The effect of protein restriction on the development of protective immunity to Leishmania mexicana

Summary Protein deprived C57BL/6 mice infected with 10³ amastigotes of Leishmania mexicana showed early arrest of lesion progression during the first S weeks of infection with subsequent development of progressive non-healing lesions. In contrast, well nourished mice similarly infected developed gradual healing lesions. The early resistance of malnourished mice to 10³ amastigotes was overcome by a larger dose. After a primary inoculation with 10³ amastigotes protein deprived mice failed to express protective immunity to a challenge inoculum given at 5 weeks of infection. When challenge was delayed until 10 weeks, protein deprived mice developed lesions at the site of challenge which tended to regress but were unable to manifest the high level of protective immunity seen in normally nourished reinfected controls. A challenge infection given at 5 or 10 weeks prejudiced the control of primary lesions particularly in the group of protein deprived mice challenged at 10 weeks. Equivalent levels of specific delayed hypersensitivity responses were found in protein deprived and normally nourished uninfected mice immunized with killed parasites which imply that the impaired protective immunity observed in protein deprived mice is not due to a deleterious effect of protein deprivation on the ability of the host to develop cellular responses such as delayed type hypersensitivity to Leishmania antigens.     

The immune response and immunopathology in infection with Schistosoma mansoni: a key role of major egg antigen Sm-p40

The immune response and related granulomatous inflammation in infection with Schistosoma mansoni are ultimately dependent on SEA-sensitized CD4⁺ Th cells and comprise multiple pathways variously involving the activation and recruitment of different cell populations and the production of different inflammatory cytokines, all under the influence of regulatory genetic factors. The spontaneous down-regulation of granuloma formation (immunomodulation), in turn, is a well-known phenomenon, but the full extent of its precipitating factors is still uncertain. This review describes a pathway leading to immunomodulation that features at its centre the down-regulatory cytokine IL-10. This mechanism is attractive because it offers a cogent correlation between findings in the laboratory and those displayed by patients affected with the disease. The Sm-p40 antigen, a major component of schistosome eggs, elicits a strong CD4⁺ Th cell response in H-2^k mice that correlates with intense granuloma formation; in contrast, its immunogenicity is relatively minor during infection of other mouse strains that develop smaller granulomas. Of great interest is that the Sm-p40 antigen only elicits a Th-1 type cytokine response, a phenotype that remains constant even as the overall response to SEA shifts to a Th-2 type. The Sm-p40 molecule has a dominant epitope that is the target of CD4⁺ Th cells from infected H-2^k mice; indeed, a minimal peptide that bears the epitope binds to I-A^k. The importance of pursuing a systematic elucidation of the major egg antigens, resides in the exciting possibility of specifically desensitizing the CD4⁺ Th cells that mediate granuloma formation, which may achieve meaningful prevention or amelioration of clinical disease .     

Human immunoglobulin G mediates protective immunity and identifies protective antigens against larval Strongyloides stercoralis in mice

Protective immunity to larval Strongyloides stercoralis in mice has been shown to be dependent on antibody, complement, and granulocytes. The goals of the present study was to determine the following: (1) whether human serum could passively transfer immunity to mice, (2) the mechanism by which the serum mediated killing, and (3) whether the antigens (Ags) recognized by the protective human antibody could induce protective immunity in mice. Immunoglobulin G (IgG) from a S. stercoralis-seropositive individual passively transferred immunity to mice. The antibody required granulocytes, but not eosinophils, and complement activation to kill the larvae. Antibody-dependent cellular cytotoxicity was not required for larval killing. Immunization of mice with soluble larval Ags isolated by use of the protective immune IgG resulted in protective immunity. In conclusion, immunity could be transferred to mice by IgG from immune humans, and Ags identified by the immune human IgG induced protective immunity in mice, which thereby suggests their possible use in a vaccine against this infection.     

Blastogenic responses, interleukin-2 production and interleukin-2 receptor expression on CD4+ and CD8+ lymphocytes in rhesus monkeys experimentally inoculated with Loa loa

To better understand cellular responses in loiasis infection, in vitro blastogenesis of peripheral blood mononuclear cells (PBMC) to filarial antigen was assessed in 12 Loa loa -inoculated rhesus monkeys over a two-year period. Cellular reactivity to antigen was observed between 10–35 weeks postinoculation (WPI), but had declined by week 50. The roles of interleukin-2 (IL-2) and IL-2 receptor (IL-2R) expression on CD4⁺ and CD8⁺ T cells in regulating the response to antigen were examined during the initial (57 WPI) and late (92 WPI) time points of the observed diminished reactivity to antigen. The levels of IL-2 in antigen cultures at both time points were not significantly different from those in unstimulated cultures. Also, exogenous IL-2 partially reversed the PBMC response to antigen. The percentages of CD4⁺ and CD8⁺ T cells expressing IL-2R in antigen cultures at 57 WPI were not different from those of control animals. Likewise at 92 WPI, the percentage of CD4⁺ T cells expressing IL-2R in antigen cultures, were not increased above those of control animals. In contrast, the percentage of CD8⁺ T cells expressing IL-2R in antigen cultures were significantly increased above those of control animals ( P  < 0.0001), coinciding with an increase in CD8⁺ T cell numbers in these cultures. The data show that factors besides IL-2, and probably an imbalance in the percentages of CD4⁺ and CD8⁺ T cells bearing IL-2R in antigen cultures, may contribute to the diminished reactivity to antigen in L. loa -inoculated rhesus monkeys .     

The role of B cells in immunity against larval Strongyloides stercoralis in mice

The objective of this study was to examine the role of B cells in primary and challenge infections of larval Strongyloides stercoralis in mice. Two strains of B-cell deficient mice were used in these studies, microMT mice that lack all B cells and Xid mice that lack B-1 cells. Primary immune responses in microMT mice were sufficient to eliminate all parasites within 1 week after infection. Immunized microMT and Xid mice, however, were unable to kill challenge parasites at 24 h post infection, the time that they were eliminated in immunized wild-type mice. This was despite having a significant increase in interleukin-5 secreting cells and high numbers of eosinophils in the microenvironment of the challenge larvae. In addition, immunized Xid mice did not generate parasite-specific immunoglobulin (Ig)M but did develop a weak IgG response compared to wild-type mice. These results demonstrate a dichotomy in the requirement of B cells in immunity to S. stercoralis. B cells are not required in the primary response, yet they are required in the secondary immune response. B-1 cells are required for the secondary immune response and their role appears to be the production of IgM and not as a source of immunoregulatory molecules.     

Protective CD4+ T-cell lines raised against Plasmodium chabaudi show characteristics of either Th1 or Th2 cells

Splenic T-lymphocyte lines were established in vitro from Plasmodium chabaudi-infected NIH mice on days 16 and 20 of a primary infection, and from mice after two or three infections. Each line responded specifically to stimulation with a lysed soluble extract of P. chabaudi-infected erythrocytes (pRBC), and displayed a CD4⁺ (L3T4⁺) surface phenotype. Both the day 16 and 20 cell lines, when stimulated in vitro, secreted interleukin-2 (IL-2) and γ-interferon (IFN-γ), indicative of their belonging to the T helper 1 (Th1) CD4⁺ subset. In contrast, both lines derived from reinfected mice secreted interleukin-4 (IL-4) and provided helper activity in antibody production to P. chabaudi in vitro, and thereby had the characteristics of Th2 cells. All four T-cell lines provided significant protection to naïve mice infected with P. chabaudi. In immuno-compromised mice, the day 16 T-cell line was as protective as in naïve mice whereas the cell line from mice infected twice required the additional transfer of mature naïve splenic B cells to provide protection comparable to that seen in the immunocompetent naïve recipients. The results establish that protective immunity to P. chabaudi may be associated with the induction of CD4⁺ Tcells of either the Th1 or Th2 subset which confer protection against this malaria parasite by mechanisms independent of, and dependent upon, B-cell involvement, respectively.     

Intranasal interleukin-12 is a powerful adjuvant for protective mucosal immunity.

The use of interleukin (IL)-12 as a new vaccine adjuvant for stimulating protective antiviral mucosal immunity has been examined. Mice were immunized intranasally (in) with an influenza vaccine consisting of soluble hemagglutinin (H1) and neuraminidase (N1) plus IL-12. This treatment resulted in elevated levels of lung and splenic interferon-gamma and IL-10 mRNA. Total and IgG2a anti-H1N1 antibody levels in serum were significantly elevated, as were total, IgG1, IgG2a, and secretory IgA antibody levels in bronchoalveolar lavage (BAL) fluids compared with animals receiving vaccine alone. Mice immunized in with vaccine and IL-12 also exhibited decreased weight loss and dramatically enhanced survival after lethal challenge with infectious influenza virus. Protection was dependent upon the presence of B cells and could be transferred to naive mice by inoculation of either serum or BAL fluid from IL-12-treated mice. These findings show for the first time that soluble IL-12 delivered in serves as a powerful respiratory adjuvant for protective antiviral immunity.     

Setting in motion the immune mechanisms underlying genetically determined resistance and susceptibility to infection with Leishmania major

The murine model of infection with Leishmania major has allowed the demonstration of a causal relationship between, on the one hand, genetically determined resistance to infection and the development of a Th1 CD4⁺ cell response, and on the other hand, genetically determined susceptibility and Th2 cell maturation. Using this murine model of infection, the role of cytokines in directing the functional differentiation pathway of CD4⁺ T cell precursors, has been demonstrated in vivo . Thus, IL-12 and IFN-γ have been shown to favour Th1 cell development and IL-4 is crucial for the differentiation of Th2 responses. Maturation of a Th2 response in susceptible BALB/c mice following infection with L. major is triggered by the IL-4 produced during the first two days after parasite inoculation. This IL-4 rapidly renders parasite specific CD4⁺ T cells precursors unresponsive to IL-12. A restricted population of CD4⁺ T cells expressing the Vβ4Vα8 TCR heterodimer and recognizing a single epitope on the LACK (Leishmania Activated C-Kinase) antigen of L. major is responsible for this rapid production of IL-4, instructing subsequent differentiation towards the Th2 phenotype of CD4⁺ T cells specific for several parasite antigens .     

Major role for CD8+ T cells in the protection against Toxoplasma gondii following dendritic cell vaccination

Toxoplasma gondii is the causative agent of toxoplasmosis, a worldwide zoonosis for which an effective vaccine is needed. Vaccination with pulsed dendritic cells is very efficient but their use in a vaccination protocol is unconceivable. Nevertheless, unravelling the induced effector mechanisms is crucial to design new vaccine strategies. We vaccinated CBA/J mice with parasite extract-pulsed dendritic cells, challenged them with T. gondii cysts and carried out in vivo depletion of CD4⁺ or CD8⁺ T lymphocytes to study the subsequent cellular immune response and protective mechanisms. CD4⁺ lymphocytes were poorly implicated either in spleen and mesenteric lymph node (MLN) cytokine secretion or in mice protection. By contrast, the increasing number of intracerebral cysts and depletion of CD8⁺ cells were strongly correlated, revealing a prominent role for CD8⁺ lymphocytes in the protection of mice. Splenic CD8⁺ lymphocytes induce a strong Th1 response controlled by a Th2 response whereas CD8⁺ cells from MLNs inhibit both Th1 and Th2 responses. CD8⁺ cells are the main effectors following dendritic cell vaccination and Toxoplasma infection while CD4⁺ T cells only play a minor role. This contrasts with T. gondii infection which elicits the generation of CD4⁺ and CD8⁺ T cells that provide protective immunity.     

Demonstration of strain-specific CD8 T cell responses to Theileria annulata

The present study set out to examine the nature and specificity of the bovine CD8 T cell response at the clonal level in a group of eight animals immunized with a cloned population of Theileria annulata . The results demonstrated that immunized animals generated parasite-specific CD8 T cells that produced IFNγ in response to parasite stimulation but had highly variable levels of cytotoxicity for parasitized cells. The study also demonstrated that these parasite-specific CD8 T cells could be propagated and cloned in vitro from the memory T cell pool of cattle immunized with live T. annulata parasites. Within the small group of animals studied, there was evidence that responses were preferentially directed to antigens presented by an A10⁺ class I major histocompatibility complex (MHC) haplotype, suggesting that responses restricted by products of this haplotype may be dominant. The A10-restricted responses showed differential recognition of different parasite isolates and clones. By using a cloned population of parasites both for immunization of the animals and for in vitro analyses of the responses, we obtained unambiguous evidence that at least a proportion of CD8 T cells restricted by one MHC haplotype were parasite strain restricted.     

CD4+ T lymphocytes contribute to protective immunity induced in sheep and goats by Haemonchus contortus gut antigens

Immunization with parasite antigens derived from the gut of adult Haemonchus contortus induces significant levels of protection against the parasite in sheep and goats. However, the mechanisms of immunity involved in this protection are not clear. Here, we investigate the requirement for CD4⁺ T lymphocytes in gut antigen-induced immunity against H. contortus. Gut antigen immunized animals were depleted (>98%) of their CD4⁺ T lymphocytes in peripheral blood by intravenous injection of an anti-CD4 MoAb. Depletion in peripheral blood persisted for at least eight days, after which there was gradual recovery of CD4⁺ T lymphocytes. Serum antibody levels in gut antigen-immunized animals correlated significantly with worm parameters, suggesting a contribution by antibody to the immunity observed. By covariate analysis, using ELISA OD as the covariate, CD4⁺ T lymphocyte depletion was shown to partially abrogate immunity induced by gut antigen immunization, against challenge infection with H. contortus. The greatest effect of CD4⁺ T lymphocyte depletion was observed at 14 days post-infection, with differences between CD4⁺ T lymphocyte depleted and intact animals less apparent between days 21 and 25. Collectively, our data indicate that CD4⁺ T lymphocytes contribute to immunity induced by gut antigens. Our results also suggest that antibody works synergistically with CD4⁺ T lymphocytes to confer this immunity.     

MHC-restricted antibody responses to Trichuris muris excretory/secretory (E/S) antigen

Summary Two panels of H-2 recombinant mice were used in a detailed serological study to analyse the role of H-2-linked genes in the control of the antibody response to excretory/secretory (E/S) antigens of Trichuris muris. An apparent H-2 ^q ( 1-A ^q) restriction on the early development of high levels of IgGl antibody to E/S antigen was revealed by ELISA. No such restriction was demonstrated for the specific IgG2a response patterns. Recognition of two high molecular weight antigens (90–95 kDa, 105–110 kDa) by IgG antibodies was also shown to be almost exclusively H-2 ^q restricted and may be related at least in part to the high antibody levels seen for H-2 ^q strains of mice. Immune serum from resistant (B10.BR × B10.G) Fl hybrid mice ( H-2 ^qk) containing high levels of IgGl antibodies specific for T. muris E/S and IgG antibodies which recognized the 90–95 kDa and 105–110 kDa E/S antigens was effective in transferring protection to the non-responsive B10.BR mouse strain as seen on day 35 post-infection (p.i.). It is suggested that the IgG responses described for the generally very resistant H-2 ^q mouse strains may contribute to, but not be an absolute requirement for. protective immunity, antibody-mediated damage facilitating a subsequent cellular attack in certain strains of mice.     

Structural and molecular specificity of antibody responses in mice immune to third stage larvae of Onchocerca volvulus

Immunization of mice with irradiated Onchocerca volvulus infective stage larvae (L3) has been demonstrated to confer protection against challenge infections with these larvae. Additionally, cytokine level measurements and cytokine depletion studies have shown that both IL-4 and IL-5 are important in generating a protective immune response against O. volvulus challenge infections, thus suggesting a dependency of protective immunity on IgG1, IgE and/or eosinophils. In the present study, we examined the humoral responses of immunized mice to O. volvulus L3 antigens. ELISA measurements of total serum antibody levels indicated that IgE was the only antibody isotype elevated in mice immunized with O. volvulus L3. IgM from immunized mice was the only isotype that recognized surface antigens on intact O. volvulus L3. IgG1, IgG3, IgE and IgA recognized internal parasite antigens on O. volvulus L3 frozen sections. Western blot analysis of L3 proteins showed that in serum from mice immunized with O. volvulus L3 IgG1, IgG2a/2b, IgA, and IgE, as well as IgM, recognized unique L3 proteins. Antibodies in serum from L3 immunized mice were able to detect O. volvulus adult antigens in a pattern similar to the recognition found in O. volvulus L3. Some L3 antigens were shared by adults, while other antigens were L3 specific. The ELISA, immunohistochemistry and Western blot findings thus demonstrate a complex pattern of antigen recognition of parasite antigens by antibodies found in mice immune to the L3 of O. volvulus     

Effect of chronic ethanol consumption on protective T-helper 1 and T-helper 2 immune responses against the parasites Leishmania major and Strongyloides stercoralis in mice

BACKGROUND: Chronic alcohol consumption has been associated with significant increases in the prevalence of infectious diseases, and it has been suggested that these increases are caused by a direct effect of ethanol on the immune response. The objective of this study was to determine whether chronic ethanol consumption would affect the development of protective immunity to Leishmania major, which is controlled by the T-helper 1 (Th1) subset of CD4 cells, and Strongyloides stercoralis, which is controlled by the Th2 subset. METHODS: Mice were fed ethanol-containing liquid diet (25% ethanol-derived calories), liquid isocaloric diet without ethanol, or solid chow and then exposed to either of the two parasites. The ability of the mice chronically consuming alcohol to eliminate the infections was determined, as were the levels of parasite-specific humoral and cellular immune responses. RESULTS: Mice chronically consuming alcohol were capable of eliminating both of these infections in a manner identical to the control mice. In addition, splenocytes from mice chronically consuming alcohol infected with L. major produced nitric oxide at the same levels as in control mice. Antibody responses were altered in a manner suggesting an increase in Th2 immunity and a decrease in Th1 immunity in the mice chronically consuming alcohol. In mice chronically consuming alcohol that were infected with S. stercoralis, eosinophils migrated to the parasite's microenvironment, and antibodies were produced at levels equivalent to those seen in control mice. CONCLUSIONS: Mice maintained on an ethanol-containing liquid diet had some alteration in their ability to produce Th1 and Th2 immune responses yet were capable of generating unimpaired protective Th1 and Th2 responses.     

Polyclonal hypergammaglobulinemia in a case of B-cell chronic lymphocytic leukaemia: the result of IL-2 production by the proliferating monoclonal B cells?

Summary. CLL is typically characterized by acquired hypogammaglobulinemia. We report the case of a female patient suffering from B-CLL who developed polyclonal hypergammaglobulinaemia: 38-3 g/I polyclonal IgG, 0.97g/1 IgA and 0.33 g/1 IgM. Immunophenotyping showed a monoclonal lymphocytic population CD19⁺ CD5⁺ CD40⁺ CD23+, low slg⁺ (95%), K type in the great majority (96%). RT-PCR of immunoglobulin genes gave evidence of monoclonal rearrangement of the IgM type. Our tests showed that IL-2 was produced when leukaemic B cells were stimulated with phorbol myristate acetate, ionomycin and lipopoly-saccharide. In addition, transfections with the full IL-2 promoter or elements thereof revealed that IL-2 expression is inducible and mediated through the NF-kB-promoter element. Finally, the amount of IL-2 secreted by these cells is about 39ng/ml/10⁶ cells, which is remarkably high for non-T cells. These results suggest that the large amounts of polyclonal IgG seen in this case of B-CLL are secreted by normal B cells which are in turn stimulated by IL-2 produced by proliferating monoclonal (leukaemic) B cells. Under cyclosporin A treatment, immunoglobulin secretion and B cell count remained low.     

Eosinophils act as antigen-presenting cells to induce immunity to Strongyloides stercoralis in mice

The objective of the present study was to explore the ability of eosinophils to present Strongyloides stercoralis antigen in naive and immunized mice. Antigen-pulsed eosinophils were injected intraperitoneally into naive or immunized mice, and then mice were examined for antigen-specific immune responses. A single inoculation of antigen-pulsed eosinophils was sufficient to prime naive mice and to boost immunized mice for antigen-specific T helper cell type 2 (Th2) immune responses with increased interleukin (IL)-4 and IL-5 production. Mice inoculated 3 times with live eosinophils pulsed with antigen showed significant increases in parasite antigen-specific immunoglobulin (Ig) M and IgG levels in their serum. Antigen-pulsed eosinophils deficient in major histocompatibility complex class II molecules or antigen-pulsed dead eosinophils failed to induce immune responses, thereby demonstrating the requirement for direct interaction between eosinophils and T cells. These experiments demonstrate that eosinophils function as antigen-presenting cells for the induction of the primary and the expansion of the secondary Th2 immune responses to S. stercoralis in mice.     

Protective immunity and specificity of antibody responses elicited in cattle by irradiated Trypanosoma brucei

The level and specificity of antibody responses elicited in cattle by irradiated non-infective Trypanosoma brucei were examined and related to the development of protective immunity. These responses were compared with those induced by infection and by inoculation with purified variable surface glycoprotein (VSG) in adjuvant. It was found that 10⁷ or more irradiated trypanosomes inoculated intravenously into cattle conferred complete protection against challenge with 10³ homologous trypanosomes 14 days later. Animals immunized with 10⁶ organisms showed partial protection. Of the assays used for detection of antibody, neutralization of infectivity was slightly more sensitive than either the Farr assay or the immunofluorescence test which were both more sensitive than solid-phase radioimmunoassay (RIA). Detection of specific antibody correlated with immunity, in that all animals inoculated intravenously with 10⁶ or more trypanosomes developed neutralizing activity in their sera. The antibody responses after intravenous inoculation were consistently superior to those induced by the subcutaneous route. By carrying out blocking assays, most of the antibody elicited by irradiated trypanosomes was found to be specific for antigenic determinants on the VSG exposed on the surface of live trypanosomes. A similar specificity was found for the antibody induced during infection with T. brucei. Conversely, a large component of the antibody induced by purified VSG in adjuvant appeared to be directed against determinants on the VSG which are not exposed on live trypanosomes and are probably not involved in protective immunity.     

The immune response during a Strongyloides ratti infection of rats

A range of immune parameters was measured during a primary infection of Strongyloides ratti in its natural rat host. The immune parameters measured were interleukin-4 (IL-4) and interferon-gamma from both the spleen and mesenteric lymph node (MLN) cells; parasite-specific immunoglobulin G(1)(IgG(1)), IgG(2a) and IgG(2b) in serum and in intestinal tissue; parasite-specific IgG and total IgE in serum; parasite-specific and total IgA in intestinal tissue and rat mast cell protease II in intestinal tissue. Parasite-specific IgG(1), IgG(2a) and total IgE in serum and parasite-specific IgA and rat mast cell protease II in intestinal tissue all occurred at significantly greater concentrations in infected animals, compared with non-infected animals. Similarly, the production of IL-4 by MLN cells stimulated with parasitic female antigen or concanavalin A occurred at significantly greater concentrations in infected animals, compared with non-infected animals. In all, this suggests that there is a T-helper 2-type immune response during a primary S. ratti infection. These data also show the temporal changes in these components of the host immune response during a primary S. ratti infection.     

Comparative efficacy of the Schistosoma mansoni nucleic acid vaccine,Sm23, following microseeding or gene gun delivery

Sm23 is an integral membrane protein expressed widely in the human parasitic worm Schistosoma mansoni. Sm23 has already been shown to elicit protective immune responses following immunization with peptides or DNA constructs. In this study, we evaluated the immunogenicity and the protective efficacy of the Sm23 DNA vaccine using two different intradermal DNA delivery methods: microseeding and gene gun. Using both techniques, all mice immunized with the Sm23-pcDNA construct generated Sm23-specific immunoglobulin (Ig)G antibody, while mice immunized with the control plasmid, pcDNA, did not. Antibody isotypes analysis revealed that microseeding elicited mainly IgG2a and IgG2b antibodies, with relatively low levels of IgG1 and IgG3. The relative IgG1/IgG2a ratio was 0.03, indicative of a Th1 type immune response. In contrast, gene gun immunization resulted in significantly higher levels of IgG1 and IgG3. The relative IgG1/IgG2a ratio in this case was 11, indicative of a Th2 type immune response. No significant difference in the levels of IgG2b was observed. Coimmunization with plasmid DNA encoding either interleukin (IL)-12 or IL-4 by microseeding did not affect the levels of IgG1, while the levels of IgG2a and IgG2b were reduced. On the other hand, the levels of IgG3 were significantly increased by IL-4, but unchanged by IL-12. Importantly, in all experiments, the Sm23-pcDNA vaccine provided statistically significant levels of protection against challenge infection. Microseeding immunizations resulted in higher levels of protection (31-34% protection) than gene gun immunization (18% protection). This suggests that the Th1 type immune response elicited by microseeding immunization was responsible for the higher protection levels. However, the protective effect of the vaccine was not affected by coadministering plasmids encoding either IL-12 or IL-4 using the microseeding technique.     

Cocktail of gp63 and Hsp70 induces protection against Leishmania donovani in BALB/c mice

The 63-kDa antigen of Leishmania donovani is a membrane-anchored matrix metalloprotease that has been shown to be involved in the infection process. We have shown that this antigen alone generates a Th1 type of protective response that is partial but when the animals are primed with the antigen along with the Hsp70, the level of protection is raised significantly, which is demonstrated by a considerable reduction in parasite load of immunized animals when compared to the infected controls. Delayed-type hypersensitivity responses to leishmanin were measured as an index of cell-mediated immune response and were found to be higher in immunized animals when compared to the infected controls, the maximum being in the animals immunized with cocktail of both the antigens. Maximum IgG2a and minimum IgG1 levels were observed in this group of animals. These animals also generated maximum levels of IFN-γ and IL-2 and minimum levels of IL-4 and IL-10 pointing towards the generation of a protective Th1 response and the suppression of the Th2 type of immune response.