Parasite specific antibodies in three strains of mice after infection with Trypanosoma cruzi

Three inbred strains of mice (BALB/cJ, C3H/HeJ and NZB/BInJ) were infected with trypomastigotes of Trypanosoma cruzi. Sera were taken at different times after infection and radioimmunoprecipitation assays were used to detect antibodies against individual T. cruzi epimastigote and trypomastigote antigens. The mouse strains differed in regard to the spectrum of antibodies and the time after infection when the various epimastigote specific antibody species appeared. NZB mice had antibodies against at least 25 polypeptides ranging in molecular weight from 20,000 to 90,000 D at 3 wk after infection, and these persisted until at least 10 wk post-infection. C3H and BALB/c had antibodies against fewer than 5 antigens at 3 wk after infection; whereas by week 10, antibodies against at least 25 polypeptides were detected. C3H mice that were most susceptible to infection (but not NZB or BALB/c mice) had antibodies against a 25,000 D molecular weight epimastigote antigen. The antibody response against trypomastigote polypeptides was more uniform. Sera from all mouse strains at 3 wk after infection precipitated the same polypeptides and the radioimmunoprecipitation patterns did not change as a function of time after infection.     

Terminal beta-D-galactofuranosyl epitopes recognized by antibodies that inhibit Trypanosoma cruzi internalization into mammalian cells

Polyclonal antisera to 80 - 90-kDa and to 50 - 60-kDa polypeptides of tissue-culture trypomastigotes which inhibit the interiorization of trypomastigotes of the Y strain (up to 70%) and of metacyclic trypomastigotes of the CL-14 clone (up to 50%) in cultured mammalian cells, were obtained. Both sera immunoprecipitate surface polypeptides of 90 kDa, 80 kDa, 72 kDa and 58 kDa in trypomastigotes and of 80 kDa, 77 kDa and 74 kDa in metacyclic trypomastigotes. These antigens are glycoproteins with affinity for concanavalin A. The antibodies (IgG class) of the inhibitory sera are mainly directed against carbohydrate epitopes, which were identified as being beta-D-galactofuranosyl units by radioimmunoinhibition assays. The direct involvement of the beta-D-galactofuranosyl unit in the process of parasite infection was verified using the synthetic disaccharide beta-D-galactofuranose (1----3)-alpha-D-mannopyranose which promoted, at 1 mg/ml concentration, 50% inhibition of internalization.     

Changes in humoral responses to Trypanosoma cruzi during the course of infection in mice held at elevated temperature.

A parasite-specific, enzyme-linked immunosorbent assay and immunoblot analysis were used to examine the development of humoral immunity in Trypanosoma cruzi-infected C3H mice that survive acute infection when held at elevated environmental temperature. Both parasite-specific antibody levels and numbers of antigens identified increased during infection in mice held at 36 C, with the greatest reactivity measured in sera from mice that had resolved parasitemias. Heat shock of culture forms of T. cruzi resulted in production of different antigens, but there was no strong difference in the antigens recognized by sera from mice held at room temperature and those recognized by sera from mice held at 36 C. Immunoblot analysis using blood-form trypomastigote antigens identified a band of approximately 61 kDa produced by trypomastigotes in mice held at 36 C that was strongly detected by sera from mice held at 36 C. Little if any reactivity to this antigen was observed using sera from mice held at room temperature.     

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.     

Identification of novel Leishmania major antigens that elicit IgG2a response in resistant and susceptible mice

Experimental murine models with high, intermediate and low levels of genetically based susceptibility to Leishmania major infection reproduce almost entire spectrum of clinical manifestations of the human disease. There are increasing non-comparative studies on immune responses against isolated antigens of L. major in different murine strains. The aim of the present study was to find out whether there is an antigen that can induce protective immune response in resistant and susceptible murine strains. To do that, crude antigenic extract of procyclic and metacyclic promastigotes of L. major was prepared and subjected to SDS-PAGE electrophoresis. Western-blotting was used to search for antigen(s) capable of raising high antibody level of IgG2a versus IgG1 in the sera of both infected resistant and susceptible strains. Two novel antigens from metacyclic promastigotes of L. major (140 and 152 kDa) were potentially able to induce specific dominant IgG2a responses in BALB/c and C57BL/6 mice. The 2 antigens also reacted with IgG antibody of cutaneous leishmaniasis patients. We confirm that 140 and 152 kDa proteins of L. major promastigotes are inducing IgG production in mice and humans.     

Natural and immune human antibodies reactive with antigens of virulent Neisseria gonorrhoeae: immunoglobulins G, M, And A

Natural and immune human antibodies reactive with heat-labile and heat-stable antigens of virulent Neisseria gonorrhoeae were studied by use of an indirect fluorescent-antibody (IFA) procedure. The immunoglobulin class of the reactive antibodies was identified by using fluorescein-conjugated antisera specific for human IgG, IgA, or IgM in the IFA procedure. The effects of heat and mercaptoethanol on IFA reactivities were also studied. It appeared that antibodies of the IgG, IgM, and IgA classes present in the sera of both infected persons (immune antibodies) and normal persons with no history of gonococcal infection (natural antibodies) react with heat-stable somatic antigens. Immune IgG antibodies, however, were distinguishable from natural IgG antibodies by their ability to recognize heat-labile surface antigens. The distinction between natural and immune IgM antibodies was less obvious. IgM antibodies from both infected and normal persons appeared to react with heat-labile antigens. Some, but not all, infected persons had immune IgA antibodies to heat-labile as well as to heat-stable antigens. Treatment of sera with mercaptoethanol had no effect on IgG antibodies. The IFA activity of IgM antibodies was decreased, but not abolished. The effects of mercaptoethanol on IgA antibodies were variable. Some sera showed a decrease in IgA titer, and others showed an increase in IgA activity to certain antigens. Immune IgG antibodies were more resistant to heating than were natural IgG antibodies. Natural and immune IgM antibodies appeared equally sensitive to heating. IgA activity, on the other hand, was increased by heating sera at 60 C, but was decreased at higher temperatures. Thus, it appears that natural and immune human IgG antibodies to N. gonorrhoeae may be distinguished by their interactions with heat-labile antigens and by their resistance to heating.     

Immunological consequences of infection and vaccination in South American trypanosomiasis

Trypanosoma cruzi infection provokes a vigorous immune response that terminates the parasitaemia associated with the acute stage within two to three months of initial infection. Even so, a variable proportion of patients may develop severe Chagas' disease, often decades after initial infection. Recent experimental findings suggest that trypomastigotes of T. cruzi possess a surface bound neuraminidase and sugar binding protein by means of which they invade host cells--a mechanism very reminiscent of influenza virus. Studies of the antibody response to trypomastigotes in patients or murine models have identified a series of antibodies able to mediate lysis of live parasites in a complement mediated lysis (c.m.l.) assay. These antibodies have also been linked to resistance to infection in vivo and disappear following successful parasitological 'cure' in drug-treated animals and human patients. Immunochemical studies have shown that sera from infected patients or mice lacking this c.m.l. activity also lack those antibodies able to bind trypomastigote surface components of 85 and 160 kDa relative molecular mass. The availability of rabbit and mouse models of Chagas' disease have produced data that suggest that chronic stage pathology may have an immunological basis dependent on the known cross reactivity between host and parasite cells. Delivery of the lethal hit leading to host cell destruction is probably facilitated by the ability of parasite antigens to bind to host cells thus exposing them to the host's own anti-parasite immune response. If Chagas' disease does indeed have an immunological basis, then this might be controlled in turn by immunoregulation, in a manner analogous to that achieved in experimental allergic encephalomyelitis.     

Isotype determination of anti-Trypanosoma cruzi antibody in murine Chagas' disease.

Isotypic analysis of anti-parasite humoral responses of C57B1/6 and C3H (He) mice surviving acute Trypanosoma cruzi infection showed that both mouse strains demonstrate IgG1, IgG2a, IgG2b, and IgM enzyme-linked immunosorbent assay titers from days 21 to 300 of infection. Using the western blot technique to determine the antigen specificity of the isotypic responses, 100-day infected C3H mice showed strong IgG1, IgG2a, and IgG2b responses to many antigens, whereas C57B1/6 mice showed weak responses to fewer antigens. Isotype western blots showed that reactivity to the T. cruzi antigen of 75-77 kDa is present in the humoral response of day 21-infected mice that will survive and missing in those that will not survive. In general, surviving immunized C3H mice respond with IgG1, IgG2a, and IgG2b reactions to the 75-77-kDa and other antigens, whereas resistant B6 mice concentrate their anti-T. cruzi response in the IgG2b isotype to the 75-77-kDa antigen. Perhaps induction of ineffective antibody responses to nonprotective antigens is beneficial to the parasite and detrimental to the host.     

Trypanosoma cruzi: differences in cell surface interaction of circulating (trypomastigote) and culture (epimastigote) forms with macrophages

Characteristics of the association of circulating (trypomastigote) and cultured (epimastigote) forms of Trypanosoma cruzi with macrophages were studied. Treatment of mouse macrophages with the anti-microfilament drug cytochalasin D severely reduced the ability of these cells to bind either trypomastigotes or epimastigotes. Instead, treatment with the antimicrotubule drug colchicine or 2-deoxyglucose afforded differential effects because epimastigote but not trypomastigote association with the macrophages was significantly inhibited. Prior treatment of epimastigotes with either trypsin or neuraminidase decreased their uptake by macrophages whereas treatment of trypomastigotes with either enzyme increased it. Pretreatment of macrophages with neuraminidase did not affect epimastigote uptake but reduced that of trypomastigotes. Pretreatment of macrophages with trypsin reduced the uptake of both forms of the parasite. However, quantitative differences in the extent of such reduction were noted when varying concentrations of trypsin were used, epimastigote uptake being more drastically affected. These results suggest that the initial interaction of virulent circulating trypomastigote and the attenuated cultured epimastigote forms of T. cruzi to macrophages may involve attachment via different surface structures.     

Microarray analysis of the human antibody response to synthetic Cryptosporidium glycopeptides

Glycoproteins expressed by Cryptosporidium parvum are immunogenic in infected individuals but the nature of the epitopes recognised in C. parvum glycoproteins is poorly understood. Since a known immunodominant antigen of Cryptosporidium, the 17 kDa glycoprotein, has previously been shown to bind to lectins that recognise the Tn antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with different Tn valency and presentation were prepared. In addition, glycopeptides were synthesised based on a 40 kDa cryptosporidial antigen, a polymorphic surface glycoprotein with varying numbers of serine residues, to determine the reactivity with sera from C. parvum-infected humans. These glycopeptides and non-glycosylated peptides were used to generate a glycopeptide microarray to allow screening of sera from C. parvum-infected individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera from C. parvum-infected individuals bound to multivalent Tn antigen epitopes presented on glycopeptides, suggesting that glycoproteins from C. parvum that contain the Tn antigen induce immune responses upon infection. In addition, molecular differences in glycosylated peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation had a pronounced effect on reactivity. Lastly, pooled sera from individuals infected with either Toxoplasma or Plasmodium were also tested against the modified Cryptosporidium peptides and some sera showed specific binding to glycopeptide epitopes. These studies reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen may be useful diagnostically and in defining the roles of parasite glycoconjugates in infections.     

Genetics of resistance to the African trypanosomes. III. Variant-specific antibody responses of H-2-compatible resistant and susceptible mice

Genetically based differences in variant-specific immunity to the African trypanosomes were examined. H-2-compatible inbred mouse strains that differed in relative resistance were infected with Trypanosoma rhodesiense clone LouTat 1. Antibody responses to exposed epitopes of the LouTat 1 variant-specific surface glycoprotein (VSG) were measured. Relatively resistant B10.BR mice (H-2k) made predictable IgM antibody responses to the VSG of LouTat 1 which were associated with clearance of the LouTat 1 variant antigenic type from blood; IgG responses to LouTat 1 surface antigen appeared after clearance occurred, and were lower than peak titers of IgM. Intermediately susceptible CBA mice (H-2k) also made predictable IgM and IgG responses which followed the same pattern as the more resistant strain. Peak titers were lower for both Ig classes, however, and a delayed appearance of antibody was correlated with delayed clearance of LouTat 1. In contrast to B10.BR and CBA mice, the susceptible C3H mice (H-2k) failed to make detectable antibodies to LouTat 1 surface antigen and also failed to control the first peak of parasitemia. The absence of immunity in infected C3H mice was selective for antibody to exposed epitopes of LouTat 1 VSG because antibody was detectable to invariant VSG or internal trypanosome antigens. Also, the C3H strain was shown not to be a genetic nonresponder to LouTat 1 surface antigen because VSG-specific antibodies appeared within 1 wk after trypanocidal chemotherapy. Finally, we demonstrated that the susceptibility of C3H mice was not associated with an inability of the mononuclear phagocyte system to clear the parasites because drug cure, passive transfer of immune serum, or sensitization of trypanosomes with antibody all led to trypanosome clearance from blood by the liver. In summary, we show for the first time that major differences in variant-specific immunity occur in MHC-compatible animals after infection with the African trypanosomes.     

Trypanosoma cruzi: location of a specific antigen on the surface of bloodstream trypomastigote and culture epimastigote forms.

The nature of surface antigens of culture epimastigote and bloodstream trypomastigote forms of Trypanosoma cruzi was investigated by light and electron microscopy using indirect immunofluorescence and peroxidase labeling techniques and antisera against unique, common, and contaminant antigens. A specific antigen, identified by monospecific rabbit antiserum (anti-component 5 antiserum), is the major constituent of the cell surface and flagellar membrane of both the culture epimastigote and bloodstream trypomastigote forms. Antigens of heterologous stercorarian trypanosomes (Trypanosoma rangeli) and of culture medium proteins could not be detected on the cell surface of culture epimastigote forms and bloodstream trypomastigote forms.     

Comparison of the C-mediating killing activity and C-activating properties of mouse monoclonal and polyclonal antibodies against Trypanosoma cruzi

A Mouse polyclonal antiserum against Trypanosoma cruzi or its IgG and IgM fractions and five monoclonal antibodies (two IgM, two IgG(1) and one IgG(2a)) recognize and combine with membrane components of trypomastigote forms of the parasite as revealed by immunofluorescence. Although all these antibodies sensitize trypomastigotes and prepare them to activate the complement (C) system, as measured by consumption of total C, C4, B and C3, only the polyclonal antiserum or its IgG, IgM and Fabmu fragments were able to induce trypanosome lysis by the alternative C pathway.     

Detection of antibodies to Streptobacillus moniliformis in rats by an immunoblot procedure

An immunoblot (IB) technique for detecting antibodies to Streptobacillus moniliformis in rat sera was evaluated. Immune sera to three S. moniliformis strains showed a similar reactivity pattern with both autologous and homologous antigens in the 18-87 kDa range. Using a rat S. moniliformis strain as the antigen, a similar reactivity pattern was found with sera from rats infected experimentally with S. moniliformis and sentinels. Two to five proteins were detected in the 32-55 kDa range. Over a period of 2.5 years, 27/133 rat serum panels submitted for routine monitoring yielded one or more S. moniliformis enzyme-linked immunosorbent assay (ELISA)-positive samples. In one of these 27 panels, sera showed an IB reactivity pattern resembling that observed with immune sera and with sera from infected and exposed rats. S. moniliformis was confirmed in the colony by both culture and polymerase chain reaction (PCR). Sera from the remaining 26 ELISA-positive serum panels frequently showed activity to a 57 kDa antigen but not more than one antigen was detected in the 32-55 kDa range. We conclude that the IB can be used as a confirmatory test for the detection of S. moniliformis infection in ELISA-positive rats.     

Immunoblotting analysis of anti-rickettsial antibodies produced in patients of Tsutsugamushi disease

The reactivity of sera from patients of Tsutsugamushi disease (scrub typhus) with the antigenic polypeptides of Rickettsia tsutsugamushi was analyzed by the immunoblotting method. The reactivity varied greatly among the sera of individual patients tested. IgG and IgM antibodies of most patients reacted with the 54-56 kilodalton (54-56K) polypeptide located on the rickettsial surface, suggesting that this polypeptide is a predominant antigen in the infection. Other polypeptides of 60K, 50-52K, 46-47K, 35K, and 21-25K were reactive with some but not all sera. From the reactivity of these polypeptides, it was suggested that the 54-56K polypeptide is both strain-specific and group-specific, the 60K polypeptide is group-specific, and the 35K and 21-25K polypeptides are subgroup-specific. IgG antibodies seem to be more cross-reactive with polypeptides of multiple strains than IgM antibodies and have a tendency of increased cross-reactivity that was observed in the sera obtained at the later stage of illness.     

Specificity of the microimmunofluorescence assay for the serodiagnosis of Chlamydia pneumoniae infections.

Chlamydia pneumoniae infections are mostly confirmed using an indirect microimmunofluorescence test for which potential cross-reactions between antigens from different chlamydial species are not well documented. Using this assay, 928 sera (507 subjects) submitted for Chlamydia pneumoniae serology were tested for specific IgM and IgG to this bacteria using the TW-183 antigen. IgM and IgG reactivities to Chlamydia trachomatis serotypes C, D, E, and L2 and Chlamydia psittaci strain 6BC antigens were also tested. A sample was interpreted as positive only when evenly fluorescent elementary bodies were observed. Twenty-five subjects (4.9%) showed serological evidence of recent Chlamydia pneumoniae infection (IgM positive and (or) IgG seroconversion); 11 of them also showed serological evidence of recent infection with at least one other chlamydial species. Specificity was 50 and 63% for IgM and IgG detection, respectively. These results suggest that mixed or temporally related infections might occur, or, more likely, that some Chlamydia pneumoniae IgM or IgG reactivities might be due to heterotypic antibodies.     

Human antibody responses to Brugia malayi antigens in brugian filariasis.

Human antibody responses to Brugia malayi antigens were studied with sera from a Brugia endemic area in South India. Patients with clinical filariasis had significantly higher IgE and lower IgG4 levels to adult worm antigens than people with asymptomatic microfilaraemia. Intermediate antibody levels were observed in endemic normals. A majority of sera from each clinical group contained IgG antibodies to surface antigens of infective larvae (L3) by IFAT. IgG immunoblot studies did not reveal group differences in L3 antigen recognition. IgE antibodies bound to a subset of antigens bound by IgG. IgE antibodies in sera from clinical filariasis patients preferentially bound to L3 antigens at 200, 97, 68 and 58 kDa compared with sera from microfilaria carriers. These results are consistent with prior studies of antibody responses in filariasis and add new information on the targets of IgG and IgE antibodies to L3 antigens in brugian filariasis.     

Detection of Toxoplasma gondii Infections using Virus-Like Particles Displaying T. gondii ROP4 Antigen

Toxoplasma gondii ME49 infections are typically diagnosed by serological tests. However, serological diagnosis of RH strain-induced toxoplasmosis remains unknown. In order to develop seradiagnosis of above 2 kinds of infections, we generated recombinant virus-like particles (VLPs) displaying the T. gondii rhoptry protein 4 (ROP4) and evaluated their potential in T. gondii ME49 or RH strain infection diagnostics. Mice were orally infected with either the tachyzoites of T. gondii (RH) or cysts of T. gondii (ME49) at various dosages, and sera were collected at regular intervals. ELISA-based serological tests were performed to assess IgG, IgM, and IgA antibody responses against ROP4 VLP antigen and tissue lysate antigen (TLA). Compared to TLA, IgG, IgM, and IgA levels to ROP4 VLP antigen were significantly higher in the sera of T. gondii RH-infected mice 1 and 2 week post-infection (PI). T. gondii-specific IgG antibody was detected at 1, 2, 4, and 8 week PI in the T. gondii ME49-infected mice with infection dose-dependent manner. These results indicated that the ROP4 VLP antigen was highly sensitive antigens detecting T. gondii RH and ME49 antibodies at an early stage.     

IgM and IgG significant reactivity to Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii among Italian patients affected by Lyme arthritis or neuroborreliosis

Abstract This survey evaluates the specificity of band patterns in immunoblot of sera taken from clinically defined cases of Lyme arthritis and neuroborreliosis, towards three locally isolated strains of Borrelia burgdorferi , belonging to the three species: Borrelia sensu stricto, Borrelia garinii and Borrelia afzelii . To assess specificity, patient sera were statistically ( χ ², P ≤ 0.05) compared with blood donors sera samples. Both IgG and IgM antibodies were considered. The overall reactivity of the three Borrelia strains in IgG immunoblots indicated that ten protein bands were significant, with a different prevalence of some of them in the two groups of patient sera: bands at 60-58, 30–33, 36–37 and 28-27 kDa were markers for neuroborreliosis sera; proteins at 100-83, 72-70 and 18-17 kDa behaved like markers for Lyme arthritis. The IgM Immunoblots revealed significant bands at 100-83, 72-70, 51, 24-21 and 18-17 kDa only with neuroborreliosis sera. Though there were variable band reactivities in each strain, a correlation emerged between the three genospecies and the clinical symptoms: in fact B. afzelii and B. garinii were prevalent in Lyme arthritis sera, (IgG Immunoblots); B. garinii was associated to neuroborreliosis (IgG and IgM Immunoblots); B. sensu stricto was strongly reactive with neuroborreliosis in IgM immunoblots. These data indicate that the three locally isolated strains of Borrelia representing the three genospecies should be used together in immunoblot to detect antibodies elicited in neuroborreliosis and Lyme arthritis.     

Immunoblot analysis of serum IgG, IgA and IgE responses against larval excretory-secretory antigens of Anisakis simplex in patients with gastric anisakiasis

To increase our understanding of the immune response to Anisakis infection, antigen specific IgG, IgA and IgE responses were identified using an immunoblot technique after polyacrylamide gel electrophoresis of excretory-secretory products from the larval stage of Anisakis simplex. Nine sera were drawn from proven cases of gastric anisakiasis within 3 days after symptoms had developed. The molecular weight of the major antigenic bands were distributed between 50 kDa and 120 kDa of the antigens. In nine cases of gastric anisakiasis, three of them were positive for IgG response, five for IgE, and six for IgA, respectively. None of control sera recognized the antigenic bands in IgA and IgE responses. In contrast, two controls had IgG antibodies against 1-2 proteins in the 65-95 kDa region. The antigenicity of the excretory-secretory products was lost following treatment by 0.2% trypsin, but not by 0.2 M periodic acid. Based on the results of reactivity to lectins, antigenic bands of the ES products possessed mucin type glycoconjugate residues in their protein portion. This indicates that the humoral responses of IgA and IgE antibodies to the larval ES antigens are a more reliable index of infection than that of the IgG response.