Antigenic similarity between the coccidian parasites Toxoplasma gondii and Hammondia hammondi

The antigens that are present in the coccidian parasites Toxoplasma gondii and Hammondia hammondi were demonstrated and defined by using SDS-PAGE and immunoenzymatic techniques with 125I-labeled and unlabeled antigens of T. gondii and sera of mice infected orally or intraperitoneally with H. hammondi . All cell surface antigens of T. gondii that were labeled with 125I were recognized by antibodies in the sera of the mice infected with H. hammondi except the antigen of approximate molecular weight of 21.5 Kd. This suggests that this antigen is specific for T. gondii. Various antigens in the T. gondii-lysed antigen preparations were recognized by antibodies to H. hammondi . The number of recognized antigens increased as the infection of the mice with H. hammondi progressed. Oral infection with H. hammondi appeared to induce the formation of antibodies that recognized more T. gondii antigens than infection by intraperitoneal inoculation.     

Identification of circulating antigens, including an immunoglobulin binding protein, from Toxoplasma gondii tissue cyst and tachyzoites in murine toxoplasmosis

Here we describe the identification of Toxoplasma gondii circulating antigens in sera of BALB/c mice experimentally infected with either the virulent RH strain, or the cystogenic WTD1 strain or with an isolate from a human patient. The circulating antigens were identified by immunoblot in tachyzoite (RH strain) and in tissue cyst (ME-49 strain) crude antigens, using antibodies produced by immunisation of BALB/c mice with homologous sera from infected animals. The most relevant tachyzoite antigen identified are in the following four clusters of 109-94, 67-57, 35-31 and 28-21 kDa. Tissue cyst-specific circulating antigens, like the 18 kDa one, were detected in sera from mice infected with the cystogenic strains. These immune sera, after depletion of tachyzoite specific antibodies, recognised three tissue cysts antigens with Mr of 120, 79 and 48 kDa, and a cluster of antigens in the range of 68-53 kDa. We produced monoclonal antibodies by fusion of myeloma cells with lymphocytes from the mouse immunised with circulating antigens from the RH strain. One of the clones (3A11/H12) obtained, secretes IgG(1) and recognises a peptide epitope from a tachyzoite 67 kDa protein. This parasite protein also binds irrelevant mouse IgG(1) as well as immunoglobulins from other species. The reactivity with non-specific antibodies was inhibited by preincubation with 2% normal mouse and goat serum, while the reaction with the monoclonal antibody 3A11/H12 was not. Furthermore, a biotinylated F(ab')(2) of an irrelevant mouse IgG(1) did not show any reactivity while the F(ab')(2) of the monoclonal antibody 3A11/H12 reacts specifically with the 67 kDa antigen suggesting that this circulating antigen is a putative Fc binding protein.     

A recombinant antigen with potential for serodiagnosis of Echinococcus granulosus infection in dogs

Antibodies specific for Echinococcus granulosus were affinity purified from dog serum on transfer blots containing putative serodiagnostic antigens. These antibodies and serum pools derived from dogs with E. granulosus infections were used to screen a lambda gt11 cDNA library constructed using E. granulosus protoscolex mRNA. Nine definitive antigenic clones were isolated and characterized, of which one (c10P1) gave strong specific reactions in plaque immunoassay with sera from E. granulosus infected dogs. These clones were all subcloned into the plasmid vector pGEX-1. Antigenicity of clones was confirmed in colony immunoassay and/or immunoblot. Glutathione S-transferase (GST) fusion proteins of individual subclones were produced in Escherichia coli, purified by affinity chromatography and evaluated in ELISA using sera from dogs with infections of E. granulosus, Taenia spp. or nematodes, and helminth-free dog sera. The GST fusion protein 10P1 showed a specificity of 100% in ELISA for diagnosis of E. granulosus infection in dogs despite its relatively low sensitivity. Further investigations aim to identify additional recombinant antigens and test 10P1 expressed in alternative expression systems to increase diagnostic sensitivity of the ELISA.     

Trypanosoma cruzi: antibodies to a MAP-like protein in chronic Chagas' disease cross-react with mammalian cytoskeleton

Trypanosoma cruzi lambda gt 11 library from epimastogote derived mRNA was screened with human chagasic sera or sera from chronically infected mice. Strong reactive recombinants were detected with both sera. Two recombinant clones were studied in more detail and shown to be composed of the same 114-bp repetitive sequence coding for a 38 amino acid repetition. This repetition is the same size and shares greater than 60% homology with the reported T. brucei microtubule associated protein (MAP) p320. The insert of one of these clones, K1-7 (228 bp), was subcloned into pMSgt11 and the soluble recombinant polypeptide expressed. Antibodies against the K1-7 fusion polypeptide recognized a major 110-kDa band from cytoskeleton. Anti K1-7 monospecific antibodies detected several cytoskeletal proteins from 3T3 fibroblasts and bovine brain microtubule preparations. Reciprocally, anti-MAP1b monoclonal antibodies raised against bovine brain microtubule reacted with the K1-7 polypeptide on Western blots. The protein identified by K1-7 antibodies may be one of the parasite molecules associated to molecular mimicry.     

Epitope-selected monospecific antibodies to recombinant antigens from Toxoplasma gondii reacted with dense granules of tachyzoites.

Epitope-selected monospecific antibodies were applied to investigate the localization of antigenic molecules in Toxoplasma gondii by immunoelectron microscopy. Eighty cDNA clones encoding antigenic polypeptides were immunoscreened from lambda gt11 expression library with T. gondii infected mouse sera. Twenty different clones with no crossreactivity were selected from eighty clones. Monospecific antibodies to antigens derived from respective cDNA clones extracted from infected mouse sera by the epitope selection method were used in Western blot analysis and immunoelectron microscopy. Eleven antigens were detected with epitope-selected antibodies in lysates of T. gondii tachyzoites. Five of the antigens with molecular weights of 60, 40, 35, 28, and 27 KD were localized in the dense granules. Monospecific antibodies purified by the epitope selection method were useful for investigating the localization of antigens without preparation of a monoclonal antibody from a hybridoma.     

猪瘟病毒E2(gp55)基因的克隆表达及其DNA疫苗的初步研究

用RTPCR方法从中国标准强毒株石门毒的细胞培养物中 扩增获得了其结构蛋白E2基因cDNA,将之克隆到pGEM5Z T载体,用双脱氧链终止法测定其核苷酸序列,并推导出其对应氨基酸序列,与几个代表毒株Alfort株、Brescia株和C株相应序列进行比较,所测核苷酸序列与各株的同源性分别为84.7%、92.6%和95.2%,氨基酸序列的同源性分别为89.4%,92.6%和94.6%;将此E2片段亚克隆至真核表达载体pcDNA3.1,构建表达CSFV E2蛋白的重组质粒pcE2,用脂质体转染法将pcE2导入cos7细胞进行瞬时表达,用针对E2蛋白的特异性单抗以间接免疫荧光法检测,结果E2蛋白在cos7细胞中获得了正确表达,随之将pcE2质粒DNA进行小鼠肌内接种免疫,ELISA法检测证实在免疫后2周和4 周的小鼠体内可诱导出较为明显的阳性血清,并高于E2单抗的阳性对照,病毒中和试验也表明DNA免疫后小鼠体内可诱导产生CSFV中和抗体;同时构建了能在昆虫细胞Sf9中表达GSTE2和GSTGFPE2融合蛋白的重组杆状病毒;上述研究结果为研制针对CSFV的DNA疫苗,亚单位疫苗及其诊断试剂打下了基础。     

日本血吸虫新抗原基因Sj—Ts4的克隆、表达及免疫保护性

为探讨旋毛虫感染小鼠后抗血吸虫感染的分子机制 ,并为血吸虫病疫苗的研究提供新的抗原分子 ,用旋毛虫感染鼠血清筛选日本血吸虫成虫cDNA文库。经过 3轮筛选 ,获得 9个阳性克隆 ,其中新基因Sj Ts4有一完整的编码框 ,编码2 10个氨基酸。该蛋白质的理论分子量为 2 3kD ,等电点为 7.72。将Sj Ts4亚克隆于原核表达载体 pGEX 5X 3,以GST融合蛋白的形式在E .coliDH5α中表达。Western印迹分析显示 ,重组Sj Ts4蛋白 (rSj Ts4)能被慢性感染鼠血清识别。rSj Ts4或与弗氏完全佐剂混合后免疫小鼠 ,均可以诱导产生特异性的IgG抗体 ,抗体效价可高达 1∶2 5 6 0 0。两免疫组的减虫率分别为31.36 %和 36 .80 % ,与对照组相比都具有统计学意义     

Role of Toxoplasma gondii HSP70 and Toxoplasma gondii HSP30/bag1 in antibody formation and prophylactic immunity in mice experimentally infected with Toxoplasma gondii.

Production of antibodies against Toxoplasma gondii (T. gondii)-derived stress proteins, T. gondii HSP70 (T.g.HSP70) and T.g.HSP30/bagl, in C57BL/6 and BALB/c mice perorally infected with cysts of the avirulent Fukaya strain of T. gondii was analyzed. Production of anti-T.g.HSP70 IgG antibodies was transient, whereas production of anti-T.g.HSP30/bag1 IgG antibodies persisted after infection in both C57BL/6 and BALB/c mice. C57BL/6 mice, a susceptible strain, predominantly produced IgG antibodies specific for T.g.HSP70, whereas BALB/c mice, a resistant strain, predominantly produced IgG antibodies specific for T.g.HSP30/bag1, after T. gondii infection. Immunization with rT.g.HSP30/bag1 enhanced, whereas immunization with rT.g.HSP70 reduced host protective immunity against T. gondii infection with a cyst-forming avirulent strain, Fukaya, and a virulent strain, RH.     

Antigenic Similarity Between the Coccidian Parasites Toxoplasma gondii and Hammondia hammondi1

The antigens that are present in the coccidian parasites Toxoplasma gondii and Hammondia hammondi were demonstrated and defined by using SDS-PAGE and immunoenzymatic techniques with ¹²⁵I-labeled and unlabeled antigens of T. gondii and sera of mice infected orally or intraperitoneally with H. hammondi. All cell surface antigens of T. gondii that were labeled with ¹²⁵I were recognized by antibodies in the sera of the mice infected with H. hammondi except the antigen of approximate molecular weight of 21.5 Kd. This suggests that this antigen is specific for T. gondii. Various antigens in the T. gondii-lysed antigen preparations were recognized by antibodies to H. hammondi. The number of recognized antigens increased as the infection of the mice with H. hammondi progressed. Oral infection with H. hammondi appeared to induce the formation of antibodies that recognized more T. gondii antigens than infection by intraperitoneal inoculation.     

Delayed-type hypersensitivity to elastase-soluble lung peptides in the tight-skin (Tsk) mouse

The development of immunity to homologous connective tissue antigens was studied with respect to aging in the tight-skin (Tsk) mouse mutant. A delayed-type hypersensitivity (DTH) response to elastase-solubilized lung peptides in Tsk/+ mice, which became evident at 10 weeks of age and increased in intensity until 22 weeks, was observed. Tsk mice did not demonstrate significant DTH responses when challenged with type I or IV collagen, and normal (+/+) littermates of all ages did not respond to any of the antigens under study. DTH responses could be adoptively transferred to normal +/+ and C57BL/6 mice with spleen cells from 30-week-old Tsk/+ mice; treatment with anti-Thy 1.2 antibodies plus complement significantly reduced the ability of these Tsk/+ cells to transfer DTH reactivity. No antibody activity to the antigens under study could be detected in the sera of Tsk/+ or +/+ mice at any age. These results are discussed with regard to the pathological manifestations observed in the Tsk/+ mutant mouse.     

Construction and immunologic evaluation of a Porphyromonas gingivalis subsequence peptide fused to hepatitis B virus core antigen

Several proteins of Porphyromonas gingivalis contain multiple copies of a 47 amino acid conserved repeated sequence. A fusion protein was constructed in which the P. gingivalis peptide was fused to the carboxy terminus of the hepatitis B core protein. This fusion protein was expressed in Escherichia coli, purified, and used to vaccinate mice that were later challenged with P. gingivalis W83 using the mouse abscess model. Although the mice were not protected against bacterial challenge, Western blot analysis showed that sera from the mice and from rabbits immunized with the fusion protein reacted with a number of vesicle proteins from P. gingivalis W83. These data suggested that this peptide is recognized by the host's immune system but that the antibodies are not protective.     

抗肿瘤单抗3H11对应抗原cDNA片段的克隆

单克隆抗体３Ｈ１１能与多种肿瘤细胞特异结合,克隆其对应抗原无疑具重要意义．用胃癌细胞ＭＧＣ８０３构建ｃＤＮＡ表达文库,通过抗体３Ｈ１１对其进行原核表达筛选,获得一株能与３Ｈ１１特异反应的阳性克隆．其ｃＤＮＡ插入片段为５５４ｂｐ．ＧｅｎＢａｎｋ不含其同源序列．将此ｃＤＮＡ片段与谷胱甘肽转移酶表达质粒ｐＧＥＸ－４Ｔ重组,Ｗｅｓｔｅｒｎｂｌｏｔ和竞争抑制实验表明,表达产物依然保持同３Ｈ１１反应的特异性．可见它是３Ｈ１１对应抗原的ｃＤＮＡ．     

Induced recovery of a suppressed idiotype by immunization with anti-idiotype

Neonatal Balb/c mice were suppressed forthe H8/T15 idiotype by injection of homologous anti-H8 (D. S. Strayer, D. A. Rowley, and H. Köhler, et al J. Immunol.114, 722, 1975. Six to eight weeks later groups of these suppressed mice were immunized up to three times with isologous anti-H8 raised in Balb/c. After a rest of 4 weeks each group was challenged with R36a vaccine and bleedings were obtained before and after this challenge. All sera were assayed for total anti-PC and H8 idiotype amounts by solid-phase radioimmunoassay. After two-preimmunizations with isologous anti-H8 no increase of the H8 levels was observed though these mice responded to R36a immunization with an increase of total anti-PC antibodies. After the third preimmunization, however, the H8 idiotype was increased in sera taken before and after challenge with R36a. These findings demonstrate that the state of neonatal idiotype suppression can be broken by immunization with complementary anti-idiotype.     

Isolation of Toxoplasma gondii from capybaras (Hydrochaeris hydrochaeris) from São Paulo State, Brazil

The capybara (Hydrochaeris hydrochaeris) is a large rodent used for human consumption in certain areas of South America. In the present study, viable Toxoplasma gondii was isolated for the first time from this host. Antibodies to T. gondii were assayed in the sera of 64 capybaras from 6 counties of S?o Paulo State, Brazil, using the modified agglutination test (MAT, > or =1:25 dilution) and the indirect fluorescent antibody test (IFAT, > or =1:16 dilution), and antibodies were found in 48 (75%) by MAT, and 49 (76.6%) by IFAT. Samples of brain, heart, and tongue of 40 seropositive capybaras were pooled, digested in pepsin, and bioassayed in mice. Toxoplasma gondii was isolated from tissue homogenates of 36 capybaras, and the isolates were designated TgCyBrl-36. Most isolates were lethal to mice; 17 of the 36 isolates killed 100% of infected mice, 11 isolates caused mortality in 25-90% of infected mice, and 8 isolates were nonpathogenic to mice. Results indicate that asymptomatic capybaras can harbor mouse-virulent T. gondii, and hence they can serve as a source of infection for humans.     

First biologic and genetic characterization of Toxoplasma gondii isolates from chickens from Africa (Democratic Republic of Congo, Mali, Burkina Faso, and Kenya)

The prevalence of Toxoplasma gondii in free-ranging chickens (Gallus domesticus) is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. In the present study, prevalence of T. gondii in chickens from Democratic Republic of Congo, Mali, Burkina Faso, and Kenya is reported. The prevalence of T. gondii antibodies in sera of 50 free-range chickens from Congo was 50% based on the modified agglutination test (MAT); antibody titers were 1:5 in 7, 1:10 in 7, 1:20 in 6, 1:40 in 1, and 1:160 or more in 4 chickens. Hearts, pectoral muscles, and brains of 11 chickens with titers of 1:20 or more were bioassayed individually in mice; T. gondii was isolated from 9, from the hearts of 9, brains of 3, and muscles of 3 chickens. Tissues of each of the 14 chickens with titers of 1:5 or 1:10 were pooled and bioassayed in mice; T. gondii was isolated from 1 chicken with a titer of 1:10. Tissues from the remaining 25 seronegative chickens were pooled and fed to 1 T. gondii-free cat. Feces of the cat were examined for oocysts, but none was seen. The results indicate that T. gondii localizes in the hearts more often than in other tissues of naturally infected chickens. Genotyping of these 10 isolates using the SAG2 locus indicated that 8 were isolates were type III, 1 was type II, and 1 was type I. Two isolates (1 type I and 1 type III) were virulent for mice. Toxoplasma gondii was isolated by mouse bioassay from a pool of brains and hearts of 5 of 48 chickens from Mali and 1 of 40 chickens from Burkina Faso; all 6 isolates were avirulent for mice. Genetically, 4 isolates were type III and 2 were type II. Sera were not available from chickens from Mali and Burkina Faso. Toxoplasma gondii antibodies (MAT 100 or more) were found in 4 of 30 chickens from Kenya, and T. gondii was isolated from the brain of 1 of 4 seropositive chickens; this strain was avirulent for mice and was type II. This is the first report on isolation and genotyping of T. gondii from any source from these 4 countries in Africa.     

小鼠对重组痘苗病毒表达流感病毒血凝素的免疫反应

实验比较了小鼠对表达流感病毒A/NJ/11/76(H1N1)和A/Jap/305/57(H2N2)血凝素基因的痘苗病毒重组株HSW2和VInf1的免疫反应,两株重组病毒经静脉或静脉加鼻腔免疫小鼠后都产生相应血凝抑制抗体;用不同剂量的痘苗病毒重组株HSW2皮内接种家兔也产生相应抗体,且抗体滴度与接种的病毒量成正比关系,但用痘苗病毒野毒株免疫的家兔未测到抗体,这两株痘苗病毒重组株免疫的小鼠,能保护小鼠对流感病毒母株的攻击,HSW2和VInf1的保护指数分别为3.3和3.9个对数,但这两株病毒未能诱导细胞毒性T细胞反应。     

Schistosoma mansoni: immunoblot analysis of adult worm proteins

Proteins of adult Schistosoma mansoni were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assayed in immunoblots for reactions with individual mouse sera. Four weeks after a heavy infection with a few hundred cercariae, IgG antibodies directed predominantly against a protein of 31 kDa were detected. The protein was only weakly recognized by antibodies of mice harboring a 4-week-old light infection with about 60 cercariae. After 6 weeks or more, mice infected with either dose formed antibodies, not only against the 31-kDa protein and a 67-kDa protein, but also against a number of other components. While reactions with the 31- and 67-kDa proteins occurred with sera of all individual mice of four different strains, the reactions with other components were less consistently observed. Mice vaccinated with a heavy or light dose of 20,000-rad-irradiated cercariae did not form antibodies detectable in the blotting system. However, in immunofluorescence assays with living skin schistosomula, but not lung schistosomula, antibodies against the larval surface were detected with all sera obtained 4 weeks after infection or vaccination. In addition, immunofluorescence studies using the same sera and sectioned adult parasites demonstrated the presence of antibodies against the parasite surface in all sera except those obtained from mice exposed to a light infection with normal cercariae. Mice infected in this latter way were the only animals that did not develop a significant resistance against a challenge infection 4 weeks after exposure to normal or irradiated cercariae. The presence of an immunofluorescent reaction against the schistosome gut always coincided with a reaction of the sera with the 31-kDa protein in the immunoblots. Although a role in immune resistance could not be ascribed to any of the proteins reacting in the immunoblots, the data demonstrate important differences in the antibody specificities induced by various infection schemes.     

Production and Evaluation of Toxoplasma gondii Recombinant GRA7 for Serodiagnosis of Human Infections

The precise diagnosis of the acute toxoplasmosis in pregnant women and immunocompromsied patients has critical importance. Most of the commercially available assays use the whole Toxoplasma soluble extract as the antigen. However, the assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the lack of a purified standardized antigen. The aim of this study was production and evaluation of the usefulness of the recombinant Toxoplasma gondii GRA7 antigen for the serodiagnosis of Toxoplasma gondii IgM and IgG by ELISA. A total of 70 T. gondii IgM positive sera, 74 T. gondii IgG positive sera, and 60 sera from subjects who were not infected with T. gondii were examined. These sera were shown different absorbance values in ELISA test. To control the specificity of the rGRA7 other parasitic diseases, for example, echinococcosis, malaria, leishmaniasis, fascioliasis, and strongyloidiasis were tested of which none showed positive results. Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (com ELISA) were 89% and 90%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 96% and 90%, respectively. The results obtained here show that this antigen is useful for diagnostic purposes.     

Cardiac-oxidized antigens are targets of immune recognition by antibodies and potential molecular determinants in chagas disease pathogenesis

Trypanosoma cruzi elicits reactive oxygen species (ROS) of inflammatory and mitochondrial origin in infected hosts. In this study, we examined ROS-induced oxidative modifications in the heart and determined whether the resultant oxidized cardiac proteins are targets of immune response and of pathological significance in Chagas disease. Heart biopsies from chagasic mice, rats and human patients exhibited, when compared to those from normal controls, a substantial increase in protein 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), carbonyl, and 3-nitrotyrosine (3-NT) adducts. To evaluate whether oxidized proteins gain antigenic properties, heart homogenates or isolated cardiomyocytes were oxidized in vitro and one- or two-dimensional gel electrophoresis (2D-GE)/Western blotting (WB) was performed to investigate the proteomic oxidative changes and recognition of oxidized proteins by sera antibodies in chagasic rodents (mice, rats) and human patients. Human cardiomyocytes exhibited LD(50) sensitivity to 30 μM 4-HNE and 100 μM H(2)O(2) at 6 h and 12 h, respectively. In vitro oxidation with 4-HNE or H(2)O(2) resulted in a substantial increase in 4-HNE- and carbonyl-modified proteins that correlated with increased recognition of cardiac (cardiomyocytes) proteins by sera antibodies of chagasic rodents and human patients. 2D-GE/Western blotting followed by MALDI-TOF-MS/MS analysis to identify cardiac proteins that were oxidized and recognized by human chagasic sera yielded 82 unique proteins. We validated the 2D-GE results by enzyme-linked immunosorbent assay (ELISA) and WB and demonstrated that oxidation of recombinant titin enhanced its immunogenicity and recognition by sera antibodies from chagasic hosts (rats and humans). Treatment of infected rats with phenyl-α-tert-butyl nitrone (PBN, antioxidant) resulted in normalized immune detection of cardiac proteins associated with control of cardiac pathology and preservation of heart contractile function in chagasic rats. We conclude that ROS-induced, cardiac-oxidized antigens are targets of immune recognition by antibodies and molecular determinants for pathogenesis during Chagas disease.     

Demonstration of antigenic similarities and variations in excretory/secretory antigens of Toxoplasma gondii.

C57BL/6 mice were orally infected with different doses of cysts of ME49 strain of Toxoplasma gondii to produce groups of acutely and chronically infected mice. Sera were obtained at different periods post-infection. SDS-PAGE was ran with excretory/secretory antigens of ME49 and RH strains of T. gondii, followed by Western blot analyses using the above sera and anti- IgA, IgM, IgG as conjugates. The SDS-PAGE profiles of the two antigens were similar. However the antigenic bands showed variations in all blots, most evidently in IgA blots of chronic sera. IgG blots showed greatest similarities in reactive bands. In IgM blots, more common bands were shown in chronic sera than in acute sera. Variations and similarities in prominence of some bands and time of their appearance were also noted, especially in IgM and IgG blots of chronic sera. Thus antigenic variations and similarities are present in excretory/secretory products of different strains of T. gondii.