Kinetics of viral load and antibody response in relation to COVID-19 severity
Yanqun Wang, … , Yimin Li, Jincun Zhao
J Clin Invest. 2020. https://doi.org/10.1172/JCI138759.
Research In-Press Preview Infectious disease Virology
Graphical abstract
Find the latest version: https://jci.me/138759/pdf 1 Kinetics of viral load and antibody response in relation to COVID-19
2 severity
3 Running Title: Antibody Response in COVID-19 patients
4
5 Yanqun Wang1#, Lu Zhang2#, Ling Sang1#, Feng Ye1#, Shicong Ruan3#, Bei Zhong4#, Tie
6 Song5#, Abeer N. Alshukairi6#, Rongchang Chen7#, Zhaoyong Zhang1, Mian Gan1, Airu Zhu1,
7 Yongbo Huang1, Ling Luo1, Chris Ka Pun Mok8, Manal M. Al Gethamy9, Haitao Tan3,
8 Zhengtu Li1, Xiaofang Huang1, Fang Li1, Jing Sun1, Yanjun Zhang1, Liyan Wen1, Yuming Li1,
9 Zhao Chen1, Zhen Zhuang1, Jianfen Zhuo1, Chunke Chen1, Lijun Kuang1, Junxiang Wang1,
10 Huibin Lv8, Yongliang Jiang4, Min Li4, Yimin Lin4, Ying Deng4, Lan Tang4, Jieling Liang4,
11 Jicheng Huang10, Stanley Perlman 11, Nanshan Zhong1, Jingxian Zhao1*, J. S. Malik Peiris8*,
12 Yimin Li1*, Jincun Zhao1,2*
13
14 1State Key Laboratory of Respiratory Disease, National Clinical Research Center for
15 Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital
16 of Guangzhou Medical University, Guangzhou, Guangdong, China 510120
17 2Institute of Infectious disease, Guangzhou Eighth People's Hospital of Guangzhou Medical
18 University, Guangzhou, Guangdong, China 510060
19 3Yangjiang People’s Hospital, Yangjiang, Guangdong, China
20 4The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People’s Hospital,
21 Qingyuan, Guangdong, China
22 5Guangdong Provincial Center for Disease Control and Prevention, Guangzhou, Guangdong,
23 China
24 6King Faisal Specialist Hospital and Research Centre, Jeddah, Kingdom of Saudi Arabia
25 7Shenzhen Institute of Respiratory Disease, First Affiliated Hospital of South University of
26 Science and Technology of China (Shenzhen People's Hospital), Shenzhen, Guangdong,
27 China
1
28 8The HKU–Pasteur Research Pole, School of Public Health, Li Ka Shing Faculty of Medicine,
29 the University of Hong Kong, Hong Kong SAR, China 19406
30 9Al Nour Specialist Hospital, Makkah, Kingdom of Saudi Arabia
31 10 Technology Centre, Guangzhou Customs
32 11Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA
33
34 #These authors contributed equally to this work.
35
36 *Corresponding authors
37 Dr. Jincun Zhao, State Key Laboratory of Respiratory Disease, National Clinical Research
38 Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First
39 Affiliated Hospital of Guangzhou Medical University; Institute of Infectious disease,
40 Guangzhou Eighth People's Hospital of Guangzhou Medical University, No.195 Dongfeng Xi
41 Road, Yuexiu District, Guangzhou, China 510120. Tel: +86-20-83205059, email:
43 Dr. Yimin Li, State Key Laboratory of Respiratory Disease, National Clinical Research
44 Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First
45 Affiliated Hospital of Guangzhou Medical University, No.195 Dongfeng Xi Road, Yuexiu
46 District, Guangzhou, China 510120. Tel: +86-20-83062114, email: [email protected]
47 Dr. Peiris Malik, The HKU–Pasteur Research Pole, School of Public Health, Li Ka Shing
48 Faculty of Medicine, the University of Hong Kong; L6-42, Laboratory Block, 21 Sassoon
49 Road, Hong Kong SAR, China, 19406. Tel: +852 3917 7537, email: [email protected]
50 Dr. Jingxian Zhao, State Key Laboratory of Respiratory Disease, National Clinical Research
51 Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First
52 Affiliated Hospital of Guangzhou Medical University, No.195 Dongfeng Xi Road, Yuexiu
53 District, Guangzhou, China 510120. Tel: +86-20-83205061, email: [email protected]
54
55
2
56 Keywords: SARS-CoV-2, COVID-19, Antibody response, Cross-reactivity, Neutralizing
57 antibody
3
58 Abstract
59 The SARS-CoV-2 is the causative agent for COVID-19 pneumonia. Little is known about the
60 kinetics, tissue distribution, cross-reactivity and neutralization antibody response in COVID-
61 19 patients. Two groups of RT-PCR confirmed COVID-19 patients were enrolled in this study,
62 including 12 severe patients in ICUs who needed mechanical ventilation and 11 mild patients
63 in isolation wards. Serial clinical samples were collected for laboratory detection. Results
64 showed that most of the severe patients had viral shedding in a variety of tissues for 20~40
65 days post onset of disease (8/12, 66.7%); while the majority of mild patients had viral
66 shedding restricted to the respiratory tract and had no detectable virus RNA after 10 days
67 post-onset (9/11, 81.8%). Mild patients showed significantly lower IgM response compared
68 with that of the severe group. IgG responses were detected in most patients in both severe
69 and mild groups at 9 days post onset and remained high level throughout the study.
70 Antibodies cross-reactive to SARS-CoV and SARS-CoV-2 were detected in COVID-19
71 patients but not in MERS patients. High-levels of neutralizing antibodies were induced after
72 about 10 days post onset in both severe and mild patients which were higher in the severe
73 group. SARS-CoV-2 pseudotype neutralization test and focus reduction neutralization test
74 with authentic virus showed consistent results. Sera from COVID-19 patients, but not
75 convalescent SARS and MERS patients inhibited SARS-CoV-2 entry. Anti-SARS-CoV-2 S
76 and N IgG level exhibited moderate correlation with neutralization titers in patients’ plasma.
77 This study improves our understanding of immune response in human after SARS-CoV-2
78 infection.
79
4
80 Introduction
81 Before November 2019, six coronaviruses (CoVs) were known to infect humans and cause
82 respiratory disease, including four community-acquired CoVs OC43, 229E, HKU1, NL63 that
83 are endemic in humans (1-4), and two highly pathogenic CoVs, severe acute respiratory
84 syndrome CoV (SARS-CoV) (5) and Middle East respiratory syndrome CoVs (MERS-CoV)
85 (6) which have zoonotic transmission followed by variable transmission between humans.
86 Recently, a novel coronavirus first identified in Wuhan, Hubei province, China in late 2019
87 has spread a worldwide to cause a pandemic of pneumonia(7). The novel CoV was named
88 “SARS-CoV-2” and the disease was called “coronavirus disease 2019” (COVID-19) (8).
89 Robust human to human transmission of SARS-CoV-2 has led to a pandemic(9) involving
90 every province of China and then to more than 203 countries and territories. As of May 12
91 2020, 4,088,848 cases of COVID-19 have been reported World Health Organization (WHO),
92 including 283,153 deaths (10).
93 SARS-CoV-2 belongs to lineage B betacoronavirus and has high nucleotide
94 homology with bat SARS-like CoV and SARS-CoV (11). SARS-CoV-2 causes severe
95 respiratory illness similar to SARS-CoV. Older adults and individuals with comorbidities are
96 at higher risk for severe disease (12-14). Little is known about the immune response and
97 their relationship with the clinical outcomes in SARS-CoV-2 infected patients. Information
98 about kinetics of virus replication, neutralizing antibody responses and cross-reactivity with
99 other human respiratory CoV are also required for diagnosis, prognosis and epidemiology
100 investigations.
101 Here, by monitoring viral shedding and antibody responses in patients with severe
102 and mild disease in different tissues, we found that COVID-19 patients with different severity
103 of disease showed different patterns of viral shedding and antibody responses. Severe
104 patients had more prolonged viral shedding in a variety of tissues than mildly ill patients. IgM
105 responses in mild patients were much lower than those observed in severe patients
106 indicating IgM detection in mild patients was not sensitive and efficient. SARS-CoV-2-
107 specific antibodies were found in tissues outside respiratory tract in severe patients.
5
108 Detection of antibody responses in urine and other body fluids could be used to as a marker
109 to determine disease severity. By using plasma from SARS, MERS and COVID-19 patients,
110 strong cross-reactivities were detected between SARS-CoV-2 and SARS-CoV, but not
111 MERS-CoV which is an important information for differential diagnosis in Middle East
112 countries. Antibody against N or S protein were correlated with neutralizing antibody titers
113 which may be useful when screening of convalescent plasma for passive transfusion therapy.
6
114 Results
115 Patients and clinical information.
116 Twenty-three laboratory-confirmed SARS-CoV-2 patients were enrolled in this study. Serial
117 clinical samples were collected every 3~4 days for profiling the kinetics of antibody
118 responses and viral loads during SARS-CoV-2 infection. As showed in Table 1, a total of 12
119 severely ill patients and 11 mildly ill patients from three hospitals were enrolled in this study.
120 Most patients were older than 50 years old with a median age of 56 years (24~82 years old).
121 At the time of writing, most mild patients (8/11, 72.7%) were discharged from the hospital
122 while the majority of severe patients (10/12, 83.3%) were still in the ICU. Most of the patients
123 had visited Wuhan or had direct contact with other confirmed cases.
124
125 Prolonged viral shedding from multiple sites in severely patients.
126 A total of 461 clinical samples were obtained from 23 severe and mild patients, including 84
127 nasal swabs, 59 throat swabs, 36 sputum, 90 fecal samples, 79 urine samples, 113 samples
128 of plasma, and one biopsy of gastric juice. As showed in Fig. 1, most of the patients with
129 severe disease had viral shedding for up to 30~40 days post-onset; a majority of mild
130 patients had no detectable viral loads after 15 days post-onset. There was significant
131 difference in the peak viral load (P<0.001, T test) between severe and mild cases. The viral
132 loads in respiratory samples were higher in the severe group and gradually declined over
133 time. SARS-CoV-2 virus was mainly detected in respiratory samples (nasal swabs, throat
134 swabs and sputum) (Fig. 1A, B, C). However, in most of the severely ill patients, feces
135 remained positive for viral RNA over a prolonged time (Fig. 1D). Periodically, urine and
136 plasma were also tested positive (Fig 1E, F).
137
138 Mildly ill patients have lower IgM responses against SARS-CoV-2.
139 To understand the kinetics of the antibody responses against SARS-CoV-2 in patients, IgM
140 and IgG antibody responses against the N protein of SARS-CoV-2 in plasma were assayed
141 (Fig. 2). One hundred and twenty plasma specimens were obtained from 23 patients at
7
142 different time points. IgM responses in patients with severe disease increased within one to
143 two weeks post onset and gradually decreased after four weeks (Fig. 2A), while IgM
144 responses were much lower in mildly ill patients. Most of the mild patients (8/11) did not
145 produce significant IgM antibody throughout the whole disease course indicating IgM
146 diagnosis for mild patients was not sensitive and efficient (Fig. 2A). IgG responses emerged
147 at 10~15 days post onset (Fig. 2B). Most patients showed high levels of IgG antibodies,
148 which were maintained for at least for 6 weeks (Fig. 2B). Forty-eight plasma samples
149 collected from healthy voluntary donors (HD) in 2017-2018 were used as controls to assess
150 the specificity of the tests. As compared with positive and negative controls provided in the
151 kit, healthy donors did not have any SARS-CoV-2 specific antibodies.
152
153 Detection of IgM and IgG antibodies in respiratory specimens and other body fluids.
154 To investigate the presence of SARS-CoV-2-specific antibodies in other tissues with viral
155 shedding, 93 urine, 60 feces, 67 sputum, one BALF and one pleural effusion samples were
156 collected from severe and mild patients. IgM and IgG antibody response against SARS-CoV-
157 2 nucleocapsid protein were detected. As shown in Fig. 3A and B, viral specific IgM were
158 detected in urine (3/10) and sputum (4/10) in severe patients. While, viral specific IgG were
159 present in urine (7/10) and sputum (7/10) in 10 severe patients. In contrast, no antibody was
160 detected in the mild group indicating severe infection might result in tissue damage including
161 airways in the lung and kidneys (Fig. 3A, B). Detection rates of IgM were lower in the urine
162 and sputum of severely ill patients which was consistent with the larger size of IgM
163 pentamers than monomer IgG. Appearance of SARS-CoV-2 specific IgG in urine and
164 sputum could be a potential marker to determine disease severity. No antibody was detected
165 in fecal samples (Fig. 3C). SARS-CoV-2 specific IgM and IgG antibodies were also detected
166 in bronchoalveolar lavage fluid (BALF) and pleural effusion from the same severe patient
167 PT7 indicating various tissue damage and antibody distribution in severe patients (Fig. 3D).
168
8
169 S2 fragment of spike protein was preferentially recognized by SARS-CoV-2-specific
170 antibodies in patients.
171 To compare antigenicity of different SARS-CoV-2 structural proteins, S (aa 1-1213 of spike
172 protein), S1 (aa 1-685 of spike protein), S2 (aa 686-1213 of spike protein), RBD (receptor
173 binding domain, aa 319-514 of spike protein) and N proteins (aa 1-419 of nucleocapsid
174 protein) were used as coating antigens for ELISA assays (Fig. 4). All of the S, S1, S2, RBD
175 and N proteins were recognized by patient plasma and peaked at 3-4 weeks post-onset. The
176 seroconversion rates against S and S2 proteins reached 100% between 7~14 days after
177 illness onset. They were lower against S1 (10/20, 50%), RBD (13/20, 65%) and N (18/20,
178 90%) proteins at 7~14 days, and reached 100% after 3 weeks after illness onset (Fig. 4B, C,
179 E). S2 and S2 containing full-length S protein based ELISAs performed better than the
180 others and could detect SARS-CoV-2-spefic antibody in all patients even at the first two
181 weeks post-onset (Fig. 4A, D), while healthy donors did not have any SARS-CoV-2 specific
182 antibodies. No obvious differences in IgG response against viral proteins (S, S2, RBD and N)
183 were observed between severe and mild patients, except anti-S1 IgG response due to
184 limited samples. The correlations between IgG levels against different viral proteins (S, S1,
185 S2, N and RBD) were performed and compared as showed in Fig 4F. Most of IgG
186 responses against different viral proteins (RBD, N, S, and S1) showed moderate to strong
187 correlations with each other, except for the correlation between anti-S2 IgG and anti-S1/N
188 IgG. In addition, the anti-S and anti-S2 IgG (r2=0.6902) was better correlated than that
189 between anti-S and anti-N IgG (r2=0.4255).
190
191 Strong cross-reactive antibody responses between SARS-CoV-2 and SARS-CoV but
192 not MERS-CoV.
193 Spike (S) and nucleocapsid (N) proteins of six hCoVs were used to establish in-house ELISA
194 assays for IgG antibody detection. Ninety-six healthy donors were included as controls. As
195 shown in Fig 5A-D, plasma from both severe and mild groups recognized HCoV-229E, NL63,
196 HKU1 and OC43, which shared similar trend as healthy donors since these viruses were
9
197 prevalent worldwide, and most of adults were serologically positive for these viruses (15, 16).
198 To further investigate the cross reactivity with SARS-CoV and MERS-CoV, plasma was
199 obtained from 18 SARS convalescents in 2018, 15 years after SARS-CoV infection and 12
200 MERS convalescents in 2015, 6-18 months after MERS-CoV infection were also included in
201 this study. SARS convalescent plasma showed high-levels of cross-reactivity against SARS-
202 CoV-2 S and N proteins as predicted since SARS-CoV shared 88.6% with N and 69.2% with
203 S homologies, respectively (Fig 5E) (17). No obvious cross-reactivity was observed between
204 MERS-CoV and SARS-CoV-2-specific antibodies. MERS-CoV is still circulating in Middle
205 East countries. Patients infected with MERS-CoV represented similar clinical symptoms as
206 observed in COVID-19 patients. However, our result indicated that it was unlikely to
207 misdiagnose MERS and COVID-19 patients using serological tests. (Fig 5F).
208
209 Neutralizing activities were correlated with the magnitude of SARS-CoV-2 S and N
210 antibody responses.
211 Little is known about the kinetics of anti-SARS-CoV-2 neutralizing antibodies in patients with
212 severe or mild disease. Neutralizing antibodies against authentic SARS-CoV-2 in severe and
213 mild patients were evaluated. We found that 73.9% (17/23) of patients generated robust
214 neutralizing antibodies (FRNT50>500) 3 weeks post disease onset (Fig. 6A, 6B). Higher
215 neutralizing antibody titers were induced in the severely ill group as compared to mildly ill
216 patients. Plasma collected from patients 3 weeks post-onset were also tested to compare
217 their neutralizing activities against SARS-CoV-2 pseudotype (Fig 6C) and authentic virus
218 (Fig 6D) at a fixed dilution (1:40). Similar results were obtained with both neutralizing tests
219 indicating there was a good correlation between these two detection methods (21/23
220 pseudotype, 19/23 live virus-FRNT). SARS and MERS convalescents plasma could not
221 neutralize SARS-CoV-2 pseudotype and authentic virus indicating that SARS and MERS
222 convalescent patients could still be vulnerable to SARS-CoV-2 infection. (Fig. 6C, D). Anti-
223 SARS-CoV-2 S and N IgG level exhibited moderate correlation with neutralization titers of
224 patients’ plasma ((Pearson r=0.5393 P value<0.0001 S, r= 0.6709 P value<0.0001 N),
10
225 suggesting that monitoring S and N antibody levels could be useful to determine neutralizing
226 titer before convalescent plasma transfusion (Fig. 6E-F). No obvious correlation between
227 viral load and neutralizing titer was observed (Fig. 6G).
11
228 DISCUSSION
229 Temporal profile of serial viral loads from different tissue samples of patients indicated that
230 viral shedding was more common in respiratory and fecal material, especially in severe
231 patients, and to a much less extent in urine and blood. A recently study reported that
232 detectable SARS-CoV-2 viral RNA in blood strongly correlated with clinical severity (18).
233 A lower level of IgM response was observed in mildly ill patients compared with that
234 of the severe group. The lower level of IgM response associated with mild disease probably
235 reflects lower viral loads and viral antigens. Similar IgG responses were detected in both
236 severe and mild patients. Remarkably, virus-specific IgM and IgG were detectable in serial
237 urine and sputum samples of most of severe patients, but not in mild patients indicating
238 severe tissue damage in these patients which could be used as a marker to determine
239 disease severity.
240 Several SARS-CoV-2 proteins induced IgG responses in severe and mild patients.
241 Although all proteins including S, S1, S2, RBD and N could be used to detect antibody
242 response, S2 and S2 containing full length S proteins performed better in these ELISA
243 assays and antibodies could be detected in most infected patients in the first two weeks post
244 onset. The sensitivity of detection method was associated with abundance, conservation and
245 antigenicity of viral proteins indicating S2 region possessed more epitopes recognized by
246 viral-specific antibodies.
247 Six human coronaviruses have been identified. Although SARS-CoV disappeared,
248 HCoV-229E, NL63, HKU1 and OC43 are circulating worldwide and MERS-CoV primarily is
249 in Middle East countries (19, 20). Antigenic cross-reactivity was observed previously
250 between SARS-CoV and HCoV-229E, OC43 (21). Cross-reactivity analysis between SARS-
251 CoV-2 and other six CoVs provided essential information for diagnosis, epidemiological
252 studies and help to dissect the roles of pre-existing antibodies against other CoVs in SARS-
253 CoV-2 and MERS-CoV infected patients. Our ELISA results showed that although our
254 healthy controls were often infected with 229E, NL63, HKU1 of OC43 viruses, there was no
255 cross reaction in the SARS-CoV-2 ELISA assays, indicating that the SARS-CoV-2 S, S1,
12
256 RBD, S2 and N protein assays are specific for the virus. Our results showed that most of the
257 SARS-CoV-2 infected patients were infected with the 4 low pathogenic CoVs previously.
258 Plasma from COVID-19 patients also showed high-level of antibody binding to SARS-CoV N
259 and S proteins which was consistent with high homology between the genomes of these two
260 viruses. Of note, stronger cross-reactivities against SARS-CoV proteins were observed in
261 severe patients. Although SARS-CoV-2 and HCoV-OC43 full-length spike proteins share
262 relative low homology (27.2%), some regions in S2 fragments show high homology
263 (70%~80%), which might account for the cross-reactivity between HCoV-OC43 and SARS-
264 CoV-2. Lack of obvious cross-reactivity between MERS-CoV and SARS-CoV-2 plasma ruled
265 out the possibility of misdiagnosis using serologic tests in Middle East region where MERS-
266 CoV has been prevalent for over 7 years.
267 The plaque/focus reduction neutralization assay is considered to be “gold standard”
268 to quantity antibody neutralization titer. In this study, SARS-CoV-2 pseudotype neutralization
269 system and FRNT assay were compared. SARS-CoV-2 pseudotype expressing SARS-CoV-
270 2 spike protein was successfully used to detect neutralizing antibody with similar sensitivity
271 which could be useful for hospitals without BSL-3 labs where they could do neutralizing test
272 before convalescent plasma transfusion. In addition, anti-SARS-CoV-2 S and N IgG level
273 exhibited moderate correlation with neutralization titers of patients’ plasma, which also
274 provided an alternative method to determine neutralizing titers. Convalescent plasma from
275 SARS and MERS survivors could not inhibit SARS-CoV-2 pseudotype and authentic virus
276 entry. Although SARS-CoV shared 88.6% homology with SARS-CoV-2, a recent study
277 showed their RBD which is the major domain for neutralizing antibody induction was different
278 in structure observed under CryoEM (22).
279 In summary, this study provides comprehensive information on kinetics, tissue
280 distribution, cross-reactivities and neutralization of antibody responses in COVID-19 patients,
281 and will improve our understanding of humoral immune response in human after SARS-CoV-
282 2 infection as well as will shed light on diagnosis, prognosis, convalescent plasma
283 transfusion therapy and epidemiology studies of SARS-CoV-2 infection in human.
13
284 Methods
285 Patient enrollment and sample collection.
286 Between January 28th to February 24th, 2020, 23 patients with confirmed novel coronavirus
287 (SARS-CoV-2) infection by real-time PCR were hospitalized in the First Affiliated Hospital of
288 Guangzhou Medical University (n=13 patients), the Sixth Affiliated hospital of Guangzhou
289 Medical University, Qingyuan People’s Hospital (n=3 patients) and Yangjiang People’s
290 Hospital (n=7 patients). Respiratory swabs, sputum, body fluids and plasma specimens were
291 collected every three to four days following admission. Clinical data including patient
292 demographic information and clinical outcome was retrieved from the medical records. A
293 total of 12 severely and 11 mildly ill patients were enrolled for serological analysis. Patients
294 with severe pneumonia who were admitted to the ICU and required mechanical ventilation
295 were enrolled in severe group; the patients with a mild clinical presentation (mainly with fever,
296 cough, malaise and headache, including non-pneumonia or mild pneumonia) were enrolled
297 into mild group. Eighteen SARS convalescent plasma were collected in 2018 from
298 healthcare workers who were infected with SARS-CoV 17 years ago. Twelve MERS
299 convalescent plasma were acquired as previously described(23), Ninety-six healthy donor
300 plasma samples which were collected in 2017-2018 were used as controls in this study.
301
302 Real-time PCR detection of SARS-CoV-2.
303 Nucleic acid was extracted from respiratory samples and urine using a Viral RNA extraction
304 kit from Zybio Inc (Chongqing). RNA extraction from feces and blood were performed by
305 total RNA extraction kit from Sangon Biotech (Shanghai). A real-time PCR assay kit
306 targeting SARS-CoV-2 RdRp and N gene regions was provided by Zybio Inc. Serial
307 sampling of nasal swabs, throat swabs, sputum, anal swabs, urine and blood were used to
308 monitor viral shedding.
309
310 IgM and IgG ELISA assays.
14
311 Clinical samples, including plasma, urine, sputum, feces, bronchoalveolar lavage fluid (BALF)
312 and pleural fluid were collected and analyzed for the presence of SARS-CoV-2-specific IgM
313 and IgG antibodies to the SARS-CoV-2 nucleoprotein using a commercial antibody detection
314 kit provided by Lizhu Medicine Group Holding CO., LTD (Zhuhai). Plasma and urine were
315 used directly in ELISA assays while feces and sputum were diluted with the same volume of
316 PBS and centrifuged at 3000 rpm for 10 minutes before supernatant was harvested for
317 antibody detection. According to the manufacturer’s instructions, threshold IgM value was
318 negative control + 0.1; while IgG threshold for the presence of SARS-CoV-2 infection was
319 negative control+0.13.
320
321 Comparison of antibody response to different SARS-CoV-2 proteins.
322 To assess the antibody response to different SARS-CoV-2 proteins or different fragments of
323 the spike protein, SARS-CoV-2 S (spike protein, 1203 aa), S1 (675 aa), S2 (533 aa), RBD
324 (228 aa) and N (424 aa) proteins were obtained from Sino Biological, Inc (Beijing). and in-
325 house ELISAs for detection of SARS-CoV-2 specific IgG antibody were established. Briefly,
326 96-well plates (Jet, Biofil Co., Ltd, Guangzhou) were coated with 100 μl/well (0.5 μg/ml) of
327 SARS-CoV-2 S, S1, S2, RBD or N protein in DPBS buffer (Thermo Fisher Scientific (China),
328 Shanghai) overnight at 4oC. After blocking (DPBS, 10%FBS), 100 μl diluted plasma (1:100)
329 were added and plates were incubated at 37 oC for one hour. After washing, plates were
330 incubated with 100 μl of HRP-conjugated mouse anti-human IgG (H+L) antibody (Code: 109-
331 035-088, Jackson ImmunoResearch, West Grove, PA) at 37 oC for one hour. Reactions were
332 visualized by adding 50 μl TMB substrate solution (Biohao Biotechnology Co., Ltd., Beijing).
333 Optical densities at 450 nm were then read. The mean value of heathy donor anonymous
334 plasma (named HD group) collected in 2017-2018 plus 3 standard deviations was used as
335 the detection threshold.
336
337 Cross-reactivity between SARS-CoV-2 and other six human CoVs.
15
338 To determine the cross-reactivity between SARS-CoV-2 and other six human respiratory
339 CoVs, including SARS-CoV, MERS-CoV, HCoV-NL63, HCoV-OC43, HCoV-229E and
340 HCoV-HKU1, we purchased S and N proteins of all six human CoVs from Sino Biological
341 (Beijing), Inc and established an in-house viral protein ELISA assays for cross-reactive IgG
342 antibody detection as described above.
343
344 Pseudotype-based neutralization assay.
345 To safely and rapidly assess neutralization activity of patients’ plasma against SARS-CoV-2,
346 we developed a luciferase reporter-based pseudotype neutralization assay which has a non-
347 replicative human immunodeficiency virus backbone coated with the SARS-CoV-2 spike
348 protein. SARS-CoV-2 pseudotype neutralization assays were performed on ACE2-
349 overexpressing HEK293 cells (ATCC, Manassas, VA) (HEK293-ACE2 cells) in 96-well
350 microplates. Fifty microliters of 20-fold diluted patients’ plasma were combined with equal
351 volume of the SARS-CoV-2 pseudotype and incubated for 60 min at 37oC. The mixtures
352 were then added to 96-well plates seeded with HEK293-ACE2 cells at 3.8×104 cell/well.
353 Cells were further cultured for 40 hours at 37oC. Luciferase activities in cell lysates were
354 measured using steady-Glo luciferase assay kit (Promega, Madison, WI). Neutralizing
355 activity was defined as the ratio of inhibition of SARS-CoV-2 pseudotype (SARS-CoV-2-pp)
356 luciferase activity comparing patients’ plasma (1:40 dilution) to control.
357
358 Focus reduction neutralization test.
359 SARS-CoV-2 focus reduction neutralization test (FRNT) was performed in a certified
360 Biosafety level 3 lab. Fifty microliters plasma samples were serially diluted, mixed with 50 μl
361 of SARS-CoV-2 (100 focus forming unit, FFU) in 96-well microwell plates and incubated for 1
362 hour at 37oC. Mixtures were then transferred to 96-well plates seeded with Vero E6 cells
363 (ATCC, Manassas, VA) for 1 hour at 37oC to allow virus entry. Inoculums were then
364 removed before adding the overlay media (100 μl MEM containing 1.2%
16
365 Carboxymethylcellulose, CMC). The plates were then incubated at 37oC for 24 hours.
366 Overlays were removed and cells were fixed with 4% paraformaldehyde solution for 30 min.
367 Cells were permeabilized with 0.2% Triton X-100 and incubated with cross-reactive rabbit
368 anti-SARS-CoV-N IgG (Cat: 40143-R001, Sino Biological, Inc, Beijing) for 1 hour at room
369 temperature before adding HRP-conjugated goat anti-rabbit IgG(H+L) antibody (1:4000
370 dilution) (Code: 111-035-144, Jackson ImmunoResearch, West Grove, PA). Cells were
371 further incubated at room temperature. The reactions were developed with KPL TrueBlue
372 Peroxidase substrates (Seracare Life Sciences Inc, Milford, MA). The numbers of SARS-
373 CoV-2 foci were calculated using an EliSpot reader (Cellular Technology Ltd, Shaker
374 Heights, OH).
375
376 Statistical analysis
377 Statistical analysis was performed using Graphpad Prism software, version 7.00. Pearson’s
378 correlation coefficient was used to assess the relationship between anti-viral IgG levels and
379 neutralizing titers. A Student’s t test was used to analyze differences in mean values
380 between groups. A P value <0.05 was considered to be statistically significant. (*, P values
381 of ≤0.05. **, P values of ≤0.01. ***, P values of ≤0.001.) All values are depicted as mean ±
382 SEM. Multiple comparisons following one-way ANOVA and Kruskal-Wallis tests were
383 performed for statistical analysis in cross-reactivity and neutralizing experiments since
384 experiment for each virus was independently carried out. Bonferroni correction was used to
385 avoid inflation of experiment-wise Type I error.
386
387 Study approval
388 Institutional Review Board approval from the Health Commission of Guangdong Province as
389 well as the Ethics Committees of each of the hospitals to use patients and healthy donor
390 samples in this study was obtained. Written informed consent forms were obtained from all
391 participants.
17
392 Author contributions
393 JZ, YL, MP, and JZ conceived the study; YW, LZ, LS, FY, SR, BZ, TS, AA, RC, ZZ, MG, AZ,
394 YH and LL collected clinical specimen and executed the experiments; CM, MG, HT, ZL, XH,
395 FL, JS, YZ, LW, YL, ZC, ZZ, JZ, CC, LK, JW, HL, YJ, ML, YL, YD, LT and JL analyzed the
396 data; NZ, PS and JH contributed to critical revision of the manuscript; JZ, MP, YW and LZ
397 wrote the manuscript. All authors revised and approved the final version.
398
399 Acknowledgements
400 This study was approved by the Health Commission of Guangdong Province to use patient’s
401 specimens for this study. This study was funded by grants from The National Key Research
402 and Development Program of China (2018YFC1200100, 2018ZX10301403), the emergency
403 grants for prevention and control of SARS-CoV-2 of Ministry of Science and Technology
404 (2020YFC0841400) and Guangdong province (2020B111108001, 2018B020207013,
405 2020B111112003), the Guangdong Province Basic and Applied Basic Research Fund
406 (2020A1515010911), National Key Technology R&D Program (2018YFC1311900),
407 Guangdong Science and Technology Foundation (2019B030316028), State Key Laboratory
408 of Respiratory Disease (SKLRD-QN-201715, SKLRD-QN-201912 and SKLRD-Z-
409 202007)Guangzhou Medical University High-level University Innovation Team Training
410 Program (Guangzhou Medical University released [2017] No.159), the NIH, USA (PO1
411 060699 (SP), National Natural Science Foundation of China (NSFC)/Research Grants
412 Council (RGC) Joint Research Scheme (N_HKU737/18), the Research Grants Council of the
413 Hong Kong Special Administrative Region, China (Project no. T11-712/19-N). We thank the
414 patients who took part in this study. We thank Dr. Eric Lau at the HKU–Pasteur Research
415 Pole, School of Public Health, and University of Hong Kong for helping statistical analysis.
416
417 DISCLOSURE STATEMENT
418 No conflict of interest was reported by the authors
419
18
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Table 1. Demographic and clinical outcomes of SARS-CoV-2 infected patients
Patient Gender1 Age City, Country Visited Wuhan Direct contact Receiving Clinical number Hubei, China2 with confirmed mechanical outcome cases3 ventilation4 Group A: Severe PT1 M 67 Guangzhou, YNY Still in ICU patients (n=12) China PT2 M 49 Guangzhou, YYY Still in ICU China PT3 M 50 Guangzhou, YNAY Still in ICU China PT4 M 53 Guangzhou, NYY Still in ICU China PT5 M 61 Guangzhou, YNY Still in ICU China PT6 M 42 Guangzhou, YNAY Transferred out China of ICU PT7 M 72 Guangzhou, NYY Still in ICU China PT8 M 58 Guangzhou, NYY Still in ICU China PT9 M 42 Guangzhou, YYY Transferred out China of ICU P10 M 79 Guangzhou, NYY Still in ICU China PT11 M 61 Yangjiang, YYY Still in ICU China PT12 F 60 Yangjiang, YYY Still in ICU China Group B: mild PT13 F 56 Yangjiang, YYN Discharged patients China (n=11) PT14 F 25 Guangzhou, NYN Discharged China PT15 M 50 Guangzhou, NNNIsolationward China PT16 F 82 Guangzhou, NYNIsolationward China PT17 M 24 Yangjiang, YNN Discharged China PT18 M 35 Yangjiang, YYN Discharged China PT19 M 69 Yangjiang, YNN Discharged China PT20 M 77 Yangjiang, YYN Discharged China PT21 M 52 Qingyuan, YNAN Discharged China PT22 M 50 Qingyuan, YNAN Discharged China PT23 M 65 Qingyuan, YNANIsolationward China
1Gender: M male, F female 2Visited Wuhan Hubei, China. Y yes, N no, NA not available. 3Direct contact with confirmed cases. Y yes, N no, NA not available 4Receiving mechanical ventilation Figure 1
A Nasal swab D Feces
PT1 PT3 PT5 PT7 PT9 PT11 PT13 PT15 PT17 PT19 PT21 PT23 1 3579 11 13 15 17 19 21 23 25 27 29 31 33 35 37 Days post-onset
B Pharyngeal swab E Urine
C Sputum F Blood
Ct value
1 Figure 1. Temporal profile of serial viral load from different tissue samples. Viral loads
2 in patients in the ICU (PT1~PT12) and mild patients with mild disease (PT13~PT23) in nasal
3 swabs (A), pharyngeal swabs (B), sputum (C), feces (D), urine (E) and blood (F) were
4 measured. X-axis indicates the days post-onset, y-axis indicates patient numbers. Heat map
5 of Ct values of viral loads were shown. Ct value < 37 indicates the presence of SARS-CoV-2
6 nucleic acid in the samples. Each square represents one sample detected and gray squares
7 indicate viral nucleotide acid negative. Figure 2
A
B
1 Figure 2. Kinetics of IgM and IgG responses against SARS-CoV-2 in severe and mild
2 patients. IgM (A) and IgG (B) antibody response against the N protein of SARS-CoV-2 in
3 plasma were detected. Serial plasma samples were collected from 12 severe and 11 mild
4 SARS-CoV-2 infected patients. Forty-eight plasma previously collected from healthy
5 voluntary donors in 2017-2018 were used as healthy donor group (HD). Positive (PC) and
6 negative (NC) controls provided by detection kit were included to ensure test validity. Figure 3
A Urine
Mild patient IgM 2.5
2.0
1.5 45 0n m
D 1.0 O 0.5
0.0 0 6 12 18 24 30 36 42 Days post-onset
B Sputum
Severe patient IgM Mild patient IgM Severe patient IgG Mild patient IgG 2.5 2.5 2.5 2.5
2.0 2.0 2.0 2.0
1.5 1.5 1.5 1.5
1.0 1.0 1.0 1.0
0.5 0.5 0.5 0.5
0.0 0.0 0.0 0.0 0 6 12 18 24 30 36 42 0 6 12 18 24 30 36 42 0 6 12 18 24 30 36 42 0 6 12 18 24 30 36 42 Days post-onset Days post-onset Days post-onset Days post-onset
C Feces Mild patient IgM 2.5
2.0
m 1.5 450 n
D 1.0 O 0.5
0.0 0 6 12 18 24 30 36 42 Days post-onset
D BALF and Pleural effusion IgM detection 3.0 2.5
m 2.0 50 n 4 1.5 D
O 1.0 0.5 0.0 F n D C C L io F n D C C s H P N L io A s H P N BA ffu B u e ff l e ra l ra u Pleu le P
1 Figure 3. Kinetics of IgM and IgG responses against SARS-CoV-2 in different tissues.
2 Specimens from urine (A), sputum (B), feces (C), BALF and pleural effusion (D) from
3 COVID-19 patients were detected for the presence of IgM and IgG antibody against the N
4 protein of SARS-CoV-2. Positive (PC) and negative (NC) control provided by detection kit
5 were included to ensure test validity. Plasma from 48 health donors (HD) were also included. Figure 4
A B
C D m n 0n m 0 5 5 4 4 D D O O
E F m 50 n 4 D O
1 Figure 4. IgG antibody response against different SARS-CoV-2 proteins or fragments.
2 Plasma collected at different time points after admission were used for IgG detection in
3 different protein-coated ELISA assays, including S (1209 aa) (A), S1 (681 aa) (B), RBD (457
4 aa) (C), S2 (539 aa) (D), N (430 aa) (E). Eleven plasma from healthy donors (HD) were used
5 as controls. The correlations between IgG levels against different viral proteins were
6 analyzed and summarized. Pearson’s correlation coefficient was used to assess the
7 relationship among anti-viral IgG levels of different proteins (F). A Student’s t test was used to
8 analyze differences in mean values between groups (A-E), a P value <0.05 was considered
9 to be statistically significant. Figure 5 A B
C D
E SARS-S SARS-N *** *** F 2.5 *** 2.5 *** ns *** ns *** 2.0 2.0 ns *** ns 1.5 ** 1.5 1.0 1.0 0.5 0.5 0.0 0.0 Severe Mild SARS HD Severe Mild SARS HD
1 Figure 5. IgG cross-reactivity analysis between the other six human CoVs and
2 SARS-CoV-2. Spike (S) and nucleoprotein (N) of the other six human CoVs were used as
3 coated target antigen to establish in-house ELISA to detect IgG antibody for HCoV-229E (A),
4 HCoV-NL63 (B), HCoV-HKU1 (C), HCoV-OC43 (D), SARS-CoV (E), MERS-CoV (F). Plasma
5 from 96 healthy donors and 23 SARS-CoV-2 infected patients were used (A-F). Severe:
6 severe COVID-19 patient, mild: mild COVID-19 patients; HD: healthy donors. Plasma from 18
7 SARS (E) and 12 MERS (F) convalescents were used as control, respectively. A Student’s t
8 test was used to analyze differences in mean values between groups (A-F). Experiment for
9 each virus was independently carried out. Multiple comparisons following one-way ANOVA
10 and Kruskal-Wallis test were performed for statistical analysis. Bonferroni correction was
11 used to avoid inflation of experiment-wise Type I error. In Fig 5A-5D, a difference was
12 considered statistically significant when the p-value is lower than 0.0167 (=0.05/3) (*, P values
13 of ≤0.0167. **, P values of ≤0.0033. ***, P values of ≤0.00033). In Fig 5E-5F, a difference was
14 considered statistically significant when the p-value is lower than 0.0083 (=0.05/6) (*, P values
15 of ≤0.0083. **, P values of ≤0.0017. ***, P values of ≤0.00017). Figure 6 A Severe B 2000
1500 0 5 T
1000 PT1 PT7 N R
PT2 PT8 F PT3 PT9 500 PT4 PT10 PT5 PT11 PT6 PT12 0 0 5 10 15 20 25 30 35 40 45
Days post-onset
C D SARS-CoV-2 authentic virus
** ns 100 ** 80
% ns
e 60 t
a ns r 40 ns n 20 iti o 0 i b h
n -20 I -40 Severe Mild SARS MERS HD
E F G
2.0
1.5
1.0
0.5 r = 0.6709 P<0.0001 0.0 0 500 1000 1500 2000
FRNT50
Figure 6. Neutralizing and cross protection of antibody response against SARS-CoV-2 in severe and mild patients. Serial plasma were collected from severe (A) and mild (B) SARS-CoV-2 infected patients, and used for authentic SARS-CoV2 neutralizing test FRNT50 to evaluate kinetics of neutralizing antibodies in SARS-CoV-2 infected patients. Plasma collected three weeks post-onset were used to compare cross neutralizing antibodies between severe, mild SARS-CoV-2 patients and SARS-CoV convalescents using SARS-CoV-2 pseudotype (C) and authentic virus (D) at a fixed dilution (1:40). A Student’s t test was used to analyze differences in mean values between groups. Experiment for each virus was independently carried out. Multiple comparisons following one-way
ANOVA and Kruskal-Wallis tests were performed for statistical analysis. Bonferroni correction was used to avoid inflation of experiment-wise Type I error. There were a total of 10 pairwise comparisons among 5 groups. Hence, a difference was considered statistically significant when the p-value is lower than 0.005 (=0.05/10) (*, P values of
≤0.005. **, P values of ≤0.001. ***, P values of ≤0.0001) (C-D). Pearson’s correlation coefficient was used to assess the relationship between neutralizing titer and S and N-specific IgG level (E, F) and viral loads of respiratory specimens
(G) were analyzed.