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. IMPORTANCE Influenza 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.


Potent inhibitory activity of GRFT and GL25E against pseudotyped and authentic IAV infection in vitro
To evaluate the effectiveness of GRFT and GL25E in inhibiting IAV infection, we con structed a series of HIV-based pseudoviruses (PsVs) bearing the HA protein of H5 and H7 subtypes of IAVs, including H5N1/Thailand, H5N1/QH, H5N1/Vietnam, H5N1/HK, H5N1/AH, H5N1/XJ, and H7N9/Shanghai.We found that both GRFT and GL25E exhibited potent inhibitory activity against infection of the above IAV PsVs with half maximal inhibitory concentration (IC 50 ) ranging from 39.9 to 556 nmol/liter and from 26.6 to 528 nmol/liter, respectively (Fig. 1A through G).
We then assessed the inhibitory activity of GRFT and GL25E on infection of authentic IAV, including H1 and H3 subtypes, such as A/Puerto Rico/8/1934(H1N1), A/California/07/2009(H1N1), A/Shanghai/37T/2009(H1N1), A/WSN/1933(H1N1), and A/ Guizhou/54/1989(H3N2).As shown in Fig. 1H through L, both GRFT and GL25E were effective in inhibiting authentic IAV infection in a dose-dependent manner with the IC 50 ranging from 168 to 625 nmol/liter and from 150 to 519 nmol/liter, respectively.However, EK1 alone exhibited no inhibitory activity against infection of either pseudoty ped or authentic IAVs at the concentration up to 10,000 nmol/liter.Further investigation revealed that both GRFT and GL25E effectively inhibited authentic IAV infection as determined by indirect immunofluorescence assay (Fig. S1) and Westen blot assay on MDCK, A549, and Calu-3 cells (Fig. S2).These findings indicate that both GRFT and GL25E exhibit inhibitory activity against IAV infection in vitro, possibly by targeting the HA of two major phylogenetic subgroups: group 1 (H1 and H5) and group 2 (H3 and H7).

Prophylactic or therapeutic effect of GRFT and GL25E on the protection of mice against challenge with IAV A/PR8/H1N1
We then determined whether GRFT and GL25E were effective in protecting mice from infection of IAV A/PR8/H1N1, a mouse-adapted strain of A/Puerto Rico/8/1934(H1N1).GRFT and GL25E as prophylactic or therapeutic agents diluted in PBS were intranasally administered to C57BL/6J mice at a dose of 10 mg/kg, either 0.5 h before or after IAV challenge for prevention or treatment, respectively, while mice receiving PBS only before or after IAV challenge and those receiving PBS only without IAV challenge were tested as vehicle control group and mock infection group, respectively.In the prophylactic experiment, all mice in the vehicle control group died on day 9 after infection and showed continuous weight loss starting from day 5 post-infection.However, pretreat ment with GRFT or GL25E delayed, or reversed, body weight loss and effectively protected the mice from death (Fig. 2A and B).In the therapeutic experiment, mice administered with GRFT or GL25E exhibited a 50% survival rate with a reduced body weight loss, while no mice in the vehicle control group survived by day 8 post-infection (Fig. 2D and E).To further confirm the protective effect, we also assessed viral load in the lungs using real-time PCR.As depicted in Fig. 2C and F, treatment with GRFT or GL25E significantly reduced viral load in the lung, and prophylactic effect was superior to the therapeutic effect.Additionally, mice treated with GRFT or GL25E exhibited only minor lung damage compared with mice in the vehicle control group in prophylactic group (Fig. 2G).No histopathological changes were observed in the mock-infected group, while mice in the vehicle control group displayed significantly increased histopathological changes in the lung with indistinct outlines of large areas of alveoli in the field of vision and structural disorder (red arrow).Some epithelial cells were detached from the bronchioles (blue arrow), and a few erythrocytes and foam cells were visible in the lumen (green arrow).A significant number of inflammatory cells infiltrated the tissue (yellow arrow).In contrast, mice in the GRFT and GL25E treatment groups showed 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.mildly abnormal structure with no apparent infiltration of inflammatory cells and slight thickening of the alveolar wall in the field of vision.Pathologists performed a blinded qualitative histological assessment of lung tissues, and the mean histological scores are presented in Fig. 2H.The GRFT-and GL25E-treatment groups had significantly lower scores compared with the vehicle control group (Table S1).These results indicate that both GRFT and GL25E possess inhibitory activity against IAV infection in vivo.

Inhibition of IAV infection by GRFT and GL25E through interfering with the viral entry stage
A time-of-addition assay was conducted to determine which stage in the IAV life cycle GRFT and GL25E may target.As shown in Fig. 3A and B, GRFT and GL25E exhibited over 70% inhibition of H5N1/QH PsV infection when the inhibitor was added to cells at −0.5, 0, 0.5, 1 and 2 h after IAV PsV infection, while their inhibitory activity decreased quickly at 4 and 6 h post-IAV PsV infection, suggesting that GRFT and GL25E inhibit IAV infection at the viral entry stage.To further elucidate whether GRFT and GL25E act on virus or cells, a washout assay was performed.As shown in Fig. 3C and D, no inhibition of H5N1/QH PsV infection was observed when the virus was added to cells after inhibitor-pretreated cells were washed, indicating that GRFT and GL25E inhibit virus infection by acting on the virus, not the host cells.
We then investigated the potential interaction between these inhibitors and HA1, considering that hemagglutinin 1 (HA1) plays an important role in IAV entry into host cells.Recombinant HA1 protein was expressed and purified, as previously described (30), and then identified using SDS-PAGE (Fig. 3E).An enzyme-linked immunosorbent assay (ELISA) was performed to assess the binding ability of GRFT or GL25E to HA1.As shown in 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. 3F, GRFT or GL25E bound to HA1 in a dose-dependent manner, confirming that HA1 is a target for these inhibitors.
We have previously demonstrated that GRFT and GL25E inhibit SARS-CoV-2 infection by binding the glycans in its S1 protein, demonstrating that mannose can attenuate antiviral activity of the glycan-binding proteins GRFT and GL25E (26,27).In this study, we evaluated whether mannose could interfere with the interaction between these inhibitors and HA1.As shown in Fig. 3G and H, mannose did exhibit dose-dependent reduction in the binding of GRFT or GL25E to HA1.Similarly, mannose attenuated the inhibitory effect of GRFT and GL25E on IAV PsV infection in a dose-dependent manner (Fig. 3I and J).These findings suggest that GRFT and GL25E inhibit IAV infection by binding to the glycans presented on HA1.

Efficacy of GL25E over that of GRFT in inhibiting IAV and SARS-CoV-2 coinfection
To determine whether GL25E is effective in inhibiting IAV and SARS-CoV-2 co-infection, we performed in vitro co-infection models (Fig. 4C).Briefly, GL25E at graded concentra tion was incubated with both pseudotyped IAV and SARS-CoV-2 before the addition of the inhibitor/virus mixture to cultured A549 cells susceptible to IAV and Caco-2 cells susceptible to SARS-CoV-2, or to cultured Calu-3 cells susceptible to both IAV and SARS-CoV-2.As shown in Fig. 4D, GL25E effectively inhibited the co-infection of pseudotyped IAV and SARS-CoV-2 in A549/Caco-2 cells with the IC 50 of 164 nmol/liter, while GRFT was less effective against IAV and SARS-CoV-2 in A549/Caco-2 cells with IC 50 of 972 nmol/liter.EK1 peptide exhibited less than 50% inhibition at the concentration up to 10,000 nmol/liter.Similarly, GL25E showed significant inhibition of IAV and SARS-CoV-2 co-infection in Calu-3 cells with IC 50 of 194 nmol/liter, whereas GRFT was much less potent with IC 50 of 1,257 nmol/liter.Again, EK1 showed no significant inhibition at the concentration up to 10,000 nmol/liter (Fig. 4E).

Good safety and stability profile of GL25E
Finally, we evaluated the in vitro and in vivo safety and stability of GL25E, as previously described (31).As shown in Fig. 5A through E, the viability of MDCK, A549, Calcu-3, Caco-2, and Vero-E6 cells remained above 80% after 48 h of treatment with GL25E at concentrations up to 10 µmol/liter.We then investigated the in vitro stability of GL25E by testing its inhibitory activity against IAV PsV infection after storage at 4, 25, and 37℃ for different durations.As shown in Fig. 5F, GL25E retained remarkable antiviral activity after storage at 4, 25, and 37°C for 1-4 weeks, respectively, confirming its high in vitro stability.Subsequently, we assessed in vivo safety in C57BL/6J mice receiving intranasal administration of GL25E in PBS (10 mg/kg) or PBS as control, as previously described (32).Body weight changes were monitored for the next 2 weeks, and serum samples were collected on days 0, 1, 3, 5, and 7 to detect the levels of alanine aminotransferase (ALT) and creatinine.On day 30, mice were euthanized for collection of lungs, livers, and kidneys for hematoxylin and eosin (H&E) staining (Fig. 5G).As shown in Fig. 5H through K, no significant differences were observed in body weight loss, histopathology, or serum levels of ALT and creatinine between mice administered with GL25E in PBS and PBS control, indicating that GL25E also possesses a satisfactory in vivo safety profile.
To detect the in vivo distribution and stability of GL25E, we intranasally administered 10 mg/kg of Cy5-GL25E or PBS as a control to C57BL/6J mice and monitored the distribution of Cy5-GL25E in the lung, kidney, heart, spleen, and liver at 3, 24, 48, 72, and 96 h, respectively, using the IVIS Lumina K Series III in vivo imaging system.As shown in Fig. S3A, the fluorescence signals of Cy5-GL25E were mainly observed in the lung after intranasal administration of GL25E up to 96 h post-treatment but not in other organs.After mice were dissected at 48 h after administration, the fluorescence in various organs was measured.As shown in Fig. S3B and S3C, the Cy5-GL25E fluorescence signals were predominantly concentrated in the lungs, while those in other organs were minimal, similar to those in the PBS control group.These results suggest that intranasally adminis tered GL25E is mainly distributed in lung for up to 96 h.

DISCUSSION
Measures implemented during the COVID-19 pandemic, such as lockdowns and facemask mandates, may make individuals gain "immunity debt, " thus becoming more susceptible to influenza virus infections (33,34).For example, a significant wave of influenza virus infections with a peak infection rate of 37.3% in 2023 was reported in China (35).Simultaneously, SARS-CoV-2 Omicron subvariants have continuously emerged with high immune evasion capacity and transmissibility, such as XBB.1.5,BA.2.86, and JN.1 (11,36).Even worse, prior infection with IAV can significantly exacer bate the infectivity and pathogenesis of SARS-CoV-2 in co-infected patients (14,15,37).These recent discoveries have intensified the need to develop effective and broad-spec trum antiviral agents to combat mono-and co-infection of influenza virus and SARS-CoV-2.
Currently, numerous antiviral drugs have been approved for the treatment or prevention of SARS-CoV-2 infection, including two classes of viral replication inhibitors, main protease (M pro ) and RNA-dependent RNA polymerase inhibitors, and one class of viral entry inhibitors, S protein-specific neutralizing antibodies (nAbs) (38).Because of their extensive S protein mutations, Omicron variants and subvariants have become resistant to most nAbs under emergency use authorization (39).Besides, these anti-SARS-CoV-2 nAbs are ineffective against IAV infection.Therefore, it is essential to develop broad-spectrum viral entry inhibitors with activity against co-infection of divergent IAV and SARS-CoV-2 strains.
We observed that the presence of mannose attenuated inhibitory activity against IAV infection, suggesting that the glycans displayed on HA1 were the target of GRFT.These findings highlight the potential of GRFT and GL25E as therapeutics and prophylactics for controlling influenza and COVID-19 pandemics.Interestingly, GL25E exhibited stronger inhibitory activity against SARS-CoV-2 variants in a carbohydrate-dependent manner.This confirms our hypothesis that the glycans displayed on S protein and HA could be promising targets for the development of antiviral drugs (Fig. 6).Antiviral strat egies commonly involve the utilization of vaccines and antiviral medications.Influenza virus vaccines can be effective in reducing morbidity and mortality, but their clinical use is limited because of the high variability of influenza viruses.For antiviral drugs, the Centers for Disease Control and Prevention recommends Oseltamivir phosphate, Zanamivir, Peramivir, and Baloxavir marboxil to treat influenza virus infection.However, the emergence of drug-resistant influenza strains can render these drugs ineffective (40,41).
Mechanistic studies have shown that GRFT and GL25E inhibit IAV and SARS-CoV-2 infection by blocking viral entry into the host cells in a mannose-dependent manner, suggesting that both GRFT and GL25E bind to and cross-link glycans in the HA of IAV, particularly HA1, and S protein of SARS-CoV-2, especially S1 subunit, on the surface of viral particles, preventing them from undergoing structural changes or accessing the cell surface receptor.The effectiveness of this approach is supported by the fact that a glycan-targeted nAb 2G12 has been shown to inhibit both HIV and IAV infection (42).GL25E is significantly more effective than GRFT in inhibiting the mono-and co-infection of IAV and SARS-CoV-2 because GL25E can target the glycans mainly in HA1 of IAV, S1 subunit of SARS-CoV-2, and HR1 domain in S2 subunit of SARS-CoV-2.Its inhibitory potency is generally unaffected by protein sequence variations in divergent IAV and SARS-CoV-2 strains.This unique characteristic of GL25E enhances its broad-spectrum activity against IAV and SARS-CoV-2 infection.Additionally, GL25E is cost-effective because it can be produced at a large scale using an Escherichia coli expression system.Moreover, GL25E exhibits excellent in vitro and in vivo safety and stability, along with good druggability.Therefore, GL25E is a promising candidate for further development as a broad-spectrum antiviral drug for the treatment and prevention of mono-or coinfection of IAV and SARS-CoV-2.
Live influenza viruses were propagated in MDCK cells, and virus titers were determined using a plaque formation assay.Authentic SARS-CoV-2 Omicron variants BA.2.2 and BA.5 were maintained in the BSL-3 Laboratory of Shanghai Medical College, Fudan University.The EK1 peptide used in this study was synthesized by Chengdu Shengnuo Biotechnol ogy Co., Ltd.(Chengdu, China), and its sequence was previously described (31).

Plaque reduction assay
Antiviral activity of GRFT and GL25E on IAV infection was measured using a PRA (47).Briefly, IAVs were treated with GRFT, GL25E, or EK1 peptide at the indicated concentra tion for 1 h at 37°C, and inhibitor-virus mixture was transferred to MDCK cells.At 2 h post-infection, supernatants were removed and covered with DMEM containing 1% low melting point agarose (Invitrogen, Carlsbad, CA, USA) and 1 µg/mL TPCK-trypsin.After 2-3 days, the cell monolayer was fixed and stained with 4% paraformaldehyde containing 0.5% crystal violet for 4 h, and the number of plaques was counted.

Inhibition of IAV PsV or SARS-CoV-2 PsV infection
The inhibition of infection by IAV PsVs or SARS-CoV-2 PsVs was performed as described previously (48,49).In brief, pseudotyped IAV or SARS-CoV-2 Omicron variant was mixed with an equal volume of the tested proteins or peptide at the indicated concentration and incubated for 30 min before being transferred to the target cells (MDCK cells for IAV PsVs and Caco-2 cells for SARS-CoV-2) for 12 h.Supernatants were removed, and target cells were cultured with fresh DMEM medium for 48 h.Target cells were lysed, and inhibitory activity was determined using a Luciferase Assay System (Promega, Madison, WI, USA).

Enzyme-linked immunosorbent assay
The interaction between HA1 protein and GRFT or GL25E was evaluated using ELISA as previously described (50).Plates were coated with GRFT, GL25E, and BSA in PBS and incubated at 4°C overnight and blocked with 2% gelatin at 37°C for 2 h.HA1 was added and incubated at 37°C for 1 h.Detection was done using HRP-conjugated goat anti-human IgG at 37°C for 1 h.Inhibition of GRFT/GL25E binding to HA1 by mannose was assessed by pre-treating GRFT or GL25E with mannose at indicated concentrations and then adding the mixture to plates coated with HA1 for 1 h.Detection was performed using HRP-conjugated anti-6 His antibody at 37°C for 1 h (27).

In vivo inhibition of authentic IAV infection
Female C57BL/6J mice (6-8 weeks old) were purchased from Charles River (Beijing, China) and divided into four groups (15 mice/group): mock-infection, vehicle, GRFT, and GL25E.Mice in the mock-infection group were not exposed to IAV, while those in the other groups were challenged with IAV as previously described (51).To evaluate the prophylactic or therapeutic effects of GRFT and GL25E, intranasal administration of GRFT or GL25E at 10 mg/kg, or PBS (vehicle) was performed before or after challenge with A/Puerto Rico/8/1934 (4.4 × 10 3 PFU/mL).Changes in body weight and survival rate of the mice were recorded daily up to 15 days after infection (n = 6).Three mice from each group were euthanized 7 days post-infection, and the lungs were removed and fixed in 4% paraformaldehyde at 4℃ for H&E staining and histological analysis based on the following indicators: alveolar edema, hemorrhage, neutrophil infiltration, hyaline membrane formation, thickness of the alveolar wall, and atelectasis.The scoring system assessed the level of damage on a scale ranging from 0 (not damage observed) to 3 (severe damage).Six mice from each group were euthanized for examination of viral titer in the lungs 7 days post-infection.Total RNA was extracted from the lung homogenates, and the levels of viral RNA were determined using a One-Step qRT-PCR kit (Takara Bio, Shiga, Japan) with primer sequences listed in Table S3.

Time-of-addition assay and washout assay
Time-of-addition assay was performed as described previously (52).Briefly, MDCK cells were seeded into wells of a 96-well plate.GRFT or GL25E was added at a final concentra tion of 10 µmol/liter either 0.5 h before or at 0, 0.5, 1, 2, 4, 6, and 8 h after the addition of H5N1/QH PsV.The inhibitory activity of GRFT or GL25E on virus infection was determined as described above.In the washout assay, MDCK cells were incubated with GRFT or GL25E at 10 µmol/liter at 37°C for 1 h.Cells were then washed with PBS to remove unbound GRFT or GL25E, followed by the addition of H5N1/QH PsVs.In the control group, the cells preincubated with GRFT or GL25E were not washed before the addition of virus.After 12 h, the medium was changed, and luciferase activity was measured as described above.

Inhibition of GRFT or GL25E on IAV and SARS-CoV-2 co-infection
To assess the inhibitory activity of GRFT or GL25E (EK1 as a control) on IAV PsV and SARS-CoV-2 PsV co-infection, A549 cells and Caco-2 cells were mixed at a 1:1 ratio and seeded in 96-well plates at 1 × 10 4 /well, while Calu-3 cells alone were seeded at 2 × 10 4 / well.After 24 h, a mixture of IAV H5N1/Thailand PsV and SARS-CoV-2 D614G PsV at a 1:1 ratio was added to the cells in the presence or absence of GRFT or GL25E.The inhibitory activity of GRFT, GL25E or EK1 on IAV and SARS-CoV-2 PsV infection was detected as described above.

Immunoblotting and immunofluorescence assays
Immunoblotting and immunofluorescence analysis were performed as previously (46,53).Briefly, MDCK, A549, and Calu-3 cells were seeded in wells of 6-well plates and 96-well plates, respectively.Followed by the addition of the mixture IAV at 0.2 MOI and an inhibitor at the indicated concentrations.For the immunoblotting, cells were lysed in 1 × RIPA buffer after washing with PBS.The protein concentration of total cell lysate was measured with BCA kit (Beyotime, Shanghai, China), separated via 12% SDS-PAGE, then and subjected to Western blot analysis.Commercial antibodies used for detecting IAV nucleoprotein (NP) included a rabbit polyclonal antibody against IAV NP (GeneTex, Irvine, CA, USA), a murine HRP-conjugated GAPDH monoclonal antibody (Proteintech, Wuhan, China), and an HRP-conjugated goat-anti-rabbit IgG (Dako, Copenhagen, Denmark).Image development was performed using an ECL Substrate kit (Tanon, Shanghai, China).For the immunofluorescence assay, cells were fixed and incubated with a primary antibody against IAV NP.A secondary antibody, goat-anti-rabbit IgG Alexa Fluor 488 (Abcam, Cambridge, UK), and DAPI (Invitrogen) were added sequentially according to the standard protocol.Cell imaging was observed and photographed using a fluorescence microscope (Invitrogen).

Inhibition of authentic SARS-CoV-2 Omicron variants
Briefly, Caco-2 and Vero-E6 cells were used as target cells for RT-qPCR (54) and plaquereduction assays (53), respectively.Cells were seeded in wells of a 96-well plate for 24 h before the addition of BA.2.2 or BA.5 variants (100 TCID 50 ) in the presence or absence of GRFT, GL25E, or EK1 at indicated concentrations for 30 min.Supernatants were collected from Caco-2 cell culture after 48 h for RT-qPCR analysis using specific primers of nucleoprotein (N) genes, while Vero-E6 cells were stained with rabbit anti-SARS-CoV-2 N antibody and goat-anti-rabbit IgG Alexa Fluor 488.Plaques were visualized and calculated using a CTL ImmunoSpot S6 Ultra-V analyzer.

Assays for cytotoxicity in vitro and safety in vivo
The CCK-8 kit (Dojindo, Kumamoto, Japan) was used to determine the cytotoxicity of GL25E.In brief, GL25E at a serial dilution was mixed with various cell lines (MDCK, A549, Calu-3, Caco-2, and Vero-E6) seeded in wells of 96-well plates.After 48 h, 10-fold diluted CCK-8 solution was added and incubated for 4 h.The absorbance at 450 nm was measured using a microplate reader (Tecan, USA).
The in vivo safety of GL25E in mice was assessed as previously described (32).Briefly, 8-week-old female C57BL/6J mice were randomly divided into two groups (n = 6) and received intranasal administration of PBS or a single-dose of GL25E diluted in PBS (10 mg/kg).Body weight changes were monitored for the next 2 weeks.ALT and creatinine levels in the serum of each group were measured using ALT and creatinine assay kits (NJJCBIO, Nanjing, China) at days 0, 1, 3, 5, and 7.After 30 days of administra tion, the mice in each group were euthanized, and their lungs, livers, and kidneys were collected for H&E staining.

In vivo fluorescence imaging
GL25E protein was labeled with NHS ester following instructions in the manual of APExBIO (Houston, TX, USA).Eight-week-old female C57BL/6J mice were randomly divided into two groups.The first group (n = 6) received 10 mg/kg of Cy5-GL25E in PBS through nasal administration, while the second group (n = 3) received PBS as a control for measuring background fluorescence.The distribution of Cy5-GL25E was monitored at different time points (3,24,48,72, and 96 h) using the IVIS Lumina K Series III in vivo imaging system (PerkinElmer, Waltham, MA, USA).After 48 h, mice were euthanized using isoflurane inhalation, and their lungs, livers, kidneys, spleens, and hearts were obtained for imaging (n = 3).The relevant radiant efficiency (Ps −1 cm −2 sr −1 ) (μW −1 cm 2 ) was calculated using Living Image 4.4 software (55).

Statistical analysis
Data were analyzed using GraphPad Prism 8.0, and the results were presented as the mean ± standard deviation.Statistical analysis was conducted using GraphPad Prism 8.0 with one-way ANOVA and a Neuman-Keuls post hoc analysis or Student's t-test, depending on the situation.Statistical significance was defined as P < 0.05.

FIG 2
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).

FIG 3
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.

FIG 4
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
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
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).

TABLE 1
Inhibitory activity of GRFT, GL25E, and EK1 against infection of SARS-CoV-2 Omicron variant PsV a a IC 50 : half maximal inhibitory concentration.