key: cord-0025670-lny93wvz
authors: Maghsood, Faezeh; Shokri, Mohammad-Reza; Jeddi-Tehrani, Mahmood; Rahvar, Monireh Torabi; Ghaderi, Abbas; Salimi, Vahid; Kardar, Gholam Ali; Zarnani, Amir-Hassan; Amiri, Mohammad Mehdi; Shokri, Fazel
title: Identification of immunodominant epitopes on nucleocapsid and spike proteins of the SARS-CoV-2 in Iranian COVID-19 patients
date: 2022-01-05
journal: Pathog Dis
DOI: 10.1093/femspd/ftac001
sha: 07ff0b3e17c9fd1bed40a862f00669d761678a60
doc_id: 25670
cord_uid: lny93wvz

Given the emergence of SARS-CoV-2 virus as a life-threatening pandemic, identification of immunodominant epitopes of the viral structural proteins, particularly the nucleocapsid (NP) protein and receptor binding domain (RBD) of spike protein, is important to determine targets for immunotherapy and diagnosis. In this study, epitope screening was performed using a panel of overlapping peptides spanning the entire sequences of the RBD and NP proteins of SARS-CoV-2 in the sera from 66 COVID-19 patients and 23 healthy subjects by enzyme-linked immunosorbent assay (ELISA). Our results showed that while reactivity of patients' sera with reduced recombinant RBD protein was significantly lower than the native form of RBD (p<0.001), no significant differences were observed for reactivity of patients' sera with reduced and non-reduced NP protein. Pepscan analysis revealed weak to moderate reactivity towards different RBD peptide pools, which was more focused on peptides encompassing aa 181-223 of RBD. NP peptides, however, displayed strong reactivity with a single peptide covering aa 151-170. These findings were confirmed by peptide depletion experiments using both ELISA and Western blotting. Altogether, our data suggest involvement of mostly conformational disulfide bond-dependent immunodominant epitopes in RBD-specific antibody response, while the IgG response to NP is dominated by linear epitopes. Identification of dominant immunogenic epitopes in NP and RBD of SARS-CoV-2 could provide important information for the development of passive and active immunotherapy as well as diagnostic tools for the control of COVID-19 infection.

COVID-19 has remained a major health concern since the World Health Organization (WHO) declared it a pandemic on 11 March 2020 (Cohen & Normile, 2020 . As of 16 May 2021, severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has infected more than 161 million people worldwide, with over 3 million deaths (World-Health-Organization, 2021) . This has imposed a huge burden on health care systems worldwide and provoked a serious economic crisis following inevitable social lockdown (McKee & Stuckler, 2020) . Thus, there is a desperate need for developing sensitive and robust diagnostic tests and potentially effective vaccines for SARS-CoV-2 to alleviate the growing socioeconomic impacts. To this goal, obtaining illuminating insights into the immune response, particularly the antibody response, to SARS-CoV2 is obviously crucial.

From the experience with SARS-CoV virus, it is postulated that neutralizing antibodies elicited against SARS-CoV-2 in convalescent individuals may be important correlates of protection (induced either by natural infection or vaccination) , Shen et al., 2020 , and detection of virus-specific antibodies may provide a robust diagnostic tool for epidemiological purposes. Moreover, early seroconversion and high antibody titers could be used as a prognostic marker for disease severity and ARDS development in patients with SARS-CoV-2 (Shen et al., 2020) .

Like other coronaviruses infecting humans, SARS-CoV-2 is an enveloped positive-sense RNA virus, composed of four structural proteins known as spike (S), envelope (E), membrane (M), and nucleocapsid (NP) proteins . Apart from NP which is associated to the viral RNA genome, the other three, S, E, and M proteins organize the viral envelope and are directly accessible to the host immune system . The S protein is a large type I transmembrane glycoprotein that is responsible for receptor binding and membrane fusion. With a total length of 1273 amino acids, S protein has two functional domains, S1 and S2, located in the N-and C-terminal, respectively (Walls et al., 2020 , Wrapp et al., 2020 . S1 subunit itself consists of an N-terminal domain (14-305 residues) and a receptorbinding domain (RBD, 319-541 residues) which binds to the host receptor angiotensin converting enzyme 2 (ACE2), while the S2 subunit which plays an important role in SARS-CoV-2 fusion with the target cells, harbors the fusion peptide (FP) (788-806 residues), heptapeptide repeat sequence 1 (HR1) (912-984 residues), HR2 (1163-1213 residues), transmembrane (TM) domain (1213-1237 residues), and cytoplasmic domain (1237-1273 residues) , Walls et al., 2020 .

It has become evident that SARS-CoV-2 like other human-infecting coronaviruses (e.g. SARS-CoV, MERS-CoV) can elicit IgM and IgG antibodies directed against the S and NP proteins. after initial infection (Ni et al., 2020) . This antibody response is initially used in serological assays for diagnostic purposes (Krammer & Simon, 2020) . However,

due to the presence of several cross-reactive epitopes in NP and S with SARS-CoV and MERS-CoV (Grifoni et al., 2020) , the detected antibody response may not represent only anti-SARS-CoV-2 antibodies and could be a reflection of previous exposures to other human coronaviruses . Moreover, these serology-based assays do not offer any clue about antibody functionality, including neutralization potential which is a prerequisite in vaccine development.

On the other hand, antibody-dependent enhancement (ADE) through low affinity, low quantity, and non-neutralizing antibodies is a concern with human coronaviruses (Iwasaki & Yang, 2020) , which further necessitates epitope dissection in order to resolve virus-specific and favorable antibodies from unfavorable cross-reactive antibodies.

Since SARS-CoV-2 virus largely depends on the spike glycoprotein for binding to ACE2 receptor and cell entry , several studies have focused on detecting antibodies against S protein and its RBD to assess their neutralizing effect. However, antibodies against nucleoprotein appear earlier in the course of seroconversion (Meyer et al., 2014) and could be more indicative for diagnostic applications. In the current study, we present several immunodominant epitopes on the RBD and NP proteins by Pepscan analysis on the sera from Iranian SARS-CoV-2

patients. This information may be useful for designing COVID-19 diagnostics and vaccines.

For primary screening, the total number of 120 cases, including 97 COVID-19 patients and 23 healthy controls, were recruited in this study. Healthy control samples were collected from the Blood Transfusion Organization of Iran 5 months before the COVID-19 epidemic in Iran and stored at -20 °C. All patients were laboratory-confirmed positive for SARS-CoV-2 by real time PCR using throat or nasopharyngeal swab specimens. All the samples were collected in April and

May 2020 before the emergence of new variants of concern or interest. At that time, the original Chinese isolate (Wuhan- 

The sequences we used for the design of linear peptides of the RBD and NP of SARS-CoV-2 (starin Wuhan-Hu-1) were obtained from GenBank under accession numbers NC_045512.2 and MN908947.3. For epitope screening of RBD and NP, we designed panels of linear peptides spanning the entire sequence of the RBD and NP protein of SARS-CoV-2 (each peptide contains 20 amino acid residues with 5 residues overlapping with the adjacent peptides). All the peptides were synthesized by Pepmic company (China). Peptide synthesis was performed using a standard solid-phase Fmoc method. Peptides were purified to homogeneity (purity more than 90%) by HPLC and identified by laser desorption mass spectrometry. Peptides were used individually or as pooled sets. 

For ELISA assays, we used recombinant SARS-CoV-2 NP protein expressed in Baculovirus-insect cells ( nm using a microplate reader (Biotek, USA). The quantitative cut-off value for seropositivity for COVID-19 was defined as the mean OD of healthy samples plus 2 SDs.

Preliminary peptide-based ELISA was performed using pooled sets of three peptides. An ELISA assay was initially performed using serial dilutions of a number of samples to exclude background signals and determine a dilution in which negative and positive samples could be differentiated. Based on these preliminary results, we selected a single 1:200 dilution for all samples to be able to compare the results. ELISA was performed according to the protocol described in section 2.3. with the following modifications. In brief, flat-bottom 96-well Maxisorp plates (Nunc) were coated with 3.5 µg/ml of each peptide in 3 peptide pool sets, native proteins (1.0 µg/ml of RBD or 1.5 µg/ml NP protein) or 1% 2-Mercaptoethanol (2-ME, Sigma Aldrich) reduced proteins in PBS and incubated overnight at 4 °C. After washing three times with 0.05% PBST, the plates were blocked using blocking buffer for 1 h at 37 °C, before the addition of serum samples. in the blocking buffer for 1 h at 37°C. HRP-conjugated mouse monoclonal anti-human IgG antibody was used 

To verify reactivity of RBD-specific and NP-specific antibodies to the identified peptides from both proteins, a depletion assay was performed. Using principles similar to the previous section, we performed peptide adsorption as follows.

P151-170 of NP and P204-223 of RBD were selected as the immunodominant peptides. P136-155 of NP and P196-215 of RBD were selected as non-reactive control peptides. 2.0 µg/ml of selected peptides were incubated with the patient's sera for 3 h at 37 °C to adsorb peptide-specific antibodies. Peptide-adsorbed patients' sera were then incubated for 1h at 37°C in NP or RBD pre-coated ELISA plates. ELISA analysis was performed as described in section 2.4. Non-adsorbed sera were added as control.

To analyze reactivity of peptide-adsorbed sera with SARS-CoV-2 RBD and NP proteins, 2 µg of each protein in native or 1% 2-ME reduced form were subjected to10%. Polyacrylamide gel in SDS sample buffer. Proteins were separated by electrophoresis at 100 V for 1h, then transferred to a 0.45 μm hydrophilic polyvinylidene fluoride (PVDF) membrane at 

proteins and depletion assays analysis. Differences were considered to be significant at P<0.05(*), P<0.01(**), P<0.001(***), and P<0.0001(****).

Serum levels of IgM and IgG against RBD and NP proteins were determined in patients' sera (n=79) 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.

We next assessed the linear dominant antigenic determinants within the RBD domain of the spike protein (319-541 aa), and NP protein (1-419 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.

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) .

While all 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) .

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 ).

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 nonadsorbed sera against RBD was slightly, but non-significantly, higher in comparison with that of P204-223 adsorbed sera (p=0.274).

Native 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.

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 136-185 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 (Fig. 6a) , which may reflect the contribution of other potential conformational epitopes in native NP.

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".

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 sera adsorbed with peptide P151-170 have lower reactivity (p=0.0156) with native NP in comparison with non-adsorbed sera.

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 using serum samples collected from COVID-19 Iranian patients by Pepscan analysis employing 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 , we observed low reactivity to peptides covering 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) by Ladner et al. (Ladner et al., 2021) .

Meng Poh and colleagues using a pepscan analysis, also failed to find a linear immunodominant epitope exactly localized to RBD region with high reactivity to patients' sera (Poh et al., 2020) . This weak/ lack of reactivity in peptide-based approaches implies that antibodies reactive to RBD region are largely directed against conformational epitopes and/or blind to epitopes generated in the secondary or tertiary structure of proteins, we speculate that RBM possesses conformational epitopes, so that we found no reactivity in patient's sera against linear peptides corresponding to RBM region (P106-125, P121-140, P136-155, P151-170, P166-185, and P181-200) . 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 that some residues that are distal in the linear sequence of RBD and their glycosylation state contribute to antibody binding (Pinto et al., 2020 .

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 (Mu et al., 2020) . Like SARS-CoV, NP protein of SARS-CoV-2 consists of three distinct domains: an N-terminal RNAbinding 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 (Kang et al., 2020 , Khan et al., 2020 .

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) . 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 (Buchholz et al., 2004 , Pang et al., 2004 , Liang et al., 2005 ; however, significant cytotoxic T lymphocyte (CTL) response against NP has been reported using vector-based vaccines containing NP protein (Gao et al., 2003 , Buchholz et al., 2004 , Zhu et al., 2004 . 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 (Kim et al., 2004) . 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 (Dutta et al., 2020) . A recently published data by Ahlén et al. (Ahlén et al., 2020) , 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 summary, we studied the B cell linear epitopes on RBD and NP proteins of SARS-CoV-2 using serum samples from COVID-19 patients. While the antibody response to NP was mainly directed against linear epitopes as evidenced by reactivity to synthetic peptides and reduced native protein, RBD specific antibodies seem to largely recognize disulphid-bond dependent conformational epitopes. These findings propose that peptide-based vaccines may not be able to elicit virus neutralizing antibodies against SARS-CoV-2. Furthermore, profiling of the NP specific antibody response by Pepscan suggests that the immunodominant peptides could be employed for immunodiagnosis of the infection. samples (Patient #1 (star shape) and Patient #2 (bullet shape)) were also tested by Western blot on NP protein (45 kD).

P<0.05(*). NP: Nucleocapsid.

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The authors would like to thank Jalal Khoshnoodi and Mohammad Ali Judaki for their technical assistance.

This study was partially supported by TUMS and a grant from the National Institute for Medical Research Development (NIMAD) of Iran (Grant No. 993421).

This study was approved by the Ethical Committee of the National Institute for Medical Research Development (NIMAD) of Iran (IR.NIMAD. REC.1399.194). Written consent was obtained from all patients included in this study or their legal representatives.

The authors declare no competing interests.

The original data sets of this study are available from the corresponding author, upon reasonable request.