(T0930-04-22) Evaluating Immunization Efficacy of Self-administrable Subunit Microparticulate COVID-19 Vaccine Delivered by Nasal, Buccal, and Dermal Routes

Purpose: The SARS-CoV-2 virus continues to cause havoc with more than 512 million COVID-19 cases and 6.23 million deaths worldwide, pertaining the need for yearly vaccination. Despite the rapid development and approval of COVID-19 vaccine, the need for cold chain storage impedes the global distribution of vaccines in developing countries. Moreover, approved vaccines utilize invasive, non-patient compliant, intramuscular routes of immunization. Thus, there is an urgency for a globally available, non-invasive, self-administrable vaccination strategy for mass immunization against COVID-19. Firstly, we developed highly stable microparticulate vaccine that aids in avoiding cold chain storage. Our antigen of interest was receptor-binding domain (RBD) of the spike protein on the virus as it is highly immunogenic in nature, playing a critical role in the entry of the virus. Furthermore, increasing the molecular size (MS) of the low MS RBD with biodegradable polymer prevents RBD from escaping immune system leading to better uptake by APCs. Additionally, MPs protect RBD and provide sustained release. We also utilized adjuvants such as Alum, AddaVax™ and CpG as MPs to boost the immune response. Secondly, we investigated non-invasive routes for vaccine delivery since they are patient compliant due to needle-free approach. This enables possibility of self-administration offering added advantages from immunological perspective by induction of systemic and mucosal responses. Mucosal routes like intranasal and buccal are promising because of high vasculature and tissue-resident immune cells. Moreover, intradermal route is favorable due to the presence of Langerhans cells that efficiently drain to lymph nodes inducing a robust immune response. Thus, we compared non-invasive routes like intranasal, buccal, and intradermal with invasive intramuscular route. We explored immunogenicity of innovative vaccine formulations such as quick-dissolving oral films (ODF) and “band-aid-like” microneedles (MN) patches incorporating MPs. To summarize, we compared the immunogenicity of our combined vaccination strategy (non-invasive routes and RBD MPs) to the invasive intramuscular microparticulate vaccine.

Methods: For formulation of MPs, RBD and adjuvants were encapsulated in poly-(lactic-co-glycolic acid) matrix with double-emulsion method following lyophilization. The MPs were characterized for morphology, size, zeta potential. For formulation of ODFs, the MPs were suspended in a polymer base, dispensed precisely to in-house designed PDMS molds using 3D-printer to automate the process. For formulation of MNs, MPs were suspended in hyaluronic acid gel and formed into an 8*8 array using PDMS molds. The formulations were visually characterized for morphology using SEM. For in vitro studies, immunogenicity of RBD MPs with adjuvant MPs in APCs was assessed to release nitrite: innate immune marker using Griess’s assay. We evaluated the potential of these MPs to induce adaptive immune response by the expression of autophagosomes and the co-expression of antigen-presenting molecules (MHC I, MHC II), their co-stimulatory molecules (CD86, CD40) using flow cytometry. For in vivo studies, mice were immunized with these formulations with prime and 3 booster doses. Mice were administered MPs suspension intramuscularly and intranasally, ODFs through buccal mucosa, and MNs intradermally. Serum samples were collected bi-weekly to assess the humoral response against the vaccines by presence of antibodies such as IgG, IgG1, IgG2a and IgA using ELISA. Mice were sacrificed after week 12 and secondary lymphoid organs such as spleen and lymph nodes were isolated for evaluating presence of adaptive immune response by production of RBD specific CD4+ and CD8+ T cell and IL-4 and IFN-γ secreting cells using flow cytometry.

Results: We successfully formulated RBD MPs of 712.2 ± 50.6 nm size with surface charge of -20.03± 0.80 mV representing stability. RBD MPs+Adj MPs were able to induce extremely significant levels (****p < 0.0001) of nitrite in APCs, indicating the presence of innate immune marker. Moreover, MPs were able to express very significant levels (**p < 0.01) of autophagosomes in APCs, indicating ability of MPs to aid in expression of antigen-presenting molecules. We further observed extremely significant (****p < 0.0001) co-expression of these molecules in APCs, exhibiting potential of adaptive immune response as these are connecting links between innate and adaptive immune response. In vivo studies revealed the presence of humoral and mucosal responses characterized by presence of extremely significant levels (****p < 0.0001) of IgG, IgG1, IgG2a and IgA respectively in the serum as compared to naïve group. Results indicated comparable immune responses by non-invasive routes to invasive route especially in the terminal week (post-sacrifice). The presence of significantly high (*p < 0.05) RBD specific-helper, cytotoxic T cells, IL-4 and IFN- γ secreting cells indicated induction of cellular immune response providing viral clearance.

Conclusion: Our vaccine formulations induced balanced Th1 and Th2 immune responses indicated by humoral (IgG1, IgG2a) and cellular responses, essential for rapid clearance of virus. In vitro, MPs induced innate and adaptive immune markers. In vivo, formulations administered with non-invasive routes were able to induce comparable immune responses to invasive intramuscular route exhibiting presence of humoral, mucosal, and cellular immune responses. Thus, our vaccine formulations exhibit potential for self-administrable vaccine candidates conferring yearly protection against COVID-19.


Graphical abstract representing formulation of microparticles followed by the administration through invasive: intramuscular and non-invasive: intranasal, buccal with Oral dissolving films (ODFs), and intradermal with microneedles (MNs)


Fig: In vitro immunogenicity of RBD loaded microparticles (MPs) with adjuvant MPs a) Nitrite release as obtained by Griess’s assay in Dendritic cells b) Percent cell count of cells expressing autophagosomes in dendritic cells obtained by CYTO-ID® Autophagy detection assay c) Co-expression of antigen-presenting molecules MHC I, MHC II and their co-stimulatory molecules CD86 and CD40 were analyzed using BD Accuri C6 Plus flow cytometer upon staining with APC and FITC markers in dendritic cells. Data are expressed as mean ± SEM analyzed using one-way ANOVA followed by post hoc Tukey’s multiple comparison test. **p < 0.01 very significant,***p < 0.001 highly significant,****p < 0.0001 extremely significant​

Fig: Spike RBD MP specific antibody levels of IgG were analyzed using ELISA in bi-weekly collected serum samples in different routes of administration. Data are expressed as mean ± SEM analyzed using two-way ANOVA followed by post hoc Tukey’s multiple comparison test. *p < 0.05 significant, ** < 0.01 very significant,***p < 0.001 highly significant, ****p < 0.0001 extremely significant.
MPs- Microparticles, RBD- receptor binding domain, Adj- Adjuvants, ODF-Oral dissolving films, MN-Microneedles