Leupeptin alters the proteolytic processing of P126, the major parasitophorous vacuole antigen of Plasmodium falciparum

Among several protease inhibitors tested, only leupeptin was found to modify qualitatively the processing of P126, a major antigen of the parasitophorous vacuole of Plasmodium falciparum, and to inhibit the release of merozoites. Whereas P126 is normally processed upon merozoite release into 2 polypeptides of 50 and 73 kDa which are discharged in the culture medium, leupeptin treatment led to the recovery of a 56 kDa fragment which was recognized by a monoclonal antibody specific for the 50 kDa polypeptide and of a 73 kDa fragment comigrating with the one obtained in normal culture conditions. Mild trypsinization of the 56 kDa polypeptide gave rise to a 50 kDa product the tryptic fragments of which comigrated with those of the 50 kDa antigen obtained from untreated cultures.     

A component of an antigenic rhoptry complex of Plasmodium falciparum is modified after merozoite invasion

Human antibodies affinity purified on an adsorbent prepared from a cDNA clone (Ag44) expressing a portion of a rhoptry antigen were used to characterize the synthesis and fate of the antigen in the asexual blood stages of Plasmodium falcipartum. The rhoptry antigen is synthesized in the mature trophozoite-stage parasites as a 103 kDa polypeptide, is present in the schizonts and merozoites as a 105 kDa polypeptide, is discharged from the rhoptries and found in the newly invaded red cells as a 110 kDa polypeptide. Anti-Ag44 antibodies immunoprecipitate the antigen and two additional polypeptides of 135 and 150 kDa from lysates of infected cells and from culture supernatants. The three polypeptides are associated in a non-covalent complex that persists in the newly invaded red cells. All the components of the high molecular weight rhoptry complex are antigenic and can be precipitated with immune human serum. The 135 kDa polypeptide is identical to a 140 kDa rhoptry antigen previously identified by a monoclonal antibody.     

A component of an antigenic rhoptry complex of Plasmodium falciparum is modified after merozoite invasion

Human antibodies affinity purified on an adsorbent prepared from a cDNA clone (Ag44) expressing a portion of a rhoptry antigen were used to characterize the synthesis and fate of the antigen in the asexual blood stages of Plasmodium falcipartum. The rhoptry antigen is synthesized in the mature trophozoite-stage parasites as a 103 kDa polypeptide, is present in the schizonts and merozoites as a 105 kDa polypeptide, is discharged from the rhoptries and found in the newly invaded red cells as a 110 kDa polypeptide. Anti-Ag44 antibodies immunoprecipitate the antigen and two additional polypeptides of 135 and 150 kDa from lysates of infected cells and from culture supernatants. The three polypeptides are associated in a non-covalent complex that persists in the newly invaded red cells. All the components of the high molecular weight rhoptry complex are antigenic and can be precipitated with immune human serum. The 135 kDa polypeptide is identical to a 140 kDa rhoptry antigen previously identified by a monoclonal antibody.     

Identification and purification of novel internalin-related proteins in Listeria monocytogenes and Listeria ivanovii.

Monoclonal antibodies were generated against a 30-kDa protein fraction derived from culture supernatants of a Listeria monocytogenes strain complemented with additional copies of the prfA regulator gene. Several of the antibodies reacted specifically with a hitherto unidentified, secreted 30-kDa polypeptide. By immunoblot analysis, the expression of this 30kDa polypeptide was found to be dependent on the presence of the PrfA regulator protein. Microsequencing of peptides derived from the partially purified 30-kDa protein revealed homologies to the InlA and InlB polypeptides of L. monocytogenes, which are required for the internalization of the bacteria into nonphagocytic cell lines. This prompted us to term the 30-kDa polypeptide internalin-related protein (Irp). Irp-specific monoclonal antibodies cross-reacted with a 24-kDa polypeptide present in culture supernatants of Listeria ivanovii, indicating the existence of an Irp-related protein in this pathogenic Listeria species.     

Identification of a Trypanosoma cruzi antigen that is shed during the acute phase of Chagas' disease

A Trypanosoma cruzi antigen which is shed into the culture medium by the trypomastigote stage of the parasite and detected in blood of acutely infected mice was cloned and characterized. We designate this antigen shed acute phase antigen (SAPA). Five protein bands with apparent molecular masses ranging from 160 to 200 kDa were detected by immunoblotting of plasma from infected mice and in supernatants of cultured trypomastigotes upon reaction with antibodies against SAPA. A serum obtained from a patient acutely infected with Chagas' disease revealed a similar set of polypeptides in supernatants of cultured trypomastigotes when tested by immunoblotting. SAPA seems thus to be a major shed protein during the acute period of the disease. Twenty-six of 28 sera from human acute cases of Chagas' disease tested reacted with SAPA. Conversely, only 8-10% of sera from chronic cases of the disease contained detectable levels of antibody against SAPA. Sera from rabbits infected with six different parasite strains all contained antibodies against SAPA. Antibodies against SAPA are detectable 15 days after the manifestation of acute Chagas' disease symptoms in humans and 15 days post-infection in sera from mice and rabbits. The nucleotide sequence of a genomic clone encoding the 3' end of the SAPA gene revealed the presence of 14 tandemly arranged 12-amino acid-long repeats. A 39-amino acid-long region that is very hydrophobic precedes the stop codon. Due to its early appearance it might be possible to design diagnostic assays which are based on SAPA for identification of recently infected cases of Chagas' disease.     

Stage and strain specific expression of the tandemly repeated 90 kDa surface antigen gene family in Trypanosoma cruzi

A recombinant cDNA library constructed in the expression vector lambda gtll using mRNA from the trypomastigote stage of Trypanosoma cruzi was screened with two monoclonal antibodies that have been shown to react with a 105 kDa and a 90 kDa surface antigen in trypomastigotes of the Peru and Y strains of T. cruzi. One recombinant lambda phage, designated Tcc-20, was reactive to both monoclonals. The beta-galactosidase/T. cruzi hybrid protein encoded in Tcc-20 is recognized by the monoclonal antibodies and by serum antibodies from mice infected with strains of T. cruzi which contain the 90 kDa antigen. Antibodies immunoselected from serum of mice infected with the Peru strain by adsorption to Tcc-20 fusion protein react specifically with a 90 kDa polypeptide in trypomastigote but not epimastigote lysates of T. cruzi. The mRNA complementary to the DNA insert in Tcc-20 is present only in those stages and strains of T. cruzi which express the 90 kDa surface antigen. These characteristics are strong evidence that the T. cruzi DNA fragment cloned into Tcc-20 encodes a portion of the 90 kDa surface antigen. The gene(s) which encodes this polypeptide is shown to be present in approximately 20 copies per haploid genome and most, and possibly all, of the copies are found in a tandemly linked multigene family.     

Intramolecular mapping of Plasmodium falciparum P126 proteolytic fragments by N-terminal amino acid sequencing.

Protein P126 (also called P140, P113, SERA, SERP1) is a major parasitophorous vacuole antigen of Plasmodium falciparum. This protein is processed upon merozoite release into 2 fragments of 73 kDa (P73) and 50 kDa (P50), which are found in the culture medium. P73 is composed of 2 polypeptides of 47 and 18 kDa linked by disulfide bridges. In the presence of leupeptin, an inhibitor of serine and cysteine proteases which inhibits merozoite release, a 56-kDa intermediate product (P56) is recovered in the culture medium instead of P50. In order to map these proteolytic fragments on the 126-kDa precursor, we purified them from Plasmodium falciparum culture medium by immunoadsorption, SDS-electrophoresis and Western blotting on PVDF membrane and determined the N termini of P126, P73 (P47 and P18), P50 and P56. Comparison of these sequences with the amino acid sequence deduced from the P126 gene allowed the mapping of the different fragments on the precursor. P47 was at the N-terminal and P18 at the C-terminal end of P126. P56 and P50 had the same N-termini and were located in the middle of P126. This latter result indicates that the proteolysis of P56-P50 occurs at the C-terminus of P56. The peptide bonds cleaved by leupeptin-insensitive activities are Glu-Thr and Gln-Asp; C-terminal sequencing of P50 will be needed to identify the leupeptin-sensitive cleavage site.     

Characterization of a Plasmodium falciparum polypeptide associated with membrane vesicles in the infected erythrocytes

A Plasmodium falciparum polypeptide (46 kDa) associated with the infected erythrocytes of all asexual stages as well as immature gametocytes was identified by the monoclonal antibody (Mab) 30B8.3. The expression of this protein was not dependent upon the knobby phenotype and was detected in parasites grown either in human or Aotus erythrocytes. The antigen was heatstable, did not label with [14C]glucosamine, and was not sensitive to periodate oxidation. Immunofluorescent staining patterns of Mab 30B8.3 on in vitro cultured parasites varied from punctate (rings and trophozoites) to patchy (trophozoites and schizonts) fluorescence. The Mab 30B8.3 antigen was not detected on the infected erythrocyte surface by conventional wet-mount IFA procedure. However, when parasites were cultured in the presence of Mab 30B8.3, the epitope was detected by the monoclonal antibodies present in the culture medium. Differential extraction of the polypeptide from infected erythrocytes and immune electron microscopy of cryosectioned parasites localized the 30B8.3 epitope primarily on membranes of Maurer's clefts within the infected erythrocyte's cytosol. This 46 kDa polypeptide is unique because it seemed to be an integral membrane protein of the Maurer's clefts/vesicles and it was not secreted into the culture medium nor deposited on the infected erythrocyte membrane. Previous studies indicate that several parasite proteins, excreted extracellularly or deposited on infected erythrocyte membrane, are found to be associated with Maurer's cleft membranes and vesicles. The 46 kDa polypeptide described in this study may play an important role in the transport of the parasite antigens.     

Onchocerca volvulus larval antigen, OvB20, induces partial protection in a rodent model of onchocerciasis.

OvB20 is an antigen of Onchocerca volvulus preferentially recognized by sera from cattle vaccinated with irradiated infective larvae of Onchocerca lienalis. Antibodies raised against the recombinant protein were used to characterize the expression of the native protein in different developmental stages of O. volvulus and the rodent filaria Acanthocheilonema viteae. In O. volvulus, antibodies reacted to a polypeptide of 42 kDa in microfilariae and with proteins of 52 and 65 kDa in third-stage larvae. No products were detected in adult stages. Immunogold electron microscopy localized the native protein to discrete patches of the hypodermis and cuticle of infective larvae. Characterization of a homologous protein in A. viteae confirmed the stage-specific expression in infective larvae of the 65-kDa protein, which was secreted during in vitro culture. Vaccination of rodents against A. viteae with a B20-maltose-binding-protein fusion protein resulted in a 49 to 60% reduction in adult worm recoveries with a corresponding 97% reduction in microfilaremia.     

Enhanced production of monoclonal antibodies against zona pellucida-free, unfertilized mouse oocytes by intrasplenic immunization.

Monoclonal antibodies were obtained by intrasplenic insertion of a total of 40-50 zona pellucida-free, unfertilized mouse oocytes into each mouse. The oocytes were prepared by a combined enzyme-mechanical method without impairing the fertility of the oocyte or inducing parthenogenesis. Of a total of 1063 hybrids obtained, 15 yielded supernatants with positive reactions to unfertilized oocytes. Tests for preimplantation stage specificity demonstrated that 9 of the supernatants detected antigen epitopes in unfertilized oocytes only. The other supernatants showed positive reaction patterns to various embryonal stages of the preimplantation period. Two supernatants recognized materials in the culture medium shedded by zona pellucida-free oocytes. Thus, by applying intrasplenic immunization, the production of antibodies against zona pellucida-free mouse oocytes is enhanced. The procedures described outline a rational way of selecting antibodies of interest for research in the mechanism of fertilization.     

Expression of HLA-DR and common acute lymphoblastic leukemia antigens on glioma cells

The expression on several established human glioma cell lines of two well-defined differentiation antigens, HLA-DR and the common acute lymphoblastic leukemia antigen (CALLA) has been demonstrated. Rabbit anti-CALLA antiserum and monoclonal anti-la antibodies specifically lysed glioma cells in the presence of complement. Absorption of anti-CALLA antiserum and anti-Ia antibodies by glioma cells abolished their cytotoxicity against blasts isolated from a common acute lymphoblastic leukemia. Immunoprecipitation of solubilized glioma cells by monoclonal anti-Ia antibodies revealed two polypeptide chains of 28 and 33 kDa, whereas the anti-CALLA antiserum precipitated a single polypeptide chain of 100 kDa.     

The biosynthesis of the knob protein and a 65 000 dalton histidine-rich polypeptide of Plasmodium falciparum

Previous studies have shown the association of an 80 kDa polypeptide (KP) with the knobs which develop on the membranes of erythrocytes infected with Plasmodium falciparum. KP was also found to share antigenic determinants with the histidine-rich protein of Plasmodium lophurae. In this study, ring stages of knobby (K+) and knobless (K-) variants of P. falciparum were used in pulse-chase experiments to elucidate the temporal sequence of the biosynthesis of KP. Analysis of radiolabeled parasite-polypeptides on SDS-polyacrylamide gels indicated that pulse-labeled KP has the electrophoretic mobility of a 75 kDa polypeptide and is subsequently chased to an apparently 80-85 kDa form. In addition to KP, antibodies raised against HRP immunoprecipitated a 65 kDa histidine-rich polypeptide from K- as well as K+ parasites. Differential incorporation of selected amino acids into KP and the 65 kDa polypeptide revealed some distinct differences between these two polypeptides as well as from HRP.     

Monospecific antibodies to Marek's disease virus antigen B dimer (200 kDa) and monomer (130 and 60 kDa) glycoproteins neutralize virus infectivity and detect the antigen B proteins in infected cell membranes

Summary Monospecific antibodies were prepared by nitrocellulose blot immunoaffinity to 3 polypeptide components of the host-membrane associated B antigen of Marek's disease herpesvirus (MDV) and to its soluble A antigen. The B antigen comprised a 200 kDa dimer which is 2-mercaptoethanol (2-ME) labile, a monomer of 130 kDa and a 60 kDa protein, both of which are 2-ME resistant. Cross-immunoblotting studies showed that the anti-dimer antibody recognized the dimer protein as well as the 130 and 60 kDa components. In contrast, the anti-130 kDa antibody gave the strongest signal on blots of reducing gels indicating that the monomer is largely formed by in vitro reduction with 2-ME. All four antibodies recognized membrane antigens on chicken embryo fibroblasts infected with MDV vaccine viruses representative of the three serotypes and in addition, neutralized the homologous MDV isolate. The anti-dimer antibody was greatest, the anti-monomer antibody was the weakest and the anti-60 kDa antibody intermediate in neutralizing efficacy to all four viruses. We conclude from these studies that the B antigen presents at least two classes of neutralizing epitopes: one is discontinuous and of broad specificity on the intact dimer molecule and the other, on the 130 and 60 kDa proteins, is continuous and of lower avidity.     

Cross-reactive antigens shared by Pseudomonas aeruginosa, Helicobacter pylori, Campylobacter jejuni, and Haemophilus influenzae may cause false-positive titers of antibody to H. pylori.

Cystic fibrosis (CF) patients suffer from many of the gastrointestinal conditions which occur in non-CF individuals, e.g., dyspepsia and peptic ulceration. These symptoms may be caused by Helicobacter pylori but could also be due to either pancreatic insufficiency or the intensive antibiotic treatment used in CF patients. Since CF patients chronically infected with Pseudomonas aeruginosa produce antibodies against a wide range of antigens, including antigens common to many other bacteria, e.g., GroEL and lipopolysaccharide, we studied, by the Western blot (immunoblot) technique, the specificity of immunoglobulin G antibodies to H. pylori in Danish CF patients chronically infected with P. aeruginosa, CF patients without P. aeruginosa infection but with Haemophilus influenzae infection, patients with dyspeptic ulcers associated with H. pylori, and patients recovering from acute Campylobacter jejuni or Campylobacter coli infection. Sera from CF patients with chronic P. aeruginosa or H. influenzae infection and patients recovering from acute C. jejuni infection cross-reacted with H. pylori antigens. A strong cross-reacting protein antigen at approximately 14 kDa and minor cross-reactive antigens at approximately 27, 30, and 60 kDa (the heat shock protein GroEL is equivalent to the common antigen of P. aeruginosa) could be demonstrated. The results of this study show that high immunoglobulin G antibody titers against H. pylori in CF patients cannot be regarded as indicating present or past H. pylori infection unless their specificity is proven by absorption studies.     

Identification of circulating antibodies in fasciolosis and localization of 66 kDa antigenic target using monoclonal antibodies.

We identified three specific circulating antibodies in serum of cattle naturally infected with Fasciola gigantica. Two of the antibodies were found to react specifically to 97 and 66 kDa antigenic molecules of adult worm tegumental membrane extract. The third antibody was identified by the reaction with 26-28 kDa molecule of the excretory/secretory antigens. Monoclonal antibody against 66 kDa protein was developed and used for localization of its antigenic target in adult worm frozen sections. The experiment demonstrated that 66 kDa protein is a component on the outer surface membrane and on the membrane lining of the caecal epithelial of adult worm. The 66 kDa antigen was considered as a promising candidate for immunodiagnosis and vaccine.     

Accumulation of the 126 kDa protein of tobacco mosaic virus during systemic infection analysed by immunocytochemistry and ELISA

Summary Systemic infection of tobacco with tobacco mosaic virus (TMV) strain WU1, is accompanied by massive accumulation of the virus-coded non-structural 126 kDa protein in X-bodies. The development of X-bodies and the time course of the increase in 126 kDa protein in systemically infected leaves were analyzed by immunocytochemistry and ELISA, respectively, using an antiserum raised against a fusion protein of -galactosidase and part of the 126 kDa protein. The ELISA assay developed enabled routine detection of viral 126 kDa (as well as 183 kDa) protein in samples of less than 5 mg of systemically infected leaves. Plants were inoculated by differential temperature treatment, whereafter the accumulation of 126 kDa protein was related to viral multiplication, the development of X-bodies and the formation of symptoms. Both 126 kDa protein and coat protein became detectable between 40 and 66 h after transfer of the plants and increased in parallel up to 200 h. Vein clearing was visible at 66 h, followed by mosaic in the newly developed leaves at 112 h. By electron microscopical analysis small X-bodies, weakly labelled with antibodies against the 126 kDa protein, were detected as early as 24 h after transfer. At this stage they were not associated with nuclei. Thereafter, however, X-bodies increased in size and 126 kDa labelling density, and were increasingly often observed attached to nuclei. In emerging leaves that developed mosaic symptoms, X-bodies were associated with nuclei already at an early stage. These observations are consistent with the hypothesis that association of X-bodies with nuclei may lead to symptom induction, when the leaf is invaded by the virus early in its development.     

A 50 kilodalton exoantigen specific to the merozoite release-reinvasion stage of Plasmodium falciparum

The immunoglobulins G of a human plasma inhibiting in vitro Plasmodium falciparum merozoite reinvasion have been purified and used to immunoprecipitate the antigens released into the culture medium by an [35S]methionine-labeled synchronous culture. Several of the major exoantigens identified were found throughout the entire life cycle; they were also immunoprecipitated from the labeled parasitized cells. Some antigens were found only after the reinvasion stage, and especially a major one of molecular mass 50 kDa and pI 5.5. The latter was not found in the parasitized cells but derived most likely from the processing of a major 126 kDa antigen which disappeared from the parasites during the reinvasion period and which was immunoprecipitated by an anti-50 kDa monoclonal antibody.     

Expression of 120 kilodalton protein and cytotoxicity in Helicobacter pylori.

Antral biopsy culture supernatants from 14 subjects with chronic gastritis, known to have IgA antibodies to the 120 kilodalton protein, showed positive recognition of this antigen in western blots against a cytotoxin positive strain of Helicobacter pylori but gave negative reactions with two cytotoxin negative strains. Control immunoblots with culture supernatants from 13 non-responders to the protein were all negative. This indicates a direct association between expression of the 120 kilodalton protein in H pylori strains and cytotoxicity.     

The 56-kilodalton major protein antigen of Rickettsia tsutsugamushi: molecular cloning and sequence analysis of the sta56 gene and precise identification of a strain-specific epitope.

Lasting immunity against Rickettsia tsutsugamushi, the causative agent of scrub typhus fever, has been demonstrated to be strain specific. Two protein antigens of 110 and 56 kilodaltons (kDa) have been shown to exhibit strain-specific epitopes. The 56-kDa scrub typhus antigen (Sta56) is an abundant outer membrane protein of R. tsutsugamushi and is an antigen often recognized by humans infected with this obligate intracellular bacterium. In this study the complete gene encoding Sta56 (strain Karp) was cloned into pBR322 on a 2.3-kilobase genomic HindIII DNA fragment and the complete 56-kDa polypeptide was expressed in Escherichia coli. DNA sequence analysis of the 2.3-kilobase HindIII fragment revealed an open reading frame large enough to encode a 56-kDa polypeptide. A putative signal sequence was identified at the deduced amino terminus of the Sta56 polypeptide, and pulse-chase analysis of maxicells labeled with [35S]methionine demonstrated that a higher-molecular-weight precursor matures into the 56-kDa polypeptide. Epitope scanning analysis with synthetic peptides derived from the deduced amino acid sequence identified an octapeptide (located from amino acid residues 117 to 124) that was reactive with a Karp strain-specific monoclonal antibody (K13F88A). Other epitopes recognized by different monoclonal antibodies, including another Karp strain-specific monoclone (K1E106), were localized to different regions of the protein based on their reactivities with lambda gt11 recombinants expressing various portions of the sta56 gene.     

Characterization of a surface antigen of Eimeria tenella sporozoites and synthesis from a cloned cDNA in Escherichia coli

An antigenic surface protein of Eimeria tenella sporozoites has been identified that is the target of two neutralizing monoclonal antibodies Ptn 7.2A4/4 and Ptn 9.9D12. The antigen as isolated from the parasite is composed of a 17 kDa polypeptide and a 8 kDa polypeptide linked by a disulfide bridge. De novo synthesis of the antigen does not begin until approximately 16-20 h after the initiation of oocyst sporulation. A cDNA library was constructed using mRNA from sporulated oocysts and a clone encoding the antigen was isolated. The Ta4 gene encodes a single polypeptide of 25 kDa which contains the 17 and 8 kDa polypeptides. The protein has been synthesized in Escherichia coli either directly or as part of a beta-galactosidase fusion protein. The products synthesized in E. coli are single polypeptides and are not cleaved to two polypeptides as is seen in the parasite. The products accumulate in bacteria in an insoluble form which can be solubilized and renatured to an immunoreactive form.