Fig 1.
Structure of the HC19 antibody in complex with the HA1 domain of Aichi H3N2.
A) 3D structure three fragments of antibody (Fab, blue) bound to the HA1 domains of trimeric HA (brown) of Aichi H3N2 (PDB: 2vir) [26]. B) Zoom in on the CDR containing Fab subdomain. Peptides containing the CDR sequences 26–32 (CDR1), 53–55 (CDR2) and 99–110 (CDR3) were entitled PeA (green), PeC (black) and PeB (red) [27]. C) PeB interacting with HA in proximity to the sialic acid binding pocket (yellow). Amino acids beneficial for binding are shown in blue, non-favorable amino acids (see Results) are shown in black. D) Primary and secondary structures of VH of the Fab fragment. Antibody derived peptides are colored as in B.
Table 1.
Total free energy of binding of CDR-related peptides to HA of Aichi H3N2.
CDR sequences are underlined.
Fig 2.
MD simulation of peptide-hemagglutinin complexes.
A) Time evolution of root-mean-square deviations (RMSDs) of backbone atoms relative to their initial configurations for Aichi H3N2 HA complexed with PeB (black) and PeBGF (gray). N- and C-terminal flexible tails were excluded for RMSD calculation. B) Contributions of the individual residues of PeB (black) and PeBGF (gray) to the total binding free energy change.
Fig 3.
Binding of influenza virus to antibody derived peptides.
Aichi H3N2 virus was injected on surface immobilized peptides. PeA-Lys (0.77 pmol mm-²), PeB-Lys (0.57 pmol mm-²) and PeC-Lys (1.93 pmol mm-²). The SPR signal at 10 s after terminating injection was taken as binding response.
Fig 4.
Specificity of virus-peptide binding.
A) Dose-response curves for binding of Aichi H3N2 to immobilized PeB-Lys and three monosubstituted derivatives of the peptide. B) Binding of Aichi H3N2 virus to immobilized PeB-Lys compared with a set of well-known proteins (S1 Table). All proteins were diluted to a concentration of 10 μg ml-1. Columns represent the mean value of duplicate experiments. The error bars show the SEM.
Fig 5.
Binding of PeB to immobilized HA from Aichi H3N2.
A) SPR sensorgrams of PeB binding to Aichi H3N2 HA. Peptide injections range from 0.27–70 μM. B) SPR response at equilibrium plotted against injected peptide. Data points were fitted by a steady state affinity fit curve.
Fig 6.
Enhancement of peptide binding affinity to HA by site-directed substitution.
A) Inhibition of Aichi H3N2 binding to immobilized fetuin by PeB-Lys and PeBGF-Lys as determined via SPR (n = 2). Error bars indicate the SEM. Dashed lines represent a four parameter logistic fit. B) Microarray based substitutional analysis of PeB using fluorescently labeled Aichi H3N2 viruses as analytes (for further virus strains see S7 Fig). Amino acids in the first row represent that of PeB, while amino acids in the first column show the substitution. The values represent the contrast relative to fetuin as positive control (see Material and Methods). C) Binding of different viruses to selected immobilized single and double amino acid substituted variants of peptide PeB-Lys. The binding response from SPR measurements is shown (as in Fig 3).
Table 2.
Mean values of IC50 [μM] from inhibition experiments using SPR (left column), together with its SEM (n≥2).
SPR binding of virus Aichi H3N2 to fetuin is shown in Fig 6A. Inhibitor constant Ki(HAI) [μM] from hemagglutination inhibition experiments with its SEM (n≥3). Data is plotted in Fig 7. Mean values of IC50 [μM] from infection inhibition (Fig 7B and 7C) and microneutralization assays (Fig 7D and 7E) are presented together with its SEM (n≥3).
Fig 7.
Peptide mediated inhibition of virus binding and infection.
A) Inhibition of Aichi H3N2 and Rostock H7N1 mediated hemagglutination by PeB and PeBGF. Ki(HAI) values represent the minimum concentration for full hemagglutination inhibition. B and C) Infection inhibition of MDCK II cells with PeB, PeBGF and PeBP (MOI 0.05) with Aichi H3N2 (B) or Rostock H7N1 (C). Infection was followed by measuring the decrease of absorption of the MTS reagent. Error bars represent the SEM with n≥3. D and E) Neutralization of Aichi H3N2 (D) and Rostock H7N1 (E) by PeB and PeBGF, while PeBP served as a control. Error bars represent the SEM with n = 3.
Fig 8.
Inhibitory potency of PeB and PeBGF as compared with known anti-influenza peptides.
Inhibition of hemagglutination based on their Ki(HAI) of different peptides as compared to 2,6‘-sialyllactose (EB: RRKKAAVALLPAVLLALLAP [45], s2(1–5): ARLPRTMVHPKPAQP [17], Phage 1 (P1) H5N1: HAWDPIPARDPF [18], Phage 1 (L-P1) H9N2: NDFRSKT [16]. Error bars represent the SEM (n = 3).