Modulation of humoral immunity to a ricin vaccine antigen by wild-type and mutant type II heat-labile enterotoxins
Greene, Christopher John
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As incidents of bioterrorism increase across the globe, the need to develop safe and effective vaccines against potential biological agents is high. A large effort is currently underway to develop a safe and effective vaccine against ricin toxin, which has been identified as a weapon of mass destruction and potential bioterror weapon. One of the top candidate antigens (Ag) for the ricin vaccine is RiVax, an engineered detoxified ricin A-subunit. There is a concurrent search for novel adjuvants to augment the ricin vaccine for administration specifically in the skin. The type II heat-labile enterotoxins (HLT) expressed by enterotoxigenic Escherichia coli were initially identified as potent mucosal adjuvants when administered by the intranasal (i.n.) route. Due to inherent toxicity, neuronal trafficking along the olfactory bulb, and neuroinflammatory propensities of HLT when administered intranasally, their use as adjuvants by this route has been discouraged. Nevertheless, this setback has fostered exploration of new, alternative routes of administration of HLT and for the development of detoxified, mutant HLT. LT-IIb and LT-IIb(T13I) are the best studied members of the type II subfamily of HLT. In this study, the ability of LT-IIb and LT-IIb(T13I), a mutant HLT with alterations in receptor-binding affinity, to augment humoral immunity to a co-administered Ag (RiVax) by the intradermal (i.d.) route was evaluated. Results of this study demonstrated that LT-IIb and LT-IIb(T13I) possess potent i.d. adjuvant properties and enhance the production of RiVax-specific serum IgG antibodies (Ab). LT-IIb(T13I) is significantly less inflammatory than LT-IIb when administered into the skin. Strikingly, both LT-IIb and LT-IIb(T13I), when employed as i.d. adjuvants, enhanced the ability of RiVax to produce critical ricin-neutralizing Ab. Yet, when employed as intranasal adjuvants with RiVax, LT-IIb and LT-IIb(T13I) failed to enhance production of ricin-neutralizing Ab. Importantly, when subjected to ricin challenge, significantly more mice survived challenge when immunized by the i.d. route with RiVax and LT-IIb(T13I) in comparison to mice that received RiVax alone. Taken together, these data highlight the potent i.d. adjuvant properties of LT-IIb and LT-IIb(T13I) toward RiVax. Further development of these adjuvants as clinical agents requires an identification of the mechanism(s) that underlie their immunoenhancing properties. Analysis of tissue fluid and serum from mice intradermally immunized with RiVax in combination with LT-IIb or LT-IIb(T13I) showed robust production of IL-6, a cytokine known to drive humoral immunity. I.d. immunization of IL-6 knockout mice with RiVax and LT-IIb or LT-IIb(T13I) resulted in significantly decreased RiVax-specific IgG Ab in comparison to their wild-type counterparts. I.d. administration of recombinant IL-6 with RiVax produced Ag-specific and ricin-neutralizing Ab to levels that were similar to those produced by i.d. immunization of mice with RiVax and LT-IIb. Finally, a cellular source of IL-6 induced by LT-IIb and LT-IIb(T13I) was identified as skin infiltrating neutrophils. Ablation of neutrophils was correlated with a decrease in RiVax-specific IgG Ab. These data provided strong evidence that IL-6 and infiltrating neutrophils are important components of the i.d. adjuvant response of LT-IIb enterotoxins. In summation, the capacity of LT-IIb and LT-IIb(T13I) to function as i.d. adjuvants to enhance B cell activity and stimulate Ag-specific humoral immune responses is critical for subsequent development of effective protein subunit vaccines designed for administration into the skin. Overall, the non-toxic, low inflammatory nature of LT-IIb(T13I) combined with its potent adjuvant properties indicates that this mutant type II HLT is a novel, safe, and effective i.d. adjuvant with potential clinical utility.