Biochemical and immunochemical characterization of surface and excretory-secretory antigens of Loa loa microfilariae

Detergent solubilized extracts of 125Iodogen surface-labelled Loa loa microfilariae revealed a relatively simple profile of two strongly labelled molecules of 23 and 67 kDa for blood microfilariae and several strongly labelled molecules of 23, 40, 42-67 kDa for in vitro born microfilariae. In addition, there were other weakly labelled molecules which were resolved after prolonged autoradiographic exposure. Surface molecules of 28, 29, and 33 kDa were unique to blood microfilariae, a 14.4 kDa molecule was unique to in vitro born microfilariae and molecules of 23, 40, and 75-84 kDa were common to both forms of microfilariae. The profile of excretory-secretory products consisted of molecules of 14.4-198 kDa. Human albumin was a predominant component of surface molecules and excretory-secretory products from blood microfilariae. Immunoprecipitation with occult and microfilaremic loaiasis sera demonstrated that the 23 kDa surface molecule and excretory-secretory products of 14.4 and 33 kDa were only recognized by occult loaiasis sera whereas surface molecules of 40 and 75-84 kDa and excretory-secretory products of 28 and 67 kDa were recognised by both sera. Studies with heterologous sera demonstrated that with the exception of the 75-84 kDa antigens, all the L. loa microfilarial surface antigens contained epitopes which were restricted to filarial parasites. Further studies revealed that the 23 kDa antigen was a protein which contained neither asparagine-N-linked oligosaccharides nor interchain disulfide-linkages.     

Filarial surface antigens: the major 29 kilodalton glycoprotein and a novel 17-200 kilodalton complex from adult Brugia malayi parasites

Adult Brugia malayi nematode parasites possess a range of cuticular and epicuticular molecules which may be defined by various surface-labelling techniques. We present here evidence that at least two distinct antigens are associated with the surface, a glycoprotein of 29 kDa, and a complex of twelve components forming a regular series or 'ladder' between 17 and 200 kDa (17/200 kDa) which have not previously been described. Each of these products is antigenic in infected hosts, although responses in infected humans to the 17/200 kDa are relatively weak. Digestion of the 29 kDa antigen with proteases and endoglycosidases indicates that it is closely conserved between B. malayi and B. pahangi, and that it carries at least two N-linked oligosaccharide chains each of 1.5-2 kDa. By contrast, a smaller surface-labelled antigen of 15 kDa shows no glycosylation by either lectin adherence or endoglycosidase digestion assays. Trypsin treatment of intact, labelled parasites results in cleavage of 29 kDa molecules isolated 17/200 kDa 'ladder' to trypsin abolishes all bands except the 17 kDa base unit. Both the 29 kDa and 17/200 kDa antigens can be recovered as water-soluble molecules by homogenisation of the parasite in the absence of detergent, or by disruption of the cuticle with reducing agents such as 2-mercaptoethanol. In the presence of such agents, both the 17/200 kDa series and the 29 kDa glycoprotein are shed rapidly from intact parasites. Finally, two-dimensional electrophoretic analysis shows that while the 29 kDa glycoprotein is strongly basic and the 15 kDa acidic, the 17/200 kDa antigens form a related series of neutral pI.     

A novel 26 kilodalton antigen expressed on the surface membrane of activated T cells

We have identified and characterized the tissue distribution of the antigen recognized by a novel monoclonal antibody (mAb) 1B10, raised against an activated gammadelta T cell clone. Immunohistochemistry of tissue sections, and analysis of single cell suspensions by flow cytometry revealed that mAb 1B10 weakly reacted with <6% of normal human peripheral blood mononuclear cells (PBMC). After 5-6 days of in vitro culture of PBMC activated with phytohemagglutinin (PHA), 55% of the CD4+ and 25% of the CD8+ T cells became 1B10+. 1B10 expression was maintained on long term cultured interleukin 2 (IL-2)-dependent T cell receptor (TCR) alphabeta+ and gammadelta+ clones, and importantly, in contrast to resting T cells, the majority of in vivo activated synovial T lymphocytes from a patient with rheumatoid arthritis were 1B10+. In addition, myelo-monocytic U927 cells, tissue macrophages and some epithelia and fibroblasts were found to react with mAb 1B10. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of molecules immuno-precipitated by mAb 1B10 from radio-iodinated cell surface membrane lysates of T lymphocyte and U937 cells revealed 26 and 29 kiloDalton (kDa) glycoproteins respectively. In conclusion, mAb 1B10 recognizes a novel appearing 26 kDa T cell activation antigen that may be useful for further studies of activated T cells in health and disease.     

Purification and immunochemical characterization of a 22 kilodalton surface antigen from Schistosoma mansoni

A 22 kDa antigen (Sm22) was purified from schistosomula membrane extracts by immunoaffinity chromatography with monoclonal antibody M.2. Western blotting suggested that the epitope bound by M.2 required a specific conformational folding of the molecule, which was sensitive to reducing agents. Two-dimensional electrophoresis of purified Sm22 demonstrated that the single 22 kDa protein recognized by M.2 on one-dimensional gel analysis was composed of at least two isomorphs. Additional Western blotting showed that Sm2 was one of the major antigens recognized by mouse anti-irradiated cercariae serum, and that this same serum recognized at least one epitope which was not sensitive to reducing agents. The mice vaccinated with irradiated cercariae were shown to be 75% protected from cercarial challenge. Sera from a rabbit immunized with Sm22 contained antibodies which bound to the surface of schistosomula and detected a single protein at 22 kDa by immunoprecipitation or Western blot. The rabbit anti-Sm22 sera also immunoprecipitated a 22 kDa in vitro translation product, indicating that at least one epitope on Sm22 is not dependent on glycosylation.     

Purification and characterization of an immunodominant 36 kDa antigen present on the cell surface of Clostridium difficile.

The 36 kDa antigen represents the major EDTA-extracted protein of Clostridium difficile strains belonging to electrophoretic group 2. Antibodies to this antigen are found in sera of patients with C. difficile-associated diarrhoea. The 36 kDa antigen was extracted from C. difficile C253 by EDTA and purified by gel filtration (Sephacryl S300) and ion exchange chromatography (DEAE-Trisacryl M). The molecular weight of the purified protein was 36 kDa as determined by SDS-PAGE, also in non-reducing conditions. By gel filtration, the molecular size appeared to be 72 kDa and was not modified by the presence of EDTA or SDS in the column buffer. Since IEF showed a single isoelectric form of pI 4.6, the protein could be a homodimeric molecule. Immunofluorescence demonstrated that the 36 kDa antigen was located on the surface of the C. difficile cell. Monospecific antiserum raised in rabbits reacted positively with the 36 kDa protein of most group 2 C. difficile strains isolated from antibiotic-associated diarrhoea (AAD) outbreaks in Italy and other European countries.     

Immunoblot evaluation of the specificity of the 29-kDa antigen from young adult female worms Angiostrongylus cantonensis for immunodiagnosis of human angiostrongyliasis

The antigenic components of Angiostrongylus cantonensis young adult female worm somatic extract (FSE) were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The sera tested were from patients with proven angiostrongyliasis, other parasitic diseases, and healthy adults. Both the sera and cerebrospinal fluid (CSF) were tested from patients with clinical angiostrongyliasis. The CSF from patients with other neurological diseases were also included. Using SDS-PAGE, we found that the FSE comprised more than 30 polypeptides. Immunoblot analysis revealed at least 12 or 13 antigenic bands in patients with proven or clinical angiostrongyliasis, respectively. The patterns of reactivity recognized by the serum and CSF antibodies against FSE were similar. These antigenic components had molecular masses ranging from less than 14.4 to more than 94 kDa. The prominent antigenic band of 29-kDa might serve as a reliable marker for the diagnosis of angiostrongyliasis. The sensitivity, specificity, positive and negative predictive values of immunoblot analysis in this antigenic band were 55.6%, 99.4%, 83.3% and 97.4%, respectively.     

Isolation and partial characterisation of a 26 kilodalton antigen from Plasmodium falciparum recognised by an inhibitory monoclonal antibody

A 26 kDa protein, present in trophozoites and schizonts of Plasmodium falciparum, has been identified as the target of a monoclonal antibody that weakly inhibits parasite growth in vitro. The antigen has been purified to homogeneity by immuno-affinity chromatography and electrophoresis. The sequence of 19 amino acids at the N-terminus of the protein has been determined.     

The biochemical and immunochemical characterisation of the 30 kilodalton surface antigen of Brugia pahangi

The major surface antigen (30 kDa) of Brugia pahangi has been characterised by a number of biochemical and immunochemical means. The 30 kDa polypeptide is a glycoprotein which can be extracted from the worm surface by homogenization in the absence of detergents. The 30 kDa polypeptide can be metabolically labelled with [35S]methionine in adult male and female parasites. In addition small amounts of the 35S-labelled 30 kDa antigen can be detected in the medium of worms cultured in vitro. 125I labelling of the excretory-secretory (ES) products of adult male and female parasites followed by immunoprecipitation and peptide mapping has confirmed the relationship between the surface located 30 kDa polypeptide and that released in vitro.     

Cloning and characterisation of an immunodominant major surface antigen of Echinococcus multilocularis.

A lambda gt11 cDNA expression library from mRNA of Echinococcus multilocularis protoscolices has been constructed in Escherichia coli Y1090. Immunoscreening with pooled sera obtained from patients suffering from E. multilocularis disease revealed 5 reactive clones. By partial DNA sequence comparison all clones proved to encode the same gene. The complete cDNA sequence of the clone pEM10 with the largest insert of 2.2 kb was determined and an open reading frame of 1.7 kb could be described. The derived amino acid sequence shares 42.6% identity with human microvillar cytovillin found in the membranes of placenta and carcinoma tissues. The coding region of the cDNA of pEM10 was amplified by polymerase chain reaction (PCR) and cloned in frame into expression vector pGEX-3X. Immunoblot analysis revealed the expression of a recombinant antigen of 65 kDa and a protein with the same molecular weight was also found in the lysate of E. multilocularis protoscolices. In contrast, the protein was absent from hydatid fluid or larvae of Echinococcus granulosus. By means of immunofluorescence studies this immunodominant antigen could be located in the germinal layer of brood capsules and in the tegument of E. multilocularis protoscolices. The fusion protein was purified and used for diagnostic purposes in immunoblot. The diagnostic value of this antigen is discussed.     

T-Cell epitopes and human leukocyte antigen restriction elements of an immunodominant antigen of Blastomyces dermatitidis

Humans infected with the dimorphic fungus Blastomyces dermatitidis develop strong T-lymphocyte responses to WI-1, an immunodominant antigen that has been shown to elicit protective immunity in mice. In the present study, the T-cell epitopes of WI-1 and human leukocyte antigen (HLA) restricting elements that display them were investigated. Peripheral blood mononuclear cells (PBMC) from 37 patients with a confirmed history of blastomycosis were tested for a response to WI-1 in primary proliferation assays; PBMC from 35 (95%) responded. Six patients whose PBMC proliferated strongly in response to WI-1 (defined as a stimulation index greater than 50) were tested further for responses to subcloned, recombinant fragments of the antigen. These patients responded chiefly to sequences within the N terminus and the 25-amino-acid tandem repeat. Cloned CD4(+) T cells from an infected individual were used to delineate more precisely the peptide epitopes in the fragments and HLA restricting elements that present them. A majority of the T-cell clones recognized an epitope spanning amino acids 149 to 172 within the N terminus, displayed by HLA-DR 15. A minority of the clones, which have been shown to perform a cytolytic function in vitro, recognized an epitope in the tandem repeat displayed by HLA-DPw4, an uncommon restricting element. Tandem repeat epitopes required display by the beta chain of DPw4 heterodimers. Thus, human T cells with different functions in vitro also recognize distinct regions of WI-1, raising the possibility that HLA restricting elements that present them could modulate immunity during blastomycosis by selection and display of WI-1 peptides.     

Analysis of the antigen specificity of influenza haemagglutinin-immune human T lymphocyte clones: identification of an immunodominant region for T cells

Human T lymphocyte clones specific for the haemagglutinin (HA) molecule of A/Texas/1/77 were maintained in long-term culture with T cell-growth factor. The clones were analysed for their viral antigen specificity using serologically defined type A influenza subtypes and chemically synthesized peptides of the HA-1 molecule. With the exception of one T cell clone that recognized only the HA molecule used for immunization, the clones responded to determinants that showed partial or complete cross-reactivity amongst the strain A subtypes. The cross-reactive population of T cell clones were specific for peptides distinct from the antibody binding sites of HA. Furthermore, one peptide located at the carboxyl terminus of the HA-1 molecule appeared to be immunodominant, although the critical residues for T cell antigen recognition within that could not be identified.     

Procyclin: an unusual immunodominant glycoprotein surface antigen from the procyclic stage of African trypanosomes

An immunodominant species-specific surface glycoprotein antigen was purified from procyclic culture forms of Trypanosoma brucei rhodesiense using lectin affinity chromatography and a monoclonal antibody immunoadsorbent. The purified molecule appears on a 10% polyacrylamide gel as a wide, dark silver staining band having an apparent molecular mass of between 30 and 40 kDa, identical to that revealed by immunoblotting using anti-procyclic lysates. The molecule, which we have named procyclin, was shown by immunofluorescence and immunoelectron microscopy to be exposed on the surface of procyclic trypanosomes. Gasphase protein microsequencing and micro-amino acid analysis revealed an unusual acidic polypeptide with an amino-terminal amino acid sequence which matched portions of previously published sequences predicted from two different cDNAs obtained using mRNA from procyclic trypanosomes. The procyclin molecules contained a large glutamic acid-proline repeat and the form we isolated was highly water soluble. Ten different monoclonal antibodies were used in ELISA with synthetic peptides to localize parasite surface epitopes to various portions of procyclin. The results showed that surface epitopes were spread throughout most of the procyclin molecule, including the glutamic acid-proline repeat portion. Procyclin is distributed over the surface of both culture form and tsetse fly midgut form procyclic trypanosomes, is developmentally regulated and is immunologically species-specific.     

Molecular cloning and primary sequence of a cysteine protease expressed by Haemonchus contortus adult worms

We have cloned cDNAs encoding a 35-kilodalton cysteine protease that is a major component of protective extracts isolated from blood-feeding Haemonchus contortus adult worms. Near full-length cDNAs for the protease were isolated by immunoscreening an adult worm cDNA expression library with a rabbit antiserum prepared against the protein eluted from preparative SDS gels and by rescreening the library with oligonucleotide probes. The protein predicted from the nucleotide sequence of the cDNAs and of a genomic DNA clone comprises 342 amino acids and contains an N-terminal signal sequence, 16 cysteine residues and four potential N-linked glycosylation sites. The enzyme appears to be glycosylated in vivo. The H. contortus protease, called AC-1, displays an overall 42% sequence identity with the human lysosomal thiol protease cathepsin B. The similarities between cathepsin B and AC-1 are localized primarily to regions of cathepsin B that comprise the mature, active form of the enzyme. A stretch of six amino acids that includes the active site cysteine of cathepsin B is conserved, and is present in the same relative location in AC-1, suggesting that this region comprises the active site of the H. contortus enzyme.     

Isolation and partial characterization of I antigen from I adult erythrocyte membranes.

The i-active substance was isolated from i adult and I int erythrocyte membranes by butanol-water extraction and was purified by SDS-gel filtration and thin layer chromatography of peracetylated samples. The i-active substances from both types of erythrocytes showed also IF activity. In antigen isolated from i adult red cells the IF activity was lower than i activity. On the other hand, the antigen of I int red cells showed higher IF than i activity. The chemical characterization of isolated antigens indicated their glycosphingolipid character.     

Cloning and sequence comparisons of four distinct cysteine proteases expressed by Haemonchus contortus adult worms.

Three new members of a developmentally regulated cysteine protease gene family of the parasitic nematode Haemonchus contortus have been isolated and characterized. One of the new genes, AC-3, was found to be linked in tandem to the previously characterized AC-2 gene. Nucleotide sequence analyses revealed that the first 90 amino acids of AC-3 are organized into four exons, similar to the situation in AC-2. A cDNA that appears to be a near full-length copy of the AC-3 gene was isolated using the polymerase chain reaction (PCR) technique to amplify cDNAs from adult worm poly(A)+ mRNAs. In addition to AC-3, a distinct cysteine protease cDNA, AC-4, was amplified by the same oligonucleotide primers. cDNAs encoding a fifth cysteine protease, AC-5, were isolated from an adult worm cDNA expression library using specific rabbit antisera and by PCR. Comparison of the predicted amino acid sequences of AC-3, AC-4 and AC-5 reveal that they share 64-77% identity with one another and with the previously reported AC-1 and AC-2 sequences. The amino acids surrounding the active site cysteine are highly conserved, as are the positions of other cysteine residues in the mature protein sequences. The H. contortus proteases are more similar to one another than they are to human cathepsin B (38-44% amino acid identity) or to the Sm31 cysteine protease of Schistosoma mansoni (36-40% identity). Our studies indicate that H. contortus adult worms express mRNAs for several distinct cysteine proteases. The significant primary sequence differences between the proteases suggest that they differ in their substrate specificities and precise physiological functions.     

K99 surface antigen of Escherichia coli: purification and partial characterization.

K99, a presumed colonizing factor of enterotoxigenic Escherichia coli of calf origin, has been purified. K99 was removed from K99+ bacteria by salt extraction and subsequently purified by ammonium sulfate precipitation and column chromatography on diethylaminoethyl-Sephadex. The purified material was homogenous in size, having an s20,w of 13 to 15 S. It was composed of two subunits: a major component with a molecular weight of 22,500 and a minor component of 29,500. When observed in the electron microscope, K99 appeared to be rod-shaped, with a strong tendency for self-aggregation. At concentrations where aggregation was minimized, individual rods were observed with diameters of 8.4 nm and mean lengths of 130 nm. Based on the subunit structure, exterior location, and rod-like shape of K99, it is concluded that K99 is a pilus or pilus-like structure. Chemically, K99 is composed primarily of protein and has an isoelectric point of greater than 10. Purified K99 did not hemagglutinate guinea pig erythrocytes.     

应用痘苗病毒载体表达的重组甲肝抗原特性的研究

目的 探讨以痘苗病毒载体表达的甲肝病毒培养合适的细胞系和重组病毒灭活以及甲肝病毒抗原保存条件.方法 将构建成功的表达甲肝抗原的重组痘苗病毒分别接种于K4、143、HEL、Hep-2和Vero等细胞,用ELISA测定重组甲肝抗原的表达产量和不同方法灭活重组病毒后的抗原活性.结果 在K4、143和HEL细胞表达重组甲肝抗原的产量略优于Hep-2和Vero细胞,重组病毒经β-丙内酯和甲醛灭活后,甲肝抗原活性不变,制备的重组甲肝抗原稳定性好.结论 痘苗病毒表达载体可以提高甲肝抗原表达效率,具有十分广阔的应用前景.     

Identification and characterization of an immunodominant 58-kilodalton antigen of Aspergillus fumigatus recognized by sera of patients with invasive aspergillosis.

Sera from 38 patients with invasive aspergillosis were tested by Western immunoblotting for the presence of antibodies to antigens of Aspergillus fumigatus present in a mycelial extract of the organism. All of the sera contained antibodies to an antigen of molecular weight 58,000, which was which was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was the only antigen recognized in approximately 90% of the sera tested. The 58-kDa antigen is an abundant component of mycelial extracts composing approximately 50% of the Coomassie blue-stained protein. The antigen also contains carbohydrate, since it is stained by the carbohydrate stain periodic acid-Schiff and it binds to the lectin concanavalin A. It was purified by immunoaffinity chromatography employing a monoclonal antibody directed against an epitope on the 58-kDa antigen. Analysis of the purified antigen by gas-liquid chromatography revealed the presence of mannose, galactose, and glucose residues in a 2:1:2 ratio. The ratio of protein to carbohydrate is 1.16:1. The protein is slightly acidic, containing relatively high quantities of glutamic and aspartic acids, glycine, alanine, serine, and threonine. The 58-kDa antigen also contains phosphate groups as part of its structure. Serological activity was totally destroyed after treatment with sodium metaperiodate and was partially destroyed after treatment with pronase. The 58-kDa antigen was not able to hydrolyze protein.     

Conformational dependence and conservation of an immunodominant epitope within the babesia equi erythrocyte-stage surface protein equi merozoite antigen 1

Equi merozoite antigen 1 (EMA-1) is an immunodominant Babesia equi erythrocyte-stage surface protein. A competitive enzyme-linked immunosorbent assay (ELISA), based on inhibition of monoclonal antibody (MAb) 36/133.97 binding to recombinant EMA-1 by equine anti-B. equi antibodies, detects horses infected with strains present throughout the world. The objectives of this study were to define the epitope bound by MAb 36/133.97 and quantify the amino acid conservation of EMA-1, including the region containing the epitope bound by MAb 36/133.97. The alignment of the deduced amino acid sequence of full-length EMA-1 (Florida isolate) with 15 EMA-1 sequences from geographically distinct isolates showed 82.8 to 99.6% identities (median, 98.5%) and 90.5 to 99.6% similarities (median, 98.9%) between sequences. Full-length and truncated recombinant EMA-1 proteins were expressed and tested for their reactivities with MAb 36/133.97. Binding required the presence of amino acids on both N- and C-terminal regions of a truncated peptide (EMA-1.2) containing amino acids 1 to 98 of EMA-1. This result indicated that the epitope defined by MAb 36/133.97 is dependent on conformation. Sera from persistently infected horses inhibited the binding of MAb 36/133.97 to EMA-1.2 in a competitive ELISA, indicating that equine antibodies which inhibit binding of MAb 36/133.97 also recognize epitopes in the same region (the first 98 residues). Within this region, the deduced amino acid sequences had 85.7 to 100% identities (median, 99.0%), with similarities of 94.9 to 100% (median, 100%). Therefore, the region which binds to both MAb 36/133.97 and inhibiting equine antibodies has a median amino acid identity of 99.0% and a similarity of 100%. These data provide a molecular basis for the use of both EMA-1 and MAb 36/133.97 for the detection of antibodies against B. equi.     

Complement regulation on the surface of cultured schistosomula and adult worms of Schistosoma mansoni

Cercaria and freshly prepared schistosomula of Schistosoma mansoni are highly sensitive to complement. However, early in their maturation, the schistosomula become resistant to complement killing. This conversion is preceded by a rapid and massive release of several acetabular proteases and of the glycocalyx coat. Thus, shedding of the glycocalyx which is a major immunogen and a strong activator of the alternative pathway of complement permits the parasite to escape immune damage. Mechanically transformed schistosomula, which were cultured in a defined synthetic medium and developed complement resistance, could be converted by proteolysis to complement sensitivity. Trypsin and pronase markedly increased the susceptibility of cultured schistosomula to complement. The trypsin-induced complement sensitivity persisted for at least 19 h without recovery of resistance. Similar treatment with trypsin produced complete killing of adult worms by complement in absence of antibodies. Efficient killing was obtained with normal human serum (NHS), with normal guinea pig serum (GpS), and with C4-depleted HS and C4-deficient GpS indicating that the killing was mediated by the cytolytic alternative pathway of complement. Larger quantities of C3b with intact alpha' chain could be demonstrated on trypsin-treated than on non-treated schistosomula. Antibodies which were raised in rabbits by immunization with the trypsin-released material bound to cultured (non-treated) schistosomula and to adult worms, and induced their killing in GpS and C4-deficient GpS. These results suggest that following release of the glycocalyx, the transforming schistosomula of S. mansoni spontaneously express a complement regulatory protein(s). A similar regulator is postulated to be present on the surface of adult worms. Such regulatory molecules may serve as good targets for immunotherapy, since antibodies directed to them will inhibit their regulatory activity and thus potentiate in vivo the lytic action of complement.