Effector functions are required for broad and potent protection of neonatal mice with antibodies targeting HSV glycoprotein D

Figures

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  Figure 1. Biophysical characterization of HSV gD-specific mAbs (A) Visualization of the Fc domains of mAbs used in this study. For reference purposes, mutated positions in the HSV mAbs are superimposed on the crystal structure of the Fc domain of the HIV-specific mAb b12 (PDB: 1HZH). Reported neutralization potencies of each mAb and the expected ability of each Fc domain to bind FcγRs are indicated. (B) FcγR binding profiles of the mAbs used in this study. Bar graphs present the area under the curve (AUC) for the binding of each mAb to recombinant human (left) and mouse (right) Fc receptors. Orthologous human and mouse Fc receptors are color matched. See also Figure S1 and Table S1.

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  Figure 2. In vitro functional characterization of HSV gD-specific mAbs (A and B) Ability of the HSV gD mAbs to bind recombinant HSV-2 gD (A) or cell-surface-expressed HSV-1 gD (B) via ELISA or flow cytometry, respectively. (C and D) Ability of the HSV gD mAbs to neutralize HSV-1 (C) or HSV-2 (D) by plaque reduction assay. (E–H) Effector function of HSV gD mAbs, including human FcγRIIIA stimulation of a reporter cell line in the context of antibody-bound HSV-2 gD on a microtiter plate (E), or HSV-1 gD-expressing cells (F) as surrogates for ADCC activity, phagocytosis using anti-human Fab beads (G), or complement deposition using HSV-1 gD-expressing cells (H). Error bars represent standard deviation from the mean. OD, optical density; MFI, mean fluorescent intensity; RLU, relative light units; APC, allophycocyanin. Unless otherwise labeled, the dashed lines represent no-antibody controls. Assays were performed in technical and biological replicates. See also Figure S2.

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  Figure 3. Both neutralization and effector function contribute to mAb-mediated protection from lethal HSV-1 challenge Immediately before lethal intranasal (i.n.) challenge with 1 × 104 plaque-forming units (PFU) HSV-1, 2-day-old pups were administered 40 μg mAb by intraperitoneal (i.p.) injection. (A–C) Survival of C57BL/6J pups receiving neutralizing mAbs UB-621, HSV8, or HSV8 LALAPG (A), non-neutralizing mAbs CH42 AAA or CH42 NA (B), or isotype control mAb VRC01 (C). (D–F) Survival of FcγR−/− pups receiving neutralizing (D), non-neutralizing (E), or isotype control mAb (F). Number of mice in each condition and statistical significance as compared to isotype control in matched mouse strain determined by the log-rank (Mantel-Cox) test (∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001) are reported in inset. Significance between HSV8 and HSV8 LALA PG or CH42 AAA and CH42 NA are reported in the top legend as determined by the log-rank (Mantel-Cox) test. See also Figure S3.

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  Figure 4. Fc functions are protective in the absence of complete viral neutralization One hour before i.n. challenge of 2-day-old pups, immune complexes were formed by incubation of 1 × 104 PFU HSV-1 with mAb at 37°C (20 μg unless otherwise noted). (A–C) Survival of C57BL/6J pups following immune complex challenge with virus opsonized with neutralizing mAbs UB-621 or HSV8 (A), non-neutralizing mAb CH42 AAA (20 or 100 μg) (B), or isotype control mAb (C). (D–F) Survival of FcγR−/− pups following immune complex challenge with virus opsonized with neutralizing mAbs UB-621 or HSV8 (D), non-neutralizing mAb CH42 AAA (100 μg) (E), or isotype control mAb (F). Number of mice in each condition and statistical significance as compared to isotype control in matched mouse strain determined by the log-rank (Mantel-Cox) test (∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001) are reported in inset. See also Figure S4.

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  Figure 5. Relative contributions of neutralization and effector functions to protection from lethal challenge depending on antibody dose Immediately before lethal i.n. challenge with 1 × 104 PFU HSV-1, 2-day-old pups were administered 10 μg mAb by i.p. injection. (A–C) Survival of C57BL/6J pups receiving neutralizing mAb HSV8 or HSV8 LALA PG (A), non-neutralizing mAb CH42 AAA or CH42 NA (B), or isotype control mAb (C). Number of mice in each condition and statistical significance as compared to isotype control determined by the log-rank (Mantel-Cox) test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001) are reported in inset. Statistical significance between HSV8 and HSV8 LALA PG or CH42 AAA and CH42 NA is reported in the top legend as determined by the log-rank (Mantel-Cox) test (∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001). (D–F) Viral titers were determined 5 days post-infection (DPI). Data are shown as viral burden in perfused organs from surviving pups following 10 μg mAb treatment on DPI 0. Statistical significance was determined by two-way ANOVA with Bonferroni’s test for multiple comparisons (∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001). See also Table S2. Geometric mean…

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  Figure 6. Effector functions accelerate control of viral replication after non-lethal HSV-1 challenge One day post-i.n. infection with a luciferase-expressing HSV-1, 2-day-old pups were administered 10 μg mAb i.p., and viral replication, as represented by bioluminescence, was quantified daily. (A) Representative bioluminescence images of viral infection and replication following mAb treatment are presented for the same two pups over time. (B) Quantification of virally derived bioluminescence over time for HSV8 and HSV8 LALA PG (top), CH42 AAA and CH42 NA (middle), and isotype control (bottom). Lines and shaded regions represent the mean luminescence and standard error of the mean across pups (number listed in inset). (C) Heatmap depicting statistical significance (two-way ANOVA with Tukey’s test for multiple comparisons) between groups treated with indicated mAbs over time after infection.

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  Figure 7. Antibody functions contributing to protection differ between HSV serotypes Immediately before lethal i.n. challenge with 300 PFU HSV-2, 2-day-old pups were administered 40 μg mAb by i.p. injection. Survival of C57BL/6J pups receiving neutralizing mAb HSV8 or HSV8 LALAPG (A), non-neutralizing mAb CH42 AAA or CH42 NA (B), or isotype control mAb (C). Number of mice in each condition and statistical significance as compared to isotype control determined by the log-rank (Mantel-Cox) test (∗p < 0.05 and ∗∗∗p < 0.001) are reported in inset. Statistical significance between HSV8 and HSV8 LALA PG or CH42 AAA and CH42 NA is reported in the top legend as determined by the log-rank (Mantel-Cox) test (∗p < 0.05 and ∗∗∗p < 0.001). See also Figure S6.