Fig 1.
Attenuated replication of LAVNsp16 in K18-hACE2 mice.
B6.Cg-Tg(K18-ACE2)2Prlmn/J mice were infected intranasally with 1,000 PFU of wt or LAVNsp16 virus in 30 µl PBS (n = 4). Animals were sacrificed on day seven postinfection, or when predefined welfare endpoints (clinical score ≥ 20) were reached. Viral genome titers in bronchoalveolar lavage (BAL) (A), lung- (B) and brain tissue (C) were determined by RT-qPCR and are depicted as mean + /- s.d. overlaid with individual data points. Samples with undetectable viral load were set to 1. (A-C) Statistical analysis was done using Kruskal-Wallis tests and corrected for multiple comparison using the Dunn’s test. (D) As a surrogate for tissue damage, protein concentration in the bronchoalveolar lavage fluid was determined by BCA assay. Concentrations are depicted as mean + /- s.d. overlaid with individual data points. Statistical analysis was performed using ordinary one-way ANOVA, followed by Tukey’s correction for multiple comparison. (E) Survival of wt- or LAVNsp16-infected K18-ACE2 mice (n = 4) was monitored over the course of seven days. (F) Weight of wt and LAVNsp16 infected animals (n = 4) is normalized to the initial bodyweight.
Fig 2.
Generation of a novel chimeric ACE2 mouse model by forward directed mutagenesis.
Structural analysis of the interactions formed between the SARS-CoV-2 RBD and (A) murine (m) ACE2 or (B) human (h) ACE2. The SARS-CoV-2 RBD and ACE2 are shown in cyan and blue, respectively. The four residues, which differ between hACE2 and mACE2, as well as their spatially adjacent residues within the RBDs are explicitly labeled. In hACE2, the four residues form strong polar intermolecular interactions (thick green lines). In mACE2 these polar interactions are either weaker (thin green lines) or completely absent. In addition, an unfavorable steric clash is observed between N31 and F456 of RBD and mACE2 (red arrow). (C) Alignment of the human, murine and chimeric ACE2 amino acid sequences, created with the UniProt Align tool. Blue numbers indicate the amino acid position within mACE2. Grey boxes highlight the amino acids, which have been changed to create a human/mouse chimeric receptor (chACE2). (D) Immunoblot analysis of ACE2 expression in lung and brain tissue of eight weeks old chACE2, K18-ACE2, and C57BL/6 mice.
Fig 3.
Chimeric ACE2 supports SARS-CoV-2 wt and LAVNsp16 replication in vivo.
Eight weeks old chACE2 mice were infected intranasally with 7x104 PFU of wt or LAVNsp16 virus in 30 µl PBS. Clinical scores and bodyweight of infected mice were monitored daily. Animals were sacrificed on days two, four, and seven postinfection (n = 4). Bodyweight (A) and survival status (B) of animals sacrificed on day seven postinfection are depicted. Weight measurements of individual animals were normalized to the initial weight and are shown as mean + /- s.d. At each endpoint, bronchoalveolar lavage (BAL) (C), lung (D), and brain (E) samples were collected and viral RNA copy numbers were determined by RT-qPCR. Bars represent the mean + /- s.d. overlaid with individual data points. Statistical analysis of bodyweight and viral genome titers was performed by conducting separate two-tailed t-tests for each timepoint.
Fig 4.
Lung infiltration and expression of interferon stimulated genes upon wt virus and LAVNsp16 infection in chimeric ACE2 mice.
(A-E) Lung infiltration by monocytes (CD45+CD11b+CD11c-), neutrophils (CD45+Gr-1high), natural killer cells (CD45+CD49b+), macrophages (CD45+CD11b+CD11c+), and CD8 + T cells (CD45+CD8+) was determined by antibody staining of cells present in bronchoalveolar lavage (BAL) and subsequent flow cytometry. Bars represent the mean + /- s.d. Statistical analysis were preformed using one-way ANOVA followed by Tukey’s test (A, B, D) or Kruskal-Wallis tests followed by Dunn‘s correction (C). (F) Transcript levels of the indicated interferon-stimulated genes in lung tissue of infected animals were determined by RT-qPCR and normalized to GAPDH (ΔΔCt). Relative mRNA levels compared to uninfected controls are plotted. Statistical analysis was performed using unpaired two-tailed t-tests. Samples originate from infected animals analyzed in Fig 3 at two, four, or seven days postinfection.
Fig 5.
LAVNsp16 immunization induces local and systemic adaptive immune responses.
Eight weeks old chACE2 mice were infected with 7x104 PFU of wt virus or LAVNsp16. Bronchoalveolar lavage (BAL), serum, and lymphocytes derived from lung and spleen tissue were collected on day 21 postinfection. Spike-specific antibody levels in serum (A) and BAL fluid (B) were determined by ELISA. Mean luminescence + /- s.d upon HRP-coupled secondary antibody incubation is shown overlayed by data points of individual animals. S-specific B cells (C) and T cells (E, F) present in spleen or lung tissue were identified by antibody staining and subsequent flow cytometric analysis. Cell counts are displayed as mean + /- s.d. overlayed by individual data points. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test (B, D, E) or Kruskal-Wallis analysis followed by Dunn’s test (A, C). (D) Neutralization capacity of S-specific antibodies from sera of wt- or LAVNsp16-infected animals was analyzed in pseudotyped virus neutralization assays. Neutralization capacity is shown as mean inhibition of quadruplicate infections + /- s.d. (G, H) Spike-specific CD8+ T cells were further discriminated into KLRG1+ CD127- effector T cells (TEFF), KLRG1+ CD127+ effector memory T cells (TEM), KLRG1- CD127+ central memory T cells (TCM), and single or double positive CD69+ CD103+ tissue resident T cells (TRM). (I, K) Lung and spleen-derived T cells were restimulated ex vivo with S-derived peptides (VNFNFNGL and VTWFHAIHVSGTNGT). Resulting production of CD107a, IFNγ, TNFα, and IL2 by CD8+ T cells was determined by intracellular cytokine staining and analyzed by flow cytometry. Results are plotted as mean counts/million + /- s.d. Statistical analysis was done by two-way ANOVA followed by Bonferroni correction.
Fig 6.
SARS-CoV-2 infection results in high viral loads and severe disease in aged chACE2 mice.
Forty-six to fifty-five weeks old chACE2 mice were infected with 7x104 PFU of wt virus or LAVNsp16 in 30 µl PBS (n = 4). Mice were sacrificed on day 2 (n = 4) or day 4 (n = 5) postinfection. Weight loss (A) and clinical score (B) of all animals were determined over time and shown as mean + /- s.d.. C) Infectious viral titers in BAL and lung tissue of aged mice at day 2 postinfection were assessed by infection of Caco-2 cells followed by immunofluorescence analysis. Titers are shown as mean of triplicate infections + /- s.d. overlayed with data points of individual animals. (D) Viral genome copies BAL and lung tissue was determined by RT-qPCR and is shown as mean of quadruplicates + /- s.d. overlayed with data points of individual animals. (E, F, G) For clarity reasons, weight, clinical, and viral titers from young chACE2 mice (as shown in Fig 3) were included. Viral load in BAL (G) of young and aged chACE2 mice two days postinfection with wt and LAVNsp16 was determined by RT-qPCR and is shown as mean + /- s.d. overlayed with data points of individual animals. (A-G) Statistical analysis was performed by unpaired two-tailed t-tests. (H) ACE2 and GAPDH protein expression in lung tissue lysates of uninfected, young (8 weeks), or old (42 weeks) chACE2 and C57BL/6 (B6) mice is shown.
Fig 7.
Strong immune cell infiltration, enhanced ISG expression, and enhanced tissue damage in the lung of aged chACE2 mice.
Immune responses were analyzed in 46 to 55 weeks old chACE2 mice at the indicated time points postinfection with 7x104 PFU of wt virus or LAVNsp16 (as shown in Fig 6). (A-E) Lung infiltration by CD45+CD11b+ monocytes, CD45+Gr-1high neutrophils, CD45+CD49b+ natural killer cells, CD45+CD11b+CD11c+ macrophages, and CD45+CD8+ T cells was determined by antibody staining of cells in BAL followed by flow cytometry. The mean cell number + /- s.d. as well as individual data points are shown. Statistical analysis was done using unpaired two-tailed t-tests (F) mRNA levels of Ifi44, Oas1 and Ifnb1 present in lung tissue of infected animals were determined by RT-qPCR and normalized to GAPDH (ΔΔCt). For statistical analysis, unpaired two-tailed t-tests were performed.
Fig 8.
Lung pathology upon infection.
(A) Lungs of young and aged chACE2 mice infected with wt virus or LAVNsp16 were resected at four days postinfection (n = 3). Upon fixation with paraformaldehyde (PFA), tissues were embedded in paraffin. To visualize lung infiltration, 5 µm tissue sections were stained with hematoxylin and eosin (H&E). Slides were scanned by S210 digital slide scanner from Hamamatsu (0.23 µm/pixel; S210, Hamamatsu, Japan). Pictures were generated using the Case Viewer software (3DHistech, Hungary). One representative slide per condition is shown. (B) H&E score of lungs of naïve, WT-, and LAVNsp16 -infected mice as shown in (A). Histopathology was assed by scoring perivascular, peribronchial, intrabronchial, or parenchymal inflammatory events (total score 0 -16). Data are presented as mean ± s.d. (n = 3). (C) As surrogate for epithelial barrier damage, protein concentration in BAL samples of aged animals sacrificed on day two and day four postinfection was determined by BCA assay. Results are shown as mean + /- s.d.
Fig 9.
LAVNsp16 immunized aged animals are protected from homologous or heterologous SARS-CoV-2 challenge.
Aged chACE2 mice were immunized with 7x104 PFU of LAVNsp16. At 21 days postinfection, immunized and naïve mice were challenged with 7x104 PFU virus of either the B.1 (WT, homologous) or the Delta (heterologous) variant. Mice were sacrificed at four days postchallenge, or when predefined welfare endpoints were reached (clinical score ≥ 20) at day 3 (♦). Weight measurements, normalized to the initial bodyweight of wt virus (A) or Delta virus (B) infected animals are shown as mean + /- s.d. Survival rates of animals challenged with wt (C) or Delta (D) virus are shown. Viral loads in bronchoalveolar lavage (BAL) (E) and lung tissue (F) were determined by RT-qPCR and plotted as mean + /- s.d. overlayed by individual data points. Statistical analysis was performed by two-way ANOVA followed by Bonferroni’s correction for multiple comparison.