The self-assembled nanoparticle-based trimeric RBD mRNA vaccine elicits robust and durable protective immunity against SARS-CoV-2 in mice

As COVID-19 continues to spread rapidly worldwide and variants continue to emerge, the development and deployment of safe and effective vaccines are urgently needed. Here, we developed an mRNA vaccine based on the trimeric receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein fused to ferritin-formed nanoparticles (TF-RBD). Compared to the trimeric form of the RBD mRNA vaccine (T-RBD), TF-RBD delivered intramuscularly elicited robust and durable humoral immunity as well as a Th1-biased cellular response. After further challenge with live SARS-CoV-2, immunization with a two-shot low-dose regimen of TF-RBD provided adequate protection in hACE2-transduced mice. In addition, the mRNA template of TF-RBD was easily and quickly engineered into a variant vaccine to address SARS-CoV-2 mutations. The TF-RBD multivalent vaccine produced broad-spectrum neutralizing antibodies against Alpha (B.1.1.7) and Beta (B.1.351) variants. This mRNA vaccine based on the encoded self-assembled nanoparticle-based trimer RBD provides a reference for the design of mRNA vaccines targeting SARS-CoV-2.


ELISA
Ninety-six-well ELISA plates (Corning, USA) were coated with the RBD protein (1 µg/mL, Sino Biological) overnight at 4°C and blocked with 4% bull serum albumin. The serum was twofold diluted and added to each well, and the plates were incubated with goat anti-mouse IgG-HRP and developed by the addition of 100 µL of 3,3',5',5-tetramethylbenzidine (TMB) to each well. Finally, 100 µL of 2 mmol/L H 2 SO 4 was added to terminate the reaction, and the light absorption of the plate was measured at 450 nm using a microplate reader (Thermo Scientific).
The OD value of the highest dilution was 2.1-fold higher than that of the negative control at the same dilution and was used as the serum endpoint dilution titer. Each experiment was performed three times.

ELISPOT
To detect specific T lymphocyte responses, the IFNγ ELISPOT assay was performed. The mice were euthanized, and their spleens were removed under aseptic conditions. Spleen cell (4×10 5 /well) suspensions and SARS-CoV-2 RBD protein diluents (10 µg/mL, Sino Biological, China) were added to 96-well ELISPOT plates (DAKEWE, China) precoated with IFNγ antibodies, and ConA was added as a positive control. Cells incubated without stimulation were employed as the negative control. After 18 h of incubation, the biotinylated antibody, streptavidin-HRP, and fresh substrate were added to the plates. Finally, the reaction was stopped by rinsing the plate with deionized water. The number of spots was determined using a CTL Spot Reader (CTL, USA).
HEK293T/hACE2 cells were inoculated before the experiment. Starting with a 1:10 dilution, each sample was continuously diluted twice in a 96-well plate. Equal volumes of pseudovirus and variant were mixed with each diluted serum sample and incubated for 1.5 h at 37 °C. The mixture of virus-serum was added to the cells. After 72 h, the cells-supernatant mixture was collected, the firefly luciferase activity in the cells was detected by chemiluminescence, and the luciferase activity was quantified to measure the transduction efficiency. To calculate the neutralization efficiency, pseudovirus without antibody was used as a positive control. Each sample was assessed in three repeat wells. Positive values were determined to be relative luminescence unit (RLU) values that were tenfold higher than that of only the cell background.
The half-maximum neutralization titer (NT 50 ) value was the reciprocal of the dilution of half of the mean RLU value of the positive control.