Phosphorylcholine and KR12-Containing Corneal Implants in HSV-1-Infected Rabbit Corneas

Abstract

Severe HSV-1 infection can cause blindness due to tissue damage from severe inflammation. Due to the high risk of graft failure in HSV-1-infected individuals, cornea transplantation to restore vision is often contraindicated. We tested the capacity for cell-free biosynthetic implants made from recombinant human collagen type III and 2-methacryloyloxyethyl phosphorylcholine (RHCIII-MPC) to suppress inflammation and promote tissue regeneration in the damaged corneas. To block viral reactivation, we incorporated silica dioxide nanoparticles releasing KR12, the small bioactive core fragment of LL37, an innate cationic host defense peptide produced by corneal cells. KR12 is more reactive and smaller than LL37, so more KR12 molecules can be incorporated into nanoparticles for delivery. Unlike LL37, which was cytotoxic, KR12 was cell-friendly and showed little cytotoxicity at doses that blocked HSV-1 activity in vitro, instead enabling rapid wound closure in cultures of human epithelial cells. Composite implants released KR12 for up to 3 weeks in vitro. The implant was also tested in vivo on HSV-1-infected rabbit corneas where it was grafted by anterior lamellar keratoplasty. Adding KR12 to RHCIII-MPC did not reduce HSV-1 viral loads or the inflammation resulting in neovascularization. Nevertheless, the composite implants reduced viral spread sufficiently to allow stable corneal epithelium, stroma, and nerve regeneration over a 6-month observation period.

Modified host defence peptide GF19 slows TNT-mediated spread of corneal herpes simplex virus serotype I infection

“…Alternatively, the lower the MPr score, the higher is its preference (stability) in an aqueous environment. KR12, the smallest active fragment of LL37 with weak anti-HSV-1 activity 23 , GF17 and GF19 all had ~ 1.5 times higher average MPr scores (MPr, averaged over all residues pertaining to the corresponding peptides) compared to that of LL37, which further suggests that all designed peptides (except GF19 scrambled ) are more vulnerable (or energetically frustrated) in an aqueous solution than LL37 and are therefore more reaction-prone. GF17 and KR12 had similar degrees of instability, while GF19 was slightly less unstable in an aqueous environment.…”

“…An initial IC50 test done to obtain the concentration where 50% of cells are killed. Together with previous studies of the bioactive KR12 region 23 within GF19 and the full-length LL37 parent peptide 21 , these early results helped to determine the test concentrations to be tested. HCECs were incubated with varying concentration of the peptides (0, 5, 10, 25 and 50 µM) for 24 h. Six samples were analyzed for each concentration.…”

“…We tested its smallest bioactive fragment, KR12, against HSV-1 in an infected rabbit corneal model. While KR12 was biocompatible, promoted epithelial healing and showed good antiviral activity in culture, its in vivo antiviral activity was weak 23 . KR12 released from silica nanoparticles (SiNP) delivered through an implantable biosynthetic cornea could not effectively block virus activity in the rabbits, but was able to nevertheless promote in situ corneal tissue regeneration 23 .…”