HSV-2 VP13/14

What is HSV-2 VP13/14 and what is its role in the viral lifecycle?

HSV-2 VP13/14 is a tegument protein encoded by the UL47 gene. It serves as a structural component in the virus particle, positioned between the viral capsid and envelope. In the viral lifecycle, VP13/14 plays roles in virion assembly and may contribute to the regulation of viral gene expression. Research indicates that VP13/14 is recognized as an immunodominant antigen by the host immune system, eliciting strong T cell responses in both mice and humans . As a tegument protein, VP13/14 is delivered into the host cell immediately upon viral entry, making it an early target for immune recognition. The protein has a molecular weight of approximately 31kDa when produced as a recombinant protein covering amino acids 1-280 .

How are VP13/14 epitopes identified and characterized?

Identification of VP13/14 epitopes employs both computational and experimental approaches. Initially, potential epitopes are predicted through bioinformatics analysis using platforms like the Immune Epitope Database (IEDB), which assesses binding affinity to specific MHC molecules . These predictions are then validated experimentally through several methods:

• MHC binding assays - Measuring the stabilization of MHC molecules on T2 cells or similar assays

• ELISpot assays - Detecting IFN-γ release upon peptide stimulation of T cells

• Flow cytometry - Assessing CD107a mobilization as a marker of T cell activation

• Tetramer staining - Directly identifying peptide-specific T cells

Studies have demonstrated that VP13/14 epitopes can be identified from both infected individuals and vaccinated subjects. Specific epitopes like VP13/14 242-250 and VP13/14 551-559 have been characterized as high-affinity binders to human HLA-A*02 and mouse MHC molecules . Experimental validation typically involves testing peptides in ELISpot assays using splenocytes from infected mice or peripheral blood mononuclear cells (PBMCs) from human donors.

What is the prevalence of immune responses to VP13/14 in HSV-2 infected individuals?

Clinical studies have demonstrated that VP13/14 is highly immunogenic in naturally infected humans. In one clinical study, 82% of HSV-2-infected persons had positive T cell responses to the VP13/14 551-559 epitope, compared to 50% responding to another HSV-2 epitope (gB2 442-451) . This indicates that VP13/14 is recognized by the immune system in a majority of infected individuals, making it a promising candidate for vaccine development.

The prevalence data shows:

This higher prevalence of responses to VP13/14 suggests it may be more consistently presented during infection or elicit stronger recall responses than other viral antigens. Importantly, sequencing of circulating HSV-2 strains has not revealed coding polymorphisms in VP13/14 epitopes, suggesting strong conservation of these regions .

What delivery platforms have been evaluated for VP13/14-based vaccines?

Multiple delivery platforms have been investigated for VP13/14-based vaccines, with DNA vaccines and viral vectors showing particular promise. The search results highlight several approaches:

• DNA Vaccination: Full-length VP13/14 DNA vaccines delivered using proprietary gold particle mediated epidermal delivery (PMED™) have successfully elicited strong CD8+ T cell responses in mice . This approach allows for direct expression of the viral protein in host cells, facilitating MHC class I presentation.

• Adenovirus Vectors: Replication-defective adenovirus vectors expressing UL47 (encoding VP13/14) have been used to stimulate both CD4+ and CD8+ T cell responses in humans and mice . These vectors effectively deliver the gene of interest to antigen-presenting cells.

• Protein-based approaches: Recombinant VP13/14 protein can be produced in expression systems like E. coli , though the search results don't specifically mention its use in vaccination.

How does VP13/14 perform as a standalone immunogen versus in combination with other HSV-2 antigens?

The efficacy of VP13/14 differs significantly when used alone versus in combination with other HSV-2 antigens. As a standalone immunogen, VP13/14 (UL47) delivered via adenovirus vectors generated T cell and antibody responses but failed to provide adequate protection against HSV-2 challenge in guinea pigs, with animals developing severe acute disease .

In contrast, when combined with other antigens, particularly those that induce neutralizing antibodies, protection improves. In one study, researchers tested:

• Mock immunization

• gC2/gD2 with CpG and alum as adjuvants

• UL19/UL47 (VP5/VP13/14) adenovirus vectors alone

• Combination of gC2/gD2-CpG/alum and UL19/UL47 adenovirus vectors

These findings suggest that VP13/14 may be more valuable as part of a multi-component vaccine that includes both T cell and antibody-inducing antigens, rather than as a standalone immunogen.

How do immune responses to VP13/14 differ between natural infection and vaccination?

Natural infection and vaccination elicit qualitatively different immune responses to VP13/14. In natural HSV-2 infection, a broad T cell response is generated against multiple epitopes within VP13/14 and other viral proteins. In contrast, vaccine-driven responses tend to be more focused .

The search results indicate that "Vaccine driven T cell responses displayed a more focused immune response than those induced by viral infection" . This focused response may be advantageous if directed against protective epitopes but might provide less coverage against viral variants.

In terms of magnitude, natural infection generally produces strong responses to VP13/14. In one study, HSV-2-infected persons had a mean response of 348 ± 346 spot-forming units (sfu) per 10^6 CD8+ cells when stimulated with whole UV-treated HSV-2, indicating robust T cell recognition . Vaccination studies have also demonstrated induction of VP13/14-specific responses, though direct comparisons of magnitude are difficult due to methodological differences.

What methods are optimal for detecting T cell responses to VP13/14 epitopes?

Multiple complementary methods have proven effective for detecting T cell responses to VP13/14 epitopes, each with distinct advantages:

• IFN-γ ELISpot: This technique quantifies antigen-specific T cells by measuring cytokine secretion after peptide stimulation. It's highly sensitive and can detect low-frequency responses, making it valuable for monitoring responses in vaccination studies and natural infection . The method involves overnight stimulation of splenocytes or PBMCs with peptide, followed by detection of IFN-γ production.

• CD107a Mobilization Assay: This flow cytometry-based method detects degranulation of CD8+ T cells upon peptide stimulation, providing a functional readout of cytotoxic activity. Studies have successfully used this approach to demonstrate "high frequencies of peptide-specific CD8+ T cell responses" elicited by DNA vaccination with VP13/14 .

• MHC-Peptide Tetramer Staining: This technique directly identifies epitope-specific T cells through binding of fluorescently labeled MHC-peptide complexes. It's highly specific and allows for phenotypic characterization of responding cells . In clinical studies, tetramer staining revealed that 82% of HSV-2-infected persons had positive responses to VP13/14 551-559 .

• In vitro T Cell Expansion: For low-frequency responses, in vitro expansion of T cells through repetitive stimulation with cognate peptides can amplify signals. This approach was necessary to identify specific responses in some human donors .

For comprehensive assessment, combining multiple methods is recommended. For example, initial screening with ELISpot followed by functional and phenotypic characterization using flow cytometry provides both quantitative and qualitative information about VP13/14-specific responses.

What factors influence the immunodominance of VP13/14 epitopes?

Several factors contribute to the immunodominance of VP13/14 epitopes:

• MHC Binding Affinity: Epitopes with high affinity for MHC molecules are more likely to be immunodominant. For example, VP13/14 242-250 demonstrated high affinity binding (AI > 1.5) to HLA-A*02 . The stability of peptide-MHC complexes on the cell surface influences the strength of T cell activation.

• Protein Abundance and Processing: As a tegument protein, VP13/14 is delivered into host cells immediately upon infection, potentially allowing for early presentation of its epitopes. The efficiency of proteasomal processing and the context of flanking sequences also impact epitope presentation.

• Conservation Across Viral Strains: Highly conserved epitopes may elicit stronger responses due to consistent presentation. Sequencing of circulating HSV-2 strains has not revealed coding polymorphisms in or flanking VP13/14 epitopes, suggesting strong conservation of these regions .

• Pre-existing Immunity: Cross-reactive T cells from prior exposures (e.g., HSV-1 infection) may influence immunodominance patterns. The search results note that some VP13/14 peptides from HSV-2 show close homology with sequences from HSV-1 .

The high prevalence of responses to VP13/14 (82% of HSV-2-infected persons responding to VP13/14 551-559) compared to other viral antigens suggests it contains particularly immunodominant epitopes . This makes VP13/14 a promising candidate for inclusion in T cell-targeting vaccines against HSV-2.

What expression systems are optimal for producing recombinant VP13/14 protein?

Based on the search results, several expression systems have been used successfully to produce recombinant VP13/14 protein:

For optimal expression, several considerations are important:

• Codon optimization for the host expression system

• Addition of purification tags (e.g., His-tag) for downstream processing

• Removal of hydrophobic domains that may affect solubility

• Expression of specific domains rather than the full-length protein if solubility issues arise

The choice of expression system should be guided by the intended application. For structural studies or antibody production, large quantities of purified protein from E. coli may be preferred. For immunological studies focusing on T cell responses, expression systems that ensure proper processing and presentation of epitopes (such as DNA vaccines or viral vectors) may be more appropriate.

What animal models are most appropriate for studying VP13/14-based vaccines?

Several animal models have been utilized for studying VP13/14-based vaccines, each with distinct advantages and limitations:

• BALB/c Mice: This model has been extensively used for identifying VP13/14 epitopes and evaluating T cell responses. BALB/c mice allow for detailed immunological studies due to well-characterized MHC molecules (H-2Kd, Ld, and Dd) . The search results describe using this model to identify T cell epitopes through infection studies and to evaluate DNA vaccines encoding VP13/14.

• Guinea Pigs: This model is particularly valuable for HSV-2 vaccine studies as it allows for assessment of both acute and recurrent genital disease following intravaginal challenge . Guinea pigs develop genital lesions and establish latency, making them useful for evaluating protection against disease and viral shedding. The search results describe immunizing guinea pigs with UL19/UL47 adenovirus vectors alone or in combination with gC2/gD2.

• HLA-A*02 Transgenic Mice: Although not explicitly mentioned in the search results, HLA transgenic mice expressing human MHC molecules are valuable for studying epitopes restricted by human HLA, such as the HLA-A*02-restricted VP13/14 epitopes identified .

The choice of animal model should be driven by the specific research questions:

• For mechanistic studies of T cell responses: BALB/c or HLA transgenic mice

• For evaluating protection against disease: Guinea pig model

• For assessing viral shedding and latency: Guinea pig model

It's important to note that no single animal model perfectly recapitulates human HSV-2 infection. The search results highlight that while the addition of UL19 and UL47 to a gC2/gD2 vaccine did not significantly improve protection in the guinea pig model, these findings may not necessarily translate to humans . Therefore, a comprehensive evaluation might require using multiple animal models before advancing to human clinical trials.

How do VP13/14-specific T cell responses correlate with clinical outcomes in HSV-2 infection?

While the search results don't provide direct information about correlations between VP13/14-specific T cell responses and clinical outcomes, they do offer insights into the prevalence and magnitude of these responses in infected individuals. A high percentage (82%) of HSV-2-infected persons demonstrated positive responses to VP13/14 551-559, suggesting this is a common target of the immune response .

To establish correlations between VP13/14-specific responses and clinical outcomes, researchers would need to:

• Measure VP13/14-specific T cell responses using methods like ELISpot or tetramer staining

• Collect detailed clinical data including:

  • Frequency and severity of recurrences

  • Viral shedding patterns

  • Duration of lesions

  • Time to first recurrence after primary infection

• Perform longitudinal studies to track changes in immune responses and clinical patterns over time

• Analyze data for statistical correlations between immune parameters and clinical outcomes

Such studies would be valuable for identifying immune correlates of protection, which could guide vaccine development. If strong VP13/14-specific responses correlate with better clinical outcomes, this would further support its inclusion in vaccine candidates.

What are the challenges in translating VP13/14 vaccine studies from animal models to humans?

Translating VP13/14 vaccine findings from animal models to humans faces several challenges:

• Differences in Immune Recognition: Despite conservation of VP13/14 sequences, the immunodominant epitopes may differ between species due to differences in MHC molecules and antigen processing. The search results mention the need to adapt epitope predictions for both mouse H-2 molecules and human HLA alleles .

• Disease Manifestation Differences: Animal models, while useful, don't perfectly recapitulate human HSV-2 infection patterns. For example, the guinea pig model shows similarities to human infection but has important differences in recurrence patterns and immune responses .

• Vaccination Response Variability: The search results indicate that responses to VP13/14 in naturally infected humans vary, with 82% showing responses to certain epitopes . This variability may be even greater in vaccine responses, complicating efficacy assessments.

• Adjuvant Selection: Finding appropriate adjuvants that are both effective and safe for human use remains challenging. Animal studies may use adjuvants that aren't approved for humans.

• Evaluation Endpoints: While animal studies can use challenge models to assess protection, human trials initially rely on immune correlates before efficacy studies can be conducted. Establishing reliable correlates of protection is therefore crucial.

To address these challenges, a stepwise approach is necessary:

• Conduct studies in HLA transgenic mice to better predict human epitope recognition

• Perform detailed immunological studies in both animals and humans to identify cross-species correlates

• Design human trials with appropriate immunological endpoints

• Consider heterologous prime-boost strategies to enhance both breadth and magnitude of responses

This methodical approach can help bridge the gap between promising preclinical findings and successful human applications of VP13/14-based vaccines.