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.     

Vaccination with the Leishmania infantum acidic ribosomal P0 protein plus CpG oligodeoxynucleotides induces protection against cutaneous leishmaniasis in C57BL/6 mice but does not prevent progressive disease in BALB/c mice

We have examined the efficacy of the administration in mice of a molecularly defined vaccine based on the Leishmania infantum acidic ribosomal protein P0 (rLiP0). Two different challenge models of murine cutaneous leishmaniasis were used: (i) subcutaneous inoculation of L. major parasites in susceptible BALB/c mice (a model widely used for vaccination analysis) and (ii) the intradermal inoculation of a low infective dose in resistant C57BL/6 mice (a model that more accurately reproduces the L. major infection in natural reservoirs and in human hosts). First, we demonstrated that C57BL/6 mice vaccinated with LiP0-DNA or rLiP0 protein plus CpG oligodeoxynucleotides (ODN) were protected against the development of dermal pathology and showed a reduction in the parasite load. This protection was associated with production of gamma interferon (IFN-γ) in the dermal site. Secondly, we showed that immunization with rLiP0 plus CpG ODN is able to induce only partial protection in BALB/c, since these mice finally developed a progressive disease. Further, we demonstrated that LiP0 vaccination induces a Th1 immunological response in both strains of mice. In both cases, the antibodies against LiP0 were predominantly of the immunoglobulin G2a isotype, which was correlated with an rLiP0-stimulated production of IFN-γ in draining lymph nodes. Finally, we demonstrated that LiP0 vaccination does not prevent the Th2 response induced by L. major infection in BALB/c mice. Taken together, these data indicate that the BALB/c model of cutaneous leishmaniasis may undervalue the potential efficacy of some vaccines based on defined proteins, making C57BL/6 a suitable alternative model to test vaccine candidates.     

Immunization with Leishmania donovani protein disulfide isomerase DNA construct induces Th1 and Th17 dependent immune response and protection against experimental visceral leishmaniasis in Balb/c mice

In the present study, the efficacy of Leishmania donovani protein disulfide isomerase (LdPDI) as a DNA vaccine was evaluated in BALB/C mice. Mice immunized with the LdPDI-DNA construct were found to be the most immuno-reactive, as the construct induced higher T-cell proliferation. The increased T-cell proliferation was associated with a substantial rise in Th1 and Th17+ CD4 cell response and triggered a higher proportion of CD8+ T cells for the release of interferon-gamma along with a reduced splenic parasite load on Days20 and 60 post challenge (PC). Furthermore, the vaccine construct triggered increased interferon (IFN)-γ, interleukin(IL)-17A, and IL-22 release accompanied by decreased extracellular signal-regulated kinases (ERK) 1/2 signaling and increased mitogen-activated protein kinase (MAPK) signaling coinciding with an increase in the amount of nitrite and reactive oxygen species (ROS)in vaccinating the splenocyts. We summarize from our data that the PDI-DNA construct of Leishmania donovani has the potential to elicit protective immunity through the pro-inflammatory cytokines of CD8+ and CD4+(Th1 and Th17) following an intervention in the downstream signaling event of ERK1/2 (probably through p38MAPK signaling). Therefore, the study suggests a new control against visceral leishmaniasis in the future.     

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.     

Immune induction by adjuvanted Leishmania donovani vaccines against the visceral leishmaniasis in BALB/c mice

Visceral leishmaniasis (VL) is a neglected tropical disease caused by Leishmania donovani or Leishmania infantum. Currently, the patients are treated with chemotherapeutic drugs; however, their toxicity limits their use. It would be desirable to develop a vaccine against this infection. In this study, we assessed the efficacy of different vaccine formulations at variable time points. Heat-killed (HK) antigen of Leishmania donovani was adjuvanted with two adjuvants (AddaVax and Montanide ISA 201) and three immunizations at a gap of 2 weeks (wk) were given to BALB/c mice. After 2 weeks of the last booster, mice were given challenge infection and sacrificed before challenge and after 4wk, 8wk, and 12 wk post-challenge. Significant protective immunity was observed in all the immunized animals and it was indicated by the notable rise in delayed-type hypersensitivity (DTH) response, remarkably declined parasite burden, a significant increase in the levels of interferon-gamma (IFN-γ), interleukin-12, interleukin-17 (Th1 cytokines), and IgG2a in contrast to infected control mice. Montanide ISA 201 with HK antigen provided maximum protection followed by AddaVax with HK and then HK alone. These findings elaborate on the importance of the tested adjuvants in the vaccine formulations against murine visceral leishmaniasis.     

Induction of host protective Th1 immune response by chemokines in Leishmania donovani-infected BALB/c mice

The resolution from leishmanial infection is dependent on the coordinated interactions between the components of the cell mediated immune system and the activation of T-cell population into appropriate cytokine production and the activation of macrophages. Earlier reports established that C-C chemokines particularly macrophage inflammatory protein (MIP)-1alpha and macrophage chemoattractant protein (MCP)-1 restrict the parasitic burden via the regulation of impaired protein kinase C (PKC) signalling and induction of free-radical generation in murine leishmaniasis. This study explored the role of MIP-1alpha and MCP-1 in the induction of T helper 1 (Th1) immune response and suppression of T helper 2 (Th2) response in Leishmania donovani-infected BALB/c mice. These chemokines induced the known pro-inflammatory cytokine interleukin (IL)-12 secretion and inhibited the secretion of anti-inflammatory cytokines IL-10 and transforming growth factor-beta in infected macrophages. Impaired antigen presentation capability of infected macrophages was also restored by the chemokine treatment. C-C chemokine treatment resulted in reduced levels of mRNA expression of IL-10, but increased levels of mRNA expression of IL-12p40, interferon (IFN)-gamma, tumour necrosis factor-alpha and inducible nitric oxide synthase in both liver mononuclear cells as well as in splenocytes, reflecting a switch of CD4+ differentiation from Th2 to Th1. Flow cytometric analysis of infected spleen cells suggested that C-C chemokine treatment enhances the CD4+ T cells to produce increased levels of IFN-gamma. These studies hypothesize a promising immuno-prophylactic effect of chemokines against leishmaniasis by induction of Th1 cytokine release imparting a long-term resistance.     

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.     

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.     

BALB / c 小鼠免疫重组相思子毒素B 链蛋白的免疫应答

目的:评价重组相思子毒素B 链蛋白(rATB)免疫雌性BALB/ c 小鼠的免疫效果,并初步探讨其免疫机制。方法:以rATB 为免疫原,腹腔免疫雌性BALB/ c 小鼠,间接ELISA 方法检测小鼠血清中IgG、IgG1 、IgG2a,流式细胞技术检测IFN-γ、IL-4,并进行天然毒素攻毒实验和体外中和实验。结果:血清IgG、IgG1 效价达到1 ‘106 以上,且攻毒后产生二次免疫应答,与PBS 对照组差别有统计学意义(P<0.05);而IgG2a的血清效价未发生明显变化。细胞因子检测结果表明IFN-γ的表达量与PBS 组差异无统计学意义,而IL-4 的表达量显著提高(P<0.05)。小鼠攻毒保护实验和体外中和实验结果说明,腹腔注射rATB 可以抵抗最高剂量为5‘LD50 的天然AT 毒素中毒,保护率为100%。结论:rATB 蛋白免疫小鼠后显示出良好的免疫原性,能诱导以IgG1 为主的抗体和Th2 优势应答,为候选疫苗的研制奠定基础。     

Vaccine-induced immunity against cutaneous leishmaniasis in BALB/c mice.

Partially purified antigens, derived from Leishmania infantum or L. major promastigotes and isolated under reducing conditions, were used to immunize BALB/c mice. Three subcutaneous injections of the 64- to 97-kilodalton preparation in conjunction with muramyl dipeptide conferred long-lasting immunity against L. mexicana subsp. mexicana and L. major infection; they led to the development of antibodies neutralizing the infectiousness of promastigotes, induced specific delayed-hypersensitivity reactions, and generated populations of peritoneal macrophages capable of killing amastigotes. Vaccination resulted in no harmful effects, since these antigen neither exacerbated preexisting Leishmania infection nor impeded the formation of antibodies to other antigens administered concomitantly.     

Specific immune response and pathological findings in BALB/c mice inoculated with recombinant BCG expressing HIV-1 antigen

Recombinant BCGs (rBCGs) containing extrachromosomal plasmids with different HIV-1 insert sequences: nef, env (V3J1 and E9Q), gag p17 or whole gag p55 were evaluated for their immunogenicity, safety and persistent infection in BALB/c mice. Animal injected with, rBCG-plJKV3J1, rBCG-pSO gag p17 or rBCG-pSO gag p55 could elicit lymphocyte proliferation as tested by specific HIV-1 peptides or protein antigen. Inoculation with various concentration of rBCG-pSO gag p55 generated satisfactory specific lymphocyte proliferation in dose escalation trials. The rBCG-pSO gag p55 recovered from spleen tissues at different time interval post-inoculation could express the HIV protein as determined by ELISA p24 antigen detection kit. This result indicated that the extrachromosomal plasmid was stable and capable to express Gag protein. It was also demonstrated that rBCGs did not cause serious pathological change in the inoculated animals. The present study suggested the role of BCG as a potential vehicle for using in HIV vaccine development.     

Pretreatment with recombinant Flt3 ligand partially protects against progressive cutaneous leishmaniasis in susceptible BALB/c mice

Dendritic cells are potent antigen-presenting cells that also produce interleukin-12 (IL-12) during innate and adaptive cellular immune responses and that thereby promote the differentiation of gamma interferon (IFN-gamma)-producing Th1-type CD4(+) T lymphocytes. We hypothesized that expanded dendritic-cell populations in mice pretreated with the hematopoietic cytokine Flt3L would protect against cutaneous Leishmania major infection. Pretreatment of disease-susceptible BALB/c mice with 10 microg of recombinant Flt3L (rFlt3L) for 9 to 10 days before infection increased lymph node IL-12 p40 productive capacity 20-fold compared to that of saline-injected controls. Furthermore, 9 of 22 (40.9%) rFlt3L-pretreated BALB/c mice resolved their cutaneous infections, whereas none of the 22 control BALB/c mice healed. Healed, rFlt3L-pretreated mice did not develop disease following reinfection. Flt3L pretreatment also reduced parasite numbers 1,000-fold in the cutaneous lesions at 2 weeks after infection relative to numbers in lesions of untreated controls. However, Flt3L pretreatment did not significantly alter L. major-induced IFN-gamma and IL-4 production in lymph node culture at 1, 2, and 4 weeks after infection. Despite the lack of Th immune deviation, Flt3L ligand-pretreated lymph nodes expressed up to 10-fold higher levels of IL-12 p40 and inducible (type 2) nitric oxide synthase mRNA at 7 days after infection. In contrast, treatment with rFlt3L after infection failed to protect against disease despite comparable expansions of dendritic cells and IL-12 p40 productive capacity in both infected and uninfected BALB/c mice treated with rFlt3L. We conclude that rFlt3L pretreatment before infection with L. major reduces parasite load and promotes healing of cutaneous lesions without stable cytokine deviation towards a dominant Th1 cytokine phenotype.     

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.     

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.     

Modulation of the immune response of BALB/C mice against Leishmania Major by imuvert

This study investigated the effect of Imuvert, a biological response modifier derived from Serratia marcescens, on the progression of Leishmania major infection in Balb/c mice.A single 100 μg Imuvert injection was significantly protective in Balb/c mice when challenged 28 days later in the footpad with 5 × 10⁵ stationary phase L. major promastigotes. Intraperitoneal (i.p.) and intravenous (i.v.) immunization of mice with heat-killed stationary phase L. major promastigotes significantly reduced lesion development following challenge with L. major promastigotes. Subcutaneous (s.c.) immunization had no protective effect. A single 100 μg Imuvert injection significantly reduced lesion development in s.c. immunized mice, but had a lesser effect in mice immunized by i.p. and i.v. routes. Balb/c mice receiving four Imuvert injections 14, 7, 2 and 1 day prior to footpad challenge with L. major promastigotes were not protected, but rather displayed significant exacerbation of infection.Our results suggest the possibility that Imuvert could be useful in stimulating a protective response against L. major when given along with s.c. vaccine, a realistic route for vaccinating humans in contrast to either i.v. or i.p. routes. Since the protective response in Balb/c mice against L. major is dependent on the stimulation of T_hl cells, it is suggested that the observed adjuvant effect of Imuvert given with s.c. vaccine perhaps is due to changes in immunological responses in such a direction.     

Priming of a beta-galactosidase (beta-GAL)-specific type 1 response in BALB/c mice infected with beta-GAL-transfected Leishmania major

To determine whether an ongoing response to Leishmania major would affect the response to a non-cross-reacting, non-leishmanial antigen, susceptible BALB/c mice and resistant C3H mice were infected with L. major parasites expressing Escherichia coli beta-galactosidase (beta-GAL); this parasite was designated L. major-betaGAL. BALB/c and C3H mice responded to infection with L. major-betaGAL by mounting a CD4 T-cell response to both parasite antigens and to the reporter antigen, beta-GAL. The phenotypes of these T cells were characterized after generating T-cell lines from infected mice. As expected, BALB/c mice responded to infection with L. major-betaGAL by producing interleukin 4 in response to the parasite and C3H mice produced gamma interferon (IFN-gamma) in response to the parasite and beta-GAL. Interestingly, however, BALB/c mice produced IFN-gamma in response to beta-GAL. Taken together, these results demonstrate that priming of IFN-gamma-producing cells can occur in BALB/c mice despite the fact the animals are simultaneously mounting a potent Th2 response to L. major.     

Humoral immune response of BALB/c mice immunized with chimer HBcAg proteins carrying the epitopes of surface hepatic B virus protein

Chimeric HBcAg proteins carrying epitopes from surface hepatitis B virus (HBV) protein (regions 137-147 a.o. HBsAg, 27-37 a.a. region preS1 and 131-145 a.a. region preS2) have been early constructed. This paper presents the data of an investigation of a humoral immune response in mice immunized with obtained by chimeric HBcAg proteins. The findings suggest that the chimeric HBcAg proteins carrying the epitopes of surface HBV protein are able to induce an immune response to both inserted epitopes and carrying protein (HBcAg). Immunization with a mixture of chimeric proteins taken in equivalent quantities induces the synthesis of antibodies to hybrid proteins. The use of aluminum hydroxide considerably enhances a humoral immune response during immunization with chimeric bovine proteins.     

BALB / c 小鼠免疫重组蓖麻毒素B 链蛋白的免疫机制研究

目的:探讨重组蓖麻毒素B 链蛋白(rRTB)免疫雌性BALB/ c 小鼠的免疫机制。方法:雌性BALB/ c 小鼠分为rRTB 组和PBS 组,间隔14 d 皮下多点注射,共4 次。间接ELISA 分析小鼠血清抗体效价和抗体分型,采用流式细胞仪检测脾细胞因子IFN 和IL-4。结果:血清IgG 效价达到1 107 以上,且攻毒后产生二次免疫应答,与PBS 对照组差别有统计学意义(P<0.05);IgG1 达到1 105 以上,与PBS 对照组差别有统计学意义(P<0.05);IgG2a未发生明显变化。细胞因子检测结果表明IFN 的表达量与PBS 组差异无统计学意义,而IL-4 的表达量显著提高(P<0.05)。结论:rRTB 蛋白免疫可刺激小鼠产生高水平的特异性抗体和细胞因子,诱导以IgG1 为主的抗体和Th2 优势应答,为候选疫苗的研制奠定基础。     

Treatment with cathepsin L inhibitor potentiates Th2-type immune response in Leishmania major-infected BALB/c mice

Prior to the activation of CD4 (+) T cells, exogenous proteins must be digested by endo/lysosomal enzymes in antigen-presenting cells (APC) to produce antigenic peptides that are able to be presented on class II molecules of the MHC. Studies described here inspect the functional significance of cathepsin L inhibition for antigen processing and T (h) 1/T (h) 2 differentiation in experimental leishmaniasis. We first demonstrated using in vitro systems that cathepsin L is one of the candidate endo/lysosomal enzymes in processing of soluble Leishmania antigen (SLA) and that its specific inhibitor, CLIK148, modulated the processing of SLA. BALB/c mice are known to be susceptible to infection with Leishmania major. Interestingly, treatment of BALB/c mice with CLIK148 exacerbated the infection by enhancing the development of SLA-specific T (h) 2-type response such as production of IL-4 and generation of T (h) 2-dependent specific IgE/IgG1 antibodies. Moreover, addition of CLIK148 in incubation of a SLA-specific CD4 (+) T cell line with APC up-regulated the production of IL-4. However, CLIK148 did not exert any direct influence on the function of T cells themselves. Taken together, these findings suggest that treatment of host mice with CLIK148 affects the processing of SLA in APC, resulting in the potentiation of T (h) 2-type immune responses and thus leading to exacerbation of the infection. Furthermore, endo/lysosomal cathepsin L was found to be functionally distinct from previously described cathepsins B and D.