Abbreviations
ACE2
angiotensin‐converting enzyme 2
COVID‐19
coronavirus disease
ELISA
enzyme‐linked immunosorbent assay
HRP
horseradish peroxidase
IgG, IgA, IgM
immunoglobulin G, A, M
MERS
middle east respiratory syndrome
OD
optical density
RBD
receptor‐binding domain
RNA
ribonucleic acid
RT‐PCR
reverse transcription polymerase chain reaction
RV
rhinovirus
S
spike protein
S1
spike protein receptor‐binding subunit
S2
spike protein membrane fusion subunit
SARS
severe acute respiratory syndrome
To the Editor,
After the appearance of first cases in Wuhan, China in December 2019, the novel human
coronavirus disease, COVID‐19, has become the first coronavirus pandemic in history.
1
On 16 July 2020, more than 13.5 million patients worldwide have been infected with
the novel coronavirus, SARS‐CoV‐2, and more than 584.000 global deaths related to
COVID‐19 have been reported (see: The Center for Systems Science and Engineering (CSSE)
at Johns Hopkins University, Baltimore: https://gisanddata.maps.arcgis.com/apps/opsdashboard/index.html#/bda7594740fd40299423467b48e9ecf6).
The first descriptions of coronaviruses date back to the 1930s when they were isolated
from chickens. Originally, coronaviruses were associated with important diseases in
cattle, poultry, pigs and cats. They are large, enveloped, positive‐stranded RNA viruses
with round structure and long, petal‐shaped spikes protruding from their surface.
Coronaviruses can be divided into three serogroups of which groups I and II have been
isolated from mammals and group III from birds. Members from groups I and II (Group
I: HCo‐229E, HCoV‐NL63; Group II: HCoV‐OC43, HCoV‐HKU1) have been known for decades
as causes for relatively mild common colds in humans. However, in 2002, severe acute
respiratory syndrome (SARS) (Group IIb) and, in 2012, Middle East respiratory syndrome
(MERS) (Group IIc) were shown to be caused by the novel coronaviruses, SARS‐CoV and
MERS‐CoV, respectively, which caused high death rates in up to 10% of infected people.
1
Like SARS‐CoV, SARS‐CoV‐2 uses angiotensin‐converting enzyme 2, ACE2 on human cells
as its receptor
2
and binds to it with its receptor‐binding domain (RBD). The RBD is located in the
spike protein S within S1, the receptor‐binding subunit close to the C‐terminal S2
membrane fusion subunit.
2
The clinical course of COVID‐19 has a tri‐phasic pattern with fever, cough, fatigue
in week 1, dyspnoea, lymphopenia and pneumonia in week 2 and resolution in week 3.
However, in severe cases, thrombocytopenia, coagulopathy, acute kidney injury, myocardial
injury, respiratory distress syndrome and deteriorating multi‐organ dysfunction can
occur.
3
Acute infection can be diagnosed by demonstrating the presence of virus‐derived nucleic
acid by RT‐PCR in nasopharyngeal swabs in patients. However, there is currently no
specific and effective treatment for COVID‐19. Accordingly, quarantine, social distancing
and enhanced hygiene precautions are the only measures to prevent virus spread.
It has been shown that COVID‐19 patients develop SARS‐CoV‐2‐specific antibodies but
it is not known if and in how many infected subjects the virus‐induced antibodies
are protective.
In order to investigate whether COVID‐19 convalescent patients have developed antibodies
that may protect from reinfection, we collected sera from COVID‐19 convalescent patients
approximately 10 weeks after confirmation of COVID‐19 by qRT‐PCR (Table S1) (group
B, n = 25, 11 females, 14 males, age range: 18‐70 years, median age 52.2) and included
for control purposes sera from subjects obtained before the COVID‐19 pandemic (historic
control group P, n = 24, 13 females, 11 males, age range: 18‐68 years, median age
43.2) (Table S1). The course of COVID‐19 in the PCR‐confirmed convalescent subjects
(group B) was relatively mild and did not require hospitalization but the duration
of COVID‐19‐related symptoms varied considerably among patients (ie from 1 to 23 days)
(Table S1). COVID‐19 convalescent patients showed a quite strong and distinct IgG
reactivity to S and RBD whereas no RBD‐specific IgG was found in all but one (ie P014)
of the historic control sera (group P) of whom few showed some S‐specific IgG (Figure 1).
IgA anti‐RBD and anti‐S responses measured in a subset of COVID‐19 convalescent patients
were low and not detectable in a subset of historic controls (Figure S1, Methods in
the Appendix S1). Strong S‐ and RBD‐specific IgM responses were found in convalescent
patients but we found also frequent and distinct IgM responses in the historic controls
(Figure 1). In this context, it must be mentioned that S and RBD contain several glycosylation
sites (Figure S2) (see reference in the Appendix S1). S and RBD used in our ELISA
were expressed in eukaryotic cells and hence were glycosylated which would explain
the occasional and weak recognition by IgG and the more frequent recognition by IgM,
an isotype frequently reacting with glycan moieties, by the presence of anti‐carbohydrate
antibodies in the sera. It is therefore quite possible that anti‐glycan antibodies
may give “false” positive test results when glycosylated RBD or spike proteins are
used in serological assays for COVID‐19. RBD‐specific IgG levels determined by ELISA
were highly correlated with SARS‐CoV‐2‐specific antibodies determined with the fully
automated Siemens, Atellica IM SARS‐CoV‐2 Total (COV2T) test (see methods in Appendix
S1, Figure S3A, Table S2). We also found a significant correlation of RBD‐specific
IgM levels measured by ELISA and the Siemens test (Figure S3B).
FIGURE 1
IgG (upper panel) and IgM (lower panel) reactivity (y‐axis: OD values corresponding
to bound immunoglobulin) to S and RBD determined for COVID‐19 convalescent patients
(group B: B001‐B032, right) and for individuals from a historic control group before
the pandemic (group P: P001‐P023, left). The threshold for background has been subtracted
Receptor‐binding domain‐specific IgM responses in COVID‐19 convalescent patients were
not always associated with corresponding IgG responses (Figure 1). For example, subjects
B003 and B00X showed RBD‐specific IgM reactivity whereas they mounted almost no RBD‐specific
IgG and subject B004 contained S‐ and RBD‐specific IgG but no specific IgM was detected
(Figure 1). We found no correlation between S‐specific IgM and IgG responses and a
significant correlation between RBD‐specific IgM and IgG responses (Figure S4; Methods
in the Appendix S1). While we could not find any correlation between age and S‐ and
RBD‐specific IgM or IgG levels (Figure S5), it was interesting to note that RBD‐specific
IgG and IgM levels were significantly correlated with the duration of COVID‐19 symptoms
suggesting that prolonged disease and thus virus‐load may lead to increased virus‐specific
antibody production (Figure S6).
In a subset of sera, we could analyse antibody reactivity to 25 synthetic overlapping
25‐30 amino acids long peptides spanning the complete receptor‐binding subunit S1,
including RBD (Table S3, Figure S2 and Methods in the Appendix S1) indicating that
there is no relevant peptide‐specific IgG or IgA reactivity detectable (Figures S7,
S8
). Sera from five tested convalescent COVID‐19 subjects and, to a lower degree, sera
from subjects of control group P showed some IgM reactivity to peptides from the N‐
and C‐terminus of S1 and to distinct RBD‐derived peptides (Figures S7, S8). The amino
acid sequences of the larger part of S1‐derived peptides from SARS‐CoV‐2 are highly
conserved in SARS‐CoV but not in the other corona viruses known to cause common colds
in humans (Figures [Link], [Link], [Link], [Link], [Link]) indicating, that the latter
had not induced the peptide‐specific IgM responses. It is a limitation of our study
that our ethics permission did not allow obtaining sputum or nasal secretion for the
analysis of SARS‐CoV‐2‐specific secretory antibodies.
However, the interesting question for us was to study if and how many COVID‐19 convalescent
patients develop antibodies which can inhibit the binding of the virus via RBD to
the corresponding receptor ACE2 which would protect them from a recurrent infection.
Since there is currently no accepted/standard virus neutralization assay authorized
(FDA, July 3, 2020: https://www.cdc.gov/coronavirus/2019‐ncov/lab/resources/antibody‐tests‐guidelines.html),
we developed a molecular interaction assay mimicking SARS‐CoV‐2 binding to its receptor
ACE2 to investigate if COVID‐19 convalescent patients develop antibodies that can
inhibit the binding of the virus‐derived receptor‐binding domain (RBD) to its receptor
ACE2. This ELISA assay is based on plate‐bound recombinant ACE2 which is allowed to
bind to recombinant His‐tagged RBD (Figure 2A). Bound RBD is then detected with a
mouse monoclonal anti‐His antibody followed by a secondary HRP‐labelled anti‐mouse
IgG1 antibody (Figure 2A and methods in this article´s Online Repository). This assay
is similar to an interaction assay which recently became available (https://www.creative‐diagnostics.com/sars‐cov‐2‐inhibitor‐screening‐eia‐kit‐278105‐466.htm;
https://www.researchsquare.com/article/rs‐24574/v1).
FIGURE 2
Molecular interaction assay based on ACE2 and SARS‐CoV‐2 RBD. (A) Scheme of the molecular
interaction assay. ELISA plate‐bound recombinant ACE2 is incubated with His‐tagged
recombinant SARS‐CoV‐2 RBD which is detected with a mouse monoclonal anti‐His‐tag
antibody followed by HRP‐labelled anti‐mouse antibodies. (B) Specific binding of three
different concentrations of RBD vs a control protein (Par j 2) (y‐axis: OD values
correspond to bound RBD) to ACE2. Reactants and concentrations in ng/ml are summarized
below the x‐axis. (C) Inhibition of RBD binding (y‐axis: OD values) to plate‐bound
ACE2 by soluble ACE2 (ACE2 + RBD) vs a control protein (Bet v 1 + RBD). (D) Effects
of serum antibodies from COVID‐19 convalescent subjects (group B) and (E) from subjects
obtained before the COVID pandemic (group P, historic controls) on the ACE2‐RBD interaction.
Shown is the binding of RBD to ACE2 (y‐axis: OD values correspond to amounts of ACE2‐bound
RBD) which had been pre‐incubated with sera or buffer without serum (Co) (x‐axis).
Each result is an average of duplicate determinations with <5% difference between
the two values. The grey bar indicates the area of no alteration of RBD binding to
ACE2 including the 10% variability of the assay. The arrows pointing downwards from
the grey bar indicate the extent of inhibition and the red line marks 50% inhibition
of RBD binding to ACE2. The arrows point upwards of the grey bars show enhancement
of RBD binding to ACE2
Figure 2B shows that RBD binds to ACE2 in a dose‐dependent and specific manner whereas
a negative control protein, the cysteine‐containing, His‐tagged recombinant Parietaria
allergen, Par j 2, did not bind to ACE2 (Methods, Appendix S1). Next, we investigated
whether binding of RBD to ACE2 can be blocked specifically by pre‐incubation with
soluble ACE2 (Figure 2C and Methods in the Appendix S1). We found that pre‐incubation
of RBD with ACE2 almost completely inhibited RBD binding to plate‐bound ACE2 whereas
pre‐incubation with a negative control protein, recombinant major birch pollen allergen,
Bet v 1, did not affect binding of RBD to ACE2 (Figure 2C).
We then studied the effects of antibodies in serum samples of COVID‐19 convalescent
patients on the binding of RBD to ACE2. Figure 2D and Table S2 show the optical density
(OD) values corresponding to the binding of RBD after pre‐incubation with sera from
the 25 COVID‐19 convalescent patients to ACE2. A more than 50% inhibition was found
for six sera (B013, B017, B018, B019, B029, B030), an up to 50% inhibition was found
for nine sera (B003, B014, B015, B020, B024, B025, B027, B031, B032), no inhibition
was found for five sera (B00X, B016, B021, B022, B023) and for five sera (B001, B002,
B004, B026, B028) we noted even an enhancement of RBD binding to ACE2 (Figure 2D,
Table S2). No relevant inhibition was observed for the 24 historic control sera obtained
before the COVID‐19 pandemic indicating a high specificity of our assay (100%) (Figure 2E).
One serum (ie P0014) from the control group which contained elevated S‐ and RBD‐specific
IgM antibodies caused an enhancement of RBD binding to ACE2 (Figure 2E, Table S2)
pointing to the existence of “immune‐enhancing” natural anti‐glycan antibodies. Interestingly,
neither the levels of S‐ nor RBD‐specific IgG or IgM antibodies were correlated with
the inhibition of the binding of RBD to ACE2 in the inhibition assay (Figure S14).
There were also no significant correlations between the percentages of inhibition
of RBD binding to ACE2 and the duration of COVID‐19 symptoms and the age of the subjects,
respectively (Figure S15).
There is a need for assays that can inform about characteristics of SARS‐CoV‐2‐specific
antibodies such as possible protective effects and to detect potentially immune‐enhancing
antibodies. The assay developed by us like another recently described similar assay
(https://www.creative‐diagnostics.com/sars‐cov‐2‐inhibitor‐screening‐eia‐kit‐278105‐466.htm;
https://www.researchsquare.com/article/rs‐24574/v1) would be simple and robust ELISA‐based
molecular interaction assays which may allow testing for antibodies and compounds
capable of inhibiting the binding of SARS‐CoV‐2 RBD to its receptor. This is important
because certain molecules such as ACE2 derivatives or recombinant antibodies are being
considered for treatment of COVID‐19 infections and there is a need to identify more
and distil out the most efficient compounds for treatment.
4
,
5
Once these tests can be validated they may be also useful to characterize and identify
COVID‐19 convalescent subjects producing antibodies capable of inhibiting the virus‐receptor
interaction for obtaining therapeutic convalescent plasma and validating polyclonal
immunoglobulin and monoclonal antibody preparations. Furthermore, once validated these
assays could be suitable for a mass screening of COVID‐19 convalescent subjects regarding
the presence of antibodies which prevent binding of the spike protein to the ACE2
receptor considering the possibility of future outbreaks of the virus. Our data, although
limited, would rather indicate that the natural SARS‐CoV‐2 infection does not establish
an antibody response in all infected subjects which can prevent the receptor‐virus
interaction. Only 60% of COVID‐19‐convalescent patients had produced antibodies that
inhibited the binding of RBD to ACE2. Since we could not perform additional virus
neutralization tests, we cannot exclude the possibility that COVID‐19 convalescent
subjects produce other types of protective antibodies besides those inhibiting RBD‐ACE2
binding. For example, there may be antibodies that may inhibit the fusion of the virus
with the cell membrane or such contributing to virus clearance via Fc‐receptors. However,
our study is the first to provide evidence for an increase in RBD binding to ACE2
caused by sera from patients who produced RBD‐specific IgG antibodies. This could
be explained by the formation of immune complexes consisting of RBD and antibodies
that bind to RBD without blocking the receptor interaction and eventually may be directed
to carbohydrate epitopes of the virus. Such a mechanism of immune complex‐enhanced
SARS‐CoV‐2 receptor binding would explain earlier findings of immune enhancement in
COVID‐19.
6
It is also conceivable that such an immune complex‐mediated cross‐linking of infected
cells or cells containing ACE2‐bound virus could be responsible for the inexplicably
high incidence of thromboembolic events as observed in patients suffering from severe
COVID‐19 despite massive anticoagulation.
7
In this context, it should be mentioned that ACE2 is expressed on vascular endothelial
cells.
8
However, studies are needed to investigate whether antibody‐mediated increases of
RBD binding to ACE2 have a clinical relevance.
In summary, our findings suggest that a natural SARS‐CoV‐2 infection, similar to that
observed previously for rhinovirus (RV) infections,
9
does not induce a protective antibody response inhibiting the virus‐receptor interaction
in all infected patients and therefore underline the urgent need for the development
of a SARS‐CoV‐2 vaccine. The molecular interaction assays could be useful for identifying
subjects having developed protective antibodies and for screening candidate vaccines
to induce antibodies that inhibit the RBD‐ACE2 interaction once they have been validated.
CONFLICT OF INTEREST
Rudolf Valenta has received research grants from HVD Life‐Sciences, Vienna, Austria,
and from Viravaxx, Vienna, Austria. He serves as consultant for Viravaxx. Rainer Henning
and Renata Kiss are employees of Viravaxx, Vienna, Austria. The other authors have
no conflict of interest to declare.
Supporting information
App S1
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Fig S1
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Fig S2
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Fig S3
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Fig S4
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Fig S5
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Fig S6
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Fig S7
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Fig S8
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Fig S9
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Fig S10
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Fig S11
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Fig S12
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Fig S13
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Fig S14
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Fig S15
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Tab S1
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Tab S2
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Tab S3
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