Helix pomatia hemocyanin - a novel bio-adjuvant for viral and bacterial antigens.

BACKGROUND: New generated subunit vaccines are characterized by increased safety and lack of side effects, however they suffer from weak immunogenicity. The adjuvants are substances that have the ability to enhance the magnitude and duration of the immune response and to increase vaccine efficacy, but the different vaccines may require diverse adjuvants. The urgent need of novel adjuvant formulations occurs, thus ensuring protective cellular and humoral responses against infectious pathogens. The hemocyanins, oxygen binding copper proteins in the hemolymph of molluscs and arthropods, are widely used as peptide carriers and vaccine adjuvants. RESULTS: In the present study we promote the hemocyanin isolated from the terrestrial gastropod Helix pomatia (HPH) as bio-adjuvant, combined with standard antigens. The purified HPH combined with influenza virus hemagglutinin intersubunit peptide (IP) or with tetanus toxoid (TT) were used for immunization. Administration of tetanus toxoid combined with HPH in mice resulted in an increased number of anti-TT IgG producing plasmocytes and induced a significant increase of B and T cell proliferation. The level of the anti-TT IgG antibodies in mice sera was comparable to the group administered with TT+Al(OH)3. An immunization of experimental animals with IP combined with H. pomatia hemocyanin led to generation of strong anti-influenza cytotoxic response. CONCLUSION: The vaccination of mice demonstrates that the HPH is acceptable as a potential bio-adjuvant for subunit vaccines and it could be used as a natural adjuvant or protein carrier.

Anti-cancer properties of gastropodan hemocyanins in murine model of colon carcinoma.

BACKGROUND: Various immunotherapeutic approaches have been used for the treatment of cancer. A number of natural compounds are designed to repair, stimulate, or enhance the immune system response. Among them are the hemocyanins (Hcs) - extracellular copper proteins isolated from different arthropod and mollusc species. Hcs are oxygen transporter molecules and normally are freely dissolved in the hemolymph of these animals. Hemocyanins are very promising class of anti-cancer therapeutics due to their immunogenic properties and the absence of toxicity or side effects. KLH (Megathura crenulata hemocyanin) is the most studied molecule of this group setting a standard for natural carrier protein for small molecules and has been used in anti-tumor clinical trials. RESULTS: The Hcs isolated from marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix pomatia (HpH) express strong in vivo anti-cancer and anti-proliferative effects in the developed by us murine model of colon carcinoma. The immunization with RtH and HpH prolonged the survival of treated animals, improve humoral anti-cancer response and moderate the manifestation of C-26 carcinoma symptoms as tumor growth, splenomegaly and lung metastasis appearance. CONCLUSION: Hemocyanins are used so far for therapy of superficial bladder cancer and murine melanoma models. Our findings demonstrate a potential anti-cancer effect of hemocyanins on a murine model of colon carcinoma suggesting their use for immunotherapy of different types of cancer.

Suppression of dsDNA-specific B lymphocytes reduces disease symptoms in SCID model of mouse lupus.

Self-specific B cells play a main role in the pathogenesis of lupus. This autoimmune disease is characterized by the generation of autoantibodies against self antigens, and the elimination of B and T cells involved in the pathological immune response is a logical approach for effective therapy. We have previously constructed a chimeric molecule by coupling a DNA-mimotope peptides to an anti-CD32 antibody. Using this protein molecule for the treatment of lupus-prone MRL/lpr mice, we suppressed selectively the autoreactive B-lymphocytes by cross-linking B cell receptors with the inhibitory FcγRIIb receptors. This approach was limited by the development of anti-chimeric antibodies in MRL mice. In order to avoid this problem, we established a murine severe combined immunodeficiency lupus model, allowing a long-term chimera therapy. Elimination of the double-stranded DNA-specific B cells by chimera therapy in MRL-transferred immunodeficient mice resulted in inhibition of T cell proliferation and prevented the appearance of IgG anti-DNA antibodies and of proteinuria.

Generation of gene-engineered chimeric DNA molecules for specific therapy of autoimmune diseases.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the development of self-reactive B and T cells and autoantibody production. In particular, double-stranded DNA-specific B cells play an important role in lupus progression, and their selective elimination is a reasonable approach for effective therapy of SLE. DNA-based vaccines aim at the induction of immune response against the vector-encoded antigen. Here, we are exploring, as a new DNA-based therapy of SLE, a chimeric DNA molecule encoding a DNA-mimotope peptide, and the Fv but not the immunogenic Fc fragment of an FcγRIIb-specific monoclonal antibody. This DNA construct was inserted in the expression vector pNut and used as a naked DNA vaccine in a mouse model of lupus. The chimeric DNA molecule can be expressed in eukaryotic cells and cross-links cell surface receptors on DNA-specific B cells, delivering an inhibitory intracellular signal. Intramuscular administration of the recombinant DNA molecule to lupus-prone MRL/lpr mice prevented increase in IgG anti-DNA antibodies and was associated with a low degree of proteinuria, modulation of cytokine profile, and suppression of lupus nephritis.

Marine gastropod hemocyanins as adjuvants of non-conjugated bacterial and viral proteins.

Killed viral vaccines and bacterial toxoids are weakly immunogenic. Numerous compounds are under evaluation as immunological adjuvants and peptide-carriers to improve the immune response. The hemocyanins, giant extracellular copper proteins in the blood of many mollusks, are widely used as immune stimulants. In the present study we investigated the adjuvant properties of hemocyanins isolated from marine gastropods Rapana thomasiana and Megathura crenulata. An immunization with Influenza vaccine or tetanus toxoid combined with Rapana thomasiana hemocyanin (RtH) and Keyhole limpet hemocyanin (KLH) in mice induced an anti-influenza cytotoxic response lasting at least 5 months and an antibody response to viral proteins. The IgG antibody response to the tetanus toxoid (TT) combined with RtH or KLH was comparable to the response of the toxoid in complete Freund's adjuvant. The results obtained demonstrate that the both hemocyanins are acceptable as potential bio-adjuvants for subunit vaccines.

Re-establishing tolerance to DNA in humanized and murine models of SLE.

DNA-specific B cells in SLE represent a logical target for therapeutic intervention. We hypothesize that it is possible to re-establish tolerance to native DNA in SCID mice with cells transferred from SLE patients or from lupus-prone MRL/lpr mice by administering chimeric molecules, containing a monoclonal antibody against inhibitory B cell receptors coupled to a peptide that antigenically mimics DNA. These protein-engineered molecules are able to co-crosslink selectively the antigen receptors of B cells possessing anti-native DNA specificity with the inhibitory surface receptors, thus delivering a strong suppressive signal.