Enhanced structural stability of Concholepas hemocyanin increases its immunogenicity and maintains its non‐specific immunostimulatory effects

Abstract

Hemocyanins, which boost the immune system of mammals, have been used as carrieradjuvants to promote Ab production against haptens and peptides, as immunostimulants during therapy for bladder carcinoma and as a component in therapeutic vaccines for cancer. These biomedical applications have led to growing interest in obtaining hemocyanins with high immunogenicity. Here, we study the immunological properties of a modified oxidized Concholepas concholepas hemocyanin (Ox-CCH) obtained by the oxidation of its carbohydrates using sodium periodate. We assessed the internalization of Ox-CCH into DCs and its immunogenicity and antitumor effects. Transmission electron microscopy showed no changes in Ox-CCH quaternary structure with respect to native CCH, although proteolytic treatment followed by SDS-PAGE analysis demonstrated that Schiff bases were formed. Interestingly, DCs internalized Ox-CCH faster than CCH, mainly through macropinocytosis. During this process, Ox-CCH remained inside endosome-like structures for a longer period. Mouse immunization experiments demonstrated that Ox-CCH is more immunogenic and a better carrier than CCH. Moreover, Ox-CCH showed a significant antitumor effect in the B16F10 melanoma model similar to that produced by CCH, inducing IFN-γ secretion. Together, these data demonstrate that the aldehydes formed by the periodate oxidation of sugar moieties stabilizes the CCH structure, increasing its adjuvant/immunostimulatory carrier effects.

Hemocyanins Stimulate Innate Immunity by Inducing Different Temporal Patterns of Proinflammatory Cytokine Expression in Macrophages

“…2B). Therefore, these results strongly suggest that the slow Ag processing in macrophages, which also occurs in DCs (8,14), allows the sequestration of these proteins for presentation to T lymphocytes several days later, which is a key feature of Ags that enhance immunogenicity (45).…”

“…However, the response was sufficient to induce the secretion of several proinflammatory cytokines, including IL-6, IL-12p40, IL-23, and TNF-a (58). It has been shown that the lysosomal activity in DCs is less efficient in macrophages (59), and this activity cannot significantly degrade large proteins (60), such as KLH or CCH (14). It is unlikely that these structures hydrolyze these proteins to smaller peptide fragments equivalent to those found in macrophages (Fig.…”

“…Because mannose is the major sugar found in these proteins, we proposed that APCs might recognize mollusk hemocyanins as a "highly mannosylated infectious agent" (6). This assumption is supported by the promotion of human DC maturation by KLH in vitro through its engagement of the membrane C-type lectin mannose receptor (MR) (80), a phenomenon that is not observed in murine DCs cultured in vitro with KLH and CCH (14,81). However, FLH promotes the secretion of several proinflammatory cytokines, although this ability ceases when DCs are treated with deglycosylated FLH, suggesting a crucial role for oligosaccharides in the immunomodulatory effects of FLH (8).…”

“…To determine the processing pattern of CCH, FLH, and KLH by macrophages, we used the general procedure previously described by Arancibia et al (14). Briefly, the macrophages were incubated with 100 mg/ml each protein at 37˚C for 4, 24, 72, 120, and 144 h. For the electrophoretic analysis of macrophage extracts, the cells were washed with PBS, harvested with trypsin/EDTA for 30 min at 37˚C, and lysed with a low-detergent buffer (20 mM Tris/HCl [pH 7.5], 2 mM EDTA, 150 mM NaCl, and 0.5% Triton X-100).…”

“…We propose that when hemocyanins are incorporated by APCs, they induce a proinflammatory milieu that produces a bystander effect, which is a key event in their nonspecific immunostimulatory activity (14). Indeed, macrophages play an essential role in both innate and adaptive immunity as regulatory and effectors cells, and as one of the primary danger sensors in the host (15,16).…”