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.
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.
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 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.
1. Virus and viral particle assembly
2. Viral infectivity assay
3. RNA extraction of Ha-CoV-2 and quantitative reverse transcriptase PCR (RT-qPCR)
4. Neutralizing antibody assay
5. Quantification and statistical analysis
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 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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 µm. Please click here to view a larger version of this figure.
Supplementary Figure 1. Graphical Abstract. The Ha-CoV-2 pseudovirus structure and application. Image created with Biorender.com. Please click here to download this File.
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). Please click here to download this File.
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 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.
The authors have nothing to disclose.
This work was supported by George Mason University internal research fund.
27VB1 20 µg SARS-CoV-2 Standard Neutralizing Antibody | Virongy Biosciences | 27VBI-01 | |
500 mL – US Origin FBS | Neuromics | FBS001 | |
AB Mixing Plate: Olympus 96-Well PCR Plate, Non-Skirted UltraThin Wall, Natural, 25 Plates/Unit | Genesee Scientific | Cat# 24-300 | |
Allegra 6R Centrifuge | Beckman Coulter | 2043-30-1158 | |
DMEM (1x) | ThermoFisher | 11995-073 | |
GenClone 25-209, TC Treated Flasks, 250ml, Vent Growth Area: 75.0cm2, 5 per Sleeve, 100 Flasks/Unit | Genesee Scientfic | 25-209 | |
GlowMax Discover Microplate reader | Promega | GM3000 | |
Ha-CoV-2 E Vector | Virongy Biosciences | pCoV2_E | |
Ha-CoV-2 M Vector | Virongy Biosciences | pCoV2_M | |
Ha-CoV-2 N Vector | Virongy Biosciences | pCoV2_N | |
Ha-CoV-2 WT S Vector | Virongy Biosciences | pCoV2_WT S | |
Hek293T cells | ATCC | CRL-3214 | |
Illumination Firefly Luciferase Enhanced Assay Kit 1000 assays | Gold Bio | I-930-1000 | |
Infection Plate: 96-Well Tissue Culture Plate, Greiner Bio-One (With Lid, μClear White Flat Round, Chimney) | VWR | Cat# 82050-758 | |
pAlphaPro-Luc-GFP-PreΨ (Ha-CoV-2 Genome) Vector | In house | ||
PEI-based Transfection Reagent | Virongy Biosciences | Transfectin | |
Penicillin-Streptomycin-Glutamine (100X) | Invitrogen | 10378016 | |
Polyethylenimine, branched | Millipore Sigma | 408727-100ML | |
QuantStudio 7 Pro Real-Time PCR System | ThermoFisher | A43163 | |
Ready to use (HEK293T)(ACE2/TMPRSS2) Cells | Virongy Biosciences | Ready-To-Use-Cells | |
SARS-CoV-2 S Omicron (B.1.1.529) Vector | Virongy Biosciences | pCoV2-B.1.1.529 | |
SARS-CoV-2 S Delta (B.1.617.2) Vector | Virongy Biosciences | pCoV2- B.1.617.2 | |
Syringe Filters, PES, 0.22µm | Genesee Scientfic | 25-244 | |
TaqMan Fast Virus 1-Step Master Mix | ThermoFisher | 4444432 | |
Trypan Blue Solution, 0.4% | ThermoFisher | 15250061 |