HSV-2 gD

Advanced Research Questions

• Why have subunit vaccines based on HSV-2 gD consistently failed in clinical trials?

  The disappointing performance of gD-2 subunit vaccines in clinical trials appears to stem from multiple factors:

  First, despite eliciting measurable neutralizing antibody responses in vitro, these vaccines have failed to provide meaningful protection against HSV-2 genital disease in human trials . In the most recent efficacy trial, an adjuvanted gD-2 subunit vaccine provided no protection against HSV-2 genital disease, despite generating neutralizing antibodies (titers of 1:97 against HSV-2) .

  Second, the limited antigenic breadth of subunit vaccines may be insufficient to generate the diverse antibody responses needed for protection. The observation that HSV-2 ΔgD-/+gD1 vaccination induces antibodies against multiple viral proteins suggests that a broader antigen profile may be necessary .

  Third, the immunodominance of gD may actually be counterproductive, potentially masking other protective antigens and directing the immune response toward neutralizing antibodies rather than the non-neutralizing Fc-mediated responses that appear crucial for protection .

  Fourth, gD's interaction with HVEM on immune cells may have immunomodulatory effects that skew the response in suboptimal directions, potentially limiting the effectiveness of vaccines containing intact gD .

  Finally, subunit vaccines may not generate sufficient mucosal immunity, whereas live attenuated approaches (like HSV-2 ΔgD-/+gD1) induce antibodies that effectively translocate to mucosal sites .

• How does deletion of gD in HSV-2 affect viral pathogenesis and immune responses?

  Deletion of gD-2 fundamentally alters viral pathogenesis and generates distinctive immune responses with significant vaccine implications:

  The HSV-2 ΔgD-/+gD1 virus (with gD-2 deleted and complemented with HSV-1 gD) grows to high titers (10^8 pfu/ml) when phenotypically complemented but is completely unable to spread from cell to cell in non-complementing conditions . This renders the virus remarkably safe - even severe combined immunodeficiency (SCID) mice showed no disease signs when inoculated with high doses (10^7 pfu) of HSV-2 ΔgD-/+gD1, while much lower doses (10^4 pfu) of wild-type virus were lethal .

  Immunologically, the deletion mutant appears to act as an antigenic "factory" - infected cells generate viral proteins and defective particles that are highly immunogenic, with heat-killed preparations failing to provide similar protection . This suggests the importance of active viral protein production rather than just antigen presentation.

  Vaccination with HSV-2 ΔgD-/+gD1 induces remarkably high serum antibody titers (1:800,000) that effectively translocate to mucosal sites, with antibodies detectable in vaginal washes after challenge . These antibodies recognize multiple viral proteins beyond gD and predominantly mediate protection through Fc receptor-dependent mechanisms rather than direct neutralization .

  Most significantly, this vaccine approach provides complete protection against lethal challenges and prevents both acute replication and establishment of latency, as evidenced by the inability to detect HSV DNA in dorsal root ganglia of vaccinated mice or to reactivate virus from these tissues .

• What immune mechanisms explain the protection provided by HSV-2 ΔgD-/+gD1 vaccination?

  The protective immunity generated by HSV-2 ΔgD-/+gD1 vaccination appears to rely primarily on non-neutralizing antibody-mediated mechanisms rather than traditional neutralization:

  Immunized mice develop high-titer HSV-specific antibodies (1:800,000) that are detected in both serum and vaginal washes after challenge . Despite their abundance, these antibodies demonstrate minimal virus-neutralizing activity in traditional neutralization assays .

  Instead, the protective antibodies function through Fc-mediated effector mechanisms. This was conclusively demonstrated through passive transfer experiments where immune serum completely protected wild-type mice but failed to protect Fcγ-receptor knockout or neonatal Fc-receptor knockout mice . This indicates that antibody binding to viral antigens on infected cells, followed by Fc receptor engagement on immune cells, is the primary protective mechanism.

  The vaccine also generates antibodies against multiple HSV-2 proteins beyond gD, suggesting that a broad, polyantigenic antibody response contributes to protection . This polyantigenic response may overcome viral immune evasion strategies that might circumvent immunity to a single protein.

  Additionally, the vaccine appears to induce antibodies that efficiently translocate to mucosal sites, as evidenced by their detection in vaginal washes after challenge . This mucosal antibody presence likely contributes to the rapid viral clearance observed in vaccinated animals after challenge .

• How can epitope binning and surface plasmon resonance imaging (SPRi) advance our understanding of HSV-2 gD antigenic structure?

  Epitope binning using array-based SPRi represents a significant methodological advancement for understanding complex antigen structures like HSV-2 gD:

  This high-throughput technique enables simultaneous measurement of multiple protein-protein interactions, allowing comprehensive analysis of antibody-antigen binding patterns and competition relationships . In recent studies, this approach permitted cross-competition analysis with 39 anti-gD monoclonal antibodies against four different soluble forms of gD in a single experiment - a scale impossible with traditional methods .

  The technique organizes antibodies into "epitope communities" based on their competition patterns, revealing structural relationships between different antigenic regions . For HSV gD, this approach identified four distinct epitope communities and demonstrated that relationships within and between these communities differed significantly depending on the specific form of gD tested (varying by serotype and protein length) .

  SPRi also enables quantitative off-rate analysis, revealing differences in antibody-gD avidity depending on gD serotype and length . These avidity differences provide insights into the structural basis for differential neutralization of HSV-1 versus HSV-2.

  By comparing epitope binning patterns between different forms of gD, researchers can identify subtle structural differences that may explain serotype-specific neutralization and protection . This approach revealed that despite binding to similar epitopes on both gD1 and gD2, certain antibodies only neutralize HSV-1, providing a potential explanation for the Herpevac trial results .

• What explains the paradoxical finding that gD2 vaccination protected against HSV-1 but not HSV-2 in clinical trials?

  The unexpected observation from the GSK Herpevac trial - that gD2(284t) vaccination protected against genital HSV-1 (86% protection) but not HSV-2 - can be explained by recent epitope mapping studies:

  Advanced SPRi analysis revealed that gD1 and gD2 differ significantly in their structural topography despite sequence homology . Several monoclonal antibodies were identified that bind both gD1 and gD2 yet only neutralize HSV-1, not HSV-2 . This suggests that while these antibodies recognize epitopes present on both proteins, subtle conformational differences affect their neutralizing capacity in a serotype-specific manner.

  The truncated gD2(284t) vaccine likely induced antibodies targeting conserved epitopes between gD1 and gD2, but the specific conformational presentation of these epitopes in HSV-1 facilitated more effective neutralization of this serotype . Off-rate analysis further demonstrated differences in antibody-gD avidity depending on serotype, supporting this hypothesis .

  These findings suggest that antibody-mediated protection against HSV may depend more on precise epitope conformation than on simple binding affinity, explaining how a gD2 vaccine could preferentially protect against HSV-1 . This highlights the importance of understanding the three-dimensional antigenic structure of viral immunogens rather than focusing solely on sequence conservation or antibody binding alone.

• How do mutations in the nectin-1 binding domain of gD affect neural tropism of HSV-2?

  Mutations in the nectin-1 binding domain of HSV-2 gD can selectively impair neural infection while preserving epithelial cell tropism:

  The HSV2-gD27 mutant, which contains specific alterations in the nectin-1 binding domain of gD2, demonstrates a selective deficit in neural cell infection while maintaining normal infection of epithelial cells . This selective impairment demonstrates that distinct domains within gD mediate interactions with different cellular receptors or have different functional requirements depending on cell type.

  To verify the stability of these mutations during in vivo replication, researchers recovered HSV2-gD27 from dorsal root ganglia of infected mice, passaged it once in ARPE-19 cells, and sequenced the gD gene using primers gDF103, gDF106, gDR103, and gDR105 . This methodological approach ensured that the observed phenotype was due to the engineered mutations rather than reversion or compensatory changes.

  The differential tropism resulting from nectin-1 binding domain mutations has important implications for both understanding HSV pathogenesis and developing attenuated vaccine candidates. By selectively impairing neurotropism, such mutations could potentially create live vaccine candidates that maintain immunogenicity while reducing the risk of establishing latent infection in the nervous system .