Engineering PVX vectors harboring heterologous VSRs to enhance Recombinant vaccine protein expression in plants.

Plant-based systems offer a sustainable platform for production of recombinant proteins, but their utility remains limited by host RNA silencing and low yields. Potato virus X (PVX)-derived vectors have shown promise in transient expression systems; however, their efficiency can be further improved by incorporating viral suppressors of RNA silencing (VSRs). Here, we engineered optimized PVX-based expression vectors harboring heterologous VSRs (P19, P38, and NSs) from distinct plant viruses, systematically evaluating the effects of VSR type, insertion position, and transcriptional orientation. Reversing the VSR cassette orientation relative to the target gene alleviated transcriptional interference, significantly improving both target protein and VSR expression. Among the tested VSRs, NSs conferred the highest expression, followed closely by P38. The best-performing pP3-based vectors achieved GFP accumulation of up to 0.50 mg/g fresh weight (FW), representing a 3 ~ 4-fold increase compared to the parental PVX vector (0.13 mg/g FW). For vaccine antigens, maximum yields reached 0.016 mg/g FW for VP1 and 0.017 mg/g FW for S2, exceeding the parental vectors' yields by over 100-fold. These findings highlight the potential of integrating VSRs into PVX-derived vectors to enhance transient expression of recombinant proteins in Nicotiana benthamiana, providing a robust platform for vaccine antigen production.