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According to the results of the Rosetta Cartesian_ddG and Flex_ddG, we manually checked the PV2 and PV3 variants predicted to be highly stable, exhibit low binding free energy and are located outside the CDR regions, and those having pAcF facing outward were selected for experimental characterization, including PV2 variants S8pAcF, S26pAcF, S71pAcF, S74pAcF, Q82pAcF, S85pAcF and PV3 variants S8pAcF, S26pAcF, S71pAcF, N77pAcF, S85pAcF, K87pAcF (Fig