key: cord-308310-wtmjt3hf
authors: Zha, Lisha; Zhao, Hongxin; Mohsen, Mona O.; Hong, Liang; Zhou, Yuhang; Li, Zehua; Yao, Chuankai; Guo, Lijie; Chen, Hongquan; Liu, Xuelan; Chang, Xinyue; Zhang, Jie; Li, Dong; Wu, Ke; Vogel, Monique; Bachmann, Martin F; Wang, Junfeng
title: Development of a COVID-19 vaccine based on the receptor binding domain displayed on virus-like particles
date: 2020-05-14
journal: bioRxiv
DOI: 10.1101/2020.05.06.079830
sha: 
doc_id: 308310
cord_uid: wtmjt3hf

The recently ermerging disease COVID-19 is caused by the new SARS-CoV-2 virus first detected in the city of Wuhan, China. From there it has been rapidly spreading inside and outside China. With initial death rates around 4%, COVID-19 patients at longer distances from Wuhan showed reduced mortality as was previously observed for the SARS coronavirus. However, the new coronavirus spreads more strongly, as it sheds long before onset of symptoms or may be transmitted by people without symptoms. Rapid development of a protective vaccine against COVID-19 is therefore of paramount importance. Here we demonstrate that recombinantly expressed receptor binding domain (RBD) of the spike protein homologous to SARS binds to ACE2, the viral receptor. Higly repetitive display of RBD on immunologically optimized virus-like particles derived from cucumber mosaic virus resulted in a vaccine candidate (RBD-CuMVTT) that induced high levels of specific antibodies in mice which were able to block binding of spike protein to ACE2 and potently neutralized the SARS-CoV-2 virus in vitro.

onset of symptoms or may be transmitted by people without symptoms. Rapid development of a protective vaccine against COVID-19 is therefore of paramount importance. Here we demonstrate that recombinantly expressed receptor binding domain (RBD) of the spike protein homologous to SARS binds to ACE2, the viral receptor. Higly repetitive display of RBD on immunologically optimized virus-like particles derived from cucumber mosaic virus resulted in a vaccine candidate (RBD-CuMVTT) that induced high levels of specific antibodies in mice which were able to block binding of spike protein to ACE2 and potently neutralized the SARS-CoV-2 virus in vitro.

COVID-19 is caused by a novel coronavirus closely related to viruses causing SARS and MERS. As the disease caused by the other two viruses, COVID-19 mainly manifests symptoms in the lung and causes cough and fever 1 . The disease COVID-19 is less severe than SARS and MERS, which is beneficial per se but leads to easier and wider spread of the virus, in particular due to infected individuals with very little symptoms ("spreaders") and a long incubation time (up to 3 weeks) combined with viral shedding long before disease onset 2 .

A vaccine with rapid onset of protection is therefore in high demand for the control of the pandemic that is currently taking its course.

The spike protein of COVID-19 is highly homologous to the spike protein of SARS and both viruses share the same receptor, which is angiotensin converting enzyme 2 (ACE2) 3, 4 . The receptor binding domain (RBD) of the SARS spike protein binds to ACE2 and is an important target for neutralizing antibodies [5] [6] [7] . By analogy, the RBD of COVID-19 spike protein may be expected to similarly be the target of neutralizing antibodies, blocking the interaction of the virus with its receptor. We have previously shown that antigens displayed on virus-like particles (VLP) induce high levels of antibodies in all species tested, including humans 8 .

More recently, we have developed an immunologically optimized VLP platform based on cucumber mosaic virus. These CuMVTT VLPs (hereafter CuMVTT) incorporate a universal T cell epitope derived from tetanus toxin providing pre-existing T cell help. In addition, during the production process these VLPs package bacterial RNA which is a ligand for toll-like receptor 7/8 and serves as potent adjuvants 9 . Using antigens displayed on these VLPs, it 4 was possible to induce high levels of specific antibodies in mice, rats, cats, dogs and horses and treat diseases such as atopic dermatitis in dogs or insect bite hypersensitivity in horses [9] [10] [11] . To generate a COVID-19 vaccine candidate, we therefore attempted to display the RBD domain on CuMVTT (Fig. 1a) . To this end we gene-synthesized the COVID-19 RBD domain and fused it to an Fc molecule for better expression. As expected, the protein bound efficiently to the viral receptor ACE2 as determined by Sandwich ELISA (Fig. 1b) . In a next step, the protein was chemically coupled to the surface of CuMVTT using the well established chemical cross-linkers SATA and SMPH (ref. 9). SDS-Page and Western Blotting confirmed efficient coupling of the RBD-fusion molecule to CuMVTT, resulting in the vaccine candidate RBD-CuMVTT (Fig. 1c,d) .

To test immunogenicity of the vaccine candidate, mice were immunized three times (weekly schedule) with the RBD-fusion molecule alone or conjugated to the surface of CuMVTT formulated in Montanide adjuvants. As shown in Fig. 2a -c, coupling to VLPs dramatically increased the immunogenicity of the RBD. As shown by ELISA on recombinant RBD, RBD-CuMVTT showed strongly increased immunogenicity at all time-points tested (one week after the vaccine injection time-points). To assess the potential for anti-viral activity, we assessed whether the induced antibodies were able to block binding of the RBD protein to the viral receptor ACE2. As shown in Fig. 3 , immune sera obtained after two boosts (day 21) were able to strongly inhibit RBD binding to ACE2.

The best correlate of protection is viral neutralization. To this end, we generated pseudotyped retroviruses 12 expressing the SARS-CoV-2 spike protein and luciferase for quantification of infection (Fig. 4a ). Using these viruses, the neutralizing capacity of the sera from immunized mice was assessed on ACE2-transfected cells (Fig. 4b) , directly demonstrating high anti-viral neutralizing activity of the induced antibodies. Hence, the RBD-CuMVTT vaccine candidate is able to induce high levels of SARS-CoV-2 neutralizing antibodies. Furthermore, the CuMVTT based vaccine is based on highly efficient expression systems and chemical conjugation technologies, rendering it an attractive candidate for large scale production under cGMP. Previous studies with a similar VLP-based conjugate vaccine has demonstrated that high levels of specific antibodies can be mounted within a week 13, 14 (see also Fig. 2a) , offering the additional possibilities to rapidly immunize individuals that have been exposed to infected humans or those that are kept in quarantine. Thus, vaccines based on the SARS-CoV-2 RBD domain displayed on VLPs may have the potential to critically interfere with global spread of the virus. 5 

The SARS-CoV-2 receptor-binding domain (RBD) and the N-terminal peptidase domain of human ACE2 were expressed using 293F cells (Invitrogen). The SARS-CoV-2 RBD (residues Arg319-Phe541) with an N-terminal IL-2 signal peptide for secretion and a Cterminal Fc tag for purification was inserted into pFUSE-mIgG1-Fc2 vector (Invitrogen). The construct was transformed into bacterial DH5α competent cells, and the extracted plasmid was then transfected into 293F cells at a density of 3×10 6 cells/ml using PEI (Invitrogen). The cell culture supernatant containing the secreted RBD was harvested 96 h after infection, concentrated and buffer-exchanged to HBS (10 mM HEPES, pH 7.2, 150 mM NaCl). RBD was captured by protein A resin (GE Healthcare) and eluted with Gly-HCl buffer pH 2.2.

Fractions containing RBD were collected and neutralized to pH 7.0 with 1M Tris. For ELISA coating, ACE2 was cleaved from the Fc part using thrombin as described in the manufacturer's manual.

The human ACE2 (residues Ser19-Ser741) with an N-terminal IL-2 signal peptide for secretion and a C-terminal 6×His tag for purification was inserted into pFUSE-vector (Invitrogen). The human ACE2 was expressed by essentially the same protocol used for the SARS-CoV-2 RBD. ACE2 was captured by Ni-NTA resin (GE Healthcare) and eluted with 500 mM imidazole in HBS buffer. RBD was then purified by gel filtration chromatography using the Superdex 200 column (GE Healthcare) pre-equilibrated with HBS buffer. Fractions containing ACE2 were collected.

The antibody competitive binding activities of the serum were assayed by ELISA. ACE2 (1ug/ml) was incubated in 96-well plate overnight at 4°C. After incubation, the plate was blocked with 2% BSA for 2h at 37°C and then washed five times with PBS containing 0.05% Tween 20. BSA was used as negative control followed by the addition of a mixture of 40-fold diluted serum and RBD-mFc (0.15ug/ml) followed by incubation for 30 min with gentle shaking at 37°C. Plates were washed five times with PBS containing 0.05% Tween 20 (PBT) followed by 100 µl of horseradish peroxidase/anti-mFc antibody conjugate (diluted 1:5000 in PBT buffer), incubated 30 min with gentle shaking. Plates were washed five times PBT buffer and developed with 100 µl of freshly prepared 3,3',5,5'-Tetramethylbenzidine (TMB) substrate. Reaction was stopped with 100 µl of 1.0 M H3PO4 and read spectrophotometrically at 450 nm in a microtiter plate reader. 6 

The production of CuMVTT was described in detail in Zeltins et al. 9 Briefly, E coli C2566 cells 

The RBD was conjugated to CuMVTT using the cross-linker Succinimidyl 6-(betamaleimidopropionamido) hexanoate (SMPH) (Thermo Fisher Scientific, 10-molar excess, 60 minutes, 23°C). The coupling reactions were performed with 0.3x molar excess of RBD, 0.3x RBD, or equal molar amount of RBD regarding the CuMVTT (shaking at 23°C for 3 hours at 1200 rpm on DSG Titertek; Flow Laboratories, Irvine, United Kingdom). Unreacted SMPH and RBD proteins were removed using Amicon-Ultra 0.5, 100K (Merck-Millipore, Burlington, Mass). VLP samples were centrifuged for 2 minutes at 14,000 rpm for measurement on ND-1000. Coupling efficiency was calculated by densitometry (as previously described for IL17A-CuMVTT vaccine 9 ), with a result of approximately 20% to 30%. 

Pseudovirus expressing the SARS-CoV-2 spike protein was produced by lentivirus second- 

The 293T-ACE2 cells which stably express ACE2 receptors on the cell membrane were prepared by transfection of ACE2 gene into 293T cells using lentivirus system.

Pseudoviruses prepared above were added to the 293T-ACE2 cells (3 × 10 4 cells/well) with 100 μl polybrene (16μg/ml). After 48 h, the infection was monitored using the Luciferase Assay System (Promega). Titer was calculated based on serial dilutions of pseudovirus.

The mouse serum samples (2 μl) were diluted to 1:10, 1:40, 1:160, 1:640 and 1:2560 respectively, and then mixed with an equal volume of pseudovirus stock. After incubation at 37°C for 1 h, the mixture was inoculated on the 293T-ACE2 cells (3 x 10 4 cells/well). At the same time, pseudovirus+DMEM medium was set as a positive control and DMEM medium only was set as a negative control. After the cells were incubated for 72 hours, serum neutralization was measured by luciferase activity of infected pseudovirus. A cut-off of >80% was used as to determine neutralizing titer. 

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