Engineered Intranasal Virus Trap Provides Effective Protection Against SARS‐CoV‐2 Infection in Hamsters

As face masks are no longer required in many public areas, SARS‐CoV‐2 continues to spread and pose health risks to vulnerable populations such as children, the elderly, and immunocompromised individuals. This study presents the development of an Fn‐LCB1‐based engineered intranasal virus trap (EIVT) designed to capture and neutralize multiple SARS‐CoV‐2 variants, limiting viral infection and transmission. Fn‐LCB1, a fusion protein consisting of an Fn domain that binds to fibronectin and an LCB1 domain with high affinity for the Spike protein receptor‐binding domain (RBD) of SARS‐CoV‐2 viruses, can be produced on a large scale and purified in soluble form with high thermal stability. In vitro experiments demonstrated the efficient neutralization of SARS‐CoV‐2 wildtype and several variants, including Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529) by Fn‐LCB1. Additionally, Fn‐LCB1 effectively protected against SARS‐CoV‐2 Delta infection in a noncontact viral transmission Syrian hamster model. This study establishes Fn‐LCB1 as a potent prophylactic agent against SARS‐CoV‐2 in vitro and in vivo, and serves as a proof‐of‐concept for the application of intranasal proteins to capture respiratory viruses and reduce live cell infections by competing with viral receptors.


Introduction
While shifting to the endemic stage of Coronavirus Disease 2019 (COVID- 19), governments have loosened the regulations on infectious disease control.As societies return to normality, masks are no longer worn in public areas in many countries.As attitudes of the general population have changed, less tests would be done and fewer cases would be reported.Nevertheless, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus continues to transmit among people and possibly mutates to other dangerous variants. [1]Many are still dying from the viral infection and re-infections can lead to ongoing viral transmission. [2]ence, it is believed that the high-risk population, especially the elderly and the immunocompromised, should take protective measures under the pandemic background.In order to establish a prophylactic intranasal spray against SARS-CoV-2 Figure 1.Design of Fn-LCB1-based EIVT against SARS-CoV-2.A) Schematic of Fn-LCB1 design.Fn-LCB1 can be applied to the nasal cavity as an EIVT against SARS-CoV-2.B) Fn-LCB1 consists of an Fn domain that binds to fibronectin, and a LCB1 domain with high affinity to RBD region of SARS-CoV-2 viruses.
infection, we designed a fusion protein Fn-LCB1 to serve as an engineered intranasal virus trap (EIVT).The protein comprises of a fibronectin-binding domain taken from Staphylococcus aureus (S. aureus) and a SARS-CoV-2 spike protein targeting miniprotein, LCB1 (Figure 1A).
Fibronectin binding protein A (FnBPA) is one of the virulence factors of S. aureus. [3]It consists of 11 repeats and facilitates bacterial adherence to the epithelium. [4]The Fn part of Fn-LCB1 is composed of the first 3 domains of FnBPA, allowing the fusion protein to bind strongly to fibronectin in the epithelial extracellular matrix. [5]Fibronectin is abundant in the extracellular matrix of epithelial cells [6] and on the cell surface. [7]Fibronectin was also previously proven to be expressed along the respiratory tract. [8]As the nasal epithelium has the highest expression of angiotensin-converting enzyme 2 (ACE2) receptors, [9] it is an ideal site to block viral infection and transmission.It is therefore hypothesized that intranasal application of Fn-LCB1 can coat the nasal and respiratory epithelium to serve as an effective barrier for reducing viral infection.Meanwhile, LCB1 is an ultrapotent miniprotein that mimics the structure of ACE2 receptors. [10]LCB1 was proved to be able to neutralize wildtype SARS-CoV-2 viruses as well as multiple mutants at a picomolar concentration. [10,11]We proposed that by coating the nasal epithelium with Fn-LCB1, viruses can be captured and inactivated in the upper respiratory tract before they infect the host.On the other hand, LCB1 can also compete with the ACE2 receptors present along the respiratory tract to minimize live cell infection.We hence hypothesized that applying a Fn-LCB1 barrier at the primary infection site of SARS-CoV-2 can limit its replication, and therefore reduce viral transmission among the population by controlling viral shedding (Figure 1B).
In this study, we demonstrated the effectiveness of the engineered intranasal virus trap (EIVT) in neutralizing multiple SARS-CoV-2 variants in vitro.Furthermore, we showed that the EIVT provided efficient protection against SARS-CoV-2 infection in the golden Syrian hamster model.These findings highlight the potential of EIVT as a valuable prophylactic agent against the ongoing spread of SARS-CoV-2 and its emerging variants.

Production and Characterization of Fn-LCB1
Fn-LCB1 is a fusion protein with an Fn domain and an LCB1 domain connected by a GS linker (Figure 2A).The entire protein has a molecular weight of ≈17 kDa.The protein can be expressed and purified from E. Coli BL21 with a three-step chromatography strategy.The majority (> 95%) of Fn-LCB1 was produced in a soluble form at a high yield of at least 5 mg per 1 l of bacterial culture and can be purified with low levels of endotoxins (LPS) (≈1.5 EU mg −1 ).However, it usually exists as dimers of ≈35 kDa (Figure 2B).
As the Fn domain of Fn-LCB1 is a selected section from FnBPA, it is important to ensure its binding affinity to fibronectin proteins.To achieve this, we performed AlphaFold Multimer predictions to build models of the complex.Type 1 modules (F1) from fibronectin have previously been demonstrated to exhibit high affinity to S. aureus FnBPA proteins. [12]Therefore, we focused on the binding of the Fn domain of the Fn-LCB1 fusion protein to the F1 regions of human fibronectin proteins in our prediction model.We chose a specific model with the highest confidence in the pLDDT plot (model 1) for further analysis (Figure S1, Supporting Information).The interactions between the Fn and F1 regions were organized in modular networks.The Fn domain primarily consists of a chain of -strands, in which each segment can conjugate to individual F1 modules arranged in tandem in fibronectin (Figure 2C, left).To gain molecular insights into this modular interaction, we analyzed a partial segment with two modules.We observed extensive hydrogen bonding (Figure 2C, right upper panel) and abundant hydrophobic residues (Figure 2C, right lower panel) in the interaction interface, indicating strong affinity and high specificity within the modular interactions.This specific interaction network between fibronectin and FnBPA thus allows for the firm attachment of Fn-LCB1 in vivo.

Thermal Stability of Fn-LCB1
To test the stability of Fn-LCB1, we placed the protein solution at room temperature (RT) for ≈30 days with and without the addition of protease inhibitor, phenylmethylsulphonyl fluoride (PMSF).Surprisingly, from the Coomassie staining result, Fn-LCB1 exhibited great thermal stability, as it did not degrade after being placed at room temperature for 29 days (Figure 2D), even without PMSF (Figure 2E).We also performed Circular Dichroism Spectroscopy (CD) to investigate the secondary structure stability of the protein under different temperatures.After placing Fn-LCB1 in 4 °C, RT, and 37 °C for 24 h, respectively, CD spectra from 195 to 260 nm were measured.It was shown that the secondary structure of the protein did not change after being placed in different temperature conditions and could therefore sustain its functions for real-life applications (Figure 2F).Thermal denaturation assay also showed that Fn-LCB1 has a melting temperature higher than 95 °C (Figure 2G).Hence, our data demonstrated that Fn-LCB1 can be easily produced and stored, and is structurally stable to maintain its function in the real-life scenario.
To gain a better understanding of the interactions between Fn-LCB1 and SARS-CoV-2 viruses, we performed PDBePISA calculations [13] and Ligplot + analysis. [14]The interface area between LCB1 and the RBD was measured to be 973.3square angstroms, which is larger than the interface area between the ACE2 receptor and RBD proteins at 855.0 square angstroms.
The LCB1-RBD interface contains both hydrophilic and hydrophobic residues, contributing to dominant polar interactions (cyan dashed lines) involving hydrogen bonds, salt bridges, and auxiliary hydrophobic interactions (Figure 3G).In contrast, the ACE2-RBD interface consists of fewer hydrophilic interactions, with weaker hydrophobic interactions being dominant, rendering the two protein structures more spatially distant in the complex (Figure 3H).Considering the spatially closer organization of LCB1 and RBD compared to that of ACE2 and RBD (Figure 3I), we hypothesized that the binding affinity of LCB1 to wildtype RBD would be higher than that of ACE2 to RBD, leading to high competence of LCB1 in virus neutralization.Consequently, we propose that the intranasal application of Fn-LCB1 can compete with ACE2 receptors along the respiratory tract to neutralize incoming viruses and limit live cell infection.
To further evaluate the effect of temperature on Fn-LCB1 functions, we placed the protein at room temperature for an extended period and measured its viral neutralization ability against Delta pseudoviruses and authentic viruses.Fn-LCB1 was able to neutralize Delta pseudovirus at a low concentration (EC50 = 11.4 fM) after being stored at 25 °C for 1 week (Figure 3J).Its neutralization ability against Delta authentic virus was also retained (EC50 = 0.194 μg mL −1 ) after being stored at room temperature for 3 days (Figure 3K).Thus, consistent with the CD spectra analysis, Fn-LCB1 exhibits high thermal stability for storage and usage in real-life scenarios.

Intranasal Application of Fn-LCB1 Reduces SARS-CoV2 Delta Transmission
To test the prophylactic effectiveness of Fn-LCB1 in vivo, we performed a SARS-CoV-2 Delta noncontact transmission  experiment with the Golden Syrian hamster model.The Delta variant was used as it showed 100% transmission without protection in our transmission condition.For the experimental setting, we subdivided each hamster cage with an acrylic board.Multiple small holes were opened on the top part of the board for airflow communication between the two cage compartments.One infected hamster was placed on one side of the cage 24 h after infecting with 2 × 10 4 TCID 50 of Delta variant in the nasal turbinates (Figure 4A).Two naïve hamsters were inoculated intranasally with 20 μL of Fn-LCB1 (1.5 mg mL −1 ) or 20 μL of PBS.The hamsters were then cohoused with the infected hamsters in the other compartment of the cage.After 6 h of exposure, the naïve hamsters were transferred to a new clean cage.Hamsters were then sacrificed 3 days later to retrieve nasal turbinates and lungs for further analysis (Figure 4A).
In line with the in vitro data, we showed that the intranasal application of Fn-LCB1 can effectively protect the host against infection by SARS-CoV-2 Delta variant, as evidenced by reduced viral titers in the nasal turbinate and lung tissues.As shown in Figure 4B, there were significantly lower viral spike gene copies in the nasal turbinates of naïve hamsters with the Fn-LCB1 compared with the PBS control (p = 0.0013).We also found lower levels of viral spike gene copies in the lungs of naïve hamsters protected by Fn-LCB1 compared with the PBS control (p = 0.0079) (Figure 4C).
To confirm if the viruses became inactivated after being captured by Fn-LCB1 intranasally, we measured the live virus titer from nasal turbinate and lung tissues using plaque assay.It was shown that infectious virus titers were significantly lower in nasal turbinates (p = 0.006) and lung (p < 0.0001) of naïve hamsters with Fn-LCB1 application compared with the PBS control (Figure 4D,E).
Additionally, the Haematoxylin and Eosin stain (H&E stain) showed reduced epithelial shedding and better-maintained histological structures in the nasal turbinate of naïve hamsters treated with Fn-LCB1 as compared to the PBS group (Figure 4F left).The H&E stain analysis further revealed that the lung tissues of PBS control hamsters had severe mononuclear inflammatory cell infiltration and distorted alveolar structures with diffuse loss of alveolar space (Figure 4G left).In contrast, the lung tissues of hamsters treated with Fn-LCB1 showed minimal histopathological changes, with largely preserved alveolar structures and less inflammatory cell infiltration (Figure 4G right).This suggests that Fn-LCB1 administration helps in protecting the lung tissues from the damaging effects of the virus.
Immunofluorescence staining assay detected abundant SARS-CoV-2 nucleocapsid (N) protein expression in the nasal turbinates of all hamsters (Figure 4H), but a decreased expression of N proteins in the lungs of hamsters protected by Fn-LCB1 compared to the PBS group (Figure 4I).Since viruses are captured in the nasal cavity, N proteins can still be detected even after the viruses become inactive, which is why they were stained in the nasal turbinates of the Fn-LCB1-protected groups.However, the reduced viral titers in both lungs and nasal turbinates of hamsters protected by the Fn-LCB1 spray suggest that the viruses lose their ability to infect live cells and replicate after being captured.This is consistent with the reduced cellular damage seen on the H&E stains of the protein groups, despite the detection of N protein signals.These results indicate that Fn-LCB1 is effective in blocking viral transmission and infection in the nasal turbinate of hamsters.
To further visualize the distribution of Fn-LCB1 in vivo after intranasal application, we stained hamster lungs and nasal turbinate with FITC-labelled anti-His antibodies to detect the His-tagged Fn-LCB1.The fluorescence signal demonstrated that Fn-LCB1 strongly adhered to the epithelial layer in the nasal turbinate and did not enter the lungs (Figure 4J,K).The fluorescence signal in the nasal epithelium also matches the locations of the N proteins.Together with the much-reduced live virus titer in the protein-protected group, this data indicates that SARS-CoV-2 viruses are captured and remain adhered to the connective tissue layer, which then likely loses their ability to infect live cells for replication and transmission.Overall, the result demonstrates that by coating the upper respiratory tract with virus-neutralizing proteins, respiratory viruses can be firmly captured and hence neutralized to limit live cell infection, viral replication, and shedding.

Discussion
With a higher vaccination percentage and prevalence of the Omicron variant, SARS-CoV-2 is less lethal than it was when the pandemic first started.Nevertheless, infection cases are still growing worldwide and there are still a lot of uncertainties about the progression of the pandemic.SARS-CoV-2 still presents great health risks, especially during flu seasons, and surges of respiratory cases can overwhelm healthcare facilities. [15]While living with SARS-CoV-2, the high-risk population should still take measures to avoid infections.
In this study, we proposed the use of a Fn-LCB1-based intranasal spray for SARS-CoV-2 prophylaxis.By combining an S. aureus-derived protein Fn [5a] and a de novo mini-peptide LCB1, [10] we produced Fn-LCB1, which has high binding affinity to fibronectin and RBD region of SARS-CoV-2.As the major component of Fn-LCB1 comes from a naturally-existing bacterial protein FnBPA, the fusion protein demonstrated high thermal stability and can be conveniently produced in large quantities from the BL21 strain.
Although Fn-LCB1 exists as dimers in the soluble state, the structure did not hamper its viral neutralization efficiency.Pseudovirus experiments showed that Fn-LCB1 has a high binding affinity towards wildtype SARS-CoV-2 and several variants like Alpha, Delta, and Omicron BA.1.Fn-LCB1 also demonstrated high neutralization ability against SARS-CoV-2 Delta authentic virus.The viral neutralization ability was also retained after being stored at room temperature for a prolonged time.As LCB1 was first designed to neutralize wildtype SARS-CoV-2, it displayed better binding affinity against the older viral mutants.Although Fn-LCB1 can still neutralize Omicron BA.1 at picomolar concentrations, a higher working concentration is required for further Omicron sublineages like BA.5 and BF.7, as the neutralization efficiency only reached 45.76% for BA.5 and 74.34% for BF.7 pseudoviruses when 25.16 pmol of Fn-LCB1 was applied (Supplementary Figure S2, Supporting Information).This suggests that while Fn-LCB1 is effective against several SARS-CoV-2 variants, its neutralization ability may vary for different sublineages within a given variant, and optimization may be needed to improve its efficacy against specific sublineages.Nevertheless, this study demonstrated that miniproteins can provide protection against a wide spectrum of SARS-CoV2 mutants.Future de-novo proteins can be designed to target newer variants with RBD regions that have mutated further away from the wildtype strain.The LCB1 domain can also be replaced by other miniproteins to target other respiratory viruses.
Importantly in this study, we demonstrated that Fn-LCB1 can capture incoming SARS-CoV-2 viruses in aerosol particles and reduce their transmission in vivo.The intranasal application of 30 μg of Fn-LCB1 can significantly reduce the live virus titer in both nasal turbinate and lung tissues in a Syrian hamster noncontact transmission model.With a lower infectious titer, viruses lead to much-reduced tissue damage and infiltration of inflammatory cells seen in both lungs and nasal turbinates of hamsters protected by Fn-LCB1.
The findings from the fluorescence staining data show that Fn-LCB1 can effectively attach to fibronectin tissue in the nasal cavity, competing with ACE2 receptors.Additionally, while live viruses are captured in the respiratory tract, most of them may be locked in the extracellular matrix and thus cannot infect the host cells.This is supported by the colocalization of Fn-LCB1 and N proteins in the nasal turbinates.These results demonstrate the effectiveness of Fn-LCB1 in preventing viral infection by both competing with ACE2 receptors and capturing live viruses in the respiratory tract, preventing them from infecting host cells.
Nevertheless, some viruses can enter the lower respiratory tract without being in contact with the nasal epithelium, leading to some infection in the lung.Yet, as the intranasal proteins are able to filter a majority of the viruses, viral damage to nasal turbinate and lung tissues can be minimized.With fewer live viruses replicating, both viral transmission and clinical symptoms can be reduced.
In conclusion, the study demonstrates that Fn-LCB1 can serve as an inhaled antiviral agent to prevent SARS-CoV-2 infection and reduce viral transmission.Given a recent study showed that intranasal application of Remdesivir can reduce viral titer in a nonhuman primate model, [16] this study provides proof of concept that the intranasal route can also be targeted for reducing live virus replication and cellular damage using a protein spray like Fn-LCB1.This suggests that Fn-LCB1 could be a promising therapeutic option for preventing and controlling the spread of COVID-19.

Experimental Section
Production of Fn-LCB1: The Fn-LCB1 DNA sequence was synthesized (BGI Group) and cloned into pET28a (+) to generate the pET28a-Fn-LCB1 plasmid.To facilitate purification, a His tag was added to the C-terminal of Fn-LCB1.The pET28a-Fn-LCB1 plasmid was subsequently transformed into E. coli BL21 (DE).The bacteria from 1 L of bacterial culture was harvested by centrifugation.After sonication, pellets were resuspended in binding buffer (Tris-HCl pH 8.0, 20 mM imidazole) and centrifuged at 15000 × g for 30 min at 4 °C.Soluble cell extracts were collected and filtered in a 0.22 μm membrane.The filtrate was loaded on a nickel-NTA Sepharose column (GE Healthcare) and bound proteins were eluted with imidazole (400 mM) Tris-HCl (pH 8.0) buffer.The eluted His-tagged proteins were further purified by size exclusion chromatography.Purified proteins were identified by SDS-PAGE and then subject to endotoxin removal (Pierce).
Evaluation of Protein Binding Affinity: To assess the interaction between FnBPA and fibronectin, the AlphaFold2 Multimer predicted model was analyzed.Briefly, protein sequences of FnBPA and fibronectin F1 modules were uploaded for AlphaFold2 Multimer prediction, generating five models and corresponding pLDDT plots for model evaluation.The model with the highest pLDDT scores was selected for LigPlot+ analysis, which identified specific residues involved in the interaction.These residues were further visualized in PyMOL.
The interactions of LCB1 and ACE2 receptors with the RBD were assessed using LigPlot+ analysis of the structure of the LCB1-RBD complex (PDB ID: 7JZU) and the ACE2-RBD complex (PDB ID: 6M0J).Residues involved in the interaction interface were visualized in PyMOL.This approach allowed for a detailed evaluation of the binding affinities and specific residues involved in the interactions between these proteins, providing insights into their molecular mechanisms and potential for viral neutralization Circular Dichroism Spectra: CD measurements were done on Jasco J-815 Circular Dichroism (CD) Spectropolarimeter. 1 mL of Fn-LCB1 in PBS buffer at a concentration of 0.1 mg mL −1 was placed at 4 °C, room temperature, and 37 °C for 24 h.To evaluate the effect of temperature on the secondary structure of the fusion protein, wavelength was evaluated from 195 to 260 nm at 20 °C.For the temperature melt, dichroism signal was monitored from 20 °C to 95 °C at 222 nm in steps of 2 °C min −1 with 30 s of equilibration time.Wavelength scans and temperature melts were performed with a 1 mm path-length cuvette.
Pseudovirus-Based Virus Neutralization Test: A modified pseudovirusbased viral neutralization test (pVNT) was performed as previously described. [17]In brief, pVNT was carried out in HEK293-hACE2 stable cells.The Fn-LCB1 was serially diluted four times in PBS, and 10 μL of the protein was incubated with 50 μL of pseudoviruses on ice for 1 h.Next, HEK293T cells were infected with the mixture and incubated at 37 °C for 48 h.Firefly luciferase signal was detected by the Steady-Glo Luciferase Assay System (Promega, E2520) to evaluate pseudovirus infection.The fluorescent signal of plate was obtained by Varioskan LUX Multimode Microplate Reader (Thermo Scientific) and analyzed by the Thermo Scientific SkanIt Software.Before the experiment, Fn-LCB1 was stored at 4 °C or incubated at room temperature for 7 days.This was done to evaluate Figure 4.In vivo testing of the efficiency of the Fn-LCB1 against SARS-CoV-2 Delta variant.A) Schematic of in vivo testing of prophylactic ability of Fn-LCB1 intranasal spray against SARS-CoV-2 Delta.Index hamsters were infected with SARS-CoV-2 Delta 24 h prior to cohousing with naïve hamsters that were intranasally applied with PBS or Fn-LCB1.An acrylic board with air holes were used to separate the hamster cage to ensure airflow between the two compartments for noncontact viral transmission.The naïve hamsters were moved to a new cage for 6 h of cohouse.Hamsters were then sacrificed 3 days later for retrieval of nasal turbinates and lung tissues.B-C) Viral spike gene copies in hamster nasal turbinates and lungs were quantified using probe-specific RT-qPCR.(n = 4).D-E) The live virus titer in hamsters' nasal turbinates and lung tissues were measured by plaque assay.(n = 4).F-G) H&E staining of nasal turbinate (F) and lung tissues (G)of hamsters in PBS (left) and Fn-LCB1 (right) groups.H-I) SARS-CoV-2 nucleocapsid proteins (N proteins) in nasal turbinates and lung tissues were detected with immunofluorescence for PBS (left) and Fn-LCB1 (right) groups (white arrows).SARS-CoV-2 was identified using an antibody against N proteins (green signal).Cell nuclei were identified with the DAPI staining (blue signal).J-K) His-tagged Fn-LCB1 proteins were detected with FITC-labelled anti-His antibodies for PBS (left) and Fn-LCB1 (right) groups.Green signals indicate the location of Fn-LCB1 in the nasal turbinate and lung tissues, while blue signals indicate the DAPI staining.the effect of temperature on the stability and functionality of the protein.By testing the Fn-LCB1 protein after storage under different temperature conditions, we can better understand its potential for use in real-life scenarios and determine the optimal storage conditions for maintaining its viral neutralization capabilities.
Virus: SARS-CoV-2 Delta (B.1.617.2) were isolated from the nasopharyngeal aspirates of laboratory-confirmed COVID-19 patients in Hong Kong, [1,18] which were available at the Department of Microbiology of The University of Hong Kong (HKU).The virus was propagated and titrated in VeroE6-TMPRSS2 cells by plaque assay as previously described. [19]In vitro and in vivo experiments involving infectious SARS-CoV-2 were performed in a biosafety level 3 laboratory and strictly followed approved standard operating procedures. [20]ive Virus Neutralization Assay: To measure the neutralization of live SARS-CoV-2 delta variant, VeroE6-TMPRSS2 cells were seeded in 96-well plates in complete Medium (DMEM+ 10% foetal bovine serum (FBS) + 1% penicillin-streptomycin) overnight at 37 °C under 5% CO 2 .After 24 h, Fn-LCB1 was serially diluted in DMEM in 96-well plates in triplicate and then incubated with 0.01 multiplicity of infection of SARS-CoV-2 Delta variant for 0.5 h at 37 °C.The Fn-LCB1 treated virus was added to cells and further incubated at 37 °C under 5% CO 2 .The supernatant virus was measured by RT-qPCR at 24 h post-infection and IC 50 values were derived by fitting a nonlinear five-parameter dose-response curve to the data in GraphPad Prism v.9.0.Before the experiment, Fn-LCB1 was stored at 4 °C or incubated at room temperature for 3 days.
In Vivo Virus Challenge: The animal experiments were approved by the HKU Committee on the Use of Live Animals in Teaching and Research (CULATR).In brief, 6-to 8-week-old female Syrian hamsters were obtained from the HKU Centre for Comparative Medicine Research (CCMR). [21]amsters were housed under a 12-h day per night cycle at 65% humidity and 21 °C−23 °C ambient temperature with standard pellet food and water. [22]20a] The SARS-CoV-2 Delta stock was diluted in PBS to the desired concentration.Naïve hamsters were given 40 uL of either Fn-LCB1 or PBS solution intranasally.On day 1 after the virus challenge, hamsters infected with the SARS-CoV-2 (index) were cohoused with noninfected hamsters (naïve) separated by acrylic boards installed with different mask specimens.6 h after the virus challenge, all index hamsters were sacrificed and all naïve hamsters were moved to new iso-cages.On day 3 after the virus challenge, all naïve hamsters were euthanized and nasal turbinates and lungs were harvested for viral load titration and histological staining.
RNA Extraction and Quantitative RT-PCR: Harvested nasal turbinates and lungs were homogenized in Dulbecco's minimal essential medium (DMEM), followed by centrifugation at 10 000 rpm for 5 min.The tissues were lysed with RLT buffer at room temperature for 10 min followed by RNA extraction.Extracted viral RNA was reverse transcribed to cDNA by PrimeScript II 1 st Strand cDNA Synthesis Kit (Takara, Cat # RR036A).The cDNA was then amplified by using specific primer Spike-Forward: 5'-CCTACTAAATTAAATGATCTCTGCTTTACT-3' and Spike-Reverse: 5'-CAAGCTATAACGCAGCCTGTA-3' for detecting SARS-CoV-2 in LightCycle 480 SYBR Green I Master (Roach, USA).For quantitation, 10fold serial dilutions of standard plasmid were prepared to generate the calibration curve.The qPCR experiments were performed using LightCycler 96 system (Roche, USA).
Infectious Virus Titration by Plaque Assay: Infected hamsters were euthanized at 3 days post-infection.The harvested nasal turbinates and lungs were homogenized in DMEM and centrifuged.To measure infectious virus titer, supernatant from tissue homogenates was 10-fold serially diluted with DMEM and applied to Vero-TMPRSS2 cells for 1 h at 37 °C.After inoculation, cells were washed by PBS before overlaying with 1% low-melting agarose, and further incubated for 72 h.Cells were fixed with 4% (wt/vol) paraformaldehyde, and plaque formation was visualized with 0.5% crystal violet in 25% ethanol. [23]mmunofluorescence and H&E Staining: After fixing for 24 h in 10% neutral-buffered formalin, mouse tissues were processed, paraffinized, and sectioned to prepare 4 μm tissue sections on glass slides.After de-waxing, antigen retrieval was performed.SARS-CoV-2 nucleocapsid (N) protein was detected by an in-house rabbit polyclonal anti-SARS-CoV-2 N antibody followed by detection with the Alexa488 goat anti-rabbit antibody (Thermo Fisher Scientific).Nuclei were stained with the DAPI dye (Thermo Fisher Scientific) before the sections were mounted with the Diamond Prolong Antifade mounting buffer (Thermo Fisher Scientific) as we previously described. [24]For H&E staining, tissue sections were stained with Gill's hematoxylin and eosin Y (Thermo Fisher Scientific) as we previously described. [25]Images were acquired with the Olympus BX53 light microscope.
Immunofluorescence Staining for Fn-LCB1 Localization: To visualize the localization of Fn-LCB1 in nasal turbinate and lung sections, the following steps were taken: 1) Dewaxing: Nasal turbinate and lung sections were dewaxed to remove any paraffin from the tissue samples.2) Antigen retrieval: Antigen retrieval was performed to unmask the epitopes in the tissue samples, allowing for better antibody binding and staining.3) Blocking: The tissue samples were blocked with a suitable blocking solution to reduce nonspecific binding of the antibodies.4) Primary antibody incubation: FITCtagged anti-His antibodies (LSBio) were applied to the tissue sections and incubated overnight at 4 °C.This allowed the antibodies to bind specifically to the His-tagged Fn-LCB1 protein.5) Nuclei staining: The nuclei in the tissue sections were stained with DAPI dye (Thermo Fisher Scientific) to visualize the cellular structures.6) Mounting: The tissue sections were mounted using Diamond Prolong Antifade mounting buffer (Thermo Fisher Scientific) to preserve fluorescence and prevent photobleaching.Following these steps, the localization of Fn-LCB1 in the nasal turbinate and lung sections can be observed under a fluorescence microscope. [24]tatistical Analysis: Data visualization and analyses were performed in GraphPad prism 9.

Figure 2 .
Figure 2. Purification and thermal characteristics of Fn-LCB1.A) Schematic depiction of Fn-LCB1 gene sequence.The miniprotein LCB1 is connected to the Fn by a GS-linker.The sequence is inserted on pET-28a (+) plasmid, B) Fn-LCB1 can be produced with >95% purity and exists as dimers with ≈35 kDa in soluble form.C) The predicted structure of the Fn-fibronectin complex was obtained using AlphaFold Multimer prediction.In this model, individual -sheets of the Fn domain interact with F1 modules of the fibronectin protein (left).Extensive hydrogen bonds (right upper panel) and abundant hydrophobic residues (right lower panel) can be observed between the two proteins, indicating a strong interaction between the Fn domain and the fibronectin F1 modules.D-E) Fn-LCB1 was placed at room temperature (RT, 25 °C) with or without protease inhibitor PMSF for ≈30 days.Coomassie Blue staining demonstrated that the proteins remain stable even without PMSF in RT.F) Circular dichroism spectra of Fn-LCB1 after standing at 4 °C, 25 °C, and 37 °C for 24 h, respectively.The depression peaks represent the alpha-helical structures of Fn-LCB1.G) Circular dichroism signal at 222 nm wavelength as a function of temperature.

Figure 3 .
Figure 3. Neutralization efficiency of Fn-LCB1 against wildtype and mutant SARS-CoV-2 pseudoviruses and real viruses.A) Schematic of in vitro SARS-CoV2 virus neutralization experiment.(B-D) Fn-LCB1 is able to neutralize pseudoviruses infections (n = 2): wildtype SARS-CoV2 (EC50 = 16.20 fM) and multiple variants including Alpha (EC50 = 129.8 fM), Delta (EC50 = 0.0342 fM) and Omicron BA.1 (EC50 = 88.1 fM).(F) Fn-LCB1 is able to neutralize real virus infection: SARS-CoV2 Delta variant (EC50 = 0.00012 ug mL −1 ).(n = 3).G) Abundant polar interactions like hydrogen bonds, salt bridges, and auxiliary hydrophobic interactions can be seen between LCB1 domain and WT RBD protein.H) Hydrophobic interactions are dominant between ACE2 receptor protein and WT RBD protein.I) LCB1 and RBD have spatially closer interactions compared to those between the ACE2 receptor and RBD proteins.J) Fn-LB1 is able to neutralize Delta pseudovirus after being placed at room temperature for 1 week (EC50 = 11.4 fM).(n = 2).K) Fn-LB1 is able to neutralize Delta authentic virus after being placed at room temperature for 3 days (EC50 = 0.194 ug mL −1 ).(n = 3) 0. All statistical analyses in each experiment are specified in the respective figure legends.Statistical comparison between different groups was performed by one-way ANOVA, two-way ANOVA, or unpaired two-tailed Student's t-test as appropriate.Differences were considered statistically significant for *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.Study Approval: The animal protocols were approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of The University of Hong Kong (CULATR No.5610-21).