Plasmodium berghei: Specific stimulation of rat lymphocytes by soluble antigens released in vitro from infected erythrocytes

Soluble components released in vitro from rat erythrocytes parasitized with Plasmodium berghei have been shown to stimulate nonadherent spleen lymphocytes from rats convalescent from homologous infection. The active product is released from the infected erythrocytes either spontaneously during incubation, or as the result of artificial rupture of infected erythrocytes by freezing and thawing or sonication. This antigen produced precipitation lines with antiplasmodial antibodies from sera of convalescent and hyperimmune rats and from immunized rabbits.     

Isolation of a polysaccharide antigen from Schistosoma mansoni eggs.

A polysaccharide antigen was isolated from Schistosoma mansoni egg homogenates by the phenol procedure. The crude preparation (CPEA) contained at least two antigens. The more purified antigen (PEA) was isolated by sequential enzymatic treatment of CPEA with DNase, RNase, Pronase, and alpha-amylase. PEA was resistant to boiling, freezing and thawing, mild acid and alkali, and chloroform, but was destroyed with periodate. It gave a positive reaction with anthrone reagent. PEA was eluted in the wash fraction from a DEAE cellulose collumn and in the void volume of a Sephacryl 200 column. After immunoelectrophoresis and polyacrylamide electrophoresis there was little or no migration. Amino acid analysis failed to reveal ninhydrin-positive material in the a hydrolyzate of PEA. These resluts suggested that PEA is a neutral polysaccharide with a m.w. of more than 200,000 and contains no amino acids or hexosamine. Antibodies against PEA were detected in sera obtained from mice infected with S. mansoni. PEA is different from previously described antigens derived from schistosome eggs.     

Immunization against Taenia taeniaeformis in mice: studies on the characterization of antigens from oncospheres

Mature eggs of Taenia taeniaeformis hatched readily in the presence of sodium hypochlorite and no loss in infectivity of oncospheres for mice was observed after hatching. Crude and sodium deoxycholate-solubilized antigens (termed TtO-DOC) prepared from such oncospheres stimulated high levels of protection against T. taeniaeformis infection in immunized mice similar to those described previously for oncospheres prepared by other methods. Mice immunized with TtO-DOC antigens that had been exposed to potassium metaperiodate remained significantly protected against infection. Exposure of TtO-DOC antigens to pronase and thermolysin, or to trypsin, significantly reduced the ability of these antigens to protect mice against infection. These data suggest that the antigens which immunize mice against infection include protein components. ¹²⁵I-labelled TtO-DOC antigens were immunoprecipitated with sera from mice infected with T. taeniaeformis and the immunoprecipitates analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoprecipitation with sera from C3H/He mice infected for 28 days revealed a single major labelled protein antigen having a relative molecular mass (M_r) of 31,000. Sera from 5-month infected C3H/He mice immunoprecipitated at least thirteen labelled antigens, including one at M_r 31,000. Attempts to use SDS-PAGE separated proteins to immunize mice showed that oncosphere antigens exposed to the reducing conditions prior to SDS-PAGE lost their ability to protect mice against infection. It was concluded that SDS-PAGE was an unsatisfactory technique for the isolation of a host protective fraction of TtO-DOC antigens. TtO-DOC proteins were resolved by PAGE performed in the presence of sodium deoxycholate (DOC-PAGE) and mice were vaccinated with cut-outs from the gel. A fraction of the DOC-polyacrylamide gel was found to be effective in immunizing mice against infection. Thus, although the characteristics of the protein antigens in this DOC-PAGE fraction have yet to be determined, an important fractionation technique has been identified. It was shown that partial removal of DOC from oncosphere antigen preparations solubilized in 1% DOC was required for the antigen to stimulate protective immunity. These findings will facilitate further antigen characterization studies towards the development of a defined-antigen vaccine in murine cysticercosis. This is particularly so as attempts to raise anti-oncospheral monoclonal antibodies capable of passively transferring protection to mice by using crude antigen preparations to immunize donor mice have not been successful.     

Reactivity of Two Selected Antigens of Neisseria gonorrhoeae

Two antigen preparations, the soluble antigen and a fraction 1 thereof, isolated in the course of a systematic study of the various antigens of the virulent gonococcus, have been investigated for their ability to serve as antigens for the detection of antibody in patients infected with the gonococcus. The soluble antigen was reactive with 88.2% of the sera from infected females, and fraction 1 was reactive with 71.6% of the sera. Of sera from infected males, only 27.6% reacted with the soluble antigen and only 20.4% with fraction 1. Of sera from individuals presumed free of gonococcal infection, approximately 4% reacted with the soluble antigen; none reacted with fraction 1. This study suggests that these antigens might be adaptable to the detection of human gonococcal antibody, especially in the female.     

Trypanosoma lewisi: effects of immunosuppression on the serological reactivities of exoantigens and cellular antigens of bloodstream parasites.

Groups of rats were immunosuppressed with antithymocyte serum (ATS) and infected with Trypanosoma lewisi. Immunodiffusion studies were performed which demonstrated that trypanosome exoantigens, present in the plasma of these animals, were precipitated by antibodies in the sera of rats undergoing a typical primary T. lewisi infection; extracts of trypanosomes which had been collected from ATS-treated rats contained antigens which also were precipitated by antibodies in these sera. These precipitating antibodies could not be detected using either the plasma of untreated infected rats or extracts of trypanosomes which had been collected from untreated rats. With the exoantigens, precipitating antibodies were detected in serum samples collected from rats 14 to 250 days after infection. With the extract, precipitating antibodies were found as early as 5 days after infection and could be detected as late as 90 days after infection. Antigens of trypanosome extracts partially blocked the precipitin reactions between antisera and exoantigens, suggesting the presence of common antigens in the two preparations. Intact trypanosomes were serologically more reactive when collected from immunosuppressed rats. Trypanosomes collected from ATS-treated rats were agglutinated by antisera at titers fourfold higher than trypanosomes collected from untreated hosts. Absorption with exoantigens from immunosuppressed infected rats blocked trypanosome agglutination, indicating that these antigens are of cell surface origin. The experiments suggest that a likely result of immunosuppressing the host is a trypanosome antigen preparation that is a more reactive serodiagnostic reagent.     

Characterization of Plasmodium berghei antigens inducing blast transformation in immune rat lymphocytes

Rat erythrocytes, infected with Plasmodium berghei, were disrupted by freezing and thawing or parasites were released by mechanical breaking of the agglutinated erythrocytes. The released soluble antigens, inducing blast transformation in non-adherent immune rat spleen cells, were fractionated by a sequence of salting out, ion exchange chromatography and gel filtration. At the final step a purification factor of 83.7-fold was achieved. This fraction contained three components discernible by SDS-PAGE of molecular weights 56, 66 and 72 kD respectively. The free parasites, also induced blast transformation in immune rat spleen cells. After disruption by freeze-thawing the majority of the active antigens was found in the sediment after centrifugation at 17,300 g.     

Stability of rubella complement-fixing antigens

Various types of rubella complement-fixing (CF) antigen preparations derived from infected BHK-21 cell cultures were examined for stability to certain chemical and physical agents. Antigens studied included: infected culture fluids concentrated 100-fold, ether-treated fluid concentrates, alkaline buffer extracts of infected cells, and large- and small-particle CF antigens separated by Sephadex gel filtration. All preparations were stable at -20 C for months, and were unaffected by three cycles of freezing and thawing. Ether treatment rendered antigens highly susceptible to thermal inactivation at 56 C; untreated antigens were inactivated slowly and showed a persistent fraction of activity even after 2 hr. Large-particle antigen was found to be slightly more susceptible than small-particle antigen to thermal inactivation and ether treatment. CF activity in alkaline buffer extracts was more labile than that in untreated or ether-treated fluid concentrates upon dialysis, Sephadex gel filtration, and sucrose density gradient centrifugation.     

Schistosoma mansoni: antigenic heterogeneity of excretions and secretions

The excretory-secretory antigens of adult Schistosoma mansoni were obtained by in vitro cultivation of worms in a chemically-defined medium. The protein output in this system was low, 0.2–0.4 μg of proteinworm/48 hr. The composition of the crude culture antigen (CA) was approximately 80% protein, 15% carbohydrate and 5% nucleic acid. Disc gel electrophoresis of CA revealed the presence of at least 15 protein components, many with carbohydrate moieties. Three major fractions were obtained by gel filtration on Sephadex G-200. Fraction I contained the bulk of the glycoprotein material. Immunoelectrophoresis of CA with hyperimmune rabbit serum indicated the presence of at least 6 antigens, most of which eluted in Fraction II. Serum from infected mice and monkeys, but not from rabbits and rats, reacted with CA and its fractions, especially Fraction II, on immunodiffusion analysis. Comparison of CA with other adult worm extracts by immunodiffusion techniques showed that most of the excretory-secretory antigens could be obtained by either freezing and thawing or by extraction with 3 M KCl. The P.K.-type activity of CA was considerably greater than that of a lipid-free adult worm antigen. Both Fractions I and II had the P.K.-type activity. An antigen capable of eliciting macrophage migration inhibition factor from infected rat lymphocytes was detected in CA, although the lymphocyte toxicity of CA was high at concentrations above 10 μg/ml.     

Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development

The development of effective diagnostic tools will be essential in the continuing fight to reduce schistosome infection; however, the diagnostic tests available to date are generally laborious and difficult to implement in current parasite control strategies. We generated a series of single-chain antibody Fv domain (scFv) phage display libraries from the portal lymph node of field exposed water buffaloes, Bubalus bubalis, 11–12 days post challenge with Schistosoma japonicum cercariae. The selected scFv-phages showed clear enrichment towards adult schistosomes and excretory-secretory (ES) proteins by immunofluorescence, ELISA and western blot analysis. The enriched libraries were used to probe a schistosome specific protein microarray resulting in the recognition of a number of proteins, five of which were specific to schistosomes, with RNA expression predominantly in the adult life-stage based on interrogation of schistosome expressed sequence tags (EST). As the libraries were enriched by panning against ES products, these antigens may be excreted or secreted into the host vasculature and hence may make good targets for a diagnostic assay. Further selection of the scFv library against infected mouse sera identified five soluble scFv clones that could selectively recognise soluble whole adult preparations (SWAP) relative to an irrelevant protein control (ovalbumin). Furthermore, two of the identified scFv clones also selectively recognised SWAP proteins when spiked into naïve mouse sera. These host B-cell derived scFvs that specifically bind to schistosome protein preparations will be valuable reagents for further development of a cost effective point-of-care diagnostic test.     

Comparison of Various Methods for Preparation of Viral Serological Antigens from Infected Cell Cultures

In efforts to prepare more potent and sensitive viral serological antigens, several aspects of the production of antigens from infected cell cultures were studied. Antigens derived from whole, infected culture material and from the cellular and fluid phases were compared. Freezing and thawing, sonication, and alkaline buffer extraction were compared for effectiveness in releasing antigen from host cells. The effect of the multiplicity of infection on titers of viral antigens produced in cell cultures was studied. Generally, higher titered antigens were derived from the infected cells than from the culture fluids, but for certain viruses complement-fixing (CF) antigens derived from the culture fluids gave higher antibody titers than did cell-associated antigens. With each virus-host cell system studied, treatment with alkaline buffers extracted appreciable amounts of CF antigen from the host cells, but in some instances more antigen was released by freezing and thawing or by sonication. Extraction of infected cells with alkaline buffers was not a satisfactory method for preparation of hemagglutinating (HA) antigens for any of the viruses studied. The highest-titered HA antigens were produced from infected cells disrupted by freezing and thawing or sonication. The highest titered CF and HA antigens were produced from cell cultures infected at multiplicities of one or greater. Complement-fixing antigens produced by infecting cells in suspension and then planting had lower titers than antigens produced in parallel by infecting developed monolayers. Optimal methods are summarized for preparation of serological antigens to a variety of viruses of man.     

Identification and the role of soluble antigens detected in bile from Eimeria stiedai-infected rabbits

Antibodies against Eimeria stiedai sporozoites and merozoites were detected in the sera of rabbits immunized with bile obtained from infected rabbits on the 15th day post-infection. The trails made by gliding sporozoites were also detected by the sera. After penetration into the host cell, an antibody-binding region was observed on the parasitophorous vacuole membranes of the parasites. Rabbits administered a combination of the bile and cholera toxin shed fewer oocysts in the feces after infection than control rabbits. The immunized rabbits developed a high level of IgA antibody against soluble antigens in the bile. By immunoblotting, antigens with molecular masses of 32, 37, and 49 kDa were detected in the bile obtained from infected rabbits on the 15th day postinfection. Absorption treatment with sporozoites reduced or abolished the antibody reactivity to the 32-kDa antigen of merozoites and the bile antigens. However, antibody reactivity to the 37- and 49-kDa antigens still remained. These results indicate that soluble antigens are present in the bile of rabbits in the acute phase of infection, and these may be produced and released by merozoites during the host cell invasion process.     

Comparison of Schistosoma mansoni Soluble Cercarial Antigens and Soluble Egg Antigens for Serodiagnosing Schistosome Infections

A Schistosoma mansoni cercarial antigen preparation (cercarial transformation fluid – SmCTF) was evaluated for detection of anti-schistosome antibodies in human sera in 4 collaborating laboratories. The performance of SmCTF was compared with that of S. mansoni egg antigens (SmSEA) in an indirect enzyme-immunoassay (ELISA) antigen assay, the latter being used routinely in 3 of the 4 participating laboratories to diagnose S. mansoni and S. haematobium infections. In the fourth laboratory the performance of SmCTF was compared with that of S. japonicum egg antigens (SjSEA) in ELISA for detection of anti-S. japonicum antibodies. In all 4 laboratories the results given by SmCTF in ELISA were very similar to those given by the antigen preparation routinely used in the respective laboratory to detect anti-schistosome antibodies in human infection sera. In so far as the ELISA results from SmCTF are thus so little different from those given by schistosome egg antigens and also cheaper to produce, the former is a potentially useful new diagnostic aid for schistosomiasis.     

Fasciola hepatica: Comparative studies on fascioliasis in rats and mice

Chapman C. B. and Mitchell G. F. 1982. Fasciola hepatica: comparative studies on fascioliasis in rats and mice. International Journal for Parasitology12: 81–91. Certain characteristics of infection differ between rats and mice exposed to metacercariae of the trematode parasite, Fasciola hepatica. Rats develop a degree of age-related resistance (and infected older females contain fewer parasites than older males), resistance to reinfection in infected rats is demonstrated readily though is partial, and a comparable degree of resistance can be obtained in recipients of infected rat serum provided the serum is given at about the time of challenge. None of these features of F. hepatica infection is seen in mice. Rats also differ from mice in that they can be vaccinated against infection (although again, resistance is incomplete) using larval antigen mixtures in adjuvants. Mice do respond to infection by production of antilarval antibodies and a slight IgG₁ hypergammaglobulinaemia and larvae will sensitize mice for delayed hypersensitivity. The results of this study indicate that sera from infected rats versus infected mice will be useful in pinpointing antigens of F. hepatica larvae which are involved in expression of partial host protection.     

Anti-idiotypic T lymphocyte responsiveness in murine Schistosomiasis mansoni

Pooled sera from CBA/J mice infected for greater than or equal to 16 weeks with the blood fluke Schistosoma mansoni were immunoaffinity purified using soluble schistosome egg antigens (SEA) coupled to Sepharose 4B. The bound and then eluted fraction was shown to contain only immunoglobulins and to have anti-SEA activity. These anti-SEA antibodies stimulated proliferation of lymph node cells from mice infected with S. mansoni for 8, 12, or greater than or equal to 16 weeks but not from uninfected mice. The cells stimulated by anti-SEA antibodies were nylon wool adherent, Thy-1.2+, L3T4+, Lyt-2-lymphocytes. Immunoglobulins without anti-SEA activity isolated from the sera of syngeneic uninfected mice were not stimulatory for cells from normal or infected animals. Thus the responding T cells appear to be stimulated by the idiotypes expressed on the syngenic anti-SEA antibodies. These data present evidence for anti-idiotypic cellular reactions in murine schistosomiasis that could play important immunoregulatory roles in this disease.     

Trichinella spiralis: immunization of pigs with newborn larval antigens

The potential of crude Trichinella spiralis newborn larval antigens for pig immunization was investigated. A preparation of whole newborn larvae killed by freezing and thawing, and combined with Freund's complete adjuvant, induced a high level of protection against challenge (78%), compared to a 40% resistance level in pigs immunized with excretory secretory antigens of muscle larvae. Sera from pigs immunized with newborn larvae contained antibodies which bound to the surface of the newborn larvae, as determined by immunofluorescence. In a second trial, the freeze thawed newborn larvae preparation was compared with a soluble and insoluble fraction prepared by sonication of whole newborn larvae. Pigs receiving whole newborn larvae or the insoluble fraction developed strong immunity to challenge (88.2 and 85.5%, respectively); the soluble fraction was ineffective. Immunization with all preparations induced antibody to newborn larval antigens, but not to adult or muscle larvae excretory secretory antigens. Polyacrylamide gel electrophoresis of the soluble and insoluble fractions indicated that sonication was ineffective in solubilizing the larger molecular weight components. These results demonstrate that newborn larval antigens are highly protective in pigs, but that their further development as a vaccine will require more efficient procedures for antigen solubilization and large-scale production.     

ELISA of rat sera infected with Paragonimus iloktsuenensis

Enzyme-linked immunosorbent assay (ELISA) of paragonimiasis iloktsuenensis rat sera was performed using crude antigens of Paragonimus iloktsuenensis (PIA), P. westermani (PWA) and Clonorchis sinensis (CSA). Three crude antigens (PIA, PWA, CSA) were prepared to saline homogenated supernatants of whole adult worms. Infected rat sera were obtained biweekly from the albino rats fed 50-80 metacercariae of P. iloktsuenensis through gastric catheter. Experimental groups were divided into 4 groups: GI (controls), GII, GIII and GIV according to 1-7 worms as GII, 10-19 worms as GIII and 22-40 worms as GIV, respectively. In ELISA, the mean OD values of each group for the homologous antigen (PIA) were increased significantly compared to the control sera at the 4th week of infection. With the progress of duration of infection, the mean OD values of infected sera of GII & GIV continuously increased up to the 12th week (last week), but in GIII the mean OD value increased until the 10th week. No significance was noted among the infection dose groups (GII, GIII and GIV), after the 6th week of infection. Also, the OD values of all infected rats did not show any proportional relationships to the number of worms recovered. In brief, the antibody productivity of individual rats were strongly different. The rat sera infected with P. iloktsuenensis cross-reacted with those infected with P. westermani or C. sinensis, as identified by OD values.     

Schistosoma mansoni host-exposed surface antigens characterized by sera and recombinant antibodies from schistosomiasis-resistant rats

Antibodies from Schistosoma mansoni-infected rats, unlike mice, show a higher titer for schistosome apical tegumental antigens compared with non-apical membrane antigens. These antibodies bind to the surface of living lung-stage worms and to formaldehyde-fixed adult worms. We produced a single-chain antibody Fv domain (scFv) phage library displaying the antibody repertoire of rats highly immune to schistosome infection and we selected for scFvs that recognize the host-exposed surface of worms. Five unique rat scFvs (Teg1, Teg4, Teg5, Teg20 and Teg37) were obtained which recognize schistosome surface epitopes. Each of the scFvs recognizes the surface of living schistosomula and lung-stage schistosomules and/or the surface of formaldehyde-fixed adult worms. None of these scFvs reproducibly stained living adult worms. This suggests that a change occurs during the transition from lung schistosomules to 4-week adults such that at least some surface antigens, although remaining on the surface in living adult worms, can no longer be immunologically stained. Teg1 and Teg4 scFvs both recognize specific bands on Western blots. No bands were observed for the other three scFvs, suggesting that these scFvs may recognize non-protein or conformationally-dependent epitopes. Teg1 was unambiguously identified as recognizing the S. mansoni tetraspanin antigen, SmTSP-2, within the large extracellular domain. Teg4 recognizes a 35 kDa band tentatively identified as Sm29 by proteomic analysis. These scFvs can now be used to characterize schistosome epitopes at the host-parasite interface, to target worms in vivo, and to study the mechanisms by which these worms naturally evade immune damage to the tegument within permissive hosts.     

Schistosoma japonicum Soluble Egg Antigens Attenuate Invasion in a First Trimester Human Placental Trophoblast Model

Background

Schistosomiasis affects nearly 40 million women of reproductive age, and is known to elicit a pro-inflammatory signature in the placenta. We have previously shown that antigens from schistosome eggs can elicit pro-inflammatory cytokine production from trophoblast cells specifically; however, the influence of these antigens on other characteristics of trophoblast function, particularly as it pertains to placentation in early gestation, is unknown. We therefore sought to determine the impact of schistosome antigens on key characteristics of first trimester trophoblast cells, including migration and invasion.

Methods

First trimester HTR8/SVneo trophoblast cells were co-cultured with plasma from pregnant women with and without schistosomiasis or schistosome soluble egg antigens (SEA) and measured cytokine, cellular migration, and invasion responses.

Results

Exposure of HTR8 cells to SEA resulted in a pro-inflammatory, anti-invasive signature, characterized by increased pro-inflammatory cytokines (IL-6, IL-8, MCP-1) and TIMP-1. Additionally, these cells displayed 62% decreased migration and 2.7-fold decreased invasion in vitro after treatment with SEA. These results are supported by increased IL-6 and IL-8 in the culture media of HTR8 cells exposed to plasma from Schistosoma japonica infected pregnant women.

Conclusions

Soluble egg antigens found in circulation during schistosome infection increase pro-inflammatory cytokine production and inhibit the mobility and invasive characteristics of the first trimester HTR8/SVneo trophoblast cell line. This is the first study to assess the impact of schistosome soluble egg antigens on the behavior of an extravillous trophoblast model and suggests that schistosomiasis in the pre-pregnancy period may adversely impact placentation and the subsequent health of the mother and newborn.     

Macrophages as effector cells of protective immunity in murine schistosomiasis

Mice infected with Schistosoma mansoni develop a dramatic (five- to eightfold) increase in numbers of peritoneal leukocytes, and approximately 65% of these cells are macrophages. By several biochemical and cytochemical criteria, these cells were comparable to resident peritoneal macrophages of normal mice. However, macrophages from schistosome-infected mice exhibited significant nonspecific tumoricidal activity in vitro, a function associated with immunologically activated cells. The time course for development of activated macrophages in the peritoneal cavity was dependent upon the route of infection. Cytotoxic cells were present in the peritoneal cavity by 3 weeks after intraperitoneal infection, but were not evident until several weeks later in animals infected percutaneously, subcutaneously, or intravenously. However, by 3 weeks after subcutaneous infection, tumoricidal macrophages appeared in the peritoneal cavity after intraperitoneal challenge with soluble schistosome antigens. Macrophage activation was independent of the development of egg granulomas, since tumoricidal cells could be found prior to the onset of egg production and were also present in mice infected with only male worms. Development of activated macrophages in these instances is thus consistent with previous observations on induction of T lymphocyte reactivity toward schistosomula. Since other manipulations known to activate macrophages have been shown to induce partial resistance to schistosome infection, the finding that macrophage activation results from primary S. mansoni infection itself suggests that these cells may play a major role in acquired immunity to this parasite.     

Dirofilaria immitis: identification and partial characterization of parasite antigens in the serum of infected dogs

We have recently developed a sensitive and specific immunodiagnostic test for canine Dirofilaria immitis infection based on detection of soluble parasite antigens in dog sera by monoclonal antibody-based enzyme immunoassay. In addition to their importance as markers of infection, these antigens may contribute to the pathogenesis of heartworm disease in dogs. In the present study, a variety of methods were used to identify and characterize circulating D. immitis antigens. Two antigens were identified in infected dog sera that formed lines of identity in rocket-line immunoelectrophoresis with soluble antigens extracted from adult D. immitis. Circulating D. immitis antigens were also demonstrated in infected dog sera by immunoblot analysis with polyclonal and monoclonal antibodies. These antigens had apparent molecular weights that ranged from 50 to 250 kDa. Most of the circulating D. immitis antigens contained the epitope defined by monoclonal antibody 1418BF2.1 which is used in our enzyme immunoassay for circulating D. immitis antigen. Studies of parasite antigens released during in vitro culture indicated that the circulating D. immitis antigens in dog sera that are detected by our enzyme immunoassay are primarily derived from adult female worms.