Fig 7. Improvement in the thermal stability of H1N1 IAV vaccine by surface charge engineering.
(A) Heat map showing the percentage distribution of attachment glycoprotein sequences by charge (2.0 per unit, horizontal axis) for each enveloped virus (vertical axis). (B) Multi-sequence alignment of HA proteins from H1N1 IAV strains SC18, PR8-E, and FAV09, representing high, middle, and low negative charge at pH 7.4, respectively. The amino acid substitutions that can greatly influence the charge of HA protein are indicated with color letters. The dots represent the identical amino acid residues, or the substitutions that do not greatly influence the charge of HA protein. (C) Schematic representation showing the construction of recombinant H1N1 IAVs with changed negative charge of HA proteins via introducing charge-associated amino acid mutations. (D) Scatter diagram showing the relationship between thermostability of rIAVs at 56°C and their corresponding HA charges. (E-G) Molecular surfaces of HA proteins are colored according to electrostatic potentials with a range of red (- 5.0 V) to blue (+ 5.0 V). The names of IAV strains are indicated at the bottom of structures. The structures of mutated HA proteins are obtained by homology modeling, using the HN structure of P1/1951 strain (PBD ID code 6N41) as a template. (H) Survival of inactivated IAV vaccines. The inactivated vaccines are prepared by inactivating the allantoic fluids infected with rIAVs by using 0.05% BPL at 37°C for 2 h, followed by diluting to 107.5 EID50/ml with Tris-HCl (pH 7.8). (I, J) Animal test of stored vaccines. The inactivated IAV vaccines are stored at 37°C for indicated times, and used to immunize BALB/c mice with a volume of 0.1 ml. At 4 weeks post-vaccination, mice are challenged with 103.0 EID50 IAV strain rPR8-E, then monitored daily for clinical signs and mortality for 14 days. HI titers from each group (I) are determined prior to the challenge (n = 5). The percentages of survival of mice from each group (J) is calculated.