key: cord-0868458-ihqujl81
authors: Kocher, Jacob; Arwood, Leslee; Roberts, Rachel C.; Henson, Ibrahim; Annas, Abigail; Emerson, David; Stasko, Nathan; Fulcher, M. Leslie; Brotton, Marisa; Randell, Scott H.; Cockrell, Adam S.
title: Visible blue light inactivates SARS-CoV-2 variants and inhibits Delta replication in differentiated human airway epithelia
date: 2022-01-31
journal: bioRxiv
DOI: 10.1101/2022.01.25.477616
sha: 9d43eb7bc696c76df500ed38760ed8662b979f50
doc_id: 868458
cord_uid: ihqujl81

The emergence of SARS-CoV-2 variants that evade host immune responses has prolonged the COVID-19 pandemic. Thus, the development of an efficacious, variant-agnostic therapeutic for the treatment of early SARS-CoV-2 infection would help reduce global health and economic burdens. Visible light therapy has the potential to fill these gaps. In this study, visible blue light centered around 425 nm efficiently inactivated SARS-CoV-2 variants in cell-free suspensions and in a translationally relevant well-differentiated tissue model of the human large airway. Specifically, 425 nm light inactivated cell-free SARS-CoV-2 variants Alpha, Beta, Delta, Gamma, Lambda, and Omicron by up to 99.99% in a dose-dependent manner, while the monoclonal antibody bamlanivimab did not neutralize the Beta, Delta, and Gamma variants. Further, we observed that 425 nm light reduced virus binding to host ACE-2 receptor and limited viral entry to host cells in vitro. Further, the twice daily administration of 32 J/cm2 of 425 nm light for three days reduced infectious SARS-CoV-2 Beta and Delta variants by >99.99% in human airway models when dosing began during the early stages of infection. In more established infections, logarithmic reductions of infectious Beta and Delta titers were observed using the same dosing regimen. Finally, we demonstrated that the 425 nm dosing regimen was well-tolerated by the large airway tissue model. Our results indicate that blue light therapy has the potential to lead to a well-tolerated and variant-agnostic countermeasure against COVID-19.

In late 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the 34 causative agent of Coronavirus Disease 2019 , emerged in Wuhan, China and rapidly 35 spread around the globe, resulting in nearly 237 million confirmed cases and five million deaths 36 (Ritchie et al., 2020) . Due to hot spots of uncontrolled spread, novel variants have emerged 37 displaying various combinations of increased replication, increased virulence, increased 38 transmission, and the ability to evade immune response from previous infections or vaccination 39 (Harvey et al., 2021; Krause et al., 2021) . For example, the Beta variant was notable for its ability 40 to evade monoclonal antibodies and serum neutralizing antibody responses (Wang et al., 2021) . 41

Similarly, the SARS-CoV-2 Delta and Omicron variants demonstrated combinations of increased 42 transmission, virulence, and immune evasion and rapidly became the dominant global strains in 43 early and late 2021, respectively, unleashing fresh "waves" of infections, hospitalizations, 44 mortality, and economic instability (Y. Liu et al., 2021; Planas et al., 2021; Pouwels et al., 2021; 45 Ren et al., 2022; Xu et al., 2021) . Rising COVID-19 cases in regions across the world provides 46 ample opportunity for new variants (e.g. Lambda and Mu) to emerge and further threaten global 47 2021). Thus, bamlanivimab was included as a comparison to illustrate the evasion ability of 163 replication-competent SARS-CoV-2 variants in our laboratory. In the present study, we observed 164 that Beta, Delta, and Gamma evaded neutralization by bamlanivimab, but WA1 and Alpha did 165 not, which is consistent with previous reports (Planas et al., 2021; Widera et al., 2021) . When 166 comparing the PRNT50 and PRNT90 titers for 425 nm light and bamlanivimab ( 

To investigate the mechanism of 425 nm light inactivation of cell-free SARS-CoV-2, cell-173 free SARS-CoV-2 Beta was illuminated with two doses of 425 nm light and its ability to bind ACE-174 2 and enter host cells was assessed ( Figure 4 ). We selected a non-virucidal dose (15 J/cm 2 ) and 175 a high dose 50% above the reported virucidal dose (90 J/cm 2 ) to ensure complete inactivation for 176 these assays. To determine if illuminated virus maintained ACE-2 binding integrity, we conducted 177 a human ACE-2 receptor-ligand binding assay ( Figure 4A ). Illumination with 425 nm light reduced 178 SARS-CoV-2 Beta binding to ACE-2 in a dose-dependent manner, as 15 J/cm 2 reduced binding 179 by ~80% and 90 J/cm 2 eliminated all ACE-2 binding. 180

Using the same light doses, we inoculated Vero E6 cells with virus that had been 181 previously exposed to blue light and evaluated cell-associated SARS-CoV-2 viral replication via 182 N1 qRT-PCR at 3 hpi ( Figure 4B ) and 24 hpi ( Figure 4C ). At 3 hpi, both doses significantly reduced 183 detectable viral RNA compared to the unilluminated control. However, at 24 hpi, viral RNA from 184 15 J/cm 2 had similar amounts of viral RNA as cells inoculated with unilluminated virus 185 suspensions, suggesting that, while viral binding is reduced, these virions are still capable of 186 undergoing replication. Conversely, SARS-CoV-2 illuminated with 90 J/cm 2 of 425 nm light had significantly lower amounts of detectable viral RNA and did not change significantly from 3 hpi to 188 24 hpi, indicating impaired viral entry into the host cell following complete inactivation. Gene 189 expression normalized to host RNaseP confirmed these results; 15 J/cm 2 reduced detectable 190 RNA by 2 logs at 3 hpi and 90 J/cm 2 reduced detectable RNA by 2 logs and 6 logs at 3 hpi and 191 24 hpi, respectively ( Figure 4D ). We observed similar trends with the N2 qRT-PCR 192 2 replicated more consistently and to higher peak titers in the DD065Q model (UNC MLI) than in 201 the AIR-100 and TBE-14 EpiAirway TM models. Additionally, the inhibition of WA1 replication 202 following exposure to 425 nm light was evaluated in two separate model systems . In the TBE-14 model, 32 J/cm 2 reduced WA1 titers below the limit of detection compared 204 to the 2 log10 reduction observed in the DD065Q model. Since SARS-CoV-2 variants have 205 demonstrated increased replication in vitro and in vivo (Arora et al., 2021; Cheng et al., 2021; 206 Plante et al., 2021; Touret et al., 2021b) , potentially narrowing the therapeutic window of new 207 countermeasures, we selected the more stringent DD065Q model for the studies described 208

herein. Thus, we treated SARS-CoV-2 Beta-infected tissues (MOI 0.1) with 32 J/cm 2 of 425 nm 209 light once daily (QD) or twice daily (BID) starting at 3 hpi for three days ( Figure 5A ). While the QD 210 regimen reduced titers by >1 log10 at 72 hpi, the BID regimen reduced titers >4 log10 at 72 hpi 211 ( Figure 5B ). Importantly, the SARS-CoV-2 titers in BID-treated tissues decreased from 24 hpi to 212 72 hpi, indicating the inhibition of the SARS-CoV-2 Beta replication. Additionally, we observed no light-induced cytotoxicity in time-matched, uninfected tissues after 3 days of repeat dosing ( Figure  214 5C). These results demonstrate that a BID, but not QD, dosing regimen with 32 J/cm 2 of 425 nm 215 light is sufficient to inhibit SARS-CoV-2 Beta in a well-differentiated airway tissue model. 216

We next investigated whether the 32 J/cm 2 BID therapeutic approach was sufficient to 219 control SARS-CoV-2 Delta infection at multiple starting infectious titers (MOIs 0.1, 0.01, and 220 0.001) in the same model ( Figure 6 ). Concordant with Beta, 32 J/cm 2 BID reduced Delta (MOI 221 0.1) infectious titers by >4 log10 at 72 hpi; infectious Delta titers also declined from 24 hpi to 72 222 hpi ( Figure 6A ). At the lower MOIs, 425 nm light dramatically reduced infectious SARS-CoV-2 223

Delta after 3 days of twice daily repeat dosing (below limit of detection) ( Figures 6B and 6C) . 224

The effects observed following administration of blue light during early infection (3 hpi The rapid development and deployment of vaccines, improved standards of care, and 237

increased focus on therapeutics have helped slow the spread of SARS-CoV-2 and the resulting 238 worldwide economic burden. However, pockets of uncontrolled viral spread have led to the 239 emergence of novel variants that are able to evade exiting vaccines and therapeutics (Arora et 240 al., 2021; Cele et al., 2021; Wang et al., 2021) . It is expected that more will 241 arise. Accordingly, novel therapeutics that will work broadly against all variants, including those 242 that have not yet emerged, are needed. 243

The disease state at which the novel therapeutic would be most effective must also be 244 considered. SARS-CoV-2 infects the oral cavity, upper respiratory tract, and large airway (Hou et 245 al., 2020; Huang et al., 2021) prior to spread to the lower respiratory tract and the late-stage 246 development of acute respiratory distress. Sustained replication in the oral and nasal cavities is 247 likely a key contributor to the increased transmissibility of SARS-CoV-2 compared to other 248 coronaviruses (Hou et al., 2020; Huang et al., 2021; Marchesan et al., 2021) . For these reasons, 249 a targeted approach for acute SARS-CoV-2 infection of the upper airway epithelia to halt 250 progression via the oral-lung transmission axis is an attractive aim. A therapeutic that works 251 during the early stages of infection is not only essential to reduce disease burden in the treated 252 individual, but also to limit person-to-person transmission. 253

Previously, we showed that 425 nm light inhibited SARS-CoV-2 WA1 in human tissue 254 models of both oral and large airway epithelia with no damage to healthy tissue (Stasko et al., 255 2021b (Stasko et al., 255 , 2021a . In this report, we show that 425 nm light can not only inactivate all SARS-CoV-2 256 variants of concern in cell-free suspensions, but targeted energy densities inhibit SARS-CoV-2 257 infections at multiple stages of infection in tissue models of human airway epithelia. Three-258 dimensional, differentiated primary cell culture models of human airway epithelia are more 259 effective systems to evaluate therapeutic efficacy and safety than conventional two-dimensional 260 immortalized cell culture systems (e.g. Vero cells) (Do et al., 2021; Heinen et al., 2021; Touret et 261 al., 2021a) . Similar models have been used in the evaluation of several anti-SARS-CoV-2 262 therapeutics, including molnupiravir (Sheahan et al., 2020) , remdesivir (Pruijssers et al., 2020) , 263

and AT-511 (Good et al., 2021) . Further, detection of infectious viruses in the apical washes can 264 serve as a surrogate for virus shedding during infection. To this end, the proper model must be 265 selected as different patient donors, primary cell culture conditions, and differentiation protocols 266 can impact model development (Fulcher and Randell, 2013; Sellgren et al., 2014) . For example, 267 primary cells cultured on PTFE surfaces are more differentiated and more ciliated than primary 268 cells cultured on PET surfaces (Sellgren et al., 2014) . In our previous study, we demonstrated 269 that the EpiAirway TM model (AIR-100) can withstand a single dose of 120 J/cm 2 of 425 nm (Stasko 270 et al., 2021b) , but this model does not support sufficient SARS-CoV-2 WA1 replication for 271 therapeutic exploration (Supplementary Figure 3A) . The most effective model screened for these 272 studies was developed at the Marsico Lung Institute at the University of North Carolina-Chapel 273

Hill (Fulcher and Randell, 2013) where SARS-CoV-2 replication in the model displayed more 274 consistent replication kinetics and higher peak titers than in the widely available AIR-100 275 to-moderate " n.d.) . 294

We thank BEI Resources for providing the SARS-CoV-2 variants. We thank Dr. Omicron and (H) human adenovirus type 5 were illuminated with 425 nm light and enumerated 323 via plaque assay. Data presented are mean viral titers +/-SD (n = 5-6). Statistical significance 324 was determined via Mann-Whitney ranked sum test and is indicated by * (p<0.05) and ** (p<0.01). 325 Whitney ranked sum test and is indicated by * (p<0.05) and ** (p<0.01). 342 human airway tissue models were infected with SARS-CoV-2 Beta or Delta (MOI 0.001) and illuminated twice daily for 3 days with 32 J/cm 2 of 425 nm light starting at 24 hours post-infection. 364

Apical rinses were collected daily and enumerated via plaque assay. Data presented are mean 365 viral titer (PFU/mL) +/-SD (n = 6) for Beta (A) and Delta (B). Statistical significance was 366 determined with the Mann-Whitney ranked sum test and is indicated by ** (p<0.01). 367 

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