Synthetic peptides based on conserved Plasmodium falciparum antigens are immunogenic and protective against Plasmodium yoelii malaria

Two synthetic polypeptides containing multiple B- and T-cell epitopes derived from the conserved regions of two vaccine candidate antigens namely MSA-1 and RESA of human malarial parasite P. falciparum were studied for immunogenicity and protectivity. Both constructs elicited strong antibody and lymphocyte proliferation responses in BALB/c mice immunized with the carrier-free peptides. In an ELISA, these peptides also bound antibodies present in the sera from the P. vivax infected humans as well as from the P. yoelii infected mice. Significantly, our data showed that immunization of mice with these P. falciparum peptide could impart partial protection against P. yoelii challenge infection. Our finding that synthetic peptides representing portions of P. falciparum antigens were capable of stimulating protective immune responses against rodent malaria suggests that murine malaria model P. yoelii may provide a suitable system for primary screening of potentially protective synthetic immunogens.     

Monoclonal antibodies identify the protective antigens of sporozoites of Plasmodium knowlesi.

Nine monoclonal antibodies against surface antigens of sporozoites of the simian malaria parasite Plasmodium knowlesi were produced by fusion of plasmacytoma cells with spleen cells of a mouse immunized with the parasites. Immunoprecipitation of extracts of [35S]methionine-labeled sporozoites with seven of the monoclonals identified the same three polypeptides with apparent molecular weights of 52,000 (Pk52), 50,000 (Pk50) and 42,000 (Pk42). These antigens also were recognized by serum of a rhesus monkey immunized with and protected against P. knowlesi sporozoites. Pulse--chase experiments indicated that the higher molecular weight proteins are precursors of Pk42. As shown by trypsin treatment of viable sporozoites, Pk42 is a surface antigen whereas Pk52 and Pk50 appear to be intracellular. Three of the monoclonal antibodies also reacted with a membrane antigen of sporozoites of another simian malaria, P. cynomolgi, and one monoclonal antibody reacted with sporozoites of human malaria, P. falciparum. When assayed for sporozoite neutralizing activity, most of the antibodies and their Fab fragments, which recognize Pk52, Pk50, and Pk42, abolished parasite infectivity.     

Detection of blood stage antigens of Plasmodium vivax by sandwich ELISA using pan-species monoclonal antibodies and polyclonal antibodies.

This paper reports an improved PcAb-McAb-ELISA test to detect blood stage Plasmodium vivax antigen in which the plates were coated with rabbit anti-P. cynomolgi polyclonal antibody to capture the antigens in test samples and two monoclonal antibodies, M26-32 and 3F9, were added together to react with the captured antigens. The coincidence rate with this test was 93% with microscopically confirmed P. vivax cases, 97% with normal samples, 95% with microscopically negative fever cases from nonendemic areas and 86% from endemic areas, respectively. The sensitivity was greater than 1 parasite/10(5) RBC.     

Transformation-related antigens identified by monoclonal antibodies.

Tumor-cell proteins that were antigenic in a syngeneic animal were identified by immunoprecipitation with monoclonal antibodies. Spleen cells of BALB/c mice immunized with plasma membranes of Kirsten RNA sarcoma virus-transformed BALB/3T3 cells were fused with NS-1 myeloma cells. Antibodies secreted into the culture fluid from these hybridomas were distinguished by their reactivity against proteins of different target cells. A total of 191 cultures were established; 143 produced antibodies that bound to BALB/3T3 cells transformed by the RNA sarcoma virus, of which antibodies from 82 bound to BALB/3T3 transformed with simian virus 40, and antibodies from 56 bound to BALB/3T3 cells. Thus, more than 50% of the cultures produced antibodies that possibly were specific to antigens of the transformed cell. Twenty different hybridomas have been cloned, and antibodies, from eight of these were found to immunoprecipitate five different proteins. A protein of approximately 32,000 daltons was precipitated from BALB/3T3 cells transformed by the RNA sarcoma virus, simian virus 40, or methylcholanthrene but not from untransformed BALB/3T3 cells. A protein of about 300,000 daltons was precipitated from all four cell lines; precipitation was enhanced in the viral transformed cells. Proteins of approximately 57,000, 54,000, and 8500 daltons were immunoprecipitated from all four cell lines.     

Purification of antigens and monoclonal antibodies

Clinical Rheumatology -     

Plasmodium falciparum-inhibitory monoclonal antibodies produced by human hybridomas

Stable human hybridomas were generated that produced inhibitory anti-Plasmodium falciparum monoclonal antibodies. Peripheral blood lymphocytes, obtained from adults in Liberia, a malaria endemic area, were immortalized with Epstein-Barr virus and then fused with KR4, a human, lymphoblastoid cell line. Stable hybridomas that produced anti-P. falciparum monoclonal antibody were identified by an ELISA assay that used the trophozoite and schizont antigens of both the Honduras I and FCR3 parasite strains. Monoclonal antibodies produced by selected hybridomas derived from lymphocytes of two individuals were subsequently studied. The anti-parasite antibodies were produced at 1-3 micrograms/ml in culture supernatants. All of the monoclonal antibodies bound specifically to trophozoites and schizonts of both strains of parasite in an indirect immunofluorescence assay and inhibited production of ring stage parasites by more than 90% when added to trophozoite or schizont containing erythrocytes in culture. Western immunoblot analysis of antigens obtained from trophozoites and schizonts (parasite age span of 36 to 48 h) was performed using either affinity purified or ammonium sulfate-concentrated monoclonal antibody. Antibody from three hybridomas which bound primarily to antigens of the Honduras 1 strain had Mr of approximately 140,000, 130,000 and 123,000.     

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.     

Circumsporozoite protein of Plasmodium gallinaceum characterized by monoclonal antibodies

Monoclonal antibodies (MoAb) were produced against both salivary gland sporozoites (SGS) and oocyst sporozoites (OS) of Plasmodium gallinaceum, an avian malaria parasite. By indirect immunofluorescence, all of the MoAbs reacted with both SGS and OS of P. gallinaceum and two of the MoAbs cross-reacted weakly with P. berghei sporozoites. None of the MoAbs reacted with sporozoites of six additional species of mammalian plasmodia. In Western blot analysis of extracts of either SGS or OS of P. gallinaceum, these MoAbs identified two polypeptides with molecular weights of approximately 76,000 and 64,000 D. The results of a MoAb inhibition of binding assay and a two-site one-antibody immunoradiometric assay indicate that the circumsporozoite protein of P. gallinaceum, like those of mammalian malaria parasites, contains a repetitive immunodominant epitope. Two of the anti-P. gallinaceum MoAbs were tested in a sporozoite neutralization assay and decreased, but did not abolish, the infectivity of sporozoites for chickens, indicating that the polypeptide of P. gallinaceum identified by immunoblot is probably the protective antigen.     

Platelet surface antigens: Analysis by monoclonal antibodies

Summary Monoclonal antibodies were produced against human platelets. Four antibodies (PA1, PA2, PA3 and PA4) reacted specifically with platelets and megakaryocytes, but not with peripheral blood lymphocytes, granulocytes, erythrocytes or monocytes. The antibodies belonged to the mouse IgG subclass 2a (PA1, PA2, PA3), or 1 (PA4) respectively. PA1 and PA4 did not precipitate, their antigens have not yet fully been characterized. PA3 was directed against the glycoprotein (Gp) complex IIb/IIIa; PA2 precipitated Gp IIb/IIIa, and, in addition, Gp Ia. PA4 revealed specificity against the human platelet alloantigen Zw(a).     

Ultrastructural localization of bronchial antileukoprotease in central and peripheral human airways by a gold-labeling technique using monoclonal antibodies

The present report describes the ultrastructural localization of bronchial antileukoprotease (ALP) in human central and peripheral airways by using polyclonal as well as monoclonal ALP-specific antibodies in a two-step gold-labeling procedure. In the serous cells of bronchial glands, ALP could be demonstrated in secretory granules. These granules, among which 4 phenotypes could be distinguished morphologically in ultrathin sections, showed the following labeling patterns: phenotype I, which had an electron lucent, fine granular content, and phenotype II, which was homogeneously electron dense, both showed gold label over their entire area. The granules expressing zonal differences in electron density (phenotype III) showed only label in their electron-dense cores and the electron-lucent granules (phenotype IV) showed a minimal labeling. Sometimes gold particles could be observed in the rough endoplasmic reticulum and nuclear envelope, suggesting that ALP is present in these cell organelles. In the bronchiolar epithelium, ALP could be localized only in the secretory granules of Clara cells and goblet cells. These findings indicate that ALP is also synthesized in bronchioli. To our knowledge this is the first time that a well-defined protein has been described that is produced and secreted by human Clara cells.     

Cell-surface antigens of melanoma recognized by human monoclonal antibodies

Human monoclonal antibodies (mAbs) were derived from lymph node lymphocytes and peripheral blood lymphocytes (PBL) from patients with melanoma. Four methods for generating human mAbs were compared: fusion with human [LICR-LON-HMy-2 (LICR-2)] or mouse (NS-1) cells; transformation by Epstein-Barr virus (EBV); and EBV transformation followed by NS-1 fusion. NS-1 fusion with lymph node lymphocytes resulted in a higher number of growing hybrids than LICR-2 fusion. Virtually no hybrids were obtained from NS-1 or LICR-2 fusions with PBL. EBV transformed lymphocytes from lymph node and peripheral blood with equal efficiency, and the yield of proliferating cultures for antibody screening was more than 10- to 30-fold greater than that obtained by fusion techniques. However, once antibody-producing cultures had been identified, stability and clonability of EBV-transformed cells were poorer than that of NS-1 hybrid cells. To combine the strengths of both methods, cultures of EBV-transformed cells were fused with NS-1; and hybrid clones were isolated that showed vigorous growth, clonability, and stable antibody secretion. Detailed specificity analysis of the mAbs produced by six of these clones indicated detection of a class 1 (unique) melanoma antigen, a class 3 melanoma antigen, and four ganglioside antigens (GD3, GM3, and two other, as yet uncharacterized, heterophile antigens).     

Recognition of Giardia lamblia cyst-specific antigens by monoclonal antibodies

Immunization of BALB/c mice with a sonicated extract of in vitro-generated Giardia lamblia cysts produced six cystspecific monoclonal antibodies (MoAbs). Two MoAbs (8C5.C11 and 5A4.G6), which recognize proteinaceous cyst antigens, were selected for further study. In indirect immunofluorescence (IFA), MoAb 8C5.C11 reacted with encystation-specific vesicles in trophozoites beginning 3 h after the induction ofencystation in vitro. This MoAb also recognized cysts which began to appear at 12 h. In contrast, MoAb 5A4.G6 stained only cyst walls. In Western blots, both MoAbs also reacted with cyst antigens, but not trophozoite antigens. MoAb 8C5.C11 first recognized cyst antigen from 3 h encysting cultures, reacting with 26, 28, 42 and 46 kD bands. MoAb 5A4.G6 reacted with a 38 kD band, beginning with 12 h encysting cultures. When added to G. lamblia encysting cultures before the appearance of cysts (0 to 9 h) and in the presence of a source of complement, MoAb 8C5.C11 caused a significant reduction in the numbers of water-resistant cysts produced in vitro compared to the control. MoAb 5A4.G6 did not affect in vitro encystation. These findings confirm the heterogeneity of cyst antigens, and also indicate that the process of encystation in vitro can be interrupted by antibodies and complement.     

Human trophoblast cell-surface antigens defined by monoclonal antibodies.

A series of monoclonal antibodies has been raised against the human choriocarcinoma cell-line, BeWo. Four antigens, Trop-1, -2, -3, and -4, are defined on normal and malignant trophoblast cells. Trop-1 and Trop-2 appear to be specifically expressed on syncytio- and cytotrophoblasts, whereas Trop-3 and Trop-4 are also detected on various tumor cell lines, normal lymphocytes, and monocytes. Anti-Trop-1 and anti-Trop-2 antibodies might prove useful for detection and isolation of fetal trophoblast cells circulating in pregnant women's blood and for diagnosis and therapy in patients having choriocarcinomas and other germ-cell neoplasms.     

Ultrastructural localization of the 150/130 Kd antigens in sexual and asexual blood stages of Plasmodium falciparum-infected human erythrocytes

The subcellular localization of the 150/130 Kd antigen in Plasmodium falciparum-infected erythrocytes was determined by electron microscopy using monoclonal antibody 9B11 and immuno-gold labeling. We now find that this antigen may be associated with the membrane of newly-infected human erythrocytes and the cytoplasm of ring stage parasites. During differentiation of the parasite to the trophozoite stage, the antigens are no longer detectable on the erythrocyte membrane, while gold particles become more numerous within the parasite and in the erythrocyte cytoplasm adjacent to the parasite. As the parasites develop into schizonts, more antigen appears within the parasites, and some of it appears in the erythrocyte cytoplasm. At the segmented schizont stage, many intraparasitic gold particles are associated with rhoptries and micronemes of developing merozoites. Likewise, gold particles are associated with elements of the rhoptry-microneme complex in free merozoites. No gold particles are detected on the surface of merozoites. These antigens are found most abundantly in erythrocytes infected with gametocytes, revealing a localization pattern similar to that of mature trophozoite-infected erythrocytes. These subcellular localization patterns are similar to those described for the ring-infected erythrocyte surface antigen.     

Supplement of L-Arg improves protective immunity during early-stage Plasmodium yoelii 17XL infection

L-arginine (L-Arg), the precursor of nitric oxide (NO), plays multiple important roles in nutrient metabolism and immune regulation. L-Arg supplement serves as a potential adjunctive therapy for severe malaria, because it improves NO bioavailability and reverses endothelial dysfunction in severe malaria patients. In this study, we investigated the effect of dietary L-Arg supplement on host immune responses during subsequent malaria infection using the Plasmodium yoelii 17XL - BALB/c mouse model. We have shown that pretreatment of mice with L-Arg significantly decreased parasitemia and prolonged the survival time of mice after infection. L-Arg supplement led to significant increases in activated CD4(+) T-bet(+) IFN-γ(+) T cells and F4/80(+) CD36(+) macrophages during early-stage infection, which were accompanied by enhanced synthesis of IFN-γ, TNF-α and NO by spleen cells. Moreover, L-Arg-pretreated mice developed more splenic myeloid and plasmacytoid dendritic cells with up-regulated expression of MHC II, CD86 and TLR9. In comparison, L-Arg treatment did not change the number of regulatory T cells and the level of anti-inflammatory cytokine IL-10. Taken together, our results showed that L-Arg pretreatment could improve the protective immune response in experimental malaria infection in mice, which underlines potential importance of L-Arg supplement in malaria-endemic human populations.     

Two-site sandwich ELISA for detection of Plasmodium vivax blood stage antigens using monoclonal and polyclonal antibodies.

Two systems of sandwich enzyme-linked immunosorbent assay (ELISA), a two-site monoclonal antibody sandwich ELISA MAb-MAb sandwich ELISA) and a two site polyclonal-monoclonal antibody sandwich ELISA (PAb-MAb sandwich ELISA) for the detection of Plasmodium vivax antigens were developed. The assays showed good correlation with the level of parasitemia when tested against serially diluted P. vivax parasites (r = 0.937, and 0.997 for MAb-MAb and PAb-MAb sandwich ELISA, respectively), with the ability to detect as few as 6.68 parasites/10(6) erythrocytes and 2.69 parasites/10(3) erythrocytes, respectively. The MAb-MAb sandwich ELISA was specific, since it was positive only with P. vivax-infected erythrocytes from vivax malaria patients and negative when erythrocytes from 34 healthy individuals and 30 falciparum malaria cases were tested. In contrast, cross-reaction was found in the PAb-MAb sandwich ELISA when the plates were coated with polyclonal IgG and tested against the serially diluted P. falciparum SO strain antigen prepared from in vitro cultures. Comparison between the two systems of two-site sandwich ELISA showed that the MAb-MAb sandwich ELISA was superior to the PAb-MAb sandwich ELISA: (1) it gave a higher sensitivity when tested with serially diluted P. vivax antigen preparations from vivax malaria patients; (2) it gave a higher specificity when tested with the SO strain of P. falciparum from in vitro cultures, (3) it gave a lower absorbance value when tested with erythrocytes from healthy individuals. All 281 cases of vivax malaria already proven by microscopic examination were positive by MAb-MAb sandwich ELISA.(ABSTRACT TRUNCATED AT 250 WORDS)