Immunopotentiation by linking Hsp70 T-cell epitopes to Gag-Pol-Env-Nef-Rev multiepitope construct and increased IFN-gamma secretion in infected lymphocytes.

Therapeutic human immunodeficiency virus (HIV) vaccines can boost the anti-HIV host immunity to control viral replication and eliminate viral reservoirs in the absence of anti-retroviral therapy. In this study, two computationally designed multiepitope Gag-Pol-Env-Nef-Rev and Hsp70-Gag-Pol-Env-Nef-Rev constructs harboring immunogenic and highly conserved HIV T cell epitopes were generated in E. coli as polypeptide vaccine candidates. Furthermore, the multiepitope gag-pol-env-nef-rev and hsp70-gag-pol-env-nef-rev DNA vaccine constructs were prepared and complexed with MPG cell-penetrating peptide. The immunogenicity of the multiepitope constructs were evaluated using the homologous and heterologous prime/boost strategies in mice. Moreover, the secretion of IFN-γ was assessed in infected lymphocytes in vitro. Our data showed that the homologous polypeptide regimens could significantly induce a mixture of IgG1 and IgG2a antibody responses, activate T cells to secret IFN-γ, IL-5, IL-10, and generate Granzyme B. Moreover, IFN-γ secretion was significantly enhanced in single-cycle replicable (SCR) HIV-1 virions-infected splenocytes in these groups compared to uninfected splenocytes. The linkage of heat shock protein 70 (Hsp70) epitopes to Gag-Pol-Env-Nef-Rev polypeptide in the homologous regimen increased significantly cytokines and Granzyme B levels, and IFN-γ secretion in virions-infected splenocytes. Briefly, both designed constructs in the homologous regimens can be used as a promising vaccine candidate against HIV infection.

Improvement of Certolizumab Fab' properties by PASylation technology.

Certolizumab pegol is a Fab' antibody fragment for treatment of rheumatoid arthritis and Crohn's disease which is conjugated to a 40 kDa PEG molecule in order to increase the protein half-life. PEGylation may have disadvantages including immunogenicity, hypersensitivity, vacuolation, decreased binding affinity and biological activity of the protein. To overcome these problems, PASylation has been developed as a new approach. The nucleotide sequence encoding 400 amino acid PAS residues was genetically fused to the corresponding nucleotide sequences of both chains of certolizumab. Then, the bioactivity as well as physicochemical and pharmacokinetic properties of the recombinant PASylated expressed protein was assayed. Circular dichroism spectroscopy demonstrated that the random coil structure of PAS sequences did not change the secondary structure of the PASylated Fab' molecule. It was observed that PASylation influenced the properties of the Fab' molecule by which the hydrodynamic radius and neutralization activity were increased. Also, the antigen binding and binding kinetic parameters improved in comparison to the PEGylated Fab' antibody. Pharmacokinetic studies also showed prolonged terminal half-life and improved pharmacokinetic parameters in PASylated recombinant protein in comparison to the PEGylated and Fab' control molecules. The results reconfirmed the efficiency of PASylation approach as a potential alternative method in increasing the half-life of pharmaceutical proteins.