HVEM signaling promotes protective antibody-dependent cellular cytotoxicity (ADCC) vaccine responses to herpes simplex viruses

Abstract

Herpes simplex virus (HSV) glycoprotein D (gD) not only is required for virus entry and cell-to-cell spread but also binds the host immunomodulatory molecule, HVEM, blocking interactions with its ligands. Natural infection primarily elicits neutralizing antibodies targeting gD, but subunit protein vaccines designed to induce this response have failed clinically. In contrast, preclinical studies demonstrate that an HSV-2 single-cycle strain deleted in gD, ΔgD-2, induces primarily non-neutralizing antibodies that activate Fcγ receptors (FcγRs) to mediate antibody-dependent cellular cytotoxicity (ADCC). These studies were designed to test the hypothesis that gD interferes with ADCC through engagement of HVEM. Immunization of Hvem−/− mice with ΔgD-2 resulted in significant reduction in HSV-specific IgG2 antibodies, the subclass associated with FcγR activation and ADCC, compared with wild-type controls. This translated into a parallel reduction in active and passive vaccine protection. A similar decrease in ADCC titers was observed in Hvem−/− mice vaccinated with an alternative HSV vaccine candidate (dl5-29) or an unrelated vesicular stomatitis virus–vectored vaccine. Unexpectedly, not only did passive transfer of immune serum from ΔgD-2–vaccinated Hvem−/− mice fail to protect wild-type mice but transfer of immune serum from ΔgD-2–vaccinated wild-type mice failed to protect Hvem−/− mice. Immune cells isolated from Hvem−/− mice were impaired in FcγR activation, and, conversely, addition of gD protein or anti-HVEM antibodies to in vitro murine or human FcγR activation assays inhibited the response. These findings uncover a previously unrecognized role for HVEM signaling in generating and mediating ADCC and an additional HSV immune evasion strategy.

Antibodies Elicited in Response to a Single Cycle Glycoprotein D Deletion Viral Vaccine Candidate Bind C1q and Activate Complement Mediated Neutralization and Cytolysis

“…Overnight at 4 °C, 96-well plates (96 Well Flat Bottom Immuno Plate MaxiSorp (Thermo Fisher Scientific, Waltham, MA, USA) were coated with either 450 ng/mL of HSV infected (HSV-1 B 3 x1.1 or HSV-2 G) or uninfected Vero cell lysates or with 250 ng/mL of HSV-1 recombinant gD-1 or gB-1 (produced in HEK293 cells by the Einstein Protein Core Facility), as described [ 18 ]. The plates were washed 4 times with PBS-0.5% Tween (washing buffer, WB) and then blocked with 5% Non-Fat Dry Milk (Bio-Rad, Hercules, CA, USA) in phosphate buffered-saline (PBS) with 0.1% Igepal-CA 630 (Millipore Sigma, St. Louis, MO, USA) (blocking buffer) for 1 h at room temperature (RT).…”

“…Neutralization was assessed using a plaque reduction assay with minor modifications [ 18 ]. Serial dilutions of heat-inactivated immune sera were incubated for 1 h at 37 °C with HSV-1 or HSV-2 (~100 PFU/well) in the absence or presence of 10% v/v rabbit complement (Cedarlane Labs, Burlington, NC, USA).…”

“…Pooled serum containing 750 µg of total IgG (quantified by IgG ELISA, Invitrogen, Carlsbad, CA) harvested from intramuscularly ΔgD-2 or control (VD60) vaccinated C57Bl/6 mice was inoculated intraperitoneally into naïve WT or C1q-knockout BL/6 mice 24 h prior to skin challenge with HSV-2(4674) (5 × 10 PFU/mouse [ 13 ] ), as previously described [ 18 ]. Mice were then monitored daily for epithelial and neurological disease and scored as follows: (1) erythema at inoculation site; (2) spread to distant site, zosteriform lesions, edema; (3) ulcerations, epidermal spread, limb paresis; (4) hind limb paralysis and (5) death.…”

“…Rather, protection mapped to the ability of the Fc portion of the antibodies to engage Fc receptors and trigger antibody-dependent cellular cytotoxicity (ADCC), and to facilitate viral clearance by phagocytic cells [ 16 , 17 ]. The central role of the Fc receptor in mediating protection was confirmed by the observation that passive protection was lost when immune serum was transferred into FcγRIV knockout mice [ 18 ]. In contrast, adjuvanted recombinant gD protein vaccines elicited a predominant neutralizing response in the murine model, little ADCC and significantly less protection against disease following active or passive immunization when mice were challenged with clinical isolates.…”

“…In contrast, adjuvanted recombinant gD protein vaccines elicited a predominant neutralizing response in the murine model, little ADCC and significantly less protection against disease following active or passive immunization when mice were challenged with clinical isolates. Moreover, the recombinant gD protein vaccine failed to prevent latency [ 15 , 18 ].…”