RESUMO
Interleukin (IL)-15 plays an important role in several inflammatory diseases. We have previously identified an IL-15 antagonist called P8 peptide, which binds specifically to IL-15 receptor alpha subunit. However, the P8 peptide rapidly degraded by proteases, limiting its therapeutic application. Thus, we replaced each P8 peptide l-amino acid by its corresponding d-isomers. First, we determined the biological activity of the resulting peptides in a proliferation assay by using CTLL-2 cells. The substitution of l-Ala by d-Ala ([A4a]P8 peptide) increased the inhibitory effect of the P8 peptide in CTLL-2 cells in five-fold. In addition to that, the [A4a]P8 peptide dimer showed the most inhibitory effect. To protect the [A4a]P8 peptide and its dimer against exopeptidase activity, we acetylated the N-terminal of these peptides. At least a three-fold reduction in antagonist activity of acetylated peptides was exhibited. However, the substitution of the N-terminal l-Lys residue of [A4a]P8 peptide and its dimer by d-Lys ([K1k;A4a]P8 peptide) did not affect the antagonist effect of the aforementioned peptides. The [K1k;A4a]P8 peptide dimer was stable to the degradation of trypsin, chymotrypsin, and pepsin up until 48 min. Also, the safety and immunogenicity studies in healthy BALB/c mice demonstrated that the administration of this peptide did not affect the clinical parameters of the animals nor generated antipeptide antibodies. Our findings reveal that two distinct d-amino acid substitutions and dimerization increase the biological activity and stability of P8 peptide. The resulting peptide constitutes a novel IL-15 antagonist with potential applicability in inflammatory diseases.
Assuntos
Interleucina-15/antagonistas & inibidores , Peptídeos/farmacologia , Substituição de Aminoácidos/efeitos dos fármacos , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Dimerização , Feminino , Interleucina-15/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/síntese química , Peptídeos/químicaRESUMO
A preformulation study was performed for the evaluation of a vaccine candidate against HIV-1. Aluminium hydroxide was used in the preformulation. However, this adjuvant is not a good adsorbent for basic proteins since it is positively charged at a physiological pH. In the present study, we determined the adsorption of TAB9 (basic protein, pI: 11.3) by treating Alhydrogel with different ions. The immunogenicity of the vaccine candidate against HIV was also evaluated using three batches, 9801-A, 9802-A and 9803-A, and a placebo P-001. The evaluation was performed twice (0 and 9 months). Each batch was tested using groups of 10 mice that had a single inoculation. The results showed that the protein was totally adsorbed to the aluminium gel. Seroconvertion was attained in all analysed batches, indicating the potentiality of TAB9 as a vaccine candidate.