Properties of cross-linked toxoid vaccines made with hyperantigenic forms of synthetic Escherichia coli heat-stable toxin

Abstract

The ability of hyperantigenic preparations of synthetically produced Escherichia coli heat-stable toxin (ST) to provide an immunogenically more potent vaccine when cross-linked by the glutaraldehyde reaction to the heat-labile toxin B subunit was assesed. Three synthetic ST preparations were evaluated: ST(S) had the same antigenicity and toxicity (secretory potency in the suckling mouse assay) as native ST, ST 1056 had 3.5-fold more antigenicity and 1% toxicity, and ST(C) had 15-fold greater antigenicity and 31% toxicity. Vaccines that contained equal antigenic proportions of ST and B subunit, as determined by enzyme-linked immunosorbent assays, consisted by weight of 52% ST(S), 25% ST 1056, and 9% ST(C). The initially lower toxicity and smaller proportions by weight of hyperantigenic ST preparations yielded vaccines that had nearly 10-fold less residual ST toxicity than the ST(S) vaccine. Immunization of rats with graded dosages of vaccines containing 9% ST(C) and 51% ST(S) by weight, but equal amounts of ST(S) antigenicity, raised to the same degree dose-dependent increases in mucosal immunoglobulin A antitoxin titers to ST(S) which correlated with the amount of protection against challenge with a viable LT-/ST' strain. These observations indicate that hyperantigenic synthetic ST preparations provide immunologically more potent vaccines than those obtained with the previously used synthetic ST(S) preparation, which has the same biological properties as native ST.

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

“…Assay for antigenicity. The antigenicities of the synthetic B fragment-ST peptides were determined by previously de-scribed techniques (18,19), with double-sandwich ELISAs, which used the following for the solid-phase/second antibody: (i) GM1 ganglioside (Supelco Inc., Bellefonte, Pa.) -crude goat hyperimmune antiserum to LT B subunit (GM1/B), (ii) crude rabbit and goat hyperimmune antisera to LT B subunit (B/B), or (iii) crude rabbit and goat hyperimmune antisera to synthetic ST (ST/ST). Serial dilutions of the synthetic B fragment-ST peptides were assayed concomitantly with either native B subunit or synthetic ST, and the antigenicities of the B fragment-ST peptides were expressed as a percentage of those of the respective toxins based on a comparison of those doses that yielded an optical density of 0.600 at 405 nm (13).…”

“…p.o. administration of this chemically cross-linked toxoid vaccine to rats or rabbits raises intestinal IgA antitoxin titers to both toxin components of the vaccine, thereby providing strong protection against challenge with either the ST and LT toxins themselves or viable E. coli strains which produce these toxins (13,14,17,18). Despite this apparent effectiveness in experimental animals, the chemically cross-linked toxoid vaccine does have two shortcomings.…”

“…We have not observed any evidence of toxicity in rats, rabbits, guinea pigs, or dogs given effective p.o. doses of the chemically cross-linked toxoid vaccine (18), and administration of the vaccine p.o. at a dose containing the equivalent of 192 ng of nonattenuated ST toxicity has not produced any adverse effect in human volunteers (F. A. Klipstein and R. F. Engert, unpublished observations).…”

“…The factors responsible for the marked enhancement of LT B subunit antigenicity in some but not all of these segments by the addition of the ST amino acid sequence to B subunit fragment peptides are unknown. The enhancement could possibly be related to changes in the configuration of the molecule; altering the stereochemistry of the ST molecule, for example, has a profound effect on its antigenicity and other properties (18). It is now recognized that the composition of the ST sequence that we used is in error in that the tyrosine at position 11 and the asparagine at position 18 should be interchanged (R. Giannella, personal communication).…”

“…This has been accomplished in the past by chemically cross-linking the heat-stable toxin (ST) to the nontoxic B subunit of the heat-labile toxin (LT) (13). Cross-linked toxoid vaccines prepared in this manner raise dose-dependent intestinal immunoglobulin A (IgA) antitoxin titers to both toxoid components in experimental animals, thereby providing strong protection against challenge with viable enterotoxigenic E. coli strains that produce either toxin (13,14,17,18).…”