key: cord-0818708-sgsqscpy authors: Zhang, Li; Cao, Lei; Gao, Xing-Su; Zheng, Bin-Yang; Deng, Yong-Qiang; Li, Jing-Xin; Feng, Rui; Bian, Qian; Guo, Xi-Ling; Wang, Nan; Qiu, Hong-Ying; Wang, Lei; Cui, Zhen; Ye, Qing; Chen, Geng; Lu, Kui-Kui; Chen, Yin; Chen, Yu-Tao; Pan, Hong-Xing; Yu, Jiaping; Yao, Wenrong; Zhu, Bao-Li; Chen, Jianping; Liu, Yong; Qin, Cheng-Feng; Wang, Xiangxi; Zhu, Feng-Cai title: A proof of concept for neutralizing antibody-guided vaccine design against SARS-CoV-2 date: 2021-03-27 journal: Natl Sci Rev DOI: 10.1093/nsr/nwab053 sha: 29c1be3e0fed499f1ae0b24c41ee43dcd99fa7a9 doc_id: 818708 cord_uid: sgsqscpy Mutations and transient conformational movements of receptor binding domain (RBD) that make neutralizing epitopes momentarily unavailable, present immune escape routes to SARS-CoV-2. To mitigate viral escape, we developed a cocktail of neutralizing antibodies (NAbs) targeting epitopes located on different domains of spike (S) protein. Screening of a library of monoclonal antibodies generated from peripheral blood mononuclear cells of COVID-19 convalescent patients yielded potent NAbs, targeting N-terminal domain (NTD) and RBD domain of S, effective at nM concentrations. Remarkably, combination of RBD-targeting NAbs and NTD-binding NAb, FC05, enhanced the neutralization potency in cell-based assays and animal model. Results of competitive SPR assays and cryo-EM structures of Fabs bound to S unveil determinants of immunogenicity. Combinations of immunogens, identified in NTD and RBD of S, when immunized in rabbits and macaques elicited potent protective immune responses against SARS-CoV-2. More importantly, two immunizations of this combination of NTD and RBD immunogens provided complete protection in macaques against SARS-CoV-2 challenge, without observable antibody-dependent enhancement of infection. These results provide a proof-of-concept for neutralization-based immunogen design targeting SARS-CoV-2 NTD and RBD. The coronavirus spike (S) protein is a multifunctional molecular machine that facilitates viral entry into target cells by engaging with cellular receptors and determines to a great extent cell tropism and host range (9) . Coronaviruses S proteins are processed into S1 and S2 subunits by host proteases, among which S1 is responsible for receptor binding, while the S2 subunit mediates membrane fusion (10) . The S1 subunit typically possesses two types of domains capable of binding to host cell receptors. For instance, some betacoronaviruses use the N-terminal domain (NTD) of their S1 subunit to bind sialic acids located on the glycosylated cell-surface receptor (11) . Similarly, the betacoronavirus murine hepatitis virus uses its NTD for binding the protein receptor CEACAM1 (12) . In contrast to this, SARS-CoV and SARS-CoV-2 use the C-terminal domain (named also as receptor binding domain, RBD) of their S1 subunit for binding to their protein receptor hACE2 (13) . Recently, the NTD of SARS-CoV-2 has also been shown to be involved in ACE2-dependent entry into host cells by targeting the high-density lipoprotein (HDL) scavenger receptor B type 1 (SR-B1) (14) . In line with this, a number of antibodies targeting the NTD exhibit potent neutralizing activities against SARS-CoV-2 and MERS-CoV infections (15, 16 To address these issues related to SARS-CoV-2 neutralization, administration of a cocktail of NAbs targeting both the RBD and non-RBD regions, rather than using a single NAb, could potentially increase the potency of protection via binding of NAbs to multiple domains of S, thereby preventing escape of the viral particles from the NAbs. In context with this, the immunogenic characteristics of the antigens targeted by potential NAb cocktails and their structural features can inform strategies for the development of vaccines and therapeutics against COVID-19. One of the prospective goals of this study was to generate a large and diverse collection of human NAbs targeting multiple domains of S so as to allow for the formulation of a cocktail of highly potent antibodies that could simultaneously bind to the various regions of S. For this, we first Table S1 ). Among those selected, 3 (named FC01, FC08 and FC11) target the RBD, 3 (named FC05, FC06 and FC07) recognize the NTD and 4 (named FC118, FC120, FC122 and FC124) are S2-specific mAbs ( Fig. 1C and 1D ). To find out whether these antibodies crossreact with SARS-CoV and MERS-CoV, we firstly assessed the binding capacity of these mAbs to RBDs, NTDs and S2s from SARS-CoV-2, SARS-CoV and MERS-CoV by ELISA (Fig. 1C ). that, in prophylactic settings, a treatment with either individual NAbs or the cocktail led to a 3-4 log reduction of viral loads in both lungs and tracheas at day 3 when compared to the PBS-treated group. A modest synergistic protective efficacy was observed for the cocktail ( Fig. 2B and 2C ). The estimated viral loads from the lungs of groups belonging to therapeutic settings showed similar levels as those observed for the groups of the prophylactic settings, however, the viral loads from the tracheas differed for both the groups. A ~10-fold higher titer was observed for the groups in therapeutic settings ( Fig. 2B and 2C) . Notably, all mice from FC05/FC08/FC05& FC08treated group no longer had infectious virus in the lungs at day 3 as measured by a plaque assay of lung tissue homogenates (Fig. 2D ). More importantly, histopathological examination revealed a typical interstitial pneumonia, including widening of alveolar septum, vasodilation, hyperemia and edema, accompanied by a large number of monocytes and lymphocytes and a small number of lobulated granulocytes and other inflammatory cell infiltration in mice belonging to the PBS control group (Fig. 2E ). In contrast, no obvious lesions of alveolar epithelial cells or focal hemorrhage were observed in the lung sections from either of the antibody-treated groups at day 3 ( Fig. 2E ). Although numerous antibody cocktails with various synergistic neutralization potencies exhibited at cellular levels have been reported (22, 24, 25) , none of these cocktails have been reported to exhibit substantial synergistic protection in animals, indicative of a challenge to observe genuine synergy. This is more likely due to the lack of ideal animal models. To gain a better understanding of the synergy observed during the neutralization of SARS-CoV-2 by a cocktail of NAbs, we performed competitive SPR assays. The results of the assays were expected to reveal whether the NAbs recognize the same or different patches of the epitopes. As expected, the binding of the NTD-specific FC05 does not affect the attachment of any of the three RBD-specific NAbs to the SARS-CoV-2 S trimer, explaining the cooperativity between the antibodies of the cocktail as they bind simultaneously to distinct domains (Fig. 3A) . Conversely, the 3 RBD-targeting NAbs competed with each other for binding to the SARS-CoV-2 S trimer ( Fig. 3B) , which may imply that these RBD-targeting antibodies recognize similar epitopes or their epitopes overlap partially. Lastly, none of the above 4 NAbs were capable of blocking the interactions between soluble hACE2 and the SARS-CoV-2 S trimer, suggesting a distinct neutralizing mechanism (Fig. 3C) . To decipher the nature of the epitopes and the mechanism of neutralization at the atomic level, we determined cryo-EM structures of a prefusion stabilized SARS-CoV-2 S ectodomain trimer in complex with the Fab fragments of the NAbs. Surprisingly, the structural studies revealed that all the three RBD-targeting NAbs are capable of breaking the S trimer into monomers or irregular pieces. A similar perturbation of the S trimer was observed previously in the studies conducted on the CR3022 antibody (26) . The NTD-binding FC05 however did neither exhibit any such ability of disrupting the S trimer nor did it affect the viral stability (Fig. S5) . To further map epitope clusters of these RBD-binding NAbs, we used a representative antibody, FC08, for performing a competitive SPR-based epitope binding assay. Recently, we mapped the antigenic sites of 3 well characterized RBD-targeting NAbs, H014, HB27 and P17 (22, 27, 28) , which bind epitopes located on one side of the RBD, the apical head of the RBD and the receptor binding motif (RBM), respectively. Using these previously characterized antibodies along with FC08 in the assays revealed that only P17 competes with FC08 for binding to the SARS-CoV-2 S trimer (Fig. S6 ). Taking into consideration of the observation that FC08 does not seem to target the RBM due to the simultaneous bindings of FC08 and the hACE2 to SARS-CoV-2 S trimer (Fig. S6) , FC08 probably targets a cryptic epitope that partially overlaps with P17 and probably lies towards the interior of the S trimer. To gain an understanding of the molecular basis of the destruction of S trimer by FC08, structural investigations of the SARS-CoV-2 RBD in complex with FC08 were carried out. One noncompeting Fab and hACE2 that recognize the RBD beyond FC08 binding sites were used to increase the molecular weight of this complex for pursuing a high resolution (up to 3.6 Å) structure by cryo-EM reconstruction (Fig. S7 -S8 and table S2). As revealed by competitive SPR analysis, FC08 recognizes a cryptic epitope on the other side of the RBD, adjacent to hACE2binding site (Fig. 3D ). FC08 epitope is inaccessible in the prefusion S trimer (neither the open nor the closed RBDs), suggesting that FC08 binding would facilitate conversion to the disassembly states (Fig. S9) , which explains its neutralization of SARS-CoV-2 via destruction of the prefusion S trimer. FC08 uses both heavy and light chains, and five of six CDR loops for interaction with the RBD (Fig. 3E) . The buried surface area on the epitope is 1,057 Å 2 and SARS-CoV-2 recognition by FC08 is largely driven by hydrophobic interactions (Fig. 3E) . The SARS-CoV, explaining its specificity for SARS-CoV-2 for binding and neutralization (Fig. S10) . Notably, the residue 501 identified as a key mutation site in recently isolated SARS-CoV-2 variants (such as N501Y) and shown to substantially enhance interaction with hACE2, is not involved in the interactions with FC08. These results indicate that FC08 has the potential to neutralize the recent more contagious SARS-CoV-2 variants. Cryo-EM characterization of the S-FC05 complex showed full occupancy where one Fab is bound to each NTD of the homotrimeric S (Fig. 3F) . 3D classification revealed that the S trimer adopts a 3-fold symmetrical structure with all three RBDs closed albeit without imposing any symmetry. By applying a C3 symmetry, we reconstructed the cryo-EM structure of the complex at an overall resolution of 3.4 Å. However, the electron potential map for binding interface between NTD and FC05 is relatively weak due to conformational heterogeneity. To solve this problem, focusing classification and refinement by using a "block-based" reconstruction approach (29) were used to improve the local resolution to 3.9 Å (Fig. 3F, fig. S11 -S13 and table S2) of the regions of S1 as well as dispersing the S trimer (Fig. 3I) . These structural studies provide clues for unraveling the molecular basis of the synergic neutralization of SARS-CoV-2 by the cocktail of antibodies that are capable of simultaneously targeting both NTD and RBD domains. In addition, this combination of FC05 and FC08 could be complemented in synergy with an antibody that blocks hACE2 attachment, such as HB27 further reinforcing the cocktail as a SARS-CoV-2 therapeutic. Most of the potent neutralizing antibodies reported till date target the RBD of CoV (20, 27, 30) . We next evaluated the immunogenicity and protective efficacy of ReCovR+N in rhesus macaques. Macaques were immunized two times via the intramuscular route with ReCovR+N (20 μg RBD + 20 μg NTD) mixed by AS01B adjuvant or placebo (PBS + AS01B) at day 0 and 14 (n=4) (Fig. 5A ). SARS-CoV-2 specific NAbs emerged at week 2 and rose up to ~2,000 at week 5 (before virus challenge) in vaccinated group, whose titer was ~20-fold higher than that of serum from PiCoVacc immunized group (Fig. 5B ). Subsequently 5C-5E). In contrast, viral loads decreased significantly during early infection and were not detected any more by 4-7 dpi in all vaccinated macaques (Fig. 5C-5E ). In addition, no antibodydependent enhancement of infection (ADE) was observed for any vaccinated macaques. Collectively, ReCovR+N can confer effective protection in non-human primates against SARS-CoV-2 strains by eliciting potent humoral responses. COVID-19 vaccine development is moving at an unprecedented speed, with more than 250 candidates under development worldwide. S protein constitutes the main immunogen of a COVID-19 vaccine. While, S protein is relatively metastable when produced as a recombinant protein, it is prone to conversion from the prefusion to the postfusion state, losing its immunogenicity (36) . The S protein can be cleaved into S1 and S2 subunits, the former consists of the NTD, RBD and two subdomains (SD1 and SD2) (28) . Interestingly, most potent NAbs target either the RBD or the NTD, rather than other domains, indicating that the NTD and RBD might be bona fide immunogens for eliciting neutralizing antibodies. Selection of the target immunogen is critical for the success of a vaccine, since eliciting a large amount of antibody that binds, but does not neutralize, may lead to low protection or even immunopathology (37) . The ideal immunogen should elicit high-quality, functionally neutralizing antibodies while avoiding induction of nonneutralizing antibodies. 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Chunyun Sun, and Changfa Fan for providing critical reagents. We wish to thank Drs. Author contribution: F-C.Z., X.W. and C-F.Q conceived, designed and supervised the study and