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.