HSV2 gB Antibody

What is the immunological significance of HSV2 gB in the context of natural infection?

Glycoprotein B (gB) is one of the primary targets of the humoral immune response during HSV-2 infection. Research shows that gB, along with glycoproteins gD and gC, is recognized by sera from all HSV-seropositive individuals, although the intensity of response varies between patients . gB is an integral component of the multicomponent fusion system required for virus entry and cell-cell fusion . The antibody response against gB specifically contributes to virus neutralization, although surprisingly, studies have shown that only some individuals develop strong neutralizing antibodies targeting gB epitopes. In a study examining the antibody repertoire of HSV-seropositive individuals, only three out of ten samples contained neutralizing IgGs to gB epitopes, suggesting potential variability in how the immune system targets this protein during natural infection .

What methodologies are most effective for detecting and quantifying HSV2 gB-specific antibodies?

Several methodologies have proven effective for detecting and quantifying HSV2 gB-specific antibodies, each with specific applications in research settings:

• Western blotting with purified glycoproteins: This approach allows detection of antibodies against denatured epitopes of gB from both HSV-1 and HSV-2. Researchers typically use purified recombinant gB or viral lysates separated by SDS-PAGE .

• Biosensor competition assays using surface plasmon resonance: This sophisticated technique determines whether and to what extent human antibodies compete with characterized monoclonal antibodies for binding to specific epitopes on gB. The approach involves capturing soluble gB on a biosensor chip, flowing human IgG samples across the chip surface, and then measuring binding of specific monoclonal antibodies .

• Affinity chromatography: For isolation and quantification of gB-specific antibodies, tandem affinity chromatography using immobilized recombinant gB allows researchers to separate gB-specific IgGs from total serum IgGs, enabling determination of their specific contribution to neutralization .

• ELISA-based assays: While not explicitly detailed in the provided materials, ELISA represents a standard approach for quantifying antibody titers against specific viral proteins.

How does antibody valency impact the neutralization capacity of anti-gB antibodies?

The valency of anti-gB antibodies significantly influences their neutralization capacity. Research with the gB-specific monoclonal antibody MAb 2c has demonstrated that neutralization is critically dependent on cross-linkage of gB trimers, which can only be accomplished by bivalent antibody formats .
In detailed studies comparing monovalent formats (Fab and scFv) with bivalent formats (IgG and F(ab')2) of MAb 2c, researchers found that only the bivalent derivatives exhibited high neutralizing activity in vitro . This finding suggests that the mechanism of neutralization involves structural stabilization or interference with conformational changes in gB trimers rather than simple epitope blocking. The requirement for bivalency indicates that effective antibodies may function by cross-linking adjacent gB trimers or by bridging domains within a single trimer, preventing the protein from undergoing conformational changes necessary for the fusion process.
This mechanistic insight has important implications for vaccine design, as it suggests that eliciting antibodies capable of similar cross-linking effects may be crucial for protective immunity.

What is the relationship between epitope targeting on gB and neutralization effectiveness?

The relationship between epitope targeting on gB and neutralization effectiveness is complex and depends on several factors including epitope accessibility, conservation, and functional relevance. Analysis of the HSV-1 gB crystal structure in relation to neutralizing antibodies reveals that some key neutralizing epitopes are only partially accessible within the observed multidomain trimer conformation of gB, which likely represents its postfusion conformation .
Mapping studies have shown that certain epitopes are more effective targets for neutralization than others. For example, the discontinuous epitope recognized by MAb 2c is only partially accessible within the observed multidomain trimer conformation of gB . Nevertheless, antibodies targeting this region demonstrate potent neutralizing activity when in bivalent format, suggesting that they may interact with gB at stages during the fusion process when this region becomes more accessible.
Studies using biosensor competition assays with well-characterized neutralizing mouse monoclonal antibodies have also shown that during natural infection, not all neutralizing epitopes on gB induce consistent human antibody responses . This variability in epitope targeting may partially explain differences in neutralizing capacity observed among individuals.

How do fusion constructs of gB with immune modulators enhance vaccine efficacy?

Fusion constructs combining gB with immune modulators represent an innovative approach to enhance vaccine efficacy by directing the immunogen to responsive immune cells and modulating the immune response. A notable example is the HSV-2 gB-CCL19 fusion construct, which demonstrates several advantages over traditional approaches:

• Enhanced antibody production: gB-CCL19 fusion constructs induce stronger gB-specific IgG and IgA responses in both sera and vaginal fluids compared to gB alone .

• Improved neutralizing activity: The antibodies elicited by these fusion constructs show increased neutralizing activity against HSV-2 in vitro .

• Balanced immune response: gB-CCL19 fusion constructs induce balanced Th1 and Th2 cellular immune responses, which may be optimal for controlling HSV-2 infection .

• Improved protection: Mice vaccinated with the fusion constructs demonstrated superior protection from intravaginal lethal challenge with HSV-2 .

• Enhanced mucosal immunity: Compared to co-administration of gB and CCL19, fusion constructs increased mucosal surface IgA+ cells, as well as CCL19-responsive immunocytes in spleen and mesenteric lymph nodes .
  The mechanistic basis for these improvements likely relates to the ability of CCL19 to direct the immunogen to CCL19-responsive cells, improving the efficiency of antigen presentation and subsequent immune response development. This approach may be particularly valuable for developing vaccines against mucosal infections like HSV-2.

What are the optimal methods for purifying and characterizing recombinant HSV2 gB for immunological studies?

The purification and characterization of recombinant HSV2 gB for immunological studies requires careful consideration of protein structure, folding, and epitope preservation. Based on established methodologies, the following approach is recommended:

• Expression system selection: Baculovirus-infected insect cells have proven effective for expressing recombinant HSV gB. For example, a truncated form of gB consisting of amino acids 31-724 with a histidine tag (gB(724tHis)) has been successfully expressed and purified from this system .

• Protein engineering considerations:

  • Truncation at residue 724 or 730 removes the transmembrane domain while preserving the extracellular portion containing major antigenic sites

  • Addition of a histidine tag facilitates purification via metal affinity chromatography

  • Engineering a GCN4-based isoleucine zipper motif can promote proper oligomerization

• Purification protocol:

  • Metal affinity chromatography using the histidine tag

  • Size exclusion chromatography to ensure homogeneity and proper oligomerization

  • Verification of purity by SDS-PAGE and Western blotting

• Characterization methods:

  • Circular dichroism to assess secondary structure

  • Western blotting with conformation-specific antibodies to confirm proper folding

  • ELISA with a panel of monoclonal antibodies to verify preservation of key epitopes

  • Limited proteolysis, such as chymotrypsin digestion, which can provide information about domain structure and accessibility
    For detailed epitope mapping studies, chymotrypsin digestion can be particularly informative. Research has shown that chymotrypsin cleaves gB between residues Leu97/Arg98 and Gln472/Ser473, effectively removing residues Ala31 to Leu97 and yielding a stable homogeneous product that can be further analyzed .

How can biosensor competition assays be optimized for mapping HSV2 gB antibody epitopes?

Biosensor competition assays using surface plasmon resonance (SPR) represent a powerful approach for mapping HSV2 gB antibody epitopes. Based on published methodologies, the following optimization strategies are recommended:

• Immobilization strategy:

  • Capture recombinant gB on the biosensor chip via its histidine tag using an anti-histidine antibody or NTA surface

  • This orientation-specific immobilization helps preserve native epitope presentation

  • Avoid direct covalent coupling which may alter protein conformation or block epitopes

• Experimental design:

  • Use a parallel flow design with test and control surfaces

  • Capture soluble gB on the test surface

  • Expose the test surface to human IgG samples to allow binding of gB-specific antibodies

  • In parallel, expose a control surface containing gB to buffer alone

  • Flow characterized monoclonal antibodies with known epitopes across both surfaces

• Data analysis:

  • Calculate percent inhibition of monoclonal antibody binding by comparing test surface (pre-exposed to human IgG) with control surface

  • Reduced MAb binding indicates the presence of human antibodies targeting the same or overlapping epitope

  • Establish threshold values for positive competition based on signal-to-noise ratios

• Validation controls:

  • Include non-competing antibody pairs to confirm specificity

  • Use dose-response experiments with purified antibodies to establish competition sensitivity

  • Include mouse MAbs that target distinct, non-overlapping epitopes as specificity controls
    This approach allows researchers to determine whether and to what extent the binding of characterized monoclonal antibodies to gB is diminished by preincubation with human IgG samples, providing a map of the epitopes targeted during natural infection or following vaccination.

What are the key considerations for designing in vivo experiments to evaluate gB-based vaccine candidates?

Designing rigorous in vivo experiments to evaluate gB-based vaccine candidates requires careful consideration of multiple factors to ensure reliable, translatable results:

• Animal model selection:

  • C57BL/6 or BALB/c mice are commonly used for initial immunogenicity and protection studies

  • SCID mice should be included for safety evaluation of attenuated vaccines

  • Consider Fcγ-receptor or neonatal Fc-receptor knock-out mice to investigate the mechanism of antibody-mediated protection

• Immunization protocol:

  • Route: Subcutaneous immunization has been effective for gB-based vaccines

  • Schedule: Prime-boost regimens with 2-3 immunizations at 2-3 week intervals

  • Dose: Titrate vaccine dose to determine minimum effective concentration

  • Controls: Include appropriate controls (adjuvant alone, irrelevant antigen, positive control)

• Challenge model:

  • Route: Intravaginal or skin challenge models are most relevant for HSV-2

  • Dose: Determine challenge dose through titration studies (typically 10-100x LD50)

  • Timing: Challenge 3-4 weeks post-final immunization to assess peak immunity

• Endpoints and evaluation criteria:

  • Clinical scoring: Monitor animals daily for disease signs (lesions, neurological symptoms)

  • Viral shedding: Collect vaginal swabs for viral quantification

  • Latency: Examine dorsal root ganglia for viral DNA to assess protection against latent infection

  • Immune correlates: Collect serum and mucosal samples for antibody and T-cell response analysis

• Mechanistic studies:

  • Passive transfer experiments with immune serum to demonstrate antibody-mediated protection

  • Depletion of specific immune cell populations to assess contribution of cellular immunity

  • Ex vivo neutralization assays to correlate in vitro activity with in vivo protection

• Ethical considerations:

  • Implement humane endpoints based on clinical scoring systems

  • Ensure proper approval from institutional animal care and use committees

  • Follow the 3Rs principle (Replacement, Reduction, Refinement)
    These considerations will help ensure that experiments generate robust, reproducible data that can guide the development of effective HSV-2 vaccines.

How should researchers interpret discrepancies between in vitro neutralization and in vivo protection for anti-gB antibodies?

Discrepancies between in vitro neutralization and in vivo protection for anti-gB antibodies represent a complex challenge in HSV-2 research. Several key considerations can help researchers interpret these differences:

• Fc-mediated mechanisms: Research shows that non-neutralizing antibodies can provide complete protection through Fc-mediated mechanisms. Studies with HSV-2 vaccines deleted in glycoprotein D demonstrated that protection was lost when immune serum was transferred to Fcγ receptor and FcRn knockout mice, despite minimal neutralizing activity in vitro . This suggests that:

  • Antibody-dependent cellular cytotoxicity (ADCC)

  • Complement-dependent cytotoxicity

  • Fc-mediated phagocytosis
    May be critical in vivo mechanisms not captured by standard neutralization assays.

• Epitope accessibility differences: Some epitopes may be more accessible in vivo than in the virion preparations used for in vitro assays. For example, the discontinuous epitope recognized by the potent neutralizing MAb 2c is only partially accessible within the observed multidomain trimer conformation of gB, likely representing its postfusion conformation . In vivo, this epitope may become more accessible during the dynamic fusion process.

• Mucosal vs. serum antibodies: Standard neutralization assays typically use serum antibodies, but protection at mucosal surfaces may depend on locally produced IgA with different functional properties. Studies with gB-CCL19 fusion constructs showed enhanced mucosal IgA production that correlated with protection .

• Antibody valency effects: The neutralization capacity of antibodies like MAb 2c is dependent on cross-linkage of gB trimers, with bivalent formats showing significantly higher neutralizing activity than monovalent formats . Standard neutralization assays may not fully capture these valency-dependent effects that are relevant in vivo.
  To address these discrepancies, researchers should:

• Incorporate multiple assays beyond standard neutralization, including ADCC and complement fixation

• Evaluate protection in relevant animal models with intact immune systems

• Consider passive transfer experiments to determine the protective capacity of antibodies in vivo

• Examine mucosal antibody responses separately from systemic responses

What approaches can be used to determine the relative contribution of anti-gB antibodies versus other glycoprotein-specific antibodies in polyclonal serum?

Determining the relative contribution of anti-gB antibodies versus other glycoprotein-specific antibodies in polyclonal serum requires sophisticated methods to isolate and functionally characterize specific antibody populations. Based on published methodologies, the following approaches are recommended:

• Tandem affinity chromatography separation:

  • Sequential affinity chromatography using immobilized recombinant glycoproteins (e.g., gD followed by gB) can be used to fractionate serum IgGs into glycoprotein-specific populations

  • This approach has been successfully used to isolate gD-specific and gB-specific IgGs from human sera

  • The isolated antibody fractions can then be tested individually for neutralizing activity

• Quantitative depletion studies:

  • Deplete specific antibody populations using immobilized recombinant glycoproteins

  • Compare neutralizing activity of depleted versus non-depleted serum

  • The reduction in neutralizing activity represents the contribution of the depleted antibody specificity

• Competitive inhibition assays:

  • Pre-incubate virus with excess recombinant glycoproteins to block binding of specific antibody populations

  • Compare neutralizing activity with and without competitive inhibition

  • The reduction in neutralizing activity indicates the contribution of antibodies targeting the competing glycoprotein

• Western blot analysis with glycoprotein-specific fractions:

  • Test glycoprotein-specific antibody fractions against Western blots of purified individual glycoproteins

  • This confirms the specificity of the fractionated antibodies and can reveal cross-reactivity

• Biosensor epitope mapping:

  • Use surface plasmon resonance to map which epitopes on each glycoprotein are targeted by the serum

  • This provides information on both the breadth and focus of the antibody response
    A study examining the IgGs from HSV-seropositive individuals demonstrated the application of these approaches by showing that in some individuals, gD and gB together accounted for all neutralizing activity against HSV-2, with some HSV-1 neutralization directed against gC . This type of comprehensive analysis provides important insights into the protective antibody response against HSV glycoproteins.

How do differences in gB sequence and structure between HSV-1 and HSV-2 impact cross-reactive antibody responses?

The differences in gB sequence and structure between HSV-1 and HSV-2 have significant implications for cross-reactive antibody responses, which is critical for understanding cross-protection and developing broadly protective vaccines. Although the search results don't provide comprehensive details on this specific topic, we can infer several important considerations based on the available information:

• Test neutralization against both HSV-1 and HSV-2

• Use recombinant glycoproteins from both virus types in binding and competition assays

• Consider structural modeling to identify conserved and variable epitopes

• Examine whether antibodies that bind both HSV-1 and HSV-2 gB have similar functional activities against both viruses

How does deletion or modification of other viral glycoproteins affect the immune response to gB?

The deletion or modification of other viral glycoproteins can significantly alter the immune response to gB, potentially unmasking protective epitopes or changing the immunodominance hierarchy. The most striking example comes from studies of HSV-2 virus deleted in glycoprotein D (ΔgD−/+gD1):

• Unmasking of protective antigens: Deletion of gD, which is immunodominant in wild-type HSV infection, appears to unmask viral antigens important in generating a protective humoral response . Western blot analysis showed that the predominant antigen recognized by control-immunized, HSV-2-challenged mice was gD, while ΔgD−/+gD1-vaccinated mice developed stronger responses to other antigens, including gB .

• Shift in antibody functionality: The antibodies elicited by ΔgD−/+gD1 vaccination showed little neutralizing activity but demonstrated strong antibody-dependent cellular cytotoxicity (ADCC) . This suggests that modification of the viral glycoprotein composition can fundamentally alter the functional profile of the antibody response.

• Change in protective mechanisms: Protection conferred by ΔgD−/+gD1 vaccination was dependent on Fc-mediated antibody functions rather than direct neutralization. This protection was lost when immune serum was transferred to FcγR and FcRn knock-out mice, highlighting the critical role of Fc receptor interactions .

• Potential immunomodulatory effects: The deletion of gD may alter immune responses through mechanisms beyond simple antigen unmasking. gD interacts with herpesvirus entry mediator (HVEM) on immune cells, and this interaction may skew the immune response . In the absence of the gD-HVEM interaction, the host may mount a polyantigenic IgG2-dominant response capable of eliciting FcR-mediated protection.
  These findings suggest that strategic modification of viral glycoproteins could be a promising approach for developing more effective HSV vaccines. By altering the composition or expression levels of specific glycoproteins, it may be possible to direct the immune response toward more protective targets or functional mechanisms.

What role do non-neutralizing anti-gB antibodies play in protection against HSV-2 infection?

Non-neutralizing anti-gB antibodies appear to play a significant role in protection against HSV-2 infection through Fc-mediated effector functions. This represents a paradigm shift from the traditional focus on neutralizing antibodies as the primary correlate of protection. Evidence for the protective role of non-neutralizing antibodies includes:

• Fc receptor dependency: Studies with the HSV-2 ΔgD−/+gD1 vaccine demonstrated that protection was mediated by antibodies with little neutralizing activity but was completely dependent on Fc receptors. Protection was lost when immune serum was transferred to FcγR knock-out mice, despite the presence of HSV-specific antibodies in vaginal washes .

• Antibody-dependent cellular cytotoxicity (ADCC): Serum from mice immunized with ΔgD−/+gD1 elicited cell-mediated cytotoxicity, suggesting that ADCC is an important protective mechanism . This function can be mediated by non-neutralizing antibodies that recognize viral antigens on the surface of infected cells.

• FcRn-mediated transport: The neonatal Fc receptor (FcRn) plays a crucial role in transporting antibodies from serum to mucosal surfaces. Protection was lost when immune serum was transferred to FcRn knock-out mice, and HSV-specific antibodies were not detected in vaginal washes of these mice . This highlights the importance of mucosal antibody delivery for protection.

• Complement activation: While not explicitly discussed in the provided materials, complement activation represents another mechanism by which non-neutralizing antibodies can contribute to protection.
  These findings suggest that vaccine strategies targeting gB should aim to elicit not only neutralizing antibodies but also antibodies capable of mediating efficient Fc-dependent effector functions. This might be achieved through appropriate adjuvant selection, antigen formulation, or vaccine delivery strategies that promote particular antibody isotypes or subclasses associated with enhanced effector functions.
  The protective role of non-neutralizing antibodies also has implications for evaluating vaccine candidates, suggesting that standard neutralization assays alone may not predict protective efficacy and should be complemented with assays measuring Fc-mediated functions.

What are the challenges in developing standardized assays for measuring anti-gB antibody functionality?

Developing standardized assays for measuring anti-gB antibody functionality presents several technical challenges that researchers must address to ensure reliable and comparable results across different laboratories:

• Neutralization assay variability:

  • Cell type differences: Neutralization efficiency can vary depending on the cell line used

  • Virus strain differences: HSV-1 and HSV-2 strains may show variable susceptibility to neutralization

  • Assay format: Plaque reduction versus colorimetric/fluorometric readouts may yield different results

  • Incubation conditions: Temperature, time, and medium composition can affect outcomes

• Beyond neutralization functionality:

  • ADCC assays require appropriate effector cells and target cells expressing physiologically relevant levels of viral antigens

  • Complement-dependent cytotoxicity assays need standardized complement sources

  • Fc-mediated phagocytosis assays require consistent phagocytic cell preparation

• Epitope accessibility issues:

  • Key neutralizing epitopes on gB may be only partially accessible in the postfusion conformation observed in the crystal structure

  • Different assay conditions may affect protein conformation and epitope exposure

  • Antibodies may recognize epitopes that are only transiently exposed during the fusion process

• Recombinant protein quality:

  • Variation in recombinant gB preparation (truncation sites, tags, expression systems) affects epitope presentation

  • Protein aggregation or misfolding can expose non-native epitopes

  • Post-translational modifications may differ between expression systems
    Recommended solutions:

• Standardized reagents:

  • Develop and distribute reference antibodies with defined specificities and activities

  • Establish common recombinant protein preparations with defined characteristics

  • Create standardized virus stocks for neutralization assays

• Comprehensive assay panels:

  • Implement multiple complementary assays measuring different aspects of antibody functionality

  • Include both neutralization and Fc-mediated function assays

  • Validate assays with antibodies of known functionality

• Detailed protocols:

  • Publish comprehensive methodologies including all critical parameters

  • Conduct inter-laboratory comparison studies to identify and address variables affecting reproducibility

  • Establish accepted positive and negative controls for each assay

• Data normalization approaches:

  • Develop statistical methods to compare results across different assay formats

  • Establish reporting standards that include all relevant experimental parameters

  • Create conversion factors to allow comparison between different assay readouts
    By addressing these challenges, researchers can develop more reliable and comparable methods for measuring the diverse functional activities of anti-gB antibodies, facilitating more effective vaccine development and evaluation.

How can researchers effectively distinguish between type-common and type-specific antibody responses to HSV gB?

Distinguishing between type-common and type-specific antibody responses to HSV gB requires systematic approaches that can identify antibodies recognizing conserved versus variable epitopes. The following methodological approaches are recommended:

• Comparative binding assays:

  • ELISA using purified recombinant gB from both HSV-1 and HSV-2

  • Compare binding curves and calculate relative affinities

  • Antibodies with similar binding to both proteins likely target conserved epitopes

  • Western blotting against purified glycoproteins from both serotypes can reveal type-specific recognition patterns

• Cross-neutralization testing:

  • Test antibody samples against both HSV-1 and HSV-2 in neutralization assays

  • Calculate neutralization titers against each virus type

  • Compare neutralization efficiency to identify type-biased versus broadly neutralizing antibodies

  • Studies have shown that some human sera contain antibodies that neutralize both HSV-1 and HSV-2, while others show type-preference

• Epitope mapping strategies:

  • Biosensor competition assays using well-characterized type-common and type-specific monoclonal antibodies

  • Peptide mapping with overlapping peptides covering variable regions of gB

  • Domain-swapping experiments with chimeric gB proteins containing domains from HSV-1 and HSV-2

  • Chymotrypsin digestion of gB followed by epitope mapping can provide information about domain-specific recognition

• Absorption/depletion studies:

  • Sequential absorption with recombinant gB from one virus type followed by testing reactivity against the other type

  • Depletion with type-specific peptides to remove antibodies targeting variable regions

  • Analysis of the remaining antibody fraction for cross-reactivity

• Structural analysis:

  • Computational mapping of conserved and variable regions on the gB structure

  • Correlation of antibody binding sites with these regions

  • Prediction of accessible epitopes that might elicit type-common versus type-specific responses
    These approaches can be combined to provide a comprehensive analysis of the type-specificity of anti-gB antibody responses, which is crucial for understanding cross-protection and developing broadly protective vaccines. The data obtained can be presented in tables comparing binding and neutralization against both virus types, and mapping results can be visualized on structural models highlighting conserved and variable epitopes.

What are the prospects for developing gB-based universal vaccines against multiple herpesvirus infections?

The development of gB-based universal vaccines against multiple herpesvirus infections represents an ambitious but potentially achievable goal, given certain characteristics of this glycoprotein across the herpesvirus family:

• Structural and functional conservation:

  • gB is one of the most conserved glycoproteins across the herpesvirus family

  • It plays a critical role in the fusion mechanism required for virus entry across all herpesviruses

  • The core fusion machinery likely maintains similar structural features despite sequence divergence

• Evidence for cross-reactive immunity:

  • Some studies have demonstrated cross-reactivity between antibodies targeting gB from different herpesviruses

  • The success of HSV-2 gB-based vaccines in providing protection against both HSV-1 and HSV-2 challenges suggests potential for broader protection

  • The identification of conserved neutralizing epitopes could form the basis for universal vaccine design

• Strategies for enhancing cross-protection:

  • Structure-based design of immunogens focusing on conserved regions of gB

  • Multivalent constructs incorporating gB from multiple herpesviruses

  • Fusion with immune modulators like CCL19 to enhance both local and systemic immunity

  • Deletion of immunodominant variable regions to focus the immune response on conserved epitopes

• Challenges to overcome:

  • Sequence divergence between gB from distant herpesvirus family members

  • Different tissue tropism and transmission routes of various herpesviruses

  • Potential for type-specific neutralizing epitopes dominating the immune response

  • Need for different immune effector mechanisms for controlling different herpesviruses

• Innovative approaches:

  • Computational design of "consensus" gB sequences representing conserved features

  • Structure-based engineering of stabilized prefusion conformations to expose conserved neutralizing epitopes

  • Nanoparticle presentation of multiple conserved epitopes from different herpesvirus gB proteins

  • Combination with other conserved herpesvirus antigens to broaden protection
    The successful development of gB-CCL19 fusion constructs that provide protection against HSV-2 challenge and the demonstration that antibody-mediated protection can occur through non-neutralizing Fc-mediated mechanisms provide promising platforms upon which to build universal herpesvirus vaccine strategies. Future research should focus on identifying the most conserved functional epitopes across herpesvirus gB proteins and developing immunization strategies that elicit antibodies targeting these regions.

How might emerging technologies in antibody engineering and analysis advance HSV2 gB research?

Emerging technologies in antibody engineering and analysis have the potential to significantly advance HSV2 gB research, offering new tools for understanding antibody responses and developing therapeutic approaches:

• Single B cell analysis and antibody repertoire sequencing:

  • Next-generation sequencing of B cell receptors from HSV-infected or vaccinated individuals

  • Identification of expanded B cell clones targeting specific gB epitopes

  • Tracking of antibody maturation pathways to understand how protective responses develop

  • Comparison of repertoires between individuals with different levels of protection

• High-throughput antibody isolation and characterization:

  • Single-cell sorting of gB-specific B cells followed by antibody gene cloning

  • Microfluidic platforms for rapid screening of antibody binding and neutralization

  • Deep mutational scanning to map critical residues for antibody recognition of gB

  • Creation of comprehensive panels of monoclonal antibodies targeting different gB epitopes

• Advanced structural techniques:

  • Cryo-electron microscopy to visualize gB-antibody complexes in different conformational states

  • X-ray crystallography of gB bound to novel neutralizing antibodies

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes induced by antibody binding

  • Computational modeling of antibody-antigen interactions to guide vaccine design

• Antibody engineering applications:

  • Creation of bispecific antibodies targeting multiple epitopes on gB or multiple glycoproteins

  • Fc engineering to enhance ADCC, complement activation, or tissue penetration

  • Development of antibody-drug conjugates for targeted delivery to HSV-infected cells

  • Engineering of antibodies with extended half-lives for prophylactic applications

• In vivo antibody imaging:

  • Labeled antibodies for tracking biodistribution and tissue penetration

  • Intravital microscopy to visualize antibody interactions with infected cells

  • PET imaging with radiolabeled antibodies to monitor infection sites and antibody targeting

• Novel therapeutic modalities:

  • Antibody-based CAR-T cells targeting gB-expressing infected cells

  • mRNA-encoded antibodies for direct in vivo expression

  • Nanobodies and alternative scaffold proteins with enhanced tissue penetration These technologies will facilitate a more comprehensive understanding of protective anti-gB antibody responses and enable the development of novel therapeutic and prophylactic approaches. The ability to rapidly isolate and characterize potent neutralizing antibodies from infected individuals could lead to new passive immunotherapy options, while structural insights gained from antibody-antigen complexes will inform rational vaccine design strategies.