Modular Recombinant Nanocarriers for Enhanced Delivery of Antiviral Mini-Proteins

Introduction: : Viral infections, exemplified by the recent SARS-CoV-2 pandemic, emphasize the urgent need for therapeutic strategies that address post-infection treatment, complementing prophylactic methods like vaccination. Current vaccines face limitations in rapid deployment, storage, and inability to treat active infections. Recent advances in structural biology have enabled the design of potent mini-proteins and peptide inhibitors that disrupt key viral mechanisms. Despite high specificity and strong in vitro efficacy, their clinical translation has been hindered by rapid degradation, short circulation time, and delivery barriers. To address these issues, this study introduces a protein-based nanotechnology platform using Oleosin—an amphiphilic plant-derived protein known to self-assemble into micelles—as a modular scaffold for displaying antiviral peptides. This approach provides structural versatility, and capacity for functionalization and biocompatibility of the platform itself. The recombinant Oleosin micelles allow genetic fusion of therapeutic mini-proteins directly at the DNA level, ensuring uniform presentation and avoiding chemical conjugation inconsistencies. Here, we engineered Oleosin variants to display two antiviral inhibitors targeting distinct phases of SARS-CoV-2 entry: (1) LCB3, a mini-protein that binds and blocks the Spike-ACE2 interface, and (2) lHR2, a peptide that impedes membrane fusion by stabilizing a fusion-incompetent conformation of the Spike protein. By presenting both inhibitors simultaneously, the micelles aim to synergistically prevent viral attachment and entry. This protein-only approach eliminates the challenges of synthetic nanoparticles and enhances bioactivity through multivalency. We demonstrate that dual-functionalized Oleosin micelles significantly outperform their monofunctional or free peptide counterparts in SARS-CoV-2 inhibition assays.

Materials and

Methods: : Recombinant protein design began with the 25-30G-30(−) variant of Oleosin, engineered for optimal yield and stability. Using gene synthesis (gBlocks) and In-Fusion cloning, we appended the mini-protein lHR2 at the N-terminus and LCB3 at the C-terminus to create multivalent and multifunctional lHR2-Oleosin-LCB3 constructs. All constructs were cloned into pBamUK vectors and verified by sequencing. Proteins were expressed in E. coli BL21(DE3) using IPTG induction and purified via His-tag affinity chromatography. Purity and molecular weights were validated by SDS-PAGE.
To confirm micelle formation, dynamic light scattering (DLS) was performed on 10 µM solutions of each Oleosin variant in PBS. All variants—including the control Oleosin, Oleo-lHR2, Oleo-LCB3, and lHR2-Oleo-LCB3—self-assembled into nanoscale structures with diameters ranging from 15–21 nm, reflecting the incremental addition of functional domains. The dual-functional variant formed the largest and most uniform micelles.
Antiviral efficacy was evaluated using a focus forming assay (FFA) with VeroE6+TMPRSS2 cells infected by SARS-CoV-2 Omicron variant. Cells were pre-treated with varying concentrations of micelle formulations or control treatments, followed by virus exposure. Post-infection, cells were fixed, stained with antibodies to visualize infection foci. In order to assess cytotoxicity, a viability assay was performed under identical treatment conditions.

Results, Conclusions, and Discussions:: SDS-PAGE confirmed the successful expression of all Oleosin variants, including the dual-functional lHR2-Oleo-LCB3 with a molecular weight of approximately 21.7 kDa. DLS analysis revealed that functionalization increased micelle size incrementally, with the control Oleosin forming ~15.4 nm structures, and dual-functional variants averaging ~20.0 nm. These size distributions are consistent with effective multivalent display of fused peptides on the micelle surface.
Functionally, the dual-functional Oleosin exhibited markedly superior antiviral activity. In the FFA, Oleo-lHR2 and Oleo-LCB3 each reduced infection with IC₅₀ values of 462.7 nM and 324.5 nM respectively. However, the dual-functional lHR2-Oleo-LCB3 demonstrated a synergistic effect with an IC₅₀ of just 4.83 nM—representing more than 60-fold enhancement. This remarkable potency is likely due to the simultaneous and/or progressive inhibition of Spike-ACE2 interaction and membrane fusion, targeting two key stages of viral entry.
Comparative treatments using equimolar blends of mono-functional Oleosin variants did not replicate this level of inhibition, emphasizing the necessity of co-localized display for synergy. Control experiments also revealed that non-functionalized Oleosin micelles had minimal effect on infection, ruling out nonspecific effects.
In parallel cytotoxicity assessments, all Oleosin variants, including the dual-conjugate, maintained cell viability above 92%, indicating the constructs are not only potent but also safe for cellular applications. The recombinant nature of this platform allows precise genetic incorporation of multiple antiviral agents, making it highly adaptable for mutating viral strains.
In conclusion, this study validates the Oleosin micellar system as a scalable, protein-based platform for multivalent antiviral delivery. By genetically encoding antiviral peptides into the micelle structure, we eliminate the need for chemical modifications and achieve uniform, functional presentation. The multi-targeting capability of the system enables potent, stage-specific viral inhibition with minimal cytotoxicity, offering a promising post-infection treatment strategy. This technology represents a modular and biocompatible solution capable of rapid adaptation, with potential utility against a broad spectrum of viral pathogens.

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