Antibody response of Echinococcus granulosus infected mice: Protoscolex specific response during infection is associated with decreasing specific IgG1/IgG3 ratio as well as decreasing avidity

The antibody response was followed during 68 weeks in 17 Balb/c mice intraperitoneally (i.p.) infected with Echinococcus granulosus protoscoleces (PSC) and in three mice i.p. immunized with dead PSC. Titres of antibodies recognizing peptidic and glucidic PSC epitopes, as well as their isotypic and avidity profiles were followed by ELISA. In addition, antigen recognition patterns were analysed by immunoblot. The response against carbohydrate epitopes was dominant in infected and immunized mice but stronger in the first group. Infected mice showed similar profiles of specific IgG and IgM with maximum titres from week 38 to 53. Although IgG1 and IgG3 were the predominant antibody subclasses, the ratio of IgG1/IgG3 antibody titres as well as antibody avidity decreased during the experiment, encompassing a decrease in recognition of peptidic epitopes. Immunized mice did not show significant levels of specific IgM and, after week 15, showed IgG titres lower than the infected mice. IgG1 was the predominant IgG subclass during all the experiment with background levels of IgG3. The mean Ab avidity was high and showed no significant changes during immunization. Different patterns of response were thus produced by dead and developing live parasites. Although high avidity IgG1 antibodies were early found in both cases, lower avidity IgG3 antibodies were increasingly produced afterwards only in infected animals. The isotype switch and avidity decrease observed only during infection are consistent with a possible parasitic mechanism to evade host immunity .     

Antibody response of Echinococcus granulosus infected mice: recognition of glucidic and peptidic epitopes and lack of avidity maturation

The antibody response was followed weekly during 68 weeks in 17 Balb/c mice intraperitoneally (i.p.) infected with 2000 Echinococcus granulosus protoscoleces (PSC) and in three mice i.p. immunized with 2000 dead PSC. Antibodies against hydatid cyst fluid (HCFA) and its peptidic (periodate-resistant) and carbohydrate (periodate-sensitive) epitopes were titrated by ELISA. Avidity and the antigen recognition pattern of antibodies were also analysed during infection and immunization by ELISA and immunoblot, respectively.
The antibody response of infected mice showed quantitative and qualitative variations during infection, since both titre as well as recognition of peptide and carbohydrate epitopes in HCFA depended on time post infection. No avidity maturation was evident during the course of infection. Sera from infected mice recognized the 38 kDa subunit of Ag5 but did not react with the 8 kDa subunit of AgB. On the contrary, the antibody response of immunized mice showed only one peak of antibodies that recognized both peptidic and carbohydrate epitopes of HCFA. In addition, sera from these mice recognized mainly 60 and 110 kDa bands. Our results suggest that: a) avidity and antigen recognition patterns of antibodies in mice treated with live PSC are different from those treated with dead PSC; b) antibodies against HCFA glucidic or peptidic epitopes appear at different times post infection.     

Carbohydrates on the surface of Echinococcus granulosus protoscoleces are immunodominant in mice

A carbohydrate enriched soluble fraction (CHP) was prepared by mild treatment of viable Echinococcus granulosus protoscoleces (PSC) with the enzyme endoglycosidase-F (endo-F) and characterized by SDS-PAGE, glycoside- inhibition ELISA, and immunoblotting. Three groups of four BALB/c mice were immunized with the CHP, with the remaining deglycosylated PSC (DGP) and with dead PSC (DPSC) to analyse the relative immunogenicity of carbohydrates on the surface of PSC. A fourth sentinel group was not immunized. The antibody response was analyzed during primary and hyperimmune responses. Specific antibody titres (IgG and IgM) against somatic PSC antigens (PSA) were evaluated by ELISA and their antigen recognition pattern by immunoblotting, discriminating carbohydrate and peptide specific antibody responses by periodate treatment of PSA. The avidity index of those antibodies and the titer of non-specific immunoglobulins during the whole protocol were evaluated by ELISA. In vitro mitogenic activity of CHP was also evaluated. The results indicated: 1) immunodominance of surface carbohydrate epitopes from PSC, 2) predominant IgM and low avidity response against these epitopes, 3) a dramatic increase of non-specific antibody titres and 4) in vitro mitogenic activity of CHP.     

Fc-binding molecules specific for human IgG1 and IgG3 are present in Echinococcus granulosus protoscoleces

In this work we describe the presence of Fc-binding activity on the suckers and tegument of E. granulosus protoscoleces. A fraction (PSA-Fc⁺) from protoscolex somatic antigens was isolated by affinity chromatography on human Fc-γ1-Sepharose. Analysis by SDS-PAGE of PSA-Fc⁺ showed that it contained two major components. Using mouse F(ab′)2-human Fc chimaeric monoclonal antibodies we verified that PSA-Fc⁺ bound mainly to human Fc-γ1 and Fc-γ3 isotypes. In addition, one of the components of PSA-Fc⁺ showed proteolytic activity. Both, Fc-binding and proteolytic activities localized on the protoscolex surface, may play a relevant role in the host-parasite interaction.     

Antibody response in CD4-depleted mice after immunization or during early infection with Echinococcus granulosus

The aims of this work were to investigate the existence of T-independent antigens in Echinococcus granulosus protoscoleces and to evaluate the relative contribution of T-independent stimulation to the overall antibody response in early infection. Mice depleted of CD4(+)-cells were immunized with protoscolex somatic antigens (PSA) or infected with E. granulosus protoscoleces (PSC). Results showed that the response of CD4-depleted immunized mice had the expected characteristics of a T-independent stimulation and that such T-independent stimulation was important mainly during primary response. During infection absence of CD4(+)-cells affected mainly the secretion of all IgG subclasses with the exception of IgG3 and IgM. To carry out a preliminary isolation of PSC T-independent antigens we prepared a carbohydrate enriched fraction from protoscolex antigens, using a monoclonal antibody specific for the carbohydrate moiety Gal alpha(1,4)Gal highly expressed in PSC. This fraction was mitogenic for naive mouse splenocytes and was recognized by a high percentage of the specific antibodies secreted by CD4-depleted immunized or infected mice. In summary, these results suggest that E. granulosus protoscoleces contain immunogenic T-independent antigens. Primary antibody responses to protoscolex somatic antigens and the production of IgM and IgG3 in early infection would be mainly stimulated by a T-independent mechanism.     

Immunological responses to antigen B from Echinococcus granulosus cyst fluid in hydatid patients

The immunological reactivity of Echinococcus granulosus antigen B was evaluated in 30 hydatid patients. Antigen B was purified from sheep hydatid cyst fluid by electroelution from a non-reducing SDS-PAGE gel (AgB). In ELISA and immunoblotting (IB), determining antibody production in sera from patients with hydatid disease and with other parasitic infections, purified AgB showed higher specificity than a partially purified antigen named pH₅PPT (100% vs 83% in pH5PPT-ELISA and 58% in pH₅PPT-IB). AgB-IB achieved higher sensitivity than AgB-ELISA (80% vs 63%). All AgB-IB positive sera recognized the 12 kDa subunit. Qualitative AgB-IB assessment of IgG isotype responses identified IgG4 as the predominant isotype (87%). The other isotypes showed a lower percentage of positive reactions: IgG1, 33%; IgG2, 21%; and IgG3, 17%. PBMC proliferative assay revealed a cellular response to AgB in 100% of patients' PBMC. These findings confirm antigen B, especially its smallest subunit, as a good diagnostic molecule.     

Cytokine response and outcome of infection depends on the infective dose of parasites in experimental infection by Echinococcus granulosus

We here analysed whether the cytokine responses in early and late experimental infection with Echinococcus granulosus depend on the dose of parasites to which the host is exposed. To this purpose Balb/c mice were inoculated intraperitoneally (i.p.) with either 500 or 2000 protoscoleces. Splenocytes of mice were obtained at days 3, 7, 14 and 21 and also on week 37 post-infection and cultured in vitro with protoscolex antigens. Type-1 and type-2 cytokines were analysed in supernatants by ELISA. Results showed that the inoculation of 500 protoscoleces induced an early type-0 and a late type-2 cytokine response, whereas the inoculation of 2000 protoscoleces induced an early type-2 and a late type-0 cytokine response. Parasite growth was lower in the group inoculated with the low infective dose. These results indicate that the cytokine response during the infection by the helminth E. granulosus depends on the dose of parasites to which the host has been exposed.     

Antibody and Th1/Th2-type responses in BALB/c mice inoculated with live or dead Echinococcus granulosus protoscoleces

The aims of this study were to investigate whether a Th1- or a Th2-type response is stimulated in the first stages of experimental infection with Echinococcus granulosus, and to determine whether live or dead protoscoleces equally contribute to such Th1/Th2-type polarization. Live parasites stimulated the production of IL-10, IL-4 and IL-5 as early as week 1 postinoculation. The levels of IL-10 and IL-4 decreased towards week 4 p.i. and that of IFN gamma increased. The production of specific antibodies was characterized by high levels of systemic IgG1 and local IgM and IgG3 (measured in peritoneal lavages). In contrast, dead parasites induced elevated levels of IL-4, IFN gamma, IL-10 and IL-5 on week 1 postinoculation followed by a decrease of IFN gamma and an increase of IL-4. Low levels of specific antibodies were stimulated by dead parasites both systemically and in the peritoneal cavity. These results show that E. granulosus infection induced an early Th2-type response and that live parasites stimulated stronger antibody responses than dead parasites. In addition, they strongly suggest that both phenomena were modulated by live protoscoleces.     

Analysis of cytokine and specific antibody profiles in hydatid patients with primary infection and relapse of disease

We studied in vitro cytokine production by peripheral blood mononuclear cells (PBMC) from patients with primary and recurrent hydatid disease when cells were incubated with mitogen (PHA) and antigen from hydatid cyst fluid (HCFAg); levels of specific IgE, IgG4 and eosinophil counts were also measured in sera. When specifically stimulated, PBMC from patients produced higher levels of IL-2 (P < 0.02), IFN-γ (P < 0.0028) and IL-5 (P < 0.01) than those from uninfected donors, whereas IL-10 levels were comparable. Notably, IL-5 was also produced in higher levels (P < 0.01) by PBMC from patients when incubated with PHA. The IL-5:IFN-γ ratio was significantly greater (P < 0.02) when measured in response to specific stimulation than it was for PHA-stimulated cultures. These cytokine data suggest a bias towards a Th2-response which is in agreement with the high levels of IgG4 and IgE observed. The polarized response appears to be related to clinical status, as differences between patients with primary infection and those with relapse of disease were demonstrated, with significantly higher levels of IgE (P < 0.003), IgG4 (P < 0.04) titres and eosinophil counts (P < 0.04) in the latter; in addition a tendency to an increased production of IL-5 buy lower IFN-γ was also observed in this group. These results merit further study as they are suggestive of a putative role of Th2-like responses in susceptibility to reinfection by E. granulosus.     

Further characterization of the 38 kDa antigen from Echinococcus granulosus (hydatid disease) cyst fluid: evidence for antigenic heterogeneity and reactivity with anti-P1 antibodies

A panel of 5 IgM and 6 IgG1 monoclonal antibodies (MoAbs) was produced against a band, eluted from a reducing SDS-PAGEgel, containing the 38 kDa subunit of antigen 5 (Ag5) from Echinococcus granulosus cyst fluid; seven of the MoAbs were shown subsequently to bind epitopes on Ag5 but none recognizedphosphorylcho-line or periodate-sensitive carbohydrate epitopes. Differences in the fine specificity of the MoAbs were apparent and, upon reduction, heterogeneity in 38 kDa components from hydatid cyst fluids of different intermediate host origin was revealed by peptide fingerprinting and immuno-blotting using the MoAbs. One of the IgGl MoAbs (ED9) was able to distinguish a reduced 38 kDa molecule in cyst fluids from two distinct genotypes–the horse/dog and sheep/dog strains–of E. granulosus and this may have implications for hydatid serology, immunoepidemiology and strain typing. Furthermore, epitopes on this 38 kDa component or on a different molecule with the same or similar M_r are reactive with anti-P1 blood group antigen antibodies and this could result in false-positive reactions where sera from P2 patients with suspected hydatid disease are tested by immunoblot or immunoprecipitation analysis.,     

棘球蚴感染绵羊并诱发休克期间特异性IgG、IgE抗体对特异性抗原的识别

目的 探讨细粒棘球蚴(E.g.)囊液粗制抗原和B抗原(EgB)识别绵羊感染E.g.后及诱发过敏性休克期间特异性IgG和IgE抗体的特异性反应,并对抗原特性及分子量进行描述。 方法 制备E.g.囊液粗制抗原及EgB,应用免疫印迹技术检测经剖杀证实感染E.g.并诱发过敏性休克的20只绵羊血清特异性IgG和IgE抗体对两种抗原的抗体反应性,并对抗原特性和分子量进行描述。 结果 特异性IgE抗体与耐热、低分子量的EgB(8、12和16 kDa)抗原未见明显的反应条带,而与E.g.囊液粗制抗原在43 kDa处可见明显的反应条带。IgG抗体与E.g.囊液粗制抗原未见明显的反应条带,但与EgB抗原反应后在31、43和66.2 kDa处可见较为明显的反应条带。 结论 EgB可能不是特异性IgE抗体的特异性抗原,而是IgG抗体的特异性抗原。E.g.粗制囊液抗原中含有特异性IgE抗体的特异性抗原组分,其分子量大于43 kDa,可能是导致棘球蚴病患者过敏性休克的主要致敏原。     

转Eg95-EgA31融合基因苜蓿疫苗免疫BALB/c小鼠IgG及其亚类和IgE的动态观察

目的动态观察转Eg95-EgA31融合基因苜蓿疫苗免疫BALB/c小鼠后IgG及其亚类和IgE的变化。方法热絮凝法提取转Eg95-EgA31基因苜蓿疫苗叶蛋白,配成浓度为20μg/μl。88只BALB/c小鼠随机分为2组,分别用100μl(约含1μg融合抗原)口服灌胃和10μl(约含0.1μg融合抗原)滴鼻接种分别免疫小鼠,每3 d 1次,连续免疫2个月。末次免疫后0、2、4、6、8、10、12、14、16、18和20周各组随机剖杀4只小鼠,眼球取血,ELISA法检测血清IgG及其亚类和IgE水平。结果口服灌胃组小鼠血清IgGI、gG2aI、gG2bI、gG1I、gG3和IgE水平分别在末次免疫后2~14、2~14、4~20、6~12、6~12和4~16周升高,分别在末次免疫后4、4、6、8、8和10周达最高水平;滴鼻接种组小鼠的血清IgGI、gG2aI、gG2bI、gG1I、gG3和IgE水平分别在末次免疫后2~14、2~18、4~10、6~14、8和6~12周升高,分别在末次免疫后4、4、6、12、8和6周达最高水平。结论细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗在免疫早期(2~12周)可诱导小鼠产生Th1和Th2混合型免疫应答。     

Identification and diagnostic value of a major antibody epitope on the 12 kDa antigen from Echinococcus granulosus (hydatid disease) cyst fluid

An IgG1 monoclonal antibody (MoAb), designated C9E7H8, has been produced against an epitope on the 12 kDa antigen of Echinococcus granulosus cyst fluid, believed to represent the smallest subunit of antigen B. This MoAb, raised against purified 12 kDa antigen eluted from a reducing SDS-PAGE gel, demonstrated strong binding to native sheep cyst fluid in ELISA and recognition of all three subunits of antigen B (at 12, 16, 23 kDa) by immunoblot under both reducing and non-reducing conditions. Immunoblot analysis also indicated that the complementary epitope is conserved amongst cyst fluids from different intermediate hosts of E. granulosus, including fluids from cysts of two distinct strains, and is present in cyst fluid from E. multilocularis. The monoclonal displays binding to a cDNA clone, EgPS-3, which we have previously shown expresses part of the 12 kDa molecule. EgPS-3, expressed as a glutathione-S-transferase fusion protein, was successful in positive detection of 74% of cystic hydatid patients, although cross-reactions were observed with 25% of sera from alveolar hydatid and 22% of sera from schistosomiasis japonica patients. Three peptides, based on the predicted amino acid sequence of EgPS-3. showed increased specificity but slightly reduced sensitivity in the detection of antibody from E. granulosus patients. The predominant epitope recognized by human antibody occurs in the Nterminal 27 amino acids (peptide 65) of EgPS-3 which also correlates with the location of the monoclonal antibody epitope.     

''Arc 5'' antibodies in sera of sheep infected with Echinococcus granulosus, Taenia hydatigena and Taenia ovis

The 'arc 5' precipitin band, formed when test human serum is reacted against immunoelectophoresed hydatid cyst fluid antigen, has provided a positive diagnosis of Echinococcus granulosus infection. These antibodies to 'arc 5' antigen have now been found in the sera of sheep. They appear 2 weeks after infection with Taenia ovis, after 3 weeks with T. hydatigena and after 16 weeks with E. granulosus. An antigen similar to the 'arc 5' antigen of E. granulosus cyst fluid was also demonstrated in cyst fluid from T. hydatigena, but it would not be positively identified in T. ovis cyst fluid. The presence of 'arc 5' in immunoelectrophoresis tests is not suitable for specific immunodiagnosis of E. granulosus infections in sheep in New Zealand. 'Arc 5' antibodies were only associated with living E. granulosus cysts and were not present if cysts were dead. The location of the cysts in either liver or lungs and the onset of brood capsule production did not influence the presence of 'arc 5' antibodies.