Preparation of monoPEGylated Cyanovirin-N's derivative and its anti-influenza A virus bioactivity in vitro and in vivo

Influenza A virus (IAV) has been raising public health and safety concerns worldwide. Cyanovirin-N (CVN) is a prominent anti-IAV candidate, but both cytotoxicity and immunogenicity have hindered the development of this protein as a viable therapy. In this article, linker-CVN (LCVN) with a flexible and hydrophilic polypeptide at the N-terminus was efficiently produced from the cytoplasm of Escherichia coli at a > 15-l scale. PEGylation at the N-terminal alpha-amine of LCVN was also reformed as 20 kDa PEGylated linkered Cyanovirin-N (PEG(20k)-LCVN). The 50% effective concentrations of PEG(20k)-LCVN were 0.43 +/- 0.11 A mu M for influenza A/HK/8/68 (H3N2) and 0.04 +/- 0.02 A mu M for A/Swan/Hokkaido/51/96 (H5N3), dramatically lower than that of the positive control, Ribavirin (2.88 +/- 0.66 x 10(3) A mu M and 1.79 +/- 0.62 x 10(3) A mu M, respectively). A total of 12.5 A mu M PEG(20k)-LCVN effectively inactivate the propagation of H3N2 in chicken embryos. About 2.0 mg/kg/day PEG(20k)-LCVN increased double the survival rate (66.67%, P = 0.0378) of H3N2 infected mice, prolonged the median survival period, downregulated the mRNA level of viral nuclear protein and decreased (attenuated) the pathology lesion in mice lung. A novel PEGylated CVN derivative, PEG(20k)-LCVN, exhibited potent and strain-dependent anti-IAV activity in nanomolar concentrations in vitro, as well as in micromolar concentration in vivo.