Individuals suffering from severe viral respiratory tract infections have recently emerged as “at risk” groups for developing invasive fungal infections. Influenza virus is one of the most common causes of acute lower respiratory tract infections worldwide.

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.

Hemocyanins are used as immunomodulators in clinical applications because they induce a strong Th1‐biased cell‐mediated immunity, which has beneficial effects. They are multiligand glycosylated molecules with abundant and complex mannose‐rich structures. It remains unclear whether these structures influence hemocyanin‐induced immunostimulatory processes in human APCs. We have previously shown that hemocyanin glycans from Concholepas concholepas (CCH), Fissurella latimarginata (FLH), and Megathura crenulata (KLH), participate in their immune recognition and immunogenicity in mice, interacting with murine C‐type lectin receptors (CLRs). Here, we studied the interactions of these hemocyanins with two major mannose‐binding CLRs on monocyte‐derived human DCs: MR (mannose receptor) and DC‐SIGN (DC‐specific ICAM‐3–grabbing nonintegrin). Diverse analyses showed that hemocyanins are internalized by a mannose‐sensitive mechanism. This process was calcium dependent. Moreover, hemocyanins colocalized with MR and DC‐SIGN, and were partly internalized through clathrin‐mediated endocytosis. The hemocyanin‐mediated proinflammatory cytokine response was impaired when using deglycosylated FLH and KLH compared to CCH. We further showed that hemocyanins bind to human MR and DC‐SIGN in a carbohydrate‐dependent manner with affinity constants in the physiological concentration range. Overall, we showed that these three clinically valuable hemocyanins interact with human mannose‐sensitive CLRs, initiating an immune response and promoting a Th1 cell‐driving potential.

Vaccine adjuvants play a crucial role in the field of vaccinology, yet they remain one of the least developed and poorly characterized components of modern biomedical research. The limited availability of clinically approved adjuvants highlights the urgent need for new molecules with well-defined mechanisms and improved safety profiles. Hemocyanins, large copper-containing metalloglycoproteins found in mollusks, represent a unique class of natural immunomodulators. Hemocyanins serve as carrier proteins that help generate antibodies against peptides and hapten molecules. They also function as non-specific protein-based adjuvants (PBAs) in both experimental human and veterinary vaccines. Their mannose-rich N-glycans allow for multivalent binding to innate immune receptors, including C-type lectin receptors (e.g., MR, DC-SIGN) and Toll-like receptor 4 (TLR4), thereby activating both MyD88- and TRIF-dependent signaling pathways. Hemocyanins consistently favor Th1-skewed immune responses, which is a key characteristic of their adjuvant potential. Remarkably, their conformational stability supports slow intracellular degradation and facilitates dual routing through MHC-II and MHC-I pathways, thereby enhancing both CD4+ and CD8+ T-cell responses. Several hemocyanins are currently being utilized in biomedical research, including Keyhole limpet hemocyanin (KLH) from Megathura crenulata, along with those from other gastropods such as Concholepas concholepas (CCH), Fissurella latimarginata (FLH), Rapana venosa (RvH), and Helix pomatia (HpH), all of which display strong immunomodulatory properties, making them promising candidates as adjuvants for next-generation vaccines against infectious diseases and therapeutic immunotherapies for cancer. However, their structural complexity has posed challenges for their recombinant production, thus limiting their availability from natural sources. This reliance introduces variability, scalability issues, and challenges related to regulatory compliance. Future research should focus on defining the hemocyanin immunopeptidome and isolating minimal peptides that retain their adjuvant activity. Harnessing advances in structural biology, immunology, and machine learning will be critical in transforming hemocyanins into safe, reproducible, and versatile immunomodulators. This review highlights recent progress in understanding how hemocyanins modulate mammalian immunity through their unique structural features and highlights their potential implications as potent PBAs for vaccine development and other biomedical applications. By addressing the urgent need for novel immunostimulatory platforms, hemocyanins could significantly advance vaccine design and immunotherapy approaches.

While Fc-CTLRs probes are useful tools to study the interaction of CTLRs with ligands, their use is limited by the lack of appropriate negative controls in assays involving fungi and potentially other pathogens. We have developed and characterized 2 negative controls for Fc-CTLRs assays: Fc-control and a Fc-Dectin-1 mutant.