TLR Tri-Agonist and TLR-Antigen Conjugates as Modulators of Innate Immunity and as Adjuvants in a Q-Fever Vaccine
- Author(s): Albin, Tyler James
- Advisor(s): Esser-Kahn, Aaron P
- Prescher, Jennifer A
- et al.
Although one of the greatest achievements of modern medicine, traditional vaccination strategies have failed to generate effective vaccines for many infections including global diseases, like tuberculosis, and regional diseases, like Q-Fever. New approaches are needed for each type of disease. The protective immunity and distinct responses of many successful vaccines come from activating multiple Toll-like Receptors (TLRs). Vaccines with multiple TLR agonists as adjuvants have proven effective in preclinical studies, but current research has not explored two important elements. First, few multi-TLR systems explore spatial organization – a critical feature of whole-cell vaccines. Secondly, no multi-TLR systems to date provide systematic analysis of the combinatorial space of three TLR agonists. This work describes the effects of conjugation between combinations of three TLR agonists and between TLR agonists and antigens on immunological activity. Here, we present the first examination of the combinatorial space of several spatially defined triple-TLR adjuvants, by synthesizing a series of five triple-TLR agonists and testing their innate activity both in vitro and in vivo. The combinations were evaluated by measuring activation of immune stimulatory genes (Nf-B, ISGs), cytokine profiles (IL12-p70, TNF-, IL-6, IL-10, CCL2, IFN-, IFN-, IFN-) and in vivo cytokine serum levels (IL-6, TNF-, IL12-p40, IFN-, IFN-). We demonstrate that linking TLR agonists substantially alters the resulting immune response compared to their unlinked counterparts and that each combination results in a distinct immune response, particularly between linked combinations. We show that combinations containing a TLR9 agonist produce more TH1 biasing immune response profiles, and that the effect is amplified upon conjugation. However, combinations containing TLR2/6 agonist are skewed toward TH2 biasing profiles despite the presence of a TLR9 agonist. To assess their efficacy in a vaccine, we formulated antigens from C. burnetii, the causative agent of Q-Fever, with TLR tri-agonists and evaluated the outcomes of vaccination in vivo. We found that the Tri-agonists elicited unique, antigen-specific immune responses in vivo which matched our initial in vitro analysis. We evaluated our top candidates in a live C. burnetii aerosol challenge model in mice and found two of our TLR tri-agonist containing formulations conferred partial protection to the challenged animals. Our findings characterize a novel adjuvant platform and offer an alternative approach to generating protective and effective vaccine candidates against C. burnetii. These results demonstrate the profound effects that conjugation and combinatorial administration of TLR agonists can have on immune responses, a critical element of vaccine development. In addition, numerous studies have shown that conjugation of TLR agonists to antigens can beneficially influence their potency, toxicity, pharmacokinetics, and function. Here, we present our work in developing site-specific conjugation approaches of TLR agonists to antigens.