Serologic Responses To Sars-Cov-2 Infection Among Hospital Staff With Mild Disease in Eastern France

medRxiv preprint doi: https://doi.org/10.1101/2020.05.19.20101832.this version posted May 22, 2020.
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Serologic responses to SARS-CoV-2 infection among hospital staff with mild disease
in eastern France
Samira FAFI-KREMER1,2,*,, Timothée BRUEL3,*, Yoann MADEC4, Rebecca GRANT4, Laura

TONDEUR4, Ludivine GRZELAK3,5, Isabelle STAROPOLI3, François ANNA6, Philippe
SOUQUE7, Catherine SCHMIDT-MUTTER8, Nicolas COLLONGUES8,14, Alexandre BOLLE8,
Aurélie VELAY1,2 , Nicolas LEFEBVRE9, Marie MIELCAREK10, Nicolas MEYER10,11, David
REY 12, Pierre CHARNEAU6,7, Bruno HOEN13, Jérôme De SEZE8,14, Olivier SCHWARTZ3,**
and Arnaud FONTANET4,15,**
1
CHU de Strasbourg, Laboratoire de virologie, F-67091 Strasbourg, France
2
Université de Strasbourg, INSERM, IRM UMR_S 1109, Strasbourg, France.
3
Virus & Immunity Unit, Department of Virology, Institut Pasteur, Paris, France; CNRS UMR
3569, Paris, France; Vaccine Research Institute, Creteil, France
4
Institut Pasteur, Emerging Diseases Epidemiology Unit, Paris, France
5
Université de Paris, Sorbonne Paris Cité, Paris, France
6
Pasteur-TheraVectys joined unit
7
Molecular Virology & Vaccinology Unit, Department of Virology, Institut Pasteur, Paris,
France.
8
Centre d’investigation Clinique INSERM 1434, CHU Strasbourg, France
9
CHU de Strasbourg, Service des infectieuses et tropicales, F-67091 Strasbourg, France
10
CHU de Strasbourg, Service de santé Publique, GMRC, F-67091 Strasbourg, France
11
Université de Strasbourg, CNRS, iCUBE UMR 7357, Strasbourg, France
12
CHU de Strasbourg, Pôle SMO, le Trait d’Union, F-67091 Strasbourg, France
13
Direction de la recherche médicale, Institut Pasteur, Paris, France
14
CHU de Strasbourg, Service de Neurologie, F-67091 Strasbourg, France
15
Conservatoire National des Arts et Métiers, PACRI Unit, Paris, France
* Co-first authors
** Co-last authors
Correspondence to [email protected] and [email protected]
1
medRxiv preprint doi: https://doi.org/10.1101/2020.05.19.20101832.this version posted May 22, 2020. The copyright holder for this preprint
Abstract
Background: The serologic response of individuals with mild forms of SARS-CoV-2 infection
is poorly characterized.
Methods: Hospital staff who had recovered from mild forms of PCR-confirmed SARS-CoV-2
infection were tested for anti-SARS-CoV-2 antibodies using two assays: a rapid
immunodiagnostic test (99.4% specificity) and the S-Flow assay (~99% specificity).The
neutralizing activity of the sera was tested with a pseudovirus-based assay.
Results: Of 162 hospital staff who participated in the investigation, 160 reported SARS-CoV-
2 infection that had not required hospital admission and were included in these analyses. The
median time from symptom onset to blood sample collection was 24 days (IQR: 21-28, range
13-39). The rapid immunodiagnostic test detected antibodies in 153 (95.6%) of the samples
and the S-Flow assay in 159 (99.4%), failing to detect antibodies in one sample collected 18
days after symptom onset (the rapid test did not detect antibodies in that patient). Neutralizing
antibodies (NAbs) were detected in 79%, 92% and 98% of samples collected 13-20, 21-27
and 28-41 days after symptom onset, respectively (P=0.02).
Conclusion: Antibodies against SARS-CoV-2 were detected in virtually all hospital staff
sampled from 13 days after the onset of COVID-19 symptoms. This finding supports the use
of serologic testing for the diagnosis of individuals who have recovered from SARS-CoV-2
infection. The neutralizing activity of the antibodies increased overtime. Future studies will
help assess the persistence of the humoral response and its associated neutralization
capacity in recovered patients.
2
Introduction
A novel human coronavirus that is now named severe acute respiratory syndrome coronavirus
2 (SARS-CoV-2) emerged in Wuhan, China, in late 2019. In response, many countries have
implemented large scale public health and social measures in an attempt to reduce
transmission and minimize the impact of the outbreak. As the benefits of these measures are
now becoming apparent in terms of a reduction in the daily incidence of SARS-CoV-2
infections and associated deaths, countries are looking for ways to lift these measures and
resume economic and social activities. Ideally, the lifting of measures would occur if the
population had built sufficient collective immunity, known as herd immunity, to the point that
any reintroduction of the virus would not trigger a new epidemic wave. In this context, it is
important to understand the extent to which infection has spread in communities, and to which
those who have been infected may be protected from re-infection. This requires further
understanding of antibody kinetics following SARS-CoV-2 infection.
Numerous serologic assays are now available, which provide information on extent of infection
and estimates of protective immunity – that is, protection against re-infection. To date, it is
thought that for hospitalised patients with COVID-19, seroconversion occurs within the second
week following onset of symptoms, with a median time of 5-12 days for IgM antibodies and 14
days for IgG and IgA [1–6]. However, it remains unclear whether time to seroconversion may
differ according to disease severity, and early reports suggest that individuals with mild
infection may have delayed or absent seroconversion [4]. Further, the correlation between
detection of antibodies generated in response to SARS-CoV-2 infection and protective
immunity has not yet been established.
The first three COVID-19 cases identified in France were reported on 24 January 2020 in
travellers returning from Wuhan, China [7]. Between 17 and 24 February, an annual religious
gathering attended by 2500 people took place in Mulhouse, eastern France and resulted in a
3
SARS-CoV-2 superspreading event. Infected individuals went to regional hospitals, and this
led to a cluster of infected staff at the Strasbourg University Hospitals from the first week of
March. Most of them are young individuals who developed mild forms of disease.
The epidemic in Strasbourg, and specifically, the cluster of infected hospital staff, provides the
opportunity to use serologic assays, to assess antibody kinetics in individuals who had
recovered from COVID-19 and to understand how this correlates with protective immunity.
4
Methods
Participants
Between 6 April and 8 April 2020, all hospital staff from Strasbourg University Hospitals with
PCR-confirmed SARS-CoV-2 infection were invited to participate in the investigation. This
invitation included doctors, nurses, physiotherapists, dentists, medical students, orderlies,
hospital assistants, and hospital administrative staff.
Following informed consent, participants completed a questionnaire which covered
sociodemographic information, underlying medical conditions, and details related to SARS-
CoV-2 infection, including date of testing, date of symptom onset and a description of
symptoms. A 5 mL blood sample was taken from all participants.
Serologic response measurement
All serum samples were tested for antibody responses to SARS-CoV-2 using two serologic
assays: 1) a rapid immunodiagnostic assay detecting SARS-CoV-2 spike protein receptor
binding domain (RBD) developed by Biosynex®; 2) the S-Flow assay, a flow-cytometry based
assay that measures antibodies binding to the Spike protein (S) expressed at the surface of
target cells [8]. Two parameters can be calculated with this assay: the first is the percentage
of cells having captured antibodies, defining the seropositivity. The second is the mean
fluorescence intensity (MFI) of this binding, which provides a quantitative measurement of the
amount of antibodies and their efficacy [8]. As a control for the S-Flow tests, we included
samples from pre-epidemics individuals, providing cut-offs for the the S-Flow >99% specificity
([8] and Fig. 1A). The rapid immunodiagnostic assay has a specificity estimated at 99.4% for
the IgM, 100% for the IgG, and 99.4% for the combined IgM/IgG results (S.F.K., personal
communication). Samples were also tested for neutralization activity using a viral pseudotype-
based assay [8]. Briefly, single cycle lentiviral pseudotypes coated with the S protein and
encoding for a luciferase reporter gene were preincubated with the serum to be tested at a
dilution of 1:100, and added to 293T-ACE2 target cells. The luciferase signal was measured
5
after 48h. The percentage of neutralization was calculated by comparing the signal obtained
with each serum to the signal generated by control negative sera. In some analyses, we
categorized the samples according to the extent of neutralization observed at the 1:100
dilution. Neutralizing activities >50% and >80% corresponded to inhibitory dilution 50% (ID50)
>100 and ID80 >100, respectively.
Statistical analyses
Seropositivity was defined as the presence of detectable anti-SARS-CoV-2 antibodies. The
proportion of seropositive samples was compared by time between onset of symptoms and
collection of blood sample using chi-square test.
Antibody neutralizing activity was compared by age, gender, underlying medical conditions,
time from symptom onset and type of symptoms using chi-square or Fisher’s exact test where
appropriate. Logistic regression was used for multivariable analysis.
The S-Flow MFI and neutralization of sera were compared by delay since onset of symptoms
using the Kruskall-Wallis non-parametric test. The S-Flow MFI of sera with ID50 and ID80
above or below 100 were compared using Student’s t-test. The chi-2 test was used to evaluate
the association between investigated factors and neutralization levels.
All analyses were performed using Stata (Stata Corp., College Station, Texas, USA) or
GraphPad Prism 8 (GraphPad Software, LLC).
Ethical considerations
This study was registered with ClinicalTrials.gov (NCT04325646) and received ethical
approval by the Comité de Protection des Personnes Ile de France III. Informed consent was
obtained from all participants.
Results
Between 6 April and 8 April 2020, 162 hospital staff from Strasbourg University Hospitals who
had recovered from RT-PCR confirmed SARS-CoV-2 infection participated in the
6
investigation. Two individuals who were hospitalized for COVID-19 were excluded from these
analyses to determine serologic responses in those with mild forms of COVID-19. Table 1
indicates the characteristics of these 160 hospital staff. The median age was 32 years (inter
quartile range (IQR): 26-44) and 50 (31.2%) were males. The majority of participants were
medical students (28.1%), doctors (20.0%) or nurses (19.4%).
In terms of possible sources of SARS-CoV-2 infection, 74 (46.2%) reported having had contact
with a COVID-19 patient either in the ward or in the emergency room. A further 38 (23.7%)
reported having had contact with a COVID-19 case outside the health care setting.
One hundred and fifty five (96.9%) had symptoms consistent with COVID-19 (dry cough, fever,
dyspnea, anosmia or ageusia). The median time between onset of symptoms and PCR testing
was 2 days (IQR:1-4), and the median time from onset of symptoms to blood sampling was
24 days (IQR: 21-28, range 13-39).
Figure 1 and Table 2 indicate the seropositivity rates detected by the three assays and
categorized by the delay between onset of symptoms and collection of samples. Across all
160 participants, 159 had detectable anti-SARS-CoV-2 antibodies by S-Flow (99.4%
sensitivity). The only participant whose serology was negative with all assays was a 58-year-
old male with a body mass index of 32 kg/m2 and no other risk factors for severe COVID-19
disease. His blood was sampled 18 days after onset of symptoms which persisted at the time
of blood collection. The S-Flow MFI displays a significantly higher signal in individuals sampled
at days 28-41 compared to those sampled at days 13-20 (Figure 1A). These results suggest
that the overall amount or the affinity of the antibodies improved with time since onset of
symptoms.
7
The IgM rapid test appeared more sensitive than IgG (overall sensitivity: 88.1% vs 71.2%,
repectively), especially at the earlier timepoints (Table 2). The combination of IgG and IgM
rapid test data increased the sensitivity to 95.6%.
Figure 1B and Table 3 show the proportion of individuals with a neutralizing activity, using the
pseudovirus neutralization assay. The proprotion of samples with neutralizing activity
increased over time (Figure 1B), reflecting the increase of antibody titers observed with the S-
Flow. The proportion of individuals with an ID50 ≥100 were 79 %, 92% and 98% at 13-20, 21-
27 and 28-41 days after symptom onset, respectively (P=0.02) (Figure 1B).
The associations between the neutralizing activity and the type of symptoms, age, underlying
medical conditions and tobacco use are summarized in Table 3. The characteristics
associated with neutralizing activity (ID50 > 100) were time since onset of symptoms (P=0.02),
absence of asthma (P=0.02), and absence of a flu vaccine (P=0.02). In a multivariable model
including the three variables, none remained associated with neutralizing activity. We also
analysed the association of high neutralizing activity (ID80 ≥100) with patients characteristics.
High neutralizing activity was associated with time since onset of symptoms (P = 0.004),
having a dry cough (P = 0.04), male gender (P=0.07), high BMI (P=0.02), and high blood
pressure (P = 0.03). All these characteristics remained independently associated with high
neutralizing activity in multivariable analysis except for high blood pressure (P = 0.11). There
was no association between neutralizing activity and ageusia, anosmia, or fever.
We next examined the relationship between the extent of antibody response and the
neutralizing capacity of the sera. Regardless of the time post-symptom onset, samples with
ID50 and ID80 ≥100 displayed significantly higher signals in the S-Flow assay (Figure 1C).
8
Discussion
In this investigation, we described the serologic responses of 160 hospital staff who recovered
from PCR-confirmed mild SARS-CoV-2 infection. Most studies published to date have been
based on hospitalized patients, and therefore have not been able to evaluate serologic
responses in individuals with mild or subclinical infection. Since these individuals are currently
understood to represent at least 80% of all SARS-CoV-2 infections [9], it is crucial to assess
antibody responses in those with mild disease. In our study, we were able to show that all but
one (99.4%) participant had detectable levels of anti-SARS-CoV-2 antibodies from 13 days
after onset of symptoms. The differences observed between time to seroconversion across
the different assays reflect their sensitivity. The S-Flow assay, which displays a high
sensitivity, detected seroconversion in all but one sample. The rapid immunodiagnostic test
performed well 21 days after onset of symptoms. The rapid test therefore has utility as a tool
for diagnosis in the recovery phase of infection. The neutralization assay was positive in 91%
of the samples, and the extent of neutralization paralleled the levels of signal obtained with
the S-Flow and.
At the community level, countries that have implemented public health and social measures
to limit transmission are now lifting some of these measures. Most of the evidence to date
suggests that herd immunity after the first wave of the epidemic will be far from sufficient to
provide protection against a second epidemic wave [10]. In our study, neutralizing ID50 ≥100
were found in 91% of the individuals. We further report that the neutralization activity of the
serum increases with time, reaching 97% four weeks after the onset of symptoms. Therefore,
it is a fair assumption that the majority of individuals with mild COVID-19 generate neutralizing
antibodies within a month after onset of symptoms. Although not yet demonstrated, several
lines of evidence suggest that the presence of neutralizing antibodies may be associated with
protective immunity for SARS-CoV-2 infection. In humans, passive immunotherapy based on
transfer of antibodies from recovered COVID-19 patients decreases disease severity [1,3,6].
In a monkey model, protection from a second SARS-CoV-2 infection is associated with the
presence of neutralizing antibodies in the serum [11]. SARS-CoV-2 NAbs are known to be
9
present in symptomatic individuals [5,12–14]. In a study of 175 convalescent patients with mild
symptoms, NAbs were most often detected 10-15 days after symptom onset [14]. However,
about 30% of recovered patients generated low titers of NAbs (≤1:500), even at a later time
point [14]. Our results are in line with this observation and indicate that recovery from mild
cases is generally, but not always, associated with high titers of NAbs in the serum. Indeed,
we report here that one month after the onset of symptoms, 98% and 77% of individuals
display Nabs with an ID50 and ID80 ≥100, repectively. Antibody titers are generally higher in
patients with severe or critical diseases [6,14]. Interestingly, in our study, individuals with
factors associated with more severe disease (e.g., male sex, high body mass index and high
blood pressure), were more likely to have high titers of neutralizing antibodies compared to
others. This may be due to a higher antigenic burden in such individuals, which will generate
a stronger humoral response, or may, on the contrary, suggest that some antibodies may play
a deleterious role during infection [15]. Future studies are warranted to characterize the
beneficial or detrimental role of specific antibodies in COVID-19 patients and the minimal titer
required for protection.
For patients with SARS-CoV-1, antibodies persist for at least 2 years after symptomatic
infection [16]. In the case of Middle East Respiratory Syndrome (MERS)-CoV, the antibody
response is variable, not robust, and often undetectable when disease is mild [17–20]. Future
studies will help evaluating the persistence of antibodies upon SARS-CoV-2 infection. The
cohort of hospital staff described here provides the opportunity to study the duration of the
humoral response and the dynamics of the neutralization capacity of the sera. A clinical and
virological assessment of potential reinfections will also help establishing the links that may
exist between the antibody response and immune protection.
10
Acknowledgments
We thank the patients and individuals who donated their blood and the ICAReB team for
management and distribution of the samples.
Authors contribution
Conceptualization and Methodology: SFK, TB, YM, OS, AF
Cohort management and sample collection: SFK, YM, RG, LT, CSM, NC, AB, AV, NL, MM,
NM, DR, BH, JDS, AF
Serological and seroneutralisation assays: TB, LG, IS, FA, PS, SVDW, PC, OS
Data assembly and manuscript writing: SFK, TB, YM, RG, LT, OS, AF
Funding acquisition: PC, TR, BH, JDS, OS, AF
Supervision: OS, AF
Conflicts of interest
SFK, TB, YM, RG, LT, LG, IS, FA, PS, CSM, NC, AB, AV, NL, MM, NM, DR, BH, JDS, OS
and AF have no competing interest to declare.
PC is the founder and CSO of TheraVectys.
Fundings
OS lab is funded by Institut Pasteur, ANRS, Sidaction, the Vaccine Research Institute (ANR-
10-LABX-77), Labex IBEID (ANR-10-LABX-62-IBEID), “TIMTAMDEN” ANR-14-CE14-0029,
“CHIKV-Viro- Immuno” ANR-14-CE14-0015-01 and the Gilead HIV cure program. LG is
supported by the French Ministry of Higher Education, Research and Innovation. SFK lab
receives funding from Strasbourg University Hospitals (COVID-HUS ; CE-2020-34)
11
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Tables and Figures
Table 1. Characteristics of the 160 hospital staff with PCR-confirmed SARS-CoV-2 infection
Characteristic N (%)
Male 50 (31.2)
Age (years), median (IQR) 32 (26-44)
Age group (years)
≤29 66 (41.3)
30-39 40 (25.0)
40-49 26 (16.2)
≥50 28 (17.5)
Occupation
Physician 32 (20.0)
Nurse 31 (19.4)
Medical student 45 (28.1)
Orderly 17 (10.6)
Hospital assistant 4 (2.5)
Administrative staff 17 (10.6)
Other 14 (8.8)
Contact with COVID-19 patients
No 80 (50.0)
Yes 74 (46.3)
Missing 6 (3.7)
Level of potential exposure to COVID-19 patients*
None 10 ( 13.5)
Some exposure 27 (36.5)
High exposure 37 (50.0)
Types of care activities**
Mouth care 15 (40.5)
Intubation 13 (35.1)
Other contact with tracheo-bronchial sputum 16 (43.2)
Nasopharyngeal smear 7 (18.9)
Other 10 (27.0)
Symptoms
Minor only 5 (3.1)
Major (cough, fever, dyspnoea, anosmia and 155 (96.9)
ageusia)
Number of major symptoms
0 5 (3.1)
1 41 (25.6)
2 33 (20.6)
3 35 (21.9)
4 32 (20.0)
5 14 (8.8)
Reported symptoms
Ageusia 89 (55.6)
Anosmia 76 (47.5)
Asthenia 137 (85.6)
Dry cough 93 (58.1)
Diarrhea 44 (27.5)
Dyspnoea 55 (34.4)
Fever 97 (60.6)
Fever, feeling of 53 (33.1)
Headache 120 (75.0)
Chest pain 46 (28.7)
Abdominal pain 27 (16.9)
Myalgia 112 (70.0)
Nasal obstruction 57 (35.6)
Nausea 21 (13.1)
Pharyngitis 44 (27.5)
Rhinitis 70 (43.7)
Shivers 45 (28.1)
Sweats 55 (34.4)
Vomiting 3 (1.9)
Other 29 (18.1)
14
Time between onset of symptoms and positive PCR 2 (1-4)

test result (days), median (IQR)
Time from onset of symptoms to blood sample 24 (21-28)
collection (days), median (IQR)
Time from onset of symptoms to blood sample
collection (days)
7-13 1 (0.6)
14-20 28 (17.5)
21-27 83 (51.9)
³ 28 48 (30.0)
* based on 75 participants who reported having contact with COVID-19 patients
** based on the 37 participants who reported having a care activity with high exposure
15
Table 2. Seropositivity with the different assays (Rapid test, S-Flow, and pseudoneutralisation)
according to the time after onset of symptoms
Time from onset of 13-20 21-27 ³28 Total P value

symptoms (days) (n=29) (n=83) (n=48)
Rapid test IgM 26 (89.7) 75 (90.4) 40 (83.3) 141 (88.1) 0.47
Rapid test IgG 14 (48.3) 59 (71.1) 41 (85.4) 114 (71.2) 0.002
Rapid test IgG or IgM 27 (93.1) 80 (96.4) 46 (95.8) 153 (95.6) 0.76
S-Flow 28 (96.5) 83 (100) 48 (100) 159 (99.4) 0.18
Pseudoneutralisation ID50 23 (79.3) 76 (91.6) 47 (97.9) 146 (91.2) 0.020
>100
Pseudoneutralisation ID80 11 (37.9) 44 (53.0) 37 (77.1) 92 (57.5) 0.002
>100
16
Table 3. Proportion of 160 participants with protective immunity according to time since onset of
symptoms, type of symptoms, age, underlying medical conditions and tobacco use.
N Neutralization P value Neutralization P value
ID50 > 100 ID80 > 100
Time between onset of 0.02 0.004
symptoms and collection
of blood sample (days)
13-20 29 23 (79.3) 11 (37.9)
21-27 83 76 (91.6) 44 (53.0)
³28 48 47 (97.9) 37 (77.0)
Number of participants 0.87 0.44
with major symptoms
0 5 4 (80.0) 3 (60.0)
1 41 38 (92.7) 29 (70.7)
2 33 30 (90.9) 16 (48.5)
3 35 35 (94.3) 20 (57.1)
4 32 33 (94.3) 16 (50.0)
5 14 12 (85.7) 8 (57.1)
Ageusia 0.85 0.39
No 84 77 (91.7) 51 (60.7)
Yes 76 69 (90.8) 41(53.9)
Anosmia 0.21 0.48
No 71 67 (94.4) 43 (60.6)
Yes 89 79 (88.8) 49 (55.1)
Dry cough 0.22 0.04
No 67 59 (88.1) 45 (67.2)
Yes 93 87 (93.5) 47 (50.5)
Fever 0.15 0.29
No 63 55 (87.3) 33 (52.4)
Yes 97 91 (93.8) 59 (60.8)
Gender 0.41 0.07
Male 50 47 (94.0) 34 (68.0)
Female 110 99 (90.0) 58 (52.7)
Age group 0.92 0.17
≤29 66 59 (89.4) 33 (50.0)
30-39 40 37 (92.5) 23 (57.5)
40-49 26 24 (92.3) 15 (57.7)
≥50 28 26 (92.9) 21 (75.0)
BMI 0.22 0.02
<18.5 10 7 (70.0) 3 (30.0)
18.5-25 105 97 (92.4) 55 (52.4)
25-30 27 25 (92.6) 19 (70.4)
≥30 17 16 (94.1) 14 (82.4)
Missing 1 1 (100) 1 (100)
Arterial hypertension 0.31 0.03
No 150 136 (90.7) 83 (55.3)
Yes 10 10 (100) 9 (90.0)
Asthma 0.02 0.67
No 149 138 (92.6) 85 (57.1)
Yes 11 8 (72.7) 7 (63.6)
Flu vaccine 0.02 0.46
No 104 99 (95.2) 62 (59.6)
Yes 56 47 (83.9) 30 (53.6)
Blood group 0.26 0.96
A 55 50 (90.9) 31 (56.4)
B 18 18 (100) 9 (50)
AB 3 2 (66.7) 2 (66.7)
O 50 44 (88.0) 30 (60.0)
Not specified 34 32 (94.1) 20 (58.8)
Tobacco use 0.57 0.97
No 141 128 (90.8) 81 (57.5)
Yes 19 18 (94.7) 11 (57.9)
Exposure to patients 0.32 0.49
None 96 85 (88.5) 52 (54.2)
Low 27 26 (96.3) 18 (66.7)
High 37 35 (94.6) 22 (59.5)
17
18
A *
100 105
80
S-Flow (MFI)
S-Flow (%)
104
60
40
103
20
n.a
0 102
pre
13-20
21-27
28-41
pre
13-20
21-27
28-41
B 100
*
100
P = 0.02
100
P = 0.004
% of neutralisation
% of individuals
% of individuals
80 80 80
60 60 60
40 40 40
20 20 20
13-20 n=29
21-27 n=83
28-41 n=48
13-20 n=29
21-27 n=83
28-41 n=48
0 0 0
13-20
21-27
28-41
ID50 <100
< 50% >100
> 50 % ID80 <100
< 80% >100
> 80 %
C **** ****
105 105
S-Flow (MFI)
S-Flow (MFI)
104 104
103 103
102 102
<50% >100
<100 >50% <80% >100
<100 >80%
neutralisation
ID50 neutralisation
ID80
Figure 1. Analysis of SARS-Cov-2 antibody response. (A) Sera from the 160 HCW were surveyed for
anti-SARS-Cov-2 antibodies. S- Flow data are represented by the frequency of S+ cells (n=160, left
panel) and the median Fluorescence intensity (MFI) in positive samples (n=159, middle panel).
Historical pre-epidemic samples (pre) were included to determine backgrounds of S Flow (n=140).
Each dot represents a sample. Samples were grouped according to the number of days after
symptom onset. Statistical analyses were performed using Kruskall-Wallis with Dunn’s multiple
comparisons test or chi-2 test. * p<0.005. n.a.: not applicable (B) Neutralizing activity of the 160 sera.
The ability of each serum to neutralize lentiviral S- pseudotypes was assessed at a serum dilution of
1:100 (left panel). Samples are grouped according to the number of days after symptom onset. For
each time group, the frequencies of samples displaying a ID50 >100 (middle panel) or a ID80 >100
(left panel) were determined. Each dot represents a sample. ** p<0.005 Kruskall-Wallis with Dunn’s
multiple comparisons test. (C) Relationship between serological measurement and neutralizing
activity. The S-Flow MFI of samples displaying ID50 and ID80 above or below 100 are depicted.
Each dot represents a sample. **** p<0.0001 Unpaired t-test. The statistically significant differences
are depicted.
19
20
100
80
60
40
20
0
13 to 20 days 21 to 27 days 28 to 41 days
RDT IgM RDT IgG RDT IgM/IgG S-Flow Pseudo ID50 > 100 Pseudo ID80 > 100
Figure 2. Seropositivity by serologic assay used (Rapid test, S-Flow, and pseudoneutralisation)
according to the time between onset of symptoms and collection of blood sample.
21

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medRxiv preprint doi: https://doi.org/10.1101/2020.05.19.20101832.this version posted May 22, 2020.

The copyright holder for this preprint

Samira FAFI-KREMER1,2,*,, Timothée BRUEL3,*, Yoann MADEC4, Rebecca GRANT4, Laura

Correspondence to [email protected] and [email protected]

neutralizing activity of the sera was tested with a pseudovirus-based assay.

and 28-41 days after symptom onset, respectively (P=0.02).

capacity in recovered patients.

now becoming apparent in terms of a reduction in the daily incidence of SARS-CoV-2

understanding of antibody kinetics following SARS-CoV-2 infection.

detection of antibodies generated in response to SARS-CoV-2 infection and protective

immunity has not yet been established.

PCR-confirmed SARS-CoV-2 infection were invited to participate in the investigation. This

invitation included doctors, nurses, physiotherapists, dentists, medical students, orderlies,

hospital assistants, and hospital administrative staff.

Following informed consent, participants completed a questionnaire which covered

sociodemographic information, underlying medical conditions, and details related to SARS-

symptoms. A 5 mL blood sample was taken from all participants.

Serologic response measurement

assays: 1) a rapid immunodiagnostic assay detecting SARS-CoV-2 spike protein receptor

>100 and ID80 >100, respectively.

Seropositivity was defined as the presence of detectable anti-SARS-CoV-2 antibodies. The

collection of blood sample using chi-square test.

appropriate. Logistic regression was used for multivariable analysis.

the association between investigated factors and neutralization levels.

GraphPad Prism 8 (GraphPad Software, LLC).

obtained from all participants.

had recovered from RT-PCR confirmed SARS-CoV-2 infection participated in the

medical students (28.1%), doctors (20.0%) or nurses (19.4%).

24 days (IQR: 21-28, range 13-39).

160 participants, 159 had detectable anti-SARS-CoV-2 antibodies by S-Flow (99.4%

rapid test data increased the sensitivity to 95.6%.

pseudovirus neutralization assay. The proprotion of samples with neutralizing activity

was no association between neutralizing activity and ageusia, anosmia, or fever.

the S-Flow and.

protective immunity for SARS-CoV-2 infection. In humans, passive immunotherapy based on

required for protection.

exist between the antibody response and immune protection.

management and distribution of the samples.

Conceptualization and Methodology: SFK, TB, YM, OS, AF

NM, DR, BH, JDS, AF

Funding acquisition: PC, TR, BH, JDS, OS, AF

and AF have no competing interest to declare.

PC is the founder and CSO of TheraVectys.

10-LABX-77), Labex IBEID (ANR-10-LABX-62-IBEID), “TIMTAMDEN” ANR-14-CE14-0029,

“CHIKV-Viro- Immuno” ANR-14-CE14-0015-01 and the Gilead HIV cure program. LG is

receives funding from Strasbourg University Hospitals (COVID-HUS ; CE-2020-34)

1. Amanat F, Stadlbauer D, Strohmeier S, et al. A serological assay to detect SARS-CoV-2

6. Zhao J, Yuan Q, Wang H, et al. Antibody responses to SARS-CoV-2 in patients of novel

8. Grzelak L, Temmam S, Planchais C, et al. SARS-CoV-2 serological analysis of COVID-19

12. Hoffmann M, Kleine-Weber H, Krüger N, Müller M, Drosten C, Pöhlmann S. The novel

14. Wu F, Wang A, Liu M, et al. Neutralizing antibody responses to SARS-CoV-2 in a COVID-19

16. MO H, ZENG G, REN X, et al. Longitudinal profile of antibodies against SARS-coronavirus in

Tables and Figures

Time between onset of symptoms and positive PCR 2 (1-4)

Time from onset of 13-20 21-27 ³28 Total P value

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Samira FAFI-KREMER1,2,,, Timothée BRUEL3,, Yoann MADEC4, Rebecca GRANT4, Laura