key: cord-0959859-aesw50tz
authors: Dai, Lianpan; Zheng, Tianyi; Xu, Kun; Han, Yuxuan; Xu, Lili; Huang, Enqi; An, Yaling; Cheng, Yingjie; Li, Shihua; Liu, Mei; Yang, Mi; Li, Yan; Cheng, Huijun; Yuan, Yuan; Zhang, Wei; Ke, Changwen; Wong, Gary; Qi, Jianxun; Qin, Chuan; Yan, Jinghua; Gao, George F.
title: A universal design of betacoronavirus vaccines against COVID-19, MERS and SARS
date: 2020-06-28
journal: Cell
DOI: 10.1016/j.cell.2020.06.035
sha: 0d52f7f90227e0e21b180bd383ee4c4624458e34
doc_id: 959859
cord_uid: aesw50tz

Summary Vaccines are urgently needed to control the ongoing pandemic COVID-19 and previously-emerging MERS/SARS caused by coronavirus (CoV) infections. The CoV spike receptor-binding domain (RBD) is an attractive vaccine target but is undermined by limited immunogenicity. We describe a dimeric form of MERS-CoV RBD that overcomes this limitation. The RBD-dimer significantly increased neutralizing antibody (NAb) titers compared to conventional monomeric form and protected mice against MERS-CoV infection. Crystal structure showed RBD-dimer fully exposed dual receptor-binding motifs, the major target for NAbs. Structure-guided design further yielded a stable version of RBD-dimer as a tandem repeat single-chain (RBD-sc-dimer) which retained the vaccine potency. We generalized this strategy to design vaccines against COVID-19 and SARS, achieving 10-100-fold enhancement of NAb titers. RBD-sc-dimers in pilot scale production yielded high yields, supporting their scalability for further clinical development. The framework of immunogen design can be universally applied to other beta-CoV vaccines to counter emerging threats.

To determine the molecular basis of the observed dimer immunogen at an atomic level, we 170 determined the crystal structure of MERS-CoV RBD-dimer at a resolution of 2.9 Å ( Table  171   S1 ). Interestingly, RBD-dimer is arranged as a "bilateral-lung"-like structure with axial 172 symmetry ( Figure 2A ). The dual RBMs are located at the lower lobes of both "lungs" facing 173 outwards against the axis (Figure 2A ). Both RBMs are fully exposed (Figure 2A and Figure  174 S1), suggesting the potential to elicit antibodies interfering with receptor binding. The core 175 subdomains of both RBD protomers stack on each other via α3 helices and loops surrounding 176 the helices. The N-and C-termini are invisible in the electron density maps, indicating the 177 flexibility of these regions. Accordingly, in the previously determined structure of the 178 full-length MERS-CoV-S, these regions consist of flexible loops which facilitate the 179 hinge-like conformational movements of RBD for receptor engagement (Yuan et al., 2017) . 180 Each RBD contains 9 cysteine residues, with 8 of them forming 4 intra-molecule disulfide 181

bonds. An additional invisible C-terminal cysteine residue (C603) is proposed to build the 182 inter-molecule disulfide bond covalently linking the RBD-dimer (Figure 2A ). Given the 183 number of flexible, unresolved residues that link the two subunits of the dimer together, and 184 the small non-covalent interface observed between the two subunits of the dimer, it is far 185 from certain that the crystallographically observed structure corresponds to a unique structure 186 the protein adopts in solution. 187 188 Structure-guided design of the RBD-sc-dimer 189 Since the RBD forms a monomer-dimer equilibrium in solution, the yields of dimer protein 190 varied from batch to batch. To facilitate downstream vaccine development, we sought to 191 further engineer the RBD-dimer in a more stable form. The structure of RBD-dimer showed both N-and C-termini of two RBD protomers were juxtaposed and 193 closed to each other (Figure 2A ), which inspired us to link these two RBD as a tandem repeat 194 single chain. In order to avoid introducing any exogenous sequences, we sought to link them 195 by their own flexible terminal residues. Both RBDs were truncated at N602, the position just 196 before C603 to avoid potential instability caused by cysteine residue, and then connected in 197 tandem ( Figure 2B ). RBD expressed in a mammalian cell system was previously reported to 198 be able to induce stronger neutralizing antibody response than those expressed in insect cells 199 (Du et al., 2009b) . We, therefore, constructed and expressed immunogens switching from 200 insect cells to mammalian cells. The RBD-sc-dimer was expressed in mammalian HEK293T 201 cells. The supernatant of the transfected cells was collected for further purification. Analytical 202 gel filtration showed that the new construct was expressed as a single peak with a size of ~60 203 kDa, indicating the dimeric form of RBD ( Figure 2C ). The molecular weight was further 204 corroborated as 60.7 kDa by analytical ultracentrifugation ( Figure 2D ). Surface plasmon 205 resonance (SPR) assay demonstrated the RBM is exposed in the RBD-sc-dimer as in 206 conventional RBD-monomer, supported by the comparable binding affinities of RBD to its 207 receptor hCD26 ( Figure 2E ). 208 12 The RBD construct for SARS-CoV-2 started at R319 (the E367 position of MERS-CoV S 231 protein) and was truncated at C-terminal residue K537, the amino acid just before C603 232 position of MERS-CoV S protein ( Figure S2 and Figure 3A ). Two copies of RBD were then 233 dimerized in tandem as the strategy described for MERS-CoV RBD-sc-dimer ( Figure 3A) . 234 SARS-CoV-2 RBD was expressed as a single dimer-sized protein, as verified by analytical 235 gel filtration and gel electrophoresis ( Figure 3A ). Further analytical ultracentrifugation 236 determined its molecular weight as 61.7 kDa, suggesting a stable dimer formation ( Figure 3B) . 237

The SPR assay demonstrated RBD-sc-dimer bound to receptor hACE2 with comparable 238 affinity as its monomer counterpart, implying the exposure of the RBMs ( Figure 3C and Table  239 S2). Interestingly, the hACE2 protein expressed by baculovirus from insect cells showed 240 weaker affinities to bind SARS-CoV-2 RBDs (two orders of magnitude lower) compared to 241 mammalian-derived one, suggesting different glycosylation patterns of the hACE2 proteins 242 may account for the distinct binding affinities (Table S2) . 243

To further assess the immunogenicity, we first immunized BALB/c mice with the 244 SARS-CoV-2 RBD-sc-dimer or conventional RBD-monomer (10 µg dose of immunogen plus 245 AddaVax TM adjuvant). PBS formulated with adjuvant was given as control. Three-dose 246 regimens were performed to assess the response dynamics. Serum samples were collected 247 after each immunization and measured for the humoral responses. As expected, the 248 RBD-sc-dimer of SARS-CoV-2 induced a significantly higher antigen-specific IgG compared 249 to the conventional RBD-monomer after each immunization (P<0.0001) ( Figure 3D ). 250

Consistently, RBD-sc-dimer elicited ~10-100-fold higher titer of NAb compare to monomer 251 in an assay with pseudotyped virus ( Figure 3E ). Two immunizations almost maximized the 252 activities after the second immunization against live SARS-CoV-2 (2020XN4276 strain) 254 infection. Impressively, all serum samples from RBD-sc-dimer group showed high NAb titers, 255 with the 50% neutralization titer (NT 50 ) of most samples reaching 4096 and above ( Figure 3F ). 256

In contrast, only 2 out of 8 serum samples from RBD-monomer-vaccinated mice reached 257 NAb titers beyond 16, to a maximum of 256 ( Figure 3F ). To characterize the cellular immune 258 responses, enzyme-linked immunospot (ELISPOT) and intracellular cytokine staining (ICS) 259 assays were performed. We could not detect substantial induction of T-cell responses in 260 RBD-sc-dimer-vaccinated mice compared to the PBS-vaccinated ones after the last 261 vaccination ( Figure S3 ). 262

In order to design RBD-sc-dimer for SARS-CoV, the RBD construct was started at R306 263 (the E367 positon of MERS-CoV S protein) and truncated at Q523, one residue ahead of the 264 C603 position in the MERS-CoV S protein ( Figure S2 ). Two copies of RBD were further 265 linked as tandem repeat. The stable dimer was detected in analytical gel filtration and gel 266 electrophoresis ( Figure 4A ), with a molecular weight of 54.5 kDa as determined by analytical 267 ultracentrifugation ( Figure 4B ). SARS-CoV receptor hACE2 was found to bind 268 RBD-sc-dimer with affinity comparable to its binding to RBD-monomer, suggesting the 269 exposure of RBMs ( Figure 4C and Table S3 ). We also evaluated the immunogenicity for 270 RBD-sc-dimer of SARS-CoV compared with its monomeric form using the same three-dose preventing diseases such as hepatitis B and herpes zoster (Syed, 2018; Valenzuela et al., 1982) . 299

Here we reported the design of CoV RBD-sc-dimer as a protein subunit vaccine, representing 300 a promising pathway for CoV vaccine development. 301

Structure-guided antigen design is an important tool to make vaccines with speed and 302 precision (Graham, 2020). Full-length S protein is another common choice as CoV antigen 303 subunit. Full-length trimeric S protein is usually highly immunogenic due likely to its large 304 size (~600 kDa). It contains not only RBD, the major target for potent neutralizing antibodies, 305 but also non-RBD regions that can also induce neutralizing or protective antibodies, for Figure 4D -E). Thereby, a two-dose vaccination regimen will be applied to evaluate the 324 protective efficacy in animal models and humans for the RBD-sc-dimer-based CoV vaccines. 325

Of note, after three immunizations, the monomeric RBD is similar in terms of 326 immunogenicity as two vaccinations with the sc-dimer. In particular, for SARS-CoV vaccine, 327 RBD-sc-dimer showed only marginally higher antibody response (** P<0.01) and NAb titer 328 (* P<0.05) after three immunizations ( Figure 4D -E). 329

The enhanced immunogenicity of RBD-sc-dimer could be explained by (i) doubling the 330 molecular weight of antigen from ~30 kDa to ~60 kDa, (ii) dual RBMs, by which the dimer 331 works bivalently, that may cross-link B cell receptors in B cells for a better stimulation, (iii) 332 non-RBM epitopes on dimer-interface of RBD are likely occluded to further improve immune 333 focusing and (iv) exposure of the immunodominant epitopes. 334 We provided a universal strategy to design beta-CoV vaccines and proved the concept in 335 vaccine development against MERS, COVID-19 and SARS. The resulting immunogens could 336 be applied to other expression systems, such as yeast, insect cells, and also to other vaccine 337 platforms, like DNA, messenger RNA and vaccine vectors. RBD-sc-dimer engineered without 338 introduction of any exogenous sequence highlighted the feasibility for clinical development of here are of promise for further development from bench to clinic. The antigen yields at g/L 341 level highlight the scale-up production capacity to meet the urgently global demands, in 342 particular, for the pandemic COVID-19. 343

It is known that CoV RBD is the major target for NAbs interfering with viral receptor 346 binding and we were focusing on the humoral response induced by the RBD-based vaccines. 347

An extended study, by e.g. passive transfer experiment, should be performed to further 348 confirm whether the humoral response is sufficient to protect against CoV challenge, though The values shown in (C-F) are the mean ± SEM. The horizontal dashed line indicates the limit 408 of detection. P values were analyzed with one-way ANOVA (ns, P > 0.05; *, P < 0.05; **, P 409 < 0.01; ***, P < 0.001; ****, P < 0.0001). producing key cytokines (IFN-γ, IL-2, TNF-α and IL-4) following stimulation with 10 µg/mL 520 peptide pool (SARS-CoV-2 RBD). Shown are the frequencies of respective 521 cytokine-producing cells. 522

The values are the mean ± SEM. P values were analyzed with unpaired t-test (ns, P > 0.05). 523

Lead Contact. 527

Further information and requests for resources and reagents should be directed to and will 528 be fulfilled by the Lead Contact, George F. Gao (gaof@im.ac.cn). 529

All requests for unique/stable reagents generated in this study should be directed to and 531 will be fulfilled by the Lead Contact author with a completed Materials Transfer Agreement. 532

The accession number for the atomic coordinates and diffraction data reported in this study 534 is PDB code 7C02. All the other data supporting the finding of this study are available within 535 the paper and are available from the corresponding author upon request. of SARS-CoV-2 was two RBD (S protein residues 319-537) connected as tandem repeat. 584 RBD-sc-dimer of SARS-CoV was two RBD (S protein residues 306-523) connected as 585 tandem repeat. For each construct, signal peptide sequence of MERS-CoV S protein (S 586 protein residues 1-17) was added to the protein N terminus for protein secretion, and a 587 hexa-His tag was added to the C terminus to facilitate further purification processes. These The TCID 50 was determined by infection of Huh7 cells (Li et al., 2005b) . To evaluate the 640 pseudovirus neutralization activity of mouse serum, heat-inactivated serum was 2-fold serially 641 diluted and incubated with an equal volume of 100 TCID 50 pseudovirus at 37 °C. The medium 642 was also mixed with pseudovirus as control. Then the mixture was transferred to pre-plated 643 Huh7 cell monolayers in 96 well plates. After incubation for 24 or 48 hours, the cells were 644 lysed and luciferase activity was measured by the Luciferase Assay System (Promega, USA) 645 according to the manufacturer's protocol. NT 90 was defined as the highest reciprocal serum 646 dilution at which the relative light units (RLUs) were reduced by greater than 90% compared 647 with virus control wells. NT 90 below the limit of detection was determined as half the limit of plate and incubated overnight at 37°C in a CO 2 incubator. Then, 100 µL of 10-fold serially 666 diluted suspension was added to each well in quadruplicate. The virus was allowed to adsorb 667 to the cells at 37°C for 1 hour. After adsorption, the viral inocula were removed, and 0.1 mL 668 medium (DMEM, 2% FBS) was added to each well. The plates were incubated in a CO 2 32 magnification. The virus titer of each specimen, expressed as the TCID 50 , was calculated by 671 the Reed-Muench method (Reed and Muench, 1938) . Wang, C., Horby, P.W., Hayden, F.G., and Gao, G.F. (2020a). A novel coronavirus outbreak of 941 global health concern. Lancet 395, 470-473.

A dimeric form of MERS-CoV RBD is highly immunogenic and protective in mice RBD-dimer structure guides further design of a homogeneous dimer by tandem repeat

The strategy is generalizable to design beta-CoV vaccines against COVID-19 and SARS CoV RBD-dimer immunogens can be produced at high yields in pilot scale production

Gao et al. present the structure-guided design of a coronavirus immunogen comprised of two protein subunits each containing the of virus spike receptor binding domain fused together via a disulfide link. The immunogen elicits strong immunogenicity in mice and protects them against viral challenge. The vaccine design strategy can be universally applied to SARS, MERS, COVID-19 and other CoV vaccines to counter emerging threats.

RBD-sc-dimer) and hACE2 (from 0.78125nM to 200nM for SARS-CoV-2 RBD and 739 RBD-sc-dimer, and from 3.125nM to 400 nM for SARS-CoV RBD and RBD-sc-dimer) were

then used to flow over the chip surface at 30 µL/min and the real-time response was recorded

After each cycle, the sensor surface was regenerated using 7 µL of 10 mM NaOH

Sedimentation velocity experiments were carried out on three samples

MERS-CoV RBD-sc-dimer and SARS-CoV RBD-sc-dimer) using the

A volume of 749 380 µL of protein sample (A 280 = 0.6 -0.8) and 400 µL of matching buffer

150mM NaCl, pH8.0) were injected into appropriate channels of 12 mm double sector 751 aluminium epoxy cells with sapphire windows

Python-based system for macromolecular structure solution

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Health Organization consultation

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ENDscript server

Structural basis of receptor recognition by SARS-CoV-2

A human neutralizing antibody targets the receptor binding site of SARS-CoV-2. 918

Novel chimeric virus-like particles vaccine displaying MERS-CoV 944 receptor-binding domain induce specific humoral and cellular immune response in mice

Structural definition of a neutralization-sensitive 948 epitope on the MERS-CoV S1-NTD

Subunit vaccines against emerging 950 pathogenic human coronaviruses

Structure of MERS-CoV spike receptor-binding domain complexed with human 953 receptor DPP4

Structural and functional basis of SARS-CoV-2 entry by using human ACE2

Antibody-dependent SARS coronavirus infection is 959 mediated by antibodies against spike proteins

MolProbity: More and better reference data 967 for improved all-atom structure validation

Global epidemiology of bat 969 coronaviruses

Comparative analysis of complete genome sequences of three 972 avian coronaviruses reveals a novel group 3c coronavirus

A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to 975 its receptor ACE2

dimerization of the N-terminus and trimerization of the ectodomain

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Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the 983 dynamic receptor binding domains

Receptor-binding domain-based domain of MERS-CoV spike glycoprotein

Rapid generation of a mouse model for 991

Middle East respiratory syndrome

Structural definition of a neutralization epitope on the N-terminal domain of 994

Advances in MERS-CoV 996 vaccines and therapeutics based on the receptor-binding domain

A novel coronavirus from patients with pneumonia in China