A Modular Biomaterial Scaffold-Based Vaccine Elicits Durable Adaptive Immunity to Subunit SARS-CoV-2 Antigens

Abstract

The coronavirus disease 2019 (COVID-19) pandemic demonstrates the importance of generating safe and efficacious vaccines that can be rapidly deployed against emerging pathogens. Subunit vaccines are considered among the safest, but proteins used in these typically lack strong immunogenicity, leading to poor immune responses. Here, a biomaterial COVID-19 vaccine based on a mesoporous silica rods (MSRs) platform is described. MSRs loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF), the toll-like receptor 4 (TLR-4) agonist monophosphoryl lipid A (MPLA), and SARS-CoV-2 viral protein antigens slowly release their cargo and form subcutaneous scaffolds that locally recruit and activate antigen-presenting cells (APCs) for the generation of adaptive immunity. MSR-based vaccines generate robust and durable cellular and humoral responses against SARS-CoV-2 antigens, including the poorly immunogenic receptor binding domain (RBD) of the spike (S) protein. Persistent antibodies over the course of 8 months are found in all vaccine configurations tested and robust in vitro viral neutralization is observed both in a prime-boost and a single-dose regimen. These vaccines can be fully formulated ahead of time or stored lyophilized and reconstituted with an antigen mixture moments before injection, which can facilitate its rapid deployment against emerging SARS-CoV-2 variants or new pathogens. Together, the data show a promising COVID-19 vaccine candidate and a generally adaptable vaccine platform against infectious pathogens.

Keywords: COVID-19; SARS-CoV-2; antibodies; cytotoxic T-cells; mesoporous silica rods; monophosphoryl lipid A (MPLA); recombinant proteins; vaccines.

Figure 1

Schematics for the use of MSRs for vaccination. MSRs are loaded with a recruiting factor, adjuvant, and antigen by adsorption, before being lyophilized. The vaccine can be stored in powder form until ready to be administered. After reconstitution in an aqueous buffer, MSR vaccine can be injected subcutaneously and create a scaffold for DC recruitment and homing, allowing their local activation and antigen presentation. Recruited and activated DCs migrate to the draining lymph node where they participate in the generation of an adaptive immune response against the desired antigens by priming cognate T‐cells. B‐cell, activation, in conjunction with DC‐induced CD4⁺ T cells participate in the generation of antigen‐specific B‐cell response.

Figure 2

MSR vaccine formulated with SARS‐CoV‐2 antigens attracts immune cells and sustainably releases vaccine adjuvants. a) Composition of SARS‐CoV‐2 MSR vaccine. Cumulative in vitro release of b) GM‐CSF (n = 3), c) MPLA (n = 2), and d) N, S1, and S2 antigens from MSRs (n = 3). Data represent mean ± SD. SEM images of e) unloaded MSRs before injection and f) SARS‐CoV‐2 MSR vaccine explanted 7 days after injection into the skin of BALB/c mice. Scalebar = 200µm and 20µm, respectively. g) Scaffold volume over time (n = 3). Data represent mean and SD.

Figure 3

Two doses of N/S1/S2 MSR vaccine generate persistently high antibody responses to SARS‐CoV‐2 antigens. Six to 8 weeks old BALB/c mice were immunized on day 0 and boosted on day 28 with MSRs loaded with 1 µg GM‐CSF, 25 µg MPLA, 1 µg N protein, 1 µg S1 protein, and 1 µg S2 protein (N/S1/S2 MSR, n = 10) or a Sham vaccine containing GM‐CSF and MPLA only (n = 10). Serum antibody concentration against the full‐length N protein, the S1, S2, and RBD were measured over time by ELISA. Total IgG (n = 10; a), IgG1 (n = 8; b), and IgG2a (n = 8; c) are reported. The dotted line indicates peak concentration. P‐values represent the results of Friedman test with post hoc Wilcoxon for repeated measures comparing all timepoints to day 0 concentration.

Figure 4

N/S1/S2 MSR vaccine induces adaptive immunity with anti‐SARS‐CoV‐2 activity. Six to 8 weeks old BALB/c mice were immunized with MSRs loaded with 1 µg GM‐CSF, 25 µg MPLA, 1 µg N protein, 1 µg S1 protein, and 1 µg S2 protein (N/S1/S2 MSR vaccine, n = 10) or a Sham vaccine containing GM‐CSF and MPLA only (n = 10). All animals received a second injection on day 28 and serum was used to determine neutralization titers against a SARS‐CoV‐2 pseudovirus load over 180 days (a). The solid line represents mean, P‐values represent the results of Friedman test with post hoc Wilcoxon for repeated measures comparing all timepoints to day 0 values. Splenocytes of animals having received a single injection on day 0 were restimulated with peptides spanning the S‐protein on day 28 and CTLs were analyzed for IFN‐γ production by Flow Cytometry. Kruskal–Wallis test with post hoc Dunn test. **P < 0.01.

Figure 5

Add‐in‐time MSR vaccine induces persistent humoral response with neutralization potential. Six to 8 weeks old BALB/c mice were immunized on day 0 and boosted on day 28 with MSRs loaded with 1 µg GM‐CSF, 25 µg MPLA with 1 µg N protein, 1 µg S1 protein, and 1 µg S2 protein added minute injections (N/S1/S2 MSR vaccine, n = 10) or a Sham vaccine containing GM‐CSF and MPLA only (n = 10). Serum IgG concentration against the S2 domain (a), S1 domain (b), and RBD (c) were measured over time by ELISA. P‐values represent the results of Friedman test with post hoc Wilcoxon for repeated measures comparing all timepoints to day 0 concentration. d) Neutralization titer – line represents data mean (n = 10). P‐values represent the results of Friedman test with post hoc Wilcoxon for repeated measures comparing all timepoints to day 14 titer.

Figure 6

Single‐shot MSR vaccine formulations induce anti‐SARS‐CoV‐2 activity. Six to 8 weeks old BALB/c mice were single immunized with vaccine formulations shown in (a) on day 0 (n = 10). Total S1‐ (b), S2‐ (c), and RBD‐specific (d) serum IgG was measured using ELISA. P‐values (black) represent the results of Wilcoxon signed‐rank test between day 0 and day 233 for each group (in all 4 groups, P < 0.01). In (b–d), all groups were compared against each other at the final timepoint using Kruskal–Wallis test with P < 0.05 for (b), P < 0.01 for (c), and P < 0.0001 for (d), the P values of the post hoc analysis of the comparisons at day 233 are indicated in orange. The neutralization potential of immunized sera was assessed against SARS‐CoV‐2 pseudovirus. e) Kruskal–Wallis test P‐values were P < 1 × 10⁻⁷, 10⁻⁴, 10⁻⁴, and 10⁻⁵ for days 28, 56, 104, and 135, respectively. Post hoc Conover test P‐values for the comparison of each group with each other are depicted in the plot.