key: cord-0895522-9fjm9rqv
authors: Varese, Augusto; Ceballos, Ana; Palacios, Carlos; Figueroa, Juan Manuel; Dugour, Andrea Vanesa
title: Iota-carrageenan prevents the replication of SARS-CoV-2 on an in vitro respiratory epithelium model
date: 2021-04-27
journal: bioRxiv
DOI: 10.1101/2021.04.27.441512
sha: f527e0c3d49a009c89b079d09c83e0fcf2f89d35
doc_id: 895522
cord_uid: 9fjm9rqv

There are, except for remdesivir, no approved antivirals for the treatment or prevention of SARS-CoV-2 infections. Iota-carrageenan formulated into a nasal spray has already been proven safe and effective in viral respiratory infections. We explored this antiviral activity in Calu-3, a human respiratory model cell line. A formula of iota-carrageenan and sodium chloride, as a nasal spray, already approved for human use, effectively inhibited SARS-CoV-2 infection in vitro, providing a more substantial reference for further clinical studies or developments.

Introduction 20

The severe acute respiratory coronavirus 2 (SARS-CoV-2) is responsible for the currently ongoing 21 pandemic coronavirus disease (COVID-19), counting more than 144.878.978 confirmed cases and 22 more than 3.075.042 deaths worldwide by April 23, 2021 . There are still no 23 adequate therapeutic or preventive medicines for COVID-19; repurposing established medications 24 with recognized safety profiles is a possible approach for preventing or treating the disease and 25

shortening the time-consuming drug development stages. 26

During the first days of the infection, the virus replicates mainly in the nasal cavity and the 27 nasopharynx; therefore, nasal sprays with antiviral activity would reduce the viral load in these 28

cavities. 29

Marine-derived polysaccharides, such as carrageenans, are a family of linear sulfated polysaccharides 30 extracted from red seaweeds, widely used as thickening agents and stabilizers for food. Besides these 31

properties, the iota-carrageenan demonstrated antiviral activity against several viruses, including 32 respiratory viruses such as human rhinovirus, influenza A H1N1, and common cold coronavirus 33 (Grassauer et The kinetics of SARS-CoV-2 infection show differences between Vero E6 and Calu-3 cells, most 58

probably related to the differential expression of the angiotensin-converting enzyme 2 (ACE2) entry 59 receptor and other facilitating molecules like the cellular serine protease TMPRSS2. Calu-3 cells 60 express ACE2 on the apical membrane domain and are infected by SARS-CoV-2 via this route. The 61

higher number of Vero E6 infected cells is also associated with the differences in viral entry pathway 62 and the expression of pro-apoptotic proteins, which increased drastically in these cells but not in 63

Calu-3 cells ( Aires. 84

Solutions with iota-carrageenan and sodium chloride were prepared using a sterile nasal spray for 86 therapeutic use. All the formulations and placebos were prepared at Laboratorio Pablo Cassará S.R.L. 87

(Argentina) under aseptic conditions. The composition of active and placebo formulations is depicted 88

in Table 1 . 89

To determine antiviral efficacy of formulations by titer reduction assay, sample formulations were 90 used at a final iota-carrageenan concentration of 600 µg/ml; 60 µg/ml, 6 µg/ml, 0.6 µg/ml and 0,06 91 µg/ml. An equivalent concentration of placebos was used for titer reduction assay as controls. 92

Calu-3 cells were seeded in 96-well tissue culture microplates at 3x10 4 

In the cellular viability assays, One-way ANOVA followed by Dunnett's multiple comparisons test 115 was performed, and in the infection assays, the results were analyzed statistically by two-way 116 ANOVA, both using GraphPad Prism version 9.1.0, GraphPad Software, San Diego, California USA, 117 www.graphpad.com. 118

3

Results 119

The antiviral effects of iota-carrageenan on SARS-CoV-2 were tested in a dose-dependent manner. In 120 the first set of experiments, Vero E6 cells were pre-treated with different iota-carrageenan 121 concentrations (600 µg/ml to 0.06 µg/ml), and cell viability was quantified ( Figure 1A ). No 122 difference in cell viability was observed in iota-carrageenan treated cells compared to vehicle-treated 123 control cells. 124

Next, Vero cells were pre-treated with iota-carrageenan (600 µg/ml to 0.06 µg/ml) for 2 h and then 125

infected with SARS-CoV-2 (MOI: 0.01), after that, cells were washed to remove the viral inoculum, 126

and fresh medium was added. Forty-eight hours later, supernatants were harvest. The SARS-CoV-2 127 production was evaluated by adding the supernatants to Vero E6 cells for 1 hour. After incubation, 128 the inoculum was removed, and monolayers were incubated at 37 °C for 72 hours. Then cells were 129 fixed and stained with crystal violet. Virus endpoint titer was determined by Reed-Muench formula 130

and expressed as TCID50/ml. Our results showed that iota-carrageenan markedly inhibits SARS-131

CoV-2 production in a dose-dependent manner (Figure 1 , B and C). No antiviral activity was only 132 observed at the lowest concentration of iota-carrageenan (0.06 µg/ml). Lastly, there was no reduction 133 in virus production with vehicle formulation, suggesting that the iota-carrageenan, not the sample 134 excipient components, inhibited the SARS-CoV-2 replication ( Figure 1B) . Finally, Figure 1C shows 135 that inhibition of viral production is also observed when pretreatment with carrageenan is applied to 136 the cells, followed by an infection at a higher MOI (0.1). 137 Calu-3 studies. 148

When considering Vero E6 as a model, it should consider their deficient expression ACE2 and 149 TMPRSS2 and the non-specific endocytic viral uptake mechanisms responsible for viral entry in this 150 cell line. Calu-3 cells, besides of the exposed before, are very similar to primary cultures of bronchial 151 epithelium obtained by biopsy or surgery; it develops the characteristic tight junctions present in the 152 respiratory epithelium, expression of cystic fibrosis transmembrane conductance regulator (CFTR) 153 chloride channels, capacity for the secretion of mucus and proteins towards the apical end and 154 exchange of water and electrolytes (Duszyk, 2001; Zhu et al., 2008) . 155

In our research, we used dilutions of a commercially available iota-carrageenan spray. The 156

concentrations found to be active in vitro, as shown in previous (Bansal et al., 2020) , and in the 157 current work, are those that would be achieved using the spray according to the approved dosage. 158

These results are supporting the efficacy of this spray in the prevention of COVID-19 in a clinical 159 trial recently carried out with frontline healthcare personnel exposed to SARS-CoV-2 (Figueroa et  160 al., 2021). 161

In summary, our results confirm that a formulation of iota-carrageenan and sodium chloride available 163 as a nasal spray effectively inhibited SARS-CoV-2 infection in vitro in human respiratory epithelial 164 cell line culture, strengthening the hypothesis that a nasal spray with iota-carrageenan may be helpful 165

in the prevention or treatment of COVID-19 and reinforces the interest in the development of clinical 166 trials on this topic. 167

6

Conflict of Interest 168

The authors declare that the research was conducted in the absence of any commercial or financial 169 relationships that could be construed as a potential conflict of interest. (600 µg/mL to 0 µg/mL) for 48 h at 37°C. After incubation, cellular viability was analyzed, and no 185 statistically significant difference was found between the groups compared to the vehicle control 186

group. Data are expressed as mean ± SD derived from three independent experiments. B & C) 187

Infection assays. Calu-3 cells were pre-treated with iota-carrageenan or placebo (600 µg/mL to 0 188 µg/mL) for 1 h. After one hour of pretreatment, cells were infected, in two different conditions MOI: Iota-carrageenan 1.7 mg/mL -Sodium Chloride 9 mg/mL 9 mg/mL pH adjusted to 6.00 -7.00 6.00 -7.00

Replication Kinetics of Severe Acute Respiratory Syndrome 198 Coronavirus 2

Iota-carrageenan and Xylitol inhibit SARS-CoV-2 in cell culture

Vero cell technology 203 for rapid development of inactivated whole virus vaccines for emerging viral diseases

Application prospect of polysaccharides in the 206 development of anti-novel coronavirus drugs and vaccines

An interactive web-based dashboard to track COVID-19 in 209 real time

CFTR and lysozyme secretion in human airway epithelial cells

Inhibition of SARS-CoV-2 by type I and type III interferons

Efficacy of a nasal spray containing Iota-Carrageenan in the prophylaxis of COVID-19 in 217 hospital personnel dedicated to patients care with COVID-19 disease A pragmatic multicenter, 218 randomized, double-blind, placebo-controlled trial

Antiviral Activity of Sulfated Polysaccharides 221 Carrageenan from Some Marine Seaweeds

Iota-Carrageenan is a potent inhibitor of rhinovirus infection

IL-6: 227 relevance for immunopathology of SARS-CoV-2

Antiviral activity of sulfated polysaccharides from marine 230 algae and its application in combating COVID-19: Mini review

Chloroquine does not inhibit infection of human lung cells with SARS-CoV-2

Antiviral Compound from the Pandemic Response Box that Efficiently Inhibits SARS-CoV-2 237 Infection In Vitro

Carrageenan nasal spray in virus confirmed common cold: individual patient data analysis 240 of two randomized controlled trials

Iota-Carrageenan Is a Potent Inhibitor of Influenza A Virus Infection

SARS-CoV-2 in-245 vitro neutralization assay reveals inhibition of virus entry by iota-carrageenan

The Intranasal Application of Zanamivir and Carrageenan Is Synergistically Active against 249 Influenza A Virus in the Murine Model

SARS-CoV-2 tropism, entry, replication, and propagation: Considerations for drug discovery and 253 development

The Genome 255 Landscape of the African Green Monkey Kidney-Derived Vero Cell Line

Differential Signaling 258 and Virus Production in Calu-3 Cells and Vero Cells upon SARS-CoV-2 Infection

Tropism of SARS-CoV-2 in commonly used laboratory cell lines and their proteomic landscape 262 during infection

Suramin Inhibits SARS-CoV-2 Infection in Cell Culture by Interfering with Early Steps of 265 the Replication Cycle

Inhibitory activities of 267 marine sulfated polysaccharides against SARS-CoV-2

The Anticoagulant Nafamostat Potently Inhibits SARS-CoV-2 S Protein-Mediated Fusion 271 in a Cell Fusion Assay System and Viral Infection In Vitro in a Cell-Type-Dependent Manner

Cultured Human Airway Epithelial Cells (Calu-3): 274 A Model of Human Respiratory Function, Structure, and Inflammatory Responses

Effects of oxygen 277 concentration and exposure time on cultured human airway epithelial cells