In Silico Redesign of Epstein-Barr Virus Entry Glycoproteins to Redirect Cellular Tropism as a Foundation for a Novel B Cell Oncolytic Therapy.

B cell acute lymphoblastic leukemia (B-ALL) affects thousands of patients each year, with relapse occurring in some cases due to residual malignant B cells following chemotherapy or immunotherapy. Relapsed disease has a median survival of only a few months under conventional salvage therapy and a markedly elevated mortality rate.Oncolytic viruses offer an alternative treatment that works by specifically replicating within cancer cells, inducing cytolysis, and replicating and amplifying the viral particles at the site of infection until the tumor is eliminated, a mechanism valuable for eliminating residual disease that traditional therapies fail to eliminate. Epstein-Barr virus (EBV) is an ideal oncolytic candidate because of its natural tropism to B cells, where the virus enters through gp42 (glycoprotein 42, a viral protein) binding to HLA (human leukocyte antigen) class II molecules on B cells before entering the cell through gH/gL (glycoprotein H/glycoprotein L) viral glycoprotein complex on the viruses' outer membrane)-mediated membrane fusion. However, EBV can also infect epithelial cells through an alternative pathway in which the gH/gL complex binds to the EphA2 (ephrin type-A receptor 2) receptor before triggering membrane fusion, creating an off-target pathway with potential for toxicity, uncontrolled viral replication, and inflammatory response that prevents clinical translation. If the gH/gL-EphA2 interaction is disrupted, however, then this off-target concern would be addressed. We therefore computationally reengineered the viral gH/gL complex to selectively disrupt the gH/gL-EphA2 interaction while avoiding disruption of the gH/gL/gp42 complex stability required for B cell entry. The residues to be mutated were chosen through quantitative analysis on PDBePISA; mutant complexes were modeled using AlphaFold-Multimer (DeepMind Technologies, London, UK), refined through HADDOCK docking (Bonvin Lab, Utrecht University, Netherlands), and validated through 20-ns molecular dynamics simulations in OpenMM (Simbios Center, Stanford University, CA, USA). Analysis of the complexes showed success in selective disruption: the mutant gp42-HLA class II complex showed stability with a root mean square deviation (RMSD) of 2.29 ± 0.18 Å and mean root mean square fluctuation (RMSF) of 1.31 Å when compared to the mutant gH/gL-EphA2 interaction, which showed significant instability with an RMSD of 4.77 ± 0.22 Å and mean RMSF of 2.11 Å, with 46% of residues exceeding the 2.0 Å flexibility. In summation, this paper intends to lay an initial computational framework for a B cell-selective oncolytic EBV variant that eliminates epithelial off-target effects while maintaining therapeutic efficacy against leukemic B cells.