(M1130-01-09) Development of an In Vitro Cell Culture Model for Evaluation of Influenza Vaccine Formulated Using Spray-Dried Albumin Nanoparticles

Purpose: Vaccines formulated as aqueous solutions complicate the distribution process as they are required to be maintained at cold temperatures due to their higher susceptibility to physical and chemical degradation. This adds to the financial burden of storing vaccines after their successful development. Also, administering an injection of the vaccine solution requires assistance from a trained professional, and causes a risk of needle injuries, pain at the site of administration, patient distress along with low compliance. With the urge to find an alternative for administering and distributing vaccines effectively on a larger scale, we intended to formulate them in the form of spray-dried solid nanoparticles (SNPs). These solid nanoparticles can either be reconstituted with sterile water or can be administered through the mucosal route (nasal). Vaccine evaluation using animal models is a costly and time-consuming process. Many animals are required during the screening stage to assess the toxicity, potency, safety, and efficacy of the candidate. Given the limit, there is a need to develop models and methods for efficient and inexpensive evaluation of vaccines that would increase the chances of success in the real world. Dendritic cells are potent antigen-presenting cells and act as mediators of innate and adaptive immunity. The mature DC present the processed peptides of the invading antigen on their surfaces thus releasing signals in the form of cytokines to naïve T cells. This characteristic of DC can be used for screening vaccines at the initial stage.

Methods: Preparation of Albumin nanoparticles - To prepare bovine serum albumin (BSA) nanoparticles, 1% w/v of BSA solution was prepared by dissolving it in 18 Ω ultrapure water. This solution was then cross-linked with 0.5% w/v of glutaraldehyde for 12 hours followed by constant magnetic stirring at low speed. The marketed vaccine formulation was then added to the crosslinked BSA and sprayed using a BUCHI B90 HP spray dryer.
Evaluation of Albumin nanoparticles on in vitro cell models - HeLa cells were used to perform preliminary studies as they grow fast. These cells were incubated with BSA nanoparticle suspension and the commercial nanoparticle suspension for 4 h, 8 h, 24 h, and 48 h and visualized for uptake using a Keyence fluorescence microscope.
To further confirm the uptake of nanoparticles in dendritic cells, they were stained with cell mask orange dye followed by counterstaining with DAPI. To study the activation of DC cell line, they were cultured at 37°C with 5% CO₂ in Alpha Minimum Essential media supplemented with penicillin/streptomycin, glutamine, and murine GM-CSF. Cells were seeded at a density of 1.5*10⁶ cells/ well in 12 well plates, treated as per the study groups listed below. Cytokines were quantified at 24h using ELISA.

Results: The particle size of blank nanoparticles was 650nm - 800nm and vaccine-loaded nanoparticles were 950nm - 1050nm. The intensity of fluorescence gradually increased from 4 h to 48 h further demonstrating the association of the nanoparticles with the cells. HeLa cells incubated with commercial nanoparticles also showed a similar trend thus confirming the uptake of developed nanoparticles. We then proceeded towards incubating the nanoparticles with dendritic cells and staining them to differentiate the location of nanoparticles and we found that they clustered around the nucleus indicating their presence within the cell. Commercial Flu vaccine in the liquid form was encapsulated in BSA nanoparticles via spray drying and evaluated for its ability to stimulate the dendritic cell line to release cytokines which are essential chemicals in the activation of the immune system. Vaccine-loaded nanoparticles were able to stimulate the dendritic cells to release cytokines in significantly higher quantities than the commercial Flu vaccine formulation.

Conclusion: DC cells treated with vaccine-loaded nanoparticles secreted cytokines at a level higher than the marketed formulation indicating the efficiency of solid nanoparticles at delivering the vaccine over the traditional liquid formulation. Also, the dendritic cell-based in-vitro cell culture model mimicked the characteristic of the immune system to respond to external stimuli by releasing cytokines and thus can be used for the initial screening of vaccine candidates.

References: 1. Jiang X, S.C., Rey-Ladino J, Yu H, Brunham RC, Characterization of murine dendritic cell line JAWS II and primary bone marrow-derived dendritic cells in Chlamydia muridarum antigen presentation and induction of protective immunity. Infect Immun., 2008. 76(6): p. 2392-2401.
2. Ryan SL, B.A., Vaz G, et al, Drug Discovery Approaches Utilizing Three-Dimensional Cell Culture [published correction appears in Assay Drug Dev Technol. 2016 Apr;14(3):213]. Assay Drug Dev Technol., 2016. 14(1): p. 19-28.


Table 1- Treatment Groups


Results