Severe respiratory disease with substantial morbidity and mortality was associated with influenza viral infection 
. Continuous evolution of antigenic glycoproteins requires annual reconfiguration of vaccine, specifically matching the new strain, to maintain protective efficacy. As a result of seasonal influenza, an annual average of 250,000 to 500,000 deaths occurred worldwide 
, and 90% of which are elderly persons (≥65 years old) 
. The annual health costs due to influenza epidemics in the U.S. alone are estimated to be $87.1 billion 
. The fact that vaccination is the most efficient strategy to prepare for the continuing threat of influenza calls attention to the developments of alternate vaccine, advanced vaccine delivery method, and dose sparing technologies 
Currently, the inactivated and live attenuated trivalent vaccines produced in embryonated hen’s egg have been used for intramuscular (IM)/intradermal (ID) and intranasal (IN) administration in the U.S. 
. The use of hypodermic needles in universal mass vaccination is intrinsically limited due to the need of highly skillful healthcare workers, the risk of biohazard waste, and safety concerns about needle reuse. As a result, there has been extensive research on the development of needle-free vaccine delivery methods and formulations, thereby enabling easier, safer, and cheaper vaccinations in a timelier manner 
. While injection-based transcutaneous methods are dominantly employed for current vaccination, mucosal immunization has been gaining attention due to advantages including the ability to induce both mucosal and systemic immune responses 
. Unlike IM injection, which mainly produces virus-neutralizing IgG antibodies, mucosal vaccination can induce the specific immune response of mucosa-associated lymphoid tissue (e.g., secretory IgA antibodies) to prevent pathogen entry into epithelial cells 
. Among various mucosal vaccination methods, IN administration with the live attenuated influenza vaccine is currently allowed for use in healthy persons aged 5–49 years 
. However, an inactivated IN vaccine formulation containing adjuvant was withdrawn from the market due to the safety issues such as causing Bell’s palsy 
In terms of oral influenza immunization, considerable efforts have been devoted to develop optimal formulations using inactivated virus vaccine due to potential advantages including safety, cost-effectiveness, and ease of production. Because administration through oral route does not require trained personnel, oral vaccine provides a promising platform for the people in developing countries as well as for pandemic influenza preparation. However, in vivo
animal studies have demonstrated that orally administered influenza vaccine did not produce satisfactory levels of immunogenicity compared with other routes of administration, mainly due to the destabilization of oral vaccines in the stomach 
. Thus, to induce a similar level of protective immunogenicity, the vaccine must contain a larger quantity of antigens 
, resulting in the decrease of economic benefits of inactivated oral influenza vaccine. As a result, despite tremendous efforts, there is still no commercially available oral influenza vaccine. Therefore, it is believed that a mechanistic understanding of the effects of the physicochemical gastric environment as a whole on vaccine stability is recognized as a critical step for successful development of oral influenza vaccines.
Ingested vaccine is exposed to low stomach pH at about 37°C during digestion in the stomach. Although gastric digestion rate exhibits a significant level of person-to-person/meal-to-meal variation 
, average transit time for half gastric emptying is estimated to be about 80 min or 127 min for fluid or solid meal, respectively 
. This indicates that oral vaccines are exposed to gastric juice with a low pH (about 2.0) for about 2 h 
. Furthermore, enveloped live/inactivated influenza vaccines are susceptible to osmotic stress present in the stomach. Osmotic pressure gradient is generated from osmolarity differences across the lipid membrane, which has been known to influence structural/functional integrity of organisms 
. Thus, osmotic pressure-induced functional activity loss of vaccine has been recognized as a major obstacle to the development of vaccines 
. Considering oral vaccines are subjected to these harsh conditions, systematic studies on time-dependent vaccine stability change at low pH and physiological temperature in the presence of osmotic stress are essential to understand vaccine destabilization mechanisms. Since pH-induced antigen denaturation has been previously reported 
, the same principle has been exclusively adopted to explain lower level of immunogenicity of orally administered influenza vaccine. Interestingly, stability of oral vaccine has been studied with over-simplified conditions and a time-dependent detailed analysis has not been carried out so far under conditions similar to gastric environments. Thus, other major gastric conditions such as temperature, osmotic stress, and gastric residence time were neglected in the analysis. It is unlikely that only pH-induced irreversible conformational change of hemagglutinin (HA) contributes to activity loss of oral vaccine during the whole gastric digestion process.
Therefore, we performed experiments using stopped-flow light scattering (SFLS) analysis to observe time-dependent, osmotic stress-induced morphological change of the whole inactivated influenza virus vaccine under physiological gastric conditions. The underlying hypothesis is that low pH-induced conformational change of HA protein leads to a decrease of vaccine efficacy at the initial stage, while osmotic stress, combined with other gastric environmental factors (temperature, osmotic stress, dilution effect, and retention time) results in further decrease of vaccine efficacy. SFLS is commonly used to examine reactions kinetics after small volumes of solutions are mixed. For this purpose, a correlation between osmotic swelling/shrinking behavior and scattered light intensity change was established for liposomes made of phosphatidylcholine, i.e. one of major lipid components of the egg-grown viruses, as a model system for influenza vaccine 
. This relationship was then used in quantitative analysis of morphological variation in terms of influenza vaccine size change at different test conditions. Transmission electron microscopy (TEM), intrinsic fluorescence, and hemagglutination activity were subsequently measured to determine how functional activity of HA can be related to the time-dependent membrane perturbation of the virus.