The development of antitumor drugs and therapy requires new approaches and molecules, and products of natural origin provide intriguing alternatives for antitumor research. Gastropodan hemocyanins-multimeric copper-containing glycoproteins have been used in therapeutic vaccines and antitumor agents in many cancer models. Materials and Methods: We established a murine model of melanoma by challenging C57BL/6 mice with a B16F10 cell line for solid tumor formation in experimental animals. The anticancer properties of hemocyanins isolated from the marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix aspersa (HaH) were evaluated in this melanoma model using various schemes of therapy. Flow cytometry, ELISA, proliferation, and cytotoxicity assays, as well as histology investigations, were also performed. Results: Beneficial effects on tumor growth, tumor incidence, and survival of tumor-bearing C57BL/6 mice after administration of the RtH or HaH were observed. The generation of high titers of melanoma-specific IgM antibodies, pro-inflammatory cytokines, and tumor-specific CTLs, and high levels of tumor-infiltrated M1 macrophages enhanced the immune reaction and tumor suppression. Discussion: Both RtH and HaH exhibited promising properties for applications as antitumor therapeutic agents and future experiments with humans.

Objectives: SARS-CoV-2 caused the COVID-19 pandemic, which overwhelmed global healthcare systems. Over 776 million COVID-19 cases and more than 7 million deaths were reported by WHO in September 2024. COVID-19 vaccination is crucial for preventing infection and controlling the pandemic. Here, we describe the design and development of a next-generation multi-epitope vaccine for SARS-CoV-2, consisting of T cell epitopes. Methods: Immunoinformatic methods were used to derive models for the selection of MHC binders specific for the mouse strain used in this study among a set of human SARS-CoV-2 T cell epitopes identified in convalescent patients with COVID-19. The immunogenicity of the vaccine prototype was tested on humanized-ACE2 transgenic B6.Cg-Tg(K18-ACE2)2Prlmn/J mice by in vitro, in vivo, and ex vivo immunoassays. Results: Eleven binders (two from the Envelope (E) protein; two from the Membrane (M) protein; three from the Spike (S) protein; and four from the Nucleocapsid (N) protein) were synthesized and included in a multi-epitope vaccine prototype. The animals were immunized with a mix of predicted MHC-I, MHC-II, or MHC-I/MHC-II peptide epitopes in Complete Freund’s Adjuvant, and boosted with peptides in Incomplete Freund’s Adjuvant. Immunization with SARS-CoV-2 epitopes remodeled the lymphocyte profile. A weak humoral response and the significant production of IL-4 and IFN-γ from T cells were found after the vaccination of the animals. Conclusions: The multi-epitope vaccine prototype presented in this study demonstrates immunogenicity in mice and shows potential for human vaccine construction.

Hashimoto’s thyroiditis is one of the most common endocrine disorders, affecting up to 20% of the adult population. No treatment or prevention exists except hormonal substitution for hypothyroidism. We hypothesize that it may be possible to selectively suppress anti-thyroglobulin (Tg) IgG antibody-producing B lymphocytes from HT patients by a chimeric protein molecule containing a monoclonal antibody specific for the human inhibitory receptor CR1, coupled to peptide epitopes derived from Tg protein. We expect that this treatment will down-regulate B-cell autoreactivity by delivering a strong inhibitory signal. Three peptides—two epitope-predicted ones derived from Tg and another irrelevant peptide—were synthesized and then coupled with monoclonal anti-human CR1 antibody to construct three chimeric molecules. The binding to CD35 on human B cells and the effects of the chimeric constructs on PBMC and TMC from patients with HT were tested using flow cytometry, ELISpot assay, and immunoenzyme methods. We found that after the chemical conjugation, all chimeras retained their receptor-binding capacity, and the Tg epitopes could be recognized by anti-Tg autoantibodies in the patients’ sera. This treatment downregulated B-cell autoreactivity and cell proliferation, inhibited Tg-specific B-cell differentiation to plasmablasts and promoted apoptosis to the targeted cells. The treatment of PBMCs from HT patients with Tg-epitope-carrying chimeric molecules affects the activity of Tg-specific autoreactive B lymphocytes, delivering to them a strong suppressive signal.

Finding new effective compounds of natural origin for composing anti-tumor vaccines is one of the main goals of antitumor research. Promising anti-cancer agents are the gastropodan hemocyanins–multimeric copper-containing glycoproteins used so far for therapy of different tumors. The properties of hemocyanins isolated from the marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix aspersa (HaH) upon their use as carrier-proteins in conjugated vaccines, containing ganglioside mimotope GD3P4 peptide, were studied in the developed murine melanoma model. Murine melanoma cell line B16F10 was used for solid tumor establishment in C57BL/6 mice using various schemes of therapy. Protein engineering, flow cytometry, and cytotoxicity assays were also performed. The administration of the protein-engineered vaccines RtH-GD3P4 or HaH-GD3P4 under the three different regimens of therapy in the B16F10 murine melanoma model suppressed tumor growth, decreased tumor incidence, and prolonged the survival of treated animals. The immunization of experimental mice induced an infiltration of immunocompetent cells into the tumors and generated cytotoxic tumor-specific T cells in the spleen. The treatment also generates significantly higher levels of tumor-infiltrated M1 macrophages, compared to untreated tumor-bearing control mice. This study demonstrated a promising approach for cancer therapy having potential applications for cancer vaccine research.

Melanoma is one of the most common tumors worldwide, and new approaches and antitumor drugs for therapy are being investigated. Among the promising biomolecules of natural origin for antitumor research are gastropodan hemocyanins—highly immunogenic multimeric glycoproteins used as antitumor agents and components of therapeutic vaccines in human and mouse cancer models. A murine melanoma model established in C57BL/6 mice of the B16F10 cell line was used to study anticancer modified oxidized hemocyanins (Ox-Hcs) that were administered to experimental animals (100 μg/mouse) under different regimens: mild, intensive, and with sensitization. The solid tumor growth, antitumor response, cell infiltration in tumors, and survival were assessed using flow cytometry, ELISA, and cytotoxicity assays. Therapy with Ox-RtH or Ox-HaH resulted in the generation of enhanced specific immune response (increased levels of tumor-infiltrated mature NK cells (CD27+CD11b+) in sensitized groups and of macrophages in the intensively immunized animals) and tumor suppression. Beneficial effects such as delayed tumor incidence and growth as well as prolonged survival of tumor-bearing animals have been observed. High levels of melanoma-specific CTLs that mediate cytotoxic effects on tumor cells; tumor-infiltrating IgM antibodies expected to enhance antibody-dependent cellular cytotoxicity; type M1 macrophages, which stimulate the Th1 response and cytotoxic cells; and proinflammatory cytokines, were also observed after Ox-Hcs administration. The modified Hcs showed strong antitumor properties in different administration regimens in a murine model of melanoma with potential for future application in humans.

The potential of plant-based bioactive peptides to be used as an alternative or complementary therapy for the safe management of metabolic diseases such as diabetes is garnering increasing attention. In this in vivo study, the hypoglycemic effect of bromelain hydrolysates of hemp seed cake protein is analyzed in streptozotocin-induced diabetic mice. The results demonstrate that treatment of mice with the hydrolysate at a dose of 200 mg/kg/day for 4 weeks leads to a significant decrease in blood glucose levels (by 39%), serum triglyceride levels (16.44%), total cholesterol (13.50%), and low-density lipoprotein LDL (23.60%) compared to the untreated streptozotocin (STZ) group. The obtained values are comparable to those of the group treated with acarbose, which is used as a reference drug in the study. The applied hydrolysate significantly reduces serum alanine aminotransferase levels (by 39.38%) compared to the values of the untreated STZ group, while the change in superoxide dismutase levels was comparable to that of the group treated with acarbose. This study demonstrates the capacity of the applied hydrolysate to improve hyperglycemic conditions and reveals opportunities for future research into its potential as a functional ingredient.