Specificities of antibodies that inhibit merozoite dispersal from malaria-infected erythrocytes

When malaria schizont-infected erythrocytes are cultured with immune serum, antibodies prevent dispersal of merozoites, resulting in the formation of immune clusters of merozoites (ICM) and inhibition of parasite growth. Antigens recognized by these antibodies were identified by probing two dimensional immunoblots of Plasmodium falciparum antigens with antibodies dissociated from immune complexes present at the surface of merozoites in ICM. Total immune serum recognized 88 of the 135 protein spots detected by colloidal gold staining, but antibodies dissociated from immune complexes recognized only 15 protein spots attributable to no more than eight distinct antigens. Antigens recognized by antibodies that inhibit merozoite dispersal include the precursor to the major merozoite surface antigens (gp195), a 126-kDa serine-repeat antigen (SERA), the 130-kDa protein that appears to bind to glycophorin (GBP130), and the approx. 45-kDa merozoite surface antigen. One other antigen (230/215-kDa doublet) was identified by using antibodies affinity purified from recombinant expression proteins. The identities of the other three antigens (150 kDa, 127 kDa and less than 30 kDa) were not determined. This approach provides a strategy for identifying epitopes accessible at the merozoite surface which may be important components of a multivalent vaccine against blood stages of P. falciparum.     

Characterization of surface antigens ofEimeria nieschulzi (Sprozoa,Eimeriidae) merozoites

Two surface antigens ofEimeria nieschulzi merozoites were characterized by monoclonal antibodies (Mcab) as follows: an antigen of 31 (reducing conditions), 25 and 31 kDa (nonreducing conditions), by Mcab A1F6; and an antigen of 52 and 30 kDa (reducing conditions) or 42 kDa (nonreducing conditions), by Mcab H7. The localization of the antigens in fluorescence and electron micrographs is presented. Despite being accessible to antibodies on live (nonpermeabilized) parasites, neither antigens were significantly labelled using lactoperoxidase radioiodination.     

Antibodies Reactive with the N-Terminal Domain of Plasmodium falciparum Serine Repeat Antigen Inhibit Cell Proliferation by Agglutinating Merozoites and Schizonts

The serine repeat antigen (SERA) is a vaccine candidate antigen of Plasmodium falciparum. Immunization of mice with Escherichia coli-produced recombinant protein of the SERA N-terminal domain (SE47') induced an antiserum that was inhibitory to parasite growth in vitro. Affinity-purified mouse antibodies specific to the recombinant protein inhibited parasite growth between the schizont and ring stages but not between the ring and schizont stages. When Percoll-purified schizonts were cultured with the affinity-purified SE47'-specific antibodies, schizonts and merozoites were agglutinated. Indirect-immunofluorescence assays with unfixed parasite cells showed that SE47'-specific immunoglobulin G (IgG) bound to SERA molecules on rupturing schizonts and merozoites but the IgG did not react with the schizont-infected erythrocytes (RBC). Furthermore, double-fluorescence staining against SE47'-specific IgG and anti-human RBC membrane IgG showed that the RBC membrane disappeared from SE47'-specific-IgG-bound schizonts after cultivation. These observations suggest that the SE47'-specific antibodies inhibit parasite growth by cross-linking SERA molecules that are associated with merozoites in rupturing schizonts with partly broken RBC and parasitophorous vacuole membranes, blocking merozoite release.     

Identification of the sporozoite antigens of Eimeria tenella

The surface membranes of Eimeria tenella sporozoites were labelled with 125I and polypeptides resolved by polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS-PAGE). The most heavily labelled polypeptides were 47, 26, 21 and less than or equal to 18 kDa but significant amounts of 125I were incorporated into a number of polypeptides with molecular weights ranging from greater than 200 000 to less than 18 000. Similar 125I-polypeptide profiles were observed after the surface labelling of sporozoites of E. acervulina, E. maxima and E. nieschulzi. Sporozoites of E. tenella were also radiolabelled by incubation in medium containing [35S]methionine and SDS-PAGE resolved more than 35 radiolabelled polypeptides with molecular weights from greater than 200 000 to less than 18 000. 125I and 35S-labelled sporozoites of E. tenella were solubilised in the detergents Triton X-100 or sodium deoxycholate and immunoprecipitated with serum from chickens immunized by infection with this species. Polypeptides of unlabelled E. tenella sporozoites, resolved by SDS-PAGE, were blotted onto nitrocellulose and the antigens, which reacted with the chicken serum, identified by immunoperoxidase staining. There was some variation between different sporozoite preparations in the number and molecular weights of antigens identified by these techniques but, consistently, the major surface polypeptides that were specifically immunoprecipitated were 104, 47 and 43 kDa. Specifically immunoprecipitated 35S-labelled antigens were of 123-94 kDa, 54-42 kDa and 32-25 kDa and antigens detected on Western blots were within the following ranges: 113-96 kDa, 73-67 kDa, 54-42 kDa, 37-32 kDa and 18-14 kDa.     

Fragments of the polymorphic Mr 185,000 glycoprotein from the surface of isolated Plasmodium falciparum merozoites form an antigenic complex

Merozoites of the human malaria parasite Plasmodium falciparum express on their surface several antigens derived from a polymorphic glycoprotein precursor of Mr 185,000 synthesised earlier on by trophozoites and schizonts. A panel of 18 monoclonal antibodies against a range of different specificities of the precursor was used to characterise its mature products in spontaneously released merozoites. Merozoites released by [35S]methionine or [14C]glucosamine-labelled schizonts, or surface 125I-labelled purified merozoites, were extracted in detergents, and the antigens were detected by immunoprecipitation or Western blotting. We show that a nonglycosylated peptide of Mr 80,000 and two glycosylated fragments of Mr 40,000 and Mr 16,000, all derived from the precursor, are exposed on the surface of the mature merozoite. Precipitations from extracts in different detergents indicate that the 80 and 40 kDa fragments can form a non-covalent complex with each other and two additional major surface antigens of 36 and 22 kDa. Several antibodies react strongly with the complex but not with its dissociated subunits, thus indicating presence of conformational epitopes. Other epitopes are positively mapped on different dissociated subunits by immunoprecipitation and Western blotting. The 80 and 40 kDa antigens each carry a different polymorphic marker epitope, and both of these markers are absent on the 16 kDa fragment. The 40 and 16 kDa glycoproteins share common epitopes, and the latter may be derived from the former fragments. Only epitopes present on the 16 kDa antigen, but not those specific for the larger fragments, are detectable by immunofluorescence in the ring-stage. This indicates that the whole or a part of the 16 kDa antigen remains on the parasite through the invasion process.     

Sheep lymphocyte antigens (OLA). I. Major histocompatibility complex class I molecules

Three monoclonal antibodies, SBU.I 41-17, 41-19 and 41-28, have been produced which recognize sheep Class I major histocompatibility complex antigens (OLA). All three antibodies are able to precipitate a heavy chain of 44,000 MW and a smaller beta 2-microglobulin of 12,000 MW from 125I-surface labelled lymphocytes. The antibodies have been used to localize OLA Class I antigens in lymphoid and non-lymphoid tissues using indirect immunoperoxidase histological staining and cytofluorograph analyses. Evidence suggests that the three antibodies are directed against monomorphic determinants but that they recognize different epitopes.     

cDNA encoding an immunogenic region of a 22 kilodalton surface protein of Eimeria acervulina sporozoites

cDNA encoding an immunogenic region of a 22 kDa surface protein of Eimeria acervulina sporozoites was cloned and expressed in the bacteriophage lambda gt11 vector. The recombinant beta-galactosidase fusion protein, designated MA1, has an apparent molecular size of 125 kDa. Immunofluorescence staining of intact E. acervulina sporozoites and merozoites and immunoblotting of 125I-surface labeled protein from both stages revealed exclusive expression of the cloned cDNA in the sporozoite stage. The gene encoding the 22 kDa surface protein appears to exist as a single copy sequence as revealed by Southern blot hybridization utilizing the cDNA insert as a probe. Although not recognized by immune serum, purified recombinant MA1 antigen induced significant in vitro activation of T lymphocytes obtained from chickens immune to E. acervulina. DNA sequencing and hydropathic analysis of the predicted amino acid sequence revealed a central hydrophilic region surrounded by two hydrophobic areas which may represent exposed and transmembrane regions of the protein.     

Intraerythrocytic development and antigenicity of Plasmodium falciparum and comparison with simian and rodent malaria parasites

Using sorbitol-synchronised cultures and metabolic labelling with [35S]methionine, the stage specificity of polypeptides synthesised by the intraerythrocytic stages of Plasmodium falciparum was studied. We confirmed that the synthesis of many polypeptides is restricted to defined morphological stages of parasite development, while other polypeptides are synthesised more or less throughout the cycle. The synthesis of at least 6 polypeptides was confined to the period of differentiation of mature trophozoites to schizonts and merozoites. Polypeptides synthesised by a cloned long-term passage isolate were very similar to those of a recently cultured uncloned isolate. Comparison of polypeptides synthesized during differentiation of mature trophozoites to schizonts and merozoites by P. falciparum with those of P. chabaudi and P. knowlesi showed that while P. chabaudi and P. knowlesi synthesised a 250 000 molecular weight polypeptide at this stage the apparently equivalent polypeptide of P. falciparum was of significantly lower molecular weight being 200 000. Using a surface immunoprecipitation technique, it was shown that this 200 000 mol. wt. polypeptide was accessible to antibodies on the surface of erythrocytes infected with mature trophozoites and schizonts. A 150 000 mol. wt. polypeptide was also accessible to antibodies. By comparing polypeptides synthesised during the differentiation of mature trophozoites to schizonts and merozoites with those recovered in the ring stage parasites after schizogony and erythrocyte invasion, it was shown that this 200 000 mol. wt. polypeptide and 140 000 and 120 000 mol. wt. polypeptides were not taken into the erythrocyte by the invading merozoite. The importance of these polypeptides in terms of the parasite biology and in the induction and expression of immunity to malaria is discussed.     

Immunoglobulin synthesis by human peripheral lymphocytes and thymocytes in vitro. Specificity of immunochemical methods and stimulation by phytohaemagglutinin

Antibody–antigen precipitates were formed in solutions of radioactive products of human lymphocytes. The amount of radioactive material co-precipitating with non-specific, apparently unrelated, antibody–antigen precipitates was of the same magnitude as that co-precipitating with related, specific precipitates of IgG–anti-IgG or Tg–anti-Tg. Treatment of the solutions with an unrelated EA–anti-EA precipitate readily removed material capable of reacting with IgG–anti-IgG or Tg–anti-Tg precipitates.

By contrast the evidence obtained with the technique of autoradiography of immunoelectrophoresis patterns indicated that the labelling of immunoglobulin arcs, and of a specific antibody–antigen precipitate, was specific. Only products of the lymphocyte from a subject with Hashimoto's disease labelled a Tg–anti-Tg line, although the control preparations from normal lymphocytes labelled the immunoglobulin arcs on the other side of the pattern. When mouse serum was used as carrier, and the immunoelectrophoresis was developed with rabbit anti-mouse serum, the immunoglobulin lines were not labelled. Products of human lymphocytes did not label an EA–anti-EA line in the immunoelectrophoresis.

The anodal ends of gels were eluted after electrophoresis of radioactive products synthesized by lymphocytes. The amount of material co-precipitating in these eluates with specific precipitates was 1·8 times greater than that adhering to non-specific precipitates. These data raise the possibility that immunoglobulin molecules may be non-specifically adsorbed to unrelated antigen–antibody precipitates.

The data indicated that phytohaemagglutinin stimulated synthesis of immunoglobulin was less than the overall increase of protein synthesis. Phytohaemagglutinin sometimes stimulated the total protein synthesis while inhibiting IgG synthesis.

     

Identification of a novel allergen molecule from Dermatophagoides by monoclonal antibodies

Six different monoclonal antibodies (MAb) have been obtained which bind to components contained in extracts from Dermatophagoides. One out of the six MAb recognized molecules displaying IgE binding ability. This MAb (MADP-1) immunoprecipitated allergenic polypeptides of 42 kDa and 30 kDa from 125I-labeled extracts of D. pteronyssinus and D. farinae respectively. Purified allergen preparations from both extracts have been obtained by affinity chromatography using a column of purified MADP-1 coupled to Sepharose. The purified fractions partially compete the binding of specific IgE contained in sera from sensitized patients to the whole extracts, in a radioallergosorbent inhibition test.     

Interaction between the CD45 antigen and phytohemagglutinin. Inhibitory effect on the lectin-induced T cell proliferation by anti-CD45 monoclonal antibody

Immunoprecipitation studies from 125I-labeled T cells, previously activated with the lectin phytohemagglutinin (PHA) from Phaseolus vulgaris, showed a preferential association between the CD45 glycoprotein family containing members of 220, 205, 190 and 180 kDa, and a protein of 33 kDa. This 33-kDa protein, with an identical molecular mass as PHA, was not associated with other highly expressed molecules such as class I histocompatibility antigens (HLA-A,B,C), transferrin receptor, CDw44 and CD11a. Further immunoprecipitation analysis from peripheral blood lymphocytes incubated with 125I-labeled PHA confirmed the identification of the CD45-associated 33-kDa protein as the lectin PHA. These results prompted us to analyze the possible involvement of the CD45 molecules in the T cell activation process induced by PHA. Thus, two monoclonal antibodies specific for CD45 antigens were able to inhibit the T cell proliferation induced by the lectin. This inhibitory ability was exerted by affecting both the interleukin 2 production and the interleukin 2 receptor expression.     

Active release of surface proteins: a mechanism associated with the immune escape of Acanthocheilonema viteae microfilariae

Living Acanthocheilonema viteae microfilariae obtained from peripheral blood of parasitised Meriones unguiculatus were surface-labelled with 125I. Four major surface exposed proteins of approximately 14.50, 14.55, 17.5, 19 kDa and one less abundant protein of 40 kDa were identified. Under non-reducing conditions the low-molecular-weight (LMW) proteins were isolated as multimers suggesting the presence of intermolecular disulphide linkages. In gels containing Triton X-100 the labelled epicuticular proteins behaved lipophilically. By cultivation of surface-labelled and metabolically labelled microfilaria in vitro, a continuous shedding of two LMW proteins was demonstrated. These proteins were produced in large amounts and released into the culture supernatant as monomeric and pentameric molecules. Concomitant with this release, one of the proteins appeared to lose its lipophilic character, giving rise to a hydrophilic 14.50-kDa entity. Although most of the extracted surface proteins reacted with sera from patent jirds, these sera failed to recognise the surface of living microfilariae. However, microfilariae pretreated with glutaraldehyde or attenuated with Na-azide could be labelled with surface specific antibodies.     

Identification of a molecule of Porphyromonas gingivalis that binds to Streptococcus gordonii

The molecules that mediate the adherence of Porphyromonas gingivalis, a periodontal pathogen, to Streptococcus gordonii, a commensal plaque organism, were investigated. Outer membrane proteins of P. gingivalis were labelled with biotin, extracted by EDTA and reacted with S. gordonii cells. Interactive porphyromonas components were identified by SDS-PAGE of the S. gordonii cells followed by electroblotting and visualization of the adsorbed porphyromonas molecules with streptavidin-alkaline phosphatase. A P. gingivalis molecule of 35 kDa bound to S. gordonii. Monospecific polyclonal antibodies to the 35 kDa protein inhibited binding of P. gingivalis to S. gordonii by 71%. The antibodies also reacted with the P. gingivalis fimbriae, indicating that the 35 kDa molecule is antigenically related to, or associated with, the fimbriae.     

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.     

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.     

The synthesis and fate of stage-specific proteins in Plasmodium falciparum cultures

Cultured ring, trophozoite and schizont stages of Plasmodium falciparum were metabolically labeled with [35S]methionine. After labeling, cultures were incubated for varying times in the presence of non-radioactive methionine. Triton-soluble proteins from different stages of growth were analysed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Most proteins were synthesized by every stage of growth and remained unchanged throughout the cycle through to the ring stage following merozoite invasion of erythrocytes. At least 15 proteins, most of high molecular weight, were synthesized solely or predominantly by schizonts. Eight proteins (approx. 177, 170, 158, 87, 83, 47, 41 and 24 kDa) appeared in schizonts but not merozoites. Eight proteins (approx. 240, 203, 106, 80, 35, 19, 15 and 14 kDa) appeared in merozoites, but not in rings following merozoite invasion. Some proteins appeared to be modified after synthesis.     

The identification and partial characterization of an immunodominant 29-31 kilodalton surface antigen expressed by adult worms of the human filaria Loa loa

Detergent solubilized extracts of 125Iodogen surface labelled adult Loa loa revealed a relatively simple profile consisting of a strongly labelled molecule at 29-31 kDa and weakly labelled molecules at 14.5, 17, 21, 23, 34, 58, and 86 kDa. Residents of a L. loa endemic zone were assessed clinically and parasitologically and classified as microfilaremic, amicrofilaremic with documented ocular passage of adult worms, or 'resistant' subjects without any signs of infection. Sera from these subjects were used to identify L. loa adult surface antigens. All 'resistant' sera immunoprecipitated the 29-31 kDa antigen although some were more strongly reactive than others. The amicrofilaremic sera strongly immunoprecipitated the 29-31 kDa antigen, whereas microfilaremic sera reacted weakly or not at all with this antigen. Longer exposures of immunoprecipitates of strongly reactive sera revealed the recognition of additional antigens of 86, 44, 34, 23, 21, 17 and 14.5 kDa. Studies with heterologous sera demonstrated that these antigens contain cross-reactive epitopes which are restricted to filarial parasites. Biochemical characterization of the predominant 29-31 kDa antigen showed that it bound concanavalin A, was sensitive to proteases, and its antigenicity was resistant to heat but sensitive to periodate and endo-beta-N-acetylglucosaminidase H. These observations suggest that it is a glycoprotein containing mannose and N-acetylglucosamine residues and that the carbohydrate moiety is important for antibody binding. The importance of the 29-31 kDa glycoprotein in the immunobiology of loaiasis is suggested by the finding that resistant and infected amicrofilaremic individuals have strongly reactive IgG antibodies to this antigen.     

Use of immunoblotting to characterize the mitochondrial antigens recognized by anti-mitochondrial autoantibodies

Sera with anti-mitochondrial autoantibodies detected by indirect immunofluorescence and/or enzyme-linked immunosorbent assay (ELISA) were examined by immunoblotting against pig heart mitochondria. Seven types of reactions were defined, according to the pattern of the labelled bands. Type I sera reacted with 12 bands located within four zones. The most intensively labelled bands were located at 70, 67, 58, 63 and 43 kDa. Other types gave decreasing band numbers. When beef heart mitochondria were used, sera belonging to each of the above types had a profile of labelled bands which sometimes differed from those obtained with pig heart mitochondria. When the chloroform extracted F1-ATPase from beef heart mitochondria was used to prepare the immunoblots, primary biliary cirrhosis (PBC) sera with anti-mitochondria antibodies reacted with all the bands although zone A bands were less labelled. Rat liver mitochondria gave seven bands with type I sera among which the 57 and 35 kDa bands were specific for rat liver mitochondria, as shown by absorption tests. Sera of PBC patients were also tested in immunoblotting against rat liver subcellular fractions including mitoplasts, submitochondrial particles, inner membrane, outer membrane, matrix proteins and inter-membrane proteins. Antigenic bands of A and B zones were localized in the inner membrane and/or in the matrix proteins and the 35 kDa band in inter-membrane proteins. The outer membrane gave no reaction. The most frequent anti-mitochondrial autoantibody types in PBC were type II, then I, whilst for chronic active hepatitis type III was the most common. Type V was only seen in a patient suffering from a typical PBC. Some sera from patients with syphilis, collagenous colitis or progressive systemic sclerosis labelled one or two bands distinct from those labelled by the PBC sera. Sera from patients with drug-induced hepatitis with endoplasmic reticulum antibodies and with systemic lupus erythematosus were generally found negative by immunoblotting.