Characterization of anti-viral immunity in recovered individuals infected by SARS-CoV-2-广东盛普生命科技有限公司

首页 ꄲ 盛普前沿 ꄲ COVID-19 ꄲ Characterization of anti-viral immunity in recovered individuals infected by SARS-CoV-2

Characterization of anti-viral immunity in recovered individuals infected

by SARS-CoV-2

Ling Ni

1, *

, Fang Ye

2, *

, Meng-Li Chen

3, *

, Yu Feng

, Yong-Qiang Deng

, Hui Zhao

, Peng

Wei

, Jiwan Ge

, Xiaoli Li

, Lin Sun

, Pengzhi Wang

, Peng Liang

, Han Guo

, Xinquan

Wang

, Cheng-Feng Qin

, Fang Chen

4,#

, Chen Dong

1,7,#

1. Institute for Immunology and School of Medicine, Tsinghua University, Beijing 100084,

China

2. Department of Hematology, Chui Yang Liu Hospital affiliated to Tsinghua University,

Beijing 100022, China

3. Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Institute of

Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China

4. Department of Cardiology, Chui Yang Liu Hospital affiliated to Tsinghua University,

Beijing100022, China

5. School of Life Sciences, Tsinghua University, Beijing 100084, China

6. Department of Orthopedics, Chui Yang Liu Hospital affiliated to Tsinghua University,

Beijing 100022, China

7. Beijing Key Lab for Immunological Research on Chronic Diseases, Beijing 100084,

China

*These authors contributed equally to this work.

#To whom correspondence should be addressed: Chen Dong, chendong@tsinghua.edu.cn;

or Fang Chen, anzhenchenfang@163.com

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Abstract

The WHO has declared SARS-CoV-2 outbreak a public health emergency of international

concern. However, to date, there was hardly any study in characterizing the immune

responses, especially adaptive immune responses to SARS-CoV-2 infection. In this study, we

collected blood from COVID-19 patients who have recently become virus-free and therefore

were discharged, and analyzed their SARS-CoV-2-specific antibody and T cell responses. We

observed SARS-CoV-2-specific humoral and cellular immunity in the patients. Both were

detected in newly discharged patients, suggesting both participate in immune-mediated

protection to viral infection. However, follow-up patients (2 weeks post discharge) exhibited

high titers of IgG antibodies, but with low levels of virus-specific T cells, suggesting that they

may enter a quiescent state. Our work has thus provided a basis for further analysis of

protective immunity to SARS-CoV-2, and understanding the pathogenesis of COVID-19,

especially in the severe cases. It has also implications in designing an effective vaccine to

protect and treat SARS-CoV-2 infection.

Keywords: SARS-CoV-2, COVID-19 patients, humoral immunity, cellular immunity

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At the end of 2019, patients with Coronavirus Disease 2019 (COVID-19) were identified

in Wuhan, China

, infected by a novel coronavirus, now named as severe acute respiratory

syndrome coronavirus 2, SARS-CoV-2. The WHO then declared this outbreak a public health

emergency of international concern

. The genome sequence of SARS-CoV-2 bears 96%

and 79.5% identity to that of a bat coronavirus and SARS-CoV, respectively

. Like SARS-

CoV and MERS-CoV, SARS-CoV-2 belongs to the beta genus Coronavirus in the

Corornaviridae family

. Clinically, several papers showed that most COVID-19 patients

developed lymphopenia as well as pneumonia with higher plasma levels of pro-inflammatory

cytokines in severe cases

6-8

, suggesting that the host immune system is involved in the

pathogenesis

9,10

. Patients infected by SARS-CoV or MERS-CoV were previously reported to

have antibody responses

11-14

, but exhibited defective expression of type I and II interferon

(IFN), indicative of poor protective immune responses

15-17

. However, to date, there was hardly

any study in characterizing the immune responses, especially adaptive immune responses to

SARS-CoV-2 infection. Only one COVID-19 patient was shown with nucleocapsid protein

(NP)-specific antibody response, in which IgM peaked at day 9 after disease onset and then

switched to IgG by week 2

, suggesting involvement of humoral immunity. SARS-CoV-2-

specific T lymphocyte response was unclear. In this study, we collected blood from COVID-

19 patients who have recently become virus-free and therefore were discharged, and

analyzed their SARS-CoV-2-specific antibody and T cell responses.

Clinical and pathological characteristics of the 12 COVID-19 patients in this study were

shown in Table 1. All the patients initially showed mild symptoms via CT scan and were

positive during SARS-CoV-2 nucleic acid testing. Of them, 6 (patients #1-6) were newly

discharged, while the remaining 6 were 2 weeks post discharge (follow-up patients, patients

#7-12). In line with the previous reports, 2 patients (#5, 8) showed lymphopenia (normal range

is 1.1-3.2X10e9 cells per L). However, only two travelled to Wuhan city within the past 3

months. One serum was obtained before the SARS-CoV-2 outbreak (healthy donor #1). 3

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additional healthy donors (#2-4) who had been in close contacts with the patients were

recruited in this study. Human AB serum (GemCell, CA) was used as a negative control.

In order to detect anti-viral immune responses, we first constructed recombinant pET28-

N-6XHis by linking 6 copies of His tag to the C-terminus of NP in the pET28-N vector (Biomed,

Cat. number: BM2640). Escherichia coli transformed with pET28-N-6xHis was lysed and

tested by Coomassie blue staining to confirm NP expression at 45.51 kDa. NP was further

purified by Ni-NTA affinity chromatography and gel filtration. The purity of NP was

approximately 90% (Figure S1A). The presence of NP was subsequently confirmed by anti-

Flag antibody (Figure S1B). The RBD region of S protein (S-RBD) and main protease (doi:

https://doi.org/10.1101/2020.02.19.956235) were produced by a baculovirus insect

expression system and purified to reach the purity of 90% (Figure S1A).

Using sera from patients and healthy donors, IgG and IgM against SARS-CoV-2 NP, main

protease and S-RBD antigens were analyzed. There was no significant antibody response to

main protease in sera from several patients (data not shown), suggesting that it may not serve

as an antigen for humoral immunity. We thus focused on NP and S-RBD. The serum from a

patient and human AB serum were titrated in order to determine optimal dilutions (data not

shown). Dilution of 1:50 was used for IgM and 1:150 for IgG. NP- and S-RBD-specific IgM and

IgG antibodies were both detected in the sera of newly discharged patients, compared with

healthy donor groups (Figure 1A). Anti-SARS-CoV-2 IgG antibodies were also more obviously

observed than IgM in the follow-up patients (#7-12), when compared with healthy donors

(Figure 1A). Taken together, these findings indicate that COVID-19 patients mounted IgG and

IgM responses to SARS-CoV-2 proteins, especially NP and S-RBD, and also suggest that

infected patients could maintain their IgG levels, at least for two weeks.

Since RBD domain of the S protein has been shown to bind to human receptor ACE2

the existence of antibodies against it may suggest neutralization of SARS-CoV-2 infection. To

assess this, we performed pseudovirus particle-based neutralization assay. As shown in

Figure 1B, patients #1, 2, 4 and 5, all within the discharged group, had high levels of

neutralizing antibody titers. These results demonstrate that most recently discharged patients

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had protective humoral immunity to SARS-CoV-2. All except patient #11, the follow-up

patients had lower levels of neutralizing antibody titers than recently discharged patients,

though all positive with the exception of patient #7 being negative. Whether protective humoral

immunity can be maintained needs further investigation.

To explore cellular immune responses to SARS-CoV-2, we isolated PBMCs from the

whole blood and phenotypically analyzed them by flow cytometry (Figure 2A). We found that

compared to discharged patients, there was a trend towards an increased frequency of NK

cells in the follow-up patients (Figure S2). However, there was no significant difference in

terms of the percentages of T cells among those two groups and the healthy donors.

To assess virus-specific cellular immunity, we then treated PBMCs with recombinant NP,

main protease and S-RBD, followed by IFN-g ELISpot analysis. The results were considered

positive if there were at least 1-fold increase in the numbers of IFN-g-secreting T cells in the

subject than in the healthy donors. As shown in Figure 2B, compared with healthy donors, the

numbers of IFN-g-secreting NP-specific T cells in patients #1, 2, 4 and 5 were much higher

than other patients, suggesting that they had developed SARS-CoV-2-specific T cell

responses. Of note, patients #1, 2, 4 and 5 developed both strong humoral and cellular

immune responses. Main protease-specific T cells were detected in patient #1, 2 and 5, while

patients # 1, 2, 4, 5 and 6 showed S-RBD-specific T cells. Although the numbers of IFN-g-

secreting S-RBD specific T cells were much lower than those of NP-specific T cells, they could

be detected in more patients than those for other viral proteins. S-RBD thus not only elicited

humoral immunity that may result in blockade of receptor binding during viral entry in host

cells, but also induced T cell immune responses, suggesting S-RBD is a promising target for

SARS-CoV-2 vaccines. In the follow-up patients, only patient #8 who showed lymphopenia

before treatment still had a high number of IFN-g-secreting T cells in response to NP, main

protease and S-RBD (Figure 2B), which suggests that anti-viral T cells may not be maintained

at high numbers in the PBMCs in the recovered patients. More interestingly, when combining

all 12 patients in our analysis, there was a significant correlation between the neutralizing

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antibody titers and the numbers of NP-specific T cells (Figure 2C), indicating that the

development of neutralizing antibodies may be correlated with the activation of anti-viral T

cells. Thus, effective clearance of virus may need collaborative humoral and cellular immune

responses.

In summary, we provided the first analysis of SARS-CoV-2-specific humoral and cellular

immunity. Both were detected in newly discharged patients, suggesting both participate in

immune-mediated protection to viral infection. However, follow-up patients exhibited high titers

of IgG antibodies, but with low levels of virus-specific T cells, suggesting that they may enter

a quiescent state.

Our work has thus provided a basis for further analysis of protective immunity to SARS-

CoV-2, and understanding the pathogenesis of COVID-19, especially in the severe cases. It

has also implications in designing an effective vaccine to protect and treat SARS-CoV-2

infection.

perpetuity.

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Materials and Methods

Ethics statement

All procedures followed were in accordance with the ethical standards of the responsible

committee on human experimentation (the institutional review board at Tsinghua University)

and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained

from all subjects for being included in the study. All patient data were anonymized before study

inclusion. The blood samples of COVID-19 patients and healthy donors were obtained from

Chui Yang Liu Hospital affiliated to Tsinghua University in Beijing.

Expression and Purification of recombinant proteins

The recombinant His-tagged NP of SARS-CoV-2 was expressed in E. coli by a T7

expression system, with 1 mM IPTG induction at 37 ºC for 4 h. pET-28aHis-tagged S-RBD

(amino acids 319-541) was expressed by a Baculovirus system in insect cells (doi:

https://doi.org/10.1101/2020.02.19.956235). Purified proteins were identified by SDS-PAGE

gels and stained with Coomassie blue. Western blot was performed to confirm their

antigenicity by mouse anti-His monoclonal antibody (Proteintech, HRP-66005).

Isolation of PBMC

PBMCs were isolated from anti-coagulant blood using Ficoll-Hypaque gradients (GE

Healthcare Life Sciences, Philadelphia, PA) as previously described

under the BSL-3 facility

in AMMS.

Anti-SARS-CoV-2 IgG/IgM ELISA

For IgM/IgG testing, 96-well ELISA plates were coated overnight with recombinant NP

and S-RBD (100 ng/well). The sera from COVID-19 patients were incubated for 1 h at 37°C.

An anti-Human IgG-biotin conjugated monoclonal antibody (Sino Biological Inc., Wayne, PA)

and streptavidin-HRP were used at a dilution of 1:500 and 1:250, respectively, and anti-human

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IgM-HRP conjugated monoclonal antibody (Inova Diagnostics, Inc., San Diego, CA). The OD

value at 450 was calculated.

Neutralizing antibody assay

Pseudovirus expressing the SARS-CoV-2 S protein was produced as described

previously

. pNL43Luci and GP-pCAGGS were co-transfected into 293T cells. 48 hours later,

SARS-CoV-2 pseudovirus-containing supernatants were mixed with at least 6 serially diluted

serum samples from the COVID-19 patients at 37°C for 1 hour. Then the mixtures were

transferred to 96-well plates containing monolayers of Huh-7 cells. 3 hours later, the medium

was replaced. After incubation for 48 h, the cells were washed, harvested in lysis buffer and

analyzed for luciferase activity by the addition of luciferase substrate. Inhibition rate = [1-(the

sample group- the cell control group) / (the virus control group- the cell control group)] x 100%.

The neutralizing antibody titer were calculated by performing S-fit analysis via Graphpad Prism

7 software.

Interferon Gamma (IFN-γ) ELISpot

IFN-g-secreting T cells were detected by Human IFN-g ELISpot

pro

kits (MABTECH AB,

Sweden) according to the manufacture protocol. Fresh PBMCs were plated in duplicate at

150k per well and then incubated 48h with 1uM of recombinant proteins. Spots were then

counted using an ELIspot Reader System (AT-Spot2100, atyx). The number of spots was

converted into the number of spots per million cells and the mean of duplicate wells plotted.

FACS staining

PBMCs were washed with PBS plus 2% FBS (Gibco, Grand Island, NY), and then Fc

blocking reagent (Meltenyi Biotec, Inc., Auburn, CA) was added followed by a wash with PBS

plus 2% FBS. Cells were then incubated for 30 min on ice with anti-CD45 (H130) (BioLegend),

anti-CD3 (OKT3) (BioLegend), anti-CD8 (SK1) (BD), anti-CD56 (HCD56) (BioLegend), anti-

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CD38 (HIT2) (BioLegend) and live/dead fixable aqua dye (eF660, eBioscience), washed twice

with PBS plus 2% FBS and then stored at 4 °C until acquired by FACS Verse (BD Biosciences,

San Jose, CA). Data were analyzed using FlowJo software (Version 10.0.8, Tree Star Inc.,

Ashland, Or).

Statistical analysis

Prism 7 software is used for statistical analysis. Student’s t test was performed for two-

group analysis. Pearson’s correlation coefficients were calculated. P values less than 0.05

were considered to be statistically significant.

ACKNOWLEDGEMENT

This work was supported in part by grants from Natural Science Foundation of China

(NSFC, Grant No. 31991173 to CD), National key research (Grant No. 2016YFC130390 to

LN) and an award from Zhejiang University Foundation (to CD).

Conflict of interest

LN, YF, WP and CD have filed a provisional patent on the methodology of detecting

SARS-CoV-2-specific antibody responses.

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Figure legends

Figure 1. Detection of antibody responses to recombinant SARS-CoV-2 proteins in

COVID-19 patients.

(A) Serological responses of 12 COVID-19 patients to recombinant NP (top) and S-RBD

(bottom). The experiment was performed in duplicates. (B) Measurement of neutralizing

antibody titers by pseudovirus-based assay. The experiment was performed in triplicates. NP,

nucleocapsid protein. S-RBD, receptor binding domain of spike protein. HD, healthy donor. Pt,

patient. HD#1, the serum was collected in 2018. HD#2-4, the sera were from close contacts.

*P<0.05, 0.05<**P<0.001, ***P<0.001.

Figure 2. T cell responses to recombinant SARS-CoV-2 proteins in COVID-19 patients.

(A) Phenotypic analysis of PBMCs from representative COVID-19 patients. (B) IFN-g ELISpot

analysis of COVID-19 patients to recombinant proteins. The experiment was performed in

duplicates. (C) Correlation analysis of the neutralizing antibody titers with the numbers of NP-

specific T cells (n=12). M protease, main protease. NP, nucleocapsid protein. S-RBD, receptor

binding domain of spike protein.

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Table 1 Clinical and pathological characteristics of the COVID-19 patients

Pt#

Sex

Age

Travel in

Wuhan

Fever

Fatigue

Lymphocyte

Count

Days in

hospital

Before treatment

Discharge

CT scan

NA test

CT scan

NA test

yes

1.1X10

Patchy ground

glass shadows on

both lungs

improvement

2.5X10

Multiple patchy

ground glass and

high-density

shadows in both

lungs

improvement

yes

1.7X10

Exudative lesion

of the right lower

lung

improvement

yes

1.5X10

Patchy ground

glass shadows on

both lungs

significant

improvement

yes

0.8X10

Patchy ground

glass shadows on

both lungs

significant

improvement

yes

2.1X10

Multiple ground

glass shadows in

both lungs

significant

improvement

yes

2.9X10

Right lung

inflammation

normal

yes

0.7X10

Multiple patchy

ground glass

shadows are seen

in the left lung,

and the upper

lobe of the left

lung is obvious

significant

improvement

yes

1.9X10

Double lung veins

thickened

normal

#10

yes

1.9X10

Ground glass in

the pleura of the

lower lobe of both

lungs

normal

#11

yes

1.1X10

Patchy ground

glass shadows on

both lungs

significant

improvement

#12

yes

2.3X10

Multiple ground

glass shadows in

both lungs

normal

Notes: pt, patient; F, female; M, male; P, positive; N, negative; NA, nucleic acid

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Figure 1

NP-based ELISA

S-RBD-based ELISA

IgM

IgG

IgM

IgG

IgG NP 1:50

AB serum

HD from Donglab

HD from hospital

Pts #1-6

Pts #7-12

0.0

0.5

1.0

1.5

2.0

2.5

Serum

450

AB serum

HD from Donglab

HD from hospital

Pts #1-6

Pts #7-12

***

AB serum

HD#1

HD#2-5

Pts#1-6

Pts#7-12

#10

#11

#12

Discharged pt.

Follow-up pt.

Neutralizing antibody titer

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none

main protease

S-RBD

1000

2000

3000

4000

Spot counts per million PBMCs

Pt#7

Pt#8

Pt#9

Pt#10

Pt#11

none

RBD

none

main protease

S-RBD

1000

2000

3000

4000

Spot counts per million PBMCs

Pt#7

Pt#8

Pt#9

Pt#10

Pt#11

Pt#12

M protease

Gated on live

lymphocytes

Gated on live

CD3

CD56

cells

Gated on live

CD3

CD56

cells

CD56

CD3

CD8

CD3

CD38

CD3

Discharged

Follow-up

Figure 2

Spot counts per million PBMCs

none

M protease

Neutralizing antibody titer

NP-specific T cell No.

none

S-RBD

500

1000

1500

2000

Spot counts per million PBMCs

HD#1

HD#2

HD#3

Pt#1

Pt#2

Pt#3

Pt#4

Pt#5

Pt#6

none

RBD

none

main protease

500

1000

1500

2000

2500

3000

3500

4000

Spot counts per million PBMCs

HD#1

HD#2

HD#3

Pt#1

Pt#2

Pt#3

Pt#4

Pt#5

Pt#6

none

main protease

S-RBD

500

1000

1500

2000

2500

3000

3500

4000

Spot counts per million PBMCs

HD#1

HD#2

HD#3

Pt#1

Pt#2

Pt#3

Pt#4

Pt#5

Pt#6

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