A completely synthetic toxoid vaccine containing Escherichia coli heat-stable toxin and antigenic determinants of the heat-labile toxin B subunit

Abstract

The immunodeterminant regions of the Escherichia coli heat-labile toxin B subunit were identified by determining the antigenicity, by using enzyme-linked immunosorbent assays, of synthetically produced peptides corresponding to various segments of its 124-amino-acid sequence. The addition of the 18-amino-acid sequence of heat-stable toxin (ST) to some of these peptides enhanced their B subunit antigenicity. Peptide residues containing the 26 amino acids of B subunit sequence 58 to 83 joined to the 18-amino-acid sequence of ST yielded a 44-amino-acid peptide whose antigenicity was 50% that of both native B subunit and ST. This peptide was completely nontoxic when tested in Chinese hamster ovary tissue culture, suckling mouse, and rat ligated ileal loop assays. Peroral immunization of rats with the polymeric form of this peptide yielded a dose-dependent response of intestinal immunoglobulin A antitoxin titers to both the ST and B subunit components and provided strong protection against challenge with viable STand heat-labile toxin-producing E. coli strains. The immunogenicity of the synthetic peptide in rats was the same as that of ST and about 50% that of native B subunit. The completely synthetic peptide vaccine has the following advantages over previously described toxoid vaccines that consist of synthetic ST chemically cross-linked to native B subunit derived from bacterial cultures: it is produced by a single synthetic process, it is completely nontoxic, and it is immunogenic for both ST and B subunit.

Opsonic antibodies evoked by hybrid peptide copies of types 5 and 24 streptococcal M proteins synthesized in tandem.

“…Recently, Houghten et al (20) reported the stimulation of protective immune responses against both the heat-labile and heat-stable toxins of Escherichia coli by peroral administration of a hybrid peptide containing a 26 amino acid-residue copy of the labile toxin joined in tandem to an 18-residue copy of stable toxin. However, we believe our studies are the first to show that a synthetic hybrid peptide can raise antibodies that promote the phagocytosis and killing of two different bacterial pathogens.…”

“…Synthetic copies of the COOH-terminus, 23-35, of the cyanogen bromide-derived fragment 7 (CB7) of type 24 M protein (17), and the NH~-terminus, 1-20, of pep M5 (9) were the same as those used and reported in previous studies (9,10). They are designated S-CB7(23-35)C and S-M5 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), respectively. In addition, two hybrid peptides, S-M5(1-20)-S-CB7(23-35)C and S-M5(1-20)-S-CB7 (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34) were synthesized by the solid-phase method of Merrifield (18) as previously described (10).…”

Escherichia coli heat-labile toxin subunit B fusions with Streptococcus sobrinus antigens expressed by Salmonella typhimurium oral vaccine strains: importance of the linker for antigenicity and biological activities of the hybrid proteins

“…LT-I and CT and their B subunits were described as very strong immunogens which elicit high-titer serum and secretory antibodies when administered into the gut via the oral route in microgram amounts of protein (21,45) or when delivered to the lymphoid tissue by attenuated Salmonella strains (7,8,11). The B subunits of both enterotoxins also enhance the immunogenicity of relatively poor immunogens when mixed or coupled (chemically or genetically) and given orally (13,15,21,31,40,41). Czerkinsky et al (13) demonstrated that intragastric immunization of mice with Streptococcus mutans antigen I/II covalently coupled to the B subunit of CT elicits a significant immune response (mucosal and serum immunoglobulin A [IgA] and IgG), although subclinical doses of free CT were required to obtain the optimal antibody response.…”

Optimal conditions for the toxoiding of pertussis toxin with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide · HCl

“…When carbodiimides are used in protein modification, reaction conditions can be more easily controlled and side reactions and the formation of undesirable side-products may be effectively suppressed [31]. Examples of successful carbodiimide-mediated conjugations include meningococcal capsular polysaccharide-tetanus toxoid vaccines [32,33] and E. coli enterotoxin vaccines [34]; other work has shown that EDAC toxoids are stable at 4 ° C and highly immunogenic in mice [15]. In view of these findings, such toxoids should be given further consideration for use in human vaccines and a reappraisal of toxoiding reagents for first and second generation acellular pertussis vaccines is long overdue.…”