Effective oncolytic virotherapy may require systemic delivery, tumor targeting, and resistance to virus-neutralizing (VN) antibodies. Since herpes simplex virus (HSV) glycoprotein D (gD) is the viral attachment/entry protein and predominant VN target, we examined the impact of gD retargeting alone and in combination with alterations in dominant VN epitopes on virus susceptibility to VN antibodies. We compared the binding of a panel of anti-gD monoclonal antibodies (mAbs) that mimic antibody specificities in human HSV-immune sera to the purified ectodomains of wild-type and retargeted gD, revealing the retention of two prominent epitopes. Substitution of a key residue in each epitope, separately and together, revealed that both substitutions (1) blocked retargeted gD recognition by mAbs to the respective epitopes, and, in combination, caused a global reduction in mAb binding; (2) protected against fusion inhibition by VN mAbs reactive with each epitope in virus-free cell-cell fusion assays; and (3) increased the resistance of retargeted HSV-1 to these VN mAbs. Although the combined modifications of retargeted gD allowed bona fide retargeting, incorporation into virions was partially compromised. Our results indicate that stacking of epitope mutations can additively block retargeted gD recognition by VN antibodies but also that improvements in gD incorporation into virus particles may be required.,? 2020 The Authors.