Stability and antiviral activity of SP40 peptide in human serum

•SP40 peptide demonstrated a good in vitro stability and anti-EV-A71 activity in human serum.•SP40 peptide is mainly cleaved by exopeptidases.•Four amino acids at the N and C terminals of the SP40 peptide were dispensable for its in vitro anti-EV-A71 activity. Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). SP40 peptide was previously identified to inhibit EV-A71 strains from genotypes A, B and C. However, the stability and antiviral activity of SP40 peptide in human serum are yet to be established. To address this, we evaluated the stability and anti-EV-A71 activity of SP40 peptide after incubation in 25 % human serum. Reverse-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC/MS) were utilized to evaluate serum stability and cleavage patterns of SP40 peptide after incubation in human serum. Cell protection assay was used to evaluate the anti-EV-A71 activity of SP40 peptide after incubation in human serum and to identify the minimal active sequence of SP40 peptide that retained antiviral activity. The results showed that the SP40 peptide was stable in human serum with 56 % of the full-length SP40 peptide being detected after 48 h incubation in human serum. The SP40 peptide was mainly cleaved by exopeptidases and no endoprotease recognition sites were identified within the SP40 peptide. Cell protection assays revealed that the SP40 peptide retained substantial activity after 24 and 48 h incubation in human serum. Furthermore, the data revealed that three amino acids at the N-terminus and one amino acid at the C-terminus of the SP40 peptide were dispensable for its antiviral activity. Importantly, the four truncated peptides displayed better potency than the full-length SP40 peptide. Overall, this study provided insights into the stability and activity of SP40 peptide in human serum and will facilitate the development of SP40 peptide as an anti-EV-A71 agent.