INFECrION AND IMMUNrrY, Sept. 1992, p. 3947-3951

Vol. 60, No. 9

0019-9567/92/093947-05$02.00/0 Copyright X 1992, American Society for Microbiology

A Conjugated Synthetic Peptide Corresponding to the C-Terminal Region of Clostridium perfiringens Type A Enterotoxin Elicits an Enterotoxin-Neutralizing Antibody Response in Mice TIMOTHY A. MIETZNER, JOHN F. KOKAI-KUN, PHILIP C. HANNA,t AND BRUCE A. McCLANE* Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261-2072 Received 9 April 1992/Accepted 26 June 1992

A synthetic peptide homolog corresponding to the C-terminal 30 amino acids of Clostridium perfringens type A enterotoxin (CPE) was conjugated to a thyroglobulin carrier and used to immunize mice. Conjugateimmunized mice produced antibodies which neutralized native CPE cytotoxicity, at least in part, by blocking enterotoxin binding. This peptide may be useful for the development of a vaccine to protect against CPE-mediated disease.

Clostridium perfringens type A food poisoning is among the most common human food-borne diseases in the United States (14). All symptoms associated with this illness are caused by C. perfringens enterotoxin (CPE), a 319-residue polypeptide (35 kDa) with a unique amino acid sequence (14, 15). Besides having a proven role in C. perfringens food poisoning (14), CPE has been implicated in other human gastrointestinal illnesses, including antibiotic-associated diarrhea, chronic diarrhea, and infantile diarrhea (14, 15). CPE also appears to be involved in serious veterinary gastrointestinal disease (1, 2). Recent studies have focused on the molecular basis of CPE cytotoxic action. The unique mechanism of CPE action involves the following, in sequence: (i) specific binding of enterotoxin to a proteinaceous receptor on mammalian membranes, (ii) insertion of CPE into plasma membranes of mammalian cells, (iii) formation of a membrane complex between CPE and two eucaryotic proteins of 70 and 50 kDa, and (iv) production of membrane permeability alterations leading to rapid changes in intracellular levels of ions and other small molecules (5, 10, 12-19, 22-24). These primary events lead to secondary effects which culminate in cell death. Structure-versus-function studies (5-9) have localized a 30-amino-acid sequence at the C terminus of CPE which possesses most, if not all, receptor-binding activity associated with enterotoxin (Fig. 1). Recombinant and synthetic peptide homologs corresponding to the 30-mer sequence (termed CPE290-319) have been shown to block specific binding of native CPE to the enterotoxin receptor (6). These CPE290_319 homologs are not cytotoxic, but preincubation of Vero cells with recombinant or synthetic CPE29-319 prior to CPE challenge blocks native CPE cytotoxicity through receptor competition. Recent studies (7) have also shown that there is at least one (and possibly more) neutralizing epitope associated with the CPE29,-319 region, since polyclonal antibodies prepared against native CPE were shown to (i) react with CPE290_319 recombinant and synthetic peptide homologs, (ii) block the binding of both CPE290319 and *

native enterotoxin to mammalian receptors, and (iii) neutralize native enterotoxin cytotoxicity. Production of antitoxin immunity is an important component of several approved and effective bacterial vaccines (e.g., the diphtheria-pertussis-tetanus vaccine). Vaccines which protect against toxin-mediated bacterial diseases often work by eliciting a strong antitoxin response involving antibodies which block toxin binding to mammalian cells. Similar results (24) demonstrate that CPE cytotoxicity also can be neutralized by antibodies which abolish enterotoxin binding. Since CPE29,_319 contains a binding-neutralizing epitope and is not cytotoxic itself, these results collectively suggest that CPE290-319 might serve as a candidate for the development of a toxoid vaccine to protect against CPEmediated disease. However, if CPE29,a319 is to be useful for vaccine development, it must elicit a strong immune response, involving antibodies capable of neutralizing native enterotoxin. The present study demonstrates that a CPE290_319 synthetic peptide homolog conjugated to a thyroglobulin carrier can elicit a neutralizing antienterotoxin response in mice. Short synthetic peptides such as CPE29,-319 are often poorly immunogenic (11). However, the immune response to a peptide can routinely be improved by conjugating the peptide to a protein carrier (e.g., thyroglobulin) (4). A synthetic peptide homolog of CPE290_319 (Fig. 1) containing one additional cysteine residue at its N terminus was prepared by previously described methods (6). The sulfhydryl side chain of this cysteine was used as a target for conjugation to the epsilon amino groups of the carrier protein thyroglobulin by using the heterobifunctional cross-linker

m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (Pierce Chemical, Rockford, Ill.). MBS was chosen as a cross-linker for several reasons, including the following: (i) it has been used previously in similar conjugation procedures which elicited antipeptide antibody responses (4, 20), (ii) it is a mild cross-linker causing relatively few side reactions, and (iii) it can be used in a stepwise fashion to minimize the formation of homopolymers. Briefly, 10 mg of bovine thyroglobulin (Sigma, St. Louis, Mo.) was dissolved in 2 ml of phosphate-buffered saline (PBS), pH 7.4, and combined with 5 mg of MBS dissolved in 0.5 ml of dimethylformamide. After 1 h at 24°C with sample stirring, the activated carrier

Corresponding author.

t Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115. 3947

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INFECT. IMMUN.

NOTES

l NH3

Insertion & Cytotoxicity 13~LI~I 26 71

Binding

=

319

236

SLDAGQYVLVMKANSSYSGNYPYSEIFQKF-cooH FIG. 1. Molecular map of CPE functions. The locations of regions designated as being involved in insertion, cytotoxicity, and binding were determined in previous studies (5-8). The binding region sequence shown for CPE290 319 was determined previously (6). This sequence was used to construct a synthetic peptide for conjugation to thyroglobulin for the immunization of mice (see text). The asterisk denotes the binding region of CPE, which contains at least one neutralizing epitope (5-8).

protein was separated from unbound cross-linker by gel filtration over a Sephadex G-10 column (Pharmacia LKB, Piscataway, N.J.) by using 0.1 M phosphate, pH 6.0, as an eluent. The CPE290319 homolog containing an additional N-terminal cysteine was dissolved in 0.1 M borate, pH 9.1, at a concentration of 1 mg/ml, and the sulfhydryl group on the cysteine was reduced by exposure to 5 ,uM fresh NaBH4. After 5 min, the NaBH4 was inactivated by acidification with HC1. The peptide solution was neutralized with NaOH, and 5 ml of solution was combined with the activated thyroglobulin carrier. After overnight incubation of this mixture at 24°C with sample stirring, free peptide was removed from the thyroglobulin-peptide conjugate by gel filtration over a Sephadex G-25 column (Pharmacia LKB) with PBS as an eluent. The efficacy of coupling was assessed by amino acid composition analysis, and it was determined that approximately 30 mol of peptide was conjugated per mol of thyroglobulin. These levels of peptide coupling are typical for thyroglobulin conjugates which have been used successfully to obtain antipeptide antibodies (4). Female BALB/c mice (Zivic-Miller Laboratories, Pittsburgh, Pa.) each received three intraperitoneal injections given at 2-week intervals. Three mice were immunized throughout the study with CPE290319-thyroglobulin conjugate. Each mouse in this group received 0.2-ml injections containing conjugate (25 p,g) dissolved in 100 ,u of sterile PBS plus 100 ,u of either Freund's complete adjuvant (first injection only) or Freund's incomplete adjuvant (last two injections). Similarly, three mice received a series of three 0.2-ml injections of thyroglobulin (25 ,ug) dissolved in 100 ,ul of PBS plus 100 ,u of either Freund's complete or Freund's incomplete adjuvant, given as described above for conjugate. Mock-immunized mice received three injections, each containing 100 ,ul of PBS plus 100 RI of Freund's complete or incomplete adjuvant, also given as for conjugate. Ten days after the third immunization, all mice were periorbitally bled. The resultant sera were heated at 56°C for 30 min to inactivate complement prior to use. To detect an antibody response following this immunization protocol, immuno-dot blot analysis was performed with each mouse serum by previously described methods (6, 7). These experiments (Fig. 2) indicate that sera from mockimmunized mice did not react with any of the test antigens. Sera from the thyroglobulin-immunized mice reacted with thyroglobulin but not with either CPE, CPE290319, or CPE290-319-thyroglobulin conjugate. These results suggest that the thyroglobulin-immunized mice are not recognizing the thyroglobulin component of the conjugate. This may be due to the antigenic masking of thyroglobulin during the conjugation procedure. Sera from all three mice immunized with CPE290-319thyroglobulin conjugate recognized conjugate. Importantly, these sera also reacted with native CPE and nonconjugated CPE290319 synthetic peptide but did not react with either thyroglobulin or a negative-control peptide corresponding to a sequence from hen egg lysozyme (6). Also, preimmune

sera from the conjugate-immunized mice did not react with any of the test antigens (data not shown). The combination of an antibody response to CPE290319 in conjugate-immunized sera and the lack of reactivity of these sera with thyroglobulin is consistent with the previously stated hypothesis suggesting that the thyroglobulin component of the conjugate is not well presented to the immune system because of antigenic masking during the course of the conjugation process. Collectively, these results confirm that the CPE29, 319 synthetic peptide has an epitope(s) present in native CPE and indicate that peptide homologs corresponding to sequences in the C-terminal CPE290 319 region can be engineered to elicit an antibody response which recognizes native CPE. To assess whether the antibodies in the conjugate-immunized sera were capable of specifically neutralizing native CPE cytotoxicity, a 86Rb release Vero cell assay of CPE cytotoxicity was used (6, 13, 18). Native enterotoxin (5 jig) was preincubated for 15 min at 37°C in 2 ml of Hanks' balanced salt solution with or without 5, 20, or 50 ,ul of the specified mouse serum. Each preincubation mixture was then added to a confluent Vero cell culture which had been 86Rb labeled, as described previously (6, 13, 18). After 15 min at 37°C, the culture supernatant was carefully with-

Mouse Sera:

Conjugate Thyroglobulin Mock Antigen: Conjugate * *

*

Native CPE

a

*

S

CPE290319

*

v

*

Thyroglobulin

*

*

4

Neg Peptide

1

2 3

1

2 3

1

FIG. 2. Immuno-dot blot analysis of sera from immunized mice. Mice were immunized with either CPE2 9319-thyroglobulin conjugate, thyroglobulin, or PBS (mock), as described in the text. Antigens (either [i] 500 ng of CPE290-319 synthetic peptide or a negative-control peptide [Neg peptide] corresponding to residues 46 through 61 of lysozyme or [ii] 5 p,g of CPE290-319-thyroglobulin conjugate, native CPE, or native thyroglobulin) were immobilized on nitrocellulose strips. Each strip was then reacted with a 1/500 dilution of serum from a mouse immunized with conjugate, thyroglobulin, or PBS (Mock). After 2 h, the serum was removed, the blots were washed, and bound mouse antibodies were detected as described previously (6), with a goat anti-mouse immunoglobulin G-alkaline phosphatase (Bio-Rad Laboratories, Richmond, Calif.) detection system. Results are shown for sera from three conjugate-immunized and three thyroglobulinimmunized mice. Results for only one mock-immunized mouse are shown, but identical results were obtained with two other immunized mice. Preimmune sera from none of the mice reacted with any of the test antigens (not shown).

VOL. 60, 1992

NOTES

3949

100 100

* Mouse 1

80

c

:i

M Mouse 2

a

-

80 -

C

l0 Mouse 3

g

-0 z4-

*

Mouse

M El

Mouse 2 Mouse 3

60 -

0

z 40-

:a4=

20-

200-

0

Thyroglobulin

Conjugate

Conjugate

Mock

FIG. 3. Neutralization of native CPE cytotoxicity by serum from mice immunized with CPE29-319-thyroglobulin conjugate. CPE (5 jLg) was preincubated for 15 min at 37°C with 20 p.1 of the specified mouse serum (for designations, see the legend to Fig. 2), and the preincubation mixture was then added to 8Rb-labeled Vero cells for 15 min. The CPE-induced percentage of maximal 8sRb release in the presence and absence of each serum and the serum-mediated neutralization were determined as described in the text. Results shown are means + standard errors from three experiments with duplicate samples for each serum. Means without error bars had standard errors too small to depict. The CPE-induced percentage of 8sRb release in the absence of test serum was greater than 99% of available cytoplasmic label (i.e., 3 x 103 to 4 x 103 cpm). Spontaneous release (i.e., no CPE added to sample) after 15 min was 0.5 x 103 to 0.7 x 103 cpm.

drawn, and nonadherent cells in the supernatant were removed by microcentrifugation. Radioactivity in the culture supematant was determined with a Packard gamma counter. Spontaneous release (i.e., background 86Rb release from cultures not treated with CPE) and maximal 86Rb release (i.e., total cytoplasmic 86Rb available before CPE challenge) were determined as described previously (18). The CPEinduced percentage of maximal 86Rb release was calculated as follows (21): % of maximal release = [(86Rb release from CPE-treated samples - spontaneous release)/ (maximal release spontaneous release)] x 100 -

Serum-mediated neutralization

was

calculated

as

follows:

% neutralization = [(CPE-induced percentage of maximal 86Rb release without test serum CPE-induced percentage of maximal 86Rb release with test serum)/(CPE-induced percentage of maximal 86Rb release without test serum)] x 100 -

Results shown in Fig. 3 indicate that 20 RI of each serum from all mice immunized with CPE290-319-thyroglobulin conjugate was able to neutralize CPE cytotoxicity in this assay. Complete neutralization was also observed with either 5 or 50 pl of sera from conjugate-immunized mice (data not shown). However, 20 RI of each serum from thyroglobulinimmunized or mock-immunized mice had no significant effect on CPE cytotoxicity (Fig. 3). Similarly, neither 5 nor 50 p,l of these two types of serum affected enterotoxin cytotoxicity (data not shown). Additional control experiments (data not shown) demonstrated that up to 50 p,l of preimmune serum from conjugate-immunized mice did not neutralize CPE cytotoxicity. Sera (20 pl each) from rabbits immunized with native CPE also neutralized CPE under the

Thyroglobulin

Mock

FIG. 4. Inhibition of 125I-CPE binding by serum from mice immunized with CPE290-319-thyroglobulin conjugate. 1"I-CPEspecific binding to rabbit intestinal brush border membranes in the presence or absence of 40 ,ul of the specified serum was determined and binding inhibition was calculated as described in the text. Results shown are means t standard errors from two experiments with duplicate samples for each serum. Means without error bars had standard errors too small to depict. '25I-CPE-specific binding in this system was approximately 1 x 105 to 2 x 107cpm per sample and represented approximately 80% of total binding.

assay conditions used for Fig. 3 experiments (data not shown). These results demonstrate that the sera from the conjugate-immunized mice could specifically neutralize CPE cytotoxicity. As mentioned, the CPE29,_319 sequence has been implicated in enterotoxin binding, and antibodies which react with CPE29,-319 block native enterotoxin binding (6), so it might be expected that the sera from conjugateimmunized mice would block native CPE binding. This prediction was tested experimentally b using an 1I-CPEspecific binding assay (6). Briefly, 1 I-CPE (1 ,ug) was preincubated in the presence or absence of the specified serum (either 4 or 40 ,ul) for 30 min at 37°C. Half of each preincubation mixture then received a 50-fold excess of unlabeled CPE (to determine nonspecific binding; see below). All mixtures were then added to rabbit intestinal brush border membranes (100 ,ug of protein), prepared as described previously (6). After 30 min at 24°C, membranes were pelleted and washed twice to remove unbound CPE. Sample radioactivity was determined with a Packard gamma counter. Specific binding is calculated as the difference between radioactivity in total binding samples (i.e., no unlabeled CPE added) and nonspecific binding samples (i.e., samples receiving excess unlabeled CPE). The inhibition of 1"I-CPE-specific binding by each serum was calculated as follows: % inhibition = [(specific binding in the absence of test serum - specific binding in the presence of test serum)/ (specific binding in the absence of test serum)] x 100 Results shown in Fig. 4 confirm the prediction that sera (40 p,l each) from the conjugate-immunized mice would block CPE-specific binding. Complete inhibition of enterotoxin binding was also observed with 4 p,l of serum from these mice (not shown). This effect was limited to the conjugateimmunized sera since neither 4 p,l (data not shown) nor 40 p,l (Fig. 4) of each serum from thyroglobulin-immunized or mock-immunized mice affected 1"I-CPE binding. Preimmune sera (40 p.l each) from conjugate-immunized mice also did not affect '"I-CPE binding (data not shown). As a

3950

INFECr. IMMUN.

NOTES

positive control, sera (20 ,ul each) from rabbits immunized with native CPE also completely abolished 1251I-CPE specific binding (data not shown). From these results it can be concluded that CPE290_319 conjugated to a thyroglobulin carrier can be used to obtain antibodies which react with native enterotoxin. These antibodies neutralize native CPE cytotoxicity, at least in part, by blocking the binding of enterotoxin to its receptor. These studies support the use of the noncytotoxic sequence CPE290319, or conjugated CPE290_319 toxoids, in vaccine development for protection against CPE-mediated disease. It is apparent that additional research is required before a successful CPE vaccine will be available. Types of needed studies include (i) titration studies with conjugate-immunized sera to determine whether all serum-mediated neutralization of CPE cytotoxicity results from binding inhibition, (ii) fine mapping of the neutralizing epitope(s) in CPE290319 to obtain optimal epitope presentation for a vaccine, (iii) development of a strategy for delivery of this epitope to intestinal lymphoid tissue to obtain a protective immune response against oral CPE challenge, and (iv) demonstration of vaccine effectiveness against oral challenge with viable enterotoxigenic C. perfringens in an appropriate experimental model. Experiments to address these problems are under

3.

4.

5.

6.

7. 8.

way.

Besides its potential use for protection against CPEmediated disease, CPE290-319 (or CPE290_319 derivatives) also may prove useful as a prototype or marker for evaluation and development of new vaccine strategies. CPE290319 represents one of the most defined toxin sequences known to retain both binding activity and a neutralizing epitope. CPE290_319 is nontoxic and antigenic and is easily assayed by both serologic and biologic assays. The efficacy of CPE290-319-based vaccines in human or animal models could be easily tested, since C. perfringens food poisoning is a relatively uncomplicated disease involving only a single known virulence factor (CPE), implying that development of immunity to challenge with viable bacteria should require only anti-CPE immunity. Collectively, these facts suggest that CPE290-319 may be used as a prototype to develop a new generation of specifically engineered toxoid vaccines against enteric disease. Additionally, since there is considerable interest in the development of intestinal vaccines by using live vaccine vectors and foreign gene expression systems (3), CPE290319 may be a useful marker for evaluating such vaccine parameters as foreign antigen expression by live vaccine vectors, vector survival and delivery to the immune system, adjuvant effects on the intestinal immune response, and efficacy of vaccine administration protocols for maximizing the intestinal immune response. Future experimentation using CPE290319-based vaccine candidates as markers could help address these basic problems in vaccine research. This research was supported by Public Health Service grant AI-19844-10 from the National Institute of Allergy and Infectious Diseases. We thank Maja Stefanovic-Racic for technical assistance and Patricia Swanson for typing the manuscript.

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600:974-985. 17. McClane, B. A., and A. P. Wnek. 1990. Studies of Clostridium perfringens enterotoxin action at different temperatures demonstrate a correlation between complex formation and cytotoxicity. Infect. Immun. 58:3109-3115. 18. McClane, B. A., A. P. Wnek, K. I. Hulkower, and P. C. Hanna. 1988. Divalent cation involvement in the action of Clostridium perfringens type A enterotoxin: early events in enterotoxin action are divalent cation independent. J. Biol. Chem. 263:24232435. 19. McDonel, J. L. 1980. Binding of Clostridium perfringens "1enterotoxin to rabbit intestinal cells. Biochemistry 19:48014807. 20. Rothbard, J. B., R. Fernandez, and G. K. SchoolniL 1984. Strain-specific and common epitopes of gonococcal pili. J. Exp. Med. 160:208-221. 21. Thelestam, M., and R. Mollby. 1975. Sensitive assay for detection of toxin-induced damage to the cytoplasmic membrane of

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human diploid fibroblasts. Infect. Immun. 12:225-232. 22. Wnek, A. P., and B. A. McClane. 1986. Comparison of receptors for Clostridium perfringens type A and cholera enterotoxins in rabbit intestinal brush border membranes. Microb. Pathog. 1:89-100. 23. Wnek, A. P., and B. A. McClane. 1989. Preliminary evidence

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that Clostridium perfringens type A enterotoxin is present in a 160,000-Mr complex in mammalian membranes. Infect. Immun. 57:574-581. 24. Wnek, A. P., R. J. Strouse, and B. A. McClane. 1985. Production and characterization of monoclonal antibodies against Clostridium perfringens type A enterotoxin. Infect. Immun. 50:442-448.