Using Split Luciferase Assay and Anti-Herpes Simplex Virus Glycoprotein Monoclonal Antibodies To Predict a Functional Binding Site between gD and gH/gL

Abstract

Herpes simplex virus (HSV) entry and cell-cell fusion require glycoproteins gD, gH/gL, and gB. HSV entry begins with gD binding its receptor (nectin-1), which then activates gH/gL to enable the conversion of pre-fusion gB to its active form to promote membrane fusion. Virus-neutralizing monoclonal antibodies (Mabs) interfere with one or more of these steps and localization of their epitopes identifies functional sites on each protein. Utilizing this approach, we have identified the gH/gL binding face on gD and the corresponding gD binding site on gH/gL. Here, we used combinations of these Mabs to define the orientation of gD and gH/gL relative to each other. We reasoned that if two Mabs, one directed at gD and the other at gH/gL, block fusion more effectively than when either were used alone (additive), then their epitopes would be spatially distanced and binding of one would not directly interfere with binding of the other during fusion. However, if the two Mabs blocked fusion with equal or lesser efficacy that when either were used alone (indifferent), we propose that their epitopes would be in close proximity in the complex. Using a live cell fusion assay, we found that some Mab pairings blocked the fusion with different mechanisms while other had a similar mechanisms of action. Grouping the different combinations of antibodies into indifferent and additive groups, we present a model for the orientation of gD vis-à-vis gH/gL in the complex. Importance: Virus entry and cell-cell fusion mediated by HSV require four essential glycoproteins, gD, gH/gL, gB and a gD receptor. Virus-neutralizing antibodies directed against any of these proteins bind to residues within key functional sites and interfere with essential steps in the fusion pathway. Thus, the epitopes of these Mabs overlap and point to critical, functional sites on their target proteins. Here, we combined gD and gH/gL antibodies to determine whether they work in an additive or non-additive (indifferent) fashion to block specific events in glycoprotein-driven cell-cell fusion. Identifying combinations of antibodies that have additive effects will help in the rational design of an effective therapeutic “polyclonal antibody” to treat HSV disease. In addition, identification of the exact contact regions between gD and gH/gL can inform the design of small molecules that would interfere with the gD-gH/gL complex formation, thus preventing the virus from entering the host cell.

Antibodies to Crucial Epitopes on HSV-2 Glycoprotein D as a Guide to Dosing an mRNA Genital Herpes Vaccine

“…We again detected the lowest antibody titers in the 0.3-μg group. (4) We next evaluated prototype MAbs MC2, which blocks gD2 activation thereby preventing its interaction with gH2/gL2 [ 17 , 18 ]. The antibody titers to the MC2 epitope followed a similar pattern as ID3 in that the 10-μg group had lower titers than the 1-μg group, suggesting possible changes to epitope conformation or interference by other antibodies in the sera ( Figure 3 d).…”

“…The antibody titers to the MC2 epitope followed a similar pattern as ID3 in that the 10-μg group had lower titers than the 1-μg group, suggesting possible changes to epitope conformation or interference by other antibodies in the sera ( Figure 3 d). (5) We then assessed prototype MAb MC5, which blocks gD2 binding to gH2/gL2 [ 18 ]. The antibody titers to MC5 plateaued at doses ≥ 1 μg ( Figure 3 e).…”

Herpes Simplex Virus 1 Entry Glycoproteins Form Complexes before and during Membrane Fusion

“…Therefore, gD-gH/gL and gH/gL-gB interactions through their TMDs and CTDs are greater than those through their ectodomains. Whereas previous studies focused mainly on ectodomain interactions ( 38 , 39 , 62 ), our study highlights the previously overlooked yet important contributions of the TMD and CTDs to HSV-1 glycoprotein interactions. Indeed, our recent study proposed a mechanism by which the cytoplasmic tail of gH activates gB through its CTD ( 63 ).…”

Herpes simplex virus 1 entry glycoproteins form stable complexes prior to and during membrane fusion

“…Therefore, gD-gH/gL and gH/gL-gB interactions through their TMDs and CTDs are greater than through their ectodomains. Whereas previous studies focused mainly on ectodomain interactions [35,36,61], our study highlights the previously overlooked yet important contributions of the TMD and CTDs to HSV-1 glycoprotein interactions.…”