3.1. IgM and IgG responses to RBD and NP proteins
Serum levels of IgM and IgG against RBD and NP proteins were determined
in patients’ sera (n=97) by ELISA (Fig. 1). Sera from 23 healthy
individuals were used as controls. Our data demonstrated that based on
assigned cut-off OD values (cut-off =mean ±2SD of normal individuals)
anti-RBD and anti-NP IgG were positive in 94% and 92%. and anti-RBD
and anti-NP IgM were positive in 90% and 80% of patients’ sera,
respectively. Subsequently, 66 serum samples from patients with IgG
titer higher than the cut-off level (0.33 for anti-RBD IgG and 0.43 for
anti-NP IgG) were selected for further Pepscan experiments.
Identification of linear epitopes on the SARS-CoV-2 RBD and NP
proteins
We next assessed the linear dominant antigenic determinants within the
RBD domain of the spike protein (319-541 aa), NP protein (1-419 aa), and
three different fragments within the S2 domain of the spike protein,
namely FE-18 (802-819 aa), FI-22
(888-909 aa) and CK-16 (1254-1269
aa), by Pepscan analysis. A total of 66 serum samples from COVID-19
patients were selected for epitope mapping which had been verified to be
reactive to the target proteins by ELISA. Serum samples from 23 healthy
subjects were also included as negative controls. A set of 20-aa
overlapping peptides spanning the entire RBD domain and NP protein in
pools of 3 adjacent peptides were used as coating antigens in ELISA
(Table. 1), and serum samples were tested against each of the peptides.
To test whether the reactivity is proportional to linear epitopes or
conformational ones, reduced as well as native proteins were also used
as coating antigens, and all sera were assessed with these antigens as
well.
- Reactivity against native and reduced SARS-CoV-2 RBD
proteins
Native and reduced SARS-CoV-2 RBD proteins were employed to assess the
reactivity of RBD-specific IgG antibodies with cysteine bonds
dependent and independent epitopes. Significantly higher reactivity
was observed with native non-reduced protein as compared to the
reduced preparation (p=0.0002 ) (Fig. 2).
- Reactivity against RBD peptide pools
While all of the 66 serum samples from COVID-19 patients were reactive
against native RBD protein, less than 40% of these samples recognized
peptide pools A to D, and pool E alone reacted with about 40% of
patients’ sera. On the other hand, reactivity with all RBD peptide
pools was significantly higher in patients’ sera than healthy controls
(Fig. 3A).
- Identification of the immunodominant peptide in pool E
Since patients’ sera showed better reactivity with pool ”E” of RBD
peptides, we evaluated the reactivity of 25 pool ”E” positive serum
samples with individual peptides of this peptide pool. Most samples
reacted either with peptide P204-223 or 181-200 of RBD (Fig. 3B).
- Depletion assay confirmed specific reactivity with
P204-223 on SARS-CoV-2 RBD protein
Further assessment was performed to verify specific reactivity of
RBD-specific antibodies from 10 patients to P204-223 peptide from RBD
protein (Fig. 4). Patients’ sera were adsorbed with P204-223 as the
most reactive peptide and P196-215 as the non-reactive peptide, and
evaluated by ELISA against native RBD (Fig. 4A). Two of these samples
were also tested by Western blotting (Fig. 4B). The reactivity of
non-adsorbed sera against RBD was slightly higher in comparison with
that of P204-223 adsorbed sera (p=0.274 ). It confirms that the
antibody response against RBD is largely related to the conformational
epitopes.
- Identification of linear epitopes on the SARS-CoV-2 NP protein
- Reactivity against native and reduced SARS-CoV-2 NP
proteinsNative and reduced SARS-CoV-2 NP proteins were employed to assess
the reactivity of NP-specific antibodies with cysteine bonds
dependent and independent epitopes. The reactivity to native NP was
similar to the reduced NP (p=0.206 ) (Fig. 5), suggesting that
denaturation does not significantly alter recognition of the NP
protein by patients’ antibodies.
- Reactivity against NP peptide pools
Pepscan analysis of peptide pools covering the entire NP protein
against sera obtained from COVID-19 and healthy controls also
revealed significantly higher reactivity of patients’ sera than
healthy controls in each block of peptides (Fig. 6A). Most pools of
peptides were recognized by less than 50% of patients’ sera, while
one distinct antigenic site corresponding to aa 136 to 185 in the
N-terminal domain of NP protein (pool I) reacted very strongly with
more than 75% of the serum samples from COVID-19 patients. This
suggests that the identified region, namely pool I, contains at
least one of the major linear immunodominant epitopes that induces
the antibody response in COVID-19 patients. The results also
indicate that the reactivity against pool I was collectively weaker
than reactivity to native NP protein (p<0.001) (Fig. 6A),
which may reflect the contribution of other potential conformational
epitopes in native NP.
- Identification of the immunodominant peptide in pool
I
Next, we further assessed individual peptides within pool I from NP
protein which includes aa 136-155, 151-170, and 166-185, to
precisely determine the exact 20-mer epitope which attributes to the
highest reactivity of COVID-19 patients’ sera (Fig. 6B). Sera
obtained from 25 patients were used in this experiment. The data
revealed that peptide P151-170 is the dominant hit from pool ”I”.
- Depletion assay confirmed specific reactivity with
P151-170 on SARS-CoV-2 NP protein
Further assessment was performed to verify the specific reactivity
of NP-specific antibodies from 10 patients to the dominant P151-170
peptide from NP protein (Fig. 7). Patients’ sera were individually
adsorbed with P151-170, as well as P136-155 as the non-reactive
peptide, and evaluated by ELISA against native NP (Fig. 7A). Two of
these samples were also tested by Western blotting (Fig. 7B). The
data revealed that peptide P151-170 adsorbed sera have decreased
reactivity (p=0.0156 ) with native NP in comparison with
non-adsorbed sera.
- Identification of linear epitopes on the SARS-CoV-2 S2 proteinThree peptides of S2 domain, including FE-18 (aa 802-819), FI-22 (aa
888-909), and CK-16 (aa 1254-1269)
were selected to assess serum reactivity of 66 patients and 23 normal
subjects. Almost 50% of patients’ sera reacted with these three
peptides (Fig. 8), among which CK-16 displayed the highest reactivity.
Discussion
Identification of major antigenic determinants of SARS-CoV-2 proteins
which provoke remarkable antibody response in COVID-19 patients may
provide valuable information for understanding the virus-neutralizing
antibody response and developing efficient vaccines and serological
assays.
Here, we have mapped the linear immunodominant epitopes on SARS-CoV-2 NP
and RBD proteins by Pepscan analysis using 20-aa overlapping peptides
spanning the whole sequence of both proteins. We showed that Iranian
patients with COVID-19 develop significant anti-RBD and anti-NP IgG and
IgM antibody responses. However, while the antibody response against RBD
seems to be largely raised against the S-S bond-dependent conformational
determinants, the antibody response against NP protein is mostly
directed linear epitopes.
Despite strong serological reactivity of convalescent patients against
RBD reported in studies using full-length RBD antigen of SARS-CoV-2 (16,
17), reports on serological reactivity to ”linear” immunodominant sites
on SARS-CoV-2 RBD are very limited in the literature. Although Zhang et
al. reported four linear immunodominant sites on RBD detected by sera
from COVD-19 patients (18), we and others observed low reactivity to
peptides designed from the RBD of SARS-CoV-2. Weak serological
reactivity to peptides within the RBD was also observed in a recent
study using a highly multiplexed peptide assay (PepSeq) platform by
Ladner et al. (19). Meng Poh and colleagues using a pepscan analysis,
failed to find a linear immunodominant epitope exactly localized to RBD
region with high reactivity to patients’ sera (20). This weak/ lack of
reactivity in peptide-based approaches implies that antibodies reactive
to RBD region are largely directed against conformational epitopes
and/or epitopes generated by post-translational modifications. Pepscan
analysis in our assay and peptide-based antibody assays exclusively
detects linear epitopes created by the primary structure of proteins and
are blind to epitopes generated in the secondary or tertiary structure
of proteins. Interestingly, the reduction of S-S bonds within the native
RBD molecule by a reducing agent resulted in a significant reduction of
antibody reactivity in patients’ sera (Fig. 2).
To assess the linear epitopes which provoke anti-RBD antibody response,
we performed Pepscan analysis using 20-aa long overlapping peptide
covering the entire RBD domain. Although antibody reactivity against 5
different peptide pools, each consisting of 3 peptides, was
significantly higher in sera from patients compared to healthy subjects,
it was significantly lower when compared with reactivity to the native
RBD protein (Fig. 3A). Of the 5 peptide pools, pool ”E” which covers the
C-terminal residues of RBD (aa 181-223) displayed the highest
reactivity. Thus, we investigated the antibody response to the three
peptides of this pool in 25 selected patients’ sera to identify the most
immunodominant one. Both peptides P181-200 and particularly P204-223,
but not peptide P196-215, displayed modest reactivity with some of the
samples (Fig. 3B). Preincubation of serum samples from 10 of these
patients with peptide P204-223 resulted in slightly lower reactivity of
these samples to native RBD by ELISA and Western blot (Fig. 4). All
these findings suggest that the antibody response to RBD is dominated by
S-S bond-dependent conformational epitopes. The fact that the
immunogenicity of RBD mainly relies on the conformational structures
and/or post-translational modifications (i.e., glycosylation) is proved
by studying the binding footprint of neutralizing monoclonal antibodies
that inhibit RBD binding to ACE2. Studies revealed residues that are
distal in the linear sequence of RBD and their presence and
glycosylation state contributed to antibody binding (21, 22).
We adopted a similar approach to investigate the antibody response
against the NP protein. SARS-CoV-2 NP is a 419 aa phosphoprotein that
associates with the viral RNA genome as well as other proteins to form
the ribonucleoprotein core (23). Like SARS-CoV, NP protein of SARS-CoV-2
consists of three distinct domains: an N-terminal RNA-binding domain
(NTD) which associates with the RNA genome, an intrinsically disordered
central Ser/Arg (SR)-rich linker and a C-terminal domain (CTD) which
allows dimerization of NP proteins (24, 25).
Our results revealed that in contrast to RBD, NP-specific antibody
response is mainly directed against linear epitopes. No significant
difference was observed between serum levels of antibody against reduced
and non-reduced NP protein (Fig. 5). This was also supported by the
Pepscan data which showed substantially higher reactivity of the anti-NP
antibodies from patients’ sera with all peptide pools, particularly
peptide pool ”I”, which covers aa 136-185 (Fig. 6A). When the three
peptides within this pool were dissected and tested in a number of
patients’ sera, the antibody response was almost entirely directed
against one of these three peptides which encompasses aa 151-170 (Fig.
6B).
Amino acids 151-170 are located on RNA-binding terminal domain of NP
protein which has been previously reported by Amruna et al. as an
immunodominant epitope in SARS-CoV-2 and as a disease severity correlate
in COVID-19 patients (26). Sequence homology analysis demonstrated that
the epitope we identified in SARS-CoV-2 NP protein is highly conserved
among human coronavirus strains, including SARS-CoV, MERS-CoV, HKU1-CoV,
and OC43-CoV. Although most studies only address the diagnostic value of
nucleocapsid protein and its epitopes for detection of seroconversion in
COVID-19 patients, the highly conserved nature of this epitope in NP
protein would be an advantage in developing epitope-based vaccines
against SARS-CoV-2 which can develop cross-protection against other
human coronaviruses. The majority of vaccines which are under clinical
investigation against SARS-CoV-2 are developed based on viral spike
protein and mostly rely on the ensuing neutralizing antibody response
which blocks viral entry rather than killing infected cells. This could
be largely attributed to the localization of NP protein. While the three
other structural proteins of the virus, S, M, and E reside at the
interface of the virus to the external environment, the NP protein is
located inside the viral particles and is not accessible to the antibody
during the course of infection. Therefore, although elevated
anti-SARS-CoV-2 NP IgG and IgM antibody titers were observed in our
study and have been reported by other investigators (27, 28), they do
not seem to have neutralization potential.
Since most of our current knowledge about SARS-CoV-2 NP protein comes
from previous studies on SARS-CoV, better evidence in this regard could
be obtained from SARS-CoV literature. Several studies during the SARS
outbreak clearly showed that SARS-CoV NP protein could not induce strong
neutralizing antibody responses neither in human nor in animal (29-31);
however, significant cytotoxic T lymphocyte (CTL) response against NP
has been reported using vector-based vaccines containing NP protein (29,
32, 33) . Kim and colleagues introduced an effective DNA vaccine using
SARS-CoV NP protein as a target antigen fused to calreticulin (CRT) to
enhance MHC class I presentation of linked antigen to CD8(+) T cells.
Their results showed that the NP-based vaccine generated strong
NP-specific humoral and T-cell immune responses in mice (34). Recently,
it has been suggested that SARS-CoV-2 NP could be considered as an
advantageous vaccine target owing to its conserved nature and strong
immunogenicity (35). A recently published data by Ahlén et al. (36),
also showed a DNA vaccine based on a codon-optimized SARS-CoV-2 NP gene
induced high titers of anti-NP antibodies in immunized rabbits, but most
interestingly, they showed that immunization of mice with a DNA vaccine
expressing the SARS-CoV-2 NP protein induced the strongest T cell
response against a peptide region spanning our P151-170 peptide.
In this study, we also observed a substantial IgG response against CK-16
peptide fragment from the S2 domain of spike protein. The S2 subunit,
composed of a fusion peptide, HR1, HR2, transmembrane and cytoplasmic
domains, is responsible for viral fusion and host cell entry and thus
plays a crucial role in the virus pathogenicity (8). The CK-16 peptide
(aa 1254-1269) is located at the very C-terminal end of the spike
protein (in the cytoplasmic domain of S2). It is surprising to see the
high immunogenicity of CK-16 because it is located in the cytoplasmic
domain of S2 and after viral entry, is not accessible to B cells.
Whether the antibodies that recognize this epitope are neutralizing or
not necessitates further study. However, it is interesting that CK-16
peptide shares a high sequence identity with C-terminus end of spike
protein from all other coronaviruses, and the CK-16 peptide region is
identical among SARS-CoV-2, SARS-CoV-1, SARS-CoV S Urbani, SARSr-CoV
ZXC21and SARSr CoV-RaTG13 (8, 37). It implies that this epitope may
serve a functional role for the virus. It has been shown that C-terminus
of S2 subunit contributes to the stabilization of the trimeric structure
of the spike protein (38), and destabilization of this area in the spike
protein of SARS-CoV interferes with spike trimerization and reduces
fusion and infectivity of SARS-CoV (39). A similar mechanism could be
postulated for antibodies directed against CK-16 in SARS-CoV-2 that may
destabilize the trimeric structure of spike protein and reduces viral
pathogenicity. Yang Li et al. have also reported IgG antibodies against
this epitope in the sera from some convalescent COVID-19 patients,
however, the neutralization assay was not reported for this epitope
(40).
On the other hand, due to high homology with other coronaviruses, it is
possible that some patients have preexisting antibodies to this region,
and the corresponding B cell clones against this region are expanded
after SARS-CoV-2 infection. This epitope has also been identified in 2
other publications studying antibody response in convalescent sera from
SARS patients (41, 42); hence the functional importance of this epitope
also remains to be elucidated. Most recently, in a comprehensive linear
epitope landscape using sera from 1,051 COVID-19 patients, Yang Li and
colleagues revealed an immunodominant region at the C-terminal domain of
S2 subunit rich in linear epitopes which covers our CK-16 (43). Although
the clinical and functional significance of antibodies against this
region were not proved in their study, the reactivity of a large number
of COVID19 patients’ sera against this region reinforces its
immunogenicity, making it more deserving for further investigation.
In summary, we identified the dominant B cell linear epitopes on RBD and
NP proteins of SARS-CoV-2 in COVID-19 patients. These epitopes may serve
as a vaccine candidate after being confirmed for their neutralization
function using appropriate assays.