A Neutralization Assay Based on Pseudo-Typed Lentivirus with SARS Cov-2 Spike Protein in ACE2-Expressing CRFK Cells

pathogens

Article A Neutralization Assay Based on Pseudo-Typed Lentivirus with SARS CoV-2 Spike Protein in ACE2-Expressing CRFK Cells

Yalçın Pısıl 1,2, Hisatoshi Shida 3 and Tomoyuki Miura 1,*

1 Laboratory of Primate Model, Research Center for Infectious Diseases, Institute for Frontier Life and Medical Science, Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan; [email protected] 2 Graduate School of Human and Environmental Studies, Department of Interdisciplinary Environment, Dynamics of Natural Environment, Dynamics of Biological Environment, Kyoto University, Kyoto 606-8501, Japan 3 Division of Molecular Virology, Institute of Immunological Science, Hokkaido University, Sapporo 060-0808, Japan; [email protected] * Correspondence: tmiura@virus.kyoto-u.ac.jp

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic zoonotic virus that spreads rapidly. In this work, we improve the hitherto existing neutralization assay system to assess SARS-CoV-2 inhibitors using a pseudo-typed lentivirus coated with the SARS-CoV-2 spike protein (LpVspike +) and angiotensin-converting enzyme 2 (ACE2)-transfected cat Crandell–Rees feline kidney (CRFK) cells as the host cell line. Our method was 10-fold more sensitive compared to the typical human embryonic kidney 293T (HEK293T) cell system, and it was successfully applied to quantify the titers of convalescent antisera and monoclonal anti-spike antibodies required for pseudo virus neutralization. The 50% inhibition dilution (ID50) of two human convalescent sera, SARS-CoV-2 immunoglobulin G (IgG) and SARS-CoV-2 immunoglobulin M (IgM), which were 1:350 (±1:20) and 1:1250 (±1:350), respectively. The 50% inhibitory concentration (IC50) Citation: Pısıl, Y.; Shida, H.; Miura, T. of the IgG, IgM and immunoglobulin A (IgA) anti-SARS-CoV-2 monoclonal antibodies (mAbs) A Neutralization Assay Based on against LpVspike(+) were 0.45 (±0.1), 0.002 (±0.001) and 0.004 (±0.001) µg mL−1, respectively. Pseudo-Typed Lentivirus with SARS We also found that reagents typically used to enhance infection were not effective in the CFRK CoV-2 Spike Protein in ACE2-Expressing CRFK Cells. system. This methodology is both efﬁcient and safe; it can be employed by researchers to evaluate Pathogens 2021, 10, 153. https:// neutralizing monoclonal antibodies and contribute to the discovery of new antiviral inhibitors against doi.org/10.3390/pathogens10020153 SARS-CoV-2.

Academic Editor: Theresa Chang Keywords: SARS-CoV-2; Covid-19; HIV; CRFK Cell; ACE2; spike; monoclonal antibody; neutraliza- Received: 7 December 2020 tion assay; pseudo-typed lentivirus Accepted: 29 January 2021 Published: 2 February 2021

Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in An outbreak of severe acute respiratory syndrome (SARS) from 2002 to 2003 in Guang- published maps and institutional afﬁl- dong, China, resulted in over 8000 infections and almost 800 deaths. This was caused by a iations. coronavirus (CoV), known as SARS-associated CoV (SARS-CoV) [1]. In December 2019, a novel infectious disease caused by a related coronavirus, SARS-CoV-2, was reported in Wuhan, China. It is widely referred to as coronavirus disease 2019 (COVID-19) [2]. SARS- CoV-2 belongs to the same species as SARS-CoV and other SARS-related bat CoVs [3]. Copyright: © 2021 by the authors. COVID-19 was declared a worldwide pandemic by the World Health Organization (WHO), Licensee MDPI, Basel, Switzerland. and has infected over 51 million people and killed more than 1.2 million (December 2020). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Pathogens 2021, 10, 153. https://doi.org/10.3390/pathogens10020153 https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, 153 2 of 13

SARS-CoV-2 is a β-coronavirus, as characterized by its envelope and non-segmented, positive-sense single-stranded RNA genome [4]. It has a genome size of 29,881 bp, which encodes 9860 amino acids [5]. Two-thirds of the viral RNA encodes two polyproteins and 16 non-structural proteins, whereas the remainder of the viral genome encodes four essential structural proteins: the spike (S) glycoprotein, a small envelope protein, a matrix protein, and a nucleocapsid protein. Several accessory proteins, which interfere with the host innate immune response, are also encoded within the viral genome [4,6,7]. The S protein is 1273 amino acids in length and is coated with polysaccharide molecules, providing camouflage that enables the virus to evade the host immune system during entry [8]. During its biosynthesis, the S protein is cleaved by a furin-like protease in the Golgi apparatus into two subunits, S1 and S2, which form the bulbous head and stalk regions of the S protein. The S proteins of both SARS-CoV-2 and SARS-CoV bind to the angiotensin-converting enzyme 2 (ACE2) receptor, which they use for cell entry [1,9,10]. Following binding of the S protein to the ACE2 receptor, transmembrane protease serine 2 (TMPRSS2), which is located on the host cell membrane, further cleaves the S2 subunit into S2’ and S2” subunits, activating the membrane fusion domain [11]. Vero and VeroE6 cells, which express the ACE2 receptor at high levels, have been used to isolate and study SARS-CoV-2. SARS-CoV-2 is categorized as a biosecurity level (BSL) 3 agent, according to WHO guidelines, which impedes the rapid development of new protocols and medicines related to the virus [12–14]. To lower the biosecurity risk of viruses, non-replicative pseudo-typed viruses carrying viral S proteins on their surfaces have been developed; these pseudoviruses can infect host cells but cannot replicate, meaning that they can be dealt with using less stringent BSL-2 facilities [15]. Pseudoviruses have recently been developed for SARS-CoV-2 using human immunodeficiency virus (HIV)-based lentiviral particles, murine leukemia virus-based retroviral particles and the vesicular stomatitis virus (VSV) [15]. VSV pseudo-types expressing the SARS-CoV-2 S protein are currently being used to study viral entry [9]; however, the construction of VSV-based pseudo-typed viruses requires a complex procedure. By contrast, pseudo-typed lentiviral viruses can be produced using a comparatively simple method, involving the co-transfection of host cells with multiple plasmids [10]. Human embryonic kidney 293T (HEK293T) cells are currently the main cell line used for the production of pseudo-typed SARS-CoV-2 and to assay its infectivity due to its high transfection efficiency. However, further improvements to this system could be made [3]. Here, we report the development of a simple assay that is 10-fold more sensitive than the commonly-used HEK293T cell assay, using a pseudo-typed lentivirus coated with SARS-CoV-2 S protein (LpVspike(+)). Using our system, we successfully measured the titer of anti-CoV antibodies required for neutralization. Figure1 shows an illustration outlining our new system. Pathogens 2021, 10, x FOR PEER REVIEW 3 of 14

Pathogens 2021, 10, 153 3 of 13

FigureFigure 1. Generation 1. Generation of ofa pseudo a pseudo-typed-typed lentivirus lentivirus coated coated with with severe severe acute acute respiratory syndromesyndrome coronavirus coronavirus 2 2 (SARS- (SARS- CoVCoV-2)-2) Spike Spike protein protein using using human human embryonic embryonic kidney kidney 293T 293T ( (HEK293T)HEK293T) cells, andand illustrationillustration of of the the neutralization neutralization assay assay principleprinciple using using angiotensin angiotensin-converting-converting enzyme enzyme 2 (ACE2)-expressing 2 (ACE2)-expressing Crandell–Rees Crandell– felineRees kidney feline kidney(CRFK) cells. (CRFK The) cells. pseudo- The pseudotyped-typed lentivirus lentivirus coated coated with SARS-CoV-2with SARS-CoV Spike-2 (LpVspike(+))Spike (LpVspike and(+ harboring)) and harboring the 2019-nCov_pcDNA3.1(+)-P2A-eGreen the 2019-nCov_pcDNA3.1(+)-P2A- eGreenFluorescent Fluorescent Protein Protein (GFP) (GFP) plasmid plasmid was prepared was prepared by co-transfecting by co-transf 5ecting× 105 cell5 × mL105 −cell1 of mL HEK293T−1 of HEK293T cells with cells the with HIV-1 the ◦ HIVNL4-3-1 NL4∆-Env3 ΔEnv∆Vpr ΔVpr Luciferase Luciferase Reporter Reporter Vector. Vector. Transfected Transfected HEK293T HEK293T cells were cells incubated were atincubated 37 C under at 37 5% °C CO under2 for 25% days. CO2 for 2The days. supernatant The supernatant medium medium containing containing LpVspike(+) LpVspike(+ was harvested) was andharvested ﬁltered and through filtered a 0.45- throughµm pore a 0.45 size-µm ﬁlter. pore To generatesize filter. To generateACE2-expressing ACE2-expressing cell lines, cellscell lines, were cells transfected were transfected with pcDNA3.1+/C-(K) with pcDNA3.1+/C DYK-ACE2-(K) DYK and- incubatedACE2 and at incubated 37 ◦C under at 37 5% °C

underCO 5%2 for CO 1 day.2 for The 1 day. transfected The transfected cells were cells then were able tothen transiently able to transiently express the express ACE2 receptor the ACE2 on theirreceptor membrane on their surface membrane and surfacecould and hence could be hence infected be by infected LpVspike(+). by LpVspike(+) Approximately. Approximately 2.3 × 104 ACE2-expressing 2.3 × 104 ACE2-expressing cells were infected cells were per infected well in 96-well per well in 96plates,-well andplates, luciferase and luciferase expression expression was measured was measured at 48 h post-infection. at 48 h post-infection. This ﬁgure This was figure created was by created biorender.com. by biorender.com.

2. Results 2.1. Measuring LpVspike (+) Infectivity in Various ACE2-Expressing Cell Types Although ACE2-expressing HEK293T cells are typically used with the pseudo-typed lentiviral system, the sensitivity of infectivity assays is low. With the goal of identifying a Pathogens 2021, 10, x FOR PEER REVIEW 4 of 14

more sensitive cell line, we measured the infectivity of a SARS-CoV-2-S-coated pseudovirus, or LpVspike(+), in the following ACE2-expressing cell lines: Cos7, TZM-bl, Cran- dell–Rees feline kidney (CRFK), and Vero cells. The various cell types were infected with 100 µL of viral solution in 96-well plates. The luciferase readings, which indicated pseudoviral infection, were > 10-fold higher in ACE2-expressing CRFK (ACE2–CRFK) cells compared to ACE2-expressing HEK293T (ACE2–HEK293T) cells; ACE2–CRFK cells and ACE2–HEK293T cells produced readings of 8 × 104 and 5 × 103 relative luciferase units (RLU), respectively. A reading of approximately 5 RLU was detected in uninfected control cells (Figure 2). We also examined LpVspike(+) infectivity in VeroE6/TMPRSS2 cells, which have pre- Pathogens 2021, 10, 153 4 of 13 viously been shown to be 10-fold more sensitive to infection than parental VeroE6 cells [16]. LpVspike(+) infected VeroE6/TMPRSS2 cells less efficiently compared to ACE2– CRFK and ACE2–HEK293T cells (Figure 2). A reading of only 103 RLU was detected when VeroE6/TMPRSS22. Results cells were infected with 100 µL of viral solution. LpVspike(+) did not appear2.1. Measuring to infect LpVspike(+) ACE2-expressing Infectivity Cos7 in Variouscells (Figure ACE2-Expressing 2). Cell Types AlthoughThe TZM- ACE2-expressingbl cell line (also called HEK293T JC.53bl cells-13) are is typicallya HeLa cell used derivative with the pseudo-typedengineered to expresslentiviral CD4, system, CCR5 the and sensitivity CXCR4, as of well infectivity as Tat-responsive assays is low.firefly With luciferase the goal reporter of identify- genes. Theseing a morefeatures sensitive make TZM cell- line,bl cells we highly measured susceptible the infectivity to HIV-1 infection of a SARS-CoV-2-S-coated and enables quan- tificationpseudovirus, of viral or LpVspike(+), infection and in neutralizing the following antibody ACE2-expressing titers [17]. Because cell lines: TZM Cos7,-bl TZM-bl,already hasCrandell–Rees an integrated feline copy kidney of the (CRFK), luciferase and reporter Vero cells. gene, The we various constructed cell types a second were infectedpseudo- withtyped 100 lentivirusµL of viral, named solution LpVs inpike2 96-well(+); this plates. was The essentially luciferase the readings, same as LpVspike(+) which indicated, ex- ceptpseudoviral it did not infection, contain were the >luciferase 10-fold higher gene. inACE2 ACE2-expressing-expressing TZM CRFK-bl (ACE2–CRFK)cells infected with cells undilutedcompared LpVspike2(+) to ACE2-expressing produced HEK293T a reading (ACE2–HEK293T) of 2 × 104 RLU, which cells; was ACE2–CRFK comparable cells to andthat ofACE2–HEK293T the background cells cell line produced that ha readingsd not been of virally 8 × 10 infected4 and 5 × (110 × 103 relative4 RLU; Figure luciferase 2). There- units fore,(RLU), the respectively. TZM-bl cell A line reading was deemed of approximately an unsuitable 5 RLU host was for detected our S-covered in uninfected pseudo control-typed lentivircells (Figureus system.2).

Figure 2. Titers of the LpVspike(+)Figure 2. were Titers determined of the LpVspike(+) by measuring were determined relative luciferase by measuring units (RLU) relative at 48 luciferase h after infection. units (RLU) Approximately 2.3 × 104 ACE2-expressingat 48 h after infection. cells were Approximately seeded per well 2.3 × in 10 96-well4 ACE2 plates.-expressing Cells cells were were infected seeded with per 100 wellµL ofin 96- undiluted LpVspike(+), thenwell two-fold plates. dilutions Cells were were infected made. with 100 µL of undiluted LpVspike(+), then two-fold dilutions were made. We also examined LpVspike(+) infectivity in VeroE6/TMPRSS2 cells, which have previously been shown to be 10-fold more sensitive to infection than parental VeroE6 cells [16]. LpVspike(+) infected VeroE6/TMPRSS2 cells less efficiently compared to ACE2– CRFK and ACE2–HEK293T cells (Figure2). A reading of only 10 3 RLU was detected when VeroE6/TMPRSS2 cells were infected with 100 µL of viral solution. LpVspike(+) did not appear to infect ACE2-expressing Cos7 cells (Figure2). The TZM-bl cell line (also called JC.53bl-13) is a HeLa cell derivative engineered to express CD4, CCR5 and CXCR4, as well as Tat-responsive firefly luciferase reporter genes. These features make TZM-bl cells highly susceptible to HIV-1 infection and enables quantifi- cation of viral infection and neutralizing antibody titers [17]. Because TZM-bl already has an integrated copy of the luciferase reporter gene, we constructed a second pseudo-typed lentivirus, named LpVspike2(+); this was essentially the same as LpVspike(+), except it did not contain the luciferase gene. ACE2-expressing TZM-bl cells infected with undiluted PathogensPathogens 20212021,, 1010,, 153x FOR PEER REVIEW 55 ofof 1314

2.2.LpVspike2(+) The compar producedison of LpVspike(+) a reading Infectivity of 2 × 10 in4 HEK293TRLU, which and wasCRFK comparable Cell Lines with/without to that of the ACE2background Transfection cell line that had not been virally infected (1 × 104 RLU; Figure2). There- fore,The the TZM-blS protein cell of lineSARS was-CoV deemed-2 uses an ACE2 unsuitable as its point host of for entry, our S-covered and hence pseudo-typed requires host ACE2lentivirus expression system. to initiate infection. We confirmed the specificity of this process by examining the infectivity of pseudo-typed lentiviruses containing the SARS-CoV-2 S protein (2.2.LpVspike The Comparison(+)) and those of LpVspike(+) that did not Infectivity (LpVspike in HEK293T[−]) in four and cell CRFK lines: Cell CRFK Lines, ACE2 with/without–CRFK, ACE2 Transfection HEK293T and ACE2–HEK293T. Neither LpVspike(+) nor LpVspike(−) infected the HEK293TThe S cells protein. LpVspike(+) of SARS-CoV-2 and a small uses amount ACE2 asof itsLpVspike( point of−) entry, were able and to hence infect requires ACE2– HEK293host ACE2T cell expressions, with LpVspike(+) to initiate infection.- and LpVspike( We confirmed−)-infected the ACE2 specificity–HEK293T of this cells process pro- ducingby examining readings the of infectivity 2000 and 5 of00 pseudo-typed RLU with 50 μL lentiviruses of viral solution, containing respectively the SARS-CoV-2 (Figure 3a)S protein. These (LpVspike(+))results suggest and that those LpVspike( that did−) can not infect (LpVspike[ ACE2−–HEK]) in293T four cells lines: non-specifi- CRFK, − ACE2–CRFK,cally, without HEK293Tbinding to andthe ACE2 ACE2–HEK293T. receptor. Neither LpVspike(+) nor LpVspike( ) in- − fectedN theeither HEK293T LpVspike(+) cells. nor LpVspike(+) LpVspike( and−) infect a smalled CRFK amount cell ofs. LpVspike( Whereas LpVspike(+)) were able in- to infect ACE2–HEK293T cells, with LpVspike(+)- and LpVspike(−)-infected ACE2–HEK293T fected ACE2–CRFK cells (104 RLU with 50 µL of viral solution), LpVspike(−) did not (Fig- cells producing readings of 2000 and 500 RLU with 50 µL of viral solution, respectively ure 3b). These results indicate that only LpVspike(+) can infect ACE2-transfected CRFK (Figure3a). These results suggest that LpVspike( −) can infect ACE2–HEK293T cells non- cells, indicating a S-ACE2 specific interaction. specifically, without binding to the ACE2 receptor.

Figure 3. (a) Titers of LpVspike(+) and LpVspike(−) as determined by measuring relative luciferase units (RLU) at 48 h after infection in 293 T cell/ACE2Figure 293 3. ( Ta) cell. Tite (rsb )of Titers LpVspike(+) of LpVspike(+) and LpVspike( and LpVspike(−) as determined−) as determined by measuring by RLU relative at 48 h afterluciferase infection in CRFK cell/ACE2-expressingunits (RLU) at CRFK 48 h after cell. Cellsinfection at concentrations in 293 T cell/ACE2 of approximately 293 T cell. (b 2.3) Titers× 104 ofcell LpVspike(+) of each cell and line (HEK293T, ACE2-expressingLpVspike( HEK293T,−) CRFK,as determined and ACE2-expressing by RLU at 48 h CRFK) after infection were seeded in CRFK per wellcell/ACE2 in 96-well-expressing plates. CRFK The initial volume of LpVspike(+)cell. used Cells for at infection concentrations was 50 ofµL approximately of undiluted virus, 2.3 × then104 cell three of each 1:2 dilutions cell line (HEK293T, were made. ACE2-expressing HEK293T, CRFK, and ACE2-expressing CRFK) were seeded per well in 96-well plates. The initialNeither volume LpVspike(+) of LpVspike(+) nor LpVspike(used for infection−) infected was 50 CRFKµL of undiluted cells. Whereas virus, then LpVspike(+) three 1:2 dilutionsinfected were ACE2–CRFK made. cells (104 RLU with 50 µL of viral solution), LpVspike(−) did not (Figure3b). These results indicate that only LpVspike(+) can infect ACE2-transfected CRFK 2.3.cells, Effect indicating of Diethylaminoethyl a S-ACE2 speciﬁc-Dextran interaction. on Infectivity TZM-bl cells are maximally sensitive to HIV infection in diethylaminoethyl-dextran (Deax)2.3. Effect-supplemented of Diethylaminoethyl-Dextran medium [17]. on We Infectivity therefore examined the effects of Deax on LpVspike(+)TZM-bl cells and are LpVspike( maximally− sensitive) infectivity to HIV in infection CRFK in diethylaminoethyl-dextranand ACE2–CRFK cells. (Deax)- Both LpVspike(supplemented+) and medium LpVspike( [17].− We) infected therefore the examined CRFK cells the in effects the presence of Deax onof Deax, LpVspike(+) producing and readingsLpVspike( of− )10 infectivity4–105 RLU in per CRFK 50 andμL ACE2–CRFKof viral solution cells. (Figure Both LpVspike(+) 4a); this was and in LpVspike( contrast −to) CRFKinfected cells the CRFKwithout cells Deax, in the which presence were of Deax,only infected producing by readings LpVspike(+). of 104–10 The5 RLU ACE2 per–CRFK 50 µL cellsof viral were solution infected (Figure by both4a); LpVspike(+) this was in contrastand LpVspike( to CRFK−) in cells the without presence Deax, of Deax, which whereas were infectiononly infected only by occurred LpVspike(+). in the The ACE2 ACE2–CRFK–CRFK line cells with were LpVspike(+) infected by infection both LpVspike(+) (1 × 104 RLU and withLpVspike( 50 μL− of) inviral the solution) presence in of the Deax, absence whereas of Deax. infection Deax only can therefore occurred ind in theuce ACE2–CRFK non-specific 4 LpVspikeline with LpVspike(+)infection in infectionCRFK cells. (1 × Notably,10 RLU only with LpVspike(+) 50 µL of viral efficiently solution) infected in the absenceACE2– Pathogens 2021, 10, x FOR PEER REVIEW 6 of 14

CRFK cells, LpVspike(−) could not infect CRFK or ACE2–CRFK cells without Deax (Figure 4b). Pathogens 2021, 10, 153 The addition of Deax increased the infection efficiency of both LpVspike(+)6 of 13 and LpVspike(−) 1000-fold in CRFK cells. While Deax increased the infection efficiency of LpVspike(+) 10-fold in ACE2–CRFK cells, it increased the infection efficiency of ofLpVspike( Deax. Deax−) can1000 therefore-fold. These induce results non-specific suggestLpVspike that Deax infection can cause in CRFKnon-specific cells. Notably,infections onlyin LpVspike(+)HEK293T, ACE2 efficiently–HEK infected293T, CRFKACE2–CRFK, and ACE2 cells, LpVspike(–CRFK cell−) line coulds. notDeax infect was CRFK therefore or ACE2–CRFKruled out as cells a supplement without Deax for (Figure enhancing4b). LpVspike infection efficiency for our system.

Figure 4. a b ( ) RLU measurementsFigure were 4. taken (a) RLU at 48 measurements h after infection were in CRFKtaken cell.at 48 ( h) after RLU infection measurements in CRFK were cell taken. (b) RLU at 48 measure- h 4 after infection in ACE2 expressingments CRFK were cell. taken Approximately at 48 h after infection 2.3 × 10 incells ACE2 of eachexpressing cell line CRFK (CRFK cell. and Approximately ACE2-expressing 2.3 × 104 CRFK) were seeded per well incells 96-well of each plates. cellThe lineinitial (CRFK volume and ACE2 of LpVspike(+)-expressing usedCRFK for) were infection seeded was per 50 wellµL of in undiluted 96-well plates. virus, then three 1:2 dilutions wereThe initial made. volume Dextran of (Deax) LpVspike(+) was added used to for the infection medium was to a 50 ﬁnal µL concentrationof undiluted virus, of 8 µ gthen mL −three1. 1:2 dilutions were made. Dextran (Deax) was added to the medium to a final concentration of 8 µg mLThe−1. addition of Deax increased the infection efficiency of both LpVspike(+) and LpVspike(−) 1000-fold in CRFK cells. While Deax increased the infection efficiency of LpVspike(+)2.4. Effect of 10-fold Polybrene in ACE2–CRFK on Infectivity cells, it increased the infection efficiency of LpVspike(−) 1000-fold.Hexadimethrine These results bromide suggest that, also Deax known can as cause polybrene non-specific, is a cationic infections polymer in HEK293T, capable of ACE2–HEK293T,enhancing in vitro CRFK, HIV and-1 infection ACE2–CRFK by reducing cell lines. electrostatic Deax was repulsion therefore ruledbetween out virion as a s supplementand sialic foracid enhancing on the cell LpVspike surface [18] infection. Polybrene efficiency has forbeen our widely system. used in neutralization 2.4.and Effect infectivity of Polybrene assay ons Infectivity examining HIV and pseudo-typed lentiviruses coated with the SARS-CoV-2 S protein [1,15,19,20]. Therefore, we examined the effect of polybrene in our pseHexadimethrineudovirus system. bromide, Following also LpVspike(+) known as polybrene, infection, isACE2 a cationic–CRFK polymer cells produced capable a of enhancing in vitro HIV-1 infection by reducing electrostatic repulsion between virions reading of approximately 104 RLU with 50 μL of viral solution both with and without and sialic acid on the cell surface [18]. Polybrene has been widely used in neutralization polybrene supplementation, suggesting that polybrene did not provide any enhancement and infectivity assays examining HIV and pseudo-typed lentiviruses coated with the effect (Figure 5b). For both CRFK and ACE2–CRFK cells, no LpVspike(−) infection was SARS-CoV-2 S protein [1,15,19,20]. Therefore, we examined the effect of polybrene in detected following polybrene addition (Figure 5a,b). Therefore, polybrene did not induce our pseudovirus system. Following LpVspike(+) infection, ACE2–CRFK cells produced non-specific infections. a reading of approximately 104 RLU with 50 µL of viral solution both with and without polybrene supplementation, suggesting that polybrene did not provide any enhancement effect (Figure5b). For both CRFK and ACE2–CRFK cells, no LpVspike( −) infection was detected following polybrene addition (Figure5a,b). Therefore, polybrene did not induce non-speciﬁc infections.

2.5. Stability of ACE2 Expression in CRFK Cells and LpVspike(+) We found that CRFK cells were the most sensitive of the cell lines tested, producing a 10-fold higher RLU reading compared to the HEK293T cells. In this experiment, ACE2 was expressed in CRFK cells using a transient system, as opposed to a stable genome-integrated system. We therefore set out to determine the stability of transient ACE2 expression in CRFK cells, with the goal of optimizing transfection conditions. Following seeding and harvesting, ACE2-transfected CRFK cells were incubated for three time periods: 1 h, 1 day, and 2 days. At each time point, the ACE2-transfected CRFK cells were exposed to LpVspike(+) in 96-well plates, then incubated for a further 48 h. The luciferase reporter measurements were similar for each time point, suggesting that the CRFK cells stably Pathogens 2021, 10, 153 7 of 13

Pathogens 2021, 10, x FOR PEER REVIEW 7 of 14 express the ACE2 receptor throughout the 48-h period following transfection with the ACE2 vector (Figure6a).

Figure 5. (a) RLU measurements were taken at 48 h after infection in CRFK cell. (b) RLU measurements were taken at 48 h Figure 5. (a) RLU measurements were taken at 48 h after infection in CRFK cell. (b) RLU measure- after infection in ACE2 expressing CRFK cell. Approximately 2.3 × 104 cells of each cell line (CRFK and ACE2-expressing ments were taken at 48 h after infection in ACE2 expressing CRFK cell. Approximately 2.3 × 104 CRFK) were seeded per well in 96-well plates. The initial volume of LpVspike(+) used for infection was 50 µL of undiluted Pathogens 2021, 10, x FOR PEER REVIEW cells of each cell line (CRFK and ACE2-expressing CRFK) were seeded per well in 96-well8 plates. of 14 µ −1 virus, then three 1:2 dilutions wereThe made.initial Polybrenevolume of wasLpVspike(+) added to used the medium for infection to a ﬁnalwas 50 concentration µL of undiluted of 10 virus,g mL then. three 1:2 dilutions were made. Polybrene was added to the medium to a final concentration of 10 µg mL−1.

2.5. Stability of ACE2 Expression in CRFK Cells and LpVspike(+) We found that CRFK cells were the most sensitive of the cell lines tested, producing a 10-fold higher RLU reading compared to the HEK293T cells. In this experiment, ACE2 was expressed in CRFK cells using a transient system, as opposed to a stable genome- integrated system. We therefore set out to determine the stability of transient ACE2 expression in CRFK cells, with the goal of optimizing transfection conditions. Following seeding and harvesting, ACE2-transfected CRFK cells were incubated for three time periods: 1 h, 1 day, and 2 days. At each time point, the ACE2-transfected CRFK cells were exposed to LpVspike(+) in 96-well plates, then incubated for a further 48 h. The luciferase reporter measurements were similar for each time point, suggesting that the CRFK cells stably express the ACE2 receptor throughout the 48-h period following transfection with the ACE2 vector (Figure 6a). We then examined the stability of LpVspike(+) following incubation at 37 °C. After 1 h of incubation, luciferase expression had not changed, indicating that LpVspike(+) was stable (Figure 6b).

Figure 6. (a) Effect of varying the incubation period following ACE2 transfection on LpVspike(+) infectivity. Open circles: infectivityFigure 6. (a of) Effect CRFK of cells varying at 1 the h after incubation ACE2 transfection.period following Black ACE2 triangles: transfection infectivity on LpVspike(+) of CRFK cellsinfectivity. at 24 h Open after circles: ACE2 transfection.infectivity of CRFK Open invertedcells at 1 h triangles: after ACE2 infectivity transfection. of CRFK Black cells triangles: at 48 hinfectivity after ACE2 of CRFK transfection. cells at 24 The h after initial ACE2 volume trans- of LpVspike(+)fection. Open used inver forted infection triangles: was infectivity 50 µL of of undiluted CRFK cells virus, at 48 then h after three ACE2 1:2 dilutionstransfection. were The made. initial Approximately volume of LpVspike(+) 2.3 × 104 4 ACE2-expressingused for infection CRFKwas 50 cells µL wereof undiluted seeded pervirus, well then in 96-wellthree 1:2 plates. dilutions Relative were luciferase made. Approximately unit (RLU) measurements 2.3 × 10 ACE2 were-expressing CRFK cells were seeded per well in 96-well plates. Relative luciferase unit (RLU) measurements were taken at 48 taken at 48 h after infection. (b) Effect of varying the viral incubation period at 37 ◦C on LpVspike(+) infectivity. Following h after infection. (b) Effect of varying the viral incubation period at 37 °C on LpVspike(+) infectivity. Following infection, infection, cells were incubated with the virus for 10 min (open squares), 30 min (black triangles), or 60 min (open circles). cells were incubated with the virus for 10 min (open squares), 30 min (black triangles), or 60 min (open circles). The initial Thevolume initial of LpVspike(+) volume of LpVspike(+) used for infection used forwas infection 50 µL of wasundiluted 50 µL virus, of undiluted then three virus, 1:2 dilutions then three were 1:2 made. dilutions Approximately were made. 4 Approximately2.3 × 104 ACE2-expressing 2.3 × 10 ACE2-expressing CRFK cells were seeded CRFK cells per well were in seeded 96-well per plates. well inRLU 96-well measurements plates. RLU were measurements taken at 48 h wereafter takeninfection. at 48 h after infection.

2.6. Inhibition of LpVspike(+) in ACE2-Expressing CRFK Cells with Plasma and Monoclonal Antibodies We next assessed whether our LpVspike(+)/ACE2–CRFK cell system could be applied to assess antibody neutralization. We first calculated ID50 of two human convalescent sera, SARS-CoV-2 IgG and SARS-CoV-2 IgM, which were 1:350 (±1:20) and 1:1250 (±1:350), respectively (Figure 7). The luciferase readings at ID50 of LpVspike(+) against IgM and IgG were both approximately 1 × 104 RLU (Figure 7a).

Pathogens 2021, 10, x FOR PEER REVIEW 8 of 14

Figure 6. (a) Effect of varying the incubation period following ACE2 transfection on LpVspike(+) infectivity. Open circles: infectivity of CRFK cells at 1 h after ACE2 transfection. Black triangles: infectivity of CRFK cells at 24 h after ACE2 transfection. Open inverted triangles: infectivity of CRFK cells at 48 h after ACE2 transfection. The initial volume of LpVspike(+) Pathogensused2021, for10, 153infection was 50 µL of undiluted virus, then three 1:2 dilutions were made. Approximately 2.3 × 104 ACE2-ex-8 of 13 pressing CRFK cells were seeded per well in 96-well plates. Relative luciferase unit (RLU) measurements were taken at 48 h after infection. (b) Effect of varying the viral incubation period at 37 °C on LpVspike(+) infectivity. Following infection, cells were incubated with the virus for 10 min (open squares), 30 min (black triangles), or 60 min (open circles). The initial volume of LpVspike(+) used forWe infection then examinedwas 50 µL of the undiluted stability virus, of LpVspike(+) then three 1:2 dilutions following were incubation made. Approximately at 37 ◦C. After 2.3 × 104 ACE2-expressing1 CRFK h of incubation, cells were seeded luciferase per well expression in 96-well had plates. not RLU changed, measurements indicating were that taken LpVspike(+) at 48 h after was infection. stable (Figure6b).

2.6.2.6. Inhibition Inhibition of of LpVspike(+) LpVspike(+) in in ACE2-Expressing ACE2-Expressing CRFKCRFK CellsCells withwith PlasmaPlasma andand Monoclonal MonoclonalAntibodies Antibodies WeWe next next assessed assessed whether whether our our LpVspike(+)/ACE2–CRFK LpVspike(+)/ACE2–CRFK cell cell system system could could be applied be ap- toplied assess to antibody assess antibody neutralization. neutralization We ﬁrst. We calculated first calculated ID50 of twoID50 human of two convalescent human convales- sera, SARS-CoV-2cent sera, SARS IgG- andCoV SARS-CoV-2-2 IgG and SARS IgM,-CoV which-2 IgM were, which 1:350 were (±1:20) 1:35 and0 (± 1:12501:20) and (± 1:350),1:1250 respectively(±1:350), respectively (Figure7). The(Figure luciferase 7). The readingsluciferase at readings ID50 of LpVspike(+)at ID50 of LpVspike(+) against IgM against and IgGIgM were and both IgG approximatelywere both approximately 1 × 104 RLU 1 ×(Figure 104 RLU7a). (Figure 7a).

Pathogens 2021, 10, x FOR PEER REVIEW 9 of 14

Figure 7. (a–b) Neutralization of LpVspike(+) with human plasma extracted from patients infected with SARS-CoV-2. After pre-incubating 100 TCID50 of LpVspike(+) with human plasma, the virus- Figure 7. (a,b) Neutralization ofplasma LpVspike(+) mixture withwas humanadded to plasma ACE2- extractedexpressing from CRFK patients cells and infected incubated with SARS-CoV-2.at 37 °C for 48 After h, after pre-incubating 100 TCID50 ofwhich LpVspike(+) luciferase with activity human was plasma, measured. the virus-plasma Plasma was mixturesubjected was to serial added two to- ACE2-expressingfold dilutions from an CRFK cells and incubated at 37initial◦Cfor dilution 48 h, afterof 1:60 which down luciferase to 1:15,360 activity (X axis) was. X axis measured. and Y axis Plasma are indicated was subjected with log to serialscale. Fig- two-fold dilutions from an initialure a dilution show row of 1:60data, down figure to b 1:15,360 is calculated (X axis). by figure X axis a and. Y axis are indicated with log scale. Figure a show row data, ﬁgure b is calculated by ﬁgure a. Next, we examined the effect of monoclonal antibodies (mAbs) raised against SARS- CoVNext,-2. The we IC50 examined of the theIgG, effect IgM of, and monoclonal IgA anti-SARS antibodies-CoV- (mAbs)2 mAbs raisedagainst against LpVspike(+) SARS- CoV-2.were The0.45 IC50(±0.1) of, 0.0 the02 IgG, (±0. IgM,001), andand IgA0.004 anti-SARS-CoV-2 (±0.001) µg mL− mAbs1, respectively against LpVspike(+) (Figure 8b). wereEach 0.45of (the±0.1), anti 0.002-SARS (±-CoV0.001),-2 mAbs and 0.004 was (able±0.001) to neutralizeµg mL−1, p respectivelyseudo-typed (Figure lentiviru8b).ses Each coated of thewith anti-SARS-CoV-2 SARS-CoV-2 S mAbsprotein was during able the to neutralizeinfection of pseudo-typed ACE2-expressing lentiviruses CRFK c coatedells (Figure with SARS-CoV-28a,b). S protein during the infection of ACE2-expressing CRFK cells (Figure8a,b).

Figure 8. (a,b) NeutralizationFigure of LpVspike(+) 8. (a–b) Neutralization with anti-SARS-CoV-2 of LpVspike(+) monoclonal with anti antibodies-SARS-CoV (mAbs).-2 monoclonal After pre-incubating antibodies 100 TCID50 of LpVspike(+) with(mAbs). each After anti-SARS-CoV-2 pre-incubating neutralizing 100 TCID50 mAb, of LpVspike(+) the mAb-virus with mixtures each anti were-SARS added-CoV- to2 neutralizing ACE2- expressing CRFK cells and culturedmAb, the for mAb 48 h,-virus after whichmixtures luciferase were added activity to ACE2 was measured.-expressing TheCRFK IgG, cells IgM, and and cultured IgA mAbs for 48 h, were subjected to serial three-foldafter dilutions, which luciferase from an activity initial concentration was measured. of The 10 µ IgG,g mL IgM-1 down, and toIgA 0.016 mAbsµg mLwere−1 subjected. X axis and to serial three-fold dilutions, from an initial concentration of 10 µg mL-1 down to 0.016 µg mL−1. X axis Y axis are indicated with log scale. Left figure show row RLU data, right figure is calculated by left figure. and Y axis are indicated with log scale. Left figure show row RLU data, right figure is calculated by left figure.

3. Discussion In this study, we found that feline CRFK cells were 10-fold more sensitive to infection with S protein-coated HIV-1 pseudoviruses compared to human HEK293T cells. Given that both pseudoviral S-protein expression and CRFK ACE2 expression were required for infection with LpVspike(+), we conclude that our system reflects the natural SARS-CoV-2 infection process. Moreover, this system was tested by measuring the neutralizing activities of convalescent sera and anti-spike mAbs, thus demonstrating its practicality. In contrast to the high LpVspike(+) infectivity observed in CRFK cells, the infection rates of Cos7 and Vero cells, which are derived from macaque kidneys, were low. The LpVspike(+) pseudovirus was based on HIV-1, meaning that completion of the infection process following entry into the cytoplasm is dependent on whether the host cellular environment facilitates viral uncoating, reverse transcription, movement of the virus into the nucleus, and integration of the viral genome into chromosomes. Macaque cells possess restriction proteins such as APOBEC3 and TRIM5α/TRIM5CypA, which disrupt the HIV-1 genome and core [21], whereas cat cells express a truncated, non-functional version of Trim5α. CRFK cells may lack both functional APOBEC3 and Trim5α. Although human APOBC3 and Trim5α cannot prevent HIV-1 infection, human Trim5α exhibits weak restriction activity [22]. Our attempts to improve infection efficiency by adding Deax and polybrene were not successful. Deax caused non-specific infection in all combinations of cell types and pseudoviruses tested, regardless of the presence of the S protein or its ACE2 receptor. However, Deax specifically enhanced the infection of TZM-bl cells expressing human CD4/CCR5 with a HIV-1 envelope-coated pseudovirus. Although we do not have a definitive explanation for these results, we propose that Deax may disturb the membranes of both the cell and virus. Another unexpected result is that LpVspike(−) infected ACE2– HEK293T cells, but not ACE2–CRFK cells. ACE2 transfection might therefore perturb the membranes of HEK293T cells, causing non-specific infection. The neutralization assay developed in this work using modified cat CRFK cells is an efficient and safe method that could be a useful research tool, enabling the examination of the binding mechanism between the SARS-CoV-2 S protein and the ACE2 receptor, as well as the development of antiviral drugs. As demonstrated above (Figure8), this system could be employed to assess SARS-CoV-2 inhibitors and evaluate the neutralizing activity of convalescent antisera and anti-S protein mAbs; anti-S protein antibodies may be useful in treating critically ill COVID-19 patients. Although vaccine development is progressing at an unprecedented rate, this neutralization assay could still be a useful tool for determining the robustness of the B-cell response elicited by newly developed SARS-CoV-2 vaccines.

4. Materials and Methods 4.1. Cell Culture HEK293T cells (American Type Culture Collection (ATCC) CRL 3216), CRFK cells (ATCC CCL-94), African green monkey kidney clone of Vero-E6 (VeroE6) cells (ATCC CRL 1586), monkey kidney fibroblast-like cell lines (Cos7; ATCC CRL 1651) and TZM-bl cells (National Institute of Allergy and Infectious Diseases (NIH) ARP #8129) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM sodium pyruvate (MP Biomedicals Inc., Santa Ana, CA, USA) and 4 mM L-glutamine (Fujifilm Wako Pure Chemical Corporation). Cells were harvested and passaged using trypsin/ethylenediaminetetraacetic acid solution (Nacalai Tesque, Kyoto, Japan). All cell ◦ lines were maintained at 37 C under 5% CO2 in a humidified environment.

4.2. Generation of Pseudo-Typed Lentivirus with SARS-CoV-2 S Particles Cloning of all DNA plasmids was conducted by transformation into Escherichia coli Stbl3 cells, as described previously [23]. Lentiviral-based pseudo-type viruses were constructed as described previously [24]. In brief, pseudo-type lentiviruses coated with the Pathogens 2021, 10, 153 10 of 13

SARS-CoV-2 S protein harboring the vector 2019-nCov_pcDNA3.1(+)-P2A-eGFP spike (1 µg mL−1) was prepared by co-transfecting 5 × 105 cells mL−1 of HEK293T cells with the HIV-1 NL4-3 ∆Env∆ Vpr Luciferase Reporter Vector (1 µg mL−1)[25]. Additionally, 200 µL of Opti-MEM and 8 µL of X-Treme HP transfection reagent were ◦ added. Transfected HEK293T cells were incubated at 37 C under 5% CO2 for 2 days, after which the supernatant containing pseudo-typed lentivirus particles coated with SARS-CoV- 2 S protein was harvested and ﬁltered through a 0.45-µm ﬁlter (Millipore, Burlington, MA, USA); 400-µL aliquots were stored in 1.5-mL tubes at −80 ◦C. The 1 µg mL−1 HIV-1 NL4-3 ∆Env Vpr Luciferase Reporter Vector (pNL4-3. Luc. R-E-) (catalogue number 3418) was kindly provided by Dr. Nathaniel Landau (NIH AIDS Reagent Program, Division of AIDS). The 2019-nCov_pcDNA3.1(+)-P2A-eGFP spike vector (catalogue number MC_0101087) was purchased from Nacalai Tesque from Molecular Cloud. Pseudo-typed lentiviruses with SARS-CoV-2 S protein without the luciferase gene were also constructed to transfect the TZM-bl cell line. Pseudo-type lentivirus particles coated with SARS-CoV-2 S that harbored the 2019-nCov pcDNA3.1 plasmid (1 µg mL−1) or 2019-nCoV pCMV plasmid (1 µg mL−1) were generated by co-transfecting HEK293T cells with the pSG∆env vector (1 µg mL−1). The pSG∆env (catalogue number 11051) vector was kindly provided by John C. Kappes and Xiaoyun Wu (NIH AIDS Reagent Program, Division of AIDS).

4.3. Generation of Transient ACE2-Expressing Cell Lines HEK293T, CRFK, Cos 7, and TZM-bl cells were seeded at concentrations of 2.5 × 105 cells mL−1 in six-well plates, using DMEM supplemented with 10% fetal bovine serum, 2% L-glutamine and 2% pyruvate. Cell lines were incubated for 1 day, at which point cell confluency reached 60–80%. The pcDNA3.1+/C-(K) DYK-ACE2 (2 µg mL−1) plasmid was transfected into the cell lines with 200 µL of Opti-MEM and 8 µL of X-Treme HP transfection reagent. The pcDNA3.1+/C-(K) DYK-ACE2 plasmid (catalogue number MC_0101086) was purchased from Nacalai Tesque (Molecular Cloud). ACE2-expressing cell lines were seeded at a cell concentration of 2.5 × 105 cells mL−1 in six-well plates and incubated at 37 ◦C under 5% CO2 for 1 day. HEK293T cells were seeded in poly-L-lysine-coated 96-well plates (Sumitomo Bakelite Celltight Poly-L-lysine-coated flat bottom 96-well plates, catalogue no: ms-0096L). All other cell types were seeded in standard 96-well plates (Falcon tissue culture plate, 96 wells, flat bottom with low evaporation lid).

4.4. Neutralization Assays ACE2-expressing CRFK cells were seeded into 100 µL of medium to a concentration of 2.5 × 105 cell mL−1 in 96-well plates 1 day before infection. Previously harvested samples containing pseudo-typed lentiviral particles coated with the SARS-CoV-2 S protein were thawed. To perform the virus titration, pseudo-typed virus samples were serially diluted two-fold a total of nine times and transferred to a 96-well plate. Control wells contained only supplemented medium and no virus sample. Each dilution was made in triplicate. For infection, 100 µL of each LpVspike(+) or LpVspike2(+) dilution was added to the ◦ cell-seeded 96-well plates, which were incubated at 37 C under 5% CO2 for 2 days. Luciferase activity was measured in HEK293T, CRFK, Cos7, Vero, and TZM-bl cells. The details of the neutralization assays performed in this study have been described previously [26,27]. Brieﬂy, to measure luciferase activity, 50 µL of cell lysate solution (Toyo B-Net, Tokyo, Japan) was added to each well, and the plate was agitated for 15 min. An aliquot of 30 µL of lysate was transferred to a Nunc F96 MicroWell white plate (Thermo Fisher Scientiﬁc, Waltham, MA, USA), and luminescent substrate (30 µL) was added to each well. Luciferase activity was measured using a TriStar LB 941 reader (Berthold Tech- nologies, Bad Wildbad, Germany) and MikroWin software. ID50 values were calculated as previously described [28]. The neutralization assays were performed using adjusted viral doses that yielded equivalent infectivity levels for each cell line. The neutralization assays were performed in Pathogens 2021, 10, 153 11 of 13

a 96-well format as described previously. ID50 was reported as either the concentration or sample dilution at which the RLU readout was reduced by 50% compared to the RLU readings measured in the virus control wells (cells plus virus without test sample) after subtracting background RLU values from cell control wells (cells only, no virus or test sample) [29]. To test the stability of ACE2-expression in CRFK cells, ACE2-expressing CRFK cells were seeded to a concentration of 2.5 × 105 mL−1 in 96-well plates and incubated for three different time periods: 1 h, 1 day and 2 days. Pseudo-typed viruses were then added to a ﬁnal concentration of 6000 RLU mL−1 to the ACE2-expressing CRFK cells and incubated ◦ for 48 h at 37 C under 5% CO2. To evaluate the stability of LpVspike(+) when incubating at 37 ◦C, diluted pseudo- typed viruses were incubated in triplicate for 10, 30 and 60 min. To test viral infectivity after incubation at 37 ◦C, virus samples were added to ACE2-expressing CRFK cells in ◦ 96-well plates and incubated for 48 h at 37 C under 5% CO2. SARS-CoV-2 plasma serum of COVID-19 patients with varying IgM and IgG antibody levels was purchased from RayBiotech (Peachtree Corners, GA, USA). The serum plasma was subjected to serial three-fold dilutions, from an initial dilution of 1:60 down to 1:1,180,980. We added 120 µL of supplemented medium to each well in a 96-well plate, with the exception of the outer edge wells, to which 250 µL of phosphate-buffered saline was added. Next, 180 µL of 1/20- or 1/40-diluted plasma was added to the ﬁrst row of wells and subjected to three-fold serial dilutions. Control wells contained no plasma, only cells and virus. Then, 60 µL of pseudo-typed virus at a concentration of 6000 RLU mL−1 was added to wells containing the diluted plasma. The total volume of the diluted plasma and virus was approximately 180 µL. The plasma-virus mixtures were incubated at 37 ◦C under 5% CO2 for 1 h. After incubation, 150 µL of the mixture was added to the seeded ACE2-expressing CRFK cells in 96-well plates. The plates were incubated at 37 ◦C under 5% CO2 for 2 days, and then the luciferase activity of the samples was measured. ID50 was calculated as described previously [28]. Three anti-SARS-CoV-2 neutralizing mAbs were used, including human IgG (catalogue number E-AB-V1021), IgM (catalogue number E-AB-V1026), and IgA (catalogue number E-AB-V1027) isotypes with clone 8A5 Fab that was elicited using recombinant 2019-nCoV S-trimer Protein (His Tag) as the immunogen. The mAbs were diluted 10-fold three times from 1 to 0.001 µg mL−1. The experimental method for testing neutralizing mAbs was the same as the method used for assessing the neutralizing ability of plasma. IC50 values were calculated as described previously [28]. N mAbs were purchased from Elabscience (Houston, TX, USA).

5. Conclusions ACE2-expressing CRFK cells were 10 times more sensitive to viral infection compared to the typically used HEK293T cells. We tested our system with a neutralization assay and found that the pseudo-typed lentiviruses coated with SARS-CoV-2 S protein were neutralized by anti-SARS-CoV-2 IgG, IgM, and IgA mAbs and sera. We also evaluated the influence of Deax and polybrene on infectivity. Deax caused non-specific viral infection in the absence of ACE2 receptors and polybrene did not have a significant impact on infectivity or sensitivity. Our neutralization assay using pseudo-typed lentiviruses coated with SARS-CoV-2 S protein and ACE2-expressing CRFK cells is both easier and safer than the methods currently available and can aid researchers in understanding SARS-CoV-2 pathogenesis as well as discovering new drugs and vaccines.

Author Contributions: T.M. and Y.P. were involved in planning. T.M. supervised the research. T.M. and Y.P. conceived of the presented idea, Y.P. developed the theory and performed the computations. Y.P. carried out the experimental methods. T.M. and H.S. encouraged Y.P. to investigate for speciﬁc substitutions. Y.P. contributed to the design and implementation of the research, to the analysis of the results, and the writing of the manuscript. All authors discussed the results and commented on the manuscript. All authors have read and agreed to the published version of the manuscript. Pathogens 2021, 10, 153 12 of 13

Funding: This work was supported by a Research on HIV/AIDS grant from The Ministry of Health, Labour, and Welfare of Japan, by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (Grant No. 16H04682), and by a grant from the Japan Agency. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We thank Zafer Yazici, Komatsu Tadahiko, Yoshida Atsushi, Otsuki Mai, Yumi Nakamura, Ahmed Lugman Abdul Fatah, Theventhiran Mahandoran, and Soner Ulusoy for com- ments and assistance during the study. Conflicts of Interest: The authors declare no conflict of interest.

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