The recombinant receptor-binding domain of a pangolin coronavirus spike by prokaryotic expression can partially induce neutralizing antibodies

doi:10.21203/rs.3.rs-2501181/v1

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The recombinant receptor-binding domain of a pangolin coronavirus spike by prokaryotic expression can partially induce neutralizing antibodies

https://doi.org/10.21203/rs.3.rs-2501181/v1

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We previously reported that a SARS-CoV-2 related pangolin coronavirus GX_P2V can induce neutralizing antibodies against SARS-CoV-2 in a golden hamster model. The two viruses GX_P2V and SARS-CoV-2 have genomes with high homology and both use ACE2 as their receptor, but have distinct receptor-binding domains (RBDs) of their spike proteins. The shared neutralizing epitopes of these two viruses are unknown. Here we describe a novel method of soluble expression of GX_P2V RBD in E. coli by tagging RBD with a carboxy-terminal fragment of the SADS coronavirus nucleocapsid protein. The recombinant GX_P2V RBD have human ACE2-binding activities. Mice immunized with this recombinant RBD produced significant titers of neutralizing antibodies against GX_P2V pseudoviruses, not SARS-CoV-2 pseudoviruses. Our data suggest that the GX_P2V RBD has none or limited neutralizing epitopes against SARS-CoV-2, thus neutralizing antibodies against SARS-CoV-2 from GX_P2V infected golden hamsters is likely RBD-independent.

COVID-19, an ongoing pandemic caused by SARS-CoV-2, was firstly reported in Wuhan, China [1]. As of September 2022, the world health organization has reported more than 605 million COVID-19 cases with more than 6.5 million deaths. The origin of SARS-CoV-2 remains unknown, but was likely from bats or pangolins [1, 2]. We previously reported a SARS-CoV-2 related pangolin coronavirus isolate GX_P2V. The genome of this GX_P2V isolate shares high homology (~ 90% nucleotide identity) with that of SARS-CoV-2 [2]. Subsequent animal experiments found that GX_P2V infections in golden hamsters could induce high titrations of neutralizing antibodies against SARS-CoV-2 [3]. Findings from animal experiments combined with the fact GX_P2V is not a human pathogen prompt us to propose that the GX_P2V virus is a live vaccine candidate against COVID-19. However, the mechanism of GX_P2V inducing cross-reacting neutralizing antibodies against SARS-CoV-2 is unknown.

The receptor binding domain (RBD) of the coronavirus spike protein is the major target of neutralizing antibodies [4]. Candidate COVID-19 vaccines based on SARS-CoV-2 RBDs have been developed and tested [4, 5]. Especially, a subunit vaccine based on a tandem-repeat dimeric RBD protein–ZF2001 for COVID-19 prevention has been approved in China [6].

As SARS-CoV-2 variants continue to surge, RBD is also a target immunogen for designing pan-SARS-CoV-2 vaccines. SARS-CoV-2 and pangolin coronavirus GX_P2V use a same receptor ACE2 for infection, while their RBDs have only 86% amino acid identity [7]. We intend to test whether RBD of GX_P2V can induce neutralizing antibodies against SARS-CoV-2 in animals, which might provide ideas for the design of RBD-based COVID-19 vaccines.

Here we report a novel method of soluble expression of GX_P2V RBD in E. coli by tagging RBD with a portion of nucleocapsid protein from the swine acute diarrhea syndrome coronavirus (SADS-CoV). Recombinant GX_P2V RBD is capable of binding to human ACE2 in a binding assay. BALB/c mice immunized with recombinant GX_P2V RBDs produced a significant titration of neutralizing antibodies against GX_P2V pseudoviruses. However, no neutralizing antibodies against SARS-CoV-2 pseudoviruses were detected in the immunized sera. Our data suggest that the capability of GX_P2V inducing cross-reacting neutralizing antibodies against SARS-CoV-2 is largely RBD-independent.

Protein expression

E. coli BL21(DE3) transformants containing the expression vector of GX_P2V RBD was constructed by Beijing Diagreat biotechnology company. Single colonies of transformants were used to inoculate 2 mL of LB-ampicillin medium, and the cell suspension was incubated at 37°C for 2h under constant shaking (220 rpm). These pre-cultures were diluted (1:1000) in fresh LB-ampicillin medium and grown at 37°C for 3 h. Finally, overexpression of GX_P2V RBD was induced with 0.15 mM IPTG (Cat. No. M15211, MERYER) for 20 h under constant shaking (120 rpm), at 15°C. E. coli cells were collected by centrifugation (8,000 g, 15 min, 4°C).

Protein Purification

The E. coli cells were resuspended in buffer A (20 mM Tris-HCl, pH7.4, 30 mM imidazole, and 500mM NaCl). Cells were broken by a high-pressure cell homogenizer (JN-Mini Pro, JNBIO, Guangzhou, China) and supernatants were collected after centrifugation for 60 min at 8,000 ×g. Recombinant GX_P2V RBDs were purified by binding to Ni-NTA agarose (Cat. No. BN26070, BIORIGIN), washed with Ni wash buffer (Ni lysis buffer with 50 mM imidazole), and eluted with Ni elution buffer (Ni lysis buffer with 250 mM imidazole). Proteins were collected in the presence of protease inhibitors (Cat. No. IA0110, Solarbio) and protein buffers were changed to PBS by using Amicon Ultra centrifugal filters.

ELISA

For direct ELISA, ELISA plate (Corning) was coated with hACE2 proteins (Cat. No. P2325, Beyotime) at 100ng per well in 100µl Tris-HCl buffer A (50mM, pH 7.5, containing 150 mM NaCl) overnight at 4°C. Then plates were washed with Tris-HCl buffer B (12mM, pH 7.5, containing 140 mM NaCl, 3 mM KCl, 0.05% Tween-20) and incubated with 200 µl/well of a blocking solution of Tris-HCl buffer C (2% BSA (w/v) in Tris-HCl buffer B) for 2 h at room temperature. HRP-conjugated GX_P2V RBD (MD4001, MDTK-BIO) was added to the hACE2-coated plate at different concentrations in 100µl of sample diluent for 1 h at room temperature. Unbound HRP-conjugated antigens were removed by five washes with Tris-HCl buffer B. A colorimetric signal was developed on the enzymatic reaction of HRP with a chromogenic substrate, 3,3',5,5'-tetramethylbenzidine (TMB) (Cat. No. PR1210, Solarbio). An equal volume of TMB stop solution (2M H₂SO₄) was added to stop the reaction, and the absorbance readings at 450nm were acquired using a synergistic H1 hybrid reader (Biotek, USA).

For indirect ELISA, 100ng recombinant GX_P2V RBD was coated onto the ELISA plate using Tris-HCl buffer A and incubated overnight at 4°C. Sera from BALB/c mice immunized GX_P2V RBD were tested at a dilution of 1:10 to 1:10,000,000 and detected by goat anti-mouse IgG (H + L) HRP conjugate (Cat. No. HS201-01, Trans) at 1:1,000 dilution. Wells were blocked using Tris-HCl buffer C for 2 h at room temperature and heat-inactivated sera at 1:10 dilution were added and incubated for 1 h at 37°C. The chromogenic reaction was quantified following the addition of TMB substrate and stop solution (2M H₂SO₄). The absorbance of the samples was measured at 450 nm. Luciferase activity was then measured using a synergistic H1 hybrid reader (Biotek, USA).

Mice Immunization

Specific pathogen-free 6-week-old female young BALB/c mice were purchased from Charles River. The immunogen was prepared by mixing up GX_P2V RBD and aluminum hydroxide adjuvant (Cat. No. C07-01013, Bioss). 56 µg of GX_P2V RBD in 50 µl were mixed with triple volume of aluminum hydroxide adjuvant (Cat. No. C07-01013, Bioss) for boosts. The mixture protein was shaken for 30 minutes at 4°C before intraperitoneal injection in mice. Intraperitoneal injections were performed at each indicated time point (1, 14, and 28 d). At the 7, 14, and 28th day, caudal blood was sampled before immunization. The blank control group was injected with only aluminum hydroxide adjuvant. On day 35, mice were anesthetized intraperitoneally with pentobarbital sodium and orbital blood sampling was performed. All fresh blood was kept overnight at 4°C and centrifuged at 3,000 g for 5 min the following day for splitting and freezing.

Pseudotyped Virus Neutralization Assay

The SARS-CoV-2 pseudoviruses expressing a luciferase reporter gene were generated in an approach similar to the previous description [8]. Serum samples were diluted into five 3-fold serial dilutions and incubated with 1000 TCID₅₀ of pseudovirus in a 96-well plate to make the initial dilution at the appropriate dilution. After incubation at 37°C for 1 hour, cells (2.5 x 10⁴ cells/well) were added and incubated for a further 24 hours in a CO₂ incubator at 37°C. Cells were lysed by the addition of luciferase substrate (Cat.No.11401ES76, Yeason). Luciferase activity was then measured using a synergistic H1 hybrid reader (Biotek, USA). Using the Reed-Muench method, a 50% neutralization titer (NT₅₀) was calculated for each serum sample.

Design and Soluble expression of recombinant GX_P2V RBD in E. coli

The vector pGEX-4T-1 was used for the recombinant expression GX_P2V RBD (Fig. 1A). The recombinant protein consists of three tandem segments -GST (glutathione-S-transferase), NC188 (C-terminal 188 amino acids of SADS-coronavirus nucleocapsid) and GX_P2V RBD (319–541 amino acids of GX_P2V spike protein). The GST gene fusion system is a method for high-level protein expression, and has been reported for the soluble expression of SARS-CoV-1 RBD. The C-terminal SADS-coronavirus nucleocapsid was also used as a tag in an attempt to increase the solubility and immunogenicity of recombinant GX_P2V RBD. This NC188 tag is chosen as its gene sequence is different from the analogous sequence of SADS-CoV-2 and cannot cause false positive results of SARS-CoV-2 PCR detection. The soluble recombinant GX_P2V RBD was expressed, and purified by utilizing a nickel ion affinity resin (Fig. 1B, supplementary Fig S1). Its predicted molecular weight is 71kDa. Whether and how these two tags contribute to the solubility of recombinant GX_P2V RBD is not studied.

Recombinant Gx_p2v Rbd Possesses Hace2 Binding Activity

Cellular entry of SARS-CoV-2 into human cells is mediated via RBDs binding to cell-surface-expressed ACE2 molecules [9]. Mutations in the RBD region that promote stronger ACE2 binding activities tend to be selected [10]. Next, we tested the potential of recombinant GX_P2V RBD binding to human ACE2 (hACE2) by using horseradish peroxidase (HRP)-tagged GX_P2V RBD in a direct ELISA assay. Our results show that the GX_P2V RBD protein has a dose-dependent hACE2 binding property, can bind to hACE2 at a concentration of ~ 27 nM –a similar concentration reported with SARS-CoV-2 RBDs [11] (Fig. 2). Thus, our results confirm that GX_P2V RBD has a strong hACE2-binding activity, although the amino acid sequence of GX_P2V RBD is divergent from those of SARS-CoV-1 and SARS-CoV-2. The native spike proteins of SARS-CoV-2 form trimers that functions as attachment proteins binding to hACE2. Whether recombinant GX_P2V RBDs form multimers is unknown. We tried protein cross-linking assays, but the results did not suggest the likelihood of forming multimers.

Mice Immunized With Recombinant Gx_p2v Rbds Can Produce Neutralizing Antibodies Against Gx_p2v, But Not Sars-cov-2

To test whether recombinant GX_P2V RBDs can induce neutralizing antibodies against GX_P2V and SARS-CoV-2, antisera were prepared by three consecutive immunizations in BALB/c mice with recombinant GX_P2V RBDs (Fig. 3A). After the final immunization, ELISA titers of the antisera were bigger than 1:10⁶, indicating a good immunogenicity of the recombinant proteins. Then, the antisera were analyzed by using a pseudovirus neutralization assay (Fig. 3B). Clearly, the mouse antisera have neutralizing antibodies against GX_P2V pseudoviruses, but no significant neutralizing activities against SARS-CoV-2 pseudoviruses. In contrast, the golden hamster sera collected from GX_P2V infected animals have a high neutralizing titer against both GX_P2V and SARS-CoV-2. It is noted that the mouse antisera have a lower neutralizing titer against GX_P2V than golden hamster sera prepared by viral infections, suggesting that the recombinant GX_P2V RBD or mouse immunization are not optimal. In conclusion, the above results suggest that the capability of GX_P2V inducing cross-reacting neutralizing antibodies against SARS-CoV-2 is largely RBD independent.

The nature of pangolin coronavirus GX_P2V inducing a significant titer of cross-reactive neutralizing antibodies against SARS-CoV-2 is unknown. The RBDs of these two viruses are the major neutralizing targets, as they can unequivocally bind to hACE2 receptors. Thus, it is logical to test whether the recombinant RBD of GX_P2V has shared neutralizing epitopes with that of SARS-CoV-2. The recombinant GX_P2V RBDs have normal function of binding to its receptors, and can induce neutralizing antibodies against GX_P2V, but not SARS-CoV-2. These results suggest that the odds of shared neutralizing epitopes in GX_P2V RBDs are low. The caveat of this study is the use of prokaryotic proteins as these proteins have no mammalian glycosylation modifications.

Acknowledgements

We thank Pro. Zheng Zhang at Shenzhen Third People's Hospital for kindly providing stable hACE2-293T cells.

Author contributions

Lihua Song conceived and designed the research. Shuiqing Liu, Chen Chen, Shanshan Lu, Dongxiao Cheng, and Shengdong Luo conducted experiments. Lihua Song, Yigang Tong, Zeliang Chen, and Huahao Fan performed analyses. Shuiqing Liu and Lihua Song wrote the paper. All of us reviewed and approved the manuscript.

Funding

This research was supported by NSFC-MFST projects (China-Mongolia) (No. 32161143027), Inner Mongolia Key Research and Development Program (No. 2019ZD006) and Foundation of State Key Laboratory of Pathogen and Biosecurity (No. SKLPBS2250).

Availability of data and materials

The materials used in the current study are available from the corresponding author on reasonable request.

Ethics approval

All animals involved in this study were housed and cared for in an AAALAC (Association for the Assessment and Accreditation of Laboratory Animal Care)-accredited facility. The procedure of animal experiments (IACUC-2019-0027) was approved by the Institutional Animal Care and Use Committee of Fifth Medical Center, General Hospital of Chinese PLA, which complies with IACUC standards.

Consent for publication

Not applicable.

Competing interests

Authors have no competing interests to declare.

Author details

¹Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China. ²Research Center for Clinical Medicine, The Fifth Medical Center of PLA General Hospital, Beijing, China. ³Department of Epidemiology, School of Public Health, Sun Yat-sen University, Guangzhou, Guangdong Province, China.

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No competing interests reported.

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Supplementary Information The online version contains supplementary material available at. Additional file 1: Original images for the construction of figure 1B.

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The recombinant receptor-binding domain of a pangolin coronavirus spike by prokaryotic expression can partially induce neutralizing antibodies

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Version 1

Abstract

Figures

Introduction

Materials And Methods

Protein expression

Protein Purification

ELISA

Mice Immunization

Pseudotyped Virus Neutralization Assay

Results

Recombinant Gx_p2v Rbd Possesses Hace2 Binding Activity

Mice Immunized With Recombinant Gx_p2v Rbds Can Produce Neutralizing Antibodies Against Gx_p2v, But Not Sars-cov-2

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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