Recurrent herpes stromal keratitis (HSK) remains the major cause of infectious blinding disease in the US, with the only effective treatment being corneal transplantation. HSK is triggered by reactivation of herpes simplex virus type 1 (HSV-1) from a latent state that is established in sensory neurons during the primary infection. The most likely means of preventing HSK will be to block the triggering HSV-1 reactivation from nerves at the trigeminal ganglia. The once widely-held concept that HSV-1 latency is antigenically silent (and thus invisible to the immune system) is now yielding to overwhelming evidence for active immunosurveillance of latently infected neurons by CD8+T cells, that prevent full reactivation leading to virus production. The latent state is associated with a persisting cellular immune infiltrate that contains virus-specific CD8+T cells which bear hallmarks of recent contact with viral antigen. These also have the remarkable ability to block reactivation from latency. Our overlying hypothesis is that boosting the ganglionic CD8+T cell infiltrate to the correct viral targets during latency will afford protection against reactivated infections. The three specific aims in this proposal address the characteristics of the viral targets of the CD8+T cell infiltrate and the means to augment it. Our studies use the C57Bl6 mouse model, where the viral glycoprotein gB encodes an immunodominant target. Our first specific aim tests the hypothesis that CD8+T cell recognition of viral antigens made before viral DNA replication is critical to effectively block reactivation in a non-cytolytic manner. We have developed a recombinant HSV-1 that expresses gB only after viral DNA replication has initiated. We will determine if the delay of gB expression influences the specificity of the lymph node and ganglionic CD8+ T cell response, the activation state of CD8+ T cells in the TG, the viral load during latency, or the functional capacity of gB-specific CD8+ T cells to block HSV-1 reactivation in ex vivo TG cultures. This will establish the importance of "true late" viral antigens in development of reactivation-blocking vaccines.
Specific Aim 2 will seek to optimize strategies that augment the CD8+T cell infiltration of the TG. In the background of HSV-1 lacking the immunodominant gB target for CD8+ T cells, we will test lytic and latency active promoters to express a multimeric form of the immunodominant peptide and induce the infiltration of gB-specific CD8+ T cells in the latently infected ganglia. The optimal strategy will be evaluated for the ability to augment the natural CD8+Tcell response and afford a higher level of protection against reactivation.
The third Aim will test the hypothesis that a broader HSV- specific CD8+ TCR repertoire induced by HSV-1 with a mutation that disrupts the dominant gB epitope will not improve CD8+ T cell immunosurveillance. We will determine the reactivation blocking ability of the alternative response, and the breadth of the viral targets of CD8+T cell infiltrate in the HSV latently infected ganglia. This could indicate those HSV-1 proteins that could be potentially exploited in vaccine design to block reactivation when immunological pressure causes loss of immunodominant epitopes.

Public Health Relevance

The work detailed in this project is designed to augment the protection by the immune system against infections by herpes simplex virus, particularly the repeated infection of the cornea, where the infection repeatedly triggers a response that clouds corneal clarity, causes scarring and eventually leads to blindness. The work has important implications in vaccine design aimed to prevent the triggering infections.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Anterior Eye Disease Study Section (AED)
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Mckie, George Ann
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University of Pittsburgh
Schools of Medicine
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Bertke, Andrea S; Swanson, Sophia M; Chen, Jenny et al. (2011) A5-positive primary sensory neurons are nonpermissive for productive infection with herpes simplex virus 1 in vitro. J Virol 85:6669-77
Ramachandran, Srividya; Davoli, Katherine A; Yee, Michael B et al. (2010) Delaying the expression of herpes simplex virus type 1 glycoprotein B (gB) to a true late gene alters neurovirulence and inhibits the gB-CD8+ T-cell response in the trigeminal ganglion. J Virol 84:8811-20
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