\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[T2A]{fontenc}
\usepackage[polish,ngerman,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{Identification of SARS-CoV-2 Neutralizing Potency of Human Convalescent
Plasma Using Pseudovirus Neutralization Assay}



\author[1]{Yeşim Tok}%
\author[2]{Gizem Çelebi Torabfam}%
\author[2]{Atike Nur Çimen}%
\author[3]{Gülen Esken}%
\author[1]{EBRU YÜCEBAĞ}%
\author[1]{Neşe Arslan}%
\author[1]{Devrim Saribal}%
\author[3]{Özlem Doğan}%
\author[1]{Mert Ahmet Kuskucu}%
\author[1]{Bilgül Mete}%
\author[1]{Gökhan Aygün}%
\author[1]{Fehmi Tabak}%
\author[3]{Füsun Can}%
\author[3]{Önder Ergönül}%
\author[2]{Özlem Kutlu}%
\author[2]{Sibel Çetinel}%
\author[1]{Kenan Midilli}%
\affil[1]{Istanbul University-Cerrahpaşa}%
\affil[2]{Sabanci Holding}%
\affil[3]{Koc Holding AS}%


\vspace{-1em}



  
  \date{June 14, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Convalescent plasma samples that can be collected from individuals after
the resolution of infection and vaccination are an invaluable source of
neutralization antibodies against the virus. Although plaque reduction
assay with replicative virus is the gold standard of analyzing
neutralization potency of convalescent plasma, it is a technically
demanding procedure requiring high biosafety level (BSL-3/4) laboratory
and equipment. The abundance of neutralizing antibodies varies among
individuals, therefore fast and reliable methods to identify
neutralization potency of plasma samples are needed. In this paper,
G-protein deficient vesicular stomatitis virus (VSV-\selectlanguage{greek}Δ\selectlanguage{english}G) carrying a
C-terminal 21 amino acid truncated severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) Spike protein was generated for pseudovirus
neutralization assay. We analyzed SARS-CoV-2 neutralizing potency of
vaccinated human convalescent plasma samples (n=13) and plasma samples
of healthy people (n=2). Human convalescent plasma samples were examined
against the ancestral Wuhan strain and two SARS-CoV-2 variants (B.1.1.7,
and B.1.351) using a VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 pseudovirus and Vero E6 cell line.
Neutralization values against pseudotyped virus were compared to those
of plaque reduction assay against authentic virus. The serum
neutralizing titer of convalescent plasma measured by pseudovirus assay
has a good correlation with that measured by plaque reduction assay (R
\textsuperscript{2}= 0.7). The pseudovirus assay is safer and timesaving
than the replicative virus-based plaque reduction assay, and has several
advantages in evaluating a new vaccine, newly emergent variants, and
approved vaccine efficacy against variants of concern as well as in
viral fusion-focused treatment analysis that can be performed under
BSL-2 conditions.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Serum neutralization corresponds protection against viral infection
after vaccination or natural infection. However, high levels of mutation
observed in viral glycoprotein of different variants could prevent
effective protection provided by neutralization serum. Therefore,
immediate determination of serum neutralization potency against
different variants is crucial (Wu et al., 2020). Particularly, new
variants have begun to emerge for disease caused by global infection
with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since
2019. It has been reported that mutations on the major neutralization
target of these variants alter serum-neutralizing activity induced by
early strains or vaccines (Kuzmina et al., 2021; Robbiani et al., 2020).
Some of these mutations can provide an escape from immune response by
decreasing monoclonal antibody (mAb) neutralization (Liu et al., 2021).

Due to the high risk of SARS-CoV-2 infection, cultivation of authentic
viruses requires laboratories with at least level 3 biological safety
(BSL3) equipped with negative pressure systems. This limitation prevents
to perform neutralization assays in many research centers, which only
has BSL2 laboratories. Pseudotyped virus neutralization assay is an
alternative method based on packaging a reliable replication-defective
pseudovirus with Spike protein to mimic the entry of the authentic
virus. Thereby, pseudoviruses can be contained in BSL2 conditions to
perform neutralization assays (Chen and Zhang, 2021; Li et al., 2018).
In the broad extent, pseudoviruses can be utilized in
seroepidemiological studies, vaccine and monoclonal antibody
development, screening of viral entry inhibitors, and fundamental
virological methods (Ou et al., 2020). Several convenient pseudotyped
virus for SARS-CoV-2 have been reported, such as human immunodeficiency
virus (HIV)-based lentiviral particles (Donofrio et al., 2021), murine
leukemia virus (MLV)-based retroviral particles (Chen and Zhang, 2021),
or vesicular stomatitis virus (VSV)-based systems (Nie et al., 2020;
Salazar-Garc\selectlanguage{ngerman}ía et al., 2022) with higher performance for evaluating the
efficacy of therapeutic drugs and vaccines (Salazar-García et al.,
2022). The results of such pseudovirus neutralization tests are very
closely correlated to those of authentic virus measurements. Also,
tracking the changes in Spike glycoprotein using pseudovirus
neutralizing assays is relatively easy (Schmidt et al., 2020).

From the beginning of the pandemic, DNA sequencing data of virus
glycoproteins is utilized to identify individual mutations in SARS-CoV-2
(Schrörs et al., 2021). One of the first variants called B.1.1.7 was
first emerged in the United Kingdom and had multiple mutations in the
target regions of neutralizing antibodies such as receptor binding
domain and N-terminal domain of Spike. SubsequentlyB.1.351 variant has
been identified in South Africa with additional mutations. B.1.1.7 and
B.1.351 variants share key mutations in the RBD (N501Y, D614G), but
B.1.351 has additional changes causing widespread escape from mAbs
(E484K, K417N) (Harvey et al., 2021; Zhou et al., 2021).

In this paper, we evaluated human convalescent plasma with different
serum neutralizing activities using pseudotyped VSV-\selectlanguage{greek}Δ\selectlanguage{english}G virus carrying
Spike variants (\emph{Wuhan} strain, B.1.1.7, and B.1.351). First, we
made a series of point mutations in Spike sequence of SARS-CoV-2 to
obtain global variants. Second, we generated pseudotyped viruses
usingSpike of ancestoral Wuhan strain and two variants and utilized to
evaluate neutralization activity of human serum samples. Neutralization
assay using Spike pseudotyped VSV-\selectlanguage{greek}Δ\selectlanguage{english}G virus was found to correlate with
plaque assay using SARS-CoV-2 authentic virus. Additionally, Spike
pseudotyped VSV-\selectlanguage{greek}Δ\selectlanguage{english}G virus was sufficient to discriminate serum responses
against different variants of the virus.

\textbf{Materials and Methods}

\textbf{Plasmid construction}

The expression vector for SARS-CoV-2 Spike
(pTWIST-EF1-alpha-SARS-CoV-2-S-2xStrep vector) was kindly provided by
Dr. Nevan Krogan. Spike gene was codon optimized to improve the
expression efficiency in mammalian cells (Genbank: MN985325). A pMD2.G
lentiviral plasmid containing Spike gene was generated. Briefly, forward
primer 5'-ATAGAATTCGCCGCCACCATG-3' and reverse primer
5'-ATAGAATTCTCATCAACTACCGCAAGAACAACAACC-3' were used to amplify mutant
Spike protein with 21 amino acid deletion in C-terminal. To clone Spike
gene successfully, TA cloning was applied. In the first step, PCR
reaction was set up to add adenine bases to 3'OH terminal of Spike gene.
The amplified product was ligated into pGEM-T easy vector system. In the
second step, Spike gene was restricted by \emph{EcoRI} and ligated into
pMD2.G vector. Spike gene with C-terminal 21 amino acid deletion
(pmD2.SpikeCdel21) was confirmed with Sanger DNA sequencing.

B.1.1.7 (\emph{Alpha} ) variant including \selectlanguage{greek}Δ\selectlanguage{english}H69-V70, N501Y, D614G, \selectlanguage{greek}Δ\selectlanguage{english}Y144
mutations and B.1.351 (\emph{Beta} ) variant including E484K, N501Y,
D614G, K417N mutations of SARS-CoV-2 were constructed in
pmD2.SpikeCdel21 plasmid by using site-directed mutagenesis. PCR
conditions were set up by following procedures of Phusion High-Fidelity
Polymerase (Thermo Sci., F530L) and Quick-change Site-directed
Mutagenesis Kit (Startagene, Agilent, 200518). Next, the template was
digested with \emph{DpnI} to eliminate parental DNA. The constructed
vector was transferred into \emph{E. coli} \emph{XL-1 Blue} competent
cells. Following the picking up colonies, plasmids were sequenced, and
mutations were confirmed. Sequences of mutation primers and sequences of
plasmid constructs are given in \textbf{Supporting Information File.}

\textbf{Cell culture}

HEK (Human Embryonic Kidney) 293T cells (ATCC, CRL-321) and Vero E6
(Monkey African Green Kidney) (ATCC, CRL-1586) cells were cultured in
Dulbecco's modified Eagle medium (DMEM, PAN Biotech, P04-3500)
supplemented with 10\% fetal bovine serum (PAN Biotech, FBS standard,
P30-3306) and \%1 L-glutamine (200 mM; PAN Biotech, P04-80100 ) and 1\%
penicillin/streptomycin (Pan Biotech, P06-07100) at 5\% (v/v)
CO\textsubscript{2}, 37 \selectlanguage{ngerman}°C incubator.

\textbf{Plasma samples}

Human convalescent plasma samples were obtained from volunteer donors
after 2 months post-vaccination \textbf{(Table 1)} . Plasma samples were
heat-inactivated at 56\textsuperscript{o}C for 30 minutes before using
for neutralization assays. Sinovac and BioNTech-Pfizer mRNA vaccine
BNT162b2 were used for vaccination in Turkey.

\textbf{Table 1.} Vaccination status of human plasma samples used for
neutralization assays\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Plasma samples & Vaccination\tabularnewline
\midrule
\endhead
1 & Sinovac, 2 dose\tabularnewline
2 & Sinovac, 2 dose\tabularnewline
3 & Sinovac, 2 dose\tabularnewline
4 & Sinovac, 2 dose; BioNTech, 1 dose\tabularnewline
5 & Sinovac, 2 dose; BioNTech, 1 dose\tabularnewline
6 & Sinovac, 2 dose; BioNTech, 1 dose\tabularnewline
7 & Sinovac, 2 dose\tabularnewline
8 & Sinovac, 2 dose; BioNTech, 1 dose\tabularnewline
9 & BioNTech, 2 dose\tabularnewline
10 & BioNTech, 2 dose\tabularnewline
11 & BioNTech, 2 dose\tabularnewline
12 & Sinovac, 2 dose\tabularnewline
13 & Sinovac, 2 dose\tabularnewline
14 & Healthy control\tabularnewline
15 & Healthy control\tabularnewline
\bottomrule
\end{longtable}

\textbf{Plaque reduction neutralization assay}

Serum samples (non-denatured) were diluted in DMEM from 1:10 to 1:320.
SARS-CoV-2 virus was diluted in DMEM to a final concentration of
10\textsuperscript{-3} pfu/ml. 300 \selectlanguage{greek}µ\selectlanguage{english}l serum and 300 \selectlanguage{greek}µ\selectlanguage{english}l diluted virus
were mixed and incubated in37 \selectlanguage{ngerman}°C incubator at 5\% (v/v)
CO\textsubscript{2} for 1 hr. Then, the serum-virus mixture was added to
Vero E6 cells and incubated in, 37 °C incubator at 5\% (v/v)
CO\textsubscript{2} for 1 hr. Serum-virus mixture was discarded from the
plate and the plate was covered with 2\% methylcellulose and DMEM at 1:2
ratio and incubated for 4 days. Methylcellulose was discarded and cells
are washed with 1xPBS. Then, 4\% PFA was used for cell fixation at room
temperature for 20 minutes. After fixation, cells were stained with
crystal violet solution for 30 minutes at room temperature. Plaques were
counted after washing with PBS to remove the excessive stain. The number
of plaques were used to calculate virus neutralization titers.

\textbf{Generation of SARS-CoV-2 pseudotyped variants}

rVSV-\selectlanguage{greek}Δ\selectlanguage{english}G-G* (G*: VSV glycoprotein) and VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Spike (carrying Spike
protein of coronavirus instead of VSV glycoprotein) virus were produced
according to the protocol provided by Whitt in 2010 (Whitt, 2010).
BHK-21/WI-2 cells (Baby Hamster Kidney-21/ Clone WI-2, Kerafast, EH1011)
were infected with vaccinia virus (ATCC, strain vTF7-3,
VR-2153\textsuperscript{TM}) for 1 h, followed by co-transfection of
five plasmids: VSV-\selectlanguage{greek}Δ\selectlanguage{english}G, together with VSV accessory plasmids encoding for
VSV-N, P, L, and G proteins (Kerafast), all of which were under T7
promoter control. The primary transfection was performed using
Lipofectamine 2000. BHK-21/WI-2 cells were transfected with Spike
plasmid to assist in creating passage 1 (P1). Forty-eight hours
following primary transfection, supernatant containing the recovered
VSV-Spike was collected, centrifuged at 1300 \selectlanguage{ngerman}× g × 5 min to remove cell
debris. Total supernatant was collected, centrifuged, and used for
sequential passaging in Vero E6 cells to eliminate parental virus
(Yahalom-Ronen et al., 2020), as shown in \textbf{Figure 1} .
VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Spike was propagated in DMEM containing 5\% FBS (reduced serum
media), MEM NEAA, 2mM L-glutamine, and 1\% P/S.

\textbf{SARS-CoV-2 pseudovirus infection for viral entry and titration}

To determine viral titer, Vero E6 cells were seeded in a 24-well-plate
before the day of infection. 100 \selectlanguage{greek}µ\selectlanguage{english}l VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Spike pseudotyped virus was
diluted 3-fold serial dilutions up to 1:243 using DMEM 5\% FBS as
diluent. After 24 hours, GFP positive cells were counted in the last
wells to calculate the TU/ml.

For viral entry assay, medium was replaced with 200 \selectlanguage{greek}µ\selectlanguage{english}l cell culture
medium including Hydroxychloroquine (20-50 \selectlanguage{greek}µ\selectlanguage{english}M) as a SARS-CoV-2 blocker
for 20 minutes. Then, treated wells were inoculated with SARS-CoV-2
Spike protein pseudotyped VSV-\selectlanguage{greek}Δ\selectlanguage{english}G (VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21). 48 hours of post
infection, the plate was scanned with Axio Observer Fluorescence
Microscope (Zeiss).

\textbf{Immunocytochemistry (ICC)}

Indirect immunofluorescence analysis was performed for cellular Spike
protein and ACE-2 protein detection. Vero E6 cells which are infected
with Spike pseudotyped VSV-\selectlanguage{greek}Δ\selectlanguage{english}G virus were fixed with \%4 Paraformaldehyde
(PFA) and stained with anti-Spike antibody (SinoBiological 2019-nCoV
Spike S1 Rabbit mAb, 40150-R007) (1:80 dilution), then incubated with
1:1000 diluted Alexa Fluor 568-conjugated goat anti-rabbit IgG secondary
antibody. ACE-2 protein was detected with anti-hAce2 (R\&D Human Ace2,
AF933) (1:120 dilution), incubated with Alexa Fluor 568-conjugated
anti-goat IgG secondary antibody (1:500 dilution).

\textbf{Western Blot (WB)}

Western Blot analysis was performed to validate SARS-CoV-2 Spike protein
expression in HEK293T cells. Total protein was subjected to SDS-PAGE
followed by immunoblotting to nitrocellulose membranes (250 mA for 1.15
hours). Rabbit SARS-CoV-2 (2019-nCoV) Spike S1 monoclonal antibody
(40150-R007, SinoBiological) and a horseradish peroxidase-conjugated
secondary antibody (1/10,000) were used for detection. Antibodies were
used at 1/500 for Spike S1 subunit and at 1/10,000 for \selectlanguage{greek}β-\selectlanguage{english}actin.

\textbf{Pseudovirus based neutralization assay}

Serum samples were diluted in DMEM-5 (reduced serum media; 5\% FBS, 1\%
L-glutamine, 1x MEM NEAA) from 1:30 to 1:2430. SARS-CoV-2 Spike
pseudotyped VSV-\selectlanguage{greek}Δ\selectlanguage{english}G virus was diluted in DMEM-5 to a final concentration
of 10\textsuperscript{-4} TU/ml. 100 \selectlanguage{greek}µ\selectlanguage{english}l diluted serum and 50 \selectlanguage{greek}µ\selectlanguage{english}l diluted
virus were mixed in a 96 well plate and incubated in
37\textsuperscript{0}C incubator for 1 hr. Then, serum-virus mixture was
added to Vero E6 adherent cells (80\% confluent) and incubated in a 5\%
(v/v) CO\textsubscript{2}, 37 \selectlanguage{ngerman}°C incubator for 2 days. Cells were fixed
with 4\% PFA and analyzed by using Tecan plate reader (Infinite M200
Pro, Life Science). The GFP fluorescence intensity was microscopically
detected at 48 hours post-infection by Axio Observer Fluorescence
Microscope (Zeiss).

\textbf{Statistical Analysis}

Data were analyzed using GraphPad Prism 8 (GraphPad Software, La Jolla,
CA, USA) and expressed as mean ± standard deviation (SD). One-way
analysis of variance (ANOVA) was used to compare differences between
groups, and P values are indicated by asterisks (****p\textless{}
0.0001, **p \textless{} 0.01, and *p \textless{} 0.05).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} Generation of VSV pseudotyped viruses bearing
truncated SARS-CoV-2 Spike proteins. \textbf{(A)} Preparation of
rVSV-\selectlanguage{greek}Δ\selectlanguage{english}G-G* in BHK21/W1 cell line. \textbf{(B)} Packaging of
rVSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 in HEK293T cell line. \textbf{(C)} Infection of
pseudovirus carrying GFP reporter and sequential passaging of
pseudovirus to eliminate parental virus (rVSV-\selectlanguage{greek}Δ\selectlanguage{english}G-G*) using plasmids
encoding Sdel21 and T7 polymerase transfected Vero E6.
\textbf{(D)}SARS-CoV-2 Spike pseudovirus neutralization assay using
human convalescent plasma samples and GFP signal reading.

\textbf{RESULTS}

G-protein deficient VSV bearing C-terminal 21 aminoacid truncated Spike
protein were packaged in HEK293T cell line (\textbf{Figure 1} ). To
fully eliminate ancestral VSV-G, Spike pseudoviruses were serially
passaged in pmD2.SpikeCdel21 transfected Vero E6 cells.

Hydroxychloroquine (HCQ) effectively inhibits viral entry and has been
proposed for prophylaxis/treatment of SARS-CoV-2 infection. Therefore,
HCQ was used to display inhibition of pseudovirus entry and construction
of reliable pseudovirus assays. Harvested VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 pseudotyped
virus were analyzed for viral entry using HCQ (20-50 \selectlanguage{greek}µ\selectlanguage{english}M). Virus
infectivity was decreased with increasing concentration of
hydroxychloroquine based on percentage of GFP-positive
cell\textbf{(Figure 2A and 2B)} . The number of GFP-positive cells in
VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21-infected Vero E6 cells were significantly reduced from
30\% to 10\% in highest concentration of HCQ compared to positive
control (p\textless{}0.0001). Representative image of positive control
was given in Figure 2S.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2.} Viral entry assay in Vero E6 cell line.
\textbf{(A)}Vero-E6 cells were inoculated with VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 pseudotyped
virus. Cells were treated with hydroxychloroquine at 20 min before
inoculation. After 24h, GFP signal detected under fluorescence
microscope is shown in representative cellular images. \textbf{(B)} For
quantification, GFP signal from 25 images per sample was analyzed by
using Image J at 24h of post-inoculation. Values are presented as \% of
total population. Scale bar: 100\selectlanguage{greek}µ\selectlanguage{english}m. Data are represented as means of
\selectlanguage{ngerman}±S.D. of at least three independent experiments. *p \textless{} 0.05;
****p \textless{} 0.0001 - using One-Way ANOVA with Bonferroni's
post-hoc test.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3.} Colocalization of pseudovirus with hACE-2 receptor or
SARS-CoV-2 Spike protein in Vero E6 cell line. Cells were incubated with
anti-SARS-CoV-2 Spike, human anti-ACE2 and DAPI.
\textbf{(A--D)}Mock-treated VeroE6 cells with hACE2 staining.
(\textbf{E-H)}VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 infected Vero E6 cells with hACE2 staining.
\textbf{(I-L)}VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 infected Vero E6 cells with SARS-CoV-2 Spike
staining. Yellow color shows viral S protein (\textbf{J} ), and red
colors represents human ACE2 (\textbf{B, F} ). Scale bars, 20 \selectlanguage{greek}μ\selectlanguage{english}m. Data
in\textbf{A--L} are representative images of two independent
experiments.

To validate Spike and ACE2 protein expression of infected cells,
immunostaining was performed using VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 pseudotyped virus.
Meanwhile, colocalization of VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 and human ACE2 receptor was
investigated. \textbf{(Figure 3E-H)} . Co-localization was not found in
mock-treated hACE2 \textbf{(Figure 3A--D),} indicating that SARS-CoV-2
uses human ACE2 as a receptor for entry. In addition, colocalization of
VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 and Spike protein confirmed the existence of Spike as a
surface glycoprotein in our VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 pseudotyped particles

\textbf{(Figure 3I-L).}

The convalescent plasma samples were analyzed with VSV-\selectlanguage{greek}Δ\selectlanguage{english}G bearing
truncated SARS-CoV-2 Spike protein (VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21). Spike pseudotyped
viruses acquire the host spectrum of coronavirus and a rapid read out of
the results is likely by the transfer of the \emph{GFP} gene as a
measure of infection. Administration of neutralizing antibodies against
Spike pseudotyped VSV-\selectlanguage{greek}Δ\selectlanguage{english}G virus reduce the number of GFP-positive cells.
Neutralization activity of plasma was calculated based on GFP signal.
Neutralization activities of plasma samples were depicted in

\textbf{Figure 4} . With this assay, samples 2, 3, 4, 6, 7, 8, 9, 10,
and 11 showed high neutralization activity with 50\% inhibition above
1:270 dilutions.

To confirm data obtained by pseudovirus neutralization assay, we
performed Plaque Reduction Assay (PRA) against SARS-CoV-2 Wuhan variant
by using the same convalescent plasma samples. PRA is the gold standard
phenotypic method to determine antiviral activity of plasma samples.
This assay relies on quantifying the titer of neutralizing antibody
through the number of plaques forming units (pfu) generated in a
monolayer of virus-infected cells. Isolated SARS-CoV-2 was used to
infect Vero E6 cells in the presence of decreasing concentrations of
convalescent plasma and the 50\% plaque reduction titer was determined.
Several samples (\# 7, 8, 9, and 11) showed high neutralization activity
over a PRA50 of 1:320 (\textbf{Table 2} ).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4.} In vitro pseudovirus neutralization activity of human
plasma samples (1-13) against SARS-CoV-2 Spike variants;
\emph{Wuhan}(\textbf{A} ), \emph{Alpha} (\textbf{B} ), \emph{Beta}
(\textbf{C} ). Neutralization curves of human convalescent plasma
samples against pseudotyped SARS-CoV-2 variants; (\textbf{A)}
VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 \emph{Wuhan}type, (\textbf{B)} VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 \emph{Alpha}
variant carrying \selectlanguage{greek}Δ\selectlanguage{english}H69-V70, N501Y, D614G, \selectlanguage{greek}Δ\selectlanguage{english}Y144 and (\textbf{C)}
VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 \emph{Beta} variant carrying E484K, N501Y, D614G, K417N
mutations were measured as GFP signal in Vero E6 cells, and the
percentage of neutralization was calculated. Data are represented as
means of \selectlanguage{ngerman}±S.D. of at least two independent experiments.

\textbf{Table 2.} PRA Titers and Vaccination Profile of Human Plasma
Samples\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\#Plasma samples & Vaccination & Neutralization titer by plaque
assay\tabularnewline
\midrule
\endhead
1 & Sinovac, 2 dose & 1:10\tabularnewline
2 & Sinovac, 2 dose & 1:20\tabularnewline
3 & Sinovac, 2 dose & 1:20\tabularnewline
4 & Sinovac, 2 dose; BioNTech, 1 dose & 1:80\tabularnewline
5 & Sinovac, 2 dose; BioNTech, 1 dose & 1:80\tabularnewline
6 & Sinovac, 2 dose; BioNTech, 1 dose & 1:80\tabularnewline
7 & Sinovac, 2 dose & 1:320\tabularnewline
8 & Sinovac, 2 dose; BioNTech, 1 dose & 1:1280\tabularnewline
9 & BioNTech, 2 dose & 1:640\tabularnewline
10 & BioNTech, 2 dose & 1:160\tabularnewline
11 & BioNTech, 2 dose & 1:320\tabularnewline
12 & Sinovac, 2 dose & 1:40\tabularnewline
13 & Sinovac, 2 dose & 1:10\tabularnewline
14 & Healthy control & 0\tabularnewline
15 & Healthy control & 0\tabularnewline
\bottomrule
\end{longtable}

In \textbf{Figure 5} , pseudovirus neutralization assay results were
plotted against PRA results (\emph{Wuhan} type) to compare their
reliability. Plots showed linearity with a measure of certainty of
R\textsuperscript{2} = 0.7 (p= 0.0006) for pseudovirus neutralization
against PRA with isolated authentic virus (\textbf{Figure 5A)} . The
measure of certainty (R\textsuperscript{2}) was decreased for
pseudovirus bearing Spike protein of \emph{Alpha} and
\emph{Beta}variants against the PRA with \emph{Wuhan} strain
(\textbf{Figure 5B and 5C} ). Linearity test of pseudovirus bearing
Spike protein of\emph{Alpha} variant was less significant for the
comparison of pseudovirus bearing Spike protein of \emph{Wuhan} strain
versus PRA (R\textsuperscript{2} = 0.31, p= 0.048). In general, the
pseudovirus neutralization assay overestimated the samples with low
neutralizing activities determined by PRA. It might be less reliable for
plasma samples with low neutralizing capacity.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5} . \textbf{Comparison of pseudovirus neutralization
assays with the plaque reduction assay.} Pseudovirus neutralization
assays of \emph{Wuhan} \textbf{(A)} , \emph{Alpha} \textbf{(B)}
and\emph{Beta} variants \textbf{(C)} were plotted against the virus
neutralization activities determined by PRA with authentic
\emph{Wuhan}virus. The R\textsuperscript{2} value indicates the
certainty of the values to be at the trend line.

\textbf{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-\selectlanguage{greek}Δ\selectlanguage{english}G 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-\selectlanguage{greek}Δ\selectlanguage{english}G-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-\selectlanguage{greek}Δ\selectlanguage{english}G-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-\selectlanguage{greek}Δ\selectlanguage{english}G-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-\selectlanguage{greek}Δ\selectlanguage{english}G-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-\selectlanguage{greek}Δ\selectlanguage{english}G-Spike can
mimic the entry of authentic SARS-CoV-2. The serum neutralizing titer of
vaccinated convalescent patients measured by the VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-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, \emph{Alpha} and \emph{Beta} variants of Spike protein were
constructed and tested against human convalescent plasma samples listed
in \textbf{Table 1} . It is shown that the pseudovirus assay based on
the VSV-\selectlanguage{greek}Δ\selectlanguage{english}G 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 \emph{Alpha} (B.1.1.7), \emph{Beta}
(B.1.351), and \emph{Wuhan} (Wild type) viruses. The pseudovirus
neutralization assay showed no loss of neutralizing activity against
Spike \emph{Alpha}variant, whereas every sample lost activity against
Spike \emph{Beta}variant. Overall, the neutralizing activity against
\emph{Alpha} Spike pseudotyped virus was essentially unchanged, but
significantly lower against \emph{Beta} Spike pseudotyped virus. The
main contributing factor is suggested to be E484K mutation in
\emph{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 \emph{Alpha} variant pseudovirus (Muik
et al., 2021). The largely preserved neutralization of pseudovirus
bearing the \emph{Alpha} Spike by BNT162b2-immune plasma makes it
unlikely that the \emph{Beta} variant virus will escape
BNT162b2-mediated protection. However, pseudovirus neutralization assay
showed a substantial drop-in neutralization activity against
the\emph{Beta} 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.

\textbf{Acknowledgement}

This project is financially supported by Sabanci University
Nanotechnology Research and Application Center (SUNUM) and Scientific
Research Projects Coordination Unit of Istanbul University-Cerrahpasa
(Project ID: TSG-2020-34933).

\textbf{Disclosure statement}

No potential conflict of interest was reported by the authors.

\textbf{Author contribution}

GCT conducted the experiments, performed the statistical analysis,
wrote, and edited the original draft. ANC and YTT participated in
experiments. OK and SC reviewed the manuscript. Study concept and
design: YTT, EY, NA, DS, GE, OD, MAK, BM, GA, FT, FC, OE, KM, OK, SC.
All authors contributed to and have approved the final version of the
manuscript.

\textbf{References}

Almahboub, S.A., Algaissi, A., Alfaleh, M.A., ElAssouli, M.Z., Hashem,
A.M., 2020. Evaluation of Neutralizing Antibodies Against Highly
Pathogenic Coronaviruses: A Detailed Protocol for a Rapid Evaluation of
Neutralizing Antibodies Using Vesicular Stomatitis Virus
Pseudovirus-Based Assay. Front. Microbiol. 11, 2020.
https://doi.org/10.3389/FMICB.2020.02020/BIBTEX

Chen, M., Zhang, X.-E., 2021. Construction and applications of
SARS-CoV-2 pseudoviruses: a mini review. Int. J. Biol. Sci. 2021,
1574--1580. https://doi.org/10.7150/ijbs.59184

Condor Capcha, J.M., Lambert, G., Dykxhoorn, D.M., Salerno, A.G., Hare,
J.M., Whitt, M.A., Pahwa, S., Jayaweera, D.T., Shehadeh, L.A., 2021.
Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and
Neutralization Assays: A 1-Week Protocol. Front. Cardiovasc. Med.
https://doi.org/10.3389/fcvm.2020.618651

Crawford, K.H.D., Eguia, R., Dingens, A.S., Loes, A.N., Malone, K.D.,
Wolf, C.R., Chu, H.Y., Tortorici, M.A., Veesler, D., Murphy, M., Pettie,
D., King, N.P., Balazs, A.B., Bloom, J.D., 2020. Protocol and Reagents
for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for
Neutralization Assays. Viruses 2020, Vol. 12, Page 513 12, 513.
https://doi.org/10.3390/V12050513

Donofrio, G., Franceschi, V., Macchi, F., Russo, L., Rocci, A.,
Marchica, V., Costa, F., Giuliani, N., Ferrari, C., Missale, G., 2021. A
simplified sars-cov-2 pseudovirus neutralization assay. Vaccines 9,
1--12. https://doi.org/10.3390/vaccines9040389

Garcia-Beltran, W.F., Lam, E.C., St. Denis, K., Nitido, A.D., Garcia,
Z.H., Hauser, B.M., Feldman, J., Pavlovic, M.N., Gregory, D.J.,
Poznansky, M.C., Sigal, A., Schmidt, A.G., Iafrate, A.J., Naranbhai, V.,
Balazs, A.B., 2021. Multiple SARS-CoV-2 variants escape neutralization
by vaccine-induced humoral immunity. Cell 184, 2372-2383.e9.
https://doi.org/10.1016/J.CELL.2021.03.013

Harvey, W.T., Carabelli, A.M., Jackson, B., Gupta, R.K., Thomson, E.C.,
Harrison, E.M., Ludden, C., Reeve, R., Rambaut, A., Peacock, S.J.,
Robertson, D.L., 2021. SARS-CoV-2 variants, spike mutations and immune
escape. Nat. Rev. Microbiol. 2021 197 19, 409--424.
https://doi.org/10.1038/s41579-021-00573-0

Hoffmann, M., Arora, P., Gro\selectlanguage{ngerman}ß, R., Seidel, A., Hörnich, B.F., Hahn,
A.S., Krüger, N., Graichen, L., Hofmann-Winkler, H., Kempf, A., Winkler,
M.S., Schulz, S., Jäck, H.M., Jahrsdörfer, B., Schrezenmeier, H.,
Müller, M., Kleger, A., Münch, J., Pöhlmann, S., 2021. SARS-CoV-2
variants B.1.351 and P.1 escape from neutralizing antibodies. Cell 184,
2384. https://doi.org/10.1016/J.CELL.2021.03.036

Kuzmina, A., Khalaila, Y., Voloshin, O., Keren-Naus, A., Boehm-Cohen,
L., Raviv, Y., Shemer-Avni, Y., Rosenberg, E., Taube, R., 2021.
SARS-CoV-2 spike variants exhibit differential infectivity and
neutralization resistance to convalescent or post-vaccination sera. Cell
Host Microbe 29, 522-528.e2. https://doi.org/10.1016/J.CHOM.2021.03.008

Li, Q., Liu, Q., Huang, W., Li, X., Wang, Y., 2018. Current status on
the development of pseudoviruses for enveloped viruses. Rev. Med. Virol.
28, 1--10. https://doi.org/10.1002/rmv.1963

Liu, Z., VanBlargan, L.A., Bloyet, L.M., Rothlauf, P.W., Chen, R.E.,
Stumpf, S., Zhao, H., Errico, J.M., Theel, E.S., Liebeskind, M.J.,
Alford, B., Buchser, W.J., Ellebedy, A.H., Fremont, D.H., Diamond, M.S.,
Whelan, S.P.J., 2021. Identification of SARS-CoV-2 spike mutations that
attenuate monoclonal and serum antibody neutralization. Cell Host
Microbe 29, 477. https://doi.org/10.1016/J.CHOM.2021.01.014

Muik, A., Wallisch, A.K., Sänger, B., Swanson, K.A., Mühl, J., Chen, W.,
Cai, H., Maurus, D., Sarkar, R., Türeci, Ö., Dormitzer, P.R., \selectlanguage{polish}Ş\selectlanguage{english}ahin, U.,
2021. Neutralization of SARS-CoV-2 lineage B.1.1.7 pseudovirus by
BNT162b2 vaccine--elicited human sera. Science (80-. ). 371, 1152--1153.
https://doi.org/10.1126/science.abg6105

Neefjes, M., Housmans, B.A.C., van den Akker, G.G.H., van Rhijn, L.W.,
Welting, T.J.M., van der Kraan, P.M., 2021. Reporter gene comparison
demonstrates interference of complex body fluids with secreted
luciferase activity. Sci. Rep. 11, 1--13.
https://doi.org/10.1038/s41598-020-80451-6

Nie, J., Li, Q., Wu, J., Zhao, C., Hao, H., Liu, H., Zhang, L., Nie, L.,
Qin, H., Wang, M., Lu, Q., Li, X., Sun, Q., Liu, J., Fan, C., Huang, W.,
Xu, M., Wang, Y., 2020. Establishment and validation of a pseudovirus
neutralization assay for SARS-CoV-2. Emerg. Microbes Infect. 9,
680--686. https://doi.org/10.1080/22221751.2020.1743767

Ou, X., Liu, Y., Lei, X., Li, P., Mi, D., Ren, L., Guo, L., Guo, R.,
Chen, T., Hu, J., Xiang, Z., Mu, Z., Chen, X., Chen, J., Hu, K., Jin,
Q., Wang, J., Qian, Z., 2020. Characterization of spike glycoprotein of
SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV.
Nat. Commun. 11. https://doi.org/10.1038/s41467-020-15562-9

Robbiani, D.F., Gaebler, C., Muecksch, F., Lorenzi, J.C.C., Wang, Z.,
Cho, A., Agudelo, M., Barnes, C.O., Gazumyan, A., Finkin, S., H\selectlanguage{ngerman}ägglöf,
T., Oliveira, T.Y., Viant, C., Hurley, A., Hoffmann, H.H., Millard,
K.G., Kost, R.G., Cipolla, M., Gordon, K., Bianchini, F., Chen, S.T.,
Ramos, V., Patel, R., Dizon, J., Shimeliovich, I., Mendoza, P.,
Hartweger, H., Nogueira, L., Pack, M., Horowitz, J., Schmidt, F.,
Weisblum, Y., Michailidis, E., Ashbrook, A.W., Waltari, E., Pak, J.E.,
Huey-Tubman, K.E., Koranda, N., Hoffman, P.R., West, A.P., Rice, C.M.,
Hatziioannou, T., Bjorkman, P.J., Bieniasz, P.D., Caskey, M.,
Nussenzweig, M.C., 2020. Convergent antibody responses to SARS-CoV-2 in
convalescent individuals. Nature 584, 437--442.
https://doi.org/10.1038/S41586-020-2456-9

Salazar-García, M., Acosta-Contreras, S., Rodríguez-Martínez, G.,
Cruz-Rangel, A., Flores-Alanis, A., Patiño-López, G., Luna-Pineda, V.M.,
2022. Pseudotyped Vesicular Stomatitis Virus-Severe Acute Respiratory
Syndrome-Coronavirus-2 Spike for the Study of Variants, Vaccines, and
Therapeutics Against Coronavirus Disease 2019. Front. Microbiol. 12,
4276. https://doi.org/10.3389/FMICB.2021.817200/BIBTEX

Sanders, D.A., 2002. No false start for novel pseudotyped vectors. Curr.
Opin. Biotechnol. 13, 437--442.
https://doi.org/10.1016/S0958-1669(02)00374-9

Schmidt, F., Weisblum, Y., Muecksch, F., Hoffmann, H.H., Michailidis,
E., Lorenzi, J.C.C., Mendoza, P., Rutkowska, M., Bednarski, E., Gaebler,
C., Agudelo, M., Cho, A., Wang, Z., Gazumyan, A., Cipolla, M., Caskey,
M., Robbiani, D.F., Nussenzweig, M.C., Rice, C.M., Hatziioannou, T.,
Bieniasz, P.D., 2020. Measuring SARS-CoV-2 neutralizing antibody
activity using pseudotyped and chimeric viruses. J. Exp. Med. 217.
https://doi.org/10.1084/JEM.20201181

Schrörs, B., Riesgo-Ferreiro, P., Sorn, P., Gudimella, R., Bukur, T.,
Rösler, T., Löwer, M., Sahin, U., 2021. Large-scale analysis of
SARS-CoV-2 spike-glycoprotein mutants demonstrates the need for
continuous screening of virus isolates. PLoS One 16, e0249254.
https://doi.org/10.1371/JOURNAL.PONE.0249254

Von Rhein, C., Scholz, T., Henss, L., Kronstein-Wiedemann, R., Schwarz,
T., Rodionov, R.N., Corman, V.M., Tonn, T., Schnierle, B.S., 2021.
Comparison of potency assays to assess SARS-CoV-2 neutralizing antibody
capacity in COVID-19 convalescent plasma. J. Virol. Methods 288, 114031.
https://doi.org/10.1016/j.jviromet.2020.114031

Wang, P., Nair, M.S., Liu, L., Iketani, S., Luo, Y., Guo, Y., Wang, M.,
Yu, J., Zhang, B., Kwong, P.D., Graham, B.S., Mascola, J.R., Chang,
J.Y., Yin, M.T., Sobieszczyk, M., Kyratsous, C.A., Shapiro, L., Sheng,
Z., Huang, Y., Ho, D.D., 2021. Antibody resistance of SARS-CoV-2
variants B.1.351 and B.1.1.7. Nature 593, 130--135.
https://doi.org/10.1038/S41586-021-03398-2

Whitt, M.A., 2021. Generation of VSV pseudotypes using recombinant
\selectlanguage{greek}Δ\selectlanguage{english}G-VSV for studies on virus entry, identification of entry inhibitors,
and immune responses to vaccines. Journal of Virological Methods 169(2),
365-374. https://doi.org/10.1016/j.jviromet.2010.08.006

Wu, N.C., Yuan, M., Bangaru, S., Huang, D., Zhu, X., Lee, C.C.D.,
Turner, H.L., Peng, L., Yang, L., Burton, D.R., Nemazee, D., Ward, A.B.,
Wilson, I.A., 2020. A natural mutation between SARS-CoV-2 and SARS-CoV
determines neutralization by a cross-reactive antibody. PLoS Pathog. 16,
1--18. https://doi.org/10.1371/journal.ppat.1009089

Yahalom-Ronen, Y., Tamir, H., Melamed, S., Politi, B., Shifman, O.,
Achdout, H., Vitner, E.B., Israeli, O., Milrot, E., Stein, D.,
Cohen-Gihon, I., Lazar, Shlomi, Gutman, H., Glinert, I., Cherry, L.,
Vagima, Y., Lazar, Shirley, Weiss, S., Ben-Shmuel, A., Avraham, R.,
Puni, R., Lupu, E., Bar-David, E., Sittner, A., Erez, N., Zichel, R.,
Mamroud, E., Mazor, O., Levy, H., Laskar, O., Yitzhaki, S., Shapira,
S.C., Zvi, A., Beth-Din, A., Paran, N., Israely, T., 2020. A single dose
of recombinant VSV-[?]G-spike vaccine provides protection against
SARS-CoV-2 challenge. Nat. Commun. 11, 1--13.
https://doi.org/10.1038/s41467-020-20228-7

Yuan, Z., Pavel, M.A., Wang, H., Kwachukwu, J.C., Mediouni, S.,
Jablonski, J.A., Nettles, K.W., Reddy, C.B., Valente, S.T., Hansen,
S.B., 2022. Hydroxychloroquine blocks SARS-CoV-2 entry into the
endocytic pathway in mammalian cell culture. Commun. Biol. 2022 51 5,
1--12. https://doi.org/10.1038/s42003-022-03841-8

Zhou, D., Dejnirattisai, W., Supasa, P., Liu, C., Mentzer, A.J., Ginn,
H.M., Zhao, Y., Duyvesteyn, H.M.E., Tuekprakhon, A., Nutalai, R., Wang,
B., Paesen, G.C., Lopez-Camacho, C., Slon-Campos, J., Hallis, B.,
Coombes, N., Bewley, K., Charlton, S., Walter, T.S., Skelly, D., Lumley,
S.F., Dold, C., Levin, R., Dong, T., Pollard, A.J., Knight, J.C., Crook,
D., Lambe, T., Clutterbuck, E., Bibi, S., Flaxman, A., Bittaye, M.,
Belij-Rammerstorfer, S., Gilbert, S., James, W., Carroll, M.W.,
Klenerman, P., Barnes, E., Dunachie, S.J., Fry, E.E., Mongkolsapaya, J.,
Ren, J., Stuart, D.I., Screaton, G.R., 2021. Evidence of escape of
SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera. Cell
184, 2348. https://doi.org/10.1016/J.CELL.2021.02.037

\textbf{Figure 1.} Generation of VSV pseudotyped viruses bearing
truncated SARS-CoV-2 Spike proteins. \textbf{(A)} Preparation of
rVSV-\selectlanguage{greek}Δ\selectlanguage{english}G-G* in BHK21/W1 cell line. \textbf{(B)} Packaging of
rVSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 in HEK293T cell line. \textbf{(C)} Infection of
pseudovirus carrying GFP reporter and sequential passaging of
pseudovirus to eliminate parental virus (rVSV-\selectlanguage{greek}Δ\selectlanguage{english}G-G*) using plasmids
encoding Sdel21 and T7 polymerase transfected Vero E6.
\textbf{(D)}SARS-CoV-2 Spike pseudovirus neutralization assay using
human convalescent plasma samples and GFP signal reading.

\textbf{Figure 2.} Viral entry assay in Vero E6 cell line.
\textbf{(A)}Vero-E6 cells were inoculated with VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 pseudotyped
virus. Cells were treated with hydroxychloroquine at 20 min before
inoculation. After 24h, GFP signal detected under fluorescence
microscope is shown in representative cellular images. \textbf{(B)} For
quantification, GFP signal from 25 images per sample was analyzed by
using Image J at 24h of post-inoculation. Values are presented as \% of
total population. Scale bar: 100\selectlanguage{greek}µ\selectlanguage{english}m. Data are represented as means of
\selectlanguage{ngerman}±S.D. of at least three independent experiments. *p \textless{} 0.05;
****p \textless{} 0.0001 - using One-Way ANOVA with Bonferroni's
post-hoc test.

\textbf{Figure 3.} Colocalization of pseudovirus with hACE-2 receptor or
SARS-CoV-2 Spike protein in Vero E6 cell line. Cells were incubated with
anti-SARS-CoV-2 Spike, human anti-ACE2 and DAPI.
\textbf{(A--D)}Mock-treated VeroE6 cells with hACE2 staining.
(\textbf{E-H)}VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 infected Vero E6 cells with hACE2 staining.
\textbf{(I-L)}VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 infected Vero E6 cells with SARS-CoV-2 Spike
staining. Yellow color shows viral S protein (\textbf{J} ), and red
colors represents human ACE2 (\textbf{B, F} ). Scale bars, 20 \selectlanguage{greek}μ\selectlanguage{english}m. Data
in\textbf{A--L} are representative images of two independent
experiments.

\textbf{Figure 4.} In vitro pseudovirus neutralization activity of human
plasma samples (1-13) against SARS-CoV-2 Spike variants;
\emph{Wuhan}(\textbf{A} ), \emph{Alpha} (\textbf{B} ), \emph{Beta}
(\textbf{C} ). Neutralization curves of human convalescent plasma
samples against pseudotyped SARS-CoV-2 variants; (\textbf{A)}
VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 \emph{Wuhan}type, (\textbf{B)} VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 \emph{Alpha}
variant carrying \selectlanguage{greek}Δ\selectlanguage{english}H69-V70, N501Y, D614G, \selectlanguage{greek}Δ\selectlanguage{english}Y144 and (\textbf{C)}
VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21 \emph{Beta} variant carrying E484K, N501Y, D614G, K417N
mutations were measured as GFP signal in Vero E6 cells, and the
percentage of neutralization was calculated. Data are represented as
means of \selectlanguage{ngerman}±S.D. of at least two independent experiments.

\textbf{Figure 5} . \textbf{Comparison of pseudovirus neutralization
assays with the plaque reduction assay.} Pseudovirus neutralization
assays of \emph{Wuhan} \textbf{(A)} , \emph{Alpha} \textbf{(B)}
and\emph{Beta} variants \textbf{(C)} were plotted against the virus
neutralization activities determined by PRA with authentic
\emph{Wuhan}virus. The R\textsuperscript{2} value indicates the
certainty of the values to be at the trend line.

\textbf{Table 1.} Vaccination status of human plasma samples used for
neutralization assays

\textbf{Table 2.} PRA Titers and Vaccination Profile of Human Plasma
Samples

\textbf{Figure 1S.} Spike expressing construct map

\textbf{Figure S2} . Positive control images of viral entry assay in
Vero E6 cell line. Vero-E6 cells were inoculated with VSV-\selectlanguage{greek}Δ\selectlanguage{english}G-Sdel21
pseudotyped particles. After 24 h, the GFP signal was observed using
Axio Observer Fluorescence Microscope. The representative cellular
images were given.

\textbf{Table S1.} The sequences of the mutation primers

\selectlanguage{english}
\FloatBarrier
\end{document}

