Griffithsin and Carrageenan Combination Results in Antiviral Synergy against SARS-CoV-1 and 2 in a Pseudoviral Model

Over 182 million confirmed cases of COVID-19 and more than 4 million deaths have been reported to date around the world. It is essential to identify broad-spectrum antiviral agents that may prevent or treat infections by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) but also by other coronaviruses that may jump the species barrier in the future. We evaluated the antiviral selectivity of griffithsin and sulfated and non-sulfated polysaccharides against SARS-CoV-1 and SARS-CoV-2 using a cytotoxicity assay and a cell-based pseudoviral model. The half-maximal cytotoxic concentration (CC50) and half-maximal effective concentration (EC50) were determined for each compound, using a dose-response-inhibition analysis on GraphPad Prism v9.0.2 software (San Diego, CA, USA). The therapeutic index (TI = CC50/EC50) was calculated for each compound. The potential synergistic, additive, or antagonistic effect of different compound combinations was determined by CalcuSyn v1 software (Biosoft, Cambridge, UK), which estimated the combination index (CI) values. Iota and lambda carrageenan showed the most potent antiviral activity (EC50 between 3.2 and 7.5 µg/mL). Carrageenan and griffithsin combinations exhibited synergistic activity (EC50 between 0.2 and 3.8 µg/mL; combination index <1), including against recent SARS-CoV-2 mutations. The griffithsin and carrageenan combination is a promising candidate to prevent or treat infections by SARS-CoV-1 and SARS-CoV-2.


Introduction
The emergence of a novel coronavirus, quickly escalated into the current pandemic that was declared on 11 March 2020, by the World Health Organization. The pandemic has placed healthcare systems under extreme pressure; globally, over 182 million confirmed cases of COVID-19 and more than 4 million deaths have been reported to date [1]. The novel virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belongs to the Coronaviridae family. This family includes SARS-CoV-1 and MERS-CoV, two zoonotic viruses that emerged in 2003 and 2012, respectively [2]. Although highly effective vaccines targeting SARS-CoV-2 spike proteins have been approved for emergency use by multiple stringent regulatory authorities, accumulation of mutations in the spike protein may allow the virus to be transmitted more effectively and, in the worst-case scenario, evade the immune response triggered by vaccines [3][4][5][6].
The coronaviruses have shown an impressive ability to jump the species barrier, and more coronaviruses may migrate from animal reservoirs to humans in the future. For these reasons, it is essential to identify broad-spectrum antiviral agents that inhibit different coronaviruses and help to prevent/treat the diseases caused by these viruses. SARS-CoV-1 and SARS-CoV-2 have a similar natural history of infection; both enter the upper respiratory tract and infect the epithelial cells lining the respiratory tract. They enter target cells by binding of their surface spike proteins to human angiotensin-converting enzyme 2 (ACE2), the primary viral receptor, present on the surface of target cells [7]. Therefore, antiviral agents that target SARS-CoV-1 and SARS-CoV-2 entry have the potential to prevent/treat these infections.
Naturally occurring antiviral agents, such as griffithsin (GRFT, Figure 1A), an antiviral lectin, and carrageenan (CG, Figure 1B), a sulfated polysaccharide, can target a wide range of enveloped viruses [8]. Importantly, GRFT has previously been reported to have broadspectrum in vitro activity against Coronaviridae and in vivo activity against SARS-CoV-1 in a mouse model system following intranasal administration [9]. Similarly, the activity of sulfated polysaccharides against SARS-CoV-2 is documented in the literature [10,11]. GRFT is a homodimeric lectin of 121 aminoacids and six carbohydrate-binding sites with high affinity for high mannose arrays. These mannose arrays are frequently found in viral spikes of important pathogens like HIV, HSV, hepatitis C virus (HCV), ebola virus, and members of the Coronaviridae family [13]. The ability to block HIV in vitro, at picomolar concentrations, makes GRFT one of the most potent molecules inhibiting HIV replication [14]. Although EC 50 values are higher against coronaviruses, GRFT is potent enough to make this naturally occurring agent a promising candidate to fight the current SARS-CoV-2 pandemic. In addition to the drug potency, GRFT is poorly immunogenic, and several studies in animal models have shown its excellent safety profile [15]. CGs are polysaccharides isolated from red seaweed; its broad antiviral spectrum includes viruses like HSV, rhinoviruses, and coronaviruses. The antiviral activity is well-documented against HPV and is probably the most potent anti-HPV agent reported in the literature [16,17]. Several preclinical and clinical studies have shown its excellent safety profile after vaginal and respiratory routes of administration [18,19]. Additionally, CG is Generally Recognized as Safe (GRAS) by the Food and Drug Administration and is a common food additive [20]. Herein, we further explore the potential antiviral selectivity of GRFT, non-sulfated and sulfated polysaccharides, and combinations thereof against SARS-CoV-1 and SARS-CoV-2. Table 1 shows the half-maximal cytotoxic concentration (CC 50 ), half-maximal effective concentration (EC 50 ), and therapeutic index (TI) values for all the compounds tested. GRFT showed broad-spectrum antiviral activity against SARS-CoV-1 and 2. While the polysaccharides with selective antiviral activity were all sulfated, the sulfate group's presence is not a determining factor of the efficacy; some sulfated polysaccharides, such as heparin, heparan sulfate, fucoidan, and chondroitin sulfate did not show antiviral activity against SARS-CoV-1 or SARS-CoV-2. Among the polysaccharides, ι-CG and λ-CG had the best activity, with EC 50 values below 7.5 µg/mL. Therefore, we focused on ι-CG and λ-CG, evaluating their combination with GRFT. Previous studies have shown the GRFT and CG combination results in synergistic or additive effects against different viruses including HPV and HSV [8]. The potential synergistic effect of the GRFT and CG combination against SARS-CoV-1 and SARS-CoV-2 was determined by CalcuSyn software (Biosoft, Cambridge), which estimated the combination index (CI) values. As shown in Table 2, ι-CG and λ-CG inhibited entry of SARS-CoV-1 and all SARS-CoV-2 PsVs with lower EC 50 values (between 3.2 and 7.5 µg/mL) than those shown by GRFT (between 12.5 and 37.6 µg/mL). The EC 50 of GRFT against all three SARS-CoV-2 PsVs was larger when compared to SARS-CoV-1 PsV. However, some of the most relevant mutations recently identified in the SARS-CoV-2 spike protein (D614G, K1417N/E484K/N501Y), did not significantly impact the susceptibility of these PsVs to GRFT. Interestingly, GRFT/CG combinations showed more potent antiviral activity, specially at a 5:1 ratio, as indicated by the lower EC 50 values in Table 2. The data also revealed their synergistic activity, based on CI values below 1.

Discussions
This synergistic in vitro activity of GRFT and CG suggests that formulations delivering combinations of GRFT and CG might be useful in preventing or treating infections caused by SARS-CoV-1 or SARS-CoV-2. In addition to the potent activity of GRFT and CG against SARS-CoV-2, CG has been shown to have activity against the common cold [21], which is caused by a variety of respiratory viruses, including coronaviruses. Clinical studies show significant reduction in severity of common cold symptoms in subjects that administered CG nasally. Furthermore, data in mice inoculated intranasally with influenza A virus in the presence of ι-CG, demonstrated potent activity of CG against influenza. This activity is mediated through direct binding of CG to viral particles, which inhibits viral adsorption and internalization [22].
Importantly, in vivo studies will still be required to confirm this in vitro synergistic activity in animal models. Pharmacokinetic and pharmacodynamic studies will be essential to understand the potential of this combination [23]. Our formulation will require topical administration through the upper respiratory tract contrary to other antivirals currently used to treat SARS-CoV-2 infections that require intravenous (IV) administration. One of these antivirals is remdesivir, an inhibitor of the viral RNA-dependent RNA polymerase (RdRp) that requires daily IV doses of 100 or 200 mg [24]. The GRFT and CG combination has the advantage of using two naturally occurring agents whose safety has been thoroughly studied in different animal models and clinical studies [13,15,18,25,26]. The mode of action of these molecules does not require systemic adsorption, and topical application in the upper respiratory tract will interfere with the coronaviruses' attachment and entry to epithelial cells.

Conclusions
These results strongly suggest that a GRFT/CG combination could provide broadspectrum antiviral activity targeting different respiratory viruses, including a range of coronaviruses. The extensive preclinical safety data available for the GRFT and CG combination [8,25] and the results shown herein support testing this combination in preclinical efficacy and toxicology models and its subsequent clinical evaluation for delivery in the upper (and possibly lower) respiratory tract.

Cytotoxicity and Antiviral Assay
The cytotoxicity and antiviral activity were determined using the XTT [26] and the cell-based pseudoviral entry assay, respectively. Briefly, different concentrations of each compound (Table 1) were added in triplicate to HeLa ACE-2 cells seeded in clear bottom 96well microplates and then incubated at 37 • C, 5% CO 2 , and 98% humidity for 72 h. Tween 20 (Sigma Aldrich, St. Louis, MO, USA) was used as a positive control for cytotoxicity. XTT (ThermoFisher Scientific, Waltham, MA, USA) was added to all wells after 72-h incubation, and the absorbance was measured at 450 nm using a Spectramax iD3 (Molecular Devices, San Jose, CA, USA). The antiviral activity of the same compounds tested in the XTT assay was evaluated using the cell-based pseudoviral entry assay with SARS-CoV-1 PsV and SARS-CoV-2 Wuhan PsV. The same concentrations/replicates of each compound tested in the XTT assay were challenged (co-treatment) with SARS-CoV-1 PsV or SARS-CoV-2 Wuhan PsV in HeLa ACE-2 cell monolayers seeded in white opaque 96-well microplates. The plates were incubated at 37 • C, 5% CO 2 , and 98% humidity for 72 h. The TurboLuc™ Luciferase One-Step Glow Assay (ThermoFisher Scientific, Waltham, MA, USA) was used to determine the percentage entry of the PsVs in the presence of each compound concentration versus the virus control. The half-maximal cytotoxic concentration (CC 50 ) and half-maximal effective concentration (EC 50 ) were determined for each compound, using a dose-response-inhibition analysis on GraphPad Prism v9.0.2 software (San Diego, CA, USA). The therapeutic index (TI = CC 50 /EC 50 ) was calculated for each compound.

Combination Studies
The potential synergistic, additive, or antagonistic effect of different compounds combinations was determined by CalcuSyn v1 software (Biosoft, Cambridge, UK), which estimated the combination index (CI) values. For this purpose, equipotential combinations of GRFT and CG were evaluated in this experiment. Based on the EC 50 values of each compound, ratios of 1:3 or 1:5 (CG:GRFT) were determined as optimal. Each compound alone or in combination was tested against SARS-CoV-1 PsV, SARS-CoV-2 Wuhan PsV, SARS-CoV-2 D614G PsV, and SARS-CoV-2 K1417N/E484K/N501Y PsV. The cytotoxicity and antiviral assay described in the previous section were used to evaluate the toxicity and efficacy of these combinations. The inhibitory effects for each drug alone or in combination were transferred to CalcuSyn software to estimate CI values. All compounds and combinations were tested in at least two independent experiments.