(M1030-01-02) Biomimetic SARS-CoV-2 Spike Protein Nanoparticles as Viral Inhibitors against COVID-19

Purpose: Coronavirus disease 2019 (COVID-19) is a serious, infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One FDA-approved COVID-19 treatment is the antiviral drug remdesivir. However, this drug is marginally effective in shortening the recovery time and mortality rate of patients hospitalized with COVID-19. Due to the dire need for effective treatments for COVID-19, disrupting the SARS-CoV-2 internalization mechanism into host cells would be a potent therapeutic strategy. One emerging method to deliver recombinant proteins is via attachment to recombinant polypeptides, such as elastin-like polypeptides (ELPs). Derived from the human tropoelastin protein, ELPs are genetically engineered biopolymers that self-assemble into protein-based nanoparticles. ELPs consist of repeated pentameric motifs with the amino acid sequence (Val-Pro-Gly-Xaa-Gly)n where the guest residue, Xaa, controls the hydrophobicity, and n determines the peptide molecular weight. This study explores a novel COVID-19 treatment by developing temperature-sensitive recombinant proteins composed of the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and a thermally responsive ELP. These fusions self-assemble into colloidally stable nanoparticles. Herein, the biophysical properties of this novel “spike-A192” protein are characterized and its internalization into a lung epithelial cell line is assessed.

Methods: The encoding sequence of the spike RBD was designed using Escherichia coli (E. coli) biased codons, ligated to the A192 sequence (VPGAG)_192, that was synthesized in a modified pET-25b(+) vector. The spike-A192 plasmid was expressed in E. coli for protein expression. Following protein purification, the thermal responsiveness property of spike-A192 was characterized using UV-visible spectrophotometry, whereby the optical density at 350 nm (OD­₃₅₀) was measured over a range of temperatures. The ELP’s transition temperature (T_t) was obtained from the following equation:T_t = b - mlog₁₀(C_ELP), where b is the y-intercept representing the extrapolated T_t at a concentration of 1 μM and m is the slope representing the change in °C per 10-fold change in the ELP concentration (C_ELP). Next, the hydrodynamic radius (R_h) and polydispersity of the spike-ELPs were measured by dynamic light scattering (DLS). Size exclusion chromatography-multiangle light scattering (SEC-MALS) was used to determine the absolute molar mass of the spike-A192 nanoparticles and the degree of oligomerization. Lastly, we used negative-staining transmission electron microscopy (TEM) to visualize spike-A192’s morphology. Following biophysical characterization, we evaluated the effects of spike-A192 on the binding interaction between recombinant SARS-CoV-2 spike RBD and hACE2 using an in vitro enzyme-linked immunosorbent assay (ELISA) and the half-maximal inhibitory concentration (IC₅₀) for the inhibitory activity was estimated from non-linear regression. Subsequently, live cell imaging was performed to determine whether spike-A192 can be efficiently internalized into A549 lung epithelial cells, more specifically into lysosomal compartments. The A549 cells were incubated with NHS-Rhodamine-conjugated spike-A192 and Lysotracker Green at 37 °C for 1 hour and then imaged at 37 °C using confocal microscopy. To quantify the degree of colocalization between the Rho-ELPs and LysoTracker Green, the Pearson’s correlation coefficient (PCC) was calculated.

Results: UV-visible spectrophotometry analysis shows that the T_t of spike-A192 is 43.7 °C, while A192 phase separates above 57 °C at 50 μM concentration. For both proteins, the T_t is sensitive to the ELP concentration; however, the extent of T_t sensitivity is less in spike-A192 (m = 1.2±0.174 °C/Log₁₀(μM)) than A192 (m = 8.4±0.557 °C/Log₁₀(μM)). Both fusions remain soluble at physiological temperatures. DLS results demonstrate that the R_h of spike-A192 (25 μM) is 71.4±0.15 nm, representative of monodispersed, large nanoparticles. The R_h of spike-A192 does not change significantly over a 72-hr, suggesting colloidal stability at 37 °C. As obtained from SEC-MALS, the absolute molar mass of the spike-A192 nanoparticles is 5.4 x 10⁷ Da, suggesting that on average 550 spike-A192 monomers oligomerize to form a nanoparticle. Moreover, TEM images reveal a distribution of elongated nanostructures. Using an adapted ELISA, we demonstrated that spike-A192 inhibits the binding of the recombinant SARS-CoV-2 spike RBD to hACE2 in an ELP concentration-dependent manner. A sigmoidal model generated an IC₅₀ of 13.1 μM. ­Live cell images show that there was colocalization of the red fluorescence of both Rho-ELPs and the green fluorescence of the lysosomal compartments. However, Rho-spike-A192 shows a stronger intracellular fluorescence than Rho-A192. The mean PCC of cells treated with Rho-A192 and Rho-spike-A192 was 0.47 and 0.55 (p >0.05). These findings suggest that both ELPs are internalized into lysosomes to a moderate extent following endocytosis into A549 cells. However, spike-A192 exhibits higher internalization efficiency.

Conclusion: SARS-CoV-2 spike RBD can be fused with an ELP to generate stable, multivalent nanoparticles (spike-A192). These nanoparticles effectively interfere with the SARS-CoV-2 spike RBD binding to hACE2 and can be efficiently internalized into A549 lung epithelial cells.

Acknowledgments: This work was made possible by Dr. Paul Seidler at the USC School of Pharmacy for his contribution in acquiring and processing TEM images, the University of Southern California (USC) to AP, the Gavin S. Herbert Professorship to JAM, National Institutes of Health R01 GM114839 and R01EY026635 to JAM, R01 GM114839-04S1 to HA, P30 CA014089 to the USC Norris Comprehensive Cancer Center, and P30 EY029220 to the USC Ophthalmology Center Core Grant for Vision Research. The authors have no conflicts to report.

Figure 1. Design of the spike-ELP to inhibit cellular SARS-CoV-2 entry



Figure 3. Spike-A192 is trafficked into the lysosomal compartment of A549 cells



Figure 2. Spike-A192 monomers oligomerize to form stable, elongated nanostructures and effectively inhibit the binding interaction btw. recombinant SARS-CoV-2 spike RBD and hACE2