Fig. 1. Prophylactic, neutralization, and therapeutic efficacy of HPAC.

(A) Fluorescence imaging of green fluorescent protein (GFP)–HSV-1– and GFP–HSV-2–infected human corneal epithelial cells (HCEs) or HeLa cells treated with HPAC (1 mg/ml) prophylactically. (B) GFP HSV-1 and HSV-2 viruses were neutralized with HPAC (1 mg/ml) before its application to HeLa cells, and the viral entry was measured. Blue, 4′,6-diamidino-2-phenylindole; red, phalloidin staining of actin; green, GFP virus. (C) HCEs and HeLa cells were infected with HSV-1 and HSV-2, respectively, for a period of 2 hours before the addition of mock phosphate-buffered saline (PBS) or HPAC at 1 mg/ml. Twenty-four hours after infection, fluorescence images were taken to understand the extent of viral spread in HPAC-treated samples compared to mock. Green, 17 GFP HSV-1 or HSV-2 333 GFP virus. Viral entry for prophylactic (D) and neutralization treatments (E) was quantified using a β-galactosidase reporter virus. (F) Intracellular viral load for HPAC therapeutic treatment was quantified using a plaque assay. PFU, plaque-forming units. Representative immunoblots of HSV-1 (G) or HSV-2 gB (H) protein and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from infected HCEs or HeLa cells treated with varying concentrations of HPAC. (I) Toxicity of HPAC in vitro was evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay after the incubation of multiple concentrations of HPAC with HCEs, HeLa cells, VK2s (vaginal epithelial cells), and HFFs (human foreskin fibroblasts) for 24 hours. (J) Optical density measurements were performed by serially diluting HPAC in a 96-well plate and measuring the optical absorbance at 650 nm. Lower absorbance corresponds to a clearer solution. Data are presented as means ± SD. Significance to mock-treated cells was determined by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test (n = 3 replicates). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.