key: cord-0691700-d1ra9jzd
authors: Madan, Taruna; Biswas, Barnali; Varghese, Praveen M.; Subedi, Rambhadur; Pandit, Hrishikesh; Idicula-Thomas, Susan; Kundu, Indra; Rooge, Sheetalnath; Agarwal, Reshu; Tripathi, Dinesh M.; Kaur, Savneet; Gupta, Ekta; Gupta, Sanjeev K.; Kishore, Uday
title: A recombinant fragment of Human surfactant protein D binds Spike protein and inhibits infectivity and replication of SARS-CoV-2 in clinical samples
date: 2020-12-18
journal: bioRxiv
DOI: 10.1101/2020.12.18.423415
sha: bf326ea75ff36f70642c78ffbb7d12b68be9cc81
doc_id: 691700
cord_uid: d1ra9jzd

Rationale COVID-19 is an acute infectious disease caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Human surfactant protein D (SP-D) is known to interact with spike protein of SARS-CoV, but its immune-surveillance against SARS-CoV-2 is not known. Objective This study aimed to examine the potential of a recombinant fragment of human SP-D (rfhSP-D) as an inhibitor of replication and infection of SARS-CoV-2. Methods rfhSP-D interaction with spike protein of SARS-CoV-2 and hACE-2 receptor was predicted via docking analysis. The inhibition of interaction between spike protein and ACE-2 by rfhSP-D was confirmed using direct and indirect ELISA. The effect of rfhSP-D on replication and infectivity of SARS-CoV-2 from clinical samples was studied by measuring the expression of RdRp gene of the virus using qPCR. Measurements and Main Results In-silico interaction studies indicated that three amino acid residues in the RBD of spike of SARS-CoV-2 were commonly involved in interacting with rfhSP-D and ACE-2. Studies using clinical samples of SARS-CoV-2 positive cases (asymptomatic, n=7 and symptomatic, n=8 and negative controls n=15) demonstrated that treatment with 5μM rfhSP-D inhibited viral replication by ~5.5 fold and was more efficient than Remdesivir (100 μM). Approximately, a 2-fold reduction in viral infectivity was also observed after treatment with 5μM rfhSP-D. Conclusions These results conclusively demonstrate that the calcium independent rfhSP-D mediated inhibition of binding between the receptor binding domain of the S1 subunit of the SARS-CoV-2 spike protein and human ACE-2, its host cell receptor, and a significant reduction in SARS-CoV-2 infection and replication in-vitro.

The COVID-19 pandemic, caused by the Severe acute respiratory syndrome Coronavirus-2 107 (SARS-CoV-2) (1, 2) , has affected ~ 58 million people across the globe and has claimed more 108 than a million lives within its first year (3). The SARS-CoV-2 spike protein (S protein) is 109 cleaved into S1 subunit, which is involved in host receptor binding, and S2 subunit, which is 110 involved in membrane fusion, by the host's transmembrane Serine Protease 2 (TMPRSS2) (4). 111

This priming of the S protein by host proteases enables it to bind with the angiotensin-112

converting enzyme 2 (ACE2) receptor on the nasopharyngeal epithelial cells, leading to its 113 entry into the host cell (4). While vaccines against the virus are being developed and trialled, 114 the current therapeutic strategy is empirical and comprises of anti-viral medications and 115

immunosuppressants (5). 116 The innate immune system plays a crucial role against SARS-CoV-2 infection; majority of 117 infected individuals purge the virus within a few days with minimal involvement of adaptive 118 immune response (6). Collectins are a group of humoral pattern recognition receptors, of which 119 human lung surfactant protein D (SP-D), is known to act as a potent viral entry inhibitor, 120

including HIV-1 and influenza A virus (7, 8) . The primary structure of SP-D is characterised 121 by an N-terminus that is involved in multimerization; a triple-helical collagenous region made 122 up of Gly-X-Y repeats, an α-helical coiled-coil neck region, and a C-terminal C-type lectin or 123 carbohydrate recognition domain (CRD) (9). The protective effects of SP-D against a range of 124 bacterial, viral, and fungal pathogens leading to their agglutination, growth inhibition, 125 enhanced phagocytosis, neutralisation, and modulation of immune responses are well 126 documented (9, 10). 127

During the SARS-CoV epidemic in 2002, elevated levels of SP-D were reported in the serum 128 of the patients infected with highly pathogenic β-CoV, SARS CoV (11). Purified SP-D has 129 been shown to bind to the receptor-binding domain (RBD) of the glycosylated Spike protein 130 of SARS-CoV, which shares 74% homology with the RBD of SARS-CoV-2 (12). In addition, 131 SP-D also binds α-CoV, HCoV-229E, and inhibits infection in human bronchial epithelial cells 132 (13) . These mounting pieces of evidence encouraged exploration of the therapeutic potential 133 of SP-D in In this study, we used a well-characterised recombinant fragment of human SP-D (rfhSP-D) 135 comprising homotrimeric neck and CRD regions to study its protective effect against SARS-136

CoV-2 infection. As the recombinant form has the advantage of a smaller size to reach the 137 distal lung locations and higher resistance to proteases and collagenases over the full-length 138 SP-D, we evaluated the interaction of rfhSP-D with RBD and Spike of SARS-CoV-2 and its 139 inhibitory potential against infection and replication of SARS-CoV-2 in clinical samples. 140

Clinical Samples 143

The clinical samples (Table 1) , nasopharyngeal (NP) and oropharyngeal (OP) swabs (n=30) 144 used in this study, were stored at the BSL-3 facility of the Institute of Liver and Biliary 145

Sciences, Delhi. These clinical samples (n=15) were from symptomatic contact of lab-146 confirmed cases (Cat 2), hospitalised severe acute respiratory infections (SARI) case-patients 147 (Cat 4), asymptomatic direct and high-risk contacts of lab-confirmed case (Cat 5a) and 148 hospitalised symptomatic influenza-like illness (ILI) case-patients (Cat 6) that had tested 149 positive by RT-PCR test for SARS-CoV-2. The samples obtained were placed in the viral 150 transport medium (Hanks Balanced Salt Solution (HBSS) Neck and CRD Regions 179

The rfhSP-D used was expressed and purified from E. coli as described previously (18, 19) . 180

Briefly, the pUK-D1 plasmid that codes for the 8 Gly-X-Y repeats, neck and CRD regions of 181 human SP-D was transformed into Escherichia coli BL21 (λDE3) pLysS (Invitrogen). The 182 transformed colonies (selected by ampicillin resistance) were grown in Luria-Bertani media 183

supplemented with a final concentration of 100 µg/ml ampicillin and 34 µg/ml 184 chloramphenicol (Sigma-Aldrich) to an OD600 of 0.6. The bacterial culture was then induced 185

to produce the recombinant protein by the addition of 0.5 M isopropyl β-d-1-186 thiogalactopyranoside (IPTG) (Sigma-Aldrich) and was allowed to grow for a further 3 h. Post 187 incubation, the bacteria were harvested and lysed using lysis buffer (50 mM Tris-HCl pH7.5, 188

200 mM NaCl, 5 mM EDTA pH 8, 0.1% v/v Triton X-100, 0.1 mM phenyl-methyl-sulfonyl 189 fluoride, 50 µg/ml lysozyme) and sonicated (five cycles, 30 s each). The sonicate was harvested 190 via centrifugation at 12,000 × g for 30 min. This was followed by solubilisation of inclusion 191 bodies in refolding buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM 2-192

Mercaptoethanol) containing 8 M urea. and stepwise dialysis of the solubilised fraction against 193 refolding buffer containing 4 M, 2 M, 1 M, and no urea. rfhSP-D was purified from the 194 dialysate by affinity chromatography using a maltose agarose column (5 ml; Sigma-Aldrich). 195

The bound rfhSP-D to the maltose was eluted using elution buffer (50 mM pH 7.5, 196 100 mM NaCl, and 10 mM EDTA) and passed through a PierceTM High-Capacity Endotoxin 197

Removal Resin (Thermofisher) to remove endotoxin. Finally, the endotoxin levels were 198 measured via the QCL-1000 Limulus amoebocyte lysate system (Lonza) and found to be <5 199 pg/µg of rfhSP-D. The purified rfhSP-D was subjected to western blotting after running on 200 12% w/v acrylamide SDS-PAGE to assess purity and immunoreactivity (18). 201

Assays to determine the binding of the S protein or its RBD of SARS-CoV-2 was performed 203

using the SARS-CoV-2 (COVID-19) Inhibitor Screening Kit from Acrobiosystems (EP-105) 204

as per the manufacture's protocol. Briefly, S protein diluted in Coating Buffer (15 mmol/L 205 sodium carbonate (Na2CO3), 35 mmol/L sodium hydrogen carbonate (NaHCO3), pH 9.6) to a 206 final concentration of 0.3 µg/ml were added to 96 well plates and incubated overnight (~16 h) 207

at 4 o C. The uncoated protein was removed by washing the wells with Wash Buffer (PBS with 208 0.05% (v/v) Tween-20, pH 7.4) three times. The wells were then blocked using the Blocking 209

Buffer (PBS with 0.05% (v/v) Tween-20 and 2% (w/v) bovine serum albumin (BSA), pH 7.4) 210 for 1.5 h at 37 o C. 211

To assess the direct binding of rfhSP-D to S protein, rfhSP-D (20, 10 and 5 µg/ml) were added 212

to the wells. The plate was then incubated for 1 h at 37 o C, and any unbound protein was 213 removed by washing the wells three times with the wash buffer. The wells were probed using 214 either polyclonal or monoclonal antibodies against SPD at a dilution of 1:5000 for 1 h at 37ºC 215 to detect S protein-rfhSP-D binding. Unbound antibodies were removed by washing three times 216 using the wash buffer. Anti-mouse IgG-Horseradish peroxidase (HRP) (Cat # 31430, 217

Invitrogen), anti-rabbit IgG HRP (Cat # 31466, Invitrogen) or Protein A HRP 218 Merck) at 1: 5000 dilution was used secondary antibodies by adding them to the respective 219 wells of the appropriate primary antibodies and incubating them for 1 h at 37ºC. Following 220 washes with wash buffer three times, the binding was detected using 3,3′,5,5′-221

Tetramethylbenzidine (TMB) substrate (100 µl/well) (DuoSet ELISA Ancillary Reagent Kit, 222 R&D Systems) as per the manufacturer's instruction, followed by stopping the reaction using 223

1M sulphuric acid (100 µl/well) (Cat # Q29307, Thermofisher). The plate was read at 450 nm 224 using a microplate absorbance reader (Synergy H1 multimode plate reader, Biotek). Full-length 225

Surfactant Protein D (FL SP-D) (20 µg/ml) was also used in a similar manner to assess the 226 binding of S protein to FL SP-D. A similar experiment was carried out in parallel using rfhSP-227 D (20, 10 and 5 µg/ml), supplemented with 10mM EDTA and probed with polyclonal 228 antibodies against SPD (1:5000) to evaluate if the S protein-rfhSP-D binding was calcium-229 dependent. 230

The binding of rfhSP-D or FL SP-D to ACE-2 was evaluated using a similar experiment as 231 above. Briefly, FL SP-D (0.1 µg/ml) or rfhSP-D (0.1 µg/ml) were coated in a 96 well plate and 232 probed with decreasing concentration of ACE-2 hACE-2 (0.12, 0.06 and 0.00 µg/ml). The 233

binding was detected using streptavidin tagged with HRP (1:5000) (EP-105, Acrobiosystems), 234 and the colour was developed as described above. 235

In a separate experiment to assess if rfhSP-D inhibited the interaction between the S protein 236 and biotinylated human Angiotensin-converting enzyme 2 (hACE-2), decreasing concentration 237 of rfhSP-D (5, 1 and 0 µg/ml) preincubated with (hACE-2), were added to wells coated with S 238 protein (0.3 µg/ml) and blocked as described above. The plate was incubated for 1h at 37 o C 239 and washed with the wash buffer the times to remove any unbound proteins. The S protein-240 hACE-2 binding was measured by probing the wells with the HRP tagged Streptavidin 241 antibody (1:5000) for 1h at 37 o C. Colour was developed using 3,3′,5,5′-Tetramethylbenzidine 242

(TMB) substrate. The reaction was stopped using 1 M H2SO4, and the absorbance was read at 243 450 nm using a microplate absorbance reader. rfhSP-D (5 µg/ml) supplemented with either 244 with 10mM EDTA was used in a similar manner to evaluate if the rfhSP-D mediated inhibition 245 of the interaction between the S protein and biotinylated hACE-2 occurred in a calcium-246 independent manner. rfhSP-D mediated inhibition of the interaction between the RBD of 247 SARS-CoV-2 S protein and biotinylated hACE-2 was also assessed in a similar manner. 248

Vero cell line (derived from African green monkey epithelial Kidney cells) (ATCC® CCL-250 81™) (5x10 4 ) were cultured for 16 h in each well of a 12 well plate in serum-free medium 251

(MEM Glutamax, containing 1% v/v Penicillin-Streptomycin and 1%v/v sodium pyruvate 252 [Gibco, Thermofisher] ). SARS-CoV-2 clinical samples (100 TCID50/ well, MOI 0.01) were 253 preincubated with rfhSP-D [0 µg/ml (0 µM), 50 µg/ml (~2.5µM) or 100 µg/ml (~5µM)] in 254 MEM containing 5mM CaCl2 for 1h at RT and 1h at 4 o C. This pre-treated or untreated virus 255 was added to the cells (Cells + rfhSP-D + Virus). After 1h incubation at 37ºC, 5% CO2, the 256 medium was removed, and cells were washed with PBS to remove any unbound CoVs. 257

Infection medium (MEM+0.3% BSA) was added to the cells and incubated for 24 h to assess 258

replication. The cells were then harvested by scraping with a sterile disposable cell scraper 259 and centrifuged at 1500 x g for 5 minutes. Total RNA was extracted using the Perkin Elmer 260 automated extractor and subjected to Real-time RT-PCR for SARS-CoV-2 using Pathodetect 261 kits from MyLabs, as per manufacture's protocol. For the replication analysis of SARS-CoV-262 2, Ct value for SARS-CoV-2 RNA dependent RNA polymerase (RdRp) gene was used for 263

analysis. Cells incubated with rfhSP-D, without virus was used protein control (Cells + rfhSP-264 D) and cells incubated with BSA (100µg/ml), and the virus was used as non-specific protein 265 control (Cells + Virus). Sterile PBS with the virus was used as negative control. 266

The effect of rfhSP-D on viral infection was assessed by culturing Vero cells (5x10 5 ) in a 12 267

well plate in serum-free MEM. SARS-CoV-2 clinical samples (500 TCID50/ well, MOI 0.05) 268

were treated with rfhSP-D and added to the cells as described above. However, after the 269 addition of the infection medium, the cells were incubated only for 2h, after which they were 270 harvested, and Real-time RT-PCR was performed using the same controls and parameters 271 described above. 272

Graphs were generated using GraphPad Prism 8.0 software, and the statistical analysis was 274 performed using a two-way ANOVA test. Significant values were considered based on *p < 275 0.1, **p < 0.05, ***p < 0.01, and ****p < 0.001 between treated and untreated conditions. 276

Error bars show the SD or SEM, as indicated in the figure legends. 277

rfhSP-D interacts with the Spike protein of SARS-CoV-2 and human ACE-2 in silico 280 S protein is known to interact via the receptor binding motif (RBM:455-508) in the receptor 281 binding domain (RBD: aa 319-527) with virus binding hotspot residues comprising of Lys31, 282

Glu35 and Lys353 of dimeric hACE2 (14-16). The structure of hACE2 receptor, co-283 crystallized with Spike S protein of SARS-CoV-2, is available in RCSB (pdb id: 6VW1). The 284 receptor (ACE2) and ligand (Spike S) were separated and docked to validate the docking 285

protocol. The redocked complex of ACE2 and S protein had root mean square deviation 286

(RMSD) of 7.9 Å. The close agreement between the docked and crystal structures validated 287 the docking protocol used in the study. with RBM residues Tyr449, Gln493 Gln498, implying that rfhSP-D could bind to Spike protein 291 in a manner that can inhibit ACE2-S protein interaction (Table 2; Figure 1 ). To ascertain this 292 hypothesis, the complex of S protein with rfhSP-D was docked to ACE2. S protein and rfhSP-293 D bound to ACE2 via common interacting residues. 294

The top ranked docked structure of ACE2 and rfhSP-D had binding energy of -24.30 kcal/mol. 295

In this pose, rfhSP-D interacted with the virus-binding hotspot residues Ser19, Lys31, His34 296

and Glu35 of ACE2, implying that rfhSP-D could bind to ACE2 in a manner that can inhibit 297 ACE2-S protein interaction (Table 2, Figure 1 ). To corroborate this postulation, the complex 298 of ACE2 with rfhSP-D was docked to Spike S. Top ranked pose of ACE2-rfhSP-D complex 299 docked with open S protein had binding energy of -33.01 kcal/mol and several common 300

interactions between rfhSP-D and ACE2 with S protein (Supplementary Figure 1) . The 301

docking experiments led us to infer that rfhSP-D could bind to both ACE2 and Spike S and 302 prevent ACE2-S protein interaction. 303 rfhSP-D binds to the immobilised S protein of the SARS-CoV-2 as well as hACE-2 304

The possible binding between rfhSP-D and S protein hinted by the docking analysis was 305 confirmed in vitro via an indirect ELISA. rfhSP-D was found to bind the immobilised S protein 306 in a dose-dependent manner ( Figure 2a ). However, a significant difference in the absorbance 307 was observed based on the specificity of the primary antibody used. S protein-rfhSP-D binding 308 that was probed with the polyclonal antibody against SP-D reported a significantly higher 309

absorbance when compared to the wells that were probed with a monoclonal antibody directed 310 against the CRD of SP-D. This difference suggests involvement of CRD of rfhSP-D with the 311 spike protein and therefore, the CRD was not available for interaction with the monoclonal 312 antibody. S protein was also found to bind to the FL SP-D. The treatment of rfhSP-D with 313

10mM EDTA did not significantly alter the binding of rfhSP-D to S protein ( Figure 2b ). 314

Hence, rfhSP-D binds to the S protein in a dose-dependent but a calcium-independent manner. 315

A similar parallel experiment revealed that rfhSP-D bound ACE2 in a dose-dependent manner 316

( Figure 2c ). 317 rfhSP-D inhibits the interaction of S protein and its RBD with biotinylated hACE-2 in a 318 calcium-independent manner 319

Since rfhSP-D was found to bind to the S protein and ACE-2, and as both rfhSP-D and ACE-320 2 were predicted to share the same binding site on S protein, rfhSP-D mediated inhibition of 321 the interaction between the RBD of S protein of SARS-CoV-2 and ACE-2 was assessed using 322 a colorimetric ELISA. 323

The wells were coated with either the S protein or its RBD domain that was preincubated with 324 rfhSP-D followed by biotinylated hACE-2. The functionality and the range of the assay were 325

initially assessed by verifying if the assay could detect the binding of hACE-2 at a 326 concentration of 0.12 µg/ml and 0.06 µg/ml. The binding occurred in a dose-dependent manner, 327

confirming that the assay can detect binding between S protein or its RBD domain with hACE-328 2 at a concentration as low as 60 ng/ml (Supplementary Figure 2) . A decrease in binding 329 between S protein and hACE-2 was observed as the concentration of rfhSP-D increased 330

( Figure 3 and Figure 4 ). Approximately 50% decrease in S protein-hACE-2 binding was 331 observed as rfhSP-D concentration increased 5-fold ( Figure 3a) . A similar result was observed 332

between the binding of the RBD of S protein and hACE-2. An 8-fold increase in the 333 concentration of rfhSP-D was found to decrease RBD-hACE-2 interaction by ~25% ( Figure  334 4a). No significant difference was observed between the samples with 10mM EDTA and 335

without EDTA in terms of rfhSP-D mediated S protein/RBD-hACE-2 binding (Figure 3b ; 336 Figure 4b ). Hence, rfhSP-D mediated inhibition of the interaction between the RBD of S 337 7 protein or the S protein itself with biotinylated hACE-2 occurred in a calcium-independent 338 manner. 339

As rfhSP-D is known to induce apoptosis in cancer and immortalised cells (18, (20) (21) (22) , the 341 effect of rfhSP-D on Vero cells was assessed using MTT assay. rfhSP-D treatment had no 342 significant effect on the viability of Vero cells (Supplementary figure 3) . At the outset, the 343 TCID50 values of the clinical samples were obtained by evaluating the cytopathic effects using 344

MTT assay. As expected, when Vero cells were challenged with 100 TCID50, or 50 TCID50 of 345 viral samples from SARS-CoV-2 clinical samples, a 50% or 25% reduction in cell viability 346 was observed, respectively, compared to the viability of uninfected Vero cells, confirming the 347 assayed TCID50 values ( Figure 5) . with rfhSP-D, led to a reduction in RdRp levels in a dose-dependent manner ( Figure 6 ; Table  354 S1). The pre-treatment of samples from all categories of SARS-CoV-2 positive cases [as 355

representatives, the figure 6 shows the data for 1S (Cat 2) and 3S (Cat 6) The present study explored the likely protective effect of a recombinant fragment of human 380 lung surfactant protein D, rfhSP-D, against SARS-CoV-2. As predicted by the docking study, 381

rfhSP-D interacted with the spike protein of SARS-CoV-2, its receptor binding domain (RBD) 382 as well as ACE-2. Importantly, these interactions may have contributed to significant inhibition 383 of infectivity and replication of SARS-CoV-2 virus present in the clinical samples derived from 384 asymptomatic, symptomatic and severe patients of One of the first steps of the SARS-CoV-2 infection is the binding of the S protein to the host 386 cell via, ACE-2 receptor (23). S1 protein is known to interact with ACE-2 receptor via the 387 receptor-binding motif (RBM:455-508) in the receptor-binding domain (RBD: aa 319-527) 388

with virus binding hotspot residues comprising of Lys31, Glu35 and Lys353 of dimeric ACE2 389

(24-26). Since SP-D interaction with spike protein of SARS-CoV has been reported, which 390

shares ~74% homology with the RBD of SARS-CoV-2 (12) and rfhSP-D is known to bind to 391 viral surface proteins such as haemagglutinin and neuraminidase of influenza A virus, gp120 392 of human immunodeficiency virus 1 (7, 27), and S protein of SARS-CoV (12), the possibility 393 of rfhSP-D binding to the S protein of SARS-CoV-2 was examined. 394

In-silico interaction of rfhSP-D with RBD of Spike protein of SARS-CoV-2 revealed that 395

Tyr449, Gln493 and Gln498 of RBD overlapped with the residues that are essential for the 396 binding of S protein to the target protein ACE-2. The binding of S protein to rfhSP-D or FL 397 SP-D was confirmed using an indirect ELISA. A comparatively lower absorbance with the 398 monoclonal antibodies raised against the CRD region of human SP-D than the polyclonal 399 antibodies could be attributed to the fact that the binding between rfhSP-D and S protein inhibitor against SARS-CoV-2. These results suggest that rfhSP-D is a potential candidate to 425 be used as an S protein-based inhibitor against SARS-CoV-2 infections. With established 426 safety in vivo and therapeutic efficacy against several respiratory pathogens, rfhSP-D will 427 effectively combat the nosocomial co-infections in COVID-19 patients. 428

There is dysregulated pro-inflammatory cytokine response without protective IFNs in response 429

to SARS-CoV-2 mediated lung tissue damage leading to Acute Respiratory Distress Syndrome 430 (ARDS). The levels of SP-D were significantly altered in bronchoalveolar lavage of patients 431 of ARDS and were strong predictors of poor prognosis (31, 32 The interactions between S protein and ACE-2 are deduced from the crystal structure (PDB 620 ID: 6VW1) and between rfhSP-D, and ACE-2 protein are based on docked complexes. 621

Individual intermolecular interactions between (C) S protein (Green) and ACE-2 (Blue); (D) S 622 protein (Green) and rfhSP-D (Red) and (E) rfhSP-D (Red) and ACE-2 (Blue). The S protein 623 residues, Tyr449, Gln493 and Gln498, participate in intermolecular interactions with both 624 ACE-2 and rfhSP-D. 625 points. The data were expressed as the mean of triplicates ± SD. 639 incubated with biotinylated human Angiotensin-converting enzyme 2 (hACE-2) was added to 643 the wells. To assess the effect of calcium in the rfhSP-D-mediated inhibition of S protein-644 hACE-2 interaction (B), 5 µg/ml of rfhSP-D with/without 10 mM EDTA. S protein-hACE-2 645 binding was detected with Streptavidin-HRP. Background was subtracted from all data points. 646

The data were normalised with 100% S protein: hACE-2 binding being defined as the mean of 647 the absorbance recorded from the control sample (0 µg/ml of rfhSP-D). The data were 648

presented as the mean of the normalised triplicates ± SEM for inset A and B. The data were 649

presented as the mean of the triplicates ± SEM for inset C. Significance was determined using 650 the two-way ANOVA (n = 3); no significant difference was observed between the samples 651 with 10mM EDTA and without EDTA in terms of rfhSP-D-mediated S protein:hACE-2 652 binding. 653 Angiotensin-converting enzyme 2 (hACE-2) was added to the wells. To assess the effect of 658 calcium in the rfhSP-D-mediated inhibition of S protein RBD: hACE-2 interaction (B), 5 µg/ml 659 of rfhSP-D with/without 10mM EDTA was used. S protein RBD-hACE-2 binding was detected 660

with Streptavidin-HRP. Background was subtracted from all data points. The data obtained 661

were normalised with 100% S protein RBD-hACE-2 binding being defined as the mean of the 662 absorbance recorded from the control sample (0 µg/ml of rfhSP-D). The data were presented 663 as the mean of the normalised triplicates ± SEM for inset A and B. The data were presented as 664 the mean of the triplicates ± SEM for inset C. Significance was determined using the two-way 665 ANOVA (n = 3) and no significant difference was observed between the samples with 10mM 666

EDTA and without EDTA in terms of rfhSP-D mediated S protein RBD: hACE-2 binding. 667 for 96 h. Viability of the cells was evaluated using MTT assay. MTT (0.5 mg/ml) containing 675 medium was added to the wells for 4h. The supernatants were removed, and cells were lysed 676 using DMSO. Absorbance was measured at 590nm. The data obtained were normalised with 677

100% cell viability being defined as the mean of the absorbance recorded from the control 678 sample (0 TCID50/well) and TCID50 units were evaluated in each sample. The same assay was 679 used to validate the cytopathic effects of 100TCID50 and 50TCID50 units of the samples. The 680

representative data for cases (n=2) and controls (n=2) are presented as the mean of the 681 normalised triplicates ± SEM Significance was determined using the two-way ANOVA (n = 682

3) test (**p < 0.01, and ****p < 0.0001). 683 The pre-treated or untreated virus in the sample was added to the cells and incubated for 1h at 689

37°C under 5% CO2. The wells were washed with PBS twice, and infection medium 690 (MEM+0.3% BSA) was added to the cells and incubated for 24h at 37 o C. The supernatants 691

were collected, RNA was extracted by Perkin Elmer automated extractor, and subjected to 692

qRT-PCR for SARS-CoV-2. For control samples, the volume of the sample taken was 693 equivalent to the volume of the case sample (100 TCID50) where no RdRp expression was 694 detected. The relative expression of RdRp was calculated using rfhSP-D untreated cells (0 µM  695 rfhSP-D), infected with respective samples as the calibrator. Data of representative cases (n=2) 696 is presented as the mean of triplicates (n=3). Error bars represent ± SEM. Significance 697 (compared to 100µM Remdesivir) was determined using the two-way ANOVA test (*p < 0.05, 698 ***p < 0.01, and ****p < 0.0001). 699 

Tyr505, Gly502 Gly354 Tyr505 2 rfhSP -D S protein (Open) -20.63

Ala274, Tyr314 752 *The S protein residues in bold are predicted to be part of the common binding site for ACE2 753 and rfhSP-D. Decreasing concentration of hACE-2 (0.12, 0.06 and 0.00 µg/ml) were added to the wells. S 813 protein or RBD: hACE-2 binding was detected with Streptavidin-HRP. Background was 814 subtracted from all data points. The data were expressed as the mean of triplicates ± SD. for 24 h. 0.5 mg/ml MTT containing medium was added to the wells for 4h. The supernatants 829

were removed, and cells were lysed using DMSO. Absorbance was measured at 590nm. 830

Background was subtracted from all data points. The data obtained were normalised with 100% 831 cell viability being defined as the mean of the absorbance recorded from the control sample (0 832 µg/ml of rfhSP-D). The data were presented as the mean of the normalised triplicates ± SEM. 833

Significance was determined using the two-way ANOVA test and no significant reduction in 834 cell viability was observed. 

Coronaviridae Study Group of the 477 International Committee on Taxonomy of V. The species Severe acute respiratory 478 syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. 479

A Novel Coronavirus from Patients with Pneumonia in China

World Health 485 Organization

Targeting TMPRSS2 in 487 SARS-CoV-2 Infection

Emerging treatment 490 strategies for COVID-19 infection

Clearance and persistence of SARS-CoV-2 RNA in patients with COVID-494 19

Entry Inhibition and Modulation of

Immune Response Against Influenza A Virus by a Recombinant Truncated Surfactant 498 Protein D

Chakraborty 502 T. Surfactant proteins SP-A and SP-D in human health and disease

Collectins: Innate Immune Pattern Recognition 505 Molecules

Protein D (SP-D) Levels and a Direct Correlation with Anti-severe Acute Respiratory 509

Syndrome Coronavirus-specific IgG Antibody in SARS Patients

The SARS coronavirus spike 512 glycoprotein is selectively recognized by lung surfactant protein D and activates 513 macrophages

Infection of 515 human alveolar macrophages by human coronavirus strain 229E

Efficient unbound docking of rigid molecules

PatchDock and SymmDock: 520 servers for rigid and symmetric docking

Serial changes in 573 surfactant-associated proteins in lung and serum before and after onset of ARDS

Plasma surfactant protein-D as a diagnostic biomarker for 577 acute respiratory distress syndrome: validation in US and Korean cohorts

Role of surfactant proteins D, D, and C1q in the 581 clearance of apoptotic cells in vivo and in vitro: Calreticulin and CD91 as a common 582 collectin receptor complex

Truncated recombinant human SP-D attenuates 585 emphysema and type II cell changes in SP-D deficient mice

Animal models for COVID-19

3% BSA) was added to the cells and incubated for 2h at 37 o C under 5%

RNA was 708 extracted and subjected to RT-PCR for SARS-CoV-2. For control samples, the volume of the 709 sample taken was equivalent to the volume of the case sample (500 TCID50); no RdRp 710 expression was detected the relative expression of RdRp was calculated by using rfhSP-D 711 untreated cells (0 µM rfhSP-D), infected with respective samples as the calibrator. Data for 712 representative cases (n=2) is presented as the mean of triplicates (n=3). Error bars represent ± 713 SEM. Significance

Supplementary Figure S1: Docked poses of S protein (Green) and rfhSP-D (Red) complex 800 with ACE2 (Blue) (A-C) and ACE2 and rfhSP-D