(M0930-03-17) Heterologous Microneedle Vaccine Strategy for COVID-19

Purpose: The pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has severely impacted the public health. Despite the availability of current vaccines, there is a need for extensive research to develop broader, cross-reactive immunity that can effectively combat emerging variants of SARS-CoV-2. In our proof-of-concept study, we explored a heterologous approach with two strains of SARS-CoV-2 administered at prime and booster dosing strategy. Gamma irradiated whole inactivated delta strain of SARS-CoV-2 was used at prime dosing due to it being dominant strain in first wave and in booster immunization omicron variant was used as it was the most transmitted strain. We formulated Microneedle microparticulate vaccine by encapsulating the antigen in biodegradable polymer. This dual approach will provide protection, eliminate the need for cold chain storage and tackle the problem of vaccine hesitancy among individuals. Microneedles are minimally invasive as penetration onto pain receptors from skin is limited. Additionally, the skin acts as a defensive barrier equipped with immune surveillance cells like Langerhans cells (LCs) and dermal dendritic cells, which play crucial roles in detecting and responding to foreign substances.

Methods: Gamma irradiated whole cell inactivated SARS-CoV-2 from delta variant (GIWCIS-D) and gamma irradiated whole cell inactivated SARS-CoV-2 from omicron variant (GIWCIS-O) was used as the model antigen, with Alum as the adjuvant. 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 determine the percent encapsulation efficiency. The safety and effectiveness of microneedles were evaluated in vitro using the MTT assay to assess safety and the Griess assay to measure efficacy. The vaccine and adjuvanted microparticles were suspended in sodium hyaluronic acid base to form microneedles. The microparticulate microneedles were delivered to mice as one prime and one booster dose, two weeks apart. In the prime dosing and booster dosing, the treatment group received gamma irradiated whole cell inactivated SARS-CoV-2 of delta variant-based microparticulate microneedle and gamma irradiated whole cell inactivated SARS-CoV-2 of omicron variant-based microparticulate microneedle vaccine, respectively. Vaccine microparticles were administered intramuscularly to the mice. Study groups include (1) naïve/no treatment, (2) Intra-muscular microparticulate vaccine, and (3) Microneedle microparticulate vaccine. Mouse sera were tested for antibodies (IgG, IgG1, IgG2a, and IgA) using an enzyme-linked immunosorbent assay. At the end of the study, 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). All immune responses were tested against the following antigens: (1) inactivated SARS-CoV-2 of delta strain, (2) inactivated SARS-CoV-2 of omicron strain. To determine if the vaccine was providing cross-reactive immune responses, antibodies were analyzed antigen-antibody response with the present variants of SARS-CoV-2 namely (1) inactivated SARS-CoV-2 of alpha and, (2) inactivated SARS-CoV-2 of beta strain.

Results: The vaccine and adjuvant MPs had a successful recovery yield of > 90% w/w. The vaccine and adjuvant MPs ranges for characterization were a) size 500-900 nm, b) surface charge: -40 to -50 mV, and c) PDI: 0.5-0.7. The percent encapsulation efficiency of vaccine microparticles was greater than 80% indicating that majority of antigen was encapsulated into polymeric matrix. In vitro assessment revealed that MPs, even at doses (500 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 delta and omicron strains, alpha and beta strains.

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 immunogenic. In vivo testing indicated that an adjuvanted-heterologous microparticulate vaccine produced robust humoral (antibody), cellular (helper and cytotoxic T cells) immune response. Immune responses were specific to the vaccine antigens and other variants of SARS-CoV-2 which are beta and alpha variants. Our vaccination strategy could have the potential to offer cross-protection against mutating strains. Therefore, our minimally invasive vaccination strategy will be a pioneer in providing a broader immune response.

References: 1. Sue Ann Costa Clemens et al., “Heterologous versus Homologous COVID-19 Booster Vaccination in Previous Recipients of Two Doses of CoronaVac COVID-19 Vaccine in Brazil (RHH-001): A Phase 4, Non-Inferiority, Single Blind, Randomised Study,” The Lancet 399, no. 10324 (February 2022): 521–29, https://doi.org/10.1016/S0140-6736(22)00094-0.
2. Sharon Vijayanand et al., “Microneedle Delivery of an Adjuvanted Microparticulate Vaccine Induces High Antibody Levels in Mice Vaccinated against Coronavirus,” Vaccines 10, no. 9 (September 7, 2022): 1491, https://doi.org/10.3390/vaccines10091491.
3. Peng He, Yening Zou, and Zhongyu Hu, “Advances in Aluminum Hydroxide-Based Adjuvant Research and Its Mechanism,” Human Vaccines & Immunotherapeutics 11, no. 2 (February 2015): 477–88, https://doi.org/10.1080/21645515.2014.1004026.
4. R. Sealy et al., “Robust IgA and IgG-Producing Antibody Forming Cells in the Diffuse-NALT and Lungs of Sendai Virus-Vaccinated Cotton Rats Associate with Rapid Protection against Human Parainfluenza Virus-Type 1,” Vaccine 28, no. 41 (September 2010): 6749–56, https://doi.org/10.1016/j.vaccine.2010.07.068.

Acknowledgements: 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: 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. Mice sera were collected biweekly and evaluated for antibodies against inactivated SARS-CoV-2 (delta and omicron strains) and inactivated SARS-CoV-2 (beta and alpha strains). Significant levels of IgG were observed in all four 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: MTT assay – cytotoxicity assessment

Determination of cytotoxicity of MPs at increasing concentration. DC 2.4 was exposed to increasing amounts of vaccine antigen-loaded microparticles and incubated for 24 hrs. prior to being assessed for cytotoxicity of MPs. Data expressed as mean ± SEM was analyzed using one-way ANOVA followed by post hoc Tukey’s multiple comparison test. ns- not significant, ns-non significant, ****p < 0.0001.