The HRA2pl fusion peptide exerts in vitro antiviral activity against human respiratory paramyxoviruses and pneumoviruses

Abstract

Acute respiratory infections are a group of diseases caused by viruses, bacteria, and parasites that mainly affect children until the age of 5 and immunocompromised senior adults. In Mexico, these infections are the main cause of morbidity in children, with more than 26 million cases of respiratory infections reported by the Secretariat of Health, in 2019. The human respiratory syncytial virus (hRSV), the human metapneumovirus (hMPV), and the human parainfluenza-2 (hPIV-2) are responsible for many respiratory infections. Currently, palivizumab, a monoclonal antibody against the fusion protein F, is the treatment of choice against hRSV infections. This protein is being studied for the design of antiviral peptides that act by inhibiting the fusion of the virus and the host cell. Therefore, we examined the antiviral activity of the HRA2pl peptide, which competes the heptad repeat A domain of the F protein of hMPV. The recombinant peptide was obtained using a viral transient expression system. The effect of the fusion peptide was evaluated with an in vitro entry assay. Moreover, the effectiveness of HRA2pl was examined in viral isolates from clinical samples obtained from patients with infections caused by hRSV, hMPV, or hPIV-2, by evaluating the viral titer and the syncytium size. The HRA2pl peptide affected the viruses' capacity of entry, resulting in a 4-log decrease in the viral titer compared to the untreated viral strains. Additionally, a 50% reduction in the size of the syncytium was found. These results demonstrate the antiviral potential of HRA2pl in clinical samples, paving the way toward clinical trials.

Keywords: acute respiratory infections; fusion peptide; human respiratory virus; paramyxovirus; pneumovirus; syncytium size.

Figure 1

The HRA2pl peptide does not affect cell viability of (A) HEp-2 and (B) Vero cultures. For the assay, a confluent monolayer of HEp-2 and Vero cells (95%–100%) was treated with 90 μL of HRA2pl purified in DMEM (B1: 172.31 ng/μL; B2: 182.9 ng/μL) and 10 μL of AlamarBlue to measure the absorbance at 0 h, 24 h, and 48 h. NS: Nonsignificant; *p< 0.05;**p< 0.01; ***p< 0.001.

Figure 2

HRA2pl reduces the viral titer. For the assay, a confluent monolayer of HEp-2 (A, B) and Vero (C, D) cells (95%–100%) was treated with HRA2pl purified in DMEM at three different concentrations (C1: 3.2 µg/mL; C2: 2.1 µg/mL; C3: 1.8 µg/mL). The monolayers were treated with a mixture of C1, C2, or C3 and hRSV (1.9 × 10⁴ TCID₅₀/ml) and hPIV-2 (1 × 10⁵ TCID₅₀/ml). The reduction in the viral titer was established by TCID₅₀/ml.***p< 0.001.

Figure 3

The syncytium size was reduced after treatment with HRA2pl B2. In HEp-2 cultures infected with (A) hRSV or (B) hMPV viral isolates, and (C) in Vero cultures infected with hPIV-2 viral isolates, treatment with HRA2pl significantly reduced the syncytium size. ***p< 0.001.

Figure 4

The changes in the number of syncytia after HRA2pl B2 treatment varied according to the viral infection. HRA2pl treatment increased the number of syncytia in (A) HEp-2 cells infected with hRSV viral isolates. (B) Some, but not all, HEp-2 cell cultures infected with hMPV viral isolates showed an increased number of syncytia after treatment with HRA2pl. (C) Treatment with the HRA2pl peptide decreased the number of syncytia in Vero cells infected with hPIV-2 viral isolates. NS, Nonsignificant; *p< 0.05; **p< 0.01; ***p< 0.001.