This work was supported by George Mason University internal research fund.

1. Virus and viral particle assembly
<ol>
	<li>Vectors: Purchase expression vectors for SARS-CoV-2 M, E, or N as well as the SARS-CoV-2 S (Wild-type,Wt), Delta (B.1.617.2), and Omicron (B.1.1.529) commercially. 
	NOTE: Protein sequences of the expression vectors are provided in Supplementary File 1. The vendor of these expression vectors can also be found under the Table of Materials.</li>
	<li>Cells and cell culture: Maintain HEK293T cells in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), 50 units/mL of penicillin, and 50 &#181;g/mL of streptomycin. 
	&#8203;NOTE: All cell culture work needs to be done in a laminar airflow biosafety cabinet. The polyethyleneimine (PEI) transfection typically requires 5-6 h incubation. Start times need to be considered carefully beforehand.</li>
	<li>Virus assembly and PEI-based cotransfection: Assemble Ha-CoV-2 particles by cotransfection of HEK293T cells. Seed cells in a 10 cm cell culture Petri dish (4-5 x 106 cells per dish) in 10 mL of complete DMEM medium the day prior to cotransfection. For this study, three Petri dishes are used to seed cells for the assembly of Ha-CoV-2 wild-type, Delta, and Omicron, respectively.</li>
	<li>Incubate Petri dishes overnight in a CO2 incubator at 37 &#176;C. Check the dishes next morning to ensure that cells are 80% confluent. Remove complete medium and replace it with 9 mL of DMEM serum-free media.</li>
	<li>For each dish, prepare a cotransfection mixture with 2.5 &#181;g of each of the SARS-CoV-2 structural protein expression vectors (N, E, M), 10 &#181;g pAlphaPro-Luc-GFP-Pre&#936; (HaCoV2 Genome), and 2.5 &#181;g of the S protein expression vector, either the Delta or the Omicron S variants and 45 &#181;L of PEI-based transfection reagent. Allow the cotransfection mixture to form complexes by incubating for 13 min (do not incubate for longer than 30 min).</li>
	<li>After incubation, add the cotransfection mixture to each Petri dish slowly, drop by drop. Place the dishes inside a CO2 incubator at 37 &#176;C for 6 h. After 6 h, remove serum-free DMEM and replace it with complete DMEM medium. Harvest virus at 48-60 h post-cotransfection.</li>
	<li>Virus harvesting and storage: Harvest particles at 48 h post cotransfection. Detach cells by pipetting repeatedly over the surface of the monolayer and collect cells from each dish, put into 15 mL centrifuge tubes, and centrifuge at 400 x g for 5 min. Collect the supernatant and pass it through a 0.22 &#181;M filter. Store the Ha-CoV-2 pseudovirus at -80 &#176;C.</li>
</ol>
2. Viral infectivity assay
<ol>
	<li>Cells and cell culture: Maintain HEK293T(ACE2/TMPRSS2) cells in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) containing 10% heat-inactivated FBS, 50 units/mL of penicillin and 50 &#181;g/mL of streptomycin.</li>
	<li>Seeding HEK293T(ACE2/TMPRSS2) cells: The day before viral infectivity assay, seed HEK293T(ACE2/TMPRSS2) cells in a 96-well plate in 50 &#181;L of complete DMEM media. For each 96-well plate, seed 2.5 x 104 cells into each well, and a total of 2.5 x 106 cells are needed for one plate. Place the 96 well plate in a CO2 incubator at 37 &#176;C overnight.</li>
	<li>Infection of HEK293T(ACE2/TMPRSS2): Use Ha-CoV-2 variants particles to infect HEK293T(ACE2/TMPRSS2) cells. On the morning of infection, remove 50 &#181;L of DMEM from pre-seeded 96 well plate. Replace media with 50 &#181;L of either Ha-CoV-2 Wild-type, Delta, or Omicron for 18 h at 37 &#176;C. 
	NOTE: The protocol can be stopped here until the infection has reached 18 h of incubation and the plate is ready to be analyzed via Luciferase Assay. The success of the infection is determined by the Luciferase assay resulting from the Luciferase reporter gene expressed in the infected cells, therefore the more signal is produced, the more successful the infection of the Ha-CoV-2 variant is.</li>
	<li>Luciferase assay: After 18 h of incubation, add 7.5 &#181;L of cell lysis buffer directly to each well and mix by orbital shaking for 2 min. Lyse cells in lysis buffer for at least 5 min at room temperature.</li>
	<li>Prepare the Firefly luciferase assay solution by mixing the D-luciferin solution with the Firefly luciferase assay solution in a 1:50 ratio. For a whole 96-well plate, combine 3 mL of the luciferase substrate solution with 60 &#181;L of the D-luciferin solution with 2940 &#181;L of the Firefly luciferase buffer solution.</li>
	<li>Add 25 &#956;L of the Firefly luciferase assay solution to the cell lysates and mix the plate by orbital shaking for 1 min. Analyze the luciferase activity by using a commercial luciferase microplate reader.</li>
</ol>
3. RNA extraction of Ha-CoV-2 and quantitative reverse transcriptase PCR (RT-qPCR)
<ol>
	<li>Viral RNA extraction: Extract the viral RNA from Ha-CoV-2 Wild-type and the Ha-CoV-2 Delta and Omicron variant particles using a commercial viral RNA extraction kit, following the manufacturer&#39;s instructions. Store the extracted viral RNA at -80 &#176;C or use it immediately for RT-qPCR.</li>
	<li>RT-qPCR: Perform RT-qPCR on viral RNA using a one-step master mix. Perform the reaction in a commercial PCR machine. Please note that the target of amplification is the genomic RNA of Ha-CoV-2. Use Ha-CoV-2 vector DNA as a standard for creating a standard curve and calculating the RNA copy number of each variant.</li>
</ol>
4. Neutralizing antibody assay
<ol>
	<li>Seeding HEK293T(ACE2/TMPRSS2) cells: The day before the assay, seed HEK293T(ACE2/TMPRSS2) cells in a 96 well plate in 50 &#181;L of complete DMEM media. HEK293T(ACE2/TMPRSS2) cells are commercially purchased.</li>
	<li>For counting cells, obtain 20 &#181;L cells from a T75 flask containing HEK293T(ACE2/TMPRSS2) cells, and mix with 20 &#181;L of trypan blue solution. Add 20 &#181;L of this mixture to cell counting chamber and count the number of cells per mL. To seed a 96 well plate for infection, use 2.5 x 104 cells per well, and 2.5&#160; x 106 cells will be needed in total. Place 96 well plate in a CO2 incubator at 37 &#176;C overnight.</li>
	<li>Neutralizing antibody assay: In a sterile polypropylene 96-well plate, prepare the standard 27BV neutralizing antibody and Ha-CoV-2 mixture. Add 8 &#181;L of 27BV (45 mg/mL) to the plate and perform serial dilutions of the antibody with 6 &#181;L of serum-free DMEM. 
	NOTE: Be sure to exchange pipette tips between well transfers and ensure the antibody and serum-free medium are mixed thoroughly to produce accurate results.</li>
	<li>To the serially diluted antibody, add 54 &#181;L of Ha-CoV-2 particle and mix the virus and the antibody. Pre-incubate Ha-CoV-2 particles with serially diluted 27BV for 1 h at 37 &#176;C at 5% CO2. After the 1 h incubation, apply 50 &#181;L of the antibody and Ha-CoV-2 mixture to the 96-well plate containing the HEK293T(ACE2/TMPRSS2) cells (2.5 x 104 cells per well) seeded the day before.</li>
	<li>For controls, leave at least three wells containing only the HEK293T(ACE2/TMPRSS2) cells. Add 50 &#181;L of complete medium to these wells to serve as non-infected wells for background signal of the luciferase assay readings. 
	&#8203;NOTE: The protocol can be stopped here until the infection has reached 18 h of incubation at 37 &#176;C, and then the plate is ready to be analyzed via luciferase assay.</li>
	<li>Luciferase assay: After 18 h of incubation, add 7.5 &#181;L of lysis buffer directly to each well and mix by orbital shaking for 2 min. Lyse cells in lysis buffer for at least 5 min at room temperature.</li>
	<li>Prepare the Firefly luciferase assay solution by mixing the D-luciferin solution with the Firefly luciferase assay solution in a 1:50 ratio. For a whole 96-well plate, combine 3 mL of the luciferase substrate solution with 60 &#181;L of the D-luciferin solution with 2940 &#181;L of the firefly luciferase buffer solution.</li>
	<li>Add 25 &#956;L of the Firefly luciferase assay solution to the cell lysates and mix the plate by orbital shaking for 1 min. Analyze the luciferase activity by using a commercial luciferase microplate reader.</li>
</ol>
5. Quantification and statistical analysis
<ol>
	<li>Data collection: Perform infection and luciferase assays in triplicate as indicated (Figure 1). Quantify luciferase expression with luciferase assay readings. The mean is the average value of the three luciferase assay readings. Background signal readings from non-infected wells are subtracted from this mean value. Standard deviation (SD) is determined from the average value of the luciferase assay readings.</li>
	<li>Data analysis: Plot antibody neutralization activity and calculate the ID50 (50% inhibition dosage) values using commercial graphing software. The ID50 value is defined as the antibody inhibitory dilutions at which a 50% reduction in the infection of HEK293T(ACE2/TMPRSS2) cells (based on luciferase assay readings) is attained.</li>
</ol>

Quantifying Vaccine-Induced Neutralizing Antibodies in SARS-CoV-2 Variants

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A patent has been filed by George Mason University and licensed to Virongy Biosciences Inc. for product development. Y.W. is a founder and member of the advisory board of Virongy Biosciences. B. H is currently the CEO and Chief Scientific Officer of Virongy Biosciences. The authors have no other conflicts of interest.

The Ha-CoV-2 platform provides a rapid, robust, and simple workflow to quantify viral variants and neutralize antibodies. However, there are a few critical steps that need attention. The production of The Ha-CoV-2 pseudovirus should be performed using HEK293T cells with high viability. The cotransfection efficiency can be monitored 24 h post-transfection using the GFP reporter gene from the Ha-CoV-2 genome. The Ha-CoV-2 genome can contain two reporters (GFP and Luc), and GFP can be expressed during cotransfection and following Ha-CoV-2 infection of target cells12. The GFP+ cells from infection are normally at a low percentage (1% to 5%), but each infected cell expresses strong GFP signals (Figure 3). This low GFP percentage may limit the use of GFP as a robust readout for quantifying antibody neutralization, as compared with the Luc reporter, which quantifies the whole population of infected cells.
When performing the neutralization assay, it is essential to change pipette tips between well transfers and to ensure the antibody and serum-free medium are mixed thoroughly to produce accurate results. Additionally, when conducting the luciferase assay protocol, cells must be fully lysed for at least 3 min to ensure complete lysis of cells and the release of the luciferase enzyme. This will ensure the accuracy of the assay. Additionally, once the Firefly luciferase assay solution is added to the optical white-walled 96 well plates, the plate must be analyzed within 10 min as the initial light emission is high but decreases over time as the ATP is depleted21.
As more SARS-CoV-2 variants continue to evolve, there is an increased need for platforms like Ha-CoV-2 to rapidly screen for variant infectivity and variant sensitivity to vaccine-induced neutralizing antibodies. The Ha-CoV-2 platform offers faster speed, a higher signal-to-noise ratio, and a simple protocol compared to existing pseudovirus-based neutralization assays8,9,10,11. The Ha-CoV-2 platform also offers the advantage that it can be used in BSL-2 laboratories and does not require the use of BSL-3 facilities. This allows SARS-CoV-2 to research to be pursued in common research and clinical laboratories. Furthermore, the Ha-CoV-2 platform produces rapid results in comparison with other systems. For instance, the study of neutralizing antibodies against infectious SARS-CoV-2 virus often makes use of the plaque reduction neutralization test (PRINT)22. Although PRINT produces reliable results, manual counting of plaque-forming units (PFUs) is slow and requires 3-5 days to obtain results23,24. Other pseudotype systems, such as the lentivirus-pseudovirus need 24-72 h to produce a&#160; detectable reporter signal12. In comparison, the Ha-CoV-2 neutralization assay can generate results within 18 h. The Ha-CoV-2 provides a convenient tool for rapid screening and quantification of viral variants and neutralizing antibodies for pandemic monitoring.
Monitoring the infectivity of SARS-CoV-2 is essential as more variants of concern (VOCs) continue to emerge. Ha-CoV-2 offers the advantage of quickly determining the infectivity of VOCs. Previous studies have used artificial intelligence (AI)-based modeling to quantitatively analyze the infectivity of the Omicron subvariant and the other SARS-CoV-2 variants, such as the Delta variant25. These studies have shown that the Omicron variant is more contagious than the original virus, and more likely to escape neutralizing antibodies25. In these studies, using Ha-CoV-2, similar phenotypes were observed. Additionally, in the antibody neutralization assays, the Omicron variant is ten times less likely to be neutralized by 27BV than the Wuhan and Delta strains. These results are also consistent with the reported higher transmissibility of the Omicron variant, which has at least 15 mutations on its receptor binding domain (RBD), likely enhancing viral binding affinity to the ACE2 receptor for higher transmissibility and greater immune escape26.

As of May 2023, there have now been more than 766 million COVID-19 cases1. Despite worldwide vaccination campaigns, SARS-CoV-2 continuously circulates and infects people, largely due to the emergence of new variants such as Delta (B.1.617.2) and Omicron (B.1.1.529) that drive new waves of infection2,3,4. Given that SARS-CoV-2 is constantly evolving, it is important to develop quick assays that can accurately measure the infectivity of emerging variants and the effectiveness of vaccine-induced neutralizing antibodies against these variants. These assays are essential for pandemic surveillance and for determining the efficacy of vaccines and their variant-specific boosters.
Due to the highly contagious nature of SARS-CoV-2, the Center for Disease Control and Prevention (CDC) requires that the study of SARS-CoV-2 and its variants is conducted in biosafety level (BSL) 3 facilities5,6. This BSL-3 requirement limits the use of live viruses for quantifying the infectivity of viral variants and their neutralizing antibodies in common research and clinical laboratories. In addition, traditional SARS-CoV-2 neutralization assays, such as the plaque- or cytopathic effect-based assays using replication-competent live viruses, are time-consuming and require long incubation periods7. Several spike (S) protein-pseudotyped SARS-CoV-2 pseudoviruses have been developed to quantify the effectiveness of neutralizing antibodies8,9,10,11,12. In SARS-CoV-2, the S protein is the major protein that mediates viral entry13, and is the main antigen used in SARS-CoV-2 vaccines9,10,14,15,16. The S protein-pseudotyped virions, such as those of the vesicular stomatitis virus (VSV-G) or lentivirus, have been used for the quantification of neutralizing antibodies17,18,19. Nevertheless, the lentivirus-based pseudovirus normally requires 2 to 3 days of infection in order to quantify reporter signals. VSV-based pseudovirus systems often contain residual VSV viruses, which can result in high rates of false-positive results and typically require 24 h of infection20.
A novel SARS-CoV-2 pseudovirus system, the hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2), has been recently developed by Hetrick et al12. Ha-CoV-2 provides a new tool for the rapid quantification of virus infectivity and virus sensitivity to neutralizing antibodies in common BSL-2 laboratories. Structurally, Ha-CoV-2 resembles the SARS-CoV-2 virion particle, consisting of SARS-CoV-2 structural proteins including the S protein (S), the membrane (M), the nucleocapsid (N), and the envelope (E), and there is no structural protein from other viruses. Additionally, Ha-CoV-2 particle contains a rapid-expressing RNA genome from an alphavirus for fast reporter expression in cells. Ha- CoV-2 has been shown to quickly measure the neutralizing activity of antibodies in the sera of vaccinated and convalescent individuals12. As demonstrated by Hetrick et al., when compared with lentivirus-based SARS-CoV-2 pseudovirus in a time course assay, Ha-CoV-2 expressed the Luc reporter as early as 2-4 h post-infection while the lentivirus-pseudovirus expressed Luc after 24 h12. In addition, the potential application of Ha-CoV-2 variants for quantifying neutralizing antibodies is further demonstrated by using a&#160; standard monoclonal neutralizing antibody, 27BV (See Supplementary Figure 1)12. This work details the use of the Ha-CoV-2 platform for rapid quantification of the infectivity of SARS-CoV-2 variants, using the Delta (B.1.617.2) and the Omicron (B.1.1.529) variants as examples. In addition, the potential application of Ha-CoV-2 variants for quantifying neutralizing antibodies is further demonstrated by using a standard monoclonal neutralizing antibody, 27BV12.

<table><tbody><tr><td>27VB1 20 &amp;micro;g SARS-CoV-2 Standard Neutralizing Antibody</td><td>Virongy Biosciences</td><td>27VBI-01</td><td></td></tr><tr><td>500 mL - US Origin FBS</td><td>Neuromics</td><td>FBS001</td><td></td></tr><tr><td>AB Mixing Plate: Olympus 96-Well PCR Plate, Non-Skirted UltraThin Wall, Natural, 25 Plates/Unit&amp;nbsp;</td><td>Genesee Scientific</td><td>Cat# 24-300&amp;nbsp;</td><td></td></tr><tr><td>Allegra 6R Centrifuge</td><td>Beckman Coulter</td><td>2043-30-1158</td><td></td></tr><tr><td>DMEM (1x)&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>11995-073</td><td></td></tr><tr><td>GenClone 25-209, TC Treated Flasks, 250ml, Vent Growth Area: 75.0cm2, 5 per Sleeve, 100 Flasks/Unit</td><td>Genesee Scientfic</td><td>25-209</td><td></td></tr><tr><td>GlowMax Discover Microplate reader</td><td>Promega&amp;nbsp;</td><td>GM3000&amp;nbsp;</td><td></td></tr><tr><td>Ha-CoV-2 E Vector&amp;nbsp;</td><td>Virongy Biosciences</td><td>pCoV2_E</td><td></td></tr><tr><td>Ha-CoV-2 M Vector&amp;nbsp;</td><td>Virongy Biosciences</td><td>pCoV2_M</td><td></td></tr><tr><td>Ha-CoV-2 N Vector&amp;nbsp;</td><td>Virongy Biosciences</td><td>pCoV2_N</td><td></td></tr><tr><td>Ha-CoV-2 WT S Vector</td><td>Virongy Biosciences</td><td>pCoV2_WT S</td><td></td></tr><tr><td>Hek293T cells</td><td>ATCC</td><td>CRL-3214</td><td></td></tr><tr><td>Illumination Firefly Luciferase Enhanced Assay Kit 1000 assays</td><td>Gold Bio&amp;nbsp;</td><td>I-930-1000</td><td></td></tr><tr><td>Infection Plate: 96-Well Tissue Culture Plate, Greiner Bio-One (With Lid, &amp;mu;Clear White Flat Round, Chimney)&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>Cat# 82050-758&amp;nbsp;</td><td></td></tr><tr><td>pAlphaPro-Luc-GFP-Pre&amp;Psi; (Ha-CoV-2 Genome) Vector&amp;nbsp;</td><td>In house</td><td></td><td></td></tr><tr><td>PEI-based Transfection Reagent</td><td>Virongy Biosciences</td><td>Transfectin</td><td></td></tr><tr><td>Penicillin-Streptomycin-Glutamine (100X)</td><td>Invitrogen</td><td>10378016</td><td></td></tr><tr><td>Polyethylenimine, branched</td><td>Millipore Sigma</td><td>408727-100ML</td><td></td></tr><tr><td>QuantStudio 7 Pro Real-Time PCR System</td><td>ThermoFisher&amp;nbsp;</td><td>A43163</td><td></td></tr><tr><td>Ready to use (HEK293T)(ACE2/TMPRSS2) Cells</td><td>Virongy Biosciences</td><td>Ready-To-Use-Cells</td><td></td></tr><tr><td>SARS-CoV-2 S&amp;nbsp; Omicron (B.1.1.529) Vector</td><td>Virongy Biosciences</td><td>pCoV2-B.1.1.529</td><td></td></tr><tr><td>SARS-CoV-2 S Delta (B.1.617.2)&amp;nbsp;Vector</td><td>Virongy Biosciences</td><td>pCoV2- B.1.617.2</td><td></td></tr><tr><td>Syringe Filters, PES, 0.22&amp;micro;m</td><td>Genesee Scientfic</td><td>25-244</td><td></td></tr><tr><td>TaqMan Fast Virus 1-Step Master Mix</td><td>ThermoFisher&amp;nbsp;</td><td>4444432</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>ThermoFisher&amp;nbsp;</td><td>15250061</td><td></td></tr></tbody></table>

application of ha-cov-2 pseudovirus for rapid quantification of sars-cov-2 variants and neutralizing antibodies

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 variants and the effectiveness of vaccine-induced neutralizing antibodies against viral variants. These assays are essential for pandemic surveillance and validating vaccines and variant-specific boosters. This manuscript demonstrates the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) for quick quantification of SARS-CoV-2 variant infectivity and vaccine-induced neutralizing antibodies to viral variants. Ha-CoV-2 is a SARS-CoV-2 virus-like particle consisting of viral structural proteins (S, M, N, and E) and a fast-expressing RNA genome derived from an alphavirus, Semliki Forest Virus (SFV). Ha-CoV-2 also contains both green fluorescent protein (GFP) and luciferase reporter genes that allow for quick quantification of viral infectivity. As an example, the infectivity of the SARS-CoV-2 Delta (B.1.617.2) and the Omicron (B.1.1.529) variants are quantified, and their sensitivities to a neutralizing antibody (27VB) are also measured. These examples demonstrate the great potential of Ha-CoV-2 as a robust platform for rapid quantification of SARS-CoV-2 variants and their susceptibility to neutralizing antibodies.

Ha-CoV-2 particles were assembled using five different DNA vectors that express the Ha-CoV-2 RNA genome and the structural proteins (M, N, E, and S) of SARS-CoV-2 in HEK293T cells. The S protein vector varies depending on the S variant. The S protein from the original Ha-CoV-2 Wuhan strain (Wild-type, Wt) was used as a positive control, and it was assembled along with the S protein from each of the other two variants: the Delta (B.1.617.2) or the Omicron (B.1.1.529). The same M, N, E were used in all variants. Ha-CoV-2(Wt) and variant particles were collected 48 h post-cotransfection and then used to infect HEK293T(ACE2/TMPRSS2) cells. Infectivity was measured by expression of luciferase at 18 h post-infection. In this system, higher expression levels of luciferase signal reflect a higher infection of cells by Ha-CoV-2. The luciferase signal was normalized with genomic RNA copies by RT-qPCR for each variant. As shown in Figure 1, the Ha-CoV-2&#160;Omicron variant generated 4- to 10-fold higher signal than the original Ha-CoV-2(Wt), suggesting a higher infectivity.
Furthermore, the capacity of 27BV to neutralize HaCoV-2(Wt), Delta, and Omicron variants was quantified. 27BV is a rabbit monoclonal antibody that was developed against the RBD domain of the SARS-COV-2 S1 protein. For neutralization assays, serial dilutions of 27BV were performed in a 96-well plate, pre-incubated with Ha-CoV-2, and then added to HEK293T(ACE2/TMPRSS2) target cells. The results demonstrated that 27BV had neutralizing activity against all the variants tested (Figure 2). Interestingly, the ID50 of 27VB for Omicron was approximately 10 times less potent than the ID50 for Ha-CoV-2(WT) and Ha-CoV-2(Delta; Figure 2). These results demonstrate that the Ha-COV-2 platform can be used as a rapid method for quantifying vaccine-induced neutralizing antibodies in emerging variants.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65793/65793fig01.jpg" /> 
Figure 1. Assembly and quantification of Ha-CoV-2 variants. (A) Illustration of the assembly of the Ha-CoV-2 and variant particles. The vectors expressing the Ha-CoV-2 reporter genome and structural proteins (M, S, N, and E) are cotransfected in HEK293T cells. Particles were harvested 48 h post-cotransfection (the imaging of virion particle and HEK293T cells were created with Biorender.com). (B) Quantification of the infectivity of Ha-CoV-2 variants. The relative infectivity of the two variants (Delta and Omicron) is quantified and normalized using genomic RNA copies of individual Ha-CoV-2 (Luc) variants. Wild-type is the Ha-COV-2 (Wt), which is used as a control for comparison. Infection and luciferase assays were performed 3x. RU, relative unit. The mean and standard deviation (SD) are shown. <a href="https://www.jove.com/files/ftp_upload/65793/65793fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65793/65793fig02.jpg" /> 
Figure 2. Quantification of 27BV neutralization activity against Ha-CoV-2(Luc) variants Neutralization activity of 27BV was analyzed 18 h post infection of HEK293T(ACE2/TMPRSS2) cells. The ID50 was calculated using relative infection rate (luciferase activity) versus 27BV concentration. <a href="https://www.jove.com/files/ftp_upload/65793/65793fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65793/65793fig03.jpg" /> 
Figure 3. Infection of HEK293T(ACE2/TMPRSS2) with Ha-CoV-2(GFP). Ha-CoV-2(GFP) particles were assembled and then used to infect HEK293T(ACE2/TMPRSS2) cells. GFP expression was observed at 48 h post-infection using fluorescent microscopy. The white field of infected cells is shown on the left, and GFP imaging is shown on the right. The white bar represents 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65793/65793fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1. Graphical Abstract. The Ha-CoV-2 pseudovirus structure and application. Image created with Biorender.com. <a href="https://www.jove.com/files/ftp_upload/65793/Graphical Abstract_RE.pdf" target="_blank">Please click here to download this File.</a>
Supplementary File 1. Protein Sequences. List of the sequences of SARS-CoV-2 S, M, N, and E proteins. The S protein sequences also include the SARS-CoV-2 variants Omicron (B.1.1.529) and Delta (B.1.617.2). <a href="https://www.jove.com/files/ftp_upload/65793/Protein Sequences_joVE_RE.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies at JoVE.com

Author Spotlight: A Pseudotype Virus System for Assessing Omicron Subvariants and Neutralizing Antibodies in SARS-CoV-2 Research

This protocol describes the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) as a platform for rapid quantification of infectivity of SARS-CoV-2 variants and their sensitivity to neutralizing antibodies.

Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 ...

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JoVE Journal

Immunology and Infection

1.1K Views.  George Mason University. This protocol describes the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) as a platform for rapid quantification of infectivity of SARS-CoV-2 variants and their sensitivity to neutralizing antibodies.

Video: Author Spotlight: A Pseudotype Virus System for Assessing Omicron Subvariants and Neutralizing Antibodies in SARS-CoV-2 Research

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can result in disease. Understanding the underlying molecular mechanisms surrounding cell membrane repair is, therefore, an important objective to the development of novel therapeutic strategies for diseases associated with dysfunctional cell membrane dynamics. Many in vitro and in vivo studies aimed at understanding cell membrane resealing in various disease contexts utilize two-photon laser ablation as a standard for determining functional outcomes following experimental treatments. In this assay, cell membranes are subjected to wounding with a two-photon laser, which causes the cell membrane to rupture and fluorescent dye to infiltrate the cell. The intensity of fluorescence within the cell can then be monitored to quantify the cell's ability to reseal itself. There are several alternative methods for assessing cell membrane response to injury, as well as great variation in the two-photon laser wounding approach itself, therefore, a single, unified model of cell wounding would beneficially serve to decrease the variation between these methodologies. In this article, we outline a simple two-photon laser wounding protocol for assessing cell membrane repair in vitro in both healthy and dysferlinopathy patient fibroblast cells transfected with or without a full-length dysferlin plasmid.

Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

Assessing Serum Neutralization Against SARS-CoV-2 with Pseudotyped Viruses

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses.
Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 &#176;C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.

Author Spotlight: Studying Host-Virus Interactions with Pseudotyped Viruses 

Pseudotyped viruses (PVs) are replication-defective virions that are used to study host-virus interactions under safer conditions than handling authentic viruses. Presented here is a detailed protocol that shows how SARS-CoV-2 PVs can be used to test the neutralizing ability of patients' serum after COVID-19 vaccination.

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...

Assessing Functional SARS-CoV-2 Neutralizing Antibodies

This video showcases a neutralization assay measuring serum efficacy in neutralizing SARS-CoV-2 viruses. It involves pseudotyped viruses, serum dilution, and luminescence-based quantification, revealing the neutralization titer.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Pseudotype-based neutralization ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Assessing Functional Neutralizing Antibody Responses Against a Panel of SARS-CoV-2 Variants of Concern Using Standardized, Quantitative Pseudotype Neutralization Assays

To begin, add 50 microliters of DMEM with 10 percent FBS and 1 percent Penicillin-Streptomycin to rows B to H, columns 1 to 12 of a white 96-well plate.
Add 5 microliters of convalescent sera into wells of row A, columns 2 to 10, in a total volume of 100 microliters DMEM with 10 percent FBS and 1 percent Penicillin-Streptomycin. Add 5 microliters of positive and negative antisera into wells A11 and A12 as controls.
Remove 50 microliters of the solution from the wells of row A and perform two-fold serial dilutions of all the wells beneath it. For each dilution, use a multichannel pipette to mix the solutions 5-10 times by pipetting up and down without creating air bubbles.
After completing the serial dilution, discard the final 50 microliters from the last well of each column.
Centrifuge the plate for 1 minute at 500 rotations per minute to ensure no liquid is left on the walls of the well. Calculate the amount of DMEM required to dilute the SARS-CoV-2 PV as described in the text manuscript.
Mix the diluted PV solution in a pipette basin and aliquot 50 microliters into each well, except for wells A6 to A12 which comprise the cell-only control. The A1 to A6 wells serve as PV-only control.
Centrifuge plate for 1 minute at 500 rotations per minute to ensure no virus is left on the walls of the well. Then, incubate the plates at 37 degrees Celsius with 5 percent carbon dioxide for 1 hour to allow the serum to bind with the SARS-CoV-2 glycoprotein.
Next, prepare a plate of HEK 293T/17 target cells transfected 24 hours earlier with ACE2 and TMPRSS2 plasmids. Remove the culture media from the plate, and wash it twice with 2 milliliters of PBS, by adding it to one side of the dish to avoid cell detachment.
Discard the PBS and add 2 milliliters of trypsin to the plate. Place the plate in an incubator until the cells are detached.
After cells have detached, add 6 milliliters of DMEM with 10 percent FBS and 1 percent Penicillin-Streptomycin to the plate to quench trypsin activity, and resuspend cells gently.
Count the cells using an automated cell counter or by counting the chamber slide. Add 1 x 104 cells aliquoted in a total volume of 50 microliters to each well and incubate the plate at 37 degrees Celsius with 5 percent carbon dioxide for 48 to 72 hours.
After incubation, remove the culture media from all wells, and add 25 microliters of a 1:1 mix of PBS and a commercial luciferase assay reagent. Read the plate using a commercial luciferase assay system with a microplate luminometer.
Using the raw data generated by the luminometer, calculate the IC50 neutralizing antibody titers as described in the text manuscript.

Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies

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Chapters in this video

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Generation of Monoclonal Antibodies Against Natural Products

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Assessing Functional SARS-CoV-2 Neutralizing Antibodies

Assessing Functional Neutralizing Antibody Responses Against a Panel of SARS-CoV-2 Variants of Concern Using Standardized, Quantitative Pseudotype Neutralization Assays