Coexistence of antigen-specific TH1 and TH2 cells in genetically susceptible BALB/c mice infected with Leishmania major

CD4-positive T cell clones with specificity for the protozoan parasite Leishmania major (L. major) of both the protective TH1 and the disease-exacerbating TH2 subtype were isolated from a diseased L. major-infected mouse of the susceptible BALB/c strain. In addition, TH2 cells were isolated from the lesion-draining lymph nodes of an animal clinically healed nine months after sublethal irradiation and subsequent infection. Our data support the notion that the differential outcome of the disease in non-irradiated versus irradiated BALB/c mice reflects the regulation of the two CD4+ T cell subsets. These data also argue against the possibilities that: 1) TH2 cells and their precursors are totally eliminated by irradiation and that 2) TH2 cells are capable of completely hindering the expansion of TH1 cells in diseased animals.     

Establishment of resistance to Leishmania major infection in susceptible BALB/c mice requires parasite-specific CD8+ T cells

Although CD4⁺ T cells are generally accepted to be responsiblefor the determination of resistance to infection in experimentalmurine cutaneous leishmanlasis, a contribution of CD8⁺ lymphocytesto immunity can be demonstrated under certain well-defined conditions.Normally highly susceptible BALB/c mice can be rendered resistantto infection with Leishmania major promastigotes by a singleinjection of monoclonal anti-CD4 antibodies at the beginningof infection. Mice treated in such a way can heal their primarycutaneous lesions and acquire immunity to subsequent challengeinfection. Both the resolution of the primary infection andthe induced state of immunityto reinfection in these mice isshown to be dependent upon the anti-leishmanial effector functionsof CD8⁺ T cells. Furthermore, in contrast to control infectedBALB/c mice, which are unable to mount a delayed-type hypersensitivity(DTH) response to viable parasites, mice cured as a result oftreatment with anti-CD4 antibodies in vivo exhibit a strongDTH response, which can be significantly reduced by injectionof either anti-CD4 or anti-CD8 monoclonal antibodies prior toantigenic challenge with viable promastigotes. Moreover, increasednumbers of specific CD8⁺ T cells, able to transferLeishmania-specificDTH responses, were found in lymphold organs of BALB/c micerendered resistant to infection by immunointervention with anti-CD4monoclonal antibodies at the beginning of infection. Neutralizationin vivo of interleukin 4 during the course of infection in BALB/cmice also enables these otherwise susceptible mice to resolvetheir cutaneous lesions and to decrease the parasite burdenin infected tissues. CD8⁺ T cells are required for both of thesebeneficial effects. Taken together, these results indicate thatin the immune BALB/c mouse, as in the normally resistant CBAmouse, CD8⁺ lymphocytes are involved in the elimination of L.major and in the establishment and maintenance of immunity againstinfection with this parasite.     

Senescent BALB/c mice are able to develop resistance to Leishmania major infection

Aging has been associated with a decline in immunocompetence and resistance to infections, partially due to dysregulated NO production by macrophages and deficits in mounting Th2 cell responses. We wondered if these alterations would reverse the immune response in experimental leishmaniasis. Bone-marrow-derived macrophages from 2- and 18-month-old (senescent) C57BL/6 or BALB/c mice showed no marked difference in leishmanicidal functions. In vivo infections of resistant C57BL/6 mice with Leishmania major revealed no difference between senescent and young mice. However, among susceptible BALB/c mice, senescent animals showed less foot-pad swelling than young mice, and 40 to 60% of them even showed healing of ulcers, reduced parasite dissemination, and a Th1 cell response. These changes were associated with a spontaneous release of interleukin-12 (IL-12) by macrophages from aged but not from young mice. Since exogenous microbial stimulation can influence immune responses during aging, we also infected senescent mice who were raised under specific-pathogen-free (SPF) conditions. They showed neither resistance nor a Th1 response, but their macrophages still spontaneously released IL-12. A microbiological analysis showed that conventionally kept mice, but not SPF mice, had experienced infection with murine hepatitis virus (MHV), an infection associated with a Th1-like response. We conclude that for the reversal of the immune response, senescence is the premier requirement but needs to be completed by another mandatory event such as microbial stimulation. One of the age-related, but not environment-related, factors is the spontaneous release of IL-12 by macrophages, while confrontation with MHV presents an environment-related difference, with both having the potential to support a Th1 response.     

Protective effect of isoprinosine in genetically susceptible BALB/c mice infected with Leishmania major.

The effects of an immunopotentiating drug Inosine Pranobex (isoprinosine) were investigated in an experimental cutaneous leishmaniasis model. The highly susceptible BALB/c mice treated orally with isoprinosine developed significantly delayed onset of disease when infected with Leishmania major compared to untreated mice. The drug itself is not toxic to the parasite up to millimolar levels in vitro. The increase in resistance to L. major infection is accompanied by a marked decrease in the CD4+/CD8+ ratio and the leishmanial antigen-specific proliferative response of the spleen cells of isoprinosine-treated mice compared to untreated mice. There was a significant increase in the production of IFN-gamma but a decrease in the secretion of IL-3 and IL-4 by the spleen cells of isoprinosine-treated mice in response to concanavalin A with or without L. major infection compared to untreated controls. There was, however, no significant difference in the level of IL-2 production by the spleen cells between mice with or without isoprinosine treatment. These data are consistent with the interpretation that isoprinosine potentiates the resistance to leishmanial infection by up-regulating the host-protective Th1 cells and down-regulating the disease-promoting Th2 cells or, alternatively, by increasing CD8+ T-cell function.     

Immunization with Leishmania major exogenous antigens protects susceptible BALB/c mice against challenge infection with L. major

The potential of Leishmania major culture-derived soluble exogenous antigens (SEAgs) to induce a protective response in susceptible BALB/c mice challenged with L. major promastigotes was investigated. Groups of BALB/c mice were immunized with L. major SEAgs alone, L. major SEAgs coadministered with either alum (aluminum hydroxide gel) or recombinant murine interleukin-12 (rmIL-12), L. major SEAgs coadministered with both alum and rmIL-12, and L. major SEAgs coadministered with Montanide ISA 720. Importantly and surprisingly, the greatest and most consistent protection against challenge with L. major was seen in mice immunized with L. major SEAgs alone, in the absence of any adjuvant. Mice immunized with L. major SEAgs had significantly smaller lesions that at times contained more than 100-fold fewer parasites. When lymphoid cells from L. major SEAg-immunized mice were stimulated with leishmanial antigen in vitro, they proliferated and secreted a mixed profile of type 1 and type 2 cytokines. Finally, analyses with Western blot analyses and antibodies against three surface-expressed and secreted molecules of L. major (lipophosphoglycan, gp46/M2/PSA-2, and gp63) revealed that two of these molecules are present in L. major SEAgs, lipophosphoglycan and the molecules that associate with it and gp46/M2/PSA-2.     

Intravenous injection of irradiated Leishmania major into susceptible BALB/c mice: immunization or protective tolerance

It is well established that BALB/c mice can be protected fromfatal infection with Leishmania major by prophylactic intravenous(i.v.) immunization with irradiated parasites. Protection iscritically dependent on the route of injection with i.v. injectionbeing protective and subcutaneous injection not protective.We used this BALB/c-L major model system to investigate thisphenomenon. We analyzed quantitatively the parasite-specific,CD4⁺ T cell mediated immune responses by limiting dilution.Subcutaneous vaccination resulted in priming of CD4⁺ precursorT cells, whereas i.v. vaccination was ineffectual. Moreover,i.v. injection prevented the increase in the number of specificprecursor cells induced by infection of normal mice during thefirst weeks post-challenge with virulent parasites. We showhere that this was not due to the elimination of the virulentchallenge parasites as a result of immunity nor to inefficientantigen presentation of the irradiated organisms after i.v.injection, The data presented here suggest that i.v. injectionresults in tolerizatlon rather than immunization. Tolertzationas a mechanism of host protection is consistent with earlierobservations that transient immunosuppresslon results in cureof L. major infection in BALB/c mice. Transfer of antigen presentingcells (APC) isolated from spleens of mice injected previouslywith irradiated parasites mimicked to some extent the effectof i.v. immunization with irradiated parasites. The possibleinvolvement of these APC in decreasing the parasite-specificT cell response is discussed.     

Effect of glucan on Leishmania major infection in BALB/c mice

The effect of glucan on Leishmania major infection was studied in BALB/c mice, which are highly susceptible to leishmania infection. Glucan (0.45 mg), or isovolumetric dextrose, was administered intraperitoneally 7, 5, 3 and 1 day before infection with L. major promastigotes. At 3, 5, 6, 8 and 10 weeks after infection, animals were killed; the liver and spleen of each animal were weighed and the parasite burden was calculated. A significant (p less than 0.01) reduction in amastigote proliferation in liver and spleen of animals pretreated with glucan was demonstrated 4, 6 and 8 weeks after infection.     

Vaccination of BALB/c mice with Leishmania major amastigote-specific cysteine proteinase

Cellular immune mechanisms resulting in interferon-gamma (IFN-gamma) production are essential for protection against cutaneous leishmaniasis. Antigens of the intracellular amastigote form of the parasite, found in mammalian hosts, are likely to be good candidates for the induction of T cell response and protection from development of leishmaniasis. We purified a stage-specific antigen from amastigote soluble antigen (A-SLA) of Leishmania major by immunoaffinity chromatography. The purified protein was characterized as a cysteine proteinase with enzymatic activity which is inhibited by E-64, and it was named the amastigote cysteine proteinase (ACP). BALB/c mice were immunized by two intraperitoneal injections, at a month interval, of 5 microg of ACP or A-SLA in Freund's complete adjuvant (FCA). Animals were challenged 4 weeks later with 106 L. major promastigotes and examined 4 months after the last injection. The immunized animals developed significantly smaller or no lesions compared with controls. Spleen cells from immunized mice showed a significant proliferative response and produced a high level of IFN-gamma in response to ACP, suggesting the induction of Th1 cells after immunization. These results make 24-kD ACP a possible component for an eventual cocktail vaccine against L. major infection.     

Vaccination with plasmid DNA encoding TSA/LmSTI1 leishmanial fusion proteins confers protection against Leishmania major infection in susceptible BALB/c mice

We have recently shown that a cocktail containing two leishmanial recombinant antigens (LmSTI1 and TSA) and interleukin-12 (IL-12) as an adjuvant induces solid protection in both a murine and a nonhuman primate model of cutaneous leishmaniasis. However, because IL-12 is difficult to prepare, is expensive, and does not have the stability required for a vaccine product, we have investigated the possibility of using DNA as an alternative means of inducing protective immunity. Here, we present evidence that the antigens TSA and LmSTI1 delivered in a plasmid DNA format either as single genes or in a tandem digene construct induce equally solid protection against Leishmania major infection in susceptible BALB/c mice. Immunization of mice with either TSA DNA or LmSTI1 DNA induced specific CD4(+)-T-cell responses of the Th1 phenotype without a requirement for specific adjuvant. CD8 responses, as measured by cytotoxic-T-lymphocyte activity, were generated after immunization with TSA DNA but not LmSTI1 DNA. Interestingly, vaccination of mice with TSA DNA consistently induced protection to a much greater extent than LmSTI1 DNA, thus supporting the notion that CD8 responses might be an important accessory arm of the immune response for acquired resistance against leishmaniasis. Moreover, the protection induced by DNA immunization was specific for infection with Leishmania, i.e., the immunization had no effect on the course of infection of the mice challenged with an unrelated intracellular pathogen such as Mycobacterium tuberculosis. Conversely, immunization of BALB/c mice with a plasmid DNA that is protective against challenge with M. tuberculosis had no effect on the course of infection of these mice with L. major. Together, these results indicate that the protection observed with the leishmanial DNA is mediated by acquired specific immune response rather than by the activation of nonspecific innate immune mechanisms. In addition, a plasmid DNA containing a fusion construct of the two genes was also tested. Similarly to the plasmids encoding individual proteins, the fusion construct induced both specific immune responses to the individual antigens and protection against challenge with L. major. These results confirm previous observations about the possibility of DNA immunization against leishmaniasis and lend support to the idea of using a single polygenic plasmid DNA construct to achieve polyspecific immune responses to several distinct parasite antigens.     

Leishmania tarentolae secreting the sand fly salivary antigen PpSP15 confers protection against Leishmania major infection in a susceptible BALB/c mice model

Cutaneous leishmaniasis is a zoonotic, vector-borne disease causing a major health problem in several countries. No vaccine is available and there are limitations associated with the current therapeutic regimens. Immune responses to sand fly saliva have been shown to protect against Leishmania infection. A cellular immune response to PpSP15, a protein from the sand fly Phlebotomus papatasi, was sufficient to control Leishmania major infection in mice. This work presents data supporting the vaccine potency of recombinant live non-pathogenic Leishmania (L.) tarentolae secreting PpSP15 in mice and its potential as a new vaccine strategy against L. major. We generated a recombinant L. tarentolae-PpSP15 strain delivered in the presence of CpG ODN and evaluated its immunogenicity and protective immunity against L. major infection in BALB/c mice. In parallel, different vaccination modalities using PpSP15 as the target antigen were compared. Humoral and cellular immune responses were evaluated before and at three and eight weeks after challenge. Footpad swelling and parasite load were assessed at eight and eleven weeks post-challenge. Our results show that vaccination with L. tarentolae-PpSP15 in combination with CpG as a prime-boost modality confers strong protection against L. major infection that was superior to other vaccination modalities used in this study. This approach represents a novel and promising vaccination strategy against Old World cutaneous leishmaniasis.     

Previous exposure to a low infectious dose of Leishmania major exacerbates infection with Leishmania infantum in the susceptible BALB/c mouse

The geographic distribution of Leishmania major overlaps with several other species of Leishmania. This study seeks to examine what effect previous exposure to L. major has on the outcome of infection with Leishmania infantum, the agent of virulent visceral leishmaniasis. The L. major immune response is well characterized by a strong Th1 response leading to resolution and protection against subsequent re-infection. A contrasting Th2 immune response leads to disseminated disease, while the role Th17 cytokines may play in Leishmania infection is still being explored. The cytokine profile, antibody titer, and parasite burden were evaluated in the susceptible BALB/c mouse after L. infantum infection in either naïve mice or those previously infected with a low/self-healing dose of L. major. Only IL-4 expression in mice previously exposed to L. major was found to be significantly increased over controls, a cytokine with an ambiguous role in L. infantum infection. However, disease exacerbation, with a notably higher parasite burden, was observed in the L. major exposed mice compared to the L. infantum only. Cross-reactive antibodies were seen in both groups of infected mice regardless of their immune history. Studies have shown a role for opsonizing antibodies leading to increased disease in visceral leishmaniasis. We speculate that cross-reactive antibodies may be playing a role in augmenting visceral disease in mice with immunological memory to L. major.     

T cells that react to the immunodominant Leishmania major LACK antigen prevent early dissemination of the parasite in susceptible BALB/c mice

Susceptibility of BALB/c mice to Leishmania major depends on the early production of IL-4 by CD4(+) T cells which react to the parasite LACK antigen. Here, we show that LACK-specific cells are rapidly recruited to the site of infection and favor the early dissemination of L. major to the internal organs.     

An avirulent lipophosphoglycan-deficient Leishmania major clone induces CD4+ T cells which protect susceptible BALB/c mice against infection with virulent L. major.

An avirulent clone of Leishmania major was used to immunize susceptible BALB/c mice against challenge with virulent L. major. By using the immunized animals as a source of cells, CD4+ parasite-specific T-cell lines could be generated in vitro which, when adoptively transferred to naive BALB/c recipients, conferred marked protection against challenge with virulent L. major. Compared with CD4+ parasite-specific T-cell lines generated from nonimmunized BALB/c mice infected with L. major, the protective T-cell lines generated from immunized mice produced substantially less interleukin-4 and substantially more tumor necrosis factor and interleukin-2. Interestingly, the protective CD4+ T cells did not mediate L. major-specific delayed-type hypersensitivity in vivo and proliferated in vitro only in response to living L. major and not to frozen-and-thawed antigen preparations of the parasite. Finally, the avirulent clone of L. major was found to express the major surface glycolipid of L. major, lipophosphoglycan, at a level that was sixfold less than expression of this molecule by virulent L. major. In addition, lipophosphoglycan of the avirulent parasite failed to mature into the larger, or metacyclic, form of the molecule.     

Combined treatment with interleukin-12 and indomethacin promotes increased resistance in BALB/c mice with established Leishmania major infections

Following infection of susceptible BALB/c mice with Leishmania major, early production of interleukin-4 (IL-4) is associated with the development of a nonprotective Th2 response and the development of progressive disease. Treatment of mice with IL-12 at the time of infection can promote the activation of a protective Th1 response; however, IL-12 treatment of mice with established infections has little effect on the progress of lesion development. This may be due to a down-regulation of the IL-12 receptor beta2 chain (IL-12Rbeta2) that accompanies the expansion of IL-4-producing Th2 cells. We have examined whether prostaglandins function to regulate in vivo responsiveness to IL-12. Mice treated with indomethacin are responsive to treatment with exogenous IL-12 through at least the first 2 weeks of infection and, unlike control mice treated with IL-12, develop an enhanced Th1-type response associated with increased enhanced resistance to infection. Cells from indomethacin-treated mice also exhibit enhanced production of gamma interferon (IFN-gamma) following in vitro stimulation with IL-12. Although in vivo indomethacin treatment did not appear to influence IL-12 production in infected mice, cells from indomethacin-treated mice did express higher levels of IL-12Rbeta2, suggesting that prostaglandins may play a role in the loss of IL-12 responsiveness observed during nonhealing L. major infections.     

Depletion of interleukin-4 in BALB/c mice with established Leishmania major infections increases the efficacy of antimony therapy and promotes Th1-like responses.

Whereas most inbred mouse strains mount a protective Th1 helper T-cell response following infection with Leishmania major, an ineffective Th2 response develops in BALB/c mice, leading to the development of disseminated, ultimately fatal disease. Interleukin-4 (IL-4) production is required for the initiation of the Th2 response, though little is known about the requirements for the long-term maintenance of this response. In order to investigate the role of the expanding parasite population on the Th2 response, mice infected for 2 weeks with L. major, which exhibited a Th2-like cytokine profile, were treated with a leishmanicidal agent (Pentostam) and/or various doses of anti-IL-4 antibody. Untreated mice, mice treated with Pentostam alone, or mice treated with 2.5 mg of anti-IL-4 antibody given at days 13 and 21 of infection developed progressive disease. However, in 8 of 10 mice treated with this dose of anti-IL-4 antibody plus Pentostam lesion development was arrested and lesions were either controlled or eventually healed. Healing was associated with the production of high levels of gamma interferon by spleen cells, and low levels of immunoglobulin E in serum compared with levels for control animals, indicating that a Th1-like response had developed in mice receiving both treatments. Thus, depletion of IL-4 only in combination with a reduction in the parasite burden allowed the expression of a Th1 response. When the dose of anti-IL-4 antibody was increased to 5 mg per injection, all mice treated with this dose of antibody, with or without Pentostam therapy, healed. However, combined therapy with Pentostam in mice treated with this dose of antibody had an additional protective effect. As expected, a Th1 response developed in mice treated with this dose of anti-IL-4 antibody with or without combined therapy with Pentostam, whereas a Th2 response developed in control mice. Thus, a significant effect on the course of disease is noted when mice with established L. major infections are treated with anti-IL-4 antibody in combination with Pentostam, suggesting that the combined effect of inhibiting IL-4 and reducing the parasite burden has a dramatic effect on the development of resistance to L. major.     

gp63 in stable cationic liposomes confers sustained vaccine immunity to susceptible BALB/c mice infected with Leishmania donovani

Visceral leishmaniasis is deadly if not treated, and development of a vaccine with long-term immunity remains a challenge. In this study, we showed that cationic distearoyl phosphatidylcholine (DSPC) liposomes, when used as vaccine adjuvant with the immunodominant 63-kDa glycoprotein (gp63) of Leishmania donovani promastigotes, induced significant protection against progressive visceral leishmaniasis in susceptible BALB/c mice. gp63 used without adjuvant elicited partial protection but in association with liposomes exhibited marked resistance in both the livers and spleens of the mice challenged 10 days after the last vaccination. The protective efficacy of liposomal gp63 vaccination was dose dependent, with 2.5 μg of protein showing optimal protection. The immunity conferred by this vaccine formulation was durable, as mice challenged 12 weeks after immunization were still protected, and the infection was controlled for at least 3 months postchallenge. Production of gamma interferon (IFN-γ) and interleukin-4 (IL-4) by splenic T cells, and of serum immunoglobulin G1 (IgG1) and IgG2a following immunization, suggested that a mixed Th1/Th2 response had been induced following immunization. However, control of disease progression and parasitic burden in mice vaccinated with gp63 in cationic DSPC liposomes was associated with enhancement of antigen-specific IFN-γ and downregulation of IL-4, demonstrating a Th1 bias. Long-term immunity elicited by this vaccine corresponded to, in addition to the presence of antigen-specific Th1, CD8⁺ T-cell responses. Our results demonstrated that stable cationic liposomes containing gp63 acted as a potent adjuvant for protein antigen to induce long-term protection against L. donovani that represents an alternative to DNA vaccination.     

Overexpression of miniexon gene decreases virulence of Leishmania major in BALB/c mice in vivo

During the construction of a physical map for Leishmania major (LV39) chromosome 2 we have rescued and characterized a L. major (LV39) derived genomic clone bearing solely as insert a long stretch of the miniexon gene array. The recombinant was devised as a tool to study the effect of miniexon overexpression on virulence and growth advantage. Such clone, 32D05, contains approximately 40 kb of the miniexon tandem array. We have examined the course of infection in susceptible BALB/c mice inoculated with transfectants carrying 32D05 as an episome. The study was carried out in two different clonal lines of L. major: virulent line LV39 (clone 5) and avirulent LT252 (CC1 clone). The results presented here indicate that high levels of miniexon expression affect negatively the ability of once virulent lines to induce lesions when injected in susceptible mice.     

IL-4 depletion enhances host resistance and passive IgA protection against tuberculosis infection in BALB/c mice

The influence of Th2 cytokines in tuberculosis has been a matter of dispute. Here we report that IL-4 has a profound regulatory effect on the infection of BALB/c mice with Mycobacterium tuberculosis. Depletion of IL-4 with a neutralizing mAb caused only evanescent reduction of lung infection, but when combined with i.n. inoculations of IgA anti-mycobacterial alpha-crystallin mAb and mouse rIFN-gamma, we observed a 40-fold reduction of the bacterial counts in the lungs at 3 wks following i.n. infection (p<0.001). In genetically deficient IL-4-/- BALB/c mice, infection in both lung and spleen was substantially reduced for up to 8 wks without further treatment. Reconstitution of IL-4-/- mice with rIL-4 increased bacterial counts to wild-type levels and made the mice refractory to protection by IgA/IFN-gamma. Analysis of the lungs showed increased granulomatous infiltration and proinflammatory mediators in anti-IL-4/IgA/IFN-gamma-treated and infected mice. We conclude that the action of IL-4 in tuberculosis is targeted at macrophages and that it may include an antagonistic effect on their IgA/IFN-gamma-induced activation and nitric oxide production. The described novel immunotherapy, combining treatments with anti-IL-4, IgA antibody and IFN-gamma, has potential for translation toward the passive immunoprophylaxis of tuberculosis.     

Characterization of Leishmania major antigen-liposomes that protect BALB/c mice against cutaneous leishmaniasis.

Leishmania major antigen-liposomes prepared as dehydration-rehydration vesicles (DRV) and composed of equimolar amounts of L-alpha-distearoyl phosphatidylcholine and cholesterol confer high-level host-protective immunity against virulent homologous challenge to susceptible BALB/c mice. Physical and antigenic characterization of these protective liposomes is described. Both empty and L. major antigen-DRV were multilamellate and heterogeneous in size, ranging from 0.10 to 2.00 microns. Although the liposomes were made by using a crude mixture of promastigote antigens, lipophosphoglycan covered the liposome surface; this was demonstrated by immunogold electron microscopy. Application of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis revealed preferential entrapment of the 63-kilodalton promastigote protease (gp63) into the DRV. We suggest that our L. major antigen-DRV merit further study because of their preferential entrapment of these two host protective antigens together with their long in vivo half-life. In addition, this report illustrates that intravenous or subcutaneous immunization of BALB/c mice with the same limited subset of protective antigens, predominantly lipophosphoglycan and gp63, within DRV liposomes leads to either protection and low splenic interleukin-3 production or to nonprotection and high splenic interleukin-3 production, respectively. This was consistent with our hypothesis that differential antigen presentation after administration of the same immunogen by the intravenous or the subcutaneous route results in differential T-cell activation.