key: cord-0302270-ykyc07go authors: Yu, Jiaping; Yao, Wenrong; Hu, Yingsong; Wu, Shuang; Li, Jiao; Zhou, Hongjun; Hong, Kunxue; Chen, Jianping; Liu, Longding; Lan, Ke; Zhu, Feng-Cai; Liu, Yong title: A trimeric NTD and RBD SARS-CoV-2 subunit vaccine induced protective immunity in CAG-hACE2 transgenic mice and rhesus macaques date: 2021-11-05 journal: bioRxiv DOI: 10.1101/2021.11.03.467182 sha: d9a041defc0acff6825d057eae2a19dd9c6c437d doc_id: 302270 cord_uid: ykyc07go The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to significant public health, economic and social problems. Development of effective vaccines is still a priority to contain the virus and end the global pandemic. In this study, we reported that ReCOV, a recombinant trimeric NTD and RBD two-component SARS-CoV-2 subunit vaccine adjuvanted with BFA03 (an AS03-like squalene adjuvant), induced high levels of neutralizing antibodies against SARS-CoV-2 and the circulating variants in mice, rabbits and rhesus macaques. Notably, two-dose immunizations of ReCOV provided complete protection against challenge with SARS-CoV-2 in hACE2 transgenic mice and rhesus macaques, without observable antibody-dependent enhancement of infection. These results support further clinical development of ReCOV and the vaccine is currently being evaluated in a phase I clinical trial in New Zealand (NCT04818801). RBD-targeting NAbs act synergistically to confer protection against SARS-CoV-2, 75 suggesting that NTD is a promising immunogenic partner of the SARS-CoV-2 RBD. 76 In fact, the combined immunogens of the NTD and RBD, elicited more robust 77 neutralization activity compared with a single immunogen consisting of either the RBD 78 or NTD [14] . In addition, accumulating evidence suggests multimerized antigens are 79 better in engaging interactions with B cell receptors thereby facilitating generation of 80 high-affinity antibodies compared to monomeric antigens [15] . Therefore, we assume 81 that trimeric display of SARS-CoV-2 NTD and RBD protein as vaccine candidates may 82 represent a promising strategy to induce potent neutralizing antibody responses to 83 prevent SARS-CoV-2 spread. In this study, we demonstrated that ReCOV, a recombinant trimeric NTD and RBD The affinity of NTD-RBD-foldon binding Human ACE2 was measured by Biacore 8K 130 (GE Healthcare) according to the manufacturer's instrument instructions. In brief, NTD-131 RBD-foldon was diluted with 10 mM NaAc (pH 5.5) to 6.0μg/mL, respectively, and The TMB was used as the substrate. After reaction stopping, plates were read at 450 171 nm wavelength. corresponding to the monomer, dimer and trimer respectively) ( Figure 1C ). The molecular weight of the NTD-RBD-foldon main peak analyzed by SEC-MALS is 249 264.3kD, which is 3 times of the predicted monomer molecular weight, indicating that 250 NTD-RBD-foldon is a trimer as designed. While the molecular weight of peak 2 was 251 189.6kD, which corresponds to 2 copies of NTD-RBD-foldon and 1 copy of RBD 252 molecule, assumed to be a degradation product ( Figure 1D ). The Surface Plasmon Resonance (SPR) assay was performed to determine the binding accounted for 50% to 75% of the total tissue. In contrast, the damage of lung tissue of 372 RECOV inoculated mice were significantly alleviated 50% to 25% at day 6 post 373 infection, and further to 25% or even recovered at day14 post infection. The neutralizing activity against the authentic virus was evaluated using sera taken at To ensure the right glycosylation, the NTD-RBD-foldon was expressed in CHO cells. 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