DISCUSSION
Viral epidemics can easily spread around the worldas recently witnessed for COVID-19 epidemic. Efficient viral tools are necessary for developing vaccines or therapeutic drugs; however, handling infectious SARS-CoV-2 requires biosafety level-3 (BSL-3) facilities, which limits the research and development of therapeutic approaches. Therefore, developing safer and easier viral assays for SARS-CoV-2 became crucial to grapple with the viral epidemic in a widespread manner. The pseudovirus system is a promising approach to evaluate viral inhibitors and neutralizing antibodies against SARS-CoV-2, in convalescent plasma (Chen and Zhang, 2021). Although, both Lentivirus and Vesicular Stomatitis Virus (VSV) are commonly used as pseudovirus systems, the pseudotyped virus titer obtained in the VSV-ΔG system is generally higher than in the retrovirus systems (Li et al., 2018).
VSV is a glycoprotein (G)-enveloped negative-stranded RNA virus that infects a wide range of animals and rarely humans causing mild symptoms of flu (Rodriguez, 2002; Tani et al., 2011). VSV can effectively integrate and express irrelevant transmembrane proteins onto the surfaces of recombinant virus particles. In addition, their easily modifiable small genome (11 kb) and abundant replication in a wide range of cell lines have favored the use of VSV in pseudovirus systems (Almahboub et al., 2020). On the other hand, VSV pseudovirus system might not reflect the density and distribution of Spike protein, because viral surface geometry of the authentic spherical SARS-CoV-2 virus is different from bullet shape VSV (Chen and Zhang, 2021).
Besides, there may be residual VSV-ΔG-G* virus interfere with the pseudovirus due to its packaging process in the generation of pseudovirus. Parental VSV-G may cause additional infection apart from VSV-ΔG-Spike pseudovirus and lead to false‐positive results (Li et al., 2018). To solve this issue, serial passaging during pseudovirus generation and antibodies against G protein could be utilized (Condor Capcha et al., 2021; Yahalom-Ronen et al., 2020). Yahalom-Ronen et al. showed that at least 5 passages of recombinant VSV-ΔG-Spike virus ensure elimination of residual VSV-G. They also reported that sequential passaging of pseudotyped virus result in increased prevalence of Spike protein structures per single particle (Yahalom-Ronen et al., 2020). Therefore, in wet-laboratory processes, we applied 6 serial passaging of VSV-ΔG-Spike pseudovirus with Spike protein in Vero E6 cells to remove virions bearing G protein.
In advance of pseudovirus neutralization assay, we showed SARS-CoV-2 Spike based pseudovirus entry by using HCQ, a blocker for SARS-CoV-2 (Yuan et al., 2022). Increasing concentration of HCQ led to a decrease in GFP signal in Vero E6 cells. GFP fluorescence signal was utilized as a measurement of infectivity encoded by the main VSV plasmid. GFP signaling is a rapid and inexpensive tool, allowing to follow by live imaging systems and flow cytometry. There are many alternative reporters described in literature for the pseudovirus system such as Red Fluorescence Protein (RFP/dsRed), firefly Luciferase (fLuc), and secreted embryonic alkaline phosphatase (SEAP). Although both fLuc and SEAP require additional steps and reagents, fLuc is the most preferred reporter for both VSV and LV pseudovirus generation in literature (Donofrio et al., 2021; Nie et al., 2020). Several studies demonstrated slower kinetics and sensitivity for fluorescent proteins (GFP or DsRed) when compared to Firefly luciferases (Neefjes et al., 2021). However, GFP reporter provides robust expression in the cell and ease to follow both generation and infectivity processes. We used the intensity of GFP fluorescence to determine VSV titer and inhibition. On the other hand, it might lead to more accurate results, if both GFP and fLuc reporter encoding plasmid backbone are used for pseudovirus generation (Crawford et al., 2020).
The infection model consisting of Vero E6 cells and VSV-ΔG-Spike can mimic the entry of authentic SARS-CoV-2. The serum neutralizing titer of vaccinated convalescent patients measured by the VSV-ΔG-Spike pseudovirus assay has a good correlation with that measured by the authentic SARS-CoV-2 PRA assay. In addition, the pseudovirus assay is safer and time-efficient than the wild type SARS-CoV-2 PRA assay. More importantly, this method can be used to determine the neutralization titer of serum against the SARS-CoV-2 variants. In our pseudovirus study, Alpha and Beta variants of Spike protein were constructed and tested against human convalescent plasma samples listed in Table 1 . It is shown that the pseudovirus assay based on the VSV-ΔG system was successful in discrimination of different SARS-CoV-2 variants. In consistence with literature, pseudovirus neutralization assay exaggerated the potency of samples with low neutrality activity, but increased the inhibition limit, enabling the identification of samples with high neutrality potency (von Rhein et al., 2021). Each vaccinated human convalescent serum sample was assayed for neutralization against Alpha (B.1.1.7), Beta (B.1.351), and Wuhan (Wild type) viruses. The pseudovirus neutralization assay showed no loss of neutralizing activity against Spike Alphavariant, whereas every sample lost activity against Spike Betavariant. Overall, the neutralizing activity against Alpha Spike pseudotyped virus was essentially unchanged, but significantly lower against Beta Spike pseudotyped virus. The main contributing factor is suggested to be E484K mutation in Beta variant which provides neutralization resistance of the virus. Studies indicate that this mutation in receptor binding motif is in an immunodominant epitope of Spike protein (Garcia-Beltran et al., 2021; Wang et al., 2021).
When Sinovac and BioNTech-Pfizer mRNA vaccine BNT162b2 were compared, BioNTech-vaccinated plasma samples were highly neutralizing according to PRA and pseudovirus neutralization assay. In consistent with literature, the human plasma had slightly reduced but overall, largely preserved neutralizing titers against the Alpha variant pseudovirus (Muik et al., 2021). The largely preserved neutralization of pseudovirus bearing the Alpha Spike by BNT162b2-immune plasma makes it unlikely that the Beta variant virus will escape BNT162b2-mediated protection. However, pseudovirus neutralization assay showed a substantial drop-in neutralization activity against theBeta variant, consistent with conclusions being reached by others (Hoffmann et al., 2021; Wang et al., 2021).
Even though pseudoviruses only contain the envelope proteins of the authentic virus, pseudovirus‐based assays have demonstrated a good correlation with the infectious virusbased assay. Besides, the pseudovirusbased neutralization assays are usually highhroughput procedures requiring less amounts of serum samples (Sanders, 2002). Our pseudovirus neutralization assay can be used to evaluate the neutralization potency of vaccines or antibodies. Taken together, this convenient and reliable pseudovirus system based on a VSV packaging system could be greatly beneficial for developing SARS-CoV-2 vaccines and therapeutic drugs as well as for testing the inhibition potency of convalescent plasma samples.