Abstract
Enveloped virus vaccines can be damaged by high osmotic strength solutions, such as those used to protect the vaccine antigen during drying, which contain high concentrations of sugars. We therefore studied shrinkage and activity loss of whole inactivated influenza virus in hyperosmotic solutions and used those findings to improve vaccine coating of microneedle patches for influenza vaccination. Using stopped-flow light scattering analysis, we found that the virus underwent an initial shrinkage on the order of 10% by volume within 5 s upon exposure to a hyperosmotic stress difference of 217 milliosmolarity. During this shrinkage, the virus envelope had very low osmotic water permeability (1 – 6×10−4 cm s–1) and high Arrhenius activation energy (E a = 15.0 kcal mol–1), indicating that the water molecules diffused through the viral lipid membranes. After a quasi-stable state of approximately 20 s to 2 min, depending on the species and hypertonic osmotic strength difference of disaccharides, there was a second phase of viral shrinkage. At the highest osmotic strengths, this led to an undulating light scattering profile that appeared to be related to perturbation of the viral envelope resulting in loss of virus activity, as determined by in vitro hemagglutination measurements and in vivo immunogenicity studies in mice. Addition of carboxymethyl cellulose effectively prevented vaccine activity loss in vitro and in vivo, believed to be due to increasing the viscosity of concentrated sugar solution and thereby reducing osmotic stress during coating of microneedles. These results suggest that hyperosmotic solutions can cause biphasic shrinkage of whole inactivated influenza virus which can damage vaccine activity at high osmotic strength and that addition of a viscosity enhancer to the vaccine coating solution can prevent osmotically driven damage and thereby enable preparation of stable microneedle coating formulations for vaccination.
Highlights
This study focused on virus particle stability in hyperosmotic conditions is motivated by the need to stabilize vaccines during preparation of microneedle (MN) patches
Permeability of inactivated influenza virus under osmotic stress To understand the effects of osmotic stress on influenza virus stability, we first determined viral membrane permeability by measuring changes in virus size as a function of osmotic strength
Our research was performed only on influenza virus, we propose that the general idea of osmotic pressure-induced viral deactivation and the stabilizing effect of a viscosity enhancer can be used in the future development of both solid and liquid drug formulations for other enveloped vaccines, as exemplified by influenza vaccine-coated MNs
Summary
This study focused on virus particle stability in hyperosmotic conditions is motivated by the need to stabilize vaccines during preparation of microneedle (MN) patches. Previous studies have shown that MN patches coated with influenza and other vaccines provide effective skin vaccination that generates protective immune responses that are at least as potent as conventional intramuscular or subcutaneous vaccination [1,2,3]. Advantages of MNs include the potential for vaccine dose-sparing and improved immunogenicity via alternative administration routes [4,5,6,7]. Because solid MN vaccines provide a promising platform for long-term stability and maintenance of protective immunogenic potency, considerable efforts have been devoted to the fabrication of various types of MNs and demonstrating their in vivo benefits. Among many factors involved in this problem (e.g., phase transformation, dehydration effects, interaction between vaccine and substrate, osmotic stress, pH change, etc.) we hypothesize that osmotic stress is a significant underlying problem for MN coating with enveloped vaccines/viruses
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