Effectiveness of convalescent plasma therapy in severe
COVID-19 patients
Kai Duan
a,b,1
, Bende Liu
c,1
, Cesheng Li
d,1
, Huajun Zhang
e,1
, Ting Yu
f,1
, Jieming Qu
g,h,i,1
, Min Zhou
g,h,i,1
,
Li Chen
j,1
, Shengli Meng
b
, Yong Hu
d
, Cheng Peng
e
, Mingchao Yuan
k
, Jinyan Huang
l
, Zejun Wang
b
, Jianhong Yu
d
,
Xiaoxiao Gao
e
, Dan Wang
k
, Xiaoqi Yu
m
,LiLi
b
, Jiayou Zhang
b
, Xiao Wu
d
, Bei Li
e
, Yanping Xu
g,h,i
, Wei Chen
b
,
Yan Peng
d
, Yeqin Hu
b
, Lianzhen Lin
d
, Xuefei Liu
g,h,i
, Shihe Huang
b
, Zhijun Zhou
d
, Lianghao Zhang
b
, Yue Wang
d
,
Zhi Zhang
b
, Kun Deng
d
, Zhiwu Xia
b
, Qin Gong
d
, Wei Zhang
d
, Xiaobei Zheng
d
, Ying Liu
d
, Huichuan Yang
a
,
Dongbo Zhou
a
, Ding Yu
a
, Jifeng Hou
n
, Zhengli Shi
e
, Saijuan Chen
l
, Zhu Chen
l,2
, Xinxin Zhang
m,2
,
and Xiaoming Yang
a,b,2
a
China National Biotec Group Company Limited, 100029 Beijing, China;
b
National Engineering Technology Research Center for Combined Vaccines, Wuhan
Institute of Biological Products Co. Ltd., 430207 Wuhan, China;
c
First People’s Hospital of Jiangxia District, 430200 Wuhan, China;
d
Sinopharm Wuhan
Plasma-derived Biotherapies Co., Ltd, 430207 Wuhan, China;
e
Key Laboratory of Special Pathogens, Wuhan Institute of Virology, Center for Biosafety
Mega-Science, Chinese Academy of Sciences, 430071 Wuhan, China;
f
WuHan Jinyintan Hospital, 430023 Wuhan, China;
g
Department of Respiratory and
Critical Care Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 200025 Shanghai, China;
h
National Research Center for
Translational Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 200025 Shanghai, China;
i
Institute of Respiratory Diseases, Ruijin
Hospital, Shanghai Jiao Tong University School of Medicine, 200025 Shanghai, China;
j
Clinical Research Center, Department of Gastroenterology, Ruijin
Hospital North, Shanghai Jiao Tong University School of Medicine, 200018 Shanghai, China;
k
Wuhan Blood Center, 430030 Wuhan, China;
l
State Key
Laboratory of Medical Genomics, Shanghai Institute of Hematology, National Research Center for Translational Medicine, Ruijin Hospital, Shanghai Jiao
Tong University School of Medicine, 200025 Shanghai, China;
m
Research Laboratory of Clinical Virology, Ruijin Hospital and Ruijin Hospital North, National
Research Center for Translational Medicine, Shanghai Jiao Tong University School of Medicine, 200025 Shanghai, China; and
n
National Institute for Food
and Drug Control of China, 102629 Beijing, China
Contributed by Zhu Chen, March 18, 2020 (sent for review March 5, 2020; reviewed by W. Ian Lipkin and Fusheng Wang)
Currently, there are no approved specific antiviral agents for novel
coronavirus disease 2019 (COVID-19). In this study, 10 severe pa-
tients confirmed by real-time viral RNA test were enrolled prospec-
tively. One dose of 200 mL of convalescent plasma (CP) derived
from recently recovered donors with the neutralizing antibody
titers above 1:640 was transfused to the patients as an addition
to maximal supportive care and antiviral agents. The primary end-
point was the safety of CP transfusion. The second endpoints were
the improvement of clinical symptoms and laboratory parameters
within 3 d after CP transfusion. The median time from onset of
illness to CP transfusion was 16.5 d. After CP transfusion, the level
of neutralizing antibody increased rapidly up to 1:640 in five cases,
while that of the other four cases maintained at a high level
(1:640). The clinical symptoms were significantly improved along
with increase of oxyhemoglobin saturation within 3 d. Several
parameters tended to improve as compared to pretransfusion, in-
cluding increased lymphocyte counts (0.65 × 10
9
/L vs. 0.76 × 10
9
/L)
and decreased C-reactive protein (55.98 mg/L vs. 18.13 mg/L). Ra-
diological examinations showed varying degrees of absorption of
lung lesions within 7 d. The viral load was undetectable after
transfusion in seven patients who had previous viremia. No severe
adverse effects were observed. This study showed CP therapy was
well tolerated and could potentially improve the clinical outcomes
through neutralizing viremia in severe COVID-19 cases. The opti-
mal dose and time point, as well as the clinical benefit of CP ther-
apy, needs further investigation in larger well-controlled trials.
COVID-19
|
convalescent plasma
|
treatment outcome
|
pilot project
S
ince December 2019, a pneumonia associated with severe
acute respiratory syndrome coronavirus 2 (SARS-CoV-2),
named as coronavirus disease 2019 (COVID-19) by World
Health Organization (WHO), emerged in Wuhan, China (1–3).
The epidemic spread rapidly worldwide within 3 mo and was
characterized as a pandemic by WHO on March 11, 2020. As of
March 12, 2020, a total of 80,980 confirmed cases and 3,173
deaths had been reported in China. Meanwhile, a total of 44,377
confirmed cases and 1,446 deaths was reported in another 108
countries or regions. Currently, there are no approved specific
antiviral agents targeting the novel virus, while some drugs are
still under investigation, including remdesivir and lopinavir/
ritonavir (4, 5). Although remdesivir was reported to possess
potential antiviral effect in one COVID-19 patient from the
United States, randomized controlled trials of this drug are on-
going to determine its safety and efficacy (6). Moreover, the
corticosteroid treatment for COVID-19 lung injury remains
controversial, due to delayed clearance of viral infection and
complications (7, 8). Since the effective vaccine and specific
antiviral medicines are unavailable, it is an urgent need to look
Significance
COVID-19 is currently a big threat to global health. However,
no specific antiviral agents are available for its treatment. In
this work, we explore the feasibility of convalescent plasma
(CP) transfusion to rescue severe patients. The results from 10
severe adult cases showed that one dose (200 mL) of CP was
well tolerated and could significantly increase or maintain the
neutralizing antibodies at a high level, leading to disappear-
ance of viremia in 7 d. Meanwhile, clinical symptoms and par-
aclinical criteria rapidly improved within 3 d. Radiological
examination showed varying degrees of absorption of lung
lesions within 7 d. These results indicate that CP can serve as a
promising rescue option for severe COVID-19, while the ran-
domized trial is warranted.
Author contributions: Z.C., X. Zhang, and X. Yang designed research; S.M., Yong Hu, C.P.,
M.Y., Z.W., J.Y., X.G., D.W., L. Li, J.Z., X.W., B. Li, W.C., Y.P., Yeqin Hu, L. Lin, S.H., Z. Zhou,
L.Z., Y.W., Z. Zhang, K. Deng, Z.X., Q.G., W.Z., X. Zheng, Y.L., H.Y., D.Z., D.Y., J. Hou, and
Z.S. performed research; J. Huang, X. Yu, Y.X., X.L., S.C., and Z.C. analyzed data; and
K. Duan, B. Liu, C.L., H.Z., T.Y., J.Q., M.Z., L.C., and Z.C. wrote the paper.
Reviewers: W.I.L., Columbia University; and F.W., Beijing 302 Hospital.
The authors declare no competing interest.
This open access article is distributed under Creative Commons Attribution License 4.0
(CC BY).
Data deposition: Detailed data on patients that support the findings of this study have
been deposited in the Open Science Framework (https://osf.io/gahz5).
1
K. Duan, B. Liu, C.L., H.Z., T.Y., J.Q., M.Z., and L.C. contributed equally to this work.
2
To whom correspondence may be addressed. Email: zchen@stn.sh.cn, zhangx@shsmu.
edu.cn, or yangxiaoming@sinopharm.com.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/
doi:10.1073/pnas.2004168117/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.2004168117 PNAS Latest Articles
|
1of7
MEDICAL SCIENCES
Downloaded by guest on April 27, 2020
for an alternative strategy for COVID-19 treatment, especially
among severe patients.
Convalescent plasma (CP) therapy, a classic adaptive immu-
notherapy, has been applied to the prevention and treatment of
many infectious diseases for more than one century. Over the
past two decades, CP therapy was successfully used in the
treatment of SARS, MERS, and 2009 H1N1 pandemic with
satisfactory efficacy and safety (9–12). A meta-analysis from 32
studies of SARS coronavirus infection and severe influenza
showed a statistically significant reduction in the pooled odds of
mortality following CP therapy, compared with placebo or no
therapy (odds ratio, 0.25; 95% confidence interval, 0.14–0.45)
(13). However, the CP therapy was unable to significantly improve
the survival in the Ebola virus disease, probably due to the absence
of data of neutralizing antibody titration for stratified analysis
(14). Since the virological and clinical characteristics share simi-
larity among SARS, Middle East Respiratory Syndrome (MERS),
and COVID-19 (15), CP therapy might be a promising treatment
option for COVID-19 rescue (16). Patients who have recovered
from COVID-19 with a high neutralizing antibody titer may be a
valuable donor source of CP. Nevertheless, the potential clinical
benefit and risk of convalescent blood products in COVID-19
remains uncertain. Hence, we performed this pilot study in three
participating hospitals to explore the feasibility of CP treatment in
10 severe COVID-19 patients.
Results
Neutralizing Activity of CP against SARS-CoV-2. The neutralizing
activity against SARS-CoV-2 was evaluated by classical plaque
reduction test using a recently isolated viral strain (1). Among
the first batch of CP samples from 40 recovered COVID-19
patients, 39 showed high antibody titers of at least 1:160, whereas
only one had an antibody titer of 1:32. This result laid the basis
for our pilot clinical trial using CP in severe patients.
General Characteristics of Patients in the Trial. From January 23,
2020, to February 19, 2020, 10 severe COVID-19 patients (six
males and four females) were enrolled and received CP transfusion.
The median age was 52.5 y (interquartile range [IQR], 45.0 y to
59.5 y) (Table 1). None of the patients had direct exposure to
Huanan Seafood Wholesale Market. The median time from onset
of symptoms to hospital admission and CP transfusion was 6 d
(IQR, 2.5 d to 8.5 d) and 16.5 d (IQR, 11.0 d to 19.3 d), re-
spectively. Three patients were affected by clustering infection. The
most common symptoms at disease onset were fever (7 of 10 pa-
tients), cough (eight cases), and shortness of breath (eight cases),
while less common symptoms included sputum production (five
cases), chest pain (two cases), diarrhea (two cases), nausea and
vomiting (two cases), headache (one case), and sore throat (one
case). Four patients had underlying chronic diseases, including car-
diovascular and/or cerebrovas cular diseases and essential hyperten-
sion. Nine patients received arbidol monotherapy or combination
therapy with remdesivir (in one case not included in the current
clinical trial), or ribavirin, or peramivir, while one patient received
ribavirin monotherapy (Table 2). Antibacterial or antifungal treat-
ment was used when patients had coinfection. Six patients received
intravenous (i.v.) methylpredni solone (20 m g every 24 h).
On computer-assisted tomography (CT), all patients presented
bilateral ground-glass opacity and/or pulmonary parenchymal
consolidation with predominantly subpleural and bronchovascular
bundles distribution in the lungs. Seven patients had multiple lobe
involvement, and four patients had interlobular septal thickening.
Effects of CP Transfusion.
Improvement of clinical symptoms. All symptoms in the 10 patients,
especially fever, cough, shortness of breath, and chest pain, dis-
appeared or largely improved within 1 d to 3 d upon CP trans-
fusion. Prior to CP treatment, three patients received mechanical
ventilation, three received high-flow nasal cannula oxygenation, and
two received conventional low-flow nasal cannula oxygenation.
After treatment with CP, two patients were weaned from me-
chanical ventilation to high-flow nasal cannula, and one patient
d iscontinued hig h-flow nasal cannula . Besides, in one patient
treated with conventional nasal cannula oxygenation, continuous
oxygenation was shifted to intermittent oxygenation (Table 2).
Reduction of pulmonary lesions on chest CT examinations. According to
chest CTs, all patients showed different degrees of absorption of
pulmonary lesions after CP transfusion. Representative chest CT
Table 1. Clinical characteristics of patients receiving CP transfusion
Patient no. Sex Age, y
Clinical
classification
Days of
admission
from symptom
onset
Days of CP
therapy from
symptom onset
Clustering
infection Principal symptoms Comorbidity
1 M 46 Severe 8 11 No Fever, cough, sputum production,
shortness of breath, chest pain
Hypertension
2 F 34 Severe 0 11 Yes Cough, shortness of breath, chest
pain, nausea and vomiting
None
3 M 42 Severe 8 19 Yes Fever, cough, sputum production,
shortness of breath, sore
throat, diarrhea
Hypertension
4 F 55 Severe 10 19 No Fever, cough, sputum production,
shortness of breath
None
5 M 57 Severe 4 14 No Fever, shortness of breath None
6 F 78 Severe 8 17 Yes Fever, cough, sputum production,
shortness of breath, muscle ache
None
7 M 56 Severe 4 16 No Fever, cough, sputum production,
arthralgia
None
8 M 67 Severe 10 20 No Fever, cough, headache,
diarrhea, vomiting
Cardiovascular and
cerebrovascular
diseases
9 F 49 Severe 1 10 No Cough, shortness of breath None
10 M 50 Severe 3 20 No Shortness of breath Hypertension
M, male; F, female.
2of7
|
www.pnas.org/cgi/doi/10.1073/pnas.2004168117 Duan et al.
Downloaded by guest on April 27, 2020
images of patient 9 and patient 10 are shown on Fig. 1. Patient 9,
a 49-y-old female admitted 1 day postonset of illness (dpoi),
showed the most obvious pulmonary image improvement. At 10
dpoi, one dose of 200-mL transfusion of CP was given. The
SARS-CoV-2 RNA converted to negative at 12 dpoi. Compared
with the result at 7 dpoi, massive infiltration and ground-glass
attenuation disappeared on CT image performed at 13 dpoi, ac-
companied by a much better pulmonary function. Patient 10, a
50-y-old male, was admitted 3 dpoi and was given a 200-mL trans-
fusion of CP at 20 dpoi. His chest CT presented massive infiltration
and widespread ground-glass attenuation on admissio n an d
started to s how a gradual absorption of lung lesions 5 d after CP
transfusion. The SARS-CoV-2 RNA became negative at 25 dpoi.
Amelioration of routine laboratory criteria and pulmonary function.
Lymphocytopenia, an important index for prognosis in COVID-
19 (2), tended to be improved after CP transfusion (median:
0.65 × 10
9
per L vs. 0.76 × 10
9
per L), 7 out of 10 patients showing
an increase of lymphocyte counts (Fig. 2). Concerning other lab-
oratory tests, we observed a tendency of decrement of parameters
indicative of inflammation and/or liver dysfunction as compared to
the status before CP therapy. These included C-reactive protein
(CRP) (median : 55.98 mg/L vs. 18.13 mg/L), alanine amino-
transferase (median: 42.00 U/L vs . 34.30 U/L), and aspartate
aminotransferase (median: 38.10 U/L vs. 30.30 U/L) (Table 3).
The total bilirubin (median: 12.40 μmol/L vs. 13.98 μ mol/L)
remained unchanged, except for an obvious incr ement in patient
1(Fig.2).AnincreaseofSaO
2
(median: 93.00% vs. 96.00%), a
measurement constantly performed in most patients in our trial,
was found, which could indicate recovering lung function. This
temporal relationship was notable despite the provision of maxi-
mal supportive care and antiviral agents.
Remarkably, patient 1, a 46-y-old male admitted 8 dpoi, had a
very quick recovery, with much improved result of laboratory
tests. He received antiviral drugs (arbidol and ribavirin) treatment
and high-flow nasal cannula on admission. Mechanical ventilation
was given at 10 dpoi for critical care support. CP transfusion was
performed at 11 dpoi. At 12 dpoi, the SARS-CoV-2 test turned to
negative, with a sharp decrease of CRP from 65.04 mg/L to 23.57
mg/L and increment of SaO
2
from 86% to 90% (Fig. 3). The me-
chanical ventilation was successfully weaned off 2 d after CP
transfusion. At 15 dpoi, a steady elevation of lymphocyte count and
a drop of aminopherase level were observed, indicating improve-
ment of immunological and hepatic function.
Increase of neutralizing antibody titers and disappearance of SARS-CoV-2
RNA.
We determined neutralizing antibody titers before and after
CP transfusion in all patients except one (patient 2) (Table 4).
The neutralizing antibody titers of five patients increased and four
patients remained at the same level after CP transfusion. SARS-
CoV-2 RNA, assayed by RT-PCR, was positive in seven patients
and negative in three cases before CP transfusion. Of note is that
SARS-CoV-2 RNA was decreased to an undetectable level in
three patients on day 2, three patients on day 3, and one patient
on day 6 after CP therapy. These results are in support of a
neutralizing effect of CP on serum SARS-CoV-2.
Outcome of patients treated with CP as compared to a recent historic
control group. A histori c control group was formed by random
selection of 10 p atients from the cohort treated in the same
hospitals and matched by age, gender, and severity of the dis-
eases to the 10 cases in our trial. Ba se li ne ch ar ac te ris ti cs of
patients between CP treatment group and c ontrol group
showed no significant differences, while clinical outcomes of
these two groups were different: three cases discharged while
seven cases in much improved status and ready for discharge
in CP group, as compared to three deaths, six cases in stabilized
status, and one case in improvement in the control group (P <
0.001; SI Appendix,TableS1).
Table 2. Other treatments of ten patients receiving CP transfusion
Drugs administered Oxygen support
Patient no.
Antiviral
treatment
Antibiotic or antifungal
treatment
Corticosteroids
treatment Before CP therapy After CP therapy
1 Arbidol 0.2 g q8h po.
Ribavirin 0.5 g qd i.v.
Cefoperazone Sodium i.v. None High-flow nasal cannula,
mechanical ventilation
Mechanical
ventilation
2 Arbidol 0.2 g q8h po. Cefoperazone Sodium i.v. None None None
3 Arbidol 0.2 g q8h po. Moxifloxacin i.v. Methylprednisolone
i.v.
High-flow nasal cannula,
mechanical ventilation
High-flow nasal
cannula
4 Ribavirin 0.5 g qd i.v. Linezolid i.v.
Imipenem-Sitastatin
Sodium i.v.
Methylprednisolone
i.v.
Mechanical ventilation High-flow nasal
cannula
5 Arbidol 0.2 g q8h po. Moxifloxacin i.v. Methylprednisolone
i.v.
Low-flow nasal cannula Low-flow nasal
cannula
Remdesivir 0.2 g qd i.v. Cefoperagone Sodium and
Tazobactam Sodium i.v.
IFN-ɑ 500MIU qd inh.
6 Arbidol 0.2 g q8h po. Cefoperazone Sodium i.v.
Levofloxacin i.v.
Methylprednisolone
i.v.
High-flow nasal cannula High-flow nasal
cannula
7 Arbidol 0.2 g q8h po. Cefoperagone Sodium and
Tazobactam Sodium i.v.
Methylprednisolone
i.v.
High-flow nasal cannula None
Fluconazole i.v.
8 Arbidol 0.2 g q8h po. None None None None
Ribavirin 0.5 g qd i.v.
9 Arbidol 0.2 g q8h po.
Oseltamivir 75 mg q12h po.
None None Low-flow nasa l cannula Low-flow nasal
cannula (Intermitte nt)
Peramivir 0.3 g qd i.v.
10 Arbidol 0.2 g q8h po.
IFN-ɑ 500 MIU qd inh.
Cefoperazone Sodium i.v.
Caspofungin i.v.
Methylprednisolone i.v. High-flow nasal cannula High-flow nasal
cannula
po., per os; i.v., i.v. injection; inh., inhalation; q8h, every 8 h; qd, per day; q12h, every 12 h; MIU, million IU.
Duan et al. PNAS Latest Articles
|
3of7
MEDICAL SCIENCES
Downloaded by guest on April 27, 2020
Adverse Effects of CP Transfusions. Patient 2 showed an evanescent
facial red spot. No serious adverse reactions or safety events were
recorded after CP transfusion.
Discussion
Our study explores the feasibility of CP therapy in COVID-19.
All enrolled severe COVID-19 patients achieved primary and
secondary outcomes. One dose of 200-mL CP transfusion was
well tolerated, while the clinical symptoms significantly improved
with the increase of oxyhemoglobin saturation within 3 d, ac-
companied by rapid neutralization of viremia.
Severe pneumonia caused by human coronavirus was charac-
terized by rapid viral replication, massive inflammatory cell in-
filtration, and elevated proinflammatory cytokines or even
cytokine storm in alveoli of lungs, resulting in acute pulmonary
injury and acute respiratory distress syndrome (ARDS) (17).
Recent studies on COVID-19 demonstrated that the lymphocyte
counts in the peripheral blood were remarkably decreased and
the levels of cytokines in the plasma from patients requiring
intensive care unit (ICU) support, including IL-6, IL-10, TNF-ɑ,
and granulocyte-macrophage colony-stimulating factor, were
Fig. 1. Chest CTs of two patients. (A) Chest CT of patient 9 obtained on February 9 (7 dpoi) before CP transfusion (10 dpoi) showed ground-glass opacity with
uneven density involving the multilobal segments of both lungs. The heart shadow outline was not clear. The lesion was close to the pleura. (B) CT Image of
patient 9 taken on February 15 (13 dpoi) showed the absorption of bilateral ground-glass opacity after CP transfusion. (C) Chest CT of patient 10 was obtained
on February 8 (19 dpoi) before CP transfusion (20 dpoi). The brightness of both lungs was diffusely decreased, and multiple shadows of high density in both
lungs were observed. (D) Chest CT of patient 10 on February 18 (29 dpoi) showed those lesions improved after CP transfusion.
Before CP treatment After CP treatment
0
50
100
150
CRP
Before CP treatment After CP treatment
0
20
40
60
80
100
ALT
Before CP treatment After CP treatment
0
1
2
3
4
Lym
Before CP treatment After CP treatment
0
20
40
60
AST
Before CP treatment After CP treatment
80
85
90
95
100
105
SaO
2
Before CP treatment After CP treatment
0
20
40
60
80
TBIL
patient 3
patient 4
patient 5
patient 6
patient 7
patient 9
patient 1
patient 2
patient 8
patient 10
Fig. 2. Dynamic changes of laboratory parameters in all patients. The dotted horizontal line represents the refere nce value range. SaO
2
, oxyhemoglobin
saturation; TBIL, total bilirubin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Lym, lymphocyte.
4of7
|
www.pnas.org/cgi/doi/10.1073/pnas.2004168117 Duan et al.
Downloaded by guest on April 27, 2020
significantly higher than in those who did not require ICU con-
ditions (2, 18). CP, obtained from recovered COVID-19 patients
who had established humoral immunity against the virus, con-
tains a large quantity of neutralizing antibodies capable of neu-
tralizing SARS-CoV-2 and eradicating the pathogen from blood
circulation and pulmonary tissues (19). In the present study, all
investigated patients achieved serum SARS-CoV-2 RNA neg-
ativity after CP transfusion, accompanied by an increas e of
oxygen saturation and lymphocyte counts, and the improve-
ment of liver function and CRP. The results suggest that the
inflammation and overreaction of the immune system were
alleviated by antibodies contained in CP. The case fatality rates
(CFRs) in the present study were 0% (0/10), which was com-
parable to the CFRs in SARS, which varied from 0% (0/10) to
12.5% (10/80) in four noncomparative studies usi ng CP treat-
ment (9, 20 – 22). Based on our preliminary results, CP therapy
can be an easily accessible, promising, and safe rescue option
for severe COVID-19 patie nts. It is, nevertheless, worth men-
tioning that the absorption of pulmonary lesions often lagge d
behind the improvement of clinical symptoms, as shown in pa-
tients 9 and 10 in this trial.
The first key factor associated with CP therapy is the neu-
tralizing antibody titer. A small sample study in MERS-CoV
infection showed that the neutralizing antibody titer should ex-
ceed 1:80 to achieve effective CP therapy (12). To find eligible
donors who have high levels of neutralizing antibody is a pre-
requisite. Cao et al. (23) showed that the level of specific neu-
tralizing antibody to SARS-CoV decreased gradually 4 mo after
the disease process, reaching undetectable levels in 25.6% (IgG)
and 16.1% (neutralizing antibodies) of patients at 36 mo after
disease status. A study from the MERS-CoV−infected patients
and the exposed healthcare workers showed that the prevalence
of MERS-CoV IgG seroreactivity was very low (2.7%), and the
antibodies titer decreased rapidly within 3 mo (24). These studies
suggested that the neutralizing antibodies represented short-
lasting humoral immune response, and plasma from recently
recovered patients should be more effective. In the present
study, recently recovered COVID-19 patients, who were infected
by SARS-CoV-2 with neutralizing antibody titer above 1:640 and
recruited from local hospitals, should be considered as suitable
donors. The median age of donors was lower than that of re-
cipients (42.0 y vs. 52.5 y). Among the nine cases investigated,
the neutralizing antibody titers of five patients increased to 1:640
within 2 d, while four patients kept the same level. The antibody
titers in CP in COVID-19 seem thus higher than those used in
the treatment of MERS patient (1:80) (12).
The second key factor associated with efficacy is the treatment
time point. A better treatment outcome was observed among
SARS patients who were given CP before 14 dpoi (58.3% vs.
15.6%; P < 0.01), highlighting the importance of timely rescue
therapy (9). The mean time from onset of illness to CP transfusion
was 16.5 d. Consistent with previous research, all three patients
receiving plasma transfusion given before 14 dpoi (patients 1, 2,
Table 3. Comparison of laboratory parameters before and after
CP transfusion
Clinical factors
Before CP
transfusion
After CP
transfusion
CRP (mg/L, normal range
0to6)
55.98 (15.57
to 66.67)
18.13 (10.92
to 71.44)
Lymphocyte (10
9
per L, normal
range 1.1 to 3.2)
0.65 (0.53
to 0.90)
0.76 (0.52
to 1.43)
Alanine aminotransferase (U/L,
normal range 9 to 50)
42.00 (28.25
to 61.85)
34.30 (25.75
to 53.90)
Aspartate aminotransferase
(U/L, normal range 15
to 40)
38.10 (28.50
to 44.00)
30.30 (17.30
to 38.10)
Total bilirubin (μmol/L, normal
range 0 to 26)
12.40 (11.71
to 22.05)
13.98 (12.20
to 20.80)
SaO
2
(%, normal range
≥ 95)
93.00 (89.00
to 96.50)
96.00 (95.00
to 96.50)
SaO
2
, oxyhemoglobin saturation.
Day 0 Day 1
0
20
40
60
80
CRP
Day 0 Day 1 Day 3 Day 4
0
20
40
60
80
ALT
Day 0 Day 1 Day 3 Day 4
0
1
2
3
4
Lym
Day 0 Day 1 Day 2 Day 3 Day 4
0
10
20
30
40
AST
Day 0 Day 1
80
85
90
95
100
105
SaO
2
Day 0 Day 1 Day 3 Day 4
0
20
40
60
80
TBIL
Fig. 3. Change of laboratory parameters in patient 1. The x axis represents the day post-CP transfusion. The dotted horizontal line represents the reference
value range.
Duan et al. PNAS Latest Articles
|
5of7
MEDICAL SCIENCES
Downloaded by guest on April 27, 2020
and 9) in our study showed a rapid increase of lymphocyte counts
and a decrease of CRP, with remarkable absorption of lung lesions
in CT. Notably, patients who received CP transfusion after 14 dpoi
showed much less significant improvement, such as patient 10.
However, the dynamics of the viremia of SARS-CoV-2 was un-
clear, so the optimal transfusion time point needs to be deter-
mined in the future.
In the present study, no severe adverse effects were observed.
One of the risks of plasma transfusion is the transmission of the
potential pathogen. Methylene blue photochemistry was applied
in this study to inactivate the potential residual virus and to
maintain the activity of neutralizing antibodies as much as pos-
sible, a method known to be much better than ultraviolet (UV) C
light (25). No specific virus was detected before transfusion.
Transfusion-related acute lung injury was reported in an Ebola
virus disease woman who received CP therapy (26). Although
uncommon in the general population receiving plasma trans-
fusion, this specific adverse reaction is worth noting, especially
among critically ill patients experiencing significant pulmonary
injury (27). Another rare risk worth mentioning during CP
therapy is antibody-dependent infection enhancement, occurring
at subneutralizing concentrations, which could suppress innate
antiviral systems and thus could allow logarithmic intracellular
growth of the virus (28). The special infection enhancement also
could be found in SARS-CoV infection in vitro (29). No such
pulmonary injury and infection enhancement were observed in our
patients, probably owing to high levels of neutralizing antibodies,
timely transfusion, and appropriate plasma volume.
There were some limitations to the present study. First, except for
CP transfusion, the patients received other standard care. All patients
received antiviral treatment despite the uncertainty of the efficacy of
drugs used. As a result, the possibility that these antiviral agents could
contribute to the recovery of patients, or synergize with the therapeutic
effect of CP, could not be ruled out. Furthermore, some patients re-
ceived glucocorticoid therapy, which might interfere with immune
response and delay virus clearance. Second, the median time from
onset of symptoms to CP transfusion was 16.5 d (IQR, 11.0 d to 19.3
d). Although the kinetics of viremia during natural history remains
unclear, the relationship between SARS-CoV-2 RNA reduction and
CP therapy, as well as the optimal concentration of neutralizing an-
tibodies and treatment schedule, should be further clarified. Third, the
dynamic changes of cytokines during treatment were not investigated.
Nevertheless, the preliminary results of this trial seem promising, jus-
tifying a randomized controlled clinical trial in a larger patient cohort.
In conclusion, this pilot study on CP therapy shows a potential
therapeutic effect and low risk in the treatment of severe COVID-19
patients. One dose of CP with a high concentration of neutralizing
antibodies can rapidly reduce the viral load and tends to improve
clinical outcomes. The optimal dose and treatment time point, as
well as the definite clinical benefits of CP therapy, need to be
further investigated in randomized clinical studies.
Materials and Methods
Patients. From January 23, 2020, to February 19, 2020, 10 patients in three
participating hospitals (Wuhan Jinyintan Hospital; the Jiangxia District
Hospital of Integrative Traditional Chinese and Western Medicine, Wuhan;
and the First People’s Hospital of Jiangxia District, Wuhan) were recruited in
this pilot study. All patients were diagnosed as having severe COVID-19
according to the WHO Interim Guidance (30) and the Guideline of Di-
agnosis and Treatment of COVID-19 of National Health Commission of China
(version 5.0) (31), with confirmation by real-time RT-PCR assay. The enroll-
ment criteria were one of the conditions 2 to 4 plus condition 1: 1) age ≥ 18 y;
2) respiratory distress, RR ≥30 beats/min; 3) oxygen saturation level less
than 93% in resting state; and 4) partial pressure of oxygen (PaO
2
)/oxygen
concentration (FiO
2
) ≤ 300 mmHg (1 mmHg = 0.133 kPa). The exclusion
criteria were as follows: 1) previous allergic history to plasma or ingredients
(sodium citrate); 2) cases with serious general conditions, such as severe
organ dysfunction, who were not suitable for CP transfusion. Written in-
formed consent according to the Declaration of Helsinki was obtained from
each patient or legal relatives. This study was approved by the Ethics Com-
mittee of the Chi na National Biotec Group Co., Ltd. (Approval number 2020-
0001). The registration number of this trial is ChiCTR2000030046.
Donors for Convalescent Plasma Transfusion. Ten donor patients who re-
covered from COVID-19 were recruited from three participating hospitals.
The recovery criteria were as follows: 1) normality of body temperature for
more than 3 d, 2) resolutio n of respiratory tract symptoms, and 3) two
consecutively negative results of sputum SARS-CoV-2 by RT-PCR assay (1-d
sampling interval). The donor’s blood was collected after 3 wk pos tonset
Table 4. Comparison of serum neutralizing antibody titers and SARS-CoV-2 RNA load before and after CP therapy
Patient no.
CP transfusion
date
Before CP transfusion After CP transfusion
Date
Serum n eutralizing
antibody titers
Serum
SARS-CoV-2 RNA load
(Ct value) Date
Serum neutralizing
antibody titers
Serum
SARS-CoV-2 RNA load
(Ct value)
1 February 9 February 8 1:160 37.25 February
10
1:640 Negative
2 February 9 February 8 Unavailable 35.08 February
11
Unavailable Negative
3 February 13 February 12 1:320 38.07 February
14
1:640 Negative
4 February 13 February 12 1:160 37.68 February
14
1:640 Negative
5 February 12 February 11 1:640 Negative February
14
1:640 Negative
6 February 12 February 11 1:640 Negative February
14
1:640 Negative
7 February 12 February 11 1:320 34.64 February
14
1:640 Negative
8 February 12 February 11 1:640 35.45 February
14
1:640 Negative
9 February 12 February 11 1:160 Negative February
14
1:640 Negative
10 February 9 February 8 1:640 38.19 February
14
1:640 Negative
6of7
|
www.pnas.org/cgi/doi/10.1073/pnas.2004168117 Duan et al.
Downloaded by guest on April 27, 2020
of illness and 4 d postdischarge. Written informed consent was obtained from
each patient.
Plasma Preparation Procedure and Quality Control. Apher esis was performed
using a Baxter CS 300 cell separator (Baxter). Convalescence plasma for
treatment was col lected from 40 donors. The median age was 42.0 y
(IQR, 32.5 y to 49 y). A 200- to 400-mL ABO-compatible plasma sample
was harve sted fro m each donor depending on ag e and body weight,
and each sample was divided and stored as 200-mL aliquots at 4 °C
without a ny detergent or heat treatment. The CP was then treated
with methylene blue and light treatment for 30 min i n the medical
plasma viru s inactivation cabinet (Shandong Zhongbaokang Medical
Appliance Co., Ltd).
Serology Test and Real-Time RT-PCR Detection of SARS-CoV-2 and Other
Pathogens. The neutralizing activity of plasma was determined by plaque
reduction neutralization test using SARS-CoV-2 virus in the high biosafe level
(BSL-4) laboratory of Wuhan Institute of Virology, Chinese Academy of Sci-
ences. Neutralization titer was defined as the highest serum dilution
with 50% reduct ion in t he number of plaques, as compared with the
number of plaques in wells in the absence of novel coronavirus antibody
as blank control. The neutralizing activity of the receptor-binding do-
main of antibody in the CP was detected by a sandwich e nzyme-linked
immunosorbent assay (ELISA). SARS-CoV-2 IgG antibody titer was tested
by ELIS A. SARS-C oV-2 RNA was detected by RT-PCR assa y, and the r esult
was presented as cycle threshold (Ct) value (Shanghai Bi oGerm Medical
Biotechnol ogy Co., Ltd ). Methylene blue residue was detected by the
verified UV method. The serology s creening for hepat itis B and C vir us,
HIV, and syphilis spirochete was negative. The protocols for neutraliza-
tion assay, serological test, and real -time RT-PCR detection of SARS-CoV
RNA are present ed in SI Appendix.
Treatment. All patients were admitted to the ICU and received antiviral
therapy and other supportive care, while some patients received antibiotic
treatment, antifungal treatment, glucocorticoid, and oxygen support at
the appropriate situation. One dose of 200 mL of inactivated CP with neu-
tralization activity of >1:640 was transfused into the patients within 4 h
following the WHO blood transfusion protocol.
Data Collection. Clinical information of all enrolled patients was retrieved
from the hospital electronic history system, including the baseline de-
mographic data, days of illness duration, presenting symptoms, different
kinds of examinat ion, and methods of treatment. Bacterial coinfection was
identified by a positive culture from respiratory, urinary, or blood cultur e
within 48 h of hospit al admission. Complications, including acute renal
failure, acute coronary syndrome, myocarditis, AR DS, and nosocomial in-
fection, were recorded. The applications of assisted mechanical v entila-
tion, intranasal oxygen inhalation, and medication regimen were
recorded. The SARS-CoV-2 RNA from the ser um sample was monitored
during treatment.
Outcome Measures and Definitions. The clinical symptoms were recorded by
attending physicians daily. The blood test and biochemical tests were carried
out every 1 d to 2 d. SARS-CoV-2 RNA was detected every 2 d to 3 d. CT scan
was repeated every 3 d to 5 d. The primary endpoint was the safety of CP
transfusion. The second endpoints were the improvement of clinical symp-
toms and laboratory and radiological parameters within 3 d after CP
transfusion. Clinical symptoms improvement was defined as temperature
normalization, relief of dyspnea, and oxygen saturation normalization, and
radiological improvement was defined as different degrees of absorption of
lung lesions.
Statistical Analysis. Continuous variables were presented as the median and
IQR. Graphs were plotted using GraphPad Prism 7.0. Statistical software used
included SPSS 24.0.
Data Availability Statement. All data relevant to this manuscript and avail-
able to the authors at the time of publication are included in the main text
or SI Appendix. Further detailed data on patients that support the find-
ings of this study have been deposited in the Open Science Framework
(https://osf.io/gahz5).
ACKNOWLEDGMENTS. This study was funded by Key projects of the Ministry
of Science and Technology China “Preparation of specific plasma and specific
globulin from patients with a recovery period of COVID-19 infection” (Proj-
ect 2020YFC0841800). This work was also supported by Shanghai Guangci
Translational Medicine Development Foundation. We thank all patients and
donors involved in this study.
1. P. Zhou et al., A pneumonia outbreak associated with a new coronavirus of probable
bat origin. Nature 579, 270–273 (2020).
2. N. Chen et al., Epidemiological and clinical characteristics of 99 cases of 2019 novel
coronavirus pneumonia in Wuhan, China: A descriptive study. Lancet 395, 507–513
(2020).
3. World Health Organization, Coronavirus disease (COVID-19) Pandemic. https://www.
who.int/emergencies/diseases/novel-coronavirus-2019. Accessed 11 March 2020.
4. H. Lu, Drug treatment options for the 2019-new coronavirus (2019-nCoV). Biosci.
Trends 14,69–71 (2020).
5. M. Wang et al., Remdesivir and chloroquine effectively inhibit the recently emerged
novel coronavirus (2019-nCoV) in vitro. Cell Res. 30, 269–271 (2020).
6. M. L. Holshue et al.; Washington State 2019-nCoV Case Investigation Team, First
case of 2019 novel coronavirus in the United States. N.Engl.J.Med.382, 929–936
(2020).
7. C. D. Russell, J. E. Millar, J. K. Baillie, Clinical evidence does not support corticosteroid
treatment for 2019-nCoV lung injury. Lancet 395, 473–475 (2020).
8. L. Shang, J. Zhao, Y. Hu, R. Du, B. Cao, On the use of corticosteroids for 2019-nCoV
pneumonia. Lancet 395, 683–684 (2020).
9. Y. Cheng et al., Use of convalescent plasma therapy in SARS patients in Hong Kong.
Eur. J. Clin. Microbiol. Infect. Dis. 24,44–46 (2005).
10. B. Zhou, N. Zhong, Y. Guan, Treatment with convalescent plasma for influenza A
(H5N1) infection. N. Engl. J. Med. 357, 1450–1451 (2007).
11. I. F. Hung et al., Convalescent plasma treatment reduced mortality in patients with
severe pandemic influenza A (H1N1) 2009 virus infection. Clin. Infect. Dis. 52, 447–456
(2011).
12. J. H. Ko et al., Challenges of convalescent plasma infusion therapy in Middle East
respiratory coronavirus infection: A single centre experience. Antivir. Ther. 23 , 617–
622 (2018).
13. J. Mair-Jenkins et al.; Convalescent Plasma Study Group, The effectiveness of conva-
lescent plasma and hyperimmune immunoglobulin for the treatment of severe acute
respiratory infections of viral etiology: A systematic review and exploratory meta-
analysis.
J. Infect. Dis. 211,80–90 (2015).
14. J. van Griensven et al.; Ebola-Tx Consortium, Evaluation of convalescent plasma for
Ebola virus disease in Guinea. N. Engl. J. Med. 374,33–42 (2016).
15. P. I. Lee, P. R. Hsueh, Emerging threats from zoonotic coronaviruses-from SARS and
MERS to 2019-nCoV. J. Microbiol. Immunol. Infect., in press.
16. L. Chen, J. Xiong, L. Bao, Y. Shi, Convalescent plasma as a potential therapy for
COVID-19. Lancet Infect. Dis. 20, 398–400 (2020).
17. R. Channappanavar, S. Perlman, Pathogenic human coronavirus infections: Causes
and consequences of cytokine storm and immunopathology. Semin. Immunopathol.
39, 529–539 (2017).
18. C. Huang et al., Clinical features of patients infected with 2019 novel coronavirus in
Wuhan, China. Lancet 395, 497–506 (2020).
19. G. Marano et al., Convalescent plasma: New evidence for an old therapeutic tool?
Blood Transfus. 14, 152–157 (2016).
20. V. W. Wong, D. Dai, A. K. Wu, J. J. Sung, Treatment of severe acute respiratory
syndrome with convalescent plasma. Hong Kong Med. J. 9, 199–201 (2003).
21. K. M. Yeh et al., Experience of using convalescent plasma for severe acute respiratory
syndrome among healthcare workers in a Taiwan hospital. J. Antimicrob. Chemother.
56, 919–922 (2005).
22. L. K. Kong, B. P. Zhou, Successful treatment of avian influenza with convalescent
plasma. Hong Kong Med. J. 12, 489 (2006).
23. W. C. Cao, W. Liu, P. H. Zhang, F. Zhang, J. H. Richardus, Disappearance of anti-
bodies to SARS-as sociated c oronav irus af ter rec overy. N.Engl.J.Med.357, 1162–
1163 (2007).
24. Y. M. Arabi et al., Feasibility of using convalescent plasma immunotherapy for MERS-
CoV infection, Saudi Arabia. Emerg. Infect. Dis. 22, 1554–1561 (2016).
25. M. Eickmann et al., Inactivation of Ebola virus and Middle East respiratory syndrome
coronavirus in platelet concentrates and plasma by ultraviolet C light and methylene
blue plus visible light, respectively. Transfusion 58, 2202–2207 (2018).
26. M. Mora-Rillo et al.; La Paz-Carlos III University Hospital Isolation Unit, Acute re-
spiratory distress syndrome after convalescent plasma use: Treatment of a patient with
Ebola virus disease contracted in Madrid, Spain. Lancet Respir. Med. 3,554–562 (2015).
27. A. B. Benson, M. Moss, C. C. Silliman, Transfusion-related acute lung injury (TRALI): A
clinical review with emphasis on the critically ill. Br. J. Haematol. 147, 431–443 (2009).
28. S. B. Halstead, Dengue antibody-dependent enhancement: Knowns and unknowns.
Microbiol. Spectr. 2, AID-0022-2014 (2014).
29. S. F. Wang et al., Antibody-dependent SARS coronavirus infection is mediated by anti-
bodies against spike proteins. Biochem. Biophys. Res. Commun. 451, 208–214 (2014).
30. World Health Organization, Clinical management of severe acute respiratory infection
when Novel coronavirus (nCoV) infection is suspected: Interim guidance. https://www.
who.int/publications-detail/clinical-management-of-severe-acute-respiratory-infection-
when-nove l-coronavirus-(ncov)-infection-is-suspected. Accessed 13 March 2020.
31. National Health Commission of China, Guideline for diagnosis and treatment for
novel coronavirus pneumonia (fifth edition). http://www.nhc.gov.cn/xcs/zhengcwj/
202002/3b09b894ac9b4204a79db5b8912d4440.shtml. Accessed 4 February 2020.
Duan et al. PNAS Latest Articles
|
7of7
MEDICAL SCIENCES
Downloaded by guest on April 27, 2020
