Mollusk hemocyanins have biomedical uses as carriers/adjuvants and nonspecific immunostimulants with beneficial clinical outcomes by triggering the production of proinflammatory cytokines in antigen-presenting cells (APCs) and driving immune responses toward type 1 T helper (Th1) polarization. Significant structural features of hemocyanins as a model antigen are their glycosylation patterns. Indeed, hemocyanins have a multivalent nature as highly mannosylated antigens. We have previously shown that hemocyanins are internalized by APCs through receptor-mediated endocytosis with proteins that contain C-type lectin domains, such as mannose receptor (MR). However, the contribution of other innate immune receptors to the proinflammatory signaling pathway triggered by hemocyanins is unknown. Thus, we studied the roles of Dectin-1, Dectin-2, and Toll-like receptor 4 (TLR4) in the hemocyanin activation of murine APCs, both in dendritic cells (DCs) and macrophages, using hemocyanins from Megathura crenulata ( KLH), Concholepas concholepas (CCH) and Fissurella latimarginata (FLH). The results showed that these hemocyanins bound to chimeric Dectin-1 and Dectin-2 receptors in vitro ; which significantly decreased when the glycoproteins were deglycosylated. However, hemocyanin-induced proinflammatory effects in APCs from Dectin-1 knock-out (KO) and Dectin-2 KO mice were independent of both receptors. Moreover, when wild-type APCs were cultured in the presence of hemocyanins, phosphorylation of Syk kinase was not detected. We further showed that KLH and FLH induced ERK1/2 phosphorylation, a key event involved in the TLR signaling pathway. We confirmed a glycan-dependent binding of hemocyanins to chimeric TLR4 in vitro . Moreover, DCs from mice deficient for MyD88-adapter-like (Mal), a downstream adapter molecule of TLR4, were partially activated by FLH, suggesting a role of the TLR pathway in hemocyanin recognition to activate APCs. The participation of TLR4 was confirmed through a decrease in IL-12p40 and IL-6 secretion induced by FLH when a TLR4 blocking antibody was used; a reduction was also observed in DCs from C3H/HeJ mice, a mouse strain with a nonfunctional mutation for this receptor. Moreover, IL-6 secretion induced by FLH was abolished in macrophages deficient for TLR4. Our data showed the involvement of TLR4 in the hemocyanin-mediated proinflammatory response in APCs, which could cooperate with MR in innate immune recognition of these glycoproteins.

Hemocyanins are widely used as carriers, adjuvants, and nonspecific immunostimulants in cancer because they promote Th1 immunity in mammals. Hemocyanins also interact with glycan-recognizing innate immune receptors on antigen-presenting cells, such as the C-type lectin immune receptors mannose receptor (MR), macrophage galactose lectin (MGL), and the Toll-like receptors (TLRs), stimulating proinflammatory cytokine secretion. However, the role of N-linked oligosaccharides on the structural and immunological properties of hemocyanin is unclear. Mollusk hemocyanins, such as Concholepas concholepas (CCH), Fissurella latimarginata (FLH), and Megathura crenulata (KLH), are oligomeric glycoproteins with complex dodecameric quaternary structures and heterogeneous glycosylation patterns, primarily consisting of mannose-rich N-glycans. Here, we report that enzyme-catalyzed N-deglycosylation of CCH, FLH, and KLH disrupts their quaternary structure and impairs their immunogenic effects. Biochemical analyses revealed that the deglycosylation does not change hemocyanin secondary structure but alters their refolding mechanism and dodecameric structure. Immunochemical analyses indicated decreased binding of N-deglycosylated hemocyanins to the MR and MGL receptors and TLR4 and reduced endocytosis concomitant with an impaired production of tumor necrosis factor α, and interleukins 6 and 12 (IL-6 and IL-12p40, respectively) in macrophages. Evaluating the function of N-deglycosylated hemocyanins in the humoral immune response and their nonspecific antitumor effects in the B16F10 melanoma model, we found that compared with native hemocyanins N-deglycosylated hemocyanins elicited reduced antibody titers, as well as partially diminished antitumor effects and altered carrier activities. In conclusion, the glycan content of hemocyanins is, among other structural characteristics, critically required for their immunological activities and should be considered in biomedical applications.

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.

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