Antagonizing Viral MicroRNAs Reduces Ocular HSV-1 Pathogenesis and Enhances Mucosal Immune Homeostasis.

Purpose: Herpes simplex virus 1 (HSV-1) is a globally prevalent pathogen that causes recurrent lesions at mucosal and cutaneous sites, including the cornea, leading to herpetic keratitis, a major cause of infectious blindness. While HSV-1-encoded microRNAs (v-miRs) are known to regulate viral latency and immune evasion, their role in acute mucosal infection remains unclear. This study investigates the function of v-miRs during acute HSV-1 infection of the cornea. Methods: Using a murine model of corneal HSV-1 infection, we performed RNA sequencing and in situ hybridization to identify v-miRs enriched in the cornea during acute infection. Topical locked nucleic acid (LNA)-modified inhibitors targeting individual v-miRs (miR-H1-5p, miR-H3-3p, miR-H6-3p, and miR-H27) were administered, and effects on disease severity, viral replication, immune response, and lymphoid cell activation were assessed. Results: Inhibition of select v-miRs significantly attenuated corneal keratitis, reduced viral titers, and suppressed Th1/Th17-mediated inflammation. v-miR inhibition also decreased immune cell infiltration in draining lymph nodes and enhanced the frequency of IL-10-producing CD4⁺ T cells. Expression of immunoregulatory genes, including Arg1 and CD25, was increased, and T-cell proliferation was reduced ex vivo, indicating the establishment of a tissue-protective immune environment. Conclusions: HSV-1 v-miRs act as key immunoregulatory effectors during acute corneal infection. Their targeted inhibition using LNA-based therapy mitigates inflammation and promotes immune regulation, highlighting a novel therapeutic strategy for ocular HSV-1 disease.