Fourth dose of microneedle array patch of SARS-CoV-2 S1 protein subunit vaccine elicits robust long-lasting humoral responses in mice

Abstract

The COVID-19 pandemic has underscored the pressing need for safe and effective booster vaccines, particularly in considering the emergence of new SARS-CoV-2 variants and addressing vaccine distribution inequalities. Dissolving microneedle array patches (MAP) offer a promising delivery method, enhancing immunogenicity and improving accessibility through the skin's immune potential. In this study, we evaluated a microneedle array patch-based S1 subunit protein COVID-19 vaccine candidate, which comprised a bivalent formulation targeting the Wuhan and Beta variant alongside a monovalent Delta variant spike proteins in a murine model. Notably, the second boost of homologous bivalent MAP-S1(WU + Beta) induced a 15.7-fold increase in IgG endpoint titer, while the third boost of heterologous MAP-S1RS09Delta yielded a more modest 1.6-fold increase. Importantly, this study demonstrated that the administration of four doses of the MAP vaccine induced robust and long-lasting immune responses, persisting for at least 80 weeks. These immune responses encompassed various IgG isotypes and remained statistically significant for one year. Furthermore, neutralizing antibodies against multiple SARS-CoV-2 variants were generated, with comparable responses observed against the Omicron variant. Overall, these findings emphasize the potential of MAP-based vaccines as a promising strategy to combat the evolving landscape of COVID-19 and to deliver a safe and effective booster vaccine worldwide.

Keywords: Boost; COVID-19; Forth dose; Humoral immunity; Microneedle array patch; S1 protein subunit; SARS-CoV-2; Third dose; Vaccine.

Fig. 1.. Fabrication of dissolving microneedle array patch (MAP)

(A) A shuttle vector carrying the codon-optimized two variants of SARS-CoV-2-S1 gene encoding N-terminal 1-661 with a c-tag (EPEA) was designated as shown in the diagram. Amino acid changes in the SARS-CoV-2-S1 region of in this study are shown. ITR: inverted terminal repeat; RBD: receptor binding domain. (B) Silver stained SDS-PAGE of purified SARS-CoV-2 S1 proteins from Wuhan strain (lane1), Beta strain (lane 2), and S1RS09 of Delta strain (lane 3) (C and D) Image of MAP. Each MAP was approximately 700 μm in length, occupying a 5 × 5 array, 1.76 cm² circle area size of the patch with rS1WU and rS1Beta (E) Delivery efficiency of rS1 by MAP into mouse skin. Recovered protein was analyzed via sandwich ELISA with S1-specific monoclonal antibody pair. Percent recovery is relative to the original control prepared fresh on the day of each assay, calculated as [(initial dose–residual dose)/initial dose × 100%]. Data are representative of the mean with standard error of mean (SEM).

Fig. 2.. Antigen-specific antibody responses in mouse immunized with MAP of SARS-CoV-2 rS1 protein subunit vaccine after the 2^nd booster.

(A) Schedule of immunization and blood sampling for IgG end point titration. BALB/c (N=5) were immunized with MAP-rS1(WU+Beta) at week 0, 3, and 12. The red drops denote times at which blood was drawn. (B) Sera were diluted, and SARS-CoV-2-S1-specific antibodies were quantified by ELISA to determine the IgG endpoint titer. The IgG titers at each time points were showed for each mouse. The bars represent the geometric mean. Statistical comparisons with the pre-immunized sera for ELISA were determined by an unpaired nonparametric t-test (Mann-Whitney test, grey asterisks). Differences among more than two groups were compared by the Kruskal-Wallis test, followed by Dunn’s multiple comparisons (black asterisks). Significant differences are indicated by *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant.

Fig. 3.. Long-lasting immune response after the 3^rd boost.

(A) Schedule of immunization and blood sampling for IgG end point titration. BALB/c (N=5) were immunized with MAP-rS1(WU+Beta) at week 0, 3, and 12 and MAP-rS1RS09Delta at week 31. The red drops denote times at which blood was drawn. Sera at weeks 0, 6, 9, 15, 31, 34, and 83 were diluted, and SARS-CoV-2-S1WU-specific (B) and SARS-CoV-2-S1Delta-specific (C) antibodies were quantified by ELISA to determine the IgG endpoint titer. The IgG titers at each time points were showed in each mouse. The bars represent geometric mean. (D) Fold change of IgG endpoint titer after prime (W3/W0), 1^st(W9/W3), 2^nd (W15/W9), and 3^rd (W34/W15) boost. Data are representative of the mean with SEM. (E) The SARS-CoV-2-S1WU-specific IgG titers at all time points until week 111 post-prime (BALB/c (N=5) except at week 102 (N=4) and at week 111 (N=3)). Arrows along the X-axis illustrate the time points of boost vaccinations (grey arrows; MAP-rS1(WU+Beta), blue arrow; MAP-rS1RS09Delta). Statistical comparisons with pre-immunized sera for ELISA were determined by unpaired nonparametric t-test (Mann-Whitney test, grey asterisks). Differences among more than two groups were compared by Kruskal-Wallis test, followed by Dunn’s multiple comparisons (black asterisks). Significant differences are indicated by *p < 0.05, **p < 0.01.

Fig. 4.. IgG subclasses after boosters.

BALB/c (N=5) were immunized with MAP-rS1(WU+Beta) at week 0, 3, and 12, and MAP-rS1RS09Delta at week 31. Sera at weeks 0, 6, 9, 15, 31, 34, and 83 were diluted, and SARS-CoV-2-S1WU-specific IgG1 (A), IgG2a (B), IgG2b (C), and IgG3 (D) were quantified by ELISA to determine each IgG subclasses endpoint titer. The titers at each time points were showed for each mouse. The bars represent geometric mean. (E) S1-specific IgG1/IgG2a+IgG2b+IgG3 ratios of individual mice at weeks 9, 15, 34, and 83 as mean values with SEM. Groups were compared by the Kruskal-Wallis test at each time point, followed by Dunn’s multiple comparisons. Significant differences are indicated by *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant.

Fig. 5.. Neutralization of SARS-CoV-2 variants.

Serum from mice immunized with SARSCoV-2 S1 via MAP intradermal delivery was assessed using a microneutralization assay (VNT₉₀) for neutralization against SARS-CoV-2 variants (A) Wuhan, (B) Delta variant (B.1.617.2), and (C) Omicron variant (BA.1). Data are representative of the geometric mean with error bars representing geometric standard deviation. Groups were compared by the Kruskal-Wallis test at each time point, followed by Dunn’s multiple comparisons. Significant differences are indicated by *p < 0.05, n.s., not significant.