Sustained Delivery of SARS-CoV-2 RBD Subunit Vaccine Using a High Affinity Injectable Hydrogel Scaffold

Abstract

The receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein that mediates viral entry into host cells is a good candidate immunogen for vaccine development against coronavirus disease 2019 (COVID-19). Because of its small size, most preclinical and early clinical efforts have focused on multimerizing RBD on various formats of nanoparticles to increase its immunogenicity. Using an easily administered injectable hydrogel scaffold that is rationally designed for enhanced retainment of RBD, an alternative and facile approach for boosting RBD immunogenicity in mice is demonstrated. Prolonged delivery of poly (I:C) adjuvanted RBD by the hydrogel scaffold results in sustained exposure to lymphoid tissues, which elicits serum IgG titers comparable to those induced by three bolus injections, but more long-lasting and polarized toward T_H 1-mediated IgG2b. The hydrogel scaffold induces potent germinal center (GC) reactions, correlating with RBD-specific antibody generation and robust type 1 T cell responses. Besides being an enduring RBD reservoir, the hydrogel scaffold becomes a local inflammatory niche for innate immune cell activation. Collectively, the injectable hydrogel scaffold provides a simple, practical, and inexpensive means to enhance the efficacy of RBD-based subunit vaccines against COVID-19 and may be applicable to other circulating and emerging pathogens.

Keywords: cellular immunity; germinal center; humoral immunity; injectable hydrogel scaffolds; severe acute respiratory syndrome coronavirus 2 receptor binding domain; sustained release; vaccines.

Figure 1

Schematic and selection of the high affinity injectable hydrogel scaffold for RBD and poly (I:C) loading. a) Scheme of the injectable hydrogel for sustained delivery of SARS‐CoV‐2 RBD and poly(I:C). b) Affinities of SARS‐CoV‐2 RBD with various polymer side chains: APMA, single hydroxyl group functionalized, two hydroxyl group functionalized, THMA and APMA‐THMA.

Figure 2

Schematic and characterization of the injectable hydrogel. a) Scheme of the hydrogel preparation. b) Frequency‐dependent (strain = 0.1%, 37 °C) oscillatory shear rheology, and c) steady shear rheology of two hydrogel formulations designated as “6% gel” and “12% gel”. d) Step‐shear measurements of 6% gel and 12% gel over two cycles with alternating high shear (100 s⁻¹) and low shear (0.05 s⁻¹) rates. e) Images of 12% gel injection through an 18‐gauge needle during injection (left) and after injection (right) (the hydrogel was mixed with a black food dye for easier visualization). f) In vivo degradation of the hydrogels over time following subcutaneous injection into C57BL/6 mice. g) In vivo RBD release from the hydrogels over time. h) Antigenicity profiles of RBD released from the 12% gel, as evaluated by ELISA analysis with rabbit‐anti‐RBD polyclonal antibodies. Binding was normalized to released RBD concentration. In (f,g), n = 3 mice per group. In (h), n = 3 per time point. Mean ± SD was shown.

Figure 3

The hydrogel scaffolds serve as a local inflammatory site to recruit innate immune cells. Mice were immunized on day 0, and the hydrogels were collected on day 14 after immunization (n = 4 mice for 6% gel, and n = 5 mice for 12% gel). a) Absolute counts of total immune cells infiltrating into the hydrogels. b–e) Frequency and absolute counts of different innate immune cell subsets in the hydrogel scaffolds, including DC b), macrophage c), monocyte d), and neutrophil e). f) Frequency of each DC subset of the total DCs in the hydrogel scaffolds. Data were presented as mean ± S.E.M., and p‐values were determined by a two‐tailed t test.

Figure 4

Hydrogel formulations drive strong and durable SARS‐CoV‐2 RBD‐specific humoral immune responses. a) Timeline of mice immunization and blood sampling. For all groups, mice were immunized on day 0 (n = 7 mice for 1, 2, and 3 bolus, n = 9 mice for 6% and 12% gels). The 2 and 3 bolus groups were boosted on day 14, and day 14 and 28, respectively. Blood was harvested on day 0, 14, 28, 42, 63, 77, and 100 for RBD‐specific titer determination. b) RBD‐specific serum IgG titers 14 days after 1 bolus, the second of 2 bolus, the third of 3 bolus, 6% gel, and 12% gel injections. c) RBD‐specific serum IgG1 and IgG2b titers 14 days after the third of 3 bolus, 6% gel, and 12% gel injections. d) RBD‐specific serum IgG titers over time. p values were calculated for 6% gel and 12% gel vs three bolus injections, respectively. In (b–d), mean ± S.E.M. was shown, and one‐way ANOVA with Turkey's post hoc test was conducted. Titers below 10², were displayed as 10².

Figure 5

Hydrogel vaccine formulations promote GC responses. Mice were immunized on day 0, and the inguinal LNs were collected on day 14 (a, b, d, e, g, h) and 28 (c, f, i) for analysis. a) Representative FACS plots of GC B cells, defined as live CD45⁺CD19⁺Fas⁺GL7⁺ cells. b) Frequency of GC B cells as defined in (a). c) Kinetics of GC B cell frequency. d–f) Representative FACS plots of RBD‐specific GC B cells d), their frequency on day 14 e), and their kinetics over the course of vaccination f). RBD‐specific GC B cells are defined as live CD45⁺CD19⁺Fas⁺GL7⁺RBD‐PE⁺RBD‐APC⁺ cells. g–i) Representative FACS plots of T_FH cells g), their frequency on day 14 h), and their kinetics i). Cells were pre‐gated on live CD45⁺CD3⁺CD4⁺CD44^hiCD62L⁻ cells. For each immunization group, n = 5 mice were analyzed on day 14 and 28, respectively. In (b,c,e,f,h,i), data were presented as mean ± S.E.M., and p values were determined by one‐way ANOVA with Turkey's post hoc test.

Figure 6

Hydrogel formulations induce SARS‐CoV‐2 RBD‐specific T cell immune responses. Mice were immunized on day 0, and the spleens were harvested, processed to single cells, and stimulated with SARS‐CoV‐2 RBD peptide pools 14 days post‐immunization. a) Representative FACS plots of IFN‐γ ⁺TNF‐α ⁺ CD4⁺ T cells, which were further gated to characterize IFN‐γ ⁺IL‐2⁺TNF‐α ⁺ cells. b) Representative FACS plots of IL‐2⁺TNF‐α ⁺ CD8⁺ T cells, as determined by intracellular staining. c,d) Frequencies of each category of cells in CD4⁺ and CD8⁺ subsets. n = 5 mice per immunization group were analyzed. mean ± S.E.M. was shown, and one‐way ANOVA with Turkey's post hoc test was conducted.