key: cord-0323623-c82gze35
authors: Tan, Shudan; Hu, Xue; Li, Yufeng; Wu, Zihan; Zhao, Jinghua; Lu, Guoliang; Yu, Zhaoli; Du, Binhe; Liu, Yan; Li, Li; Chen, Yuchen; Li, Ye; Yao, Yanfeng; Zhang, Xiaoyu; Rao, Juhong; Gao, Ge; Peng, Yun; Liu, Hang; Yuan, Zhiming; Liu, Jia; Wang, Qianran; Hu, Hengrui; Gao, Xiaobo; Zhou, Hui; Yu, Hang; Xu, Yingjie; Yu, Wei; Feng, Lin; Wang, Manli; Shan, Chao; Lu, Jing; Lin, Jinzhong
title: Preclinical evaluation of RQ3013, a broad-spectrum mRNA vaccine against SARS-CoV-2 variants
date: 2022-05-10
journal: bioRxiv
DOI: 10.1101/2022.05.10.491301
sha: 11a6ac06315571458d7895c66739d7fbf8fca202
doc_id: 323623
cord_uid: c82gze35

The global emergence of SARS-CoV-2 variants has led to increasing breakthrough infections in vaccinated populations, calling for an urgent need to develop more effective and broad-spectrum vaccines to combat COVID-19. Here we report the preclinical development of RQ3013, an mRNA vaccine candidate intended to bring broad protection against SARS-CoV-2 variants of concern (VOCs). RQ3013, which contains pseudouridine-modified mRNAs formulated in lipid nanoparticles, encodes the spike(S) protein harboring a combination of mutations responsible for immune evasion of VOCs. Here we characterized the expressed S immunogen and evaluated the immunogenicity, efficacy, and safety of RQ3013 in various animal models. RQ3013 elicited robust immune responses in mice, hamsters, and nonhuman primates (NHP). It can induce high titers of antibodies with broad cross-neutralizing ability against the Wild-type, B.1.1.7, B.1.351, B.1.617.2, and the omicron B.1.1.529 variants. In mice and NHP, two doses of RQ3013 protected the upper and lower respiratory tract against infection by SARS-CoV-2 and its variants. We also proved the safety of RQ3013 in NHP models. Our results provided key support for the evaluation of RQ3013 in clinical trials.

In the early effort to combat the COVID-19 pandemic, mRNA vaccines with the lipid nanoparticle (LNP) delivery system have achieved unprecedented success.

Two mRNA vaccines from Moderna (mRNA-1273) and Pfizer-BioNTech (BNT162b2), encoding the prefusion stabilized full-length spike (S) protein of SARS-CoV-2, are now being widely used 1,2 , and several other mRNA vaccine candidates are in clinical trials. However, the effectiveness of the two mRNA vaccines is waning significantly as the virus continues to evolve, generating new variants. Studies have shown that sera from BNT162b2 or mRNA-1273 recipients exhibit more than a 30-fold reduction in neutralization titers against the latest Omicron variant compared to against Wild-type 3 .

Previously, we developed three mRNA vaccine candidates against the wildtype SARS-CoV-2 expressing the receptor-binding domain(RBD), S, and viruslike particles(VLP). Both the VLP and S immunogen elicited potent immune responses in mice 4 . We generated a pool of optimized mRNA sequences of assorted modifications from which the optimal one carrying the pseudouridine modification was identified. As the SARS-CoV-2 variants of concern (VOCs) kept posing new waves of global health threats, we began to focus on developing a broad-spectrum mRNA vaccine. One candidate, RQ3013, adapted from the previous S-based mRNA vaccine, showed encouraging results in the preclinical development. The mRNA sequence and modification of RQ3013 are inherited from its predecessor, but several changes have been made to the S immunogen. The furin cleavage site was mutated to prevent proteolysis of the S protein. The C-terminal eighteen residues were truncated for enhanced antigen expression. Most importantly, the S antigen carries mutations from the B.1.1.7 and B.1.351 variants and shares seven of them with the S protein from B.1.1.529. Here we characterized the structure of expressed immunogen of RQ3013 and reported a comprehensive evaluation of the immunogenicity, efficacy, and safety of RQ3013 in animal models of mice, hamsters, and NHPs. We provided evidence that RQ3013 is a promising vaccine candidate against SARS-CoV-2 VOCs, adding support for its clinical development.

RQ3013 encodes a near full-length S protein lacking the eighteen cysteine-rich residues at the extreme C-terminus. The To characterize the S protein encoded by RQ3013, we overexpressed and affinity-purified the antigen and investigated its 3D structure by single-particle cryo-EM analysis. Initial 2D classification revealed two distinct populations corresponding to prefusion and postfusion S, from which two mass density maps were produced at a nominal resolution of 3.87 Å and 3.49 Å, respectively analysis. This indicates that the structural transition of SARS-CoV-2 S protein from pre-to postfusion may occur in the absence of cleavage, which has been reported for other class I fusion proteins 8 . Whether such transition occurs in vivo, or to what extent, remains to be determined as the postfusion structure could result from the purification process in the presence of detergent.

To assess the immunogenicity of RQ3013 in BALB/c mice. The two groups of mice (n = 18) were immunized intramuscularly with 2 µg or 5 µg RQ3013 on day 0 (Fig. 2a) . A control group of mice (n = 18) received PBS as the placebo.

All groups were boosted on day 21. No local inflammation or other adverse effects were observed throughout the experiment. We evaluated sera collected on day 28 for binding IgG to RBD and S. Data from enzyme-linked immunosorbent assay (ELISA) showed that the 2 µg RQ3013 induced in mice equally high titers of IgG binding to Wild-type RBD and S from the B.1.1.7, B.1.351, and B.1.617.2 variants (Fig. 2b) . There was no difference between the low-dose group (2 μg) and the high-dose group (5 μg) (Fig. 2b) (Fig. 2c) . To investigate whether RQ3013 activates a T cell response in mice, we collected peripheral blood mononuclear cells (PBMCs) on day 41 and re-stimulated them with the S peptide mix. Using an enzymelinked immunosorbent spot (ELISPOT) assay, high levels of IFN-γ secreting in Th1 cells were detected in all RQ3013-vaccinated mice but not in the mice received PBS (Fig. 2d) . days after the boost. The lung tissues were collected at 5 days post-infection (dpi) for viral load determination (by qRT-PCR) and histopathological analysis ( Fig. 2a) . Compared to the PBS control, both 2-μg and 5-μg doses of RQ3013 prevented weight loss of mice beginning at 2 dpi (Fig. 2e ). All mice in the placebo group developed a high load of viral sgRNAs (~10 6 sgRNA copies/µL) in the lungs, which were not detectable in both groups of immunized mice (Fig.   2f ). Accordingly, lung pathology showed intact alveolar structures in immunized mice, while mice in the control group developed pathological characteristics of typical lung lesions (Fig. 2g) . These results demonstrated that RQ3013 could effectively prevent replication of the B.1.351 variant in the lower respiratory tract and provides rapid protection in BALB/c mice from lung lesions.

In the K18-hACE2 transgenic mouse model, B.1.1.7 and B.1.351 are 100 times more lethal than the original virus 10 . We next assessed the immunogenicity and efficacy of RQ3013 in K18-hACE2 transgenic mice. Both 2-μg and 5-μg doses of RQ3013 elicited high titers of RBD-and S-specific IgG antibodies in sera of K18-hACE2 mice at 7 days post-boost in a dose-dependent manner (Fig. 3a, b). We also measured inhibition of cell entry of viruses pseudotyped with the wild-type and B.1.1.529 S protein. 5-μg dose elicited neutralizing antibodies in all mice for Wild-type (GMT 434) and B.1.1.529 (GMT 173) (Fig. 3c) . We next challenged the mice with the variant B.1.351 on day 61 (1×10 3 PFU). Mice in the control group showed weight losses at 4 dpi, which were prevented in both dose groups of RQ3013-vaccinated mice (Fig. 3d ). Mice were euthanized at 4 dpi, and a large amount of viral RNAs was detected in the lung (10 6 copies per μg of RNA), trachea (10 5 copies per μg of RNA), and brain (10 9 copies per μg of RNA) of the control mice. RQ3013 effectively prevented viral replication in these tissues. A decrease in viral load (> 3 logs) in the lung was observed in all RQ3013-vaccinated mice. Viral replication was completely prevented in the brain except for two mice in the 2-μg dose group (Fig. 3e) . 

The Syrian hamster having the ACE2 receptor is highly susceptible to SARS-CoV-2 and develops similar pneumonia to that in COVID-19 patients, making them a suitable model for evaluating vaccines 11 . Three groups of hamsters were vaccinated on day 0 and day 21 with PBS or RQ3013 ( 5 or 25 µg ) (Fig.   4a ), and sera were collected and evaluated for immunogenicity on day 28. High titers of binding antibodies against RBD and S (wild-type and B.1.617.2) were detected (Fig. 4b) . We monitored the level of S-specific IgG over the course of 33 weeks post-immunization, and a reduction in titer was observed in the first 13 weeks and then sustained at a high level for the rest time ( for 5 μg and 25 μg doses, respectively (Fig. 4d) . We also collected PBMCs and re-stimulated them with the S1 and S2 peptide pools. Using an enzyme-linked immunosorbent spot (ELISPOT) assay, we detected high levels of IFN-g secreting cells in RQ3013-immunized hamsters in a dose-dependent manner (Fig. 4e) . These data confirmed that the mRNA vaccine could induce a strong T cell response in hamsters.

We next evaluated the immunogenicity of RQ3013 in rhesus macaques, an The PRNT50 data agree reasonably well with the antigen design of RQ3013 which is more specific to B.1.351 than Wild-type and B.1.617.2. Interestingly, the level of cross-neutralizing antibody against B.1.1.529 is encouragingly high in NHP, near the level for the Wild-type virus (Fig. 5d ).

At 7 weeks following the second vaccination, rhesus macaques were challenged with the Wild-type virus (1×10 5 TCID50). All animals exhibited no noticeable behavioral differences and no apparent changes in body weight and body temperature during the experimental period (Fig. S4a, b ).

In the low-dose group of animals, viral RNA was not detected since day 1 postinfection in the nasal and anal swabs and became absent at 6 dpi in the oropharyngeal swabs. A similar trend was observed for the high-dose group. In contrast, the PBS-vaccinated animals experienced a continuous viral replication as seen in anal and oropharyngeal swabs. Viruses were not detected in blood samples of all animals (Fig. S4c-f ). These data showed rapid control of viral replication within 2 days in both the upper and lower airways conferred by RQ3013 vaccination.

After sacrifice, no viral RNA was detected in the lungs of all RQ3013-vaccinated animals. One from each dose group has viral RNA in trachea-bronchus detected. A high load of viruses was seen in the lung, trachea, and bronchus of animals in the control group (Fig. 5e ).

X-ray images showed no noticeable lung shadow in all infected animals ( Fig.   S5 ).

Pathological changes characterized by viral pneumonia and pulmonary fibrosis were moderate in the low-dose group and generally mild in the high-dose group, significantly mitigated compared with the control group ( Fig. 5f ).

We evaluated the safety of RQ3013 in NHP cynomolgus macaques. Fifty cynomolgus macaques were divided into five groups (n = 10), each on day 0 received intramuscularly one dose of PBS or RQ3013 (60 µg and 240 µg) or empty LNPs ( 1.2 mg or 4.8 mg of total lipid) (Fig. 6a ). All groups were doubleboosted on days 14 and 28. Neither fever nor weight loss was observed in all animals (Fig. S6) . The blood samples of all animals were collected at different time points for hematological and biochemical analysis. All animals displayed no appreciable changes in hematological indices and the percentage values of lymphocyte subsets, including CD3 + , CD4 + , and CD8 + T cells ( Fig. S7 and Fig.   6b ). For key cytokines, no notable changes in Th1 cytokines (interferon-g, tumor necrosis factor a, interleukin-2) or Th2 cytokines (interleukin-4, 5) secretion was observed in all animals ( Fig. 6c and Fig. S8 ). The IL-6 level increased following each injection of either RQ3013 or empty LNP in a dose-dependent manner.

All recovered in a week. (Fig. S8 ). We detected high levels of IFN-γ secreting Th1 cells and interleukin-4 (IL-4) secreting Th2 cells in animals immunized with RQ3013 but not PBS or empty LNP (Fig. S9 ).

Finally, three groups of animals (PBS, high-dose groups for RQ3013 and empty LNP) were euthanized on day 31. Lungs, brains, hearts, livers, spleens, and kidneys were harvested for histopathological analysis and safety assessment of RQ3013. Vaccine-associated immunopathologic changes were not observed in any of the sections examined in these tissues of all animals ( Fig. 6d and Fig.   S10 ). Together, these data provided evidence that a high dose application of RQ3013 is safe in NHP, supporting the development of clinical trials.

The mRNA vaccines mRNA-1273 and BNT162b2 were developed at the beginning of the COVID-19 pandemic and have shown greater than 90% efficacy during the early phase of the pandemic 13 In addition to variant-related mutations, we also changed the furin cleavage site in S to eliminate post-translational proteolysis, thus stabilizing S in the prefusion state. However, structural characterization revealed trimeric S in both prefusion and postfusion states. Although we could not locate the S1 subunit in the postfusion structure due to its flexibility, it most likely remained in the structure.

A previous study used the same strategy to determine the S structure only to report the prefusion conformation 6 . The structural transition observed in our study may be induced by harsh detergent conditions during purification. It is evident that prevention of proteolysis can stabilize the prefusion conformation but not to the extent conferred by proline substitutions 18 . On the other hand, the exposure of the S2 subunit may be beneficial for vaccine development against SARS-CoV-2. Since S2 is more conserved than S1, antibodies targeting S2 have shown great cross-neutralization activity against b-coronaviruses 19,20 .

Therefore, it is possible that the S2-binding antibodies may partly contribute to the broad spectrum of RQ3013. In summary, we have conducted a broad-spectrum assessment of RQ3013 immunogenicity, efficacy, and safety, providing support for the evaluation of this vaccine in clinical trials. 

Virus titrations were performed by endpoint titration in Vero E6 cells. Cells were inoculated with 10-fold serial dilutions of cell supernatant. One hour after inoculation of cells, the inoculum was removed and replaced with 100 μL of DMEM supplemented with 2% FBS, 1 mM L-glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin. Three days after inoculation, CPE was scored, and the TCID50 was calculated.

Production of RQ3013 mRNA and LNP used in this study was conducted under current good manufacturing practice (cGMP). The optimized DNA template for RQ3013 RNA was cloned into the plasmid with backbone elements (T7 promoter, 5′ and 3′ UTR, poly(A) tail). RQ3013 mRNA was in vitro-transcribed by T7 RNA polymerase in the presence of the Cap1 analog (B8176, APExBIO) and nucleotides with a global substitution of uridine with pseudouridine (Ψ, APExBIO). RNA was purified through two chromatographic procedures. RNA integrity was assessed by microfluidic capillary electrophoresis, and the concentration, pH, osmolality, and endotoxin level were determined. Purified RNA was formulated into lipid nanoparticles containing four lipids( an ionizable lipid, PEG2000-DMG, DSPC, and cholesterol ) as previously described.

Particle sizes were measured using dynamic light scattering on a Malvern Zetasizer Nano-ZSP (Malvern).

The protein expression of mRNAs was tested in HEK 293A cells. mRNA 

To prepare cryo-EM grids, 3.5 μL of freshly purified spike proteins at ~0.3 mg/mL was applied to Quantifoil R1. The sera of immunized mice, hamsters and rhesus macaques were collected and inactivated at 56˚C for 0.5h to detect the SARS-CoV-2-specific IgG and neutralizing antibodies as described below.

SARS-CoV-2 S-specific antibody responses in immunized sera were determined by enzyme-linked immunosorbent assay (ELISA) assay, as previously described. Briefly, 96-well plates were coated with 50 μL of coating buffer containing 100 ng/well recombinant SARS-CoV-2 spike or RBD antigens (Sino Biological, Arco) at 4˚C overnight. Plates were blocked with 2% bovine serum albumin solution in PBST at room temperature for 1 hour. Immunized mice sera were diluted 100-fold as the initial concentration, and then a 5-fold serial dilution of a total of 11 gradients in PBS buffer. PBST washed plates were incubated with serially diluted sera at room temperature for 2 hours. For determination of S-specific antibody response, plates were incubated with goat anti-mouse IgG HRP (for mouse sera, Proteintech Cat: SA00001-1) or goat anti-Syrian hamster IgG HRP (for hamster sera, abcam Cat: ab6892) or goat anti-monkey IgG HRP (for NHP sera, Invitrogen Cat: PA1-84631) at 37˚C for 1 hour and then substrate tetramethylbenzidine (TMB) solution (Invitrogen) was used to develop. The color reaction was quenched with 1N sulfuric acid for about 10 minutes, and the optical density was measured at a wavelength 450 nm by Synergy H1 microplate reader (BioTek).

The pseudovirus-based neutralization assay was performed at Genescript.

Briefly, serum samples collected from immunized animals were serially diluted with the cell culture medium. The diluted serums were mixed with a pseudovirus suspension in 96-well plates at a ratio of 1:1, followed by 1 hours incubation at RT. Opti-HEK293/ACE2 cells were then added to the serum-virus mixture, and the plates were incubated at 37˚C in a 5% CO2 incubator. 48 hr later, the luciferase activity, reflecting the degree of SARS-CoV-2 pseudovirus transfection, was measured using the Luciferase Assay kit. The NT50 was defined as the fold-dilution, which emitted an exceeding 50% inhibition of pseudovirus infection compared to the control group.

Cytopathic effect (CPE). Serum samples collected from immunized animals were inactivated at 56˚C for 30min and serially diluted with cell culture medium in two-fold steps. The diluted serums were mixed with a virus suspension of 100 TCID50 in 96-well plates at a ratio of 1:1, followed by 1 hours of incubation at 37˚C in a 5% CO2 incubator. Vero E6 cells were then added to the serumvirus mixture, and the plates were incubated for 4 days at 37˚C in a 5% CO2

incubator. Cytopathic effect (CPE) of each well was recorded under microscopes, and the neutralizing titer was calculated by the dilution number of 50% protective condition.

was performed in a 24-well plate. The serum samples from immunized animals were heat-inactivated at 56˚C for 30 min. The serum samples were diluted at 1:30, 1:90, 1:270, 1:810, 1:2430 and 1:7290, and then an equal volume of virus stock was added and incubated at 37˚C in a 5% CO2 incubator. After 1 hour of incubation, 100 μL mixtures were inoculated onto monolayer Vero cells in a 24well plate for 1 hour with shaking every 15 minutes. The inocula were removed, and cells were incubated with DMEM supplemented with 2% FBS containing 0.9% methylcellulose for 4 days before fixation. The cells were then fixed with 8% formaldehyde for 1 hour. The formaldehyde solution was removed and the cells were washed with tap water, followed by crystal violet staining. The plaques were counted for calculating the titer.

The mouse and hamster elispot analysis was performed ex vivo using PBMCs with commercially available Mouse IFN-γ ELISpot assay kit (Dakewe) and

Hamster IFN-γ ELISpotPLUS kit (Mabtech), respectively. The T cell immune responses in Cynomolgus macaques were detected using PBMCs with commercially available Monkey IFN-γ ELISpot assay kit and a Monkey IL-4

ELISpot assay kit (Mabtech). A pool of 15-mer peptides that overlapped by 11 amino acids and covered the whole sequence of the SARS-CoV-2 spike protein (Genscript) was used for ex vivo stimulation of PBMCs for ELISpot assay, which was divided into S1 peptide pool and S2 peptide pool. For the mouse and hamster IFN-γ ELISpot assays, 2. Analysis of lymphocyte subset percent (CD3 + , CD4 +, and CD8 + ), key cytokines (TNF-α, IFN-γ, IL-2, IL-4, IL-5, and IL-6), and biochemical blood tests are also performed in collected blood samples. 60% of monkeys were euthanized at day 31 post-immunization, and the left 40% were euthanized on day 42. Organs of lung, brain, heart, liver, spleen, and kidney were collected for pathologic analysis.

All statistics data were performed and graphed using GraphPad Prism8.0. The EC50 values were calculated by non-linear regression. Statistical analyses were carried out by Student's t-test when two groups were analyzed and by ANOVA when more than two groups were analyzed. The cryo-EM map and the refined atomic model of the S antigen in the prefusion conformation. One RBD in the upright state was indicated. c The cryo-EM map and the refined model of the postfusion S antigen. The unseen S1 domains were sketched. Protomers were colored in pink, cyan, and orange, and glycosylations were colored in green.

The immunization scheme of RQ3013 in mice. Mice (n = 18 per group) were intramuscularly immunized with PBS(Placebo, blue circle) or low dose (2 μg, red square) or high dose (5 μg, green triangle) of RQ3013. Time points of vaccination, bleeding, viral challenge, and sera collection are indicated by arrows. b ELISA analysis of binding IgG to the wild-type RBD antigen and S proteins from SARS-CoV-2 variants with sera collected on day 28. Values are GMT mean ± SD. c Neutralizing antibody titers in sera collected on day 28, analyzed by the lentiviral luciferase-based pseudovirus assay. The black dashed line indicates the assay's detection limit (reciprocal titer of 40). Any measurement below the detection limit was assigned a value of half the detection limit for plotting and statistical purposes. Values are GMT mean ± SD. Statistical analyses were carried out by Student's t-test when two groups were analyzed, and by ANOVA when more than two groups were analyzed (*P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0001). Statistical analyses were carried out by Student's t-test when two groups were analyzed, and by ANOVA when more than two groups were analyzed (ns, Not Significant; *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0001). Binding IgG to the wild-type RBD and S proteins from the wild-type and B.1.617.2 viruses, analyzed with sera collected in week 4 by ELISA. Values are GMT mean ± SD. c Neutralizing antibody titers in week-4 sera, analyzed by the lentiviral luciferase-based pseudovirus assay. The black dashed line indicates the assay's limit of detection (reciprocal titer of 40). Any measurement below the detection limit was assigned a value of half the limit of detection for plotting and statistical purposes. Values are GMT mean ± SD. d The CPE-based live virus micro-neutralization assay of week-5 sera against SARS-CoV-2 B.1.617.2 and B.1.1.529. The black dashed line indicates the assay's detection limit (reciprocal titer of 8). Any measurement below the detection limit was assigned a value of half the limit of detection for plotting and statistical purposes. Values are GMT mean ± SD. e The IFN-γ ELISpot assay with PBMCs obtained in week 13 and restimulated with overlapping peptide pools of S1 and S2 domains. SFU, spot-forming units. Data are presented as mean ± SEM. Statistical analyses were carried out by Student's t-test when two groups were analyzed, and by ANOVA when more than two groups were analyzed (ns, Not Significant; *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0001). IgG to the wild-type RBD antigen and S proteins from variants, analyzed with sera collected on days 28 (Wk4) and 35 (Wk5) by ELISA. Values are GMT mean ± SD. c Neutralizing antibody titers in sera from days 28 and 35, analyzed by the lentiviral luciferase-based pseudovirus assay. The black dashed line indicates the assay's detection limit (reciprocal titer of 20). Any measurement below the detection limit was assigned a value of half the limit of detection for plotting and statistical purposes. Values are GMT mean ± SD. d The PRNTbased neutralization assay of day-42 sera against SARS-CoV-2 variants. The black dashed line indicates the assay's limit of detection (reciprocal titer of 30). Any measurement below the detection limit was assigned a value of half the detection limit for plotting and statistical purposes. Values are GMT mean ± SD. e Viral RNA loads in aminal lungs and trachea-bronchus at 7 dpi, determined by qRT-PCR. The black dashed line indicates the assay's detection limit (500 Copies/g). Any measurement below the detection limit was recorded as '2' for plotting and statistical purposes. Data are presented as mean ± SEM. f Histopathological examinations of aminal lungs at day 7 dpi. Lung tissues were collected and stained with hematoxylin and eosin. Black scale bar, 100 μm; blue scale bar, 60 μm. Data are presented as mean ± SEM. Statistical analyses were carried out by Student's t-test when two groups were analyzed, and by ANOVA when more than two groups were analyzed (*P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0001). 

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high dose RQ3013 (240 μg, green triangle), low dose (purple diamond) and high dose (orange inverted triangle) of empty LNP. Time points of vaccination, bleeding, and sample collection are indicated by arrows. (b, c) Hematological analysis in all five groups of macaques

TNF-α, and IL-2 were monitored on days indicated. Values are mean ± SEM. The black dashed line indicates the assay's detection limit (IFN-γ, 3.3 pg/mL; TNF-α 0.3 pg/mL

The atomic coordinates and cryo-EM maps of the S immunogen have been deposited in the Protein Data bank (PDB) and Electron Microscopy Data Bank (EMDB). Accession numbers are 7L7F and EMD-33346 for the prefusion state, and 7XOG and EMD-33347 for the postfusion state.