Evaluation of Neutralizing Antibodies against Highly Pathogenic Coronaviruses: A Detailed Protocol for a Rapid Evaluation of Neutralizing Antibodies Using Vesicular Stomatitis Virus (VSV) Pseudovirus-based Assay

1 Vaccines and Immunotherapy Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia 2 Department of Medical Laboratories Technology, College of Applied Medical Sciences, Jazan University, Jazan, Saudi Arabia 3 Medical Research Center, Jazan University, Jazan, Saudi Arabia 4 Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia 5 Department of Medical Microbiology and Parasitology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia 6 Corresponding author: amhashem@kau.edu.sa Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2020 doi:10.20944/preprints202005.0379.v1


INTRODUCTION
Coronaviruses (CoVs) are the largest group of enveloped positive-sense RNA viruses that primarily infect the respiratory and gastrointestinal tracts of birds and mammals (Fehr and Perlman, 2015). Many CoVs could be zoonotic viruses capable of crossing the species barrier and infecting humans (Kandeel et al., 2020). Human CoVs mainly cause mild respiratory tract infections and no highly pathogenic CoVs were recognized until the beginning of the 21 st century (Cui et al., 2019). Since 2002, a number of highly pathogenic human CoVs have emerged including the severe acute  (Ksiazek et al., 2003, Zaki et al., 2012. In December 2019, a novel human highly pathogenic CoV known as severe acute respiratory syndrome-CoV-2 (SARS-CoV-2) has emerged in Wuhan, China causing the coronavirus disease-19 (COVID-19) pandemic , Zhu et al., 2020. While SARS-CoV has disappeared, both MERS-CoV and SARS-CoV-2 continue to be a major global threat especially that no clinically proven treatments or vaccines are available for human use to date (Al-Amri et al., 2017, Liu et al., 2018, Padron-Regalado, 2020, Tse et al., 2020. The spike (S) proteins of CoVs are surface glycoproteins that facilitate viral entry into host cells. The S1 subunit at the N terminal end of the S protein contains the receptorbinding domain (RBD) responsible for the attachment to cellular receptors, while the S2 subunit at the C-terminus contains the necessary machinery that mediates the fusion with the host membranes. The S protein of SARS-CoV and SARS-CoV-2 attaches to the angiotensin-converting enzyme 2 (ACE2) receptor, and the S protein of MERS-CoV recognizes the dipeptidyl peptidase 4 (DPP-4) (Liu et al., 2018, Hoffmann et al., 2020. The S protein, particularly the RBD, is considered as a major immunogenic component of CoVs and a target for most neutralizing antibodies (nAbs).
Live virus-based neutralization methods are the gold standard serological assays to detect and measure nAbs levels, however, they require working under strict biocontainment conditions in biosafety level-3 (BSL-3) laboratories when working with highly pathogenic CoVs (Algaissi and Hashem, 2020). While conventional enzymelinked immunosorbent assay (ELISA) and immunofluorescence assay (IFA) have been used for CoVs antibodies screening, cross-reactivity with other common CoVs may lead to false-positive results (Lester et al., 2019, Degnah et al., 2020.
Additionally, positivity in these assays does not necessarily reflect the presence of nAbs in samples, requiring other confirmatory functional assays. Thus, replicationincompetent pseudotyped viruses bearing S proteins from highly pathogenic CoVs could represent an alternative safe and convenient method for CoVs nAbs detection and quantification in serum samples under biosafety level-2 (BSL-2) conditions (Almasaud et al., 2020). Several studies have shown encouraging results by utilizing vesicular stomatitis virus (VSV) as a platform to generate pseudoviruses that can be used in seroepidemiological studies, vaccine development, monoclonal antibodies and entry inhibitors screening, and basic research investigations of CoVs (Fukushi et al., 2005, Lester et al., 2019, Nie et al., 2020.
VSV is a zoonotic enveloped negative-stranded RNA virus that infects a wide range of animals and less frequently humans causing mild flu-like illness symptoms (Rodriguez, 2002, Tani et al., 2011. Its small 11 kb genome, simple structure, and ability to grow in different types of mammalian cells with high-titer made VSV a promising virus vector and a valuable tool in molecular biology and virology fields (Ruedas and Connor, 2017). Interestingly, recombinant VSV (rVSV) with G gene being replaced by reporter luciferase gene (rVSV-ΔG-luciferase) can normally bud from cells transfected with mammalian expression plasmid encoding VSV G protein or heterologous envelope protein from other viruses (Whitt, 2010, Tani et al., 2011. Thus, rVSV-ΔG-luciferase system could be used to produce single round replicationincompetent VSV pseudoviruses bearing any viral envelope glycoprotein specially from those requiring work under BSL-3 containment in BSL-2 laboratories (Whitt, 2010).
In these detailed protocols, we explain step by step how to generate VSV pseudoviruses bearing CoVs S proteins from MERS-CoV and SARS-CoV-2 using transient expression in BHK-21/WI-2 cells (Basic Protocol 1). This is followed by pseudovirus titration method (Basic Protocol 2) and pseudovirus-based neutralization assay (Basic Protocol 3) relying on reading quantitative luciferase luminescence signals. We utilized this system to conduct seroprevalence studies and measure nAbs in MERS-CoV and SARS-CoV-2 infected patients. Notes and comments have been added to overcome any difficulties. We believe that the described platform (Figure 1) can be adapted and used for research studies as well as diagnostic purposes.
To maintain the sterility and ensure success in all cell culture experiments in all basic protocols listed here, some precautions have to be taken, which include: • All experiments related to cell culture should be performed in BSL-2 cabinet in a tissue culture room under suitable antiseptic techniques.
• Sterile materials and solutions must be used throughout the protocol.
• All solutions should be warmed up in 37°C water bath or room temperature as specified in the protocol.
• All cells growth and incubation should be carried out in a humidified 37°C incubator with 5% CO2. 6. Incubate the transfection mixture for 20 min at room temperature.
3.5 ml is the total volume of the transfection mixture.
7. Take out the BHK-21/WI-2 cells in the T175 flask form the incubator and transfect the cells by adding the transformation mixture dropwise on the cells monolayer using 5 ml sterile serological pipette while swirling the flask gently to ensure even dispersal.

Day 2: Infection of transfected cells with rVSV-ΔG/G*-luciferase
9. In a 15 ml polypropylene sterile tube prepare the virus inoculation mixture by adding 5 ml DMEM-5 containing an amount of rVSV-ΔG/G*luciferase equivalent to multiplicity of infection (MOI) of 4 using the working stock virus form Kerafast.
Thaw the virus stock on ice before preparing the inoculum. It is recommended to generate additional working stocks by amplifying an aliquot of the rVSV-ΔG/G*-luciferase stock form Kerafast in BHK-21/WI-2 cells transfected with pCAGGS-G (expression plasmid encoding VSV-G protein). For rVSV-ΔG/G*luciferase titration, viral plaque assay can be used as previously described (Whitt, 2010).
10. Take out the transfected BHK-21/WI-2 cells in the T175 flask from Day 1 experiment from the incubator and remove the growth medium.
11. Infect the cells with the 5 ml media containing the rVSV-ΔG/G*luciferase and make sure to distribute equally over the cells monolayer. 14. After 1 hr incubation, remove the virus inoculum and wash the cells twice with 12 ml of pre-warmed 1x PBS.
Washing with 1x PBS helps to remove the rest of the rVSV-ΔG/G*-luciferase that do not inter the cells.
15. Add the prepared 15 ml DMEM-5 medium supplemented with anti VSV-G antibodies to the cells monolayer.

Avoid drying the cells by working quickly between removing the growth medium and adding the medium that supplemented with the antibodies.
16. Incubate the flask for 24 hr at 37°C in 5% CO2 humidified incubator.

Day 3: Collection and storage of the generated VSV pseudovirruses
17. Next day, collect the supernatant that contains the VSV pseudoviruses in a 50 ml polypropylene sterile conical tube.
18. Remove the cells debris by centrifugation the supernatant at 600 x g for 5 min.
Avoid thawing and freezing the virus stocks as this well affect virus titer.

Basic protocol 2 Titration assay of the generated VSV pseudoviruses by measuring luciferase activity
This protocol is based on the use of luciferase activity as a main readout of the system to titrate the produced rVSV pseudoviruses. The measured luciferase activity is defined as relative luminescence unit (RLU). This protocol is summarized in Figure 3. Different luciferase assay systems or in house made luciferase substrate and buffer could be used. It is preferred to subculture confluent Vero E6 at 1:4 ratio 48 hr before use.

Huh-7 cells could be used instead.
2. Prepare 11 ml of Vero E6 cells suspension at a density of 2 x 10 5 cells/ml in a 50 ml polypropylene sterile conical tube.

Pre-warmed DMEM-5 is used to prepare the cells suspension.
3. Seed the Vero E6 cells suspension in a 96-well white or black cell culture plate with clear bottom by distributing 100 μl of the cells/well using multichannel pipette, sterile filtered tips and sterile reservoir.
To save time, steps 1-4 can be done in the last day of Basic Protocol 1 after collecting the pseudovirus.

Day 2: Cell infection with generated VSV pseudoviruses bearing CoV S gene
5. In a sterile U shape 96-well cell culture plate, add 60 μl of pre-warmed DMEM-5 to all wells in column 12 as a negative cell control; cell only control (CC).
6. Add 60 μl of pre-warmed DMEM-5 to all wells in columns 1 to 11 in rows B to H using multichannel pipette, filtered tips and sterile reservoir. 7. Thaw the supernatant containing generated VSV pseudotyped viruses on ice.
10. Remove 60 μl from virus-containing wells in row A (A1-A11) and perform 1:2 serial dilution downward to all wells below using multichannel pipette and filtered tips.
Other dilutions such as 1:3 or 0.5 log could be used.
11. During each dilution step, mix well by pipetting eight times up and down.
12. Continue the dilution until row H and discard the final 60 μl from the last wells in row H.
13. Remove the media from the plated Vero E6 cells in 96-well plate that was seeded on Day 1.
14. Using multichannel pipette and filtered tips, transfer 50 μl from all wells in the U shape 96-well cell culture plate to corresponding wells in the 96-well plate of Vero E6 cells.

Day 3: Luciferase assay
17. Prepare 1x lysis buffer from 5x CCLR in a 15 ml polypropylene sterile conical tube by adding 4 volumes of water to 1 volume of 5x CCLR. A total of 2.5 ml of 1x lysis buffer will be enough for each 96-well plate (20 μl/ well).
Equilibrate 5x CCLR to room temperature before preparing the 1x lysis buffer.
18. By using luciferase assay system, prepare the luciferase assay reagent by adding 10 ml luciferase assay buffer to a vial containing lyophilized luciferase assay substrate.
Equilibrate luciferase assay buffer to room temperature before preparing the reagent. Avoid exposure of the luciferase assay reagent to multiple freezethaw cycles. We use a dilution of the generated pseudovirus that yields 5 x 10 4 RLU.

Basic protocol 3 Neutralization assay to determine CoVs specific nAbs titers in serum samples
As in Basic protocol 2, measuring of nAbs titers depends on using luciferase-based assay. Inhibition of the generated pseudovirus entry into Vero E6 cells by CoV antibodies is correlated with the deceased levels of luciferase expression signals. This assay could be used to measure nAbs titers from different species including humans and animals as well as testing monoclonal antibodies. Figure 4 illustrates the workflow.

Materials
All materials as in Basic Protocol 2

Day 2: Pseudovirus neutralization assay
2. In a sterile U shape 96-well cell culture plate, add 60 μl of pre-warmed DMEM-5 to all wells in columns 1 to 12 in rows B to G.
3. Add 120 μl of pre-warmed DMEM-5 to wells H1 to H6 to serve as negative cell control; cell only control (CC). 4. Add 60 μl of pre-warmed DMEM-5 to wells H7 to H12 to be used as virus control (VC). 5. Add 120 μl of 1:10 dilution of heat-inactivated serum samples in wells in row A, add each sample in duplicate.
Heat-inactivate serum samples at 56°C for 30 min.
Other dilutions such as1:3 or 0.5 log could be used. 11. Incubate the plate for 1 hr at 37°C in 5% CO2 humidified incubator.
12. Take out the plated Vero E6 cells in 96-well cell culture plate form the incubator that was seeded on Day 1 and remove the growth medium.
13. Using a multichannel pipette and filtered tips, transfer 100 μl from all wells in the U shape 96-well cell culture plate to corresponding wells in the 96-well plate of Vero E6 cells.

Day 3: Luciferase assay
15. Follow the same 17-23 steps that have been described in the luciferase assay in Basic Protocol 2 to measure luciferase activity. An example of neutralization assay results is shown in Figure 5.  Store at 4°C for a month.

Background Information
The detailed protocols described here can serve as convenient methods to detect MERS-CoV and SARS-CoV-2 nAbs in serum samples under BSL-2 conditions. The pseudovirus assays detailed here could also be used to evaluate the immunogenicity of vaccines and potency of monoclonal antibodies, other biologics and small molecules. These assays rely on a well-established technique using rVSV-ΔG/G*luciferase pseudovirus system. Most reagents required for this system are commercially available and could be adopted and used by researchers and laboratories around the world. Such assay provides a number of advantages over standard serological assays including ability to test for nAbs in serum samples under BSL-2 conditions with minimal equipment and relatively low cost.

Low/ poor transfection efficiency
A number of important parameters should be considered to increase the transfection efficiency including: BHK-21/WI-2 cells: Low transfection efficiency could lead to low pseudovirus titers. Using healthy and low passage BHK-21/WI-2 cells at ~70% confluency enhances the experiment outcomes. Starting with low or large number of cells could lead to reduction in the uptake of foreign DNA.
Plasmid DNA: It is highly recommended to use high-quality expression DNA plasmid encoding the gene of interest for cells transfection. A number of commercial kits can be used to obtain purified plasmid free of RNA, nucleases, proteins, chemicals and microbial contamination.
Transfection reagent: For preparing the transfection mixture, it is required to use serum-free cell culture medium such as Opti-MEM ® I (1x) reduced-serum medium. We used Lipofectamine TM 2000 transfection reagent and it worked well in our hands, however, other reagents or methods could be used.

Luciferase assay background readings
Some steps need to be duly done to avoid the luciferase reading errors, which are: Removal of rVSV-ΔG/G*-luciferase pseudovirus after infection: One of the most common problems that could occur during the generation of pseudoviruses (Basic Protocol 1) is having residual or background rVSV-ΔG/G*-luciferase pseudovirus in the collected supernatant ( Figure 2). Complete removal of excess rVSV-ΔG/G*luciferase that do not infect the cells could be done by washing the cell monolayer twice with 1x PBS as well as using DMEM-5 supplemented with anti VSV-G antibodies.
Dilution of pseudovirus and serum samples: Changing pipette tips between steps (Basic Protocols 2 and 3) is important to get accurate luciferase activity results.
Additionally, complete removal of residual liquid after washing wells and before adding the luciferase reagent helps reducing the background noise.

Understanding Results
Measuring nAbs titers using pseudoviruses depends on luminescence signal reads obtained from the activity of the expressed luciferase. The recommended amount of VSV pseudovirus to be used in neutralization assays should have a signal above cellonly control and in the linear part of the curve. It is recommended to use a dilution of the pseudovirus that yields 5x10 4 RLU. The nAb titers (NT50) in serum samples could be calculate as the reciprocal of the serum dilution that reduced luciferase activity by 50%. The inhibition rate is calculated as 100 -[(mean RLU from each sample -mean RLU from CC) / (mean RLU from VC -mean RLU from CC) ×100].

Time Considerations
Basic Protocols 1 and 2 can be completed in about 7 consecutive days. However, it is possible to do it in 6 days if seeding of Vero E6 cells in 96-well plates for titration is prepared in the same day of collecting the pseudovirus supernatant. In details, it takes 4 days to produce VSV pseudoviruses bearing CoV S protein (Basic Protocol 1).
Once done successfully, the produced pseudoviruses can be stored at -80°C for long time. Titration of generated pseudovirus (Basic Protocol 2) is usually done in 3 days.
Pseudovirus neutralization assay (Basic Protocol 3) takes 3 days, and up to 6 serum samples could be tested per plate.