Matundan, Jaggi and Ghiasi (2022) showed that nine major glycoproteins of herpes simplex virus type 1 (HSV-1) differentially regulate the promoter activities of the immune genes CD4, CD8, CD80, and CD86 in transfected cells: gB, gC, gE, and gI stimulate all four promoters, gD, gK, and gL suppress all four, and gG and gH suppress the CD4 promoter selectively. Two source-faithful questions remain open: (i) which common host transcription factors or signaling pathways underlie this differential regulation, knowledge that could permit therapeutic modulation of costimulatory-molecule and T cell responses even in the absence of viral infection; and (ii) whether a rationally designed vaccine cocktail containing only the stimulatory glycoproteins (gB, gC, gE, gI) - excluding the suppressive gD, gK, gL and the mixed-action gG/gH - protects against ocular HSV-1 disease and death better than individual glycoproteins or prior cocktails, and whether the glycoprotein-specific cytokine milieus observed in splenocytes and dendritic cells translate into superior protection.

Background

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus that establishes lifelong latent infection in sensory ganglia and causes recurrent disease, including herpetic keratitis, a leading infectious cause of corneal blindness. No HSV vaccine has been licensed; in particular, a large human efficacy trial of a vaccine based on the viral glycoprotein D (gD) failed to protect against infection (Belshe et al. 2012). The HSV-1 virion carries about a dozen glycoproteins - membrane-anchored proteins that mediate viral entry, cell-to-cell spread, and immune evasion, and which are the main inducers and targets of antibody and T cell responses to the virus.

Effective T cell activation requires costimulation in addition to antigen recognition: CD80 and CD86 (also called B7-1 and B7-2) are ligands on antigen-presenting cells that engage the CD28 receptor on T cells, delivering signals required for productive CD4 and CD8 T cell responses. How much CD4, CD8, CD80, and CD86 protein is made is governed substantially at the transcriptional level, and the activity of a gene's promoter (the DNA control region where transcription initiates) can be quantified with a reporter assay, in which the promoter sequence is placed upstream of a luciferase gene so that light output reports promoter activity.

Matundan, Jaggi and Ghiasi (2022) used such reporter assays in transfected 293 cells (a human embryonic kidney cell line that is readily transfectable and widely used for promoter-reporter studies) to ask how each of nine major HSV-1 glycoproteins (gB, gC, gD, gE, gG, gH, gI, gK, gL) affects the promoter activities of CD80, CD86, CD4, and CD8. gB, gC, gE, and gI stimulated all four promoters; gD, gK, and gL suppressed all four; and gG and gH suppressed only the CD4 promoter while stimulating the other three. Parallel profiling of secreted cytokines and chemokines (immune signaling proteins) using a Luminex multiplex assay in transfected mouse splenocytes and bone-marrow-derived dendritic cells (BMDCs; antigen-presenting immune cells generated from bone marrow precursors in culture) showed that each glycoprotein induces a distinct cytokine milieu. These in vitro findings echo the authors' prior vaccination results: immunizing mice with any of gB, gC, gD, gE, or gI individually protected against lethal ocular challenge (gD being the one that induces no cytotoxic T lymphocyte responses), while a cocktail of seven glycoproteins (gB, gC, gD, gE, gG, gH, gI) protected better than any single glycoprotein (Ghiasi et al. 1996), and a five-glycoprotein mixture that dropped gG and gH (gB, gC, gD, gE, gI; the gG/gH genes had proven ineffective or harmful) performed better still as a DNA vaccine (Osorio et al. 2004) - though every cocktail tested to date still includes gD, which the promoter map classifies as suppressive on all four promoters. One established innate signaling entry point is Toll-like receptor 2 (TLR2, a cell-surface pattern-recognition receptor for microbial molecules), which gH/gL and gB bind, and soluble gH/gL suffices to activate the NF-kB inflammatory transcription pathway (Leoni et al. 2012). What is not known is which host transcription factors or signaling pathways the individual glycoproteins actually engage to produce their distinctive stimulatory or suppressive effects on the CD4, CD8, CD80, and CD86 promoters - and, on the applied side, whether antigen selection based on this stimulator/nonstimulator map (choosing gB, gC, gE, gI and excluding gD, gK, gL, and the mixed-action gG/gH) improves protection in vivo.

Problem Statement

The problem posed by Matundan, Jaggi and Ghiasi (2022, mSphere 7(5):e00382-22) has two strands, each raised in the source itself and each resting on its promoter-regulation map.

  1. Mechanism. Identify the common host transcription factor(s) and/or signaling pathway(s) through which individual HSV-1 glycoproteins differentially regulate the promoter activities of CD4, CD8, CD80, and CD86: gB, gC, gE, and gI stimulating all four promoters, gD, gK, and gL suppressing all four, and gG and gH suppressing CD4 selectively. What is sought is which factor(s)/pathway(s) are responsible, whether perturbing them abolishes or reverses the glycoprotein-specific promoter effects in the reporter system (and, where relevant, in primary antigen-presenting cells), and whether a single shared mechanism or several distinct ones operate across the nine glycoproteins, so that the stimulator/nonstimulator classification is explained. The source motivates this with a therapeutic payoff: such factors could be used to regulate costimulatory-molecule and T cell responses even in the absence of viral infection.

  2. Rational vaccine evaluation. Evaluate, in an in vivo ocular HSV-1 infection model, the protective efficacy of a vaccine cocktail constructed according to the promoter map - containing only the stimulatory glycoproteins (gB, gC, gE, gI) and excluding the promoter-suppressive glycoproteins (gD, gK, gL) and the mixed-action gG/gH - against ocular HSV-1 disease and death, with appropriate comparators (individual glycoproteins and/or prior cocktails such as the seven-glycoprotein mixture or the five-glycoprotein gB/gC/gD/gE/gI mixture of Osorio et al. 2004), and with immunological readouts (neutralizing antibody, glycoprotein-specific CD4/CD8 T cell responses, and cytokine milieus of the kind measured in splenocytes and BMDCs) that test whether the predicted immunological correlates of the promoter map translate into superior protection.

The two strands were posed separately in the source: the vaccine strand selects antigens directly from the empirical stimulator/nonstimulator classification, while the mechanistic strand asks which host factors and pathways generate that classification. Neither strand alone resolves the problem, and the three sub-asks within the mechanism strand - which factor(s)/pathway(s) are responsible, whether perturbing them abolishes or reverses the glycoprotein-specific promoter effects, and whether one shared mechanism or several distinct mechanisms operate - are jointly posed.

References

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