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

To generate a SARS-CoV-2 mouse-adapted strain, the human clinical isolate of SARS-CoV-2 (BetaCov/human/CHN/Beijing_IME-BJ05/2020, abbreviated as IME-BJ05) was serially passaged by means of intranasal inoculation in aged mice (Fig. 1A), as previously described for SARS-CoV (12). Briefly, 9-month-old BALB/c mice were intranasally inoculated with 7.2 × 10⁵ plaque forming units (PFU) of SARS-CoV-2, and the lung tissues were collected from each passage for viral RNA load analysis at 3 days after inoculation. Substantial viral RNAs (10^8.32 copies/g) were readily detected after a single passage, which was defined as passage 0 (P0), in the lung homogenate (Fig. 1B). Subsequently, the viral RNA copies in the lung approached 10^10.68 RNA copies/g at passage 3 (P3), which was about 250-fold higher than those at P0 and remained at a similar level during the following passages (Fig. 1B). The final viral stock at passage 6 (P6) was titrated by means of plaque assays (fig. S1A) and called MASCp6 for further characterization.

To determine whether the increased viral RNA loads in mouse lungs could be attributed to the enhanced infectivity of the virus in mice, we examined the replication kinetics and tissue tropism of MASCp6 in both aged (9 months old) and young (6 weeks old) BALB/c mice. After intranasal inoculation with 1.6 × 10⁴ PFU of MASCp6, high amounts of viral RNAs in the lungs and tracheas were detected at 3, 5 and 7 days after inoculation in all aged mice (Fig. 1C), with peak viral RNA loads of ~10¹⁰ copies/g at 3 days after inoculation, which was comparable with the results from the human ACE2 transgenic mice (10). Viral RNAs were also detected in heart, liver, spleen, and brain, as well as in feces. Marginal viral RNA was detected in the kidney and serum from individual infected mice (Fig. 1C). Similar tissue distribution of SARS-CoV-2 RNA was also seen in the MASCp6-infected young mice (Fig. 1C). Immunostaining of lung section from MASCp6-infected mice showed robust expression of S protein along the airways and at the alveolus in both young and aged mice at 3 and 5 days after inoculation (fig. S1B). To identify the major cell types infected by SARS-CoV-2 in our model, lung sections were further analyzed by means of multiplex immunofluorescence staining for SARS-CoV-2 S protein and specific lung epithelium cell markers. As shown in Fig. 1D, colocalization of CC10⁺ club cells and SARS-CoV-2 S protein were observed predominantly in the bronchi and bronchioles as well as the bronchioalveolar-duct junction (BADJ) of the lungs. Furthermore, SPC⁺ alveolar type 2 (AT2) cells were also costained with S protein in the BADJ and alveoli. However, SARS-CoV-2 S protein was not detected in all β-IV-tubulin⁺ ciliated cells and PDPN⁺ alveolar type 1 (AT1) cells. Thus, club cells and AT2 cells are the major target cells that support SARS-CoV-2 replication in mouse lung in our model.

To further characterize pathological features in the MASCp6-infected BALB/c mice, lung tissues were collected at 3 or 5 days after inoculation, respectively, and subjected to histopathological analysis by means of hematoxylin and eosin (H&E) staining. Both aged and young BALB/c mice presented with mild to moderate pneumonia after MASCp6 infection (Fig. 2, A and C). In the aged mice, MASCp6 infection caused interstitial pneumonia at 3 days after inoculation, characterized with denatured and collapsed epithelial cells, thickened alveolar septa, alveolar damage, focal exudation and hemorrhage, and activated inflammatory cell infiltration. Vessels were obviously injured, with adherent inflammatory cells and damaged basement membrane (Fig. 2A). At 5 days after inoculation, the lung damage was much milder than that seen at 3 days after inoculation (Fig. 2A), suggesting a self-recovering process. In addition, multiplex immunofluorescence staining demonstrated that SARS-CoV-2 infection led to massive cell death, as evidenced with cleaved caspase-3 staining, at 3 days after inoculation, as well as remarkable inflammatory cell infiltration in CD103⁺ dendritic cells, CD163⁺ macrophages, and CD3⁺ T lymphocytes in the lung of aged mice (fig. S2A). Serum concentrations of inflammatory cytokines—including interleukin-1β (IL-1β), IL-6, and IL-5—were up-regulated upon MASCp6 challenge (Fig. 2B and fig. S3A). As expected, the young mice developed similar but much milder lung damage than that of the aged mice after MASCp6 challenge (Fig. 2C). Similarly, inflammatory cell infiltration (fig. S2B) and serum cytokine response (Fig. 2D and fig.S3B) in the young mice was much weaker than that of aged mice. Additionally, MASCp6 infection caused no obvious changes in the body weight of aged or young mice (fig. S4). Taken together, these results demonstrate that MASCp6 can productively replicate in the lower respiratory tract of wild-type BALB/c mice, resulting in a more severe interstitial pneumonia phenotype in the aged mice.

To decipher the underlying mechanism for the increased virulence of MASCp6, the complete genome of MASCp6 was subjected to deep sequencing with an Ion Torrent S5Plus sequencer. Compared with the full genome of the original SARS-CoV-2 strain IME-BJ05, MASCp6 contains five nucleotide mutations that are distributed within the ORF1ab, S, and N genes, respectively (Fig. 3A and table S1). The A23063T mutation resulted in a N501Y amino acid substitution in the RBD of the S protein, which is assumed to be responsible for receptor recognition and host range of SARS-CoV-2 (13, 14). (A, alanine; T, threonine; N, asparagine; Y, tyrosine. In the mutants, other amino acids were substituted at certain locations; for example, A23063T indicates that alanine at position 23063 was replaced by threonine.) Structural remodeling also suggested that the N501Y substitution in the RBD of SARS-CoV S protein increased the binding affinity of the protein to mouse ACE2 (Fig. 3B). Immunofluorescence staining supported colocalization of mouse ACE2 and SARS-CoV-2 S protein in the lungs of MASCp6-infected mice (Fig. 3C). To further trace the adaption history of MASCp6, the emergence of N501Y substitution was further analyzed by means of deep sequencing. As expected, all reads from the original IME-BJ05 isolate were pure A23063. T23063 readily emerged after a single passage in one of the three mouse lung homogenates (table S2), and the proportion of A23063T mutation gradually increased during subsequent passages (Fig. 3D). Thus, the increased virulence of SARS-CoV-2 MASCp6 in mice was likely attributed to the rapid emergence of N501Y substitution in the RBD of SARS-CoV-2 S protein.

To validate the utility of this mouse challenge model, we tested the protection efficacy of a recombinant subunit vaccine candidate against COVID-19. Briefly, SARS-CoV-2 RBD (aa 331-524) fused with a human immunoglobulin G (IgG) Fc at the C terminus (fig. S5A) was expressed in CHO-K1 cells and purified through affinity chromatography and anion exchange chromatography in a good laboratory practice (GLP) laboratory. As expected, the molecular weight of recombinant RBD-Fc was about 47.98 kD, as detected with mass spectroscopy (fig. S4B), and flow cytometry analysis confirmed that RBD-Fc, not the Fc control, specifically bound to human ACE2 expressed in the stable ACE2/293T cells (fig. S4C) (15).

Female BALB/c mice were then subcutaneously immunized with two doses of recombinant RBD-Fc (10 μg/mouse) at a 2-week interval, and mice immunized with phosphate-buffered saline (PBS) were set as controls. As expected, high levels of SARS-CoV-2–specific IgG antibodies (Fig. 4A) and neutralization antibodies (Fig. 4B) were elicited in all of the RBD-Fc immunized mice at 2 weeks after boost immunization. All immunized mice were then intranasally challenged with MASCp6 (1.6 × 10⁴ PFU), and lung tissues were collected for virological and histopathological analysis at 5 days after challenge. As expected, all the PBS-treated mice sustained high amounts of viral RNA loads in the lung at 5 days after challenge. By contrast, a significant reduction in viral RNA loads (approximately 0.1%) were seen in the lung of RBD-Fc immunized mice compared with the control animals (Fig. 4C). Moreover, immunofluorescence staining for SARS-CoV-2 S protein showed that only a small population of positive cells was detected in the lung from the RBD-Fc–immunized mice, whereas abundant viral proteins were seen in the lung from PBS-immunized mice (Fig. 4D). No apparent pathological damage was observed in the lung of RBD-Fc immunized mice, whereas inflammatory lung injury—with focal perivascular and peribronchiolar inflammation, as well as thickened alveolar septa—were found in the lung of the control mice (Fig. 4E). Taken together, these data indicate that our newly developed mouse model with MASCp6 represents a useful tool for testing the efficacy of COVID-19 vaccine candidates.