HSV-2 gD (525-578)

What is the functional significance of glycoprotein D (gD) in HSV-2 infection?

Glycoprotein D (gD-2) serves as a critical viral envelope protein required for both HSV-2 entry into host cells and cell-to-cell spread during infection. Research demonstrates that gD-2 plays a fundamental role in viral attachment and fusion with host cell membranes through interactions with cellular receptors, including herpesvirus entry mediator (HVEM) . This protein is so essential that when deleted from the viral genome, the virus can only undergo a single round of replication when complemented with HSV-1 gD, making it highly attenuated .

Beyond its role in viral entry, gD-2 exhibits immunomodulatory functions through interactions with HVEM on immune cells, potentially skewing immune responses in ways that may limit protective immunity . This dual functionality makes gD-2 particularly significant for understanding HSV-2 pathogenesis and developing preventive strategies.

Methodologically, researchers can investigate gD functions through:

• Complementation studies using cell lines expressing gD (such as VD60 cells)

• Site-directed mutagenesis to identify functional domains

• Receptor binding assays to characterize interaction partners

• Viruses with deleted or modified gD regions to assess effects on viral replication and pathogenesis

How can researchers effectively study the specific HSV-2 gD (525-578) region?

To effectively study the HSV-2 gD (525-578) region, researchers should employ multiple complementary approaches:

• Sequence analysis: Compare this region across HSV strains and related viruses to identify evolutionary conservation, suggesting functional importance. Researchers should normalize extracted DNA to 10 ng per reaction when performing molecular analyses .

• Structure-function studies: Generate recombinant HSV-2 viruses with specific mutations or deletions in the 525-578 region using techniques similar to those used for creating the HSV-2 ΔgD−/+gD−1 virus . These viruses can be propagated on complementing cell lines like VD60 cells that express HSV-1 gD under control of the gD-1 promoter .

• Domain mapping: Create fusion proteins or peptides containing this specific region to test for receptor binding, fusion activity, or antibody recognition. Western blotting techniques can verify expression using specific antibodies .

• Functional assays: Assess how modifications to this region affect viral entry, cell-to-cell spread, and immune evasion. Plaque assays on Vero cells can quantify these effects, with titers reported as plaque-forming units (pfu) per milliliter .

• Immunological analysis: Determine whether this region contains important epitopes recognized by neutralizing or non-neutralizing antibodies using sera from infected or vaccinated subjects .

What experimental models are most appropriate for studying HSV-2 gD functions?

Based on research findings, several complementary experimental models are recommended for studying HSV-2 gD functions:

• In vitro cell culture systems:

  • VD60 cells expressing HSV-1 gD under the control of the gD-1 promoter for complementing and propagating gD-deleted HSV-2 (achieving titers of 10^8 pfu/ml)

  • Vero cells for assessing viral entry, replication, and plaque formation

  • Cell-based fusion assays for studying gD-mediated membrane fusion

• Mouse models:

  • Immunocompetent strains (C57BL/6 or BALB/c) for vaccination and challenge studies

  • Immunodeficient (SCID) mice for safety assessment of attenuated viruses

  • Fc receptor knockout mice to study antibody effector functions

  • Intravaginal challenge models using clinical isolates (such as HSV-2 strain 4674) to assess mucosal protection

  • Subcutaneous challenge models to evaluate systemic protection

• Ex vivo systems:

  • Dorsal root ganglia cultures for latency and reactivation studies, maintained for up to 21 days to assess viral reactivation

  • Tissue explants for studying viral spread in complex tissue environments

• Immune transfer studies:

  • Passive transfer of serum (250 μl containing 750 μg of total IgG) or purified antibodies to naive recipients

  • Adoptive transfer of T cells (3 × 10^6 cells) to delineate cellular immune contributions

What methods should be used to characterize the structure and function of HSV-2 gD (525-578)?

To thoroughly characterize the structure and function of HSV-2 gD (525-578), researchers should implement a multifaceted methodological approach:

• Structural analysis:

  • X-ray crystallography or cryo-electron microscopy to determine three-dimensional structure

  • Molecular modeling based on homologous proteins

  • Circular dichroism spectroscopy to assess secondary structure elements

  • Nuclear magnetic resonance (NMR) for dynamic structural information

• Protein-protein interaction studies:

  • Surface plasmon resonance to measure binding kinetics with potential receptors

  • Co-immunoprecipitation to identify interaction partners

  • ELISA-based binding assays using purified proteins or peptides

  • Yeast two-hybrid screening for novel interaction partners

• Functional assessment:

  • Site-directed mutagenesis of specific residues within the 525-578 region

  • Cell-cell fusion assays to assess fusion-triggering capacity

  • Viral entry assays using pseudotyped viruses

  • Competitive inhibition assays with peptides derived from this region

• Immunological characterization:

  • Epitope mapping using overlapping peptides or display libraries

  • Assessment of antibody recognition using sera from infected or vaccinated subjects (using ELISA techniques with cell lysates at 45 μg/well)

  • Evaluation of T cell responses to peptides from this region

  • Analysis of how mutations affect immune recognition and evasion

Why have subunit vaccines based on gD-2 failed in clinical trials despite promising preclinical results?

The failure of gD-2 subunit vaccines in clinical trials despite promising preclinical results presents a significant scientific contradiction that research has begun to address:

• Limited immune response profile: gD-2 subunit vaccines primarily elicited neutralizing antibodies and CD4+ T cell responses but failed to generate the broad, polyantigenic immune response necessary for protection . This narrow response profile may be insufficient against the complex pathogenesis of HSV-2 infection.

• Immunodominance masking protective antigens: Evidence suggests that gD-2 is immunodominant, potentially directing the immune response away from other viral antigens that might elicit more protective responses . The deletion of gD-2 in experimental vaccines unmasks these protective antigens, allowing for more effective immunity.

• Immunomodulatory effects of gD-HVEM interaction: The research indicates that interactions between gD and HVEM on immune cells may skew immune responses in counterproductive ways . Specifically, this interaction may favor the development of neutralizing antibodies rather than the Fc receptor-mediated antibody functions that appear more protective.

• Inadequate mucosal immunity: Clinical trials measured neutralizing antibodies in serum but not at mucosal sites . The most recent efficacy trial showed neutralizing antibody titers of 1:422 against HSV-1 and 1:97 against HSV-2, yet these did not correlate with protection against HSV-2 .

• Methodological implications: Future subunit vaccine designs should consider including multiple viral antigens beyond gD-2, specifically targeting epitopes that elicit antibodies with effective Fc-mediated functions rather than focusing solely on neutralizing activity .

What mechanistic insights have been gained from studying HSV-2 variants with deleted gD regions?

Research on HSV-2 variants with deleted gD regions has provided several critical mechanistic insights:

• Novel mechanism of protection: HSV-2 ΔgD−/+gD−1 immunization elicited antibodies that provided protection through Fc-mediated effector functions rather than neutralization . This protection was completely dependent on Fcγ receptors and neonatal Fc receptors, as demonstrated by passive transfer experiments in knockout mice .

• Safety through attenuation: Deletion of gD-2, which is required for viral entry and cell-to-cell spread, restricts the virus to a single round of replication . This creates a highly attenuated vaccine candidate that showed no disease in SCID mice even at doses 1000-fold higher than wild-type virus doses that proved lethal .

• Prevention of latency establishment: Unlike natural infection or other vaccine approaches, immunization with HSV-2 ΔgD−/+gD−1 prevented the establishment of latency following challenge . No HSV DNA was detected by qPCR in neural tissue (limit of detection: 3 HSV-2 genome copies), and no virus could be reactivated from dorsal root ganglia in ex vivo cultures maintained for 21 days .

• Unmasking of protective antigens: The deletion of immunodominant gD-2 appears to unmask other viral antigens important for generating protective immune responses . This represents a novel vaccine design principle that could be applied to other pathogens with immunodominant antigens that may misdirect the immune response.

• Altered antibody isotype profile: The gD-deleted virus likely elicits an IgG2-dominant antibody response capable of effective Fc receptor-mediated protection, contrasting with the response to wild-type virus or gD subunit vaccines .

How should researchers design experiments to evaluate novel HSV-2 vaccine candidates targeting gD?

Researchers designing experiments to evaluate novel HSV-2 vaccine candidates targeting gD should implement a comprehensive methodological approach:

• Immunogen design and characterization:

  • Generate constructs with specific modifications to gD (mutations, truncations, fusions)

  • Verify protein expression and folding using Western blot analysis with specific antibodies

  • Confirm antigenic integrity using monoclonal antibodies to key epitopes

  • For gD-deleted constructs, ensure complementation in appropriate cell lines like VD60

• Preclinical immunogenicity evaluation:

  • Use multiple mouse strains (e.g., C57BL/6 and BALB/c) to account for genetic variation

  • Implement prime-boost protocols (e.g., initial immunization followed by boost after 3 weeks)

  • Collect serum at multiple timepoints: 1 week post-prime, at boost, and 3 weeks post-boost

  • Sample mucosal sites (vaginal washes) to assess local immunity

  • Characterize antibody responses by:

    • Total HSV-specific antibody titers using ELISA with infected cell lysates

    • Antibody isotype analysis (IgG1, IgG2a, IgG2b, IgG3)

    • Functional assays including neutralization and ADCC

    • Western blot immune-target profiling to identify recognized antigens

• Challenge studies:

  • Challenge via clinically relevant routes (intravaginal for genital herpes)

  • Use clinical isolates (e.g., HSV-2 strain 4674) rather than laboratory strains

  • Test protection against different challenge doses (e.g., LD90 and 10× LD90)

  • Monitor:

    • Survival and disease symptoms using standardized scoring systems

    • Viral shedding in mucosal secretions over time

    • Viral titers in relevant tissues using plaque assays

    • Establishment of latency using qPCR and ex vivo reactivation assays

• Mechanistic studies:

  • Conduct passive transfer experiments with immune serum (250 μl containing 750 μg total IgG)

  • Test protection in receptor knockout mice (Fcγ-receptor, neonatal Fc-receptor)

  • Perform antibody depletion studies using protein L columns

  • Adoptive transfer of T cells (3 × 10^6 cells) to assess cellular immunity

What immunological parameters should be measured when assessing HSV-2 vaccine candidates targeting the gD protein?

When assessing HSV-2 vaccine candidates targeting the gD protein, researchers should measure a comprehensive set of immunological parameters:

• Antibody responses:

  • Quantity: Total HSV-specific antibody titers in serum (reported up to 1:800,000 for effective vaccines) and mucosal secretions using ELISA with infected cell lysates (45 μg/well)

  • Quality: Antibody isotype distribution (IgG1, IgG2a, IgG2b, IgG3) which correlates with Fc receptor binding properties

  • Specificity: Western blot immune-target profiling to identify which viral proteins are recognized

  • Functionality:

    • Neutralizing activity (traditional focus)

    • Antibody-dependent cellular cytotoxicity (ADCC) measured by flow cytometry using infected target cells and effector cells at 25:1 ratio

    • Complement activation

    • Fc receptor binding assays

• Cellular immunity:

  • T cell responses to gD and other viral antigens

  • CD4+ vs CD8+ T cell activation

  • Cytokine production profiles

  • T cell functionality (proliferation, cytotoxicity)

  • Tissue-resident memory T cell generation in relevant anatomical sites

• Mucosal immunity:

  • Antibody levels in vaginal washes collected in 150 μl PBS containing protease inhibitor

  • Tissue-resident immune cells in genital mucosa

  • Local cytokine environment before and after challenge

• Protective efficacy measures:

  • Survival following lethal challenge

  • Disease scores for epithelial and neurological symptoms

  • Viral shedding quantified by plaque assay in vaginal washes

  • Viral loads in tissues (genital tract, skin, dorsal root ganglia) measured by plaque assay and reported as log10 pfu per gram of tissue

  • Establishment of latency assessed by:

    • qPCR for viral DNA (using primers for US6 and UL30 genes) with a detection limit of 3 HSV-2 genome copies

    • Ex vivo reactivation from dorsal root ganglia cultured for up to 21 days

How do antibodies against HSV-2 gD mediate protection through non-neutralizing mechanisms?

Research has revealed that antibodies against HSV-2 gD can mediate protection through several non-neutralizing mechanisms, challenging the traditional focus on neutralizing antibodies in vaccine development:

• Fc receptor-dependent effector functions: Studies with HSV-2 ΔgD−/+gD−1 vaccines demonstrated that protection was completely dependent on Fc receptors . Passive transfer of immune serum (250 μl containing 750 μg of total IgG) protected wild-type mice but failed to protect Fcγ-receptor or neonatal Fc-receptor knockout mice, definitively proving the importance of these receptors in antibody-mediated protection .

• Antibody-dependent cellular cytotoxicity (ADCC): Antibodies elicited by HSV-2 ΔgD−/+gD−1 vaccination demonstrated significant cell-mediated cytotoxicity against HSV-2-infected cells . This was measured using a flow cytometry-based assay with PKH-26-labeled target cells, effector cells at a 25:1 ratio, and Live/Dead Red fixable dye to quantify cell death .

• IgG subclass profile: Research suggests that deletion of gD-2 leads to an IgG2-dominant antibody response, which is particularly effective at engaging Fc receptors and mediating effector functions . This contrasts with the response to wild-type virus or gD subunit vaccines, which may induce different IgG subclass distributions.

• Mucosal immunity: HSV-specific antibodies were detected in vaginal washes following challenge, indicating that local antibody responses at mucosal surfaces contribute to protection even without neutralizing activity . These antibodies likely function through local ADCC or other Fc-mediated mechanisms.

• Rapid viral clearance: Vaccinated mice cleared virus by day 4 post-challenge, while control mice showed persistent virus . This rapid clearance, before establishment of latency, suggests effective elimination of infected cells through non-neutralizing antibody mechanisms.

What methodologies are most effective for analyzing Fc receptor-dependent protection against HSV-2?

To effectively analyze Fc receptor-dependent protection against HSV-2, researchers should implement these complementary methodological approaches:

• In vivo models with receptor knockouts:

  • Utilize Fcγ-receptor knockout mice to directly assess the requirement for these receptors in protection

  • Include neonatal Fc-receptor knockout mice to evaluate the role of this receptor in antibody transport and half-life extension

  • Compare protection in wild-type versus knockout mice following vaccination or passive antibody transfer

• Passive transfer studies:

  • Transfer immune serum (standardized to 750 μg of total IgG in 250 μl) to naive recipients 24 hours before viral challenge

  • Isolate different antibody fractions or isotypes to determine which contribute most to protection

  • Deplete specific antibodies using antigen columns or Protein L to confirm their role

  • Quantify protection by measuring survival, disease scores, viral loads, and establishment of latency

• Antibody functional assays:

  • ADCC assays: Co-culture HSV-infected target cells labeled with PKH-26, effector cells from naive mice, and serum antibodies at an effector:target ratio of 25:1 for 4 hours, then assess killing by flow cytometry using Live/Dead Red fixable dye

  • Antibody-dependent cellular phagocytosis (ADCP): Measure uptake of antibody-opsonized viral particles or infected cell material by phagocytes

  • Complement-dependent cytotoxicity: Assess complement activation and subsequent lysis of infected cells

• Antibody engineering and modification:

  • Generate modified antibodies with altered Fc regions to enhance or eliminate specific Fc receptor interactions

  • Create Fab or F(ab')2 fragments to confirm the requirement for the Fc portion

  • Introduce specific mutations that affect complement activation versus Fc receptor binding

• Tissue-specific protection assessment:

  • Evaluate protection at mucosal surfaces versus systemic compartments

  • Quantify viral loads in different tissues (genital tract, skin, dorsal root ganglia) using plaque assays (reported as log10 pfu per gram of tissue)

  • Measure viral DNA in tissues using qPCR with primers for US6 or UL30 genes (reported as log10 HSV-2 copy numbers per gram of tissue)

How does the interaction between HSV-2 gD and HVEM affect immune responses?

The interaction between HSV-2 gD and herpesvirus entry mediator (HVEM) has significant immunological consequences beyond facilitating viral entry:

• Immune response modulation: Research suggests that gD-HVEM interactions may skew immune responses in ways that limit protection . Specifically, these interactions may favor the development of neutralizing antibodies over antibodies with effective Fc-mediated effector functions .

• T cell signaling alteration: HVEM naturally interacts with LIGHT, BTLA, and CD160 to regulate T cell activation. When gD binds HVEM, it can disrupt these normal interactions, potentially interfering with appropriate T cell responses. This mechanism represents a potential viral immune evasion strategy.

• Potential immunosuppressive functions: The research references "yet undiscovered immunosuppressive functions of gD," suggesting additional mechanisms by which gD-HVEM interactions might dampen effective immunity . These could include effects on antigen-presenting cells or regulatory T cells.

• Experimental approaches to study this interaction:

  • Compare immune responses to wild-type virus versus gD-deleted virus (HSV-2 ΔgD−/+gD−1)

  • Examine responses in HVEM-deficient mice

  • Test immunity induced by HSV-2 complemented with mutant forms of gD that cannot interact with HVEM

  • Analyze differences in antibody isotype profiles, as the hypothesis suggests that absence of gD-HVEM interactions may promote an IgG2-dominant response capable of effective Fc receptor-mediated protection

• Implications for vaccine design: Understanding how gD-HVEM interactions affect immune responses can inform the development of improved vaccines. Approaches might include creating modified gD proteins that maintain protective epitopes but lack immunomodulatory functions, or completely removing gD as in the HSV-2 ΔgD−/+gD−1 vaccine, which provided 100% protection against lethal challenge .

What experimental designs best evaluate the role of HSV-2 gD-specific antibodies in protection?

To rigorously evaluate the role of HSV-2 gD-specific antibodies in protection, researchers should implement these experimental designs:

• Passive immunization studies:

  • Isolate gD-specific antibodies from immune serum using affinity purification

  • Transfer defined amounts (e.g., 750 μg in 250 μl) to naive recipients 24 hours before challenge

  • Include appropriate controls: total immune IgG, IgG depleted of gD-specific antibodies, and non-immune IgG

  • Assess protection by monitoring survival, disease scores, viral shedding, and establishment of latency

  • Test protection in wild-type versus Fc receptor knockout mice to determine mechanism

• Comparative vaccination protocols:

  • Immunize separate groups with:

    • Wild-type HSV-2 (inactivated)

    • HSV-2 ΔgD−/+gD−1 vaccine

    • Purified gD protein with appropriate adjuvants

    • Other viral antigens as controls

  • Use standardized immunization protocols (prime and boost 3 weeks apart) with 5 × 10^6 pfu administered subcutaneously

  • Challenge with clinical isolates of HSV-2 (e.g., strain 4674) at LD90 (5 × 10^4 pfu/mouse) or higher doses

  • Comprehensively assess immune responses and protection

• Epitope-specific approaches:

  • Generate monoclonal antibodies targeting different epitopes within gD

  • Map these epitopes through techniques like peptide scanning or site-directed mutagenesis

  • Test individual monoclonal antibodies for protective capacity through passive transfer

  • Correlate protection with specific functions: neutralization, ADCC, complement activation

• Competitive inhibition experiments:

  • Pre-incubate immune serum with purified gD protein or specific peptides

  • Assess whether absorbing gD-specific antibodies reduces protective capacity

  • Perform similar experiments with other viral antigens for comparison

• Functional characterization:

  • Compare neutralizing versus non-neutralizing anti-gD antibodies for protective capacity

  • Conduct ADCC assays using HSV-infected target cells and effector cells at a 25:1 ratio

  • Analyze Fc glycosylation patterns, which affect Fc receptor binding and effector functions

  • Determine antibody isotype distributions (IgG1, IgG2a, IgG2b, IgG3) using isotype-specific detection antibodies in ELISA

What cutting-edge approaches should be used to study HSV-2 latency in the context of gD-targeted interventions?

Studying HSV-2 latency in the context of gD-targeted interventions requires sophisticated methodological approaches:

• Sensitive viral detection methods:

  • Quantitative PCR: Extract DNA from neural tissue using commercial kits (e.g., DNeasy Blood and Tissue) and perform qPCR using primers for multiple viral genes (US6/gD and UL30/polymerase) . Normalize to 10 ng of DNA per reaction and use HSV-2 viral DNA as a standard curve to determine absolute copy numbers . This approach can detect as few as 3 HSV-2 genome copies .

  • Digital droplet PCR: For even greater sensitivity and absolute quantification without standard curves

  • RNA sequencing: To detect and quantify latency-associated transcripts and other viral RNAs

  • In situ hybridization: To visualize viral nucleic acids within specific cell types in tissue sections

• Ex vivo reactivation assays:

  • Harvest neural tissue (dorsal root ganglia including sciatic nerve from hind limb to spinal cord) at day 5 post-challenge

  • Cut tissue into 3-4 pieces and co-culture with confluent Vero cell monolayers in serum-free DMEM

  • Observe cultures daily for up to 21 days for cytopathic effect, exchanging media every 2 days

  • Harvest supernatants every other day to measure viral plaque-forming units by standard plaque assay

  • Compare reactivation rates between different intervention groups

• Single-cell approaches:

  • Single-cell RNA sequencing of neurons from dorsal root ganglia to identify viral transcripts and host response signatures

  • Single-cell proteomics to detect viral proteins in latently infected cells

  • Laser capture microdissection to isolate specific infected neurons for molecular analysis

• In vivo latency and reactivation models:

  • Establish latent infection through controlled challenge protocols

  • Test interventions targeting gD during establishment of latency or during reactivation

  • Induce reactivation through physiological stressors (UV exposure, hormonal changes)

  • Monitor viral shedding following reactivation triggers

• Neuronal culture systems:

  • Primary neuronal cultures from dorsal root ganglia

  • Human induced pluripotent stem cell (iPSC)-derived neurons for human-relevant models

  • Compartmentalized chamber systems to study axonal transport and retrograde signaling

• Immunofluorescence and imaging:

  • Multiplex immunofluorescence to simultaneously detect viral proteins and host factors

  • Tissue clearing techniques for 3D visualization of infected neurons within intact ganglia

  • Live cell imaging to observe dynamics of viral reactivation in real-time

What are the most precise methods for quantifying HSV-2 in tissues following experimental interventions?

For precise quantification of HSV-2 in tissues following experimental interventions, researchers should employ these state-of-the-art methods:

• Viral plaque assays for infectious virus:

  • Weigh and homogenize tissue samples (genital tract, skin, dorsal root ganglia) in serum-free DMEM using RNase-free pestles

  • Sonicate homogenates for 30 seconds at maximum strength and centrifuge at 10,000×g for 5 minutes

  • Overlay supernatants on confluent Vero cell monolayers (2 × 10^5 cells/well in 48-well plates) for 1 hour

  • Wash with PBS, add 199 medium containing 1% heat-inactivated FBS with 0.5% methylcellulose overlay

  • Incubate at 37°C for 48 hours, then fix with 2% paraformaldehyde and stain with crystal violet

  • Quantify plaque-forming units and report as log10 pfu per gram of tissue

• Quantitative PCR for viral genome:

  • Extract DNA from weighed tissue samples using commercial kits (e.g., DNeasy Blood and Tissue)

  • Normalize extracted DNA to 10 ng per reaction

  • Perform real-time quantitative PCR using validated primers for HSV-2 genes:

    • US6 (gD) primers (IDT cat: 117920498)

    • UL30 (polymerase) primers (IDT cat: 1179200494)

  • Use HSV-2 viral DNA to create a standard curve for absolute quantification

  • Consider samples with three or fewer copy numbers as negative

  • Report results as log10 HSV-2 copy numbers per gram of tissue

• Digital droplet PCR:

  • Provides absolute quantification without standard curves

  • Higher sensitivity and precision, especially for low copy number samples

  • Partitions the sample into thousands of nanoliter-sized droplets

  • Each droplet represents an individual PCR reaction

  • Results are reported as absolute copies per sample

• In situ hybridization combined with quantitative image analysis:

  • RNAscope or DNAscope technologies for highly specific detection of viral nucleic acids

  • Automated whole-slide scanning and quantitative image analysis

  • Provides spatial information about infected cells within tissue context

  • Can be multiplexed to simultaneously detect viral and host markers

• Viral protein quantification:

  • Western blot analysis with standardized loading controls

  • ELISA-based methods for soluble viral proteins

  • Mass spectrometry for unbiased protein quantification

  • Multiplexed immunoassays for simultaneous detection of multiple viral proteins

How can researchers comprehensively characterize antibody responses to HSV-2 gD following vaccination or infection?

To comprehensively characterize antibody responses to HSV-2 gD following vaccination or infection, researchers should implement this multifaceted analytical approach:

• Antibody quantification:

  • ELISA for HSV-2 specific antibody detection:

    • Prepare cell lysates from mock-infected or HSV-2-infected Vero cells (24 hours post-infection)

    • Coat 96-well MaxiSorp ELISA plates with 45 μg cell lysate/well

    • Permeabilize with PBS/0.1% Triton X-100 and fix with 1% formaldehyde

    • Incubate with serially diluted serum or vaginal wash samples

    • Detect bound antibodies using biotin-conjugated anti-mouse Ig κ or isotype-specific antibodies (IgG1, IgG2a, IgG2b, IgG3)

    • Develop with HRP-conjugated streptavidin and TMB substrate

    • Subtract values from uninfected lysates to determine specific binding

• Epitope mapping:

  • Peptide scanning using overlapping peptides covering the entire gD sequence

  • Competitive binding assays with known monoclonal antibodies

  • Phage display techniques to identify linear and conformational epitopes

  • HDX-MS (hydrogen-deuterium exchange mass spectrometry) for conformational epitope mapping

• Antibody functionality:

  • Neutralization assays using standard plaque reduction neutralization tests

  • ADCC assays:

    • Infect Vero cells with HSV-2 at MOI of 5