(M0930-02-09) Combating SARS-CoV-2 Variants: Pain-Free Bivalent Intranasal Vaccination Delivery

Purpose: Emerging SARS-CoV-2 strains continue to threaten global health, and the effectiveness of current vaccines targeting the rapidly mutating spike protein is a concern against these new strains. Consequently, vaccines inducing cross-reactive immunity are urgently needed. The nucleoprotein, conserved across different strains of SARS-CoV-2, is a promising vaccine target. This “proof-of-concept” study investigated nucleoprotein and spike protein as a bivalent vaccine in the microparticulate form delivered as an intranasal vaccine. We hypothesized that a vaccine formulation with both spike and nucleoprotein could induce a cross-reactive systemic and mucosal immune response against various strains of SARS-CoV-2. Our vaccination strategy involves encapsulating the antigens in a polymeric matrix to form microparticles, thereby enhancing immunogenicity while protecting the antigen. Furthermore, administration through intranasal route has multiple advantages, such as i) specialized immune cells in nasal mucosal layers, ii) neutralizing antibodies at the virus entry sites (nose,mouth), iii) unified mucosal immunity (the entire mucosal layer is capable of inducing an immune response, even though only one mucosal site is exposed) iii) compliant than painful intramuscular route

Methods: Spike and nucleoprotein of the SARS-CoV-2 Wuhan-Hu-1 strain were used as vaccine antigens. Alhydrogel®, AddaVax™, and CpG 7909 were chosen as adjuvants to enhance immunogenicity. The vaccine and adjuvant microparticles were formulated with poly-(lactic-co-glycolic acid) polymer using the double emulsion solvent evaporation method and lyophilized. The microparticles were characterized for size, surface charge, and polydispersity index using Malvern Zetasizer. The protein content in the vaccine microparticles was analyzed to measure percent encapsulation efficiency using Micro bicinchoninic (BCA) assay. Microparticles were evaluated in vitro in dendritic cells for safety profile and immunostimulatory effect using cytotoxicity and Griess assays, respectively. The adjuvanted-bivalent microparticles were suspended in appropriate buffer to be administered intranasally without irritation to the nasal mucosa. The vaccine was delivered to mice (n=5) as one prime and two booster doses, three weeks apart. The mice were randomly assigned to the following groups: i) naïve/no treatment, and ii) intranasal bivalent microparticulate vaccine. Mouse sera were tested for antibodies (IgG, IgG1, IgG2a, and IgA) using an enzyme-linked immunosorbent assay. Mice were euthanized and the lungs and secondary lymphoid organs, such as the spleen and lymph nodes, were harvested to test for cellular immune responses (CD4+ and CD8a+ T cells). The lung supernatant samples were tested for secretory antibody (IgA). All immune responses were tested against the following antigens: i) spike protein (Wuhan-Hu-1 strain), ii) nucleoprotein (Wuhan-Hu-1 strain), iii) inactivated SARS-CoV-2 (delta strain), and iv) inactivated SARS-CoV-2 (omicron strain). The objective of testing for immune responses against the different variants was to test for cross-reactive immune responses produced by the bivalent vaccine against the variants of concerns of SARS-CoV-2.

Results: The vaccine and adjuvant MPs had a successful recovery yield of > 85% w/w. The vaccine and adjuvant MPs ranges for characterization were a) size 500-1200 nm, b) surface charge: -15 to 15 mV, and c) PDI: 0.2-0.8. The percent encapsulation efficiency of vaccine microparticles was greater than 89% (both spike and nucleoprotein), indicating that vaccine antigen was not lost to a great extent during formulation process. In vitro assessment revealed that MPs, even at higher doses (1000 µg/mL), were non-cytotoxic to dendritic cells. Moreover, the immunostimulatory activity (nitric oxide release) was significantly higher (***p < 0.0001) in the adjuvanted-bivalent vaccine MPs group when compared to no treatment group. In vivo immunization revealed significantly higher IgG and IgA antibodies in the serum and lung supernatants for vaccinated mice than the no-treatment group. Moreover, significantly high(***p < 0.0001) IgG1 and IgG2a levels were observed in the vaccinated mice, indicating a balanced Th1 and Th2 immune response, respectively. Furthermore, helper (CD4+) T cells and cytotoxic (CD8a+) T cells in the lungs(**p < 0.01), lymph nodes, and spleen were significantly higher in all vaccine treatment groups than in the untreated groups. The immune responses were significantly higher against not only the spike and nucleoprotein of the Wuhan-Hu-1 strain but also against the delta and omicron strains, indicating a cross-reactive immune response. Interestingly, the immune responses observed were relatively higher against the delta strain than the omicron strain.

Conclusion: The vaccine and adjuvant microparticles were successfully formulated. The characterizations indicated that MPs could appear more immunogenic to immune cells. In vitro testing revealed that MPs were safe and immunostimulatory. In vivo testing indicated that an adjuvanted-bivalent microparticulate vaccine could produce robust humoral (antibody), cellular (helper and cytotoxic T cells), and mucosal (secretory IgA) immune responses. Immune responses were specific to the bivalent vaccine antigens (spike and nucleoprotein) and SARS-CoV-2 (delta and omicron variants). Thus, our vaccination strategy produced a potent and cross-reactive immune response against the various strains of SARS-CoV-2. Therefore, our vaccination strategy will pioneer in providing a broader immune response and pain-free vaccination alternative against the emerging strains of SARS-CoV-2. This vaccine candidate is critical for the development of a universal vaccine against COVID-19.

References: 1. Dutta, N. K., Mazumdar, K., & Gordy, J. T. (2020). The Nucleocapsid Protein of SARS–CoV-2: A Target for Vaccine Development. Journal of Virology, 94(13), e00647-20. https://doi.org/10.1128/JVI.00647-20
2. Jearanaiwitayakul, T., Seesen, M., Chawengkirttikul, R., Limthongkul, J., Apichirapokey, S., Sapsutthipas, S., Phumiamorn, S., Sunintaboon, P., & Ubol, S. (2021). Intranasal Administration of RBD Nanoparticles Confers Induction of Mucosal and Systemic Immunity against SARS-CoV-2. Vaccines, 9(7), 768. https://doi.org/10.3390/vaccines9070768

Acknowledgements: 1. NR-52946- The following reagent was produced under HHSN272201400008C and obtained through BEI Resources, NIAID, NIH: Spike Glycoprotein Receptor Binding Domain (RBD) from SARS-Related Coronavirus 2, Wuhan-Hu-1 with C-Terminal Histidine Tag, Recombinant from HEK293T Cells, NR-52946.
2. NR-53246- The following reagent was obtained through BEI Resources, NIAID, NIH: Nucleocapsid Protein N-Terminal RNA Binding Domain from SARS-Related Coronavirus 2, Wuhan-Hu-1 with N-Terminal Histidine Tag, Recombinant from Escherichia coli, NR-53246.

Figure 1: IgG and IgA antibody levels in vaccinated and unvaccinated mice serum in week 5(after one prime and two booster doses) and terminal week (4 weeks after the second booster dose). The mice were vaccinated with an adjuvanted-bivalent microparticulate vaccine. The vaccine treatment groups were vaccinated intranasally with one prime and two booster doses. Mice sera were collected biweekly and evaluated for antibodies against the spike and nucleoprotein (Wuhan-Hu-1 strain) and inactivated SARS-CoV-2 (delta and omicron strains). A) and B) IgG and IgA levels were significantly high in vaccinated mice against the spike and nucleoprotein of the Wuhan-Hu-1 strain. C) and D) Significant levels of IgG and IgA were also observed against the delta and omicron 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 2: (A-D) Mucosal antibody levels in the lung supernatant of vaccinated and unvaccinated mice obtained at terminal week (4 weeks after second booster dose). The secretory IgA antibody was tested against specific antigens such as A) spike protein B)Nucleoprotein C) Delta variant D) Omicron variant. The mice were vaccinated with adjuvanted-bivalent microparticulate vaccine intranasally with one prime and two booster doses. Post sacrifice, lung samples were used to obtain lung supernatant and evaluated for secretory IgA against the spike and nucleoprotein (Wuhan-Hu-1 strain) and inactivated SARS-CoV-2 (delta and omicron strains). IgA levels were significantly high in vaccinated mice against all four antigens. (E-F) Cellular immune response in the spleen and lymph nodes of vaccinated mice. Post sacrifice, lungs, spleen, and lymph node samples were collected and evaluated for CD4+ T cells and CD8a+ T cells. The cellular response was significantly high in vaccinated groups against the E) spike protein and F) nucleoprotein of the Wuhan-Hu-1 strain. Data is expressed as Mean ± SEM and analyzed with Unpaired t-test * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001