key: cord-0822307-wp5d8l5f authors: Liang, Qingtai; Wang, Yifeng; Zhang, Shuyuan; Sun, Jing; Sun, Wenbo; Li, Jizhou; Liu, Yaping; Li, Mingxi; Cheng, Lin; Jiang, Yuhang; Wang, Ruoke; Zhang, Rui; Yang, Zihan; Ren, Yifei; Chen, Peng; Gao, Peng; Yan, Huayuan; Zhang, Zheng; Zhang, Qi; Shi, Xuanling; Wang, Jianbin; Liu, Wanli; Wang, Xinquan; Ying, Bo; Zhao, Jincun; Qi, Hai; Zhang, Linqi title: RBD trimer mRNA vaccine elicits broad and protective immune responses against SARS-CoV-2 variants date: 2022-03-11 journal: iScience DOI: 10.1016/j.isci.2022.104043 sha: 00c4ba41816302f001398e6033c068d82c36b782 doc_id: 822307 cord_uid: wp5d8l5f With the rapid emergence and spread of SARS-CoV-2 variants, development of vaccines with broad and potent protectivity has become a global priority. Here, we designed a lipid nanoparticle-encapsulated, nucleoside-unmodified mRNA (mRNA-LNP) vaccine encoding the trimerized receptor binding domain (RBD trimer) and showed its robust capability in inducing broad and protective immune responses against wildtype and major variants of concern (VOCs) in the mouse model of SARS-CoV-2 infection. The protectivity was correlated with RBD-specific B cell responses especially the long-lived plasma B cells in bone marrow, strong ability in triggering BCR clustering and downstream signaling. Monoclonal antibodies isolated from vaccinated animals demonstrated broad and potent neutralizing activity against VOCs tested. Structure analysis of one representative antibody identified a novel epitope with a high degree of conservation among different variants. Collectively, these results demonstrate that the RBD trimer mRNA vaccine serves as a promising vaccine candidate against SARS-CoV-2 variants and beyond. The current vaccines that were developed by targeting the prototype SARS-CoV-2 Figure 3F) . Importantly, at that point, the RBD trimer group attained a much higher 216 level of RBD-specific plasma cells in the bone marrow. Although the precise origin of 217 this superiority of the RBD trimer mRNA vaccine is currently unknown, the fact that 218 the difference appeared after the boost suggests memory B cells induced by the RBD 219 trimer mRNA vaccine immunization may be functionally superior. J o u r n a l P r e -p r o o f Next, we found that all three vaccines were able to induce strong S protein-specific 221 T cell responses on day 35 after the initial immunization. Interestingly, the RBD trimer 222 vaccine tended to elicit more antigen-specific polyfunctional CD4 T cells than CD8 T 223 cells expressing type 1 (Th1) immune response cytokines such as interferon gamma 224 (IFNγ), tumor necrosis factor (TNF)-α, and interleukin (IL)-2 ( Figure 3G and 3H). This 225 biased CD4 helper response may help to explain the broad and potent neutralizing 226 antibody response observed in animals that were immunized with the RBD trimer 227 mRNA vaccine (see above). In contrast, the native spike vaccine tended to trigger a 228 higher CD8 T cell response than the RBD trimer mRNA vaccine, as determined by to WT D614G, these mAbs showed either comparable activity or enhanced activity 304 against the major variants. In particular, like that observed for immune serum, many 305 mAbs demonstrated improved neutralizing activity largely due to 242-244del mutation 306 in the NTD (Fig. 5B) . Furthermore, mAb T6 was able to exert at least a 3-fold increase 307 in neutralization to 3 of the 4 variants of concern, thus suggesting that its epitope might 308 have become more exposed in some of these variants. Examination of the enhancement 309 patterns across the single and triple mutant pseudoviruses further showed that the 242- Table S1 ). In both models, the CL and CH1 of the T6 Fab were not built due to weak 343 densities. As the T6 Fab is only bound to the "up" RBD, it follows that the T6 epitope 344 must be buried when RBD is in the "down" conformation in the spike trimer. 345 We further determined the crystal structure of the T6 Fab bound to the RBD of Figure 6C and Table S2 ). The T6 Fab buries a surface area of 661.4 348 Å 2 on the RBD. Of this, 358.8 Å 2 was composed of the heavy chain and 302.6 Å 2 by 349 the light chain ( Figure 6D ). A total of 16 residues from the T6 Fab, and 11 residues from Figure 6F ). The mutated residues N417, K484, and Y501, 353 shared among several SARS-CoV-2 variants were not located within the epitope of the 354 T6 Fab, although N417 and K484 were both located in a proximate position; Y501 has 355 located some distance away ( Figure 6D ). The L452R mutation, found in several SARS- that the mechanism underlying its neutralization ability was to compete with ACE2 to 365 bind to the RBD, thereby disrupting the very first step of viral entry ( Figure 6H ). Finally, 366 analysis of T6 epitope residues in the GISAID database revealed a high degree of 367 conservation, thus providing a sequence and structural basis for its broad and potent 368 neutralizing activity against a wide range of SARS-CoV-2 variants ( Figure 6D ). The RBD trimer mRNA vaccine protects K18-hACE2 mice from death after CoV-2 infection. In terms of viral load in lungs, WT D614 infected control animals had 396 J o u r n a l P r e -p r o o f 9.6×10 6 copies/mg for sgRNA and 8.0×10 7 copies/mg for gRNA, which were about 397 two-logs higher than that in the vaccinated animals (9.6×10 4 copies/mg for sgRNA and Students' t-test (two-tailed) (*P < 0.05; **P < 0.01; ****P < 0.0001). Schematic of variant spike of SARS-CoV-2 used in this study are shown in Figure S1 . Figure S5D . "+++" >60% competition; "+" 30-60%; and "-" <30% T1 T4 T6 T9 T11 T20 T26 T34 T35 T38 T41 T42 T45 T47 T48 T50 T51 T53 T54 T55 Log μg/ml % Neutralization Figure S4 . Neutralization of SARS-CoV-2 variants by each antibody, related to Figure 5B . Pseudoviruses bearing the indicated mutations were tested against serial dilutions of each mAb. Neutralizing activity was defined as the percent reduction in luciferase activities compared to no antibody controls. Levels of resistance were calculated as the fold change in IC50 between each mutant and WT D614G, as presented in Figure 5B . Results were calculated from three independent experiments. J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f Table S1 . Cryo-EM data collection, refinement, and validation statistics, related to Figure 6A , 6B. Data collection and processing J o u r n a l P r e -p r o o f Table S2 . Crystallization data collection and refinement statistics, related to Figure 6C . New SARS-CoV-2 Variants -Clinical, Public Health, 1118 and Vaccine Implications PHENIX: a comprehensive Python-based system for 1121 macromolecular structure solution SARS-CoV-2 neutralizing antibody structures 1124 inform therapeutic strategies Resistance of SARS-CoV-2 variants to neutralization by monoclonal and 1127 serum-derived polyclonal antibodies Tackling COVID-19 with neutralizing 1132 monoclonal antibodies Viral targets for vaccines against COVID-19 Coot: model-building tools for molecular graphics Multiple SARS-CoV-2 variants escape 1139 neutralization by vaccine-induced humoral immunity Antibody neutralization of SARS-CoV-2 through ACE2 receptor mimicry SARS-CoV-2 variants, spike mutations and immune escape PyMod 2.0: improvements in protein 1146 sequence-structure analysis and homology modeling within PyMOL Human neutralizing antibodies elicited by SARS-CoV-2 infection Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor Automated acquisition of cryo-electron micrographs for single particle 1153 reconstruction on an FEI Tecnai electron microscope Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike Intrinsic properties of 1158 immunoglobulin IgG1 isotype-switched B cell receptors promote microclustering and the initiation of 1159 signaling Functional and genetic analysis of viral receptor ACE2 orthologs reveals a broad potential host range of 1162 SARS-CoV-2 Efficacy of the ChAdOx1 nCoV-19 Covid-19 Vaccine against 1165 the B.1.351 Variant N-terminal domain antigenic mapping reveals a site of 1168 vulnerability for SARS-CoV-2 Phaser crystallographic software Lethal infection of K18-hACE2 mice infected with severe acute 1173 respiratory syndrome coronavirus UCSF Chimera--a visualization system for exploratory research and analysis UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein 1179 Sci Full-1181 length RNA-seq from single cells using Smart-seq2 Cross-neutralization of SARS-CoV-2 by a human monoclonal 1184 SARS-CoV antibody Reduced sensitivity of SARS-CoV-2 variant Delta to 1187 antibody neutralization Convergent antibody responses to SARS-CoV-2 in convalescent 1190 individuals Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from 1193 disease in a small animal model From vaccines to memory and back mRNA vaccination boosts cross-variant neutralizing 1198 antibodies elicited by SARS-CoV-2 infection Neutralizing and protective human monoclonal antibodies 1201 recognizing the N-terminal domain of the SARS-CoV-2 spike protein Ultrapotent human antibodies protect against SARS-CoV-1204 2 challenge via multiple mechanisms Sequencing and cloning of antigen-specific antibodies from mouse memory B cells Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7 Analysis of SARS-CoV-2 variant mutations reveals neutralization escape mechanisms and the ability to 1212 use ACE2 receptors from additional species mRNA vaccine-elicited antibodies to SARS-1215 CoV-2 and circulating variants Memory B Cells of Mice and Humans SARS-CoV-2 501Y.V2 escapes 1220 neutralization by South African COVID-19 donor plasma Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Quantitative Detection and Viral Load Analysis of SARS-CoV-2 in Infected Patients Gctf: Real-time CTF determination and correction A Thermostable mRNA Vaccine against COVID-19 MotionCor2: 1231 anisotropic correction of beam-induced motion for improved cryo-electron microscopy Evidence of escape of SARS-CoV-2 variant B.1.351 from 1235 natural and vaccine-induced sera Estimation of high-order aberrations and anisotropic 1237 magnification from cryo-EM data sets in RELION-3.1 Potently neutralizing and protective human antibodies against 1240 SARS-CoV-2 A mRNA vaccine encoding the RBD trimer of wildtype SARS-CoV-2 was designed and studied 2. The vaccine elicited strong RBD-specific memory and plasma B cell responses 3. The vaccine induced broadly serum and monoclonal neutralizing antibodies in mice The vaccine induced strong and protective immunity against major SARS-CoV-2 variants *** *** * ** (Zhang, 2016 ) and non-templated particle 1045 picking (Gautomatch v.0.56) were automatically executed by TsingTitan.py program. 1046 Sequential data processing was carried out on RELION-3.1 (Zivanov et al., 2020) . 1047 Initially, ~1,491,000 particles were subjected to 2D classification. After three additional 1048 2D classification, the best selected 1,156,670 particles were applied for initial model 1049 and 3D classification. A subset of 286,245 particle images from state 1 (2 RBD up and 1050 1 RBD down) and 258,463 particle images from state 2 (3 RBD up) were further subject 1051 to 3D auto-refine and post-processing. The final resolution for state 1 and state 2 are 1052 3.25 Å and 3.34 Å, respectively. The interface between spike protein and T6 Fab was 1053 subject to focused refinement with mask on the region of the RBD-Fab complex to 1054 improve the map quality. The 306,208 good particles after 3D classified focused on