Adaptation of SARS-CoV-2 in BALB/c mice for testing vaccine efficacy

Abstract

The ongoing coronavirus disease 2019 (COVID-19) pandemic has prioritized the development of small-animal models for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We adapted a clinical isolate of SARS-CoV-2 by serial passaging in the respiratory tract of aged BALB/c mice. The resulting mouse-adapted strain at passage 6 (called MASCp6) showed increased infectivity in mouse lung and led to interstitial pneumonia and inflammatory responses in both young and aged mice after intranasal inoculation. Deep sequencing revealed a panel of adaptive mutations potentially associated with the increased virulence. In particular, the N501Y mutation is located at the receptor binding domain (RBD) of the spike protein. The protective efficacy of a recombinant RBD vaccine candidate was validated by using this model. Thus, this mouse-adapted strain and associated challenge model should be of value in evaluating vaccines and antivirals against SARS-CoV-2.

Fig. 1. Generation and characterization of a mouse-adapted strain of SARS-CoV-2 in BALB/c mice.

(A) Schematic diagram of the passage history of SARS-CoV-2 in BALB/c mice. The original SARS-CoV-2 viruses are shown in black, and the adapted viruses are in red. (B) SARS-CoV-2 genomic RNA loads in mouse lung homogenates at P0 to P6. Viral RNA copies were determined by means of quantitative reverse transcription polymerase chain reaction (RT-PCR). Data are presented as means ± SEM (n = 2 to 4 mice per group). (C) Tissue distribution of SARS-CoV-2 viral RNAs in mice infected with MASCp6. Groups of aged and young mice were inoculated with 1.6 × 10⁴ PFU of MASCp6 and sacrificed at 3, 5, or 7 days after inoculation, respectively. Feces, sera, and the indicated tissue samples were collected at the specified times and subjected to viral RNA load analysis by means of quantitative RT-PCR. Dashed lines denote the detection limit. Data are presented as means ± SEM (n = 3 mice per group). (D) Multiplex immunofluorescence staining of mouse lung sections. SARS-CoV-2 S protein (green), CC10 (red), β-IV-tubulin (cyan), PDPN (magenta), SPC (gold), and nuclei (blue). The dash box is magnified at the bottom right corner of the same image. Yellow arrowheads indicate SARS-CoV-2⁺/CC10⁺ cells, redarrow heads indicate SARS-CoV-2⁺/CC10⁺/SPC⁺ cells, and the white arrowheads indicate SARS-CoV-2⁺/SPC⁺ cells.

Fig. 2. MASCp6 infection causes pathological lung lesions and inflammatory responses in both aged and young BALB/c mice.

(A) H&E staining of lung sections from aged (9 months old) BALB/c mice infected with MASCp6. Blue arrows indicate the normal areas, and yellow arrows indicate damaged areas. Data from semiquantitative analysis of histopathological changes of lung tissues are presented as means ± SEM (n = 3 mice per group). Statistical significance was analyzed by means of Mann-Whitney test. (B) Serum cytokine and chemokine heatmap in MASCp6-infected aged mice. Data are presented as fold change relative to mock infection (n = 5 mice per group). (C) H&E staining of lung sections from MASCp6-infected young mice (n = 3 mice per group). (D) Serum cytokine and chemokine heatmap in MASCp6-infected young mice (n = 5 mice per group). *P < 0.05, ***P < 0.001.

Fig. 3. MASCp6 carries a distinct amino acid substitution in the RBD of the Spike (S) protein.

(A) Schematic diagram of SARS-CoV-2 genome and all the adaptive mutations identified in MASCp6. Amino acid sequences of the parental IME-BJ05 strain and the MASCp6 strain adjacent to the N501Y mutation are shown. (B) Homology modeling of mouse ACE2 (pink) in complex with SARS-CoV-2 RBD (green) with N501 (left) or Y501 residue (right). (C) Colocalization of SARS-CoV-2 S protein (green) and mouse ACE2 (red) in the lung from SARS-CoV-2–infected mice. The dashed box in the left image is magnified in the three images at the right. (D) The proportion of A23063T mutation in each passage. The mutation threshold was defined as 1% according to the average quality score of sequenced base.

Fig. 4. Protection efficacy of the recombinant RBD-Fc vaccine candidate against MASCp6 challenge in mice.

(A) SARS-CoV-2–specific IgG antibody titers were detected with enzyme-linked immunosorbent assay at 2 weeks after primary and boost immunization, respectively (n = 10 mice per group). Statistical significance was analyzed by means of one-way analysis of variance. (B) Neutralizing antibody titers against SARS-CoV-2 were determined with the microneutralization assay at 2 weeks after boost immunization (n = 10 mice per group). (C) Viral RNA loads in lung of vaccinated mice were detected at 5 days after MASCp6 challenge (n = 5 mice per group). Statistical significance was analyzed by means of Student’s t test. (D) Immunofluorescence staining of mouse lung sections for S protein (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). The dotted boxes are magnified at the bottom of the same image. (E) H&E staining of mouse lung sections. Focal perivascular (green square) and peribronchiolar (yellow square) inflammation and thickened alveolar septa (blue arrow) are indicated. n.s., not significant; **P < 0.01, ***P < 0.001, ****P < 0.0001.