key: cord-286466-scokdxp2
authors: Tani, Hideki; Tan, Long; Kimura, Miyuki; Yoshida, Yoshihiro; Yamada, Hiroshi; Fukushi, Shuetsu; Saijo, Masayuki; Kawasuji, Hitoshi; Ueno, Akitoshi; Miyajima, Yuki; Fukui, Yasutaka; Sakamaki, Ippei; Yamamoto, Yoshihiro; Morinaga, Yoshitomo
title: Evaluation of SARS-CoV-2 neutralizing antibodies using a vesicular stomatitis virus possessing SARS-CoV-2 spike protein
date: 2020-08-23
journal: bioRxiv
DOI: 10.1101/2020.08.21.262295
sha: 
doc_id: 286466
cord_uid: scokdxp2

SARS-CoV-2 is a novel coronavirus that emerged in 2019 and is now classified in the genus Coronavirus with closely related SARS-CoV. SARS-CoV-2 is highly pathogenic in humans and is classified as a biosafety level (BSL)-3 pathogen, which makes manipulating it relatively difficult due to its infectious nature. To circumvent the need for BSL-3 laboratories, an alternative assay was developed that avoids live virus and instead uses a recombinant VSV expressing luciferase and possesses the full length or truncated spike proteins of SARS-CoV-2. Furthermore, to measure SARS-CoV-2 neutralizing antibodies under BSL2 conditions, a chemiluminescence reduction neutralization test (CRNT) for SARS-CoV-2 was developed. The neutralization values of the serum samples collected from hospitalized patients with COVID-19 or SARS-CoV-2 PCR-negative donors against the pseudotyped virus infection evaluated by the CRNT were compared with antibody titers determined from an immunofluorescence assay (IFA). The CRNT, which used whole blood collected from hospitalized patients with COVID-19, was also examined. As a result, the inhibition of pseudotyped virus infection was specifically observed in both serum and whole blood and was also correlated with the results of the IFA. In conclusion, the CRNT for COVID-19 is a convenient assay system that can be performed in a BSL-2 laboratory with high specificity and sensitivity for evaluating the occurrence of neutralizing antibodies against SARS-CoV-2.

Coronavirus with closely related SARS-CoV. SARS-CoV-2 is highly pathogenic in humans and is 25 classified as a biosafety level (BSL)-3 pathogen, which makes manipulating it relatively difficult 26 due to its infectious nature. To circumvent the need for BSL-3 laboratories, an alternative assay was 27 developed that avoids live virus and instead uses a recombinant VSV expressing luciferase and 28 possesses the full length or truncated spike proteins of SARS-CoV-2. Furthermore, to measure 29 SARS-CoV-2 neutralizing antibodies under BSL2 conditions, a chemiluminescence reduction 30 neutralization test (CRNT) for SARS-CoV-2 was developed. The neutralization values of the serum 31 samples collected from hospitalized patients with COVID-19 or SARS-CoV-2 PCR-negative 32 donors against the pseudotyped virus infection evaluated by the CRNT were compared with 33 was designated as pCAG-SARS-CoV-2. The plasmid, which contains the S protein gene with a 19 89 aa truncation at the C-terminus, was constructed using the cDNA of pCAG-SARS-CoV-2. The S 90 proteins with the 19 aa deletion of coronaviruses were previously reported to show increased 91 efficiency regarding incorporation into virions of VSV (9, 10). 92 93

Human (Huh7 and 293T), monkey (Vero), hamster (BHK and CHO), and mouse (NIH3T3) cell 95 lines were obtained from the American Type Culture Collection (Summit Pharmaceuticals 96

International, Tokyo, Japan). All cell lines were grown in Dulbecco's modified Eagle's medium 97 (DMEM; Nacalai Tesque, Inc., Kyoto, Japan) containing 10% heat inactivated fetal bovine serum 98 (FBS). 99 100

Pseudotyped VSVs bearing the S protein, the 19 aa-truncated S protein of SARS-CoV-2, or VSV-G 102 were generated as described below. Briefly, 293T cells were grown to 80% confluence on 103 collagen-coated tissue culture plates and then transfected with each expression vector: 104 pCAG-SARS-CoV-2 S-full, pCAG-SARS-CoV-2 S-t19, and pCAG-VSV-G. After 24 h of 105 incubation, the cells transfected with each plasmid were infected with G-complemented (*G) 106 VSV∆G/Luc (*G-VSV∆G/Luc)(11) at a multiplicity of infection (MOI) of 0.5 per cell. Then, the 107 virus was adsorbed and extensively washed four times with 10% FBS DMEM. After 24 h of 108 incubation, to remove cell debris, the culture supernatants containing pseudotyped VSVs were 109 centrifuged, and then, they stored at −80°C until ready for use. The pseudotyped VSV bearing 110 SARS-CoV-2 S protein or SARS-CoV-2 truncated S protein are referred to as Sfullpv or St19pv, 111

respectively. The infectivity of Sfullpv, St19pv, or VSVpv to 293T cells was assessed by measuring 112 the luciferase activity. The value of the relative light unit (RLU) of luciferase was determined using 113 a PicaGene Luminescence Kit (TOYO B-Net Co., LTD, Tokyo, Japan) and GloMax Navigator 114 System G2000 (Promega Corporation, Madison, WI), according to the manufacturer's protocol. (PBS) containing 1% NP40. Then, the lysates were centrifuged to separate insoluble pellets from 120 supernatants. The supernatants were used as samples. The Sfullpv or St19pv, which were generated 121 as described above, were pelleted through a 20% (wt/vol) sucrose cushion at 25,000 rpm for 2 h in 122 an SW41 rotor (Beckman Coulter, Tokyo, Japan). Then, the pellets were resuspended in PBS. Each 123 sample that was boiled in loading buffer was subjected to 10% sodium dodecyl 124 sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). According to the manufacturer's protocol, 125 the proteins in the gel were stained with CBB Stain One (Nacalai Tesque, Inc.). Next, the proteins 126 in another gel were electrophoretically transferred to a methanol-activated polyvinylidene difluoride 127 (PVDF) membrane (Millipore, Billerica, MA) and reacted with COVID-19 hospitalized patient sera 128 (#12). Then, immune complexes were visualized with SuperSignal West Dura Extended Duration 129 Substrate (Pierce, Rockford, IL) and detected by an LAS3000 analyzer (Fuji Film, Tokyo, Japan). 130 131

Twenty-three serum samples were collected from hospitalized patients with COVID-19 who were 133 admitted to the University of Toyama Hospital, Toyama, Japan. In addition, nineteen serum 134 samples were collected from COVID-19 PCR-negative donors at the University of Toyama 135

Hospital. The diagnosis of COVID-19 in all patients or donors was assessed using the real-time 7 PCR method with specific primers, which were developed at the National Institute of Infectious 137

Diseases, Japan (12). 138

By using a blood collection tube containing EDTA, whole blood samples were obtained from 5 139 hospitalized patients (the University of Toyama Hospital) with COVID-19. 140 141

The patient sera used in this study were collected from participants after obtaining informed consent. 143

To examine neutralization of the human serum or whole blood samples against pseudotyped viruses, 144

Vero cells were treated with serially diluted sera or whole blood of convalescent patients with 145 COVID-19 or PCR-negative donors and then inoculated with Sfullpv, St19pv, or VSVpv. To 146 remove hematopoietic cells from whole blood samples, centrifugation was performed at 2,000 × g 147 for 5 min. Infectivity of the pseudotyped viruses were determined by measuring luciferase activities 148

after 24 h of incubation at 37 °C. and ∆Gpv by CBB staining (Fig. 1A, left panel) . Overall, the amount of St19 proteins incorporated 173 was higher than that of Sfull proteins, although the amount of structural proteins of VSV was 174 almost the same level among all the virions. These results indicate that the incorporation of 175 truncated S proteins into VSV particles was more efficient than the full-length S protein. 176

Next, ∆Gpv, Sfullpv, St19pv, and VSVpv were inoculated into the indicated cell lines to examine 177 the infectivity of pseudotyped viruses to various mammalian cell lines (Fig. 1B) . Among the tested 178 cell lines, Huh7, 293T, Vero, and BHK cells were susceptible to Sfullpv and St19pv infection. 179

showed no susceptibility. Notably, the infectivity of St19pv was higher than that of Sfullpv in Vero 181

To determine the specificity of infection of Sfullpv and St19pv, a neutralization assay of the 183 pseudotyped viruses was performed using sera of two hospitalized COVID-19 patients. The 9 infectivity of Sfullpv and St19pv, but not that of VSVpv in Vero cells, were clearly inhibited by 185 sera of the patients in a dose-dependent manner (Fig. 2) . These data indicated that Sfullpv and 186

St19pv infection exhibited an S protein-mediated entry. 187 188

To examine the neutralization of COVID-19 hospitalized patients or COVID-19 PCR-negative 191 donors against St19pv, Vero cells with each serum were infected with St19pv and VSVpv. 192 Neutralization of St19pv was observed based on the sixteen sera of COVID-19 hospitalized patients 193 at a rate of more than 99% (Fig. 3A) . Sera, which did not show the neutralization of St19pv, were 194 derived from COVID-19 hospitalized patients who were hospitalized for a short period before 195 antibody production (such as within 3 days after onset). No neutralization was observed in the 196

VSVpv infection by any of the sera of COVID-19 PCR-negative donors ( Fig. 3A and B) . The dot 198 plot graph shows a classification of each pseudotyped virus from Fig. 3A and 3B graphs (Fig. 3C) . 199

Due to the presence of convalescent and non-convalescent patient sera, the degree of neutralization 200 activity of St19pv by COVID-19 hospitalized patient sera was variable. 201

To examine the correlation of the antibody titers using CRNT compared to those determined by the 204 IFA, the IFA was also performed using COVID-19 hospitalized patients or COVID-19 205 PCR-negative donors. The fluorescence intensity of IFA was correlated with the sera, which exhibit 206 a high neutralizing activity in the CRNT (Fig. 3 and Table 1 ). Sera with low neutralizing activity in 207 the CRNT showed weak fluorescence intensity, and sera that demonstrated no neutralizing activity 208 in the CRNT were also negative by IFA. 209 210

Also, we compared the neutralizing effect of pseudotyped viruses between the sera and whole blood 212 of COVID-19 hospitalized patients with or without centrifugation (Fig. 4) . After centrifugation of 213 whole blood, hematopoietic cells, including red blood cells, may be removed. As a result, the 214 neutralization of both sera and whole blood against St19pv infection was observed with or without 215 centrifugation (Fig. 4) . Although the neutralizing activities of whole blood were higher than those 216 of the sera in St19pv infection, the infectivity of VSVpv was reduced by approximately 1/10 217 without centrifugation (Fig. 4A) . The reduction of infectivity of both St19pv and VSVpv was 218 suppressed, by removing hematopoietic cells in whole blood after centrifugation (Fig. 4B) . 219

Therefore, some inhibitory factors, such as hematopoietic cells, may be involved in pseudotyped 220 virus infection in whole blood. 221 222 223

A rapid, safe, and highly sensitive CRNT system using VSV-based pseudotyped viruses with 225 SARS-CoV-2 S or truncated S proteins was developed. Because this system utilizes replication and 226 translation of VSV, neutralization against pseudotyped virus infection can be determined within 12-227 16 hours. Another pseudotyped viral system that uses retroviral or lentiviral vectors takes 228 approximately 48 h to obtain results. Therefore, the VSV-based pseudotyped viral system is 229 considered more useful. In addition, since measurement of luciferase activity is a quantitative 230 method, it is not necessary to count GFP-positive cells. Therefore, this CRNT system permits a 231 simple and objective evaluation for the neutralization. 232

For many viral species, CRNT systems were developed using VSV-pseudotyped viruses with 233 their own envelope proteins (11, (13) (14) (15) . In SARS-CoV-2, researchers recently demonstrated the 234 construction of pseudotyped viruses and evaluation of the presence of neutralizing antibodies (16). 235

In this study, we prepared a pseudotyped virus that possesses a truncated SARS-CoV-2 S 236 protein, which showed higher infectivity. Furthermore, the neutralizing activity of the test sera and 237 whole blood against the pseudotyped virus was quantitatively detected in a convalescent patient 238 with COVID-19, while the donor sera of the COVID-19 PCR-negative patient showed a negative 239 reaction. In the CRNT of St19pv, the infectivity of St19pv was reduced by 99% or more by the 240 convalescent phase patient sera. This demonstrated that the convalescent phase patient sera of 241 COVID-19 exhibited a high neutralizing antibody activity. The results determined by the CRNT 242 also correlated with those determined by the IFA. Antibodies against the S protein of SARS-CoV-2 243 in COVID-19 convalescent patient sera were capable of neutralizing the viral infection. 244

If using whole blood in the CRNT becomes possible, the work of separating serum will no 245 longer be necessary. The CRNT can be performed with an extremely small amount of blood sample 246

(only a few microliters). Therefore, when we confirmed that the CRNT with whole blood of the 247 convalescent phase patient of COVID-19 was possible, a high neutralizing activity by the CRNT 248 should be observed. However, the infectivity of the control VSVpv was also reduced by the whole 249 blood control. Since many hematopoietic cells, including red blood cells, are contained in whole 250 blood, these cells are present on the Vero cells used in the CRNT with whole blood. The possibility 251 highly exists that this inhibition is due to the presence of hematopoietic cells because removal of the 252 cells with centrifugation suppressed non-specific reduction of the pseudotyped viral infection. 253

However, inhibition of St19pv infection shows a stronger neutralizing activity compared to VSVpv 254 infection. Therefore, evaluating the CRNT using whole blood is possible. 255

The neutralizing antibody measurement system using pseudotyped viruses for SARS-CoV-2 is 256 an effective tool for evaluating the presence or duration of the neutralizing antibody in convalescent 257 patients and to screen for those who present with the neutralizing antibody among suspected 258

populations. In addition, this CRNT system does not require the use of infectious viruses to measure 259 neutralizing antibodies. Therefore, once the pseudotyped virus system is established, it can be made 260 available at many laboratories without BSL-3 facilities. Furthermore, because of the measuring 261 system by chemiluminescence, the results can be obtained safely and quickly. Finally, the CRNT 262 using whole blood is a simpler and safer method because it can be measured with only a very small 263 amount of blood from an eligible person. 

The authors declare no conflicts of interest in association with the present study. 

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