A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2

Abstract

The rapid expansion of the COVID-19 pandemic has made the development of a SARS-CoV-2 vaccine a global health and economic priority. Taking advantage of versatility and rapid development, three SARS-CoV-2 mRNA vaccine candidates have entered clinical trials with a two-dose immunization regimen. However, the waning antibody response in convalescent patients after SARS-CoV-2 infection and the emergence of human re-infection have raised widespread concerns about a possible short duration of SARS-CoV-2 vaccine protection. Here, we developed a nucleoside-modified mRNA vaccine in lipid-encapsulated form that encoded the SARS-CoV-2 RBD, termed as mRNA-RBD. A single immunization of mRNA-RBD elicited both robust neutralizing antibody and cellular responses, and conferred a near-complete protection against wild SARS-CoV-2 infection in the lungs of hACE2 transgenic mice. Noticeably, the high levels of neutralizing antibodies in BALB/c mice induced by mRNA-RBD vaccination were maintained for at least 6.5 months and conferred a long-term notable protection for hACE2 transgenic mice against SARS-CoV-2 infection in a sera transfer study. These data demonstrated that a single dose of mRNA-RBD provided long-term protection against SARS-CoV-2 challenge.

Fig. 1. Construction and characterization of mRNA-RBD vaccine.

a Schematic of the mRNA-RBD vaccine design. The SARS-CoV-2 mRNA encodes the signal peptide (SP), receptor-binding domain (RBD) from SARS-CoV-2 strain Wuhan/IVDC-HB-01/2019. b mRNA-RBD was transfected into HEK293T cells. RBD expression in the cell lysate and supernatant was analyzed by western blotting. c Particle size of LNPs by dynamic light scattering. d A representative cryo-electron microscopy image of a LNPs solution following mRNA encapsulation. Scale bar, 100 nm. e Zeta potential for LNPs at pH 4.0 and 7.4. For b and d, two independent experiments were carried out with similar results. For c and e, one representative result from three independent experiments is shown. Source data are provided as a Source Data file.

Fig. 2. Immunogenicity evaluation of a single mRNA-RBD vaccination.

a–c Groups of BALB/c mice (n = 6) were immunized with a single injection of mRNA-RBD at different doses or with a placebo via the i.m. route. Sera at 4 weeks post immunization were collected. SARS-CoV-2 RBD-specific IgG (a) and neutralizing antibody titers in sera against pseudovirus (b) and live virus (c) infection were determined. d–h C57BL/6 mice (n = 6) were inoculated with a single mRNA-RBD vaccination or a placebo. Serum samples were collected from mice at 4 weeks following vaccination. RBD-specific IgG titers and pseudovirus-neutralizing antibodies were measured as shown in d and e, respectively. f An ELISPOT assay was performed to evaluate the capacity of splenocytes to secrete IFNγ following re-stimulation with SARS-CoV-2 RBD peptide pools. g, h An ICS assay was conducted to quantify the proportions of IFNγ-secreting CD8⁺ (g) and CD4⁺ (h) T cells. mRNA-RBD-L indicates the low dose (2 μg). mRNA-RBD-H indicates the high dose (15 μg). HCS represents human convalescent sera. Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t-test (unpaired, two tailed). Placebo animals = black circles; mRNA-RBD-L vaccinated animals = blue triangles; mRNA-RBD-H vaccinated animals = red squares; HCS = brown circles; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

Fig. 3. Protection efficacy of mRNA-RBD in hACE2 transgenic mice against SARS-CoV-2.

a-d Groups of hACE2 transgenic mice (n = 6) received one (prime group) or two (boost group) doses of mRNA-RBD-H or placebo via the i.m. route. Four weeks post initial vaccination, mice were challenged with 1 × 10⁵ FFU of SARS-CoV-2 virus. a Mice immunization and challenge schedule. The blue arrows indicate the time of vaccination. b, c Sera collected at 4 weeks post initial vaccination were examined for IgG (b) and neutralizing antibody (c) titers. d Mice weight change after challenge. e Virus titers in lungs of challenged mice (n = 4). f Representative histopathology (H&E) of lungs in SARS-CoV-2-infected hACE2 mice (5 dpi). Infiltration of lymphocytes within alveolar spaces is indicated by yellow arrows. Scale bar, 100 μm. g Representative immunohistochemistry (IHC) of lung tissues with SARS-CoV-2 N-specific monoclonal antibodies. Virus is indicated by yellow arrows. Scale bar, 100 μm. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t-test (unpaired, two tailed). Placebo animals = black circles; one injection-animals = blue triangles; two injections-vaccinated animals = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

Fig. 4. Duration and long-term protection of humoral response induced by mRNA-RBD.

a Passive immunization and challenge schedule. The blue and red arrow indicates the time of vaccination and sera transfer, respectively. b, c Groups of BALB/c mice (n = 10) received 15 μg of mRNA-RBD or a placebo. Half of the mice per group were euthanized at 8 weeks (short term) post vaccination, and massive sera were collected for further passive immunization. The other mice of the group were bled as desired and eventually euthanized at 26 weeks (long term) post vaccination to collect massive sera for further passive immunization. All serum samples were detected for IgG (b) and neutralizing antibodies (c) titers. d–e hACE2 transgenic mice (n = 5) were administered 350 μl per mouse of pooled short- and long-term immune sera and one day later were challenged with 1 × 10⁵ FFU of SARS-CoV-2 via the i.n. route. d The hACE2 mice weight change was recorded after challenge. e Virus titers in lung. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t-test (unpaired, two tailed). Placebo animals = black circles; animals for long-term study = blue triangles; animals for short-term study = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.