(M1330-04-24) Dual Immunization Against COVID-19 and Influenza Using Fast-Dissolving Buccal Films

Purpose: SARS-CoV-2 and influenza are transmissible respiratory viruses that frequently mutate, impacting the efficacy of currently available vaccines. Thus, vaccination against SARS-CoV-2 variants and seasonal influenza strains may be required every year. This proof-of-concept study investigates the immunogenicity of an adjuvanted microparticulate SARS-CoV-2-influenza dual vaccine administered as one 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 both viruses when administered as a combination using an ODF. The vaccine approach encapsulates the vaccine antigens and adjuvants 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. A ‘dual vaccine’ is essentially one vaccine targeting two diseases, thereby eliminating separate immunizations for each disease. Moreover, a buccal vaccine can be self-administered and an alternative to the more traditional and potentially painful intramuscular (IM) injection.

Methods: Inactivated SARS-CoV-2 (i-SARS-CoV-2) and inactivated Influenza A H1N1 virus (i-Influenza A H1N1) were used as the vaccine antigens in polymeric MPs. The adjuvants Alhydrogel®, AddaVax™, and CPG 7909 were selected to enhance the immunogenicity of the antigen. The antigen and adjuvant PLGA MPs were prepared separately 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 appearance of the MPs was visualized using a scanning electron microscope (SEM). % 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+adjuvant MPs with the gel base, then added to a 96-well Polydimethylsiloxane (PDMS) template. The films formed after 8 hours of drying in a vacuum desiccator. The ODFs were characterized for dissolution time, diameter(mm), and thickness(μm). The adjuvanted vaccine ODFs were administered to mice (N=6) as one prime (w0) and two boosts (w2,w4) 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 recovery yield of MPs was greater than 80%w/w. The characterization assessments revealed the following 1.) size: less than 1μm. 2.) PDI range: 0.1 to 0.5. 3.) Charge: -20mV to -30mV 4.) %EE: 80% to 90% (values are expressed within a range to account for the different MP preparations). SEM observations showed that the particles were spherical with smooth surfaces and evenly distributed. The in vitro nitric oxide (NO) released by the DCs upon exposure to the vaccine MPs was assessed as a marker for an innate immune response which was enhanced upon the addition of adjuvant MPs (Alhydrogel®+AddaVax™+CPG 7909). The in vitro cytotoxicity assessment showed that the MPs 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. The cross-sectional thickness of the ODFs was measured as 190μm. Following in vivo immunization, the anti-SARS-CoV-2 and anti-Influenza A H1N1 serum IgG and IgA levels increased significantly (p≤0.0001) when compared to the No treatment group. The IgG subtype analyses showed an equal distribution and significantly high levels of serum IgG1 (Th-2 mediated humoral response) and IgG2a (Th-1 mediated cytotoxic response) antibodies specific to both viruses (p≤0.01). Serum IgM levels were predominant in the initial weeks (p≤0.0001). The increased serum IgG and IgA levels specific to both viruses may suggest that the isotype IgM undergoes a balanced class switch to IgG and IgA.

Conclusion: The vaccine and adjuvant MPs and the vaccine ODFs were successfully prepared and characterized. The values of the characteristic parameters were within the desired range. The in vitro assessment suggests that the vaccine was safe and immunogenic. The in vivo assessment evaluated the immunogenicity of the dual vaccine for its ability to generate anti-SARS-CoV-2 and anti-Influenza A H1N1 antibodies, which increased significantly following immunization. The study has yielded promising results thus far. Our future directions include assessing neutralization titers, cellular immune response, and markers for mucosal immunity. Thus, a dual vaccine for COVID-19 and Influenza and buccal immunization using ODFs has great potential to cause a fundamental breakthrough in vaccine development.

References: 1.) Gala RP, Popescu C, Knipp GT, et al. Physicochemical and Preclinical Evaluation of a Novel Buccal Measles Vaccine. AAPS PharmSciTech. 2017;18(2):283-292. doi:10.1208/s12249-016-0566-3
2.) Braz Gomes, K., D’Souza, B., Vijayanand, S., Menon, I., & D’Souza., MJ. A Dual-Delivery Platform for Vaccination using Antigen-loaded Nanoparticles in Dissolving Microneedles, International Journal of Pharmaceutics, 2021, 121393, ISSN 0378-5173

Acknowledgements: 1.) The following reagent was obtained through BEI Resources, NIAID, NIH: CpG 7909 Adjuvant, NR-52393.
2.) The following reagent was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA/CA_CDC_5574/2020, Heat Inactivated, NR-55245.
3.) The following reagent was obtained through BEI Resources, NIAID, NIH: Influenza A Virus, A/Brisbane/59/2007 (H1N1), BPL-Inactivated, NR-19322.


Figure 1: Graphical Abstract. The inactivated viruses (SARS-CoV-2 and Influenza A H1N1) were encapsulated separately in PLGA matrices to form the MPs. The MPs were then characterized for size, PDI, %EE, and charge. The MPs were tested in vitro for immunogenicity and safety. The ODF were characterized for their size, thickness, and dissolution time. The final adjuvanted vaccine MP loaded ODFwere administered to mice to test the in vivo vaccine efficacy. Following immunization, the serum antibody levels (IgG, IgG1, IgG2a, IgM, and IgA) were assessed and reported.


Figure 2: Characterization of Microparticles (MPs) and Oral Dissolving Films (ODFs). (A) Scanning electron microscopy (SEM) image of the MPs. The size of the MPs was less than 1μm. (B) SEM image of an entire ODF. The diameter was measured to be 4mm. (C) Image of ODF compared to the one-cent coin. (D) SEM image of the cross-section of ODF. The thickness was measured to be around 190μm.


Figure 3: SARS-CoV-2 and Influenza A H1N1 specific serum IgG and IgA levels in vaccinated mice: The MP (Vaccine + Adjuvants) in ODF group contained the vaccine MPs (i-SARS-CoV-2 + i-Influenza A H1N1) and Adjuvants MPs (Alhydrogel®+AddaVax™+CPG 7909) in ODF. The adjuvanted vaccine ODFs were administered to mice via the buccal route as one prime dose (w0) and two booster doses (w2 and w4). The virus-specific serum IgG and IgA levels were assessed bi-weekly using an enzyme-linked immunosorbent assay (ELISA). Both Anti-SARS-CoV-2 and Anti-Influenza A H1N1 serum IgG and IgA levels increased significantly (****,p≤0.0001) in vaccinated mice following immunization and remained as such up to w10 (significance as compared to No treatment group). Data are expressed as Mean ± SEM, N=6.