(T1330-06-37) 3D Printed Buccal Film Having Microparticulate COVID-19 Vaccine Delivered by Heterologous Strategy

Purpose: The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in over 776 million cases and 7.1 million deaths worldwide. Various vaccines targeting different viral components such as inactivated virus, spike proteins, nucleoproteins, mRNA, and vector-based platforms have been developed. However, these vaccines often show limited effectiveness against emerging variants due to ever mutating nature and it’s antigen drift. Variants such as Delta, Omicron, Alpha, and Beta have caused widespread disruption due to their transmissibility and immune evasion. Because of the virus’s continual evolution, developing a vaccine with broad-spectrum immunity and cross-reactivity is crucial. Heterologous vaccination, which involves administering different viral variants in the prime and booster doses, is a promising strategy. This method promotes a broader immune response and facilitates mass immunization over a short period. We investigated this approach by formulating microparticles having whole heat-inactivated SARS-CoV-2 strains—Delta for the prime and Omicron for the booster administered and delivering through oral dissolving film (ODF) Microparticles provide antigen stability by encapsulating them in a polymeric matrix, acts as controlled released that prolong antigen presentation, biodegradable and non-toxic system of delivery. Moreover, delivery through buccal route will provide (1) ease and pain-free immunization as compared to traditional intra-muscular route (2) highly vascularized delivery - vaccine particles are rapidly uptaken due to coming in direct contact with epithelial layer of buccal cavity having mucosa associated lymphoid tissue which is packed with antigen presenting cells to generate immune response (3) unified mucosal delivery – capable of inducing mucosal response

Methods: Microparticles containing vaccine and adjuvant were prepared using the double emulsion solvent evaporation method with poly-(lactic-co-glycolic acid) (PLGA) polymer, followed by lyophilization. Whole inactivated SARS-CoV-2 Delta and Omicron strains were used as antigens, with alum as the adjuvant. The particles were characterized for size, surface charge, polydispersity index (PDI), morphology, and encapsulation efficiency. Safety and immunogenicity were assessed using MTT and Griess assays in vitro. Oral dissolving films were formulated by suspending the microparticles in the ODF biodegradable polymer like PEG2000 along with plasticizers and film forming agents in ethanol to form a gel base and was then transferred into the mold and dried it overnight. The ODFs were tested for pH, disintegration time, thickness, and Diameter. Mice were immunized with either intramuscular microparticulate vaccine or ODF-based microparticulate vaccine. Samples were collected biweekly post-immunization, and serum antibody titers (IgG, IgG1, IgG2a) and mucosal IgA levels were measured using ELISA. Cellular immune responses (CD4+ and CD8a+ T cells) were evaluated in the spleen, lymph nodes. To test for cross-reactivity, responses were also assessed against Alpha and Beta strains.

Results: Microparticles showed a recovery yield above 90%, with particle sizes ranging from 650–750 nm, surface charge between −40 to −55 mV, and a PDI of 0.5–0.7. Encapsulation efficiency exceeded 85%, and scanning electron microscopy revealed a donut-shaped morphology favorable for uptake by antigen-presenting cells. MTT assays confirmed non-cytotoxicity up to 500 µg/mL, while the Griess assay demonstrated significantly elevated nitric oxide release in the adjuvanted vaccine group. Average pH of the ODF was found to be of 7.5 and disintegrated time was 2-3 mins; diameter was found to be 0.170 and the average thickness was 0.197± 0.08   In vivo, vaccinated mice showed significantly higher IgG levels in serum and lung samples compared to controls. High IgG1 and IgG2a levels suggested a balanced Th1/Th2 response. Cellular analysis indicated increased CD4+ and CD8a+ T cell populations in vaccinated groups across spleen. Notably, robust immune responses were detected against both Delta and Omicron strains, and significant cross-reactivity was observed against Alpha and Beta strains. Overall, partial cross-reactivity was achieved against most of the strains of COVID-19. The Beta strain showed limited cross-reactivity due to mutations in the receptor-binding domain and antigenic sites, leading to the antigenic drift that makes these sites less recognizable by antibodies. Moreover, these mutations alter the structure and differentiate Beta from other variants like Omicron and Delta, thus reducing binding with the antibodies Alpha, on the other hand, has several mutations, but it is less divergent compared to the Delta and Omicron variants. This is reflected in the cross-reactive response against Delta- and Omicron-specific antibodies.

Conclusion: This proof-of-concept study successfully demonstrated that an adjuvanted microparticulate vaccine delivered via oral dissolving films can elicit strong systemic and mucosal immune responses in mice. The vaccine formulation was characterized by favorable particle size, charge, and morphology, and was shown to be safe and immunogenic in vitro. In vivo experiments confirmed the ability of this heterologous vaccine to induce humoral and cellular immunity, with cross-reactive responses to multiple SARS-CoV-2 variants. The use of a buccally delivered ODF platform offers advantages in vaccine storage, administration, and public acceptance, presenting a promising strategy for broader, more accessible immunization against evolving viral threats.

References: 1. World Health Organization. WHO Coronavirus (COVID-19) Dashboard. 2023. Available online: https://data.who.int/dashboards/covid19/cases
2. Arte, T.M.; Patil, S.R.; Adediran, E.; Singh, R.; Bagwe, P.; Gulani, M.A.; Pasupuleti, D.; Ferguson, A.; Zughaier, S.M.; D’Souza, M.J. Microneedle Delivery of Heterologous Microparticulate COVID-19 Vaccine Induces Cross Strain Specific Antibody Levels in Mice. Vaccines 2025, 13, 380. https://doi.org/10.3390/vaccines13040380
3. Bhatnagar, N.; Kim, K.-H.; Subbiah, J.; Muhammad-Worsham, S.; Park, B.R.; Liu, R.; Grovenstein, P.; Wang, B.-Z.; Kang, S.-M. Heterologous Prime-Boost Vaccination with Inactivated Influenza Viruses Induces More Effective Cross-Protection than Homologous Repeat Vaccination. Vaccines 2023, 11, 1209
4. Vijayanand, S.; Patil, S.; Joshi, D.; Menon, I.; Braz Gomes, K.; Kale, A.; Bagwe, P.; Yacoub, S.; Uddin, M.N.; D’Souza, M.J. Microneedle Delivery of an Adjuvanted Microparticulate Vaccine Induces High Antibody Levels in Mice Vaccinated against Coronavirus. Vaccines 2022, 10, 1491.
5. Lin, B.; Cheng, L.; Zhang, J.; Yang, M.; Zhang, Y.; Liu, J.; Qin, X. Immunology of SARS-CoV-2 Infection and Vaccination. Clin. Chim. Acta 2023, 545, 117390.

Acknowledgements: We acknowledge BEI resources for providing antigens for this study.
1. NR-56496 SARS-Related Coronavirus 2, Isolate hCoV-19/USA/GA-EHC-2811C/2021 (Lineage B.1.1.529; Omicron Variant), Gamma-Irradiated
2. NR-56129 SARS-Related Coronavirus 2, Isolate hCoV-19/USA/MD-HP05285/2021 (Lineage B.1.617.2; Delta Variant), Gamma-Irradiated
3. NR-55277 Spike Glycoprotein Receptor Binding Domain (RBD) from SARS-Related Coronavirus 2, Alpha Variant with C-Terminal Histidine Tag, Recombinant from HEK293 Cells
4. NR-55278 Spike Glycoprotein Receptor Binding Domain (RBD) from SARS-Related Coronavirus 2, Beta variant with C-Terminal Histidine Tag, Recombinant from HEK293 Cells


figure 1: (A) Scanning electron microscopy at the magnification of 5200X (B) SDS pages analysis. Column (a) with a blue arrow represents bands formed by Delta 328 suspension and column (b) with an orange arrow represents bands formed by omicron 329 suspension. Column (c) with a blue arrow represents bands formed by Delta microparticles and 330 column (d) with an o range arrow represents bands formed by Omicron microparticles. (C) Nitrite release determination using Griess assay. Cells were seeded at a density of 1 × 10⁴ cells per well and exposed to various treatment groups for 24 h. The treatment groups included untreated cells (no treatment), blank microparticles (2 µg), whole inactivated virus Delta suspension (2 µg), microparticulate vaccine with whole inactivated virus Delta variant (2 µg), microparticulate vaccine with WIV Delta variant + adjuvant microparticles (2 µg), whole inactivated virus Omicron suspension (2 µg), microparticulate vaccine with whole inactivated virus Omicron variant (2 µg), and microparticulate vaccine with whole inactivated virus Omicron variant + adjuvant microparticles (2 µg). Following incubation, supernatants were collected and analyzed for nitrite release using the Griess assay. Data are presented as mean ± SEM and were analyzed using one-way ANOVA, followed by Tukey’s post hoc multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001


Figure 2: IgG specific antigen antibody response. IgG antibody levels in vaccinated and unvaccinated mice serum in week 2, week 5 and terminal (after prime and booster dose). The mice were vaccinated with an adjuvanted- microparticulate vaccine following the heterologous dosing. Mice sera were collected biweekly and evaluated for antibodies against inactivated SARS-CoV-2 (delta and omicron strains) and inactivated SARS-CoV-2 (beta and alpha strains). Significant levels of IgG were observed in all four variants of SARS-CoV-2, indicating a cross-reactive immune response. Data are expressed as Mean ± SEM and analyzed with Two-way ANOVA, Dunnett’s multiple comparisons test * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001




Figure 3 – Cellular responses in vaccinated mice. Spleen was harvested, antigen specific cellular responses and cross-reactive cellular responses were observed for the variants of antigens used in this study (A) WIV-Omicron specific CD4+ cells Tcell response (B) WIV-Omicron specific CD8a cells Tcell response (C) WIV- Delta specific CD4+ Tcell response (D) WIV- Delta specific CD4+ Tcell response (D) WIV- Delta specific CD8a cells Tcell response. Cross -reactivity was also analyzed using Spike RBD of Alpha and Beta specific antigens (E) WIV- Alpha specific CD4+ Tcell response (F) WIV- Alpha specific CD8a Tcell response (G) WIV- Beta specific CD4+ cells Tcell response (H) WIV- Beta specific CD8a Tcell response. The responses obtained are compared to the naïve group. Data expressed as Mean ± SEM n = 5 and two-way ANOVA test. ns: non-significant, ** p ≤ 0.01, and **** p ≤ 0.0001.