SAG1 is a host-targeted antigen for protection against toxoplasma gondii infection.

We previously reported that SAG1 transgenic (tg) mice have an elevated susceptibility resulting from their inability to elicit strong Th1-based protection against Toxoplasma gondii infection. Here, we demonstrate that SAG1 tg mice were protected against T. gondii infection, characterized by a decline in IFN-gamma levels, following administration of a lethal dose of T. gondii. Moreover, immunization with T. gondii homogenate conferred protection and induced production of IgG, with IgG1 and IgG2a subclasses driven by Th2 and Th1 responses, respectively, in both SAG1 tg and wild-type (wt) mice. IgG titers were significantly higher from day 10 after immunization in wt mice compared to those in SAG1 tg mice. There were no significant differences observed in levels of IgG1 in both groups. However, significantly lower IgG2a titers were measured in the sera from SAG1 tg mice on days 10, 15 and 20. IFN-gamma levels in sera were significantly lower in SAG1 tg mice compared to those in wt mice on day 20 after immunization. When challenged with a lethal dose of the Beverley strain of T. gondii, 80 and 100% survival rates were observed in SAG1 tg and wt mice, respectively, indicating that SAG1 tg mice were protected to a lesser extent from challenge due to the decrease in protective immunity. These results suggest that SAG1 plays a critical role in eliciting protection, hence a target antigen for the development of protective Th1-based responses against T. gondii infection in mice.     

Intranasal immunization with SAG1 and nontoxic mutant heat-labile enterotoxins protects mice against Toxoplasma gondii

Effective protection against intestinal pathogens requires both mucosal and systemic immune responses. Intranasal administration of antigens induces these responses but generally fails to trigger a strong protective immunity. Mucosal adjuvants can significantly enhance the immunogenicities of intranasally administered antigens. Cholera toxin (CT) and heat-labile enterotoxin (LT) are strong mucosal adjuvants with a variety of antigens. Moreover, the toxicities of CT and LT do not permit their use in humans. Two nontoxic mutant LTs, LTR72 and LTK63, were tested with Toxoplasma gondii SAG1 protein in intranasal vaccination of CBA/J mice. Vaccination with SAG1 plus LTR72 or LTK63 induced strong systemic (immunoglobulin G [IgG]) and mucosal (IgA) humoral responses. Splenocytes and mesenteric lymph node cells from mice immunized with LTR72 plus SAG1, but not those from mice immunized with LTK63 plus SAG1, responded to restimulation with a T. gondii lysate antigen in vitro. Gamma interferon and interleukin 2 (IL-2) production by splenocytes and IL-2 production by mesenteric lymph node cells were observed in vitro after antigen restimulation, underlying a Th1-like response. High-level protection as assessed by the decreased load of cerebral cysts after a challenge with the 76K strain of T. gondii was obtained in the group immunized with LTR72 plus SAG1 and LTK63 plus SAG1. They were as well protected as the mice immunized with the antigen plus native toxins. This is the first report showing protection against a parasite by using combinations of nontoxic mutant LTs and SAG1 antigen. These nontoxic mutant LTs are now attractive candidates for the development of mucosally delivered vaccines.     

脂质体介导弓形虫SAG1与SAG3复合基因诱导小鼠免疫应答研究

目的评价弓形虫SAG1与SAG3基因真核表达质粒DNA免疫小鼠的免疫应答效果。方法以PCR方法扩增出SAG1与SAG3目的基因片段,并插入载体pEGFP-N3以构建重组质粒pE- SAG1／SAG3,瞬时转染细胞Cos-7。质粒pESAG1／SAG3以10μg剂量,脂质体介导肌肉注射BALB／c小鼠,免疫3次,最后一次免疫后4周,观察体液和细胞免疫。RT-PCR方法检测注射部位目的基因的转录;斑点杂交方法检测目的基因是否与小鼠染色体基因组整合。结果质粒pESAG1／SAG3转染细胞后观察到绿色荧光;Western blot显示pESAG1/SAG3表达识别条带在相对分子质量(Mr)66．2×103附近。小鼠免疫后,血清产生抗弓形虫速殖子抗体,诱导IFN-γ及IL-2水平高于对照组。RT-PCR显示首次免疫15 d后,目的基因在肌肉组织中仍有转录,斑点杂交显示目的基因未与小鼠的基因组发生整合,混合质粒注射的小鼠抗弓形虫感染的存活时间延长。结论脂质体介导弓形虫SAG1与SAG3复合编码基因质粒接种小鼠能明显诱导体液和细胞免疫应答。     

Protection against Toxoplasma gondii in mice immunized with Toxoplasma cell fractions, RNA and synthetic polyribonucleotides

Mice immunized with fractions obtained by centrifugation of disrupted Toxoplasma gondii trophozoites as well as with 200 μg of Toxoplasma ribonucleic acid (RNA) were resistant (as measured by time to death and total mortality) to challenge with Toxoplasma 30 days later. When mice were challenged at 15 days no protection was noted. A dose of 50 μg of Toxoplasma RNA was effective in protecting mice against lethal challenge only when incorporated into Freund's incomplete adjuvant. In studies performed to determine the specificity of the resistance observed, resistance was also noted in mice immunized with 200 μg of RNA extracted from normal mouse peritoneal macrophages, as well as in mice immunized with 100 μg of the synthetic polyribonucleotide polycytidylic acid. Polyadenylicuridylic acid conferred protection only when incorporated into Freund's incomplete adjuvant and polyinosinic—cytidylic acid had no effect. The protection induced by Toxoplasma RNA was eliminated by prior treatment of the preparation with ribonuclease but not by treatment with pronase, suggesting that the moiety responsible for the protective effect was RNA. In experiments designed to explore the mechanism of resistance in the vaccinated mice, macrophages harvested from mice which had been injected with Toxoplasma RNA 15 days earlier were found to be activated in that they resisted challenge with Listeria monocytogenes.     

DNA vaccination with genes encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2 induces partially protective immunity against lethal challenge in mice

C57BL/6, C3H, and BALB/c mice were vaccinated with plasmids encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2, previously described as strong inducers of immunity. Seroconversion for the relevant antigen was obtained in the majority of the animals. T. gondii lysate stimulated specific T-cell proliferation and secretion of gamma interferon (IFN-gamma) in spleen cell cultures from vaccinated BALB/c and C3H mice but not in those from control mice. Although not proliferating, stimulated splenocytes from DNA-vaccinated C57BL/6 mice also produced IFN-gamma. No interleukin-4 was detected in the supernatants of lysate-stimulated splenocytes from DNA-vaccinated mice in any of the mouse strains evaluated. As in infected animals, a high ratio of specific immunoglobulin G2a (IgG2a) to IgG1 antibodies was found in DNA-vaccinated C3H mice, suggesting that a Th1-type response had been induced. For BALB/c mice, the isotype ratio of the antibody response to DNA vaccination was less polarized. The protective potential of DNA vaccination was demonstrated in C3H mice. C3H mice vaccinated with plasmid encoding GRA1, GRA7, or ROP2 were partially protected against a lethal oral challenge with cysts of two different T. gondii strains: survival rates increased from 10% in controls to at least 70% after vaccination in one case and from 50% to at least 90% in the other. In vaccinated C3H mice challenged with a nonlethal T. gondii dose, the number of brain cysts was significantly lower than in controls. DNA vaccination did not protect BALB/c or C57BL/6 mice. Our results demonstrate for the first time in an animal model a partially protective effect of DNA vaccination against T. gondii.     

弓形虫复合基因SAG1-BAG1的构建、表达及重组抗原的免疫反应性分析

目的构建并表达弓形虫复合基因SAG1-BAG1及分析重组抗原的免疫反应性。方法利用PCR扩增弓形虫SAG1基因,利用RT-PCR扩增弓形虫BAG1基因,通过酶切连接分别将SAG1、BAG1基因克隆到pET28a表达载体中构建复合基因SAG1-BAG1。将质粒pET28a-SAG1-BAG1转入大肠杆菌BL21（DE3）中进行诱导表达,Western-blot分析该重组蛋白的免疫反应性。结果复合基因SAG1-BAG1全长约1 407 bp,重组蛋白相对分子质量约为50 000,表达量约占菌体总蛋白的10%,Western-blot分析显示重组蛋白具有良好的免疫反应性。结论成功构建并表达弓形虫复合基因SAG1-BAG1。     

Protection against anthrax lethal toxin challenge by genetic immunization with a plasmid encoding the lethal factor protein

The ability of genetic vaccination to protect against a lethal challenge of anthrax toxin was evaluated. BALB/c mice were immunized via gene gun inoculation with eucaryotic expression vector plasmids encoding either a fragment of the protective antigen (PA) or a fragment of lethal factor (LF). Plasmid pCLF4 contains the N-terminal region (amino acids [aa] 10 to 254) of Bacillus anthracis LF cloned into the pCI expression plasmid. Plasmid pCPA contains a biologically active portion (aa 175 to 764) of B. anthracis PA cloned into the pCI expression vector. One-micrometer-diameter gold particles were coated with plasmid pCLF4 or pCPA or a 1:1 mixture of both and injected into mice via gene gun (1 microg of plasmid DNA/injection) three times at 2-week intervals. Sera were collected and analyzed for antibody titer as well as antibody isotype. Significantly, titers of antibody to both PA and LF from mice immunized with the combination of pCPA and pCLF4 were four to five times greater than titers from mice immunized with either gene alone. Two weeks following the third and final plasmid DNA boost, all mice were challenged with 5 50% lethal doses of lethal toxin (PA plus LF) injected intravenously into the tail vein. All mice immunized with pCLF4, pCPA, or the combination of both survived the challenge, whereas all unimmunized mice did not survive. These results demonstrate that DNA-based immunization alone can provide protection against a lethal toxin challenge and that DNA immunization against the LF antigen alone provides complete protection.     

Hematopoiesis during acute Toxoplasma gondii infection in mice

We studied hematopoiesis in bone marrow and blood of CBA mice following infection with Toxoplasma gondii. Our data showed that acute infection with the virulent RH strain was associated with leucopenia, thrombocytopenia and bone marrow hypoplasia while, in spite of the infection-induced damage of the granulocyte cell lineage, in bone marrow stimulated production of granulocytes was revealed. In peripheral blood, T. gondii infection caused a significant decrease in the total number of white blood cells, reticulocytes and platelets. However, the relative proportion of granulocytes and lymphocytes was changed in favor of granulocytes, as compared to pre-infection levels. The functional activity of granulocytes was also increased. The bone marrow alterations were characterized by a decrease in the total number of nucleated cells due to the reduced numbers in all cell compartments of erythroid and megakaryocytic lineage, as well as in the number of mature granulocytes and lymphocytes. In contrast, femoral granulocytic proliferative compartments, colony forming unite granulocyte-macrophage (CFU-GM) and morphologically recognizable proliferative granulocytes (PG), exhibited stimulated granulopoiesis, while the number of mature monocytes was close to the control value. In summary, we have shown that acute T. gondii infection results in profound alterations of the hematopoietic system that markedly contribute to the clinical onset of the disease and the, ultimately lethal, outcome.     

Multicomponent DNA vaccine-encoding Toxoplasma gondii GRA1 and SAG1 primes: anti-Toxoplasma immune response in mice

A multicomponent DNA vaccine, encoding Toxoplasma gondii GRA1 and SAG1, was constructed and tested for its ability to confer protection. BALB/c mice were challenged with tachyzoites of the virulent T. gondii RH strain at 4?weeks following the last immunization, and immune responses and survival times were observed. The results show that vaccination by the multicomponent vaccine prolonged survival of mice challenged with the T. gondii RH strain (from average 4.50?±?0.22 to 7.60?±?0.74?days); induced high levels of IgG antibody (from 0.252?±?0.080 to 0.790?±?0.083), IFN-gamma (from 598.74?±?67.50 to 853.77?±?66.74?pg/ml), and IL-2 (from 89.44?±?10.66 to 192.24?±?19.90?pg/ml); changed the CD4⁺/CD8⁺ lymphocyte ratio (from 1.81?±?0.14 to 1.09?±?0.19); and stimulated NK cell-killing activity (from 46.81?±?3.96 to 64.15?±?7.71?%). These findings demonstrate that a multicomponent DNA vaccine, encoding GRA1 and SAG1, primes a strong humoral and cellular immune response and enhances protection against T. gondii challenge. The new, combined DNA vaccine provides another means to combat T. gondii infection.     

The SAG1 Toxoplasma gondii surface protein is not required for acute ocular toxoplasmosis in mice

The SAG1 Toxoplasma gondii surface protein stimulates acute ileitis. To determine whether SAG1 is also important in the eye, wild-type or SAG1 knockout parasites were injected intravitreally into mice. No differences in retinal damage or parasite growth were observed, indicating that unlike the case for the intestine, factors besides SAG1 are important for retinal damage.     

Regulation of immunoglobulin E production in mice immunized with an extract of Toxoplasma gondii.

Repeated infection with Toxoplasma gondii could not induce immunoglobulin E (IgE) antibody production. When mice were injected intraperitoneally twice over a 3-week interval with an extract of tachyzoites of T. gondii and Al(OH)3 as adjuvant, antitoxoplasma IgE antibody was produced. Antitoxoplasma IgE antibody titers were low and diminished after a short time in B10.S, BALB/c, C3H, and C57BL/6 mice. This tendency was more evident in IgE low-responder SJL mice. IgE-inducing activity of Toxoplasma antigen was weaker than those of keyhole limpet hemocyanin, ovalbumin, and an extract from Nippostrongylus brasiliensis. The antitoxoplasma IgE antibody production was enhanced by and persisted after whole-body irradiation (150 R) following secondary immunization. The enhanced antitoxoplasma IgE antibody production was suppressed by transferring spleen cells from Toxoplasma antigen-immunized mice. The suppressive effect of the spleen cells was Toxoplasma antigen specific and was removed by treatment with anti-Thy-1.2 and complement. These results indicate that the low IgE production induced by Toxoplasma antigen is the result of irradiation-sensitive and antigen-specific suppressor T cells. These findings might explain the lack of IgE antibody response in mice with Toxoplasma 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.     

Use of a recombinant antigen,SAG2, expressed as a glutathione-S-transferase fusion protein to immunize mice against Toxoplasma gondii

 The capacity of Toxoplasma gondii surface protein SAG2 to induce protective immunity against the parasite in mice was studied using recombinant SAG2 expressed as a glutathione-S-transferase (GST) fusion protein incorporated into immune stimulating complexes (iscoms). Immunization with the iscoms resulted in the production of antibodies recognizing SAG2 as well as GST. After oral challenge infection with T. gondii oocysts or tissue cysts, no protective effect was observed. On the contrary, mice immunized with fusion SAG2 or with GST iscoms died earlier than non-immunized control mice. Received: 2 March 1996 / Accepted: 9 July 1996     

Pinocytic rates of macrophages from mice immunized against Toxoplasma gondii and macrophages stimulated to inhibit toxoplasma in vitro.

The rate of pinocytosis by macrophages when measured by uptake of horseradish peroxidase was significantly reduced during toxoplasma infection of the cells in vitro when the macrophages were from toxoplasma-immune mice and when control cells were stimulated in vitro to inhibit toxoplasma multiplication. There was, however, no direct correlation between reduced pinocytosis in this model and inhibition or enhancement of toxoplasma multiplication. We conclude that a reduced pinocytic rate is a feature of the unstimulated toxoplasma-immune macrophage, but this change in rate alone does not correlate with the cell's ability to inhibit toxoplasma. In addition, we observed that enhanced pinocytosis as seen in the elicited macrophage was not a requirement for inhibition of toxoplasma multiplication.     

Toxoplasma gondii antigen-pulsed-dendritic cell-derived exosomes induce a protective immune response against T. gondii infection

It was previously demonstrated that immunizing mice with spleen dendritic cells (DCs) that had been pulsed ex vivo with Toxoplasma gondii antigens triggers a systemic Th1-biased specific immune response and induces protection against infection. T. gondii can cause severe sequelae in the fetuses of mothers who acquire the infection during pregnancy, as well as life-threatening neuropathy in immunocompromised patients, in particular those with AIDS. Here, we investigate the efficacy of a novel cell-free vaccine composed of DC exosomes, which are secreted antigen-presenting vesicles that express functional major histocompatibility complex class I and II and T-cell-costimulatory molecules. They have already been shown to induce potent antitumor immune responses. We investigated the potential of DC2.4 cell line-derived exosomes to induce protective immunity against toxoplasmosis. Our data show that most adoptively transferred T. gondii-pulsed DC-derived exosomes were transferred to the spleen, elicited a strong systemic Th1-modulated Toxoplasma-specific immune response in vivo, and conferred good protection against infection. These findings support the possibility that DC-derived exosomes can be used for T. gondii immunoprophylaxis and for immunoprophylaxis against many other pathogens.     

Mast cells are crucial in the resistance against Toxoplasma gondii oral infection

During oral infection, mucosal immunity assumes a predominant role. Here, we addressed the role of mast cells (MCs), which are mainly located in mucosa during oral infection with Toxoplasma gondii, using MC‐deficient (W/W^v) mice. We show that in the absence of MCs the resistance of W/W^v mice to oral infection was considerably reduced. W/W^v mice uniformly succumbed within 15 days of infection after administration of cysts of the ME49 strain of T. gondii. The rapid lethality of T. gondii in W/W^v mice correlated with a delayed Th1‐cell response, since IFN‐γ and IL‐12 levels peaked in the later phase of the infection. In vitro, BM‐derived MCs were able to recognize parasite lysate in a MyD88‐dependent way, reaffirming the role of this TLR adapter in immune responses to T. gondii. The importance of MCs in vivo was confirmed when W/W^v mice reconstituted with BM‐derived MCs from control mice retrieved an early strong Th1‐cell response and specially a significant IL‐12 production. In conclusion, MCs play an important role for the development of a protective immune response during oral infection with T. gondii.