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

The immunogenicity of the surface polysaccharides of bacterial pathogens is improved when these antigens are bound covalently to a carrier protein (conjugate) (33, 35). Most carriers have been medically useful proteins, including inactivated tetanus, diphtheria, pertussis, and Pseudomonas aeruginosa toxins (1, 8, 9, 11, 12, 28, 33–35, 37). Thus, conjugate vaccines may confer immunity against pathogens whose protective antigens are the carrier proteins, including those that cause toxin-mediated diseases. One variable affecting serum antibody (Ab) responses to the saccharide component is the carrier protein. For example, a genetically inactivated diphtheria toxin (CRM197) was a more effective carrier than the formalin-treated toxoid (1). In addition, treatment of two genetically inactivated medically important antigens, diphtheria toxin (CRM9) 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 polysaccharide-protein 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.