(W1130-07-41) Inhale It: Heterologous Intranasal Delivery of a SARS-CoV-2 Microparticle Vaccine

Purpose: The COVID-19 pandemic, caused by SARS-CoV-2 and its different variants has resulted in over 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 as most of this vaccine uses homologous one platform strategy. This leads to failure of immunity to generate broader immune response against various variants. To better handle this, in this study we propose heterologous vaccination, which involves administering different viral variants and different platforms in the prime and booster doses. This method promotes a broader immune response and facilitates mass immunization over a short period. We investigated this approach by formulating microparticles having a sub-unit part of the virus that is spike glycoprotein (most immunogenic part) of Delta strain for the prime and whole inactivated virus (WIV) of Omicron for the booster and administered through pain-free intranasal route of administration. 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 nasal route will provide (1) ease and pain-free immunization as compared to traditional intra-muscular route (2) highly perfused vascular - vaccine particles are rapidly up taken by coming in direct contact with epithelial layer of buccal cavity having nasal associated lymphoid tissue (NALT) 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, zeta potential, polydispersity index (PDI) using zeta sizer, morphology by Scanning electron microscopy (SEM), and encapsulation efficiency by BCA assay. Safety and immunogenicity were assessed using MTT and Griess assays in vitro. Following groups were considered for immunization in mice. (1) Naïve group – no treatment (control); (2) Intramuscular-suspension microparticle group; (3) Intranasal microparticulate group. Intramuscular administration was executed in the thigh muscle of the mice using phosphate buffer saline as diluent. Intranasal dose was resuspended in sterile de-ionized water administered in nasal cavity. Serum and mucosal samples were collected biweekly post-immunization. Antibody titers (IgG, IgG1, IgG2a) and mucosal IgA levels were measured using Enzyme linked immunosorbent assay (ELISA). Animals were sacrificed at the end of the study. Cellular immune responses (CD4+ and CD8a T cells) were evaluated in the secondary lymphoid organ samples like spleen, lymph node using flow cytometry analysis. Mucosal antibody levels were analyzed in the lung samples.

Results: Spike glycoprotein – Delta variant microparticles showed a recovery yield was with particle sizes ranging from 799nm ± 33.7; zeta potential was -25.1 ± 1.01 mV, and PDI was 0.55 ± 0.32. Encapsulation efficiency exceeded 88.03 ± 0.72. Whole inactivated virus – omicron variant microparticles shows recovery yield was 89.12 ± 0.77; particle size was 731nm ± 21.3 with the PDI of 0.6 ± 0.26 and zeta potential was -43 mV ± 15.2. Scanning electron microscopy revealed a donut-shaped morphology favorable for uptake by antigen-presenting cells. MTT assays performed in dendritic cells (DCs) confirmed non-cytotoxicity up to 500 µg/mL, thus confirming the safety of the microparticulate vaccine. Moreover, Griess assay demonstrated significantly elevated nitric oxide release vaccine group which demonstrated that the vaccine particles are capable to trigger immune response in DCs. In vivo, vaccinated mice showed significantly higher IgG levels in serum samples in all the weeks as compared to the naïve group. High IgG1 and IgG2a levels suggested a balanced Th1/Th2 response and was also observed in all weeks especially in the terminal week. Mucosal IgA was also significant in the treatment group as compared to no treatment group. Cellular response indicated increased CD4+ and CD8a T cell populations in vaccinated groups across spleen indicating generation of robust immune response.

Conclusion: This proof-of-concept study successfully demonstrated that an adjuvanted microparticulate vaccine delivered via intranasal route can elicit 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. In future study we plan to evaluate neutralization activity of the vaccine and analyze memory responses.

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Acknowledgements: Special thanks to BEI resources for providing the antigen needed for the 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: Characterization of nanoparticles


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. Significant levels of IgG were observed in mice against Omicron and delta variants of SARS-CoV-2. 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 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. The responses obtained are compared to the naïve group. Data expressed as Mean ± SEM, n=6 and two-way ANOVA test. ns: non-significant, *** p ≤ 0.001 ** p ≤ 0.01, and * p ≤ 0.05.