key: cord-0793042-8kjfzz3e
authors: Sun, Shihui; He, Lei; Zhao, Zhongpeng; Gu, Hongjing; Fang, Xin; Wang, Tiecheng; Yang, Xiaolan; Chen, Shaolong; Deng, Yongqiang; Li, Jiangfan; Zhao, Jian; Li, Liang; Li, Xinwang; He, Peng; Li, Ge; Li, Hao; Zhao, Yuee; Gao, Chunrun; Lang, Xiaolin; Wang, Xin; Fei, Guoqiang; Li, Yan; Geng, Shusheng; Gao, Yuwei; Wei, Wenjin; Hu, Zhongyu; Han, Gencheng; Sun, Yansong
title: Recombinant Fc-fusion vaccine of RBD induced protection against SARS-CoV-2 in non-human primate and mice
date: 2020-11-30
journal: bioRxiv
DOI: 10.1101/2020.11.29.402339
sha: d7fafd8b3d1836845861e939b4be7a74e608e746
doc_id: 793042
cord_uid: 8kjfzz3e

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to infect people globally. The increased COVID-19 cases and no licensed vaccines highlight the need to develop safe and effective vaccines against SARS-CoV-2 infection. Multiple vaccines candidates are under pre-clinical or clinical trails with different strengths and weaknesses. Here we developed a pilot scale production of a recombinant subunit vaccine (RBD-Fc Vacc) with the Receptor Binding Domain of SARS-CoV-2 S protein fused with the Fc domain of human IgG1. RBD-Fc Vacc induced SARS-CoV-2 specific neutralizing antibodies in non-human primates and human ACE2 transgenic mice. The antibodies induced in macaca fascicularis neutralized three divergent SARS-CoV2 strains, suggesting a broader neutralizing ability. Three times immunizations protected Macaca fascicularis (20ug or 40ug per dose) and mice (10ug or 20ug per dose) from SARS-CoV-2 infection respectively. These data support clinical development of SARS-CoV-2 vaccines for humans. RBD-Fc Vacc is currently being assessed in randomized controlled phase 1/II human clinical trails. Summary This study confirms protective efficacy of a SARS-CoV-2 RBD-Fc subunit vaccine.

The COVID-19 pandemic, caused by a novel Coronavirus SARS-CoV-2, has infected more than 60 million individuals and killed more than 1.4 million people globally 1 . Because of the high prevalence, longer incubation, no preexisting immunity to SARS-CoV-2 in human and even no effective treatment or drugs currently available, it is urgent to develop safe and efficient vaccines to control and prevent the further dissemination of SARS-CoV-2. 4 . Compared with other vaccines, recombinant subunit protein vaccine shows many advantages such as safety, higher cost efficiency and easy to be handled 3 . The main concern about the recombinant protein vaccine is that whether it can induce enough neutralizing antibodies and provide protection. Fortunately, recombinant subunit vaccine targeting SARS-CoV-2 RBD induced higher neutralizing antibodies without evident antibody dependent enhancement effects (ADE) 5 and may protect animals from SARS-CoV-2 attack 6, 7 .

SARS-CoV-2, homologous to SARS-CoV and the Middle East respiratory syndrome coronavirus, also contains four structural proteins, including spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. Among them, S protein plays the most important role in viral attachment, fusion and entry, and induces neutralizing antibodies with virus infection, so serves as a target for development of vaccines. The immunogenicity of SRAS-CoV-2 RBD, lies in S1 subunit by binding to the receptor ACE2 and is crucial in mediating viral entry into host cells, has been studied to induce neutralizing antibodies, which has been verified in samples from patients recovered from COVID-19 8 . These data indicate that RBD-based subunit proteins vaccine is a competitive candidate.

The Fc fusion protein is recently used as an important backbone for drug development, owing to its advantages including an expedient for rapid purification, longer half-life, increasing immunogenicity of target antigens and eliciting neutralizing antibody response 9 .In addition, Fc promote the correct folding of fusion protein and to enhance binding to antigen-presenting cells (APCs) and cell lines expressing Fc receptors (FcR) 10, 11 . For example, Enbrel, TNF Receptor-Fc fusion protein used for the treatment of rheumatoid arthritis or other human diseases, is a therapeutic biological drug without any known side-effect 12 . We have previously developed Fc-fused protein vaccines against MERS, SARS-CoV and H5N1 influenza, and found that Fc fused protein is more immunogenic than those lacking fused Fc [13] [14] [15] .

In this study, we designed a similar recombinant subunit vaccine (RBD-Fc Vacc) with the Receptor Binding Domain of SARS-CoV-2 S protein fused the Fc domain of human IgG1. The RBD-Fc Vacc showed immunogenicity and protective efficacy in non-human primate and human ACE2 transgenic (hACE2 Tg) mice with broader neutralizing ability against divergent SARS-CoV-2 virus strains. This novel COVID-19 subunit vaccine is currently being evaluated in phase I/II clinical trials. And for our best knowledge, this is the first Fc fusion protein-based vaccine tested in clinical trials.

Here a dimer-based RBD-Fc Vacc against SARS-CoV-2 was developed by fusing RBD (aa331-524) with human IgG1 Fc. The predicted dimer structure is shown in Fig 1a ( left panel) by protein structure prediction server version 3.0 16 : Two RBD domains are fused through Fc fragment to form the Y-shaped structure. The RBD-Fc fusion protein was expressed in mammalian CHO cells and the antigenicity and the dimer conformation were identified by western blot analysis using anti-serum from COVID-19 recovered patients and commercial antibody respectively. (Fig.1a right panel) . The expressed protein is about 120 kDa under non-reduced condition and 60 kDa after reduced. The calculated molecular weight of RBD-Fc protein is about 95.9 kDa while the purified recombinant protein appears to be 120kDa on the western blot owning to protein glycosylation. Three Nglycosylation sites on asparagine, one O-glycosylation on serine and one O-glycosylation on threonine were identified using mass spectrum (Fig.1b up panel) . These identified glycosylation sites were further mapped on the complex structure of SARS-CoV-2 RBD-Fc bound to ACE2 predicted by ZDOCK server 17 . The glycosylation sites were located on the RBD core subdomain and were found to be distant from the area bound to ACE2 (Fig.1b down panel) , indicating that the decorated glycopeptides may not interfere with receptor recognition.

The binding of RBD-Fc protein with ACE2 was then confirmed via surface plasmon resonance (SPR) Biacore and potent interactions were observed in concordance with a previous study 7 . Results from the SPR assay demonstrated that RBD-Fc protein bound to receptor hACE2 with much higher avidity compared with RBD-His monomer (74.13nM vs. 8.26nM), indicating the better conformation of our RBD-Fc protein (Fig.1c) . The immunogenicities of RBD-Fc or RBD-His protein were tested in BALB/c mice after two immunizations using aluminum as the adjuvant.

Compare to those from RBD-His vaccinated mice, serum from RBD-Fc vaccinated mice showed significantly higher RBD specific IgG antibody titers (with median peak of 1/40765 vs 1/7845) and live virus neutralizing antibody titers (with median peak of 1/61.8 vs. 1/24.4) (Fig.1d&e) . These data suggested that RBD-Fc fusion protein is more antigenic than monomer RBD protein.

We evaluated the immunogenicity of RBD-Fc Vacc in 3-year-old Macaca fascicularis, a non-human primate model that was susceptible to SARS-CoV-2 infection [18] [19] [20] Although there is a moderate increase for RBD-specific IgG titers in serum collected at day 42 compare to those at day 28 ( Fig.2b) , there is a much higher NT50 in sera from three dose-RBD-Fc Vacc-immunized macaca fascicularis (day 42, boost twice) compare to those from two dose-RBD-Fc Vacc-immunized animals (d28, boost once) (Fig.2c) , which indicated that three dose of immunization may induce a much better protective response. The variability in immunogenicity in this study facilitated an analysis of correlations of different examining methods. NAb titers measured by live virus neutralization assay correlated with RBD specific IgG titers (Fig.2d) .

Because of the novel epidemic SARS-CoV-2 strains with specific mutations was recorded 20,21 , we further tested whether Macaca fascicularis sera post RBD-Fc vaccination could cross neutralize different epidemic strains of SARS-CoV-2. Here three epidemic strains BJ08, BJ05 and BJ01, which shared the same RBD sequence as our RBD-Fc vaccine 22 , were used to test the cross neutralization effects. All the sera from RBD-Fc Vacc immunized Macaca fascicularis showed similar neutralizing capability against three SARS-CoV-2 epidemic strains, and there was no significant difference in NT50 titers (Fig.2e ). In addition, the NT50 in sera from non-human primates receiving RBD-Fc Vacc immunization was comparable to those from 23 COVID-19 convalescent patients' serum. To compare the cellular immune response, IFN-g ELISPOT assay and flow cytometry (d42) were performed in Macaca fascicularis at 2 weeks post 2 nd boost. There are comparable S1 or RBD specific IFN-g response among splenocytes from non-vaccinated or from RBD-Fc Vacc vaccinated macaca fascicularis (Fig. S3a) ,and the percentages of S1-specific IL-4 + CD4 + T cells, IL-4 + CD8 + T cells; TNF-a + CD4 + T cells, TNF-a + CD8 + T cells were at the same level (Fig. S3b) , suggesting that RBD-Fc Vacc mainly induces humoral immune response in vaccinated animals.

We next evaluated whether RBD-Fc Vacc induces protective immunity against SARS-CoV-2 in Macaca fascicularis. Four weeks after the third injection of 40ug RBD-Fc Vacc (d56), macaques were challenged with 10 6 TCID50/mL SARS-CoV-2 as follows: 2 mL inoculation by the intratracheal route, 1 mL by the intranasal route and 0.2 mL by the intraocular route. The substantial fraction of viral RNA which represent input challenge virus in Nasal, throat and anal swabs were examined at different times following challenge by quantitative real-time reverse transcription-PCR (qRT-PCR). Peak viral loads occurred variably following challenge. All control Macaca fascicularis showed excessive copies of viral genomic RNA in the nasal, throat, anal and lung by day 2-6 post inoculation (Fig.3a , b and Table S1 ) and severer interstitial pneumonia (Fig.3c) . By contrast, all vaccinated Macaca fascicularis were largely protected against SARS-CoV-2 infection with much lower or absence of viral RNA copies and very mild histopathological changes in a few lobes of lung. These data demonstrated that RBD-Fc Vacc induced potent immune response and efficiency protected Macaca fascicularis from SARS-CoV-2 attack.

The protective efficiency of RBD-Fc Vacc against SARS-CoV-2 was also confirmed in hACE2-Tg mice. Four weeks after the second boost immunization, mice were challenged intranasally with 1.8 x 10 7 PFU/ml SARS-CoV-2. Five days following challenge, viral RNA levels in lung were assessed by RT-PCR. RBD-Fc Vacc immunization in hACE2-Tg mice lead to a median reduction of 2.10 Log10RNA copies/g and 2.13 in 10ug and 20ug group respectively (Fig.S4a ). In addition, all vaccinated animals were largely protected against SARS-CoV-2 infection with very mild histopathological changes in the vaccinated animals, while moderate interstitial pneumonia in sham group. (Fig.S4b) .

The variability in protective efficiency in this study facilitated an analysis of immune correlations of protection. The live virus neutralization antibody titers at day49 inversely correlated with viral RNA in lung of hACE2-Tg mice (Fig. 4a ). In addition, the pseudovirus neutralization antibody titers and the IgG titers at day 49 also indicated inversely correlation with viral RNA in lung of hACE2-Tg mice (Fig. 4b&c) . The vaccine induced immune response and protection also showed some inverse correlation in Macaca fascicularis, however, due to the limitation of sample numbers，the data cannot be statistically analyzed. These data demonstrated that serum IgG titers and neutralization antibody titers elicited by RBD-Fc may be immune correlates of protection against SARS-CoV-2 infection. The data also suggested that these parameters would be simple and useful benchmark for clinical development of SRAS-CoV-2 vaccine.

The serious pandemic of current COVID- 19 showed that RBD is highly conserved among ten pandemic SARS-CoV-2 strains worldwide. Our data showed that serum from RBD-Fc Vacc immunized Macaca fascicularis neutralized three representative SARS-CoC-2 strains in microneutralization assay (Fig.3d) . RBD-Fc-Vacc-elicited serum IgG titers and NAb titers also correlates negatively with the virus copies of immunized hACE2-Tg mice, demonstrating IgG and NAb titers as an immune correlate of protection. These data suggested that RBD is an excellent target for SARS-CoV-2 vaccine.

The antibody mediated enhancement effects (ADE) is the most important safety concerns in the development of vaccines. A recent report also showed that nonhuman primates infected with SARS-CoV-2 are protected from reinfection with the virus 29 , which indicated that virus infection induced a protective immune response. Reports showed that non-neutralizing antibodies is likely to promote more efficient ADE than neutralizing antibodies 30 . It has been noted that non-neutralizing coronavirus antibodies may cause ADE in feline infectious peritonitis. Such efforts have prompted investigators to focus on the RBD as a lead vaccine candidate while remove potential ADEpromoting S protein epitopes outside the RBD 13,31 . Indeed, a recent data showed that RBD of SARS-CoV-2 raised potently neutralizing antisera in immunized rats and these sera without ADE 5 . Our reports showed that RBD-Fc Vacc immunized macaca fascicularis and mice were protected from SARS-CoV-2 attack without evident side effects, which suggested that RBD is a safe and efficient target for vaccine use.

Fusion proteins based on immunoglobulin Fc domain have received considerable attention over the past two decades because of its capability to promote protein expression, purification, and improve the immunogenicity of the fused proteins. Many therapeutic biological drugs based on Fc protein fragment had been used for treatment of human diseases 32 . The application of Fc-fusion protein as vaccine delivery platform was first reported in 1989 in which gp120-Fc was used as a potential candidate for AIDS therapy 33 found that Fc fragment enhances the immunogenicity of RBD as RBD-Fc induced much higher titer of S1 protein specific IgG antibodies and neutralization antibodies than RBD-His (Fig.1d&e) , which further demonstrated that Fc-fusion protein is a promising vaccine delivery platform.

In summary, our data demonstrated that RBD-Fc Vacc, with adjuvant of aluminum, induced a robust humoral immune response and protected non-human primate and hACE2 Tg mice from SARS-CoV-2 infection. Importantly, our data demonstrate the immune correlates of protection and protective efficacy and support the immunogenicity of the RBD-Fc Vacc candidate in clinical use.

For the best of our knowledge, this is the first Fc fusion protein-based vaccine tested in clinical trials.

Further research will need to address the important questions of the durability of protective immunity and the optimal vaccine strategy for a SARS-CoV-2 vaccine for humans.

All animal studies were performed in strict accordance with the guidelines set by the Chinese

Housing Facilities. All animal procedures were reviewed and approved by the Animal Experiment 

The recombinant RBD-Fc protein was purified using affinity chromatography and anion exchange chromatography. The purified RBD-Fc protein was then reduced by incubation with 10 mM Tris 

SPR-based measurements were performed by Biacore T200 (GE Healthcare, Uppsala, Sweden), as described previously 16 The association and dissociation rate constants ka and kd were monitored respectively and the avidity value KD was determined.

African green monkey kidney cell Vero (ATCC, CCL-81), were maintained in ulbecco's minimal essential medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and penicillin (100 /ml)-streptomycin (100 µg/ml) (Thermo 

Eight-week old human ACE2 transgenic mice (hACE2-Tg, obtained from National Institutes for Food and Drug Control, Beijing, China) , Balb/C mice and 3-year old Macaca fascicularis (all from Laboratory animal center of the academic of the military medical sciences ) were immunized with SARS-CoV-2 RBD-Fc Vacc as described in Fig.1a . Four weeks after the second boost immunization, macaca fascicularis were inoculated with 10 6 TCID50/ml BetaCoV(BJ01) as following:

intratracheal 2ml, intranasal 0.5ml, intraocular 0.2ml; hACE2-Tg mice were inoculated intranasally with 40 μL of BetaCoV(BJ05). All mice or Macaca fascicularis were observed daily. On day 5 post infection (for mice), or on day 7 post infection (for Macaca fascicularis), eight mice in each group and four Macaca fascicularis in each group were sacrificed, and their lungs were removed for detection of viral load or were embedded for pathological analysis as described below.

ELISA was performed to detect SARS-CoV-2 RBD and S1-specific IgG antibodies in the immunized mouse or Macaca fascicularis sera. Briefly, ELISA plates were precoated with SARS-CoV-2 S1 or RBD protein (2.0μg/ml) overnight at 4℃ and blocked with 3% BSA in PBS for 2 h at 37℃. Serially diluted sera were added to the plates and incubated for 45 min at 37℃. After four washes, the bound antibodies were detected by incubation with horseradish peroxidase (HRP)- 

The SARS-CoV-2 pseudovirus based neutralization assay was performed as described previously 42 .

In brief, mice or Macaca fascicularis sera at 3-fold serial dilutions were incubated with 650 TCID50 of the pseudovirus for 1 hour at 37 °C, and then 20000 Huh7 cells were added into each well. DMEM was used as negative control. After 24h incubation in 37℃, the supernatant was then removed and luciferase substrate was added to each well followed by incubation for 2 minutes in darkness at room temperature. Luciferase activity was then measured using GloMax® 96 Microplate Luminometer (Promega). The 50% neutralization titer (NT50) was defined as the serum dilution at which the relative light units (RLUs) were reduced by 50% compared with the virus control wells.

A micro-neutralization assay was carried out to detect neutralizing antibodies against SARS-CoV-2 infection. Briefly, mouse or Macaca fascicularis sera at 2-fold serial dilutions were incubated with SARS-CoV-2 (BetaCoV/BJ01); 100 TCID50) for 1 h at 37℃ and added to Vero cells. The cells were observed daily for the presence or absence of virus-induced Cytopathic Effect (CPE)and recorded at 72 h. Neutralizing antibody titers were determined as the highest dilution of sera that can completely inhibit virus-induced CPE in at least 50% of the wells (NT50).

Tissue homogenates were clarified by centrifugation at 6,000 rpm for 6 

Five-or seven-days post virus challenge, animals were sacrificed and lung tissues were obtained, and paraffin-embedded in accordance with the standard procedure. Sections at 5 μm thickness were stained with hematoxylin and eosin (H&E), and examined by light microscopy. Lung tissue lesions

were assessed according to the extent of denatured and collapsed bronchiole epithelial cells, degeneration of alveoli pneumocytes, infiltration of inflammatory cells, edema, hemorrhage, exudation and expansion of parenchymal wall. The semi-quantitative assessment was scored for the severity of lung damage according to the above criteria (0, normal; 1, mild; 2, moderate; 3, marked).

The cumulative scores of the severity provided the total score per animal, and the average of six to eight animals from each group was taken as the total score for that group.

Statistical analyses were carried out using Prism software (GraphPad). All data are presented as means ± standard error of the means (SEM). Statistical significance among different groups were calculated using the Student's t test, Fisher's Exact test, two-sided Spearman rank-correlation test，or Mann-Whitney test. *, **, and *** indicate P < 0.05, P < 0.01, and P < 0.001, respectively. specific IgG titers (c) prior to challenge with log peak mRNA copies/g in lungs following challenge in hACE2-Tg mice. Red lines reflect the best-fit relationship between these variables. P

and R values reflect two-sided Spearman rank-correlation tests. SEM. Significance was calculated using unpaired t-test (n.s., not significant). b). SARS-CoV-2

fascicularis PBMCs were detected by flow cytometry.

The gender, the marked number for each animal and the Log10 gRNA copies/g values for each lung lobes of vaccinated or non-vaccinated macaca fascicularis were shown. 

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SARS-Cov2 RBD-Fc vaccine Design. a) Two RBD domains are fused through Fc fragment to form the Y-shaped structure via protein structure prediction server

The docking between ACE-2 and RBD-Fc predicted by ZDOCK server. An overview of the glycosylation sites illustrated based on the solved complex structure of SARS-CoV-2 RBD-Fc bound to ACE2 (PDB code: 1R42). The identified sites, colored red for Nglycosylation, purple for O-glycosylation are shown as spheres and labeled. The right panel (surface representation) was generated by rotating the structure in the Left panel (cartoon representation) around a vertical axis for about 90° (lower panel). c) The real-time binding profile between our purified RBD-Fc protein and ACE2 characterized by SPR Biacore. d&e) Balb/C mice were immunized with RBD-his or RBD-Fc(10ug/mouse)in the presence of aluminum at d0 and d14

Macaca fascicularis (n=5) were immunized on day 0, day 14, d28 with 20ug and 40ug doses of RBD-Fc Vacc or PBS and the serum were collected at the indicated time. The SARS-CoV-2 RBD specific IgG were examined by ELISA. c) Neutralizing antibodies were determined by microneutralization assay using the SARS-CoV-2 (NT50). d). Correlation analysis of antibody titers tested by ELISA and microneutralization assay. P and R values reflect two-sided Spearman rank-correlation tests. e) Serum cross neutralization against SARS-CoV-2 epidemic strains in RBD-Fc immunized macaca fascicularis. NT50 against the SARS-CoV-2 epidemic strains (BJ08, BJ05, BJ01) were performed using macaca fascicularis sera collected at 21 days post third immunization