The combination of DNA vectors expressing IL-12 + IL-18 elicits high protective immune response against cutaneous leishmaniasis after priming with DNA-p36/LACK and the cytokines,followed by a booster with a vaccinia virus recombinant expressing p36/LACK

Protocols of immunization based on the DNA prime/vaccinia virus (VV) boost regime with recombinants expressing relevant antigens have been shown to elicit protection against a variety of pathogens in animal model systems, and various phase I clinical trials have been initiated with this vaccination approach. We have previously shown that mice immunized with a DNA vector expressing p36/LACK of Leishmania infantum followed by a booster with VVp36/LACK induced significant protection against Leishmania major infection. To further improve this protocol of immunization, here we investigated whether the cytokines interleukin-12 (IL-12) and IL-18 could enhance protection against L. major infection in BALB/c mice. We found that priming with DNA vectors expressing p36/LACK and either IL-12 or IL-18, followed by a booster with a VV recombinant expressing the same L. infantum LACK antigen, elicit a higher cellular immune response than by using the same protocol in the absence of the cytokines. The cytokine IL-12 triggered a higher number of IFN-gamma-secreting cells specific for p36 protein than IL-18. When immunized animals were challenged with promastigotes, the highest protection against L. major infection was observed in animals primed with DNAp36 + DNA IL-12 + DNA IL-18 and boosted with VVp36. This protection correlated with a Th1 type of immune response. Our findings revealed that in prime/booster protocols, co-expressing IL-12 and IL-18 during priming is an efficient approach to protect against leishmaniasis. This combined prime/booster immunization regime could have wide use in fighting against parasitic and other infectious diseases.     

Molecular cloning, cell localization and binding affinity to DNA replication proteins of the p36/LACK protective antigen from Leishmania infantum.

The p36/LACK antigen from Leishmania, an analogue of the receptor for activated protein kinase C (PKC), induces high levels of protection against parasite infection in the BALB/c mouse model. This protection is more than twice as high as that elicited by major parasite antigens such as soluble Leishmania antigen or the main surface protease gp63. We have cloned and purified p36/LACK from Leishmania infantum, the causative agent of visceral leishmaniasis in Europe. This protein belongs to the large family of WD 40 repeat proteins confined to eukaryotes and involved in numerous regulatory functions. Differential solubilization and immunofluorescence experiments indicate that p36/LACK is present close to the kinetoplast disc in the cell cytoplasm, probably bound to multiprotein complexes but not to membrane structures. These complexes probably also include cytoplasm PKC isoforms. The use of a genetically-encoded peptide library indicates that p36/LACK binds sequences present in several proteins involved in DNA replication and RNA synthesis. The recognition and binding sequences present in vacuolar proteins and at the beta-chain of major histocompatability complex (MHC) class II suggest the involvement of this regulatory protein in the early mechanisms triggering the protective immune response of the host against the parasite infection.     

Induction of protective CD4+ T cell-mediated immunity by a Leishmania peptide delivered in recombinant influenza viruses

The available evidence suggests that protective immunity to Leishmania is achieved by priming the CD4(+) Th1 response. Therefore, we utilised a reverse genetics strategy to generate influenza A viruses to deliver an immunogenic Leishmania peptide. The single, immunodominant Leishmania-specific LACK(158-173) CD4(+) peptide was engineered into the neuraminidase stalk of H1N1 and H3N2 influenza A viruses. These recombinant viruses were used to vaccinate susceptible BALB/c mice to determine whether the resultant LACK(158-173)-specific CD4(+) T cell responses protected against live L. major infection. We show that vaccination with influenza-LACK(158-173) triggers LACK(158-173)-specific Th1-biased CD4(+) T cell responses within an appropriate cytokine milieu (IFN-γ, IL-12), essential for the magnitude and quality of the Th1 response. A single intraperitoneal exposure (non-replicative route of immunisation) to recombinant influenza delivers immunogenic peptides, leading to a marked reduction (2-4 log) in parasite burden, albeit without reduction in lesion size. This correlated with increased numbers of IFN-γ-producing CD4(+) T cells in vaccinated mice compared to controls. Importantly, the subsequent prime-boost approach with a serologically distinct strain of influenza (H1N1->H3N2) expressing LACK(158-173) led to a marked reduction in both lesion size and parasite burdens in vaccination trials. This protection correlated with high levels of IFN-γ producing cells in the spleen, which were maintained for 6 weeks post-challenge indicating the longevity of this protective effector response. Thus, these experiments show that Leishmania-derived peptides delivered in the context of recombinant influenza viruses are immunogenic in vivo, and warrant investigation of similar vaccine strategies to generate parasite-specific immunity.     

Intranasal immunization with chitosan microparticles enhances LACK-DNA vaccine protection and induces specific long-lasting immunity against visceral leishmaniasis

Development of a protective vaccine against Leishmania depends on antigen formulation and adjuvants that induce specific immunity and long-lasting immune responses. We previously demonstrated that BALB/c mice intranasally vaccinated with a plasmid DNA encoding the p36/LACK leishmanial antigen (LACK-DNA) develop a protective immunity for up to 3 months after vaccination, which was linked with the systemic expression of vaccine mRNA in peripheral organs. In this study, LACK-DNA vaccine was associated with biocompatible chitosan microparticles cross-linked with glyceraldehyde (CMC) to boost the long-lasting immunity against the late Leishmania infantum challenge. Infection at 7 days, 3 or 6 months after vaccination resulted in significantly lower parasite loads when compared with non-vaccinated controls. Besides, LACK-DNA-chitosan vaccinated mice showed long-time protection observed after the late time point challenge. The achieved protection was correlated with an enhanced spleen cell responsiveness to parasite antigens, marked by increased proliferation and IFN-γ as well as decreased IL-10 production. Moreover, we found diminished systemic levels of TNF-α that was compatible with the better health condition observed in LACK-DNA/CMC vaccinated-infected mice. Together, our data indicate the feasibility of chitosan microparticles as a delivery system tool to extend the protective immunity conferred by LACK-DNA vaccine, which may be explored in vaccine formulations against Leishmania parasite infections.     

High quality long-term CD4+ and CD8+ effector memory populations stimulated by DNA-LACK/MVA-LACK regimen in Leishmania major BALB/c model of infection

Heterologous vaccination based on priming with a plasmid DNA vector and boosting with an attenuated vaccinia virus MVA recombinant, with both vectors expressing the Leishmania infantum LACK antigen (DNA-LACK and MVA-LACK), has shown efficacy conferring protection in murine and canine models against cutaneus and visceral leishmaniasis, but the immune parameters of protection remain ill defined. Here we performed by flow cytometry an in depth analysis of the T cell populations induced in BALB/c mice during the vaccination protocol DNA-LACK/MVA-LACK, as well as after challenge with L. major parasites. In the adaptive response, there is a polyfunctional CD4(+) and CD8(+) T cell activation against LACK antigen. At the memory phase the heterologous vaccination induces high quality LACK-specific long-term CD4(+) and CD8(+) effector memory cells. After parasite challenge, there is a moderate boosting of LACK-specific CD4(+) and CD8(+) T cells. Anti-vector responses were largely CD8(+)-mediated. The immune parameters induced against LACK and triggered by the combined vaccination DNA/MVA protocol, like polyfunctionality of CD4(+) and CD8(+) T cells with an effector phenotype, could be relevant in protection against leishmaniasis.     

Vaccination with the Leishmania major ribosomal proteins plus CpG oligodeoxynucleotides induces protection against experimental cutaneous leishmaniasis in mice

In the present work we analyze the antigenicity of Leishmania major ribosomal proteins (LRP) in infected BALB/c mice. We show that BALB/c mice vaccinated with LRP in the presence of CpG oligodeoxynucleotides (CpG-ODN) were protected against the development of dermal pathology and showed a reduction in the parasite load after challenge with L. major. This protection was associated with the induction of an IL-12 dependent specific-IFN-gamma response mediated mainly by CD4(+) T cell, albeit a minor contribution of CD8(+) T cells cannot be ruled out. Induction of Th1 responses against LRP also resulted in a reversion of the Th2 responses associated with susceptibility. A marked reduction of IgG1 antibody titer against parasite antigens besides an impaired IL-4 and IL-10 cytokine production by parasite specific T cells was observed. In addition, we show that the administration of the LRP plus CpG-ODN preparation also conferred protection in the naturally resistant C57BL/6 mice. In this strain protection was associated with a LRP specific IFN-gamma production in lymph nodes draining the challenge site. We believe that these evolutionary conserved proteins, combined with adjuvants that favor Th1 responses, may be relevant components of a pan-Leishmania vaccine.     

IL-10 from regulatory T cells determines vaccine efficacy in murine Leishmania major infection

Leishmaniasis affects 12 million people, but there are no vaccines. Immunological correlates of vaccine efficacy are unclear. Polarized Th1 vs Th2 responses in Leishmania major-infected mice suggested that a shift in balance from IL-4 to IFN-gamma was the key to vaccine success. Recently, a role for IL-10 and regulatory T cells in parasite persistence was demonstrated, prompting re-evaluation of vaccine-induced immunity. We compared DNA/modified vaccinia virus Ankara heterologous prime-boost with Leishmania homolog of the receptor for activated C kinase (LACK) or tryparedoxin peroxidase (TRYP). Both induced low IL-4 and high IFN-gamma prechallenge. Strikingly, high prechallenge CD4 T cell-derived IL-10 predicted vaccine failure using LACK, whereas low IL-10 predicted protection with TRYP. The ratio of IFN-gamma:IL-10 was thus a clear prechallenge indicator of vaccine success. Challenge infection caused further polarization to high IL-10/low IFN-gamma with LACK and low IL-10/high IFN-gamma with TRYP. Ex vivo quantitative RT-PCR and in vitro depletion and suppression experiments demonstrated that Ag-driven CD4+ CD25+ T regulatory 1-like cells were the primary source of IL-10 in LACK-vaccinated mice. Anti-IL-10R treatment in vivo demonstrated that IL-10 was functional in determining vaccine failure, rendering LACK protective in the presence of high IFN-gamma/low IL-5 responses.     

Leishmania braziliensis: partial control of experimental infection by interleukin-12 p40 deficient mice

Resistance to infection by Leishmania major has been associated with the development of a Th1 type response that is dependent on the presence of interleukin 12 (IL-12). In this work the involvement of this cytokine in the response to infection by L. braziliensis, a less virulent species in the mouse model, was evaluated. Our results show that while interferon (IFN-gamma) deficient (-/-) mice inoculated L. braziliensis develop severe uncontrolled lesions, chronic lesions that remained under control up to 12 weeks of infection were observed in IL-12p40 -/- mice. IL 12p40 -/- mice had fewer parasites in their lesions than IFN-gamma (-/-) mice. Lymph node cells from IL-12p40 -/- were capable of producing low but consistent levels of IFN-gamma suggestive of its involvement in parasite control. Furthermore, as opposed to previous reports on L. major-infected animals, no switch to a Th2 response was observed in IL-12p40 -/- infected with L. braziliensis.     

MVA-LACK as a safe and efficient vector for vaccination against leishmaniasis

An optimal vaccine against leishmaniasis should elicit parasite specific CD4+ and cytotoxic CD8+ T cells. In this investigation, we described a prime/boost immunization approach based on DNA and on poxvirus vectors (Western Reserve, WR, and the highly attenuated modified vaccinia virus Ankara, MVA), both expressing the LACK antigen of Leishmania infantum, that triggers different levels of specific CD8+ T cell responses and protection (reduction in lesion size and parasitemia) against L. major infection in mice. A prime/boost vaccination with DNA-LACK/MVA-LACK elicits higher CD8+ T cell responses than a similar protocol with the replication competent VV-LACK. Both CD4+ and CD8+ T cells were induced by DNA-LACK/MVA-LACK immunization. The levels of IFN-gamma and TNF-alpha secreting CD8+ T cells were higher in splenocytes from DNA-LACK/MVA-LACK than in DNA-LACK/VV-LACK immunized animals. Moreover, protection against L. major was significantly higher in DNA-LACK/MVA-LACK than in DNA-LACK/VV-LACK immunized animals when boosted with the same virus dose, and correlated with high levels of IFN-gamma and TNF-alpha secreting CD8+ T cells. In DNA-LACK/MVA-LACK vaccinated animals, the extent of lesion size reduction ranged from 65 to 92% and this protection was maintained for at least 17 weeks after challenge with the parasite. These findings demonstrate that in heterologous prime/boost immunization approaches, the protocol DNA-LACK/MVA-LACK is superior to DNA-LACK/VV-LACK in triggering specific CD8+ T cell immune responses and in conferring protection against cutaneous leishmaniasis. Thus, MVA-LACK is a safe and efficient vector for vaccination against leishmaniasis.     

Leishmania major: immune response in BALB/c mice immunized with stress-inducible protein 1 encapsulated in liposomes

Protection against leishmaniasis is depending upon generation of a Th1 type of immune response. Field trials of first generation Leishmania vaccine showed a limited efficacy even with multiple doses mainly due to lack of an appropriate adjuvant. In this study, susceptible BALB/c mice were immunized with rLmSTI1 encapsulated in liposomes to explore the extent of protection induced by Leishmania antigen encapsulated in the liposomes against challenge with Leishmania major. The results showed that s.c. immunization of BALB/c mice with liposomal rLmSTI1 induced a significant protection against challenge and a significant lower parasite burden in spleen up to 14 weeks after challenge. The protected animals showed a significantly smaller footpad thickness after challenge, and a higher level of anti-SLA IgG antibodies before and after challenge with a predominant IgG2a titer. The data supports the possibility of using liposomal Leishmania antigens as a vaccine.     

Vaccination with Leishmania soluble antigen and immunostimulatory oligodeoxynucleotides induces specific immunity and protection against Leishmania donovani infection

In this report, we investigated the effect of ODN containing immunostimulatory CG motifs as adjuvant with soluble antigen (SA) from Leishmania donovani. BALB/c mice were vaccinated with the soluble antigen with or without CpG-ODN as adjuvant and then challenged with L. donovani metacyclic promastigotes. CpG-ODN alone resulted in partial protection against challenge with L. donovani. Immunization of mice with SA and CpG-ODN showed enhanced reduction in parasite load ( approximately 60%) when compared to SA ( approximately 40%) immunized mice. Immunization with SA by itself resulted in a mixed Th1/Th2 response whereas co-administration of SA with CpG-ODN resulted in a strong Th1 promoting isotype as they together promoted production of immunoglobulin G2a. Leishmania-specific Th1 cytokine response was induced by co-administering CpG-ODN and SA as they together promoted production of IFN-gamma and IL-12. In the present study, we demonstrate that immunostimulatory phosphorothioate-modified ODN are promising immune enhancers for vaccination against visceral leishmaniaisis.     

The potency and durability of DNA- and protein-based vaccines against Leishmania major evaluated using low-dose, intradermal challenge

DNA- and protein- based vaccines against cutaneous leishmaniasis due to Leishmania major were evaluated using a challenge model that more closely reproduces the pathology and immunity associated with sand fly-transmitted infection. C57BL/6 mice were vaccinated s.c. with a mixture of plasmid DNAs encoding the Leishmania Ags LACK, LmSTI1, and TSA (AgDNA), or with autoclaved L. major promastigotes (ALM) plus rIL-12, and the mice were challenged by inoculation of 100 metacyclic promastigotes in the ear dermis. When challenged at 2 wk postvaccination, mice receiving AgDNA or ALM/rIL-12 were completely protected against the development of dermal lesions, and both groups had a 100-fold reduction in peak dermal parasite loads compared with controls. When challenged at 12 wk, mice vaccinated with ALM/rIL-12 maintained partial protection against dermal lesions and their parasite loads were no longer significantly reduced, whereas the mice vaccinated with AgDNA remained completely protected and had a 1000-fold reduction in dermal parasite loads. Mice vaccinated with AgDNA also harbored few, if any, parasites in the skin during the chronic phase, and their ability to transmit L. major to vector sand flies was completely abrogated. The durable protection in mice vaccinated with AgDNA was associated with the recruitment of both CD8(+) and CD4(+) T cells to the site of intradermal challenge and with IFN-gamma production by CD8(+) T cells in lymph nodes draining the challenge site. These data suggest that under conditions of natural challenge, DNA vaccination has the capacity to confer complete protection against cutaneous leishmaniasis and to prevent the establishment of infection reservoirs.     

Requirements for the maintenance of Th1 immunity in vivo following DNA vaccination: a potential immunoregulatory role for CD8+ T cells

Protective immunity against Leishmania major generated by DNA encoding the LACK (Leishmania homologue of receptor for activated C kinase) Ag has been shown to be more durable than vaccination with LACK protein plus IL-12. One mechanism to account for this may be the selective ability of DNA vaccination to induce CD8+ IFN-gamma-producing T cells. In this regard, we previously reported that depletion of CD8+ T cells in LACK DNA-vaccinated mice abrogated protection when infectious challenge was done 2 wk postvaccination. In this study, we extend these findings to study the mechanism by which CD8+ T cells induced by LACK DNA vaccination mediate both short- and long-term protective immunity against L. major. Mice vaccinated with LACK DNA and depleted of CD8+ T cells at the time of vaccination or infection were unable to control infection when challenge was done 2 or 12 wk postvaccination. Remarkably, it was noted that depletion of CD8+ T cells in LACK DNA-vaccinated mice was associated with a striking decrease in the frequency of LACK-specific CD4+ IFN-gamma-producing T cells both before and after infection. Moreover, data are presented to suggest a mechanism by which CD8+ T cells exert this regulatory role. Taken together, these data provide additional insight into how Th1 cells are generated and sustained in vivo and suggest a potentially novel immunoregulatory role for CD8+ T cells following DNA vaccination.     

Dendritic cells (DC) activated by CpG DNA ex vivo are potent inducers of host resistance to an intracellular pathogen that is independent of IL-12 derived from the immunizing DC

We used the model of murine leishmaniasis to evaluate the signals enabling Ag-pulsed dendritic cells (DC) to prime a protective Th1 response in vivo. Bone marrow-derived DC (BMDC) that had been activated by TNF-alpha or CD40 ligation were not able to induce protection against leishmaniasis in susceptible BALB/c mice. In contrast, all mice vaccinated with a single dose of Leishmania major Ag-pulsed BMDC stimulated by prior in vitro exposure to CpG-containing oligodeoxynucleotides (ODN) were completely protected, had a dramatic reduction in parasite burden, and developed an Ag-specific Th1 response. Importantly, systemic administration of CpG ODN was not required. Protection mediated by ex vivo CpG ODN-activated and Ag-pulsed DC was solid, as documented by resistance to reinfection with a higher parasite dose, and long-lasting, as immunized mice were still protected against L. major challenge 16 wk after vaccination. A significantly increased level of protection could also be elicited in resistant C57BL/6 mice. Surprisingly, IL-12 expression by the immunizing BMDC was not required for induction of host resistance. In contrast, the availability of IL-12 derived from recipient cells was essential for the initial triggering of protective immunity by transferred BMDC. Together, these findings demonstrate that the type of stimulatory signal is critical for activating the potential of DC to induce a Th1 response in vivo that confers complete protection against an intracellular pathogen. Moreover, they show that the impact of activated DC on the initiation of a protective Th cell response in vivo may be independent of their ability to produce IL-12.     

Reciprocal CD40 signals through p38MAPK and ERK-1/2 induce counteracting immune responses

Macrophages play host to Leishmania major, a parasite that causes leishmaniasis in 500,000 people annually. Macrophage-expressed CD40, a costimulatory molecule, induces interleukin-12 (IL-12)-dependent and interferon-gamma (IFN-gamma)-dependent host-protective immune responses to Leishmania and other intracellular pathogens. Paradoxically, IL-10, another CD40-induced cytokine in macrophages, promotes Leishmania infection. How CD40 signaling regulates the secretion of these two counteractive cytokines remains unknown. Here we show that weak CD40 signals induce extracellular stress-related kinase-1/2 (ERK-1/2)-dependent IL-10 expression, whereas stronger signals induce p38 mitogen-activated protein kinase (p38MAPK)-dependent IL-12 production. p38MAPK and ERK-1/2 therefore have counter-regulatory actions. Leishmania skews CD40 signaling toward ERK-1/2, inducing IL-10, which inhibits activation of CD40-induced p38MAPK and expression of inducible nitric oxide synthase-2 (iNOS-2) and IL-12. ERK-1/2 inhibition or IL-10 neutralization restores CD40-induced p38MAPK activation and parasite killing in macrophages and the BALB/c mouse, a susceptible host. These data uncover a new immune evasion strategy, whereby Leishmania differentially modulates CD40-engaged, reciprocally functioning signaling modules, and provide a new conceptual framework for immune homeostasis.     

Induction of cellular and humoral responses by autoclaved and heat-killed antigen of Leishmania donovani in experimental visceral leishmaniasis

The potential of autoclaved and heat-killed antigen of Leishmania donovani to induce cell-mediated and humoral response has been evaluated in the present study. The vaccines were delivered thrice subcutaneously at an interval of 2 weeks. Two weeks after second booster, BALB/c mice were challenged with 10⁷ stationary phase promastigotes of L. donovani. Significant protection was achieved in immunized mice against L. donovani challenge with 69% to 76% and 59% to 64% reduction in parasite load in the liver and spleen respectively. Immunization induced significantly higher level of delayed type hypersensitivity (DTH) response in mice immunized with heat-killed antigen followed by autoclaved antigen. The immune response was assessed by quantifying Leishmania-specific antibodies and cytokine production. The antibody response was predominantly of IgG type with increased IgG2a production and lesser amount of IgM. The immunization preferentially stimulates the production of IFN-γ and IL-2 in splenocytes which suggests a Th1 type response with a concomitant down-regulation of IL-10 and IL-4. These results indicate a potential for the heat-killed and autoclaved antigen as a vaccine which could trigger cell-mediated immune response.     

A single intradermal administration of soluble leishmanial antigen and plasmid expressing interleukin-12 protects BALB/c mice from Leishmania major infection.

In murine leishmaniasis, the induction of the T-helper type 1 (Th1) response contributes to infection resistance, whereas the establishment of the Th2 response makes the mice susceptible to infection. Interleukin-12 (IL-12) plays a pivotal role in the diversification of immune responses to the Th1 type. In this study, we tested whether the co-administration of IL-12 expression plasmid which compose p35 and p40 subunits and soluble leishmanial antigen (SLA) will skew the susceptible BALB/c mice to Th1 response and protect from leishmaniasis. When the mice were intradermally injected with the combination of IL-12 plasmid and SLA 7 days prior to the challenge with 1x10(6) promastigotes of Leishmania major, the local lesions completely healed and the parasite burden in the local lymph nodes significantly decreased. The cured mice attained long-term immunity, and were resistant to any subsequent rechallenge of the lethal dose of the parasite. The protective effect was associated with the development of a Th1 response, as demonstrated by the enhanced level of antigen-specific interferon-gamma (IFN-gamma) and dominant production of IgG2a in the serum. In contrast, the administration of empty plasmid plus SLA or IL-12 plasmid alone failed to protect the disease and shape the Th1 response. Furthermore, the protective efficiency induced by the vaccination was clearly prevented by the injection of either neutralizing anti-IL-12 mAb or anti-IFN-gamma mAb. The IL-12 expression plasmid is thus an effective adjuvant for the elicitation of a protective Th1 response against leishmaniasis and is therefore, considered to be appropriate for vaccinations that require the induction of Th1 type immunity.     

Resolution of an infection with Leishmania braziliensis confers complete protection to a subsequent challenge with Leishmania major in BALB/c mice

Both Leishmania major and L. braziliensis induce cutaneous leishmaniasis in BALB/c mice. Whereas BALB/c mice die of infection with L. major, they cure an infection with L. braziliensis. We report here that after curing an infection with L. braziliensis, BALB/c mice are resistant to challenge with L. major. When challenged with L. major, L. braziliensis pre-treated BALB/c mice mounted a delayed-type hypersensitivity response to L. major and produced high amounts of interferon-gamma (IFN-gamma) but low amounts of interleukin-4. The IFN-gamma produced by the L. braziliensis pre-infected mice was involved in the protection seen against L. major challenge since treating the mice with a neutralizing anti-IFN-gamma abrogated the protection. This suggests that cross-reactive antigen epitopes exist between L. braziliensis and L. major and that pre-infection with L. braziliensis primes BALB/c mice to epitopes on L. major that can elicit a protective Th1 response to the parasite.     

Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis

Vaccination against visceral leishmaniasis has received limited attention compared with cutaneous leishmaniasis, although the need for an effective vaccine against visceral leishmaniasis is pressing. In this study, we demonstrate for the first time that a recombinant stage-specific hydrophilic surface protein of Leishmania donovani, recombinant hydrophilic acylated surface protein B1 (HASPB1), is able to confer protection against experimental challenge. Protection induced by rHASPB1 does not require adjuvant and, unlike soluble Leishmania Ag + IL-12, extends to the control of parasite burden in the spleen, an organ in which parasites usually persist and are refractory to a broad range of immunological and chemotherapeutic interventions. Both immunohistochemistry (for IL-12p40) and enzyme-linked immunospot assay (for IL-12p70) indicate that immunization with rHASPB1 results in IL-12 production by dendritic cells, although an analysis of Ab isotype responses to rHASPB1 suggests that this response is not sufficient in magnitude to induce a polarized Th1 response. Although both vaccinated and control-infected mice have equivalent frequencies of rHASPB1-specific CD4(+) T cells producing IFN-gamma, vaccine-induced protection correlates with the presence of rHASPB1-specific, IFN-gamma-producing CD8(+) T cells. Thus, we have identified a novel vaccine candidate Ag for visceral leishmaniasis, which appears to operate via a mechanism similar to that previously associated with DNA vaccination.     

Dendritic cell-derived IL-12p40 homodimer contributes to susceptibility in cutaneous leishmaniasis in BALB/c mice

Protection against Leishmania major in resistant C57BL/6 mice is mediated by Th1 cells, whereas susceptibility in BALB/c mice is the result of Th2 development. IL-12 release by L. major-infected dendritic cells (DC) is critically involved in differentiation of Th1 cells. Previously, we reported that strain differences in the production of DC-derived factors, e.g., IL-1alphabeta, are in part responsible for disparate disease outcome. In the present study, we analyzed the release of IL-12 from DC in more detail. Stimulated DC from C57BL/6 and BALB/c mice released comparable amounts of IL-12p40 and p70. In the absence of IL-4, BALB/c DC produced significantly more IL-12p40 than C57BL/6 DC. Detailed analyses by Western blot and ELISA revealed that one-tenth of IL-12p40 detected in DC supernatants was released as the IL-12 antagonist IL-12p40 homodimer (IL-12p80). BALB/c DC released approximately 2-fold more IL-12p80 than C57BL/6 DC both in vitro and in vivo. Local injection of IL-12p80 during the first 3 days after infection resulted in increased lesion volumes for several weeks in both L. major-infected BALB/c or C57BL/6 mice, in higher lesional parasite burdens, and decreased Th1-cytokine production. Finally, IL-12p40-transgenic C57BL/6 mice characterized by overexpression of p40 showed increased levels of serum IL-12p80 and enhanced disease susceptibility. Thus, in addition to IL-1alphabeta, strain-dependent differences in the release of other DC-derived factors such as IL-12p80 may influence genetically determined disease outcome.