key: cord-1034716-zdrd3u1f authors: Jia, L.; Weng, S.; Wu, J.; Tian, X.; Zhang, Y.; Wang, X.; Wang, J.; Yan, D.; Wang, W.; Zhu, Z.; Qiu, C.; Zhang, W.; Xu, Y.; Wan, Y. title: Pre-existing antibodies targeting a dominant linear antibody epitope on SARS-CoV-2 S2 cross-reacted with commensal gut bacteria and shaped immune responses elicited by a candidate vaccine date: 2021-07-15 journal: nan DOI: 10.1101/2021.07.13.21260404 sha: 9b4c8df747ea258c48ff5088f4af6d28ab132088 doc_id: 1034716 cord_uid: zdrd3u1f Pre-existing SARS-CoV-2 cross-reactive antibodies have been detected in both unexposed human and animals. However, the origins of these cross-reactive antibodies and their potential impacts on vaccine efficacy have not been completely clarified. In this study, we demonstrated that the S2 subunit was the predominant target of the pre-existing SARS-CoV-2 spike protein cross-reactive antibodies in both healthy human and naive SPF mice. Through linear epitope mapping, we identified a dominant antibody epitope on the connector domain of S2 (aa1145-aa1162), which could be recognized by antibodies pre-existed in unexposed human and mice. Six monoclonal antibodies against this linear epitope were isolated from naive SPF mice and were proved to cross-react with commensal gut bacteria collected from both human and mouse. Via immunizing mice with a candidate DNA vaccine encoding the full length of SARS-CoV-2 spike protein, we further demonstrated that high levels of pre-existing S2 cross-reactive antibodies did not impair the immunogenicity of the DNA vaccine. On the contrary, mice with high levels of pre-existing antibodies mounted stronger S2 specific binding antibody responses compared to mice with low levels of pre-existing antibodies. In addition, S1 specific T cell and binding antibody responses also tended to be enhanced in mice with high levels of pre-existing antibodies. Introduction 5 these two cohorts was described in Table 1 . Detection of SARS-CoV-2 S1 and S2 specific binding antibodies 137 In-house enzyme-linked immunosorbent assays (ELISA) were developed to 138 measure SARS-CoV-2 S1 and S2 specific binding antibodies. High-binding The data were analyzed using the FlowJo software (BD Biosciences, USA). 206 About 2g of each fecal sample was suspended with 15ml sterile 1×PBS and 207 vortexed thoroughly to obtain uniform mixtures. After centrifugation at 200×g 208 for 5 min, the supernatants were collected, and the sediments were discarded. 209 This process was repeated twice. Next, all the supernatant samples were 210 centrifuged twice at 9000×g for 5 min and the supernatants were discarded. 211 The precipitated bacteria pellets were resuspended in 500µl of 1 × PBS 212 (containing 1mM PMSF) and disrupted with an ultrasonic cell crusher (the Tris-HCl, pH 8.5, proteins were reduced by 10 mM DTT at 37 °C for 30 min and 8 filtrated out and the filter was washed twice with 15% ACN, and all filtrates 240 were pooled and vacuum-dried to reach a final concentration to 1 mg/ml. 241 LC-MS analysis was performed using a nanoflow EASYnLC 1200 system 242 (Thermo Fisher Scientific, Odense, Denmark) coupled to an Orbitrap Fusion 243 Lumos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). A 244 one-column system was adopted for all analyses. Samples were analyzed on 245 a home-made C18 analytical column (75 µm i.d. × 25 cm, ReproSil-Pur 120 246 C18-AQ, 1.9 µm (Dr. Maisch GmbH, Germany)). The mobile phases consisted 247 of Solution A (0.1% formic acid) and Solution B (0.1% formic acid in 80% ACN). The derivatized peptides were eluted using the following gradients: 2-5% B in pre-existing S2 binding antibodies were measured using the previously 269 described in-house ELISA method. According to their pre-existing S2 binding 270 antibody levels (at 1:100 dilution of serum), the mice were divided into three OD450nm-630nm≤0.750, n=6) and high (OD450nm-630nm＞0.750, n=6). All mice 273 were immunized intramuscularly with the candidate S protein DNA vaccine 274 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. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (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 15, 2021. To track the origins of the pre-existing cross-reactive antibodies to 353 SARS-CoV-2 spike protein, in this study, we first measured the levels of naïve SPF mice using the competitive ELISA assay (Fig.3 ). The P144 specific antibody responses might be engendered by 378 exposures to certain commensal gut bacteria 379 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 15, 2021. ; To explore the potential origins of the pre-existed P144 specific antibodies, we To probe the potential antigens that might induce the P144 binding antibodies, 410 we isolated 6 mAbs from two naïve SPF mice (one C57BL/6J mouse and one 411 BALB/c mouse) with high levels of pre-existing S2 specific antibody responses. Five of these mAbs recognized P144 solely, while one mAb (clone M3) bound 413 with P144 and P103 simultaneously (Supplementary Figure 3) . Results of 414 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. Figure 3A ). The neutralizing potentials of these isolated monoclonal antibodies 417 were evaluated using a pseudo virus-based neutralization assay. Our results 418 showed that these monoclonal antibodies exhibited limited neutralizing activity (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 15, 2021. ; https://doi.org/10.1101/2021.07.13.21260404 doi: medRxiv preprint 14 180KD, 100KD, 55KD-70KD and 40KD-55KD (indicated by arrows in Figure 450 5A, panel F5) were selected. For human fecal bacteria samples, protein bands 451 with molecular weights around 50KD-70KD, 70KD and 70KD-100KD 452 (indicated by arrows in Figure 5B , panel F5) were selected. The lists of 453 proteins identified in mouse and human samples were shown in Table 2 Figure 6C ). More specifically, the average level of P144 specific 477 antibody responses was also stronger in mice with high levels of pre-existing 478 S2 binding antibodies than mice with low levels of pre-existing S2 binding 479 antibodies ( Figure 6D and 6E). By comparison, both the S1 binding antibody 480 and the neutralizing antibody titers did not significantly differ among all groups, 481 despite that mice with moderate or high levels of pre-existing antibodies 482 tended to mount higher average titer of S1 binding antibodies ( Figure 6F and 483 6G). Mice without vaccination showed neither obvious S1 binding antibody 484 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 15, 2021. ; https://doi.org/10.1101/2021.07.13.21260404 doi: medRxiv preprint 15 response nor neutralizing activity (Data not shown). We further investigated 485 the influence of pre-existing antibodies on humoral immune responses in 486 mouse respiratory tract after vaccination. And our data showed that the levels 487 of S1 specific IgG in BALF were similar among the three groups after DNA 488 vaccination ( Figure 7A) , while the average level of S2 specific IgG in BALF 489 from mice with high pre-existing S2 binding antibodies was significantly higher 490 than those from mice with low pre-existing antibodies ( Figure 7B ). S protein 491 specific IgA response did not increase significantly after vaccination as The origins of pre-existing cross-reactive immunities against SARS-CoV-2 515 have been investigated vigorously since the outbreak of the pandemic (44). Accumulating data suggest that cross-reactive T cells (33, (45) (46) (47) (48) in 517 SARS-CoV-2 unexposed human might be induced by previous infections of 518 other hCoVs. While the origins of pre-existing cross-reactive antibodies could 519 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 15, 2021. ; https://doi.org/10.1101/2021.07.13.21260404 doi: medRxiv preprint 16 not be completely explained by previous infections of other coronaviruses, as 520 recent studies revealed that the magnitude of antibody responses to 521 SARS-CoV-2 S protein in the sera of patients with COVID-19 was not related 522 to HCoVs' S titers (49) and immunization with coronaviruses OC43 did not 523 induce significant SARS-CoV-2 S protein cross-reactive antibodies in mice. Moreover, it has also been observed that SARS-CoV-2 S protein specific 525 binding antibody responses were weak in SARS-CoV-2 unexposed individuals 526 with obvious binding antibody responses against S proteins of common cold 527 hCoVs (50, 51). To track the potential origins of the pre-existed cross-reactive antibodies 529 targeting SARS-CoV-2 spike protein, in this study, we first screened the 530 cross-reactive antibody responses in SARS-CoV-2 unexposed human plasma 531 collected in 2020 and 2016, respectively. In both cohorts, we found that the 532 magnitudes of S2 binding antibodies were significantly higher than those of S1 533 binding antibodies. This finding is consistent with previous studies showing 534 that pre-existing S2 cross-reactive antibody responses are stronger than S1 535 cross-reactive antibody responses in SARS-CoV-2 unexposed individuals (49, 536 52, 53). Since S2 cross-reactive antibody responses have also been observed 537 in unexposed animals (49), we continued to screen the cross-reactive antibody 538 responses in two strains of naïve SPF mice. Our data showed that the OD 539 values of S2 cross-reactive antibodies were significantly higher than those of 540 S1 cross-reactive antibodies in naïve BALB/c and C57BL/6 mice. Detections of 541 mouse sera collected from another two independent SPF animal facilities 542 confirmed this finding (Data not shown). We also tried to detect the 543 SARS-CoV-2 S protein specific T cells responses in mice with high 544 pre-existing S2 cross-reactive antibodies using the IFN-γ ELISPOT assay, 545 which showed that there was no pre-existing cross-reactive T cell response. (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 15, 2021. ; https://doi.org/10.1101/2021.07.13.21260404 doi: medRxiv preprint 2016. More interestingly, we found that the pre-existing S2 cross-reactive 555 antibodies in mice were predominantly against the single epitope. Of note, 556 although the aa sequence of P144 is highly conserved between SARS-CoV-2 557 and SARS-CoV, its similarity with four seasonal hCoVs is relatively low. A 558 recent study showed that this epitope was more frequently recognized than its 559 homologous peptides from common cold hCoVs by antibodies in plasma of 560 COVID-19 negative individuals (23) . The above evidence implied again that 561 the pre-existing S2 specific antibodies might not be necessarily elicited by 562 previous common cold coronavirus infections. As the pre-existing S2 binding antibodies in mice were predominantly against 564 P144, it provided us a good chance to unveil their origins. To do so, we first 565 labeled mouse B cells with purified S2 protein and found that the frequency of 566 S2 specific B cells was significantly higher in mesenteric LN than in spleen, 567 suggesting that the gastrointestinal tract might be the primary site where the 568 cross-reactive B cells were activated. Exposure to a certain gut microbial 569 antigen, which can promote B cell diversification and stimulate antibody 570 production in both T-dependent and -independent ways (54), might account for 571 the presence of the cross-reactive antibodies. To prove this hypothesis, we 572 next compared the levels of pre-existing S2 cross-reactive antibodies between 573 mice housed in a sterile isolation pack and mice maintained in SPF condition. 574 Our results showed that the levels of pre-existing S2 cross-reactive antibodies 575 were significantly higher in SPF mice. Through metagenomic sequencing, we 576 further demonstrated that the abundance of bacteroidaceae, prevotellaceace 577 and parabacteroides were also significantly higher in the commensal gut 578 bacteria of SPF mice. Therefore, we speculated that the S2 cross-reactive 579 antibodies might be induced by commensal gut bacteria. To prove the above speculation directly, we isolated six P144 specific 581 monoclonal antibodies from a naïve BALB/c mouse and a naïve C57BL/6 582 mouse, respectively. All the six mAbs were confirmed to be able to bind with 583 P144 and showed weak neutralizing compacities against five SARS-CoV-2 584 variants. By leveraging these mAbs, we detected the potential cross-reactive 585 antigens in mouse and human fecal microbiota through WB assays. Compared 586 with a control mouse IgG, specific bands were observed for each mAb, which 587 proved antibody cross-reactivities between SARS-CoV-2 and commensal gut 588 bacteria. The strongly recognized protein bands were further analyzed by 589 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 15, 2021. ; https://doi.org/10.1101/2021.07.13.21260404 doi: medRxiv preprint 18 LC-MS. Our data showed that antigens derived from bacteroides and 590 parabacteroides were frequently identified in fecal bacteria samples of both 591 human and mouse, which was in consistence with our metagenomic 592 sequencing data showing that the abundance of bacteroides and 593 parabacteroides was significantly higher in the commensal gut bacteria of mice 594 with high pre-existing S2 binding antibody levels. More intriguingly, five 595 potential cross-reactive microbial antigens were identified in mouse and 596 human samples simultaneously, which implied that the S2 cross-reactive 597 antibodies might naturally occur in different species of mammals. We analyzed 598 the sequence similarities between P144 and the proteins identified by LC-MS. 599 Our results showed that most identified proteins shared 40%-70% identities for 600 more than 8 residues with P144 (Data not shown). The cross-reactive epitopes 601 on the identified proteins could not specified based on current data. Besides, 602 the neutralizing mechanism(s) of P144 specific antibodies and their potential 603 influences on gut microbiota were not clarified in this study. We plan to look 604 into these issues in future. In parallel with tracking the initial antigens that induced the S2 cross-reactive 606 antibodies, we investigated the impact of pre-existing antibodies on the 607 immunogenicity of a candidate DNA vaccine as well. According to previous 608 reports, pre-existing cross-reactive antibodies may influence the effects of 609 different vaccines differentially (6, 55). In this study, our data showed that the antibody responses was primarily epitope specific. In addition to antibody 618 response, we also found that the high levels of pre-existed S2 binding 619 antibodies tended to induce higher S1 specific T cell responses while lower S2 620 specific T cell responses, which indicated that the pre-existing cross-reactive 621 antibodies might change the balance between humoral versus cellular immune 622 responses. Collectively, the above results manifested that the pre-existing 623 cross-reactive antibodies against SARS-CoV-2 S2 did not impair the 624 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. A deep understanding of pre-existing cross-reactive antibodies against 629 SARS-CoV-2 will enable better therapeutic, diagnostic and vaccine strategies. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The authors declare that they have no relevant conflicts of interest. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 788 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. 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 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 pre-existing cross-reactive antibodies against S1 and S2 were measured The pre-existing S1 and S2 cross-reactive antibody levels of 6 mice selected (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 15, 2021. Cross-reactivities between P144 specific mAbs and gut microbial antigens 897 were detected using WB assays. A purified mouse IgG was used as the control. (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 15, 2021. and IgA (C and D) against S1 or S2 were detected using in-house ELISA (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 15, 2021. ; https://doi.org/10.1101/2021.07.13.21260404 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 15, 2021. (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 15, 2021. (D614G, B.1.617, B.1.1.7, B.1.351 and P.1) (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 15, 2021. ; https://doi.org/10.1101/2021.07.13.21260404 doi: medRxiv preprint 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 15, 2021. ; Pre-existing cross-reactive antibodies against SARS-CoV-2 S protein observed in both healthy human and naive SPF mice predominantly targeted S2 subunit 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 15, 2021. ; https://doi.org/10.1101/2021.07.13.21260404 doi: medRxiv preprint Figure 2 The pre-existing S protein cross-reactive antibodies in naïve SPF mice recognized S2 exclusively and a dominant linear antibody epitope was identified on the connector domain (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 15, 2021. Cross-reactive antibodies recognizing epitope P144 widely existed in both healthy human and naïve SPF mice 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 15, 2021. ; https://doi.org/10.1101/2021.07.13.21260404 doi: medRxiv preprint Figure 4 The generation of the pre-existing S2 cross-reactive antibodies might be associated with commensal gut microbiota <0.0001 Mice in SPF 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 15, 2021. ; https://doi.org/10.1101/2021.07.13.21260404 doi: medRxiv preprint Figure 5 P144 specific monoclonal antibodies isolated from naïve SPF mice cross-reacted with commensal gut bacteria from both human and mouse 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 15, 2021. ; https://doi.org/10.1101/2021.07.13.21260404 doi: medRxiv preprint Figure 6 The impact of pre-existing antibody on the humoral immune responses elicited by a DNA vaccine encoding SARS-CoV-2 S protein Week -1 Week 0 Week 2 Week 4 Week 6 (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 15, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Pre-existing heterologous T-cell immunity and neoantigen 702 immunogenicity T-cell cross-reactivity may explain the large variation in how cancer patients respond to 704 checkpoint inhibitors Negative selection, epitope mimicry and autoimmunity. Current opinion in immunology Seasonal 708 human coronavirus antibodies are boosted upon SARS-CoV-2 infection but not associated with 709 protection Humoral Responses and Serological 711 Assays in SARS-CoV-2 Infections Cross-reactive antibody 713 responses against SARS-CoV-2 and seasonal common cold coronaviruses Viral epitope profiling of 715 COVID-19 patients reveals cross-reactivity and correlates of severity SARS-CoV-2 antibodies and protective role of pre-existing antibodies to seasonal human 719 coronaviruses on Epitope-resolved 721 profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with endemic human 722 coronaviruses Preexisting and de novo 724 humoral immunity to SARS-CoV-2 in humans Lack of antibody-mediated cross-protection 726 between SARS-CoV-2 and SARS-CoV infections Cross-reactive Antibody Response 728 between SARS-CoV-2 and SARS-CoV Infections Cross-reactive neutralization of SARS-CoV-2 730 by serum antibodies from recovered SARS patients and immunized animals Cross-reactive memory T cells and herd immunity to 733 SARS-CoV-2 Is there a link between 735 pre-existing antibodies acquired due to childhood vaccinations or past infections and COVID-19? A 736 case control study A majority of 738 uninfected adults show pre-existing antibody reactivity against SARS-CoV-2 Pre-existing immunity to SARS-CoV-2: the knowns and unknowns Interleukin-21 enhances the antibody avidity elicited by DNA prime and 742 MVA boost vaccine Antigen engineering in DNA immunization 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 15, 2021. ; https://doi.org/10.1101/2021.07.13.21260404 doi: medRxiv preprint Figure 9 The impact of pre-existing antibody on the cellular immune responses induced by a DNA vaccine encoding SARS-CoV-2 S protein (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 15, 2021. ; https://doi.org/10.1101/2021.07.13.21260404 doi: medRxiv preprint