The 3'' portion of the gene for a Plasmodium yoelii merozoite surface antigen encodes the epitope recognized by a protective monoclonal antibody.

The 230-kDa merozoite antigen of the murine malarial parasite Plasmodium yoelii provides a potential model system for the development of a protective erythrocytic stage vaccine. To characterize this antigen at the molecular level, isolated P. yoelii 17XL DNA was used to construct a genomic library in the expression vector lambda gt11. A monoclonal antibody, mAb 302, which passively protected mice against P. yoelii challenge infection, was used to identify a lambda gt11 recombinant clone encoding a portion of the 230-kDa antigen of this parasite. Using this clone as a probe, we identified an mRNA of 7.6 kilobases by RNA blot analysis. Nucleic acid sequence analysis of the clone showed that the epitope recognized by the protective mAb 302 is encoded by the 3' portion of the gene for the 230-kDa antigen. The deduced amino acid sequence revealed that this antigen also contains the tandemly repeated tetrapeptide Gly-Ala-Val-Pro, a series of 10 cysteine residues located within the terminal 110 amino acids, and a potential membrane anchor of 18 hydrophobic residues. Comparison of this C-terminal sequence with the carboxyl segment of the 195-kDa merozoite antigen of Plasmodium falciparum revealed nucleic acid and amino acid sequence similarities ranging from 40% to 70%. The localization of a B-cell epitope recognized by the protective mAb 302 to this carboxyl region of the P. yoelii antigen, combined with the limited strain variability in this region of the homologous 195-kDa antigen of P. falciparum, has implications for the development of an effective erythrocytic stage malarial vaccine.     

Low immunogenicity of a Plasmodium vivax circumsporozoite protein epitope bound by a protective monoclonal antibody.

The repeat region of the Plasmodium vivax circumsporozoite (CS) protein contains 20 copies of the nine-amino acid sequence DRA A/D GQPAG. A monoclonal antibody that passively protects monkeys against sporozoite challenge recognizes a four-amino acid linear sequence AGDR included within this nonamer, but when monkeys were immunized with a vaccine, NS1(81)V20, which contains 20 copies of the nonamer, they failed to produce antibodies to AGDR. To determine if natural exposure to sporozoites induces antibodies to AGDR, we tested sera from 176 individuals from a malaria-endemic area in Flores, Indonesia. Seventy-one percent of the adults had antibodies to the P. vivax repeat region; only 18% had detectable antibodies to AGDR. None of the subjects had antibodies to the P. vivax variant repeat ANGAGNQPG. We next tested sera from six human volunteers immunized with NS1(81)V20 and found that the vaccine, despite inducing antibodies against the nonamer, as it did in the monkeys, did not induce antibodies against AGDR. To further test our ability to raise anti-AGDR antibodies using synthetic peptides, we immunized Aotus monkeys and BALB/c mice with AGDR. Sera from the mice reacted strongly with both AGDR and a recombinant protein containing the 20 copies of the nonamer. Sera from the monkeys reacted only minimally with a protein (VIVAX-1) that contains monomeric AGDR within its sequence. Sera from the mice also bound air-dried P. vivax sporozoites, while sera from the monkeys did not.(ABSTRACT TRUNCATED AT 250 WORDS)     

A high molecular weight antigen in Plasmodium falciparum recognized by inhibitory monoclonal antibodies

Inhibitory monoclonal antibodies which bind to some isolates of Plasmodium falciparum from Papua New Guinea, but not from other areas, bound to a 220 kD antigen. By immunofluorescence microscopy this antigen was shown to be located both within the schizont cytoplasm and also within the schizont infected erythrocyte, but external to the schizont itself. Even at antibody concentrations which caused greater than 70% inhibition of parasite multiplication, accumulation of schizont stages or aggregates of merozoites were not seen, consistent with inhibition occurring at a point after the release of merozoites. While this suggests that the antigen may be present on merozoites, the quantity was below the limit of detection. It is suggested that the large amount of antigen released by rupturing schizonts may be a mechanism used by the parasite to evade immunological attack.     

Characterization of a differential immunoscreen epitope of Plasmodium falciparum using combinatorial agents

A differential serological screening of a lambdagt11 cDNA expression library has identified several clones, which react exclusively to sera samples from persons clinically immune to malaria but not to acute malaria patient sera. One such clone, IPf9, has a 315-bp cDNA insert, which was found to be conserved in different strains of the human and rodent malarial parasite Plasmodium falciparum and Plasmodium berghei, respectively. The induced expression product of IPf9 was used to generate polyclonal sera in rabbits. The IPf9 expression product was also screened with phage surface display combinatorial libraries to isolate reagents that specifically bound to the IPf9 product. The polyclonal antisera and the combinatorial reagents recognized a 50-kDa protein from P. falciparum, and a 53-kDa product from P. berghei. Immunofluorescence studies using asexual and sexual stages of P. falciparum showed the protein to be present within the parasite in each of the asexual and sexual stages. The combinatorial reagents showed a partial inhibition in the growth of P. falciparum in vitro. Mice infected with the P. berghei showed the presence of T-cells that exhibited lymphoproliferation when stimulated with the IPf9 protein. It is suggested that IPf9 protein is a conserved protein epitope, and may be relevant for a protective immune response to malaria.     

Blood-brain barrier protein recognized by monoclonal antibody.

An IgG1 mouse monoclonal antibody produced in response to immunization with rat brain homogenate reacted with endothelial cells in the central and peripheral nervous system. Because antibody reactivity was associated with endothelia that have a selective permeability barrier, the antibody was called anti-endothelial-barrier antigen (anti-EBA). Paraffin sections of Bouins'-fixed rat tissue were used for initial screening and subsequent characterization of antibody reactivity. The antibody was generally unreactive with endothelial cells in other organs and with nonendothelial cells in or outside of the nervous system. Antibody binding was greatly reduced or absent in endothelia of the area postrema and choroid plexus, sites known to possess fenestrated blood vessels. In developing rat brain, anti-EBA binding to some microvessels was seen at 3 days postnatally. Anti-EBA reactivity outside the nervous system occurred in spleen and skin. Patchy reaction with portions of some spleen blood vessels and binding to some cells in the spleen were observed. In the skin, small cells, tentatively identified as Langerhans cells, which participate in Ia presentation, were stained. On immunoblots of rat brain microvessel preparations electrophoresed in Na-DodSO4/polyacrylamide gels, anti-EBA reacted with a protein triplet of Mr 30,000, 25,000, and 23,500 components.     

An autoimmuno-dominant thyroglobulin epitope characterized by a monoclonal antibody.

It has become evident in recent years that autoimmune thyroglobulin (Tg) antibodies of Graves disease and Hashimoto's thyroiditis show a restricted epitope repertoire compared to Tg heteroantibodies. We have produced monoclonal antibodies (Mab) against human Tg by the hybridoma technique and the epitope specificity was determined by crossblocking experiments. Six noncrossreactive Mabs were used in a double determinant IRMA system for plasma Tg measurements. Sensitivity of the assays was between 1 and 2 ng/ml, intraassay variation less than 5%. Recovery experiments with added Tg were performed in 25 Graves sera with elevated Tg autoantibodies. Monoclonal antibody Tg13 showed an unusual strong interference with autoantibodies resulting in a very low recovery in all sera (median: less than 10%). In further studies Tg was digested by trypsin and after Western blotting, the resulting fragments were incubated with different Mab antibodies, a polyclonal antibody and 10 different Graves sera with high Tg autoantibodies. In contrast to all other mabs only Mab Tg13 showed several low molecular weight bands between 17 and 50 KD. The major bands recognized by Mab Tg13 corresponded to bands obtained by the autoimmune sera, which showed a very homogeneous band pattern. We conclude that Mab Tg13 is specific for an autoimmunodominant B cell epitope of human Tg.     

A Schistosoma mansoni epitope recognized by a protective monoclonal antibody is identical to the stage-specific embryonic antigen 1.

In infections with the parasitic trematode Schistosoma mansoni, a component of the host defense is directed against the invading larval form, and carbohydrates on the surface of these larvae are targets for the immune attack during the early stages of infection. To identify such carbohydrate epitopes, which may be suitable for immunization against schistosomiasis, we have previously generated monoclonal antibodies to surface antigens; some of these confer protection to naive mice when passively administered. Here we show that one of the protective antibodies recognizes a determinant present in both the parasite and its mammalian hosts. The immunohistochemical distribution of this determinant in the head of embryonic mice was found to be identical to the stage-specific embryonic antigen 1 (SSEA-1), an epitope abundant in pre-implantation embryos, several adult tissues, and malignant tumors. Oligosaccharides containing the SSEA-1 trisaccharide Gal beta 1-4(Fuc alpha 1-3)GlcNAc inhibit antibody binding to parasite antigen. SSEA-1 antibodies generated from mice immunized with rodent neural antigens bind to the surface of the schistosome larvae and mediate antibody-dependent cellular cytotoxicity. SSEA-1 antibodies are also elicited during human schistosomiasis infection, and this autoantibody response may be involved in the development of the natural immunity against the parasite.     

Studies of a human T lymphocyte antigen recognized by a monoclonal antibody.

A monoclonal antibody (designated L17F12) detects an antigen present on 95-100% of human peripheral T lymphocytes, the majority of thymocytes, and acute lymphocytic leukemia T cells but not B cells, B-cell lines, or monocytes. Examination of frozen tissue sections by the immunoperoxidase method revealed that the cells expressing this antigen were found predominantly in the medulla of thymus and in T-cell zones of lymph node and spleen. The antigen recognized by L17F12 was associated with a cell-surface glycoprotein of 67,000 daltons. L17F12 was used to isolate this molecule from human thymocytes, normal peripheral T cells, leukemic T cells, and T-cell lines. Expression of this antigen on normal T cells was not diminished by prolonged exposure in vitro to various T-cell stimuli. In the absence of complement, L17F12 bound to T cells without altering proliferative functions, thus enabling rapid purification of functionally intact T cells. In the presence of complement, L17F12 was cytolytic for T cells, providing the basis for depletion of T cells from heterogeneous populations. These data suggest that the monoclonal antibody L17F12 recognizes a specific T-cell differentiation protein. This antibody will be useful in studies of the human immune system.     

Cloning of cDNAs for M-phase phosphoproteins recognized by the MPM2 monoclonal antibody and determination of the phosphorylated epitope.

The MPM2 monoclonal antibody binds to a phospho amino acid-containing epitope present on more than 40 proteins of M-phase eukaryotic cells. We have developed a technique for cloning cDNAs encoding MPM2-reactive phosphoproteins from bacteriophage lambda expression libraries. Proteins from phage plaques were absorbed to nitrocellulose filters, phosphorylated by M-phase kinases, and screened for MPM2 binding. Partial-length cDNAs encoding two MPM2-reactive proteins termed MPM2-reactive phosphoproteins 1 and 2 (MPP1 and MPP2) were isolated. The deduced MPP1 and MPP2 amino acid sequences are not closely related to any previously described proteins. To determine which amino acid stretches contained the MPM2 epitope, sequences from a 15 amino acid peptide expression library were selected for binding to MPM2 after phosphorylation by M-phase kinases. A string of five amino acids was similar among all selected peptides, and the sequence reflecting the most frequent amino acid at each position was Leu-Thr-Pro-Leu-Lys (LTPLK). MPP1 and MPP2 proteins, respectively, contained five and nine sites closely related to LTPLK, including two that were common to both proteins, (F/T)TPLQ and SSP(I/S)D. Peptides containing LTPLK and FTPLQ were strongly phosphorylated by M-phase, but not interphase, cytosolic kinases, and the phosphorylated peptides were bound by MPM2. Thus, we have identified M-phase-specific phosphorylation sites bound by MPM2 and two putative M-phase phosphoproteins containing these sites.     

Identification of a continuous and cross-reacting epitope for Plasmodium falciparum transmission-blocking immunity.

Identification of continuous epitopes in the target antigens of Plasmodium falciparum transmission-blocking antibodies is likely to facilitate the production of a subunit peptide vaccine. Two such epitopes shared among several sexual-stage antigens were identified with murine monoclonal antibodies. An epitope recognized by four monoclonal antibodies capable of blocking infectivity of gametocytes in the mosquitoes is shared among three antigens (230, 48/45 doublet, and 27 kDa). These antigens are synthesized at different times during the development and maturation of gametocytes, and the blocking epitope appears conserved among parasites from diverse geographical locations. Immune response against such a unique epitope (continuous, cross-reacting, and conserved) is likely to be boosted by natural infection. The 27-kDa protein is reported here as a target of malaria transmission-blocking monoclonal antibodies, and the cross-reacting epitope represents an attractive candidate for a transmission-blocking vaccine.     

Synthesis and evaluation of monoclonal antibody against Plasmodium falciparum merozoite surface antigen 2

ObjectiveTo assess the quality of expressed MSP-2 and also to confirm the immune response against different domains of these proteins.MethodsMice were immunized with a schizont extract to stimulate the immune system to make antibodies against different antigens of the late stage parasite including production of antibodies against different domains of Plasmodium falciparum (P. falciparum) MSP-2. B lymphocytes of immunized mice were extracted from the spleen and the fusion was performed using NS-1 myeloma cells and the hybridoma cells were assayed by ELISA either with a schizont extract or different domains of MSP-2 and/or by IFAT with whole schizont preparation. Fusion of NS-1 and spleen cells was performed. The positive hybrids were cloned and ELISA was applied against different dilutions. The positive clones were transferred to a small tissue culture flask and after developing they were assayed against schizont extract and the different MSP-2 domains. The positive clones were expanded to large (75 cm²) flask and cultured under the same conditions, checking them using both ELISA and IFAT and the positive cells were frozen as soon as possible.ResultsA total number of 7 fusions including 26 plates (2 496 wells) were performed, of which 1 336 hybrids were produced and the overall efficiency (1 336/2496 × 100) was about 53%. ELISA was performed to detect the positive hybrids against crude schizont extract by which the highest frequency to crude schizont extract was found for the supernatant of the hybrids produced in fusion number 3 (66 out of 315 hybrids). The supernatant of both B5 and F1 hybridoma cells were more positive against domain 2 of the MSP-2 recombinant protein in Western blotting test. Western blotting results also showed that different domains of the MSP-2 recombinant protein and also the MSP-2 of the P. falciparum parasite were recognized by some of the positive clones and also immune sera.ConclusionsBringing together all the results of this study it has been confirmed that some clones have recognized both schizont extract and different domains of the MSP-2 recombinant protein and therefore confirming the quality of the MSP-2 domains.     

Characterization and tissue specificity of a monoclonal antibody against human uridine 5'-diphosphate-glucuronosyltransferase

A monoclonal antibody against human liver uridine 5'-diphosphate-glucuronosyltransferase (UDPGTase) was developed. Enzyme inhibition studies with this monoclonal antibody showed inhibition of human liver UDPGTase activity with bilirubin, 4-methylumbelliferone, and 4-nitrophenol as substrates. Testosterone, estrone, and phenolphthalein UDPGTase activity was not inhibited. The monoclonal antibody probably recognizes the uridine 5'-diphosphate-glucuronic acid binding site at 4-nitrophenol UDPGTase. Proteins with a molecular mass of 54,000 daltons were immunopurified from solubilized human liver using the monoclonal antibody as ligand coupled to Sepharose 4B beads. The distribution of UDPGTase isoforms in various human tissues was studied by immunofluorescence. The enzyme could be detected in liver, kidney, stomach, small intestine, colon, and skin. In liver, only hepatocytes contain UDPGTase. In kidney, the enzyme was localized exclusively in proximal tubuli and in stomach in epithelial mucous cells. In small intestinal epithelium, maximal fluorescence was seen at the villous tip. In colon, all lining epithelial cells were stained. In colon polyps, staining for UDPGTase was clearly decreased, and in colon carcinoma it was undetectable. In skin, the stratum corneum was intensely stained, whereas the stratum basale showed no fluorescence.     

Serotypic antigens of Plasmodium falciparum recognized by serotype-restricted inhibitory human sera

Immune sera from some Cambodian refugees contain functional serotypic antibodies that inhibit invasion of erythrocytes by the Camp strain but not by the FCR-3 strain of Plasmodium falciparum. Using a new assay, the "competitive heterologous antigen assay" (CHAA), the serotypic antibodies in a pool of three inhibitory sera were characterized by the antigens they precipitated. In the CHAA, immunoprecipitation of antigens by antibodies to common or cross-reacting antigenic determinants was blocked with excess heterologous unlabeled FCR-3 antigens before 3H-labeled Camp schizont and merozoite antigens were immunoprecipitated. The predominant Camp strain serotypic antigens revealed after electrophoresis and autoradiography were the major 195 Kd glycoprotein surface antigen (gp195) and its processed products at 150, 83, 73, and possibly 45 Kd. Additional serotypic antigens were identified at 180, 130, 65, 50, and 32 Kd. It is likely that one or more of these serotypic antigens is a target for the serotypic antibodies that inhibit invasion.     

Circumsporozoite antibody as a serologic marker of Plasmodium falciparum transmission.

In a longitudinal study of a malaria-endemic village in southeastern Thailand, circumsporozoite (CS) antibody to sporozoites of Plasmodium falciparum was measured by an enzyme-linked immunosorbent assay to determine its usefulness as a seroepidemiologic marker of malaria transmission. The CS anti-(NANP)n antibody level and prevalence during a 25-month period paralleled the pattern of seasonal transmission consistent with conventional parasitologic and entomologic measurements. The prevalence and level of antibody decreased during the non-transmission wet season, and increased over a 1-2-month transition period between the end of monsoon rains and the onset of dry conditions, an interval of maximum vector activity. Antibody increased with age in the population. The prevalence of antibody to the asexual blood stage as measured by conventional indirect fluorescent antibody assay did not coincide with changes in transmission and was sustained throughout the study period. Thus, CS antibody appeared to reflect the relative population exposure to mosquito inoculation of P. falciparum sporozoites and provided a useful measure of malaria transmission dynamics.     

Membrane antigen on Epstein--Barr virus-infected human B cells recognized by a monoclonal antibody.

This paper describes a monoclonal antibody (B532) that detects a membrane antigen present on greater than or equal to 95% of the B cells from lines carrying the Epstein-Barr virus (EBV) genome. Evidence suggesting that B532 is EBV-related was originally obtained by using a cell-binding radioassay with different cell line substrates. Immunofluorescence and cell-sorter analysis confirmed that the antigen was present in high density on all EBV-infected lymphoblastoid B-cell lines, but not on EBV-negative B-, T-, myeloid, or null cell lines. Isolated normal peripheral blood B and T lymphocytes and monocytes failed to bind B532. The monoclonal antibody did not inhibit in vitro EVB infection nor did it block the killing of EBV-infected targets by cytotoxic T lymphocytes. The cell surface antigen recognized by B532 was shown by immunoprecipitation to have a molecular weight of approximately 45,000.     

Shared polypeptides from various strains of Plasmodium falciparum recognized by Cameroon sera

Sera collected from 176 individuals residing in three malaria-endemic regions of Cameroon, West Africa, were tested against Plasmodium falciparum isolates from different areas of the world by enzyme-linked immunosorbent assay (ELISA), growth inhibition studies, and immunoprecipitation assays. A wide range of parasite proteins with apparent molecular weights of 14 to 250 kilodaltons (Kd) were immunoprecipitated by the sera. Of these, two polypeptides of 41 and 96 Kd could be associated with parasite growth inhibition in vitro.     

Transmission of mixed Plasmodium species and Plasmodium falciparum genotypes

We studied malaria transmission by comparing parasite populations in humans and mosquito vectors at the household level. Blood samples were collected from all inhabitants for microscopic detection of gametocytes and polymerase chain reaction analysis. The next morning, blood-fed resting mosquitoes were collected inside the bed nets used by the individuals surveyed the previous afternoon. After 8 days of maintenance, mosquitoes were dissected, and midguts and salivary glands were recovered for polymerase chain reaction analysis. Results showed that parasite distribution was the same in the 2 hosts when compared at each household but was different when whole populations were analyzed. Different associations of Plasmodium species seem to occur in humans (Plasmodium falciparum/Plasmodium malariae) and mosquitoes (P. falciparum/Plasmodium ovale). Regarding P. falciparum infections, a higher proportion of single-genotype infections and less allele diversity are observed in mosquitoes than in humans.     

Cross–reactivity of antibody against an epitope of the Plasmodium falciparum second merozoite surface antigen

Monoclonal antibodies directed against the 51 kD merozoite surface antigen of Plasmodium falciparum also bind to other antigens within the infected cell. The sizes of these cross-reacting antigens have been characterized. Immunofluorescence due to the reaction of one of the monoclonal antibodies with these cross-reacting antigens was localized in the intra-erythrocytic parasite and in granules in the infected red cell cytoplasm. This immunofluorescence could be distinguished from the merozoite surface antigen in parasite lines with a variant serotype of the merozoite surface antigen which fails to react with the monoclonal antibodies. It was found that the in-vitro growth inhibition caused by the presence of one of the monoclonal antibodies, 8G10/48, was dependent on the expression of the corresponding serotype of merozoite surface antigen, a finding consistent with the inhibitory effect of this antibody being primarily directed against the merozoite surface antigen and not the cross-reacting antigens. Analysis of the frequency at which epitopes occur suggests that such cross-reacting proteins will be commonly seen in malaria, without the need to postulate a selective advantage for such cross-reacting specificities.     

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In this report, the blood samples from 30 falciparum malaria patients with parasitemia 0.015-0.58% and the blood samples from 30 healthy persons were examined by monoclonal antibody (McAb) sandwich dot-immunogold silver staining assay (Dot-IGSSA). When the McAb 11G5, 13A2 and 13A1 were used for sandwich Dot-IGSSA with McAb 14D9 labeled with colloidal gold respectively, the 0.0001% of parasitemia could be detected and the McAb 11G5, 13A1 and 14D9 labeled with colloidal gold could also be used to detect the antigens of asexual blood stages of Yunnan and Anhui isolates of Plasmodium falciparum cultured in vitro. These McAbs did not cross-react with the antigens of Plasmodium knowlesi and Plasmodium berghei, however, the McAbs 13A1 and 14D9 weakly cross-reacted with the antigens of Plasmodium cynomolgi and the antigens in the infected blood samples from patients with vivax malaria.     

Characterization of antigens recognized by monoclonal and polyclonal antibodies directed against uvomorulin.

Uvomorulin is a cell surface glycoprotein involved in compaction of early mouse embryo. Antibodies, either monoclonal or polyclonal, raised against a purified tryptic fragment of uvomorulin recognize, in a detergent lysate of embryonal carcinoma cells metabolically labeled with 35S, three molecules (120, 100, and 88 kDa) that are not related, as judged by peptide mapping. Only the 120-kDa form is related to the tryptic fragment of uvomorulin and, thus, is considered as the native form of uvomorulin. Although all three products are apparently detectable at the cell surface, only the 120-kDa form is glycosylated. Coimmunoprecipitation of the three different polypeptides is probably due to shared epitopes rather than to their presence in a multimeric complex.