key: cord-0728305-zdh8gc27
authors: Singh, Sudhakar; Dahiya, Surbhi; Singh, Yuviana J.; Beeton, Komal; Jain, Ayush; Sarkar, Roman; Dubey, Abhishek; Tehseen, Syed Azeez; Sehrawat, Sharvan
title: Targeting conserved viral virulence determinants by single domain antibodies to block SARS-CoV2 infectivity
date: 2021-01-13
journal: bioRxiv
DOI: 10.1101/2021.01.13.426537
sha: 0291fd3db272db3edc8ce49caa8a284e027d1b94
doc_id: 728305
cord_uid: zdh8gc27

We selected SARS-CoV2 specific single domain antibodies (sdAbs) from a previously constructed phage display library using synthetic immunogenic peptides of the virus spike (S) protein as bait. The sdAbs targeting the cleavage site (CS) and the receptor binding domain (RBD) in S protein efficiently neutralised the infectivity of a pseudovirus expressing SARS-CoV2 S protein. Anti-CS sdAb blocked the virus infectivity by inhibiting proteolytic processing of SARS-CoV2 S protein. Both the sdAbs retained characteristic structure within the pH range of 2 to 12 and remained stable upto 65°C. Furthermore, structural disruptions induced by a high temperature in both the sdAbs were largely reversed upon their gradual cooling and the resulting products neutralised the reporter virus. Our results therefore suggest that targeting CS in addition to the RBD of S protein by sdAbs could serve as a viable option to reduce SARS-CoV2 infectivity and that proteolytic processing of the viral S protein is critical for infection.

The scale of infectivity and the spread of Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV2) caused COVID-19 pandemic continue to increase and the inflicted mortality is nearing two million worldwide 1 . There has been unprecedented progress in developing vaccines and therapeutics and some vaccines have been approved for use in some countries 2, 3 . Many aspects of vaccine induced protective immunity such as the durability of response, the involvement of key immune mediators are yet to be adequately investigated. The development of potent yet affordable therapeutics such as anti-viral monoclonal antibodies is highly desirable not only to limit the consequences of COVID-19 but also to decipher events in virus entry as well as intracellular trafficking processes. Many such steps could provide useful anti-viral targets especially because the virus generates escape mutants that might be refractory to the induced immunity contributing to the enhanced virulence or transmissibility 4 .

Passive immunotherapy with convalescent sera from recovered COVID-19 patients has limited utility and suffers from scalability issues 5 . Furthermore, the full-length conventional antibodies when administered in low abundance or those binding to the viral antigens with suboptimal avidities might contribute to antibody dependent enhancement (ADE) of infection 6, 7, 8 . Therefore, smaller variants of neutralizing antibodies such as fragment antigen binding (Fab), single chain fragment variable (scFv) or camelid single domain antibodies (sdAbs) could be considered as a promising approach 9, 10 . Such interventions could reduce the viral loads to levels, which are efficiently controlled by immune mediators to restore homeostasis. Therefore, a potentially severe COVID-19 could be converted into a mild and manageable reaction.

However, the commonly observed aggregation tendencies of Fabs and scFvs are yet to be satisfactorily resolved but the sdAbs are usually refractory to structural perturbations induced by biochemical and biophysical insults 11 . Such binders are well suited to neutralize infectious agents and toxins due to their ability to seek out cryptic epitopes 12 . Furthermore, SARS-CoV2 specific sdAbs could serve as valuable tool to investigate internalization, intracellular trafficking, replication, and assembly as well as the exocytic processes of the virus.

Membrane anchored spike (S) protein of SARS-CoV2 facilitates the virus entry in susceptible cells and is therefore considered as a major target for anti-viral manoeuvres 13, 14 . Proteases such as furin, the host cell membrane associated TMPRSS2 process S protein by recognizing its polybasic residues (RRAR) in the cleavage site (CS) to generate S1 and S2 fragments 15, 16 . This step is considered crucial for SARS-CoV2 entry, the viral fusion with endosomal membranes and to ensure the release of its genomes for translation and replication 17, 18 . The acquisition of such a site by the newly emerged SARS-CoV2 is proposed to serve as a virulent factor for enhanced infectivity 19 . Not only SARS-CoV2 but many other viruses, bacterial products and toxins enhance their pathogenicity by such acquisitions and adaptations 20 . Antibodies targeting the RBD of SARS-CoV2 S protein have been selected and shown effective in the virus neutralization but those targeting the CS of SARS-CoV2 are not yet reported 21 . We therefore selected sdAbs against the CS and the RBD of S protein from a previously generated camelid variable region of heavy chain of heavy chain antibodies (VHH) phage display library 22 .

We used synthetic peptides encompassing the cleavage site between S1/S2 as well as the linear epitopes present in the RBD of SARS-CoV2 S protein that were predicted to be immunogenic for selecting SARS-CoV2 specific sdAbs. Characteristically such sdAbs resisted structural disruptions and retained functionality even when exposed to harsh biophysical and biochemical conditions. We then demonstrated neutralizing potential of such sdAbs using a lentivirus based pseudovirus expressing surface SARS-CoV2 S protein as their entry mediator. Anti-CS sdAbs prevented the proteolytic cleavage of S protein and in so doing blocked the virus entry. Therefore, targeting multibasic CS as well as the RBD by sdAbs could be considered as a potential strategy to reduce SARS-CoV2 infectivity.

We predicted B cell epitopes of SARS-CoV2 S protein using the input amino acid sequence (PDB ID: 6VSB) described elsewhere 23 . The target regions of RBD were such selected that antibodies against such epitopes could interfere with the interactions involving the virus S protein and cellular entry receptors (Fig 1A) . We also used a peptide encompassing the polybasic CS between S1/S2 fragments of S protein to select binders that could inhibit proteolytic processing by masking the site and in so doing inhibit the virus infectivity ( Fig 1A, middle panel) . Using synthetic peptides as bait, we biopanned specific sdAbs from a previously constructed phage display library without immunizing animals with SARS-CoV2 or its derivatives (Fig 1B, 24 ). The sdAbs were expressed in E. coli (TG1 strain) and purified from the periplasmic fractions as well as inclusion bodies (Fig 1C and D , S1A-F). The purity and structural properties of the sdAbs were analysed by gel filtration, SDS-PAGE and circular dichroism (Fig 1C and D , and S1B-F). Fractions in peak 2 (P2) contained folded anti-CS sdAbs as revealed by a prominent peak at 218nm and a minor peak at 230nm in CD spectral plots, which indicated the presence of β sheets and aromatic amino acids in its secondary structure ( Fig S1B, inset) 25 . Similarly, we selected, expressed and purified anti-RBD sdAb that were fully folded as revealed by CD plots (Fig 1D, S1D -F).

We then analysed physicochemical robustness of sdAbs by exposing them to a range of pH and temperature (Fig 1E-G) . The sdAbs retained their structural attributes between the pH ranges of 2 to 12 ( Fig 1E) . The sdAbs resisted structural perturbations when exposed upto a 65°C temperature ( Fig 1F) . Furthermore, the lost structures at high temperature (70°C) were regained with a gradual reduction in the storage temperature to 20°C (Fig 1G, data not shown) . The affinity of anti-CS sdAbs with SARS-CoV2 S proteins was measured by biolayer interferometry and was found to be in the nanomolar range (Kd=2.6x10 -8 ) (Fig 1H) . The immune reactivity and the specificity of these sdAbs were determined against their selecting peptides as well as SARS-CoV2 S protein by western blotting and ELISA (Fig 1I and J, Fig 2) . Anti-CS and anti-RBD sdAbs reacted with the virus S protein (~180kDa) while anti-RBD also reacted with recombinantly expressed RBD revealing a band of ~35kDa size (Fig 1I and J) . The SARS-CoV2 S transfected HEK293T cells when probed with anti-CS sdAb revealed a specific band migrating above 180kDa molecular mass while no reactivity was observed against untransfected cells, assembled bald particles of pseudovirus, LV(BALD), or the VSV-G protein expressing pseudovirus, LV(VSV-G), resolved by a 12% SDS-PAGE followed by western blotting (Fig 1I and J) .

The CS and the RBD peptides when probed with their specific sdAbs expectedly showed a concentration dependent increase in ELISA readouts (OD405) but flipping the probing sdAbs failed to do so (Fig 2A and E , S1G and H). Furthermore, a prior incubation of both the sdAbs separately with their selecting peptides in solution followed by probing against plate bound index peptides reduced signal intensity as the concentration of respective peptides increased (Fig 2B and F) . These results showed specificity of sdAbs for their cognate peptides. We also demonstrated the concentration dependant reactivity of both the sdAbs against SARS-CoV2 S protein in ELISA (Fig 2C and G). Moreover, their prior incubation with index peptides inhibited the binding to immobilized SARS-CoV2 S protein albeit the response was less evident for anti-RBD sdAb (Fig 1D and H) . A 30µg/ml of the CS peptide and a 40µg/ml of RBD peptides preincubated with their respective sdAbs completely erased the signal (Fig 2D and H) .

These results established the specific binding of anti-CS and anti-RBD sdAbs. We also probed anti-RBD sdAb against the recombinantly expressed RBD using ELISA and observed a concentration dependent increase in OD405 values that were reduced by prior incubation with the RBD peptide (Fig 2I and J) We then attempted to map the binding sites of anti-CS sdAb in order to reveal its potential anti-viral mechanisms. Trypsin, a serine protease that recognises and cleaves basic residues, was incubated with the CS-peptide in varying concentrations at 37°C and the mix was coated to ELISA plates followed by its probing with anti-CS sdAb in ELISA ( Fig 2K and L and Fig S1I) . The signal intensity was gradually reduced as the concentration of trypsin increased (Fig 2K) . Equivalent concentrations of a non-specific protein, bovine serum albumin, incubated with the peptides under similar conditions did not affect the assay readouts ( Fig 2K) . Furthermore, a prior incubation of the CSpeptide with protease inhibitor reversed these effects ( Fig 2K) . These results suggested that the proteolytic activity of trypsin could have erased the epitope recognised by anti-CS sdAb and in so doing diminished the response. We then determined whether these results could also be due to masking of epitope by trypsin and thereby making it unavailable for binding to the antibody. To this end, we incubated the peptide and trypsin mix at low temperature (4°C), a procedure that would minimize the proteolytic activity of trypsin but leaving the binding unaffected. The mix of CS-peptide and trypsin was additionally heat inactivated at 90°C to inhibit residual proteolytic activity before coating onto the plates. The increase in OD405 values in such assays indicated the crucial role of enzymatic activity in diminishing the signal (Fig 2L) . We obtained essentially similar results when the SARS-CoV2 S protein was used (data not shown). These results could indicate that anti-CS sdAb recognized the epitope bordering S1/S2 segments in the unprocessed S protein of the SARS-CoV2. By extrapolation these experiments could suggest that anti-CS sdAb could make the CS inaccessible for proteolytic activity, a step that precedes SARS-CoV2 internalization.

Taken together, we selected SARS-CoV2 S protein specific sdAbs from a phage display library using synthetic peptides as the bait. We then established the robustness, thermostability, specificity and immune reactivity of SARS-CoV2 S protein specific sdAbs not only against their selecting peptides but also using SARS-CoV2 S protein.

Having established the biophysical and biochemical characteristics of SARS-CoV2 specific sdAbs, we measured their neutralizing activity against lentivirus (LV) based reporter pseudoviruses that express either SARS-CoV2 S or VSV-G protein (Fig 3) .

The use of pseudovirus for analysing neutralizing antibodies or other anti-viral agents could obviate the requirement of high containment facilities and the results from such assays correlate with neutralization of the virulent virus ex vivo or in vivo 26 .

Furthermore, such a system can efficiently be used for testing the neutralization of emerging mutants without necessarily isolating the virulent viruses. Fig S2B) . Upto a 50% neutralization was achieved at 100ng/ml by both the sdAbs. At 5µg/ml of either the sdAbs achieved a near complete inhibition of the virus infectivity ( Fig 3A, B and E). The inhibitory concentration (IC50) values were ~10 times lower for anti-CS sdAb as compared to anti-RBD sdAb (Fig 3E) . We also measured whether or not combining both the sdAbs enhanced the efficiency of neutralization and observed a slight improvement in the neutralization efficiencies particularly at lower concentrations when both the sdAbs (each with 0.5ng/ml) were added in similar assays ( Fig 3C) . We also observed an improved neutralization when a fixed but sub-optimal concentration (5ng/ml) of anti-CS sdAb was combined with varying concentrations of anti-RBD sdAb but a general potentiation effect was less evident (Fig 3A, B, E and Fig S3) . We also assessed the specificity of blockade by both the sdAbs using LV(VSV-G) or by their prior incubation with the cognate peptides ( Fig 3E, Fig S2C-F and Fig S4) . None of the sdAbs prevented infectivity of LV(VSV-G) even in ~10,000 molar excess values as were used for neutralizing LV(CoV2-S) (Fig 3E and Fig S2C-F) . Furthermore, a prior incubation of both the sdAbs with increasing concentrations of their cognate peptides significantly reduced the virus neutralization efficiencies (Fig S4) . These results clearly demonstrated the specificity of virus neutralization by both the sdAbs through recognition of their cognate viral epitopes displayed by SARS-CoV2 S protein in LV(CoV2-S). The heat denatured sdAbs regained structural features as the ambient temperature was reduced ( Fig 1G) .

Anti-CS and anti-RBD sdAbs upon their renaturation neutralized the virus infectivity albeit with a reduced efficiency (Fig 3C-E) . Accordingly, the IC50 concentrations for renatured anti-CS sdAb and anti-RBD sdAbs were respectively ~10 and 4 fold lower in comparisons to their native preparations ( Fig 3E) . These results demonstrated the neutralization of a reporter pseudovirus expressing SARS-CoV2 S protein by the sdAbs, which also withstood harsh biochemical and biophysical conditions. Furthermore, their lost structural and functional properties induced by a high temperature were largely regained with lowering of the storage temperature attesting to their enhanced utility.

SARS-CoV2 causes cell to cell fusion using its surface exposed S protein and in so doing infects bystander cells 27 . We therefore tested whether or not anti-CS and anti-RBD sdAbs could block fusogenic activity. HEK293T cells were used for producing LV(CoV2-S) pseudovirus particles, which could remain cell associated during the exocytic process.

The synthesized S proteins might be displayed by infected cells. HEK293T cells used for producing SARS-CoV2 expressing pseudoviruses (HEK293T +LV(CoV2-S) ) or the control particles (HEK293T +LV(BALD) ) were co-cultured with Vero E6 cells in the presence or absence of sdAbs ( Accordingly, we observed ~25% GFP +ve cells in the absence and ~5% GFP +ve cells in the presence of either of the sdAb preparation (Fig 4B-C) . Similarly, both the sdAbs either alone or in combination reduced the fusogenic activity by four-fold when transfected HEK293T cells (HEK293T +(CoV2-S) ) were incubated with Vero E6 cells (Fig 4D-F ). For such fusion events to occur, the expression of the viral S protein on cell surface is a prerequisite 28 . We therefore measured the surface expression of S protein in HEK293T cells by flow cytometry using both the sdAbs ( Fig 4G) . We used biotinylated anti-CS and anti-RBD sdAbs to detect expressed S protein by HEK293T cells. While the control cells (HEK293T +LV(BALD) ) showed no staining with either of the sdAbs, the staining for the S protein was clearly evident in the transfected cells. We observed >40% and >65% CoV2-S positive cells detected by anti-CS sdAb and anti-RBD sdAb, respectively (Fig 4G) .

Taken together our results showed that SARS-CoV2 S specific sdAbs block not only the virus entry in susceptible cells but also prevent its fusogenic activity.

We observed that a prior incubation of CS-peptide with trypsin reduced the assay readouts (Fig 2K, L and Fig S1I) . These observations led us to explore possible mechanisms by which anti-CS sdAb functions to neutralise the virus (Fig 5) . We probed the culture supernatants from Vero E6 cells infected with either control LV(CoV2-S) or anti-CS sdAb treated LV(CoV2-S) with an anti-FLAG antibody. An intense band migrating at ~180kDa molecular mass corresponding to the unprocessed spike protein (S0) was observed and its intensity increased in samples where increasing concentration of anti-CS sdAb was added (Fig 5A, Fig S6A-C) . This suggested for an abundance of LV(CoV2-S) particles in culture supernatants due to less efficient internalisation upon anti-CS antibody addition. We then tested whether or not anti-CS sdAbs inhibited the cleavage of S protein. LV(CoV2-S) were incubated with either anti-CS or anti-RBD sdAbs followed by a trypsin treatment. Four hours later the mix were analysed by immunoblotting using anti-FLAG antibodies. A pre-incubation of LV(CoV2-S) with anti-CS sdAb but not with anti-RBD sdAb prevented the processing of S protein as revealed by intense band of ~180kDa ( Fig 5B) . In other condition the band of ~180kDa was barely detected ( Fig   5B) . A prior incubation of LV(CoV2-S) with either of the sdAbs followed by trypsin treatment significantly inhibited the internalization process nonetheless. This observation suggested that both the antibodies recognised distinct epitopes in the S protein and acted independently to inhibit the virus internalization ( Fig 5C, Fig S7A) .

We also pre-treated LV(CoV2-S) with different concentrations of trypsin followed by incubation with the anti-CS sdAb (Fig 5D-F) . In these experiments, the anti-CS sdAb (100ng/ml) significantly neutralized native LV(CoV2-S) by ~50% but the trypsin treated LV(CoV2-S) particles remained refractory to the antibody (Fig 5D-F) . These results suggested that the anti-CS sdAb blocked SARS-CoV2 infectivity by recognizing the unprocessed S protein (S0) displayed by LV(CoV2-S) and prevented its proteolytic processing. Therefore, the processing of S protein is a crucial step in viral entry. We also tested whether anti-RBD sdAb by binding to a different site in the S protein could inhibit the virus internalization by independently of its proteolytic processing. A prior trypsin treatment of LV(CoV2-S) followed by its incubation with the graded concentrations of anti-RBD sdAb efficiently blocked the virus entry (Fig S7B-D) . Moreover, coated LV(CoV2-S) on the surface of plates in native form showed reactivity with anti-CS sdAb but its prior treatment with trypsin reduced the signal intensity in a dose dependent manner ( Fig S7E) . The reactivity remained unchanged with anti-RBD sdAb (Fig S7F) .

These results established not only the specificity of sdAbs to recognise epitopes displayed by S protein in the assembled LV(CoV2-S) but also hinted for the functional diversity of both the sdAbs in effecting the neutralization the pseudovirus particles.

We describe here neutralizing sdAbs selected from a phage display library of camelid VHH that target RBD and CS of SARS-CoV2 S protein. 

Competent TG1 bacterial cells were cultured in glucose supplemented 2xYT medium until an OD600 value of 0.4 was reached. TG1 cells were then infected with helper phage M13K07 under static conditions at 30°C for 40 mins. Thereafter, the medium was supplemented with kanamycin (50µg/ml) and grown overnight for the multiplication of phages. Bacterial cells were then pelleted at low temperature to collect supernatant. The supernatant was then precipitated using 20% polyethylene glycol (PEG) and 0.5M NaCl to obtain helper phages, used for infecting recombinant TG1 cells harbouring VHH sequences.

Helper phage particles expressing VHH that were then used for bio-panning as described earlier 22 . Synthetic peptides predicted to be immunogenic for B cells were coated onto ELISA plates (50µg/ml/well) at 4°C overnight followed by blocking the wells with 4% BSA for 2hrs at room temperature (RT). ELISA plates were then washed three times with freshly prepared phosphate buffer saline with 1% Tween-20 (PBST). Subsequently, 10 12 recombinant phages/well were added to the plate followed by incubation for 3 hrs at RT.

Unbound phages were removed by extensive washings (25 times) with a freshly prepared PBST. The bound phages were then eluted using freshly prepared alkaline triethylamine acetate (TEA) buffer. The eluted phages were further enriched to enhance the affinity of peptide specific VHH by performing second round of bio-panning. The eluted phages were then used for infecting TG1 bacterial cells.

Multiple bacterial colonies obtained were screened by colony PCR using VHH specific primers as described earlier 22 . The positive clones were selected to isolate phagemids.

The retrieved VHH sequences were further sub-cloned into a modified pYBNT Vector 

To purify the recombinant protein from inclusion bodies, the pellet was first resuspended in lysis buffer containing 100mM Tris base and 10mM EDTA. The bacterial suspension was sonicated on the ice at an amplitude of 40 with 8 cycles with one-minute pulse on and one-minute off. Subsequently, the cells were centrifuged at 8000rpm for 10 minutes at 4°C to obtain the pellets which were then washed twice with wash buffer A (100mM Tris base, 10Mm EDTA, 1M NaCl, pH 8.0) and once with wash buffer B (100mM Tris Base, 10Mm EDTA, 1% v/v Triton 100; pH 8.0). The pellets were finally resuspended in denaturation buffer containing 100mM NaH2PO4, 10mM Tris-HCl, 8mM urea, pH 8.0 and kept at rotation for 18-20 hours at 4°C. The denatured fractions were centrifuged at 5000 rpm at 4°C for 10 minutes to obtain the clear extract. The supernatant were subjected to Ni-NTA purification using His-trap columns preequilibrated with the denaturation buffer. The washing was done with 20mM imidazole containing denaturation buffer (pH 8.0). The bound product was eluted using 400mM imidazole in denaturation buffer (pH 7.8). The final protein yield was 20mg/liter. The protein thus obtained was mixed with an equal volume of guanidine solution (3M GuHCl, 10mM sodium acetate and 10mM EDTA, pH 4.2) and was refolded by a rapid dilution method using 100mM Tris, 1mM EDTA, 1mM GSH, 0.1mM GSSG, 400mM arginine as described earlier 39 . The refolded fraction was then subjected to size exclusion chromatography using an S200 Hiprep column and two dominant peaks were obtained. The peaks obtained were pooled separately and spectral analysis was done for both using circular dichroism.

To measure the effects of pH on the structural integrity of purified sdAbs, the preparations were incubated in the buffers with varied pH values ranging from 2 to 13 for 10 minutes and CD spectral analysis was performed. Similarly, the effect of temperature on purified sdAbs was analysed by performing thermal kinetics. The sdAbs preparations were subjected to heating at different temperatures ranging from 20°C to 70°C and then cooling from 70°C to 20°C to measure their denaturation and renaturation kinetics. In additional experiments, the anti-CS antibody was added to trypsin-treated LV(CoV2-S) for 4 hrs at 37°C, followed by incubation with anti-CS sdAb (100ng/ml) for 1 hrs on ice.

The above mixture was applied to Vero E6 cells and the infectivity was measured. Aliquots of the same sample was also for ELISA as well as western blotting to analyse to analyse the trypsin mediated cleavage of SARS-CoV2-S in the pseudovirus particles. 

After 72 hrs of transduction, Vero-E6 cells were treated with 1mM PBS-EDTA for 15 minutes at 37°C in CO2 incubator and the cells were removed from 96 well (flat bottom) plates by reverse pipetting. Cells were collected in 1.5ml micro-centrifuge tube, washed twice and acquired using flow cytometer (BD Accuri). The available data was analysed using flowJo X software (TreeStar) 44 .

The cells were analysed for GFP expression 72 hrs post-infection by fluorescence microscopy using Nikon eclipse Ti and all images were taken at 10X magnification.

Analysis and scaling of all the taken images were done using ImageJ software 44 .

Field Emission Scanning Electron Microscope (FESEM) was used to measure the surface topography of LV(CoV2-S) and LV(VSV-G). Pseudoviral particles were spread and dehydrated on glass slide overnight, followed by coating with gold nanoparticle for providing conductivity to the samples and images were acquired using JEOL JSM-7600F FESEM.

To determine the specificity and immune reactivity of the sdAbs with the SARS-CoV2 S protein purified from transfected HEK293T cells or the one displayed by the produced pseudoviruses, the polypeptides in the prepared samples were resolved using SDS-PAGE and after transferring to PVDF membrane were immunoblotted with the sdAb containing 6x(HIS)-tag or their biotinylated versions after blocking of the membrane with 5% skim milk. Anti-6x(HIS) anti-mouse antibody was used to recognize VHH 

Peptide against which sdAb was biopanned was coated at a concentration of 50µg/ml overnight at 4°C. The following day the wells were washed with 0.05% PBST and were blocked in 5% BSA in PBST for 2hrs at RT followed by washing with PBST and incubation with sdAb in different dilutions for 1.5 hrs at RT. For detection of sdAb, anti-6x(HIS) antibody was incubated for 1 hr at RT followed by washing and incubation with antimouse antibody conjugated with alkaline phosphatase for 1.5 hrs. The plate was washed and developed with 100µl of pNpp substrate from Sigma Aldrich (1mg/ml) in glycine buffer. 50µl of stopping solution (3M NaOH) after the development of colour was added and absorbance was taken at 405nm.

BLI was performed to determine the binding kinetics of anti-CS and anti-RBD antibody with the purified spike protein produced from transfected HEK293T cells ( S 20 construct with 3x FLAG-tag) using BLltz System. For loading sdAbs with 6x(HIS)-tagged, Ni-NTA probes (ForteBio) were used. 200 l of 250μg/ml of both antibodies were loaded for 5 minutes and then washed with PBS to remove non-specific binding. Different concentrations of S protein were incubated for 5 minutes to measure the binding affinity with the immobilised anti-CS and anti-RBD antibodies, and their dissociation kinetics was measured in PBS. For reusing the probes re-charging was done by placing the sensor in 10mM glycine for 1 minutes and then in PBS for 5 minutes. Finally, the probes were placed in 10mM Nickel sulphate solution for 1 minute.

One way ANOVA (and non-parametric or mixed) Dunnett's multiple comparisons test were performed for all the group analysis. The results are presented as mean ± SEM.

The p values are shown in the figures are represented as * p ≤ 0.05, * * p ≤ 0.01, or * * * p ≤ 0.001. All statistical analysis were done using Graphpad prism 8.0.2(263). J. ELISA plates were coated with CS-peptide (50µg/ml) and probed using anti-RBD sdAb pre-incubated with the indicated concentrations of its specific peptide and a fold change in the OD405 values as compared to mean OD405 values of negative controls is shown by bar diagram. K. ELISA plates were coated with 50µg/ml of CS-peptide alone or that previously incubated with different concentrations of trypsin for three hours at 37°C. In additional wells, the mix of CS-peptide was first incubated with protease inhibitors followed by addition of trypsin for three hours at 37°C. Some wells were additionally coated with the CS-peptide along with BSA under similar conditions. The plates were then probed using anti-CS sdAb and a fold change in the OD405 values as compared to mean OD405 values of negative controls is shown by bar diagram. L. ELISA plates were coated with 50µg/ml of CS-peptide alone or that previously incubated with different concentrations of trypsin for three hours at 4°C. In additional wells, the mix of CSpeptide was first incubated with protease inhibitors followed by addition of trypsin for three hours at 4°C. Some wells were additionally coated with the mix of CS-peptide and trypsin under similar conditions followed by its heating. The plates were then probed using anti-CS sdAb and a fold change in the OD405 values as compared to mean OD405 values of negative controls is shown by bar diagram. The experiments were repeated three times and representative data plots from one such experiment in the respective section is shown. One way ANOVA was used to measure the level of statistical significance. ****p<0.0001, ***p<0.001, *p<0.01 and *p<0.05. Representative overlaid histograms obtained by flow cytometry show GFP +ve cells infected with native LV (CoV2-S) infected Vero E6 cells or the pre-incubated LV (CoV2-S) with high temperature exposed anti-CS sdAb followed by its cooling at room temperature. D. Representative overlaid histograms obtained by flow cytometry show GFP +ve cells infected with native LV (CoV2-S) infected Vero E6 cells or the pre-incubated LV (CoV2-S) with high temperature exposed anti-RBD sdAb followed by its cooling at room temperature. Vertical dotted line serves as the reference for marking the peak and vertical dark line represents the marker to show GFP +ve and GFP -ve cells. E. Cumulative data obtained from neutralization experiments along with the inhibitory concentrations are depicted. The experiments were performed for more than five times with similar results. One way ANOVA was used to measure the level of statistical significance. ****p<0.0001, ***p<0.001, *p<0.01 and *p<0.05. SARS-COV 2 S protein in its homotrimeric state interacts with its cellular receptor (ACE2/neuropilin 1/ some yet to be identified receptors) through RBD. This interaction is followed by host protease mediated cleavage of S protein (S0) to yield S1 and S2 fragments. Therefore, the targeting of RBD using sdAbs could block interaction with the cellular receptor to effect neutralization but the cleavage of the S protein might still occur. As the concentration of neutralizing antibody drops to suboptimal levels the virus could infect cells followed by its membrane fusion and the release of viral genome proceeds in subcellular compartments. Targeting the cleavage site however, could serve to increase the durability of anti-viral activity as the lack of S protein processing further reduces the chances of membrane fusion and internalisation of the virus.

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We thank Professor Kartik Chandran and Dr Rohit K Jangra of Albert Einstein college of Medicine, New York and Dr Jason McLellan of the University of Texas, Austin for sharing critical reagents for the work. The initial work for preparing the phage display library was done by Dr Manpreet Kaur. We also thank Dr Indranil Banerjee, Professor Anand K Bachhawat and Dr Anant Venkatesan for discussion and additional support.