Nanobody-Functionalized Cellulose for Capturing SARS-CoV-2

Abstract

The highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 253 million people, claiming ∼5.1 million lives to date. Although mandatory quarantines, lockdowns, and vaccinations help curb viral transmission, there is a pressing need for cost-effective systems to mitigate the viral spread. Here, we present a generic strategy for capturing SARS-CoV-2 through functionalized 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 wild type 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 to capture viruses from complex fluids in a continuous fashion. By capturing and containing viruses through the Nb-functionalized cellulose, our work may find utilities in virus sampling and filtration through the development of paper-based diagnostics, environmental tracking of viral spread, and reducing the viral load from infected individuals. IMPORTANCE The ongoing efforts to address the COVID-19 pandemic center around the development of diagnostics, preventative measures, and therapeutic strategies. In comparison to existing work, we have provided a complementary strategy to capture SARS-CoV-2 by functionalized cellulose materials through paper-based diagnostics as well as virus filtration in perishable samples. Specifically, we developed 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, the fusion protein-coated cellulose substrates exhibited enhanced capture efficiency against SARS-CoV-2 pseudovirus of both the wild type 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 for binding viruses from complex biological fluids in a highly continuous and cost-effective manner. Such antigen-specific capture can potentially immobilize viruses of interest for viral detection and removal, which contrasts with the common size- or affinity-based filtration devices that bind a broad range of bacteria, viruses, fungi, and cytokines present in blood (https://ichgcp.net/clinical-trials-registry/NCT04413955). Additionally, since our work focuses on capturing and concentrating viruses from surfaces and fluids as a means to improve detection, it can serve as an "add-on" technology to complement existing viral detection methods, many of which have been largely focusing on improving intrinsic sensitivities.

Keywords: COVID-19; SARS-CoV-2; cellulose; cellulose binding domain; cellulose binding protein; nanobody.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1

Development of a bifunctional fusion protein to enable cellulose immobilization and subsequent detection and capture of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). (a) An alpaca-derived high-affinity nanobody (Nb), Ty1, for the receptor-binding domain (RBD) of SARS-CoV-2 was genetically fused with (b) a cellulose-binding domain (CBD) isolated from Clostridium thermocellum. (c) As SARS-CoV-2 is transmitted through surface contact, CBD fusion proteins or CBD-containing E. coli cell lysate was immobilized on the surface of cellulose materials, such as cellulose paper, for viral enrichment toward the development of paper-based diagnostics. (d) We also customized Nb-dependent regenerated amorphous cellulose (RAC) materials to specifically deplete the viral load of SARS-CoV-2 from virus-containing samples.

FIG 2

The fusion protein maintains its activities in binding cellulose and the RBD of SARS-CoV-2. (a) Detection of immobilized Nb (Ty1)-CBD on a cellulose paper. Nb-CBD was first spotted onto a piece of cellulose paper. Upon air drying, the paper was incubated with a rat antibody against the FLAG epitope (DYKDDDDK), followed by an anti-rat secondary antibody conjugated with HRP. The dark precipitate “Anti-COVID” was visualized after incubation with 3,3′-diaminobenzidine (DAB). (b) Quantification of maximal protein absorption on Whatman filter paper. Ten microliters of serially diluted fusion protein solutions was applied to the filter paper, followed by immunoblotting with anti-FLAG directly on the filter paper. Based on the normalized unit intensity quantified by ImageJ, protein abundance increased with concentration. We estimate that 500 ng of Nb-CBD binds to 1 mm2 of cellulose paper at saturation status. (c) Schematic of an immunoassay to evaluate the function of the fusion protein. Nb-CBD fusion proteins were immobilized on cellulose paper and then submerged in culture medium containing RBD-Fc (∼100 ng/mL) as a proxy for actual SARS-CoV-2. The capture capability was confirmed by anti-human Fc-HRP and the DAB substrate. The structure of the RBD was adapted from PDB ID no. 6ZXN. (d) Testing the capture capability of protein-coated cellulose paper discs in RBD-containing medium. Representative discs were prepared by a 6-mm biopsy punch and then coated with E. coli lysates containing the indicated recombinant fusion proteins. The functionalized discs were incubated with RBD-containing or control (no RBD) medium. The intensity of dark staining was strongest from the combination of Nb-CBD-coated disc and RBD-containing medium (∼100 ng/mL).

FIG 3

Generation of wild-type and D614G pseudoviruses for functional assays by Nb (Ty1)-CBD-functionalized cellulose. (a) Schematic overview of the pseudovirus production. HEK293T cells were transfected with a lentiviral vector expressing a green fluorescent protein (GFP), a plasmid encoding SARS-CoV-2 spike, and packaging vectors. The transfected cells produced lentiviral particles pseudotyped with the S protein of SARS-CoV-2, and the pseudovirus can transduce HEK293T expressing human angiotensin-converting enzyme 2 (hACE2) to express GFP. (b) Microscope images showing that the HEK293T-hACE2 cells expressed GFP after transduction with lentivirus pseudotyped with the wild-type (WT) SARS-CoV-2 spike protein or the D614G variant. Scale bar, 100 μm. (c) Representative flow cytometric analysis evaluating the transduction efficiency of SARS-CoV-2 WT and D614G pseudoviruses compared with two negative-control groups: HEK293T-hACE2 without any transduction and HEK293T transduced with SARS-CoV-2 WT pseudotyped lentivirus. Results are representative of three independent experiments.

FIG 4

SARS-CoV-2 pseudovirus capture by Nb (Ty1)-CBD-immobilized cellulose in two different formats. (a) Schematic of increasing surface densities of Nb-CBD through protein immobilization on cellulose materials for SARS-CoV-2 neutralization. (b) Increased neutralization efficacy of pseudovirus through protein immobilization on cellulose paper over free proteins. After incubation of the pseudovirus with 200 μL of 10 μg/mL fusion protein Nb-CBD or Nb (negative control) immobilized on cellulose paper or free protein with equal concentrations, the titers of wild-type (WT) and D614G pseudoviruses were quantified by transducing HEK293T-hACE2 cells with the remaining viruses in the supernatant. Fold changes from each treatment group were normalized to that of filter paper only. (c) An in vitro cellular assay to confirm the complete removal of bacterial toxins from the Nb-CBD-functionalized RAC column. Bacterial toxins were removed in a single step with 0.1% Triton X-114. Flowthrough factions from Nb-CBD-functionalized RAC were added to RAW-Blue cells, an engineered murine macrophage cell line that can detect trace levels of endotoxin and other bacterial toxins. The secretion of a reporter protein, phosphatase, can indicate the presence of bacterial toxins as innate immune agonists. It was found that direct flowthrough fractions were indeed contaminated with bacterial toxins, denoted by “Endotoxin contaminated,” while flowthrough fractions from Triton X-114-treated RAC (denoted by “Endotoxin removed”) did not have detectable levels of toxins compared to the culture medium control. A schematic of an Nb-CBD-functionalized RAC column is shown in the inset. (d) Capture efficacy of Nb-CBD-functionalized RAC. The flowthrough samples from functionalized RAC columns were used to transduce HEK293T-hACE2 cells to quantify viral titers for WT and D614G SARS-CoV-2 pseudoviruses, respectively. Fold changes from each treatment group were normalized to that of RAC only. Graphs are expressed as mean ± SEM (n = 4) in panel b and as mean ± SEM (n = 3) in panel d. Statistical analysis was performed by one-way analysis of variance (ANOVA) according to the following scale: **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.