High-Yield Production and Antigenic Profiling of a Stabilized SARS-CoV-2 S2-Fc Fusion Protein Using GlycoSwitch Pichia pastoris

Technical Summary: High-Yield Production and Antigenic Profiling of a Stabilized SARS-CoV-2 S2-Fc Fusion Protein

Problem Statement
The continuous mutation of the SARS-CoV-2 virus, particularly within the receptor-binding domain (RBD) of the spike (S) protein's S1 subunit, has compromised the long-term efficacy of current vaccines by facilitating immune escape. While the S2 subunit of the spike protein is highly conserved across variants and represents a promising target for broad-spectrum immunity, it has received less attention as a vaccine antigen. Developing S2-based vaccines faces significant technical hurdles: the S2 domain contains large hydrophobic regions prone to aggregation and misfolding, making the production of soluble, high-quality recombinant protein difficult. Furthermore, standard yeast expression systems often produce hyper-mannosylated proteins that can sterically hinder antigenic epitopes or induce non-protective immune responses.

Methodology
The study employed a glyco-engineered Pichia pastoris system to address these challenges.

• Construct Design: A codon-optimized gene encoding residues 686–1213 of the SARS-CoV-2 S2 subunit was fused to a human IgG1-Fc domain. The Fc region included stabilizing mutations (T250Q and M428L) to enhance FcRn affinity and resistance to proteolysis. A 18-residue Gly-Ser linker connected the S2 and Fc domains. The C-terminal transmembrane region was excluded to ensure solubility.
• Expression System: The construct was inserted into the pPICZαA vector under the AOX1 promoter and transformed into two P. pastoris strains: the wild-type KM71H and the glyco-engineered SuperMan5 (SM10) strain. The SM10 strain was selected for its ability to produce human-like core N-glycans (Man5GlcNAc2) rather than the hyper-mannosylated structures typical of wild-type yeast.
• Production and Purification: High-copy transformants were selected via zeocin resistance. Protein expression was induced with methanol over 144 hours. Secreted proteins were purified using Protein G-Sepharose affinity chromatography.
• Characterization: The purified proteins were analyzed via SDS-PAGE, Western blot, and Dot blot using human hyperimmune sera. Antigenicity was assessed through double-direct and indirect ELISAs against heat-inactivated virus and a reference vaccine (RAZI CovoPars). Functional neutralization was evaluated using pseudovirus neutralization assays.

Key Contributions

1. Optimized S2-Fc Design: The study successfully engineered a stabilized S2-Fc fusion protein that retains the conserved HR1 and HR2 domains while avoiding hydrophobic aggregation through truncation and Fc fusion.
2. Glycoengineering for Antigenicity: By utilizing the SM10 strain, the researchers demonstrated that shifting the glycosylation profile from hyper-mannosylation to human-like core structures significantly enhances the accessibility of neutralizing epitopes.
3. Scalable Production Platform: The study established a cost-effective, scalable yeast-based platform for producing high yields of the S2-Fc antigen, overcoming expression bottlenecks associated with large viral glycoproteins.

Results

• Expression and Yield: SDS-PAGE confirmed the expression of a glycosylated fusion protein in the 90–105 kDa range, consistent with the predicted molecular weight including N-glycosylation. Peak accumulation occurred between 72 and 120 hours post-induction.
• Quantitative Yield: Purified yields were 18.6 ± 0.72 µg/mL for the wild-type KM71H strain and 20.6 ± 0.85 µg/mL for the glyco-engineered SM10 strain. The increase in yield for the SM10 strain was statistically significant (p = 0.032).
• Antigenic Profiling: ELISA results showed that the SM10-derived S2-Fc exhibited a 31.4 ± 3.2% higher binding affinity compared to the KM71H counterpart (p < 0.001). Standard curves demonstrated excellent linearity (R² > 0.98), and binding reactivity strongly correlated with antibody titers (Pearson's r = 0.994).
• Neutralization: Pseudovirus neutralization assays confirmed that antibodies elicited against the recombinant S2-Fc could neutralize pseudotyped viral particles, supporting the functional integrity and proper folding of the antigen.
• Stability: The purified protein maintained structural and antigenic stability over a 144-hour time course.

Significance
The paper concludes that the S2 domain is a viable candidate for developing broad-spectrum COVID-19 vaccines capable of neutralizing diverse variants due to its genetic stability. The study highlights that the quality of glycosylation, rather than just quantity, is a critical determinant of antigenicity. By utilizing the GlycoSwitch P. pastoris system, the authors successfully produced a properly folded, glycosylated, and antigenically competent S2-Fc protein. These findings support the further preclinical evaluation of S2-based subunit vaccines and suggest that glycoengineered yeast platforms offer a robust, scalable alternative to mammalian systems for producing coronavirus spike-derived proteins. The authors note that while the study confirms in vitro antigenicity and neutralization, subsequent in vivo studies are required to determine full protective efficacy and Fc-mediated effector functions.