ORIGINAL RESEARCH

published: 28 October 2020

doi: 10.3389/fimmu.2020.587615

Immunoinformatic Analysis
of SARS-CoV-2 Nucleocapsid
Protein and Identification of
COVID-19 Vaccine Targets
Sergio C. Oliveira 1,2*†, Mariana T. Q. de Magalhães 1 and E. Jane Homan 3†
1 Departamento de Bioquı´mica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo
Horizonte, Brazil, 2 Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais (INCT-DT), Conselho Nacional de
Desenvolvimento Cientifico e Tecnologico (CNPq), Ministerio de Ciencia e Tecnologia (MCT), Salvador, Brazil,
3 ioGenetics LLC, Madison, WI, United States

COVID-19 is a worldwide emergency; therefore, there is a critical need for foundational

Edited by: knowledge about B and T cell responses to SARS-CoV-2 essential for vaccine
Katie Ewer, development. However, little information is available defining which determinants of
University of Oxford, United Kingdom
SARS-CoV-2 other than the spike glycoprotein are recognized by the host immune
Reviewed by:
Gunnveig Grødeland,
system. In this study, we focus on the SARS-CoV-2 nucleocapsid protein as a suitable
University of Oslo, Norway candidate target for vaccine formulations. Major B and T cell epitopes of the SARS-CoV-2
Salvador Iborra,
N protein are predicted and resulting sequences compared with the homolog
Universidad Complutense de Madrid,
Spain immunological domains of other coronaviruses that infect human beings. The most
*Correspondence: dominant of B cell epitope is located between 176–206 amino acids in the
Sergio C. Oliveira SRGGSQASSRSSSRSRNSSRNSTPGSSRGTS sequence. Further, we identify
[email protected]
†
sequences which are predicted to bind multiple common MHC I and MHC II alleles.
These authors share senior
authorship
Most notably there is a region of potential T cell cross-reactivity within the SARS-CoV-2 N
protein position 102–110 amino acids that traverses multiple human alpha and
Specialty section: betacoronaviruses. Vaccination strategies designed to target these conserved epitope
This article was submitted to
Vaccines and Molecular
regions could generate immune responses that are cross-reactive across human
Therapeutics, coronaviruses, with potential to protect or modulate disease. Finally, these predictions
a section of the journal can facilitate effective vaccine design against this high priority virus.
Frontiers in Immunology

Received: 26 July 2020 Keywords: severe acute respiratory syndrome coronavirus 2, Coronavirus Disease 2019, epitopes, vaccine, T cells,
Accepted: 02 October 2020 B cells, nucleocapsid
Published: 28 October 2020

Citation:
Oliveira SC, de Magalhães MTQ and
INTRODUCTION
Homan EJ (2020) Immunoinformatic
Analysis of SARS-CoV-2
The pandemic Coronavirus Disease 2019 (COVID-19) is a worldwide threat caused by the severe
Nucleocapsid Protein and
Identification of COVID-19
acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (1). By July 2020, SARS-CoV-2 had
Vaccine Targets. infected over 16 million people worldwide and killed more than 645,000 individuals. A better
Front. Immunol. 11:587615. understanding of the immunogenicity and pathogenesis of SARS-CoV-2 infections in humans is
doi: 10.3389/fimmu.2020.587615 thus urgently needed as a basis for the development of new vaccines against SARS-CoV-2 (2).

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Oliveira et al. SARS-CoV-2 T and B Cell Epitopes

The coronaviral genome encodes a relatively small number peptides. There have been several reports of bioinformatics
of proteins, classified as either structural or non-structural. analyses of SARS-CoV-2 using a variety of platforms (25–29).
Among structural proteins, the spike glycoprotein (S), and the Herein, we applied bioinformatics analysis to determine the
nucleocapsid protein (N) are the major ones, while the antigenic potential of the SARS-CoV-2 N protein. Major B and
envelope protein (E) and membrane protein (M) are smaller T cell epitopes of the SARS-CoV-2 N protein are predicted and
structural components (3, 4). The spike (S) protein is arrayed these peptides were compared to other coronaviruses that infect
on the surface of the virus particles, giving the characteristic humans. As other studies have suggested that prior exposure to
‘crown’ appearance (5). The S protein comprises two subunits: less virulent human coronaviruses may confer some protection
S1 and S2. The S1 subunit consists of an amino-terminal (24, 30–32), we focused particularly on identifying conserved
domain and a receptor-binding domain (RBD) (5, 6). The motifs which potentially could elicit cross-reacting T cell
RBD binds to ACE2 as its host cell target receptor, which responses through shared T cell exposed peptides. The epitope
allows virus entry (5, 7). Various reports related to SARS-CoV- mapping and comparison of potential cross-reactive epitopes
2 suggest a correlation between neutralizing antibodies and the presented in this study may provide an opportunity for the
number of specific T cells to viral particles (8). Some vaccine development of new vaccines and immunodiagnostic tools.
candidates have been shown to protect from infection in Finally, the sudden emergence of SARS-CoV-2 apparently from
laboratory animals models (9). Most vaccine studies so far bats is an indicator that similar betacoronaviruses could emerge in
have focused on antibody responses generated against the S the future. It is therefore of interest to determine if there are
protein, the most exposed protein of SARS-CoV-2 (10, 11). potential antigens that are conserved and could cross protect
However, antibody responses are not detectable in all infected against future zoonotic coronaviruses.
patients, especially those with less severe forms of COVID-19
(12). Previous studies with SARS-CoV-1 have also shown that
memory B cell responses tend to be short-lived after infection
(13). In contrast, memory T cell responses can persist for many MATERIAL AND METHODS
years (14), and in mice, these protect against lethal challenge
with SARS-CoV-1 (13). Additionally, the spike protein has Accession Numbers
several hotspots for mutations (15), whereas the nucleocapsid Accession numbers of the nucleocapsid proteins analyzed are as
gene is more stable and has acquired fewer mutations to follows: HKU1:YP_173242.1; 229E:NP_073556.1; MERS:
date (16). YP_009047211.1; NL63:YP_003771.1; OC43: YP_009555245.1;
In this study, we focus on the SARS-CoV-2 nucleocapsid SARS COV1:NP_828858.1; SARS-COV2: YP_009724397.2.
protein that is involved in viral pathogenesis (4, 17). The
nucleocapsid is the most abundant protein in coronaviruses,
is highly immunogenic, and its amino acid sequence is largely
conserved as previously reported (4). Therefore, this protein Determination of Predicted Epitopes for
has advantages as a candidate for vaccine development (4, 18). SARS-CoV-2 Nucleocapsid
Previous studies on SARS-CoV-1 reported N protein epitopes B cell linear epitope probability and MHC binding affinity were
as capable of eliciting massive production of antibodies in determined for all sequential peptides with a single amino acid
infected subjects (4). T cell responses to SARS-CoV-1 are in displacement, using an updated version of methods previously
some cases shown to last up to 11 years thus representing a described (33, 34). Briefly, in lieu of representing peptides
valid alternative for the design of vaccines (4, 19). Monkeys as simple alphabetic sequences, multiple physicochemical
vaccinated with an adenovirus vectored SARS-CoV-1 vaccine properties of each amino acid are transformed to mathematical
were shown to have consistent T cell responses to the N protein vectors by principal component analysis. Using a training set of
(20). Similarly in MERS the nucleocapsid has been examined known MHC binding reactions, B cell epitope binding and
as a potential vaccine candidate (21, 22). Recall responses of T cathepsin cleavage reactions, neural networks are used to derive
cells reacting with peptides of SARS-COV-2 N protein have predictive equations applicable to any peptide. Predictions are
been demonstrated in both SARS-CoV-1 recovered patients, made for 70 MHC I alleles and 65 MHC II alleles. To estimate
17 years after exposure, and those with no history of SARS- population behavior comprising multiple MHC alleles with varying
CoV-1 exposure (23, 24). Preliminary studies of SARS-CoV-2 affinities for any peptide, the LN ic50 binding data estimates were
have also demonstrated antibodies directed to the N transformed and standardized to a zero mean unit variance within
protein (2). each protein using a Johnson Sb distribution (35). To compute a
Studies involving computer simulations for the identification permuted average across human alleles, the highest predicted
of the epitopes recognized by antibodies and T cells are central to binding affinity at each peptide position was determined for every
immunological applications such as drug design and vaccine possible haplotype pairing and averaged; this was computed using
development. Bioinformatics tools offer the advantage, in predicted binding for 31 MHC IA, 31 MHC IB, and 24 DRB alleles
addition to speed and biosafety, of being unbiased by peptide as previously demonstrated (36). Predictions of the probability of
selection. Approaches which use overlapping peptides, spaced cathepsin cleavage at each dimer were similarly derived by training
other than single amino acid displacement, may exclude the key on known cleavage reactions (34). These predictive methods have

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Oliveira et al. SARS-CoV-2 T and B Cell Epitopes

been experimentally validated in proteins of multiple origins (34, RESULTS

37–40).
SARS-CoV-2 Nucleocapsid B and
Nucleocapsid Sequence Alignments T Cell Epitope Mapping
and Structural Analysis The nucleocapsid of SARS-Cov-2 exhibits both strong B and T
Several protein sequences were analyzed by using the Basic Local cell epitopes distributed across the whole protein. Figure 1
Alignment Search Tool specific for protein sequences (BLASTp) provides an overview map of both probable linear B cell
(41). Multiple sequence alignments were prepared with Clustal epitopes and regions of predicted high affinity MHC binding
Omega (multiple sequence alignment) and manually edited in for multiple alleles. Corresponding sequences of predicted
pyBoxShade 3.21 (https://github.com/mdbaron42/pyBoxshade). antigenicity are shown in Table 1. As shown in Figure 1, we
We selected statistically significant matches to calculate a predicted multiple high probability B cell linear epitopes. A 9-
similarity tree for related coronaviruses. The epitopes were mer peptide was scored as “high probability” if they were
mapped based on the amino acid physical–chemical properties predicted to be in the top 25% of probability of being in a B
and location at possible areas of cross-reactivity and antigen-
binding by using an in-house software (data not shown). Analysis
of the protein secondary structure prediction and annotation was TABLE 1 | SARS-CoV-2 predicted antigenicity of B and T cell epitopes.
carried out with PSIPRED Protein Analysis Workbench (http:// B cell epitopes
bioinf.cs.ucl.ac.uk/psipred/) (42, 43). The epitopes were identified,
built in Chimera v.1.13.1. We also used Chimera to prepare images Position Peptide sequence
and calculate RMSD between sequences (44). Distance between 21–32 SDSTGSNQNGER
76–82 TNSSPDD
residues were measured by using wizard measurement tool from 176–206 SRGGSQASSRSSSRSRNSSRNSTPGSSRGTS
PyMOL (The PyMOL Molecular Graphics System, Version 235–243 SGKGQQQQG
1.2r3pre, Schrödinger, LLC.). 249–263 KSAAEASKKPRQKRT
363–379 FPPTEPKKDKKKKADET
T Cell Exposed Motifs MHC I binding regions for multiple alleles
Position Peptide sequence
All sequential T cell exposed motif patterns were extracted from 97–137 GDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEG
each protein and ranked as previously described for each of three 209–232 RMAGNGGDAALALLLLDRLNQLES
recognition patterns of amino acids which engage T cell 261–279 KRTATKAYNVTQAFGRRGP
receptors (33, 36, 45). These T cell exposed recognition 306–335 QFAPSASAFFGMSRIGMEVTPSGTWLTYTG
MHC II binding regions for multiple alleles
patterns comprise the amino acids not hidden in pocket
Position Peptide sequence
positions. These are positions ~~~4,5,6,7,8~ within a MHC I 97–127 GGDGKMKDLSPRWYFYYLGTGPEAGLPYGANK
binding 9-mer and ~2,3,~5~7,8~ or −1~~,3,~5~7,8~ relative to 213–238 NGGDAALALLLLDRLNQLESKMSGKG
the 9-mer core of a MHC II binding 15-mer. 293–320 RQGTDYKHWPQIAQFAPSASAFFGMSRI

FIGURE 1 | Epitope mapping of nucleocapsid protein of SARS-CoV-2. The X axis indicates the index position of sequential peptides with single amino acid
displacement. The Y axis indicates predicted binding affinity in standard deviation units for the protein. The red line shows the permuted average predicted MHC-IA
and B (62 alleles) binding affinity by index position of sequential 9-mer peptides with single amino acid displacement. The blue line shows the permuted average
predicted MHC-II DRB allele (24 most common human alleles) binding affinity of sequential 15-mer peptides. Orange lines show the predicted probability of B-cell
receptor binding for an amino acid centered in each sequential 9-mer peptide. Low numbers for MHC data represent high binding affinity, whereas low numbers
equate to high B cell receptor contact probability. Ribbons (red: MHC-I, blue: MHC-II) indicate the 10% highest predicted MHC affinity binding. Orange ribbons
indicate the top 25% predicted probability B-cell binding. Horizontal dotted lines demarcate the top 5% of binding affinity for the protein (red MHC I, blue MHC II).

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Oliveira et al. SARS-CoV-2 T and B Cell Epitopes

cell epitope for the protein as a whole. The most dominant of indicates three regions of predicted high MHC II binding and
these lies between 176 and 206 in the sequence SRGGSQASSRS four regions of high affinity MHC I binding for multiple alleles,
SSRSRNSSRNSTPGSSRGTS. Additional high probability B cell which comprise the top 10% highest predicted affinity for the
epitopes are indicated in Table 1. When analyzed by the same protein. These are shown in Table 1. However, as the examples
immunoinformatic approach alongside all structural proteins in shown in Figure 2 underscore, there are differences in MHC
the virion, the nucleocapsid B cell epitope at 176–206 stands out allele-specific binding. The differences are more marked for
as dominant with respect to the epitopes in the spike MHC I, where binding is often restricted to one or two
glycoprotein (data not shown). sequential 9-mers, whereas the broader sequences identified for
Figure 1, in which consideration is given to the predicted MHC II, tend to span more alleles. For example, adjacent to the
binding of multiple common human MHC I and MHC II alleles, dominant B cell epitope we see that a DRB1_1501 has a stronger

FIGURE 2 | Predicted differential binding of example alleles. (A) MHC I and (B) MHC II. In both panels the Y axis indicates predicted binding affinity of sequential peptides.
The X axis indicates the index position of each 9 mer (MHC (I) or 15-mer (MHC II) represented by a vertical bar. Bars which are cross hatched are those peptides predicted to
be excised for binding and presentation by either cathepsin S or cathepsin L. For MHC I the cathepsin predictions are those which excise a 9 mer. For MHC II a predicted
excision of a 12–18 mer is shown. The lower tier of each panel shows the population permuted average predicted binding affinity as described for Figure 1. The top three
tiers contrast the responses of selected example alleles. For MHC I we show predicted responses of A_0101, A0201, and A1101. For MHC II we show predicted responses
of DRB1_0101, DRB1_0401, and DRB1_1501. Other alleles evaluated show a similar diversity of predicted response.

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Oliveira et al. SARS-CoV-2 T and B Cell Epitopes

predicted MHC II binding which could indicate more T cell help to this pentamer motif as the T cell exposed motif (36).
than is the case for an individual of DRB1_0101. Furthermore, Figure 3 shows the patterns of T cell exposed motif sharing
when consideration is given to probable cathepsin cleavage, not between human alphacoronaviruses 229E and NL63 with
all peptides may actually be presented. However, we appreciate betacoronaviruses HKU1, OC43, MERS, SARS-CoV-1, and
that cathepsins play a major role in generating peptides to be SARS-CoV-2. While some of the T cell exposed motifs are
presented for the vacuolar pathway (endolysosomes and conserved, the flanking regions of these peptides, comprising
phagosomes) as demonstrate by Shen et al. (46). Therefore, the groove exposed motif, differ. Most notably there is a region of
cathepsins are primarily involved in TAP-independent MHC potential T cell cross-reactivity within the SARS-CoV-2 N
class I crosspresentation. Nevertheless, this analysis suggests that protein position 102–110 that traverses the human alpha and
individuals of different immunogenetics would be expected to beta coronaviruses, except for MERS. In MERS substitution of
show differing responses. The proximity of MHC binding Leu>Thr at the SARS-CoV-2 position 113 (equivalent to the
sequences to the B cell epitopes at 76–82 and 176–206 amino MERS 103 position) removes the conservation of the T cell
acids indicates these epitopes may also receive strong epitope exposed motifs with SARS-CoV-2. The region in which the
specific T cell help. conserved motifs occur is also predicted to have high affinity
binding for multiple MHC I and II alleles. Here, we used 70
Conservation of T Cell Epitopes human MHC I and 65 MHC II alleles for our analysis of
Among Coronaviruses permuted binding that represents about 85% of human
We next compared the epitope map of SARS-COV-2 N protein population. The T cell motif sharing is further extended within
to that of other coronaviruses known to have infected humans. the betacoronaviruses. The conserved T cell exposed motifs are
Here, we focused on the T cell exposed motifs, which indicate shown in Supplementary Table 1. When the N proteins of the
where potential T cell cross-reactivity may occur. A single T-cell six viruses sharing most motifs are aligned at the peptide
receptor engages only with the few amino acids of a bound comprising the most conserved T cell exposed MHC I motif
peptide MHC that are protruding from a MHC histotope, ~~~FYYLG~ (in SARS-CoV-2 position 107), the commonality
together with contact points within the histotope. We refer of epitope patterns is evident (Figure 4).

FIGURE 3 | T cell exposed motifs conserved across coronaviruses. The cell plots show in gray where there are T cell exposed motifs shared between SARS-CoV-2
and other human coronaviruses, as shown in the X axis. The number of shared motifs indicated in the Y axis counts. The most highly conserved motifs are also
shown in Supplementary Table 1.

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Oliveira et al. SARS-CoV-2 T and B Cell Epitopes

FIGURE 4 | Comparative MHC binding patterns in human coronavirus nucleocapsid protein. X axis shows sequential peptides aligned relative to the most
conserved MHC I pentamer. Peptides are 9-mers for MHC I and 15-mers for MHC II and are indicated at their index positions. Y axis shows permuted predicted
binding in standard deviation units below the mean for the protein. (A) shows MHC I alleles. (B) shows MHC II DRB alleles.

3-D Structure Model of Nucleocapsid (RMSD) between the structures coordinates is 0.867 Å over
From Different Coronaviruses superimposed C atoms. The most dramatic differences can be
The coronavirus nucleocapsid protein consists of two folded observed in loops L1 (between b2 and b3, residues 96 to 104) and
domains (NTD and CTD) linked by an unstructured region (47). L3 (residues 119 to 128). Other authors also observed that
In more details the N protein includes the following domains: strands b2 and b3 are connected by a long flexible loop
serine–glycine–arginine-rich domain (SGRD), N-terminal composed of amino acid residues 96 to 104 protruding out of
domain (NTD), serine-rich domain (SRD), C-terminal domain the core (50, 51). We could identify and observe (Figure 5B) the
(CTD) as described in Figure 5A (48, 49). Our alignment has structure of the highly conserved twelve-residue peptide
revealed that despite the conservation of some motifs, the N corresponding to the region 1 0 7 RWYFYYLGTGPY 1 1 8
protein from various different coronaviruses often exhibit (YP_009724397.2). This peptide is located at the NTD of N
different properties, due primarily to their otherwise low protein, close to the L1 loop and has a conserved and important
sequence homology (˜50%) (Supplementary Figure 1). The epitope located in an exposed beta-strand, with two exposed
structural similarity appears to be at the whole folded level tyrosines (Figure 4B1). Both tyrosines (Y111 and Y112) have
with its five-stranded anti-parallel b-sheet sandwiched between been proposed to be involved in RNA recognition, stacking with
loops (or short 3–10 helix) on the outside (Figure 5B). Several consecutive nucleotide bases. The NTD of the N protein from the
nucleocapsid NTD domains are similar in topology and surface selected coronavirus was compared to assess the similarity level
electrostatic profiles as observed. The root mean square deviation existing between the conserved protein sequences of the human

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Oliveira et al. SARS-CoV-2 T and B Cell Epitopes

FIGURE 5 | (A) Domain organization of coronaviral N proteins. The four domains labeled are as follows: SGRD, serine–glycine–arginine-rich domain; NTD,
N-terminal domain; SRD, serine-rich domain; and CTD, C-terminal domain. (B) Superimposition of the HCoV-Sars-2 NTD in pink (pdb ID: 6M3M) with NTDs from
Sars-CoV-1 in red (pdb ID: 2OFZ), HCoV-OC43 in green (pdb ID: 4J3K), HCoV-NL63 in blue (pdb ID: 5NK4), MERS in gray (pdb ID: 4UDI). (B1) The beta-strand (b3)
region for the major conserved epitope is highlighted in blue with the two conserved tyrosines for RNA binding.

coronaviruses (4). Structural mapping of the epitopes shown in protein is being studied as the leading target antigen in vaccine
Figure 5B1 into 3D models of the NTD N protein (6M3M, development (52, 53). However, a better understanding of viral
5NK4, 4J3K, 1SSK, 4UD1 entries of PDB database) reveals a entry is required to avoid further complications with the vaccine
conserved epitope predicted in a highly immunogenic peptide immune response, similar to those observed with HIV type 1
exposed to the extracellular environment, likely, to other host (HIV-1) Env protein candidate vaccine (53, 54). Additionally,
immune system components. We were also able to demonstrate the spike protein has several hotspots for mutations (15). In
that the predicted B cell epitope in SARS-CoV-2 at contrast, the nucleocapsid gene is more conserved and stable,
176SRGGSQASSRSSSRSRNSSRNSTPGSSRGTS206 is inside the with fewer mutations over time (16). Nucleocapsid proteins of
unstructured region inside of SGRD domain of SARS-CoV-2 many coronaviruses are highly immunogenic and are expressed
(Supplementary Figure 1, sequence colored in blue). abundantly during infection (53, 55). High levels of IgG
Unfortunately, this region could not be mapped in the 3D antibodies against nucleocapsid have been detected in sera
model due to the lack of a structure model for the whole from SARS patients (53, 56), and the N protein is a
protein length. representative antigen for the T-cell response in a vaccine
setting (20, 53).
In this study, our bioinformatics analysis was able to identify
epitopes conserved in several human coronavirus N proteins.
DISCUSSION The results show that there are several overlapping conserved
peptides. When combined, our analysis could thus predict not
COVID-19 pandemic challenged the world to speed up research only high binding individual 9-mer peptides, but also highly
for a vaccine against SARS-CoV-2 infection. Despite massive exposed structural regions of immunological peptides, which
effort and many thousands of studies published within the first 8 could have potential importance as candidates for vaccines. Our
months of the pandemic, our understanding of how humans findings are consistent with the strong antigenicity previously
respond to SARS-CoV-2 is still quite limited (2). Worldwide noted in SARS N protein and prior reports for SARS-CoV-2 (24).
efforts are currently underway to map the determinants of The predicted B cell epitopes we identify are consistent with the
immune protection against SARS-CoV-2. In this study, we strong IgG, IgM, and IgA responses to the N protein in an acutely
used a bioinformatics approach to map B and T cells epitopes infected patient documented by Dahlke et al. using peptide
in the nucleocapsid protein of SARS-CoV-2. The SARS-CoV-2 S arrays (2) and with the observations of Grifoni et al. (31). We

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Oliveira et al. SARS-CoV-2 T and B Cell Epitopes

identified a strong immunodominant B cell epitope SRGGS offers protection across multiple coronaviruses. This was
QASSRSSSRSRNSSRNSTPGSSRGTS between 176 and 206 addressed for MERS by Shi et al. (21). Among the epitopes
amino acids in the nucleocapsid protein sequence. With they identified, there are several CD8+ T cell peptides in
appropriate T cell help this epitope may be a good target for homologous positions to those we have predicted in SARS-
neutralizing antibodies and long-lived immune response. CoV-2, although as noted the T cell exposed motifs conserved
Additionally, we performed an in-silico survey of the major T in SARS, SARS-CoV-2 and the other human coronaviruses do
cell epitope sequences of the nucleocapsid protein from differ from MERS. Yang et al. also proposed a nucleocapsid based
coronaviruses known to have infected humans (4). The vaccine for SARS-CoV-1 (61).
demonstration of conserved T cell exposed motifs between the In summary, the use of available information related to SARS-
N protein of multiple human coronaviruses may account for CoV-2 epitopes associated with bioinformatics predictions
the reported recall of T cell responses over decades, even in the points to specific regions of viral nucleocapsid that are targets
absence of SARS-CoV-1 exposure (23, 57). We found a region of to human immune responses (25). We understand that lack of
potential T cell cross-reactivity within the SARS-CoV-2 N biological confirmation of identified peptides may limit the
protein positions 102–110 and equivalent positions in the impact of our discovery. However, testing the antigenicity of
human alpha and beta coronaviruses, with the exception of these B and T cell epitopes will be the next step on our research
MERS. Comparison of the individual allele predicted binding program. The observation that some T cell epitopes are highly
affinities to the SARS-CoV-2 peptides shows differences in conserved between SARS-CoV-2 and other human
responses based on individual genetics. The conserved T cell coronaviruses is critical. Vaccines that target human immune
exposed motifs shared between coronaviruses are each responses toward these conserved epitopes could generate
contextualized in different flanking regions comprising pocket immunity that is cross-protective across alphacoronaviruses
positions that will bind with differing affinities. These and betacoronaviruses (25). This would be an advantage given
complexities underscore the nuanced differences in individual the potential of future novel coronavirus emergence.
patient’s responses. As much of the pathogenesis of COVID-19
disease appears linked to the immune and inflammatory
response, it is important to keep in mind that individual
differences in clinical response may be rooted in the patients
DATA AVAILABILITY STATEMENT
MHC alleles as well as in presence of the preexisting cognate The raw data supporting the conclusions of this article will be
T cell clones, which may have been primed by different peptides. made available by the authors, without undue reservation.
We also address the potential T cell epitopes by a complementary
structural bioinformatics method, which was able to assess the
conservation of these epitopes across different human
coronaviruses. We explored the fact that 89.74% of amino acid AUTHOR CONTRIBUTIONS
sequence of the N protein of SARS-CoV-1 is similar to SARS-
Conceptualization: SO, MM, EH. Methodology: SO, MM, EH.
CoV-2, with high similar 3D structures demonstrated by
Formal analysis: SO, MM, EH. Investigation: SO, MM, EH.
homology modeling, and biophysical feature comparison (58).
Writing: SO, MM, EH. All authors contributed to the article
The relevant amino acids are close to a highly dynamic loop,
and approved the submitted version.
which is important for the protein primary biological function as
the scaffolding agent for the viral genomic stability (59).
The role and diversity of the T cell response to SARS-CoV-2
was reviewed by Altmann and Boyton (60). There have been FUNDING
multiple efforts to map epitopes in the viral proteome, using both
bioinformatics and ex vivo approaches. While most of these have This work was supported by grants from Conselho Nacional de
prioritized the spike protein, several epitopes in the N protein Desenvolvimento Cientifico e Tecnologico (CNPq) grant
have been reported. Mateus et al. identify CD4+ T cell allele- #465229/2014-0, 401209/2020-2 and 302660/2015-1 (to SO)
specific epitopes encompassed in the sequences we identify from and Fundação de Amparo à Pesquisa do Estado de São Paulo
positions 213–238 to 293–320 as binding multiple MHC II alleles (FAPESP) grant #2017/24832-6 (to SO) and Coordenação de
(30). Most notably, our findings parallel those of Le Bert et al. Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES) grant
(24) who demonstrated CD4+ and CD8+ T cell responses to #88887.506611/2020-00 and 88887.504420/2020-00 and
peptides that overlap the multiallelic binding regions we National Institute of Health (NIH) grant# R01 AI 116453 (to SO).
predicted. In particular, patients who were not exposed to
SARS-CoV-2 had CD4+ T cells responsive to N101–120,
which comprises the most conserved T cell exposed motifs SUPPLEMENTARY MATERIAL
(Supplementary Table 1).
The existence of broadly conserved T cell exposed motifs in The Supplementary Material for this article can be found online
the N protein indicates that, even while peptide context is at: https://www.frontiersin.org/articles/10.3389/fimmu.2020.
different, there may be potential to develop a vaccine which 587615/full#supplementary-material

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Oliveira et al. SARS-CoV-2 T and B Cell Epitopes

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Biophys Acta (2013) 1834(6):1054–62. doi: 10.1016/j.bbapap.2013.03.003 distributed under the terms of the Creative Commons Attribution License (CC BY).
51. Kang S, Yang M, Hong Z, Zhang L, Huang Z, Chen X, et al. Crystal structure The use, distribution or reproduction in other forums is permitted, provided the
of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential original author(s) and the copyright owner(s) are credited and that the original
unique drug targeting sites. Acta Pharm Sin B (2020) 10(7):1228–38. publication in this journal is cited, in accordance with accepted academic practice. No
doi: 10.1016/j.apsb.2020.04.009 use, distribution or reproduction is permitted which does not comply with these terms.

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