key: cord-0954522-vbklsci3
authors: Ma, Xiancai; Zou, Fan; Yu, Fei; Li, Rong; Yuan, Yaochang; Zhang, Yiwen; Zhang, Xiantao; Deng, Jieyi; Chen, Tao; Song, Zheng; Qiao, Yidan; Zhan, Yikang; Liu, Jun; Zhang, Junsong; Zhang, Xu; Peng, Zhilin; Li, Yuzhuang; Lin, Yingtong; Liang, Liting; Wang, Guanwen; Chen, Yingshi; Chen, Qier; Pan, Ting; He, Xin; Zhang, Hui
title: Nanoparticle Vaccines Based on the Receptor Binding Domain (RBD) and Heptad Repeat (HR) of SARS-CoV-2 Elicit Robust Protective Immune Responses
date: 2020-11-25
journal: Immunity
DOI: 10.1016/j.immuni.2020.11.015
sha: ec34f222ea0b0286de242084928422e4dd146e80
doc_id: 954522
cord_uid: vbklsci3

Various vaccine strategies have been proposed in response to the global COVID-19 pandemic, each with unique strategies for eliciting immune responses. Here, we developed nanoparticle vaccines by covalently conjugating the self-assembled 24-mer ferritin to the receptor binding domain (RBD) and/or heptad repeat (HR) subunits of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) spike (S) protein. Compared to monomer vaccines, nanoparticle vaccines elicited more robust neutralizing antibodies and cellular immune responses. RBD and RBD-HR nanoparticle vaccinated hACE2 transgenic mice vaccinated with RBD and/or RBD-HR nanoparticles exhibited reduced viral load in the lungs after SARS-CoV-2 challenge. RBD-HR nanoparticle vaccines also promoted neutralizing antibodies and cellular immune responses against other coronaviruses. The nanoparticle vaccination of rhesus macaques induced neutralizing antibodies, and T and B cell responses prior to boost immunization; these responses persisted for more than three months. RBD- and HR-based nanoparticles thus present a promising vaccination approach against SARS-CoV-2 and other coronaviruses.Keywords: SARS-CoV-2, COVID-19, nanoparticle vaccine, RBD, HR

Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has emerged as a worldwide 53 severe pandemic and caused more than 52 million confirmed cases and more than 1 54 million deaths (as of middle of November 2020, report from https://covid19.who.int/ ) 55 (Zhu et al., 2020b) . The infection and death cases still increase rapidly for the high 56 transmissibility with a basic reproduction number (R 0 ) of 3.28 and the lack of 57 effective vaccines and therapeutic drugs Sanders et al., 2020) . 58 Developing effective, safe and preventative vaccine against SARS-CoV-2 is urgently 59 needed to end the pandemic (Lurie et al., 2020) . 60 SARS-CoV-2 is a new member of pathogenic human coronaviruses, which also 61 includes four common coronaviruses hCoV-NL63, hCoV-229E, hCoV-OC43 and 62 hCoV-HKU1, and two life-threatening beta-coronaviruses SARS-CoV and 63 MERS-CoV . Like other coronaviruses, SARS-CoV-2 harbors a large 64 single-strand positive-sense genomic RNA which encodes four major structural 65 proteins, spike (S), membrane (M), envelope (E) and nucleocapsid (N) (Shereen et al., 66 2020). The S protein, which contains S1 and S2 subunits, is embedded within viral 67 envelope and mediates the recognition and entry into human cell by binding to the 68 human angiotensin-converting enzyme 2 (hACE2) through the receptor-binding 69 domain (RBD) within S1 subunit, followed by the fusion of viral envelope and 70 cellular membrane through the participation of heptad repeat 1(HR1) and heptad 71 repeat 2 (HR2) within S2 subunit (Hoffmann et copies of RBD or RBD/HR protein subunits to the self-assembled Helicobacter pylori 109 (H. pylori) non-haem ferritin. We investigated the total antibody and the neutralizing 110 responses in both mice and rhesus macaques, and found that ferritin-based 111 nanoparticles were able to induce more neutralizing antibodies than protein subunit 112 monomers. In addition, we found that the HR subunit within S2 of S protein was also 113 able to induce notable neutralizing antibodies. The neutralizing antibody titer was 114 further increased with HR subunit conjugation to ferritin nanoparticles. We also 115 assessed the immunogenicity of RBD/HR chimeric ferritin nanoparticles. 

As S protein is a promising target to design vaccine candidates, we aligned the S 130 protein sequences within mammal coronaviruses which included three SARS-CoV-2 131 strains (SYSU-IHV, USA-IA-6399 and Wuhan-Hu-1), the other six human pathogenic 132 coronaviruses (SARS-CoV Tor2, MERS-CoV EMC, hCoV-HKU1, hCoV-OC43, 133 hCoV-NL63 and hCoV-229E), five bat coronaviruses, and two pangolin coronaviruses. 134 The alignment revealed that the heptad repeat 1(HR1) and heptad repeat 2 (HR2) 135 within S2 subunit was highly conserved across different SARS-CoV-2 strains and 136 different coronaviruses genera ( Figure S1A -S1D). The region of HR1-HR2 is more 137 conservative than the receptor-binding domain (RBD) region in the evolutionary 138 history of coronavirus ( Figure S1E ). RBD is also the ligand of the human 139 J o u r n a l P r e -p r o o f 7 angiotensin-converting enzyme 2 (hACE2) and has high immunogenicity based on the 140 experience in SARS-CoV vaccine development (Du et al., 2009 RBD/HR monomers-vaccinated mice ( Figure S2H ). 237 To further evaluate the immunogenicity, we conducted several neutralizing assays. 238 We found that neutralizing antibodies (nAbs) which induced by RBD and RBD/HR In our alignment data, we found that HR was highly conserved across different 344 coronaviruses ( Figure  S1 ). Both HR-containing monomers and 345 HR-Ferritin-containing nanoparticles were able to induce high titers of HR-specific 346 antibodies ( Figure 2C , 2E and S2B). We hypothesize that HR-containing nanoparticle 347 may also induce sufficient nAbs to neutralize other coronaviruses apart from 348 SARS-CoV-2. We constructed an HR nanoparticle which contained 100% HR-Ferritin, 349 and vaccinated mice with HR monomers or HR nanoparticles. Two weeks post boost, 350 the serum of different groups was collected and used to neutralize the authentic 351 SARS-CoV-2. The result showed that HR was able to induce nAbs which were able to 352 neutralize authentic viruses at 1:100 dilution ( Figure 5A ). HR nanoparticle vaccine 353 induced higher nAbs than monomer ( Figure 5A ). We next investigated whether 354 HR-containing vaccines induced nAbs which could neutralize other coronaviruses. 355 We found that RBD/HR nanoparticle vaccine induced higher nAbs against The efficiency of infection was determined by FRNT assay. We found that the 383 neutralizing antibodies titers of nanoparticle vaccine groups, which were represented 384 by FRNT50, were 100-fold higher than the monomer vaccine group ( Figure 6D ). The 385 FRNT50 titers of both RBD and RBD/HR nanoparticles groups were over 2. special biosafety environment concern is required for these manufacture procedures. 537 Therefore, these nanoparticle vaccines merit being further evaluated in a Phase I-II 538 clinical trial. comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. 625 See also Figure S2 and Figure S3 . See also Figure S4 and Figure S5 . See also Figure S6 and Figure S7 . 

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(SP) at the C-terminus. Subsequently, biotin was ligated to Avi-tag with biotin-protein 954 ligase BirA enzyme (GeneCopoeia), followed by incubating with BV421-tagged 955 Streptavidin (Biolegend). Target cells were incubated with RBD-BV421 to track 956 RBD-specific or RBD-binding cells. hACE2-HeLa cell line was generated through 957 infecting HeLa cells with recombinant lentiviruses which expressing human 958 angiotensin-converting enzyme 2 (hACE2). hACE2-positive cells were sorted by 959 FACS. The expression of hACE2 was confirmed by western blotting. Serum of 960 immunized BALB/c mice, which was collected at week 6, was 1:100 diluted in PBS, 961 and incubated with RBD-BV421 probe (2 ug/mL), followed by incubating with 962 hACE2-HeLa cells. Then cells were washed with PBS to remove unbound RBD probe. 963 Two hours post incubating, cells were resuspended to measure fluorescence by flow 964 cytometry (BD LSRFortessa). Recombinant SARS-CoV-2 RBD was conjugated to BV421 as we described above. 1082 The following antibodies were used to identify antigen-specific memory B cells between each group with the mean of every other group within a dataset containing 1189 more than two groups, one-way ANOVA with Tukey's multiple comparisons test was