Abstract

The development of vaccine adjuvants is needed to address persistent diseases, cancer, and future pandemics. Toll-like receptors (TLRs) have been researched as targets of vaccine adjuvants because they modulate the innate immune system and activate adaptive immunity. TLR4 signaling recruits the Myeloid Differentiation Marker 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF) adaptor proteins. MyD88‒mediated signaling leads to proinflammatory responses, and TRIF‒mediated signaling leads to an adaptive immune response. Given the role of TLR4 agonists, most studies have used ligands based on lipopolysaccharides, which have multiple side effects, including pyrogenicity and cytotoxicity. Here we propose the use of TLR4 protein-based agonists (TLR4-PBAs) which have advantages as adjuvants, including being natural biocompatible and biodegradable substances that induce minimal reactogenicity and toxicity. This work characterizes the effect of two TLR4-PBAs with proven adjuvant/immunostimulant properties on 1) downstream TLR4 signaling via MyD88 and TRIF and 2) their functional consequences in cross�presentation to CD8+ T lymphocytes. The TLR4-PBAs studied were the mollusk hemocyanin (a metaloglycoprotein) obtained from issurella latimarginata (FLH) and the recombinant Surface Immunogenic Protein from treptococcus agalactiae (rSIP). Using mouse bone marrow-derived dendritic cells, we determined that rSIP and FLH have differences in TLR4-associated signaling in MyD88 and TRIF-dependent genes. In this context, rSIP stimulated IL-6 and IP-10, dependent on the recruitment of MyD88 and TRIF, while FLH stimulated these inflammatory biomarkers with dependence on TRIF only. Despite this, rSIP and FLH require MyD88 and TRIF to activate NF-κB and IRF in the THP1 monocytic cell line. Furthermore, rSIP and FLH promote cross�presentation in CD8+ T lymphocytes, and the effect depends on TLR4 and the recruitment of MyD88 and TRIF. For the first time, these data show two TLR4-PBAs with effects dependent on the recruitment of MyD88 and TRIF. Their signaling is biased depending on the structural characteristics of each TLR4-PBA. The characterization of TLR4-PBAs with adjuvant profiles could be useful in vaccine development, allowing the use of the appropriate adjuvant depending on whether it is required to stimulate TRIF- or MyD88-dependent signaling.