key: cord-0851640-siq9xf2l
authors: Mendon, Nivya; Ganie, Rayees; Kesarwani, Shubham; Dileep, Drisya; Sasi, Sarika; Lama, Prakash; Chandra, Anchal; Sirajuddin, Minhajuddin
title: Peptide derived nanobody inhibits entry of SARS-CoV-2 variants
date: 2022-04-21
journal: bioRxiv
DOI: 10.1101/2022.04.21.489021
sha: fc77c87b3cb7b857dd6da70d28dea8252c806e0a
doc_id: 851640
cord_uid: siq9xf2l

Emergence of the new escape mutants of the SARS-CoV-2 virus has escalated its penetration among the human population and has reinstated its status as a global pandemic. Therefore, developing effective antiviral therapy against emerging SARS variants and other viruses in a short period of time becomes essential. Blocking the SARS-CoV-2 entry into human host cells by disrupting the spike glycoprotein-ACE2 interaction has been already exploited for vaccine development and monoclonal antibody therapy. Unlike the previous reports, our study used a 9 amino acid peptide from the receptor-binding motif (RBM) of Spike (S) protein as an epitope. We report the identification of an efficacious nanobody N1.2 that blocks the entry of pseudovirus containing SARS-CoV-2 spike as the surface glycoprotein. Moreover, we observe a more potent neutralizing effect against both the hCoV19 (Wuhan/WIV04/2019) and the Omicron (BA.1) pseudotyped spike virus with a bivalent version of the nanobody. In summary, our study presents a faster and efficient methodology to use peptide sequences from a protein-receptor interaction interface as epitopes for screening nanobodies against potential pathogenic targets. This approach can also be widely extended to target other viruses and pathogens in the future.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the betacoronavirus family and is the third member of the family after MERS-CoV and SARS-CoV that has infected the human population, resulting in moderate to severe pathogenic symptoms (1) . With the frequent outbreaks of new variants, these numbers are increasing every day (2) . Although several safe and effective anti-SARS-CoV-2 therapies have been recently developed which have been proven to prevent severe symptoms upon infection, none of them has been proven to be 100% effective. Partial vaccination, in turn, has put the SARS-CoV-2 virus under increased selection pressure resulting in a high mutation rate in the viral spike protein (3) . With the emergence of novel variants and escape mutants of SARS-CoV-2, there is an urgent need to develop effective antiviral therapy that can single-handedly target both the current and emerging variants.

The surface of the SARS-CoV-2 virus is decorated with homotrimer Spike (S) envelope glycoprotein, the major antigenic determinant of the host immune response and one of the main coronavirus drug targets (4) . The SARS-CoV-2 entry is orchestrated by the interaction of the receptor-binding domain (RBD) of its spike with the angiotensin-converting enzyme 2 (ACE2) receptor on the host cell membrane (5, 6) . The precise contact is established by a set of conserved residues present in the receptor-binding motif (RBM) of the spike (7) . Efforts have been made to prevent this first step of infection using antibodies or nanobodies that target the spike-ACE2 interaction (8) (9) (10) (11) (12) (13) (14) (15) (16) . Several groups have also isolated antibodies directly from convalescent SARS-CoV-2 patients that showed promising neutralization against SARS-CoV-2 in-vitro and improved clinical outcomes in tested animals in vivo (17) (18) (19) (20) (21) (22) . Nanobodies, on the other hand, offer many advantages over conventional antibodies in terms of their size, high thermal stability, solubility, and ease of expression in bacteria hence making them easily scalable and cost-effective (23) . Nanobodies, which are derived from the variable domain of the camelid heavy chain, retain specificity and affinity similar to conventional antibodies. Modularity in nanobodies allows oligomerization hence increasing their avidity and serum shelf life. (24) . Nanobodies can be easily humanized which is critical for developing antiviral therapies for humans (25) . Treatment with combinatorial nanobodies has been effective in neutralizing SARS-CoV-2 and preventing mutational escape (11) . Many of such anti-SARS-CoV-2 therapies report the use of the entire spike protein or its RBD domain as the epitope to screen active antiviral antibodies. A limitation of using the entire spike protein or RBD as an epitope is that the isolated antibodies may bind to different 3 regions of spike protein other than the ACE2 interacting domain, thus rendering them unspecific and inefficient in fully neutralizing the viral infection. Here, we report the isolation of a nanobody (N1.2) from a yeast nanobody display library using a small 9 amino acid peptide sequence as an epitope. The peptide was designed from the specific residues within the RBM showing high-affinity interaction with the spike protein with the ACE2 receptor. This nanobody, both monomer as well as tandem dimer interferes with ACE2 binding thus showing a potent virus neutralization activity in cellular models.

The trimeric spike envelope protein of SARS-CoV-2 interacts with the cellular ACE2 (angiotensin-converting enzyme 2) receptor in the host cell plasma membrane to gain access to the cytoplasm ( Figure 1A ). We designed two peptide sequences from the spike envelope protein; peptide-1, residue 482-494, and peptide-2, residues 496 -506 from the RBM of hCoV19 (Wuhan/WIV04/2019) (5) . These peptides were used as bait (epitope) against the combinatorial yeast display library of nanobodies (26) and enrich potential nanobody sequences. The screening methodology involves two rounds of magnetic screening followed by fluorescent activated cell sorting (FACS) based enrichement of library ( Figure 1B) . From the FACS analysis, only peptide-1 showed positive enrichment of the nanobody clones ( Figure 1B) . Therefore, from here onwards we focused only on the nanobody population obtained from the peptide-1 screen. After FACS, the enriched yeast clones were individually analyzed using clonal selection and sequencing. Ten unique nanobody sequences were identified and purified, named N1.1 to N1.10 (Supplementary Figure 1 and Supplementary   Table 1 ) (Methods).

The purified nanobodies were subjected to ELISA to characterise the interaction with peptide-1 and -2 (Methods). All the nanobodies showed good binding with the peptide-1 compared to the peptide-2, except N1.4 ( Figure 1C ). Suggesting that the enriched nanobody clones N1.1 to N1.10 are specific towards the peptide-1 epitope.

We then tested each of the purified nanobodies for their ability to block Spike: ACE2 interaction using a pseudovirus based neutralizing assay (Figure 2A ). Pseudoviruses 4 displaying spike envelope glycoprotein with a mCherry reporter were generated (27) (Methods). The viral infectivity was assessed using the mCherry expression, which was further quantified using fluorescence measurement (Methods).

Since the ACE2 expression in HEK293T cells is lower compared to other cell lines (28, 29) , we generated an ACE2 stable expressing HEK293T cells, herein referred to as eGFP-ACE2/HEK293T cells (Methods). The eGFP-ACE2/HEK293T stable cell lines also coexpressed eGFP as a fluorescent marker to facilitate identifying ACE2 expressing cells Interestingly, the N1.2 nanobody showed maximum efficiency in neutralizing the Spike pseudoviruses compared to the remainder of the nanobodies ( Figure 2C ). Therefore, we decided to further characterize the N1.2 nanobody as a potential nanobody against Sars-CoV2 virus entry.

In our virus titration assays, we noticed that the FACS-based quantification does not scale according to the mCherry expression (Supplementary Figure 3A and 3B) . Therefore, we simultaneously correlated the mCherry fluorescence versus virus infection using confocal microscopy-based quantification (Methods). In comparison to FACS data, the confocal microscopy based measurement, showed a sharp decline in mCherry fluorescence, i.e., >80% decrease in mCherry fluorescence when 1/10 th of the virus dilution was used for infection (Supplementary Figure 3C-E) . Therefore, hereafter, we used confocal microscopy based 5 measurement to assess the neutralizing potential of N1.2 nanobody against spike pseudovirus.

In the pseudoviral assay, we used different concentrations of N1.2 (5µM, 10µM, and 25µM) and could observe a linear reduction in the infectivity of the spike pseudoviruses towards the eGFP-ACE2/HEK293T cells ( Figure 3B , C and Supplementary Figure 5 ). To achieve a more potent inhibitory effect with N1.2, we engineered and purified a bivalent nanobody, in which the N1.2 sequence was placed in tandem separated by a glycine-serine linker (Methods) 

We also tested the efficacy of the N1. Omicron variant.

Since 2020 the SARS-CoV2 virus has evolved into many subtypes and variants that are still prevalent across the globe (30) . Therefore it is pivotal to identify broad-spectrum therapeutics that can effectively work against all SARS-CoV2 subtypes and variants. Equally important is to establish validated pipelines that can expedite identifying new therapeutics against emerging SARS-CoV2 variants or new viruses. In this study, we have addressed both the needs: first, we have identified a broad spectrum nanobody that neutralizes the original Wuhan (WIV04) and Omicron spike pseudovirus. Secondly, we have achieved the nanobody identification using a peptide as antigen, unlike the whole spike or RBD protein of SARS-CoV2 described earlier (13, 16, (31) (32) (33) . The nanobodies or antibodies obtained by using a whole spike or RBD bind to different regions of the spike protein and only a fraction of them were effective in neutralising the virus (33) . Largely because these antibodies target the regions of the spike protein other than the ones involved in spike-ACE2 interaction. Using peptides as antigens has been widely used for generating potent antibodies targeting the desired epitope, including the recent example for SARS-CoV-2 antibodies (34). However, peptides and flexible loops from a target protein have seldom been used as antigenic baits for screening display libraries. We previously reported a nanobody against tubulin posttranslation modification using the flexible carboxy-terminal tail peptide (35) . Applying a similar principle, here we show that a 9 amino acid peptide from the receptor-binding motif (RBM) of spike protein can also be employed as an epitope against a nanobody display library. This approach of using synthetic peptides as an epitope, instead of purified whole protein molecules will certainly accelerate and fine tune the entire screening process and yield nanobodies/antibodies that are efficacious in neutralizing the future potential targets /viruses. The nanobody N1.2 described in this study is effective against both the spike proteins from the original hCoV19 (Wuhan/WIV04/2019) and the recent Omicron variant. Sequence alignment of the peptide-1 and its surrounding region that was used in screening the nanobody shows the high similarity between the variants (Supplementary Figure 1) .

Suggesting that epitopes from the conserved RBM region such as the peptide-1 can yield specific nanobodies or antibodies against the SARS-CoV-2 virus, yet possess broad 7 neutralizing ability against the existing and emerging variants. Almost all the SARS-CoV2 variants reported to date utilize ACE2 receptor binding for the host cell entry and the RBM region of spike protein predominantly contributes to this interaction (36, 37) . Therefore, we anticipate that the nanobody N1.2 will remain effective in neutralizing the emerging variants as long as the SARS-CoV2 virus uses ACE2 for gaining entry into the host cells.

Additionally, the nanobodies offer advantages in engineering the valency and thereby increasing the efficacy in neutralization against the target molecules. Indeed when we engineered the nanobody N1.2 into a bivalent version, (N1.2) 2 we observed a cooperative effect in neutralizing the pseudovirus even at the highest concentrations. Together, our study offers a platform to identify broad spectrum nanobodies specific against SARS-Cov2 and its variants, which can be exploited for other therapeutically relevant targets. 

A combinatorial yeast-display library of nanobodies (NbLib) was obtained from Kerafast, Inc. The estimated diversity of the library is around 5*10ˆ8 unique nanobody clones expressed onto the surface of the yeast cells. The detailed protocol for screening nanobodies against the peptides has been adopted from the method described earlier (26) . The frozen vial of NbLib (5-fold excess of library diversity) was grown in 1L Yglc4.5-Trp media (3.8g/L of -Trp drop-out media supplement, 6.7g/L yeast nitrogen base, 10.4g/L sodium citrate, 7.4g/L citric acid monohydrate, 20g/L glucose and 10ml/L PenStrep, pH4.5) at 30°C for 24-48hr and passaged thrice before the screening. The freshly passaged NbLib (5*10ˆ9 cells, 10-fold excess of library) was induced in galactose containing media (-Trp +galactose; 3.8g/L of -Trp drop-out media supplement, 6.7g/L yeast nitrogen base, 20g/L galactose and 10ml/L PenStrep, pH6.0) at 20°C for 72hr to achieve sufficient expression in the library.

Around 5*10ˆ9 nanobody expressing yeast cells were pelleted to remove media and washed Only peptide-1 screening yielded nanobody clones from the library having sufficient binding affinity for peptide-1 therefore, we have only focused to characterise nanobodies obtained from peptide-1 screening. We have isolated plasmids from these 10 individual yeast clones from peptide-1 screening to identify the protein-coding sequences of these nanobodies for their biophysical and cellular validation.

The nanobody gene was amplified from isolated yeast colonies and cloned between HindIII and XhoI sites in a pET-22b(+) plasmid containing a C-terminal 6x histidine tag. 

Freshly passaged HEK293T cells were seeded in a 100mm cell culture plate and grown-up to 70-80% confluency. The media of the cells were changed to 10 ml complete DMEM without PenStrep before transfection. For viral particle production, 5 µg pHR lentiviral vector cloned with mCherry fluorescent protein, 3.75 µg packaging plasmid psPAX2 (Addgene; #12260), 

Omicron pseudotyped viruses were produced similarly as described above for spike pseudoviruses but instead used omicron envelope plasmid along with packaging plasmid (psPAX2) and lentiviral plasmid (pHR mCherry) in the following ratio: psPAX2 (1.3pmol), pHR mCherry: 1.64pmol, SARS-CoV-2 Omicron Strain S gene (Genscript, Cat # MC_0101274): 0.72pmol.

Human ACE2 was amplified from the mammalian Caco2 cell lines. Caco2 cells were lysed for total RNA purification using the Trizol method. Further, 2 µg of total RNA was set up for 1 3 cDNA synthesis with Verso cDNA synthesis kit (ThermoFisher Scientific, catalogue no.

AB1453A) as per the manufacturer's protocol. The protein-coding sequence of ACE2 was amplified from the cDNA with forward (5'ggaggagaaccctggacctggatccatgtcaagctcttcctggctcc-3') and reverse (5'ctcctgaccctcctcccccgtaaaaggaggtctgaacatc-3') primers using Q5 polymerase PCR reaction (NEB, catalogue no. M0492L). This amplified construct of ACE2 was cloned into a pTRIP chicken β -actin (CAG) vector (Gentili et al., 2015) . This construct contains amino-terminus EGFP followed by self-cleaving 2A peptide sequence followed by ACE2 and carboxytermini SNAP-tag and FLAG tags (eGFP-ACE2/HEK293T)

The lentiviral pTRIP vector cloned with eGFP-ACE2/HEK293T under CMV enhancer and chicken β -actin promoter (CAG promoter) flanked with 5′and 3′ long terminal repeat (LTR) sequences (39) , were used to produce lentiviral particles as per the method described before (35) . Briefly, 70-80% confluent HEK293T cells were transfected with 5 µg lentiviral construct of eGFP-ACE2/HEK293Texpression, 3.75 µg psPAX2 (Addgene; #12260), and 2.5 µg pmDG2 (Addgene; #12259) plasmids using Lipofectamine-LTX reagent. The lentiviruses collected at 48hr, 72, and 96hr were concentrated and pelleted using a lenti-X concentrator.

The white pellet of lentiviruses was resuspended in 2ml of complete DMEM media and 1ml of this virus was used to transduce HEK293T cells for stable expression of ACE2 in these cells. We could only achieve the transduction efficiency of 60-70% and therefore, we performed fluorescent activated cell sorting (FACS) experiments to enrich the eGFP expressing (a proxy for ACE2 expression) HEK293T cells in the culture. In our sorted culture, >90% of cells were found to be eGFP positive or expressing eGFP-ACE2/HEK293T

The stable HEK293T cells were seeded in ibidi glass-bottom dishes (catalogue no. 81218) precoated with poly-D-Lysine and grown to 70% confluency. The cells were washed with 1xBRB80 (80 mM Pipes, 1 mM MgCl 2 , and 1 mM EGTA, pH 6.8) buffer twice and fixed using 100% ice-cold Methanol for 10min at -20°C. Post fixation cells were washed twice with 1xBRB80 buffer and permeabilized in the same buffer having 0.1% Triton-X-100 for 10min at room temperature. Cells were blocked with 5% BSA made in 1xBRB80 + 0.1%

Triton-X-100 for 1hr at room temperature. Cells were incubated with 1:500 dilution of mouse anti-FLAG monoclonal antibody (Merck, catalogue no. F3165) overnight at 4°C. Further, 1 4 cells were washed thrice for 5min using blocking buffer, followed by secondary goat antimouse Alexa Fluor-647 antibody (Invitrogen; 1:1000 dilution) incubation in the same blocking buffer at room temperature for 2hr. Cells were stained for nucleus using DAPI (1 μ g/ml) for 10 min followed by five washes with 1xBRB80 for 5min. Cells were imaged on an H-TIRF microscope using 405nm, 488nm and 640nm laser with appropriate filter sets.

The HEK293T cells were seeded in 35mm dish grown to 70% confluency. The cells were Scientific, catalogue no. N301). Once the colour starts to develop, stop the reaction using 0.2 mM sulfuric acid. The colour will turn yellow upon sulfuric acid addition, which was measured for absorbance at 405nm using a spectrophotometer keeping appropriate controls (wells not having any immobilised peptides). The intensity of the yellow colour represents 1 5 the binding of nanobodies with the respective peptide. The relative absorbance (A405) values of individual nanobodies plotted in the graph represent A405 with peptide subtracted from A405 without peptide.

The GFP/HEK293T cells or eGFP-ACE2/HEK293T cells were grown up to 60-70% confluency in complete media before viral transduction. In a separate vial mCherry pseudoviruses/ lentiviruses were incubated with the respective purified nanobody at room temperature for 5min. The cells were transduced with the above viral mix along with 2 respectively.

All the images were acquired on an inverted confocal microscope (Olympus FV3000) equipped with six solid state laser lines (405, 445, 488, 514, 561, and 640 nm) and a 20x oil objective. For acquisition and quantification of viral transduction in the pseudoviral assay, high sensitivity spectral detectors were used marking a specific region of interest in the 2048*2048 pixel frame. For all the sets of images acquired for quantification laser power, voltage, and gain settings were kept constant. Images were analysed on Fiji software to 1 6 calculate mean fluorescence intensity (MFI) for eGFP (ACE2 expression) and mCherry (viral transduction) channel from the z-projected stacks. All the experiments were performed in triplicate sets on at least two different days. Normalised infectivity represents the mean mCherry intensity of a particular z-projected stack over the GFP intensity of the same stacks.

Representative images are the z-projected stacks of the respective condition. Respective graphs in Figure 

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The authors acknowledge the Central Imaging and Flow Facility (CIFF) at the Bangalore Life Science Cluster, India. M.S acknowledges funding support from inStem core grants from the 

The commercial usage and application related to the N1.2 nanobody sequence are patent protected.

Nivya Mendon # , Rayees Ganie # , Shubham Kesarwani # , Drisya Dileep, Sarika Sasi, Prakash Lama, Anchal Chandra and Minhajuddin Sirajuddin *