HSV-1: Promising New Treatment for Eye Infection

Researchers assessed harmol’s antiviral effectiveness, therapeutic potential, and capacity to augment the effects of acyclovir (ACV) in the treatment of herpes simplex virus type 1 (HSV-1)-induced keratitis, including drug-resistant strains. Their findings were published in the Virology Journal.

Context

A common eye illness called herpes simplex keratitis (HSK) is characterized by corneal damage that impairs vision and causes opacity. In wealthy nations, the incidence of HSK is 10–30 per 100,000, while it is higher in poorer nations.

HSK can result in blindness and perhaps lethal encephalitis if left untreated. It is mostly linked to infection with Herpes simplex virus. ACV is the usual course of treatment, but new treatments are needed due to developing medication resistance, particularly in immunocompromised people.

Several medicinal plants contain harmol, a β-carboline alkaloid that has demonstrated antiviral effects. To completely comprehend harmol’s modes of action, maximize its therapeutic potential, and assess its long-term safety and efficacy in a range of patient populations, more research is required.

About the study: 

Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) was used to maintain Vero cells from the American Type Culture Collection (ATCC). The ACV-resistant HSV-1/153 and the HSV-1 F strain were originally isolated at the Guangzhou Institutes of Biomedicine and Health and sent to Jingling Hospital in China.

After being grown in Vero cells, these strains were collected from the supernatants, centrifuged to get rid of any debris, and then stored at -80°C. Using the tissue culture infective dose (TCID50) assay, viral titers were ascertained.

Dimethyl sulphoxide (DMSO) was used to dissolve Harmol, which was obtained from MedChemExpress and extracted from Peganum harmala Linn. The National Institutes for Food and Drug Control, China, provided ACV.

502 compounds from a natural product library were tested at 10 µM for anti-Herpes simplex virus activity. After applying harmol or ACV to Vero cells, the Cell Counting Kit 8 (CCK-8) test was used to determine the viability of the cells.

According to calculations of the 50% cytotoxic concentration (CC50) and effective concentration (EC50), harmol markedly reduced HSV-1 and HSV-1/153 infections.

Bagg Albino Laboratory-bred/c (BALB/c) mice were used in a mouse HSK model. They were infected with HSV-1 or HSV-1/153 and given harmol or ACV treatment.

Using corneal staining, blepharitis scoring, Real-time optical coherence tomography (RTvue OCT) assessment, and in vivo confocal microscopy, the antiviral efficacy was assessed. P < 0.05 statistical analysis verified harmol’s significant antiviral activity.

Results of the study: Harmol strongly prevents HSV-1 replication. 22 natural compounds with greater than 75% viral suppression were found through screening with the Cytopathic Effect (CPE) inhibition assay in Vero cells. Harmol was chosen for additional research after demonstrating more than 90% inhibition. With a non-CC50 of 12.5 µM utilized for in vitro research, its CC50 was 242.54 µM. Harmol and ACV (1 µM) both decreased CPEs associated with HSV-1.

By assessing the expression of the Herpes Simplex Virus Type 1 glycoprotein D (gD-1) protein, the anti-HSV-1 impact of Harmol was verified. ACV (1 µM) and harmol (0-50 µM) both decreased the amounts of HSV-1 gD-1 protein.

With Selectivity Index (SI) values of 26.0 and 44.5 for HSV-1 F and 5.84 µM for HSV-1/153, respectively, Harmol’s EC50 values were as follows. Harmol successfully inhibited both HSV-1 strains’ ability to replicate and produce offspring.

Harmonol and ACV together showed increased anti-HSV-1 activity. More efficiently than each treatment alone, Harmol and ACV suppressed the expression of HSV-1 F gD-1 messenger ribonucleic acid (mRNA) and the development of progeny viruses.

The expression of gD protein was further decreased by increasing the doses of harmol (0, 3.12, 6.25, and 12.5 µM), with combination treatment exhibiting the best anti-HSV-1 F results. Comparable results were noted in HSV-1/153-infected cells, suggesting harmol amplifies ACV’s anti-HSV-1 activity, even against ACV-resistant strains.

To ascertain the point in HSV-1 infection at which harmol begins to exhibit its antiviral effects, a time of addition experiment was performed. At several points after infection, harmol (1 µM) was administered to Vero cells that were infected with HSV-1.

Using quantitative polymerase chain reaction (qPCR) experiments, it was demonstrated that harmol pretreatment greatly suppressed HSV-1 multiplication; however, the effectiveness of the treatment decreased with longer adding times. 

The safety of harmol applied topically was evaluated on mice corneas. In comparison to PBS therapy, mice given 5 µL harmol (0.01 mg/kg, or approximately 100 µM) on days 1, 3, and 5 did not exhibit any negative effects on body weight or corneal transparency.

Five days later, corneal fluorescein staining revealed no injury to the epithelium. Therefore, harmol did not exhibit any toxicity to mouse corneas at 0.01 mg/kg.

Harmol eyedrop therapy reduced HSK severity in a mouse model. Harmol-treated mice showed decreased corneal opacity, blepharitis scores, and early corneal damage in comparison to untreated HSK mice.

Harmol also lessened the decrease in body weight brought on by Herpes simplex virus F. The preservation of ocular structural integrity in harmol-treated animals was validated in vivo by RTvue OCT and confocal microscopy (IVCM). 

It was also shown that Harmol was effective against ACV-resistant HSV-1/153. In HSV-1/153-infected mice, weight loss was avoided and blepharitis scores and early corneal lesions were dramatically decreased by Harmol but not by ACV. Harmol together efficiently suppresses ACV-resistant HSV infections in vivo.

In conclusion

In summary, HSV-1 infection is associated with HSK development, which results in visual loss worldwide. Overuse-related drug resistance poses a serious threat to public health and calls for the development of novel therapies.

Harmol relieves early virally-induced lesions and prevents HSV-1 F and HSV-1/153 from replicating in ocular tissues. It demonstrates antiviral effectiveness against RNA viruses and amplifies the effects of ACV on HSV-1, suggesting an alternative mode of action.

Extracellular Signal-Regulated Kinases 1 and 2 (ERK1/2) and Amp-activated protein Kinasepathways (AMPK), which are critical in the fight against HSV, may be affected by Harmol’s activation of autophagy. 

For more information: Harmol used for the treatment of herpes simplex virus-induced keratitis, Virology Journal, https://doi.org/10.1186/s12985-024-02384-0.