Impact of Valency of a Glycoprotein B-Specific Monoclonal Antibody on Neutralization of Herpes Simplex Virus

Abstract

Herpes simplex virus (HSV) glycoprotein B (gB) is an integral part of the multicomponent fusion system required for virus entry and cell-cell fusion. Here we investigated the mechanism of viral neutralization by the monoclonal antibody (MAb) 2c, which specifically recognizes the gB of HSV type 1 (HSV-1) and HSV-2. Binding of MAb 2c to a type-common discontinuous epitope of gB resulted in highly efficient neutralization of HSV at the postbinding/prefusion stage and completely abrogated the viral cell-to-cell spread in vitro. Mapping of the antigenic site recognized by MAb 2c to the recently solved crystal structure of the HSV-1 gB ectodomain revealed that its discontinuous epitope is only partially accessible within the observed multidomain trimer conformation of gB, likely representing its postfusion conformation. To investigate how MAb 2c may interact with gB during membrane fusion, we characterized the properties of monovalent (Fab and scFv) and bivalent [IgG and F(ab) 2 ] derivatives of MAb 2c. Our data show that the neutralization capacity of MAb 2c is dependent on cross-linkage of gB trimers. As a result, only bivalent derivatives of MAb 2c exhibited high neutralizing activity in vitro. Notably, bivalent MAb 2c not only was capable of preventing mucocutaneous disease in severely immunodeficient NOD/SCID mice upon vaginal HSV-1 challenge but also protected animals even with neuronal HSV infection. We also report for the first time that an anti-gB specific monoclonal antibody prevents HSV-1-induced encephalitis entirely independently from complement activation, antibodydependent cellular cytotoxicity, and cellular immunity. This indicates the potential for further development of MAb 2c as an anti-HSV drug.Herpes simplex virus (HSV) is a neuroinvasive human pathogen that critically depends on efficient infection of distinct target cells within a host. At the time of primary lytic infection, HSV replicates in peripheral mucocutaneous tissues and releases virions. A decisive characteristic of HSV infections in animals and humans is the establishment of a lifelong latency. HSV spreads from infected epithelial cells to axons of sensory neurons innervating the site of the primary infection, followed by retrograde transport to the respective dorsal root ganglia (12). Recurrent infections result from reactivation in neuronal cells, followed by virus replication and anterograde transport to cells at peripheral sites innervated by the respective neurons. Transmission between cells without diffusion through the extracellular environment represents a major route for HSV to spread between tissues and is thus a very efficient way for circumventing immunological barriers of the humoral immune response. Regardless of the dissemination pathway, however, fusion of the viral envelope with host membranes for delivery of the viral genome across the cellular lipid bilayer is essential for viral replication. In contrast to that of most other enveloped viruses, entry of herpesviruses into mammalian cells requires a multicomponent sy...

Overcoming drug-resistant herpes simplex virus (HSV) infection by a humanized antibody

“…Because epitope alterations in HSV-2 vs. HSV-1 isolates could be excluded by epitope sequencing, we concluded that the different neutralization efficacy of mAb hu2c toward HSV-1 vs. HSV-2 isolates is the consequence of higher HSV-2 genome copy numbers, resulting in an increase of noninfectious virus particles, as we have reported earlier (20).…”

“…Hence, CDR coding gene segments of the murine donor-antibody 2c (i.e., 2c V L -CDR1/2/3 and 2c V H -CDR1/2/3) were grafted into acceptor frameworks coding for IGHV2-70 and IGKV2-40, respectively. Humanized or murine variable domain encoding genes (20) were subsequently cloned into Ig expression vectors containing a human constant heavy γ1 chain, and a human constant κ chain, respectively. After stable cotransfection of vectors into Sp2/0-Ag14 cells, humanized IgG hu2c and chimeric IgG ch2c were produced and purified from cell culture supernatants.…”

“…We recently mapped the epitope of the mouse antibody 2c and analyzed its binding by homology modeling based on the solved crystal structure of the gB complex (20,21). We found that the antibody binds to a discontinuous epitope comprising exposed residues 299-305 (upper section of domain I of gB) as well as residues of the sequence motif FEDF (also located in domain I).…”

“…Several attempts for developing a potent HSV vaccine have been made, but no such compound showed thus far sufficient clinical efficacy even in a large randomized trial (1). We have recently shown that the murine monoclonal antibody mAb 2c exhibits highly potent HSV-neutralizing activity through binding to a key fusogenic domain of the viral gB protein (20,21). To exploit this property for therapeutic interventions in humans we have in the present study humanized mAb 2c and characterized its potential to prevent and treat HSV infection in vitro and in severely immunocompromised mice.…”

“…Several monoclonal antibodies have been raised in mice against the viral glycoproteins gH, gD, and gB. We have previously shown that a gB-specific monoclonal antibody, designated mAb 2c, not only exhibited strong HSV-neutralizing capacity but was also capable of very effectively abrogating viral cell-to-cell spread both in vitro and in animal models (20). Interestingly, the particular sequence of the HSV-1/2 type-common gB epitope recognized by mAb 2c seemed to be indispensible for viral infectivity and fitness (21).…”