Analysis and potential of excretory-secretory antigens of Setaria cervi for immunodiagnosis of human filariasis.

The excretory-secretory (E-LS) products released by the adult Setaria cervi, a bovine filarial parasite, were used to raise polyvalent hyperimmune serum in rabbits. Analysis of E-S products, using anti-E-S serum showed the presence of 10-14 immunogenic proteins, the rabbit anti-E-S serum showed reciprocal antibody titres in the range of 100,000-250,000 by enzyme linked immunosorbent assay. The anti-E-S antibodies could detect circulating antigen in filarial patients sera by Counter immunoelectrophoresis.

Monoclonal antibodies against lactate dehydrogenase of Plasmodium knowlesi.

Lactate dehydrogenase (LDH) of malarial parasites has been demonstrated to be biochemically and immunochemically distinct from the equivalent host enzyme. The polyclonal antibodies raised against the purified plasmodial LDH showed specificity to Plasmodium spp. Six hybridoma cell lines secreting monoclonal antibodies specific to Plasmodium knowlesi LDH have been obtained. The two monoclonal antibodies (2A3B7 and 4A6A7) showed high reactivity with LDH from simian (P. knowlesi. P. cynomolgi), human (P. falciparum, P. vivax) and rodent (P. berghei, P. yoelii) malarial parasites and did not cross-react with red cell LDH as well as with isoenzymic forms of mammalian LDH (A4, B4 and C4). One monoclonal antibody (4A6A7) strongly inhibited the enzyme activity specifically of plasmodial LDH and did not have any effect on the activity of red cell LDH. The other monoclonal (2A3B7) did not show inhibitory effect on parasite LDH. These findings as well as competitive immunoassay studies suggest the presence of at least two parasite specific epitopes on plasmodial LDH.

Monoclonal antibodies against antigenic epitopes common between Setaria cervi and Brugia malayi.

Several common antigens between the bovine (Setaria cervi) and human (Brugia malayi) filarial parasites have been demonstrated [Immunol Investig, 16 (1987) 139]. Hybridoma cell lines producing monoclonal antibodies against such common antigenic epitopes were obtained by immunizing the BALB/c mice with S. cervi antigen, fusing the spleen cells with Sp2/0 myeloma cells and screening the culture supernatants for antibody against both S. cervi and B. malayi antigens by ELISA. Nine monoclonal antibodies directed against antigenic epitopes common between the bovine and human filarial parasites were identified. Two monoclonal antibodies (I3B4 and I5D6) showed reactivity with the antigen(s) present in filariasis patients serum and thus may have potential for detecting circulating antigen in filaria infected individuals.

Comparative analysis of surface components of adult, micro-filariae and infective larvae of Brugia malayi.

A comparative analysis of surface proteins of adult, microfilariae and infective larvae of Brugia malayi, the human filarial parasite, has been carried out using IODOGEN (1,3,4,6-tetrachloro-3,alpha 6 alpha-diphenyl-glycoluril) and lactoperoxidase methods. SDS-polyacrylamide gel electrophoretic and autoradiographic analyses revealed the presence of 9 proteins (15-200 kDa) in adults, while microfilariae and infective larvae showed 8 and 6 proteins (15-120 kDa), respectively. The pattern of proteins radiolabelled by IODOGEN method was very similar to that of proteins labelled by the lactoperoxidase method. Since these proteins are released by the protease treatment of whole parasites, they are likely to be present on the surface of the parasite.

Antibodies to lactate dehydrogenase of Plasmodium knowlesi are specific to Plasmodium species.

Polyclonal immune monkey serum raised against schizonts of Plasmodium knowlesi (H-strain) showed the presence of antibodies to lactate dehydrogenase (LDH) of P. knowlesi by immunodot enzyme staining method. The anti-LDH antibodies are most probably directed towards an epitope distinct from the catalytic site as shown by the specific enzyme staining of LDH after binding with antibody on nitrocellulose paper. These antibodies showed reactivity with LDH from different strains (H, P and W1 strains of P. knowlesi) and species (P. cynomolgi B, P. berghei, P. yoelii, P. falciparum and P. vivax) of malarial parasites but did not cross-react with three isoenzymic forms of mammalian LDH (A4, B4 and C4) as well as with LDH from some protozoan and helminth parasites. These findings suggest that the anti-LDH antibodies have defined specificity to Plasmodium spp.

Ancylostoma ceylanicum: I. Protein and antigenic composition of adult and larval stages.

The protein and antigenic composition of adult and larval stages of Ancylostoma ceylanicum, a human hookworm maintained in golden hamsters (Mesocricetus auratus), was studied employing immunochemical techniques. SDS-polyacrylamide gel electrophoresis revealed the presence of 47 and 43 protein bands in adult worms and infective larvae respectively in the molecular weight range of 10-170 kD. Crossed immunoelectrophoretic analysis, using immune rabbit sera, showed the presence of 32 antigenic peaks in adults and 19 in infective larval stage. Most of the antigens were common between adult and larval stage as evidenced by cross-line immunoelectrophoresis, although some stage specific antigens were also identified. These studies also demonstrate the complex nature of adult worms as compared to larvae.

Antigenic characterization of excretory-secretory products of Setaria cervi.

Excretory-secretory products (ES) of Setaria cervi, a bovine filarial parasite, were prepared by in vitro maintenance of the worms in a protein free defined medium at 37 degrees C. RPMI-1640 medium proved to be the best as the worms remained motile in it for the maximum period of time without change of medium, and approximately 12-15 mg protein per 100 adult worms could be obtained. Crossed immunoelectrophoretic analysis of ES products revealed 11-15 antigens and most of them were common to somatic antigenic preparations from both adult worms and microfilariae. The ES products were also found to contain 3-4 host proteins, including serum albumin.

Identification of antigenic proteins of setaria cervi by immunoblotting technique.

Identification and characterization of antigenic proteins of Setaria cervi (bovine filarial parasite) adults and microfilariae was done by immunoblotting technique using hyperimmune rabbit sera against S. cervi and Brugia malayi. The antigens recognized by these sera were detected by using 125I protein-A followed by autoradiography. Fifteen different antigens were observed to be common between adult and microfilarial stages of the parasite. Some stage specific antigens were also identified. Many antigens of S. cervi adults and microfilariae were also recognized by rabbit anti-B.malayi serum showing the existence of common antigenic determinants between the bovine and human filarial parasites.

Effects of transmission-blocking monoclonal antibodies on different isolates of Plasmodium falciparum.

The strain diversity in Plasmodium falciparum has been studied with respect to gamete surface antigens which are the targets of transmission-blocking antibodies. Of 12 isolates tested, 11 were positive by immunofluorescence with the three monoclonal antibodies studied. The exception was a Liberian isolate, two clones of which were found to react with only one of the three monoclonal antibodies. Antibodies IIC5-B10 and IA3-B8, which previously have been shown to act synergistically to block infectivity of 7G8, a Brazilian clone of P. falciparum, acted in an exactly similar way with another Brazilian isolate, It.D12, and an isolate from Thailand. In the presence of complement either IA3-B8 or a third antibody, IID2-A10, strongly suppressed infectivity of It.D12 as well as 7G8, but neither isolate was strongly suppressed by IIC5-B10. IA3-B8 and IID2-A10 did not react by immunofluorescence or immunoprecipitation with gametes of L.E5; IIC5-B10 reacted positively with L.E5 gametes in these tests. In the absence of complement, the combination of IA3-B8 and IIC5-B10 did not suppress infectivity of L.E5 to mosquitoes. In contrast to its effect on gametocytes of other isolates, IIC5-B10 in the presence of complement strongly suppressed infectivity of L.E5 to mosquitoes. These results imply that IA3-B8 and IIC5-B10 react with two structurally distinct epitopes on the surface of gametes of P. falciparum and that the properties of both epitopes on gametes of L.E5 differ from those on gametes of the other isolates tested.

Target antigens in malaria transmission blocking immunity.

Malaria transmission blocking immunity has been found to operate against two distinct phases of development of malaria parasites in the mosquito midgut: (i) against the extracellular gametes and newly fertilized zygotes shortly after ingestion by a mosquito of parasitized blood and (ii) against the zygotes during their subsequent development into ookinetes. Immunity is antibody-mediated and stage-specific. A set of three proteins, synthesized in the gametocytes, expressed on the surface of the gametes and newly fertilized zygotes and subsequently shed during later transformation of the zygotes, has been identified as the target antigens of anti-gamete fertilization blocking antibodies. A single protein, synthesized and expressed on the zygote surface during its development to ookinetes, has been identified as the target of antibodies which block the development of the fertilized parasites in the mosquito. Immunization of human populations against gamete or zygote antigens, while not directly protecting an immunized individual from inflection, would reduce the transfer of malaria within the population. Such immunity, in addition to reducing the overall rate of malaria transmission, would, if combined with a vaccine against the asexual (disease-causing) stages, reduce the chance of selection of parasites that are resistant to the asexual vaccine by preventing their entry into the mosquito population.

Characterization of antigens on mosquito midgut stages of Plasmodium gallinaceum. III. Changes in zygote surface proteins during transformation to mature ookinete.

Proteins expressed on the surface of zygotes of Plasmodium gallinaceum during their development from fertilization to mature ookinetes have been examined by lactoperoxidase catalysed surface radioiodination and immunoprecipitation with stage-specific immune rabbit sera and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Surface-labelled proteins of apparent Mr equal to or greater than 55 000 on the female gametes and newly fertilized zygotes were shed during transformation and were recovered quantitatively and apparently intact from the culture supernatant. Zygote surface proteins of Mr 50 000, 19 000 and 17 000 remained bound to the surface throughout the transformation. Three proteins were expressed de novo on the surface of the mature ookinete of which Mr 26 000 and 28 000 represented major surface components and Mr 52 000 a relatively minor component.

A surface protein expressed during the transformation of zygotes of Plasmodium gallinaceum is a target of transmission-blocking antibodies.

Antibodies against gametes of malarial parasites (Plasmodium spp.) have previously been shown to block infectivity of the parasites to mosquitoes by preventing fertilization of the parasites in the insect midgut. These antibodies did not have any effect on the development of fertilized parasites. We now report that a surface protein of Mr 26,000 synthesized by zygotes of P. gallinaceum is the target of antibodies which block infectivity of the fertilized parasites to mosquitoes. Identification of this target antigen offers a new stage of the parasite against which a malaria transmission-blocking vaccine could be developed.

Characterization of antigens on mosquito midgut stages of Plasmodium gallinaceum. II. Comparison of surface antigens of male and female gametes and zygotes.

Surface proteins of male and female gametes of Plasmodium gallinaceum were radioiodinated by the lactoperoxidase method, immunoprecipitated with stage specific antisera and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Stage specificity of the surface antigens was further studied by competition between surface iodinated gametes and unlabeled extracts of gametes, zygotes, or asexual parasites during immunoprecipitation reactions. These studies have identified four proteins: 250 kDa (PgZ-1), 215 kDa (PgZ-3) and 56 and 54 kDa (PgZ-13a and b), which were present in indistinguishable antigenic form on both male and female gametes. Three immunogenic proteins, 48 kDa (PgZ-14) and 19 and 17 kDa (PgZ-17a and b), were present on female but not male gametes as were several weakly labeled, non-immunogenic proteins of less than 45 kDa. A 26 kDa protein (PgZ-16) was present on male but not female gametes. Two proteins of 205 and 83 kDa (PgZ-4 and PgZ-11) were labeled on female but not male gametes. Nevertheless preparations of male gametes appeared to contain epitopes cross-reacting with these two proteins since anti-male gamete serum precipitated PgZ-4 and 11. Immune competition studies indicated that each of the surface proteins labeled on sexual stages was antigenically distinct from material present in asexual parasites.

Monoclonal antibodies against surface determinants on gametes of Plasmodium gallinaceum block transmission of malaria parasites to mosquitoes.

Monoclonal antibodies (MAb) against gametes of the chicken malaria Plasmodium gallinaceum have been derived. All reacted with the surface of extracellular gametes of the parasite in immunofluorescent antibody reactions and all agglutinated both male and female gametes. In the absence of active complement one mu isotype MAb, la 1-D5, mediated at least 95% suppression of infectivity of the parasites to Aedes aegypti mosquitoes. Individually, MAb of the gamma 1 or gamma 2a isotypes mediated only slight suppression in the absence of active complement. Certain combinations of these MAb, however, suppressed parasite infectivity by 90 to 95%. Suppression of infectivity by the MAb was shown to be mainly due to their effects on the events leading up to or including fertilization. Certain gamma 2a isotype MAb, which otherwise mediated minimal or no suppressive effect, completely abolished infectivity of the parasites if complement was present. No target antigen could be identified by immunoprecipitation of Triton X-100 extracts of surface radioiodinated zygotes or gametes of P. gallinaceum for the mu isotype MAb. All gamma isotype MAb precipitated the same three proteins of 240,000, 56,000, and 54,000 daltons under reducing conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from extracts of radioiodinated male and female gametes. These surface proteins on gametes of both sexes of P. gallinaceum thus appear to include target antigens of anti-gamete transmission blocking immunity.

Characterization of antigens on mosquito midgut stages of Plasmodium gallinaceum. I. Zygote surface antigens.

We have defined the surface protein antigens on Plasmodium gallinaceum zygotes using radioiodination methods and rabbit anti-zygote serum which blocks transmission of the parasite to Aedes aegypti mosquitoes. Fifteen protein bands (1-15) in the molecular weight range of 40 000-240 000 and one band at the bromophenol blue dye marker were labelled by the lactoperoxidase and IODOGEN (1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril) methods. The localization of these radioiodinated components on the cell surface was confirmed in two ways: (1) They were completely degraded by trypsin or Streptomyces griseus protease treatment of intact viable zygotes. (2) They were immunoprecipitated following incubation of intact zygotes with antibody prior to detergent solubilization. Reactivity with immune rabbit serum demonstrated that the major surface immunogens were components with Mr 240 000, 200 000, 180 000, 80 000, 55 000, 50 000 and the band at the dye marker. A band of Mr 180 000 was shown immunologically to be a host serum protein selectively adsorbed to the zygote surface. The identity of this protein and the significance of its adsorption to the surface of the zygote are unknown.

Radioiodination of parasite antigens with 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril (IODOGEN): studies with zygotes of Plasmodium gallinaceum.

The iodinating reagent 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril (IODOGEN) was used to label antigens on zygotes of Plasmodium gallinaceum with parallel studies using lactoperoxidase-catalyzed radioiodination for comparison. Proteins labeled by the IODOGEN method are most probably on the surface of the zygote, as the pattern of labeled proteins analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was very similar to the pattern of lactoperoxidase-labeled proteins. Furthermore, the labeled proteins represented only a subset of the total Coomassie Blue-stained proteins. The radioiodinated zygote proteins were immunoreactive after IODOGEN or lactoperoxidase labeling. The IODOGEN method is technically much more simple than the lactoperoxidase method and does not require the addition of extraneous proteins or H2O2. The advantages of IODOGEN labeling, together with the essential equivalence of results obtained by these two methods, make the IODOGEN method attractive for labeling parasite antigens in general.

Gametocytogenesis by malaria parasites in continuous culture.

Asexual proliferation of malaria parasites proceeds by multiplication of the parasites within red cells. Following rupture of the host cells the released merozoites re-invade other red cells. On re-invasion, a proportion of merozoites become, not asexual parasites but gametocytes, the sexual stages infective to the mosquito vectors. Conversion of asexual parasites to gametocytes occurs not only during natural infections but also in continuous in vitro culture as reported first by Trager and Jensen and by others. We showed previously that the proportion of early intra-erythrocytic stages (ring stages) of Plasmodium falciparum which developed into gametocytes in culture was influenced by culture conditions. Gametocyte formation was rare in conditions supporting rapid proliferation but frequent when parasite densisites were static. We now show that nearly 100% of ring stages develop into gametocytes in response to 1mM cyclic AMP in static cultures whereas in rapidly growing cultures few rings become gametocytes in response to cyclic AMP.