RBD trimer mRNA vaccine elicits broad and protective immune responses against SARS-CoV-2 variants

Figures

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  Figure 1. Design and characterization of mRNA vaccines (A) Design and construction of three mRNA vaccines expressing native receptor-binding domain (RBD), trimeric RBD with a foldon-trimerization tag (RBD Trimer), or native full-length spike (Native Spike) of SARS-CoV-2. SP: signal peptide; TM: transmembrane domain; CP: cytoplastic domain. (B) Quality control of LNP-formulated mRNA vaccines through cryo-TEM imaging and (C) dynamic light-scattering measurement. In (B), a scale bar of 200 nm is indicated. (D) Two-dimensional average of recombinant RBD trimers produced in 293F cells bound to soluble ACE2 and analyzed by Cryo-EM. Box edge, 30.4 nm. (E) Schedule for immunization and evaluation of immunogenicity in BALB/c mice (n = 160). Mice were immunized and monitored for serum binding and neutralizing antibodies, kinetics of SARS-CoV-2-specific B and T cell responses in spleen, inguinal lymph nodes, and bone marrow, and for isolating monoclonal antibodies from RBD-trimer-immunized animals. The exact date of prime and boost vaccination and sample collections are indicated. On day 10, 21, 35, and 105 after initial immunization, one group of 10 animals from each of the native RBD, the RBD…

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  Figure 2. Superior antibody response against SARS-CoV-2 variants induced by RBD trimer (A–P) (A) Serum neutralizing antibody induced by native RBD, RBD trimer, and native spike mRNA vaccines to pseudoviruses carrying full-length, single, or triple mutant spike of SARS-CoV-2 variants. Colour scheme for native RBD (blue), RBD trimer (red), and native spike (green) are consistent throughout the manuscript. Fold change in reciprocal serum neutralization (IC50) from the native RBD (B–F), RBD trimer (G–K), and native spike (L–P) mRNA vaccines immunized mice against variants B.1.1.7, B.1.351, P1, and B.1.617.2, as well as single or triple mutant pseudoviruses, relative to the WT D614G pseudovirus. (Q and R) Fold change in reciprocal serum neutralization (PRNT50 and PRNT80) in the three mRNA vaccines immunized mice against live B.1.351 variant, relative to the WT D614 live virus. The tested serum samples were collected at week 5 after initial immunization. All data are presented as the original and/or with indicated means. The fold change in ID50, PRNT50, or PRNT80 between mutant and WT D614 virus are shown by overall average at the top in (B–R). The symbol “+” indicates an increase while the…

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  Figure 3. RBD trimer mRNA vaccine elicits superior antigen-specific germinal center, memory, and long-lived plasma B cell responses (A–H) Kinetics of RBD-specific IgG+ GC cells and memory B cells in inguinal lymph nodes (A and B) or in spleen (C and D). Kinetics of RBD-specific IgG+ plasma cells in spleen (E) or in bone marrow (F). Intracellular expression of IFN-γ, TNF-α, and IL-2 in CD4+ (G) and CD8+ T (H) cells 5 week after first immunization. (I) ELISPOT analysis of IFN-γ-positive splenocytes, samples were collected at week 5 after initial immunization. All data are presented as the original with indicated means. Statistical significance was analyzed using unpaired Students’ t-test (two-tailed) (ns, not significant; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001).

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  Figure 4. RBD trimer is the most potent in triggering BCR signaling (A) Synaptic accumulation of BCRs triggered by native RBD, RBD trimer, and native spike on the surface of the J558L B cell line expressing SARS-CoV-2 antibody 2F6-IgG-BCRs captured by a confocal microscopy. The magnified insets (lower right corner) show the highlighted cells in the original image within the white boxes. The BCR molecules are labelled in red, and the scale bar in the inset window represents 1.5 μm. (B) Synaptic recruitment of phosphorylated Syk (pSyk) to the contact area of a single J558L B cell after BCR accumulation by native RBD, RBD trimer, and native spike captured by a confocal microscopy. The BCRs and pSyk molecules are respectively labelled in red and green, and their merged images are also shown. The scale bar represents 1.5 μm. (C–F) Statistical analysis for the contact area and mean fluorescence intensity (MFI) of BCRs (C and D) and pSky (E and F) recruited in the immunological synapses on the surface of J558L-2F6-IgG-BCRs B cells. Each dot in the plot represents the data of one cell with indicated means and standard deviation. (G) Fluorescence intensity profiles from (B) showing degrees of…

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  Figure 5. RBD trimer induces dominant broad and potent monoclonal antibodies against SARS-CoV-2 variants (A) Unrooted neighbor-joining tree depicting the genetic relationship among 40 monoclonal antibodies. The left panel is derived from the heavy chain variable region while the right panel from the light chain variable region. Antibody names, and their corresponding IC50, EC50, degree of somatic hyper mutation, and CDR3 sequences are indicated at the tip of the branches. The IC50 was measured against SARS-CoV-2 WT D614 pseudovirus and the EC50 was calculated against recombinant RBD trimer in the ELISA assay. (B) IC50 and their fold changes against SARS-CoV-2 variants, as well as single or triple mutant pseudoviruses, relative to that of WT D614G. The symbol “+” indicates increased while “-” decreased potency. Those highlighted in blue indicates increased potency at least threefold whereas those in red decreased potency at least threefold. Those in white change less than threefold. BDL, referring to Below Detection Limit, indicates the highest concentration (2 μg/mL) of mAbs failed to reach 50% neutralization. Results were calculated from three independent experiments. Epitope mapping…

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  Figure 6. Cryo-EM and Crystal structure of mAb T6 with spike trimer or RBD of SARS-CoV-2 variant B.1.351 (A) Cryo-EM structure of the soluble, recombinant spike trimer of B.1.351(SA) strain in complex with two variable regions of mAb T6 at 3.2 Å resolution. VH, variable region of heavy chain; VL, variable region of light chain. (B) Cryo-EM structure of the soluble, recombinant spike trimer of B.1.351(SA) strain in complex with three variable regions of mAb T6 at 3.3 Å resolution. (C) T6/RBD-3M crystal structure. T6 light chain, heavy chain, and RBD-3M are colored in violet, purple, and cyan, respectively. The three RBD-3M mutated residues (N417, K484, and Y501) are shown as red spheres. (D) The epitope of mAb T6. The epitope residues containing within 4 Å are indicated in purple. The Conservation of amino acids are labelled beside, the data are collected from GISAID database. (E) The paratope of mAb T6. The paratope residues containing within 4 Å are indicated in cyan and binding residues within are indicated in either purple or violet, depending on their origin from heavy or light chain Fab. (F) Interactions between mAb T6 Fab and SARS-CoV-2 RBD-3M. The hydrogen bonds are represented by…

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  Figure 7. RBD trimer mRNA vaccine protects K18-hACE2 mice from death after infection with live SARS-CoV-2 and its variants (A–G) (A) Timeline for vaccination, challenge with live SARS-CoV-2 WT D614, B.1.351, or B.1.617.2 and monitoring for body weight changes and survival in the ensuing 5 days, and euthanasia up to 5 dpi. Mice were challenged with 103 PFU live SARS-CoV-2 WT D614, B.1.351, or B.1.617.2 via IN route. Body weight relative to day 0 (B–D) and survival percentage (E–G) were recorded daily until death up to 5 dpi. (H–J) H&E staining of lung sections from immunized or control group. Scale bars, 50 μm. Each image is representative of each group. (K–M) Viral gRNA and sgRNA copies of N gene in lung tissue samples were quantified by droplet digital PCR (TargetingOne, China). All the samples were collected at death or at 5 dpi.