lmmunochemlstry 1975 Vol
12. pp 561-567
Pergamon Press
Prmted m Great Elrltalll
HYPERSENSITIVITY TO SCHISTOSOMA MANSONI A N T I G E N S - - I : I M M U N O C H E M I C A L A N D BIOLOGICAL CHARACTERIZATION OF AN ANTIGENIC EXTRACT* RABIA H U S S A I N , W I L T O N E. V A N N I E R a n d K DARWIN MURRELL Naval Medical Research Institute, Nahonal Naval Medical Center, Bethesda, Maryland 20014. U S A (Recewed 9 September 1974)
Abstract--An adult Schistosoma mansoni extract (FTI) was obtained in good yield (1000mg per 100,000 worms) after a single freezing and thawing cycle. This fractlon was highly reactwe by both Prausmtz-Kustner (P-K) and passive cutaneous anaphylaxts (PCA) tests with sera from schlstosome infected rats Adult, cercarlal and egg antigens were also tested by the P-K assay In general, adult antigens were more reactwe than cercanal and soluble egg antigens. Rats produced detectable levels of reagm~c antibody to FT 1 within 4 weeks after imtml mfect~on and showed a clear anamnestlc response after reinfection The ability of various antigen preparations to induce reaglnlC antibodies m rats was also studied. The FT 1 preparation was found to be composed of 10 per cent carbohydrate, 90 per cent protein and a trace of rlbonuclelc acid The fractton contained materials with a broad range of electrophoretlc moblhtles as shown by both disc gel electrophoresls and lmmunoelectrophorests
INTRODUCI'ION It has long been k n o w n that helminth infestations are associated with various manifestations of atopic sensitization such as urticaria, eosinophilia a n d elevated levels of IgE It is t h o u g h t that the sine a n d complexity of h e l m l n t h parasites a n d their intimate contact with the mucosa of the respiratory a n d gastrolntestinal tracts during the course of parasitizatlon are partly responsible for their almost universal capablhty to bring a b o u t reagln mediated hypersensitivity (Sadun, 1972). One of the major p r o b l e m s encountered in the study of h e l m i n t h antigens which induce these responses is the complexity a n d heterogeneity of antigenic extracts. M a r k e d differences have been n o t e d depending o n the extraction procedure used. Very few helminth antigens have been isolated in a highly purified form. T h e allergen from Ascarls suum has been extensively purified a n d charactermed (Hussaln et al, 1973; Ambler et al, 1974) An allergen from Nippostronyylus braslhensls has been partially purified (Ambler a n d Orr, 1972). A series of fractionatlon experiments wlth extracts of S j a p o m c u m have been carrled out a n d a n antigen fraction Isolated, which was
highly specific in skin tests in patients with sehlstosomlasis (Williams et al., 1965; Sato et al., 1969). Harris (1973) obtained fractions from adult S. mansonl by extraction with borate saline a n d precipitation at p H 4.6. Their gel filtration studies suggested t h a t the allergens of S mansonl were of a t least two types One was a glycoproteln of mol. wt 150,000 which precipitated at p H 4.6 a n d the other a glycoprotein of mol. wt 20,000-30,000 soluble at this p H We have shown that a readily soluble fraction was obtained in good yield f-om adult schistosomes by freezing a n d thawing (Vannler et al., 1974). This fraction was active In the Prausnitz-Kiistner ( P - K ) assay with sera from schlstosome infected rats a n d was able to induce the formation of reaginlc antibodies o n immunization with alum a n d B. pertussts vaccine. The present investigation was carried out in order to define the nature of antigenic extracts from adult SChlstosomes in terms of their immunologic activity a n d chemical composition. The most active materials will be used for purification a n d characterization studies of S mansoni allergens.
MATERIALS AND METHODS Buffers Borate buffered saline, pH 7 9 was prepared with 2'2 g boric acid, 0'2 g sodium hydroxide, 9 3 g sodium chloride and water to make I1 of solution
* Supported by the Bureau of Medicine and Surgery Work Unit Numbers MR011.01 01 1077, MF51.524009.0027BF6I; MR041.0901.0129B6GJ and ONR Contract Number N00014-70-C-0331 to the American Foundation for Biologic Research, Rockvdle, Maryland The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large The ammals used in this study were handled in accordance with the provisions of Pubhc Law 89-54 as amended by Pubhc Law 91-579, the 'Ammal Welfare Act of 1970" and the principles outhned in the "Guide for the Care and Use of Laboratory Animals,' U.S Department of Health, Education. and Welfare Pubhcation No (NIH) 73-23
Antu3ens Collection of adult S. mansom The S mansom hfe cycle was maintained with infected snails as described previously (Stlrewalt and Uy, 1969) NIH/NMRI Swiss mice (1520 g) of either sex were infected with 200 S mansom cercarlae from infected snails by allowing each mouse to wade for I hr in a quart jar with 30ml of water containing cercarla~ The adult worms were harvested from mice after 6-8 weeks' infection by perfuslon of the hepatic portal system with citrate buffered saline, 1 5)o w/v sodium citrate
561
562
R HUSSAIN, W E V A N N I E R a n d K D M U R R E L L
and 085% sodium chloride, by the method of Smithers a n d Terry (1965) W o r m s collected in this fashion were frozen In a small volume of citrate saline at - 2 0 = C
Preparatton of adult schzstosome freeze thaw antigen Adult worms collected over a period of several weeks were allowed to thaw over a period of 48 hr at 4°C m a small volume of borate saline The suspension was filtered through a sintered glass funnel (coarse porosity} The filtrate was designated FT l and was concentrated by Amicon ultrafiltration (UM-2 membrane, Amicon Corporauon, Lexington, Mass ) to one-tenth of its original volume and exhaustively dialyzed against borate saline (pH 8 4) The residue was then used to prepare FT 1-6 A 10-ml volume of saline was added to each 1 ml of packed worms The suspension was then frozen and thawed six times. Between each cycle of freezing a n d thawing the w o r m s were centrifuged at 3500g for 20 mIn, the supernatant removed a n d the worms resuspended in the initial volume of sahne The pooled supernatants were concentrated by ultrafiltration and dialyzed against borate saline Before use it was centrifuged for 30 m m at 10,000 g to remove any preopltated material French press anttoen I and 2 Pooled worm residues obtained after FT l preparation were also extracted after disruption by a French pressure cell A volume of 5 ml of packed worms was h o m o g e m z e d with a fine Ten Broeck tissue grinder m a volume of 50 ml until a clear suspension was obtained The homogenate was allowed to settle for 18hr at 4°C The clear supernatant was removed from the debris that settled out. The supernatant was then passed through an A m m c o French pressure cell (American Instrument C o , Silver Spring, Md.) at a pressure of 15,000 psi The effluent was centrifuged at a speed of 10,000 g for l hr and the clear supernatant dmlyzed against borate sahne The soluble products were designated French press 1 a n d 2. The French press I fraction was extracted lrom lyophlhzed worms while the French press 2 materml was prepared from freshly thawed worms Adult excretor)~secretory ant~den, cercarml antigen and egg anttgen Adult excretory-secretory antigens from adult w o r m s were obtained by an m vitro culture method (Murrell a n d Clay, 1972). W o r m s were perfused from mice with citrate sahne, washed six times with NCTC-135 (Gibco, G r a n d Island, N Y ) and placed in culture flasks (40-50 worms/flask) containing NCTC-135 supplemented with pemcilhn (200 units/m1) a n d streptomycin (200 #g/ml) The cultures were incubated 3 6 - 7 8 h r at 37~C in 95 per cent a m 5 per cent CO2 The culture fluids were pooled, filtered (0.45/~ microfilter), concentrated, a n d dialyzed in the cold against borate-buffered saline (pH 7.8) by ultrafiltratlon a n d pressure dialysis through a P M - l 0 m e m b r a n e (Amicon Corporauon, Lexington, M a s s ) The concentrated antigen was centrifuged at 10,000g (4~C) for I hr and the supernatant (CA) stored at - 7 0 ° C untd used A second procedure was also employed Fresh live w o r m s were washed s~x u m e s in Hanks' balanced salt solution (HBSS), suspended m HBSS containing penicflhn (200 unlts/ml) and streptomycin (200 #g/ml) The majority of adult worms, at the end of the incubation period, are still motile on warming to 37°C The supernatant was filtered ( 0 4 5 # microfilter) concentrated, (PM- l0 membrane) a n d centrifuged ( 10,000 g at 4~C for lhr) The supernatant was designated A x e and stored at - 7 0 ~ C A cercarial exoantigen (CXO) was prepared by a modification of a procedure described by Fife et al (1967) Cercariae were shed Into dechlorinated tap water (1000-5000/ ml) and were held at 4°C for 5-7 days The supernatant was filtered C045 # microfilter) and concentrated 5--10 x on a PM-10 m e m b r a n e The antigen was stored at - 7 0 ° C until used. Cercarml enzymes (preacetabular gland secretions) (CEI were collected according to Stirewalt a n d Austin (1973) After incubation of cercariae for l hr in the presence of
a lipid coated membrane, the water from each chamber was pooled and lyophfllzed A cercarial homogenate (CH) was prepared by grinding 3000-5000 cercanae in 1 ml HBSS in a Ten Broeck tissue homogemzer chilled in an Ice bath The homogenate was stirred at 4~C for 18hr and then centrifuged (10,000g for 30 min at 4:C) The supernatant was removed and stored at - 7 0 : C Soluble egg antigens (SEA) were prepared from schlstosome eggs obtained from the livers of infected mice, as described by Bores and Warren 11970} Eggs were homogenlzed a n d the debris removed by centrlfugation (100,000g for 2hr) The supernatant (SEA} was stored at 70°C -
Animals White, male r a n d o m bred New Zealand rabbits, weighlng 2000-3000lg, and female albino Sprague-Dawley rats bred at N M R I were used throughout the study In one experiment the ACI strain of rats (Microbiological Associates, Bethesda, M d ) was used
Antlsera Hypertmmune rabbit ann-sclustosome sera (RaSch) Rabbits received intramuscular injections of 5 mg of the schlstosome freeze thaw antigen fraction FT 1 emulsified in complete Freund's adjuvant at 7-day intervals The rabbits were bled (20ml) 7 days after every third injection A rest per~od of 4 weeks was allowed between each series of three rejections The sera were stored at - 2 0 ' C Reagnnc sera from infected rats Rats (180-200g) were infected w~th 2000 cercarlae by the method of Preston et al (1972} The rats were anesthetized with Inovar (0 36 mg per g body weight, subcutaneously} and then fitted with a 2-cm diameter glass cyhnder attached to the shaved a b d o m e n with v a c u u m grease to confine a water suspension with 2000 cercariae to a single area for skin penetration The cercarial suspensions were allowed to remain on the skin for 30 min The rats were bled (2ml) on day 28 and the serum from Individual rats frozen and stored at - 2 0 ° C One week later the rats were again challenged with approximately 2000 cercarlae, this time by allowing the rats to wade in a small tank containing cercarlae in water The rats were bled out l week after the second refection Reagmlc sera from lmmumzed rats Rats weighing 200250gin received subcutaneous injections m the inguinal region with 0 1 - 1 0 ~ g of various adult schlstosome antigen fractions Bordetella pertussts vaccine (2 x 10 l° organisms) was injected tntrapentoneally at the time of immunization The animals were bled 14 days after reJection and the sera were stored at - 2 0 : C Immunologw methods Passive cutaneous anaphylaxts (PCA) Normal nonimm u m z e d rats (160-180gin)were injected Intradermally in a shaved area on the back with 0 I ml volumes of various serum dilutions containing reaglnic antibodies. The animals were challenged 48 hr later by intravenous mjectlon of 1 5 m g of F'I" l, unless otherwise mdlcated, m 0 5 m l of 0 500 Evans blue dye (General Diagnostics, New Jersey) The results were expressed as the diameters m millimeters of the blue skin reactions measured after 30 min In some instances, a Prausnitz-Kiastner (P-K) type reaction was used m which the antigen was injected intradermally after a 48 hr latent period rote the passively sensitized skin sites (Hussain et al, 1972) Controls consisted of skin sites prepared with normal rat serum a n d challenged with an identical a m o u n t of antigen P-K assays were always done m duphcate or whenever possible quadruplicate The results are expressed as the mean difference in diameter of two or four rephcate tests The variation between tests was never greater than 2 0 m m lmmunoelectrophores~s (IEP) The method was carried out on microscope shdes with 1% agarose (Indiblose
Hypersensitivity to Schlstosoma Mansom Antigens T a b l e 2,
A 45, Fisher Scientific Co.) with barbital buffer 0 05 M, pH 8 2 The slides were washed and stained with Ponceau S.
Btochemical methods Polyacrylamlde gel electrophorests. Disc gel electrophoresls was carried out as previously described (Vanmer et al, 1974) The gels were stained for l hr with a I% w/v solution of naphthol blue black (Eastman Kodak Co, C I 20407) m 790 v'v acetic acid to visuahze the protein components Destammg was carried out with frequent changes of 7°0 acetic acid solution The Schlff penodate method (Zacharms and Zell, 1969) was used to detect carbohydrates and glycoprotems in the acrylamide d~scs Chemical analyses Protein determmatlons were performed according to Lowry et al (1951) using a bovine plasma albumin (Armour) standard and by measuring absorbance at 280nm Nitrogen analyses were carried out by digestion with sulfuric acid and hydrogen peroxide followed by Nesslerizatlon (Campbell et al, 1970) The nitrogen values were multsplled by a factor of 6'25 to estimate the protein concentration The carbohydrate content was estimated by the lndole-sulfurlc acid method of Dlsche (1955) using galactose as a standard Nucleic acid analyses were carried out after protein precipitation with cold 10~o w/v tnchloroacetic acid and heating (15 ram, 90~C) and extraction with 5% w/v trlchloroacetlc acid by the method of Schneider (1957). Rlbonuclelc acid was estimated by the orcmol method (Schneider, 1957) and deoxyribonucleic acid by reaction with diphenylamlne (Kabat, 1961) The content of combusuble solids was determined by evaporating 0-5 ml of samples of FT 1 solution in preheated crucibles and drying in a desiccator over silica gel The crucibles were heated to a constant weight in an oven (95-100°C) and then the weight loss measured after heating at 700~C for 15-20 mm RESULTS
Production of reagimc anttbodies by mfectton Both ACI and Sprague-Dawley rats, after sChlstosome infections, produced detectable levels of reagmic antibody which was reactive against antigen FT 1, as shown m Table 1. ACI rats showed a slightly better response than the Sprague-Dawley rats after a primary infection; however, both strains showed Table
I
P r o d u c t i o n o f R e a g l n l c A n t i b o d y In Rats 4 Weeks after
Primary I n f e c t i o n
and I ~eek a ~ e r
ReinfecTion with S
4 Week B l e e d i n g
I n f e c t e d w i t h S. m a n i o n i
PCX D ~ a m t e r
l~sginie Serum #b
FT 1
FT 1-6
10
1O
0
7
iO
I0
~
5
ACI 1O
IO
I0
+
8
ACI
2
15
15
5
17
$D
1
0
8
0
0
SD
4
IO
15
5
I0
aone a8 o f e a c h a n t i s e n y e s i n j e c t e d EYans b l u e dye.
PCA r e l c t l o n
~ch
Productzon of reagmtc antibodies by zmmuntzatzon with adult anttgens The adult antigen fractions were also tested for their abd~ty to reduce reaginic anUbodles when injected into rats with B pertussis vaccine. The results are shown m Table 3. All the antigen preparations PCA R e a c t i v i t y
o f A n t i g e n P r e p a r a t i o n s Testes w i t h
Rats Immunized w i t h A d u l t A n t i g e n s
5 Week B l e e d i n g
32
]
16
32
5
16
64
7
5
16
9
5
32
10
5
32
I
0
32
2
5
64
0
FT f-6
5
32
5
0
64
o f 0 . 5 p e r c e n t Evans b l u e dye
NO c f PUS a
Dose
Total
Mean PCA T l t e r b
I 0 mg
8/12
31
0 5 rng
7/8
28
Og2 mg
O"3
< 15
O [ mg
0/3
12. pp 561-567
Pergamon Press
Prmted m Great Elrltalll
HYPERSENSITIVITY TO SCHISTOSOMA MANSONI A N T I G E N S - - I : I M M U N O C H E M I C A L A N D BIOLOGICAL CHARACTERIZATION OF AN ANTIGENIC EXTRACT* RABIA H U S S A I N , W I L T O N E. V A N N I E R a n d K DARWIN MURRELL Naval Medical Research Institute, Nahonal Naval Medical Center, Bethesda, Maryland 20014. U S A (Recewed 9 September 1974)
Abstract--An adult Schistosoma mansoni extract (FTI) was obtained in good yield (1000mg per 100,000 worms) after a single freezing and thawing cycle. This fractlon was highly reactwe by both Prausmtz-Kustner (P-K) and passive cutaneous anaphylaxts (PCA) tests with sera from schlstosome infected rats Adult, cercarlal and egg antigens were also tested by the P-K assay In general, adult antigens were more reactwe than cercanal and soluble egg antigens. Rats produced detectable levels of reagm~c antibody to FT 1 within 4 weeks after imtml mfect~on and showed a clear anamnestlc response after reinfection The ability of various antigen preparations to induce reaglnlC antibodies m rats was also studied. The FT 1 preparation was found to be composed of 10 per cent carbohydrate, 90 per cent protein and a trace of rlbonuclelc acid The fractton contained materials with a broad range of electrophoretlc moblhtles as shown by both disc gel electrophoresls and lmmunoelectrophorests
INTRODUCI'ION It has long been k n o w n that helminth infestations are associated with various manifestations of atopic sensitization such as urticaria, eosinophilia a n d elevated levels of IgE It is t h o u g h t that the sine a n d complexity of h e l m l n t h parasites a n d their intimate contact with the mucosa of the respiratory a n d gastrolntestinal tracts during the course of parasitizatlon are partly responsible for their almost universal capablhty to bring a b o u t reagln mediated hypersensitivity (Sadun, 1972). One of the major p r o b l e m s encountered in the study of h e l m i n t h antigens which induce these responses is the complexity a n d heterogeneity of antigenic extracts. M a r k e d differences have been n o t e d depending o n the extraction procedure used. Very few helminth antigens have been isolated in a highly purified form. T h e allergen from Ascarls suum has been extensively purified a n d charactermed (Hussaln et al, 1973; Ambler et al, 1974) An allergen from Nippostronyylus braslhensls has been partially purified (Ambler a n d Orr, 1972). A series of fractionatlon experiments wlth extracts of S j a p o m c u m have been carrled out a n d a n antigen fraction Isolated, which was
highly specific in skin tests in patients with sehlstosomlasis (Williams et al., 1965; Sato et al., 1969). Harris (1973) obtained fractions from adult S. mansonl by extraction with borate saline a n d precipitation at p H 4.6. Their gel filtration studies suggested t h a t the allergens of S mansonl were of a t least two types One was a glycoproteln of mol. wt 150,000 which precipitated at p H 4.6 a n d the other a glycoprotein of mol. wt 20,000-30,000 soluble at this p H We have shown that a readily soluble fraction was obtained in good yield f-om adult schistosomes by freezing a n d thawing (Vannler et al., 1974). This fraction was active In the Prausnitz-Kiistner ( P - K ) assay with sera from schlstosome infected rats a n d was able to induce the formation of reaginlc antibodies o n immunization with alum a n d B. pertussts vaccine. The present investigation was carried out in order to define the nature of antigenic extracts from adult SChlstosomes in terms of their immunologic activity a n d chemical composition. The most active materials will be used for purification a n d characterization studies of S mansoni allergens.
MATERIALS AND METHODS Buffers Borate buffered saline, pH 7 9 was prepared with 2'2 g boric acid, 0'2 g sodium hydroxide, 9 3 g sodium chloride and water to make I1 of solution
* Supported by the Bureau of Medicine and Surgery Work Unit Numbers MR011.01 01 1077, MF51.524009.0027BF6I; MR041.0901.0129B6GJ and ONR Contract Number N00014-70-C-0331 to the American Foundation for Biologic Research, Rockvdle, Maryland The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large The ammals used in this study were handled in accordance with the provisions of Pubhc Law 89-54 as amended by Pubhc Law 91-579, the 'Ammal Welfare Act of 1970" and the principles outhned in the "Guide for the Care and Use of Laboratory Animals,' U.S Department of Health, Education. and Welfare Pubhcation No (NIH) 73-23
Antu3ens Collection of adult S. mansom The S mansom hfe cycle was maintained with infected snails as described previously (Stlrewalt and Uy, 1969) NIH/NMRI Swiss mice (1520 g) of either sex were infected with 200 S mansom cercarlae from infected snails by allowing each mouse to wade for I hr in a quart jar with 30ml of water containing cercarla~ The adult worms were harvested from mice after 6-8 weeks' infection by perfuslon of the hepatic portal system with citrate buffered saline, 1 5)o w/v sodium citrate
561
562
R HUSSAIN, W E V A N N I E R a n d K D M U R R E L L
and 085% sodium chloride, by the method of Smithers a n d Terry (1965) W o r m s collected in this fashion were frozen In a small volume of citrate saline at - 2 0 = C
Preparatton of adult schzstosome freeze thaw antigen Adult worms collected over a period of several weeks were allowed to thaw over a period of 48 hr at 4°C m a small volume of borate saline The suspension was filtered through a sintered glass funnel (coarse porosity} The filtrate was designated FT l and was concentrated by Amicon ultrafiltration (UM-2 membrane, Amicon Corporauon, Lexington, Mass ) to one-tenth of its original volume and exhaustively dialyzed against borate saline (pH 8 4) The residue was then used to prepare FT 1-6 A 10-ml volume of saline was added to each 1 ml of packed worms The suspension was then frozen and thawed six times. Between each cycle of freezing a n d thawing the w o r m s were centrifuged at 3500g for 20 mIn, the supernatant removed a n d the worms resuspended in the initial volume of sahne The pooled supernatants were concentrated by ultrafiltration and dialyzed against borate saline Before use it was centrifuged for 30 m m at 10,000 g to remove any preopltated material French press anttoen I and 2 Pooled worm residues obtained after FT l preparation were also extracted after disruption by a French pressure cell A volume of 5 ml of packed worms was h o m o g e m z e d with a fine Ten Broeck tissue grinder m a volume of 50 ml until a clear suspension was obtained The homogenate was allowed to settle for 18hr at 4°C The clear supernatant was removed from the debris that settled out. The supernatant was then passed through an A m m c o French pressure cell (American Instrument C o , Silver Spring, Md.) at a pressure of 15,000 psi The effluent was centrifuged at a speed of 10,000 g for l hr and the clear supernatant dmlyzed against borate sahne The soluble products were designated French press 1 a n d 2. The French press I fraction was extracted lrom lyophlhzed worms while the French press 2 materml was prepared from freshly thawed worms Adult excretor)~secretory ant~den, cercarml antigen and egg anttgen Adult excretory-secretory antigens from adult w o r m s were obtained by an m vitro culture method (Murrell a n d Clay, 1972). W o r m s were perfused from mice with citrate sahne, washed six times with NCTC-135 (Gibco, G r a n d Island, N Y ) and placed in culture flasks (40-50 worms/flask) containing NCTC-135 supplemented with pemcilhn (200 units/m1) a n d streptomycin (200 #g/ml) The cultures were incubated 3 6 - 7 8 h r at 37~C in 95 per cent a m 5 per cent CO2 The culture fluids were pooled, filtered (0.45/~ microfilter), concentrated, a n d dialyzed in the cold against borate-buffered saline (pH 7.8) by ultrafiltratlon a n d pressure dialysis through a P M - l 0 m e m b r a n e (Amicon Corporauon, Lexington, M a s s ) The concentrated antigen was centrifuged at 10,000g (4~C) for I hr and the supernatant (CA) stored at - 7 0 ° C untd used A second procedure was also employed Fresh live w o r m s were washed s~x u m e s in Hanks' balanced salt solution (HBSS), suspended m HBSS containing penicflhn (200 unlts/ml) and streptomycin (200 #g/ml) The majority of adult worms, at the end of the incubation period, are still motile on warming to 37°C The supernatant was filtered ( 0 4 5 # microfilter) concentrated, (PM- l0 membrane) a n d centrifuged ( 10,000 g at 4~C for lhr) The supernatant was designated A x e and stored at - 7 0 ~ C A cercarial exoantigen (CXO) was prepared by a modification of a procedure described by Fife et al (1967) Cercariae were shed Into dechlorinated tap water (1000-5000/ ml) and were held at 4°C for 5-7 days The supernatant was filtered C045 # microfilter) and concentrated 5--10 x on a PM-10 m e m b r a n e The antigen was stored at - 7 0 ° C until used. Cercarml enzymes (preacetabular gland secretions) (CEI were collected according to Stirewalt a n d Austin (1973) After incubation of cercariae for l hr in the presence of
a lipid coated membrane, the water from each chamber was pooled and lyophfllzed A cercarial homogenate (CH) was prepared by grinding 3000-5000 cercanae in 1 ml HBSS in a Ten Broeck tissue homogemzer chilled in an Ice bath The homogenate was stirred at 4~C for 18hr and then centrifuged (10,000g for 30 min at 4:C) The supernatant was removed and stored at - 7 0 : C Soluble egg antigens (SEA) were prepared from schlstosome eggs obtained from the livers of infected mice, as described by Bores and Warren 11970} Eggs were homogenlzed a n d the debris removed by centrlfugation (100,000g for 2hr) The supernatant (SEA} was stored at 70°C -
Animals White, male r a n d o m bred New Zealand rabbits, weighlng 2000-3000lg, and female albino Sprague-Dawley rats bred at N M R I were used throughout the study In one experiment the ACI strain of rats (Microbiological Associates, Bethesda, M d ) was used
Antlsera Hypertmmune rabbit ann-sclustosome sera (RaSch) Rabbits received intramuscular injections of 5 mg of the schlstosome freeze thaw antigen fraction FT 1 emulsified in complete Freund's adjuvant at 7-day intervals The rabbits were bled (20ml) 7 days after every third injection A rest per~od of 4 weeks was allowed between each series of three rejections The sera were stored at - 2 0 ' C Reagnnc sera from infected rats Rats (180-200g) were infected w~th 2000 cercarlae by the method of Preston et al (1972} The rats were anesthetized with Inovar (0 36 mg per g body weight, subcutaneously} and then fitted with a 2-cm diameter glass cyhnder attached to the shaved a b d o m e n with v a c u u m grease to confine a water suspension with 2000 cercariae to a single area for skin penetration The cercarial suspensions were allowed to remain on the skin for 30 min The rats were bled (2ml) on day 28 and the serum from Individual rats frozen and stored at - 2 0 ° C One week later the rats were again challenged with approximately 2000 cercarlae, this time by allowing the rats to wade in a small tank containing cercarlae in water The rats were bled out l week after the second refection Reagmlc sera from lmmumzed rats Rats weighing 200250gin received subcutaneous injections m the inguinal region with 0 1 - 1 0 ~ g of various adult schlstosome antigen fractions Bordetella pertussts vaccine (2 x 10 l° organisms) was injected tntrapentoneally at the time of immunization The animals were bled 14 days after reJection and the sera were stored at - 2 0 : C Immunologw methods Passive cutaneous anaphylaxts (PCA) Normal nonimm u m z e d rats (160-180gin)were injected Intradermally in a shaved area on the back with 0 I ml volumes of various serum dilutions containing reaglnic antibodies. The animals were challenged 48 hr later by intravenous mjectlon of 1 5 m g of F'I" l, unless otherwise mdlcated, m 0 5 m l of 0 500 Evans blue dye (General Diagnostics, New Jersey) The results were expressed as the diameters m millimeters of the blue skin reactions measured after 30 min In some instances, a Prausnitz-Kiastner (P-K) type reaction was used m which the antigen was injected intradermally after a 48 hr latent period rote the passively sensitized skin sites (Hussain et al, 1972) Controls consisted of skin sites prepared with normal rat serum a n d challenged with an identical a m o u n t of antigen P-K assays were always done m duphcate or whenever possible quadruplicate The results are expressed as the mean difference in diameter of two or four rephcate tests The variation between tests was never greater than 2 0 m m lmmunoelectrophores~s (IEP) The method was carried out on microscope shdes with 1% agarose (Indiblose
Hypersensitivity to Schlstosoma Mansom Antigens T a b l e 2,
A 45, Fisher Scientific Co.) with barbital buffer 0 05 M, pH 8 2 The slides were washed and stained with Ponceau S.
Btochemical methods Polyacrylamlde gel electrophorests. Disc gel electrophoresls was carried out as previously described (Vanmer et al, 1974) The gels were stained for l hr with a I% w/v solution of naphthol blue black (Eastman Kodak Co, C I 20407) m 790 v'v acetic acid to visuahze the protein components Destammg was carried out with frequent changes of 7°0 acetic acid solution The Schlff penodate method (Zacharms and Zell, 1969) was used to detect carbohydrates and glycoprotems in the acrylamide d~scs Chemical analyses Protein determmatlons were performed according to Lowry et al (1951) using a bovine plasma albumin (Armour) standard and by measuring absorbance at 280nm Nitrogen analyses were carried out by digestion with sulfuric acid and hydrogen peroxide followed by Nesslerizatlon (Campbell et al, 1970) The nitrogen values were multsplled by a factor of 6'25 to estimate the protein concentration The carbohydrate content was estimated by the lndole-sulfurlc acid method of Dlsche (1955) using galactose as a standard Nucleic acid analyses were carried out after protein precipitation with cold 10~o w/v tnchloroacetic acid and heating (15 ram, 90~C) and extraction with 5% w/v trlchloroacetlc acid by the method of Schneider (1957). Rlbonuclelc acid was estimated by the orcmol method (Schneider, 1957) and deoxyribonucleic acid by reaction with diphenylamlne (Kabat, 1961) The content of combusuble solids was determined by evaporating 0-5 ml of samples of FT 1 solution in preheated crucibles and drying in a desiccator over silica gel The crucibles were heated to a constant weight in an oven (95-100°C) and then the weight loss measured after heating at 700~C for 15-20 mm RESULTS
Production of reagimc anttbodies by mfectton Both ACI and Sprague-Dawley rats, after sChlstosome infections, produced detectable levels of reagmic antibody which was reactive against antigen FT 1, as shown m Table 1. ACI rats showed a slightly better response than the Sprague-Dawley rats after a primary infection; however, both strains showed Table
I
P r o d u c t i o n o f R e a g l n l c A n t i b o d y In Rats 4 Weeks after
Primary I n f e c t i o n
and I ~eek a ~ e r
ReinfecTion with S
4 Week B l e e d i n g
I n f e c t e d w i t h S. m a n i o n i
PCX D ~ a m t e r
l~sginie Serum #b
FT 1
FT 1-6
10
1O
0
7
iO
I0
~
5
ACI 1O
IO
I0
+
8
ACI
2
15
15
5
17
$D
1
0
8
0
0
SD
4
IO
15
5
I0
aone a8 o f e a c h a n t i s e n y e s i n j e c t e d EYans b l u e dye.
PCA r e l c t l o n
~ch
Productzon of reagmtc antibodies by zmmuntzatzon with adult anttgens The adult antigen fractions were also tested for their abd~ty to reduce reaginic anUbodles when injected into rats with B pertussis vaccine. The results are shown m Table 3. All the antigen preparations PCA R e a c t i v i t y
o f A n t i g e n P r e p a r a t i o n s Testes w i t h
Rats Immunized w i t h A d u l t A n t i g e n s
5 Week B l e e d i n g
32
]
16
32
5
16
64
7
5
16
9
5
32
10
5
32
I
0
32
2
5
64
0
FT f-6
5
32
5
0
64
o f 0 . 5 p e r c e n t Evans b l u e dye
NO c f PUS a
Dose
Total
Mean PCA T l t e r b
I 0 mg
8/12
31
0 5 rng
7/8
28
Og2 mg
O"3
< 15
O [ mg
0/3
