SciELO - Scientific Electronic Library Online

 
vol.105 issue6Immune response to Leishmania (Leishmania) chagasi infection is reduced in malnourished BALB/c miceNested PCR to detect and distinguish the sympatric filarial species Onchocerca volvulus, Mansonella ozzardi and Mansonella perstans in the Amazon Region author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand

Journal

Article

Indicators

Related links

Share


Memórias do Instituto Oswaldo Cruz

Print version ISSN 0074-0276

Mem. Inst. Oswaldo Cruz vol.105 no.6 Rio de Janeiro Sept. 2010

http://dx.doi.org/10.1590/S0074-02762010000600015 

ARTICLES

 

Antigenic extracts of Leishmania braziliensis and Leishmania amazonensis associated with saponin partially protects BALB/c mice against Leishmania chagasi infection by suppressing IL-10 and IL-4 production

 

 

Rafaella FQ GrenfellI, II, +; Eduardo A Marques-da-SilvaII; Miriam C Souza-TestasiccaII; Eduardo AF CoelhoIII, IV, V; Ana Paula FernandesIII; Luís Carlos C AfonsoII; Simone A RezendeII, VI

ILaboratório de Esquistossomose, Instituto de Pesquisas René Rachou-Fiocruz, Av. Augusto de Lima 1715, 30.190-002 Belo Horizonte, MG, Brasil
IICoordenadoria de Ciências Biológicas, Instituto Federal de Minas Gerais e Laboratório de Imunoparasitologia
IIIDepartamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas
IVSetor de Patologia Clínica, Colégio Técnico
VDepartamento de Microbiologia, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil
VIDepartamento de Análises Clínicas, Escola de Farmácia, Universidade Federal de Ouro Preto, Ouro Preto, MG, Brasil

 

 


ABSTRACT

This study evaluated two vaccine candidates for their effectiveness in protecting BALB/c mice against Leishmania chagasiinfection. These immunogenic preparations were composed of Leishmania amazonensisor Leishmania braziliensisantigenic extracts in association with saponin adjuvant. Mice were given three subcutaneous doses of one of these vaccine candidates weekly for three weeks and four weeks later challenged with promastigotes of L. chagasiby intravenous injection. We observed that both vaccine candidates induced a significant reduction in the parasite load of the liver, while the L. amazonensisantigenic extract also stimulated a reduction in spleen parasite load. This protection was associated with a suppression of both interleukin (IL)-10 and IL-4 cytokines by spleen cells in response to L. chagasiantigen. No change was detected in the production of IFN-γ. Our data show that these immunogenic preparations reduce the type 2 immune response leading to the control of parasite replication.

Key words:vaccine - Leishmania chagasi - Leishmania braziliensis - Leishmania amazonensis - IL-10 - IL-4


 

 

Leishmaniasis is a spectrum of diseases caused by infection with different Leishmania species that range from self-limiting cutaneous leishmaniasis to visceral leishmaniasis (VL). VL, also known as kala-azar, is fatal if not successfully treated. Human infection with Leishmania chagasi/Leishmania infantum, the protozoan causing South American VL, ranges from sub-clinical infection to progressive fatal disease (Wilson 1993). Sub-clinical infection results in the development of a cellular immune response that often results in long-term protective immunity against re-infection (Pearson & Sousa 1996), suggesting that a vaccine against leishmaniasis is feasible. Since treatment of VL is difficult and no acceptable vaccine exists against human infection, the development of an effective vaccine would be an important achievement. An ideal vaccine would be one that could offer cross-protection against diverse Leishmania species (Campbell et al. 2003).

The involvement of T helper 1 (Th1) and T helper 2 (Th2) subsets with either protection or disease exacerbation has been demonstrated in murine cutaneous leishmaniasis (Heinzel et al. 1989). A similar pattern of Th cell subsets has been shown in some studies for VL (Rhee et al. 2002), mainly because interleukin-4 (IL-4) can regulate macrophage function (Hamilton et al. 1999). Unexpectedly, some studies in animal models have proven that protection in VL is associated with the production of both type 1 and type 2 cytokines (Ghosh et al. 2001, Ramiro et al. 2003, Vilela et al. 2007).

In this study, we evaluated the potential of two freeze-thawed (FT) Leishmania antigenic extracts for protection against L. chagasi infection. Cross-species protection has been supported in many studies on leishmaniasis (Bebars et al. 2000, Misra et al. 2001, Tonui & Titus 2007). Therefore, we decided to investigate whether subcutaneous immunization with extracts of Leishmania amazonensis or Leishmania braziliensis could protect BALB/c mice against L. chagasi infection. These two antigenic extracts and a purified saponin fraction from the bark extracts of Quillaja saponaria, which has been considered a promising adjuvant in numerous prophylactic and therapeutic vaccines, were used in association (Kensil 1996). It is worth noting that the mechanism by which saponin potentiates an immune response remains unclear. Hypotheses have been raised about whether saponin, through lectin-mediated cell membrane interactions, could facilitate the uptake of the antigen into antigen-presenting cells, leading to specific cytokine profiles that enhance T and/or B-cell responses (Kensil 1996, Marciani 2003, Adams et al. 2010).

We decided to evaluate these two species because a tested vaccine composed of L. amazonensis along with Bacillus Calmette-Guérin has been effective in the treatment of cutaneous leishmaniasis patients in Venezuela (Convit et al. 2003). Further, a recent vaccine composed of L. braziliensis, sand fly gland extract and saponin was shown to be immunogenic in dogs (Giunchetti et al. 2007, 2008). These species are associated not only with localized cutaneous leishmaniasis, but also with mucocutaneous leishmaniasis and anergic diffuse cutaneous leishmaniasis in Brazil.

This study aimed at evaluating whether these two vaccine candidates, composed of antigens obtained from species responsible for cutaneous and mucocutaneous leishmaniasis, could protect against murine VL caused by L. chagasi.

 

MATERIALS AND METHODS

Leishmania parasites and antigens - The strain of L. chagasi used in this study (MHOM/BR/1974/M2682) was kindly provided by Dr Maria Norma de Melo, from the Parasitology Department, Federal University of Minas Gerais (UFMG), Brazil. Promastigotes were grown in Dulbecco's Modified Eagle Medium (DMEM; pH 6.8) supplemented with 20% heat-inactivated foetal bovine serum (FBS), 2 mM L-glutamine, 25 mM HEPES, 50 μM 2-mercaptoethanol and 20 μg/mL garamicin [DMEM 20% phosphate buffered saline (PBS)] at 25ºC. Infectivity was maintained by serial passage through mice. L. amazonensis strain PH8 (IFLA/BR/67/PH8) and L. braziliensis strain M2903 (MHOM/BR/75/M2903) were grown in Grace's Medium supplemented with 20% FBS, 2 mM L-glutamine and 20 μg/mL garamicin. Promastigotes of L. chagasi, L. amazonensis or L. braziliensis were harvested from late-log-phase cultures by centrifugation, washed three times with PBS and disrupted by three rounds of freezing and thawing. The protein contents were estimated (Lowry et al. 1951) and the antigen was frozen at -70ºC prior to use. L. amazonensis and L. braziliensis antigens were used to immunize mice.

Mice, immunizations and the quantification of parasite load - Female BALB/c mice (4-6 weeks old) were obtained from Bioterism Center, UFMG, and were maintained at the Central Biotherium, Federal University of Ouro Preto (UFOP). Four BALB/c mice per group, in three independent experiments, were injected weekly with three subcutaneous doses of 100 μg of L. amazonensis or L. braziliensis antigenic extracts together with 50 μg of saponin as an adjuvant. Control mice were inoculated with PBS or 50 μg of saponin alone. Four weeks later, mice were challenged with 1 x 107 promastigotes of L. chagasi given intravenously through the lateral tail vein. Five post challenge mice were sacrificed and the spleen and liver parasite loads were determined by quantitative limiting-dilution culture. Quantitative limiting-dilution culture was performed as previously described with some modifications (Marques-da-Silva et al. 2005). Briefly, spleen and liver were harvested and weighed. One fragment of each organ was obtained and weighed separately for parasite quantification. This fragment was homogenized in a tissue grinder, resuspended in 500 μL of DMEM containing 20% FBS and plated onto 48-well flat-bottom microtiter plates. Five-fold serial dilutions were performed and, after two weeks of incubation at 25ºC, plates were microscopically scored for parasite growth. The number of parasites was determined from the reciprocal of the highest dilution at which promastigotes could be detected and is expressed as parasites per organ.

Determination of vaccine-induced cytokine production - Single-cell suspensions of spleen were obtained by homogenization in a tissue grinder. The erythrocytes were lysed with ammonium chloride lysis buffer and the cells were washed. Cells were then cultured in DMEM (pH 7.2) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 25 mM HEPES, 50 μM 2-mercaptoethanol and 20 μg/mL garamicin (DMEM 10% FBS) at 5 x 106 cells/mL in 48-well flat-bottom plates without stimuli (ws) or stimulated with 50 μg of L. chagasiAg/mL of culture for 72 h. The production of IFN-γ and IL-4 was determined by the presence of these cytokines in cell culture supernatant, as measured by enzyme linked immunosorbent assay (ELISA) using specific purified monoclonal antibodies (Afonso & Scott 1993). The production of IL-10 was assayed using a commercial ELISA kit, according to the manufacturer's instructions (Duo Set®, R&D Systems).

Statistical analysis - Parasite burden data were logarithmically transformed to determine the homogeneity of variance. All data were analyzed using the Kolmogorov-Smirnov normality test. Data with a normal distribution were analyzed by the Student's t test.

Ethics - This study was approved by the Ethical Committee of the UFOP.

 

RESULTS

Parasite load in liver and spleen - In order to determine if L. amazonensis or L. braziliensis antigenic extracts were able to protect BALB/c mice from L. chagasi infection, we evaluated the parasite load within the liver and spleen by limiting dilution analyses. Mice that were immunized with FT L. amazonensis antigen showed significant reduction in parasite load in the liver and spleen (p < 0.05), as shown in Fig. 1. The L. braziliensis antigen was also able to reduce the parasite load in the liver but did not significantly decrease the parasite load in the spleen.

 

 

Determination of IFN-γ, IL-4 and IL-10 production by spleen cells after vaccination and challenge - In order to determine if these vaccine candidates could influence the immune response to L. chagasi, spleen cells from immunized animals were obtained and incubated with 50 μg/mL of FT L. chagasi antigen or cultured ws. Fig. 2A shows that although no change in IFN-γ production was noted following immunization, spleen cells from animals treated with either vaccine candidate exhibited significantly reduced production of IL-4 (Fig. 2B) and IL-10 (Fig. 2C) as compared to control spleen cells (p < 0.05).

 



 

DISCUSSION

In recent years, several efforts have been made to obtain a safe and efficient vaccine against leishmaniasis. Vaccination with live, attenuated parasites has been attempted (Streit et al. 2001, Nylén et al. 2006), although there are several ethical considerations regarding these vaccines. As such, attention has shifted to the use of recombinant or synthetic antigens, purified fractions or whole antigenic extracts of parasites as an alternative to live parasites. Whole antigenic extract obtained from parasites is a reasonable alternative considering its immunogenicity, cost and safety. Furthermore, any human vaccine will probably require several different antigens and adjuvant to guarantee a satisfactory response by a majority of the population, given its heterogeneity (Handman 2001).

A vaccine against cutaneous leishmaniasis was developed by Mayrink et al. (1979). It was prepared from whole parasite antigens obtained from five stocks of parasites isolated from patients with different forms of leishmaniasis. Subsequently, the same group developed a second vaccine based only on the PH8 strain of L. amazonensis. This vaccine has been used in the prevention of disease, as well as serving as an immunotherapeutic agent, thereby demonstrating that administration of the vaccine in association with antimonium salts could be therapeutic. Indeed, when compared with conventional therapy, L. amazonensis vaccine treatment reduced the time necessary for lesions in patients with cutaneous leishmaniasis to completely heal (Toledo et al. 2001). Furthermore, Mayrink et al. (2006) have shown that the association of a vaccine antigen with antimonium salts reduces both the total salt volume and the treatment length, thereby reducing the side effects otherwise associated with the use of antimonium salts.

During this study, we evaluated whether antigens from L. amazonensis and L. braziliensis could provide heterologous protection against L. chagasi infection in BALB/c mice. These antigens were used in conjunction with saponin, an adjuvant that has been used in studies involving VL or cutaneous leishmaniasis in mice and dogs (Santos et al. 2002, Nico et al. 2007, Fernandes et al. 2008, Borja-Cabrera et al. 2010). We found that both vaccine candidates were able to reduce parasite load in the liver, but that only the L. amazonensis immunogenic extract reduced the parasite load of the spleen. These data suggest that different mechanisms are utilized to afford protection by these freeze-thawed vaccines.

In mice, Vilela et al. (2007) have shown that a vaccine composed of L. amazonensis (PH8 strain) and Corynebacterium parvum is able to protect against L. chagasi infection. Although this vaccine used antigens derived from L. amazonensis, a different pattern of cytokine expression was observed, since in this case protection was associated with an increase in both type 1 and type 2 cytokines. Studies with L. braziliensis have shown that a vaccine composed of FT antigen and saponin may prevent L. chagasi infection in dogs (Giunchetti et al. 2007, 2008).

In order to assess the immune response induced by both vaccine candidates, we evaluated the production of cytokines by spleen cells. Although we detected a difference in the pattern of protection between the two organs tested, none of the immunogenic preparations led to increased IFN-γ production. In contrast both antigenic preparations resulted in suppressed IL-10 and IL-4 production by spleen cells. That the suppression of cytokines may reduce parasite load has previously been noted in spleen following the immunization of mice with Leishmania major antigens and intravenous challenge with Leishmania donovani. These mice had reduced IL-4 and IL-10 cytokine levels together with an increase in IFN-γ production. This particular cytokine pattern was not observed when the same immunization was performed together with a L. braziliensis infection. Why this occurred is a matter of debate. However, certain possibilities seem plausible: first, there may be something inherently different about L. braziliensis as compared to L. donovani and their interactions with the immune system that elicit distinct levels of protection. For instance, each of these parasites may differ in their ability to induce effector vs. regulatory T cells (Tonui & Titus 2007).

Saponin adjuvant could influence the generation of an immune response at several levels: these may include the mobilization of appropriate antigen-presenting cells to the injected site, enhancing efficient antigen processing and presentation, influencing the cytokine response, including IFN-γ and the co-stimulatory signals necessary for an optimal immune response and increasing the recruitment of effector immune cells to the inflammatory areas (Caro et al. 2003, Buendía et al. 2007). The capacity of this adjuvant to elicit a strong CD8 T cell response has also been reported (Newman et al. 1992). These studies emphasize the immunostimulatory capability of saponin that may have led to the improved level of protection found in our study, although saponin alone did not lead to enhanced IFN-γ production by spleen cells.

The effect of our vaccines on IL-10 production has been observed in other studies. Uzonna et al. (2004) had shown that vaccination with L. major mutants was associated with a decrease in IL-10 and IL-4 production. Similarly, Bhaumik et al. (2009) showed that a vaccine candidate composed of soluble antigen from attenuated L. donovani promastigotes was able to provide complete protection against experimental VL and that this protection was associated with a decrease in the production of IL-10. Gomes et al. (2007) showed that, after intranasal immunization with a plasmid expressing the Leishmania analogue of the receptors of activated C kinase, BALB/c mice developed lower parasite burdens and had a decrease in IL-10 production. In another study, it was shown that BALB/c mice immunized with a plasmid encoding the A2 gene were protected against experimental challenge with L. amazonensis or L. donovani and that this protection was associated with a reduced level of IL-10 production (Zanin et al. 2007). Finally, it is already known that human VL is associated with high levels of IL-10. As such, suppression of this cytokine might be important for disease because it is involved in the suppression of macrophage activity (Nylén & Sacks 2007).

The role of IL-4 in VL is not as well understood. Some studies have shown that IL-4 is important for granuloma maturation and anti-leishmanial activity in the murine model of L. donovani infection (Kemp et al. 1996). In contrast, another study has shown that protection from leishmaniasis is associated with a reduction of type 2 cytokines, including IL-4 (Alves et al. 2009).

The current study shows that immunogenic preparations composed of L. amazonensis or L. braziliensis partially protect BALB/c mice from intravenous challenge with L. chagasi promastigotes and that this protection is associated with a reduction in the level of IL-10 and IL-4 expression. The role played by saponin in a model whereby regulatory cytokines are suppressed is not known and should be the subject of additional investigation. Furthermore, it shows that cross-protection between Leishmania species presents a major practical implication because vaccination procedures based on the use of a vaccine from one species will likely protect against different Leishmania species.

 

ACKNOWLEDGEMENTS

To Dr Alexandre Barbosa Reis, for his assistance.

 

REFERENCES

Adams MM, Damani P, Perl NR, Won A, Hong F, Livingston PO, Ragupathi G, Gin DY 2010. Design and synthesis of potent Quillaja saponin vaccine adjuvants. J Am Chem Soc 132: 1939-1945.         [ Links ]

Afonso LC, Scott P 1993. Immune responses associated with susceptibility of C57BL/10 mice to Leishmania amazonensis. Infect Immun 61: 2952-2959.         [ Links ]

Alves CF, de Amorim IF, Moura EP, Ribeiro RR, Alves CF, Michalick MS, Kalapothakis E, Bruna-Romero O, Tafuri WL, Teixeira MM, Melo MN 2009. Expression of IFN-gamma, TNF-alpha, IL-10 and TGF-beta in lymph nodes associates with parasite load and clinical form of disease in dogs naturally infected with Leishmania (Leishmania) chagasi. Vet Immunol Immunopathol 128: 349-358.         [ Links ]

Bebars MA, el Serougi AO, Makled KM, Mikhael EM, Abou Gamra MM, el Sherbiny M, Mohareb AW, Mohammed EA 2000. An experimental vaccine providing heterologous protection for Leishmania species in murine model. J Egypt Soc Parasitol 30: 137-156.         [ Links ]

Bhaumik SK, Naskar K, De T 2009. Complete protection against experimental visceral leishmaniasis with complete soluble antigen from attenuated Leishmania donovani promastigotes involves Th1-immunity and down-regulation of IL-10. Eur J Immunol 39: 2146-2160.         [ Links ]

Borja-Cabrera GP, Santos FN, Santos FB, Trivellato FA, Kawasaki JK, Costa AC, Castro T, Nogueira FS, Moreira MA, Luvizotto MC, Palatnik M, Palatnik-de-Sousa CB 2010. Immunotherapy with the saponin enriched-Leishmune vaccine versus immunochemotherapy in dogs with natural canine visceral leishmaniasis. Vaccine 28: 597-603.         [ Links ]

Buendía AJ, Nicolás L, Ortega N, Gallego MC, Martinez CM, Sanchez J, Caro MR, Navarro JA, Salinas J 2007. Characterization of a murine model of intranasal infection suitable for testing vaccines against C. abortus. Vet Immunol Immunopathol 115: 76-86.         [ Links ]

Campbell K, Diao H, Ji J, Soong L 2003. DNA immunization with the gene encoding P4 nuclease of Leishmania amazonensis protects mice against cutaneous leishmaniasis. Infect Immun 71: 6270-6278.         [ Links ]

Caro MR, Ortega N, Buendía AJ, Gallego MC, Del Río L, Cuello F, Salinas J 2003. Relationship between the immune response and protection conferred by new designed inactivated vaccines against ovine enzootic abortion in a mouse model. Vaccine 21: 3126-3136.         [ Links ]

Convit J, Ulrich M, Zerpa O, Borges R, Aranzazu N, Valera M, Villarroel H, Zapata Z, Tomedes I 2003. Immunotherapy of American cutaneous leishmaniasis in Venezuela during the period 1990-99. Trans R Soc Trop Med Hyg 97: 469-472.         [ Links ]

Fernandes AP, Costa MM, Coelho EA, Michalick MS, de Freitas E, Melo MN, Luiz Tafuri W, Resende D de M, Hermont V, Abrantes C de F, Gazzinelli RT 2008. Protective immunity against challenge with Leishmania (Leishmania) chagasi in beagle dogs vaccinated with recombinant A2 protein. Vaccine 26: 5888-5895.         [ Links ]

Ghosh A, Zhang WW, Matlashewski G 2001. Immunization with A2 protein results in a mixed Th1/Th2 and a humoral response which protects mice against Leishmania donovani infections. Vaccine 20: 59-66.         [ Links ]

Giunchetti RC, Corrêa-Oliveira R, Martins-Filho OA, Teixeira-Carvalho A, Roatt BM, de Oliveira Aguiar-Soares RD, Coura-Vital W, de Abreu RT, Malaquias LC, Gontijo NF, Brodskyn C, de Oliveira CI, Costa DJ, de Lana M, Reis AB 2008. A killed Leishmania vaccine with sand fly saliva extract and saponin adjuvant displays immunogenicity in dogs. Vaccine 26: 623-638.         [ Links ]

Giunchetti RC, Corrêa-Oliveira R, Martins-Filho OA, Teixeira-Carvalho A, Roatt BM, de Oliveira Aguiar-Soares RD, de Souza JV, das Dores Moreira N, Malaquias LC, Mota e Castro LL, de Lana M, Reis AB 2007. Immunogenicity of a killed Leishmania vaccine with saponin adjuvant in dogs. Vaccine 25: 7674-7686.         [ Links ]

Gomes DC, Pinto EF, de Melo LD, Lima WP, Larraga V, Lopes UG, Rossi-Bergmann B 2007. Intranasal delivery of naked DNA encoding the LACK antigen leads to protective immunity against visceral leishmaniasis in mice. Vaccine 25: 2168-2172.         [ Links ]

Hamilton TA, Ohmori Y, Tebo JM, Kishore R 1999. Regulation of macrophage gene expression by pro- and anti-inflammatory cytokines. Pathobiology 67: 241-244.         [ Links ]

Handman E 2001. Leishmaniasis: current status of vaccine development. Clin Microbiol Rev 14: 229-243.         [ Links ]

Heinzel FP, Sadick MD, Holaday BJ, Coffman RL, Locksley RM 1989. Reciprocal expression of interferon gamma or interleukin 4 during the resolution or progression of murine leishmaniasis. Evidence for expansion of distinct helper T cell subsets. J Exp Med 169: 59-72.         [ Links ]

Kemp M, Theander TG, Kharazmi A 1996. The contrasting roles of CD4+ T cells in intracellular infections in humans: leishmaniasis as an example. Immunol Today 17: 13-16.         [ Links ]

Kensil CR 1996. Saponins as vaccine adjuvants. Crit Rev Ther Drug Carrier Syst 13: 1-55.         [ Links ]

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ 1951. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275.         [ Links ]

Marciani DJ 2003. Vaccine adjuvants: role and mechanisms of action in vaccine immunogenicity. Drug Discov Today 8: 934-943.         [ Links ]

Marques-da-Silva EA, Coelho EA, Gomes DC, Vilela MC, Masioli CZ, Tavares CA, Fernandes AP, Afonso LC, Rezende SA 2005. Intramuscular immunization with p36 (LACK) DNA vaccine induces IFN-gamma production but does not protect BALB/c mice against Leishmania chagasi intravenous challenge. Parasitol Res 98: 67-74.         [ Links ]

Mayrink W, Botelho AC, Magalhães PA, Batista SM, Lima A de O, Genaro O, Costa CA, Melo MN, Michalick MS, Williams P, Dias M, Caiaffa WT, Nascimento E, Machado-Coelho GL 2006. Immunotherapy, immunochemotherapy and chemotherapy for American cutaneous leishmaniasis treatment. Rev Soc Bras Med Trop 39: 14-21.         [ Links ]

Mayrink W, da Costa CA, Magalhães PA, Melo MN, Dias M, Lima AO, Michalick MS, Williams P 1979. A field trial of a vaccine against American dermal leishmaniasis. Trans R Soc Trop Med Hyg 73: 385-387.         [ Links ]

Misra A, Dube A, Srivastava B, Sharma P, Srivastava JK, Katiyar JC, Naik S 2001. Successful vaccination against Leishmania donovani infection in Indian langur using alum-precipitated autoclaved Leishmania major with BCG. Vaccine 19: 3485-3492.         [ Links ]

Newman MJ, Wu JY, Gardner BH, Munroe KJ, Leombruno D, Recchia J, Kensil CR, Coughlin RT 1992. Saponin adjuvant induction of ovalbumin-specific CD8+ cytotoxic T lymphocyte responses. J Immunol 148: 2357-2362.         [ Links ]

Nico D, Santos FN, Borja-Cabrera GP, Palatnik M, Palatnik de Sousa CB 2007. Assessment of the monoterpene, glycidic and triterpene-moieties' contributions to the adjuvant function of the CP05 saponin of Calliandra pulcherrima Benth during vaccination against experimental visceral leishmaniasis. Vaccine 25: 649-658.         [ Links ]

Nylén S, Khamesipour A, Mohammadi A, Jafari-Shakib R, Eidsmo L, Noazin S, Modabber F, Akuffo H 2006. Surrogate markers of immunity to Leishmania major in leishmanin skin test negative individuals from an endemic area re-visited. Vaccine 24: 6944-6954.         [ Links ]

Nylén S, Sacks D 2007. Interleukin-10 and the pathogenesis of human visceral leishmaniasis. Trends Immunol 28: 378-384.         [ Links ]

Pearson RD, Sousa AQ 1996. Clinical spectrum of leishmaniasis. Clin Infect Dis 22: 1-13.         [ Links ]

Ramiro MJ, Zárate JJ, Hanke T, Rodriguez D, Rodriguez JR, Esteban M 2003. Protection in dogs against visceral leishmaniasis caused by Leishmania infantum is achieved by immunization with a heterologous prime-boost regime using DNA and vaccinia recombinant vectors expressing LACK. Vaccine 21: 2474-2484.         [ Links ]

Rhee EG, Mendez S, Shah JA, Wu CY, Kirman JR, Turon TN, Davey DF, Davis H, Klinman DM, Coler RN, Sacks DL, Seder RA 2002. Vaccination with heat-killed Leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against Leishmania major infection. J Exp Med 195: 1565-1573.         [ Links ]

Santos WR, de Lima VM, de Souza EP, Bernardo RR, Palatnik M, Palatnik de Sousa CB 2002. Saponins, IL12 and BCG adjuvant in the FML-vaccine formulation against murine visceral leishmaniasis. Vaccine 21: 30-43.         [ Links ]

Streit JA, Recker TJ, Filho FG, Beverly SM, Wilson ME 2001. Protective immunity against the protozoan Leishmania chagasi is induced by subclinical cutaneous infection with virulent but not avirulent organisms. J Immunol 166: 1921-1929.         [ Links ]

Toledo VP, Mayrink W, Gollob KJ, Oliveira MA, Costa CA, Genaro O, Pinto JA, Afonso LC 2001. Immunochemotherapy in American cutaneous leishmaniasis: immunological aspects before and after treatment. Mem Inst Oswaldo Cruz 96: 89-98.         [ Links ]

Tonui WK, Titus RG 2007. Cross-protection against Leishmania donovani but not L. braziliensis caused by vaccination with L. major soluble promastigote exogenous antigens in BALB/c mice. Am J Trop Med Hyg 76: 579-584.         [ Links ]

Uzonna JE, Späth GF, Beverley SM, Scott P 2004. Vaccination with phosphoglycan-deficient Leishmania major protects highly susceptible mice from virulent challenge without inducing a strong Th1 response. J Immunol 172: 3793-3797.         [ Links ]

Vilela M de C, Gomes DC, Marques-da-Silva E de A, Serafim TD, Afonso LC, Rezende SA 2007. Successful vaccination against Leishmania chagasi infection in BALB/c mice with freeze-thawed Leishmania antigen and Corynebacterium parvum. Acta Trop 104: 133-139.         [ Links ]

Wilson ME 1993. Leishmaniasis. Curr Opin Infect Dis 6: 331-340.         [ Links ]

Zanin FH, Coelho EA, Tavares CA, Marques-da-Silva EA, Silva Costa MM, Rezende SA, Gazzinelli RT, Fernandes AP 2007. 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 9: 1070-1077.         [ Links ]

 

 

Received 23 March 2010
Accepted 20 July 2010
Financial support: FAPEMIG

 

 

+ Corresponding author: rafaella@cpqrr.fiocruz.br

Creative Commons License All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution License