Abstract
Polysaccharide-based vaccines are critical for preventing bacterial infections, yet their efficacy is often limited by weak antibody responses. Unfortunately, efficacious adjuvants for licensed native polysaccharide vaccines are lacking. The TLR4 agonist, monophosphoryl lipid A (MPL), significantly increases antibody responses to capsular polysaccharides in mice via B cell-intrinsic TLR4 and MyD88-dependent signaling. However, due to the lack of TLR4-driven adjuvant effects on polysaccharide-specific responses in non-human primates and the limited responsiveness of human B cells to TLR4 agonists, we sought to identify alternative MyD88-activating TLR agonists that could serve as suitable adjuvants to enhance humoral responses to polysaccharide vaccines in humans. In vitro assays revealed the TLR1/2 agonist Pam3CSK4 synergized with strong BCR crosslinking to optimally enhance both mouse and human B cell activation and antibody secretion. In vivo, Pam3CSK4 alone had no effect, but when paired with a squalene-based emulsion significantly increased polysaccharide-specific antibodies in both immunocompetent and PBMC-humanized mice that proved highly protective against lethal pneumococcal infections. Although a dual TLR2-7 agonist showed similar potent in vitro activity, it failed to enhance polysaccharide-specific IgG responses in vivo, mirroring the antagonistic effects observed when TLR2 and TLR7 agonists were combined both in vitro and in vivo. By contrast, inclusion of Pam3CSK4 in an adjuvant containing MPL, synthetic cord factor, and squalene emulsion further augmented protective polysaccharide-specific antibody responses in mice and rescued adjuvant effects in non-human primates. These findings reveal Pam3CSK4-containing formulations as promising adjuvants for native polysaccharide vaccines, with strong translational potential to enhance humoral immunity in humans.