Nanobody Nb1_gbHSV Targets Prefusion Glycoprotein B for Potent HSV-1/2 Neutralization

On September 3, 2025, a research team from the Center for Structural Systems Biology at the University of Hamburg published a paper in Nature titled A nanobody specific to prefusion glycoprotein B neutralizes HSV-1 and HSV-2. The study identified, for the first time, a nanobody, Nb1_gbHSV, specific to the prefusion conformation of herpesvirus glycoprotein B (gB). This nanobody exhibits potent neutralizing activity (IC50 = 1.2 nM) and cross-species efficacy against HSV-1 and HSV-2. Using cryo-EM, the team resolved the full-length, high-resolution structures of HSV-1/2 gB, revealing critical details of the membrane fusion mechanism and providing new targets and strategies for herpesvirus vaccine and antiviral therapy development.

The human herpesvirus family includes nine viruses (e.g., HSV-1, HSV-2, and human cytomegalovirus), which cause a range of diseases from oral/genital herpes to neonatal infections and cancers. Glycoprotein B (gB) on the viral envelope is critical for host cell invasion. As the most conserved glycoprotein across all herpesviruses, gB undergoes a dramatic conformational change from a prefusion to a postfusion state to facilitate membrane fusion and viral entry. However, the prefusion conformation of gB is highly unstable (metastable) and challenging to capture via structural analysis, resulting in a long-standing lack of neutralizing antibodies or targeted drugs for this conformation.

The research team innovatively used gB vesicle immunization in alpacas (preventing irreversible conversion to the postfusion conformation when gB is detached from the membrane) and constructed an immune library using phage display technology. From 17 candidate nanobodies, Nb1_gbHSV was selected. This nanobody demonstrated exceptional neutralizing activity in HSV-1 plaque reduction assays, with a half-maximal inhibitory concentration (IC₅₀) as low as 1.2 nM (Figure 1b), surpassing most conventional monoclonal antibodies. Microscale thermophoresis (MST) experiments showed a dissociation constant (K_D) of approximately 14 pM with prefusion gB, with no binding to postfusion gB, indicating high prefusion specificity. Due to the highly conserved gB epitopes between HSV-1 and HSV-2, fluorescence colocalization experiments confirmed that Nb1_gbHSV binds gB of both viruses but does not recognize gB from varicella-zoster virus (VZV) or human cytomegalovirus (HCMV), demonstrating cross-species recognition capability.

Figure 1. Generation and target stabilization of anti-gB nanobody

To capture the prefusion conformation of gB, the team designed multiple stabilizing mutations based on prior knowledge of the HSV-1 gB model. These included disulfide bond mutations to lock adjacent domains in the prefusion conformation and helix-stabilizing mutations to enhance hydrophobic interactions and prevent conformational rearrangement. Using cryo-EM, they successfully resolved the prefusion gB structure at 2.74 Å resolution. Compared to the postfusion conformation (PDB 5V2S), three key rearrangement differences were identified: (1) N-terminal residues 90-106 form a helix inserted into the DI-DII groove of the adjacent protomer, stabilized by hydrophobic interactions and salt bridges; (2) the central helix (αC) in DIII (residues 501-510) is loop-like in the prefusion state but extends into a long helix postfusion, while the DII αX helix stands upright in prefusion to avoid steric clashes and lies flat postfusion; (3) the DI fusion loop "curls" into a short 3₁₀ helix in prefusion (binding the MPR hydrophobic groove with hydrogen bonds) but fully extends postfusion, a transition critical for initiating membrane fusion.

Figure 2. Full-length prefusion structure of gB

Cryo-EM analysis of the Nb1_gbHSV-prefusion gB complex (3 Å resolution) revealed that Nb1_gbHSV binds a cross-domain epitope, simultaneously interacting with gB's domain IV (DIV), domain I (DI) of the adjacent protomer, and domain III (DIII), covering a buried surface area of 1260 Ų and forming 23 hydrogen bonds and 8 salt bridges. Since DI and DIV are adjacent in the prefusion conformation but separate postfusion, Nb1_gbHSV’s cross-domain binding causes severe steric clashes with the postfusion conformation, locking gB in the prefusion state and blocking the conformational rearrangement required for membrane fusion, clearly elucidating its neutralization mechanism. Additionally, grating-coupled interferometry identified three "prefusion-specific but non-neutralizing" nanobodies that only bind the αX helix at the gB apex, proving that "prefusion specificity" does not equate to "neutralizing activity". Neutralization requires both high affinity and binding to critical cross-domain epitopes.

Figure 3. Nb1_gbHSV binding to gB conformation

To validate the universality of the neutralization mechanism, the team co-expressed Nb1_gbHSV with wild-type HSV-2 gB, purified the protein from stably transduced HEK 293T cells, and resolved the prefusion (2.85 Å) and postfusion (2.26 Å) structures of HSV-2 gB using cryo-EM. The results showed that the prefusion conformation of HSV-2 gB is highly similar to HSV-1, with consistent features such as the N-terminal helix (residues 90-106) interaction with DII and the upright arrangement of the αX helix. Nb1_gbHSV's binding mode to HSV-2 gB is fully conserved, with binding signals detected only in the prefusion conformation, confirming that Nb1_gbHSV locks wild-type HSV-2 gB in the prefusion state. This further validates the universality of the "conformational locking" neutralization mechanism and provides structural and mechanistic support for preventing HSV-2 infections.

Figure 4. HSV-2 gB bound by Nb1_gbHSV

The gB protein is an "indispensable" target for herpesviruses, as functional mutations lead to viral inactivation. The cross-domain epitope in the prefusion conformation (e.g., the domain I–domain III–domain IV region recognized by Nb1_gbHSV) is a critical site for neutralizing interventions, unlikely to develop resistance due to viral mutations, making it highly valuable for research and applications. Nb1_gbHSV is currently the most promising gB-targeting tool: in basic research, it serves as a "conformational probe" to capture prefusion gB on live viruses or cell surfaces, aiding studies of viral invasion dynamics; in clinical translation, its small molecular weight (~15 kDa) and strong tissue penetration, combined with potential for genetic engineering to enhance half-life or multivalent binding, further boost its neutralizing activity, offering broad future applications.

abinScience, founded in Strasbourg, France, leverages the region's exceptional research and innovation ecosystem to focus on developing and producing high-quality life science reagents. Committed to its vision of  "Empowering Bioscience Discovery," abinScience provides efficient and reliable experimental solutions to researchers worldwide, driving cutting-edge life science research. The high-performing Nb1_gbHSV featured in this study is available as a targeted product from abinScience, serving as a ready-to-use research tool to advance studies on HSV invasion mechanisms, antiviral drug screening, and prefusion gB-targeted vaccine development, providing robust support for herpesvirus-related research.

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