A novel receptor-binding domain (RBD)-based mRNA vaccine against SARS-CoV-2

Dear Editor, The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the need to develop effective and safe vaccines. Similar to SARS-CoV, SARS-CoV-2 recognizes angiotensin-converting enzyme 2 (ACE2) as receptor for host cell entry. SARS-CoV-2 spike (S) protein consists of S1, including receptor-binding domain (RBD), and S2 subunits. We previously demonstrated that RBDs of SARS-CoV and MERS-CoV serve as important targets for the development of effective vaccines. To identify an mRNA candidate vaccine, we initially designed two mRNA constructs expressing S1 and RBD, respectively, of SARS-CoV-2 S protein (Fig. 1a). Both culture supernatants and lysates of cells transfected with S1 or RBD mRNA reacted strongly with a SARS-CoV-2 RBD-specific antibody (Supplementary information, Fig. S1a), demonstrating expression of the target proteins. To detect whether S1 and RBD mRNAs durably express antigens in multiple cell types, we constructed N-terminal mCherry-tagged SARS-CoV-2 S1 and RBD mRNAs, encapsulated them with lipid nanoparticles (LNPs) (Supplementary information, Fig. S1b), and tested mCherry expression. Relative to the control, both RBDand S1-mCherry mRNAs showed robust protein expression in cells for at least 160 h, with higher expression of the RBD construct (Supplementary information, Fig. S2a). In addition, these mRNAs expressed proteins efficiently in a variety of human (A549, Hep-2, HEP-G2, Caco-2, HeLa, 293 T), monkey (Vero E6), and bat (Tb1-Lu) cell lines (Supplementary information, Fig. S2b). Particularly, the expression of RBD-mCherry protein was higher than that of S1-mCherry protein in all cell lines tested (Supplementary information, Fig. S2b). These data indicate long-term and broad expression of mRNA-encoding proteins, particularly RBD, in target cells. We then characterized LNP-encapsulated S1 and RBD mRNAs for stability and subcellular localization. The mCherry-tagged S1 and RBD showed strong and stronger fluorescence intensity, respectively, irrespective of incubation temperature (4 or 25 °C) and culture time (0, 24, or 72 h) (Supplementary information, Fig. S3a). S1and RBD-mCherry proteins were not colocalized with nuclei but associated with lysosomes (Supplementary information, Fig. S3b). These results suggest that LNP-encapsulated SARS-CoV-2 S1 and RBD mRNAs are stable at various temperatures and may be resistant to lysosomal degradation. We next evaluated T follicular helper (Tfh), germinal center (GC) B, and plasma cell responses induced by SARS-CoV-2 S1 and RBD mRNA-LNPs in BALB/c mice. Mice were intradermally (I.D.) prime and boost immunized with each mRNA-LNP (30 μg/mouse) or empty LNP control, and draining lymph nodes or spleens were tested for Tfh, GC B, or plasma cells 10 days post-2nd immunization (Supplementary information, Fig. S4a). The percentages of Tfh cells (Supplementary information, Fig. S5a) and GC B cells (Supplementary information, Fig. S5b) were higher or significantly higher in the lymph nodes of RBD mRNA-LNPimmunized mice than in those of S1 mRNA-LNP-immunized mice, whereas only a background level of Tfh and GC B cells was shown in the LNP control-injected mice. Plasma cells were also significantly increased in splenocytes of the vaccinated mice, as compared to the control group (Supplementary information, Fig. S5c). These data demonstrate the recruitment of Tfh, GC B, and/or plasma cells in vivo, particularly after immunization with SARS-CoV-2 RBD mRNA-LNP vaccine. We further evaluated humoral immune responses and neutralizing antibodies induced by S1 and RBD mRNA-LNPs. Mice were immunized with each mRNA-LNP at three different schedules (Supplementary information, Fig. S4a–c), and sera were collected for detection of IgG, subtype (IgG1 and IgG2a), and neutralizing antibodies. First, ELISA results revealed that S1 and RBD mRNA-LNPs (30 μg/mouse, I.D. prime and boost) induced RBD-specific IgG (Fig. 1b), IgG1 (Th2) (Supplementary information, Fig. S5d), and IgG2a (Th1) (Supplementary information, Fig. S5e) antibodies 10 days after boost immunization and that IgG antibody titer induced by RBD was significantly higher than that by S1 (Fig. 1b). Pseudovirus neutralization assay showed that S1 and RBD mRNA-LNPs elicited neutralizing antibodies against SARS-CoV-2 pseudovirus entry into human ACE2expressing 293T (hACE2/293T) cells; particularly, RBD elicited significantly higher-titer neutralizing antibodies than S1 (Fig. 1c). Neutralizing antibodies, particularly those induced by RBD mRNALNP, also potently neutralized live SARS-CoV-2 infection (Fig. 1d). Next, both S1 and RBD mRNA-LNPs (10 μg, I.D. prime and boost) induced SARS-CoV-2 RBD-specific IgG (Supplementary information, Fig. S6a) and neutralizing antibodies against SARS-CoV-2 pseudovirus infection (Fig. 1e) 10 days after boost dose, and maintained at similarly high levels for 40 and 70 days post-boost immunization, while the titer of neutralizing antibodies elicited by RBD mRNA-LNP was always significantly higher than that by S1 mRNA-LNP (Fig. 1f, g; Supplementary information, S6b, c). Importantly, RBD mRNA-LNP induced antibody levels that potently neutralized live SARS-CoV-2 infection, reaching peak titer at 70 days post-2nd immunization and being significantly more potent than SARS-CoV-2 S1 mRNA-LNP-induced antibodies (Fig. 1h–j). Finally, RBD mRNA-LNP (10 μg, I.D. prime and intramuscular (I.M.) boost) also elicited significantly higher-titer RBD-specific IgG (Supplementary information, Fig. S6d) or neutralizing antibodies than S1 mRNA-LNP against SARS-CoV-2 pseudovirus (Supplementary information, Fig. S6g) and live SARSCoV-2 (Supplementary information, Fig. S6j) infection 10 days after boost immunization, and such antibodies maintained at similar or even higher levels for at least 70 days post-boost dose (Supplementary information, Fig. S6e, f, h, i, k, l). In contrast, empty LNP control only elicited a background, or undetectable, level of antibodies incapable of neutralizing SARS-CoV-2 infection (Fig. 1b–j; Supplementary information, Fig. S6). These data suggest that RBD mRNA-LNP vaccine immunized at different immunogen

The mRNAs were synthesized, 5'-capped and tailed with 3'-Poly-A following manufacturer's instructions. Briefly, the above S1 or RBD genes were linearized using Bgl II enzyme and Poly-A tail of about 150 base pair (bp). The mRNAs were stored at -80°C until use.

Preparation of SARS-CoV-2 S1 and RBD mRNA-LNPs
The above mRNAs were encapsulated with LNPs to further increase stability. mRNA-LNPs were prepared using GenVoy-ILM and NanoAssemblr Benchtop Instrument following manufacturer's instructions (Precision Nanosystems, Vancouver, BC). Briefly, GenVoy-ILM, which contains ionizable cationic lipid, helper lipids, and cholesterol, was dissolved in ethanol.
The lipid mixture (ethanol phase) was encapsulated with PNI Formulation Buffer (aqueous phase) (Precision Nanosystems) containing SARS-CoV S1 or RBD mRNA (0.174 mg/ml) at 1:3 ratio (ethanol:aqueous, V/V), using NanoAssemblr Benchtop. The encapsulated mRNA-LNPs were diluted in PBS, filtered through a 0.22-mm filter, and concentrated using Amicon Ultra Centrifugal Filters (EMD Millipore, Billerica, MA, USA). The empty LNP control was prepared using PNI Formulation Buffer without mRNAs as aqueous phase. To assess consistency of physical characterization of LNPs among different batches, three batches of SARS-CoV-2 S1 mRNA-LNP, RBD mRNA-LNP, and empty LNP control were encapsulated by GenVoy-ILM and NanoAssemblr Benchtop Instrument as described above following manufacturer's instructions. The endotoxin level of each formulation was < 1 EU/ml. The particle size of LNPs was analyzed by Dynamic Light Scattering (Dynapro NanoStar), which was between 80-110 nm in diameter.

mRNA transfection and protein expression
mRNAs were transfected into 293T cells using TransIT-mRNA Kit following manufacturer's instructions (Mirus Bio, Madison, WI, USA). Briefly, SARS-CoV-2 S1 or RBD mRNA (1 μg) was mixed with TransIT-mRNA and boost reagents in Opti-Minimal Essential Medium (MEM). The mixture was added to cells containing complete Dulbecco's Modified Eagle's Medium (DMEM) and cultured at 37°C with 5% CO2. 72 h after transfection, supernatants were collected, and cells were lysed in RIPA buffer (Sigma, St. Louis, MO, USA) for detection of protein expression by Western blot. Samples were incubated with 4× Laemmli buffer (Bio-Rad, Hercules, California, USA), separated on a 10% polyacrylamide gel, and transferred to PVDF membrane, which was blocked with 5% fat-free milk in PBS containing 0.5% Tween-20 (PBST). S1 and RBD protein expression was detected by sequential incubation of the membrane with mouse sera (1:1,000) immunized with SARS-CoV-2 RBD-Fc protein and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG-Fab (1:5,000) (Sigma) for 1 h at room temperature. The signals were detected using ECL Western blot substrate reagents and Amersham Hyperfilm (GE Healthcare).

Immunofluorescence staining
This was performed to detect subcellular localization of mRNA-encoding proteins as previously described with some modifications. 2,3 Briefly, mCherry-tagged SARS-CoV-2 S1 or RBD mRNA-LNP (1 μg/ml) was added to 293T cells (2×10 5 /well) pre-plated 24 h before experiments; the cells were cultured at 37°C for 48 h and harvested for immunofluorescence staining. The cells were then fixed and permed with FIX and PERM Cell Permeabilization Kit (Thermo Fisher Scientific), followed by incubation with FITC-labeled anti-human CD107a (LAMP-1, for lysosomes) antibody (1:100, BioLegend, San Diego, CA, USA) for 30 min at room temperature. After washing with PBS, the concentrated cell suspension was evenly distributed into slides, counter-stained with DAPI (4′,6-diamidino-2-phenylindole, 300 nM, Thermo Fisher Scientific) for nuclei for 5 min, and then mounted in VectaMount Permanent Mounting Medium (Vector Laboratories, Burlingame, CA, USA). The slides were imaged on a confocal microscope (Zeiss LSM 880). Images were prepared using ZEN software.

Thermal stability of mRNA-LNPs
The stability of LNP-encapsulated SARS-CoV-2 S1 or RBD mRNA was performed as described below. Briefly, mCherry-tagged SARS-CoV-2 S1 and RBD mRNA-LNPs were stored at 4℃ and 25°C for 0, 24, and 72 h and then added to 293T cells at a concentration of 1 µg/ml. The cells were cultured at 37°C for 48 h and analyzed for mCherry signal by flow cytometry (BD LSRFortessa 4 system).

Ethics statement
Six-to-eight-week-old male and female BALB/c mice were used in the study. The animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of New York Blood Center (permit number 194). All animal studies were carried out in strict accordance with the guidance and recommendations in the Guide for the Care and Use of Laboratory Animals (National Research Council Committee).
The cells were lysed using cell lysis buffer (Promega, Madison, WI, USA) 72 h post-culture and transferred into luminometer plates. Luciferase substrate (Promega) was added to the plates and measured for relative luciferase activity using the Infinite 200 PRO Luminometer (Tecan).

SARS-CoV-2 microneutralization assay
mRNA-LNP vaccine-induced serum neutralizing antibodies against live SARS-CoV-2 infection were detected using a cytopathic effect (CPE)-based microneutralization assay as previously described with some modifications. 6,8 Briefly, Vero E6 cells (10 4 /well) were pre-plated in 96-well tissue culture plates and cultured at 37°C to form a monolayer. Serially 2 or 3-fold and duplicate dilutions of mouse sera (pooled from five mice in each group) were thoroughly mixed with ~120 median tissue culture infectious dose (TCID50) of SARS-CoV-2 (US-WA-1 isolate), and incubated for 1 h at room temperature, followed by transfer of the serum-virus mixtures to the above cells and culture of the cells at 37°C for three days. Cells with or without virus were included as positive and negative controls, respectively. CPE in each well was observed under the microscopy, and recorded on day 3 post-infection.
Neutralizing antibody titer was calculated and expressed as the highest dilutions of mouse sera capable of completely preventing virus-induced CPE in at least 50% of the wells (NT50).

Inhibition of binding of SARS-CoV-2 RBD protein to hACE2 receptor
Flow cytometry was used to analyze the ability of immunized mouse sera to block the binding between SARS-CoV-2 RBD protein and cell-associated hACE2 receptor. 2 Briefly, hACE2/293T cells were incubated with SARS-CoV-2 RBD-Fc protein (5 µg/ml) in the presence or absence of serially diluted mouse sera for 30 min at room temperature. The cells were stained with FITC-labeled goat anti-human IgG-Fc antibody (1:500, Sigma) for 30 min at room temperature and measured for fluorescence by flow cytometry (BD LSRFortessa 4 system).

Isolation and analysis of lymph node cells
Lymph nodes collected at 10 days post-2 nd immunization (I.D.) with SARS-CoV-2 mRNA-LNP or empty LNP were pooled for detection as described below. 9 Briefly, lymph nodes were  Table S1) and cultured at 37°C for 72 h. At 68 h poststimulation, 1× Brefeldin A (BioLegend) was added to the cells. After stimulation, the cells were washed with PBS and stained with Fixable Viability Dye eFluor™ 780 (Thermo Fisher Scientific) for live and dead cells. The cells were stained for surface markers using anti-mouse CD45-AF700, . After fixation and permeabilization, the cells were stained for intracellular cytokine markers using IFN-γ-PE, TNF-α-BV421, and IL-4-BV711 (BioLegend). The stained cells were measured using flow cytometry (BD LSRFortessa 4 system), and the data were analyzed using FlowJo software.

Statistical analyses
All values are presented as mean plus standard error of the mean (s.e.m). Statistical differences among SARS-CoV-2 S1 mRNA-LNP, RBD mRNA-LNP and control groups, as shown in Fig.   1 and Supplementary information, Fig. S5-7, were performed using Student's two-tailed t-test. * (P < 0.05), ** (P < 0.01), and *** (P < 0.001) represent significance and high significance among different groups. All statistical analyses were performed using GraphPad Prism 5 statistical software.
a Long-term expression of mCherry protein encoded by mCherry-tagged S1 and RBD mRNAs in 293T cells. The LNP-encapsulated mRNAs encoding SARS-CoV-2 S1 or RBD protein (S1-mCherry-LNP or RBD-mCherry-LNP) were incubated with 293T cells at 37°C, and the cells were then collected at different time post-incubation for analysis of mCherry signal by flow cytometry. b Broad-spectrum expression of mCherry protein encoded by mCherry-tagged S1 and RBD mRNAs in different human, monkey, and bat cells. The LNP-encapsulated S1 or RBD mRNA (S1-mCherry-LNP or RBD-mCherry-LNP) was incubated with each cell line at a Stability of LNP-encapsulated, mCherry-tagged SARS-CoV-2 S1 and RBD mRNAs (S1-mCherry-LNP or RBD-mCherry-LNP). The mRNAs were stored at 4°C and 25°C, respectively, at the indicated time and then incubated with 293T cells at 37°C for 48 h, followed by analysis for mCherry signal by flow cytometry. Data are presented as mean MFI ± s.e.m. of triplicates (n = 3). b Detection of subcellular localization of mRNA-encoding protein. LNP-encapsulated, mCherry-tagged SARS-CoV-2 S1 and RBD mRNAs (S1-mCherry-LNP or RBD-mCherry-LNP) were incubated with 293T cells at 37°C for 48 h. Cell lysosomes (Lyso, green) and nuclei (blue) were stained, and subcellular localization of mRNA expression based on mCherry (red) signal was analyzed by immunofluorescence microscope. Representative images are shown.
Scale bar, 10 μm. Control, empty LNP. Experiments were repeated twice with similar results.

Supplementary information, Fig. S5 SARS-CoV-2 RBD mRNA-LNP elicited Tfh and GC B cell responses and subtype antibody responses specific to SARS-CoV-2 and SARS-CoV
RBDs.