Fig 1.
Time course of Stopped-flow light scattering (SFLS) analysis of inactivated influenza virus exposed to hypertonic solutions.
A virus suspension (0.2 μg/μL) was abruptly exposed to hyperosmotic solutions of trehalose (A). Hyperosmotic viral shrinkage results in an increase of light scattering intensity, believed to be a result of water efflux from the virus. (B) Data in (A) were curve-fitted using single exponentials and plotted after normalization: trehalose (T), sucrose (S), NaCl (N). (C) Rate constants (k [s–1]) of the single exponential curves and (D) corresponding osmotic water permeability coefficients (Pf [cm s–1]). Data are presented as the mean ± standard deviation (SD) for n > 30. Hypertonic osmotic differences across the viral envelope (ΔCos) are indicated within the plots of (A) and (B).
Fig 2.
Temperature dependence of the osmotic response of inactivated influenza virus.
The kinetics of osmotic water transport through the viral envelope were investigated at several temperatures while applying a hyperosmotic difference of ΔCos = 304 mOsm with trehalose. (A) Scattered light intensities and fitted curves and (B) Arrhenius plot for water transport across the viral membranes (mean ± SD, n = 8–20). The Arrhenius activation energy was calculated from the linear regression of the ln(k) vs. 1/T plot.
Fig 3.
SFLS measurement of inactivated influenza virus.
(A) Long-term course of SFLS behavior at an osmotic difference of ΔCos = 217 mOsm trehalose and (B) the corresponding relative volume calculated as the initial volume divided by the volume at time t. (C) SFLS curves in response to hyperosmotic gradients in trehalose solutions. Hypertonic osmotic differences are indicated at the right side of each curve. The same test conditions were used as in Fig 1 except for an increased monitoring time. (D) The onset time for the secondary shrinkage (t2nd) as a function of osmotic gradients by trehalose, sucrose, and NaCl. (Mean ± SD, n = 9–19.)
Fig 4.
Hemagglutinin activity change as a function of incubation time and osmotic strength.
The effect of osmotic pressures on the activity of the inactivated influenza virus was investigated by measuring HA activity change at four osmotic differences (ΔCos = 217, 420, 682, 1351 mOsm) using trehalose with the increase of incubation time (10 s, 1 min, 5 min, 10 min, and 30 min). (Mean ± SD, n = 8–24.)
Fig 5.
Effects of the viscosity enhancer CMC on the functional activity of the influenza virus.
(A) Time-course of HA activity change of the inactivated influenza virus in a vaccine formulation composed of trehalose and CMC (n = 8). (B) HA activity as a function of osmotic differences (217, 420, 682, 1351 mOsm with trehalose) in the presence of 0.5% w/v CMC after incubation for 30 min (n = 8). (C) Viral titers of live influenza virus as assessed by plaque assay (n = 3). (Mean ± SD.)
Fig 6.
Effects of viscosity enhancer on the HA activity of influenza virus after drying.
(A) Inactivated virus, (B) live virus, and (C) inactivated virus (mean ± SD, n = 8–16). In (C), HA activity of w/o viscosity enhancer was reused from (A) for better comparison.
Fig 7.
Effect of dried formulations of vaccine on in vivo immunogenicity.
Influenza vaccine-coated MNs were air-dried for one day at ambient conditions and reconstituted in DPBS for intramuscular vaccination of mice (n = 6 per group). Naïve (negative control), vaccine in iso-osmotic solution (positive control), dried vaccine-coated MNs (w/o CMC: trehalose-only, w/ 0.5% w/v CMC: trehalose plus CMC). Virus-specific IgG was assayed by ELISA with the immune sera of mice two weeks after vaccination with 0.2 μg of viral proteins. Optical density was measured at 450 nm. (Mean ± SD, n = 6.)