development against VL, focusing on well-known antigens described in the literature, as well as the discovery of new antigens by immuno-proteomic approaches.

SECOND-GENERATION VACCINES AGAINST VISCERAL LEISHMANIASIS

Advances in recombinant DNA technology have led to the extensive study of several species- or stage-specific Leishmania molecules as candidate vaccines in the form of recombinant proteins. The major advantages of these candidates are their purity and the production yields achievable. Several proteins have been frequently investigated as candidate vaccines for the cutaneous form of leishmaniasis; however, few of these have been evaluated in mammalian VL models³⁹39. Joshi J, Malla N, Kaur S. A comparative evaluation of efficacy of chemotherapy, immunotherapy and immunochemotherapy in visceral leishmaniasis-an experimental study. Parasitol Int 2014; 63:612-620.. Recombinant proteins have been evaluated as second-generation vaccines for VL with variable degrees of success, usually depending on the vaccine formulation and associated immune adjuvants, as well as the animal model used for testing. Amastigote and promastigote parasite antigens are the most common vaccine candidates tested.

Among amastigote-specific antigens tested for induction of immune protection against VL, the A2 antigen has emerged as an effective candidate. It is encoded by a multigene family that is abundantly expressed in the amastigote forms of some Leishmania species able to cause VL⁴⁰40. Fernandes AP, Costa MM, Coelho EA, Michalick MS, Freitas E, Melo MN, et al. Protective immunity against challenge with Leishmania (Leishmania) chagasi in beagle dogs vaccinated with recombinant A2 protein. Vaccine 2008; 26:5888-5895.. Studies of the administration of recombinant A2 protein associated with immune adjuvants⁴¹41. 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; 20:59-66.^{) (42}42. Coelho EAF, Tavares CAP, Carvalho FAA, Chaves KF, Teixeira KN, Rodrigues RC, et al. Immune responses induced by the Leishmania (Leishmania) donovani A2 antigen, but not by the LACK antigen, are protective against experimental Leishmania (Leishmania) amazonensis infection. Infect Immun 2003; 71:3988-3994. or as a DNA vaccine⁴³43. Zanin FHC, Coelho EAF, Tavares CAP, Marques-da-Silva EA, Costa MMS, Rezende SA, et al. Evaluation of immune responses and protection induced by A2 and nucleoside hydrolase (NH) DNA vaccines against Leishmania chagasi and Leishmania amazonensis experimental infections. Microbes Infect 2007; 9:1070-1077., as well as in attenuated non-replicative viruses⁴⁴44. Resende DM, Caetano BC, Dutra MS, Penido MLO, Abrantes CF, Verly RM, et al. Epitope mapping and protective immunity elicited by adenovirus expressing the Leishmania amastigote specific A2 antigen: correlation with IFN-γ and cytolytic activity by CD8+ T cells. Vaccine 2008; 26:4585-4593., non-pathogenic bacteria⁴⁵45. Yam KK, Hugentobler F, Pouliot P, Stern AM, Lalande J-D, Matlashewski G, et al. Generation and evaluation of A2-expressing Lactococcus lactis live vaccines against Leishmania donovani in BALB/c mice. J Med Microbiol 2011, 60:1248-1260., or non-virulent Leishmania tarentolae⁴⁶46. Mizbani A, Taheri T, Zahedifard F, Taslimi Y, Azizi H, Azadmanesh K, et al. Recombinant Leishmania tarentolae expressing the A2 virulence gene as a novel candidate vaccine against visceral leishmaniasis. Vaccine 2009, 28:53-62., have provided evidence of its protective efficacy in mammalian models. In general, anti-A2 protective immunity is associated with the generation of parasite-specific IgG2a antibodies, as well as with the production of high levels of antileishmanial IFN-γ and low levels of IL-10 by T cells in recall response to the A2 protein or parasite extracts⁴⁰40. Fernandes AP, Costa MM, Coelho EA, Michalick MS, Freitas E, Melo MN, et al. Protective immunity against challenge with Leishmania (Leishmania) chagasi in beagle dogs vaccinated with recombinant A2 protein. Vaccine 2008; 26:5888-5895.. Other amastigote-specific antigens that have been considered promising candidates for VL prevention include the cysteine proteinases (CP). These enzymes belong to the papain super-family, three classes of which (CPA, CPB, and CPC) have been identified in Leishmania parasites. Studies have shown that recombinant CPB protein, in combination with an immune adjuvant or as a DNA vaccine, induced protection against Leishmania major infection in BALB/c mice⁴⁷47. Rafati S, Kariminia A, Seyde-Eslami S, Narimani M, Taheri T, Lebbatard M. Recombinant cysteine proteinases-based vacines against Leishmania major in BALB/c mice: the partial protection relies on interferon-gamma producing CD8(+) T lymphocyte activation. Vaccine 2002; 20:2439-2447.. In another study, recombinant CPA/CPB polyprotein vaccine was administered in association with poloxamer 407 as an adjuvant, and induced a protective response against L. major in BALB/c mice, which was more robust than the response induced by recombinant CPA and CPB proteins administered as separate individual antigens⁴⁸48. Zadeh-Vakili A, Taheri T, Taslimi Y, Doustdari F, Salmanian AH, Rafati S. Immunization with the hybrid protein vaccine, consisting of Leishmania major cysteine proteinases Type I (CPB) and Type II (CPA), partially protects against leishmaniasis. Vaccine 2004; 22:1930-1940.; however, these antigens were not tested as vaccines against VL.

In an evaluation of antigens expressed in promastigote forms of Leishmania parasites as vaccine candidates against VL, parasite surface antigen-2 (PSA-2), which comprises three polypeptides with molecular weights ranging from 50.0 to 96kDa⁴⁹49. Handman E, Osborn AH, Symons F, van Driel R, Cappai R. The Leishmania promastigote surface antigen 2 complex is differentially expressed during the parasite life cycle. Mol Biochem Parasitol 1995; 74:189-200., showed satisfactory results. This immunogen was able to induce protection against a Leishmania challenge in mice, through the development of a Th1-type response, when administered associated with Corynebacterium parvum as an adjuvant⁵⁰50. Sjölander A, Baldwin TM, Curtis JM, Bengtsson KL, Handman E. Vaccination with recombinant parasite surface antigen 2 from Leishmania major induces a Th1 type of immune response but does not protect against infection. Vaccine 1998; 16:2077-2084.. Kinetoplastid membrane protein-11 (KMP-11), a highly conserved protein expressed in different Leishmania species, was also verified as protective against L. donovani infection in hamsters⁵¹51. Trujillo C, Ramírez 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; 70:203-209.^{) (52}52. Kamesipour A, Rafati S, Davoudi N, Maboudi F, Modabber F. Leishmaniasis vaccine candidates for development: a global overview. Ind J Med Res 2006; 123:423-438.. In addition, the nucleoside hydrolase 36kDa (NH36) antigen was shown to be protective against Leishmania infantum, Leishmania mexicana, and Leishmania amazonensis species in BALB/c mice, indicating its potential as a heterologous vaccine to protect against different Leishmania species⁵³53. Palatnik-de-Sousa CB. Vaccine s for leishmaniasis in the fore coming 25 years. Vaccine 2008; 26 1709-1724.^{) (54}54. Aguilar-Be I, Silva Zardo R, Paraguai-de-Souza E, Borja-Cabrera GP, Rosado-Vallado M, Mut-Martin M, et al. Cross-protective efficacy of a prophylactic Leishmania donovani DNA vaccine against visceral and cutaneous murine leishmaniasis. Infect Immun 2005; 73:812-819..

It has been postulated that a formulation containing different Leishmania proteins expressed in both parasite stages should provide better results, in terms of a more effective and protective vaccine against VL⁴²42. Coelho EAF, Tavares CAP, Carvalho FAA, Chaves KF, Teixeira KN, Rodrigues RC, et al. Immune responses induced by the Leishmania (Leishmania) donovani A2 antigen, but not by the LACK antigen, are protective against experimental Leishmania (Leishmania) amazonensis infection. Infect Immun 2003; 71:3988-3994.^{) (55}55. Martins VT, Chávez-Fumagalli MA, Lage DP, Duarte MC, Garde E, Costa LE, et al. Antigenicity, immunogenicity and protective efficacy of three proteins expressed in the promastigote and amastigote stages of Leishmania infantum against visceral leishmaniasis. PLoS One 2015; 10:e0137683.. The use of vaccines combining different proteins could provide the benefits of increased simplicity and reduced production costs, since it would only be necessary to produce a single vaccine to protect against different Leishmania species⁵⁶56. Beaumier CM, Gillespie PM, Hotez PJ, Bottazzi ME. New vaccines for neglected parasitic diseases and dengue. Transl Res 2013; 162:144-155.. However, few studies have evaluated chimeric vaccines aimed at protection against VL²⁵25. Kumari S, Kumar A, Samant M, Singh N, Dube A. Discovery of novel vaccine candidates and drug targets against visceral leishmaniasis using proteomics and transcriptomics. Curr Drug Targets 2008; 9:938-947.^{) (57}57. Saljoughian N, Taheri T, Zahedifard F, Taslimi Y, Doustdari F, Bolhassani A, et al. Development of novel prime-boost strategies based on a tri-gene fusion recombinant L. tarentolae vaccine against experimental murine visceral leishmaniasis. PLoS Negl Trop Dis 2013; 7: e2174., since the majority of reports have been of investigations of single antigens¹⁴14. Martins VT, Chávez-Fumagalli MA, Costa LE, Martins AMCC, Lage PS, Lage DP, et al. Antigenicity and protective efficacy of a Leishmania amastigote-specific protein, member of the super-oxygenase family, against visceral leishmaniasis. PLoS Negl Trop Dis 2013; 7:e2148.^{) (58}58. Rachamim N, Jaffe CL. Pure protein from Leishmania donovani protects mice against both cutaneous and visceral leishmaniasis. J Immunol 1993; 150:2322-2331.^{) (59}59. Joshi J, Kaur S. Studies on the protective efficacy of second-generation vaccine along with standard antileishmanial drug in Leishmania donovani infected BALB/c mice. Parasitology 2014; 141:554-562.^{) (60}60. Baharia RK, Tandon R, Sharma T, Suthar MK, Das S, Siddiqi MI, et al. Recombinant NAD-dependent SIR-2 protein of Leishmania donovani: immunobiochemical characterization as a potential vaccine against visceral leishmaniasis. PLoS Negl Trop Dis 2015; 9:e0003557..

The development of a multi-antigenic vaccine requires an appropriate choice of the biological targets for use in its composition. In a recent study, a polyprotein vaccine formulated with monophosphoryl lipid A, KSAC, was shown to be immunogenic and effective in inducing protection against L. infantum and L. major in mice. KSAC is a chimeric protein composed of the Leishmania homolog of the receptor for activated C kinase (LACK), glycoprotein 63 kDa (gp63), thiol-specific-antioxidant (TSA), hydrophilic acylated surface protein B (HASPB), sterol 24-c-methyltransferase (SMT), KMP-11, A2, and CPB proteins. In models challenged with both Leishmania species, the protective response was associated with the production of high levels of IFN-γ, combined with low levels of IL-4 and a decreased antileishmanial IgG1 response⁶¹61. Goto Y, Bhatia A, Raman VS, Liang H, Mohamath R, Picone AF, et al. KSAC, the first defined polyprotein vaccine candidate for visceral leishmaniasis. Clin Vaccine Immunol 2011; 18:1118-1124.. Another chimeric protein, Leish-111f, which is composed of a combination of TSA, stress inducible protein 1 (LmSTI-1), and the Leishmania homolog of the eukaryotic translation initiation factor (eIF4A), was also able to protect BALB/c mice against Leishmania infection, when administered in association with immune adjuvants⁶²62. Coler RN, Skeiky YA, Bernards K, Greeson K, Carter D, Cornellison CD, et al. Immunization with a polyprotein vaccine consisting of the T-Cell antigens thiol-specific antioxidant, Leishmania major stress-inducible protein 1, and Leishmania elongation initiation factor protects against leishmaniasis. Infect Immun 2002; 70:4215-4225..

Another field that could be developed in relation to the discovery of new candidate VL vaccines is based on vector salivary proteins. To date, evidence indicates that salivary molecules able to induce a Th1-type response in immunized animals could create a protective immunological environment at the bite site, which could influence when parasites are injected, allowing control of the disease and concomitant promotion of Leishmania-specific immunity⁶³63. Kamhawi S, Aslan H, Valenzuela JG. Vector saliva in vaccines for visceral leishmaniasis: a brief encounter of high consequence? Front Public Health 2014; 2:99.. The Th1-type immunological environment in response to these antigens at the bite site could promote a protective immune response against the parasite challenge. In this context, PdSP15, a 15-kDa salivary protein, which is a member of the family of small odorant binding proteins from Phlebotomus duboscqi, was evaluated as a candidate antigen against leishmaniasis in non-human primates⁶⁴64. Oliveira F, Rowton E, Aslan H, Gomes R, Castrovinci PA, Alvarenga PH, et al. A sand fly salivary protein vaccine shows efficacy against vector-transmitted cutaneous leishmaniasis in non-human primates. Sci Transl Med 2015; 7:290ra90.. In addition, LJM19, an 11-kDa salivary protein of unknown function and LJL143, a 38-kDa salivary protein with anticoagulant activity⁶⁵65. Collin N, Assumpcão TC, Mizurini DM, Gilmore DC, Dutra-Oliveira A, Kotsyfakis M, et al. Lufaxin, a novel factor Xa inhibitor from the salivary gland of the sand fly Lutzomyia longipalpis blocks protease activated receptor 2 activation and inhibits inflammation and thrombosis in vivo. Arterioscler Thromb Vasc Biol 2012; 32:2185-2198., both of which are present in the saliva of Lutzomyia longipalpis, were shown to be protective against VL⁶⁶66. 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 USA 2008; 105:7845-7850.. Table 1 shows a summary of relevant vaccine candidates evaluated as individual recombinant protein or polyprotein vaccines against VL⁶⁷67. Gupta R, Kumar V, Kushawaha PK, Tripathi CP, Joshi S, Sahasrabuddhe AA, et al. Characterization of glycolytic enzymes--rAldolase and rEnolase of Leishmania donovani, identified as Th1 stimulatory proteins, for their immunogenicity and immunoprophylactic efficacies against experimental visceral leishmaniasis. PLoS One 2014; 9:e86073.^{) (68}68. Poot J, Spreeuwenberg K, Sanderson SJ, Schijns VE, Mottram JC, Coombs GH, et al. Vaccination with a preparation based on recombinant cysteine peptidases and canine IL-12 does not protect dogs from infection with Leishmania infantum. Vaccine 2006; 24:2460-2468.^{) (69}69. Khoshgoo N, Zahedifard F, Azizi H, Taslimi Y, Alonso MJ, Rafati S. Cysteine proteinase type III is protective against Leishmania infantum infection in BALB/c mice and highly antigenic in visceral leishmaniasis individuals. Vaccine 2008; 26:5822-5829.^{) (70}70. Santos-Gomes GM, Rodrigues A, Teixeira F, Carreira J, Alexandre-Pires G, Carvalho S, et al. Immunization with the Leishmania infantum recombinant cyclophilin protein 1 confers partial protection to subsequent parasite infection and generates specific memory T cells. Vaccine 2014; 32:1247-1253.^{) (71}71. Jaffe CL, Rachamim N, Sarfstein R. Characterization of two proteins from Leishmania donovani and their use for vaccination against visceral leishmaniasis. J Immunol 1990; 144:699-706.^{) (72}72. Kushawaha PK, Gupta R, Sundar S, Sahasrabuddhe AA, Dube A. Elongation factor-2, a Th1 stimulatory protein of Leishmania donovani, generates strong IFN-γ and IL-12 response in cured Leishmania-infected patients/hamsters and protects hamsters against Leishmania challenge. J Immunol 2011; 187:6417-6427.^{) (73}73. Wilson ME, Young BM, Andersen KP, Weinstock JV, Metwali A, Ali KM, et al. A recombinant Leishmania chagasi antigen that stimulates cellular immune responses in infected mice. Infect Immun 1995; 63:2062-2069.^{) (74}74. Ramirez L, Villen LC, Duarte MC, Chávez-Fumagalli MA, Valadares DG, Santos DM, et al. Cross-protective effect of a combined L5 plus L3 Leishmania major ribosomal protein based vaccine combined with a Th1 adjuvant in murine cutaneous and visceral leishmaniasis. Parasit Vectors 2014; 2:3-10.^{) (75}75. Tewary P, Sukumaran B, Saxena S, Madhubala R. Immunostimulatory oligodeoxynucleotides are potent enhancers of protective immunity in mice immunized with recombinant ORFF leishmanial antigen. Vaccine 2004; 22:3053-3060.^{) (76}76. Gradoni L, Foglia-Manzillo V, Pagano A, Piantedosi D, De Luna R, Gramiccia M, et al. Failure of a multi-subunit recombinant leishmanial vaccine (MML) to protect dogs from Leishmania infantum infection and to prevent disease progression in infected animals. Vaccine 2005; 23:5245-5251.^{) (77}77. Coler RN, Duthie MS, Hofmeyer KA, Guderian J, Jayashankar L, Vergara J, et al. From mouse to man: safety, immunogenicity and efficacy of a candidate leishmaniasis vaccine LEISH-F3+GLA-SE. Clin Transl Immunology 2015; 4:e35.^{) (78}78. Molano I, Alonso MG, Mirón C, Redondo E, Requena JM, Soto M, et al. A Leishmania infantum multi-component antigenic protein mixed with live BCG confers protection to dogs experimentally infected with L. infantum. Vet Immunol Immunopathol 2003; 92:1-13.^{) (79}79. Duthie MS, Favila M, Hofmeyer KA, Tutterrow YL, Reed SJ, Laurance JD, et al. Strategic evaluation of vaccine candidate antigens for the prevention of visceral leishmaniasis. Vaccine 2016; 34:2779-2786..

THE APPLICATION OF IMMUNO-PROTEOMIC APPROACHES TO THE IDENTIFICATION OF NEW LEISHMANIA ANTIGENS WITH POTENTIAL TO BE USED AS VISCERAL LEISHMANIASIS VACCINES

Immuno-proteomic approaches have been developed to identify new Leishmania proteins with distinct biological functions, such as new diagnostic markers, vaccine candidates, and/or potential drug targets⁸⁰80. Chenik M, Lakhal S, Ben-Khalef N, Zribi L, Louzir H, Dellagi K., et al. Approaches for the identification of potential excreted/secreted proteins of Leishmania major parasites. Parasitology 2006; 132:493-509.^{) (81}81. Paape D, Barrios-Lerena ME, Le Bihan T, Mackay L, Aebischer T. Gel free analysis of the proteome of intracellular Leishmania mexicana. Mol Biochem Parasitol 2010; 169:108-114.^{) (82}82. Coelho VT, Oliveira JS, Valadares DG, Chávez-Fumagalli MA, Duarte MC, Lage PS, et al. Identification of proteins in promastigote and amastigote-like Leishmania using an immunoproteomic approach. PLoS Negl Trop Dis 2012; 6:e1430.. The use of antileishmanial antibodies obtained from infected mammalian hosts contributed to the refinement of these analyses, by assisting in the identification of antigens recognized by the immune system during active disease⁸²82. Coelho VT, Oliveira JS, Valadares DG, Chávez-Fumagalli MA, Duarte MC, Lage PS, et al. Identification of proteins in promastigote and amastigote-like Leishmania using an immunoproteomic approach. PLoS Negl Trop Dis 2012; 6:e1430.^{) (83}83. Costa MM, Andrade HM, Bartholomeu DC, Freitas LM, Pires SF, Chapeaurouge AD, et al. Analysis of Leishmania chagasi by 2-D difference gel electrophoresis (2-D DIGE) and immunoproteomic: identification of novel candidate antigens for diagnostic tests and vaccine. J Proteome Res 2011; 10:2172-2184.. Immuno-proteomic approaches usually involve protein preparation and separation by bi-dimensional electrophoresis, followed by immunoblotting experiments, and subsequent identification of protein spots by mass spectrometry (Figure 1). In a recent immuno-proteomic study, several antigenic parasite proteins were identified from serum samples of dogs developing VL⁸³83. Costa MM, Andrade HM, Bartholomeu DC, Freitas LM, Pires SF, Chapeaurouge AD, et al. Analysis of Leishmania chagasi by 2-D difference gel electrophoresis (2-D DIGE) and immunoproteomic: identification of novel candidate antigens for diagnostic tests and vaccine. J Proteome Res 2011; 10:2172-2184.. These proteins were analyzed in silico for epitope identification, and the best antigenic determinants were employed in enzyme-linked immunosorbent assay (ELISA) assays aiming to identify antigenic peptides of interest for the serodiagnosis of canine disease. The authors speculated about the use of these candidates as vaccines in future assays, owing to the existence of putative-T cell motifs in the antigens. In another immuno-proteomic approach, developed using protein extracts from the stationary promastigote and amastigote-like stages of L. infantum, several specific promastigote (Table 2) and amastigote (Table 3) hypothetical proteins were identified in serum samples from dogs with asymptomatic and/or symptomatic VL⁸²82. Coelho VT, Oliveira JS, Valadares DG, Chávez-Fumagalli MA, Duarte MC, Lage PS, et al. Identification of proteins in promastigote and amastigote-like Leishmania using an immunoproteomic approach. PLoS Negl Trop Dis 2012; 6:e1430..

Figure 1.
Experimental workflow for accurate identification of antigenic proteins using bi-dimensional (2-DE) immunoblotting assays. The technical steps usually performed when an immuno-proteomic approach is applied are: 1) Preparation of total protein extracts from the parasite. 2) Isoelectric focusing and bi-dimensional SDS-PAGE. 3) Immunoblotting experiment. 4, 5, 6) Protein digestion and peptide extraction. 7, 8) Prepare and identify spots using MALDI-TOF-TOF peptide mass mapping. 9) Database search to identify the antigenic proteins. 10) Identified proteins. SDS-PAGE: Polyacrylamide gel electrophoresis. MALDI-TOF-TOF: matrix-assisted laser desorption ionization/time-of-flight mass spectrometer.

Some of these proteins have already been validated as candidate VL vaccines (Table 4). These antigens were selected because they are conserved among different Leishmania species, but are not present in other Trypanosomatidae or in mammalian hosts. In addition, the selected antigens contain specific CD4⁺ and CD8⁺ T cell epitopes. In this context, the protective efficacy against L. infantum infection of LiHyp1, a Leishmania protein belonging to the super-oxygenase family, was evaluated in BALB/c mice. Immunization using the recombinant LiHyp1 protein plus saponin adjuvant induced a Th1 immune response in the vaccinated animals, which was primed by protein- and parasite-specific IFN-γ, IL-12, and GM-CSF production, combined with the presence of low levels of IL-4 and IL-10. In addition, the protected animals displayed significant reductions in the number of parasites in their livers, spleens, bone marrow, and draining lymph nodes, compared with that in control groups. The protection was correlated with parasite-specific and dependent IFN-γ production, mainly by CD4⁺ T cells, which were the major source of IFN-γ in these animals¹⁴14. Martins VT, Chávez-Fumagalli MA, Costa LE, Martins AMCC, Lage PS, Lage DP, et al. Antigenicity and protective efficacy of a Leishmania amastigote-specific protein, member of the super-oxygenase family, against visceral leishmaniasis. PLoS Negl Trop Dis 2013; 7:e2148..The same immune profile was found when the hypothetical LiHyD³¹31. Lage DP, Martins VT, Duarte MC, Garde E, Chávez-Fumagalli MA, Menezes-Souza D, et al. Prophylactic properties of a Leishmania-specific hypothetical protein in a murine model of visceral leishmaniasis. Parasite Immunol 2015; 37:646-656., LiHyT³²32. Martins VT, Lage DP, Duarte MC, Costa LE, Garde E, Rodrigues MR, et al. A new Leishmania-specific hypothetical protein, LiHyT, used as a vaccine antigen against visceral leishmaniasis. Acta Trop 2016; 154:73-81., LiHyp6⁵⁵55. Martins VT, Chávez-Fumagalli MA, Lage DP, Duarte MC, Garde E, Costa LE, et al. Antigenicity, immunogenicity and protective efficacy of three proteins expressed in the promastigote and amastigote stages of Leishmania infantum against visceral leishmaniasis. PLoS One 2015; 10:e0137683., and LiHyV⁸⁴84. Martins VT, Duarte MC, Chávez-Fumagalli MA, Menezes-Souza D, Coelho CS, de Magalhães-Soares DF, et al. A Leishmania-specific hypothetical protein expressed in both promastigote and amastigote stages of Leishmania infantum employed for the serodiagnosis of, and as a vaccine candidate against, visceral leishmaniasis. Parasit Vectors 2015; 8:363. proteins were evaluated as vaccine candidates. In all cases, the antigens were shown to be protective against infection, since vaccinated and challenged animals presented significantly lower parasite levels in evaluated organs compared with control groups. In addition, vaccinated and challenged animals demonstrated predominantly IL-12 driven IFN-γ production (also mediated mainly by CD4⁺ T cells) against parasite proteins, whereas unprotected controls showed high levels of anti-Leishmania IgG1 antibodies and a parasite mediated IL-4 and IL-10 response.

An aspect that should be considered when evaluating the efficacy of a vaccine is the use of adjuvants. Although recombinant protein-based vaccines offer considerable advantages in terms of safety, standardization, purity, and production costs, they generally present limited immunogenicity and require the use of immune adjuvants⁸⁵85. Santos WR, de Lima VM, de Souza EP, Bernardo RR, Palatnik M, Palatnik de Sousa CB. Saponins, IL-12 and BCG adjuvant in the FML-vaccine formulation against murine visceral leishmaniasis. Vaccine 2002; 21:30-43.. It is generally accepted that the adjuvants used in leishmaniasis vaccine formulations should be able to induce a Th1 response, and some adjuvants are capable of this, including recombinant IL-12, saponin, BCG, monophosphoryl lipid A (MPL), CpG, recombinant virus, and others⁸⁶86. Reed SG, Coler RN, Campos-Neto A. Development of a leishmaniasis vaccine: the importance of MPL. Expert Rev Vaccines 2003; 2:239-252.^{) (87}87. Stobie L, Gurunathan S, Prussin C, Sacks DL, Glaichenhaus N, Wu CY, et al. The role of antigen and IL-12 in sustaining Th1 memory cells in vivo: IL-12 is required to maintain memory/effector Th1 cells sufficient to mediate protection to an infectious parasite challenge. Proc Natl Acad Sci USA 2000; 97:8427-8432.. The induction of IL-12 is critical for vaccine efficiency and many of these adjuvants activate the innate immune response via Toll-like receptors (TLR), also influencing acquired immune responses⁸⁸88. Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol 2004; 5:987-995.. Significant protection is not usually achieved when animals are immunized with recombinant proteins in the absence of adjuvants. These findings have been corroborated by studies evaluating other well-known protective antigens against leishmaniasis³¹31. Lage DP, Martins VT, Duarte MC, Garde E, Chávez-Fumagalli MA, Menezes-Souza D, et al. Prophylactic properties of a Leishmania-specific hypothetical protein in a murine model of visceral leishmaniasis. Parasite Immunol 2015; 37:646-656.^{) (32}32. Martins VT, Lage DP, Duarte MC, Costa LE, Garde E, Rodrigues MR, et al. A new Leishmania-specific hypothetical protein, LiHyT, used as a vaccine antigen against visceral leishmaniasis. Acta Trop 2016; 154:73-81.^{) (40}40. Fernandes AP, Costa MM, Coelho EA, Michalick MS, Freitas E, Melo MN, et al. Protective immunity against challenge with Leishmania (Leishmania) chagasi in beagle dogs vaccinated with recombinant A2 protein. Vaccine 2008; 26:5888-5895.^{) (54}54. Aguilar-Be I, Silva Zardo R, Paraguai-de-Souza E, Borja-Cabrera GP, Rosado-Vallado M, Mut-Martin M, et al. Cross-protective efficacy of a prophylactic Leishmania donovani DNA vaccine against visceral and cutaneous murine leishmaniasis. Infect Immun 2005; 73:812-819..

The concept of cross-protective vaccines is based on the presence of common antigens among pathogens and on the ability of formulated vaccines to elicit cellular immunity⁸⁹89. Barbour AG, Restrepo BI. Antigenic variation in vector-borne pathogens. Emerg Infect Dis 2000; 6:449-457.. Since multiple Leishmania species are distributed in common geographical areas, it would be desirable to develop vaccines capable of inducing protection against more than one parasite species⁹⁰90. Duthie MS, Raman VS, Piazza FM, Reed SG. The development and clinical evaluation of second-generation leishmaniasis vaccines. Vaccine 2012; 30:134-141.. In this context, LiHyT, which was firstly identified, in L. infantum³²32. Martins VT, Lage DP, Duarte MC, Costa LE, Garde E, Rodrigues MR, et al. A new Leishmania-specific hypothetical protein, LiHyT, used as a vaccine antigen against visceral leishmaniasis. Acta Trop 2016; 154:73-81. as protective against this species, was also shown to confer protection in BALB/c mice against L. major and L. braziliensis. Mice immunized with LiHyT and saponin as an adjuvant, developed a robust Th1 immune response, which was responsible for the induction of significant reductions of parasite load in the tissues and organs evaluated⁸⁴84. Martins VT, Duarte MC, Chávez-Fumagalli MA, Menezes-Souza D, Coelho CS, de Magalhães-Soares DF, et al. A Leishmania-specific hypothetical protein expressed in both promastigote and amastigote stages of Leishmania infantum employed for the serodiagnosis of, and as a vaccine candidate against, visceral leishmaniasis. Parasit Vectors 2015; 8:363.. This cross-protective immunity was also found when another hypothetical protein, rLiHyD, was used as an antigen with saponin as an adjuvant⁹¹91. Lage DP, Martins VT, Duarte MC, Costa LE, Tavares GS, Ramos FF, et al. Cross-protective efficacy of Leishmania infantum LiHyD protein against tegumentary leishmaniasis caused by Leishmania major and Leishmania braziliensis species. Acta Trop 2016; 158:220-230..

As described, the use of chimeric vaccines containing multiple proteins and/or polypeptides could provide more robust protective efficacy against various Leishmania species⁶¹61. Goto Y, Bhatia A, Raman VS, Liang H, Mohamath R, Picone AF, et al. KSAC, the first defined polyprotein vaccine candidate for visceral leishmaniasis. Clin Vaccine Immunol 2011; 18:1118-1124.^{) (76}76. Gradoni L, Foglia-Manzillo V, Pagano A, Piantedosi D, De Luna R, Gramiccia M, et al. Failure of a multi-subunit recombinant leishmanial vaccine (MML) to protect dogs from Leishmania infantum infection and to prevent disease progression in infected animals. Vaccine 2005; 23:5245-5251.^{) (78}78. Molano I, Alonso MG, Mirón C, Redondo E, Requena JM, Soto M, et al. A Leishmania infantum multi-component antigenic protein mixed with live BCG confers protection to dogs experimentally infected with L. infantum. Vet Immunol Immunopathol 2003; 92:1-13.. In this context, three recombinant proteins were combined in a vaccine and tested for their protective effects against L. infantum infection. These proteins are expressed in both the promastigote and amastigote stages of the parasites, and their combination was able to induce pronounced parasite-specific IFN-γ, IL-12, and GM-CSF responses in immunized mice, which was maintained after challenge. The infected and vaccinated animals showed significant reductions in parasite burden in the various organs evaluated compared with control mice, the protection being associated with IL-12-dependent IFN-γ production against parasite extracts, and correlated with the induction of antileishmanial nitrite production. More importantly, this polyprotein vaccine was able to induce a more robust Th1 response associated with better control of parasite dissemination in the organs of the vaccinated animals, compared with the use of the individual recombinant proteins⁵⁵55. Martins VT, Chávez-Fumagalli MA, Lage DP, Duarte MC, Garde E, Costa LE, et al. Antigenicity, immunogenicity and protective efficacy of three proteins expressed in the promastigote and amastigote stages of Leishmania infantum against visceral leishmaniasis. PLoS One 2015; 10:e0137683..

CONCLUSIONS

Effective prophylactic measures to control VL are imperative. Such measures include the design of vaccines, which is the most economical way to control neglected diseases. An ideal vaccine candidate should be able to induce robust antileishmanial Th1 immunity, be parasite-specific (to avoid adverse effects in mammalian hosts), and exhibit a high degree of homology between different Leishmania species. Hypothetical proteins, considered unknown molecules until their recognition by the immune system of infected mammalian hosts, could be considered for this purpose and explored for use in the prevention of VL. In addition, the development and use of new technologies, such as reverse vaccinology, to identify novel candidate VL vaccines should be also considered.

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