DNA encoding the Trichinella spirals 53kD excretory/secretory antigen for use as immunodiagnostic reagents

Disclosed are DNA sequences which encode an amino acid sequence homologous to a segment of Trichinella spiralis 53 kilodalton excretory-secretory antigen, recombinant polynucleotide molecules containing the sequences, and transfer and replication of the sequences in a transformed host to produce antigens useful as immunodiagnostic reagents or vaccines specific for T. spiralis.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to DNA sequences which encode an amino acid 
sequence homologous to a segment of Trichinella spiralis 53 kilodalton 
(kD) excretory-secretory antigen, recombinant polynucleotide molecules 
containing the sequences, and transfer and replication of the sequences in 
a transformed host to produce antigens useful as immunodiagnostic reagents 
or vaccines specific for T. spiralis 
2. Description of the Art 
Trichinellosis (known historically as trichinosis), caused by the nematode 
parasite Trichinella spiralis, is an important zoonotic disease of world 
wide distribution. Trichinellosis results from the ingestion of raw or 
undercooked meat, generally pork, containing the infective larval stage of 
the parasite. An important component of a program to control or eradicate 
trichinellosis is a specific and sensitive diagnostic test. A variety of 
direct and indirect tests have been used for the diagnosis of 
trichinellosis in swine and other species, the most recent of which is the 
enzyme-linked immunosorbent assay (ELISA). Initially, ELISA tests were 
performed with crude extracts of T. spiralis is muscle larvae as antigen 
and resulted in a high number of false-positive reactions, apparently due 
to cross-reactions with other parasite infections. Subsequent replacement 
of the crude antigen preparation with biochemically purified stichocyte 
antigens (G. L. Seawright et al., American Journal of Tropical Med. Hyg. 
32: 1275-1284, 1983), culture-derived excretory-secretory (ES) antigens 
(H. R. Gamble et al., Veterinary Parasitology 13: 349-361, 1983), or 
antibody affinity-purified ES antigens (H. R. Gamble and C. E. Graham, 
American Journal of Veterinary Research 45: 67-74, 1984) resulted in near 
elimination of false-positive reactions; however, inconsistencies in the 
yield and purity of antigen obtained by these procedures has remained a 
problem. 
Use of naturally derived antigens to T. spiralis has several disadvantages. 
It requires continual passage of the parasites in laboratory rodents. The 
rodents must be sacrificed 30-40 days post-infection at which time the 
parasites are collected for antigen isolation, and additional rodents are 
infected for subsequent isolations. Thus, production of naturally derived 
antigens is both time consuming and costly. Further, both the yield and 
purity of material can vary significantly between preparations. What is 
needed is an economical way to provide an unlimited source of T. spiralis 
antigens having reliable and reproducible purity. 
Limited information is available about T. spiralis antigens. T. spiralis 
muscle larvae have been shown to excrete and secrete antigens in culture 
which are useful as immnodiagnostic reagents (Gamble et al., 1983, supra). 
Immunodominant antigens in ES have been identified in the 45,000 to 53,000 
molecular weight range. Similar molecular weight antigens have been 
extracted from T. spiralis stichocyte cells using biochemical procedures 
(D. S. Silberstein and D. D. Despommier, The Journal of Immunology 
132:898-904, 1984). Monoclonal antibody generated from spleen cells of 
mice infected with T. spiralis recognized an antigenic determinant unique 
to T. spiralis present on proteins of molecular weights 45,000, 49,000, 
and 53,000 (Gamble and Graham, 1984, supra; H. R. Gamble, Experimental 
Parasitology 59: 398-404, 1985, and U.S. Pat. No. 4,670,384). Two of the 
proteins (49,000 and 53,000 molecular weight) possessing this determinant 
were isolated together by monoclonal antibody-affinity chromatography, and 
the affinity-isolated antigen used successfully in an ELISA for swine 
trichinellosis (Gamble and Graham, 1984, supra). T. spiralis antigens have 
been reported to be glycoproteins (Gamble, 1985, supra, and D. S. 
Silberstein, Dissertation Abstracts International 45: 824B, 1984). A 48 kD 
antigen, presumed homologous to the 45 kD antigen of Gamble and Graham, 
1984, supra, has been partially characterized. However, no function for 
the proteins has been elucidated and biological activity has yet to be 
assigned. Further, no data has been made available indicating which stage 
of development the corresponding messenger RNA which codes for the antigen 
is produced. 
An attempt to prepare T. spiralis diagnostic antigens by recombinant DNA 
techniques has been reported (Abstract, D. S. Zarlenga and H. R. Gamble, 
Federation Proceedings 46: 2038, 1987). The researchers prepared cDNA 
clones using poly A mRNA isolated from T. spiralis muscle stage larvae; 
however, the recombinant antigens were unable to detect antibodies to T. 
spiralis using sera from experimentally infected animals (D. S. Zarlenga 
and H. R. Gamble, unpublished data). Further, none of the genes coding for 
antigens described in the Abstract were shown to code for any of the ES 
products of T. spiralis e.g., the 45, 49, and 53 kD immunodiagnostic 
antigens, (D. S. Zarlenga and H. R. Gamble, unpublished data). 
SUMMARY OF THE INVENTION 
The present invention comprises isolated DNA sequences which encode an 
amino acid sequence homologous to a segment of Trichinella spiralis 53 kD 
excretory-secretory antigen, the amino acid sequence having the capacity 
to bind antibodies made in a host to T. spiralis. Methods to obtain the 
sequences are also disclosed herein. 
A further aspect of the invention is the provision of recombinant 
polynucleotide molecules containing the sequences. Such molecules include, 
for example, recombinant vectors, such as cloning or expression vectors, 
which contain a DNA sequence encoding an amino acid sequence homologous to 
a segment of T. spiralis antigen. 
Another aspect of the invention is the provision of cells which are 
transformed by the above vectors or DNA sequences, as well as methods of 
producing T. spiralis antigen peptide molecules comprising growing such 
cells under conditions whereby a peptide comprising T. spiralis antigen is 
expressed and recovered. 
A further aspect of the present invention is provision of purified 
antigenic material which has the ability to bind antibodies made in a host 
to T. spiraliswithout cross reactivity with antibodies made to Ascaris 
suum or Trichuris suis (other common swine parasites) antigens. Use of the 
antigens of the invention in serodiagnostic tests and vaccines for 
trichinellosis is also disclosed herein. 
A still further aspect of the invention is the provision of oligonucleotide 
probes capable of detecting the gene for T. spiralis antigen or fragment 
thereof and use of the probes to isolate DNA sequences encoding T. 
spiralis antigen. The DNA sequences which hybridize to the probes are 
encompassed by the present invention. 
The invention represents the first successful cloning of an mRNA encoding a 
T. spiralis diagnostic antigen. The invention provides a means to produce 
T. spiralis antigen having desired purity and yield without the use of 
laboratory animals to maintain the parasites and without the use of 
expensive reagents needed for the isolation of the naturally derived 
protein. 
A further advantage of the invention is that it can provide an unlimited 
source of standardized T. spiralis antigen. 
Another advantage of the invention is that is provides T. spiralis antigen 
which has application as immunodiagnostic reagents or vaccines for 
trichinellosis. 
Other objects and advantages of this invention will become readily apparent 
from the ensuing description.

The following examples are intended only to further illustrate the 
invention and are not intended to limit the scope of the invention which 
is described by the claims. 
EXAMPLE 1 
The following example describes the cloning, identification, and 
characterization of a DNA sequence that encodes an amino acid sequence 
homologous to a segment of T. spiralis 53 kD ES antigen. 
A. Extraction of Total RNA. 
Trichinella spiralis were maintained in female Sprague-Dawley rats by 
serial passage. Muscle larvae (L.sub.1) were recovered 30-40 days after 
infection by pepsin-HCl (1% each) digestion of eviscerated, ground rat 
carcasses and washed by settling through several changes of water. 
Total RNA was isolated from the recovered T. spiralis muscle larvae by 
guanidinium isothiocyanate: cesium trifluoroacetate isopynic 
centrifugation as described by D. S. Zarlenga and H. R. Gamble (Analytical 
Biochemistry 162: 569-574, 1987). In brief, approximately 1.5 million 
larvae (10.sup.9 cells) recovered from four rats were concentrated by 
filtration and resuspended in 13 ml of 4M guanidinium isothiocyanate 
homogenizing solution (4M guanidinium isothiocyanate, 5 mM sodium citrate, 
10 mM EDTA, 0.5% sarkosyl, 100 .mu.M .beta.-mercaptoethanol, 0.1% antifoam 
A) . The larval suspension was homogenized and centrifuged for 10 minutes 
at 3500.times.g. The cleared supernatant was transferred to a fresh tube 
and supplemented with cesium trifluoroacetate (Pharmacia-PL Biochemicals) 
(2.0 g/ml) to a final ratio of 1:1.6 homogenate:cesium trifluoroacetate. 
The final solution was separated into 5-ml polyallomer centrifuge tubes 
and spun at 200,000.times.g for 44 hours at 15.degree. C. in an SW 55 Ti 
rotor. The DNA was removed by pipetting, immediately precipitated in 0.3M 
sodium acetate and 2 volumes of ethanol, and subsequently treated with 
proteinase K (200 .mu.g/ml) at 37.degree. C. for 30 minutes in 100 mM 
Tris, pH 7.5, 10 mM EDTA, 100 mM NaCl, and 0.5% w/v SDS. The balance of 
the gradient was aspirated down to the RNA band which was removed as a 
precipitate either by pipetting or "spooling" if sufficient amounts were 
present to do so. The RNA was washed with 70% ethanol (3.times.1.0 ml) and 
reddissolved in sterile TE (10 mM Tris, pH 7.6, 1 mM EDTA). No further 
purification was performed. 
B. Isolation of Poly A mRNA. 
Poly A mRNA was isolated by two successive passes of total RNA through an 
oligo(dT)-cellulose column essentially as described by Aviv and Leder 
(Proc. Natl. Acad. Sci. USA 69:1408-1412 (1972)). The poly A mRNA was 
eluted from the oligo(dT) cellulose column with sterile water preheated to 
65.degree. C. and then precipitated in 0.3M sodium acetate and 2.5 volumes 
of ethanol. The poly A mRNA was pelleted by centrifugation, washed with 
80% ethanol, centrifuged once again, dried in vacuo, and dissolved in 
water. 
C. Synthesis of Double Stranded cDNA. 
Double stranded cDNA was generated from 10 .mu.g of purified poly A mRNA 
according to the method of Gubler and Hoffman, Gene 25:63-269 (1983), as 
modified by Watson and Jackson, In: DNA Cloning, Volume I, Ed. D. M. 
Glover, IRL Press, Oxford, pages 79-88 (1985) using Eco RI linkers. In 
brief, first strand cDNA synthesis was carried out by priming with oligo 
dT in the presence of RNAsin, the appropriate dNTPs, and Moloney Murine 
Leukemia Virus (MMLV) reverse transcriptase. cDNA synthesis was stopped 
after 1 hour at 37.degree. C. with EDTA, and the cDNA-RNA hybrid was 
extracted with phenol-chloroform, precipitated with ethanol and sodium 
acetate, centrifuged, dried in vacuo, and resuspended in water. Second 
strand synthesis was carried out in the presence of dNTPs and RNase H by 
DNA polymerase I. After a 1 hour incubation at 15.degree. C. followed by a 
1 hour incubation at 22.degree. C., the reaction was halted and the double 
stranded cDNA was phenol-chloroform extracted, precipitated with ethanol 
and ammonium acetate, collected by centrifugation, dried in vacuo, and 
resuspended in 50 mM Tris, pH 7.6, 1 mM EDTA, 5 mM DIT. The cDNA was 
methylated with S-adenosyl-L-methionine by Eco RI methylase and the cDNA 
ends made blunt by treatment with T4 DNA polymerase in the presence of 
dNTP. After phenol-chloroform extraction, ethanol precipitation, 
centrifugation, and drying in vacuo, Eco RI linkers were ligated to the 
methylated cDNA termini using T4 DNA ligase and RNA ligase. The cDNA was 
then digested with Eco RI restriction enzyme for 1.5 hours at 37.degree. 
C. to produce Eco RI compatible ends on the cDNA. The reaction was stopped 
with EDTA, phenol-chloroform extracted, ethanol precipitated, collected by 
centrifugation, dried in vacuo, resuspended in TE, and passed over a 
"NACS" column (Bethesda Research Laboratories). The cDNA was eluted in 2M 
NaCl in TE, ethanol precipated, collected by centrifugation, dried in 
vacuo, and resuspended in TE. 
D. Preparation of Recombinant Bacteriophage .lambda. DNA Using T. spiralis 
Double-stranded cENA. 
Ten percent of the double-stranded cDNA was ligated to 1 .mu.g of 
.lambda.gt11 arms containing compatible Eco RI ends using T4 DNA ligase at 
4.degree. C. for 16 hours. 
E. Packaging of Recombinant Bacteriophage .lambda.gt11 DNA (cDNA Library 
Construction). 
T. spiralis cDNA-bacteriophage DNA was packaged into intact virus particles 
using a "Packagene" extract following the procedures described by the 
manufacturer (Promega Biotech). After packaging, E. coli strain Y1090 was 
infected with the packaged cDNA library and plated onto Luria Broth (LB) 
agar plates in LB agarose containing 0.2% X-gal, 0.1 mM IPTG, and 100 
.mu.g/ml ampicillin. Using this technique, greater than 90% of the 
bacteriophage were observed to be recombinants (i.e., contained cDNA 
inserts) as judged by the percentage of white plaques of the total plaques 
obtained. 
F. Identification of Recombinant Bacteriophages by Screening. 
1. Preparation of Immune Sera. In order to screen the cDNA libraries, 
immune sera were generated by immunizing rabbits or swine with T. spiralis 
parasites, crude worm protein extract (CWE), or ES proteins. Individual 
rabbits were immunized subcutaneously with one of the protein preparations 
emulsified in Complete Freund's adjuvant and boosted two times further at 
1 week intervals. Immune sera were collected after about 4 weeks. Swine 
were infected per os with 500 or 10,000 infective T. spiralis larvae and 
infection serum collected after 35 days. Serum from pigs with naturally 
acquired infection of T. spiralis were obtained according to Gamble et 
al., 1983, supra. 
2. Immunoscreening of Recombinant Bacteriophages. Aliquots (approximately 
10.sup.5 clones) of the T. spiralis bacteriophage libraries were used to 
infect and transform (transfect) E. coli Y1090 and plated as described in 
E above. (See R. A. Young and R. W. Davis, Proc. Natl. Acad. Sci. USA 80: 
1194-1198, 1983, and Science 222: 778-782, 1983). The plates containing 
developing phage plaques were overlaid with nitrocellulose membrane disks 
which had been soaked in 10 mM IPTG and were incubated at 42.degree. C. 
for 3 hours to induce production of the .beta.-galactosidase fusion 
protein then transfered to 37.degree. C. The nitrocellulose disks, 
impregnated with the E. coli proteins containing putative recombinant 
fusion protein, were removed from the plates after overnight incubation at 
37.degree. C., blocked in immunowash buffer (IWB) (0.15M sodium chloride, 
50 mM Tris, pH 7.8, 0.05% Tween-20, and 5% non-fat dried milk), then 
screened overnight with a 1:200 dilution of serum from a pig 
experimentally infected with T. spiralis muscle larvae. Rabbit anti-swine 
IgG (1.0 .mu.g/ml) and [.sup.125 I]-labelled goat anti-rabbit IgG 
(2.times.10.sup.6 cpm/filter) were used as second and third antibodies, 
respectively. Positive clones (antibody binding) were visualized by 
autoradiography and approximately 40-50 clones were picked and rescreened 
as described above using moderate dose swine infection sera (500 
larvae/animal). 
3. Five putative positives were picked and rescreened with rabbit antiserum 
to parasite ES antigen (diluted 1:200) followed by [.sup.125 I]-labelled 
goat anti-rabbit IgG (2.times.10.sup.6 cpm/filter). One of these clones 
showed strong hybridization in all the above screenings and was designated 
TsA-12. 
G. Plague Purification of Selected Bacteriophages in E. coli Y1090. 
Once identified and removed from the respective culture plate, positive 
bacteriophage TsA-12 identified in section F3 above was plaque purified by 
several rounds of plating, screening, and isolation as described above in 
section F2. This procedure was repeated until 100% of the plaques from the 
clone produced a positive signal upon immunoscreening. The molecular 
weight of the fusion protein was 140,000 daltons (FIG. 1). 
H. Transfer of Selected Bacteriophages into E. coli Y1089 and Production of 
Fusion Proteins. 
The titer of TsA-12 clonal bactariophage preparation prepared in section G 
above was determined by infecting E. coli Y1090 and plating on LB agar 
containing ampicillin. Individual aliquots of an overnight culture of E. 
coli Y1089 were infected at a M.O.I. equal to 10 with the bacteriophage 
cDNA clone and grown at 32.degree. C. Individual colonies were picked into 
microtiter plates in a grid design and replica plated onto two LB 
agar-ampicillin culture plates. One inoculated plate was grown at 
32.degree. C., the other grown at 42.degree. C. One colony growing at the 
lower temperature, but not at the higher (indicative of a lysogenic state) 
was isolated. The E. coli strain Y1089 containing the TsA-12 insert has 
been deposited under the terms of the Budapest Treaty in the Agriculture 
Research Culture Collection (NRRL), Northern Regional Research Center, 
Agricultural Research Service, U.S. Department of Agriculture, Peoria, 
Ill., 60164, and has been assigned the accession No. NRRL B-18503. E. coli 
,strain Y1089 containing TsA-12 insert was grown in bulk culture in LB 
broth containing ampicillin at 32.degree. C. When the O.D..sub.550 of the 
bulk culture reached 0.6-0.7, the temperature was shifted to 42.degree. C. 
and held at that level for 30 minutes to induce the lytic cycle. After a 
temperature shift to 37.degree. C., the culture was induced with 2 mM IPTG 
for 3 hours. The E. coli were then harvested by centrifugation at 3500 rpm 
for 10 minutes at 25.degree. C. and the cell pellet was washed in 
phosphate buffered saline than resuspended in a volume of 0.05M Tris, pH 
8, 10 mM MgCl.sub.2, and 0.5 mM TPCK (N-tosyl-L-phanylalanine chloromethyl 
ketone) and PMSF (phenylmethylsulfonyl fluoride). The E. coli were lysed 
by treatment with 25 .mu.g/ml lysozyme for 30 minutes on ice followed by 
freeze thawing and sonication for 20 seconds. The homogenate was treated 
with 5 .mu.g/ml DNase for 30 minutes on ice and then centrifuged at 
11,000.times.g for 15 minutes at 4.degree. C. and the resulting 
supernatant stored at -20.degree. C. for further analysis. 
I. Purification of Fusion Proteins (TsA-12 Antigen). 
The .beta.-galactosidase fusion protein produced by the procedures 
described above was purified in the following manner. Bacterial lysogens 
were grown to an O.D..sub.550 of 0.6 and induced with IPTG for 3 hours and 
treated as described in section H above (See T. V. Huynh et al., In: DNA 
Cloning, Vol. I, (Ed. D. M. Glover), 49-88,IRL Press, Oxford, (1985)). 
Cell pellets were washed twice with 10 volumes of phosphate buffered 
saline (PBS) for 2 hours each time. Bacterial debris was collected after 
each wash by centrifugation and washed two additional times with 10 ml of 
1.5% n-octyl-.beta.-D-glucopyranoside (OGP). The final pellet was 
suspended in 6M urea, 10 mM dithiothreitol and agitated overnight to 
extract the recombinant antigen. Significant antigen remained in the 
pellet which could only be solubilized with 0.5% SDS. 
J. Subcloning of Recombinant cDNA into pUC 19, Transformation of E. coli JM 
83, and Production of Cloned Sequences Therefrom. 
TsA-12 was subcloned into pUC 19 to facilitate further characterization by 
DNA mapping, DNA sequencing (J. Viera and J. Messing, Gene 19: 259-268, 
1982), and Southern and northern blot hybridization. For subcloning 
procedures, see generally, Maniatis et al., 1982, supra. In the cloning of 
TsA-12 DNA, the Eco RI restriction sites were not regenerated; 
consequently, a Kpn I: Sst I digestion of recombinant bacteriophage DNA 
prepared according to Maniatis et al., 1982, supra, was required to remove 
the TsA-12 DNA (539 base pairs) from .lambda. DNA along with approximately 
1000 base pairs of .lambda. DNA flanking each side of the insert. After 
having been purified by agarose gel electrophoresis, electroelution, and a 
"NACS" column, this entire gene segment was ligated into the expression 
plasmid pUC 19 in the Kpn: Sst I restriction site and used to transform 
competent E. coli bacterial cells, strain JM 83 using standard procedures. 
See, e.g., Hanahan, J. Molec. Biol. 166: 557, 1983. Plasmid DNA was 
generated as described by C. Sadhu and L. Gedamu, Biotechniques 6: 20-21, 
1988, are the cDNA insert isolated by restriction enzyme digestion as 
outlined above. 
K. Characterization of TsA-12 Antigen. 
1. For labeling and hybridization studies, plasmid DNA was digested with 
Eco RI, electrophoresed on LMP agarose (FMC), and the insert DNA band was 
excised, placed in electrophoresis buffer in dialysis bags and 
electroeluted for 2 hours at 100 volts. The dialysate was purified over a 
NACS column and the DNA precipitated with ethanol and sodium acetate. The 
insert DNA was labeled with 100 .mu.Ci of .sup.32 P-alpha dCTP (3000 
.mu.Ci/mMole, New England Nuclear) by nick translation, Rigby et al., J. 
Molec. Biol. 113: 237, 1977, using DNase I and DNA polymerase I (BRL). 
Labeled DNA was separated from .sup.32 P-dCTP by spun column 
chromatography as described by Maniatis et al., 1982, supra. T. spiralis 
muscle larvae DNA was purified as described by J. B. Dame and T. F 
McCutchan, Molecular and Biochemical Parasitology 8: 263-279, 1983. The 
purified DNA (10 .mu.g) was digested with 50 units of either Hind III, Pst 
I, Sal I Eco RV, Xba I or Eco RI restriction enzymes (BRL), 
electrophoresed in 0.8 % agarose (FMC) and transferred to "Nytran" 
membrane using Southern blotting procedures. See E. M. Southern, Journal 
of Molecular Biology 98: 503-517, 1975. After transfer, the DNA-blotted 37 
Nytran" paper was baked in vacuo at 80.degree. C. for 2 hours, 
prehybridized with 0.5M NaCl, 0.05M sodium citrate, pH 7.0 (6X SSC), 
Denhardt's solution, and 0.2% sodium dedecylsulfate (SDS) for 6 hours at 
65.degree. C., and hybridized with 10.sup.6 cpm of .sup.32 P-labeled probe 
for 16-20 hours at 65.degree. C. The blots were washed three times with 
0.2X SSC, 0.1% SDS at 50.degree. C. for 30 minutes per wash. The blots 
were air dried and overlaid with photographic film (Kodak XAR) to 
visualize the hybridization patterns. Northern blots were similarly 
generated using 10 .mu.g of total RNA isolated from T. spiralis infective 
muscle larvae (L.sub.1), newborn larvae (C. H. Wang and R. G. Bell, 
Parasite Immunology 10: 293-308, 1988), or adult parasite (Wang and Bell, 
supra), and separated on a 1% denaturing formaldehyde gel as described by 
L. G. Davis et al., Basic Methods in Molecular Biology, Elsevier Science 
Publishing Co., Inc., 1986. After separation on 1% agarose the RNA was 
transferred to nitrocellulose and screened with insert TsA-12 cDNA as 
described above for Southern blot analysis. 
The foregoing techniques revealed that the TsA-12 cDNA insert cloned in pUC 
19 ENA was 539 bp in length and likely contains introns within the genome. 
Northern blots of RNA from T. spiralis infective larvae, newborn larvae, 
and adult parasite indicate that the TsA-12 gene is expressed within the 
(L.sub.1) and adult stages only. 
2. Preparation of Rabbit Antisera. Rabbits were immunized with recombinant 
antigen from the induced lysogen that was separated from other bacterial 
proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis 
(SDS-PAGE). Fusion protein was excised from the gel, then emulsified in 
Freund's incomplete adjuvant and injected subcutaneously on days 1 and 8. 
A third immunization was given on day 15 in the ear vein in the absence of 
adjuvant using fusion protein that had been allowed to diffuse from gel 
slices for 24-48 hours prior to injection. Rabbits were bled 5 days later 
by heart puncture. 
3. Western Blot Analysis. The TsA-12 lysogen was induced with IPTG and the 
fusion protein purified as described above in sections H and I. Purified 
or crude protein samples (1-10 .mu.g) were boiled in SDS sample buffer for 
5 minutes and electrophoresed on 10% SDS-polyacrylamide gels (U. K. 
Laemmli, Nature 227: 680-685, 1970). Separated proteins were either 
stained with Coomassie blue or electrophoretically transferred to 
nitrocellulose (H. Towbin et al., Proc. Nat. Acad. Sci. USA 76: 4350-4354, 
1979), then blocked in IWB and incubated overnight with a 1:200 dilution 
of rabbit antiserum to either antibody-purified Ts.49, Ts.53 ES antigens, 
purified TsA-12 fusion protein or .beta.-galactosidase. The next day, the 
filters were washed in IWB and IWB containing 1% Triton-X 100 and 0.1% SDS 
then incubated with peroxidase-labelled goat anti-rabbit IgG (0.2 
.mu.g/ml) for 3-4 hours. Bound enzyme was visualized by the addition of 
H.sub.2 O.sub.2 and 4-chloro-1-napthol (Kirkegaard and Perry). 
Rabbit antibodies raised against fusion protein TsA-12 that had been 
purified by polyacrylamide gel electrophoresis were used to screen western 
blots of T. spiralis muscle larvae crude worm extract (CWE), total ES 
protein, and monoclonal antibody affinity-purified Ts.49 and Ts.53 
antigens. In all cases, rabbit anti-TsA-12 serum reacted exclusively with 
a doublet in the 53,000 molecular weight range as well as with the fusion 
protein and .beta.-galactosidase positive controls (FIG. 2). Repeat 
screenings failed to generate antibody binding to other ES antigens. In 
addition, no binding was observed when TsA-12 antiserum was used to screen 
adult CWE or ES proteins. This specificity for Ts.53 antigen with no cross 
reactivity to the Ts.49 or Ts.45 antigens was unexpected. It had been 
believed that the same gene coded for the 45, 49, and 53 kD ES antigens. 
This was based on similarities in antibody binding to the naturally 
derived antigens (Gamble and Graham, 1984, supra). Our results suggest 
that separate mRNA species may be involved in the production of the 45, 
49, and 53 kD ES antigens. Rabbit antiserum to affinity-purified antigens 
Ts.49 and Ts.53 was used to screen western blots of TsA-12 fusion protein, 
ES antigen, and .beta.-galactosidase (FIG. 3). This antiserum bound to 
antigens Ts.45, Ts.49, and Ts.53 in ES preparations; binding was also 
observed with the 140,000 molecular weight TsA-12 fusion protein with less 
intense binding to several lower molecular weight bands in the TsA-12 
preparation at 48,000, 66,000, and 95,000 molecular weight. No binding to 
purified .beta.-glactosidase was observed using antibodies to purified ES 
antigens. 
4. ELISA with TsA-12 Fusion Protein. The specificity of TsA-12 for T. 
spiralis infection was evaluated by ELISA using sera from pigs 
experimentally infected with Ascaris suum, Trichuris suis or T. spiralis. 
Purified TsA-12 fusion protein was diluted to 5 .mu.g/ml in 0.1M carbonate 
buffer (pH 9.6) and bound to microtiter plates. Sera from pigs 
experimentally infected with A. suum, T. suis, or T. spiralis were diluted 
1:100 in PBS containing 0.05% Tween-20 and 100 .mu.l was added to wells 
for 30 minutes. Rabbit anti-swine IgG (1 .mu.g/.mu.l) and peroxidase 
labelled goat anti-rabbit IgG (0.1 .mu.g/.mu.l) were used as second and 
third antibodies, respectively. Bound enzyme was quantitated by the 
addition of H.sub.2 O.sub.2 and 2',2'-azine-di[3-ethyl-benzthiazoline 
sulfate] (ABTS) (Kirkegaardand Perry). 
The results are tabulated in Table 1 below. An optical density ratio 
greater than 4:1 (0.176:0.039) was obtained for T. spiralis infection 
serum as compared to normal serum controls (0.039) whereas no optical 
density values above normal serum controls were observed with A. suum 
(0.027) or T. suis (0.037) infection sera. 
TABLE 1 
______________________________________ 
Recognition of TsA-12 by Pigs Infected With T. Spiralis 
or Other Swine Parasites 
Serum ELISA (O.D.) 
P/N Ratio 
______________________________________ 
Uninfected Pig .039 -- 
T. Spiralis Infected Pig 
.176 4.5 
Ascaris Suum Infected Pig 
.027 &lt;1.0 
Trichuris Suis Infected Pig 
.037 &lt;1.0 
______________________________________ 
To determine the kinetics of antibody responses to TsA-12, a group of 25 
Swiss-Webster mice were each orally inoculated with 150 T. spiralis muscle 
larvae. Serum was collected from 5 uninoculated mice on day 0 and 5 
infected mice on days 7, 14, 21, 28, and 35 post-inoculation. Collected 
sera was diluted 1:100 and tested by ELISA using 1 .mu.g/ml TsA-12 to coat 
plates. Goat anti-mouse IgG and IgM (1.0 .mu.g/ml ) and peroxidase 
labelled rabbit anti-goat (0.1 .mu.g/ml) (both Kirkegaard and Perry) were 
used as second and third antibody reagents. ABTS substrate was used as 
described above. 
Mice inoculated with T. spiralis L.sub.1 developed an antibody response to 
TsA-12 as early as 14 days post inoculation. Antibody levels increased 
over time and peaked at 28-35 days post inoculation (FIG. 5). 
5. Immunoperoxidase Staining. Rabbit anti-TsA-12 serum was used to localize 
the homologous T. spiralis antigen by immunoperoxidase staining. Tissue 
sections from infected mice were screened with either anti-TsA-12 serum or 
rabbit anti-.beta.-galactosidase serum as a negative control as follows. 
Tongue tissue removed from mice 30-40 days post-infection with T. spiralis 
muscle larvae was fixed in 4% glutaraldehyde and dehydrated through a 
series of washes of increasing ethanol concentration. Tissue was embedded 
in paraffin and sectioned at 5 .mu.m then mounted on slides which were 
cleared with xylene and rehydrated. Sections were incubated with a 1:1000 
dilution of either rabbit anti-TsA-12 or rabbit anti-.beta.-galactosidase 
sera. Slides were washed and processed using the "Vectastain" ABC 
immunoperoxidase kit (Vector laboratores) according to the manufacturer's 
protocol, then counterstained with Harris hemotoxylin for 30 seconds. 
Slides were washed in tap water then photographed. 
FIG. 4 shows immunoperoxidase staining of T. spiralis muscle larvae from 
infected mouse tongue. The most intense staining with TsA-12 antiserum was 
localized within the stichocyte cells of the muscle larvae (FIG. 4B). 
Control antiserum to .beta.-galactesidase did not stain parasite sections 
(FIG. 4A). 
L. DNA Sequencing of TsA-12. 
The DNA sequence of the cDNA insert TsA-12 was determined using the dideoxy 
chain termination technique (F. Sanger et al., Proc. Nat. Acad. Sci. USA 
74, 5463, 1977). For single stranded sequencing, purified cDNA insert was 
obtained in accordance with section J above, ligated into Kpn I: Sst I 
digested M13mp18 and M13mp19, and used to infect and transform JM101 cells 
(See J. Messing, Methods Enzymol. 101: 20, 1983). White colonies were 
picked and used to generate single stranded viral DNA for sequencing; 
where possible, double stranded sequencing was performed directly in pUC 
19 plasmid DNA as described by Kraft et al., BioTechniques 6:544-546 
(1988) using .sup.35 S-dATP (NEN 500 Ci/mMole) and the "Sequenase" 
Reaction kit (US Biochemicals), and analyzed on a 6% polyacrylamide 
sequencing gel. The complete TsA-12 sequence of M13 recombinant viral DNA 
and pUC double stranded plasmid DNA insert was ascartained. The sequence 
of cDNA clone TsA-12, as determined by these procedures, is shown in Table 
2 below. 
TABLE 2 
__________________________________________________________________________ 
DNA SEQUENCE OF TsA-12 CODING STRAND 
__________________________________________________________________________ 
5'TTTTTTTTTT 
TTTTTGTTTT 
TTACAGTTTG 
AAAAACTTTA 
CTGATAGATA 
GATTGCTTAA 
AGAAGCTATA 
ATTTCTGCTG 
GATTTAGAAC 
AACAACTGTA 
GTTCTGAAAA 
AACATGTTGG 
AAAAACCCTT 
TTGGGGGCTT 
TGGTTTGCGC 
TTTAAACGTA 
GTGCCGATTT 
ATCGCCTCCT 
TCCAACCAAT 
CTGAATGTTT 
CCATCACTGG 
GAAATTTATC 
AATGCTGCCA 
ATGTGCTCTT 
TGTTTTCATC 
GAACACTCCG 
GCTACTTCAG 
TTATTACGTA 
AGCATCGTCC 
GTATCATTCG 
GTGTTGCTCT 
GTGTTTAACT 
TTAGTGCTAA 
TTGTTGCATT 
GTACAGCTTT 
TGAAGTTTAT 
CTTCTTTGTC 
AGAATTGCTT 
AATTCCATGG 
TATCAAAAGC 
TTTTCTTAAA 
ATACGATCTA 
TTTCATCACC 
AAATGGCTTC 
TGAATTGGAT 
TTGTAACAAA 
AACTTCGGGA 
TTTCTATTGA 
ATCATTTGCA 
TTTATAAGAG 
ATAGAACCTC 
AGTAGTTTTA 
CCATTTTTCC 
##STR1## 
__________________________________________________________________________ 
DNA sequencing of the TsA-12 insert revealed the long poly A tail 
corresponding to the 3' end of the messenger RNA and a single Hind III 
restriction site consistent with restriction studies done on this clone. 
To the extent of any discrepancies between the sequence shown above and 
the sequence contained in the deposited clone, the latter is controlling. 
EXAMPLE2 
The following example illustrates the use of antigens of the invention as a 
diagnostic reagent. 
The ELISA test for swine or humans infected with T. spiralis uses as 
antigen a preparation of ES product or purified antigens Ts.49 and Ts.53 
(Gamble et al., 1983, supra; Gamble and Graham, 1984, supra). The 53 kD 
antigen of the invention is used in place of these other antigen 
preparations as follows: the 53 kD antigen diluted to an appropriate 
concentration in carbonate buffer (pH 9.6) is used to coat wells of a 
96-well microtiter plate. After coating and between all antibody steps 
wells are washed three times with phosphate-buffered saline (PBS) 
containing 0.05% Tween 20. Swine or human senram (diluted in PBS-Tween) is 
then added to the wells and incubated for 30 minutes. After washing, a 
second antibody (goat anti-swine or goat anti-human, conjugated to an 
enzyme) diluted in PBS-Tween is added and incubated for another 30 
minutes. After a final washing a suitable enzyme substrate is added and 
the color change read on a microtiter plate reader. Positive results are 
determined as recorded in Table 1. 
EXAMPLE3 
The following example illustrates the use of antigens of the invention as a 
vaccine. 
T. spiralis ES antigens and purified Ts.49 and Ts.53 have been used to 
immunize mice and pigs against challenge infection with T. spiralis 
(Gamble, 1985, supra). As a vaccine, the 53 kD antigen of the invention is 
used in the same protocol as follows: antigen at an appropriate 
concentration is emulsified in Freund's complete adjuvant and administered 
intraperitoneally to animals on days 0, 14, and 28. In experiments using 
ES antigens or Ts.49 and Ts.53 both mice and pigs were stimulated to 
produce an effective immune response to challenge infection. In a 
preliminary experiment, immunization of mice with 1 .mu.g doses of 
recombinant 53 kD antigen (TsA-12 antigen) prepared as described in 
Example 1 above induced a 48.3% level of immunity. In related studies, 
pigs immune to T. spiralis infection had T-cells which were immunoreactive 
to the 53 kD antigen. 
It is understood that the foregoing detailed description is given merely by 
way of illustration and that modification and variations may be made 
therein without departing from the spirit and scope of the invention.