Iota-carrageenan extracted from red algae is a potent inhibitor of SARS-CoV-2 infection in reconstituted human airway epithelia

Abstract

Iota-carrageenan (IC) nasal spray, a medical device approved for treating respiratory viral infections, has previously been shown to inhibit the ability of a variety of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), to enter and replicate in the cell by interfering with the virus binding to the cell surface. The aim of this study was to further investigate the efficacy and safety of IC in SARS-CoV-2 infection in advanced in vitro models of the human respiratory epithelium, the primary target and entry port for SARS-CoV-2. We extended the in vitro safety assessment of nebulized IC in a 3-dimensional model of reconstituted human bronchial epithelium, and we demonstrated the efficacy of IC in protecting reconstituted nasal epithelium against viral infection and replication of a patient-derived SARS-CoV-2 strain. The results obtained from these two advanced models of human respiratory tract epithelia confirm previous findings from in vitro SARS-CoV-2 infection assays and demonstrate that topically applied IC can effectively prevent SARS-CoV-2 infection and replication. Moreover, the absence of toxicity and functional and structural impairment of the mucociliary epithelium demonstrates that the nebulized IC is well tolerated.

Keywords: 3D, 3-dimensional; AE, after exposure; ALI, air–liquid interface; Air–liquid interface; BE, before exposure; Bronchial epithelium; CBF, ciliary beating frequency; COVID-19; COVID19, Coronavirus disease 2019; DMMB, Dimethylmethylene blue; IC, Iota-carrageenan; Iota-carrageenan; LDH, lactate dehydrogenase; MOI, multiplicity of infection; NHBE, normal human bronchial epithelial; Nasal epithelium; Nasal spray; PBS, phosphate-buffered saline; SARS-CoV-2; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SSPL, spike-pseudotyped lentivirus; TEER, transepithelial electrical resistance; hACE2, human angiotensin I-converting enzyme 2.

Fig. 1

Experimental design for assessing the safety of nebulized IC in bronchial cultures. TEER, transepithelial electrical resistance; CBF, ciliary beating frequency; IC, iota-carrageenan; LDH, lactate dehydrogenase.

Fig. 2

A) Mass (left y-axis) and concentration (right y-axis) of IC deposited in the apical compartment of bronchial epithelial cultures exposed to aerosolized IC. B) CBF and C) active area of ciliary beating before and 48 h after exposure to nebulized IC. Each condition was statistically compared with vehicle treatment by two-way analysis of variance with Dunnett's multiple comparison post-tests (Prism 9.0 GraphPad; *p < 0.05, **p < 0.01, ****p < 0.001). D) TEER before and 48 h after exposure to nebulized IC. E) LDH in medium of bronchial cultures 48 h after exposure to nebulized IC; 0% corresponds to the value measured in tissues exposed to PBS and 100% to the value measured in tissues incubated for 24 h with 2% Triton X-100. Data are presented as mean ± standard error of the mean for n = 3 independent inserts. BE, before exposure; AE, 48 h after exposure; IC, iota-carrageenan; PBS, phosphate-buffered saline; TEER, transepithelial electrical resistance; CBF, ciliary beating frequency; LDH, lactate dehydrogenase.

Fig. 3

Histological findings in bronchial epithelial cultures. The bronchial cultures were washed 3 times with PBS before being histologically processed and stained with hematoxylin, eosin, and Alcian blue 48 h after exposure to nebulized IC. Scale bar, 100 μm. IC, iota-carrageenan. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Antiviral activity of IC against SARS-CoV-2 infection in nasal epithelial (MucilAir™-Pool) cultures. A) Effect of IC on TEER before and 48 and 72 h after SARS-CoV-2 infection. B) Relative SARS-CoV-2 genome copy numbers on the apical side of cultures with 1-h pretreatment and repeated apical exposure to IC. As a reference, remdesivir (5 μM) was added to the basolateral medium. Data are presented as mean ± standard error of the mean for n = 12 independent inserts. Each condition was statistically compared with vehicle treatment by two-way analysis of variance with Dunnett's multiple comparison post-tests (Prism 9.0 GraphPad; **p < 0.01). IC, iota-carrageenan; TEER, transepithelial electrical resistance.