Schistosoma mansoni: Analysis of monoclonal antibodies reactive with gut-associated antigens

The analysis of a series of monoclonal antibodies (mAbs) developed in our laboratory against gutassociated antigens ofSchistosoma mansoni is described. It was found that mAbs that recognized epitopes of antigens in the gut and on the eggshell were mainly of the IgM isotype; these epitopes are likely to be carbohydrate in composition. Of a number of mAbs that were reactive with antigens important to the human humoral immune response, 75% appeared to be reactive with the circulating cathodic antigen.     

Schistosoma mansoni: Detection and characterization of schistosome derived antigens by inhibition enzyme-linked immunosorbent assay (IELISA) utilizing monoclonal antibodies

Monoclonal antibodies were used in an inhibition enzymelinked immunosorbent assay (IELISA) to detect a variety of schistosome derived antigens. Preparations were obtained from various stages ofSchistosoma mansoni and from the eggs ofS. japonicum. Using appropriate titers of monoclonal antibodies it was possible to detect less than 0.01 g/ml of schistosome antigens. The sensitivity of IELISA was dependent upon the type and concentration of the monoclonal antibody used, as well as upon the source of the antigens. Specificity studies showed that some of the monoclonal antibodies recognized species specific antigenic determinants, while others reacted against genus specific antigens. Furthermore, certain antibodies interacted with antigens which were neither genus or species specific. Fractions ofS. mansoni andS. japonicum egg antigen extracts, which have been previously considered to be relatively pure, were compared using certain monoclonal antibodies. The results indicate that these fractions are very heterogenous with regard to the unique antigenic specificities. For example, most antigenic determinants in crude soluble egg antigen are not retained on concanavalin-A lectin affinity columns and major serologic antigens have more than one determinant with different distributions. The expression of these antigenic determinants appears to be a function of both the concentration of the specific antigen and the mode of expression of the moiety within the antigenic matrix.     

Acquisition of human blood group antigens by Schistosoma mansoni.

Juvenile forms of Schistosoma mansoni (schistosomula) have been cultured in human blood of various specificities and tested for the presence of blood group substances on their surfaces. The tests employed were survival following transfer into rhesus monkeys immunized against human blood substances, mixed agglutination reactions, and immunofluorescence. A, B, H AND Lewisb+ antigens were expressed at the surface when the parasites were cultured in blood of appropriate specificities. Rhesus, M N S, AND Duffy antigens could not be detected on the parasite surface following culture. The evidence suggests that the expressed blood group antigens are of host origin and are acquired by the parasite during culture, probably in the form of glycolipids or megaloglycolipids. It is likely that these substances are also acquired by parasites in the bloodstream of man. They may serve to mask surface parasite antigens, and so enable schistosomes to evade parasite-specific humoral or cellular immune responses.     

Human immune responses to Schistosoma mansoni vaccine candidate antigens

To determine the naturally occurring immunological responses to the Schistosoma mansoni antigens paramyosin, IrV-5, Sm-23 (MAP-3), and triose phosphate isomerase (MAP-4), a total of 119 subjects from an area of endemicity for schistosomiasis, including "resistant" subjects (n = 17) were evaluated. Specific immunoglobulin G1 (IgG1), IgG2, IgG3, IgG4, and IgA levels for each of the antigens and the cytokine profile in culture supernatants from antigen-stimulated peripheral blood mononuclear cells (PBMC) were determined. Although all the subjects had a high degree of contaminated water exposure, their infection levels were variable (0 to 1,128 eggs/g of stool). There were direct correlations between infection levels and levels of SWAP- and paramyosin-specific IgG1 and IgG4 (P < 0.05). However, an inverse correlation between infection levels and specific IgG2 to IrV-5 (P < 0.01) was observed. The evaluation of the cytokine profile (interleukin 5 [IL-5], IL-10, gamma interferon [IFN-gamma], and tumor necrosis factor alpha) in response to these antigens showed inverse correlations between the degree of infection and IFN-gamma levels in PBMC supernatants stimulated with paramyosin (P < 0.05) and IrV-5 (P < 0.01). Additionally, inverse correlations between the degree of infection and IL-5 levels in MAP-3- and MAP-4-stimulated PBMC supernatants (P < 0.01) were found. Logistic regression analysis was performed to adjust the results of cytokine profile by age. IL-5 production in MAP-3-stimulated PBMC supernatants was associated with lower infection levels (odds ratio = 11.2 [95% confidence interval, 2.7 to 45.8]).     

Inhibition of HIV-1 infection by monoclonal antibodies to carbohydrates of Schistosoma mansoni

Patients infected with HIV-1 develop a potent humoral immune response against the virus, but HIV-1 primary isolates are remarkably resistant to neutralizing antibodies. Considering that the envelope glycoprotein of HIV-1 (gp120/41) is heavily glycosylated, we investigated whether anti-carbohydrate antibodies could inhibit HIV-1 infection in vitro. We studied the neutralizing activity of three monoclonal antibodies (mAbs) raised to carbohydrates of Schistosoma mansoni, against seven primary isolates of HIV-1. Assays were performed infecting peripheral blood mononuclear cells from normal donors with viral isolates previously treated with mAbs. Viral strains used were tropic for the coreceptors CCR5, CXCR4, and dual-tropic ones. We found that the anti-glycan mAbs vigorously inhibited HIV-1 infection, regardless of the preferential coreceptor usage of the isolate, in a dose-response manner. Importantly, five isolates were resistant to neutralization by two HIV-1 antibody-positive human sera endowed with potent anti-HIV-1 inhibitory activity. Our findings suggest that carbohydrates of the HIV-1 viral envelope may be a target of an effective humoral immune response elicited by vaccination.The first two authors contributed equally to this work     

Plasmodium falciparum: characterization of defined antigens by monoclonal antibodies.

Monoclonal antibodies directed against Plasmodium falciparum detect stage-specific, species-specific and common antigenic determinants of Plasmodia. These antibodies provide new tools for purification and characterization of Plasmodium falciparum antigens in relation to future procedures for immunoprophylaxis.     

Human tumor-associated antigens defined by monoclonal antibodies

Melanoma-associated antigens extracted from cultured human melanoma cells or isolated from their spent culture medium react specifically with monoclonal antibody and are represented by glycoproteins with molecular weights of 260,000 (260K), 250,000 (250K), and 100,000 (100K). The 260 and 250K antigens are present only in spent culture medium or detergent extracts of melanoma cells, respectively. The 100K antigen is an oncofetal marker present on tumor cells of different histological type. Indirect immunofluorescence data indicate that the 250K antigen is present on the surface of melanoma cells from excised melanoma tissues when the 100K antigen appears to be present in the cytosol. Subunit structure determination indicates that the 100K antigen is a single polypeptide chain whereas the 250K antigen extracted from melanoma cells is associated with a proteoglycan fragment which is important for its cell surface expression.     

Human tumor-associated antigens identified by monoclonal antibodies

Conclusions Monoclonal antibodies have been obtained that are specific for cell surface antigens of human tumors. Some of these antigens are tissue type specific tumor antigens, most strongly expressed on tumors of the same histological type. The antigens identified by the monoclonal antibodies are primarily normal differentiation antigens that are expressed more strongly on neoplastic cells than on most normal adult cells. For several antigens, the higher degree of antigen expression on neoplastic cells appear to be adequate for diagnostic and therapeutic purposes. Future work in this area is likely to have far-reaching consequences for the clinical handling of human cancer patients.This work was supported by grants CA 14135, 19148, 19149, CA 25558, CA 27841 from the National Institutes of Health, and IM 241A from the American Cancer Society. The authors wish to acknowledge collaboration with Drs. M.-Y. Yeh, R. G. Woodbury, S. M. Larson, and K. Nishiyama     

Efficient generation of stable antibody forming hybridoma cells by electrofusion

A variety of modified electrofusion protocols designed to improve the efficiency of hybridoma production have recently appeared in the literature. We undertook to maximize the number of antibody secreting murine hybridomas by optimizing the temperature and fusion strength parameters of the conventional electrofusion technique. Anti-DNP secreting hybridomas were generated by fusing SP2/0 to immunized mouse splenic lymphocytes using an unmodified electrofusion protocol consisting of washing in a weakly conducting sorbitol fusion medium supplemented with bovine serum albumin, calcium and magnesium ions. This was followed by dielectrophoretic alignment and application of 3 short duration, high intensity field pulses in helical chambers. Optimal efficiencies of hybridomas were generated by the application of 2000 V/cm pulses at 25 degrees C (2.45 hybridomas x 10(-4) splenocytes) and as many as 63% of resulting hybridomas secreted anti-DNP monoclonal antibodies, the majority of which were IgG's. These data show that modification of the electrofusion protocol by pretreatment of fusion partners with proteolytic enzymes or the use of antigen bridging is not required for the successful and efficient production of specific monoclonal antibodies by electrofusion.     

Immunoaffinity fractionation of Schistosoma mansoni worm antigens using human antibodies and its application for serodiagnosis.

A crude Schistosoma mansoni soluble worm antigen preparation (SWAP) was fractionated using an immunoaffinity column consisting of specific human anti-SWAP antibodies obtained from chronic S. mansoni-infected human sera and bound to CNBr-activated Sepharose 4B. The chromatographic separation resulted in three fractions: the unbound material (FW), and the eluted antigens with glycine-HCl (F1) and glycine-HCl-NaCl (F2). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the purified antigens F1 and F2 consisted of several bands when stained with Coomassie blue and silver stain, with molecular weights between 20 X 10(3) and 200 X 10(3). The F1 and F2 fractions in addition to FW and SWAP were used in an enzyme-linked immunosorbent assay (ELISA) to measure antibody levels in sera from schistosomiasis patients. Each individual serum assessed with the purified F2 antigen gave 100% positivity and three to four times higher optical density in comparison to SWAP with only 88% positivity. No detectable cross-reactive antibodies against F2 were found when a limited number of sera from filariasis, fascioliasis and trichinellosis patients were screened. Furthermore, F2 was also used and found to be more sensitive generally in detecting anti-adult worm antibodies than SWAP in recently schistosomiasis-infected persons. Thus, F2 appears to be a highly sensitive and specific reagent for the serodiagnosis of schistosomiasis infection.     

Detection of antibodies against ovum antigens in Schistosoma mansoni bilharziasis

Fifty-five sera from bilharziasis patients from West Indies and thirty-five control sera (from normal subjects and patients with hydatidosis and fascioliasis) were examined by ELISA for antibodies reacting with S. mansoni soluble egg antigen MSA1 . MSA1 antigen was prepared according to Pelley and coupled to isothiocyanate substituted plastic discs. Sensitivity and specificity of reaction were good. Moreover, in experimental infection in mice, sera of mice cured with praziquantel became negative. The sera were also examined by a whole egg antigen in counter immunoelectrophoresis. The results of the two different techniques correspond well although ELISA using purified antigen prove to be more specific and sensitive.     

Immunofluorescent reaction of antibodies directed against antigens from the intestinal epithelium of Schistosoma mansoni. III. Study of the reactivity of monoclonal antibodies

A monoclonal mouse antibody of IgM class was raised against an epitope of the gut epithelium of the adult worm and was applied to the detection of antigen in parasite infection. The antigen was found in urine from mice and hamsters infected with Schistosoma mansoni; a good correlation between the concentration of antigen and worm burden was observed. The antigen was thermostable, soluble in trichloracetic acid; it was not hydrolysed by proteinase K but it was destroyed by metaperiodate. The antigen was shown to be Schistosoma genus specific. It was found in different developmental stages of the parasite. High levels were detected in egg extracts.     

Human monocyte-histiocyte differentiation antigens identified by monoclonal antibodies

Two distinct differentiation antigens of human myelomonocytic cells are defined using murine monoclonal antibodies. The antigens recognized by antibodies 20.2 and 20.3 are expressed by all cells of the monocyte lineage in both peripheral blood and bone marrow. Cell-sorting experiments demonstrated that histiocytes and immature bone marrow cells with detectable alpha-naphthyl butyrate esterase activity also express both antigens. Within cells of other lineages, the antigens had distinct patterns of expression. Immature myeloid cells were 20.2 negative, but 20.3 positive; whereas mature myeloid cells were 20.2 positive, but 20.3 negative. Nucleated erythroid cells and platelets expressed only the 20.3 antigen. These results indicate that myeloid and monocytic cells share common differentiation antigens with cells of the erythroid and megakaryocytic lineages. The 20.2 and 20.3 antibodies reacted with the leukemic cells from some patients with acute nonlymphocytic leukemia (FAB, M1-M5) and with some cell lines derived from patients with nonlymphocytic leukemia, but not with blast cells from patients with lymphoid leukemia or with lymphoid leukemic cell lines. These antibodies may prove useful in studying the differentiation of bone marrow stem cells, in defining the cellular origins and classification of leukemias, and in the identification of distinct prognostic subgroups of acute nonlymphocytic leukemia.     

Immunocapture assay for quantification of human IgA antibodies to parasite antigenic enzymes. Application with the alkaline phosphatase of Schistosoma mansoni.

Conditions are described for using solid phase adsorbed jacalins in an immunocapture assay for IgA antibodies to the alkaline phosphatase of Schistosoma mansoni. Microtiter plates were activated with polylysine and jacalins were covalently adsorbed by means of glutaraldehyde. From three different jacalins, the one purified from seeds of Artocarpus tonkinensis showed the lowest non-specific adsorption and was used for further studies. Comparing solutions of bovine serum albumin, ovalbumin and Tween 20, it was shown that the latter was most successful in blocking non-specific adsorption. Low serum dilutions resulted in a less efficient IgA capture by the adsorbed jacalin than higher dilutions. Under optimal working conditions, a high correlation could be shown between the presence of specific anti-alkaline phosphatase antibodies of IgA isotype and IgG isotype.     

Immunopathology of experimental Schistosoma mansoni: immunohistochemical localization of parasite antigens in the host tissue

Schistosoma mansoni antigens play a crucial role in the induction of immunopathological processes and in modulating the host immune system. A polyclonal rabbit antiserum to an antigenic fraction of adult schistosoma worms was used to localize worm and egg antigens in tissue sections of infected mice. Most granuloma formations identified in paraffin sections of portal tracts and intestinal mucosa were vigorous with florid cellular composition, consisting of macrophages and epithelioid cells, surrounding a central nidus of S. mansoni egg. Schistosome pigments were demonstrated at the periphery of the granuloma, within sinusoidal Kupffer's cells and within macrophages of intestinal mucosa. Large amounts of schistosome antigen were found sequestered in the mesenteric lymph nodes and in the spleen. A strong reaction for locally synthesized IgG was found. These findings suggest that the schistosome antigen-antibody complex found in the host lymphoreticular system plays a major role in the immunopathological process.     

Immunochemical characterisation of Schistosoma mansoni glycolipid antigens.

The aim of this study was to investigate the occurrence, distribution and immunochemical properties of antibody-defined carbohydrate epitopes in neutral glycolipid fractions of Schistosoma mansoni eggs, cercariae and adults. The amount of extractable, antigenic, neutral glycolipids was lowest in adult worms, increasing consecutively in cercariae and eggs. The immunoreactivity of the glycolipids resided in the carbohydrate moiety in that it was periodate-sensitive. Serological reactivity, and monosaccharide component analysis, anomeric configuration and methylation-linkage analyses indicated that there were two dominant epitopes, which could be partially defined immunologically. The first epitope was detected on egg, cercarial and adult glycolipids. It was strongly recognised by mouse chronic infection sera and rabbit hyperimmune sera raised against specific egg antigens, and was defined by the monoclonal antibody M2D3H (Bickle QD, Andrews BJ. Characterisation of Schistosoma mansoni monoclonal antibodies which block in-vitro killing: failure to demonstrate blockage of immunity in vivo. Parasite Immunol 1988;10:151-168). M2D3H appeared to have the same epitope specificity as monoclonal antibody 128C3/3 (Weiss J, Magnani JL, Strand M. Identification of Schistosoma mansoni glycolipids that share immunogenic carbohydrate epitopes with glycoproteins. J Immunol. 1986;136:4275-82). The internal epitope was defined structurally by the presence of fucose 3-linked to 3,4-disubstituted N-acetylglucosamine, which was itself partially substituted by a second fucose residue, to yield the determinant -4[Fucalpha1,2Fucalpha3]GlcNAcbeta1-. The second epitope was defined by the anti-LewisX monoclonal antibody 4D1 and was found primarily on cercarial glycolipids. It was chemically characterised as the LewisX epitope of Galbeta1,4[Fucalpha1,3]GlcNAcbeta1- in a terminal position. The removal of fucose greatly diminished the binding of the anti-LewisX and M2D3H monoclonal antibodies, as well as the polyclonal chronic infection sera, to glycolipids of all three life-cycle stages and thus revealed the epitopic importance of fucose.     

Schistosoma mansoni: fractionation of polysaccharide egg antigens by lectin affinity chromatography.

Crude polysaccharide antigen was extracted from Schistosoma mansoni egg homogenate by 44% aqueous phenol. The aqueous soluble polysaccharide extract was subjected to affinity chromatography with concanavalin A-Sepharose 4B. Two fractions (bound and unbound) were obtained; both of them gave precipitin lines with serum obtained from mice infected with S. mansoni. These precipitin lines gave partial identity. Further fractionation with wheat germ agglutinin-sepharose of the unbound fraction resulted in three antigenic fractions. These different antigens were eluted with different N-acetylglucosamine molarities (0, 0.05) and 0.5) and gave lines of identity when reacted against infected mouse serum. When the four antigenic materials were subjected to polyacrylamide gel electrophoresis and stained for proteins and polysaccharides no migration bands were observed. The chemical analysis of the two initial fractions showed a small percentage of amino acids in both fractions. Sugar analysis with gas-liquid chromatography showed different sugar composition of the two initial fractions.     

Generation and characterization of human monoclonal scFv antibodies against Helicobacter pylori antigens

Infection with Helicobacter pylori is chronic despite a vigorous cellular and humoral immune response and causes severe pathology in some patients. In this study, phage display was used as a new approach in order to investigate the role of the host's humoral immune response in the pathogenesis of H. pylori gastritis. Human monoclonal single-chain Fv (scFv) antibody fragments against H. pylori cell lysate and the H. pylori urease were isolated from an immune phage display library, constructed from peripheral blood lymphocytes of an H. pylori-infected patient. After affinity selection, 23% of the clones tested showed binding activity against a lysate of the H. pylori Sydney strain in enzyme-linked immunosorbent assay (ELISA) and 9% bound the H. pylori urease. Further characterization by PCR-fingerprint analysis and sequencing revealed that two closely related H. pylori binders and one antiurease scFv could be isolated. The selected scFvs were highly specific as analyzed by ELISA and immunoblots using various bacterial lysates and recombinant proteins. Analysis of the humoral immune response following H. pylori infection using human monoclonal antibodies might contribute to a better understanding of the pathogenesis of the disease. Moreover, using immune phage display libraries, it might be possible for relevant epitopes of H. pylori antigens to be determined, which might be of use for vaccine development.