Investigating a novel approach to infect the corneal epithelium with HSV-1

Abstract

Purpose : Herpes Simplex Virus (HSV) Keratitis (HK) is the leading cause of infectious blindness in developed countries, due to recurrent corneal inflammation and resultant scarring. In line with the ethical principles of the 3Rs (replacement, reduction and refinement), corneal explant models are important to understand the pathophysiology of early viral infection. Our aim was to develop an explant model of HSV-1 infection using porcine corneas with a novel patch application method, designed to minimally disrupt the epithelium to facilitate viral entry.

Methods : The corneas of freshly enucleated porcine eyes (n=6) were excised and HSV1-GFP was delivered via novel microneedle “nanopatches” compared to a cloning cylinder. For nanopatches, corneas were dipped in a solution of HSV-1 strain F expressing GFP-US9 with a wetting agent. Tissue was cultured on a gel foam sponge that had been soaked in culture media in a 24 well plate for 24hours at 37°C and 5%CO₂. Corneas were also tested in mock and original conditions. Tissue was frozen in OCT, sectioned and imaged on the Olympus VS120 Microscope.

Results : Nanopatches were shown to penetrate deep into the anterior stroma of the porcine cornea approximately 115mm posterior to Bowman’s layer. HSV1-GFP infection was only visible in the epithelia of 2/3 corneas that received infection via patch with no infection going deeper than the Bowman’s layer into the stroma (Figure 1). This was compared to 0/3 corneas that were either infected with HSV1-GFP via a cloning cylinder or mock conditions which showed no infection.

Conclusions : Our preliminary results show that this novel approach utilising microneedle technology, is able to infect the epithelial layer of porcine corneal tissue similar to what is seen in epithelial herpes simplex keratitis in patients. Future explants using this method on human corneal tissue will be conducted to assess the early interactions between immune cells and HSV-1 during acute infection.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Figure 1. HSV1-GFP infection of porcine corneal epithelium via pretreatment of nanopatches