Recombinant Protein Nanocolloids for Inhibition of Reporter and Live SARS-CoV-2 Virus

Introduction: SARS-CoV-2 variants continue to emerge causing sustained morbidities around the world. We have demonstrated a proof-of-concept monofunctional nanocolloid (NC) system that can block the transduction of pseudovirus into cells. Interaction between the receptor binding domain (RBD) of the spike protein (S) on SARS-CoV-2 and the human ACE2 receptor is essential for virus entry into the epithelium. Following RBD-ACE2 binding, a sub-section of S (S2) gets activated by the TMPRSS2 protease or cathepsin allowing it to fuse at the plasma membrane. Our NCs are a new class of purely proteinaceous material in which a recombinant protein called oleosin forms the backbone onto which functional motifs can be genetically appended. Here, we demonstrate that oleosin NCs with dual-expressing sequences for inhibition of spike-ACE2 interaction and TMPRSS2-mediated fusion can synergistically inhibit pathways of live virus entry into cells. Oleosin is a surfactant protein found in nature as a stabilizer for fat bodies in plant seeds. We choose this platform for the following reasons: 1) recombinant proteins, unlike lipids in conventional vesicles/micelles, can be engineered precisely and do not need post formulation modification steps. 2) Sparingly soluble functional motifs can be genetically appended to our highly soluble oleosin backbone to obtain high yields of functional product, 3) Unlike free peptide inhibitors, NCs can be multifunctional, can provide a steric barrier to virus-cell interactions, and have longer in vivo half-life.

Materials and

Methods: DNA sequences for cloning were purchased from Integrated DNA Technologies. Proteins were expressed in E.Coli (BL21) and purified using Nickel-immobilized metal affinity chromatography. Proteins expressed include anti-spike proteins (Oleo-LCB1, Oleo-LCB3), andti-fusogenic proteins (Oleo-lHR2) and bifunctional proteins (LCB1-Oleo-lHR2, LCB3-Oleo-lHR2). NCs were sized using a Malvern Zetasizer, and imaged using cryo-EM. For pseudovirus proof-of-concept trials, cells (293T) and Reporter Virus Particles (RVPs) were obtained from Integral Molecular, and cell culture media and other reagents were obtained from Gibco. Per RVP manufacturer protocol, 293T cells were cultured with puromycin to express ACE2 receptors. Cells were incubated with RVPs and with and without NCs, followed by fluorescent imaging after 48 hrs. For live virus studies, Calu-3 cells (ATCC) were used. NCs were incubated with cells for 2 hrs, followed by addition of the Omicron BA.1 viral variant for 48 hrs. Cells were stained using primary antibodies against dsRNA (J2) and SPIKE (sotrovimab). The total number of cells (Hoechst-stained nuclei count) and the number of infected (dsRNA+ or SPIKE+) cells was measured using the cell scoring module (MetaXpress 5.3.3), and the percentage of infected cells (dsRNA+/SPIKE+ cells/cell number) per well was calculated. A quantitative drug combination analysis was also done to demonstrate synergistic NC efficacy. For temperature stability studies, protein NCs were stored at RT over several weeks and their inhibition efficacy was tested under the same assay conditions.

Results, Conclusions, and Discussions: We cloned various combinations of S1-binding mini-protein genes called LCBx previously designed by David Baker’s laboratory (at UW Seattle) and/or anti-fusogenic heptad repeat peptides (lHR2) to the oleosin N-terminus, which we call Oleo-LCBx or Oleo-lHR2, or LCBx-Oleo-lHR2. Proteins thus expressed showed the formation of both micellar and vesicular structure under cryo-EM confirming their nanocolloidal structure. NCs showed excellent inhibition dose response profiles against both pseudovirus (Wild type, Delta Variant) and Live Virus (Omicron BA.1), with Oleo-LCB1 showing inhibition down to nM levels. Importantly, NCs showed better inhibition efficacy than corresponding free LCBx mini-protein, likely due to both the multivalency of the NC and being a steric barrier to virus-cell interactions. This makes NCs an attractive platform for incorporating a variety of functional moieties. To validate the potential multifunctional nature of NCs, two different concentrations of Oleo-LCB1 and Oleo-LCB3 were co-mixed in the same pseudovirus sample - certain combinations performed much better than individual molecules at the same concentration. Similarly, for live virus testing, NCs Oleo-LCB3 and Oleo-lHR2 were mixed in varying combinations as antagonists for both ACE2-spike interactions and TMPRSS2-mediated virus-cell fusion. Combinatorial analysis showed certain combination of Oleo-LCB3 and Oleo-lHR2 NCs outperforming single NC treatment. Currently, NCs made of multifunctional oleosin molecules (LCBx-Oleo-lHR2) are being tested for improved efficacy that corresponding monofunctional NCs. Finally, we hypothesized that since oleosin is a unique molecule with little secondary structure - behaving like a free-chain amphiphile -, storage of oleosin-based NCs will be viable at room temperatures. Tests done with Oleo-LCB1 NCs stored for up to 6 weeks at RT validate this hypothesis showing no difference in NC inhibition efficacy against live virus. Taken together, these results demonstrate oleosin proteins as a genetically tunable, multifunctional self-assembling platform viral treatment. The strategy we employ here is modular and can easily be modified to incorporate new binding motifs that block the entry of new coronavirus variants or other emerging viral threats not yet envisioned.

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