(M1330-11-76) Triggering Mucosal Immunity: An Intranasal Combination Vaccine for COVID-19 and Influenza

Purpose: Contagious respiratory viruses such as Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and influenza frequently mutate, impacting the efficacy of currently available vaccines. Thus, like the flu shot, yearly vaccination against SARS-CoV-2 may be required. This proof-of-concept study investigates the immunogenicity of an adjuvanted microparticulate combination vaccine for COVID-19 and influenza administered via the intranasal route as nasal drops. Our central hypothesis is that the vaccine microparticles will induce strong systemic and localized mucosal immunity (IgA) against both viruses when administered via the intranasal route. Moreover, we hypothesize that the immune responses generated through the intranasal administration will be comparable to or higher than those obtained through intramuscular vaccination. The vaccine antigens and adjuvants were encapsulated in a biodegradable poly (lactic-co-glycolic acid) (PLGA) polymeric matrix, which can prevent antigen degradation and enhance the antigen's immunogenicity. The intranasal route is a mucosal route of administration that can generate localized antibodies capable of binding to and neutralizing the virus at the entry site (nasal and respiratory mucosa). Moreover, the nasal mucosa is highly vascularized, consisting of circulating immune cells that drain via the nasal cavity's lymphatic vessels to generate a prompt systemic immune response. A combination vaccine that can eliminate separate immunizations for the two diseases, administered via the non-invasive intranasal route, can significantly benefit patients with needle phobia and vaccine hesitancy.

Methods: The inactivated SARS-CoV-2 and inactivated Influenza A H1N1 viruses were used as the vaccine antigens in polymeric microparticles. Based on previous preliminary studies, the adjuvants Alhydrogel®, AddaVax™, and CPG 7909 were selected to enhance the immunogenicity of the antigen. The antigen and adjuvant PLGA microparticles were prepared separately using a double emulsion method, lyophilized, characterized for their physical properties, and assessed in vitro for their immunogenicity and cytotoxicity using cell-based assays. The adjuvanted vaccine microparticles were resuspended in buffered saline and administered to mice (N=6) as one prime (w0) and two boosts (w3, w6) via the intranasal route to test the in vivo vaccine efficacy. A no-treatment control group and an intramuscular suspension group were included as -ve and +ve controls, respectively. The virus-specific serum antibody (IgG, IgG1, IgG2a, IgM, IgA) levels were assessed 14 days after each vaccine dose using an enzyme-linked immunosorbent assay (ELISA). The virus-specific secretory antibody (SIgG, SIgA) levels in the isolated lung tissue supernatant were assessed following sacrifice and organ isolation at w12 using ELISA. The virus-specific CD4⁺ and CD8⁺ T-cells in the immune organs, such as the spleen, lymph nodes, and lung tissue, were evaluated using flow cytometry analysis.

Results: The microparticles were spherical, with smooth surfaces having a recovery yield greater than 80% w/w. The characterization assessments revealed that the particles were less than 1 μm with encapsulation efficiencies in the 80% to 90% range. The in vitro assessment showed that the microparticles were immunogenic and non-toxic to target immune cells in low doses. Following in vivo intranasal vaccination, the SARS-CoV-2-specific and Influenza A H1N1-specific serum IgG and IgA levels increased significantly compared to the control groups (Figure 2). The IgG subtype analyses showed an equal distribution and significantly high levels of serum IgG1 (Th-2/antibody mediated response) and IgG2a (Th-1/cytotoxic mediated response) antibodies specific to both viruses. Serum IgM levels were predominant in the initial weeks. The increased serum IgG and IgA levels specific to both viruses may suggest that the isotype IgM undergoes a balanced class switching to IgG and IgA. The virus-specific secretory IgG (SIgG) and IgA (SIgA) levels in the lung tissue supernatants were significantly higher when compared to the control groups (Figure 3). The virus-specific CD4⁺ T-cells and CD8⁺ T-cells in the isolated lymph node, spleen, and lung tissue were higher compared to the no-treatment control group. The overall immune response generated from the intranasal vaccination was higher than that of the intramuscular vaccine suspension group.

Conclusion: The vaccine and adjuvant microparticles were successfully prepared and characterized. The values of the characteristic parameters were within the desired range. The in vitro assessment suggests that the vaccine was safe and immunogenic. The in vivo assessment evaluated the immunogenicity of the combination vaccine for its ability to generate SARS-CoV-2-specific and Influenza A H1N1-specific serum antibodies, secretory (mucosal) antibodies, and cellular responses, which increased significantly following immunization. The intranasal vaccine induced a localized mucosal IgA response at the administration site (nasal mucosa). The study has yielded promising results thus far. Our future directions include assessing virus-specific serum and secretory neutralization titers. A combination vaccine for COVID-19 and Influenza administered via the intranasal route is an effective and non-invasive vaccination strategy with great potential to cause a fundamental breakthrough in vaccine development.

References: 1. Charles A Janeway J, Travers P, Walport M, Shlomchik MJ. The mucosal immune system. In: Immunobiology: The Immune System in Health and Disease 5th edition [Internet]. Garland Science; 2001 [cited 2023 May 2]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK27169/
2. Russell MW, Moldoveanu Z, Ogra PL, Mestecky J. Mucosal Immunity in COVID-19: A Neglected but Critical Aspect of SARS-CoV-2 Infection. Front Immunol. 2020;11:3221.
3. Vijayanand S, Patil S, Menon I, Braz Gomes K, Kale A, Bagwe P, et al. An Adjuvanted Inactivated SARS-CoV-2 Microparticulate Vaccine Delivered Using Microneedles Induces a Robust Immune Response in Vaccinated Mice. Pharmaceutics. 2023 Mar;15(3):895.

Acknowledgements:1. Graphics were created using Biorender.com 2. The following reagent was obtained through BEI Resources, NIAID, NIH: Influenza A Virus, A/California/04/2009 (H1N1)pdm09, BPL-Inactivated, NR-49450. 3. The following reagent was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-WA1/2020, Heat Inactivated, NR-52286.

Figure 1: Graphical Abstract. When administered to mice via the intranasal route, the vaccine microparticles are transported via the M-cells lining the nasal mucosa, where they are recognized and engulfed by the localized immune cells resulting in immune activation and secretion of mucosal IgA antibodies. Further, the activated immune cells drain via the lymph nodes and generate a systemic immune response. The image was created using Biorender.com.

Figure 2: SARS-CoV-2-specific and Influenza A H1N1-specific serum IgG and IgA levels in vaccinated mice. A. SARS-CoV-2-specific serum IgG B. Influenza-A-H1N1-specific serum IgG. C. SARS-CoV-2-specific serum IgA. D. Influenza-A-H1N1-specific serum IgA. Responses obtained are compared to the no treatment (-ve control) and IM suspension (+ve control) groups. Data expressed as mean ± SEM, n=6, Two-way ANOVA, Post hoc Tukey's multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Abbreviations: iSCoV-2 – inactivated SARS-CoV-2; iFlu – inactivated Influenza A H1N1; IN – intranasal; IM – intramuscular; MP – microparticles

Figure 3: SARS-CoV-2-specific and Influenza A H1N1-specific secretory IgG (SIgG) and secretory IgA (SIgA) levels in the isolated lung tissue supernatant of vaccinated mice. A. SARS-CoV-2-specific SIgG B. Influenza-A-H1N1-specific SIgG. C. SARS-CoV-2-specific SIgA. D. Influenza-A-H1N1-specific SIgA. Responses obtained are compared to the no treatment (-ve control) and IM suspension (+ve control) groups. Data expressed as mean ± SEM, n=6, One-way ANOVA, Post hoc Tukey's multiple comparisons test. ns, non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Abbreviations: iSCoV-2 – inactivated SARS-CoV-2; iFlu – inactivated Influenza A H1N1; IN – intranasal; IM – intramuscular; MP – microparticles.