Inhibitory effects on liver tumors of BALB/c mice bearing H22 cells by immunization with recombinant GnRH immunogen coupled to heat shock protein 65 (2023)

GnRH is a promising target in hormone-dependent cancer immunotherapy. In our previous study, we used a peptide vaccine GhM (GnRH₃hinge MVP) using an asparaginase-based bioprocess system. Active immunization with GhM in the presence of CFA/IFA induced a strong humoral response. In this study, the high affinity NRLLLTG motif for the nanoparticle carrier VLP HBcΔ-SBD was fused to the C-terminus of GhM to create a novel peptide vaccine GhMNR (GnRH₃hinges-MVP-NRLLLTG). The ansB-C-GhMNR fusion protein is controlled by the strong T7lac promoter and efficiently expressed as inclusion bodies after lactose induction and then purified by cell disruption, washing and cold ethanol fractionation. After 72 hours of hydrolysis, GhMNR was released from the ansB-C fusion partner and purified by CM52 cation exchange chromatography. These results suggest that the bioprocess system is suitable for large-scale expression and purification of the GhMNR peptide vaccine and even some other proteins or peptides that may be important for industrial or laboratory purposes.

Immunization with recombinant maltose binding protein gonadotropin-releasing hormone (MBP-GnRH6) altered both testicular development and pituitary GnRH receptor (GnRHR) gene transcription in boars. Scrotal measurements and blood samples were taken at 4 week intervals after immunization at 9 weeks of age. Concentrations of testosterone and anti-GnRH antibodies in serum were determined by radioimmunoassay and enzyme immunoassay. The results showed that active immunization with MBP-GnRH6 increases the serum concentration of anti-GnRH antibodies (P<0.05) and decreased serum testosterone concentration (P<0.05) compared to MBP controls. At 25 weeks of age, the boars were sacrificed and the testes were evaluated histologically. Testis development was suppressed in MBP-GnRH6 immunized animals compared to MBP immunized boars. MBP-GnRH6 immunized pigs showed grasping behavior 4 weeks later than MBP immunized boars. No mature spermatozoa were observed in animals immunized with MBP-GnRH6. Quantitative real-time PCR analysis found that the amount of GnRHR mRNA in pituitary tissue was significantly lower in MBP-GnRH6-immunized animals than in controls (P<0.05). These data demonstrate that recombinant MBP-GnRH6 was effective in immunocastration of boars.

Mucosal administration of the autoantigen HSP65 can induce an anti-inflammatory immune response and reduce organ-specific inflammation and disease in various models of autoimmunity, such as arthritis and atherosclerosis. We were interested in whether HSP65 can serve as an immunogenic carrier for the diabetogenic peptide P277 and also induce an anti-inflammatory immune response in NOD mice by mucosal administration. Thus, the dual functions of HSP65 and P277 against type 1 diabetes will be obtained. To test this hypothesis, we investigated the effect of intranasal vaccination with P277 tandem repeat sequences carried by HSP65 in the absence of adjuvant on autoimmune diabetes in NOD mice. We found a significant reduction in the incidence of diabetes, inhibition of insulitis, reduction in the secretion of isotype IgG2a antibodies against P277 and pro-inflammatory cytokines IFN-γ and IL-2, increase in subclass IgG1, IgG2b antibodies against P277 and anti-inflammatory cytokines IL-10 and IL-4 secretion, and reduced proliferation upon nasal application of the fusion protein HSP65-6×P277. Our results indicate that HSP65 may serve as a particularly beneficial carrier for P277-based vaccines, and mucosal delivery may represent a therapeutic approach for the treatment of type 1 diabetes.

The core protein (HBc) of the hepatitis B virus has been shown to be an attractive carrier for foreign epitopes and can display green fluorescent protein (GFP) on its surface. The structure of the substrate binding domain of DnaK [DnaK (394-504 aa), DnaK SBD] is similar to GFP, so we reasoned that the DnaK SBD could also be tolerated. Electron microscopic observations suggest that chimeric proteins containing truncated HBc (HBcΔ) and the DnaK SBD could self-assemble into virus-like particles (VLPs). Subsequently, availability of DnaK SBD and adjuvantity of VLP HBcΔ-SBD were demonstrated using two recombinant peptide vaccines against gonadotropin-releasing hormone (GnRH), GhM and GhMNR. The latter additionally carries the NRLLLTG peptide motif known to bind to DnaK and the DnaK SBD. The combination of VLP HBcA-SBD and GhMNR elicited stronger humoral responses and caused further testicular atrophy than the combination of VLP HBcA and GhMNR or VLP HBcA-SBD and GhM in Balb/c mice. These findings indicate that VLP HBcA-SBD could serve as an excellent carrier for GhMNR and some other peptide vaccines.

A DNA vaccine represents an attractive approach to cancer treatment by inducing active immunodeprivation of gastrin-releasing peptide (GRP) from tumor cells, whose growth depends on GRP stimulation. In this study, we developed a DNA vaccine using a plasmid vector to deliver the immunogen six copies of the B cell epitope GRP_{18–27 (display, andere).}(GRP6). Multiple strategies have been employed to increase the potency of this DNA vaccine, including DNA prime protein boost immunization and introduction of a foreign T helper epitope into the DNA vaccine. Mice vaccinated with a DNA vaccine boosted with HSP65-GRP6 protein induced high titer and relatively high avidity of anti-GRP antibodies, as well as an inhibitory effect on prostate cancer growth in mice, superior to treatment with DNA alone or BCG with initial HSP65-GRP6 protein booster. In addition, the introduction of a new foreign T helper epitope into a GRP DNA vaccine demonstrated a significantly stronger humoral immune response against GRP and tumor rejection even than a DNA prime protein boost strategy. No further enhanced immunogenicity of this modified foreign T helper epitope DNA vaccine was observed even using the modified DNA vaccine priming strategy and the HSP65-GRP6 boost method. The data presented show that improving the potency of the anti-GRP DNA vaccine with the above two possible approaches should provide useful methods in the development of a new anti-growth factor DNA vaccine for cancer immunotherapy.

Brain Research, Volume 1587, 2014, p. 40-53

Sirt3 is a mitochondrial sirtuin whose deacetylase activity regulates aspects of oxidative metabolic efficiency, antioxidant capacity and intramitochondrial signaling. In this study, we tested whether overexpression of the human Sirt3-myc transgene in differentiated PC12 cells, a model of sympathetic catecholaminergic neurons, would affect the susceptibility of these cells to oxidative stress or trophic withdrawal. Expression analysis revealed that the Sirt3-myc product is expressed as a 45 kDa proform, localized primarily in the cytosol, and as a 30 kDa processed form, primarily localized in mitochondria. When subjected to acute glucose deprivation or acute oxygen-glucose deprivation, differentiated PC12 cells overexpressing Sirt3-myc showed significantly lower levels of cytotoxicity, both at the end of injury and at various times after medium reperfusion, than cells transfected with a control plasmid. In addition, overexpression of Sirt3-myc also protected differentiated PC12 cells from apoptosis induced by trophic withdrawal. Together, these data demonstrate that upregulation of Sirt3 is sufficient to protect neuronal PC12 cells from cytotoxic insults, and add to the growing body of evidence that Sirt3 may be a target for neuroprotective intervention.

Cjepivo, volume 32, number 32, 2014, p. 4051-4058

Protein subunit vaccines as stimulatory strategies against tuberculosis (TB) infection are currently under investigation for tuberculosis vaccines. Their main limitation is their weak immunogenicity, making it necessary to combine protein subunits with auxiliary molecules. In this study, we used invasive replication-deficient drugsEscherichia colipressure to deliverMycobacterium tuberculoseof proteins in the cytoplasm of non-phagocytic eukaryotic cells using different initial and primary vaccination protocols. Our results show that intranasal administration is invasiveE coliexpressionM. tuberculoseof the protective antigen MPT64 to mice administered a recombinant BCG strain overexpressing MPT64 on the surface reduces the bacterial load in the spleen of the mouse. Our data suggest that the replication deficiency is invasiveE colimay be a suitable platform for BCG/rBCG priming followed by homologous immunization strategies.

Journal of Sexual Medicine, Volume 13, Issue 12, 2016, p. 1844-1857

The American Journal of Surgery, pagina 212, broj 4, 2016., str. 682-690.e5

Scientific Bulletin, Volume 60, Issue 8, 2015, p. 762-772

The onset and progression of hepatocellular carcinoma (HCC) is closely related to chronically diseased liver tissue. This background of diseased liver tissue is a drastically different microenvironment than a healthy liver, especially with regard to the prevalence of immune cells and the presence of mediators of immune function. To better understand the consequences of liver disease on tumor growth and interaction with its microenvironment, we used two standard methods of fibrosis induction and orthotopic tumor implantation in inflamed and fibrotic liver to mimic the liver disease of HCC patients. Compared to non-diseased controls, tumor growth was significantly improved under fibrotic conditions. The tumor-infiltrating immune cells were also drastically different, with fewer natural killer cells but significantly more immunosuppressive CD11b⁺Gr1^bokof myeloid cells in both models of fibrosis. In addition, there were model-specific differences: increased CD11b counts⁺the myeloid cell a CD4⁺CD25⁺T cells were found in tumors in the bile duct ligation model but not in the tetrachloride model. The induction of fibrosis altered the cytokine production of implanted tumor cells, which could have far-reaching implications for the immune infiltrate and its functionality. Taken together, this work demonstrates that the combination of fibrosis induction with orthotopic tumor implantation results in markedly different tumor microenvironment and tumor growth kinetics, highlighting the need for more accurate modeling of HCC progression in mice that accounts for the drastic tissue changes caused due to chronic liver disease.

Gene, Volume 530, Number 1, 2013, p. 75-82

Extracellular superoxide dismutase (EC-SOD) is the major antioxidant enzyme in the extracellular matrix. We have developed transgenic mice to analyze EC-SOD promoter activity in vivo in real time and to identify key cis elements around the 5' region of the mouse EC-SOD gene. Using this model, we have shown that luciferase reporter activity closely correlates with endogenous EC-SOD expression, although several interesting differences were also observed. Specifically, luciferase activity was detected at the highest levels in the testis, aorta and perirenal fat. Reporter expression was regulated by interferon gamma, a finding consistent with published studies of endogenous EC-SOD gene expression. Therefore, the 5' flanking region of the mouse EC-SOD gene is responsible, at least in part, for cell-specific and inducible expression.