Immunization with the receptor–binding domain of SARS-CoV-2 elicits antibodies cross-neutralizing SARS-CoV-2 and SARS-CoV without antibody-dependent enhancement

Recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic. Currently, there is no vaccine available for preventing SARS-CoV-2 infection. Like closely related severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2 also uses its receptor-binding domain (RBD) on the spike (S) protein to engage the host receptor, human angiotensin-converting enzyme 2 (ACE2), facilitating subsequent viral entry. Here we report the immunogenicity and vaccine potential of SARS-CoV-2 RBD (SARS2-RBD)-based recombinant proteins. Immunization with SARS2-RBD recombinant proteins potently induced a multi-functional antibody response in mice. The resulting antisera could efficiently block the interaction between SARS2-RBD and ACE2, inhibit S-mediated cell-cell fusion, and neutralize both SARS-CoV-2 pseudovirus entry and authentic SARS-CoV-2 infection. In addition, the anti-RBD sera also exhibited cross binding, ACE2-blockade, and neutralization effects towards SARS-CoV. More importantly, we found that the anti-RBD sera did not promote antibody-dependent enhancement of either SARS-CoV-2 pseudovirus entry or authentic virus infection of Fc receptor-bearing cells. These findings provide a solid foundation for developing RBD-based subunit vaccines for SARS-CoV2.


INTRODUCTION
In December 2019, the coronavirus disease 2019 (COVID19), caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified in Wuhan, China Zhu et al., 2020). Since then, COVID19 cases have been increasingly reported in many countries and on March 11, 2020, the World Health Organization (WHO) declared COVID19 as a pandemic.
Although the symptoms associated with COVID19 are generally mild, approximately 20% of COVID19 patients may develop severe clinical manifestations such as pneumonia, acute respiratory distress syndrome, sepsis and even death (Wu and McGoogan, 2020). According to WHO, as of April 30, 2020, there are 3,090,445 confirmed COVID19 cases and 217,769 deaths in total in the world (https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200430-sitr ep-101-covid-19.pdf?sfvrsn=2ba4e093_2). Clearly, COVID19 is a serious public health crisis. However, there is currently no vaccine available for COVID19. SARS-CoV-2 belongs to the genus Betacoronavirus of the family Coronaviridae (Gorbalenya et al., 2020). Like other coronaviruses, SARS-CoV-2 is an enveloped virus and possesses a ~30 kb single-stranded positive-sense RNA genome. This viral genome encodes 4 structural proteins including spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins, 16 nonstructural proteins, and a few accessory proteins . The S protein consists of an ectodomain, a transmembrane domain, and a short intracellular tail (Walls et al., 2020). The ectodomain can be further divided into two functionally distinct subunits, S1 and S2, which are responsible for receptor binding and membrane fusion, respectively. Like the closely related severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2 also uses human angiotensin-converting enzyme 2 (ACE2) as the key receptor to facilitate its entry into host cells . The S protein binds human ACE2 protein through its receptor-binding domain (RBD) located within the S1 subunit (Lan et al., 2020;Shang et al., 2020;Wang et al., 2020b;Yan et al., 2020) Many approaches have been tested for rapid development of SARS-CoV-2 vaccines, yielding some exciting results (Amanat and Krammer, 2020;Chen et al., 2020). For example, Gao et al. recently reported that an inactivated whole-virus vaccine provided protection in macaques against experimental SARS-CoV-2 challenge (Gao et al., 2020). Thus far, a number of SARS-CoV-2 vaccine candidates derived from different vaccine platforms, including DNA vaccine, mRNA vaccine, inactivated whole virus vaccine, and adenovirus-vectored vaccine, have rapidly progressed into clinical trials (Amanat and Krammer, 2020;Callaway, 2020;. One of the challenges in developing vaccines for coronaviruses is a potential vaccine-induced immune enhancement of disease Huisman et al., 2009). For example, immunization with inactivated whole-virus SARS-CoV vaccine was found to elicit an immune response that exaggerate disease upon viral challenges in animal models (Bolles et al., 2011;Luo et al., 2018;Tseng et al., 2012;Wang et al., 2016). Results from some other studies suggest that antibodies targeting the spike protein of coronaviruses play a major role in antibody-dependent enhancement (ADE) likely through increasing the binding/entry of antibody-bound virion to Fc receptor (FcR)-expressing cells Jaume et al., 2011;Liu et al., 2019;Olsen et al., 1992;Wan et al., 2020;Wang et al., 2014;Yip et al., 2014). So far, all the SARS-CoV-2 vaccine candidates entering clinical trials contain or express full-length or near full-length S protein and therefore bear the risk of ADE.
Thus, it is important to continue the search for a safe and effective SARS-CoV-2 vaccine.
Recombinantly produced RBD proteins of SARS-CoV and MERS-CoV have been shown to potently induce protective neutralizing antibodies against respective viruses in preclinical studies and are therefore considered promising vaccine candidates (reviewed in (Jiang et al., 2012;Zhou et al., 2018)). In the present study, we investigated the vaccine potential of SARS-CoV-2 RBD (hereafter referred as SARS2-RBD). We found that immunization of mice with recombinant SARS2-RBD elicited the production of serum antibodies that efficiently neutralized both SARS-CoV-2 and SARS-CoV but did not promote ADE.

Mice immunized with RBD-Fc fusion protein produced SARS-CoV-2 neutralizing antibodies
To rapidly evaluate the vaccine potential of SARS2-RBD, a pilot mouse immunization study was performed with a recombinant RBD fusion protein containing the Fc region of mouse IgG1 at the C-terminus (RBD-Fc) as the immunogen. The mice received three vaccine doses at days 0, 8, and 13, respectively. The first vaccine dose contained 100 μg of RBD-Fc protein, 500 μg of aluminum hydroxide and 25 μg of CpG, the second one contained 50 μg of RBD-Fc in complete Freund's adjuvant, and the third one contained 50 μg of RBD-Fc formulated with Titermax adjuvant. One week after the last immunization (day 20), serum samples were collected from the three immunized mice for antibody measurement. All three antisera dose-dependently reacted with His-tagged SARS2-RBD in ELISA, whereas the control sera from an age-matching naïve mouse did not show significant reactivity regardless of the sera doses used (Fig. 1A). The anti-RBD-Fc sera #1 that had the highest RBD-binding titer (2×10 5 ) was selected for further functional analyses. The anti-RBD-Fc sera #1 could dose-dependently inhibit the binding between recombinant ACE2-Fc fusion protein and His-tagged SARS2-RBD in competition ELISA (Fig. 1B), indicating that the antisera contain antibodies targeting the ACE2-binding motif within RBD. The anti-RBD-Fc sera #1 was then assessed for its ability to neutralize SARS-CoV-2 S protein-pseudotyped retrovirus (hereafter referred as SARS2-PV) and to neutralize live SARS-CoV-2. As shown in Fig. 1C, the antisera dose-dependently neutralized SARS2-PV entry with a calculated NT50 value of 10513. Moreover, addition of the anti-RBD-Fc sera #1 prevented the development of cytopathic effect (CPE) in SARS-CoV-2-inoculated VeroE6 cells ( Supplementary Fig. 1). Results from qRT-PCR and IFA assays showed that the anti-RBD-Fc sera #1 were highly effective, neutralizing ＞50% infection even at the serum dilution of 1:5120 ( Fig. 1D-E). Taken together, the above results demonstrate that RBD-Fc is an immunogen capable of efficiently inducing SARS-CoV-2-neutralizing antibodies.

RBD alone potently elicited cross-reactive, ACE2-binding blockade antibodies towards SARS-CoV-2 and SARS-CoV
To verify that the RBD part within the RBD-Fc fusion protein is indeed responsible for the induction of neutralizing antibodies against SARS-CoV-2, we performed a second mouse immunization study with recombinant SARS-CoV-2 RBD fused with a small 6xHis tag as the vaccine antigen. A group of six BALB/c mice were immunized by i.p. injection with 5 μg of recombinant RBD protein formulated with Alum adjuvant at days 1, 10, and 25 ( Fig. 2A). Another group of mice were injected with an irrelevant protein (HBc) plus Alum adjuvant, serving as the control. Serum samples were collected from individual mice at days 20 and 40, and analyzed for RBD-specific antibody by ELISA using SARS2-RBD as the capture antigen. As shown in Fig. 2B, neither the day-20 nor the day-40 sera in the control (HBc) group exhibited any significant binding activity; in contrast, SARS2-RBD-binding activity was readily detectable at day 20 in the sera from RBD-immunized mice and a significant increase in SARS2-RBD-binding was observed for the day-40 anti-RBD sera. Equal amount of individual antisera in the same groups were pooled for antibody titer measurement and subsequent analyses. The day-20 and day-40 pooled anti-RBD sera dose-dependently reacted with SARS2-RBD in ELISAs (Fig. 2C) and their binding antibody titers were determined to be 1.6×10 5 and 3.2×10 6 , respectively. The SARS2-RBD-binding activity of the anti-RBD sera collected at day 60 (when the mice were euthanized) was comparable to that of the day-40 anti-RBD sera (Supplemental SARS2-RBD shares high homology with the RBD of SARS-CoV (hereafter referred as SARS-RBD) in amino acid sequence, especially the regions outside the receptor-binding motif. This prompted us to evaluate the cross-reactivity of our SARS2-RBD-immunized sera towards SARS-RBD. As shown in Fig. 2D and 2E, both individual and pooled sera from the SARS2-RBD-immunized mice showed dose-dependent binding activity with SARS-RBD. The SARS-RBD-binding titers of the pooled day-20 and day-40 anti-RBD sera were determined to be 4×10 3 and 1.6× 10 5 , respectively.
The pooled day-40 antisera were assessed for their ability to block the interaction between RBDs and ACE2, the entry receptor for both SARS-CoV-2 and SARS-CoV.
Results from ACE2-binding blockade ELISA showed that the day-40 anti-RBD sera dose-dependently inhibited hACE2-Fc binding to SARS2-RBD whereas the control sera had no inhibitory effect on the SARS2-RBD/hACE2-Fc interaction regardless of the sera concentration used (Fig. 2F). The anti-RBD sera also exhibited blockade effect on the SARS-RBD/hACE2-Fc interaction, albeit with a lower efficiency (Fig.   2G).

Anti-RBD sera inhibited SARS-CoV-2 spike-mediated cell-cell fusion
The spike protein of SARS-CoV-2 has been shown to bind cell surface ACE2 and mediate cell-cell fusion, leading to syncytia formation (Wang et al., 2020a). A cell-cell fusion assay was developed to determine whether the anti-RBD sera could prevent S-mediated syncytia formation. Co-culture of the 293T cells transiently expressing S:EGFP fusion protein and the ones expressing human ACE2 protein fused with mCherry (hACE2:mCherry) for 24 hours led to the detection of dual-fluorescent cells, indicating the occurrence of cell-cell fusion (Supplemental Fig. 3). The cells solely emitting green or red fluorescence and the dual-fluorescent cells were quantified by flow cytometry. It was found that addition of the day-40 anti-RBD antisera to the co-cultures significantly inhibited cell-cell fusion in an antisera dose-dependent manner whereas the control sera did not exhibit significant inhibitory effect regardless of the antisera dose used ( Fig. 2H and Supplemental Fig. 4). This data indicate that the anti-RBD sera are able to inhibit SARS2-S-mediated cell-cell fusion.

Anti-RBD sera neutralized authentic SARS-CoV-2 infection
The antisera were next tested for neutralization of authentic SARS-CoV-2 infection. The anti-RBD sera were found to potently inhibite infection of VeroE6 cells with authentic SARS2-CoV-2 based on CPE observation (Supplemental Fig. 5), whereas the control sera did not prevent CPE development even when a sera dilution of 1:20 (the lowest dilution tested) was used. Both qRT-PCR and IFA assays revealed that the anti-RBD sera diluted 1:1280 nearly completely block the viral infection and even the 1:5120 diluted anti-RBD sera could inhibit viral infection by 83% ( Fig. 3C-D).
These results demonstrate that the anti-RBD sera possessed very strong neutralization capacity against SARS-CoV-2.

Anti-RBD mouse sera did not enhance SARS-CoV-2 infection in Fc receptor-expressing cells.
For some viruses such as dengue virus, antibodies targeting the envelope protein may increase viral infection of FcR-expressing cells -a phenomenon called antibody-dependent enhancement (ADE) (Huisman et al., 2009;Thomas et al., 2006).
Several FcR-bearing cell lines were used as the target cell in infection/neutralization assays to evaluate whether our antisera could mediate ADE in vitro, including mouse A20 cells expressing FcγRII (Antoniou and Watts, 2002), human THP-1 cells expressing both FcγRI and FcγRII (Chan et al., 2011), and K562 cells expressing human FcγRII (Block et al., 2010). Both THP-1 and K562 cells have been shown to support mouse antibody-mediated enhancement of dengue virus infection in previous studies (Block et al., 2010;Sun et al., 2017;Zhang et al., 2019). We found that SARS2-PV entry into the three FcR-expressing cell lines was minimal (＜0.02%) whereas the same amount of SARS2-PV yielded an infection rate of ~7% in VeroE6-hACE2 cells. Moreover, treatment with serially diluted (ranging from 1:10 2 to 1:10 6 ) control sera or anti-RBD sera did not significantly affect SARS2-PV entry of the three cell lines (Fig. 4A-C), indicating that the anti-RBD sera do not promote ADE of SARS2-PV. We selected K562 cells for performing ADE assay with authentic SARS-CoV-2 as the challenge virus. No significant increase in viral RNA level was observed for the antisera-treated samples as compared to the control (cells only infected with the virus) regardless of the antisera dilutions (Fig. 4D). Collectively, these results demonstrate that, in the assay system we tested, the anti-RBD antibodies do not promote ADE.

DISCUSSION
The present study was aimed to investigate the possibility of developing a RBD-based subunit vaccine for SARS-CoV-2. It was found that RBD fusion proteins with either Fc or 6 × His tag elicited high-titer neutralizing antibody responses, indicating that RBD is the antigen component responsible for the induction of neutralizing antibodies. In particular, the anti-RBD sera potently neutralized SARS2-PV entry with an NT50 value of 12764 (Fig. 3A) and its neutralization efficiency against authentic SARS-CoV-2 was ＞75% at the serum dilution of 1:5120 ( Fig. 3C). According to a recent report (Gao et al., 2020), macaques immunized with a purified inactivated SARS-CoV-2 virus vaccine candidate (which induced NT50 titers up to 3,000 in mice) produced neutralizing serum antibody titers of around 50 to 61 and were protected from authentic SARS-CoV-2 challenge. Therefore, the seemingly higher neutralizing antibody titers of our anti-RBD mouse antisera compared to that of the mouse antisera against the inactivated whole-virus vaccine (although not directly compared) strongly suggest that the RBD subunit vaccine candidate will likely be protective as well.
The anti-RBD sera not only were able to efficiently neutralize SARS-CoV-2 infections but also could inhibit SARS2-S-mediated cell-cell fusion ( Fig. 2H and Supplementary Fig. S3 and S4), demonstrating a two-layer protective potential of the antisera. Host receptor recognition and binding is required for coronavirus entry.
Previous work showed that, for SARS-CoV and MERS-CoV, RBD binding to the receptor triggers the spike to undergo conformational changes, allowing subsequent protease cleavage, virus internalization and membrane fusion (Millet and Whittaker, 2018;Pallesen et al., 2017). In this study, the anti-RBD sera were found to dose-dependently reduce the RBD binding to ACE2 in ELISAs ( Fig. 1B and 2F).
These data suggest blocking the interaction between RBD/S and the host receptor ACE2 is the main mechanism underlying the observed neutralization and cell-cell fusion inhibition by the anti-RBD sera.
Interestingly, we found that the mouse antisera raised against SARS2-RBD cross-reacted with SARS-RBD, inhibited SARS-RBD binding to ACE2, and neutralized SARS-PV entry with an NT50 of 834.8. SARS2-RBD can be divided into a core subdomain and a receptor-binding motif (RBM) which directly engages ACE2 (Lan et al., 2020;Shang et al., 2020;Yan et al., 2020). The core subdomain is highly conserved while the RBM varies significantly (~47% homology in amino acid sequence) between SARS-CoV and SARS-CoV-2. Therefore, we reason that the observed cross binding and neutralization capacity towards SARS-CoV is contributed by antibodies targeting the conserved core subdomain of SARS2-RBD. That is to say, the core subdomain contains SARS-CoV-2/SARS-CoV cross-neutralization antibody epitopes. This information will be useful for future design and development of pan-SARS-CoV vaccines.
A major concern in developing coronavirus vaccines is the risk of vaccine-induced ADE Huisman et al., 2009). ADE phenomenon has been observed for feline coronavirus (Huisman et al., 1998;Huisman et al., 2009;Weiss and Scott, 1981) and for SARS-CoV (Bolles et al., 2011;Luo et al., 2018;Tseng et al., 2012;Wang et al., 2016). Some cell culture studies suggested that anti-S antibodies mediated ADE in FcR-expressing cells likely through enhancing FcR-mediated internalization/entry of antibody-bound virions Jaume et al., 2011;Liu et al., 2019;Olsen et al., 1992;Wan et al., 2020;Wang et al., 2014;Yip et al., 2014). In the present study, we showed that the anti-RBD sera did not enhance SARS2-PV entry of the three FcR-expressing cell lines (A20, THP-1, and K562) regardless of the antisera concentration. Moreover, treatment with the anti-RBD sera did not increase authentic SARS-CoV-2 infection of K562 cell, despite the same cell line has been shown to support anti-DENV-E mouse antibody-triggered ADE of Dengue virus (DENV) infection in previous studies (Block et al., 2010;Sun et al., 2017;Zhang et al., 2019). These data clearly show that anti-RBD antibodies do not promote ADE, at least not in the assay system we used. It remains to be determined whether antibodies targeting other regions of the S protein (which presumably will be non-neutralizing or poorly neutralizing) could mediate ADE of SARS-CoV-2. Nonetheless, the extraordinary neutralization potency and lack of ADE effect observed for the anti-RBD sera indicate that SARS2-RBD is an elite antigen target for developing subunit vaccines for SARS-CoV-2.
Collectively, our results show that recombinantly expressed SARS2-RBD proteins potently elicits cross-neutralizing antibodies against both SARS-CoV-2 and SARS-CoV without induction of ADE antibodies, providing important information for further development of RBD-based SARS-CoV-2 or pan-SARS-CoV subunit vaccines.

Recombinant proteins
For mouse immunization, recombinant SARS-CoV-2 RBD fusion protein with the Fc region of mouse IgG1 at the C-terminus (RBD-Fc) was purchased from Sino Biological (Beijing, China), recombinant SARS-CoV-2 RBD with a C-terminal His-tag was purchased from Kactus Biosystems (Shanghai, China), and recombinant hepatitis B core antigen (HBc) was produced in house in E.coli as described previously (Ye et al., 2014). For biochemical and immunological assays, several mammalian cell-produced recombinant proteins were generated in house, including SARS-CoV-2 RBD (amino acids 320 to 550) fused with an N-terminal Strep-tag and a C-terminal His-tag, SARS-CoV RBD (amino acids 306-520) fused with a C-terminal His-tag, and human ACE2 ecotodomain fused with human IgG1 Fc at the C-terminus (hACE2-Fc).

Mouse immunization
All the animal experiments in this study were approved by the Institutional Animal

Serum antibody measurement
For antibody measurement, wells of 96-well microtiter plates were coated with the indicated amounts of the SARS2-RBD or SARS-RBD recombinant protein for 2 hrs at 37°C or overnight at 4°C. Then the wells were blocked with PBST containing 5% non-fat dry milk for 1 hr at 37℃, incubated with 50μl serially diluted mouse antisera for 2 hrs at 37°C and then with 50 μl of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody for 1 hr 37°C. After color development, the absorbance at 450 nm was measured in a 96-well plate reader. For a given serum sample, its endpoint titer was reported as the reciprocal of the highest serum dilution that had an absorbance ≥0.1 OD unit above the blank.

ACE2 competition ELISA
Wells of 96-well microtiter plates were coated with 25 ng/well of the SARS2-RBD or SARS-RBD recombinant protein overnight at 4°C, followed by blocking with PBST containing 5% non-fat dry milk for 1 hr at 37℃. Serially diluted mouse antisera were mixed with 20 ng of biotinylated hACE2-Fc in a final volume of 50μl and the mixtures were added to the wells, followed by incubation for 2 hrs at 37°C. Then, 50 μl of horseradish peroxidase (HRP)-conjugated streptavidin (Life Technologies, USA) was added to wells, followed by incubation for 1 hr at 37°C. After washing, TMB substrate (Life Technologies) was added into wells for color development. The plates were read for absorbance at 450 nm in a 96-well plate reader.

ADE assay
FcR-expressing cell lines, including A20, THP-1, and K562, were used to perform ADE assays. Briefly, the antisera were serially diluted, mixed with either SARS2-PV or authentic SARS-CoV-2 (6,000 PFU), and incubated at 37℃ for 1 hr. Then, the mixtures were added to the target cells. The following infection and culturing steps were carried out as described above in the pseudovirus neutralization and live virus neutralization assays. Mock-infected cells and cells only infected with SARS2-PV or authentic SARS-CoV-2 were set as the negative and positive controls, respectively.
Infection rates of the samples were determined as described above.

Statistics analysis
All statistical analyses were performed using GraphPad Prism software v5.0.