Trichinella spiralis: characterization of phage-displayed specific epitopes and their protective immunity in BALB/c mice

Trichinellosis is a global zoonosis mainly caused by Trichinella spiralis. We have previously reported that a novel Ts87 gene from the cDNA library of adult T. spiralis was cloned and expressed in a prokaryotic expression system. Vaccination with recombinant Ts87 protein (rTs87) induced a muscle larvae burden reduction in BALB/c mice by 29% in response to T. spiralis infection. In the present study, we screened a random phage-displayed peptide library using monoclonal antibody 5A3 which recognized Ts87 protein. Four positive phage clones were selected to subcutaneously immunize BALB/c mice without adjuvant. Two phage clones could effectively stimulate specific antibodies against rTs87. Mice vaccinated with these two combined phage clones showed a 28.7% worm burden reduction as compared to the control group. Therefore, the identified phage clones displayed peptides representing specific epitopes of Ts87 protein and could be considered as potential vaccine candidates for T. spiralis.     

Expression cloning of Pneumocystis carinii antigens.

We undertook expression cloning of Pneumocystis carinii antigens to overcome the difficulties encountered in purification of these antigens. Using monoclonal antibodies to the P. carinii gp120 antigen and polyclonal rabbit antiserum to rat-derived P. carinii, we have isolated cDNA clones encoding immunoreactive moieties. A cDNA clone encoding the 3' portion of a 45-55 kDa antigen of rat-derived P. carinii, was the most abundant clone isolated. The peptide encoded by this cDNA has a novel sequence with a repeated motif rich in glutamic acid residues. Affinity-purified antibodies to this peptide reacted with the 45-55 kDa band of rat-derived P. carinii. The fusion protein was recognized by serum antibodies from rats with natural exposure to P. carinii. The production of this recombinant protein should allow more detailed studies of the host-parasite relationship of this important opportunistic infection.     

Search for antibodies against Trichinella spiralis in free roaming rodents caught in a zoological park from Mexico City

A serological survey to search for antibodies against T. spiralis was performed in free roaming rats (n = 64) and mice (n = 35) caught in a zoological park from Mexico City. Serum samples were analyzed by ELISA and immunoelectrotransfer blot assay (EIBT). None serum show positive absorbance values in ELISA nor recognized T. spiralis specific antigenic fractions in EIBT. However, two rat samples recognized three antigens of 31, 37 y 55 kDa, while one of them reacted with two additional antigens of 64 and 67 kDa. As it is known that the antigen epitope profiles varied among Trichinella species, it could be possible that in rats, there is 3% of antibody prevalence against Trichinella sp.; however, due that other organisms could induce the production of cross-reacting antibodies, such conclusion can not be supported at all. These results suggest that T. spiralis was not part of helminthological fauna in these rodents.     

Identification of Trichinella spiralis early antigens at the pre-adult and adult stages

Three expression cDNA libraries from Trichinella spiralis worms 14 h, 20 h and 48 h post-infection (p.i.) were screened with serum from pigs experimentally infected with 20,000 T. spiralis muscle larvae. Twenty-nine positive clones were isolated from the 14 h p.i. cDNA library, corresponding to 8 different genes. A putative excretory-secretory protein similar to that of T. pseudospiralis was identified. Three clones corresponded to a T. spiralis serine proteinase inhibitor known to be involved in diverse functions such as blood coagulation and modulation of inflammation. Screening of the 20 h p.i. cDNA library selected 167 positive clones representing 12 different sequences. The clone with the highest redundancy encoded a small polypeptide having no sequence identity with any known proteins from Trichinella or other organisms. Fourteen clones displayed sequence identity with the heat shock protein (HSP) 70. HSPs are produced as an adaptive response of the parasite to the hostile environment encountered in the host intestine but their mechanism of action is not yet well defined. From the 48 h p.i. T. spiralis cDNA library, 91 positive clones were identified representing 7 distinct sequences. Most of the positive clones showed high similarity with a member of a putative T. spiralis serine protease family. This result is consistent with a possible major role for serine proteases during invasive stages of Trichinella infection and host-parasite interactions.     

Trichinella spiralis: light microscope monoclonal antibody localization and immunochemical characterization of phosphorylcholine and other antigens in the muscle larva

A panel of monoclonal antibodies was used to examine the structure of the muscle larva of Trichinella spiralis under the light microscope. Immunofluorescence and, in some cases, immunoperoxidase staining were used. All four antibodies reacted with the cuticle of the organism, although differences in the staining pattern were observed for some of these. Interestingly, all the antibodies also reacted with the stichosome. One of the antibodies (Ts2Ab) is specific for the hapten, phosphorylcholine. In a binding assay, this antibody also reacted with extracts of Trichuris suis, Ascaris suum, and Fasciolopsis buski, but not with extracts derived from Cysticercus cellulosae, Candida albicans, Salmonella typhi, or Escherichia coli. This crossreactivity was confirmed microscopically in which the cuticle, oviduct and eggs of T. suis, the cuticle, muscle cells, and eggs of A. suum, and the cuticle and vitelline glands of F. buski were seen to be clearly stained by the antibody. In addition, Ts2Ab also reacted with the cuticle and stichosome of the adult T. spiralis worm. In Western blot analysis, Ts2Ab recognized a 43-kDa antigen from T. spiralis muscle larvae extracts, while a previously studied antibody (7C2C5Ab) identified four major antigens (48.5, 47, 43, and 39 kDa) in this preparation. Similar results were obtained when the 24-hr excretory-secretory (ES) antigens of T. spiralis were immunoblotted with the antibodies, although the reactivity shown by Ts2Ab was relatively weak. With the 72-hr ES material, on the other hand, major antigens of lower mol wt (44, 28, and 25 kDa) were revealed by 7C2C5Ab, and no reactivity was seen with Ts2Ab. However, this antigen preparation reacted well with both antibodies in an enzyme-linked immunoassay. Taken together, the findings suggest that the 72-hr ES antigens probably result from extensive degradation of material originally secreted or excreted by the worm. Similar binding studies on the 24-hr ES preparation indicated that this source may be relatively rich in 7C2C5Ab-reactive epitopes and relatively poor in the antigen identified by Ts2Ab. Other studies performed demonstrated that the antigens recognized by these two antibodies were distinct and physically unassociated.     

Distribution and ontogeny of CD2 expression by murine T cells

We have raised a polyclonal antiserum to murine CD2 by immunization of a rabbit with a synthetic peptide corresponding to a hydrophilic sequence in the extracellular domain of the murine CD2 gene. The antiserum immunoprecipitates a 55 kDa protein, consistent with the size predicted by the cDNA sequence. Flow microfluorometric analysis of a panel of T cell tumors and clones demonstrated concordance of reactivity of intact cells with the anti-CD2 serum and the presence of CD2 mRNA. Surprisingly, although splenic T cells were found to uniformly express high levels of CD2, several of a panel of functional T cell clones were found to lack CD2 expression. This suggests that the clones lost CD2 upon in vitro cultivation, and may not be required for activation or maintenance in culture. Adult thymocytes exhibited heterogeneous expression of CD2. The majority of CD4-8- thymocytes expressed low levels and CD4+8+ thymocytes intermediate levels, whereas all CD4+8- and the majority of CD4-8+ thymocytes expressed high levels of CD2. Multiparameter analysis of CD2 expression and that of CD3, CD5, JIId, and IL-2R p55 chain showed that expression of CD2 correlates with the maturational state of thymocytes. Finally, analysis of fetal thymuses from timed pregnancies revealed that expression of CD2 is preceded by that of IL-2R p55 chain.     

Molecular analysis of the gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae.

The gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae was studied using recombinant DNA techniques. cDNA synthesized from poly(A)-rich mRNA from T. spiralis infective larvae was ligated into phage vector lambda gt11 DNA and packaged in vitro. The phages were propagated on Escherichia coli and a lambda gt11 expression library was constructed. A cDNA clone encoding a 46 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method. A clone containing nearly full-length cDNA for a 46 kDa protein was isolated. The gene encoding this 46 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the cDNA as a probe. The gene was transcribed to mRNA with approximately 1400 nucleotides and translated to 46 kDa polypeptide. The antigenic polypeptide was excreted/secreted as a 46 kDa native antigen. The antigenic beta-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera.     

Expressed sequence tags of Trichinella spiralis muscle stage larvae

In order to obtain greater insight into the relevant genomic expression patterns of Trichinella spiralis, 992 expressed sequence tags (ESTs) were collected from a cDNA library of T. spiralis muscle stage larvae and assembled into 60 clusters and 385 singletons. Of them, 445 (44.7%) ESTs were annotated to their homologous genes, and small fractions were matched to known genes of nematodes. The annotated ESTs were classified into 25 eukaryotic orthologous groups (KOG). Cytochrome C oxidase (34 clones) was found to be most frequent species.     

Trichinella spiralis: recognition of muscle larva antigens during experimental infection of swine and its potential use in diagnosis

Longitudinal studies with Trichinella spiralis experimentally infected pigs were carried out to identify muscle larva antigens recognized during infection. This was approached using Western blot analysis and ELISA assays. Immunoblots of sera from experimentally infected pigs using total parasite extracts revealed five principal parasite antigens throughout infection. A similar pattern of antigen recognition was given by sera from backyard pigs in areas of Mexico, some of them endemic for Trichinella. Four of the five antigens recognized (MW 47, 52, 67, and 72 kDa) corresponded to surface/stichosomal antigens purified by monoclonal antibody NIM-M1. In addition, Western blots of excretions-secretions of muscle larva contained three (MW 52, 67, and 72 kDa) of the four surface/stichosomal components recognized by NIM-M1. Affinity-purified surface/stichosomal components, total soluble extracts, and excretory-secretory antigens of muscle larva were then evaluated in ELISA for detection of T. spiralis infections in experimentally infected, noninfected control, and 295 backyard pigs. These assays showed that purified surface/stichosomal components and excretory-secretory antigens increased the specificity of ELISA. These results suggest that muscle larva components purified by monoclonal antibody NIM-M1 are the major antigens recognized during infection of pigs with T. spiralis and therefore potentially useful for diagnosis of swine trichinellosis.     

Trichinella spiralis shares epitopes with human autoantigens

Like other helminths, Trichinella spiralis has evolved strategies to allow it to survive in the host organism, including the expression of epitopes similar to those present in either expressed or hidden host antigens. To identify T. spiralis-derived antigens that are evolutionarily conserved in the parasite and its host and that could be responsible for its evasion of the host immune response, we examined the reactivity of six different types of autoantibodies to T. spiralis larvae from muscle. T. spiralis antigens that share epitopes with human autoantigens were identified by assessing the cross-reactivity of autoantibody-containing serum samples with T. spiralis antigens in the absence of specific anti-parasite antibodies. Of the 55 autoantibody-containing human serum samples that we analysed by immunohistological screening, 24 (43.6%) recognised T. spiralis muscle larvae structures such as the subcuticular region, the genital primordium or the midgut. Using Western blots, we demonstrated that the same sera reacted with 24 protein components of T. spiralis muscle larvae excretory-secretory L1 antigens. We found that the human autoantibodies predominantly bound antigens belonging to the TSL1 group; more specifically, the autoantibody-containing sera reacted most frequently with the 53-kDa component. Thus, this protein is a good candidate for further studies of the mechanisms of T. spiralis-mediated immunomodulation.     

Identification and characterisation of a cDNA sequence encoding a glutamic acid-rich protein specifically transcribed in Trichinella spiralis newborn larvae and recognised by infected swine serum

Presently, little is known of the mechanism by which Trichinella penetrates and modulates reprogramming of muscle cells. In light of evidence demonstrating strong protective characteristics of antigens derived from this stage, understanding this process may shed light on potential targets for effective abatement of infection. To this end, a PCR-derived cDNA expression library was constructed using 0.5 micro g of total RNA from Trichinella spiralis newborn larvae. The library consisted of >125000 insert-containing clones. Approximately 40-50 x 10(3) clones were screened immunologically using sera from pigs experimentally infected with 7000 Trichinella L1. Multiple clones reacting positively with the swine infection serum and encoding portions of a glutamic acid-rich protein were identified. Northern and Southern blots indicated at least two distinct genes that encoded the glutamic acid-rich proteins and that these genes were transcribed specifically in the newborn larvae stage. cDNA sequence data predicted open reading frames of 1497 and 1,716 bp generating proteins of 498 amino acids and 571 amino acids, respectively. Both sequences consisted of approximately 39% glutamic acid and 16% serine residues, and differed by the presence of a 219 bp fragment present in the 1716 bp sequence that was absent from the 1497 bp sequence. PCR data indicated that additional isoforms exist within this gene family that are different in length from those described above. In addition, it was found that more than one isoform can exist within a single worm and that this pattern can vary between individual worms within a population. Mouse antibodies to recombinant antigen localised the glutamic acid-rich proteins to the periphery of the developing stichocyte cells within the newborn larvae consistent with the hypothesis that the newborn larval antigens are secreted.     

Trichinella spiralis: strong antibody response to a 49 kDa newborn larva antigen in infected rats

In this work, we analyzed the kinetics of anti-Trichinella spiralis newborn larva (NBL) antibodies (Ab) and the antigenic recognition pattern of NBL proteins and its dose effects. Wistar rats were infected with 0, 700, 2000, 4000 and 8000 muscle larvae (ML) and bled at different time intervals up to day 31 post infection (p.i.). Ab production was higher with 2000 ML dose and decreased with 8000, 4000 and 700 ML. Abs were not detected until day 10, peaked on day 14 for the 2000 ML dose and on day 19 for the other doses and thereafter declined slowly from 19 to 31 days p.i. In contrast, Abs to ML increased from day 10, peaked on day 19 and remained high until the end of the study. Abs bound strongly at least to three NBL components of 188, 205 and 49 kDa. NBL antigen of 188 and 205 kDa were recognized 10-26 days p.i. and that of 49 kDa from day 10 to day 31 p.i. A weak recognition towards antigens of 52, 54, 62 and 83 kDa was also observed during the infection. An early recognition of 31, 43, 45, 55, 68 and 85 kDa ML antigens was observed whereas the response to those of 43, 45, 48, 60, 64 and 97 kDa (described previously as TSL-1 antigens) occurred late in the infection. A follow-up of antigen recognition up to day 61 with the optimal immunization dose (2000 ML) evidenced a decline of Ab production to the 49 kDa NBL antigen 42 days p.i., which suggested antigenic differences with the previously reported 43 kDa ML antigen strongly recognized late in the infection. To analyze the stage-specificity of the 49 kDa NBL antigen, polyclonal antibodies (PoAb) were obtained in rats immunized with 49 kDa NBL antigen. PoAb reacted strongly with the 49 kDa NBL component in NBL total soluble extract but no reactivity was observed with soluble antigen of the other T. spiralis stages. Albeit with less intensity, the 49 kDa component was also recognized by PoAb together with other antigens of 53, 97 and 107 kDa, in NBL excretory-secretory products (NBL-ESP). Thus, our results reveal differences in the kinetics of anti-NBL and ML Ab responses. While anti-NBL Abs declined slowly from day 19 until the end of the experiment, Abs to ML antigen remained high in the same period. It is remarkable the optimal Ab response to NBL antigens with 2000 ML infective dose and the reduced number of NBL antigens identified throughout the experimental T. spiralis infection, standing out the immunodominant 49 kDa antigen. Interestingly, this antigen, which was prominently expressed in NBL somatic proteins, was also detected in NBL-ESP.     

Identification, molecular characterisation and differential expression of caveolin-1 in Trichinella spiralis maturing oocytes and embryos

Caveolins are integral membrane proteins implicated in cholesterol homeostasis and transport, endocytosis mechanisms and regulation of signal transduction in differentiated cells. In this work a caveolin-1 gene from the nematode Trichinella spiralis (Ts-cav-1) was cloned and identified as an adult-specific antigen. For this, a cDNA library of T. spiralis 3-day-old adult worms was screened using a stage-specific cDNA-labelled probe. One positive clone contained a cDNA insert of 1427-bp and a full-length open reading frame (ORF) of 687-bp, which encodes for a 229 amino acid polypeptide with a theoretical molecular weight of 26kDa. BLAST and FASTA searches revealed a 36% and 57% identity with Caenorhabditis elegans caveolin-1, respectively. Confocal laser microscopy analysis using antibodies generated against Ts-CAV-1 protein and cross-sections of adult parasites showed that Ts-CAV-1 gradually accumulates on the surface of Trichinella oocytes and embryos, reaching a maximum at 3days p.i., and decreasing during new-born larvae (NBL) development. RT-PCR assays of parasites from 1 to 4days p.i. showed a similar gene expression profile to that observed for Ts-CAV-1 which suggests a specific developmental regulation. Free cholesterol was mainly distributed in the female germ line and it displayed increasing membrane accumulation, similar to the pattern obtained for Ts-CAV-1 protein, which suggests a temporal membrane association with Ts-CAV-1 that in turn will perform the functions mentioned above. Our results strongly indicate that Ts-cav-1 from T. spiralis plays a role in oocyte maturation and embryogenesis during development, demonstrating gender-specific expression.     

Cloning and sequencing of a dextranase-encoding cDNA from Penicillium minioluteum

Abstract A cDNA from Penicillium minioluteum HI-4 encoding a dextranase (1,6-α-glucan hydrolase, EC 3.2.1.11) was isolated and characterized. cDNA clones corresponding to genes expressed in dextran-induced cultures were identified by differential hybridization. Southern hybridization and restriction mapping analysis of selected clones revealed four different groups of cDNAs. The dextranase cDNA was identified after expressing a cDNA fragment from each of the isolated groups of cDNA clones in the Escherichia coli T7 system. The expression of a 2 kb cDNA fragment in E. coli led to the production of a 67 kDa protein which was recognized by an anti-dextranase polyclonal antibody. The cDNA contains 2109 bp plus a poly(A) tail, coding for a protein of 608 amino acids, including 20 N-terminal amino acid residues which might correspond to a signal peptide. There was 29% sequence identity between the P. minioluteum dextranase and the dextranase from Arthrobacter sp. CB-8.     

Production and properties of a mouse monoclonal IgE antibody to Schistosoma japonicum

A monoclonal IgE antibody was prepared by fusion of NS-1 myeloma cells with spleen cells of C3H/He mice immunized with an extract of adult worms of Schistosoma japonicum (Sj). The antibody was able to elicit passive cutaneous anaphylaxis in the rat skin against Sj with the highest titer of 1/256,000 in an ascitic form but did not cross-react with any of antigens extracted from S. mansoni, Fasciola hepatica, Paragoniumus westermani, or Trichinella spiralis. Western blot analysis indicated that the monoclonal IgE antibody recognized epitopes on molecules of 82 kDa, 97 kDa, 160 kDa, and 200 kDa, at least some of which were recognized by IgG antibodies of patients with chronic schistosomiasis japonica. The IgE antibody also recognized a 97-kDa antigen expressed on the surface of mechanically transformed schistosomula. Passive transfer of the antibody into mice in an early stage of challenge infection resulted in a partial but significant reduction of recovery of adult worms. However, similar treatment was not effective for the protection if the antibody was given in the postlung stage of the infection. Moreover, eosinophil-mediated damage to schistosomula was observed in vitro in the presence of the monoclonal anti-Sj IgE antibody, whereas the damage was not observed in the presence of another monoclonal IgE antibody with dinitrophenyl specificity.     

Isolation and characterization of a full-length cDNA encoding the 55-kDa rabbit zona pellucida protein.

A full-length cDNA (rc55) encoding the major rabbit zona pellucida (ZP) glycoprotein (55 kDa) has been cloned and sequenced. A lambda gt11 expression library was constructed using poly(A)+ mRNA isolated from sexually immature rabbit ovaries which contain large numbers of developing follicles. The rc55 cDNA was identified using affinity purified polyclonal antibodies specific to ZP antigens which are shared among mammalian species. The deduced amino acid sequence of the full-length rc55 clone was matched to the NH2-terminal 25-amino acid sequence obtained for this protein. The predicted amino acid sequence consists of 540 amino acids including a putative signal peptide of 18-24 residues and six potential N-glycosylation sites. The cDNA hybridizes to a 2000-base species of mRNA from rabbit ovary which is not detected in other rabbit tissues. The message is present early in ovarian follicular development and is approximately 600-fold greater in sexually immature as compared with sexually mature rabbit ovaries. This cDNA was expressed as a cro-beta-galactosidase fusion protein using the pEX expression vector. Antibodies against native rabbit ZP, affinity-purified on the recombinant 55-kDa ZP protein, were found to recognize the native rabbit ZP glycoprotein, indicating partial conservation of native epitopes in the expressed recombinant protein.     

Screening of murine monoclonal antibodies against living schistosomula of Schistosoma mansoni by radioimmunoassay

A radioimmunoassay was developed to screen supernatants of murine monoclonal antibodies against surface antigens of living schistosomula of Schistosoma mansoni. Of 196 clones screened, 10% bound schistosomula. Of these, 74% bound only schistosomula. The remaining molecules also reacted with soluble adult worm antigens and soluble egg antigens as determined by enzyme-linked immunosorbent assay. Immunoblot analysis demonstrated that monoclonal antibody 204-3E4 reacted with a 68 kDa protein, a glycoprotein that induces substantial resistance against S. mansoni infection. Recognition of an 18 kDa antigen by 204-3F1 antibody was stage-specific with the antigen being expressed in cercariae, 3- and 24-h-old parasites but not 4-day, lung stage or adult worms. Monoclonal antibody 204-4E3 reacted with purified S. mansoni paramyosin. These data indicate that radioimmunoassay using living schistosomula is a rapid alternative method to identify murine hybridomas that secrete antibodies which react with surface antigens of S. mansoni.