HSV-2 gB

What is the role of glycoprotein B in HSV-2 infection?

Glycoprotein B (gB) is one of the essential HSV entry glycoproteins required for viral infection. Together with glycoproteins gD and gH/gL, gB serves as a major stimulus for virus-neutralizing antibodies . As a class III fusion protein, gB mediates the fusion between the viral envelope and host cell membrane, a critical step in HSV-2 infection. Research indicates that gB undergoes significant conformational changes during the fusion process, working in concert with other viral glycoproteins to facilitate viral entry into host cells.

What functional domains have been identified in HSV-2 gB?

HSV-2 gB contains several functional domains essential for its role in viral entry:

• Fusion domains that directly interact with host cell membranes

• Regulatory domains that respond to signals from other viral glycoproteins

• Epitope regions recognized by neutralizing antibodies

• Transmembrane domains that anchor the protein in the viral envelope

Each domain plays specific roles in the fusion mechanism and viral entry process, making them potential targets for therapeutic intervention.

What are the most effective methods for isolating and characterizing gB-specific antibodies from human serum?

Based on current research, several effective methods have been established:

These methods allow researchers to comprehensively characterize the anti-gB antibody response in human subjects.

How can researchers effectively express and purify HSV-2 gB for structural and functional studies?

Expressing and purifying HSV-2 gB while maintaining its native conformation requires careful consideration of:

• Expression systems:

  • Mammalian expression systems (HEK293 or CHO cells) often provide proper folding and post-translational modifications

  • Baculovirus expression systems in insect cells can yield higher quantities

  • Bacterial expression systems may be useful for domain-specific studies but lack glycosylation

• Construct design:

  • Truncation strategies (removing transmembrane domains) can improve solubility

  • Addition of affinity tags facilitates purification

  • Codon optimization for the chosen expression system improves yield

• Purification strategy:

  • Multi-step chromatography including affinity, ion-exchange, and size-exclusion

  • Quality control using SDS-PAGE, Western blotting, and functional assays

These methodological considerations are critical for obtaining properly folded gB for downstream applications.

What assays best measure the neutralizing capacity of anti-gB antibodies?

To accurately assess the neutralizing capacity of anti-gB antibodies, researchers employ several complementary approaches:

• Standard plaque reduction neutralization tests (PRNT) with wild-type virus, comparing the neutralization potentials of total serum, gB-enriched, and gB-depleted fractions .

• Cell-cell fusion inhibition assays that specifically measure the ability of antibodies to block gB-mediated membrane fusion.

• Fc-dependent functional assays, as research has shown that "non-neutralizing Fc-mediated humoral responses confer protection" . This includes:

  • Antibody-dependent cellular cytotoxicity (ADCC) assays

  • Complement-dependent cytotoxicity assays

  • FcγR reporter assays

• In vivo protection studies in animal models comparing passive transfer of gB-specific versus other glycoprotein-specific antibodies.

The combination of these assays provides a comprehensive profile of antibody functionality beyond direct neutralization.

Why does HSV-2 gB appear less immunodominant than gD in human immune responses?

Research indicates that gB is typically less immunodominant than gD in natural HSV infection. This observation is supported by several findings:

• In studies of human sera, all samples contained neutralizing IgGs to gD epitopes, but "surprisingly, only three samples contained neutralizing IgGs to gB epitopes" .

• When analyzing serum from HSV-2-infected individuals using Western blots, gD was the "predominant antigen recognized by control-immunized, HSV-2-challenged" subjects, suggesting its immunodominance in natural infection .

• The deletion of gD in experimental vaccines "unmasks viral antigens important in generating a protective humoral response," including gB . This indicates that gD may normally dominate the immune response at the expense of responses to other glycoproteins.

This immunodominance pattern has significant implications for vaccine design, suggesting that strategies to shift immune focus from gD to other glycoproteins like gB may improve protective immunity.

What antibody characteristics correlate with effective neutralization of HSV-2 via gB targeting?

Research has identified several antibody characteristics that correlate with effective neutralization through gB targeting:

• Antibody subclass appears important, with protective immune sera "comprised predominantly of IgG2a and IgG2b" , suggesting these subclasses may have superior effector functions against HSV-2.

• Fc-receptor engagement is critical, as "antibody-mediated protection was lost when serum was transferred to FcγR and FcRn knock-out mice" , highlighting the importance of Fc-mediated effector functions.

• Epitope specificity matters, with antibodies targeting functional domains involved in fusion being most effective at neutralization.

• Cross-reactivity between HSV-1 and HSV-2 may be beneficial, as conserved epitopes often correspond to functionally critical regions of the protein.

These findings suggest that effective vaccines should elicit antibodies with specific characteristics rather than simply maximizing antibody titer.

How do mutations in HSV-2 gB affect viral fitness and immune evasion?

Mutations in HSV-2 gB can significantly impact viral fitness and immune evasion through several mechanisms:

Understanding these mutation effects is crucial for predicting vaccine efficacy and viral evolution under immune pressure.

How can structural biology approaches advance our understanding of HSV-2 gB to guide immunogen design?

Structural biology approaches offer several avenues to advance HSV-2 gB research:

• Cryo-electron microscopy to capture different conformational states of gB during the fusion process, which could reveal transiently exposed epitopes for targeting.

• X-ray crystallography of gB in complex with neutralizing antibodies to precisely map epitopes at the atomic level.

• Hydrogen-deuterium exchange mass spectrometry to identify flexible regions and conformational changes that occur during fusion.

• Computational modeling to predict how modifications might stabilize specific conformations that optimally present neutralizing epitopes.

These approaches could guide the design of improved immunogens that better present critical epitopes or stabilize gB in conformations that elicit more protective antibody responses.

What is the molecular mechanism of HSV-2 gB-mediated membrane fusion in concert with other glycoproteins?

The molecular mechanism of HSV-2 gB-mediated fusion involves a coordinated process with other viral glycoproteins:

• Sequential activation model: gD binding to receptors initiates conformational changes that activate gH/gL, which in turn triggers gB to execute membrane fusion .

• Conformational transitions: gB undergoes dramatic structural rearrangements from a metastable pre-fusion state to an extended intermediate and finally to a stable post-fusion conformation.

• Cooperative interactions: Direct contacts between gB and gH/gL are likely critical for transmitting activation signals between glycoproteins.

• Regulation by other viral components: Additional factors may modulate the fusion process to ensure it occurs at the right time and place.

Understanding this complex mechanism is essential for identifying new intervention points for antiviral development.

What are promising strategies for developing HSV-2 gB-based vaccines that might overcome limitations of previous approaches?

Several promising strategies emerge from recent research:

• Combination approaches: Rather than focusing solely on gD or gB, vaccines incorporating multiple glycoproteins may elicit broader protective responses. Evidence suggests that "deletion of gD, which is immunodominant...unmasks viral antigens important in generating a protective humoral response" .

• Focus on Fc-mediated protection: Research demonstrates that "non-neutralizing Fc-mediated humoral responses confer protection" , suggesting vaccines should be designed to elicit antibodies with strong effector functions rather than just neutralizing capacity.

• Structure-based design: Using structural information to stabilize gB in specific conformations might present neutralizing epitopes more effectively to the immune system.

• Altered immunodominance patterns: Strategies to shift immune focus from gD to gB and other glycoproteins could potentially elicit more diverse and protective responses.

• Adjuvant selection: Adjuvants that promote production of specific antibody subclasses (particularly IgG2a and IgG2b) may enhance protection based on findings that protective sera were "comprised predominantly of IgG2a and IgG2b" .

How can systems immunology approaches better characterize the diversity of human antibody responses to HSV-2 gB?

Systems immunology approaches offer powerful tools to comprehensively characterize human antibody responses:

• Single B-cell isolation and sequencing from HSV-infected individuals can reveal the full repertoire of gB-specific antibodies and their molecular characteristics.

• Deep serological profiling comparing high and low virus shedders (as mentioned in result ) can identify antibody signatures that correlate with better viral control.

• Epitope mapping at high resolution can determine which epitopes are consistently targeted across diverse human populations and correlate with protection.

• Longitudinal studies tracking antibody evolution from primary infection through recurrences can identify maturation patterns associated with improved viral control.

• Machine learning approaches integrating multiple antibody features (epitope specificity, isotype, glycosylation, etc.) can identify complex patterns associated with protection.

These approaches could substantially advance our understanding of protective immunity and guide more effective vaccine design.

What are the implications of HSV-2 gB research for developing therapeutics beyond preventive vaccines?

Research on HSV-2 gB has several implications for therapeutic development:

• Monoclonal antibody therapeutics targeting critical gB epitopes could provide passive protection or treat established infections, particularly in immunocompromised patients.

• Small molecule fusion inhibitors designed to block gB conformational changes could prevent viral entry and spread.

• Gene therapy approaches delivering genes encoding gB-specific antibodies could provide durable protection without requiring active immunization.

• Combination approaches targeting multiple steps in the viral entry process (involving gB, gD, and gH/gL) might provide synergistic effects and reduce the likelihood of resistance development.

• Immunomodulatory approaches to enhance existing gB-specific immune responses in infected individuals could potentially reduce recurrence frequency or severity.

These therapeutic directions represent promising avenues for translating fundamental research on HSV-2 gB into clinical applications.