Schistosoma mansoni: two-dimensional gel electrophoretic analysis of antigens uniquely immunoreactive with protective rat serum

Candidate vaccine antigens are defined by their differential immunoreactivity with antisera which are distinguishable by their capacity to confer passive resistance to infection. This "contrasting antisera" immunoassay has been successfully used in previous analyses of 4-week-old worm biosynthetically radiolabeled Schistosoma mansoni proteins to identify potentially protective antigens. Twice-infected Fischer (F-2x) and Wistar-Furth (W-2x) rat sera were the sources of protective and non-protective antibody, respectively. We have extended our original analysis by applying two-dimensional gel electrophoresis to resolve total and immunoreactive soluble proteins of the 4-week worms. Total proteins were characterized by silver staining and autoradiography. Radiolabeled protein antigens immunoprecipitated by F-2x and W-2x antisera were compared, and several were shown to be uniquely reactive with the protective immune serum. In a companion molecular approach to clone the candidate vaccine antigens, screening of a lambda gt11 adult S. mansoni cDNA expression library by the contrasting antisera assay has identified a clone (lambda 40) producing a fusion protein with epitopes uniquely reactive with F-2x. A rabbit antiserum to the lambda 40 fusion protein (anti-FP40) reacted with radiolabeled worm proteins in the 20-kDa size range. By 2D gel electrophoretic analysis, we can now demonstrate that anti-FP40 specifically immunoprecipitates most of the members of a multicomponent protein antigen subset 18-22 kDa in Mr, focusing over a pI range of 5.3-5.8, and recognized uniquely by F-2x.     

Acanthocheilonema viteae: vaccination of jirds with irradiation-attenuated stage-3 larvae and with exported larval antigens

Jirds (Meriones unguiculatus) were immunized with irradiated (35 krad) stage-3 larvae (L3) of Acanthocheilonema viteae. The induced resistance against homologous challenge infection and the antibody response of the animals were studied. Immunization with 3, 2, or 1 dose of 50 irradiated L3 induced approximately 90% resistance. Immunization with a single dose of only 5 irradiated L3 resulted in 60.8% protection while immunization with a single dose of 25 L3 induced 94.1% protection. The protection induced with 3 doses of 50 irradiated L3 did not decrease significantly during a period of 6 months. Sera of a proportion, but not all resistant jirds, contained antibodies against the surface of vector derived L3 as defined by IFAT. No surface antigens of microfilariae or adult worms were recognized by the sera. Vaccinated animals had antibody responses against antigens in the inner organs of L3 and in the cuticle and reproductive organs of adult worms as shown by IFAT. Immunoblotting with SDS-PAGE-separated L3 antigens and L3-CSN revealed that all sera contained antibodies against two exported antigens of 205 and 68 kDa, and against a nonexported antigen of 18 kDa. The 205-kDa antigen easily degraded into fragments of 165, 140, 125, and 105 kDa which were recognized by resistant jird sera. Various antigens of adult worms, but relatively few antigens of microfilariae, were also recognized. To test the relevance of exported antigens of L3 to resistance, jirds were immunized with L3-CSN together with a mild adjuvant. This immunization induced 67.7% resistance against challenge infection and sera of the immunized animals recognized the 205- and 68-kDa antigens of L3.     

Schistosoma mansoni: cloning of antigen gene sequences in Escherichia coli

Fischer rat protective antiserum (F-2x) prepared from Schistosoma mansoni-infected rats was used to screen an adult worm cDNA library constructed in a lambda gt11 bacteriophage expression vector. This led to the isolation of several clones yielding proteins reactive with antibodies in the infection serum. Counter-screening of these clones with Wistar-Furth rat nonprotective antiserum (W-2x) enabled identification of clones either uniquely or preferentially reacting with F-2x, in addition to clones of nearly equal reactivity with both antisera. Six clones were further characterized. Five expressed beta-galactosidase/S. mansoni fusion proteins which migrated more slowly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than beta-galactosidase and all were reactive in a Western immunoblot assay. The cDNA insert sizes in the clones ranged from 150 to 900 base pairs. Rabbit antibodies prepared against fusion proteins from three of the clones recognized biosynthetically radio-labeled 4-week worm proteins of sizes 20, 38, and 70 kDa, respectively. The 20- and 38-kDa proteins were among the protein antigens uniquely recognized by the F-2x protective antiserum. These proteins are therefore candidates for protective vaccine antigens and the recombinant lambda clones are now serving as useful reagents for obtaining the corresponding nucleotide gene sequences.     

Schistosoma mansoni: antigen preparations which induce antibodies to schistosomula surface antigens

To determine the easiest method of raising antibodies to antigens exposed on the surface of schistosomula of Schistosoma mansoni, several crude preparations of the parasite were used to immunize mice. Schistosomula released products, whole worm homogenate, and parasite eggs all raised antibodies which bound to the surface of live schistosomula, although the anti-egg antiserum did so less strongly. Anti-schistosomula released products antiserum recognized three schistosomula surface antigens of Mr 15,000, 20,000, and 32,000, anti-whole worm homogenate recognized 20,000, 32,000, and 38,000 Mr surface antigens, and anti-egg recognized a less than 200,000 Mr surface antigen. None of these antigens was recognized when the labeled preparation was immunoprecipitated with its homologous antiserum. When these antisera were used to immunoprecipitate cell free translation products of adult worm RNA, the antischistosomula released products and anti-whole worm homogenate recognized an 11,000 Mr doublet while the anti-egg precipitated 14,000 and 44,000 Mr antigens. Other crude preparations were used to immunize rabbits; Formalin-fixed schistosomula, denuded adult worms, and purified worm tegument all induced antibodies which recognized the 20,000, 32,000, and 38,000 Mr schistosomula surface antigens.     

The release of membrane antigens into culture by adult Schistosoma mansoni.

Antigens sharing determinants with surface membranes and soluble proteins of adult Schistosoma mansoni have been detected in culture media after incubation of radioactively labelled worms. The relative quantities of these antigens were measured with specific antisera raised in rabbits and with serum from an immune rhesus monkey. It was found that 12-16% of TCA-precipitable radioactivity in the culture medium consisted of membrane antigens and 6-8% consisted of antigens sharing determinants with proteins found in the soluble fraction of adult worms. Over half the membrane antigens were present in particulate form, while other antigens were present in solution. Surface labelling the adult worms with [125I]confirmed that some of the particles in the culture medium were derived from the surface membrane of the adult worm and electron microscope examination of such particles showed that large membrane fragments were present. These results support the hypothesis that antibodies against schistosome membrane antigens are induced by particulate membrane antigens released by the parasite.     

Brugia malayi: antibody responses to larval antigens in infected and immunized jirds.

Vaccination with irradiated third stage Brugia malayi larvae (L3) has been reported to induce partial protective immunity to L3 challenge in jirds. The purpose of this study was to identify antigens that may be targets of protective immunity in this model. Jirds were immunized by s.c. injection of irradiated L3 and challenged either s.c. or i.p. Necropsy was performed 11 wk after challenge. Partial protection was achieved in s.c. challenged animals; worm recovery was only 41% of that observed in unvaccinated controls, and worms recovered from immunized animals were stunted. Worm recoveries in immunized animals that were challenged i.p. did not differ from those of unimmunized controls. Group differences in parasite antigen levels in sera collected 2-11 wk after larval challenge were consistent with parasitological findings obtained at necropsy. Antibody studies compared prechallenge sera from immunized animals to sera from infected (unimmunized) controls. Antibody responses to L3 surface antigens (assessed by IFA) were much stronger after immunization than after infection. Immunoblot studies showed preferential recognition of several L3 antigens (97, 54, 48, and 40 kDa) by antibodies in sera from immunized animals. Additional studies are needed to determine whether immunization with such preferentially recognized antigens can induce protection to larval challenge comparable to or better than that observed with live vaccines.     

Induction of protection against Brugia malayi infection in jirds by microfilarial antigens

The development of immunologic methods to reduce transmission of human lymphatic filariasis depends on measures that will enhance the host's ability to eliminate infective larvae, adult worms, or blood-borne microfilariae (mf). The present study was designed to assess the capacity of a crude extract of Brugia malayi mf to decrease the level of microfilaremia and adult worm burden in jirds inoculated with infective larvae, and to identify the filarial antigens that elicit antibody responses in these animals. Thirty weeks after subcutaneous inoculation with 75 infective larvae, 100% of control jirds were patent (i.e., had microfilaremia) compared with 60% of the group immunized with 10 micrograms of crude microfilarial extract (p less than 0.05). In addition, microfilaremia was lower in patent immunized animals compared with controls (p less than 0.05). The mean total number of adult female B. malayi per jird recovered at necropsy in control animals was 16.0 vs 7.0 in immunized jirds (p less than 0.05). Serum of immunized jirds contained anti-mf antibodies with an end titer of 1:8000, a value similar to that of animals with chronic B. malayi infection. Microfilarial antigens of Mr approximately 150,000, 75,000, 42,000, and 25,000 were identified in immunoblotting studies by reactivity with antibodies in sera of immunized jirds. Antibodies induced by immunization with microfilarial extract were not specific for this stage of the parasite life cycle, as jird anti-mf antibodies reacted with a Mr approximately 150,000 and several Mr 50,000 to 110,000 antigens derived from immature and mature adult parasites of both sexes. These data indicate that immunization of jirds with a water soluble microfilarial extract enhances the host's ability to eliminate adult worms and blood-borne mf. The filarial antigens that induce antibodies in immunized jirds have been identified.     

Induction of protective immunity against Schistosoma mansoni by a non living vaccine. I. Partial characterization of antigens recognized by antibodies from mice immunized with soluble schistosome extracts

A single intradermal injection of frozen and thawed schistosomula in conjunction with the bacterial adjuvant Mycobacterium bovis strain Bacille Calmette Guerin, Phipps substrain (BCG) induced significant levels of resistance to challenge Schistosoma mansoni infection in C57BL/6 mice. Immunization with the aqueous fraction remaining after 100,000 X G centrifugation of the larval lysate was also protective under these conditions, suggesting that some immunogenic determinants may not be membrane associated. Frozen-thawed cercariae and soluble components of adult worms also protected against challenge infection in these experiments. These observations indicate that soluble immunogens are present in both early and late developmental stages of the parasite, and therefore may be good candidate antigens for an immunochemically defined vaccine against schistosomiasis. Induction of humoral reactivity against soluble or membrane antigens was examined in mice protected against cercarial challenge by prior exposure to frozen-thawed larvae, soluble larval, or soluble adult antigens plus BCG. Animals that were immunized with frozen-thawed larvae produced low but significant levels of antibodies against larval surface antigens when examined by indirect immunofluorescence or by immunoprecipitation of surface-labeled schistosomula. Mice immunized with soluble antigens, however, showed negligible antibody reactivity against surface membrane antigens. Because mice immunized with soluble antigens were resistant to challenge infection, these results strongly suggest that anti-surface membrane reactivity is not required in the mechanism of protective immunity in this model. Sera from mice immunized with either total freeze-thaw larval lysate or soluble schistosome extracts all showed strong reactivity against soluble antigens, as detected by ELISA. Western blot analysis showed these antisera to react with a restricted number of high m.w. antigens that were present both in schistosomula and in adult worms. These antigens are therefore likely to play a major role in the development of resistance in this model as immunogens and/or as targets of protective immune response.     

Surface antigens on cercariae,schistosomula and adult worms of Schistosoma mansoni

Shah J. and Ramasamy R. 1982. Surface antigens on cercariae, schistosomula and adult worms of Schistosoma mansoni. International Journal for Parasitology12: 451–461. The surface protein antigens of Schistosoma mansoni were radiolabelled by lactoperoxidase catalysed I¹²⁵-iodination and analysed by immune-precipitation and polyacrylamide gel electrophoresis. The results showed that regularly labelled surface antigens of mol. wts >150,000, 78,000, 45,000 and 22,000 were present on adult worms. Common surface antigens were observed on the cercariae, schistosomula and adult worms. It is suggested that surface antigens released from living adult worms can sensitise a host to react against the invading schistosomula of a secondary infection. However, the failure to vaccinate mice using material containing adult worm surface antigens suggests that the induction of protective immunity is a complex phenomenon.     

Characterization of mouse macrophage differentiation antigens by monoclonal antibodies

Monoclonal rat antibodies to mouse macrophage antigens were prepared. For immunization phagocytic cells in the spleens of mice recovering from sublethal irradiation were used. Specificities of the monoclonal antibodies obtained were determined on cells of normal mouse cell populations as well as on cells of a panel of mouse cell lines. In an attempt to monitor expression of differentiation-related antigens two models of in vitro-induced macrophage differentiation were used: differentiation of cells of the myeloblast line Ml; CSF-1-induced differentiation of bone marrow cells. The results obtained clearly show that during maturation from undifferentiated to highly differentiated cells of the macrophage lineage expression of antigens recognized by the MIV 38, MIV 55, MV 87, and MV 114 monoclonal antibodies is enhanced. At the same time, expression of antigens recognized by the MIV 52, MIV 113, and MIV 116 monoclonal antibodies diminishes at a similar rate. The suitability of these monoclonal antibodies for the characterization of differentiation states of mouse macrophages is discussed.     

Ultrastructural localization of Sm15 and Sm25, two major tegumental adult worm antigens of Schistosoma mansoni.

Sm15 and Sm25 are two of the principal tegumental antigens recognized by antibodies from mice protectively vaccinated with adult worm tegumental membranes and may therefore be potential vaccine candidate antigens. Using antibodies affinity purified from anti-tegumental membrane anti-sera, and antibodies raised against the recombinant antigens, Sm15 and Sm25 were shown to be located specifically in the tegument of adult worms being distributed throughout the syncitium but not associated with the outer membrane.     

Recognition of size and charged states of antigens by anti-peptide antibodies

The effects of the size and characteristics of peptide antigens on recognition and equilibrium between antigens and antibodies were studied by immunization of peptides with different characteristics. Four to five residues in the peptides were recognized by antibodies, and the charged states of the residues in the epitope strongly affected the immunointeraction. These results suggest a guideline for the selection of antigenic determinants to obtain antibodies suitable for immunoaffinity chromatography.     

Trichinella spiralis: immunization of mice using monoclonal antibody affinity-isolated antigens

An antigen epitope was identified from the excretory-secretory products of Trichinella spiralis first-stage larvae using monoclonal antibodies, and the glycoprotein antigens bearing this epitope (Ts.49 and Ts.53) were isolated from the crude excretory-secretory preparation by affinity chromatography. In immunization experiments carried out in mice, antigen priming with Ts.49 and Ts.53 resulted in a reduction of muscle larvae resulting from a challenge infection at a level comparable to priming with crude excretory-secretory antigens. In addition, antigens Ts.49 and Ts.53 induced an accelerated expulsion of adult worms from the intestines of immunized mice and reduced the fecundity of remaining female worms.     

Characterization of antigens recognized by new monoclonal antibodies raised against culture filtrate proteins of Mycobacterium bovis bacillus Calmette-Guérin

Effective protection against Mycobacterium tuberculosis may be achieved in experimental animals by immunization with proteins secreted by tuberculous bacilli in the extracellular milieu during growth. In this study, monoclonal antibodies were raised against Mycobacterium bovis bacillus Calmette-Guérin (BCG) culture filtrate proteins or live BCG, in an attempt to identify novel mycobacterial secretion antigens: the localization of the antigens recognized by the monoclonal antibodies within the mycobacterial cell was studied and interspecies reactivity was also investigated. The monoclonal antibodies obtained recognized proteins of molecular mass ranging from 5 to 82 kDa, with a prevailing frequency in the 30 kDa region. Three of the monoclonal antibodies recognized proteins present only in culture filtrates, one reacted with a cytoplasmic antigen, while the remaining antibodies recognized components which were mainly associated with the cell wall and the cytoplasmic membrane. The chemical nature and possible identity of the antigens was checked. Three monoclonal antibodies are likely to react with novel mycobacterial antigens of 5, 42 and 82 kDa, respectively.     

Comparative studies of the antigens recognized by sperm-immobilizing monoclonal antibodies.

Characteristic properties of the antigens recognized by sperm-immobilizing monoclonal antibodies (SI-mAbs) from different sources were compared by ELISA competitive inhibition assay, Western blot analysis, chromatographic analysis, and enzymatic digestion studies. Among 9 SI-mAbs, human mAb H6-3C4 and three mouse mAbs--2C6, 2B6, and 2E5--also possessed strong sperm-agglutinating activity. Binding of human mAb H6-3C4 to sperm was strongly inhibited by the three mouse mAbs (2C6, 2B6, and 2E5), but not by the rat or the other four mouse mAbs. SDS-PAGE revealed that mAb H6-3C4 and three mouse mAbs recognized the same antigen molecules of 15-25 kDa present in both sperm extracts and seminal plasma. Chemical treatments with trifluoromethanesulfonic acid and sodium metaperiodate destroyed the antigen determinants recognized by the above four mAbs, as detected by both ELISA and antibody absorption tests. Western blot analysis revealed that the antigens were susceptible to treatments with papain, proteinase K, and N-glycanase, but resistant to trypsin, V8 protease, and thermolysin. These results indicate that one of the major antigens recognized by mAbs with sperm-immobilizing action may be a sperm membrane-associated glycoprotein of 15-25 kDa and the epitope may involve N-linked oligosaccharides.     

Fetal antigens as tumor growth enhancing factors in Yoshida's tumor rats

Malignant neoplastic cells have been shown to have some antigenic features identical to those of embryonic cells. Since several antigens are likely to be shared by both embryonic cells and neoplastic tissue, we tried to understand the meaning of the appearance of such antigens and the type of effect that the immunization with embryonic antigens would have on the survival of Yoshida's tumor rats. Wistar rats were immunized with fetal antigens by fetal cells (1.5 x 10(6)) suspended in 0.5 ml of Hanks solution plus an equal volume of Freund adjuvant, were injected in hind footpads, i.p. and i.m., respectively, for active immunization. Rabbit antigen sera were used for passive immunization. All animals presented ascites and tumor growth. Animals immunized by means of fetal cell antigens showed a mean survival rate after neoplastic transplant of 14 days. Animals that received rabbit immune serum showed a mean survival rate after neoplastic transplant of 17 days. The immunization by means of fetal antigens elicited a scanty effect on the survival of Yoshida's tumor transplanted rats. It can be concluded that antibodies, which are able to cross react with neoplastic cells, do not have cytotoxic effect and do not interfere with the survival of the neoplastic transplanted animals. Therefore, fetal antigens are likely able to carry out an immunosuppressive action. The fact that they appear on neoplastic cells could be seen as a metabolic modification effect or as a growth enhancing factor.     

Schistosoma mansoni: A comparison of mouse and rat worms with respect to host antigens detected by the technique of transfer into hamsters

A significant proportion of schistosomes transferred from the mouse into the mesenteric veins of hamsters pre-immunized with mouse erythrocytes were rejected. The rejection was specific, could be passively transferred with immune serum and was demonstrable with mouse worms which had been ‘washed’ in normal hamsters for up to 24 h. On the contrary, schistosomes transferred from the rat into the mesenteric veins of hamsters pre-immunized with rat erythrocytes were generally not rejected to any significant extent. Rejection was not brought about by variations in the immunization procedure, or by passive transfer of immunity, or by the use of ‘older’ rat worms. Rat antigens could be detected on rat schistosomes with the use of mixed agglutination techniques, but they appeared to be present in relatively low amounts. The significance of these findings is discussed in relation to a possible protective function of host antigens.     

Breadth of humoral response and antigenic targets of sporozoite‐inhibitory antibodies associated with sterile protection induced by controlled human malaria infection

The development of an effective malaria vaccine has remained elusive even until today. This is because of our incomplete understanding of the immune mechanisms that confer and/or correlate with protection. Human volunteers have been protected experimentally from a subsequent challenge by immunization with Plasmodium falciparum sporozoites under drug cover. Here, we demonstrate that sera from the protected individuals contain neutralizing antibodies against the pre‐erythrocytic stage. To identify the antigen(s) recognized by these antibodies, a newly developed library of P. falciparum antigens was screened with the neutralizing sera. Antibodies from protected individuals recognized a broad antigenic repertoire of which three antigens, PfMAEBL, PfTRAP and PfSEA1 were recognized by most protected individuals. As a proof of principle, we demonstrated that anti‐PfMAEBL antibodies block liver stage development in human hepatocytes. Thus, these antigens identified are promising targets for vaccine development against malaria.     

Surface antigens of male worms and microfilariae of Onchocerca gibsoni

Living adult males and microfilariae of the cattle filarial parasite Onchocerca gibsoni were externally labelled with radioactive iodine using the iodogen and Bolton-Hunter procedures. Characterization of labelled surface proteins by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis revealed clear cut differences in the two life cycle stages. In addition, the two radiolabelling procedures yielded some differences in the profiles of radiolabelled surface proteins for both adults and microfilariae. Immunoprecipitation analysis revealed a number of labelled antigens recognized by antibodies in human onchocerciasis serum pools, thereby demonstrating the usefulness of O. gibsoni as a model in Onchocerca volvulus vaccine studies. The reactivity of microfilarial antigens extended to antibodies from other human nematode infections, whereas male surface antigens, particularly those of low molecular weight, were Onchocerca specific. This indicates that O. gibsoni can provide a convenient source of specific diagnostic antigen.     

The in vitro antibody response to cell surface antigens. I. The xenogeneic response to human leukemia cells.

An in vitro spleen fragment culture system has been developed for the production and analysis of xenogeneic antibody responses to cell surface antigens. Depending on the methods of immunization and in vitro stimulation employed, mouse spleen fragments can produce antibody of both IgG and IgM classes directed against human cell surface antigens for more than 30 days in culture. A saturation binding analysis of the antibody products indicates that their range of specificities was more restricted than that of serum antibody. Approximately 5% of the in vitro antibody products raised against a homogeneous population of human leukemia cells could distinguish between the antigens present on the leukemia cells and those present on normal human lymphocytes. Methods previously employed to influence the range of serum antibodies expressed against complex immunogens, such as suppression of certain responses by passive administration of antibody at the time of immunization, were tested in the in vitro spleen culture system and resulted in successful modulation of the antibody response patterns observed.