(T0930-06-38) Evaluating the Immunogenicity of Painless Microneedle Microparticulate Combination Vaccine for COVID-19 and Flu

Purpose: Respiratory viruses like SARS-CoV-2 and influenza mutate frequently, giving rise to new variants over time. To keep up, approved vaccines need regular updates, often in the form of booster shots designed to protect against the latest strains.Vaccine hesitancy, needle injections and separate vaccinations for these two viruses create inconvenience and contribute to reduced global vaccination rates. To address this, there is a need for non-invasive microneedle combination vaccine administration as an effective alternative to separate intramuscular shots. The epidermal and dermal layers of the skin consist of specialized immune cells, such as Langerhans cells and circulating dendritic cells, which can be targeted via microneedles to efficiently recognize vaccine antigens and trigger an immune response.

Methods: As a proof-of-concept, inactivated Delta and Omicron variants of SARS-CoV-2, as well as the H1N1 and H3N2 variants of Influenza were encapsulated as antigens in poly(lactic-co-glycolic acid) (PLGA) polymer matrices , achieving over 85% entrapment. The inactivated virus vaccine microparticles were formulated separately using a double emulsion method protocol. Here, heat-inactivated antigen (2% loading) was added to a pH 7.4 phosphate buffer which was further added to PLGA in DCM solution (2% w / v) and probe homogenized at 17,000 RPM using a 30 s on/off cycle for 2 min to form a primary emulsion. Next, this primary emulsion was probe homogenized with PVA solution in deionized water (0.1% w / v) for 2 min at 17,000 RPM to form a double emulsion. The final emulsion was stirred for 5 hours to remove the residual DCM via solvent evaporation. The excess PVA was removed by washing with deionized water, followed by centrifugation at 17,000 RPM for 10 min. The microparticles were then resuspended in deionized water containing trehalose (2 % w/v) as a cryoprotectant and lyophilized using a freeze dryer to obtain a freeze-dried powder. This was characterized for particle size, zeta potential and polydisperse index. The combination vaccine + Addavax (adjuvant) was administered to 6–8-week-old Swiss Webster mice as one prime and one booster dose. The mice were exposed to an intranasal challenge with Influenza A virus. The mice were then assessed for antibody levels and cellular responses.

Results: The microneedle microparticle vaccine induced high levels of antigen-specific serum IgA,IgG, IgG1 and IgG2a binding antibodies, as well as elevated IgG and IgA levels in the lung wash samples compared to naïve and intramuscular suspension group. Furthermore, the microneedle vaccine prompted increased antigen-specific CD4⁺ and CD8⁺ T-cell responses in the vaccinated mice.

Conclusion: In this study, we successfully developed a transdermal combination vaccine for SARS-CoV-2 and influenza using microparticle-loaded microneedles. When tested in mice, the vaccine triggered a strong immune response, producing high levels of antigen-specific antibodies in both the serum and lung supernatant. It also boosted the expression of CD4 and CD8a cell surface markers in immune cells from the spleen and lymph nodes. The presence of IgG2a and IgG1 antibodies, along with activated helper (CD4+) and cytotoxic (CD8a+) T cells, suggests that this vaccine can stimulate both Th1- and Th2-mediated immunity. These findings demonstrate the potential of a transdermal combination vaccine for protecting against both COVID-19 and influenza.

References: 1. Vijayanand, S.; Patil, S.; Bagwe, P.; Singh, R.; Adediran, E.; D’Souza, M.J. Evaluating the Immunogenicity of an Intranasal Microparticle Combination Vaccine for COVID-19 and Influenza. Vaccines 2025, 13, 282, doi:10.3390/vaccines13030282.
2. Vijayanand, S.; Patil, S.; Menon, I.; Braz Gomes, K.; Kale, A.; Bagwe, P.; Uddin, M.N.; Zughaier, S.M.; D’Souza, M.J. An Adjuvanted Inactivated SARS-CoV-2 Microparticulate Vaccine Delivered Using Microneedles Induces a Robust Immune Response in Vaccinated Mice. Pharmaceutics 2023, 15, 895, doi:10.3390/pharmaceutics15030895.
3. Braz Gomes, K.; Vijayanand, S.; Bagwe, P.; Menon, I.; Kale, A.; Patil, S.; Kang, S.-M.; Uddin, M.N.; D’Souza, M.J. Vaccine-Induced Immunity Elicited by Microneedle Delivery of Influenza Ectodomain Matrix Protein 2 Virus-like Particle (M2e VLP)-Loaded PLGA Nanoparticles. IJMS 2023, 24, 10612, doi:10.3390/ijms241310612


Figure 1- Antigen-specific antibody levels in vaccinated and unvaccinated mice. Delta, Omicron, H1N1 & H3N2 Serum IgG


Figure 2- Antigen-specific antibody levels in vaccinated and unvaccinated mice. Delta, Omicron, H1N1 & H3N2 Serum IgA


Figure 3- Delta specific CD4, CD8 in spleen; Omicron specific CD4 & CD8 in spleen; H1N1 specific CD4, CD8 in spleen; H3N2 specific CD4, CD8 in spleen