(595d) Recombinant Protein Nanostructures for the Inhibition of Sars-Cov-2

The emergence of the highly infectious SARS-CoV-2 virus, which is responsible for the Covid-19 pandemic, has led to an urgent need to develop therapeutics, treatments, and vaccines, both against the immediate threat, as well as developing a versatile platform for future treatments. It is known that binding between the receptor binding domain (RBD) of the spike protein (S) on SARS-CoV-2 and the human ACE2 receptor is essential for entry of the virus into the epithelium. SARS-CoV-2 can enter via endocytosis by binding to the ACE2 receptor, and/or via fusion, where, after ACE2-binding, it gets activated by the human proteases like furin, TMPRSS2, and cathepsins,allowing it to fuse at the plasma membrane. A powerful strategy to block viral infection is to use inhibitors such as short chain peptides and mini-proteins that bind to the S1 protein with high affinity. Such moieties have been designed based on either ACE2 receptor binding epitopes or designed de novoto have very low dissociation constants (K_d) with the S1 RBD. However, free peptide formulation can be difficult and non-uniform, especially with long chain lengths; additionally, free peptides and mini-proteins have a short in vivo half-life. In this work, we leverage the advantages of functional nanoparticles containing peptides/mini-proteins instead as blocking agents for the SARS-CoV-2 virion. We have developed micellar nanostructures with a functionalized recombinant protein called oleosin that will bind and inactivate the Spike S1 protein. Oleosin, found in nature as a stabilizer for fat bodies in plant seeds, is a rare protein that can act as a free-chain triblock surfactant, capable of assembling into micelles and vesicles when engineered in certain ways, as shown previously in our lab. Micelles were formed by spontaneous self-assembly of oleosin that was genetically modified with anti-S1 mini-protein and peptide sequence. Because oleosin is a protein, we can precisely incorporate into it a versatile range of functional moieties at the gene level, eliminating the need for complex post-formulation functionalization chemistry and washing steps involved in other varieties of synthetic micelles and vesicles. Unlike therapeutic antibodies, recombinant proteins can be formulated relatively easily, and unlike free peptides and mini-proteins in many cases, recombinant proteins are uniform and can be equipped with multiple functions with precision through genetic engineering. Furthermore, micelles can have a much greater in vivo half-life than free proteins.

We cloned S1-binding mini-protein genes called LCBxpreviously designed by David Baker’s laboratory (at UW Seattle) to the oleosin N-terminus, which we call Oleo-LCBx. Additionally, several ACE2-mimicking peptides were also cloned to the parent oleosin gene in the same way using In-Fusion cloning. Proteins were expressed in E.Coli(BL21) and purified using Nickel-immobilized metal affinity chromatography.These proteins formed micelles in the 10-100 nm range as verified by dynamic light scattering. To assess the inhibitory activity of these micelles, we measured their effect on the “infection” of Reporter Virus Particles (RVPs) into 293T-hsACE2 cells; the RVPs contain a gene for green fluorescent protein (GFP) and “infection” is reported by GFP-associated fluorescence. Two proteins, Oleo-LCB1 and Oleo-LCB3 were seen to have the capacity to completely block of RVP infection into cells at 10 µM, as determined from the total lack of fluorescence in the cells after co-incubation with RVPs and the protein. Based on a therapeutic dose response, these proteins were able to reduce RVP infection at a concentration as low as 5 nM, where the functional Oleo-LCBx is present in a blend of Oleo-LCBx and the non-functionalized oleosin at a total final concentration of 10 µM (above the critical micelle concentration). This is a highly promising result showing that the LCB1 conformation and affinity for the spike protein is preserved in the oleosin conjugate. Another advantage of nanoparticles instead of unassembled/free peptides/proteins is that multiple functionalities can be incorporated in the same structure by blending different oleosins, leading to a multifunctional therapeutic. To validate the potential multifunctional nature of a micellar therapeutic, two different concentrations of Oleo-LCB1 and Oleo-LCB3 were co-mixed in the same sample. We found that certain combinations performed much better than individual molecules at the same concentration, with one combination completely eliminating RVP infection. Furthermore, micelles of Oleo-LCB1 were equally effective against the Delta variant of SARS-CoV-2. In summary, we present a molecular bioengineering approach to make multifunctional nanostructures (micelles or potentially vesicles) of precise chemistry to competitively bind to virus-mimicking particles. Ongoing work involves addition of other functionalities to micellar oleosin resulting in synergistic approach to block virus entry through the ACE2 receptor. For instance, we have developed, expressed, and purified oleosin variants with anti-fusogenic peptides that will inhibit the stage of viral interaction with the cell membrane that involves the activation of membrane proteases resulting in virus-cell membrane fusion. These variants also form 10-100 nm micelles, thus allowing blending of such functionalities with Oleo-LCBxto form composite micelles. Although the immediate concern is to combat SARS-CoV-2 infection, the strategy is modular, in that domains of proteins embedded in the nanostructurescan be swapped with other bioactive domains of different specificity as required as new infectious agents, such as mutant strains, emerge.