Clostridium difficile Recombinant Toxin A Repeating Units as a Carrier Protein for Conjugate Vaccines: Studies of Pneumococcal Type 14, Escherichia coli K1, andShigella flexneri Type 2a Polysaccharides in Mice

ABSTRACT Unlike the native protein, a nontoxic peptide (repeating unit of the native toxin designated rARU) from Clostridium difficile toxin A (CDTA) afforded an antigen that could be bound covalently to the surface polysaccharides of pneumococcus type 14,Shigella flexneri type 2a, and Escherichia coliK1. The yields of these polysaccharide-protein conjugates were significantly increased by prior treatment of rARU with succinic anhydride. Conjugates, prepared with rARU or succinylated (rARUsucc), were administered to mice by a clinically relevant dosage and immunization scheme. All conjugates elicited high levels of serum immunoglobulin G both to the polysaccharides and to CDTA. Conjugate-induced anti-CDTA had neutralizing activity in vitro and protected mice challenged with CDTA, similar to the rARU alone. Conjugates prepared with succinylated rARU, therefore, have potential for serving both as effective carrier proteins for polysaccharides and for preventing enteric disease caused by C. difficile.

One variable affecting serum antibody (Ab) responses to the saccharide component is the carrier protein. For example, a genetically inactivated diphtheria toxin (CRM 197 ) was a more effective carrier than the formalin-treated toxoid (1). In addition, treatment of two genetically inactivated medically important antigens, diphtheria toxin (CRM 9 ) and P. aeruginosa exotoxin A (rEPA), with succinic anhydride improved the effectiveness of these two proteins as carriers for inducing polysaccharide Abs (28). Another variable is the total amount of a protein injected in formulations containing several conjugates sharing the same carrier: interference with the maximal level of Ab to both the polysaccharide and the protein components was related to the dose of protein administered to young children (11). Carrier protein-mediated suppression may become a problem as the number of polysaccharideprotein conjugates considered for immunization increases (33,35).
Clostridium difficile is a major cause of hospital-acquired diarrhea (15,25,27,31). Antibiotic therapy often causes this normal inhabitant of the colon to overgrow and release two toxins, A (molecular weight [MW], 308,000) and B (MW, 270,000), that cause an enteric disease ranging from diarrhea to pseudomembranous colitis (3,5,24,26,27,31). The presence of these toxins in intestinal fluids is diagnostic of this disease (24,25). Of the two, toxin A is primarily responsible for the clinical symptoms. In animal models, serum neutralizing Abs to C. difficile toxin A (CDTA) confer immunity to this pathogen (10,16,23). There is clinical evidence that serum immunoglobulin G (IgG) neutralizing Abs to these toxins confer immunity to this disease (41,42). CDTA has a series of contiguous repeating units at its COOH terminus comprising about one-third of the molecule (6,13,29). These repeating units are the region that recognizes the carbohydrate receptor of the host cells and that elicits serum Abs that neutralize the cytotoxic and lethal effects of toxin A (17,24,25,30). A recombinant nontoxic peptide, containing these repeating units (rARU), has been created and shown to elicit neutralizing Abs that can protect laboratory animals against challenge with both toxin A and C. difficile (10,16,29,30).
Based upon the high incidence of enteric disease caused by C. difficile in hospitalized patients, there is a need for an effective vaccine for this pathogen. Probably because of their high MWs, we were unable to synthesize conjugates of C. difficile toxins A and B (unpublished results). We studied rARU as a carrier protein for conjugates of the capsular polysaccharide of Escherichia coli K1 (possessing a capsular polysaccharide identical to that of group B meningococcus), pneumococcus type 14, and the O-specific polysaccharide of Shigella flexneri type 2a (2,8,9,12,28,37). Pneumococcus type 14 was chosen because it has been a major type isolated from patients over a long time span and from different diseases caused by pneumococci (2,32). S. flexneri type 2a was chosen because it is the most common Shigella type from patients in developing countries (8,9,35). E. coli K1 was chosen because there is yet no vaccine for systemic infections caused by these two pathogens (12). All three conjugates elicited high levels of Abs to their respective polysaccharides and to CDTA.

MATERIALS AND METHODS
CDTA and recombinant enterotoxin (rARU). CDTA was purified as described elsewhere (24). rARU contains the entire C-terminal repeat region (861 amino acids plus 4 amino acids upstream) and has an MW of 104. Approximately two-thirds of toxin A, including the enzymatic domain that is necessary for cytotoxicity, has been removed (13,29). Intraperitoneal (i.p.) injection of 0.5 mg of rARU (ϳ10,000 50% lethal doses of CDTA) produced no symptoms in hsd/ICR outbred mice. rARU was expressed in E. coli BL21(DE3) in the pRSET system (Invitrogen, San Diego, Calif.). rARU contains a six-histidine tag but was purified from E. coli lysate by sequential ammonium sulfate precipitation and gel filtration through CL-6B Sepharose (Sigma, St. Louis, Mo.) as described previously for the CDTA (24). The solution was concentrated to ϳ1 mg/ml, sterilely filtered, and stored at 4°C.
Polysaccharides. Pneumococcal type 14 polysaccharide, lot 40235-001, was manufactured by Lederle Laboratories, Pearl River, N.Y. S. flexneri type 2a O-specific polysaccharide and E. coli K1 polysaccharide were purified as described elsewhere (9,12,37). All preparations had less than 1% protein and nucleic acid. Analytic methods. Protein was assayed by the method of Lowry et al. (21), and saccharide was assayed by the anthrone and ninhydrin methods as described previously (8,39,43). Derivatization with adipic acid hydrazide was measured by the TNBS assay (8). The extent of succinylation was measured indirectly by the reduction in amino groups of rARU using lysine as the standard (14,28,43).
Succinylation of rARU. Preliminary experiments defined the conditions that succinylated rARU while retaining its antigenicity as measured by double immunodiffusion with goat anti-CDTA (28). Succinic anhydride was added to rARU at 1/10 (wt/wt) at room temperature with mixing; the pH was maintained at 7.2 to 7.5 with 0.5 M NaOH in a pH stat. After 20 min, the reaction mixture was passed through a 2.5-by 50-cm Sephadex G-50 column in 0.2 M NaCl, and the void volume peak was pooled and concentrated.
Conjugation of polysaccharides to rARU and to rARUsucc. Pneumococcal type 14 polysaccharide and S. flexneri type 2a O-specific polysaccharide were activated with cyanogen bromide, derivatized with adipic acid dihydrazide, and bound to rARU or rARUsucc by water-soluble carbodiimide condensation as described elsewhere with the exception that the pH of the reactants was maintained with 0.1 M MES, pH 6.0 (8,9,37). E. coli K1 polysaccharide was both derivatized with adipic acid dihydrazide and bound to rARU or rARUsucc by treatment with EDC (12). The composition of the adipic acid hydrazide-derivatized polysaccharides and of the conjugates is shown in Table 1. Note that low yields of conjugates, using rARU as the carrier, were obtained with the pneumococcal type 14 and S. flexneri type 2a polysaccharides. We were unable to synthesize a conjugate of the K1 polysaccharide with rARU.
Vaccination of mice. Female 5-week-old general-purpose Swiss Albino mice at the National Institutes of Health or outbred hsd/ICR mice (Harlan-Sprague-Dawley, Inc., Indianapolis, Ind.) at Techlab were injected subcutaneously (s.c.) with 0.1 ml containing 2.5 g of polysaccharide in the conjugate every 2 weeks. Mice (n ϭ 10) were exsanguinated 2 weeks after the first injection and 1 week after the second and third injections.
Abs to CDTA were measured by ELISA, with CDTA as the coating antigen, and by in vitro neutralization of cytotoxicity (24). Human intestinal HT-29 cells (ATCC HTB 38) were maintained in 96-well plates with McCoy's 5A medium supplemented with 10% fetal calf serum in a 5% CO 2 atmosphere (HT-29 cells were chosen because of their high sensitivity to CDTA probably because of the high density of the carbohydrate receptor on their surface). Serial twofold dilutions of sera were incubated with 0.4 g of CDTA per ml for 30 min at room temperature. CDTA-serum mixtures were added to the wells at a final concentration of 20 ng of the toxin per well (about 200 times the minimal cytotoxic dose for HT-29 cells) in a final volume of 0.2 ml. The neutralization titer is expressed as the reciprocal of the highest dilution that completely neutralized cytotoxicity.
Protection against lethal challenge of mice with CDTA. hsd/ICR mice were injected s.c. with 6.94 g of rARU. The dose of 2.5 g of polysaccharide for the conjugates contained 6.94 g of rARU for SF-rARU and 3.9 g of rARU for SF-rARUsucc (see above). Controls were mice injected with 0.1 ml of phosphate-buffered saline (PBS) by the same scheme. The mice were challenged by  a Six-week-old mice were injected s.c. with 2.5 g of pneumococcal type 14 polysaccharide as a conjugate at 2-week intervals. Mice (n ϭ 10) were exsanguinated 2 weeks after the first injection and 7 days after the second and third injections, and their sera were assayed for IgG and IgM anti-pneumococcal type 14 polysaccharide by ELISA. A hyperimmune serum, arbitrarily assigned a value of 100 EU, was the reference (37). Statistical significance of IgG results: second-and third-injection values versus first-injection values for both conjugates, P Ͻ 0.005; third-injection values versus second-injection values for both conjugates, not significant; third injection of Pn14-rARUsucc versus that of Pn14-rARU, no significance.

RESULTS
Pneumococcal type 14 Abs. Both conjugates elicited statistically significant rises of IgG Abs after the first and the second injections (P Ͻ 0.005) ( Table 2). The third injection of both conjugates elicited rises in IgG (4.38 to 6.41 ELISA units [EU] for Pn14-rARU and 6.10 to 9.76 EU for Pn14-rARUsucc) and IgM (4.82 to 7.57 EU for Pn14-rARU and 6.16 to 8.54 EU for Pn14-rARUsucc), but these were not statistically significant. Pneumococcal type 14 polysaccharide alone elicits only trace levels of Abs in mice (37); PBS did not elicit Abs (data not shown).
The correlation coefficients of Ab levels induced by both conjugates after each injection, as measured by RIA or ELISA, were statistically significant (Table 3).
S. flexneri type 2a IgG LPS Abs. Both SF-rARU and SF-rARUsucc elicited LPS Abs after the second injection compared to prevaccination levels (P ϭ 0.001) ( Table 4). Reinjection for the third time elicited a rise of IgG anti-LPS for both conjugates, but the rise was statistically significant only for SF-rARUsucc (2.48 versus 0.37 EU, P ϭ 0.04). The S. flexneri type 2a IgG anti-LPS levels induced by the two conjugates were not statistically different.  Table 5). Since the 2.5-g immunizing dose of the conjugate was based upon its polysaccharide component, the amount of rARU injected was different for each conjugate. To illustrate, on a protein weight basis, Pn14-rARU, with 1.29 g of rARU, elicited 194 g of CDTA Ab/ml (150.3 g of Ab/g of rARU injected). In contrast, Pn14-rARUsucc, which contained 7.3 g of rARU per dose, elicited 371 g of CDTA Ab/ml (50.8 g of Ab/g of rARUsucc injected). Pn14-rARU induced less anti-CDTA per g of rARU than did Pn14-rARUsucc, but the higher total amount of anti-CDTA elicited by the latter was due to its higher content of rARU. The difference between the level of anti-CDTA elicited by Pn14-rARU (194 g of CDTA Ab/ml) and that elicited by Pn14-rARUsucc (371 g of CDTA Ab/ml) was significant (P ϭ 0.01).
SF-rARU, containing 3.9 g of rARU, elicited 437 g of CDTA Ab/ml (112.0 g of Ab/g of rARU injected) compared to 518 g of CDTA Ab/ml for SF-rARUsucc (34.9 g of Ab/g of rARUsucc injected). Although the specific immunogenic activity for the rARUsucc was lower than that of the rARU in the S. flexneri type 2a conjugates, there was no statistical difference between the levels of CDTA Ab elicited by the two conjugates (437 g of Ab/ml for SF-rARUsucc versus 242 g of Ab/ml for SF-rARU).
K1-rARUsucc, which elicited 390 EU of CDTA Ab/ml, had a Five-week-old mice were injected s.c. with 2.5 g of S. flexneri type 2a O-specific polysaccharide alone or as a conjugate at 2-week intervals. Mice (n ϭ 10) were exsanguinated before and 7 days after the third injection, and their sera were assayed for IgG anti-LPS by ELISA. A hyperimmune serum pool, arbitrarily assigned a value of 100 EU, served as a reference (29). Polysaccharide alone did not elicit Abs (data not shown). Statistical significance: third-injection value versus second-injection value for IgG anti-SF-rARUsucc, P ϭ 0.04. a Six-week-old mice were injected s.c. with 2.5 g of polysaccharide as a conjugate at 2-week intervals. Groups of mice (n ϭ 10) were exsanguinated before the first injection and 7 days after the second and third injections, and their sera were assayed for anti-CDTA by ELISA. A hyperimmune serum, arbitrarily assigned a value of 100 EU, served as a reference. ND, not done.  Table 6). All sera from mice immunized with the conjugates had a neutralizing titer of Ն64 (data not shown). The geometric mean and range of neutralizing titers for each immunogen are shown in Table 6. Conjugate-induced Ab levels approached or surpassed the neutralizing activity of an affinity-purified goat Ab, containing 0.5 mg/ml, that was raised against formalininactivated CDTA.
In vivo protection of mice against CDTA. hsd/ICR mice were injected with SF-rARU, SF-rARUsucc, or rARU (see above). One week after the third injection, the mice were challenged i.p. with a lethal dose (150 ng) of CDTA. Almost all mice vaccinated with either conjugate or the rARU were protected (Table 7). Based upon the amount injected, rARU and SF-rARU elicited similar levels of anti-CDTA. As expected, SF-rARUsucc elicited lower levels of anti-CDTA than did the other two immunogens, but the recipients were comparably protected.

DISCUSSION
Although we were able to bind polysaccharides to rARU, only low yields of conjugates were obtained. Precipitation followed concentration of the rARU to 3 to 5 mg/ml. Accordingly, low concentrations of reactants had to be used for the conjugation step, reducing its efficiency and resulting in low yields of conjugates. We obtained high yields of conjugates only with the succinylated rARU. Succinic anhydride (dihydro-2,5-furandione) reacts rapidly with the ε-amino groups of lysines and with the N-amino acid termini of proteins in aqueous solutions at pH 7 to 8 by replacing the amino group with a carboxyl group (28). Carbodiimide-mediated condensation, designed to bind the hydrazide on the polysaccharide to the carboxyls of proteins, is likely accompanied by side reactions that include forming amide bonds between ε-amino groups of  a Neutralizing titers were the highest serum dilution that completely inhibited the cytotoxicity of CDTA (20 ng/well) on HT-29 cells (Materials and Methods). The titers represent the geometric means (GM) Ϯ standard deviations from general-purpose Swiss Albino mice (n ϭ 10) obtained 7 days after the third injection. Anti-CDTA was also measured by ELISA, and the individual values are expressed as micrograms of Ab per milliliter of serum.
b Affinity-purified goat Ab. lysines with adjacent carboxyls of the protein (intramolecular cross-linking) or with adjacent protein molecules (intermolecular cross-linking). Prior conversion of these amino residues by succinylation reduces the intra-and intermolecular amide formation during the conjugation step and provides additional carboxyl groups capable of binding the hydrazide-derivatized polysaccharide. Treatment with succinic anhydride has been shown to inactivate diphtheria and tetanus toxins and stabilize the resultant toxoids against aggregation (38). The by-product of succinic anhydride hydrolysis in water is the metabolite succinic acid.
The rARUsucc protein served as an effective carrier for conjugates of three medically useful and structurally different polysaccharides. Figure 1 shows that the pneumococcal type 14 polysaccharide is a neutral high-MW branched copolymer (20), S. flexneri type 2a O-specific polysaccharide is a comparatively lower-MW neutral branched copolymer (7,19), and each subunit of E. coli K1, a linear high-MW homopolymer, is negatively charged (4). Thus, the use of rARU as a carrier is likely to be applicable to all polysaccharides.
All three conjugates elicited high levels of serum CDTA Abs with in vitro neutralizing activity and in vivo protection. On a weight basis, rARU as a component of the three conjugates was as immunogenic as rARU alone. As expected, the specific immunogenic activity of rARU was greater than that of succinyl derivatives of rARU. However, the higher protein content of the succinylated derivatives compensated for this difference. The minimum protective level of CDTA Abs is, as yet, not known.
There were no significant differences between the levels of polysaccharide Abs elicited by the pneumococcal type 14 and S. flexneri type 2a O-specific polysaccharide conjugates with rARU and the levels elicited by rARUsucc. The levels of Abs to each of the three polysaccharides elicited by the conjugates prepared with rARUsucc were similar to those reported previously (12,23,37).
In summary, rARU served both as an effective carrier for conjugates of three medically useful polysaccharides and as an immunogen for neutralizing Abs to CDTA. Conjugates prepared with rARU as the carrier could induce active immunity or serve to prepare hyperimmune globulin as therapy for pseudomembranous enterocolitis. Lastly, rARU is relatively easy to produce in a clinically acceptable form and could be added to the conjugate formulation for routine immunization of infants.