Point Mutations in Retargeted gD Eliminate the Sensitivity of EGFR/EGFRvIII-Targeted HSV to Key Neutralizing Antibodies

Abstract

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.

Keywords: glycoprotein D; neutralizing antibodies; oncolytic HSV; systemic treatment; tumor targeting.

Figure 1

Retargeted gD and Viral Backbone Structures (A) Retargeted gD (gD:scEΔ38). The black box indicates replacement of residues 2–24 with anti-EGFR scFv (orange box); blue and red vertical bars represent the positions of mar mutations P54Q (blue) and T213M (red) introduced individually and in combination into gD:scEΔ38. SP, gD signal peptide; TM, transmembrane domain; Δ38, deletion of gD residue 38. (B) Genome structures of WT HSV (upper) and gD-deficient recombinant KNTc-ΔgD:GW (lower). KNTc-ΔgD:GW contains bacterial artificial chromosome (BAC) sequences between U_L37 and U_L38 for viral genome propagation and engineering in E. coli, a ubiquitin C promoter-mCherry expression cassette (UbC-mCh) between U_L3 and U_L4, two viral entry-enhancing mutations in the gB gene (gB:NT), and a GW recombination cassette in place of the gD coding sequence (ΔgD:GW) to allow rapid, orientation-specific insertion of altered gD genes under control of the gD promoter. U_L, unique long segment; U_S, unique short segment; TR_L, terminal inverted repeat flanking U_L; IR_L, internal inverted repeat flanking U_L; TR_S, terminal inverted repeat flanking U_S; IR_S, internal inverted repeat flanking U_S.

Figure 2

Effects of Retargeting and mar Mutations on mAb Binding to Purified gD Ectodomains The ectodomains of gD:scEΔ38 and its mar mutants were expressed in insect cells and purified on an anti-gD (mAb DL6) column. SPRi was used to determine the binding of 25 gD-specific mAbs to each purified protein. (A–D) Representative results show the binding of each mAb to (A) gD:scEΔ38, (B) gD:scEΔ38-P54Q, (C) gD:scEΔ38-T213M, and (D) gD:scEΔ38-P54Q/T213M as a percentage of their binding to the purified soluble ectodomain (306t) of WT gD (100%). Values are averages ± SEM of two or three independent determinations. Black triangles denote a single determination. Statistically significant differences between each gD mutant protein and the parental retargeted protein for each mAb were identified by one-way ANOVA (*p < 0.01). mAbs are named below the horizontal axes and grouped according to their designated community (yellow, green, red, blue, or brown).

Figure 3

gD Activity in Virus-Free Fusion Assays and Inhibition by mAbs (A) Inhibition of fusion by mAb MC5. (B) Inhibition by mAb MC23. B78H1 cells were transfected with expression plasmids for gB:NT, gH, gL, different versions of gD, as indicated, and split luciferase N-terminal plasmid RLuc8_1–7. EGFRvIII-expressing B78-vIII cells were transfected with split luciferase C-terminal plasmid RLuc8_8–11. Upon mixing of the cells, luminescence resulting from fusion between the two cell populations was measured over time. Antibody inhibition was performed by incubation of the transfected B78H1 cell populations with MC5 or MC23 mAb at the indicated concentrations for 1 h prior to mixing with B78-vIII cells. Data are shown relative to no antibody (red in all graphs) at 6 h (100%). Two independent experiments were performed, each in triplicate. Curves from a representative experiment are shown (averages ± SEM). For each retargeted gD protein, two-way ANOVA was used to identify statistically significant differences in fusion during the 6-h time course at each mAb concentration compared to the no antibody control. For MC5 (A), the fusion activity of the parental gD:scEΔ38 protein was significantly inhibited at 5 (p < 0.05), 10 (p < 0.01), and 20 μg/mL (p < 0.01) and the T213M mutant was inhibited at 2.5 (p < 0.05), 5 (p < 0.05), 10 (p < 0.01), and 20 μg/mL (p < 0.01). In contrast, the P54Q and T213M/P54Q mutants were not significantly impaired at any MC5 concentration. For MC23 (B), the activity of the parental gD:scEΔ38 protein was significantly inhibited at 2.5 (p < 0.05), 5 (p < 0.01), 10 (p < 0.01), and 20 μg/mL (p < 0.01), and the P54Q mutant was inhibited at all MC23 concentrations (p < 0.01). The activities of the T213M and T213M/P54Q mutants were not significantly reduced at any MC23 concentration.

Figure 4

Virus Entry Specificities and gD Abundance in Virions (A) Viruses produced by BAC DNA transfection of Vero cells were used to infect B78-H1, B78-C (nectin-1⁺), and B78-vIII (EGFRvIII⁺) cells for 20 h. Viruses are identified at the top of the columns according to their gD protein. Entry was recorded as mCherry fluorescence. Scale bars, 200 μm. (B) Western blots showing gD and VP16 contents of purified virions. Relative gD band intensities normalized to VP16 and set to gD:scEΔ38 = 1× are shown below the lanes.

Figure 5

Neutralization of Retargeted Viruses by mAbs MC5 and MC23 KNTc viruses named to the right of the panels according to their gD gene were incubated with VN mAbs MC5 (left) or MC23 (right) at a range of dilutions (x axis) prior to infection of Vero cells. Infected cell monolayers were overlaid with high-density medium, and plaques were counted 48 h later. Representative results show the percentage PFU relative to virus-only control wells (100%). (A) Neutralization of retargeted gD virus (gD:scEΔ38) and single-mar mutant derivatives. (B) Retargeted gD virus and its double-mar mutant derivative. Data represent the averages ± SEM of three independent experiments using triplicate wells each. Statistically significant differences between each gD mutant virus and the parental retargeted virus at each mAb dilution were determined by two-way ANOVA (*p < 0.01, **p < 0.001, ***p < 0.0001).

Figure 6

Retargeted and Double-mar Mutant Retargeted Virus Neutralization Viruses were incubated with a range of mAb dilutions (x axis) prior to infection of Vero cells. Infected cell monolayers were overlaid with high-density medium, and plaques were counted 48 h later. Representative results show the percentage PFU relative to virus-only control wells (100%). (A) mAb LP2; (B) mAb H162. Data are shown as the averages of triplicate wells ± SEM. Statistically significant differences between the retargeted gD double mutant virus and the parental retargeted virus at each mAb dilution were determined by two-way ANOVA (*p < 0.01, **p < 0.001, ***p < 0.0001).