Monoclonal antibodies raised in Btk(xid) mice reveal new antigenic relationships and molecular interactions among gp53 and other Trichinella glycoproteins

Tyvelose-bearing glycoproteins or Trichinella spiralis Group 1 antigens (TSL-1 antigens) are thought to be key molecules in the immunobiology of Trichinella. In the present study, we investigated the binding characteristics of several mAbs produced in Btk(xid) immunodeficient mice that recognise gp53 and some other minor glycoproteins of this parasite. The data obtained reveal the existence of an O-glycan/peptide epitope (recognised by mAb US8) common to all TSL-1 glycoproteins, as well as a specific interaction between the TSL-1 antigen gp53 and other unknown Trichinella glycoproteins in the 35-40 kDa range (these latter react with mAbs US8 and US9, but not with mAb US5). Some of the epitopes recognised by our mAbs are differentially expressed in Trichinella species: the epitope recognised by mAb US5 on gp53 (another O-glycan/peptide epitope) is present only in T. spiralis, whereas those recognised by mAbs US8 and US9 (peptide epitopes) are present in encapsulated Trichinella species. The data obtained also reveal that gp53 is synthesised and glycosylated in beta-stichocytes only. The possible relevance of these findings is discussed.     

Antigenic differences betweenTrichinella spiralis andT. pseudospiralis detected by monoclonal antibodies

Antigenic differences betweenTrichinella spiralis andT. pseudospiralis were established using two monoclonal antibodies (mAbs) that show different specificities to muscle larvae of the two variants. Enzymelinked immunosorbent assay (ELISA) revealed that mAb 3G6 reacts positively againstT. spiralis, T. nelsoni, T. nativa andT. pseudospiralis, whereas mAb 3E10 does not react withT. pseudospiralis under the same experimental conditions. These antigenic differences were confirmed after preabsorption of the antibodies with serial dilutions of extracts ofT. spiralis orT. pseudospiralis muscle larvae. The indirect immunofluorescence technique showed that the antigen corresponding to mAb 3G6 is located in the stichosomes and the cuticle surface of bothT. spiralis andT. pseudospiralis. In contrast, mAb 3E10 positively stained cryostat sections ofT. spiralis, forming a dense reaction product on the surface of the whole larvae and the surrounding capsule. This antibody can be quite useful as a specific probe for distinguishingT. spiralis fromT. pseudospiralis in taxonomic studies. Using an avidin-biotin system, we could prove that mAb 3G6 recognizes an excretory/secretory-type antigen.     

Minor interspecies variations in the sequence of the gp53 TSL-1 antigen of Trichinella define species-specific immunodominant epitopes

Among the Trichinella TSL-1 antigens, whose antigenicity is generally due mainly to tyvelose-containing epitopes, gp53 is unusual in that its antigenicity is due mainly to protein epitopes. In the present study we mapped two of these epitopes, recognized by monoclonal antibodies (mAbs) that specifically recognize gp53 from all encysting Trichinella species (mAb US9), or gp53 from Trichinella spiralis alone (mAb US5). Based on previously published sequences of this glycoprotein [Mol. Biochem. Parasitol. 72 (1995) 253], in this study, we cloned the full gp53 cDNA from a new strain, Trichinella britovi (ISS 11; AN: ), and from another T. spiralis isolate (ISS 115; AN: ). The gp53 sequence comprised an ORF of 1239bp, coding for 412 amino acids, with 61 nucleotide differences (resulting in 38 residue changes) between the two species. Mapping of US5- and US9-recognized epitopes was undertaken through the construction and expression in the pGEX4T vector of truncated gp53 peptides, and by the construction of peptides derived from the antigenic regions. The epitope recognized by mAb US9 was a linear peptide of 8 residues, 33Met- 40Ser, located in the amino-terminal region, while the corresponding epitope recognized by mAb US5 was a 47-amino acid sequence containing two alpha-helix regions flanked by random coils, 290Thr- 336Lys. Molecular modeling of these peptides seems to indicate that recognition of the US9 epitope depends on the presence of two available hydroxyl groups provided by one methionine and one serine on T. spiralis gp53 (not present on Trichinella pseudospiralis gp53). Additionally, the stability of the US5 epitope seems to depend on correct folding of the 47-amino acid sequence (only present in T. spiralis). The relevance of these findings for understanding the antigenic recognition of Trichinella TSL-1 antigens, and for further studies to investigate possible function(s) of gp53 in Trichinella, is discussed.     

Evaluation of Trichinella spiralis larva group 1 antigens for serodiagnosis of human trichinellosis

To identify Trichinella antigens suitable for high-specificity and high-sensitivity serodiagnosis of human trichinellosis, we evaluated assays using four antigens: (i) crude first-stage larval extract (CLE), (ii) O-deglycosylated CLE, (iii) tyvelose-bearing antigens (Trichinella spiralis larva group 1 [TSL-1] antigens) purified by US4 affinity chromatography and coupled directly to enzyme-linked immunosorbent assay (ELISA) plates (pTSL-1 antigens), and (iv) TSL-1 antigens immobilized on ELISA plates with the monoclonal antibody (MAb) US4 (cTSL-1 antigens). Assays using these antigens were compared by analysis of sera from healthy individuals (n = 224) (group 1), individuals with noninfectious intestinal pathologies (n = 114) (group 2), individuals with other parasitic infections (n = 107) (group 3), and individuals with confirmed trichinellosis (n = 42) (group 4). Our results indicate that capture ELISA using cTSL-1 antigens is the most effective method for serodiagnosis of human trichinellosis; this was the only method showing 100% specificity and 100% sensitivity at the patent stage of the infection, and it was also the most sensitive for sera obtained prior to patency in indirect immunofluorescence (IIF). Indirect ELISA with pTSL-1 antigens was also 100% specific but was slightly less sensitive, particularly with sera obtained before IIF patency. Inhibition ELISA with MAb US4 indicated (i) that in Trichinella-infected patients the immune response to TSL-1 antigens is directed mostly against tyvelose-containing epitopes (mean of 84.2% of total anti-TSL-1 immunoglobulin G1 [IgG1] antibody response [range, 51.3 to 97.6%]) and (ii) that in most individuals a large proportion of anti-CLE IgG1 antibodies (mean, 49.5%; range, 7.3 to 92.6%) are directed against tyvelose epitopes.     

Preotection againstTrichinella spiralis induced by purified stage-specific surface antigens of infective larvae

The stage-specific surface antigens of the infective larvae ofTrichinella spiralis, isolated using an affinity column of monoclonal antibody (Mab) NIM-M1, consisted of four components with molecular weights of 72, 65, 52, and 47 kDa, respectively. These four components may have unique as well as shared structural features and appear to be products of the stichosome. When injected i.p. as an emulsion in complete Freund's adjuvant, the purified antigens induced protection against infection of BALB/c mice withT. spiralis, as assessed by reductions in both the muscle larvae load and the number of adult intestinal worms.     

Cross-resistance betweenTrichinella spiralis andAngiostrongylus cantonensis in laboratory rats

Summary Rats were sensitized with one dose of 20, 30, or 100, or three doses of 30Trichinella spiralis larvae and then given a challenge infection with 100 third-stage larvae ofAngiostrongylus cantonensis during different periods of development ofT. spiralis. A significant reduction in the mean number of adultA. cantonensis was found in all the experimental groups. The mean number of lung worms found in the groups varied from 56 to 68, whereas a mean of 82 was found in the control. This reduction is probably caused by the non-specific inflammatory reaction and cell-mediated response induced byT. spiralis in the intestine and other organs of the host. A weaker and slower haemagglutinating antibody response againstA. cantonensis was observed in animals infected with both species of nematodes. This may be attributed to a suppressive action elicited byT. spiralis against unrelated antigens. Changes in differential and total white blood cell counts were followed in some infected animals. An increase in neutrophils 3–4 weeks after infection with either or both species of parasites was observed. Eosinophilia occurred primarily in the lung phase of the metastrongyloid infection.     

A mouse monoclonal antibody that binds to an α-stichocyte of Trichinella spiralis

Monoclonal antibodies were generated for the isolation of specific antigens from Trichinella spiralis. A monoclonal antibody (TS32D12) of the Igg₁ class was selected according to its reactivity and specificity by enzyme-linked immunosorbent assay and immunofluorescent technique. The TS32D12 antibody was purified from ascites by fast protein liquid chromatography. The purified antibody showed a sensitive reaction to the T. spiralis antigen, but not to any other heterologous parasite antigens so far examined. Western blot analysis showed that the monoclonal antibody bound to epitopes present on the 160-kDa molecule. The antigen molecule was fragmented into 56-kDa molecules by heat treatment. The epitopes seemed to be destroyed since the antibody could not bind to the 56-kDa molecule. Staining with the periodic acid-Schiff (PAS) reagent suggested that the two molecules of 160 kDa and 56 kDa were glycoproteins. The 160-kDa molecule was detected only in the -stichocyte of T. spiralis muscle larvae.     

Identification of diagnostic antigens fromTrichinella spiralis

The Western blotting technique was used to determine the antigens ofTrichinella spiralis muscle larvae that were recognized by antibodies in sera from humans and pigs displayingT. spiralis infections. This resulted in the identification of several antigens that were recognized by all sera. Some of these antigens, notably those that were recognized during the early stage of infection, cross-reacted with antibodies to other parasites. This cross-reactivity was caused by the presence of phosphorylcholine on these antigens. A large portion of the antigens that were recognized by antibodies from infected humans and pigs were found to share a singleTrichinella-specific determinant. TheTrichinella-specific antigen population could be isolated from phosphorylcholine-containing antigens by a simple two-step affinity chromatography procedure using monoclonal antibodies to both determinants. The resulting preparation consisted primarily of a single antigen showing an apparent molecular weight of 45 kDa that corresponded to a mamor constituent of excretory-secretory (ES) products of muscle larvae. When tested in an enzyme-linked immunosorbent assay (ELISA), this antigen displayed diagnostic specificity that was comparable with the ES fraction and diagnostic sensitivity comparable with the crude muscle-larvae extract.     

Serological detection of <Emphasis Type="Italic">Trichinella spiralis</Emphasis> in swine by ELISA (enzyme-linked immunosorbent assay) using an excretory–secretory (E/S) antigen

The enzyme-linked immunosorbent assay (ELISA) method is recommended for farm surveillance programs and may be useful for epidemiological studies in wildlife or for establishing Trichinella-free areas. In this study, our interest was to compare the specificity and the time of seroconversion of excretory–secretory (E/S) antigens prepared from Trichinella spiralis. A group of eight pigs was inoculated with 500 T. spiralis larvae per animal, and blood sampling was performed at 3 and 4-day intervals during all experiments. The numbers of muscle larvae were determined in four different muscles groups. The larvae per gram burden shows that the most heavily parasitized muscles were the diaphragm [mean = 43.7 larvae per gram (lpg)] and the tongue (mean = 16.9 lpg). Antibody responses were detected by any of eight infected pigs of T. spiralis. Using the ELISA method with E/S antigen, antibodies to T. spiralis were first found on the day 21st p.i. The initial detection of antibodies varied from 21st to 31st day p.i., and the peak was reported 42nd day p.i. Dynamic of antibodies was stable or increased slightly throughout the experimental period (60 days post-inoculation). Our results represent important data for validation of a serological test, especially if blood samples are taken during early stages of infection.     

The distribution of excretory/secretory antigens during the muscle phase of Trichinella spiralis and T. pseudospiralis infections

The in situ distribution of excretory/secretory (ES) antigens of the infective-stage larvae of Trichinella spiralis and T. pseudospiralis was compared at various periods of development by immunofluorescent laser confocal microscopy and the immunoperoxidase method. In the former infection, epitopes of the ES antigens were always confined exclusively within the nurse cell, i.e., in the cytoplasmic region, hypertrophic nuclei, stichocytes, and cuticular surface of worms. In the latter infection, as early as at day 15 postinfection, ES epitopes were located along the infected myofibers, in the adjacent muscles, hypertrophic nuclei, stichocytes, and cuticular surface of worms. By day 30 postinfection there was a marked increase in both the distribution and the intensity of ES antigens in infected as opposed to uninfected myofibers. A new method was also developed to reveal the number of hypertrophic nuclei, small cells, and larvae in intact nurse cells. As many as four worms could be accommodated within a single complex. The number of hypertrophic nuclei within each complex varied from 15 to 81. Received: 10 May 1999 / Accepted: 28 May 1999     

Isolation of specific antigens fromTrichinella spiralis by the rotating horizontal ampholine column method

By preparative isoelectric focusing in a rotating ampholine column, specific antigens with molecular weights of 45, 47 and 53 kDa were successfully isolated from crude somatic extracts ofTrichinella spiralis muscle larvae. These antigens, with pI 5.5, 4.3 and 4.4, were found to possess the same epitope found in specific antigens in the excretory/secretory products that were recovered by in vitro culture of muscle larvae. When the antigens obtained by the two methods were compared using enzyme-linked immunosorbent assays, they showed similar specificity and sensitivity.     

Cloning and characterization of the mitochondrial heat-shock protein 60 gene of<Emphasis Type="Italic"> Trichinella spiralis</Emphasis>

The full-length cDNA of mitochondrial heat-shock protein (hsp) 60 of the infective-stage larva of Trichinella spiralis was cloned by degenerative PCR and rapid amplification of cDNA end reactions. The 1,945 bp full-length cDNA sequence contained an open reading frame of 576 amino acids. A mitochondrial signal peptide was located at the N-terminal and a GGM motif at the C-terminal. The gene contained ten exons and nine introns. RT-PCR analysis indicated that thermal, cold, acidic and oxidative treatment did not elicit significant changes in the expression of mitochondrial hsp 60 in the larvae. Cluster analysis showed that the sequence of the hsp 60 gene of T. spiralis is closely related to that of Drosophila melanogaster.     

Development of cellular immune response of mice to infection with low doses of Trichinella spiralis, Trichinella britovi and Trichinella pseudospiralis larvae

The murine cellular immune response to the infection with ten larvae of encapsulating (Trichinella spiralis, Trichinella britovi) and non-encapsulating species (Trichinella pseudospiralis) was studied. Both T. spiralis and T. britovi stimulated the proliferation of splenic T and B lymphocytes during the intestinal phase of infection, but T. spiralis activated the proliferative response also at the muscle phase, particularly in B cells. Non-encapsulating T. pseudospiralis stimulated the proliferation of T and B cells only on day 10 post-infection (p.i.) and later at the muscle phase. The numbers of splenic CD4 and CD8 T cells of T. spiralis infected mice were significantly increased till day 10 p.i., i.e., at the intestinal phase, and then at the late muscle phase, on day 60 p.i. T. britovi infection increased the CD4 and CD8 T cell numbers only on day 30 p.i. Decreased numbers of CD4 and CD8 T cells after T. pseudospiralis infection suggest a suppression of cellular immunity. Both encapsulating Trichinella species induced the Th2 response (cytokines interleukin-5 (IL-5) and interleukin-10) at the intestinal phase and the Th2 dominant response at the advanced muscle phase. Interferon-γ (IFN-γ) production (Th1 type) started to increase with migrating newborn larvae from day 15 p.i. till the end of the experiment. IL-5 production was suppressed during the intestinal phase of T. pseudospiralis infection. The immune response to T. pseudospiralis was directed more to the Th1 response at the muscle phase, the high IFN-γ production was found on day 10 p.i. and it peaked on days 45 and 60 p.i.     

Distribution of muscle larvae and antibody dynamics in goats experimentally infected with Trichinella spiralis

Herbivorous animals can play a very important role in spreading trichinellosis. In the study presented here, the susceptibility and distribution of Trichinella spiralis infection was examined in 16 goat kids. The goats were inoculated with 10,000 T. spiralis larvae isolated by artificial digestion methods. The animals were necropsied per two animals in weekly intervals, and the larval burdens in different muscle tissue and anti-Trichinella antibodies measured with the indirect enzyme-linked immunosorbent assay (ELISA) serological method using excretory–secretory (E/S) antigen for detecting anti-Trichinella antibodies were assessed during the experiment. T. spiralis larval burden was maximal at 6 weeks postinoculation (480–5,057 larvae/g according to locality), and the larvae were also found in the myocardium (0.77 larvae/g). In this paper, our next step was to compare the specificity and the time of seroconversion by means of ELISA based on E/S antigen prepared from T. spiralis. Antibody response was detected in all 16 goats. The ELISA test carried out showed the first increments in optical density 2 weeks postinfection (p.i.), reached their peak 4 weeks p.i., and remained elevated from that day until the end of the experiment (10 weeks p.i.). These results indicated that specific anti-Trichinella antibodies in goats persist for a relatively long time.     

The intraperitoneal inoculation of Lactobacillus casei in mice induces total protection against Trichinella spiralis infection at low challenge doses

The following effects of Lactobacillus casei in NIH mice were evaluated: the establishment of Trichinella spiralis adult worms in the intestine (AWI), larvae per gram of muscle tissue (LPG), levels of IgG and IgA, and levels of IL-4 and IFN-γ. One hundred and eight mice were allocated at random into 18 groups of six mice each. Each mouse in treated or non-treated groups was inoculated intraperitoneally once a week during 6 weeks with L. casei or phosphate-buffered solution. Later each mouse was challenged either with 200, 50, or 25 T. spiralis infective larvae. When the infection dose was 200 T. spiralis infective larvae, the reductions in AWI were 78.6% at 4 days after infection (dai) and 76.7% at 10 dai; while the reduction of LPG was 80.9% with respect to control groups. When the infection dose was 50 or 25 T. spiralis infective larvae, the reductions of AWI were 100% both at 4 and 10 dai; while the reduction of LPG at 30 dai was also 100% with respect to control groups. The levels of IgG and IgA anti-T. spiralis and IL-4 were significantly higher (P < 0.01) at 4 and 10 dai in mice from groups treated with L. casei than in animals in control groups; while at 10 dai, the levels of IFN-γ were higher in control mice (P < 0.01) than in L. casei-treated animals. The results suggest that frequent treatment of mice with L. casei induces a total protection against infection with low doses of T. spiralis.     

Interferon-gamma treatment in mice experimentally infected withTrichinella spiralis

Interferon- (IFN-) treatment ofTrichinella spiralis-infected BALB/c mice was investigated. The therapeutic regimen consisted of daily intraperitoneal injection of 10⁴ U murine IFN- for 7 days, starting at 2 weeks post-infection. Striated muscle samples (diaphragm, thigh) were collected at 4, 8 and 12 weeks after infection. The muscle larval burden, the degree of encystation and the digestion ofT. spiralis larvae were investigated. Furthermore, immunohistochemical studies of the inflammatory cell infiltrate around encysted larvae were performed. The results demonstrated an influence of IFN- treatment on the CD4⁺ and CD8⁺ subset distribution during the immune response but revealed no difference in the degree of encystation or digestion of encapsulated larvae as compared with control values.Abbreviations in figures A artrficial shrinking zone (due to preparation of TEM) - CO collagen - CS cross sectioned Trichinella larvae - CU cuticle - D defence cell of host - DG degenerating laryae - HN hypertrophied host cell nucleus - TC inner cyst wall - Il inner layer of inner cyst wall - LS longitudinally sectioned larvae - NC normal host cell nucleus - NU nucleus - OC outer cyst wall - OL outer layer of inner cyst wall - SF striated muscle fibre - Z zone of host defence cells     

Activation of complement, the induction of antibodies to the surface of nematodes and the effect of these factors and cells on worm survival in vitro

This report describes the effect on various stages in the life cycle of the nematodes T. spiralis and N. brasiliensis of complement, antibodies from rats infected with these parasites and several different cell types. The cuticle of the infective larvae and adult worms of both nematode species activates complement via the alternative pathway, but the cuticle of newborn T. spiralis lacks this property initially. As newborn larvae grow, however, the newly formed cuticle in the midregion of their body is able to activate complement. Rats infected with either nematode species produce antibodies to the cuticle of all life cycle stages which show marked specificity to each stage in the life cycle. Whereas the cuticle of T. spiralis reacts evenly over the entire surface both to complement and to antibodies, the reaction of the cuticle of N. brasiliensis to either reagent is patchy. Infective larvae of N. brasiliensis were killed in vitro in the presence of complement, by neutral red-positive peritoneal macrophages which were nonadherent to plastic. The infective and newborn larvae of T. spiralis were killed by eosinophil-enriched cell populations and antibodies. The speed of eosinophil killing of the T. spiralis larvae was enhanced when the serum was freshly collected and when the eosinophil suspension also contained neutral red-positive nonadherent macrophages. Newborn larvae of T. spiralis and infective larvae of N. brasiliensis assumed a rigid appearance at death. Infective larvae of T. spiralis burst, extruding their internal organs through their cuticle weakened by antibodies and the cells.     

Immunization with Trichinella spiralis Korean Isolate Larval Excretory–Secretory Antigen Induces Protection and Lymphocyte Subset Changes in Rats

Protection and immune responses were studied in rats immunized with Trichinella spiralis muscle stage larval excretory–secretory antigen (ES Ag) without adjuvant. Protection was assessed by the degree of adult worm burden and the yield of muscle (diaphragmatic) larvae after challenge infection with live larvae. Lymphocyte subsets were identified by flow cytometry in the spleen and peripheral blood. Cytokine production and specific IgG, IgG1 and IgG2a antibody responses were measured. Immunization with ES Ag produced highly significant protection against adult stages (98.4%) and muscle larvae (82.9%). Th2 type cytokines (IL‐10, IL‐4) were predominant. Anti‐muscle stage larval ES Ag antibody was significantly elevated in the order IgG2a > IgG1 > IgG on the 2^nd day after final immunization and on the 7^th day after challenge infection. Expression of CD4⁺ and the CD4⁺/CD8⁺ ratio from spleen and blood were significantly increased compared to the control. These results demonstrate that immunization with T. spiralis antigen can elicit robust immune response, resulting in complete protection against infective larvae, and this protection can be achieved without the use of any adjuvant.     

Coordinate expression of β1 and β2 integrin “activation” epitopes during T cell responses in secondary lymphoid tissue

The monoclonal antibodies (mAb) 15/7 and 24 recognize unique activation-dependent, conformational epitopes on β1 and β 2-integrins, respectively. The expression of both of these epitopes closely correlates with the ligand binding ability of their respective integrins, and thus serves as indicators of functional integrin “activation”. Here, we have used six-parameter flow cytometry to examine the expression of these epitopes and conventional β1- and β2-integrin epitopes during human T cell activation in secondary lymphoid tissues in vivo, focusing particularly on the virgin to memory/effector cell transition. Fresh tonsil lymphocytes were stained with mAb against conventional or activation-dependent integrin epitopes, followed by staining with mAb against CD3, CD45RA, and CD45RO, thus allowing the determination of integrin epitope expression on virgin (CD3⁺) T cells (CD45RA⁺/RO^?to±), memory/effector (CD45RA^?/RO⁺⁺) T cells, and T cells undergoing the virgin to memory/effector transition: transition region-1 (T1; CD45RA^+to++/RO⁺); -2 (T2; CD45RA⁺⁺/RO⁺⁺); and -3 (T3; CD45RA⁺/RO⁺⁺). Conventional β1- and β2-integrin epitopes progressively increase during the virgin to T3 stages of the transition in tonsil, in keeping with the generally higher levels of these adhesion molecules on memory/effector vs. virgin T cells. Expression of both the β1 (15/7)-and β2 (24)-integrin activation epitopes first appears on transitional T cells, and is maintained on a relatively constant number of cells (averaging 25-30%) throughout the T1-T3 stages. These epitopes are also noted on a subset of activated memory/effector T cells. Importantly, on both transitional and activated memory/effector T cell subsets, the expression patterns of the 15/7 and 24 epitopes vs. a variety of T cell activation antigens are identical, and the expression of these epitopes relative to each other is linearly correlated, findings strongly supporting the coordinate activation of β1 and β2 integrins duringT cell activation in vivo. These results provide the first evidence of integrin activation during an in vivo immunologic response, and demonstrate the usefulness of mAb recognizing conformational epitopes and multiparameter flow cytometry in delineating the dynamic interplay of adhesion molecules during complex physiologic processes.     

Antibody response to a protease secreted byTrichinella spiralis muscle larvae

In the present study we analyzed the humoral response ofTrichinella spiralis-infected mice to a 35-kDa protease (purified from the excretory-secretory products ofT. spiralis muscle larvae) by a Western-blot procedure and an enzyme-linked immunosorbent assay (ELISA) technique using a panel of postinfection mouse anti-Trichinella sera. The results demonstrated that this response was time-dependent and that infected mice could be distinguished from controls. In addition, inhibition assays demonstrated that these antisera were capable of abolishing the proteinase activity of the 35-kDa protease in vitro.The occurrence of proteases seems to be a very common feature in parasite crude extracts and excretory-secretory products (McKerrow 1989). It is also known that these enzymes are implicated in important host-parasite interactions, and for this reason, recent reports have proposed the use of parasite proteases both as alternative targets for an induced immune response and as a rich source of antigenic material for diagnostic testing (Hotez et al. 1985; Yamasaki et al. 1989; Song et al. 1990; Frank and Grieve 1991; Britton et al. 1992; Song and Chappell 1993).We have recently purified a protease (mol. wt., 35 kDa) from the excretory-secretory (ES) products ofTrichinella spiralis (GM-1 strain) muscle larvae and established some of the biochemical properties of this protease (Armas-Serra et al. 1994). The present study was undertaken to determine whether this protease would be recognized as an antigenic molecule by the host immune system, as occurs in the case of other parasites, and which effect the host immune response could have on the biochemical activity of the enzyme.