New-generation vaccines, formulated with subunits or nucleic acids, are less immunogenic than classical vaccines formulated with live-attenuated or inactivated pathogens. This difference has led to an intensified search for additional potent vaccine adjuvants that meet safety and efficacy criteria and confer long-term protection. This review provides an overview of protein-based adjuvants (PBAs) obtained from different organisms, including bacteria, mollusks, plants, and humans. Notably, despite structural differences, all PBAs show significant immunostimulatory properties, eliciting B-cell- and T-cell-mediated immune responses to administered antigens, providing advantages over many currently adopted adjuvant approaches. Furthermore, PBAs are natural biocompatible and biodegradable substances that induce minimal reactogenicity and toxicity and interact with innate immune receptors, enhancing their endocytosis and modulating subsequent adaptive immune responses. We propose that PBAs can contribute to the development of vaccines against complex pathogens, including intracellular pathogens such as Mycobacterium tuberculosis, those with complex life cycles such as Plasmodium falciparum, those that induce host immune dysfunction such as HIV, those that target immunocompromised individuals such as fungi, those with a latent disease phase such as Herpes, those that are antigenically variable such as SARS-CoV-2 and those that undergo continuous evolution, to reduce the likelihood of outbreaks.

The development of vaccine adjuvants is of interest for the management of chronic diseases, cancer, and future pandemics. Therefore, the role of Toll-like receptors (TLRs) in the effects of vaccine adjuvants has been investigated. TLR4 ligand-based adjuvants are the most frequently used adjuvants for human vaccines. Among TLR family members, TLR4 has unique dual signaling capabilities due to the recruitment of two adapter proteins, myeloid differentiation marker 88 (MyD88) and interferon-β adapter inducer containing the toll-interleukin-1 receptor (TIR) domain (TRIF). MyD88-mediated signaling triggers a proinflammatory innate immune response, while TRIF-mediated signaling leads to an adaptive immune response. Most studies have used lipopolysaccharide-based ligands as TLR4 ligand-based adjuvants; however, although protein-based ligands have been proven advantageous as adjuvants, their mechanisms of action, including their ability to undergo structural modifications to achieve optimal immunogenicity, have been explored less thoroughly. In this work, we characterized the effects of two protein-based adjuvants (PBAs) on TLR4 signaling via the recruitment of MyD88 and TRIF. As models of TLR4-PBAs, we used hemocyanin from Fissurella latimarginata (FLH) and a recombinant surface immunogenic protein (rSIP) from Streptococcus agalactiae. We determined that rSIP and FLH are partial TLR4 agonists, and depending on the protein agonist used, TLR4 has a unique bias toward the TRIF or MyD88 pathway. Furthermore, when characterizing gene products with MyD88 and TRIF pathway-dependent expression, differences in TLR4-associated signaling were observed. rSIP and FLH require MyD88 and TRIF to activate nuclear factor kappa beta (NF-κB) and interferon regulatory factor (IRF). However, rSIP and FLH have a specific pattern of interleukin 6 (IL-6) and interferon gamma-induced protein 10 (IP-10) secretion associated with MyD88 and TRIF recruitment. Functionally, rSIP and FLH promote antigen cross-presentation in a manner dependent on TLR4, MyD88 and TRIF signaling. However, FLH activates a specific TRIF-dependent signaling pathway associated with cytokine expression and a pathway dependent on MyD88 and TRIF recruitment for antigen cross-presentation. Finally, this work supports the use of these TLR4-PBAs as clinically useful vaccine adjuvants that selectively activate TRIF- and MyD88-dependent signaling to drive safe innate immune responses and vigorous Th1 adaptive immune responses.

Introduction: Mollusk hemocyanins are known for their immunomodulatory properties in mammals. Their applications include serving as carrier glycoproteins, functioning as protein-based adjuvants, and acting as non-specific immunostimulants in cancer vaccine strategies. Their immunomodulatory effects are attributed to their xenogenicity, structural complexity, high molecular mass, and glycosylations. Recent studies have begun to clarify the immunological mechanisms by which hemocyanins induce: multiligand properties arising from interactions with C-type lectins and Toll-like receptors, and the promotion of a Th1 immune response. However, the subsequent effects of hemocyanins, particularly their intracellular targeting and degradation kinetics, remain poorly understood. The present study is the first to comprehensively examine the processing of two well-characterized hemocyanins, known for their conformational stability and clinical significance: KLH from Megathura crenulata and CCH from Concholepas concholepas . Methods: We correlated their degradation with the kinetics of the proinflammatory response they induce and their subcellular localization using the JAWS II cell line and bone marrow-derived dendritic cells (BMDCs). We utilize OVA to highlight the differences between this protein and the hemocyanins. Results: The results showed that KLH and CCH induced significant TNF levels after 24 hours and also promoted the secretion of IL-6 and IL-12p40 after 96 hours, along with the upregulation of CD80 and CD86. This delayed response corresponds with their slow intracellular degradation. Colocalization studies using LAMP-1 demonstrated that hemocyanins were localized to lysosomes only after prolonged stimulation, suggesting that they are likely stored in intracellular depots. Furthermore, hemocyanins were shown to colocalize with LMP-2 and a4, indicating that they undergo processing in the proteasome. In contrast, OVA displayed faster degradation with mild pro-inflammatory effects within 24 hours. Pharmacological inhibition of lysosomal cathepsins or the proteasome reduced the hemocyanin-dependent secretion of IL-6 and IL-12p40. Additionally, fragmented hemocyanins led to lower, less sustained cytokine levels compared to their native form. Conclusions: These findings emphasize that hemocyanins, owing to their complex oligomeric structure and high stability, are slowly processed by APCs, thereby contributing to their immunogenicity. This property is particularly relevant when hemocyanins are used as carriers for vaccine antigens because their delayed kinetics can enhance the magnitude, quality, and persistence of responses.