A Mechanistic Study on the Destabilization of Whole Inactivated Influenza Virus Vaccine in Gastric Environment

Abstract

Oral immunization using whole inactivated influenza virus vaccine promises an efficient vaccination strategy. While oral vaccination was hampered by harsh gastric environment, a systematic understanding about vaccine destabilization mechanisms was not performed. Here, we investigated the separate and combined effects of temperature, retention time, pH, and osmotic stress on the stability of influenza vaccine by monitoring the time-dependent morphological change using stopped-flow light scattering. When exposed to osmotic stress, clustering of vaccine particles was enhanced in an acidic medium (pH 2.0) at ≥25°C. Fluorescence spectroscopic studies showed that hyper-osmotic stress at pH 2.0 and 37°C caused a considerable increase in conformational change of antigenic proteins compared to that in acidic iso-osmotic medium. A structural integrity of membrane was destroyed upon exposure to hyper-osmotic stress, leading to irreversible morphological change, as observed by undulation in stopped-flow light scattering intensity and transmission electron microscopy. Consistent with these analyses, hemagglutination activity decreased more significantly with an increasing magnitude of hyper-osmotic stress than in the presence of the hypo- and iso-osmotic stresses. This study shows that the magnitude and direction of the osmotic gradient has a substantial impact on the stability of orally administrated influenza vaccine.

The osmotic stress response of split influenza vaccine particles in an acidic environment

“…2a for split vaccine particles and 2b for WIV vaccine particles in hyper-, iso-, and hypo-osmotic conditions). It is noted that gastric contents/secretions significantly influence the physiological gastric osmolarity (Hunt 1959, Choi et al 2013b). Thus, vaccine particles can be subjected to hypo- (C ex < 0.3 osM), iso- (C ex = 0.3 osM), and hyper-osmotic pressures (C ex > 0.3 osM) in the gastric environment.…”

“…Therefore, in this work we used several representative osmotic pressures to investigate the time-dependent osmotic response of the vaccine particles using SFLS. Furthermore, because the effects of temperature on the SFLS behavior and stability of WIV vaccine were reported in our previous work (Choi et al 2013b), SFLS analysis in this work was performed at 4 °C for vaccine samples to isolate the influence of osmotic stress in an acidic environment by eliminating the temperature effects.…”

“…The size of the vaccine particles was calculated using the previously reported correlation between the SFLS intensity and the size of phosphatidylcholine-liposomes (Choi et al 2013b). In this work, liposomes served as a reference for SFLS data interpretation of influenza vaccine particles under the hypothesis of a similar response to osmotic stress.…”

“…Fluorescence was measured with split vaccine particles (200 μg/ml) at the excitation wavelength of 295 nm over the scanning range of 300–500 nm (slit size: 10 nm, scanning speed: 200 nm/min) using a fluorescence spectrophotometer (Cary Eclipse; Varian Inc., Palo Alto, CA, USA) (Choi et al 2012a, 2013b, c). Split vaccine suspension in 300 mM sucrose (pH 7.0) and osmolyte sucrose solution (300 mM (iso-osmotic) and 900 mM (hyper-osmotic), pH 1.7) were mixed in an equal volume ratio to measure intrinsic fluorescence after incubation for 2 h at pH 2.0 and 4 °C.…”

“…In a recent study, we investigated destabilization mechanisms of whole inactivated influenza virus (WIV) vaccine under simulated gastric conditions, focusing on the effects of pH, temperature, gastric retention time, osmotic pressure, and dilution on the stability of vaccine (Choi et al 2013b). It was shown that vaccine particles exhibited a step-wise morphological change, and that vaccine destabilization was associated with the combined effects of low pH-induced degradation of antigens and osmotic stress-induced morphological changes of the vaccine particles.…”