Inhibition of influenza A virus and SARS-CoV-2 infection or co-infection by griffithsin and griffithsin-based bivalent entry inhibitor

Abstract

Outbreaks of acute respiratory viral diseases, such as influenza and COVID-19 caused by influenza A virus (IAV) and SARS-CoV-2, pose a serious threat to global public health, economic security, and social stability. This calls for the development of broad-spectrum antivirals to prevent or treat infection or co-infection of IAV and SARS-CoV-2. Hemagglutinin (HA) on IAV and spike (S) protein on SARS-CoV-2, which contain various types of glycans, play crucial roles in mediating viral entry into host cells. Therefore, they are key targets for the development of carbohydrate-binding protein-based antivirals. This study demonstrated that griffithsin (GRFT) and the GRFT-based bivalent entry inhibitor GL25E (GRFT-L25-EK1) showed broad-spectrum antiviral effects against IAV infection in vitro by binding to HA in a carbohydrate-dependent manner and effectively protected mice from lethal IAV infection. Although both GRFT and GL25E could inhibit infection of SARS-CoV-2 Omicron variants, GL25E proved to be significantly more effective than GRFT and EK1 alone. Furthermore, GL25E effectively inhibited in vitro co-infection of IAV and SARS-CoV-2 and demonstrated good druggability, including favorable safety and stability profiles. These findings suggest that GL25E is a promising candidate for further development as a broad-spectrum antiviral drug for the prevention and treatment of infection or co-infection from IAV and SARS-CoV-2.IMPORTANCEInfluenza and COVID-19 are highly contagious respiratory illnesses caused by the influenza A virus (IAV) and SARS-CoV-2, respectively. IAV and SARS-CoV-2 co-infection exacerbates damage to lung tissue and leads to more severe clinical symptoms, thus calling for the development of broad-spectrum antivirals for combating IAV and SARS-CoV-2 infection or co-infection. Here we found that griffithsin (GRFT), a carbohydrate-binding protein, and GL25E, a recombinant protein consisting of GRFT, a 25 amino acid linker, and EK1, a broad-spectrum coronavirus inhibitor, could effectively inhibit IAV and SARS-CoV-2 infection and co-infection by targeting glycans on HA of IAV and spike (S) protein of SARS-CoV-2. GL25E is more effective than GRFT because GL25E can also interact with the HR1 domain in SARS-CoV-2 S protein. Furthermore, GL25E possesses favorable safety and stability profiles, suggesting that it is a promising candidate for development as a drug to prevent and treat IAV and SARS-CoV-2 infection or co-infection.

Keywords: SARS-CoV-2; co-infection; griffithsin; influenza A virus; respiratory viruses.

Fig 1

Inhibition of GRFT and GL25E on in vitro infection of divergent IAVs. (A–G) Inhibition of IAV H7N9 and H5N1 PsV infection was detected by luciferase assay. (H–L) Inhibition of infection of authentic IAVs, including A/Puerto Rico/8/1934(H1N1), A/California/04/2009(H1N1), A/Shanghai/37T/2009(H1N1), A/WSN/1933(H1N1), and A/Guizhou/54/1989(H3N2), was detected by plaque-reduction assay. Samples were tested in triplicate, and the experiment was repeated at least twice. Data from a representative experiment are expressed as means ± SD.

Fig 2

Prophylactic and therapeutic effect of GRFT or GL25E against A/Puerto Rico/8/1934 (H1N1) infection in C57BL/6J mice. Female C57BL/6J mice were intranasally administered with 10 mg/kg of GRFT or GL25E 30 min before or after intranasal challenge with 176 PFU of A/Puerto Rico/8/1934 (H1N1). Each group had 15 mice, including 6 mice for detection of body weight change and survival rate, 6 mice for evaluation of viral load, and 3 mice for histological study. (A–C) Body weight change (%) (A), survival rate (%) (B), and viral load (C) in mice of the prophylactic treatment group. (D–F) Body weight change (%) (D), survival rate (%) (E), and viral load (F) in mice of the therapeutic treatment group. Data are presented as means ± SD. (G) Representative photomicrographs of lung tissue in prophylactic group (hematoxylin and eosin, H&E staining). Scale bars = 1,000 µm (1×, left) and scale bar  = 50 µm (20×, right). (H) Histologic scores. Overall histologic score is calculated by adding up the scores for each individual criterion (hemorrhage, neutrophil infiltration, thickness of the alveolar wall, and atelectasis). Each sample was tested in triplicate, and the experiment was repeated at least twice. Data from a representative experiment are presented as means ± SD. A significant difference between groups was analyzed using one-way ANOVA. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 3

Mechanism of action of GL25E in inhibiting IAV infection. (A and B) Time-of-addition assay: MDCK cells were treated with GRFT (A) or GL25E (B) at the indicated time points before or after the addition of pseudotyped H5N1/QH. (C and D) Washout assay: MDCK cells were pretreated with 10 µmol/liter of GRFT or GL25E at 37° for 1 h before cells were washed with PBS to remove unbound inhibitor. Subsequently, cells were infected with pseudotyped IAV H5N1/QH. In the other group, inhibitor-pretreated or PBS-treated cells were not washed before the addition of IAV H5N1/QH PsV. The inhibitory activity of GRFT and GL25E on IAV PsV infection was assessed using a luciferase assay. (E) Analysis of expressed recombinant HA1 protein using SDS-PAGE. (F) Measurement of the binding of HA1 protein to GRFT or GL25E using enzyme-linked immunosorbent assay (ELISA). (G and H) Measurement of the effect of mannose on the binding of GRFT or GL25E to HA1 using ELISA. (I and J) Measurement of the effect of mannose on GRFT- or GL25E-mediated inhibition of H5N1/QH PsV infection using a luciferase assay. Each sample was tested in triplicate, and the experiment was repeated at least twice. Data from a representative experiment are presented as means ± SD. Statistical analysis was performed and analyzed using one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Fig 4

Inhibition of GRFT or GL25E on the co-infection of IAV and SARS-CoV-2. (A and B) Inhibition of GRFT or GL25E (EK1 as a control) against the infection of authentic SARS-CoV-2 Omicron variants BA.2.2 (A) and BA.5 (B) in Caco-2 cells using an RT-qPCR assay for N genes. (C) Diagram illustrating the procedure for co-infection. (D and E) Inhibition of IAV and SARS-CoV-2 co-infection in the mixture of A549 and Caco-2 cells (D) or in Calu-3 cells (E). A mixture of pseudotyped IAV H5N1/Thailand and SARS-CoV-2 D614G at 1:1 ratio was added to cells in the presence of serially diluted GRFT, GL25E, or EK1. After 12 h, the culture medium was replaced, and the cells were incubated for an additional 48 h. Luciferase assay was performed to evaluate the inhibitory activity of GRFT, GL25E, or EK1 against PsV infection. Each sample was tested in triplicate, and the experiment was repeated at least twice. Data from a representative experiment are presented as means ± SD.

Fig 5

In vitro and in vivo safety and thermal stability of GL25E. (A–E) Cytotoxicity of GL25E on MDCK, A549, Calu-3, Caco-2, and Vero-E6 cells, respectively. (F) Stability of GL25E stored at 4, 25, and 37°C for 1, 2, 3, and 4 weeks, respectively, as determined by testing their its inhibitory activity against A/H5N1/Thailand PsV infection. (G) Diagram of the in vivo safety experiment procedure. (H) Monitoring of murine body weight changes once daily for 15 days after intranasal administration of GL25E (10 mg/kg). (I) Histopathological changes in murine lungs, liver, and kidneys were observed through H&E staining after intranasal administration of GL25E (10 mg/kg) or PBS (as a control) on day 30 in mice (n = 3). (J and K) The levels of ALT (U/L) (J) and creatinine (K) in sera collected from mice on days 0, 1, 3, 5, and 7, respectively. Each sample was tested in triplicate, and the experiment was repeated at least twice. Data from a representative experiment are presented as means ± SD. “ns” denotes no significance.

Fig 6

Schematic illustration of the mechanism by which GRFT and GL25E inhibit mono- or co-infection of IAV and SARS-CoV-2. GRFT or GRFT part in GL25E binds to the glycans on HA1 of IAV, thereby inhibiting IAV entry into the host cell. GL25E inhibits SARS-CoV-2 infection by binding via its GRFT part to glycans on S1 subunit of SARS-CoV-2 S protein and interacting via its EK1 part with HR1 domain in S2 subunit of SARS-CoV-2 S protein. GRFT (PDB code: 7RID).