Immunotherapeutic Potential of Mollusk Hemocyanins in Murine Model of Melanoma

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

Long-Time Cooling before Cryopreservation Decreased Translocation of Phosphatidylserine (Ptd-L-Ser) in Human Ovarian Tissue.

OBJECTIVES:To translocation (externalization) of phosphatidylserine lead at least the five negative effects observed during cells cryopreservation: hypoxia, increasing of intracellular Ca2+, osmotic disruption of cellular membranes, generation of reactive oxygen species (ROS) and lipid peroxidation. The aim of this study was to test the intensiveness of the phosphatidylserine translocation immediately after thawing and after 45 d xenografting of human ovarian tissue, which was either frozen just after operative removal from patient or cooled before cryopreservation to 5°C for 24 h and then frozen. MATERIALS AND METHODS:Ovarian fragments from twelve patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following four groups. Pieces of Group 1 (n=30) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n=30) after operation were cooled to 5°C for 24 h, then frozen after 24 h pre-cooling to 5°C, thawed and just after thawing their quality was analyzed. Group 3 pieces (n=30) were frozen immediately after operation without pre-cooling, thawed, transplanted to SCID mice and then, after 45 d of culture their quality was analyzed. Group 4 pieces (n=30) were frozen after 24 h pre-cooling to 5°C, thawed, transplanted to SCID mice and then, after 45 d their quality was analyzed. The effectiveness of the pre-freezing cooling of tissuewas evaluated by the development of follicles (histology) and by intensiveness of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide). RESULTS:For groups 1, 2, 3 and 4 the mean densities of follicles per 1 mm3 was 19.0, 20.2, 12.9, and 12.2, respectively (P1-2, 3-4 >0.1). For these groups, 99%, 98%, 88% and 90% preantral follicles, respectively were morphologically normal (P1-2, 3-4 >0.1). The FACS analysis showed significantly decreased intensiveness of translocation of phosphatidylserine after pre-cooling of frozen tissue (46.3% and 33.6% in Groups 2 and 4, respectively), in contrast with tissue frozen without pre-cooling (77.1% and 60.2 % in Groups 1 and 3, respectively, P1, 3-2, 4 <0.05). CONCLUSIONS:Long time (24 h) cooling of ovarian tissue to 5°C before cryopreservation decreased translocation of phosphatidylserine that evidences about increases the viability of the cells in the tissue after thawing

Antitumor Properties of Epitope-Specific Engineered Vaccine in Murine Model of Melanoma

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&ndash;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

A novel non-integrative single-cycle chimeric HIV lentivector DNA vaccine

International audienceNovel HIV vaccine vectors and strategies are needed to control HIV/AIDS epidemic in humans and eradicate the infection. DNA vaccines alone failed to induce immune responses robust enough to control HIV-1. Development of lentivirus-based DNA vaccines deficient for integration and with a limited replication capacity is an innovative and promising approach. This type of vaccine mimics the early stages of virus infection/replication like the live-attenuated viruses but lacks the inconvenient integration and persistence associated with disease. We developed a novel lentivector DNA vaccine "CAL-SHIV-IN-" that undergoes a single round of replication in the absence of integration resulting in augmented expression of vaccine antigens in vivo. Vaccine gene expression is under control of the LTRs of a naturally attenuated lentivirus, Caprine arthritis encephalitis virus (CAEV) the natural goat lentivirus. The safety of this vaccine prototype was increased by the removal of the integrase coding sequences from the pal gene. We examined the functional properties of this lentivector DNA in cell culture and the immunogenicity in mouse models. Viral proteins were expressed in transfected cells, assembled into viral particles that were able to transduce once target permissive cells. Unlike the parental replication-competent SHIV-KU2 that was detected in DNA samples from any of the serial passage infected cells, CAL-SHIV-IN- DNA was detected only in target cells of the first round of infection, hence demonstrating the single cycle replication of the vaccine. A single dose DNA immunization of humanized NOD/SCID/beta 2 mice showed a substantial increase of IFN-gamma-ELISPOT in splenocytes compared to the former replication and integration defective Delta 4SHIV-KU2 DNA vaccine. (C) 2015 Elsevier Ltd. All rights reserved

Targeting of Influenza Viral Epitopes to Antigen-Presenting Cells by Genetically Engineered Chimeric Molecules in a Humanized NOD SCID Gamma Transfer Model

Antiviral DNA vaccines are a novel strategy in the vaccine development field, which basically consists of the administration of expression vectors coding viral antigen sequences into the host's cells. Targeting of conserved viral epitopes by antibody fragments specific to activating cell surface co-receptor molecules on antigen-presenting cells could be an alternative approach for inducing protective immunity. It has been shown that FcRI on human monocytes enhances antigen presentation in vivo. Various DNA constructs, encoding a Single-chain variable antibodies (scFv) from mouse anti-human FcRI monoclonal antibody, coupled to a sequence encoding a T- and B-cell epitope-containing influenza A virus hemagglutinin inter-subunit peptide were inserted into the eukaryotic expression vector system pTriEx-3 Neo. The constructed chimeric DNA molecules were expressed by transfected Chinese hamster ovary cells and the ability of the engineered proteins to interact with FcRI-expressing cells was confirmed by flow cytometry. The fusion protein induced a strong signal transduction on human monocytes via FcRI. The expression vector pTriEx-3 Neo containing the described construct was used as a naked DNA vaccine and introduced directly to experimental humanized NOD SCID gamma mice with or without boosting with the expressed fusion protein. Immunization with the generated DNA chimeric molecules and prime-boost with the expressed recombinant proteins induced significant serum levels of anti-influenza immunoglobulin G antibodies and strong cytotoxic T lymphocyte activity against influenza virus-infected cells in humanized animals

Selective Silencing of Disease-Associated B Lymphocytes from Hashimoto’s Thyroiditis Patients by Chimeric Protein Molecules

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

Effect of cooling on the quality of follicles (expressed as quantity and normality of follicles).

<p>Group 1: freezing of tissue without pre-cooling, then thawing and evaluation of their quality. Group 2: freezing of tissue after 24h pre-cooling to 5°C, then thawing and evaluation of their quality. Group 3: freezing of tissue without pre-cooling, then thawing, xenografting and evaluation of their quality. Group 4: freezing of tissue without pre-cooling, then thawing, xenografting and evaluation of their quality. No statistical differences between respective groups (Group 1 <i>vs</i> Group 2 and Group 3 <i>vs</i> Group 4 (P>0.1).</p

Translocation of phosphatidylserine in ovarian tissue pre-cooled to 5°C for 24 h before the freezing and then xenografted in SCID mice: representative example of one experiment.

<p>(a, b, c, d) Group 1: pieces were frozen immediately after operation, thawed and just after thawing their quality was FACS analyzed, (e, f, g, h) Group 2: pieces after operation were cooled to 5°C for 24 h, then frozen, thawed and just after thawing their quality was FACS analyzed, (i, j, k, l), pieces were frozen immediately after operation, without pre-cooling, thawed, transplanted to SCID mice and then, after 45 d of culture their quality was FACS analyzed. (m, n, o, p) pieces were frozen after 24 h pre-cooling to 5°C, thawed, transplanted to SCID mice and then, after 45 d their quality was FACS analyzed, (a, e, i, m) forward and scatter dot plot used to select the interest population, (c, g, k, o) histograms displaying fluorescence of PE channel, used to measure fluorescence intensity of Propidium Iodide (PI), (d, h, i, p) dot plot analysis of FITC-Annexin V and PE channels, (Q1) cells negative to Annexin V (FITC A) and positive to PI (could indicate necrotic cells), (Q2) cells positive to both Annexin V and PI (could indicate late apoptotic stage), (Q3) cells negative for both Annexin V and PI (could indicate viable cells), (Q4) cells positive to FITC-Annexin V and negative to PI (could indicate early apoptotic state).</p

Xenografting of cryopreserved human ovarian tissue.

<p>(A) Ovarian pieces after thawing, (B, C, D) Transplantation of these pieces, (E, F) Ovarian pieces after 45 d xenografting. Scale bar: 2 mm.</p