(582de) Stabilization of Vaccines in Silk

There currently exists a need for new vaccine delivery systems able to improve storage stability and vaccine efficacy. Sensitive biological compounds, such as vaccines, traditionally require a time-dependent ‘cold-chain’ to maximize therapeutic activity. All vaccines require constant cold storage and special processing to ensure the necessary potency is maintained. The WHO estimates nearly 50% of all vaccines are wasted each year due to improper storage and delivery. To address these limitations, we demonstrate a novel silk-based vaccine delivery system able to entrap and stabilize the live measles, mumps and rubella (MMR) vaccine at ambient conditions and higher, eliminating the need for specialized storage needs. Using silk, a mechanically robust biomaterial with a unique block copolymer structure that provides a durable matrix, we demonstrate enhanced stabilization of these labile compounds and also provide mechanistic insight.

MMR^® II (Merck & Co., Inc., USA), a commercial lyophilized powder vaccine, was used to demonstrate the stabilization effects of silk matrices. Standard MMR-silk films were prepared by casting an aliquot of MMR-silk solution on Teflon-coated molds and air-dried. Lyophilized MMR-silk films were prepared by freeze-drying aliquot of the same solution. Vaccine potency assays were conducted via inoculation of Vero cells  (African green monkey kidney cells). The viral RNA from infected cells was extracted using TRIzol/chloroform, converted to cDNA and quantitated using qPCR. The structural characteristics of protein stability were measured using solid-state differential scanning calorimetry (DSC) and liquid-state nano-DSC. Aggregation of viral particles as a function of temperature was examined by dynamic light scattering (DLS).

Encapsulation in silk film (air-dried or lyophilized) enhanced measles, mumps, and rubella viral particle stability when stored at 25°C, 37°C and 45°C and exhibited improved residual potency over the MMR vaccine alone. For the measles component of the vaccine, after 6 months stored at 25°C, the MMR-silk films retained 83.9% potency compared to 74.5% for the MMR vaccine powder. At 37°C, silk films retained 56.5% potency compared to 9.9% from the powder. At 45°C, the vaccine powder lost all potency after 20 weeks in storage while the silk films retained 53.4% activity after 24 weeks. The mumps and rubella components displayed similar trends in potency retention. Lyophilized MMR-silk films reduced the residual moisture of the complex and after 6 months in storage at 37°C and 45°C, the lyophilized films retained ≥ 85% residual potency for all viral components of the vaccine.

The DSC thermogram of the MMR powder showed a glass transition (T_g) at 68.9°C, while the lyophilized MMR-silk films showed a T_g at 89.2°C. The nano-DSC thermogram of the purified viral particles in silk solution showed a transition point (T_p) at 68.3°C, compared to that of the viral particles in water at 16.8°C. The increase in T_g and T_p values are indicative of silk stabilizing the conformations of the protein components of the vaccine and the pure viral proteins, respectively. Conformational changes, such as protein unfolding, may affect the stability of the viral proteins by inducing aggregation and thus reducing vaccine potency. DLS results showed the purified virus solution exhibited an increase in mean effective diameter around 16°C while viral particles in silk solution did not show signs of aggregation until 70°C, indicating silk provided structural stability that prevented thermal-induced aggregation of the viral proteins. Silk is composed of large hydrophobic domains interspersed with small hydrophilic regions that form ordered crystalline domains (β-sheets) that stabilize via physical crosslinks. This assembly forms structural pockets that can immobilize bioactive molecules and improve their stability by minimizing water content and reducing protein chain mobility, thus preventing a transition from the native to denatured state. The unique chemistry, structure and assembly of silk make this protein polymer an attractive candidate for the stabilization of bioactive molecules at elevated temperatures.

Silk reduced the temperature-induced protein unfolding and subsequent aggregation by providing structural stability to the vaccine and elevated the temperature at which the viral proteins denature. These results show the potential of encapsulation of labile therapeutic molecules in silk to transform the approach to storage of these molecules and minimize the need for adherence to the ‘cold-chain’.