Multipolymer microsphere delivery of SARS-CoV-2 antigens

Abstract

Effective antigen delivery facilitates antiviral vaccine success defined by effective immune protective responses against viral exposures. To improve severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antigen delivery, a controlled biodegradable, stable, biocompatible, and nontoxic polymeric microsphere system was developed for chemically inactivated viral proteins. SARS-CoV-2 proteins encapsulated in polymeric microspheres induced robust antiviral immunity. The viral antigen-loaded microsphere system can preclude the need for repeat administrations, highlighting its potential as an effective vaccine. STATEMENT OF SIGNIFICANCE: Successful SARS-CoV-2 vaccines were developed and quickly approved by the US Food and Drug Administration (FDA). However, each of the vaccines requires boosting as new variants arise. We posit that injectable biodegradable polymers represent a means for the sustained release of emerging viral antigens. The approach offers a means to reduce immunization frequency by predicting viral genomic variability. This strategy could lead to longer-lasting antiviral protective immunity. The current proof-of-concept multipolymer study for SARS-CoV-2 achieve these metrics.

Keywords: Antiviral immunity; Multilayerpolymer; Polymeric microspheres; SARS-CoV-2; Slow-controlled antigen release.

Graphical abstract

Fig. 1

Proposed protective antiviral immune responses from MS-based SARS-CoV-2 antigen delivery. Following intramuscular injection of the MS (porous spheres) loaded with whole, inactive SARS-CoV-2 (yellow spheres) in different layers, antigen release and delivery to antigen presenting cells (APC) is anticipated to generate humoral (B-cell generation and antibody production by plasma cells) and cellular (T-cell activation, cytokine release by CD4 and CD8 T-cell subsets) responses. This leads to protective antiviral immunity. The antibodies generated by the plasma cells and the activated T-cell subsets, both aid in clearance of infection.

Fig. 2

Characterization of an “putative” inactive SARS-CoV-2 vaccine. (a) Topographic images of inactive SARS-CoV-2 was acquired by adsorbing the virions on APS modified mica and subsequent AFM image analysis. Topographical maximal (b) diameter was 25.9 ± 3.7 nm and (c) central height was 106 ± 26.5 nm (n = 89). (d) Representative negative stain TEM image of an inactive SARS-CoV-2 virion taken under magnification of 39000x, and (e) western blot analysis of SARS-CoV-2 Spike S1 (90 kDA) and Nucleocapsid (50 kDA) proteins of inactive SARS-CoV-2.

Fig. 3

Morphological depiction of the multipolymer MS. SEM images show external morphology of (a) non-porous MS, and (b) porous MS. (c) Cross section of porous MS showing the polymer distribution into distinct ‘patches’ as indicated by yellow and red arrows. (d) external morphology of porous MS with antigen (whole inactive SARS-CoV-2 virus). Scale bar: depicted at the lower bar of respective images.

Fig. 4

Spectroscopic characterization of the multipolymer MS. (a) Brightfield microscopy image of a representative sectioned multipolymer MS, and (b) overlaid spatial distribution of the component polymers generated by Raman spectroscopy, showing the ‘patchy’ polymer distribution with PCL (magenta) and PLGA (cyan) as the internal and external polymer matrices, respectively, (c-d) Comparison of the Raman spectra from the internal (magenta) and external (cyan) regions of the multipolymer MS and the individual polymers –PCL (green), PLGA (blue) and PLLA (black) within the range of 900 to 3100 wavenumber (cm⁻¹), (e) A representative virtual cross section of a few MS as measured by X-ray tomography showing porous morphology, and (f) a histogram of the 3D particle diameter was obtained from X-ray tomography (n = 5102).

Fig. 5

Microscopic analysis of microsphere (MS)-macrophage interactions. Monocyte-derived macrophages (MDMs) (2 × 10⁻⁶) were treated with 3 mg of multipolymer MS and incubated for 7 days. Light microscopy images, taken with 20x objective lenses, of (a) control MDMs, and (b) treated MDMs showed cells clustering around the multipolymer MS (blue arrows). Representative TEM images of (c) control and (d) MS SARS-CoV-2-treated macrophages showed vacant spherical compartments inside treated cells, indicating the regions where the MS resided upon intake. Scale bar: 2 µm. SEM images of (e) control MDMs, and (f-h) MS SARS-CoV-2-treated MDMs showed treated cell clusters around and the engulfed in MS (yellow arrows). (i) Confocal overlaid (MaxIP) image from 30 optical scan (Z step 1 µm) showing detected inactive SARS-CoV-2 viral particles stained with DAPI (excitation/emission: 405 nm/425 nm; panel i-1) and the merged images with MS autofluorescence (excitation/emission, 561 nm/590 nm, panel i-2). The panel i-3 is the portion of panel i-2 (the blue box) at higher magnification showing the viral particles (green/yellow) within the MS surface porous structures. Scale bar: 10 µm.

Fig. 6

Humoral immune response to MS-Ag administration in SD rats. (a) Experimental outline for the blood and organ collection, 7- and 28-days post-IM injection of SD rats with either saline, empty MS, or MS-Ag. Splenocytes were subjected to flow cytometry. Blood was analyzed for T-cell subsets and toxicity profiling, (b) Enzyme linked immunosorbent assay (ELISA) for (b) anti-SARS CoV2 S-RBD IgG and (c) Nucleoprotein (N) IgG response showed significant IgG production at day 28 for anti-SARS-CoV-2 S- RBD IgG and N-IgG. Statistical analysis was done by Two-way ANOVA and Tukey's post- hoc tests for multiple comparisons, (****P < 0.0001).

Fig. 7

Cellular immune response to MS-Ag administration in SD rats. SD rats (average body weight = 270 g) were each injected with a single intramuscular dose of 6 µg/g of body weight of chemically inactivated SARS-CoV-2 loaded MS (MS-Ag). From SD rats injected with MS-Ag, empty MS or saline, T-cell subsets of splenocytes were analyzed at 7 and 28 dpi by flow cytometry. (a-b) Percentage population of activated T-cells (CD4+CD69+ and CD8+CD69+), 28 dpi. (c-d) Percentage population of TCM - cells (CD4+CD62L+CD44+ and CD8+CD62L+CD44+), 28 dpi. Intracellular cytokine staining and flow cytometric analysis of CD4+ T-cell subset expressing (e) IFNγ at 7 dpi, (f) IFNγ at 28 dpi, (g) IL4 at 28 dpi, and (h) IL17 at 28 dpi. Statistical analysis was done by One-way ANOVA followed by Newman/Keul's post-hoc analysis for multiple comparisons, (*P < 0.05; **P < 0.01; ***P < 0.001). Abbreviations: MS, multipolymer microsphere; Ag, antigen; IFNγ, Interferon-gamma; IL, Interleukin; %, percentage of the parent population.

Fig. 8

Tissue histology of SD rats upon MS-Ag treatment. Hematoxylin and eosin staining of representative sections from liver, kidney, muscle (injection site) and spleen tissues in untreated (saline injection), empty MS and MS-Ag injected SD rats at the endpoint (28 days post-injection) of the study. No tissue pathology was observed in MS and MS-Ag groups as compared to untreated/saline injected controls. The images were captured at 20x magnification. Scale bar is 100µm.