widely circulating in the general population,
and are typically responsible for mild respiratory
symptoms ( 14 – 16 ). However, the hypothesis of
cross-reactive immunity between SARS-CoV-2
and common cold HCoVs still awaits experi-
mental trials. This potential preexisting cross-
reactive T cell immunity to SARS-CoV-2 has
broad implications because it could explain
aspects of differential COVID-19 clinical out-
comes, influence epidemiological models of
herd immunity ( 17 , 18 ), or affect the perform-
ance of COVID-19 candidate vaccines.
Epitope repertoire in
SARS-CoV-2–unexposed individuals
To define the repertoire of CD4+T cells recog-
nizing SARS-CoV-2 epitopes in previously
unexposed individuals, we used in vitro stim-
ulation of peripheral blood mononuclear cells
(PBMCs) for 2 weeks with pools of 15-mer
peptides. This method is known to be robust
for detecting low-frequency T cell responses to
allergens and bacterial or viral antigens ( 19 , 20 ),
including naive T cells ( 21 ). For screening
SARS-CoV-2 epitopes, we used PBMC samplesfrom unexposed subjects collected between
March2015andMarch2018,wellbeforethe
global circulation of SARS-CoV-2 occurred. The
unexposed subjects were confirmed to be sero-
negative for SARS-CoV-2 (fig. S1A).
SARS-CoV-2–reactive T cells were expanded,
with one pool of peptides spanning the en-
tire sequence of the spike protein (CD4-S)
and the other a nonspike“megapool”(CD4-
R) of predicted epitopes from the nonspike
regions (i.e.,“remainder”) of the viral genome
( 4 ). In total, 474 15-mer SARS-CoV-2 peptides
were screened. After 14 days of stimulation,
T cell reactivity against intermediate“meso-
pools,”each encompassing ~10 peptides, was
assayed using a FluoroSPOT assay (e.g., 22
CD4-R mesopools; fig. S2A). Positive meso-
pools were further deconvoluted to identify
specific individual SARS-CoV-2 epitopes. Rep-
resentative results from one donor show the
deconvolution of mesopools P6 and P18 to
identify seven different SARS-CoV-2 epitopes
(fig. S2B). Intracellular cytokine-staining as-
says specific for interferong(IFN-g) determined
whether antigen-specific T cells responding
to the SARS-CoV-2 mesopools were CD4+or
CD8+T cells (fig. S2C). Results from the 44
donors/CD4-R mesopool and 40 donors/CD4-S
mesopool combinations yielding a positive
response are shown in fig. S2, D and E, re-
spectively. In 82/88 cases (93.2%), the cells
responding to SARS-CoV-2 mesopool stimu-
lation were clearly CD4+T cells, as judged by
the ratio of CD4/CD8–responding cells; in four
cases (4.5%), the responding cells were CD8+
T cells; and in two cases (2.3%), the responses
were mediated by both CD4+and CD8+T cells.
The fact that CD8+T cells were rarely de-
tected was not surprising because the pep-
tides used in CD4-R encompassed predicted
class II epitopes and the CD4-S is composed
of 15-mer peptides (9- to 10-mer peptides are
optimal for CD8+T cells). Furthermore, the
2-week restimulation protocol was originally
designed to expand CD4+T cells ( 20 ). Over-
all, these results indicated that the peptide-
screening strategy used mapped SARS-CoV-2
epitopes recognized by CD4+T cells in un-
exposed individuals.
A total of 142 SARS-CoV-2 epitopes were iden-
tified, 66 from the spike protein (CD4-S) and
76 from the remainder of the genome (CD4-R)
(table S1). For each combination of epitope
and responding donor, potential human leuko-
cyte antigen (HLA) restrictions were inferred
on the basis of the predicted HLA-binding
capacity of the particular epitope for the
specific HLA alleles present in the responding
donor ( 22 ). Each donor recognized an average
of 11.4 epitopes (range 1 to 33, median 6.5; fig.
S3A). Forty of the 142 epitopes were recognized
by two or more donors (fig. S3B), accounting
for 55% of the total response (fig. S3C). These
142 mapped SARS-CoV-2 epitopes may prove90 2 OCTOBER 2020•VOL 370 ISSUE 6512 sciencemag.org SCIENCE
01 10 100202002000Positive responsesProtein sizeMNS (Non-RBD)S (RBD)EORF3a
ORF7a
ORF6ORF8nsp1nsp2nsp3nsp4
nsp5 nsp6nsp7nsp8
nsp9 nsp10nsp12
nsp13
nsp14
nsp15nsp16ORF10 r =
P =0.4170
0.038101 >1
020406080100# individuals responding% of similarity
in reference CoVs****
****
n = 332 n = 103 n = 39(^100) Non-spike Spike
10,000
100,000
1000
SFCs/ 10
6 PBMCs
High response low homology (67%)
High homology (>67%)
Other
SARS-CoV-2
proteins
Protein
length
Peptides
tested
Epitopes
identified
Positive
responses
Response
magnitude
aa n n % n % SFCs 10
(^6) /
PBMCs %
M 222 8 0 0 0 0 0 0
N 419 7 4 3 4 2 6573 2
S (Non-RBD) 1050 207 54 38 90 44 149,300 39
S (RBD) 223 46 12 8 23 11 40,873 11
E 75 8 2 1 2 1 2240 1
ORF3a 275 10 6 4 7 3 17,587 5
ORF7a 121 4 3 2 3 1 4080 1
ORF6 61 6 6 4 7 3 16,453 4
ORF8 121 3 3 2 4 2 13,553 4
nsp1 180 2 1 1 1 0 387 0
nsp2 638 14 6 4 7 3 32,673 9
nsp3 1945 34 10 7 11 5 7307 2
nsp4 500 22 8 6 10 5 5320 1
nsp5 306 6 2 1 4 2 52,907 14
nsp6 290 18 6 4 9 4 5613 1
nsp7 83 4 0 0 0 0 0 0
nsp8 198 5 2 1 2 1 6473 2
nsp9 113 2 0 0 0 0 0 0
nsp10 139 2 1 1 2 1 440 0
nsp12 932 19 7 5 9 4 6850 2
nsp13 601 14 3 2 4 2 5360 1
nsp14 527 19 3 2 4 2 2193 1
nsp15 346 4 1 1 1 0 533 0
nsp16 298 9 2 1 2 1 1853 0
ORF10 38 1 0 0 0 0 0 0
Total 474 142 100 206 100 378,570 100
AB
C
D
Fig. 1. Characteristics of SARS-CoV-2 epitopes identified in unexposed donors.Reactivity was
determined by FluoroSPOT assay after 17 days of in vitro stimulation of unexposed donor PBMCs (n= 18)
with one pool of peptides spanning the entire sequence of the spike protein (CD4-S) or a nonspike“megapool”
(CD4-R) of predicted epitopes from the nonspike (i.e.,“remainder”) regions of the viral genome. (A) Summary of the
responses as a function of the protein of origin. (B) Spearman correlation of positive responses per SARS-CoV-2
protein size. (C) Percent similarity of the identified epitopes with common cold coronavirus peptides as afunction
of the number of responding donors. (D) Each dot shows the reactivity of a donor-epitope combination derived
from either nonspike (CD4-R) or spike (CD4-S) protein. Black bars indicate the geometric mean and geometric
SD. Red indicates donor-epitope combinations with sequence identity >67% with common cold coronaviruses,
and blue indicates highly reactive donor-epitope combinations (>1000 SFCs*10^6 ) with sequence identity
≤67%. In (C) and (D), statistical comparisons were performed with a two-tailed Mann–Whitney test.
*P< 0.001, **P< 0.0001.
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