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.     

Identification and characterization of myeloma-associated antigens in Trichinella spiralis

To investigate the presence of myeloma-associated antigens in Trichinella spiralis and their anti-tumor effect, cross-immune responses between antigens of the myeloma cell SP2/0 versus positive sera to T. spiralis, and antigens of T. spiralis versus positive sera to myeloma cell SP2/0 were determined using T. spiralis and myeloma specific enzyme-linked immunosorbent assays (ELISA). The myeloma-associated antigens in T. spiralis were separated by ultrafiltration and 2-D electrophoresis, and the amino acid sequences and molecular weights were determined by spectrometry. An obvious reaction was found between a 33 kDa antigen and positive sera, and the major component of the antigen was tropomyosin (TM), which is an surface acidic protein with 284 amino acids. Mice were immunized with TM to determine the anti-tumor effect in vivo. The results showed that CD4⁺, CD8⁺ T lymphocyte, and CD19⁺ B lymphocyte were significantly increased (P < 0.05). The anti-tumor effects were significantly different between mice immunized with the antigens or adjuvant alone (P < 0.05), while the difference between mice immunized with antigens and whole T. spiralis was not significant (P > 0.05). The results indicated that TM identified in this study may play a role in eliciting cross-protective immunity.     

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.     

Immunocytolocalization study of the external covering of Trichinella spiralis muscle larva

The antibody-binding sites of the muscle larva of Trichinella spiralis were investigated by immunogold staining on the ultrathin sections of LR white resin. The antibodies, which were produced in the course of T. spiralis infection in rats, specifically bound to the inner layers of the body cuticle and the cuticle of the hindgut, but not to the cuticle of the esophagus. This is the first report that reveals the antigenic nature of the inner layers of the external coverings of T. spiralis larva.     

Comparative analysis of the excretory-secretory proteome of the muscle larva of Trichinella pseudospiralis and Trichinella spiralis

The nematodes Trichinella spiralis and Trichinella pseudospiralis are both intracellular parasites of skeletal muscle cells and induce profound alterations in the host cell resulting in a re-alignment of muscle-specific gene expression. While T. spiralis induces the production of a collagen capsule surrounding the host-parasite complex, T. pseudospiralis exists in a non-encapsulated form and is also characterised by suppression of the host inflammatory response in the muscle. These observed differences between the two species are thought to be due to variation in the proteins excreted or secreted (ES proteins) by the muscle larva. In this study, we use a global proteomics approach to compare the ES protein profiles from both species and to identify individual T. pseudospiralis proteins that complement earlier studies with T. spiralis. Following two-dimensional gel electrophoresis, tandem mass spectrometry was used to identify the peptide spots. In many cases identification was aided by the determination of partial peptide sequence from selected mass ions. The T. pseudospiralis spots identified included the major secreted glycoproteins and the secreted 5'-nucleotidase. Furthermore, two major groups of T. spiralis-specific proteins and several T. pseudospiralis-specific proteins were identified. Our results demonstrate the value of proteomics as a tool for the identification of ES proteins that are differentially expressed between Trichinella species and as an aid to identifying key parasite proteins that are involved in the host-parasite interaction. The value of this approach will be further enhanced by data arising out the current T. spiralis genome sequencing project.     

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.     

Differentiation between Trichinella spiralis and T. pseudospiralis infective larvae by a monoclonal antibody

Crude saline extracts of Trichinella spiralis and T. pseudospiralis infective larvae were studied by Western blot analysis using a monoclonal antibody, named ES/TA2 and produced against T. spiralis larvae. This monoclonal antibody recognized seven major antigenic components in T. spiralis larvae with apparent Mr: 45, 48, 50, 68, 70, 92 and 105 kDa and five in T. pseudospiralis larvae: 38, 50, 70, 72 and 92 kDa. SDS-PAGE of both extracts did not reveal appreciable differences in the range of molecular weights recognized by ES/TA2. These facts show the existence of immunological differences among proteins with apparently identical molecular weights.     

Production and immunochemical characterization of monoclonal antibody to IRR ectodomain

The insulin receptor-related receptor (IRR) is the only known metabotropic sensor of extracellular alkaline medium involved in the regulation of the acid–base balance in the body. IRR is expressed in certain cell populations of the kidney, stomach, and pancreas that can come into contact with the extracellular fluids with alkaline pH. To study IRR structure and function, we obtained a stable hybridoma cell-line-producing antibody to the extracellular portion of the receptor. The monoclonal antibody isolated from ascitic fluids showed a positive reaction with the antigen in the ELISA test. The minimum working concentration of antibodies was 12.5 ng/mL. The ability of the antibodies to specifically recognize the purified ectodomain of IRR and the fulllength receptor was confirmed by western blot, immunoprecipitation, and immunocytochemistry.     

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.     

Trichinella spiralis: a 76-kDa excretory/secretory larval antigen identified by a monoclonal antibody

Spleen cells from BALB/c mice were fused with myeloma cells following infection of the mice with Trichinella spiralis larvae and an ip booster injection with larval homogenate antigen. A monoclonal antibody (Mab), designated as TS 3G6 which did not react with sera or tissue extracts from noninfected mice, rats, and guinea pigs, was selected for further studies because of its high activity and specificity. When tested in ELISA TS 3G6 did not cross-react with Ascaris suum, A. lumbricoides, Toxocara canis, E. granulosus (larvae), Trichiuris suis, or T. ovis. Western blot analysis showed that Mab 3G6 recognized an antigen of 76 kDa located in the stichosome of the larvae as well as on the surface of the larval cuticle. Digestion of a larval extract with different enzymes suggests that the Mab TS 3G6 corresponding epitope is a polypeptide. The TS 3G6 antigen was detected in culture supernatants of Trichinella muscle larvae and in sera of experimentally infected animals using a sensitive ELISA assay. This secretory antigen also seemed to induce a specific immune response in the host since sera from infected animals could block the binding of Mab TS 3G6 to its target antigen when tested in a competitive ELISA.     

Trichinella spiralis: altered expression of muscle proteins in trichinosis

Mammalian muscle undergoes significant alterations morphologically, ultrastructurally, and biochemically following infection by Trichinella spiralis larvae. To investigate this host/parasite relationship in more detail, a new method to isolate T. spiralis-infected cells (nurse cells) in preparative quantities was developed. Nurse cells isolated by sequential protease treatments contain larvae and retain many of the characteristics of those in situ. When analyzed by SDS-PAGE, a wide range of proteins detected in nurse cells were not apparent in muscle by the methods employed. Proteins associated with the nurse cell capsule and organellar fractions appear to account for most of the dominant nurse cell proteins. In contrast, most major muscle proteins were either reduced in abundance or undetectable in nurse cells. The myofibrillar proteins myosin heavy chain, alpha-actin, and alpha- and beta-tropomyosin were identified using antibody reagents and two-dimensional PAGE analysis. None of these proteins were detectable in nurse cells and except for beta-tropomyosin, the relative abundance of these proteins was a minimum 100-fold lower compared to muscle. The data indicate that the reduction of muscle products in the nurse cell is much greater than previously reported. The inability to detect myofibrillar proteins raises the possibility that the nurse cell is not blocked in a regenerating muscle phenotype as previously suggested. Availability of isolated nurse cells in large quantity should facilitate resolution of this and other issues regarding the T. spiralis/skeletal muscle relationship.