Journal Article

Antiviral activity and mode of action of Griffithsin against HSV-2 and HPV: Preliminary studies of a potential non-ARV combination microbicide

Background
Griffithsin (GRFT) is a promising HIV microbicide candidate. Nixon et al. have shown that GRFT blocks herpes simplex 2 (HSV-2) infection in a mouse model, proposing inhibition of cell-to-cell spread as the mode of action (MOA). Using in vitro studies we further investigated the MOA of GRFT against HSV-2 and studied its antiviral activity against human papillomavirus (HPV). We also combined GRFT with zinc acetate (ZA) and/or carrageenan (CG) to render a more potent microbicide.

Methods
We used XTT assay to define non-cytotoxic concentrations of GRFT, ZA, CG or their combinations. Assays for anti-HIV, anti-HPV and anti-HSV-2 activities were performed in TZM-bl cells or PBMCs using MAGI and p24 ELISA; in HeLa cells using a luciferase assay; and in Vero cells using plaque forming units (pfu) assay. We performed time-of-addition and temperature dependence experiments to differentiate inhibition of viral adsorption from entry. Surface plasmon resonance (SPR) was used to assess GRFT binding to viral glycoproteins and immunohistochemistry was used to determine the specific glycoprotein involved. Antiviral activities of prototype GRFT/CG (GC) and GRFT/ZA/CG (GZC) gels in a vaginal HSV-2 mouse model were evaluated.

Results
GRFT shows modest in vitro antiviral activity against HSV-2 G (IC_50=5.8μg/ml) and HPV 6, 16, 18, 45 PsVs (IC_50=10.8-26.3μg/ml), compared to potent anti-HIV activity (IC_50=0.7-1.4ng/ml). As with HIV, GRFT blocks the entry but not the adsorption of HSV-2 and HPV to target cells. The combined analyses of SPR and immunohistochemistry for HSV-2 gD, suggest that GRFT binds to HSV-2 gD. GC and GZ had synergistic in vitro antiviral activity against HIV and HPV (CI<1). GC and GZC gels significantly reduced (p<0.05) HSV-2 vaginal infection in vivo when administered up to 2h before challenge with 10^6pfu/mouse.

Conclusions
GRFT blocks HSV-2 and HPV entry to target cells and combination with CG and/or ZA may result in a potent/broad-spectrum non-ARV microbicide.