Cell mediated immune response elicited in mice after immunization with the P64k meningococcal protein: epitope mapping

The P64k protein of Neisseria meningitidis has been reported as an immunological carrier for weak immunogens. This investigation was aimed at characterizing the T-cell response produced in primed mice and at identifying T helper cell epitopes within this molecule. BALB/c mice subcutaneously immunized with the recombinant antigen provided inguinal lymph node cells (LNC) that proliferated in the presence of P64k in a dose-dependent manner. Proliferating cells secreted IL-4 while the concentration of IL-12 remained unaltered in the culture supernatant. By testing a panel of 59 overlapping synthetic peptides spanning the entire sequence of the antigen a T-cell determinant was localized. Prime-boost and lymphoproliferation experiments, conducted with highly purified synthetic peptides, confirmed that the segment including amino acids 470-485 comprises a T-cell epitope within the P64k molecule

CIGB-300, a synthetic peptide-based drug that targets the CK2 phosphoaceptor domain. Translational and clinical research

CK2 represents an oncology target scientifically validated. However, clinical research with inhibitors of the CK2-mediated phosphorylation event is still insufficient to recognize it as a clinically validated target. CIGB-300, an investigational peptide-based drug that targets the phosphoaceptor site, binds to a CK2 substrate array in vitro but mainly to B23/nucleophosmin in vivo. The CIGB-300 proapoptotic effect is preceded by its nucleolar localization, inhibition of the CK2-mediated phosphorylation on B23/nucleophosmin and nucleolar disassembly. Importantly, CIGB-300 shifted a protein array linked to apoptosis, ribosome biogenesis, cell proliferation, glycolisis, and cell motility in proteomic studies which helped to understand its mechanism of action. In the clinical ground, CIGB-300 has proved to be safe and well tolerated in a First-in-Human trial in women with cervical malignancies who also experienced signs of clinical benefit. In a second Phase 1 clinical trial in women with cervical cancer stage IB2/II, the MTD and DLT have been also identified in the clinical setting. Interestingly, in cervical tumors the B23/nucleophosmin protein levels were significantly reduced after CIGB-300 treatment at the nucleus compartment. In addition, expanded use of CIGB-300 in case studies has evidenced antitumor activity when administered as compassional option. Collectively, our data outline important clues on translational and clinical research from this novel peptide-based drug reinforcing its perspectives to treat cancer and paving the way to validate CK2 as a promising target in oncology.Fil: Perea, Silvio E.. Center for Genetic Engineering and Biotechnology; CubaFil: Baladron, Idania. Center for Genetic Engineering and Biotechnology; CubaFil: Garcia, Yanelda. Center for Genetic Engineering and Biotechnology; CubaFil: Perera, Yasser. Center for Genetic Engineering and Biotechnology; CubaFil: Lopez, Adlin. Center for Genetic Engineering and Biotechnology; CubaFil: Soriano, Jorge L.. Center for Genetic Engineering and Biotechnology; Cuba. General Hospital ‘‘Hermanos Ameijeiras’; CubaFil: Batista, Noyde. Center for Genetic Engineering and Biotechnology; Cuba. General Hospital ‘‘Hermanos Ameijeiras’; CubaFil: Palau, Aley. Center for Genetic Engineering and Biotechnology; Cuba. General Hospital ‘‘Hermanos Ameijeiras’; CubaFil: Hernández, Ignacio. Center for Genetic Engineering and Biotechnology; CubaFil: Farina, Hernán Gabriel. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garcia, Idrian. Center for Genetic Engineering and Biotechnology; CubaFil: Gonzalez, Lidia. Center for Genetic Engineering and Biotechnology; CubaFil: Gil, Jeovanis. Center for Genetic Engineering and Biotechnology; CubaFil: Rodriguez, Arielis. Center for Genetic Engineering and Biotechnology; CubaFil: Solares, Margarita. Center for Genetic Engineering and Biotechnology; CubaFil: Santana, Agueda. Center for Genetic Engineering and Biotechnology; CubaFil: Cruz, Marisol. Center for Genetic Engineering and Biotechnology; CubaFil: Lopez, Matilde. Center for Genetic Engineering and Biotechnology; CubaFil: Valenzuela, Carmen. Center for Genetic Engineering and Biotechnology; CubaFil: Reyes, Osvaldo. Center for Genetic Engineering and Biotechnology; CubaFil: López Saura, Pedro A.. Center for Genetic Engineering and Biotechnology; CubaFil: González, Carlos A.. Center for Genetic Engineering and Biotechnology; CubaFil: Diaz, Alina. Center for Genetic Engineering and Biotechnology; CubaFil: Castellanos, Lila. Center for Genetic Engineering and Biotechnology; CubaFil: Sanchez, Aniel. Center for Genetic Engineering and Biotechnology; CubaFil: Betancourt, Lazaro. Center for Genetic Engineering and Biotechnology; CubaFil: Besada, Vladimir. Center for Genetic Engineering and Biotechnology; CubaFil: González, Luis J.. Center for Genetic Engineering and Biotechnology; CubaFil: Garay, Hilda. Center for Genetic Engineering and Biotechnology; CubaFil: Gómez, Roberto. Center for Genetic Engineering and Biotechnology; CubaFil: Gomez, Daniel Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; ArgentinaFil: Alonso, Daniel Fernando. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Perrin, Phillipe. No especifíca;Fil: Renualt, Jean Yves. No especifíca;Fil: Sigman, Hugo. No especifíca;Fil: Herrera, Luis. Center for Genetic Engineering and Biotechnology; CubaFil: Acevedo, Boris. Center for Genetic Engineering and Biotechnology; Cub

Gradual reduction of testosterone using a gonadotropin-releasing hormone vaccination delays castration resistance in a prostate cancer model

In a previous study aimed to design a novel prostate cancer vaccine, the authors of the present study demonstrated the advantage of combining the adjuvants Montanide ISA 51 with very small size proteoliposomes (VSSP) to promote a significant humoral immune response to gonadotropin‑releasing hormone (GnRH) in healthy animals. The present study compared the efficacy of this vaccine formulation versus the standard treatment currently available in terms of preventing the development of tumors in DD/S mice injected with Shionogi carcinoma (SC) 115 cells. The results demonstrated that 5 non‑vaccinated control mice exhibited a fast tumor growth, and succumbed to the disease within 19‑31 days. Mice immunized with the GnRH/Montanide ISA 51/VSSP vaccine exhibited a moderate decline in testosterone levels that was associated with a decrease in anti‑GnRH antibody titers, which lead to a sustained tumor growth inhibition. In total, 2 mice in the immunized group exhibited complete remission of the tumor for the duration of the present study. In addition, castrated mice, which were used as a control for standard hormonal therapy, exhibited an accelerated decrease in tumor size. However, tumor relapse was observed between days 50 and 54, and between days 65 and 85, following the injection of SC 155 cells. Therefore, these mice were sacrificed at day 90. The present study concludes that the slow and moderate reduction of testosterone levels observed using the GnRH‑based vaccine may delay the appearance of castration resistance in a Shionogi prostate cancer model. These findings suggest that this vaccine may be used to delay castration resistance in patients with prostate cancer.The authors would like to thank the Union for International Cancer Control (Geneva, Switzerland; grant no., YY1/09/008/2009) for the fellowship received to support the present study. The authors would also like to thank the researchers at the Trev and Joyce Deeley Research Centre (Victoria, Canada), British Columbia Cancer Research Centre (Vancouver, Canada) and University of Victoria (Victoria, Canada), particularly Professor Brad Nelson, Dr Julian Lum and Dr John Webb, for purchasing the mice and providing the laboratory facilities required to conduct the present study.http://www.spandidos-publications.com/or/2017-02-27am2016Mammal Research Institut

Linear polymerization of a synthetic peptide of the V3 region from HIV-1 JY1 isolate using acetamidomethyl-protected thiol groups of cysteine residues

A method to carry out the linear polymerization of a synthetic peptide using acetamidomethyl (Acm)-protected thiol groups of cysteine residues, was developed. This polymer showed to be useful in the improvement of peptide immunogenicity. To achieve the polymerization, two Cys(Acm) residues were incorporated at both ends of the peptide. The model peptide contains B and T cell epitopes placed in tandem. The B cell epitope comprises 15 amino acids of the V3 region from HIV-1 JY1 isolate, and the helper T cell epitope belongs to the region 830-844 of the tetanus toxoid. The polymerization reaction consisted in an instant process of deprotection and oxidation of thiol groups at a high monomer concentration. This reaction proceeded quickly with about 80% of conversion. The monomer and the polymers were analyzed by gel filtration chromatography, reverse phase-high performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and mass spectrometry. The sera obtained from mice immunized with the monomer and the polymer variants were assayed against a conjugated BSA-JY1 peptide in an indirect ELISA. The highest titer values corresponded to the polymer variant (p < 0.01). This result emphasizes that this strategy can be used to increase the immunogenicity of synthetic peptides in vaccines or therapeutics

REPORTE CORTO/SHORT REPORT - MOLECULES MODULATING IMMUNE/INFLAMMATORY RESPONSES, DESIGN AND DEVELOPMENT OF POTENTIAL THERAPEUTICS

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Aminocatalysis-Mediated on-Resin Ugi Reactions: Application in the Solid-Phase Synthesis of <i>N</i>‑Substituted and Tetrazolo Lipopeptides and Peptidosteroids

A new solid-phase protocol for the synthesis of <i>N</i>-substituted and tetrazolo peptides is described. The strategy relies on the combination of aminocatalysis-mediated on-resin Ugi reactions and peptide couplings for the <i>N</i>-alkylation of peptides at selected sites, including the <i>N</i>-terminal double lipidation, the simultaneous lipidation/biotinylation, and the steroid/lipid conjugation via tetrazole ring formation. The solid-phase Ugi four-component reactions were enabled by on-resin transimination steps prior to addition of the acid and isocyanide components. The strategy proved to be suitable for the feasible incorporation of complex <i>N</i>-substituents at both termini and at internal positions, which is not easily achievable by other solid-phase methods

On the utility of the extracted ion chromatograms for assigning the conjugation sites in bioconjugates synthesized by the maleimide-thiol chemistry

Maleimide-thiol chemistry is widely used to synthesize antibody-drug conjugates and conjugate vaccines. The set of MS/MS spectra automatically assigned to proteolytic type 2 peptides with a thiosuccinimide linker and its stabilized products (thiazine and hydrolyzed species), contains valuable information on the conjugation sites. Sample processing before LC-MS/MS analysis transforms the thiosuccinimide linker into its stabilized products. The MS/MS spectra of type 2 peptides must be validated based on objective criteria. The extracted ion chromatogram (XIC) has not been considered previously for this purpose. In the LC-MS/MS analysis, linear peptides eluted in a one-fraction XIC as expected. On the contrary, depending on the analyzed proteolytic digestion, the type 2 peptides with a thiazine linker were detected in 77-100 % of the cases with a two-fraction XIC pattern, probably composed by a mixture of diastereomers. Type 2 peptides with the hydrolyzed thiosuccinimide linker were detected with a mixed-pattern XIC in two, three and four elution fractions in 77 %, 15 % and 8 % of the cases, respectively. The four-fraction XIC pattern is probably composed by the full-chromatographic separation of type 2 peptides cross-linked by two positional isomers of the thiosuccinamic acid linker containing a chiral carbon. Tandem digestions increased the number of type 2 peptides detected with a multiple-fractions XIC pattern. XIC of proteolytic peptides is a relevant source of information considered for data validation. Transcyclization and hydrolysis not only stabilize the thiosuccinimide linker but also permit, together with XIC, a more reliable identification of the conjugation sites by LC-MS/MS analysis

REPORTE CORTO/SHORT REPORT - MURINE MONOCLONAL ANTIBODIES SPECIFIC FOR THE HBsAg a DETERMINANT

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