Mice humanized for MHC and hACE2 with high permissiveness to SARS-CoV-2 omicron replication

Abstract

Human Angiotensin-Converting Enzyme 2 (hACE2) is the major receptor enabling host cell invasion by SARS-CoV-2 via interaction with Spike. The murine ACE2 does not interact efficiently with SARS-CoV-2 Spike and therefore the laboratory mouse strains are not permissive to SARS-CoV-2 replication. Here, we generated new hACE2 transgenic mice, which harbor the hACE2 gene under the human keratin 18 promoter, in "HHD-DR1" background. HHD-DR1 mice are fully devoid of murine Major Histocompatibility Complex (MHC) molecules of class-I and -II and express only MHC molecules from Human Leukocyte Antigen (HLA) HLA 02.01, DRA01.01, DRB1.01.01 alleles, widely expressed in human populations. We selected three transgenic strains, with various hACE2 mRNA expression levels and distinctive profiles of lung and/or brain permissiveness to SARS-CoV-2 replication. These new hACE2 transgenic strains display high permissiveness to the replication of SARS-CoV-2 Omicron sub-variants, while the previously available B6.K18-ACE2^2Prlmn/JAX mice have been reported to be poorly susceptible to infection with Omicron. As a first application, one of these MHC- and ACE2-humanized strains was successfully used to show the efficacy of a lentiviral-based COVID-19 vaccine.

Keywords: COVID-19 animal model; HLA; Omicron variant; SARS-CoV-2; Transgenesis.

Fig. 1

Permissiveness of HHD-DR1.ACE2^Hu1 and HHD-DR1.ACE2^Hu2 transgenic mice to SARS-CoV-2 replication. hACE2⁺ offspring from the founders #8, #9 (HHD-DR1.ACE2^Hu1) or #11 (HHD-DR1.ACE2^Hu2), resulting from a conventional DNA-based transgenesis, or B6.K18-hACE2^IP-THV positive control mice were inoculated i.n. with 0.3 × 10⁵ TCID₅₀/mouse of SARS-CoV-2 Delta variant. A. Lung and brain viral RNA contents were determined by E- (top) or sub-genomic Esg- (bottom) specific qRT-PCR at 4 dpi. Red lines indicate the qRT-PCR detection limits. B. Quantification of hACE-2 mRNA in the lung and brain of the same mice. Statistical significance was evaluated by one-way Anova (*= p < 0.05, **= p < 0.01, ***= p < 0.001).

Fig. 2

Permissiveness of HHD-DR1.ACE2^Hu3 transgenic mice to SARS-CoV-2 replication and inflammatory status of their lungs and brain after SARS-CoV-2 inoculation. hACE2⁺ offspring from the founders #30 or #47 (HHD-DR1.ACE2^Hu3), resulting from a lentiviral vector-based transgenesis, were inoculated i.n. with 0.3 × 10⁵ TCID₅₀/mouse of SARS-CoV-2 Delta variant. A. Lung and brain viral RNA contents were determined by E-specific or sub-genomic Esg-specific qRT-PCR at 4 dpi. B. Quantification of hACE-2 mRNA in the lung and brain of the same mice. C. Heatmaps represent log₂ fold change in cytokine and chemokine mRNA expression in the lungs or brain of the same mice (n = 4-5/group). Data were normalized versus untreated controls. Statistical significance was evaluated by Mann-Whitney test (ns = not significant, * = p < 0.05, ** = p < 0.01). D. Pearson correlation coefficient of the analytes studied in the brain of SARS-CoV-2 Delta-inoculated HHD-DR1.ACE2^Hu3 mice.

Fig. 3

Permissiveness of HHD-DR1.ACE2^Hu1/2/3 mice to replication of SARS-CoV-2 Omicron BA.1 or BA.5 sub-variants. HHD-DR1.ACE2^Hu1/2/3 mice or B6.K18-hACE2^IP-THV positive control mice were inoculated i.n. with 0.3 × 10⁵ TCID₅₀/mouse of SARS-CoV-2 Omicron BA.1 or BA.5. A. Lung and B. brain viral RNA contents were determined by E-specific (top) or sub-genomic Esg-specific (bottom) qRT-PCR at 4 dpi with Omicron BA.1. C-D. Comparative quantification of hACE-2 mRNA in the lung and brain of individual B6.K18-hACE2^IP-THV or HHD-DR1.ACE2^Hu1/2/3 transgenic mice. E-F. Lung and brain viral RNA contents were determined by E-specific (top) or sub-genomic Esg-specific (bottom) qRT-PCR at 4 dpi with Omicron BA.5. Statistical significance was evaluated by one-way Anova (*= p < 0.05, **= p < 0.01). ND = Not determined.

Fig. 4

Spike T-cell epitope mapping in HHD-DR1 mice vaccinated with LV::S_Beta-2P. HHD-DR1 mice were primed (i.m.) at wk 0 and boosted (i.n.) at wk 8 with a control empty lentiviral vector (Ctrl LV) or LV::S_Beta-2P. A. Panels of 253 15-mers spanning the Spike protein, were organized into 32 pools (I to XXXII) containing no more than 16 individual peptides. The peptide pools were assigned to a 2D matrix system in which each peptide was represented in two different pools. The intersection of two positive pools identified potentially positive peptides. B. Splenocytes from HHD-DR1 mice (n = 5) immunized with LV::S_Beta-2P or a Ctrl LV were harvested at wk 2 post boost and were analyzed in an IFN-γ ELISPOT after stimulation with each of the 32 distinct I to XXXII pools. Heat map representing the number of IFN-γ-producing cells per million of splenocytes responding to each peptide pool. For each peptide pool, the background generated by the splenocytes from Ctrl LV-injected mice was subtracted. C. Intersection or previously identified peptides were tested individually in ELISPOT. The threshold of positivity was defined by a twofold increase in the number of spots upon stimulation with the peptide, compared to the average number of spots for the same splenocyte sample in the medium alone. D-F. The positive peptides identified were used in CD4 and CD8 surface staining and IFN-γ, IL-2, and TNF-α ICS and cytometric analysis to determine the T-subset specific to the corresponding epitopes. D. Gating strategy, E-F. IL-2/TNF-α expressing cells within the IFN-γ⁺ CD4⁺(E) or CD8⁺(F) T cells after stimulation with the indicated Spike-derived peptides or a negative control peptide. The numbers in the upper right quadrant of the dot plots indicate the percentage of the IL-2⁺ TNF-α⁺ IFN-γ⁺ population within the CD4⁺(E) or CD8⁺(F) T cells. See also Fig. S3.

Fig. 5

Use of HHD-DR1.ACE2^Hu1 mice to evaluate the protective capacity of LV::S_Beta-2P vaccine candidate against SARS-CoV-2. A. Timeline of vaccination and SARS-CoV-2 challenge. B. HHD-DR1.ACE2^Hu1 mice were untreated or primed (i.m.) at wk 0 and boosted (i.n.) at wk 3 with 1 × 10⁸ TU/mouse of LV::S_Beta-2P or Ctrl LV, before i.n. challenge at wk 5 with 0.3 × 10⁵ TCID₅₀ of SARS-CoV-2 Delta variant. Lung viral Esg-specific RNA contents was evaluated by qRT-PCR at 4 dpi. Red lines indicate the detection limits. C. Cytometric detection of neutrophils or NK cells in the lungs of untreated, Ctrl-Lenti-, or LV::S_Beta-2P-vaccinated and challenged mice at 4 dpi. D. Percentages of neutrophils or NK cells in the lungs of untreated, Ctrl LV-, or LV::S_Beta-2P-vaccinated and challenged mice at 4 dpi. Percentages were calculated versus total lung live CD45⁺ cells. Statistical significance was evaluated by one-way Anova test (*= p < 0.05, **= p < 0.01).

Fig. 6

Lung histopathology of unvaccinated or LV::S_Beta-2P-vaccinated HHD-DR1.ACE2^Hu1 mice after inoculation with SARS-CoV-2. Mice are those detailed in the Fig. 5. A. Examples of haematoxylin-eosin saffron staining of whole-lung sections (left, scale bar: 500 μm) and close-up views (right, scale bar: 100 μm), at 4 dpi. Stars mark areas of minimal (bottom) or mild-to-moderate (top) infiltration. In the blow-up panels, red arrows indicate bronchiolar epithelia. While in the bottom blow-up the epithelium is subnormal, in the top panel epithelial degenerative lesions can be seen, e.g., perinuclear clear spaces (green arrow), associated with cells remnants and proteinaceous material in the bronchiolar lumen. B. Heatmap representing the histological scores for: (i) inflammation seriousness, (ii) interstitial syndrome, and (iii) alveolar syndrome, (iv) alteration of bronchial epithelium, and (v) percent of abnormal lung zones.