Serologic cross-reactivity of SARS-CoV-2 with endemic and ...€¦ · 22/06/2020 · the four other coronaviruses, with the strongest cross-recognition between SARS-CoV-2 and SARS

Serologic cross-reactivity of SARS-CoV-2 with endemic and seasonal Betacoronaviruses

Jennifer Hicks1,2*, Carleen Klumpp-Thomas3,2*, Heather Kalish1,2*, Anandakumar

Shunmugavel2, Jennifer Mehalko4, John-Paul Denson4, Kelly Snead4, Matthew Drew4,

Kizzmekia Corbett5, Barney Graham5, Matthew D Hall3, Matthew J Memoli6, Dominic

Esposito4, Kaitlyn Sadtler2†

1Trans-NIH Shared Resource on Biomedical Engineering and Physical Science, National

Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda MD

20894

2Section on Immuno-Engineering, National Institute of Biomedical Imaging and Bioengineering,

National Institutes of Health, Bethesda MD 20892 3National Center for Advancing Translational Sciences, National Institutes of Health, Rockville

MD, 20850 4Protein Expression Laboratory, NCI RAS Initiative, Cancer Research Technology Program,

Frederick National Laboratory for Cancer Research, Frederick, MD 21702. 5Vaccine Research Center, National Institute for Allergy and Infectious Disease, National

Institutes of Health, Bethesda, MD 20892 6LID Clinical Studies Unit, Laboratory of Infectious Diseases, Division of Intramural Research,

National Institute for Allergy and Infectious Disease, National Institutes of Health, Bethesda,

MD 20894

*these authors contributed equally to this work †to whom correspondence should be addressed: [email protected]

KEYWORDS:

Infectious disease, serology, coronavirus

for use under a CC0 license. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprintthis version posted June 23, 2020. ; https://doi.org/10.1101/2020.06.22.20137695doi: medRxiv preprint

NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

https://doi.org/10.1101/2020.06.22.20137695

ABSTRACT

In order to properly understand the spread of SARS-CoV-2 infection and development of

humoral immunity, researchers have evaluated the presence of serum antibodies of people

worldwide experiencing the pandemic. These studies rely on the use of recombinant proteins

from the viral genome in order to identify serum antibodies that recognize SARS-CoV-2

epitopes. Here, we discuss the cross-reactivity potential of SARS-CoV-2 antibodies with the full

spike proteins of four other Betacoronaviruses that cause disease in humans, MERS-CoV,

SARS-CoV, HCoV-OC43, and HCoV-HKU1. Using enzyme-linked immunosorbent assays

(ELISAs), we detected the potential cross-reactivity of antibodies against SARS-CoV-2 towards

the four other coronaviruses, with the strongest cross-recognition between SARS-CoV-2 and

SARS /MERS-CoV antibodies, as expected based on sequence homology of their respective

spike proteins. Further analysis of cross-reactivity could provide informative data that could lead

to intelligently designed pan-coronavirus therapeutics or vaccines.

INTRODUCTION

The SARS-CoV-2 pandemic has reached almost every country on Earth. As with many viral

infections, our immune system responds to SARS-CoV-2 infection through a variety of cellular

and humoral effectors. These include antibodies produced by B cells, which can be formed

against various viral proteins. For SARS-CoV-2, antibodies have been detected that recognize

three of the four SARS-CoV-2 proteins exposed on the surface of the viral capsid: the

nucleocapsid (N), envelope (E), and spike (S) proteins (1). The spike protein forms as a

homotrimer and mediates receptor binding through its receptor binding domain (RBD) to host

cell ACE2 and is thus the major target of neutralizing antibody responses (2, 3). When testing for

the presence of SARS-CoV-2 antibodies, researchers have utilized the full spike ectodomain as

well as the RBD domain alone for antigens in enzyme-linked immunosorbent assays (ELISAs)

and other serologic assays (4).

The zoonotic Betacoronaviruses SARS-CoV and SARS-CoV-2 (endemic/pandemic B-lineage),

and MERS (endemic C-lineage) transferred primarily from bats, while the viruses OC43 and

HKU1 (seasonal A-lineage coronaviruses) are endemic in humans (5, 6). All of these viruses



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Figure 1: Five different Betacoronaviruses with potential for cross-reactivity. We evaluated

the serologic cross-reactivity of five betacoronaviruses in the context of ELISA-based detection

of IgG, IgM, and IgA antibodies against SARS-CoV-2.



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bear the spike protein on their surface (7, 8). As such, anti-spike antibodies are common in

response to each of the five human-infecting Betacoronaviruses (9–11). Knowledge of cross-

reactivity of anti-spike antibodies against different viruses is critical for understanding of SARS-

CoV-2 immunity of individuals who have had prior exposure to other Betacoronaviruses and of

potential future immunity of COVID-19 survivors to other coronaviruses (12). Furthermore,

knowledge of cross-reactivity is necessary to understand and properly interpret results from

serologic studies such as serosurveys and clinical antibody tests (13, 14). Previous research has

shown minimal cross-reactivity between RBD domains from differing coronaviruses; however,

these studies largely ignore the rest of the spike protein, which will be an important consideration

for identification of potential therapeutic antibodies and can be used in vitro to help identify

polyclonal responses that are not detected with RBD alone (15).

Here, we evaluated the serologic reactivity of pre-pandemic archival blood serum samples (pre-

2019) and samples collected in April 2020 from a community highly affected by SARS-CoV-2.

Utilizing twelve previously reported ELISAs (15), we tested IgG, IgM and IgA reactivity against

spike proteins from SARS-CoV-2, MERS-CoV, SARS-CoV, HCoV-OC43, and HCoV-HKU1

(Fig. 1).

RESULTS

Sequence homology between pandemic, endemic, and seasonal coronaviruses

To evaluate the potential for cross-reactivity, we first compared the spike protein sequence

homology among SARS-CoV-2, MERS-CoV, SARS-CoV, HCoV-OC43, and HCoV-HKU1

(Fig. 2, Supplementary Figure 1). The greatest homology was between SARS-CoV-2 and

SARS-CoV (76% identity, 87% similarity), followed by MERS (42% identity, 58% similarity)

and lastly OC43/HKU1 (OC43: 30% identity, 41% similarity; HKU1: 29% identity, 40%

similarity). A-lineage OC43 and HKU1 are more similar to each other (64% identity, 75%

similarity) than to the two endemic Betacoronaviruses. There is a larger fraction of homology

towards the C-terminus of the protein in all coronavirus spike proteins, which represents the

major structural regions of the protein including the heptad repeat regions responsible for



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Figure 2: Sequence homology of SARS-CoV-2 with endemic and seasonal

Betacoronaviruses. SARS-CoV-2 spike ELISA antigen protein sequence aligned with MERS-

CoV (MERS), SARS-CoV (SARS1), OC43, and HKU1 Betacoronaviruses. (A) Percent (%)

similarity to SARS-CoV-2. (B) Percent (%) identity to SARS-CoV-2.



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insertion of the fusion peptide into the host cell membrane. Homology is significantly lower in

the N-terminal regions of spike, with significant lack of similarity in the regions including the

receptor-binding domain, correlating with the difference in receptors and determinants used for

host cell entry in the different Betacoronaviruses (MERS-CoV: receptor dipeptidyl peptidase-4

(DPP4), SARS-CoV/SAR-CoV-2: ACE2, OC43/HKU1: the sugar N-Acetylneuraminic acid)(8).

Serologic reactivity of anti-spike IgG, IgM and IgA antibodies

Functional cross-reactivity was determined through the use of enzyme-linked immunosorbent

assays (ELISAs) measuring IgG, IgM and IgA subclasses, representing mature, early stage, and

mucosal specific serologic responses, respectively. We produced recombinant soluble spike

proteins of SARS-CoV-2, MERS, SARS-CoV, OC43, and HKU1 using the Expi293 expression

system, which yielded pure, intact ectodomain trimers suitable for ELISA (16). Notably, the

yields of all coronavirus spike proteins were significantly different even though all four of five

were cloned in identical vectors and contained the same modifications to the wildtype sequences

(elimination of furin cleavage site, prefusion-stabilizing proline mutations (2P), similar C-

terminal tags), none of which is expected to alter serologic recognition due to their internal

locations. The HCoV-OC43 construct has all of these features but the wild-type furin cleavage

site is present. Using similar expression conditions, SARS-CoV-2 spike was produced at a

maximum of 2 mg/L culture, while the other spike proteins were significantly easier to produce

with yields of 5, 11, 8, and 6 mg/L respectively for SARS-CoV, MERS, OC43, and HKU1. We

utilized a semi-automated ELISA protocol to detect serum antibodies from pre-2019 archival

samples and samples from a community with high SARS-CoV-2 prevalence during the 2020

pandemic (Fig. 3). In serum samples collected from healthy volunteers prior to 2019, there was

minimal reactivity with SARS-CoV-2, MERS and SARS-CoV. The majority of tested samples (n

= 114) displayed high IgG reactivity with OC43 and HKU1 spike proteins, consistent with the

extensive spread of seasonal Betacoronavirus infections within the United States (Fig. 3a,b). As

reported previously, we detected a high proportion of donors who seroconverted and were

SARS-CoV-2 IgG+ in a community in New York City, along with a significant number of IgM

and IgA seropositive donors, including several donors who were non-symptomatic (15). All

samples had low levels of IgM reactivity against MERS, SARS-CoV, OC43, and HKU1 (Fig.



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Figure 3: Serologic positivity of immunoglobulins G, M and A for five Betacoronaviruses in

pre-2019 and high prevalence SARS-CoV-2 blood donors. Signal intensity in archival

negative (pre-2019, black), hot-spot community symptomatic (pink), and hot-spot community

asymptomatic (teal) blood donors for (a-b) IgG, (c-d) IgM, and (e-f) IgA.

SARS2 MERS SARS1 OC43 HKU10

1

2

3

4Sp

ike

OD

(IgG

)

SARS-2 MERS SARS1 OC43 HKU10

1

2

3

4

Spik

e O

D (I

gM)

Archival NegativeSymptomaticAsymptomatic

SARS-2 MERS SARS-1 OC43 HKU10

1

2

3

4

Spik

e O

D (I

gA)

1

Arch

ival

Neg

ativ

e (n

= 1

14)

Sym

ptom

atic

(n =

68)

Asym

p (n

= 6

)


2

3

1

Arch

ival

Neg

ativ

e (n

= 1

14)

Sym

ptom

atic

(n =

68)

Asym

p (n

= 6

)


2

3

1

Arch

ival

Neg

ativ

e (n

= 1

14)

Sym

ptom

atic

(n =

68)

Asym

p (n

= 6

)


2

3

A

B

C

D

E

F



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3c,d). IgA antibodies were present at higher levels than IgM, but still well below levels of IgG,

correlating well with biologic prevalence of antibody classes in response to pathogens (Fig. 3e,f).

Minimal linear correlation of SARS-CoV-2 signal intensity with other Betacoronaviruses

When comparing the assay absorbance signal (optical density, OD) between SARS-CoV-2 and

the other spike proteins in the high-incidence population, we saw a stronger correlation of signal

intensity between SARS-CoV-2 and SARS-CoV IgG (Correlation = 0.711, R2 = 0.505) and the

lowest correlation with HKU1 (Correlation = 0.281, R2 = 0.079) (Fig. 4a, Supplementary

Figure 2). Though there was not a precise linear correlation for IgG, donors who represented

signal intensity in the lower 50% of SARS-CoV-2 absorbance readings did have a significantly

lower MERS and SARS-CoV signal intensity when compared to the upper 50% of SARS-CoV-2

intensity (Fig. 5). Overall, these data suggest some cross-reactivity occurs that is more easily

detectable at high titers of antibody.

Cross-reactivity of SARS-CoV-2 IgG antibodies with endemic and seasonal coronaviruses

Since we observed a difference in the IgG signal intensity of other Betacoronaviruses with high

levels of SARS-CoV-2 antibodies, we further analyzed the relationship between SARS-CoV-2

seroprevalence and antibody titer with SARS-CoV, MERS, OC43, and HKU1 in pre-pandemic

(pre-2019), high-prevalence symptomatic donors, and high-prevalence asymptomatic donors

(Fig. 6, Supplementary Figure 3). Overall, archival pre-2019 samples displayed an equivalent

low signal intensity of SARS-CoV-2, MERS, and SARS-CoV spike reactivity (Fig. 6a). One

cross-reactive donor from this group was negative for both MERS and SARS-CoV. As

previously discussed, the majority of donors were OC43 and HKU1 seropositive due to the broad

circulation of these viruses in humans. In the high incidence community, for both symptomatic

and asymptomatic individuals, there appeared to be a correlation in SARS-CoV-2 signal

intensity with MERS and SARS-CoV. To further analyze this, we directly compared the signal

intensity of archival sample controls to the high-incidence pandemic population (Fig. 6b). There

was a significant difference in signal intensity of MERS, SARS-CoV, OC43, and HKU1,



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Figure 4: SARS-CoV-2 signal intensity compared with signal intensity of other

Betacoronaviruses in pandemic hot-spot community blood draws. (a) Anti-spike IgG signal

intensity (b) Anti-spike IgM signal intensity, and (c) Anti-spike IgA signal intensity.

y = 0.5322x + 2.3931R² = 0.2664

0

1

2

3

4

0 2 4

MERS

y = 0.6897x + 1.9104R² = 0.505

0

1

2

3

4

0 2 4

SARS1

y = 1.5062x - 2.3641R² = 0.3192

0

1

2

3

4

0 2 4

OC43

y = 0.5453x + 1.2447R² = 0.0791

0

1

2

3

4

0 2 4

HKU1

y = 1.1227x + 0.6985R² = 0.2538

0

1

2

3

4

0 2 4

MERS

y = 1.2574x + 0.6306R² = 0.3797

0

1

2

3

4

0 2 4

SARS1

y = 0.5964x + 0.3509R² = 0.3058

0

1

2

3

4

0 2 4

OC43

y = 0.7332x + 0.5333R² = 0.1876

0

1

2

3

4

0 2 4

HKU1

y = 3.6353x + 0.7493R² = 0.046

0

1

2

3

4

0 2 4

MERS

y = 3.3203x + 0.6607R² = 0.3707

0

1

2

3

4

0 2 4

SARS1

y = 5.9364x + 0.4039R² = 0.1591

0

1

2

3

4

0 2 4

OC43

y = 7.7015x + 0.545R² = 0.0673

0

1

2

3

4

0 2 4

HKU1

Other Coronaviridae spike (OD)

SAR

S-C

oV-2

spi

ke (O

D)

IgG IgM IgAA B C



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Figure 5: High titers of SARS-CoV-2 spike antibodies correlate with an increase in ELISA

signal intensity for other Betacoronavirus reactivity. Comparison of the mean absorbance

(optical density, OD) of the upper (blue) and lower (red) 50% of SARS-CoV-2 signal intensity

for (a) IgG, (b) IgM, and (c) IgA.

SARS2 MERS SARS OC43 HKU10

1

2

3

4Sp

ike

OD

(IgG

)

3.81 1.91 2.59 3.81 3.65

2.42 0.80 0.90 3.47 3.21

x ̅=x ̅=

**** **** **** *** **


1

2

3

4

Spik

e O

D (I

gM)

Upper 50

Lower 50

2.02 0.12 0.23 0.15 0.09

0.28 0.10 0.07 0.10 0.07

x ̅=x ̅=

**** ns ** * ns


1

2

3

4

Spik

e O

D (I

gA)

1.50 0.28 0.36 1.21 0.67

0.28 0.06 0.05 0.60 0.31

x ̅=x ̅=

**** * ** ** **

IgG IgM IgA



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Figure 6: Anti-spike IgG signal intensity in SARS-CoV-2 seropositive and seronegative

blood samples. (a) Relationship of SARS-CoV-2 spike IgG signal intensity in archival (black),

symptomatic high exposure (pink) and asymptomatic high exposure (teal) donors. (b)

Comparison of archival sample IgG reactivity with symptomatic high exposure sample

reactivity. Students T-test.

SARS-2 MERS0

1

2

3

4

SARS-2SARS-10

1

2

3

4

SARS-2 OC430

1

2

3

4

SARS-2 HKU10

1

2

3

4

SARS-2 MERS0

1

2

3

4

SARS-2SARS-10

1

2

3

4

SARS-2 OC430

1

2

3

4

SARS-2 HKU10

1

2

3

4

SARS-2SARS-10

1

2

3

4

SARS-2 MERS0

1

2

3

4

SARS-2 OC430

1

2

3

4

SARS-2 HKU10

1

2

3

4

Arch. Symp.0

1

2

3

4

p = 0.0123

Arch. Symp.0

1

2

3

4

p < 0.0001

Arch. Symp.0

1

2

3

4

p < 0.0001

Arch. Symp.0

1

2

3

4

p < 0.0001

Spik

e Ig

G (O

D)

A

B

Spik

e Ig

G (O

D)



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suggesting potential cross-reactivity of SARS-CoV-2 IgG antibodies with MERS, SARS-CoV,

OC43 and HKU1 spike proteins.

DISCUSSION

Cross-reactivity of antibodies with multiple coronaviruses is an important consideration in

studying the SARS-CoV-2 pandemic, both technically, for identifying individuals who have

been exposed to and recovered from the virus, as well as therapeutically, to identify broadly

neutralizing antibodies or epitopes on multiple coronavirus subtypes (12, 17, 18). Accordingly,

we analyzed potential serologic cross-reactivity of antibodies with spike proteins derived from

SARS-CoV-2 as well as two endemic (MERS, SARS-CoV) and two seasonal (OC43, HKU1)

Betacoronavirus species. It is unclear, in terms of plasmid-based protein expression, why there is

so much variability in spike protein expression levels between the different viruses, but this

argues again for significant differences in the behavior of these proteins regardless of their

primary sequence homology.

Antibodies that react with the spike proteins of OC43 and HKU1 are highly prevalent in the

general population of the United States as determined by their measurement in archival pre-2019

serum samples. Previous reports of their prevalence show that the majority of children are

exposed to OC43 and seroconvert early in life (19). The detection of high serologic reactivity of

archival controls with HKU1 might, thus, be due to the strong seroprevalence of OC43

antibodies. Further studies would be needed to determine this interaction, though due to the high

level of sequence and structural homology of their spike proteins, such a cross-reactivity between

the two tested seasonal Betacoronaviruses would not be surprising.

When compared to reactivity with the SARS-CoV-2 spike protein, antibodies that react to OC43

and HKU1 have minimal cross-reactivity with the pandemic SARS-CoV-2 or two other endemic

coronaviruses, MERS and SARS-CoV. This phenotype correlates with the sequence homology

of these proteins, wherein SARS-CoV-2 spike is more similar to SARS-CoV and MERS, as

opposed to OC43 and HKU1 seasonal coronaviruses.



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When comparing serum from healthy volunteers collected pre-2019 (archival controls) to those

from a high-exposure community, we observe that SARS-CoV-2 antibodies react intermediately

with MERS and SARS-CoV spike proteins. The mean ELISA signal intensity is significantly

greater for both MERS and SARS-CoV when comparing archival controls versus the high-

incidence community. Although there is minimal linear correlation between signal intensity of

SARS-CoV-2 and MERS/SARS-CoV, the higher titer SARS-CoV-2 donors also display a

significantly higher MERS and SARS-CoV signal intensity compared to their lower titer

counterparts within the same population.

Given the low seroprevalence of SARS-CoV and MERS outside of their endemic regions, and

the significantly lower reactivity of SARS-CoV-2 patient sera to SARS-CoV and MERS spike

proteins, it is likely that any reactivity between the pandemic SARS-CoV-2 pandemic and

MERS/SARS-CoV endemic viruses would result in minimal noise between SARS-CoV-2 signal

and endemic coronavirus signal in serological assays. In countries with a higher prevalence of

MERS & SARS-CoV, researchers should include thorough analysis of archival patient sera (pre-

2019), including sera from known SARS-CoV and MERS convalescent patients, to properly

analyze the resulting data and adjust any estimates of seropositivity as needed. No clinical

serology studies of SARS-CoV-2 immunity in populations previously infected with either SARS

or MERS have yet emerged.

Additionally, individuals who have strongly seroconverted after SARS-CoV-2 infection, and

who display cross-reactivity for both MERS and SARS-CoV spike proteins, are of great interest

for translational study. These individuals could potentially harbor antibodies that are universally

reactive to multiple Betacoronaviruses and, if these antibodies are functional for neutralization,

could be important to identify to inform the development of novel therapeutics or vaccines.

MATERIALS & METHODS

Human serum samples

Archival (pre-2019) serum samples (n = 114) were collected between January 2014 and

December 2018 from healthy adults (aged 18 – 55 years) through an existing NIH study



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NCT01386424. High-incidence community samples are deidentified uncoded samples donated

from a community blood draw from donors in New York and New Jersey in April 2020. Twenty-

two (22) of these donors had a previous SARS-CoV-2 nasopharyngeal swab PCR-based

diagnosis, 46 were symptomatic but undiagnosed, and 6 were asymptomatic but had known

exposure (n = 68 symptomatic, n = 6 asymptomatic). All clinical trials were conducted in

accordance with the provisions of the Declaration of Helsinki and Good Clinical Practice

guidelines. All clinical trial participants signed written informed consent prior to enrollment.

Plasmid sourcing and preparation

SARS-CoV-2, MERS-CoV, HCoV-HKU1 and SARS-CoV spike plasmids were produced from

the McLellan lab at UT Austin and NIAID VRC and prepared as previously described (2, 20,

21). Briefly, for HCoV-OC43 S, a mammalian-codon-optimized gene encoding HCoV-OC43 S

(GI: 744516696) ectodomain with a C-terminal T4 fibritin trimerization domain, an HRV3C

cleavage site, an 8xHis-tag and a Twin-Strep-tag were synthesized and subcloned into the

eukaryotic-expression vector pαH. The S1/S2 furin-recognition site was mutated to produce a

single-chain S protein and 2 prolines were substituted, following previous-published prefusion

stabilizing mutation strategy.

Protein production and purification

Soluble spike trimers were produced by expression in Expi293 cells and purified by a

combination of tangential flow filtration, immobilized metal affinity chromatography, and

desalting, following the procedures noted in Esposito et al. Expression was carried out at 37°C

for 72 hours prior to harvest. Final purified proteins were validated by a combination of SDS-

PAGE and analytical size exclusion chromatography (AnSEC). All spike proteins produced

single peaks on AnSEC over a Superdex 200 column, and the peak elution was consistent with

the size of a trimeric spike protein. Of note, the OC43 spike protein undergoes cleavage during

SDS-PAGE leading to the appearance of two bands at 80 and 100 kDa as well as the

appropriately full-length band migrating at 180 kDa. AnSEC confirms that this is an artifact of

the SDS-PAGE process, as the protein elutes in a single trimeric peak of the appropriate size.

Enzyme-linked Immunosorbent Assays



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We performed ELISAs as previously described (15). Briefly, spike proteins were suspended at 1

ug/ml in 1x PBS. One hundred (100) microliters of protein suspension was added to each well of

a 96-well Nunc MaxiSorp ELISA plate and allowed to coat overnight at 4oC for 16 hours. Wells

were washed three times with 300 ul of 1x PBS + 0.05% Tween20 (wash buffer) followed by

blocking for 2 hours at room temperature with 200 ul of 1x PBS + 0.05% Tween20 + 5% Non-

fat dry milk (blocking buffer). Wells were washed again three times with 300 ul of wash buffer

prior to addition of 100 ul of sample diluted in blocking buffer (serum samples were heat

inactivated for 45 minutes at 56oC and diluted at 1:400 in blocking buffer). Samples were

incubated for 1 hour at room temperature, then washed three times with 300 ul of wash buffer.

One hundred (100) microliters of 1-Step Ultra TMB Substrate (ThermoFisher) was added and

the plate was incubated for 10 minutes prior to stopping the reaction with 1N sulfuric acid (Stop

Solution, ThermoFisher). Absorbance was read at 450 nm and 650 nm on a BioTek Epoch2 plate

reader. The process is semi-automated through the use of a BioTek EL406 plate

washer/dispenser and two BioStack 4 plate stackers to minimize plate-to-plate variation and

increase throughput (see Klumpp-Thomas C, Kalish H et al. 2020 for detailed automation

methods).

Data Analysis

Absorbance values (optical density) were collected at 450 and 650 nm. A650 was subtracted

from A450 to remove background signal. Data were subsequently analyzed utilizing Microsoft

Excel and GraphPad Prism.



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https://doi.org/10.1101/2020.06.22.20137695

ACKNOWLEDGEMENTS

The authors would like to thank Golan Ben-Oni, Rabbi Shua Brook, Dr. Adam Polinger, Dr. Avi

Rosenberg, and the Jewish community of New York and New Jersey for their generous donation

of blood samples use in this assay. We thank members of the FNLCR Protein Expression

Laboratory (William Gillette, Simon Messing, and Vanessa Wall) for support in DNA

production and protein purification. This research was supported in part by the Intramural

Research Program of the NIH, including the National Institute of Biomedical Imaging and

Bioengineering, the National Institute of Allergy and Infectious Disease, and the National Center

for Advancing Translational Sciences. This project has been funded in part with Federal funds

from the National Cancer Institute, National Institutes of Health, under contract number

HHSN261200800001E. Disclaimer: The NIH, its officers, and employees do not recommend or

endorse any company, product, or service.



https://doi.org/10.1101/2020.06.22.20137695

SUPPLEMENTAL FIGURES AND LEGENDS

Supplementary Figure 1: BLAST alignment of 4 coronaviruses with SARS-CoV-2.

Identities:958/1244(77%), Positives:1091/1244(87%), Gaps:24/1244(1%)

Query 12 PAYTN--SFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPV 69

P YT S RGVYYPD++FRS L+ TQDLFLPF+SNVT FH I+ F NPV

Sbjct

15 PNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTIN-------HTFGNPV 67

Query 70 LPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGV 129

+PF DG+YFA+TEKSN++RGW+FG+T+++K+QS++I+NN+TNVVI+ C F+ C++PF V

Sbjct

68 IPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAV 127

Query 130 YYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKI 189

+ ++ ++ +A NCTFEY+S F +D+ K GNFK+LREFVFKN DG+ +

Sbjct

128 ----SKPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYV 183

Query 190 YSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAA 249

Y + PI++VRDLP GF+ L+P+ LP+GINIT F+ +L + +P W AA

Sbjct

184 YKGYQPIDVVRDLPSGFNTLKPIFKLPLGINITNFRAIL----TAFSPAQDI--WGTSAA 237

Query 250 AYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPT 309

AY+VGYL+P TF+LKY+ENGTITDAVDC+ +PL+E KC++KSF ++KGIYQTSNFRV P+

Sbjct

238 AYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPS 297

Query 310 ESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVS 369

+VRFPNITNLCPFGEVFNAT+F SVYAW RK+ISNCVADYSVLYNS FSTFKCYGVS

Sbjct

298 GDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVS 357

Query 370 PTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDS 429

TKLNDLCF+NVYADSFV++GD+VRQIAPGQTG IADYNYKLPDDF GCV+AWN+ N+D+

Sbjct

358 ATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDA 417

Query 430 KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGV 489

GNYNY YR R L+PFERDIS + PC NCY+PL YGF T G+

Sbjct

418 TSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTP-PALNCYWPLNDYGFYTTTGI 476

Query 490 GYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQ 549

GYQPYRVVVLSFELL+APATVCGPK ST+L+KN+CVNFNFNGLTGTGVLT S+K+F PFQ

Sbjct

477 GYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQ 536

Query 550 QFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVA 609

QFGRD++D TD+VRDP+T EILDI+PC+FGGVSVITPGTN S++VAVLYQDVNCT+V A

Sbjct

537 QFGRDVSDFTDSVRDPKTSEILDISPCAFGGVSVITPGTNASSEVAVLYQDVNCTDVSTA 596

Query 610 IHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPGS 669

IHADQLTP WR+YSTG+NVFQT+AGCLIGAEHV+ SYECDIPIGAGICASY T +

Sbjct

597 IHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTVS----L 652

Query 670 ASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYIC 729

S + +SI+AYTMSLGA++S+AYSNN+IAIPTNF+IS+TTE++PVSM KTSVDC MYIC

Sbjct

653 LRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMYIC 712

Query 730 GDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFS 789

GDSTEC+NLLLQYGSFCTQLNRAL+GIA EQD+NT+EVFAQVKQ+YKTP +K FGGFNFS

Sbjct

713 GDSTECANLLLQYGSFCTQLNRALSGIAAEQDRNTREVFAQVKQMYKTPTLKYFGGFNFS 772

Query 790 QILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPP 849

QILPDP KP+KRSFIEDLLFNKVTLADAGF+KQYG+CLGDI ARDLICAQKFNGLTVLPP

Sbjct

773 QILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPP 832

Query 850 LLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLI 909

LLTD+MIA YT+AL++GT T+GWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQK I

Sbjct

833 LLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQI 892

Query 910 ANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSR 969

ANQFN AI +IQ+SL++T++ALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSR

Sbjct

893 ANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSR 952

Query 970 LDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFC 1029

LDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFC

Sbjct

953 LDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFC 1012

Query 1030 GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWF 1089

GKGYHLMSFPQ+APHGVVFLHVTYVP+QE+NFTTAPAICH+GKA+FPREGVFV NGT WF

Sbjct

1013 GKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWF 1072

Query 1090 VTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPD 1149

+TQRNF+ PQIITTDNTFVSGNCDVVIGI+NNTVYDPLQPELDSFKEELDKYFKNHTSPD

Sbjct

1073 ITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSPD 1132

Query 1150 VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSGYIPEAPRDGQAY 1209

VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSGYIPEAPRDGQAY

Sbjct

1133 VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSGYIPEAPRDGQAY 1192

Query 1210 VRKDGEWVLLSTFLGRSLEVLFQGPGHHHHHHHHSAWSHPQFEK 1253

VRKDGEWVLLSTFLGRSLEVLFQGPGHHHHHHHHSAWSHPQFEK

Sbjct

1193 VRKDGEWVLLSTFLGRSLEVLFQGPGHHHHHHHHSAWSHPQFEK 1236


Query 249 AAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQP 308

AA+YV LQP TFLL ++ +G I A+DC + LS+ C+ +SF VE G+Y S+F +P

Sbjct

294 AAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKP 353

Query 309 TESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGV 368

+ S+V C F + + T VY + R +NC + + L + S + F C +

Sbjct

354 SGSVVEQAEGVE-CDFSPLLSGTP-PQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQI 411

Query 369 SPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVI-

AWNSNNL 427

SP + C++++ D F ++ G I+ +NYK C+I A +NL

Sbjct

412 SPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNL 471

Query 428 DSKVGG-NYNYLYRLFRKSNLKPFERDISTEIYQAGS

----TPCNGV------EGFNCYF 476

+ Y+Y+ + R F D TE+ Q + +PC + E + Y

Sbjct

472 TTITKPLKYSYINKCSR------FLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYR 525

Query 477 ----PLQSYGFQPTNGVGYQPYRVVVLSFELLHAPAT

----VCGPKKSTNLVK-----NK 523

PL+ G+ +G + + F + T VC + N K

Sbjct

526 KQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGN 585

Query 524 CVNFNFNGLTGTGVLTESNKKFLPFQQFGRDI-ADTTDAVRDPQTLEILDITPCSFGGVS 582

CV ++ G++G GV + Q+F D + D L C VS

Sbjct

586 CVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCL--RACVSVPVS 643

Query 583 VITPGTNTSNQVAVLYQDVNCTEVPVAI--HADQLTPTWRVYSTGSNVFQTRAGCLIGAE 640

VI ++ A L+ V C + + ++ + + QT GC++G

Sbjct

644 VIYDKETKTH--ATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGL-

700

Query 641 HVNNSY---ECDIPIGAGICASYQT-QTNSPGSASSVASQSIIAYTMSLGAENSVAYSNN 696

VN+S +C +P+G +CA T T +P S SV + +A +++ V N+

Sbjct

701 -VNSSLFVEDCKLPLGQSLCALPDTPSTLTPASVGSVPGEMRLA-SIAFNHPIQVDQLNS 758

Query 697 S---IAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRAL 753

S ++IPTNF+ VT E + ++ K +VDC Y+C +C LL +YG FC+++N+AL

Sbjct

759 SYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQAL 818

Query 754 TGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGG-FNFSQILP---DPSKPSKRSFIEDLLF 809

G + QD + + +FA VK +P I FGG FN + + P S RS IEDLLF

Sbjct

819 HGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLF 878

Query 810 NKVTLADAGFIKQYGDCL--GDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGT 867

+KVT+AD G+++ Y DC+ G +ARDLICAQ G VLPPL+ M A YTS+LL

Sbjct

879 DKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSI 938

Query 868 ITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSST 927

GWT G + IPFA + YR NG+G+TQ VL ENQKLIAN+FN A+G +Q ++T

Sbjct

939 AGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQTGFTTT 998

Query 928 ASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRL 987

A K+QD VN NAQAL+ L +LS+ FGAIS+ + DI+ RLDPPE + QIDRLI GRL

Sbjct

999 NEAFHKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDPPEQDAQIDRLINGRL 1058

Query 988 QSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVV 1047

+L +V QQL+R+ SA LA K++ECV QSKR FCG+G H++SF +AP+G+

Sbjct

1059 TTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLY 1118

Query 1048 FLHVTYVPAQEKNFTTAPAICHDGKAH---FPREGVFV-SNGTH----WFVTQRNFYEPQ 1099

F+HV Y P+ +A +C P G F+ +N T W T +FY P+

Sbjct

1119 FMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYAPE 1178

Query 1100 IITTDNTFVSGNCDVVIGIVNNTVYDPLQPEL------DSFKEELDKYFKNHTSPDVDLG 1153

IT+ NT V + + L P L F++ELD++FKN ++ + G

Sbjct

1179 PITSLNTKY-----VAPQVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFG 1233

Query 1154 DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSGYIPEAPRDGQAYVRKD 1213

++ IN +++++ E+ L +V K LNES IDL+ELG Y GSGYIPEAPRDGQAYVRKD

Sbjct

1234 SLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTYGSGYIPEAPRDGQAYVRKD 1293

Query 1214 GEWVLLSTFLGRSLEVLFQGPGHHHHHHHHSAWSHPQFEKGGGSGGGGSGGSAWSHPQFE 1273

GEWVLLSTFLGRSLEVLFQGPGHHHHHHHHSAWSHPQFEKGGGSGGGGSGGSAWSHPQFE

Sbjct

1294 GEWVLLSTFLGRSLEVLFQGPGHHHHHHHHSAWSHPQFEKGGGSGGGGSGGSAWSHPQFE 1353

Query 1274 K 1274

KSbjct

1354 K 1354


Query 514 KKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFL-PFQQFGRDIADTTDAVRDPQTLEILD 572

K +T++ CVN++ G++G G+ E N + +Q D RD T

Sbjct

606 KANTDIKLGVCVNYDLYGISGQGIFVEVNATYYNSWQNLLYDSNGNLYGFRDYITNRTFM 665

Query 573 ITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTR 632

I C G VS S++ A+L++++ C V QL P N F +

Sbjct

666 IRSCYSGRVSAAFHAN--SSEPALLFRNIKCNYVFNNSLIRQLQPI--------

NYFDSY 715

Query 633 AGCLIGAEHVN--NSYECDIPIGAGICASYQTQTNSPGSASSVASQSIIAYTMSLGAENS 690

GC++ A + + CD+ +G+G C Y S + ++ Y + +

Sbjct

716 LGCVVNAYNSTAISVQTCDLTVGSGYCVDYSKNRRSRRAITT-------GYRFTNFEPFT 768

Query 691 VAYSNNS---------IAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQ 741

V N+S I IP+ FTI E + S K ++DC ++CGD C + L++

Sbjct

769 VNSVNDSLEPVGGLYEIQIPSEFTIGNMEEFIQTSSPKVTIDCAAFVCGDYAACKSQLVE 828

Query 742 YGSFCTQLNRALTGIAVEQDKNTQEVF-AQVKQIYKTPPIKDFGGFN-----FSQIL---

792

YGSFC +N LT + D +V + + + + +KD FN FS +L

Sbjct

829 YGSFCDNINAILTEVNELLDTTQLQVANSLMNGVTLSTKLKDGVNFNVDDINFSSVLGCL 888

Query 793 -PDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLL 851

+ SK S RS IEDLLF+KV L+D GF+ Y +C G RDLIC Q + G+ VLPPLL

Sbjct

889 GSECSKASSRSAIEDLLFDKVKLSDVGFVAAYNNCTGGAEIRDLICVQSYKGIKVLPPLL 948

Query 852 TDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIAN 911

++ I+ YT A + ++ WT AG +PF + + YR NG+GVT +VL +NQKLIAN

Sbjct

949 SENQISGYTLAATSASLFPPWTAAAG----VPFYLNVQYRINGLGVTMDVLSQNQKLIAN 1004

Query 912 QFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLD 971

FN+A+ IQ+ +T SAL K+Q VVN NA+ALN L++QLS+ FGAISS L +ILSRLD

Sbjct

1005 AFNNALDAIQEGFDATNSALVKIQAVVNANAEALNNLLQQLSNRFGAISSSLQEILSRLD 1064

Query 972 PPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGK 1031

PPEAE QIDRLI GRL +L YV+QQL + ++ SA A K++ECV QS R++FCG

Sbjct

1065 PPEAEAQIDRLINGRLTALNAYVSQQLSDSTLVKFSAAQAMEKVNECVKSQSSRINFCGN 1124

Query 1032 GYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDG-KAHFPREGVFVSNGTHWFV 1090

G H++S Q+AP+G+ F+H +YVP + +P +C G + P+ G FV+ W

Sbjct

1125 GNHIISLVQNAPYGLYFIHFSYVPTKYVTAKVSPGLCIAGDRGIAPKSGYFVNVNNTWMY 1184

Query 1091 TQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDV 1150

T +Y P+ IT +N V C V + + P L F+EELD++FKN TS

Sbjct

1185 TGSGYYYPEPITENNVVVMSTCAVNYTKAPYVMLNTSTPNLPDFREELDQWFKNQTSVAP 1244

Query 1151 DLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSGYIPEAPRDGQAYV 1210

DL + IN + +++Q E++RL E K LN GSGYIPEAPRDGQAYV

Sbjct

1245 DL-SLDYINVTFLDLQVEMNRLQEAIKVLN--------------GSGYIPEAPRDGQAYV 1289

Query 1211 RKDGEWVLLSTFLGRSLEVLFQGPGHHHHHHHHSAWSHPQFEKGGGSGGGGSGGSAWSHP 1270

RKDGEWVLLSTFLGRSLEVLFQGPGHHHHHHHHSAWSHPQFEKGGGSGGGGSGGSAWSHP

Sbjct

1290 RKDGEWVLLSTFLGRSLEVLFQGPGHHHHHHHHSAWSHPQFEKGGGSGGGGSGGSAWSHP 1349

Query 1271 QFEK 1274

QFEK

Sbjct

1350 QFEK 1353

Identities

:289/758(38%), Positives:419/758(55%), Gaps:70/758(9%)

Query 524 CVNFNFNGLTGTGVLTESNKKFLP-

FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVS 582

CVN++ G+TG G+ E + + +Q D +D T + I PC G VS

Sbjct

606 CVNYDLYGITGQGIFKEVSAAYYNNWQNLLYDSNGNIIGFKDFLTNKTYTILPCYSGRVS 665

Query 583 VITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHV 642

S+ A+LY+++ C+ V I + + F + GC++ A ++

Sbjct

666 A--AFYQNSSSPALLYRNLKCSYVLNNIS-

---------F

ISQPFYFDSYLGCVLNAVNL 713

Query 643 NNSYE

---CDIPIGAGICASYQTQTNSPGSASSVASQSIIAYTMSLGAENSVAYSNNS-

-697

SY CD+ +G+G C Y ++ + + + + +V++ N+S

Sbjct

714 T-

SYSVSSCDLRMGSGFCIDYALPSSGGSGSGISSPYRFVTF

-----EPFNVSFVNDSVE 767

Query 698 -

------IAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLN 750

I IPTNFTI+ E + S K ++DC+ ++C + C +LL +YG+FC +N

Sbjct

768 TVGGLFEIQIPTNFTIAGHEEFIQTSSPKVTIDCSAFVCSNYAACHDLLSEYGTFCDNIN 827

Query 751 RALTGIAVEQDKNTQEVFAQVKQ

------IYKTPPIKDFGGFNFSQILP

---DPSKPSKR 801

L + D +V + Q T D +F +L S R

Sbjct

828 SILNEVNDLLDITQLQVANALMQGVTLSSNLNTNLHSDVDNIDFKSLLGCLGSQCGSSSR 887

Query 802 SFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTS 861

S +EDLLFNKV L+D GF++ Y +C G RDL+C Q FNG+ VLPP+L++ I+ YT+

Sbjct

888 SLLEDLLFNKVKLSDVGFVEAYNNCTGGSEIRDLLCVQSFNGIKVLPPILSETQISGYTT 947

Query 862 ALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQ 921

A + W+ AG +PF++ + YR NG+GVT +VL +NQKLIAN FN A+ IQ

Sbjct

948 AATVAAMFPPWSAAAG

----VPFSLNVQYRINGLGVTMDVLNKNQKLIANAFNKALLSIQ 1003

Query 922 DSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDR 981

+ ++T SAL K+Q VVN NAQALN+L++QL + FGAISS L +ILSRLDPPEA+VQIDR

Sbjct

1004 NGFTATNSALAKIQSVVNANAQALNSLLQQLFNKFGAISSSLQEILSRLDPPEAQVQIDR 1063

Query 982 LITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQS 1041

LI GRL +L YV+QQL I+A A+ A K++ECV QS R++FCG G H++S Q+

Sbjct

1064 LINGRLTALNAYVSQQLSDITLIKAGASRAIEKVNECVKSQSPRINFCGNGNHILSLVQN 1123

Query 1042 APHGVVFLHVTYVPAQEKNFTTAPAICHDG-

KAHFPREGVFVSNGTHWFVTQRNFYEPQI 1100

AP+G++F+H +Y P K +P +C G + P++G F+ W T ++Y P+

Sbjct

1124 APYGLLFIHFSYKPTSFKTVLVSPGLCLSGDRGIAPKQGYFIKQNDSWMFTGSSYYYPEP 1183

Query 1101 ITTDNTFVSGNCDVVIG

-----IVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI 1155

I+ N +C V +NN++ P L F+ EL +FKNHTS +L

Sbjct

1184 ISDKNVVFMNSCSVNFTKAPFIYLNNSI

-----P

NLSDFEAELSLWFKNHTSIAPNLTFN 1238

Query 1156 SGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSGYIPEAPRDGQAYVRKDGE 1215

S INA+ +++ E++ + E K+LN +++ ++ G

YIPEAPRDGQAYVRKDGE

Sbjct

1239 SHINATFLDLYYEMNVIQESIKSLNSGRLEVL

----FQGPGGYIPEAPRDGQAYVRKDGE 1294

Query 1216 WVLLSTFLGRSLEVLFQGPGHHHHHHHHSAWSHPQFEK 1253

WVLLSTFLG HHHHHHSAWSHPQFEK

Sbjct

1295 WVLLSTFLGHH-

----------

HHHHHHSAWSHPQFEK

1321

MER

SSA

RS-

1O

C43

HKU

1

MER

SSA

RS-

1

OC

43H

KU1

SAR

S-C

oV-2

Spi

ke

<40

80 -

200

≥20

0BL

AST

Alig

nmen

t Sco

re:

AB

CD

E



https://doi.org/10.1101/2020.06.22.20137695

Supplementary Figure 2: Linear Correlation Statistics of Betacoronaviruses

1.000

0.516

0.711

0.565

0.281

0.516

1.000

0.760

0.422

0.299

0.711

0.760

1.000

0.470

0.406

0.565

0.422

0.470

1.000

0.154

0.281

0.299

0.406

0.154

1.000

1.000

0.215

0.609

0.399

0.259

0.215

1.000

0.288

0.872

0.862

0.609

0.288

1.000

0.404

0.245

0.399

0.872

0.404

1.000

0.866

0.259

0.862

0.245

0.866

1.000

1.000

0.504

0.616

0.553

0.433

0.504

1.000

0.948

0.523

0.341

0.616

0.948

1.000

0.579

0.483

0.553

0.523

0.579

1.000

0.864

0.433

0.341

0.483

0.864

1.000 0.4

0.6

0.8

1.0

1.000

0.266

0.505

0.319

0.079

0.266

1.000

0.577

0.178

0.090

0.505

0.577

1.000

0.221

0.165

0.319

0.178

0.221

1.000

0.024

0.079

0.090

0.165

0.024

1.000

1.000

0.046

0.371

0.159

0.067

0.046

1.000

0.083

0.760

0.744

0.371

0.083

1.000

0.163

0.060

0.159

0.760

0.163

1.000

0.750

0.067

0.744

0.060

0.750

1.000

1.000

0.254

0.380

0.306

0.188

0.254

1.000

0.900

0.273

0.116

0.380

0.900

1.000

0.335

0.234

0.306

0.273

0.335

1.000

0.746

0.188

0.116

0.234

0.746

1.000 0.2

0.4

0.6

0.8

1.0

Cor

rela

tion

Fit (

R2 )

IgG IgM IgA

SARS2 MERS SARS1 OC43 HKU1 SARS2 MERS SARS1 OC43 HKU1 SARS2 MERS SARS1 OC43 HKU1SA

RS2

MER

SSA

RS1

OC

43H

KU1

SAR

S2M

ERS

SAR

S1O

C43

HKU

1



https://doi.org/10.1101/2020.06.22.20137695

Supplementary Figure 3: Differential signal intensity of SARS-CoV-2 spike with other

Betacoronaviruses. Signal intensity displayed as SARS-CoV-2 absorbance (A450 – A650)

minus signal intensity of other Betacoronaviruses. (a) MERS, (b) SARS-CoV, (c) OC43, and (d)

HKU1. Archival negative = black, pandemic hot-spot symptomatic = pink, pandemic hot-spot

asymptomatic = teal. n =114 archival negative, n = 68 hot-spot symptomatic, n = 6 hot-spot

asymptomatic.

IgG IgM IgA-1

0

1

2

3

4SA

RS2

- M

ERS

(OD

)

IgG IgM IgA-1

0

1

2

3

4

SAR

S2 -

SAR

S1 (O

D)

IgG IgM IgA-4

-2

0

2

4

SAR

S2 -

OC

43 (O

D)

IgG IgM IgA-4

-2

0

2

4

SAR

S2 -

HKU

1 (O

D)

A

B

C

D



https://doi.org/10.1101/2020.06.22.20137695

Documents

Serologic cross-reactivity of SARS-CoV-2 with endemic and ...€¦ · 22/06/2020 · the four other coronaviruses, with the strongest cross-recognition between SARS-CoV-2 and SARS