Cloning of the gene encoding a 50 kilodalton potential surface antigen of Schistosoma mansoni

A cDNA library was constructed from the mRNA of adult worms of Schistosoma mansoni, in the expression vector lambda gt11. This library was screened with a pool of sera raised against either soluble egg antigens or purified schistosomulum tegumental membranes. An antiserum raised against the fusion protein of one clone immunoprecipitated a 45 kDa polypeptide from the in vitro translation products of adult worm mRNA and recognised a 50 kDa antigen in homogenates of adult worms. This serum gave positive fluorescence of the surface of schistosomula in indirect immunofluorescence assays and was able to mediate killing of schistosomula by human eosinophils in vitro, suggesting that this clone contained part of a gene encoding a surface antigen.     

Purification and characterization of culture-derived exoantigens of Plasmodium falciparum

An 83 kDa glycoprotein and a 100 kDa glycoprotein have been purified from the supernatant fluid of in vitro cultures of Plasmodium falciparum by conventional cation-exchange liquid chromatography, size exclusion high performance liquid chromatography, and anion-exchange high performance liquid chromatography. Both proteins exist as dimers in the native state and have been identified as parasite antigens by Western immunoblotting and by their specific reactivity in the indirect enzyme-linked immunosorbent assay. The N-terminal amino acid sequence of these two proteins has been determined and they are at least 90% homologous. The use of monospecific rabbit antisera raised against the individual pure proteins confirm their cross-reactivity. We postulate that the 83 kDa protein is a specific processing product of the larger 100 kDa protein. The presence of these proteins in the culture supernatant suggests they could both be derived from the merozoite surface coat and are potential protective antigens.     

Encystation of Giardia lamblia leads to expression of antigens recognized by antibodies against conserved heat shock proteins.

During in vitro encystation, Giardia lamblia expresses several stage-specific proteins which are recognized in immunoblots by antisera raised against antigens from three different pathogens. The antigens belong to two different families of conserved stress proteins: (i) HSP60 purified from Legionella pneumophila and recombinant HSP60 from Mycobacterium bovis BCG and (ii) recombinant HSP70 from Plasmodium falciparum.     

Identification and characterization of cDNA clones encoding antigens of Eimeria tenella

An Eimeria tenella cDNA library was constructed in the expression vector λgt11 from poly (A⁺) RNA extracted from sporulating oocysts. The library was screened with rabbit antiserum raised against antigens extracted from fully sporulated oocysts. All of the antigen-expressing plaque-purified clones were initially characterized by cross screening with antisera raised against different stages of the E. tenella life cycle, as well as with antiserum raised against sporozoites of a related species, namely E. acervulina. A selected number of clones were further characterized by antibody selection coupled with immunoblotting and DNA cross hybridization. Three different E. tenella antigens were identified. All three appear to be constitutively expressed at the protein level during sporogony.     

Cloning and characterisation of members of a family of Babesia bigemina antigen genes containing repeated sequences.

Bovine polyclonal antisera to Babesia bigemina antigens separated by phenyl-Sepharose chromatography were used to screen a B. bigemina lambda gt11 cDNA expression library. Eleven B. bigemina-specific cDNA clones were studied in detail. DNA sequencing of 2 representative clones identified open reading frames encoding polypeptides representing the carboxy-termini of 2 different proteins. Both polypeptides contained a related central motif of tandem repeats flanked by a highly conserved carboxy-terminal region, but the sequences preceding the repeats were not related. Hybridisation and restriction enzyme analysis of the cDNA clones indicated that they were derived from a family of at least nine related, but not identical genes. Four different members of the gene family have been isolated from a B. bigemina lambda EMBL3 genomic library. The genes are not closely linked and they occur on the largest and smallest B. bigemina chromosomes resolved by pulsed field gel electrophoresis (PFGE). Antibodies raised against the native antigens and purified on recombinant fusion proteins bound to multiple proteins (50-70 kDa) in the original B. bigemina antigenic fractions.     

Cloning of diagnostic 31/32 kilodalton antigens of Schistosoma mansoni

A cDNA library derived from messenger RNA of adult Schistosoma mansoni was constructed in lambda gt11 and schistosome antigens were expressed as fusions with the amino terminus of the beta-galactosidase of Escherichia coli. Using mouse and human infection sera, recombinant clones specific for a 31/32 kDa doublet having a potential in the immunodiagnosis of schistosomiasis were selected. The specificity of the clones was verified by their reactivity with monoclonal antibodies. The identity of the cloned epitopes with those of the native proteins was confirmed by Western blot analysis of total schistosome proteins, using both antibodies affinity purified from the fusion proteins and antisera raised in rabbits against the fusions. The reactivity of the cloned antigens with human infection sera suggests their usefulness for the immunodiagnosis of human schistosomiasis.     

Identification and characterization of cDNA clones encoding antigens of Eimeria tenella

An Eimeria tenella cDNA library was constructed in the expression vector λgt11 from poly (A⁺) RNA extracted from sporulating oocysts. The library was screened with rabbit antiserum raised against antigens extracted from fully sporulated oocysts. All of the antigen-expressing plaque-purified clones were initially characterized by cross screening with antisera raised against different stages of the E. tenella life cycle, as well as with antiserum raised against sporozoites of a related species, namely E. acervulina. A selected number of clones were further characterized by antibody selection coupled with immunoblotting and DNA cross hybridization. Three different E. tenella antigens were identified. All three appear to be constitutively expressed at the protein level during sporogony.     

In vitro synthesis of a 28 kilodalton antigen present on the surface of the schistosomulum of Schistosoma mansoni

Adult Schistosoma mansoni proteins were fractionated on polyacrylamide slab gels, recovered by electrophoretic elution and used for immunization of Fischer rats. Three antisera recognizing, respectively, 28, 78 and 85 kDa antigens were obtained. The 28 kDa antigen was found among the in vitro translation products from adult worm RNA, and among the 125I-labelled surface antigens of S. mansoni schistosomula. The isoelectric point of the 28 kDa antigen was 6.3-6.5. The 28 kDa antiserum mediated a cytotoxic activity against schistosomula when used in an in vitro assay in the presence of a purified eosinophil cell population.     

Cloning of cDNAs encoding a 28 kilodalton antigen of Toxoplasma gondii

By screening cDNA libraries in lambda gt11 with antibodies raised against the previously described protective F3G3 antigen of Toxoplasma gondii, and subsequently screening with nucleic acid probes, we have isolated cDNA clones that encode a 28 kDa antigen of T. gondii that is likely one of the two antigenic components of the F3G3 antigen. The gene apparently exists as a single copy in the tachyzoite haploid genome of the three strains of T. gondii examined. Northern blot analyses revealed that the cDNAs hybridize with a major T. gondii RNA species of 1.1 kb. Together the cDNAs encompass 1051 bp of cDNA sequence containing an open reading frame with the capacity to encode a 28 kDa protein. Antibodies that were affinity purified using recombinant fusion proteins produced by two of the clones reacted on protein blots of whole T. gondii lysate with a single antigen having an apparent molecular mass of 28 kDa. Both recombinant fusion proteins reacted with IgG antibodies in sera of mice and humans infected with T. gondii and therefore might be useful for the development of diagnostic assays for T. gondii infection.     

Isolation of a C (Ibc) protein from group B Streptococcus which elicits mouse protective antibody

The C (Ibc) proteins of group B Streptococcus (GBS) have been shown to induce mouse protective antibodies when present as immunogens on whole organisms. However, characterization of specific proteins responsible for inducing protection has not been reported. We have grown type Ic GBS in a dialysate of Todd Hewitt broth and analyzed the proteins extruded into the broth. Multiple proteins of varying size were visualized by SDS-PAGE. Ultrafiltration was used to separate the GBS components by molecular weight (MW) into 2 pools, those below 30,000 MW but above 10,000 MW (P10) and those above 30,000 MW (P30). The P10 contained 4 major proteins, including a 14,000 MW protein. Balb-c mice were immunized with the P10 fraction and the antisera used in mouse protection studies. This immune sera protected 100% of mice against challenge with type Ib GBS and protection was not altered by prior absorption of the sera with type Ia or Ib capsular polysaccharide. The P10 was fractionated by column chromatography and eluted proteins examined by SDS-PAGE and Western blot with the mouse protective antisera elicited to the P10. There was one major immunologically reactive protein at 14,000 MW which eluted in a partially purified form from the column. The 14,000 MW protein was reisolated from preparative SDS-PAGE gels and used to elicit antiserum in a rabbit. In mouse protection studies this rabbit antiserum protected mice against subsequent challenge with type Ib GBS (89% protection). Surface antigens were extracted from 125I-labelled type Ic GBS and immunoprecipitated with antiserum to the 14,000 MW protein. The 14,000 MW protein and multiple higher molecular weight proteins were immunologically cross-reactive suggesting the presence of shared epitopes. Thus the 14,000 MW protein from type Ic GBS that is antigenic and elicits mouse protective antibodies against the heterologous type Ib GBS fulfills the criteria for a C protein of GBS.     

Cloning of a Streptococcus sanguis adhesin which mediates binding to saliva-coated hydroxyapatite.

Chromosomal DNA from a salivary aggregating strain of Streptococcus sanguis 12 was partially digested with PstI and ligated into the plasmid vector pUC18 and transformed into Escherichia coli JM83. A total of 1,700 recombinant clones of E. coli were examined by a colony immunoassay with antisera raised against either S. sanguis 12 whole cells or S. sanguis 12 surface fibrils. Five clones which reacted with one or the other antiserum were shown to be unique by Western blotting (immunoblotting) and restriction endonuclease digestion. One recombinant plasmid pSA2 expressed two proteins with Mrs of 20,000 and 36,000. The 36,000-Mr protein has been designated SsaB. Both proteins were purified to homogeneity by Sephadex G-75 and ion-exchange chromatography. The proteins were present in mutanolysin digests of whole-cell lysates of S. sanguis 12 and in the non-saliva-aggregating variant 12na and the hydrophilic variant 12L. Polyclonal antiserum raised against the SsaB protein reacted strongly with the cell surfaces of S. sanguis 12 and 12na but not with that of 12L. SsaB inhibited the adhesion of S. sanguis 12na to saliva-coated hydroxyapatite, indicating that the adhesin mediates the binding to the pH-sensitive receptor.     

Disulfide-linked surface molecules of monoclonal antigen-specific suppressor T cells: evidence for T cell receptor structures

By two-dimensional polyacrylamide gel electrophoresis analysis under nonreducing/reducing conditions, five proteins with interchain disulfide bridges are revealed on the surface of the suppressor T cell lymphoma line LH8-105 obtained by radiation leukemia virus-induced transformation of hen egg-white lysozyme-specific suppressor T lymphocytes. Two disulfide-linked surface proteins expressed by LH8-105 cells have been positively identified by immunoprecipitation with specific antisera. The major labeled membrane protein of LH8-105 cells is the murine leukemia virus env glycoprotein gp70. The second disulfide-linked molecule identified on LH8-105 cells has a molecular mass of 84 kDa under nonreducing conditions and 42 kDa after reduction, and is immunoprecipitated by an antiserum which recognizes the T cell receptor for antigen. A disulfide-linked molecule of a similar molecular mass is also immunoprecipitated from surface-labeled LH8-105 cells by a rabbit antiserum directed against a synthetic peptide predicted from the nucleotide sequence of a cDNA clone encoding the beta chain constant region of a helper T cell hybridoma. Therefore, a dimeric structure comparable to the T cell receptor expressed by cytotoxic and helper T cells is present on the cell surface of these monoclonal antigen-specific suppressor T cells.     

Identification of lambda gt11 clones encoding the major antigenic determinants expressed by Theileria parva sporozoites.

An antiserum, C16, was raised in cattle against freeze-thawed extracts of sporozoites of Theileria parva (Muguga). This antiserum, which neutralizes sporozoite infectivity in vitro, identified theileria-specific antigens having approximate molecular masses of 105, 90, 85, 69, 67, 52, 47, and 43 kilodaltons (kDa) on Western blots (immunoblots) of infected tick salivary gland extracts. The antiserum was used to screen an expression library of T. parva (Muguga) genomic DNA fragments. Three recombinant bacteriophage clones carrying different theileria DNA inserts were studied. The expressed gene product from each clone was used to affinity purify antibodies from C16 antiserum for use in probing Western blots of uninfected and infected tick salivary gland extracts. The population of antibodies selected by each clone specifically recognized a subset of the antigens identified by C16 antiserum. The antigens fell into three distinct groups as defined by their reactivity with each set of selected antibodies. One group included antigens of 105, 90, 85, and 35 kDa, a second group included antigens of 69, 67, 52, 47, and 43 kDa, and the third group included an apparently distinct pair of antigens of 47 and 43 kDa. Thus, antibodies that reacted with determinants encoded by the three recombinant phage clones recognized all of the major antigens seen on Western blots probed with whole C16 antiserum. These results suggest that there may be only three immunodominant antigens expressed in T. parva (Muguga) sporozoites. Additionally, monoclonal antibodies have been raised which neutralize sporozoite infectivity in vitro. These antibodies react with epitopes of the antigens with Mrs of 69,000, 67,000, 52,000, 47,000, and 43,000 which are encoded in clone pgT-42 and have been used to localize these epitopes on the sporozoite surface.     

The open reading frame L2 of cottontail rabbit papillomavirus contains antibody-inducing neutralizing epitopes.

Polyclonal antisera were generated against bacterially derived fusion proteins of the open reading frames (ORFs) of the capsid proteins of cottontail rabbit papillomavirus (CRPV). The carboxy-terminal two-thirds of CRPV L1 and the carboxy-terminal half of CRPV L2 were cloned into a bacterial expression vector and induced proteins were used as antigen and immunogen. The polyclonal antisera were tested in a series of immunological assays, including ELISA, Western blot, and neutralization of CRPV. ELISA demonstrated that the polyclonal antisera raised against expressed L1 proteins reacted strongly to disrupted CRPV virion antigen and weakly both to intact CRPV virion and disrupted BPV-1 virion. Anti-CRPV L2 antisera reacted strongly only to intact and disrupted CRPV virion antigen. Viral capsid proteins of CRPV were detected in Western blots of HPV-11, BPV-1, and CRPV virus particles by these polyclonal antisera. The anti-L1 sera recognized the major capsid protein (60 kDa) and the anti-L2 sera identified a 76-kDa viral protein of CRPV. Only the antisera generated against expressed L2 neutralized CRPV. The neutralizing titer of the anti-L2 sera, however, was several orders of magnitude lower than the titer of a neutralizing polyclonal antiserum that was generated by immunizations with intact CRPV virions.     

Protection by monospecific gonococcal antisera of the chicken embryo challenged with Neisseria gonorrhoeae.

The protective effects of monospecific gonococcal antisera on 11-day chick embryos challenged with a known lethal dose of gonococci were assessed. The monospecific antisera were prepared by immunisation of rabbits with purified gonococcal antigens, and removal of trace amounts of unwanted antibodies was achieved by absorption with antigen covalently bound to cyanogen bromide-activated Sepharose beads. The antisera were standardised for IgG by solid-phase radioimmunoassay. Antiserum raised against whole outer membrane was protective and antiserum raised against the lipopolysaccharide (LPS) was moderately protective. Outer-membrane antiserum from which the LPS component was removed by absorption was less protective than either of these sera. Investigation of the protective mechanism of anti-LPS antibodies indicated that in addition to any antitoxic effect, these antibodies inhibited the multiplication of gonococci. Antisera raised against individual outer-membrane proteins offered no protection in this test. Out of five antisera tested, antipilus serum gave the strongest protection when piliate gonococci were used as the challenge in this model; antipilus serum did not protect against challenge with non-piliate gonococci.     

Autoantibodies and Autoantigens in the Urine of SLE Patients

Autoantibodies against RNA polymerase I (RNAPI), DNA, La and ribosomal P proteins were detected in the urine of systemic lupus erythematosus (SLE) patients, many with normal protein excretion rates. In a number of cases, the antibodies were detectable in the urine but not the serum sample of the same patient. The presence and relative concentrations of the urinary autoantibodies correlated with disease activity. RNAPI antigens were detected in the urine of SLE patients by radioimmunoassay and immunoblotting using rabbit antisera prepared against the purified holoenzyme. Immunoaffinity purification of the rabbit anti-RNAPI with SLE urine proteins resulted in antibodies directed primarily against the largest RNAPI subunit (S1; 194?kDa). Antibodies prepared against recombinant fusion proteins representing the DNA binding regions of human RNAPI(S1) reacted with a 35?kDa SLE urinary protein, a putative fragment of RNAPI(S1). Ribosomal protein P0 was detected in SLE patients' urine by immunoblotting, using rabbit antiserum prepared against recombinant human P1 fusion protein. The relative quantities of urinary P0 correlated with disease status. Analysis of urinary autoantibodies and corresponding antigens in conjunction with analysis of serum autoantibodies may be of value for the purpose of monitoring disease activity.     

Autoantibodies and autoantigens in the urine of SLE patients

Autoantibodies against RNA polymerase I (RNAPI), DNA, La and ribosomal P proteins were detected in the urine of systemic lupus erythematosus (SLE) patients, many with normal protein excretion rates. In a number of cases, the antibodies were detectable in the urine but not the serum sample of the same patient. The presence and relative concentrations of the urinary autoantibodies correlated with disease activity. RNAPI antigens were detected in the urine of SLE patients by radioimmunoassay and immunoblotting using rabbit antisera prepared against the purified holoenzyme. Immunoaffinity purification of the rabbit anti-RNAPI with SLE urine proteins resulted in antibodies directed primarily against the largest RNAPI subunit (S1; 194 kDa). Antibodies prepared against recombinant fusion proteins representing the DNA binding regions of human RNAPI(S1) reacted with a 35 kDa SLE urinary protein, a putative fragment of RNAPI(S1). Ribosomal protein P0 was detected in SLE patients' urine by immunoblotting, using rabbit antiserum prepared against recombinant human P1 fusion protein. The relative quantities of urinary P0 correlated with disease status. Analysis of urinary autoantibodies and corresponding antigens in conjunction with analysis of serum autoantibodies may be of value for the purpose of monitoring disease activity.     

Expression of Mycoplasma pneumoniae antigens in Escherichia coli.

A genomic library of Mycoplasma pneumoniae was generated by using bacteriophage lambda EMBL3 as the vector. Screening of the library for the expression of M. pneumoniae protein antigens with adsorbed anti-M. pneumoniae serum revealed strong reactivity from a third of those clones which contained mycoplasma DNA inserts. Three of the most highly reactive clones were analyzed in detail and found to synthesize discrete mycoplasma proteins. Two carried overlapping fragments of mycoplasma DNA which encoded a protein that was readily detected in Escherichia coli after infection with recombinant bacteriophage. The third clone contained a novel mycoplasma DNA fragment which directed the synthesis of two additional mycoplasma proteins. Further screening of the library with antiserum raised against the major M. pneumoniae adhesin protein P1 (165 kilodaltons [kDa]) yielded one clone which produced an immunologically reactive protein of 140 kDa. Adsorption of anti-P1 serum by this clone selected a population of antibodies that were reactive with M. pneumoniae adhesin P1 (165 kDa). These results demonstrate that immunologically active M. pneumoniae proteins are synthesized in E. coli.     

Purification and antigenic relatedness of proteins II of Neisseria gonorrhoeae.

Gonococcal proteins II from three strains were purified by chromatofocusing, and antisera was raised against them. These antisera were examined by immunoblotting to explore the antigenic relatedness of proteins II of seven different strains. The strongest reactions of the antisera were with the homologous proteins II. The antiserum against the proteins II of one strain also reacted with the proteins II present in all of the heterologous strains, whereas the antisera against the proteins II of two other strains showed little cross-reactivity with heterologous proteins II. Monoclonal antibodies produced against the three proteins II of strain F62 were specific for homologous proteins II and recognized epitopes unique to each individual protein II. These studies confirm the extensive intra- and interstrain variability in the antigenic structure of these proteins.