Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies
September 8th, 2023
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.
We have established two main findings in this protocol. First, our Ha-CoV-2 platform demonstrates that the newly emerged Omicron subvariant is more infectious than the original Wuhan strain. Additionally, Omicron subvariants are more resistant to neutralizing antibodies induced by previous strains.
SARS-CoV-2 research requires BSL-3 facilities, making it difficult for common laboratories to monitor SARS-CoV-2 variants and to quantify neutralizing antibodies. Our Ha-CoV-2 system is a pseudo-type virus that can be used in common laboratories and provides a faster way to quantify viral variants and their sensitivity to neutralizing antibodies. Ha-CoV-2 is a virus-like particle that has all the structural proteins of SARS-CoV-2 and resembles SARS-CoV-2.
Also, Ha-CoV-2 uses and alpha viral vector for robust and rapid gene expression, producing fast results and a high signal-to-noise ratio for great reproducibility. Begin by maintaining HEK293T cells and DMEM containing heat inactivated FBS, Penicillin, and Streptomycin in a T75 flask. For cell counting, mix 20 microliters of cell suspension with 20 microliters of Trypan Blue solution.
Add 20 microliters of dye-mixed cell suspension to the cell counting chamber and count the number of cells per milliliter. Seed HEK293T cells in 10 milliliters of complete DMEM in a 10-centimeter cell culture Petri dish. Incubate the cells overnight in a carbon dioxide incubator at 37 degrees Celsius.
The next day, remove the complete medium, and replace it with nine milliliters of DMEM serum-free medium. Using the given compounds of viral particle assembly, prepare a co-transfection mixture. Slowly add the mixture to the Petri dish, and incubate at 37 degrees Celsius for six hours.
After incubation, replace the serum-free DMEM with a complete DMEM medium, and continue incubation for co-transfection for up to 48 hours. Then detach the cells by repeatedly pipetting over the surface of the monolayer, and transfer the suspension into a 15-milliliter tube. Centrifuge the cell suspension at 400 g for five minutes.
Collect the supernatant and pass it through a 0.22 micron filter. Store the filtrate containing Ha-CoV-2 pseudo virus at 80 degrees Celsius. Begin by maintaining HEK293T ACE2/TMPRSS2 cells in DMEM containing heated activated FBS, Penicillin, and Streptomycin in a T75 flask.
For cell counting, mix 20 microliters of cell suspension with 20 microliters of Trypan Blue solution. Add 20 microliters of dye-mixed cell suspension to the cell counting chamber, and count the number of cells per milliliter. Seed 2.5 x 10 to the power of 4 HEK293T ACE2/TMPRSS2 cells in 50 microliters of complete DMEM medium into each well of a 96-well plate.
Incubate the cells in a carbon dioxide incubator overnight at 37 degrees Celsius. The next day, replace the 50 microliters of DMEM with 50 microliters of virus suspension, and incubate for 18 hours at 37 degrees Celsius. After incubation, add 7.5 microliters of cell lysis buffer to each well and mix on an orbital shaker for two minutes.
Once the cells are lysed, add freshly prepared Firefly Luciferase Assay Solution, and mix them on an orbital shaker for one minute. Analyze the luciferase activity by using a commercial luciferase microplate reader. For neutralizing antibody assay, seed HEK293T cells in 50 microliters of complete DMEM, as demonstrated previously.
In a 96-well plate, add eight microliters of 27 BV neutralizing antibody, and perform serial dilutions with six microliters of serum-free DMEM. Add 54 microliters of Ha-CoV2 virus particle to the serially diluted antibody, and mix the suspension. Incubate for one hour at 37 degrees Celsius and 5%carbon dioxide.
Then add 50 microliters of virus suspension to the wells containing HEK293T cells. For controls, add 50 microliters of complete DMEM medium into the wells containing only HEK293T cells. Place the plate in an incubator at 37 degrees Celsius for 18 hours.
After lysing the cells, as demonstrated previously, add freshly prepared Firefly Luciferase Assay Solution and mix by orbital shaking for one minute. Analyze the luciferase activity by using a commercial luciferase microplate reader Ha-CoV2 Omicron and Omicron variants generated four to tenfold higher signal than the Wild Type Ha-CoV2, suggesting higher infectivity. The antibody 27 BV demonstrated neutralizing activity against both Delta and Omicron variants.
The ID50 of 27 BV for Omicron was approximately 10 times less potent than the ID50 for Wild Type and Delta.
Overview
This protocol describes the use of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) for rapid quantification of SARS-CoV-2 variant infectivity and neutralizing antibody sensitivity. The Ha-CoV-2 platform allows for effective monitoring of variants in common laboratory settings.
Key Study Components
Area of Science
- Virology
- Immunology
- Infectious Diseases
Background
- Research on SARS-CoV-2 requires BSL-3 facilities, limiting accessibility.
- Omicron subvariants show increased infectivity and resistance to neutralizing antibodies.
- Ha-CoV-2 mimics SARS-CoV-2, facilitating safer research.
- The platform uses an alpha viral vector for efficient gene expression.
Purpose of Study
- To develop a safer method for quantifying SARS-CoV-2 variants.
- To assess the infectivity of emerging variants like Omicron.
- To evaluate the sensitivity of variants to neutralizing antibodies.
Methods Used
- Maintenance and counting of HEK293T cells.
- Co-transfection to produce Ha-CoV-2 pseudovirus.
- Luciferase assays to measure viral infectivity and neutralization.
- Serial dilution of neutralizing antibodies for efficacy testing.
Main Results
- Omicron subvariants exhibited higher infectivity compared to the original Wuhan strain.
- Neutralizing antibody 27 BV showed reduced potency against Omicron.
- Ha-CoV-2 provided a reliable platform for quantifying viral variants.
- Results indicated a significant difference in neutralization efficacy between variants.
Conclusions
- The Ha-CoV-2 platform is a valuable tool for SARS-CoV-2 research.
- It enables safer and more accessible studies on viral infectivity and antibody response.
- Findings highlight the need for ongoing monitoring of variant resistance.
Frequently Asked Questions
Executive Industry Relevance
Rapid quantification of SARS-CoV-2 variant infectivity and neutralizing antibody potency is critical for pandemic response, vaccine validation, and surveillance of emerging threats. The Ha-CoV-2 pseudovirus platform enables high-throughput, reproducible assessment of variant biology and immune escape in standard laboratory settings. This capability supports portfolio-level decisions on vaccine updates and therapeutic prioritization.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables functional interrogation of variant-specific viral entry and immune evasion mechanisms.
- Supports biological de-risking by quantifying infectivity differences among SARS-CoV-2 variants.
- Facilitates predictive confidence in target selection for vaccine and antibody development.
Screening & Assay Development
- Provides a validated, scalable system for rapid screening of neutralizing antibodies against multiple variants.
- Delivers quantitative, reproducible luciferase and GFP readouts for assay standardization.
- Enables high-throughput evaluation of compound or antibody efficacy in a BSL-2 environment.
Translational & Preclinical Research
- Aligns in vitro infectivity and neutralization data with translational biomarker strategies for variant monitoring.
- Supports continuity from discovery through preclinical validation of vaccine and therapeutic candidates.
- Reduces risk of late-stage failure by providing early, quantitative insights into variant resistance profiles.
Pipeline & Workflow Integration
The Ha-CoV-2 pseudovirus assay integrates from early discovery through lead identification and preclinical evaluation, enabling rapid hypothesis testing and portfolio triage.
- Discovery Biology: Quantifies variant infectivity and antibody neutralization to clarify mechanistic risk.
- Screening: Offers robust, reproducible outputs for compound and antibody screening workflows.
- Analytics: Provides quantitative luciferase and GFP measurements for direct comparison of variant and antibody performance.
- Translational Research: Bridges in vitro findings to preclinical decision-making for variant-specific interventions.
- Enterprise Reuse: Functions as a reusable platform for ongoing surveillance and rapid response to emerging variants.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in variant infectivity and immune escape assessment.
- Operational Value: Enables standardized, scalable, and rapid testing in BSL-2 laboratories.
- Strategic Value: Informs go/no-go decisions for vaccine and antibody candidate advancement.
- Portfolio Impact: Supports risk-adjusted prioritization of variant-targeted interventions.
Implementation Considerations
- Requires expertise in cell culture, viral pseudotyping, and quantitative assay analysis.
- Needs access to luciferase microplate readers and standard tissue culture infrastructure.
- Demands cross-team standardization of assay protocols for reproducibility.
- Adaptable to new SARS-CoV-2 variants by updating spike protein constructs.
- Dependent on validated controls and reference antibodies for benchmarking.
