HSV-2 gD (31-335)

What is HSV-2 gD (31-335) and what is its role in viral entry?

HSV-2 gD (31-335) refers to a recombinant fragment of glycoprotein D from Herpes Simplex Virus type 2, specifically spanning amino acids 31-335. This region represents the extracellular epitope-rich portion of the full glycoprotein and plays a critical role in viral entry mechanisms. Glycoprotein D is one of four glycoproteins essential for HSV entry and cell fusion .

The viral entry process mediated by gD follows specific stages: Initially, matching receptors on the virus's envelope interact with host cell membrane receptors, bringing the two together. During the transitional stage, fusion begins between the host cell and virus (hemifusion state). In the final stage, a stable pore forms through which viral particles enter the cell .

For research purposes, the 31-335 amino acid fragment is particularly valuable as it contains the functional domains necessary for receptor binding while excluding the transmembrane and cytoplasmic portions of the full protein. This specific fragment is extensively used in research because it contains the primary immunogenic epitopes and receptor-binding domains that mediate viral attachment to host cells .

What expression systems are most effective for producing HSV-2 gD (31-335) for research?

Both prokaryotic (E. coli) and eukaryotic (CHO cells) expression systems have been successfully used to produce HSV-2 gD (31-335), each offering distinct advantages depending on the intended research application.

Escherichia coli expression systems provide a straightforward and cost-effective method for producing substantial quantities of the recombinant protein. E. coli-derived HSV-2 gD recombinant protein can achieve >95% purity as determined by SDS-PAGE . This system is particularly suitable for applications requiring large protein quantities, such as structural studies or initial screening experiments.

For vaccine development and immunogenicity studies, eukaryotic expression in suspension CHO cells with serum-free medium has demonstrated superior results. Researchers have successfully established stable CHO-DG44 cell lines with high expression levels by optimizing the DNA sequence of gD2 and using plasmid pMD902 for transfection . This approach offers several advantages:

• High yield of purified gD2 (57 mg/L of serum-free culture medium)

• Excellent purity (approximately 95% as determined by HPLC analysis)

• Enhanced immunogenicity (stronger humoral immunity and higher levels of cellular immune responses compared to prokaryotically expressed gD2)

The superior immunogenicity of CHO-expressed gD2 likely results from proper post-translational modifications occurring in mammalian cells, making this expression system preferable for immunological research and vaccine development .

How does HSV-2 gD (31-335) perform as a vaccine antigen compared to other HSV-2 glycoproteins?

In comparative studies, gD-2 outperformed gC-2 in multiple protection parameters, but the gC-2-plus-gD-2 combination surpassed gD-2 alone in several critical aspects:

• Enhanced protection of dorsal root ganglia (DRG) in mice

• Reduced recurrent vaginal shedding of HSV-2 DNA in guinea pigs

• Superior viral clearance from infection sites

In mouse models, the combined gC-2-plus-gD-2 immunization resulted in the lowest viral titers on day 1 post-infection (3 log10 lower than mock-immunized) and more rapid viral clearance. Most importantly, no virus was detected in DRG from mice immunized with the combination, compared to detection in 3 of 5 DRG from gD-2-immunized mice .

Guinea pig studies demonstrated that the combined vaccination significantly reduced recurrent HSV-2 DNA vaginal shedding, as shown in this comparative data table:

These findings indicate that while HSV-2 gD (31-335) is a potent vaccine antigen independently, combination approaches with complementary antigens like gC-2 provide optimal protection through multiple immune mechanisms.

What purification methods yield the highest purity HSV-2 gD (31-335) for immunological studies?

For immunological studies requiring high-purity HSV-2 gD (31-335), a two-step chromatographic purification process has proven most effective. Research indicates that using an anion exchange column followed by a Sephadex G-25 desalting column can achieve approximately 95% purity as determined by HPLC analysis .

For researchers implementing this purification protocol, the process begins with harvesting secreted protein from serum-free culture medium of stably transfected CHO cells. The culture supernatant undergoes initial purification using anion exchange chromatography, which separates proteins based on their charge characteristics. This is followed by size exclusion chromatography using a Sephadex G-25 desalting column to remove small molecular contaminants and exchange the buffer .

The effectiveness of this purification protocol is demonstrated by:

• Achievement of >95% purity suitable for vaccination studies

• Preservation of the protein's immunogenic properties

• Yield of 57 mg/L of purified protein from serum-free culture medium

• A streamlined process compared to multi-step purification protocols

For E. coli-expressed HSV-2 gD (31-335), SDS-PAGE analysis has been used to confirm purity levels exceeding 95% . The purified protein is typically prepared in a 1X PBS buffer at concentrations of 1mg/ml for research applications .

Regardless of the expression system used, purified HSV-2 gD (31-335) should be stored below -18°C to maintain stability, with freeze-thaw cycles minimized to preserve biological activity .

What cellular and humoral immune responses are elicited by HSV-2 gD (31-335) immunization?

HSV-2 gD (31-335) immunization elicits a multifaceted immune response encompassing both humoral and cellular components, which are critical for protection against HSV-2 infection and recurrence.

Humoral immune responses include:

• Production of neutralizing antibodies capable of preventing HSV-2 infection

• Neutralizing activity that functions both in the presence and absence of complement

• Enhanced neutralizing antibody titers (up to 8-fold increase) in the presence of human complement when combined with gC-2 immunization

Cellular immune responses include:

• Significant increases in IFN-γ- and TNF-α-producing CD4+ and CD8+ T cells

• Particularly robust CD4+ T-cell responses, which are critically important for virus clearance from sensory neurons

• When combined with gC-2, restimulation with gC-2 antigen produced a significantly greater frequency of IFN-γ+TNF-α+CD4+ T cells than restimulation with gD-2 antigen

These responses can be measured through various methodological approaches:

• For humoral responses: Neutralization assays with and without complement, ELISA for antibody titers

• For cellular responses: Flow cytometry to detect cytokine-producing T cells following antigen restimulation

• For protection assessment: Challenge studies measuring viral titers, disease symptoms, and viral DNA in tissues

Research has shown that both CD4+ and CD8+ T cells are isolated from human HSV lesions and are required for viral clearance from genital epithelium. CD4+ T cells specifically play a crucial role in virus clearance from sensory neurons, highlighting the importance of vaccine formulations that elicit balanced humoral and cellular responses .

What are the advantages of combining HSV-2 gD (31-335) with gC-2 in vaccine formulations?

Combining HSV-2 gD (31-335) with glycoprotein C (gC-2) in vaccine formulations provides substantial advantages over single-antigen approaches, as demonstrated by comprehensive research in both mouse and guinea pig models.

Key advantages include:

The mechanism behind this enhanced protection involves complementary functions: while gD-2 primarily mediates direct neutralization, gC-2 blocks immune evasion domains that inhibit complement activation. Together, they create a synergistic effect that addresses multiple aspects of HSV-2 pathogenesis .

These findings strongly suggest that researchers developing HSV-2 vaccines should consider combination approaches targeting multiple viral antigens with complementary immune activation profiles rather than focusing on single antigens.

How can researchers evaluate neutralizing antibody responses to HSV-2 gD (31-335)?

Comprehensive evaluation of neutralizing antibody responses to HSV-2 gD (31-335) requires multiple methodological approaches that assess both direct virus neutralization and in vivo protection.

Based on research findings, an effective evaluation protocol should include:

• Neutralization assays with and without complement:

  • Neutralizing antibody titers in sera from animals immunized with gC-2 plus gD-2 increased up to 8-fold in the presence of human complement

  • This suggests standard neutralization assays without complement may underestimate the protective potential of anti-gD antibodies

  • Protocols should include parallel assays with heat-inactivated serum (complement-free) and with added human complement

• In vivo neutralization assessment:

  • Studies in complement-intact and C3 knockout mice revealed that antibodies were more effective when complement-mediated immunity was intact

  • Protection studies should be conducted in models with normal complement function to fully evaluate vaccine efficacy

• Titering methodology:

  • Serial dilutions of immune sera tested against standardized viral challenges

  • Results expressed as the serum dilution providing 50% protection against viral cytopathic effect

  • Both neutralizing titers and protection from disease outcomes should be measured

• Viral challenge models:

  • Vaginal challenge models in both mice and guinea pigs provide complementary information

  • Assessment should include measurement of viral titers on days 1-6 post-infection

  • Protection of dorsal root ganglia should be evaluated both by viral isolation and by PCR for viral DNA

  • Recurrent shedding should be quantified over extended periods in guinea pig models

• Quantitative PCR methodology:

  • Essential for detecting low levels of viral replication

  • Studies have quantified viral DNA in terms of copies per defined amount of host DNA (e.g., 10^4 copies of mouse adipsin DNA)

  • This approach allows detection of subclinical infection in protected animals

This multi-faceted evaluation approach provides a comprehensive assessment of vaccine-induced protection, considering not only direct virus neutralization but also prevention of latency establishment and reactivation.

What are the optimal storage conditions for maintaining HSV-2 gD (31-335) stability?

Maintaining the stability and biological activity of HSV-2 gD (31-335) requires specific storage conditions that research laboratories should strictly follow.

According to product information from multiple sources, the following guidelines are recommended:

• Long-term storage: The protein should be stored below -18°C (typically -20°C or -80°C freezers)

• Short-term stability: While HSV-2 gD (31-335) remains stable at 4°C for up to 1 week, long-term refrigeration is not recommended

• Freeze-thaw cycles: These should be minimized as they can compromise protein integrity and biological activity

Commercial preparations of HSV-2 gD (31-335) are typically supplied in 1X PBS buffer at a concentration of 1mg/ml and have an expiration date of approximately 6 months from the date of receipt when stored properly .

For researchers planning long-term studies, best practices include:

• Aliquoting the protein upon receipt to minimize the number of freeze-thaw cycles

• Preparing aliquots containing only the amount needed for a single experiment or a small series of related experiments

• Maintaining a sterile environment during handling to prevent contamination

• Using appropriate sterile containers for storage

• Clearly labeling aliquots with the date of preparation and concentration

If buffer modifications are required for specific experiments, researchers should perform these modifications immediately before use rather than storing the protein in alternative buffers unless stability in these conditions has been verified.

How can HSV-2 gD (31-335) be used to study viral entry mechanisms?

HSV-2 gD (31-335) serves as a valuable tool for investigating the molecular mechanisms of HSV-2 entry into host cells. This recombinant protein fragment contains the critical domains involved in receptor recognition and binding while being more experimentally tractable than the full-length membrane-bound glycoprotein.

The viral entry process mediated by gD follows three distinct stages:

• Initial binding: Matching receptors on the virus's envelope and host cell's membrane interact, bringing them together

• Hemifusion: A transitional stage where fusion begins between the host cell and virus

• Pore formation: A stable pore forms through which the virus's particles enter the cell

Researchers can use HSV-2 gD (31-335) to study these mechanisms through several methodological approaches:

• Receptor binding assays:

  • Using purified gD (31-335) to identify and characterize cellular receptors

  • Competition assays with soluble gD to block viral entry

  • ELISA-based binding assays to quantify receptor-ligand interactions

• Structure-function analysis:

  • Mutagenesis studies targeting specific domains within gD (31-335) to identify regions critical for receptor binding

  • Analysis of how mutations affect both binding affinity and subsequent fusion events

  • Comparison with HSV-1 gD to identify type-specific differences in receptor usage

• Inhibitor screening:

  • Using gD (31-335) as a target for screening potential entry inhibitors

  • Developing blocking antibodies that prevent gD-receptor interactions

  • Identification of small molecules that disrupt protein-protein interactions essential for viral entry

• Cell-based fusion assays:

  • Transfection of cells with gD alongside other fusion glycoproteins to recapitulate the entry process

  • Measurement of fusion using reporter systems (e.g., luciferase, GFP)

  • Assessment of how mutations in gD (31-335) affect fusion efficiency

Understanding these mechanisms is crucial for developing novel antiviral strategies targeting the entry process, including both therapeutic antibodies and small-molecule inhibitors that disrupt gD-receptor interactions.

What methods can be used to assess the quality of recombinant HSV-2 gD (31-335) preparations?

Comprehensive quality assessment of recombinant HSV-2 gD (31-335) preparations is essential for ensuring experimental reproducibility and biological relevance. Based on the research literature, multiple complementary methods should be employed to evaluate protein quality:

• Purity assessment:

  • SDS-PAGE analysis to confirm >95% purity, as documented in multiple sources

  • HPLC analysis for more precise quantification of purity (~95%)

  • Absence of contaminating proteins from the expression system

• Structural integrity:

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

  • Circular dichroism spectroscopy to assess secondary structure content

  • Mass spectrometry to confirm molecular weight and potential post-translational modifications

• Functional analysis:

  • ELISA-based binding assays to confirm receptor interaction

  • Cell-based entry inhibition assays to verify biological activity

  • Application-specific functionality tests (e.g., neutralizing antibody induction for vaccine preparations)

• Stability assessment:

  • Thermal shift assays to determine protein stability

  • Storage stability testing at different temperatures (4°C for short-term, below -18°C for long-term)

  • Freeze-thaw cycle testing to evaluate resistance to denaturation

• Endotoxin testing:

  • Limulus Amebocyte Lysate (LAL) assay to ensure endotoxin levels are below acceptable thresholds

  • Particularly important for preparations intended for immunological studies or vaccine development

For researchers developing HSV-2 gD (31-335) as a vaccine component, additional quality parameters include:

• Consistency of immunogenicity across batches

• Induction of both humoral and cellular immune responses

• Protection efficacy in appropriate animal models

Implementing these comprehensive quality control measures ensures that experimental results obtained with HSV-2 gD (31-335) are reliable and biologically relevant, particularly for critical applications like vaccine development.