key: cord-0948331-ses9hioo
authors: Parashar, Paritosh; Prabhakar, B; Swain, Sonali; Adhikari, Nisha; Aryan, Punit; Singh, Anupama; Kwatra, Mohit
title: A Novel High-Throughput Single B-Cell Cloning Platform for Isolation and Characterization of High-Affinity and potent SARS-CoV-2 Neutralizing Antibodies
date: 2022-03-20
journal: bioRxiv
DOI: 10.1101/2022.03.20.485024
sha: 78571e4a06804ebb4df52a8c8fe430d01f2e8aaf
doc_id: 948331
cord_uid: ses9hioo

Monoclonal antibodies (mAbs) that are specific to SARS-CoV-2 can be useful in diagnosing, preventing, and treating the coronavirus (COVID-19) illness. Strategies for the high-throughput and rapid isolation of these potent neutralizing antibodies are critical toward the development of therapeutically targeting COVID-19 as well as other infectious diseases. In the present study, a single B-cell cloning method was used to screen SARS-CoV-2 receptor-binding domain (RBD) specific, high affinity, and neutralizing mAbs from patients’ blood samples. An RBD-specific antibody, SAR03, was discovered that showed high binding (ELISA and SPR) and neutralizing activity (competitive ELISA and pseudovirus-based reporter assay) against Sars-CoV-2. Mechanistic studies on human cells revealed that SAR03 competes with the ACE-2 receptor for binding with the RBD domain (S1 subunit) present in the spike protein of Sars-CoV-2. This study highlights the potential of the single B cell cloning method for the rapid and efficient screening of high-affinity and effective neutralizing antibodies for Sars-CoV-2 and other emerging infectious diseases. Highlights Single B-cell cloning is a high-throughput and efficient method of generating high affinity neutralizing antibodies Single B-cell cloning method was used to screen SARS-CoV-2 receptor-binding domain (RBD) specific, high affinity, and neutralizing monoclonal antibodies from patient’s blood samples. An RBD-specific antibody, SAR03, was discovered that showed high binding and neutralizing activity against SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (Sars-CoV-2) causes the coronavirus disease that has crossed the species barrier and led to a global pandemic leading to high morbidity and mortality in humans along with significantly impacted on health, environmental, and socioeconomic status [1, 2] . At least 400 million cases of Sars-CoV-2 infections and 6 million deaths have been reported worldwide since the first case of COVID-19 was registered [3] . After the detection of the first Sars-CoV-2 variant in the Wuhan in the late year 2019, various mutants have emerged with higher infectivity rates compared to the original strain [4] . To effectively target the burden of COVID-19, the discovery and development of novel therapeutics, diagnostics, and vaccines are required. Encouragingly, several vaccine candidates against Sars-CoV-2 have emerged as timely prophylactic treatment with great success globally. However, the rapidly evolving variants of the new Sars-CoV-2 pose a threat to the COVID-19 vaccine effectiveness and hence, immediate development of alternate therapeutic interventions is needed. Although vaccine's effectiveness in blocking infectious diseases and antibody therapy is an alternative treatment strategy in the prevention of newly emerging mutant strains of Sars-CoV-2.

Passive administration of high affinity neutralizing antibodies against SARS-CoV-2 may constitute an essential role in targeting COVID-19 and complement vaccine-based prophylactic intervention. Hybridomas and phage display techniques are among the most commonly used platforms for generating antibodies for research and therapeutic purposes [5, 6] . While the hybridoma method is limited with a longer time (6-8 months) for generating library, low efficiency, and requirement of humanization step, phage display is associated with high cost, generation of biased repertoires, and loss of natural pairing information [7, 8] .

Rapid and efficient screening methods of identifying neutralizing mAbs against infectious diseases are in great demand. 4 In recent years, the emergence of Single B cell technologies has greatly accelerated the timelines for identification and further development of the natural repertoire of neutralizing antibodies against infectious diseases HIV, Ebola, and influenza [9] [10] [11] . This process involves the isolating PBMCs from recovered patient's blood, high-throughput single-cell sorting of desired antigen-specific antibody expressing B-cells, recovery of the paired variable region of antibody's heavy and light chain (V H /V L) genes through RT-PCR including two-step PCR steps, expression, and evaluation of antibody candidates. The application of single B-cell cloning has also allowed the identification of neutralizing mAbs against Sars-CoV-2, [12] [13] [14] [15] [16] .

Several neutralizing mAbs have received emergency use approval for COVID-19 diagnosis, treatment, and even prophylactic use in patients exhibiting mild-to-moderate symptoms that reduces disease progression and subsequent hospitalization [17] [18] [19] . Sars-CoV-2 acquires entry within host cells and primarily interacts through the receptor-binding domain (RBD) (within the S1 subunit of spike protein) with the angiotensin-converting enzyme 2 (ACE-2) which is a cellular receptor [20, 21] . With the critical nature of the RBD interaction with ACE-2 for viral entry, antibodies with the potential ability to bind the RBD and interfere with ACE-2 binding can have potent neutralizing activity. Therefore, RBD emerges as the primary antigen target to specifically sort antibody expression B-cells against Sars-CoV-2.

This study aimed to evaluate the utility of single B cell sorting for the isolation of potent neutralizing antibodies against SARS-CoV-2. Here, we describe an optimized and rapid system for high-throughput screening of neutralizing mAbs through separating RBD antigenspecific IgG1 + memory B cells derived from the blood of patients recovered from Sars-CoV-2 infection. Through our optimized single B-cell cloning platform, we identified a neutralizing mAb, SAR03, with high affinity and neutralizing ability against Sars-CoV-2.

Blood from the 8 selected COVID-19 hospitalized but recovered donors were collected in EDTA tubes and plasma was collected for the presence of antibodies while the cells were further processed for B-cell sorting.

For binding ELISA, 1µg/mL recombinant RBD protein (in-house expressed) was coated to Nunc MaxiSorp ELISA plates (44-2404-21; Thermo) in carbonate buffer (pH-9.5) was kept overnight at 4°C. Thereafter, plates were proceeded with washing in TBST (1X TBS +.05% Tween-20) and blocked in 3% BSA prepared in TBST for 1 hr at room temperature.

Afterward, the washing was done with TBST, followed by plasma addition to the plates at 1/10 dilution for 1 hr at 37°C. Further, washing was performed with TBST, with the addition of HRP conjugated Goat Anti-Human IgG (H+L) (109-035-088; Jackson ImmunoResearch) at 1/3000 dilution for 30 minutes at room temperature. Plates were then washed with TBST and TMB substrate (T0440; Sigma) was added for color development. The stop solution was added to terminate the reaction and absorbance was taken at 450 nm using a microplate reader.

For the neutralization activity of antibodies, competitive ELISA was used. 1µg/mL recombinant ACE-2 protein (in-house expressed) was coated onto the Nunc MaxiSorp ELISA plates (44-2404-21; Thermo) using carbonate buffer (pH-9.5) was kept overnight at 4°C. Subsequently, coated plates were washed in TBST (1X TBS +.05% Tween-20) and blocked in 3% BSA prepared in TBST for 1 hr at room temperature. Plasma at 1/10 dilution was pre-incubated with recombinant RBD-biotin (50ng/well) for 1 hr at 37°C in 100µl volume. The plasma-RBD mix was then added to ACE-2 coated plates and incubated for an additional 1 hr at 37°C. After washing the plates with TBST, HRP conjugated streptavidin (N100; Thermo) was added at 1/10000 dilution for 30 min incubated at room temperature. 6 The next step of washing was done in TBST and TMB substrate (T0440; Sigma) was added for color development. The stop solution was added to stop the reaction followed by absorbance measurement at 450 nm using a microplate reader.

Isolation of blood mononuclear cells was performed using density gradient centrifugation containing RNAse inhibitor). The plates were then stored at −80 °C for further processing.

We used an optimized heavy and light chain amplification method (in-house platform, The amplification of 2 nd PCR product was analyzed through DNA gel electrophoresis.

Samples showing the correct size band for heavy and light chain amplification in the respective wells were subsequently cloned into pAb20-hCHIgG1 (Synbio; for heavy chain) and pAb20-hCK (Synbio; for light chains) through in-fusion cloning (Takara). The ligated product was transformed to DH5-α competent cells, and the plasmid was purified through plasmid miniprep columns.

Freestyle-293F cells (R79007; Thermo Fisher) were cultured in a 96-well culture plate with a density of 10000 cells/well. 24 hours later, pAb20-hCHIgG1 and pAb20-hCK plasmids discretely expressing heavy and light chain of antibodies were transiently co-transfected into Freestyle-293F cells (R79007, Thermo Fisher) employing purefection reagent (LV750A-1;

System Bio) as per the instructions given by the manufacturer. Cells were further cultured for another 48 hours on condition of 5% CO2 at 37 °C. The supernatant was analyzed for secretion of antibodies through ELISA. Binding ELISA and neutralization activity of antibodies were performed as above. Cell culture supernatant was used at 1/10 dilution. 

Freestyle-293F cells (R79007; Thermo Fisher) cells were grown in a 6-well culture plate at a density with 2x10 6 cells/well/2ml media. 24 hours later, pAb20-hCHIgG1 and pAb20-hCK plasmids separately expressing heavy and light chain of antibodies were transiently co- The stable cells were then cultured in a shaker incubator run at 120 rpm with conditions of 8% CO 2 at 37 °C. After one week, the supernatants consisting of secreted antibodies were collected and trapped by protein G Sepharose (GE Healthcare). The bound antibodies on the Sepharose were eluted and concentrated using Centricon Plus-70 (051555; Millipore).

Successively, purified antibodies were utilized in the following binding and neutralization analysis.

The affinity of antibody-binding recombinant RBD was assessed using the Biacore T200 

Pseudovirus was created as described in an earlier report [22] . 

All grouped data are expressed as the mean ± standard deviation (SD) of a demonstrative experiment executed at least in triplicate, and almost similar data were obtained in at least three independent experiments. The statistical analyses were directed using GraphPad Prism 8.0. One-way ANOVA followed by Dunnet multiple comparison tests of individual data values were used for statistical analysis. The P < 0.05 was considered statistically significant.

To identify and clone Sars-CoV-2 neutralizing antibodies, we first obtained the plasma and PBMC from the blood of 8 convalescent patients infected with COVID-19. These isolated plasma samples were first tested for binding to recombinant RBD protein (Fig.1A) and

neutralizing activity through competition ELISA (Fig.1B and C) . Using binding ELISA, we confirmed that the antibodies present in the plasma of all the patients displayed a high binding affinity to recombinant RBD. Upon experiment for competitive ELISA, we observed that plasma samples of three patients showed high neutralizing activity (Patient 2, 3, and 5) (Fig. 1C) . With these findings, we decided to utilize these three samples for sorting of RBDspecific memory B-cells.

RBD domain to human cell surface ACE2 receptor, therefore the recombinant RBD was used as a bait to sort the specific memory B cells via flow cytometry. Pan-memory cells were first isolated using the memory-B cell isolation kit using magnetic separation. Isolated B-cells were then stained with anti-IgG-FITC and RBD-Biotin-Streptavidin-PE. RBD-specific Bcells were then gated for IgG + RBD + cells (Fig.2) . Finally, 83 RBD-specific memory B-cells were then sorted into a 96-well plate, one cell per well, for antibody gene isolation.

Immunoglobulin heavy and light (kappa) chains were acquired from the sorted single memory B-cells through RT-PCR and modified two-step nested PCR method (Fig.3A ) [23, 24] . For RT-PCR, a mix of reverse primer from the constant region of the antibody sequence were used (HC-RT for heavy chain and LC-RT for kappa light chain, unpublished data).

After RT-PCR, obtained cDNA was duplicated into two 96-well plates for amplification of heavy and light chain sequences separately. We utilized a mix of 20 nucleotides sequences clones were obtained with heavy and light chains cloned in expression plasmids.

Plasmid expressing heavy and light chains of the final 12 antibody clones were transfected into Freestyle-293F cells in a 96-well plate followed by supernatant collection after 48 hrs time interval. The supernatant was first analyzed for binding to recombinant RBD through ELISA. All 12 clones showed potent binding at 1/10 dilution of supernatant (Fig.4A) . All the clones were then tested for neutralizing activity through competitive ELISA. Three clones (SAR03, SAR09, and SAR051) showed significantly good neutralizing activity (at 1/10 13 dilution of supernatant) (Fig.4B ) and were selected for the generation of stable cell lines and large-scale protein production and purification. SAR051 (IC50-20.64 nM) showed slightly lower neutralizing activity (Fig.5A ). SAR03 was also tested for its affinity to recombinant RBD protein through surface plasmon resonance (SPR). SAR03 showed potent affinity to RBD as evident from Kd-1.2 nM (Fig.5B) . In our studied experiments, we finally identified SAR03 as the best mAb compared to other monoclones in terms of high-affinity capability and potent neutralization ability against SARS-CoV-2.

We describe here the isolation and sequencing of a high affinity and neutralizing mab against Sars-CoV-2 virus derived from a convalescent COVID-19 patient's blood through an optimized, high-throughput, and efficient single B-cell cloning.

COVID-19 pandemic has impacted the world through its devastating affected human health either directly or indirectly. This pandemic has brought the foremost attention that focuses on the development of vaccines, novel antiviral agents, and mAbs. Furthermore, the establishment of neutralizing mAbs to SARS-CoV-2 exhibits great potential for both therapeutic and prophylactic applications and can offer a framework for vaccine design and development [25] . Numerous research groups have isolated mAbs through sorting of RBD antigen-specific B cells of infected patients who have recently recovered from SARS-CoV-2 using a single B-cell cloning [12, 13, [16] [17] [18] [19] . The main target of SARS-CoV-2 neutralizing mAbs is the RBD domain of S1 surface glycoprotein (S1 protein) that mediates viral entry into host cells through interaction with ACE-2 receptors found on numerous cell types. Most studies conducted on mAbs isolated to date specifically target the RBD domain present on the spike protein. In general, for isolation of neutralizing antibodies through a single B-cell cloning method, blood samples were collected from patients recovered from SASR-CoV-2 infection. Furthermore, the RBD antigen-specific B cells are isolated from PBMC through staining with specific fluorescently labeled antibodies and sorting through FACS. Paired heavy and light chain sequences of Ab genes are then attained either using PCR or directly through a high-throughput single-cell sequencing [16, 26, 27] . In our study, we obtained the paired heavy and light chain genes using a rapid and high-throughput single B-cell screening platform for neutralizing Abs isolation and evaluated on the RT PCR platform that corroborated with the previous report [16] .

We started with sorting of Sars-CoV-2 specific memory B-cells from convalescent patient's blood using recombinant RBD protein as bait. Based on existing evidence that RBD-specific neutralizing antibodies can inhibit viral entry [22] , the RBD domain was selected as the target antigen for memory B-cell sorting for the isolation of heavy and light chains. Using a smart gating strategy, we were able to sort memory B-cells specific for Sars-CoV-2 antigen. We found promising results for both binding and competitive (neutralization) ELISA conducted on patients' plasma exhibit its high affinity towards RBD protein for the former and neutralize binding to recombinant ACE-2 protein for the latter Both of these initial studies suggested that the neutralizing activity of antibodies present in plasma can be attributed to the RBD domain, which might basically because RBD is composed with ACE-2 binding epitopes [28] . were used from a conserved region in the constant region. After RT-PCR, cDNA was divided into two plates for amplification of heavy and light chains separately. For amplification and cloning of variable regions, a two-step PCR using modified primer pairs was used. For the first PCR reaction, a mix of forward primer was designed from the 5' region of the native signal peptide of the antibody clones. A unique adaptor sequence was also added at 5' of the forward primer to be used as a forward primer in the second PCR step and for in-fusion cloning into expression plasmids. In the second PCR step, adaptor sequence was used as the forward primer and a mix of reverse primers, also containing a unique adaptor sequence, from the J-region were used to specifically amplify variable regions. B-cells showing a band for both heavy and light chain sequences were taken forward for cloning (Fig.3B) . The adaptor sequences in the forward and reverse primers were homologous to the overhangs of linearized expression plasmids. This overlap was utilized to clone the antibody genes into the expression plasmids through in-fusion cloning. The ligation mix was transformed to DH5-α competent cells in 2-ml deep well plates (total 1ml of transformed mix). 500µl of the transformed mix was cultured overnight at 37°C and the plasmid was purified the next day.

The remaining 500µl transformed mix was plated in LB-ampicillin plates for generating colonies. The plasmid was purified and transfected to freestyle-293F cells in a 96-well plate. substrate was added, and relative luminescence activity was captured as an indicator of neutralizing activity. We could observe potent neutralizing activity with one of the clones, SAR03, while low activity was observed for the remaining two clones. The affinity of SAR03 towards was also assessed with SPR and potent affinity (Kd-1.2 nM) was observed for recombinant RBD protein.

In conclusion, we have successfully established an efficient and rapid method of screening neutralizing antibodies directly from recovered patients' blood through single memory B-cell RT-PCR and 2-step PCR were carried out to amplify the variable region of heavy and light chain sequences along with their natural leader sequence. Amplified sequences were cloned into a pAb20-hCHIgG1 plasmid with hygromycin (for Gamma heavy chain) and pAb20-hCK plasmid with blasticidin (for kappa light chain). 

Impact of COVID-19 on the social, economic, environmental and energy domains: Lessons learnt from a global pandemic

A Novel Coronavirus from Patients with Pneumonia in China

Coronavirus disease (COVID-19) pandemic

The emergence and epidemic characteristics of the highly mutated SARS-CoV-2 Omicron variant

Continuous cultures of fused cells secreting antibody of predefined specificity

Phage antibodies: filamentous phage displaying antibody variable domains

Hybridoma-free generation of monoclonal antibodies

Antibody isolation from immunized animals: comparison of phage display and antibody discovery via V gene repertoire mining

Immunization-Elicited Broadly Protective Antibody Reveals Ebolavirus Fusion Loop as a Site of Vulnerability

Identification and specificity of broadly neutralizing antibodies against HIV

A broadly neutralizing anti-influenza antibody reveals ongoing capacity of haemagglutinin-specific memory B cells to evolve

Antibodies and Vaccines Target RBD of SARS-CoV-2

Neutralizing monoclonal antibodies for treatment of COVID-19

Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein

Discovery and characterization of high-affinity, potent SARS-CoV-2 neutralizing antibodies via single B cell screening

A Rapid and Efficient Screening System for Neutralizing Antibodies and Its Application for SARS-CoV-2

COVID-19 antibodies on trial

Covid-19: FDA authorises neutralising antibody bamlanivimab for non-admitted patients

Monoclonal Antibodies for Prevention and Treatment of COVID-19

Structural basis of receptor recognition by SARS-CoV-2

Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor

Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2

Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning

Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen

Monoclonal Antibodies for Emerging Infectious Diseases -Borrowing from History

A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2

Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike

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