key: cord-0328123-9tjpzv1o authors: Nitahara, Y.; Nakagama, Y.; Kaku, N.; Candray, K.; Michimuko, Y.; Tshibangu-Kabamba, E.; Kaneko, A.; Yamamoto, H.; Mizobata, Y.; Kakeya, H.; Yasugi, M.; Kido, Y. title: High resolution linear epitope mapping of the receptor binding domain of SARS-CoV-2 spike protein in COVID-19 mRNA vaccine recipients. date: 2021-07-07 journal: nan DOI: 10.1101/2021.07.03.21259953 sha: 4301f30a1e74dd6926ed5a090ad60901c7e0cd86 doc_id: 328123 cord_uid: 9tjpzv1o The prompt rollout of the coronavirus disease (COVID-19) messenger RNA (mRNA) vaccine facilitated population immunity, which shall become more dominant than natural infection-induced immunity. At the beginning of the vaccine era, the initial epitope profile in naive individuals will be the first step to build an optimal host defense system towards vaccine-based population immunity. In this study, the high-resolution linear epitope profiles between Pfizer-BioNTech COVID-19 mRNA vaccine recipients and COVID-19 patients were delineated by using microarrays mapped with overlapping peptides of the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. The vaccine-induced antibodies targeting RBD had broader distribution across the RBD than that induced by the natural infection. The relatively lower neutralizing antibody titers observed in vaccine-induced sera could attribute to less efficient epitope selection and maturation of the vaccine-induced humoral immunity compared to the infection-induced. Furthermore, additional mutation panel assays showed that the vaccine-induced rich epitope variety targeting the RBD may aid antibodies to escape rapid viral evolution, which could grant an advantage to the vaccine immunity. infection-induced immunity. In this start of the COVID-19 vaccine era, the de novo repertoire in naïve individuals, the molecular dynamics of B-cell response to SARS-CoV-2, will be the first step to build an optimal host defense system towards vaccine-based population immunity. Currently, the efficacy of vaccine-induced immunity against SARS-CoV-2 in an individual is evaluated by potential surrogate markers such as half-maximal neutralization titers (NT50) using live/pseudo viruses and total antibodies titers against the receptor binding domain (RBD) of the spike protein of the virus (1) (2) (3) (4) . Understanding the epitope profile of both vaccine recipients and COVID-19 patients shall readily elucidate the molecular basis of these markers as surrogate. Moreover, the coevolution of vaccine-induced host immunity and the virus escape will be one of the most important elements to consider in the way of achieving herd immunity against COVID-19. The RBD of the spike glycoprotein of SARS-CoV-2 is widely considered as a key protein target for designing vaccines and developing neutralizing antibodies as therapeutic agents (5, 6) . As epitope profiles of naturally infected COVID-19 patients' sera have identified several immunodominant regions in the spike protein, most linear epitopes are located outside the RBD of the spike protein, while anti-RBD antibodies consist minor proportion of the total anti-spike IgG epitopes (7) (8) (9) (10) (11) . Nonetheless, the majority of the neutralizing monoclonal antibodies (NAbs) derived from convalescent sera target the RBD, which imposes its crucial role in virus neutralization (6, (12) (13) (14) (15) (16) . Based on the steric binding orientation of the anti-RBD NAbs, four different categories of NAbs have been proposed (17, 18) . While a growing number of individuals acquire vaccine immunity, detailed epitope profile of the humoral immune response to the mRNA vaccine is not fully understood (1, 19, 20) . In this study, high resolution linear epitope profiling targeting the RBD was performed using sera of both mRNA vaccine recipients and COVID-19 patients. Comparing the detailed epitope profiling analysis and the serological markers, we sought to describe the humoral immune response elicited by mRNA vaccination and natural infection, which will be crucial in this post-vaccine era of the All rights reserved. No reuse allowed without permission. (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 preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.03.21259953 doi: medRxiv preprint COVID-19 pandemic. Two groups were analyzed in this study: (i) vaccine recipients, all received two doses of BNT162b2 mRNA vaccine (Pfizer/BioNTech) with a three-week interval (N=21, age 20s-80s years old). Blood was obtained 17-28 days after the second dose. The first mRNA vaccine approved in Japan was Pfizer/BioNTech's BNT162b2 and Moderna's mRNA-1273 was the second. (ii) COVID-19 patients confirmed by nucleic amplification testing (N=20, age 20s-80s years old). The blood collection of the patients was performed between 10 and 63 days (median 39 days) after the onset. Detailed information of the subjects and severity of the disease of the patients can be found in Supplemental Blood samples were obtained by venipuncture in serum separator tubes and the serum fraction was store at -80 . All subjects provided written consent before participating in this study. This study was approved by the institutional review board of Osaka City University (#2020-003) and the Graduate School of Life and Environmental sciences and the Graduate School of Sciences, Osaka Prefecture University. The neutralization assay was carried out as described previously (24) (1:1000, 1 hour at 37˚C). Cells were then washed and incubated with Alexa488-conjugated goat antirabbit IgG (Thermofisher scientific, USA) (1:500, 45 minutes at 37˚C). Antigen positive cells were counted under a fluorescent microscope and the percentage of neutralization was estimated as the viral infectivity under serum-treated conditions compared with that without serum. For precise linear epitope screening, overlapping 15-mer peptides (shfit by 3 amino acids) were sequentially synthesized according to the sequence of the RBD on cellulose membrane by MultiPep synthesizer (Intavis Bioanalytical Instruments, Germany) using SPOT technology (25, 26) . The sequence of the RBD was obtained by GenBank (accession: MN908947.3, S319-S541) Additional 15-mer peptides containing single mutations of variants of concerns found within the RBD were designed. Single mutations included K417N, K417T, E484K and N501Y (27) . Detailed peptide sequences used in this study can be found in Supplement Table 2 . Synthesized arrays were probed with sera at a 1:400 dilution followed by incubation with horseradish All rights reserved. No reuse allowed without permission. (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 preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.03.21259953 doi: medRxiv preprint peroxidase conjugated goat anti-human IgA +IgG +IgM polyclonal antibody at a 1:30,000 dilution. The bound of the secondary antibody on each peptide was detected and quantified by enhanced chemiluminescence. The epitopes were detected by subjective visual inspection. Chemiluminescence signal intensities of the peptide arrays were standardized in two ways: relative values to the maximum signal level of each array as 100, and z-scores considering peptide signals of individual subjects as population. Half-maximal neutralization titers (NT50) of the neutralization assay were calculated using GraphPad Prism 9. The sequence and conformational information of the RBD was obtained under the accession number 6M0J (5) and 7A94 (28) at Protein Data Bank. The images to depict the recognized epitopes are shown using The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC. All vaccine recipients (N=21) and COVID-19 patients (N=20) revealed seropositivity to anti-RBD IgG according to the manufacturer's threshold (>50 AU/mL) and the two groups did not show significant difference in their levels of titers ( Figure 1a ). However, the neutralization assay using live SARS-CoV-2 showed remarkably lower neutralizing titers in vaccine recipients' sera than the patients ( Figure 1b) . Relative anti-RBD antibody titers to NT50 values in individuals were calculated and plotted in Figure 1c , which suggested a discrepancy in epitope profiles between vaccine recipients and COVID-19 patients. None of the vaccine recipients were seropositive to anti-N IgG, ensuring that they were naïve to SARS-CoV-2 infection (Supplement Table 1 ). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Since the vaccine-elicited sera, although reactive to the RBD to similar extent, showed significantly reduced neutralization, we investigated the linear epitope profile of the anti-RBD antibodies elicited by the vaccination and the natural infection. Sera of five subjects from the vaccine recipients and ten from the patients were selected based on their anti-RBD antibody titers and NT50 (denoted as red dots in Figure 1 ). The sera were incubated with the designed microarrays arranged with 15-mer overlapping peptides of the RBD on the surface (Figure 2a ). The designated array did not show any considerable unspecific binding of the secondary antibody. We generated a heatmap according to the relative signals of the overlapping peptides ( Figure 2a ). Our epitope detection criteria were visually detectable as a significant peak in a graph depicting a mostly minimum of 0.5 z-score of the mean peptide signals ( Figure 3 ) and/or regions previously reported as neutralizing antibodies in the RBD(17,29). Comparing the epitope profiles of the two groups, two types of epitopes were identified: (1) epitopes recognized by both groups and (2) epitopes recognized only by vaccine-elicited sera. Overall, seven linear epitopes were recognized within the RBD, four within (1) A total of four linear epitopes were recognized in both groups. Three (peptide No. 33, No.47-49) of them shared the epitope regions of the RBD with neutralizing monoclonal antibodies previously reported as class1 and class 3. Two linear epitopes were identified at peptide No. 33 and peptides No.47-49, which share the epitopes with class 1 neutralizing antibodies(17). Also, peptide No. 39-40 shares epitope residues very similar to human monoclonal antibody REGN10987, categorized as Class 3 neutralizing All rights reserved. No reuse allowed without permission. (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 preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.03.21259953 doi: medRxiv preprint antibody which sterically hinders angiotensin converting enzyme 2 (ACE2) interaction (17). Linear epitope (peptide No. [26] [27] ) was reactive at the highest level in most serum samples; sera of 2/5 vaccine recipients and 7/10 patients had maximum reactivity to this peptide. The antibodies binding to this epitope do not seem to contribute to neutralizing the live virus, based on the findings we experienced. This peptide was found highly reactive in a serum of a COVID-19 patient who had undergone Rituximab treatment, whose neutralizing antibody titer was not sufficient (For details, see Supplement Figure 1 )(30). In the RBD structure, No.27 peptide is located inside the core β sheets, which is not exposed to the surface of the RBD in either an "up" or "down" position. Judging from the structural composition, this linear epitope does not seem to affect ACE2 binding (Supplement Figure 1 ). Three linear epitopes of the RBD were uniquely found in vaccine recipients' sera, two (peptide No. 6 and No.19,20) of them shared the epitope regions of the RBD with neutralizing monoclonal antibodies previously reported as class 3 and class 4. At the start of the RBD peptide, we identified an epitope region detected only in vaccine-induced sera broadly at peptide No.1-6. Especially, peptide No. 6 shall be the epitope, which class3 Nab S309 would recognize by sharing residues P337-A344 helix (31). The epitope is distinct from the receptor-binding motif and has a good accessibility both in up and down composition of the RBD (PDB, 7A49, Figure 5 ). Another identified epitope peptide No.19,20 shared epitope residues with a neutralizing monoclonal antibody CR3022, categorized as class 4, isolated from a SARS-CoV convalescent (32) . This class 4 neutralizing antibody attaches to RBD but distal from the ACE2 binding and is highly conserved between the species (18). The third epitope, located at peptide No.66-71, did not match any existing mAbs. Yi et al. detected All rights reserved. No reuse allowed without permission. (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 preprint this version posted July 7, 2021. ; the same region of the peptide (V524-F541) reactive from COVID-19 convalescent serum, although it non-specifically interacted with control sera (11). Single amino acid mutations in the RBD are reported in the variants of concerns: B. 1.1.7, B.1.351 and P.1 (PANGO lineage (33)). The mutation loci E484K and N501Y, which correspond to peptides No.52-61 in Figure 3 , did not show any significant reactivity. The peptides No.29-33 containing K417, considered as an escape mutation, was reactive to the class 1 NAb in both patients and vaccine recipients' sera as mentioned above. Additional 15-mer peptides with substituting amino acid as mentioned in the method section were incubated with both vaccine recipients' and patients' sera. Interestingly, vaccine-induced sera showed consistent signal to the mutation peptides, whereas patients' sera had almost no reaction ( Figure 6 ). This study revealed the linear epitope profile targeting RBD elicited by mRNA vaccination and natural infection of SARS-CoV-2. Our principal finding was that the variation of linear epitopes was broader in vaccine-elicited antibodies than infection-elicited antibodies, which may contribute to the potent neutralization against the variants of concerns. (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 preprint this version posted July 7, 2021. ; (14, 34) . In our study, the linear epitopes of the RBD were outlined using both infection-induced sera and the vaccine-induced sera. Both groups recognized epitopes previously described as NAbs: peptide No. (15, 20, 35, 36) . The linear epitopes, peptide No.6 and No.19-20, found only in vaccine-elicited sera revealed corresponding epitopes of the human NAbs isolated from SAR-CoV convalescent (S309 and CR3022, respectively). These cross-neutralizing antibodies readily indicate that these linear epitopes were highly conserved among the species considering the RBD is one of the most variable regions. In this context, mRNA vaccine-induced immunity, which has found to harbor broader variety in epitope profiles including class 3 and class 4 NAbs epitopes, shall be less vulnerable to the virus escaping than with infection-induced immunity (37,38). These regions are reported more conserved and resistant to mutation escape (15, 20) . Vaccine recipients' broader epitope profile across the RBD may give immunological flexibility against this evolving virus. Our mutation peptide panels showed a rather optimistic view for the efficacy of vaccine-induced immunity to capture the SARS-CoV-2 variants ( Figure 6 ). However, the linear epitope profiles in the mutation loci were not dominant in either vaccine sera or patients' sera ( Figure 2b) . Therefore, we conclude that the proportion of linear epitopes contributing to neutralization in these regions with All rights reserved. No reuse allowed without permission. (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 preprint this version posted July 7, 2021. ; mutations is limited (10, 11, 14, 19) , contrary to the conformational epitopes found abundantly in NAbs around the RBM. The specificity of the linear epitope profile was higher in each patient than in the vaccine recipients, especially within neutralizing epitopes (Figure 4 , Individual epitope distribution can be found in Supplement Figure 2 ). This phenomenon supports the idea that the infection-provoked repertoire maturation starts within as early as 10-20 days after onset, in concordance with proceeding reports noting immune repertoire change in COVID-19 patients even as early as 4-7 days after onset (39, 40) . This may indicate faster maturation of antibodies induced by natural infection when compared to vaccine-induced antibodies. Nevertheless, in this study, we captured the peak period of immune reaction in the host for both groups. Therefore, longitudinal evaluation of the epitope profile and serological markers is needed to assess the further host immune evolution of both infection and vaccine-induced epitopes. Moreover, these potentially neutralizing antibodies predominantly expressed in patients' sera might be attributed to the higher neutralizing titers observed in this study compared to vaccine recipients' sera which have broader variety in eliciting epitopes within RBD. Therefore, the discrepancy observed in serological markers, neutralizing antibody titers and anti-RBD IgG titers, between vaccine recipients and patients could be explained by the efficient epitope selection and maturation of the infection-induced humoral immunity. As for the potential use of anti-RBD antibody titers as a surrogate maker of the vaccine efficacy, the necessary anti-RBD antibody titers for vaccine-induced sera shall be higher than infection-induced sera to achieve the similar magnitude of neutralization power due to the apparent elevation of relative anti-RBD antibody titers to neutralizing titers ( Figure 1c ). To conclude the study, we evaluated the similarity and difference in humoral immunity elicited by both vaccine and natural infection of SARS-CoV-2. High resolution linear epitope profiles revealed All rights reserved. No reuse allowed without permission. (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 preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.03.21259953 doi: medRxiv preprint the unique distribution of polyclonal antibodies across the RBD in vaccine recipients' sera, which possibly accounted for the discrepancy observed in serological markers. Based on the multiple neutralizing epitopes observed in vaccine linear epitope profile, mRNA vaccine-elicited humoral immunity may be more powerful in escaping the rapidly evolving pathogen than infection-acquired immunity. There are several limitations in our study. The severity of the COVID-19 patients evaluated in this study was high (seven out of ten were critical) with comorbidities, whereas the vaccine recipients were relatively healthy without major comorbidities. The age was distributed in both groups. This analysis was focused exclusively on the linear epitope profile targeting RBD. Experimental observations on compositional epitopes nor epitopes outside the RBD region was not made in this study. Nonetheless, our results reporting the mRNA vaccine's broader RBD epitope variety are in concordance with preceding reports. This result was consistent with the recently reported immune profile of Moderna vaccines (37). All authors have read and approved the manuscript. The authors declare no competing interests to disclose. The data that supports the findings of this study are available in the supplementary material of this article. All rights reserved. No reuse allowed without permission. (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 preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.03.21259953 doi: medRxiv preprint (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 preprint this version posted July 7, 2021. ; grey lines denote peptide signals of individuals. Bold black lines denote the mean values of the peptide signals of the vaccine recipients' sera (N=5). (c) Red arrows denote epitopes recognized in the sera of both groups. Black arrows denote epitopes identified only in the vaccine recipients' sera. Designated peptide numbers are shown above the arrows. (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 preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.03.21259953 doi: medRxiv preprint calculated. Vaccine-induced sera had more variety in recognizing epitopes than infection-induced sera. Supplement Table 1 . Detailed information of the subjects included in this study. Table 2 . Sequence of the peptides on microarrays used in this study. (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 preprint this version posted July 7, 2021. 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