A tractable covalent linker strategy for the production of immunogenic antigen-TLR7/8L bioconjugates

Abstract

Despite the ease of production and improved safety profiles of recombinant vaccines, the inherently low immunogenicity of unadjuvanted proteins remains an impediment to their widespread adoption. The covalent tethering of TLR agonists to antigenic proteins offers a unique approach to co-deliver both constituents to the same cell-enhancing vaccine efficacy while minimizing reactogenicity. However, the paucity of simple and effective linker chemistries continues to hamper progress. Here, we present a modular, PEG-based linker system compatible with even extremely lipophilic and challenging TLR7/8 agonists. To advance the field and address previous obstacles, we offer the most straightforward and antigen-preserving linker system to date. These antigen-adjuvant conjugates enhance antigen-specific immune responses in mice, demonstrating the power of our approach within the context of modern vaccinology.

Fig. 1

TLR7/8Ls (A) and their linker derivatizations (B). For all synthetic details including characterization, see ESI† Section 2.

Fig. 2

Antigen-TLR7/8L bioconjugates: (A) CRM-197 or BSA conjugates produced using two-step (C1 and C2) or one-step (C3 and C4) processes; "sans NHS" refers to the loss of NHS upon lysine functionalization; (B) representative MALDI-TOF mass spectra of unmodified CRM-197 (dark blue) and a bioconjugate (C3, red, average copy # of 5.1); [M + H]⁺ (rightmost peaks) and [M + 2H]²⁺ (leftmost peaks). For bioconjugation details including characterization, see ESI† Section 2.

Fig. 3

CRM-197-specific total IgG serum antibody titers 14 days after booster injection with indicated vaccine. Mice were immunized twice, 14 days apart, with 1 or 10 μg of CRM-197 either conjugated with UM-3006, admixed with the molar equivalent of free UM-3006, or alone. 14 days after secondary immunization, serum was collected, and CRM-197-specific total IgG, IgG1, and IgG2a antibody titers were measured by ELISA. For IgG isotype titers and T cell cytokine data see Fig. S16 (ESI†).