Abstract / Summary
Abstract The continuous mutation of the SARS-CoV-2 virus drives the need for novel vaccine approaches that can effectively combat new strains. Although research has heavily focused on the spike glycoprotein's S1 domain, the S2 subunit—despite its highly conserved sequence—has received far less attention. Here, we detail the design, production, isolation, and immunological assessment of the recombinant SARS-CoV-2 S2 protein synthesized using Pichia pastoris . To maximize protein yield, a codon-adapted S2 sequence was integrated into a yeast vector driven by the AOX1 promoter. Following affinity chromatography, SDS-PAGE and Western blot analysis confirmed the expression of a ~ 95–105 kDa glycosylated fusion protein peaking at 72–120 h post-induction. Bradford quantification demonstrated purified yields of 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 (p = 0.032). Furthermore, ELISA and pseudovirus neutralization assays revealed that the human-like glycan profile of the SM10-derived S2-Fc exhibited significantly enhanced antigenicity (approximately 31.4 ± 3.2% higher binding affinity, p < 0.001) compared to the wild-type counterpart, confirming the preservation of native neutralizing epitopes. Ultimately, our results suggest that the S2 domain is a strong candidate for developing broad-spectrum COVID-19 vaccines capable of neutralizing diverse variants, highlighting the importance of subsequent preclinical evaluations to determine its full protective efficacy.