key: cord-0428257-otq80w1w
authors: Sun, Xin; Yang, Shaobo; Al-Dossary, Amal A.; Broitman, Shana; Ni, Yun; Yang, Mengdi; Li, Jiahe
title: Nanobody-Functionalized Cellulose for Capturing and Containing SARS-CoV-2
date: 2021-09-02
journal: bioRxiv
DOI: 10.1101/2021.09.01.458653
sha: f2bf7a262b5615103a2f1eaaa334dc3ea402c825
doc_id: 428257
cord_uid: otq80w1w

The highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 217 million people, claiming ~ 4.5 million lives to date. Although mandatory quarantines, lockdowns, and vaccinations help curb viral transmission, safe and effective preventative measures remain urgently needed. Here, we present a generic strategy for containing SARS-CoV-2 by cellulose materials. Specifically, we developed a bifunctional fusion protein consisting of a cellulose-binding domain and a nanobody (Nb) targeting the receptor-binding domain of SARS-CoV-2. The immobilization of the fusion proteins on cellulose substrates enhanced the capture efficiency of Nbs against SARS-CoV-2 pseudoviruses of the wildtype and the D614G variant, the latter of which has been shown to confer higher infectivity. Furthermore, the fusion protein was integrated into a customizable chromatography with highly porous cellulose for neutralizing virus from contaminated fluids in a continuous and cost-effective fashion. Taken together, our work leverages low-cost cellulose materials and recently developed Nbs to provide a complementary approach to addressing the pandemic. IMPORTANCE The ongoing efforts to address the COVID-19 pandemic center around the development of point-of-care diagnostics, preventative measures, and therapeutic strategies against COVID-19. In contrast to existing work, we have provided a complementary approach to target and contain SARS-CoV-2 from contaminated fluids and surfaces. Specifically, we present a generic strategy for the capture and containing of SARS-CoV-2 by cellulose-based substrates. This was archived by developing a bifunctional fusion protein consisting of both a cellulose-binding domain and a nanobody specific for the receptor-binding domain of SARS-CoV-2. As a proof-of-concept, our fusion protein-coated cellulose substrates exhibited enhanced capture efficiency against SARS-CoV-2 pseudovirus of both wildtype and the D614G mutant variants, the latter of which has been shown to confer higher infectivity. Furthermore, the fusion protein was integrated into a customizable chromatography with highly porous cellulose for neutralizing the virus from contaminated fluids in a highly continuous and cost-effective fashion.

shown to confer higher infectivity. Furthermore, the fusion protein was integrated into a 21 customizable chromatography with highly porous cellulose for neutralizing virus from 22 contaminated fluids in a continuous and cost-effective fashion. Taken together, our work 23 leverages low-cost cellulose materials and recently developed Nbs to provide a 24 complementary approach to addressing the pandemic. 25

The ongoing efforts to address the COVID-19 pandemic center around the development 27 of point-of-care diagnostics, preventative measures, and therapeutic strategies against 28 COVID-19. In contrast to existing work, we have provided a complementary approach to 29 target and contain SARS-CoV-2 from contaminated fluids and surfaces. Specifically, we 30 present a generic strategy for the capture and containing of SARS-CoV-2 by cellulose-31 based substrates. This was archived by developing a bifunctional fusion protein 32 consisting of both a cellulose-binding domain and a nanobody specific for the receptor-33 binding domain of SARS-CoV-2. As a proof-of-concept, our fusion protein-coated 34 cellulose substrates exhibited enhanced capture efficiency against SARS-CoV-2 35 pseudovirus of both wildtype and the D614G mutant variants, the latter of which has been 36 shown to confer higher infectivity. Furthermore, the fusion protein was integrated into a 37 customizable chromatography with highly porous cellulose for neutralizing the virus from 38 contaminated fluids in a highly continuous and cost-effective fashion.

Since the first documented coronavirus disease 2019 (COVID-19) case at the end of 2019 41

(1), the highly contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-42 adherence to these preventative measures has led to severe societal and economic 48 crises (2). Importantly, the approval and administration of several SARS-CoV-2 vaccines 49 worldwide has helped to mitigate the pandemic waves with an ever-increasing 50 immunization population (3). Nevertheless, COVID-19 poses a continued threat because 51 of constantly emerging SARS-CoV-2 variants and relatively long duration for herd 52 immunity (4). Therefore, there is a great demand for effective, low-cost, and off-the-shelf 53 agents to fast diagnose and decontaminate SARS-CoV-2 from body fluids and frequently 54 contacted environmental surfaces (5). 55 56 SARS-CoV-2 belongs to the β-coronavirus genus of the Coronaviridae family, and shares 57 the same subfamily Orthocoronaviridae with SARS-CoV, all of which lead to severe 58 respiratory tract illness in humans (6). SARS-CoV-2 is a single-stranded RNA composed 59 of 30 kb nucleotides, which encode four major structural proteins: the spike (S), the 60 membrane (M), the envelope(E), and the nucleocapsid (N) (7). Viral infections rely upon 61 cellular entry to utilize the host's machinery for replicating viral copies that are then 62 released by the host. The S protein facilitates the attachment of the virus to the host's 63 cellular receptors and promotes the fusion between host and viral membranes (8). In 64 particular, the S protein contains the receptor-binding domain (RBD), which binds to the 65

Compared to human IgG, Nbs can be produced in E. coli with high yield and purity ( Figure  136 1a). Therefore, DNA encoding the fusion protein Ty1-CBD was first cloned in a standard 137 expression vector for recombinant protein production in E. coli. Because the antigen-138 binding site of Nbs is closed to the N terminus, we placed the CBD at the C terminus of 139 Ty1 to circumvent potential steric hindrance. Meanwhile, a short 6x histidine (His-tag) was 140 attached to the N terminus for the metal affinity purification, while a FLAG epitope 141 (DYKDDDDK) was inserted between Ty1 and the CBD, simultaneously serving as a 142 hydrophilic flexible linkage and a tag for immunostaining (Figure 1b) . The yield of fusion 143 proteins was estimated to be ~50 mg per liter of bacterial culture in a shake-flask mode 144

and was purified to high homogeneity as evidenced by denaturing SDS-PAGE and size 145 exclusion chromatography (Figure 1c, Figure 1d , Figure S1a , and Figure S1b ). 146

The fusion protein is functionally active in cellulose binding and RBD detection 148

Next, we sought to evaluate whether the fusion proteins were capable of cellulose binding 149 and Nb-specific target recognition. To this end, we first spotted purified fusion proteins on 150 the surface of cellulose paper (Figure 2a) . Upon air drying, the paper was stained with a 151 rat antibody against the FLAG epitope in the fusion protein, followed by an anti-rat 152 secondary antibody conjugated with horseradish peroxidase (HRP). A dark precipitate 153 was visualized after incubation with an HRP substrate, 3,3'-Diaminobenzidine (DAB). To 154 quantify the binding efficiency of the fusion protein to the cellulose paper, we immobilized 155 serially diluted fusion proteins within a defined area on the Whatman filter paper followed 156 by immunostaining with an anti-FLAG antibody. Indeed, the extent of protein 157 immobilization correlated with the staining in a certain concentration range (Figure 2b  158 and Figure S1c ), from which we estimated that a surface area of 1 mm 2 can be saturated 159 by 500 ng (~0.02 nmol) of Ty1-CBD proteins in the Whatman filter paper. 160

Having validated the high binding capacity of purified Ty1-CBD to filter paper, we 162 speculated that because the CBD itself can act as a natural affinity ligand to cellulose, we 163 could directly immobilize E. coli cell lysate containing the CBD fusion proteins to filter 164 paper, followed by extensive washing to remove nonspecific proteins. This circumvents 165 the need to purify desired proteins beforehand, which is labor intensive and impractical 166 when it comes to the large-scale manufacturing of functionalized cellulose materials 167 (Figure 2c) . To this end, we first incubated E. coli cell lysate with filter discs and removed 168 nonspecific proteins via washing. Then we subjected the functionalized filter discs to cell 169 culture media containing secreted recombinant RBD as a proxy for actual SARS-CoV-2. 170

As shown in Figure 2d , Ty1-CBD-coated filter paper was able to capture SARS-CoV-2's 171 RBD as evidenced by the intense dark staining reflecting the detection of RBD. Of note, 172 filter discs precoated with Ty1 alone exhibited light staining, likely due to nonspecific but 173 weak absorption of Ty1 to cellulose. In comparison, in the control media without the RBD, 174

neither Ty1-nor Ty1-CBD-coatd discs displayed the dark staining. Our findings here 175 indicate that the CBD domain promoted the immobilization of Ty1-CBD on cellulose 176 substrates while Ty1 remained able to specifically recognize the target. It is worth noting that the levels of SARS-CoV-2 in COVID-19 patients range from 10 4 to 210 10 9 copies per ml depending on the type of bodily fluids and degree of the symptoms 211 (45,46). Meanwhile, we calculated the titers of wildtype or D614G pseudotyped lentivirus, 212 and estimated that ~10 5 viral particle particles per ml were present in the culture media. 213

Therefore, to demonstrate the capability of capturing SARS-CoV-2 at the lower end of the 214 viral titer range for SARS-CoV-2-containing fluids, we further diluted the media to contain 215 approximately ~10 4 pseudovirus copies per ml, and quantified the neutralization efficacy 216 of Ty1-CBD-immobilized cellulose paper (Figure 4a ). In addition, filter paper alone or filter 217 paper coated with Ty1 but lacking the CBD module served as negative controls. The 218 neutralization efficiency of Ty1-CBD-immobilized filter paper was calculated by dividing 219 the titer of media treated with Ty1-CBD-immobilized filter paper or other control groups 220 by the initial viral titer (i.e., without any treatment). Indeed, using media containing 221 wildtype or D614G pseudoviruses (Figure 4b) , Ty1-CBD-immobilized filter paper resulted 222 in ~2-fold increase of the neutralization efficacy compared to that of filter paper only, and 223 ~1.5-fold enhancement over filter paper pre-coated with Ty1 alone. Moreover, filter paper Having validated the increased capture efficiency of fusion proteins immobilized on filter 231 paper, we further sought to enhance the neutralization efficiency by incorporating the 232 fusion proteins into regenerated amorphous cellulose (RAC). Because RAC has been 233 shown to exhibit higher surface area per unit mass than filter paper (47), we reasoned 234 that using RAC can increase the immobilization density of Ty1-CBD on cellulose, which 235 in turn improves the rate and the degree of target capture based on the theoretical 236 modeling by others (23,24). Moreover, this strategy can potentially expand the utility of 237 Ty1-CBD by packing Ty1-CBD-functionalized RAC in a column, which allows for an 238 affinity chromatography system to reduce viral load from contaminated fluids (e.g., blood 239 and saliva) in a continuous mode. To test our hypotheses, we filled a 1 mL gravity-based 240 column with 0.1 mL (~50 mg dry weight) of RAC, which was subsequently saturated with 241 purified Ty1-CBD proteins or E. coli lysate containing the fusion proteins (Figure 4c) . 242

After culture media containing wildtype or D614 pseudoviruses were passed through the 243 functionalized column by gravity, viral titers were determined for different flow through 244 samples. Compared to RAC alone, Ty1-CBD-immobilized columns increased the 245 neutralization efficacy of wildtype and D614G pseudoviruses by ~ 3.5 times and ~8 times, 246

respectively. In contrast, RAC columns carrying an irrelevant Nb (caffeine specific) fused 247 with CBD or Ty1 alone failed to further enhance the degree of neutralization compared to 248 that of RAC alone (Figure 4d) . Taken together, we demonstrated that the Ty1-CBD fusion 249 protein can be integrated into an RAC column to markedly increase the neutralization 250 efficiency of SARS-CoV-2 pseudovirus in a highly specific and continuous fashion. Notably, a similar strategy has been proposed for SARS-CoV-2 detection through 261 cellulose filter paper immobilized with CBD fusion proteins (23,24). In their approach, the 262 nucleocapsid protein was fused with the CBD and the detection of SARS-CoV-2 required 263 an antibody to capture viral particles in a "sandwich" format. In comparison, we have 264 To validate the binding ability of Ty1-CBD, purified Ty1-CBD was spotted on cellulose 325

paper and dried at room temperature for ~2 min. Cellulose paper with dried Ty1-CBD 326

were incubated with 5 ml of 5% nonfat milk in Tris-buffered saline (TBS) for 30 min to 327 block non-specific binding sites. After blocking, the cellulose paper was then incubated 328 with anti-FLAG epitope (DYKDDDDK), which was diluted at 1:2000 in 3 ml TBS plus 5% 329 nonfat milk, overnight at 4°C. The paper was washed three times with 5ml 1xTBS 330 containing 0.05% Tween-20 (TBST), with 15 min per wash cycle. The paper was 331 incubated with HRP anti-rat IgG (1: 2000) for 1 hr at room temperature. After washing 332 with 1xTBS with 5 ml 0.05% Tween-20, premixed Pierce™ 3,3'diaminobenzidine (DAB) 333 substrate was directly added on the cellulose paper. The reaction was terminated with 334 water after dark spots appeared. Addgene#158762, respectively. Both plasmids were isolated following manufacturer's 341 instructions and quantified through Nanodrop. 24 hr prior to transfection, Lenti-X 293T 342 cells in logarithmic growth phase were trypsinized, and the cell density was adjusted to 343 1.0x10 6 cells/mL with complete DMEM medium. The cells were reseeded into a 10 cm 344 cell culture dishes to reach 70% confluency on the day of transfection. The plasmid 345 mixture was prepared according to Table 1 and Table 2 For capturing capability assay, cellulose filter paper discs were fitted into a 96-well plate 390 or 1.5 ml microtube followed by blocking with 1% bovine serum albumin (BSA) in 1 x TBS 391 for 1 hr. After aspirating the blocking buffer, 50-100 µl purified proteins with concentrations 392 of 100 µg/ml, 10µg/ml and 1µg/ml in 1 x TBS or 400 µl protein lysate of interest were 393 applied directly to the coated cellulose paper. After 1 hr incubation at room temperature 394 with slow shaking, purified proteins or lysates were removed from 96 well plates or 1.5 ml 395 microtube, followed by 3 times wash with 1x TBS, 10 min in between. Cellulose paper 396 was blocked with 5% nonfat milk in TBS at room temperature for 15 -30 min. 50 -100 397 µL media with spike expression were applied to the cellulose paper and incubated at room 398 temperature for 1 hr. The treated cellulose paper was washed with TBST, 4 times, with 5 399 -10 min each. The washed cellulose paper was incubated with HRP Donkey anti-human 400

IgG antibody diluted in 1 x TBS supplemented with 5% milk with antibody dilution of 401 1:2000. After 1 hr, the cellulose paper was washed with TBST for 4 times with 5 -10 402 each. Premixed DAB substrate was directly added on cellulose paper after removing the 403 TBST. The reaction was terminated with distilled water till the color development. 404

For preparing the cellulose filter paper discs to evaluate the neutralization efficiency 406 against the pseudovirus, cellulose filter paper discs were fitted into a 96-well or 48-well 407 plate followed by blocking with 1% BSA in 1 x TBS for 1 hr. After aspirating the blocking 408 buffer, 50 -100 µl of 10 µg/ml purified fusion proteins in 1 x TBS were applied directly to 409 the coated cellulose paper. After 1 hr incubation at room temperature with slow shaking, 410 diluted purified proteins were aspirated from 96-well or 48-well plates, followed by two 411 times wash with 1x TBS, 10 min in between. 100-400 µL of pseudoviruses were added 412 into each well with cellulose paper. After 1 hr incubation, the cellulose paper treated 413 pseudovirus was gently transferred to transfect HEK293T-hACE2 cells with 80% Ty1-CBD was first spotted on a piece of cellulose paper. Upon air drying, the paper was 625 incubated with a rat antibody against the FLAG epitope (DYKDDDDK), followed by an 626 anti-rat secondary antibody conjugated with HRP. The dark precipitate "Anti-COVID" was 627 visualized after incubation with 3,3'-Diaminobenzidine (DAB). b) Quantification of 628 maximal protein absorption on Waterman filter paper. 10 µL of serially diluted fusion 629

COVID-19: The first documented coronavirus pandemic 450 in history

Effect of non-452 pharmaceutical interventions to contain COVID-19 in China

Analysis of the potential impact of durability, 455 timing, and transmission blocking of COVID-19 vaccine on morbidity and mortality

CoV-2 Variants and Vaccines

Surface 463 Environmental, and Personal Protective Equipment Contamination by Severe Acute 464 Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) From a Symptomatic Patient. 465 JAMA

A pneumonia outbreak 467 associated with a new coronavirus of probable bat origin

Spike protein recognition of mammalian ACE2 predicts 470 the host range and an optimized ACE2 for SARS-CoV-2 infection

Coronavirus envelope protein: current knowledge

475 Structural analysis of full-length SARS-CoV-2 spike protein from an advanced 476 vaccine candidate

Structure of the SARS-CoV-2 spike 478 receptor-binding domain bound to the ACE2 receptor

Structural features of coronavirus SARS-CoV-2 spike 481 protein: Targets for vaccination

Cryo-EM 483 structure of the 2019-nCoV spike in the prefusion conformation

Angiotensin-486 converting enzyme 2 is a functional receptor for the SARS coronavirus

489 Stabilized coronavirus spikes are resistant to conformational changes induced by 490 receptor recognition or proteolysis

492 Nanobodies and their potential applications. Nanomedicine (Lond)

Nanobodies as therapeutics: big 495 opportunities for small antibodies

A Novel Nanobody Targeting 497 Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Receptor-Binding 498

Domain Has Potent Cross-Neutralizing Activity and Protective Efficacy against 499 MERS-CoV

An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction. Nat 502 Commun

504 Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with 505 ACE2

Structure-507 guided multivalent nanobodies block SARS-CoV-2 infection and suppress 508 mutational escape

An 511 ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike

Nanobodies from 514 camelid mice and llamas neutralize SARS-CoV-2 variants

Vertical Flow 517 Cellulose-Based Assays for SARS-CoV-2 Antibody Detection in Human Serum. ACS 518 Sens

CoV-2 Antigen Test Using Engineered Affinity Proteins

Engineering chimeric 523 thermostable GH7 cellobiohydrolases in Saccharomyces cerevisiae

Properties, production, and applications of camelid 526 single-domain antibody fragments

Cellulose affinity purification of fusion proteins 528 tagged with fungal family 1 cellulose-binding domain

531 Characterization and affinity applications of cellulose-binding domains

Cellulose-binding domains: biotechnological applications

A colorimetric paper sensor for lactate assay using a 536 cellulose-Binding recombinant enzyme

539 Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay 540 developers

Multivalent 542 Anchoring and Oriented Display of Single-Domain Antibodies on Cellulose

Enzyme-linked assay of cellulose-545 binding domain functions from Cellulomonas fimi on multi-well microtiter plate

Loading Immobilization of Proteins by Using Cohesin-Dockerin and CBM-Cellulose 549 Interactions

Coronavirus Disease 2019: Coronaviruses and Blood 551 Safety

Pathogen reduction of SARS-553

CoV-2 virus in plasma and whole blood using riboflavin and UV light

Airborne transmission of SARS-CoV-2: The world should face 557 the reality

Determinants of the Detection Limit and Specificity of Surface-Based 560

Paper-based diagnostics in 562 the antigen-depletion regime: High-density immobilization of rcSso7d-cellulose-563 binding domain fusion proteins for efficient target capture

Establishment and validation of a 566 pseudovirus neutralization assay for SARS-CoV-2. Emerg Microbes Infect

Evaluating the 569 Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity

SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity 573 of the COVID-19 Virus

SARS-CoV-2 575 spike-protein D614G mutation increases virion spike density and infectivity. Nat 576 Commun

Structural basis for the recognition of 578 SARS-CoV-2 by full-length human ACE2

Viral load of SARS-CoV-2 in clinical 580 samples. The Lancet Infectious Diseases

Virological assessment of hospitalized patients with COVID-2019

Engineering Bacillus subtilis as a 585 Versatile and Stable Platform for Production of Nanobodies

Point-of-Care Diagnostics: Present and Future

A guideline to limit indoor airborne transmission of COVID-592 19

Versatile and 595 multivalent nanobodies efficiently neutralize SARS-CoV-2. Science

Protocol 598 and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein 599 for Neutralization Assays

Engineering the Immune Adaptor 602

Advanced Therapeutics. n/a(n/a):2100066. solutions were applied to the filter paper, followed by immunoblotting with anti-630 FLAG directly on the filter paper

ImageJ, protein abundance increased with concentration. We estimate that 500 ng of 632

Ty1-CBD binds to 1 mm 2 of cellulose paper in saturation status. c) Schematic of an 633 immunoassay to evaluate the function of the fusion protein. Ty1-CBD fusion proteins were 634 immobilized on cellulose paper and then submerged in culture media containing

~100 ng/ml) as a proxy for actual SARS-CoV-2. The capturing capability was confirmed 636 by anti-human Fc-HRP and the DAB substrate. The structure of the RBD was adapted 637 from PDB: 6ZXN. d) Testing the ability of protein

Representative discs were prepared by a 6-mm biopsy punch and 639 then coated with E. coli lysates containing indicated recombinant fusion proteins. The 640 functionalized discs were incubated with RBD-containing or control (no RBD) media. The 641 intensity of dark staining was strongest from the combination of Ty1-CBD-coated disc and 642 RBD-containing media

Schematic overview of the 646 pseudotyped virus production: HEK293T cells were transfected with a lentiviral vector 647 expressing a green fluorescent protein (GFP), a plasmid encoding SARS-CoV-2 spike, 648 and packaging vectors. The transfected cells produced lentiviral particles pseudotyped 649 with the S protein of SARS-CoV-2, and the pseudovirus can transduce HEK293T 650 expressing human angiotensin-converting enzyme

Microscope images showing that the HEK293T-hACE2 cells expressed GFP after 652 transduction with lentivirus pseudotyped with the wildtype (WT) SARS-CoV-2 spike 653 protein or the D614G variant. Scale bar = 100 µm. c) Representative flow cytometric 654 analysis evaluating the transduction efficiency of SARS-CoV-2 WT and D614G 655 pseudoviruses compared with two negative control groups: HEK293T-hACE2 without any 656 transduction and HEK293T

Results are representative of three independent experiments

SARS-CoV-2 pseudovirus capture by Ty-CBD-immobilized cellulose in 660 two different formats: a) Schematic of increasing surface densities of Ty1-CBD through 661 protein immobilization on cellulose materials for SARS-CoV-2 neutralization

neutralization efficacy of pseudovirus through protein immobilization on cellulose paper 663 over free proteins

Ty1-CBD or Ty1 (negative control) immobilized on cellulose paper or free protein with 665 equal concentrations, the titers of wildtype (WT) and D614G pseudoviruses were 666 quantified by transducing HEK293T-hACE2 cells with the remaining viruses in the 667

Ty1-CBD-functionalized RAC column to capture the antigen of interest 669 in a continuous fashion. d) Neutralization efficacy of Ty1-CBD-functionalized RAC. The 670 flow through samples from functionalized RAC columns were used to transduce 671 HEK293T-hACE2 cells to quantify viral titers for WT

Fold changes from each treatment group were normalized 673 to that of RAC only. Graphs are expressed as mean ± SEM (n = 4) in b and as mean ± 674

Statistical analysis was performed by one-way analysis of variance 675 (ANOVA) according to the following scale: **P < 0.01, ***P < 0.001, and ****P < 0

The authors declare that there is no conflict of interest. 447 448