(W1230-03-16) Vaccination Against Influenza Using Fast Acting 3D Printed Oral Dissolving Films as a Delivery System

Purpose: Influenza virus is a transmissible respiratory virus that frequently mutates and is of global health concern. Although a couple of vaccines have been approved, most of these vaccines are administered via the intramuscular route which is painful and has a potential for vaccine hesitancy. The influenza virus is transmitted through the mucosal route, and we envision a robust immune response at the site of viral entry using 3D-printed oral dissolving films as a delivery system. In this proof-of-concept study, we investigated the immunogenicity of an adjuvanted microparticulate Influenza vaccine administered as oral dissolving film (ODF) via the buccal (cheek) route. Our central hypothesis is that vaccine microparticles (MPs) will induce strong systemic and mucosal immunity against the virus when administered using an ODF. The vaccine approach encapsulates the vaccine antigens in a biodegradable Poly (Lactic-co-glycolic acid) (PLGA) polymeric matrix, which can enhance antigen presentation. The vaccination strategy using ODFs administered via the buccal mucosa is promising for the following reasons 1.) The buccal mucosa is highly vascularized. 2.) It consists of circulating immune cells, which can generate a prompt immune response. 3.) Capable of inducing mucosal immunity. Moreover, a buccal vaccine can be self-administered as an alternative to the more traditional and potentially painful intramuscular (IM) injection.

Methods: Herein, we encapsulated the vaccine antigens, Inactivated Influenza A H1N1 virus (i-Influenza A H1N1) and Inactivated Influenza A H3N2 virus (i-Influenza A H3N2) in a biodegradable Poly (Lactic-co-glycolic acid) (PLGA) polymeric matrix. The adjuvant MF59(AddaVax™) was selected to enhance the immunogenicity of the antigen. The antigen PLGA MPs were prepared using a double emulsion (w/o/w) method, lyophilized, and characterized for their size (nm) and polydispersity index (PDI), and charge (mV) using a dynamic light scattering (DLS) particle size analyzer. The % encapsulation efficiency (%EE) was determined as the amount of antigen successfully encapsulated in the PLGA MPs, which was assessed using a Micro BCA™ (Bicinchoninic acid) Protein Assay. The vaccine MPs were assessed in vitro in dendritic cells (DC 2.4) for immunogenicity and safety using Griess’ nitrite release assay and MTT (3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide) reagent cytotoxicity assay respectively. The ODFs were prepared by combining the biodegradable polymers – Kollidon 90F, Kollidon VA64, and PEG2000 in ethanol to form a gel base. The final vaccine-loaded ODFs were prepared by combining the vaccine MPs with the gel base using a 3D printer. The ODFs were characterized for dissolution time, thickness(μm), and surface PH. The adjuvanted vaccine ODFs were administered to mice as one prime (w0) and one boost (w3) via the buccal route to test the in vivo vaccine efficacy. The virus-specific serum antibody (IgG, IgG1, IgG2a, IgM, IgA) levels were assessed bi-weekly using an enzyme-linked immunosorbent assay (ELISA).

Results: The yield of MPs was greater than 80%w/w±5 w/w. The characterization assessments revealed the following 1.) size: less than 2μm. 2.) PDI: 0.4±0.1. 3.) Charge: -26.4 mV ±4.609 for H1N1 microparticles and -23.8 mV ± 3.5 for H3N2 microparticles 4.) %EE: 80% to 90% (values are expressed within a range to account for the different MP preparations). The in vitro nitric oxide (NO) released by the DCs upon exposure to the vaccine MPs was assessed to determine the immunogenicity of the formulation. The in vitro cytotoxicity assessment showed that the microparticles were non-cytotoxic to cells. The vaccine-loaded ODFs quickly dissolve (less than 5 mins) in artificial saliva to release the vaccine MP. The diameter of the ODFs was measured as 4mm and the surface pH was 7.2. More importantly, following in vivo immunization, we found that the Influenza A H1N1 specific serum IgG and IgA levels and Influenza A H3N2 specific serum IgG and IgA increased significantly (p≤0.0001) compared to the control group. The IgG subtype analyses showed both significantly high levels of serum IgG1 (Th-2/antibody mediated response) and IgG2a (Th-1/cytotoxic mediated response) antibodies specific to both strains (p≤0.01) reflecting a robust humoral response. Additionally, we found significant antibody levels (IgG and IgA) in the lungs which show mucosal immunity (p≤0.0001). We also found a significant expression of CD4+ and CD8+ cells in Spleen and Lymph Node (p≤0.01) indicating a robust cellular response.

Conclusion: The in vitro assessment suggests that the vaccine was safe and immunogenic. Additionally, the in vivo assessment shows that buccal immunization generates antigen-specific antibodies, mucosal response in both serum and lung samples, and cellular response and Spleen and Lymph Node. The study has yielded promising results thus far. Our future directions include assessing lung viral titers. Overall, buccal immunization using ODFs has great potential to cause a fundamental breakthrough in vaccine development and increase vaccination coverage.

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Table 1: Characterization of Microparticles. Data are expressed as Mean ± SEM


Figure 1: Influenza A H1N1 specific and Influenza A H3N2 specific serum IgG and IgA levels in vaccinated mice: The adjuvanted vaccine ODFs were administered to mice via the buccal route as one prime dose followed by one booster dose. The virus-specific serum IgG and the serum IgA levels were assessed bi-weekly using an enzyme-linked immunosorbent assay (ELISA). Both antigen-specific serum IgG and IgA levels increased significantly (****,p≤0.0001) in vaccinated mice following immunization compared to naïve group). Data are expressed as Mean ± SEM


Figure 2: Influenza A H1N1 specific and Influenza A H3N2 specific lung IgG and IgA levels in vaccinated mice: The adjuvanted vaccine ODFs were administered to mice via the buccal route as one prime dose followed by one booster dose. The virus-specific Lung IgG and the Lung IgA levels were assessed bi-weekly using an enzyme-linked immunosorbent assay (ELISA). Both antigen-specific Lung IgG and IgA levels increased significantly (****,p≤0.0001) in vaccinated mice following immunization compared to naïve group). Data are expressed as Mean ± SEM