Comparative Adjuvant Effects of Type II Heat-Labile Enterotoxins in Combination with Two Different Candidate Ricin Toxin Vaccine Antigens

Abstract

Type II heat-labile enterotoxins (HLTs) constitute a promising set of adjuvants that have been shown to enhance humoral and cellular immune responses when coadministered with an array of different proteins, including several pathogen-associated antigens. However, the adjuvant activities of the four best-studied HLTs, LT-IIa, LT-IIb, LT-IIb T13I , and LT-IIc, have never been compared side by side. We therefore conducted immunization studies in which LT-IIa, LT-IIb, LT-IIb T13I , and LT-IIc were coadministered by the intradermal route to mice with two clinically relevant protein subunit vaccine antigens derived from the enzymatic A subunit (RTA) of ricin toxin, RiVax and RVEc. The HLTs were tested with low and high doses of antigen and were assessed for their abilities to stimulate antigen-specific serum IgG titers, ricin toxin-neutralizing activity (TNA), and protective immunity. We found that all four HLTs tested were effective adjuvants when coadministered with RiVax or RVEc. LT-IIa was of particular interest because as little as 0.03 g when coadministered with RiVax or RVEc proved effective at augmenting ricin toxin-specific serum antibody titers with nominal evidence of local inflammation. Collectively, these results justify the need for further studies into the mechanism(s) underlying LT-IIa adjuvant activity, with the long-term goal of evaluating LT-IIa's activity in humans.T he absence of effective adjuvants constitutes a critical bottleneck to the development of protein subunit vaccines for biodefense and emerging infectious diseases (1-3). RiVax, for example, is a recombinant, attenuated derivative of the enzymatic subunit of ricin toxin (RTA), which has been deemed to be safe for human use (4, 5). Unfortunately, phase I clinical trials indicated that even repeated high-dose immunizations were relatively poor at stimulating ricin-specific IgG antibodies and toxin-neutralizing activity (TNA) in sera of volunteers. Adsorption of RiVax to aluminum salts adjuvant only marginally improved the antibody response to the vaccine antigen, indicating that the future success of RiVax and other ricin toxin subunit vaccine antigens under investigation, like RVEc, may depend on the identification of morepotent adjuvants (6).The type I and type II bacterial heat-labile enterotoxins (HLTs) are among the most potent adjuvants described to date. The HLTs are AB 5 subunit toxins, consisting of a single ADP-ribosylating ("A") subunit joined noncovalently to pentamers of gangliosidebinding ("B 5 ") subunits. The well-characterized type I HLTs include cholera toxin (CT), expressed by Vibrio cholerae, and LT enterotoxin, expressed by enterotoxigenic strains of Escherichia coli. The type II enterotoxins, on the other hand, are less well characterized and more diverse than CT or LT (7,8). The beststudied type II HLTs are LT-IIa, LT-IIb, and LT-IIc, each of which has been shown to enhance antigen-specific immune responses when coadministered with a model antigen like ovalbumin (OVA) (9). Adjuvant activities of the type I and II HL...

High-Definition Mapping of Four Spatially Distinct Neutralizing Epitope Clusters on RiVax, a Candidate Ricin Toxin Subunit Vaccine

“…The loss of epitopes corresponding to clusters III and IV, as well as to part of cluster II, explains why RVEc immunization results in a slight bias for antibodies against cluster I compared to RiVax (69). Nonetheless, in spite of lacking what are seemingly important neutralizing B cell epitopes, RVEc has proven as effective as RiVax at eliciting immunity to ricin in mice across a range of doses and with different adjuvants (69,70).…”

Progress and challenges associated with the development of ricin toxin subunit vaccines

“…All vaccinated animals survived challenge, even groups that received only a prime boost regimen with the lowest dose of vaccine. Although RVEc was slightly more effective than RiVax at eliciting toxin-specific serum IgG titers and toxin-neutralizing activity (TNA) after only two immunizations, this trend was not apparent in a more recent study in which RiVax and RVEc were administered to mice by the intradermal routes [64]. While the two vaccines have not been tested head to head in the mouse model using the intramuscular route of vaccination to mimic the route by which the vaccines would be given to humans, there is no reason to expect that they would differ under such circumstances.…”

“…Investigations into the effects of different “next generation” adjuvants like AS01 on RiVax and RVEc is clearly warranted [75–77]. Towards this goal we recently demonstrated that LT-IIa and LT-IIb T13I , two related ADP-ribosylating enterotoxins from E.coli are potent adjuvants when combined with RiVax and RVEc and administered to mice by the intradermal route [64,78]. It remains to be determined whether either of these adjuvants is active in humans.…”

“…In dozens of experiments involving hundreds of mice conducted with RiVax and RVEc over the past three years, we have found that animals with detectable serum TNA are virtually guaranteed to survive a lethal dose ricin challenge [63,64,69,78]. In this respect, TNA can be considered an absolute correlate of protection, which Plotkin defines as “a quantity of a specific immune response to a vaccine that always provides near 100% protection” [90].…”

Recent advances in the development of vaccines against ricin

“…10,11 Despite structural differences between these 2 subunit vaccines, headto-head comparisons have indicated that RiVax and RVEc when adsorbed to Alhydrogel or co-administered with LT-II adjuvants have identical capacities to induce neutralizing antibodies and to protect in mice. 12,13 No head-to-head comparisons have yet been attempted in non-human primates although intramuscular administration of RiVax adsorbed to aluminum hydroxide adjuvant protects rhesus macaques against the lethality of aerosolized RT.…”