Schistosomamansoni gene GP22 encodes the tegumental antigen Sm25: (1) antibodies to a predicted...

Schistosoma mansoni gene GP22 encodes the tegumental antigen

Sm25: (1) antibodies to a predicted B-cell epitope of Sm25

cross-react with other candidate vaccine worm antigens;

(2) characterization of a recombinant product containing

tandem-repeats of this peptide as a vaccine

MARY M. PETZKE1, PARMJEET K. SURI1, RICHARD BUNGIRO1, MIRIAM GOLDBERG1, SARAH F. TAYLOR1,

SUMANT RANJI1, HARRY TAYLOR2, JOSEPH W.MCCRAY JR2 & PAUL M. KNOPF1

1Department of Molecular Microbiology and Immunology, Brown University, Providence, RI 02912, 2Department of Biology,

Morehouse College, Atlanta, GA, USA

SUMMARY

Monospeci®c antibodies against two putative epitopes of

schistosome protein encoded by gene GP22 (182 codons, no

introns) were used to probe worm extracts fractionated by

lentil-lectin af®nity chromatography or by electrophoresis.

Anti-peptide-a (codons 70±84) exclusively identi®es the N-

glycanase-sensitive, 25 kDa tegumental glycoprotein Sm25

in the lectin-bound fraction of detergent-solubilized adult

worm extract S3. In contrast, antipeptide-d (codons 151±

162) does not react with Sm25 but cross-reacts with other

schistosome proteins, including candidate vaccine antigens

paramyosin (Sm97) and glutathione-S-transferases (Sm26,

Sm28, Sj26). Recombinant protein r4 ´ 47, constructed to

express multiple copies of codon sequence 117±163 (con-

taining d), reacts with anti-d and is uniquely recognized by

protective Fischer twice-infected (F-2x) rat antiserum.

Immunization with r4 ´ 47 induces antibodies with cross

reactivities similar to anti-d, but which also recognize Sm25.

Despite these cross-reactivities with protective antigens,

rodents vaccinated with r4 ´ 47 were not protected against

cercarial infection. On the basis of these data, two hypoth-

eses are proposed: (1) antigenic epitopes other than d are

present within the r4 ´ 47 sequence which induce antibodies

reactive with Sm25 and/or (2) peptide-d assumes alternative

antigenic conformations, dependent upon the context of

neighbouring sequences, some of which mimic epitopes of

proteins encoded by other schistosome genes. These mimo-

topes are not targets of protective antibodies.

Keywords Schistosoma mansoni, recombinant antigen,

cross-reacting antigens, Sm25, expression vector pET15b,

vaccine

INTRODUCTION

S. mansoni gene GP22 was identi®ed in our laboratory

(El-Sherbeini et al. 1991) as an intronless genomic DNA

sequence which, with the exception of a single amino acid

difference (one nucleotide position1), was identical to the

cDNA sequence of the previously described tegumental

glycoprotein, Sm25 (Omer-Ali et al. 1991). Each laboratory

independently isolated cDNA clones (from different lgt11

expression libraries), containing the same 47-amino acid

insert, subsequently shown to be amino acid positions 117±

163 in the translated sequence. The two laboratories used

different screening strategies to identify candidate vaccine

antigens of the parasite. Our procedure involved screening the

cDNA expression library by the `contrasting antiserum'

protocol. The 47-amino acid schistosome sequence within

the b-galactosidase fusion protein reacted with antibodies

uniquely present in protective F-2x antiserum (from twice-

infected Fischer rats) but not with antibodies in nonprotective

immune serum W-2x (from twice-infected Wistar±Furth

rats) (El-Sherbeini et al. 1990).

Parasite Immunology, 2000: 22: 381±395

q 2000 Blackwell Science Ltd 381

Correspondence: Paul M.Knopf

Received: 13 July 1999

Accepted for publication: 23 March 2000

1 Nucleotide in position 143 was G in our sequence and C in the Mill

Hill sequence, leading to an ATG (met) versus ATC (ile) in amino acid

position 47. We have since learned of the sequence of another

independently isolated cDNA of this gene, which con®rms the G in

position 143 (Mangold B.L. & Ricardoni M., NAMRU-3, Cairo, Egypt).

However, different parasite isolates may be allelic in gene GP22.

Because of its unique immunoreactivity with antibodies

in protective serum F-2x, we decided to test the possibility

that epitopes in this sequence were targets of the protective

humoral immune response in rats. We also tested the vacci-

nation strategy in mice since Smithers et al. (1989), using

tegumental fractions of the worms, showed a correlation of

humoral anti-Sm25 activity with protection in this model.

Moreover, to improve on the immunogenicity of this

47-amino acid segment, we engineered the construction of

a tandem display of this sequence. As a screening reagent to

identify the expressed product, we developed a rabbit

polyclonal, monospeci®c antipeptide antibody against the

predicted B-cell epitope delta (d, amino acids 151±162)

present in the 47-amino acid sequence.

In these early vector expression experiments, utilizing

the pGEX vector to express gene GP22 sequence as fusion

protein with Sj26 (glutathione-S-transferase, GST), an

unexpected result emerged which stimulated our interest in

pursuing this direction of vaccine development. Anti-d anti-

bodies reacted very strongly by enzyme-linked immunosor-

bant assay (ELISA) with the puri®ed GST vector-only

control product. Not only was this cross-reactivity with

GST revealed, but immunoblotting assays revealed a strik-

ing reactivity of the anti-d with other S. mansoni macro-

molecules, the strongest of which was con®rmed to be Sm97

(paramyosin). Since both GST and paramyosin were con-

sidered prominent candidate vaccine antigens (Riveau &

Capron 1997), we proposed the following hypothesis: Gene

GP22-encoded glycoprotein products contain a short amino

acid segment capable of inducing humoral immune responses

which are cross-reactive with other antigens of the parasite.

This cross-reaction thereby expands the range of available

targets for the parasite-induced immune response, which

protects the host from becoming heavily reinfected and

vulnerable to the severe pathology of this disease.

The cross-reactivities of anti-d antibodies and of anti-

bodies to the tandem construct of the 47 amino acid sequence,

r4 ´ 47, are described, together with the results of experi-

ments using r4 ´ 47 as a vaccine in rats and mice. In addi-

tion, we report on the use of anti-d antibodies and antibodies

to alpha (a, amino acids 70±84), another predicted peptide

epitope for B cells, to characterize the expressed products of

gene GP22 in adult worms.

MATERIALS AND METHODS

Parasites

Biomphalaria glabrata snails infected with the Puerto Rican

strain of S. mansoni were provided under an NIAID supply

contract (AI052590) by Biomedical Research Institute,

Rockville, MD, USA. Juvenile or adult worms were isolated

from outbred mice exposed via the tail, 4 or 7 weeks earlier,

to 600 or 200 cercariae, respectively (Knopf et al. 1977).

Animals

Male Fischer rats (CD-F, 80±100 g) were obtained from

Charles River Breeding Laboratories (Wilmington, MA,

USA); male Wistar±Furth rats (80±100 g) were from M.

A. Laboratories (Bethesda, MD, USA). Female Balb/c mice

(23±25 g) were from Charles River. Female New Zealand

White rabbits were from Millbrook Farm (Amherst, MA,

USA).

Cloning and expression of recombinant proteins

r4 ´ 47 and r140

Restriction enzymes (New England Biolabs, Beverly, MA,

USA) were used according to manufacturer's instructions.

pET(4 ´ 47) was constructed by a series of steps utilizing

two vector systems. The 141 bp insert (codons 117±163 of

S. mansoni gene GP22, see Figure 4) was excised from the

pUC-19 cDNA clone (El-Sherbeini et al. 1991) by EcoRI

digestion and puri®ed (Maniatis et al. 1982). The insert, in

10-fold molar excess, was ligated into the EcoRI site of

pMAL-c2 vector (New England Biolabs) and ampicillin-

resistant transformants selected. Orientation of inserts in

transformants containing double inserts was con®rmed by

a mini-expression analysis of fusion proteins by sodium

dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-

PAGE) (Protein Fusion and Puri®cation System, New

England Biolabs). A clone, termed pMAL(2 ´ 47), contain-

ing two 141 bp inserts ligated in tandem (con®rmed by

sequencing), was identi®ed. Note that the EcoRI site joining

the inserts is a shared region, with a resulting sequence of

276 bp instead of 282 bp. Translation of this tandem insert

yields 92 amino acids instead of 94 a.a. (the C-terminal

glu-phe of the 50-insert is also the N-terminal glu-phe of the

30-insert). However, as a convenience of nomenclature, we

refer to this tandem amino acid sequence as 2 ´ 47.

pMAL(4 ´ 47), containing two copies of the tandem

insert, was then constructed. It consists of two 276 bp

sequences separated by ®ve codons of linker sequence

(veri®ed by DNA sequencing). This double tandem insert

was excised by cleavage with XmnI and BamHI and cloned,

utilizing a BglII linker sequence to maintain the correct

reading frame, into the pET-15b vector (Novagen, Madison,

WI, USA) to generate pET(4 ´ 47). The translated product

of pET(4 ´ 47), termed r4 ´ 47, is composed of two worm-

derived tandem sequences separated by a 5-amino acid linker,

joined at the amino terminal to a 23-amino acid `His.Tag'

vector sequence (containing six histidine residues) and a

4-amino acid linker, and followed by a 5-residue vector

M.M.Petzke et al. Parasite Immunology

q 2000 Blackwell Science Ltd, Parasite Immunology, 22, 381±395382

sequence terminating with cysteine (see Figure 4). Recom-

binant protein r4 ´ 47 was expressed and puri®ed from the

soluble fraction of sonicated BL21(DE3)pLysS cells

(Novagen) by nickel-af®nity chromatography on `His.Bind'

resin, according to manufacturer's protocol. Eluted protein

was dialysed overnight at 48C against phosphate-buffered

saline (PBS)/10% glycerol/0´01% Tween-20 using a 3´5 kDa

molecular weight cutoff dialysis tubing. The solution was

concentrated using polyethylene glycol (15 000±20 000 kDa;

Sigma Chemical Co., St Louis, MO, USA) and protein

concentration established by OD at 280 nm. In one study,

r4 ´ 47 was further puri®ed by size-fractionation chromato-

graphy over Sephacryl-200 (Sigma) and called sp-r4 ´ 47.

Vector pGEX-3X (Pharmacia Biotech, Uppsala, Sweden)

was expressed in DH5a cells. Construction of pET(140) and

expression/puri®cation of r140 (GP22 codons 43±182) is

described elsewhere (Suri et al. 1997).

Synthetic peptides

The sequences are as follows (see also Figure 4): Peptide

alpha (a): (70)NSEENSNSIITDEDY(84)GGC; peptide delta

(d): (151)TPNSANKYMKNE(162)GGC.

Peptides corresponding to codons 70±84 (a sequence)

and 151±162 (d sequence), plus a linker sequence of three

residues at the carboxy termini (GGC, single letter amino

acid code) were synthesized by Multiple Peptide Systems

(San Diego, CA, USA). For immunizations, the peptides

were each coupled to an immunogenic protein carrier via

b-maleimidopropionic acid-N-hydroxysuccinimide ester

(Sigma) linkage (McCray & Werner 1989). Further details

are given below for the peptide-carrier constructs used in the

immunization of rabbits and the puri®cation of antipeptide

antibodies by af®nity chromatography.

Other antigens and worm fractions

Recombinant S. japonicum 26 kDa GST (rSj26-GST) was

puri®ed by glutathione agarose chromatography (Torian

et al. 1990) from DH5a Escherichia coli transformed with

pGEX-3X vector. Recombinant S. mansoni 26-kDa GST

(rSm26-GST) and rSm28-GST were kindly provided by

Dr B. Doughty of Texas A & M University. Recombinant

and worm-puri®ed Sm97-paramyosin were gifts from Dr

C. Shoemaker of Harvard University and S. Heiny in Dr A.

Sher's laboratory at the NIH, respectively.

The aqueous-soluble worm fraction (S2) and the deter-

gent-solubilized worm fraction (S3) extracted from the

residual aqueous-insoluble pellet were prepared as described

(Suri et al. 1997). Protein concentrations were determined

against a bovine serum albumin standard using Bradford's

Reagent (BioRad Laboratories, Hercules, CA, USA).

Aliquots containing 2 mM phenylmethylsulphonyl ¯uoride

(PMSF) (ICN Pharmaceuticals, Inc., Costa Mesa, CA, USA)

were stored at ÿ808C.

Biosynthetically radiolabelled S2 and S3 fractions were

prepared from 4-week worms incubated in culture medium

containing [35S]-methionine (Mark et al. 1991).

Sera and antibodies

Fischer twice-infected rat serum (F-2x) was obtained

3 weeks after the second infection with S. mansoni cercariae

(Barker et al. 1985). Wistar-Furth twice-infected rat serum

(W-2x) was raised following a similar protocol.

Antisera to recombinant proteins (anti-r4 ´ 47 and anti-

r140) were prepared by immunizing Fischer rats subcuta-

neously (s.c.) with nickle-af®nity puri®ed r4 ´ 47 or r140,

emulsi®ed 1 : 1 in alum (aluminium hydroxide adsorptive

gel, Rehsorptar, Intergen Company, Purchase, NY, USA).

Rabbits were injected intradermally at intervals of 4±6

weeks with 1 mg antigen mixed 1 : 1 with alum in 1 ml.

Blood was collected 2 weeks after each injection by retro-

orbital bleeding (rats) or puncture of the ear vein (rabbits).

Serum samples with the highest ELISA antibody titres were

used.

Monospeci®c antipeptide antibodies were puri®ed from

rabbit antisera by peptide-af®nity chromatography. Rabbits

were injected s.c. at multiple sites on the back with 0´2 mg of

peptide-carrier protein conjugate mixed 1 : 1 with Freund's

complete adjuvant, in a total volume of 1 ml. A booster

injection was given s.c. 3 weeks later in Freund's incom-

plete adjuvant and repeated at intervals. For immunization

with peptide-d-conjugate, the rabbits were initially immun-

ized with an ovalbumin (OVA) conjugate, four times. We

then switched to a keyhole limpet haemocyanin (KLH)

conjugate of peptide-d when the last two boosters with

OVA±conjugate failed to increase the antipeptide titre. No

differences in the speci®cities to the antigens tested were

detectable using peptide-af®nity-puri®ed antibodies from

early or later sera from these rabbits (see Discussion).

For immunization with peptide-a-conjugate, the rabbits

were always immunized with a KLH conjugate. Serum

was prepared from blood collected 7±21 days after boost-

ing. To purify antipeptide antibodies, the peptides were

coupled to Af®-Gel 102 (BioRad) by a previously published

method (McCray & Werner 1989). Sera (5 ml) were diluted

1 : 1 with PBS and loaded onto small (3±4 ml) columns of

the peptide-Af®-Gel 102 conjugate. The columns were

washed with PBS and the bound antipeptide antibodies

were eluted with 0´1 M glycine-HCl/0´15 M NaCl buffer,

pH 2´8. Eluted fractions were collected in tubes containing

2 M Tris to raise the pH to approximately 7´0 and were tested

in an ELISA for antipeptide antibody activity. Active

Volume 22, Number 8, August 2000 Properties of antibodies to gene GP22-encoded sequences

q 2000 Blackwell Science Ltd, Parasite Immunology, 22, 381±395 383

fractions were dialysed extensively against PBS. The anti-

peptide antibodies were speci®c for the peptide used for

immunization (Petzke 1996).

Immunoassays

ELISA

This assay was utilized for preliminary determination of

the antibody-binding capacity of proteins and synthetic

peptides. Protein antigens were immobilized and antibody

titres determined as described (Suri et al. 1997). For assay-

ing peptides, each well was coated with 1 mg of synthetic

peptide, and a 1 : 5000 dilution of secondary antibody

conjugate was used (Petzke 1996).

Western immunoblots

Proteins were separated by SDS-PAGE using a Bio-Rad

Protean II vertical minigel apparatus. The gel was electro-

blotted onto nitrocellulose membrane (Hybond-ECL nitro-

cellulose, Amersham, Arlington Heights, IL, USA) using a

semidry transfer cell (BioRad) for 25 min at 10 mV. The

membrane was dried, blocked with nonfat dry milk and

incubated with primary antibody. Peroxidase-conjugated

goat anti-rat or anti-rabbit immunoglobulin heavy and

light chains was used to detect primary antibody. The

immunoblot was developed using enhanced chemilumines-

cence (ECL reagents: Amersham). Further details are pro-

vided elsewhere (Suri et al. 1997).

Competition Western immunoblots

An identical amount of protein was loaded into each well

and separated by SDS-PAGE. Following transfer to nitro-

cellulose, membranes were ®xed and proteins visualized by

Ponceau S (Sigma) staining. The membrane was cut into

strips, transferred to the wells of a mini-incubation tray

(BioRad), destained with deionized water, and blocked

overnight. Primary antibody was incubated with varying

concentrations of competitor for 1´5 h at room temperature,

transferred to a well of the mini-incubation tray and main-

tained at 378C for 1´5 h. Addition of secondary antibody

conjugate and developing are described above.

Lentil lectin sepharose chromatography of S2 and S3

Af®nity chromatography utilizing lentil lectin coupled to

beads of Sepharose 4B (Sigma) was used to adsorb and

isolate glycoproteins containing glucosyl and/or mannosyl

residues. Approximately 20 ml of lentil-lectin Sepharose 4B

suspension was packed into a glass column (2 cm ´ 22 cm,

Pharmacia) and washed with 10 bed volumes of running

buffer [9% NaCl, 10 mM CaCl2, 10 mM MnCl2, 0´1% Renex

(a nonionic detergent, Ruger Chemical Co., Irvington, NJ,

USA)]. A 2-ml volume of S2 or S3 (approximately 1´3±

1´5 mg protein/ml) was applied to the column and fractions

collected by washing with running buffer (60±80 ml) to

remove unbound sample components. The lectin-bound

components were eluted with the addition of 2´5% methyl-

a-D-mannoside (Sigma) to the running buffer. Column

¯owthrough and eluate fractions were collected assessed

for protein content by OD at 280 nm.

N-glycanase treatment of antigens

Frozen aliquots of S2 and S3 were thawed and subjected to

sonication for 10 s to resuspend particulate matter. Approxi-

mately 200 mg of each fraction containing 0´5% SDS

and 50 mM b-mercaptoethanol was denatured by boiling

5 min before addition of enzyme (3 U recombinant N-gly-

canase, Genzyme, Cambridge, MA, USA), 5 mM PMSF, and

1´1% 3-(3-cholamino-propyl)dimethylammonio-1-propane-

sulphonate (CHAPS). Reactions were for 18 h at 378C,

terminated by the addition of 5X PAGE sample buffer

followed by boiling for 5 min. Recombinant antigens r140

and r4 ´ 47 were treated in the same manner.

Immunization protocols

Three different antigens emulsi®ed 1 : 1 (v:v) in alum were

used in three separate vaccine trials with CD-F Fischer rats

(50±70 g) or female Balb/c mice (23±25 g) as indicated.

Control animals in each vaccine trial were either uninjected

or were injected with PBS emulsi®ed 1 : 1 (v:v) in alum.

Vaccine trial 1

On days 0, 24 and 51, rats received injections of 100 mg or

200 mg of nickel-af®nity-puri®ed r4 ´ 47 in a total volume of

0´2 or 0´4 ml, respectively, and mice received 75 mg in a

volume of 0´15 ml. Rats were challenged percutaneously

on day 75 by exposure of shaved abdominal skin to 520

S. mansoni cercariae (Knopf et al. 1977). Mice were

infected on the same day via tail immersion in water

containing 150 cercariae. Fluids contained < 5% residual

cercariae after 30 min exposures. Worms were collected by

portal perfusion (described below) of rats on day 103

(4 weeks postinfection) and of mice on day 124 (7 weeks

postinfection).

Vaccine trial 2

Rats were immunized s.c. on day 0 with 150 mg sp-r4 ´ 47

(Sephacryl-puri®ed r4 ´ 47), emulsi®ed in total volume

0´3 ml and were boosted on day 21 (75 mg antigen,

0´15 ml) and days 45 and 66 (150 mg antigen, 0´3 ml).

Rats were challenged percutaneously on day 76 with 500

cercariae. On day 79, one group of antigen-injected rats and

one group of PBS-injected rats (approximately 265 g)



received F-2x antiserum (0´75 ml) passively transferred by

s.c. injection. The dose of F-2x (0´25 ml/90 g rat) was

predetermined to be protective by titration but less than

the maximal amount of serum was used. Worms were

recovered by portal perfusion 4 weeks postinfection.

Vaccine trial 3

The lentil lectin-bound (LLB) material from S3 fraction of

adult worms was eluted, concentrated and used to immunize

four rats after emulsi®cation in alum. Two immuni-zations,

2 weeks apart, of 145 mg protein each of freshly prepared

lectin-bound material, was injected intramuscularly (half

into each hind leg). Two rats which yielded anti-S3 response

equivalent in titre to F-2x (positive control serum) 3 weeks

after the second injection were exsanguinated and serum

was prepared (stored at ÿ808C). The remaining two rats

were boosted again (120 mg each) and serum prepared

2 weeks later. The anti-S3 titres are given in the Results.

Rats were exposed percutaneously to 220 cercariae and on

day 3 postinfection, they received 1 ml of anti-LLB or NRS,

or 1 ml F-2x s.c. (under the scruff of the neck). On day 28

postinfection, the worm burdens in these different groups of

rats were determined after portal perfusion.

Protection assays

Worms were collected on 30 mm (rats) or 45 mm (mice)

nylon screens by perfusion of the portal-mesenteric veins

with saline (Knopf et al. 1977) and counted independently

by two researchers using a dissecting microscope and

averaged, variation < 10%. Worm pairs obtained from

mice were allowed to separate overnight, and were enum-

erated separately. The liver egg burdens were measured in

mice (Cioli et al. (1977).

RESULTS

GP22-encoded product found in S3 fraction is Sm25

glycoprotein

The amino acid sequence of gene GP22 contained two

putative N-linked glycosylation sites which could mediate

binding of the gene product(s) to lentil lectin. To character-

ize the protein product(s) of gene GP22 in adult worms, the

aqueous-soluble S2 and detergent-soluble S3 fractions were

mixed with trace amounts of the corresponding fraction

from radiolabelled worms and subjected to lentil lectin-

af®nity chromatography. The resultant chromatographic

fractions were probed for immunoreactivity by ELISA

using antiserum from twice-infected Fischer rats, F-2x (Man-

gold & Knopf 1981, Barker et al. 1985) or antiserum raised

by vaccination with GP22-encoded recombinant protein, r140

(codons 43±182) (Suri et al. 1997). In addition, radioactivity

in each column fraction was measured to monitor distribution

of proteins and glycoproteins.

The ELISA results using F-2x (Figure 1) show the

nonbound chromatographic fractions of both S2 and S3

distributing into three sharp peaks (designated 1, 2a and

2b), while the bound, mannose-containing components

distribute into a broad peak, designated peak 3. Peak 1 con-

tains residual aggregates incompletely removed by high-

speed centrifugation and excluded from the gel matrix

(> 20 000 kDa). Peak 2a represents size-fractionated material

partially excluded from the gel matrix (> 60 kDa), while

peak 2b contains all molecules < 60 kDa (con®rmed using

nonglycosylated marker proteins, data not shown). The basis



5020

2.0

1.5

60000

50000

40000

30000

20000

10000

0

1.0

0.5

0.0

OD

410

S2 lentil lectin column fraction604030100

elution

elution

13

F-2x OD

Anti-delta OD

2a

1

2a

3

2b

2b

S2 CPM

(a)

Co

un

ts per m

inu

te

50 60 7020

2.0

1.5

40000

30000

20000

10000

0

1.0

0.5

0.0

OD

410

S3 lentil lectin column fraction804030100

F-2x OD

Anti-r140 OD

Anti-alpha OD

S3 CPM

(b)

Co

un

ts per m

inu

te

Figure 1 Immunoreactivity pro®les of S. mansoni adult worm

fractions subjected to lentil-lectin af®nity chromatography. S2 (a) and

S3 (b) worm fractions from 7 to 8-week infected mice, combined

with [35S]-methionine-labelled S2 or S3 included as a marker, were

separated by af®nity chromatography and resultant column fractions

were analysed by ELISA (OD410): 100 ml aliquots of each fraction

[2´5 ml (a) or 2 ml (b)] were immobilized and probed with various

antisera, (a) with F-2x and antidelta; (b) with F-2x, anti-r140 and

antialpha. Radioactivity (counts per minute, shaded) was assayed in

other aliquots. Addition of methyl-a-D-mannoside to elute lectin-

bound material is indicated by an open arrow. Major peaks are

numbered.

for the irregular pro®le of peak 3 was not established, but

may represent differences in binding af®nity for lentil lectin

(e.g. variability in mannose residues per molecule) and/or

size-fractionation. The 35S-methionine-labelled proteins in

both fractions distributed upon chromatography (Figure 1)

into a major peak (peak 2b) and two minor peaks (peaks 1

and 3). Peak 2a contained only minimal amounts of labelled

product. For these radiolabelled products,< 5% of S2 and

approximately 10% of S3 proteins bind to the lectin; in

contrast, the relative amounts of antigen detected by F-2x

hyperinfected rat serum in the lectin-bound fractions of

S2 and S3 (Figure 1) are strikingly higher (approximately

66% and 80%, respectively). Earlier studies established

that total adult worm proteins were distributed similar to

the 35S-methionine-labelled products. We conclude that the

antigenicity of both S2 and S3 is disproportionately higher

in the lectin-bound (mannosylated) components than in the

lectin-nonbound components, when probed by antibodies in

protective F-2x.

To assess the presence of gene GP22-encoded products,

column fractions were also probed with rabbit anti-r140, the

recombinant protein containing all predicted B-cell epi-

topes. None of the chromatographic fractions of S2 showed

signi®cant reactivity with anti-r140 (data not shown), but

lectin-bound peak 3 of S3 does show strong reactivity (Figure

1b). These results establish the presence of a signi®cant

amount of GP22-encoded glycoproteins in the membrane-

associated S3 fraction.

Monospeci®c antibodies to GP22-encoded peptides

a and d identify different worm components

Computer predictions identi®ed four distinct segments of

the GP22 amino acid sequence as putative B-cell epitopes

(El-Sherbeini et al. 1991). Two of these peptide sequences,

corresponding to codons 70±84 (a sequence) and 151±162

(d sequence), were synthesized, and antisera was prepared to

protein conjugates of each in rabbits. The speci®c antipep-

tide antibodies were puri®ed by af®nity chromatography

and used to probe the S3 lectin-bound fractions. Anti-a,

similar to anti-r140, detected antigen only in the S3 bound

fraction (Figure 1b). No anti-a reactivity was observed in

the S2 bound fraction (data not shown). Surprisingly,

reactivity with anti-d was not detected with the S3 lectin-

bound fraction. Instead, its reactivity was con®ned to lectin-

nonbound peak 2, primarily peak 2a (> 60 kDa), of the S2

fraction (Figure 1a). Thus, antibodies directed against two

different peptide sequences of GP22-encoded protein

identify antigen fractions with very different characteristics

both in terms of localization in worm extracts and in the

possession of lectin-binding sugars. Anti-a, similar to anti-

r140, detects antigens in the bound glycosylated components

of S3; anti-d primarily detects antigen of > 60 kDa in the

nonbound components of S2.

To determine the sizes and con®rm the glycosylation

status of the antigenic material, peak 3 of S3 fraction was

probed with anti-a and anti-d antibodies after N-glycanase

digestion and SDS-PAGE/immunoblotting. The results

demonstrate that anti-a strongly and speci®cally reacts

with a single antigen of 25 kDa, present in both nontreated

S3 and in lectin bound peak 3 (Figure 2a). This antigen

exhibits sensitivity to N-glycanase digestion, shifting in

mobility from 25 kDa to approximately 20 kDa following

treatment. Identical results are obtained with anti-r140 (Suri



Figure 2 The 25-kDa glycoprotein encoded by gene GP22 is

detected with anti-a, but not with anti-d, ECL Western immunoblots.

(a) Samples of r140 and r4 ´ 47 (1 mg each), S2 and S3 (5 mg each)

were incubated under identical conditions with [�] or without [ÿ]

N-glycanase (N-glyc.), separated by SDS-PAGE, and antigens

detected by immunoblotting with anti-a (1 : 1000). In a separate

experiment, lentil lectin Peak 3 of S3 (de®ned in Figure 1b) was

immunoblotted with anti-a (1 : 1000). Note same shift in mobility of

anti-a-reactive antigen in S3 (open arrowheads) and peak 3 of S3.

(b) r140 (3 mg), S2 (15 mg) and S3 (10 mg [�]; 15 mg [ÿ]) were

immunoblotted with anti-d (1 : 1000). Note that mobility of 95 kDa

anti-d-reactive antigen is unaffected by N-glycanase, but 35 kDa

antigen disappears after deglycosylation (solid arrowheads). Note that

anti-d reacts strongly with r140 but not with 25 kDa antigen detected

in S3 with anti-a.

et al. 1997). These properties identify this S3 component as

Sm25, as described by Knight et al. (1989). In contrast to

anti-a, anti-d does not react with this 25 kDa protein nor

with its deglycosylated product (Figure 2b). Instead, anti-d

cross-reacts with several schistosomal proteins, present in

both S2 and S3, of approximate sizes of 35, 50 (S2 only) and

95 kDa, which are insensitive to N-glycanase digestion.

Reactivity with the 95 kDa antigen is the strongest. While

not reacting with Sm25 or its deglycosylated product, anti-d,

similar to anti-a, does react with recombinant protein r140

(Figure 2).

Worm fractions S2 and S3 each consist of a complex

mixture of proteins, when visualized by Coomassie-blue

staining of the gel (Petzke 1996), whereas lentil lectin peak

3 consists of a subset of these proteins. In contrast, only a

fraction of these proteins are antigens reacting with anti-a

and anti-d, con®rming the speci®city of these antibodies.

Characterization of de®ned schistosome antigens

cross-reacting with anti-d

The unusual immunoreactivity pro®le of antid prompted us

to attempt an identi®cation of the various cross-reactive

proteins using either recombinant or worm-puri®ed schisto-

somal proteins in the size range of the cross-reactive species.

The identity of the N-glycanase-insensitive protein detected

by anti-d at approximately 95 kDa in both S2 and S3 on

immunoblots was determined to be S. mansoni paramyosin

(Sm97). This conclusion is based on strong reactivity of

anti-d with recombinant Sm97 on both an ELISA (Figure

3a) and a Western immunoblot (Figure 3b). Additionally,

anti-d detects paramyosin puri®ed from worm extracts

(Figure 5). The identities of the weakly reacting 35- and

50-kDa antigens detected by anti-d have not yet been

determined (Figure 2b).

Cross-reactivity of anti-d to yet other schistosome anti-

gens was established in a parallel study during which we

were developing a recombinant antigen containing the delta

sequence for vaccination studies. In this initial effort, the

pGEX vector, which encodes the 26-kDa glutathione-S-

transferase of S. japonicum (Sj26), was used. Anti-d reacted

strongly on an ELISA with extracts prepared from bacteria

transformed with the pGEX vector alone (Petzke 1996).

This result suggested that anti-d cross-reacts with Sj26. The

cross-reactivity was con®rmed by ELISA (Figure 3a) and

immunoblot (Figure 3b) using recombinant Sj26. Addition-

ally, recombinant Sm26, the S. mansoni homologue of

Sj26, as well as the recombinant 28 kDa GST of S. mansoni

(Sm28), were also detected by anti-d using both assays

(Figure 3a,b). These proteins, similar to paramyosin Sm97,

are encoded by genes unrelated to GP22. However, the

reactivity of anti-d is stronger with Sm97 than with the

GSTs, and the reactivity with Sj26 GST is stronger than with

the Sm26 and Sm28 GSTs.

Construction and characterization of r4 ´ 47

Rabbit anti-d cross-reacts with paramyosin (Sm97) and

glutathione-S-transferases (Sj26, Sm26, Sm28), which are

all candidate vaccine antigens for schistosomiasis. In addi-

tion, cloning studies in two laboratories independently



Figure 3 Cross-reactivity of antipeptide-d with other schistosome

antigens. (a) (ELISA): recombinant antigens immobilized are r2 ´ 47

[4 mg (286 pmol)], Schistosoma japonicum 26-kDa GST [rSj26;

16 mg (615 pmol)], S. mansoni 26-kDa GST [rSm26; 20 mg

(770 pmol)], S. mansoni 28-kDa GST [rSm28; 20 mg (714 pmol)],

and S. mansoni 97 kDa paramyosin [rSm97; 1 mg (10 pmol)]. Anti-d

was used at 1 : 100 dilution. No reaction was observed with antia

(data not shown). (b) (ECL Western immunoblot): recombinant

antigens are (vector control pET-15b-transformed cell extracts,

50 ng); r4 ´ 47 [50 ng (7 pmol)]; rSj26 [5 mg (190 pmol)]; rSm26

[5 mg (190 pmol)]; rSm28 [5 mg (180 pmol)]; rSm97 [5 mg (50 pmol)].

Antigens were probed with anti-d (1 : 200). The lower molecular

weight moieties present in rSm97 lane represent proteolytic

breakdown products of the recombinant protein (C. Shoemaker,

personal communication).

identi®ed the same 47-amino acid peptide, containing the

delta sequence, in their screening of cDNA expression

libraries using sera from resistant mice (Knight et al. 1989)

and rats (El-Sherbeini et al. 1991). These coincidences

were suf®ciently compelling to attempt a vaccine trial

with this region of the Sm25 protein. We used the strategy

of enhancing the immunogenicity and protective capacity of

a peptide by incorporating multiple units of the peptide onto

a polymeric core (Wolowczuk et al. 1991). Multiple copies

of the 141 bp EcoR1 restriction fragment of gene GP22

(codons 117±163) were cloned by a series of steps into

the pET-15b expression vector. The predicted recombinant

protein r4 ´ 47 consists of four contiguous copies of the 47-

amino acid sequence joined to an amino terminal 2 kDa

`His.Tag' vector sequence (Figure 4). This plasmid,

pET(4 ´ 47), was constructed to include a terminal cysteine

residue at the carboxyl end of the insert sequence, allowing

the formation of a disulphide bond between two molecules

of r4 ´ 47. Analysis of r4 ´ 47 by SDS-PAGE followed by

immunoblotting with anti-d detects a major species at

approximately 26 kDa (Figure 3b; the similar MWr to

Sm25 is coincidence) which shifts to approximately

50 kDa under nonreducing conditions (Petzke 1996).

The recombinant protein r4 ´ 47 reacts strongly with anti-

d and, as expected, is not detected by anti-a (a is codons

70±84, see Figure 4). The speci®city of this reaction is

demonstrated by a competition study shown in Figure 5, in

which soluble peptide delta completely competes the bind-

ing of anti-d to r4 ´ 47, as well as to worm-puri®ed Sm97,

the cross-reacting antigen displaying the most intense reac-

tivity with anti-d.

The recombinant protein r4 ´ 47 is recognized by F-2x,

but not by W-2x serum, in a Western immunoblot analysis

(Figure 6). This unique property of F-2x is expected, since



Figure 4 Diagramatic representation of Sm25 and r4 ´ 47. Upper: Sm25 (decoded from gene GP22) consists of 182 amino acids (open

arrowheads). The locations of peptides a (amino acids 70±84) and d (amino acids 151±162) are shown in striped bars. The segment expressed

in r140 (amino acids 43±182; recombinant protein constructed by Suri et al. (1997) and in r4 ´ 47 (amino acids 117±163; this study) are shown

(solid arrowheads). Lower: Recombinant protein r4 ´ 47 consists of (B), two tandem pairs of 47 amino acid repeats (note that the last two amino

acids of the ®rst repeat are also the ®rst two amino acids of the second repeat), each tandem pair separated by (C), a 5 amino acid linker

sequence (C�GSSIS, single-letter amino acid abbreviations), and preceded by (A), a 27 amino acid N-terminal `His.Tag' sequence

(A�MGSSHHHHHHSSGLVPRGSHMLEDLIS) encoded by the pET-15b vector. The 4 amino acid C-terminal tail (D�GSGC) encoded by the

vector contains a terminal cysteine residue, resulting in formation of a disulphide bond, under nonreducing conditions, between two r4 ´ 47

molecules, producing eight copies of the same peptide sequence per molecule of antigen.

the original 47-amino acid sequence was detected as a fusion

protein with b-galactosidase in a lgt11 phage-expression

library using the identical criterion (El-Sherbeini et al. 1990).

Anti-r4 ´ 47 detects Sm25 and cross-reacts with other

worm antigens

Anti-d antibodies cross-react with several schistosome anti-

gens not encoded by gene GP22. This cross-reactivity

phenomenon is not restricted to anti-d antibodies, but is

also observed with antibodies raised against the recom-

binant protein containing the d sequence (r4 ´ 47). The

Western immunoblot shown in Figure 7 demonstrates reac-

tivity of anti-r4 ´ 47 with these schistosome antigens. Addi-

tionally, in contrast to the inability of anti-d to detect Sm25

(Figure 2b), anti-r4 ´ 47 strongly detects Sm25 in both

N-glycanase-treated and untreated S3 extracts (Figure 8)

as well as peptide delta on ELISA (Petzke 1996). This

latter ®nding suggests that either a region of the 47-amino

acid sequence other than the d sequence contains an

epitope shared with Sm25, or that the d sequences in

r4 ´ 47 are in at least two conformations, only one of

which is present in Sm25 (the conformation not present in

`free' peptide delta). Although the binding of anti-r4 ´ 47

to r4 ´ 47 in a Western immunoblot is not blocked by

peptide-d (Petzke 1996), this result does not distinguish

between the two possibilities.

Vaccine trials with r4 ´ 47: high antibody titres but no

protection

The recombinant protein r4 ´ 47, emulsi®ed in alum, was

injected into Balb/c mice (75 mg dose) and two groups of

Fischer rats, receiving either 100 mg or 200 mg doses. Three

injections were given at two or three week intervals, and an

increase in anti-r4 ´ 47 antibody titres was observed in both

rodent models following each booster injection (Figure 9A).

The anti-r4 ´ 47 antibodies in both species were mainly of

the IgG isotype (Petzke 1996). Peak antibody titres were

reached immediately prior to infection (25 000 in mice;

9 000±11 000 in rats, with little difference between the

groups receiving 100 mg or 200 mg; Figure 9a). Cercarial

challenge, however, did not induce an anti-r4 ´ 47 response

in control mice, and titres in vaccinated animals had

dropped by the time of perfusion (to 12 000 in mice; 8000

in rats). Concurrently, no protection was observed (Table 1).

A noticeable increase in worm yields, relative to the con-

trols, was detected in the vaccinated rats, consisting of a



Figure 5 Competitive Western immunoblot to de®ne anti-d

speci®city: A mixture of r4 ´ 47 [50 ng (7 pmol)/lane] and worm-

puri®ed Sm97 [2 mg (20 pmol)/lane] was separated by SDS-PAGE

and immunoblotted with anti-d (1 : 1600) which had been

preincubated with the indicated amounts of soluble peptide-d

[5 ng (2´7 pmol); 25 ng (14 pmol); 50 ng (28 pmol); 1000 ng

(560 pmol)] or no peptide [0]. Anti-d reactivity with r4 ´ 47 and

S. mansoni 97-kDa paramyosin (Sm97) is speci®c for the d epitope.

Figure 6 r4 ´ 47, but not r140, is uniquely recognized by F-2x.

Antigens, r4 ´ 47 (5 mg); extracts from pET-15b vector-transformed

cells (5 mg); r140 (3 mg) were assayed by Western immunoblotting

using F-2x (1 : 500) and W-2x (1 : 500).

Figure 7 Cross-reactivity of anti-r4 ´ 47 with schistosome antigens.

Antigens are r4 ´ 47 [50 ng (7 pmol)]; rSj26 [5 mg (190 pmol)]; rSm26

[10 mg (380 pmol)]; rSm28 [5 mg (180 pmol)]; worm-puri®ed Sm97

[1 mg (10 pmol)]. Antigens were immunoblotted with rat anti-r4 ´ 47

(1 : 200).

31% increase (P� 0´05) in the rats injected with 200 mg

doses of r4 ´ 47, and 19% increase (not signi®cant) in the

group receiving 100 mg. With mice, both liver egg burden

and worm sex ratio were measured to assess possible

antifecundity effects of the immunization. Neither para-

meter demonstrated protective effects.

A second vaccination trial was designed to establish

whether r4 ´ 47 induced blocking antibodies which inter-

fered with the host's immune response, thus serving as a

survival mechanism elicited by and aiding the parasite. To

explore this possibility, protective F-2x antiserum was

injected into vaccinated and control rats 3 days after

cercarial infection. The amount of protection transferred

by graded amounts of F-2x had been previously determined

in a separate experiment (data not shown). A dose was

chosen which conferred submaximal levels of protection,

which would enable changes in resistance to be observed.

Fischer rats were immunized with a Sephacryl-fractionated

preparation of the recombinant antigen, termed sp-r4 ´ 47

(see Methods). As in the preceding trial, vaccination elicited

high anti-r4 ´ 47 antibody titres which displayed the same

response kinetics: titres increased with each injection,

reaching maximal levels prior to challenge infection, but

fell during infection. Reduced worm burdens of approxi-

mately 35% were observed in both sp-r4 ´ 47-injected and

alum controls receiving passively transferred F-2x (Table

2). The sp-r4 ´ 47 injected rats not receiving F-2x did not

show a signi®cant change in worm yield. Therefore, vacci-

nation with sp-r4 ´ 47 elicited neither protection nor a

blocking effect on the protective activity of F-2x.

Lentil lectin-bound antigen in worm S3 fraction

induces passive protection in rats

The lentil lectin-bound (LLB) material from S3 fraction

of adult worms was eluted, concentrated, and used to

immunize four Fischer rats after emulsi®cation in alum.

Serum was assayed for anti-LLB activity 2 weeks after each



Figure 8 Anti-r4 ´ 47 detects the gene GP22 product in S3.

Immunoreactivity pro®le of rabbit anti-r4 ´ 47 is comparable to both

antia (open arrowheads, compare with Figure 2a) and anti-d (solid

arrowheads, compare with Figure 2b). Samples were incubated under

identical conditions with [�] or without [ÿ] N-glycanase (N-glyc.)

prior to SDS-PAGE separation and immunoblotting. r140, 50 ng;

r4 ´ 47 [� and ÿ], 50 ng; S2 and S3 [� and ÿ], 5 mg. Antigens were

immunoblotted with rabbit anti-r4 ´ 47 (1 : 1000). High mol wt

components in r4 ´ 47 (ÿ) are attributed to aggregation.

30000

25000

20000

15000

10000

5000

0

12000

10000

8000

6000

4000

2000

0

Tit

re

38 67 124 3814 67 103

(a)Mice

20000

15000

10000

5000

0

Tit

re

17 104765932

(b)Rats

Rats

Day of bleed

Unimmunized

Alum Only

Unimmunized

Alum Only

Alum Only

0.1 mg

0.2 mg

Figure 9 Vaccination of mice and rats with r4 ´ 47 induces high titre

antibodies, but no anamnestic response to a cercarial challenge

infection. (a) Recombinant protein r4 ´ 47, emulsi®ed in alum

adjuvant, was injected s.c. into Balb/c mice (75 mg doses) and two

groups of Fisher rats (100 mg or 200 mg doses); three injections at

2 and 3-week intervals. Controls were injected with alum only.

Antisera prepared at times indicated were assayed by ELISA against

r4 ´ 47 and titres calculated (see Methods). The animals were

challenged percutaneously on day 75 by exposure to 150 (mice) or

520 (rats) S. mansoni cercariae.(b) Fischer rats were immunized

s. c. with 150 mg sp-r4 ´ 47 (Sephacryl-puri®ed r4 ´ 47) emulsi®ed

in alum; four injections at 3-week intervals. Control animals were

either uninjected or received injections of PBS emulsi®ed in alum.

Antisera prepared at times indicated were assayed by ELISA against

r4 ´ 47 and titres calculated. Rats were challenged percutaneously on

day 76 by exposure to 500 cercariae.

injection. Anti-LLB titres equivalent to those in F-2x were

achieved after two or three immunizations. Sera from the

terminal bleeds were pooled and injected into six naive rats,

3 days after exposure to cercariae. Controls received either

normal Fischer serum or F-2x. Results (Table 3) show that

the LLB material from S3 contains antigens capable of

inducing a protective immune response, with a 29% reduc-

tion (P < 0´05) in worm burden compared to the normal

serum control. Rats passively immunized with F-2x yielded

a 44% reduction (P< 0´001).

ELISA titres of the anti-LLB pooled sera were compared

with F-2x against a variety of worm and recombinant

antigens (Table 4). Against recombinant GP22 antigens

r140 and r4 ´ 47, anti-LLB has activity comparable to

F-2x (Table 4), demonstrating that the Sm25 in the S3/LLB

fraction is immunogenic. F-2x has greater activity to r140

than anti-LLB, which may be an indication that the Sm25 in

the vaccine protocol is not as immunogenic as Sm25 in an

active infection. Anti-r140, however, is the most potent

antiserum against r140, which suggests that some epitopes

displayed in r140 are not immunogenic in the native Sm25

of LLB or worms. These may be sequences in r140 which do

not fold into the native conformation. Similarly, while F-2x

and anti-LLB have equivalent reactivities to r4 ´ 47, they are



Table 1 Effects on worm yields of animals immunized with r4 ´ 47

Treatment (n)1 Worm yields Eggs/female Preinf. Ab2

Mice

Unimmunized control (4) 60 6 8 1270 6 53 < 200

Alum only control (6) 57 6 6 1680 6 314 < 200

r4 ´ 47 (75 mg)\alum (13) 68 6 23 1310 6 108 25 000

Rats

Unimmunized control (6) 65 6 6 < 100

Alum only control (8) 64 6 7 < 100

r4 ´ 47 (100 mg)\alum (7) 76 6 64 9 300

r4 ´ 47 (200 mg)\alum (8) 84 6 65 10 600

1Vaccination protocols in Methods; mice exposed to 150 cercariae

each, perfusion after 49 days; rats exposed to 520 cercariae each,

perfusion after 28 days; worm and liver egg yields (mean 6 SEM).2Preinfection anti-r4 ´ 47 antibody titres. 319% increase in worm

yield relative to alum only (NS). 419% increase in worm yield

relative to alum only (NS). 531% increase in worm yield relative to

alum only (P� 0´05). Statistical signi®cance determined using two-

tailed Student's t-test.

Table 2 Effects on worm yields of rats actively immunized with

sp-r4 ´ 471 and passively immunized with F-2x

Treatment (n)2 Worm yields % Reduction

Alum only control (6) 96 6 5 ÿ

Alum only�F-2x3 (7) 62 6 5 35 (P< 0´001)

sp-r4 ´ 47\alum (6) 89 6 6 8 (NS)

sp-r4 ´ 47\alum�F-2x3 (6) 63 6 6 34 (P< 0´001)

1Sephacryl-puri®ed r4 ´ 47 (see Methods) 2Vaccination protocol in

Methods; rats exposed to 500 cercariae each, perfusion after 28 days;

worm yields (mean 6 SEM) 3F-2x was injected s.c., 3 days after

cercarial exposure.

Table 3 Effect on worm yields of rats passively immunized with

anti-LLB fraction of S3

Treatment (n)1 Worm yields % Reduction

Normal serum control (5) 51 6 3´5 ÿ

F-2x serum control (6) 28 6 4´0 44 (P< 0´001)

Anti-LLB serum (5) 36 6 3´9 29 (P< 0´05)

1Donor rats were immunized with lentil-lectin bound fraction of S3

(anti-LLB) or twice-infected with cercariae (F-2x) or were unexposed

(normal) and their sera (1 ml) used to passively immunize rats

exposed 3 days earlier to 220 cercariae each, perfusion after 28 days;

worm yields (mean 6 SEM).

Table 4 Comparison of immunoreactivity by ELISA of rat anti-LLB

immune serum with other rat antisera against worm and gene GP22

recombinant antigens

Rat sera (dilution) Test antigen OD410

Anti-LLB (1 : 1600)1 S3/LLB 0?33

F-2x (1 : 1600) S3/LLB 0?36

Prebleed2 S3/LLB < 0?01

Anti-LLB (1 : 1600) S3 0?12

F-2x (1 : 1600) S3 1?20

Prebleed S3 < 0?01

Anti-LLB (1 : 200) r140 0?33

F-2x (1 : 200) r140 0?59

Anti-r1403 r140 1?37

Normal4 r140 < 0?01

Anti-LLB (1 : 100) r4 ´ 47 0?16

F-2x (1 : 100) r4 ´ 47 0?19

anti-r4 ´ 47 r4 ´ 47 0?86

Normal r4 ´ 47 0?03

Anti-LLB (1 : 200) pET15b5 0?01

F-2x (1 : 200) pET15b5 0?03

Normal pET15b5 < 0?01

1Immunization of rats with the LLB fraction of S3 is described in

Methods. Data reported in this table with anti-LLB is a serum pool

from terminal bleeds of four rats. 2Prebleed are pooled sera from rats,

prior to immunization with LLB fraction. 3Anti-r140 is described in

Suri et al. 1997. 4Sera from normal Fischer rats. 5Extracts prepared

from vector-only bacterial transformants were subjected to

puri®cation by Ni-af®nity chromatography and the eluted bound

fraction represents the control for detecting contaminating bacterial

antigens.

less potent than anti-r4 ´ 47, for possibly the same reasons as

suggested for potency of anti-r140 to r140.

DISCUSSION

Antibodies to a predicted B-cell epitope of Sm25

cross-react with other candidate vaccine worm

antigens

Data presented here con®rm the identity of a predicted

glycoprotein product encoded by S. mansoni gene GP22,

cloned by our laboratory (El-Sherbeini et al. 1991], as the

Sm25 present in the tegument of adult worms, previously

described by Knight et al. (1989) and Omer-Ali et al.

(1991). Monospeci®c antibodies raised against a synthetic

peptide corresponding to a 15-codon segment termed alpha

(a) of GP22 were used to identify a glycoprotein which

possesses the following characteristics that correlate with

the properties of Sm25: (i) It is detected on Western immuno-

blots with anti-a as a 25 kDa product in a detergent-solubil-

ized fraction of worm proteins (S3, Figure 2a), consistent

with the size and localization of Sm25, which distributes

into the detergent (Triton X-114) phase of tegumental mem-

branes. (ii) It is a glycoprotein, as evidenced both by lentil-

lectin binding capacity (Figure 1b), and by mobility shift to

approximately 20 kDa following N-glycanase digestion

(Figure 2a). (iii) It reacts with anti-r4 ´ 47 and anti-r140

(Figure 8), polyspeci®c antibodies to recombinant products

of gene GP22 sequence.

Previous attempts in our laboratory to identify products of

gene GP22 had utilized an immunoprecipitation protocol

with rabbit antiserum against b-galactosidase fusion protein,

FP40, which contained one copy of the 47-amino acid

(141 bp cDNA) segment shown to be codons 117±163 of

GP22 (El-Sherbeini et al. 1991). Surprisingly, monospeci®c

antibodies to a 13-codon peptide of this segment encoding

putative B-cell epitope delta (d), do not detect Sm25 on

Western immunoblots (Figure 2b). Anti-d antibodies, how-

ever, react on Western immunoblots with proteins of approxi-

mately 35, 50 and 95 kDa in S2 and S3 (Figure 2b), in

addition to recombinant S. mansoni 26 and 28 kDa

glutathione-S-transferases (rSm26, rSm28), recombinant

26-kDa GST (rSj26) of S. japonicum (Figure 3), and

recombinant protein encoded by gene GP22, r140 (Figure

2b). Curiously, many of these antigens cross-reactive with

antipeptide-d are themselves candidate vaccine antigens

identi®ed in other studies, as follows. The most strongly

reacting antigen in worm extracts, the 95 kDa protein, was

identi®ed as S. mansoni paramyosin (Sm97): a soluble,

97 kDa schistosome protein capable of eliciting protective

immunity against S. mansoni in mice (Pearce et al. 1986,

Pearce et al. 1988). Sj26, which has been implicated in

mediating protective immunity against S. japonicum in mice,

is 70% identical in amino acid sequence (Panaccio et al.

1992) to the best characterized potential vaccine antigen of

S. mansoni, Sm28 (Auriault et al. 1988, Auriault et al.

1991), which elicits protective immunity in mice (Balloul

et al. 1987) and rats (Auriault et al. 1987, Wolowczuk et al.

1991). Genes unrelated to GP22 encode these antigens

(Sj26, Sm26, Sm28, and Sm97). The cross-reactivity of

anti-d is speci®c, as demonstrated with Sm97, and is

competed with soluble peptide-d (Figure 5).

Attempts to locate common amino acid sequences of

Sm97 and GSTs recognized by anti-d through scanning of

these schistosome proteins (performed by D. Cioli), identi-

®ed a short region of homology in peptide-d and Sm97, but

failed to detect sequence similarity between peptide-d and

GSTs. The homologous sequence consists of ®ve amino

acids, three identical and two similar: Asn±Ser±Ala±Asn±

Lys (NSANK) in peptide-d, and Asp±Thr±Ala±Asn±Lys

(DTANK) in Sm97. We attempted to determine the rele-

vance of these homologous sequences by a competition

ELISA using soluble synthetic pentapeptides NSANK and

DTANK. The pentapeptides completely failed to compete

the binding of anti-d to immobilized peptide-d, nor did anti-d

detect either of the immobilized pentapeptides (Petzke

1996).

While anti-d in Western immunoblotting does not detect

Sm25, these antibodies do recognize two recombinant

products of Sm25-encoding gene GP22 by immunoblotting:

r4 ´ 47 and r140, the latter also containing peptide-a

(Figures 3b and 2b, respectively). This result is not attribu-

table to lower af®nity of anti-d antibodies compared to anti-a

antibodies for r140, the protein which both antisera detect by

immunoblotting (Petzke 1996). However, antiserum against

recombinant protein r4 ´ 47 does detect Sm25 in addition

to r4 ´ 47 and r140 (Figure 8). Anti-r4 ´ 47 may contain

antibodies of similar speci®city as anti-d in addition to

antibodies which anti-d lacks, either to epitopes outside

of the d sequence or to alternative conformations of that

sequence within r4 ´ 47. That peptide-d does not compete

binding of anti-r4 ´ 47 to r4 ´ 47 protein (Petzke 1996) does

not resolve these possibilities. We conclude from these

results that the amino acid sequence de®ned as d can

possibly assume various conformations, some resembling

epitopes in schistosome proteins encoded by unrelated

genes (`mimotopes'), which are dependent upon the three-

dimensional context of neighbouring residues in the GP22

sequence. Alternately, multiple epitopes in addition to d are

present within the 47-amino acid segment.

Cross-reactive antibodies to the other schistosome anti-

gens (Sm97, etc.) are present in peptide-af®nity puri®ed

anti-d but are not present in peptide-af®nity puri®ed anti-a.

The carrier protein used for initial immunization of the



rabbits with peptide-d conjugate was OVA (see Methods).

Later experiments involved boosting the same rabbits with

peptide-d conjugated to KLH. The carrier protein used for

immunization of the rabbits with peptide-a conjugate was

always KLH. Since antibodies to KLH also cross-react with

schistosomal antigens (Ko & Harn 1987, Bushara et al.

1993, Thors & Linder 1998, Hamilton et al. 1999), it is

important to exclude the possibility that anti-KLH anti-

bodies were responsible for the cross reactivity observed.

Five reasons can be given which exclude a contribution

from anti-KLH in the interpretation of the results: (i)

Speci®c peptide-af®nity chromatography on a solid-phase

peptide matrix that does not include carrier protein was used

for the independent puri®cations of both anti-d and anti-a

peptide antibodies. If anticarrier antibodies were a contami-

nation and the sole source of the observed cross-reactions,

then both antipeptide antibody preparations should have

produced the observed cross-reactivity. This was obviously

not the case; as only af®nity-puri®ed antibodies to peptide-d

showed the cross reactivity (compare Figures 2 and 3). (ii)

Recombinant protein r4 ´ 47, which contains the peptide-d

sequence in four tandem copies but no KLH carrier, induced

antibodies that showed the same pattern of cross-reactivity

as antipeptide-d. Thus, con®rmation of the cross-reactivity

with a different anti-d preparation was observed indepen-

dently of KLH (see Figure 7). (iii) A competition experi-

ment was conducted using r4 ´ 47 and Sm97 as immobilized

antigens, af®nity-puri®ed anti-d antibodies, and peptide-d

(not conjugated to carrier) as the competing antigen. The

binding of anti-d antibodies to each of these antigens was

completely inhibited by addition of the free peptide only

(see Figure 5). If anti-KLH antibodies (putatively contami-

nating the af®nity puri®ed anti-d antibodies) were respon-

sible for the cross-reaction with Sm97, then peptide-d alone

should not have abolished the binding of antibodies to that

antigen. (iv) Anti-d antibodies af®nity-puri®ed from sera of

rabbits immunized with peptide-d conjugated to OVA were

used in our earliest studies. These antipeptide-d preparations

were the ones which initially provoked our excitement in

that they showed cross-reactions with a high molecular

weight antigen in S2 (see Figure 1a, peak 2a, contains

Sm97) and with sj26 GST encoded in the pGEX plasmid

vector (see Figure 3b). Thus, cross-reactivity with GST was

observed using af®nity puri®ed antipeptide-d independently

of the carrier protein used in the immunization. (v) None of

the reports involving antigens cross-reactive with anti±KLH

cite interaction with Sm97 (paramyosin) that we have

reported.

These observations eliminate the concern over anti-KLH

and clearly support the conclusion that anti-d antibodies are

responsible for the cross-reactivity. Moreover, a pilot study

with af®nity-puri®ed anti-d from a KLH-boosted rabbit

con®rms that anti-KLH is not detectable in an ELISA at

the dilutions of anti-d employed in these studies, con®rming

the success of the af®nity chromatography protocol.

Characterization of a recombinant product containing

tandem-repeats of this peptide as a vaccine

Immunization of rats and mice with r4 ´ 47 induces high

antibody titres, yet not only fails to protect against challenge

infection, but also can result in signi®cantly higher worm

burdens (Table 1). One possible explanation for this result

involves the isotype of the induced antibodies. For example,

the rat IgG2a isotype has also been associated with resist-

ance to infection, possibly via complement ®xation or

ADCC. The protective ability of IgG2a can be inhibited

by the presence of blocking antibodies of the IgG2c isotype

for a glycoprotein antigen (Grzych et al. 1982). Isotyping

studies of antigen-speci®c antibodies in r4 ´ 47 detected

IgG2a, but not IgG2c (Petzke 1996). Another approach to

testing the blocking concept was to vaccinate with the

putative inducer of blocking antibodies (r4 ´ 47) and then

to passively immunize with protective antiserum F-2x

(Table 2). Anti-r4 ´ 47 antibodies did not interfere with

the protective activity of F-2x. However, this does not

exclude the presence of blocking antibodies, if antibodies

to other antigens are also contributing to protection.

An alternative explanation to lack of protection by r4 ´ 47

and by the other recombinant product we tested, r140

(Suri et al. 1997), concerns the protective capacity of the

epitopes in these gene GP22 products. Sm25 may not be the

immunogen used by worms to produce the protective

immune response. To address this possibility, we passively

vaccinated rats with antiserum to the lentil-lectin bound S3

fraction (S3/LLB) of worms, which contains the authentic

Sm25 glycoprotein, and successfully conferred protection

(Table 3). Anti-S3/LLB and F-2x had comparable ELISA

antibody activity to the S3/LLB and to recombinant antigens

r4 ´ 47 and r140 (Table 4). While this does not demonstrate

that the anti-GP22 antibodies in either serum contribute to

the protective activity, this possibility cannot be excluded.

Since the lentil-lectin bound S3 is a mixture of products,

however, one also cannot exclude the possibilty that it was

a combination of antibodies to these other S3 antigens

(without anti-Sm25), or perhaps even anticarbohydrate anti-

bodies, which were responsible for protection. The prokary-

otic recombinant products tested in our study lacked

glycosylation.

To conclude, antibodies to peptide-a (amino acids 70±84

of Sm25) identify Sm25 in the detergent-soluble (but not the

aqueous-soluble) fraction of juvenile and adult schisto-

somes. Antibodies to peptide-d (amino acids 151±162 of

Sm25) do not react with Sm25 or its deglycosylated product



but do react with r4 ´ 47 and r140, recombinant proteins

with peptide-d as a portion of their amino acid sequence.

Anti-a reacts with the recombinant protein r140, which has

peptide-a as a portion of its amino acid sequence. Anti-d

(but not anti-a) cross-reacts with other schistosome anti-

gens: Sm97, GSTs of S. mansoni (Sm26; Sm28) and S.

japonicum (Sj26) and a 50 kDa and a 35 kDa antigen of

S. mansoni yet to be de®ned. Anti-r4 ´ 47 antibodies react

with Sm25 and both recombinant antigens, and also cross-

react with other schistosome antigens detected by anti-d.

Thus, recombinant protein r4 ´ 47 possesses the `F-2x-unique

epitope(s)' and the epitope(s)/mimotope(s) which induce

cross-reactivity. The d sequence may have multiple con-

formations dependent upon neighbouring sequences; in

r4 ´ 47, the d sequence may assume some of these conforma-

tions and possibly others which are infrequent in peptide-d.

We hypothesize that the infrequent conformations are

present in Sm25.

The lentil lectin bound fraction of detergent-solubilized

adult worms induces protective antibodies in rats, including

antibodies to r4 ´ 47. However, vaccination with r4 ´ 47 is

not protective in mice and rats, despite inducing high titres

of antibodies to r4 ´ 47. Nor do anti-r4 ´ 47 antibodies inter-

fere with protection transferred by F-2x. Two hypotheses to

account for this disparity include the requirement of antipoly-

saccharide antibodies not induced by the E. coli-expressed

fusion proteins, and the possibility that the mimotopes against

which the cross-reactive antibodies were produced are not the

target epitopes of protective antibodies.

ACKNOWLEDGEMENTS

The authors wish to thank the following individuals for their

contributions to this research: Isla Garraway, whose under-

graduate honors project generated preliminary data on the

lentil lectin fractionation of worm extracts; Frank DeSilva

and Jonathan Kurtis, who assisted with several experiments

and provided technical advice; Ken Nguyen, whose assis-

tance with immunizations and perfusions is greatly appre-

ciated; Carol Macomber and Linda Fitzgibbons for technical

assistance; and Dr Andrew Campbell, whose suggestions

regarding the competition Western immunoblotting proce-

dure and critique of the results are greatly appreciated. This

project was supported by grants to P.M.K from the UNDP/

World Bank/WHO Special Program for Research and Training

in Tropical Diseases (TDR-900293) and the US Public Health

Service, NIH (AI-31224). M.M.P. received support from an

NIH Predoctoral Training Grant (5T32 GM07601±17).

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Schistosomamansoni gene GP22 encodes the tegumental antigen Sm25: (1) antibodies to a predicted...

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