Antibodies targeting HSV glycoprotein B require effector functions to protect neonatal mice

Abstract

Glycoprotein B (gB) serves as the viral fusion protein for herpes simplex virus (HSV), mediating fusion between viral and host membranes resulting in infection. As such, gB represents a potentially critical target for the host immune system with high potential relevance for vaccine design. Here we investigated the mechanisms of protection for a panel of gB-specific monoclonal antibodies (mAb) in a mouse model of neonatal HSV (nHSV) infection. Viral neutralization contributed, but Fc effector functions were critical for mAb-mediated protection against nHSV mortality, depending on dose. Moreover, AAV-mediated in vivo expression of a gB-specific mAb in mice provided transgenerational protection against HSV-1 and HSV-2 mortality in their offspring. These findings demonstrate that antibodies targeting gB can serve as potent therapeutics and that they require diverse functional profiles to afford optimal protection, informing vaccine design.

Figure 1:. Characterization of HSV gB-specific mAbs.

A. Table of gB-specific mAbs used in this study. Reported binding domain on gB based on available crystal structures of mAbs. Neutralization potencies for each mAb. Origin mAb composition and original reference for each gB-specific mAb B. Epitopes for each mAb visualized on prefusion (left, PDB: 6z9m, 41) or postfusion (right, PDB: 2gum, 42) HSV-1 gB trimers. Hu2c: blue, HDIT102: green. Overlap between Hu2c and HDIT102 – teal, D48: orange, BMPC-23: pink.

Figure 2:. Functional characterization of WT and Fc-engineered gB-specific mAbs.

A. Binding of each gB-specific mAb variant to recombinant HSV-1 gB via multiplex assay, and as compared to an isotype control (black, left panel). Median fluorescent intensity (MFI) is reported B. Neutralization activity of each gB-specific mAb against HSV-1 st17 by plaque reduction assay. Dashed vertical line indicates EC₅₀ value. C. Human FcyR binding profiles of each gB-specific mAb variant and isotype control (black). Bar graphs represent the Area Under the Curve (AUC) values for binding to FcyRIIA (left), FcyRIIIA (center), and FcyRIIIB (right). Error bars represent standard deviation from the mean (A-B) or standard error of the mean (C). gB binding and FcyR binding experiments were performed in technical replicate. Neutralization experiments were performed in technical and 2–3 biological replicates.

Figure 3:. HSV gB-specific mAbs require effector functions to protect neonatal mice.

A-F. gB-specific mAbs were delivered intraperitoneally to 2-day-old C57BL/6J mice immediately before a lethal (1×10⁴ PFU) challenge with HSV-1 st17. (A-C) Survival of pups given 20 μg of WT IgG1 (solid line) or LALA PG KO (dashed line) forms of Hu2c (A), D48 (B), or Fd79 (C). (D-F). Survival of pups following 40 μg of WT IgG1 or LALA PG KO mAbs. Pups were given HDIT102 (D), BMPC-23 (E) or an isotype control mAb (F). Number of mice in each condition are reported in inset. Statistical significance between WT IgG1 and LALA PG KO mAbs is reported in the graph as determined by the log-rank (Mantel-Cox) test. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p <0.0001).

Figure 4:. Protection relates to dose and neutralization potency but can require effector function even at high dose.

gB-specific mAbs at the indicated dose (μg) were delivered i.p. to 2-day-old C57BL/6J mice prior to a lethal i.n. infection with 1×10⁴ PFU of HSV-1. Survival of mice following treatment with Hu2c IgG1 (A), D48 IgG1 (B), BMPC-23 IgG1 (C), or Hu2c LALA PG (D). Number of mice in each condition is reported in inset. Statistical significance between between highest and lowest doses tested as determined by the log-rank (Mantel-Cox) test.

Figure 5:. Broad intergenerational protection can be accomplished through vectored delivery of a gB-specific mAb.

A-B. Neutralization potencies of gB-specific mAbs against HSV-1 st17 (A) and HSV-2 G (B). C. Experimental overview and study design in which AAV encoding the gB-specific mAb, Fd79, on a human IgG1 backbone was administered intramuscularly to 4 female mice prior to breeding and viral challenge of pups. D. Expression of AAV-expressed Fd79 in the sera of female mice at weeks 0 through 4 post AAV administration. E. Survival of offspring of AAV-treated dams challenged with 1×10⁴ PFU of HSV-1 st17 or 300 PFU of HSV-2 G on day 2 postpartum.