Human-Immune-System (HIS) humanized mouse model (DRAGA: HLA-A2.HLA-DR4.Rag1KO.IL-2RγcKO.NOD) for COVID-19

Figures & data

Table 1. Human immune parameters of HIS-DRAGA mice.

			Peripheral blood
Mouse*	Gender	Time post-infusion with human stem cells	% human B cells (CD19^+)	% human T cells (CD3^+)	SARS-CoV-2 challenge dose (pfu)	Euthanasia (days post-infection)	Cord blood HLA haplotype^	Cord blood gender
M#1	M	21 weeks	4.5	13.5	10^4	1	A	M
F#1	F	21 weeks	48.6	9.3	10^4	14	A	M
F#2	F	21 weeks	21	33.4	2.8x10^3	14	A	M
F#3	F	24 weeks	16.1	37.8	1x10^3	25	C	M
F#4	F	16 weeks	9.0	2.5	1x10^3	25	B	F
F#5	F	24 weeks	6.0	23.8	1x10^3	25	C	M
F#6	F	30 weeks	5.9	42.6	1x10^3	25	A	M
F#7	F	16 weeks	23.6	5.0	1x10^3	25	B	F
F#8	F	16 weeks	0.6	12.5	1x10^3	25	B	F
F#9	F	16 weeks	21.5	8.0	1x10^3	25	B	F
F#10	F	16 weeks	37.0	4.7	1x10^3	25	B	F
M#2	M	30 weeks	8.3	9.3	1x10^3	4	A	M
M#3	M	16 weeks	5.7	15.0	1x10^3	4	B	F
M#4	M	16 weeks	2.0	51.1	1x10^3	4	B	F
F#11	F	16 weeks	6.1	25.4	1x10^3	4	B	F
F#12	F	24 weeks	7.0	8.8	1x10^3	4	C	M
F#13	F	24 weeks	0.6	26.8	1x10^3	4	C	M
a	M	20 weeks	18.9	28.3	—-	—-	D	F
b	M	20 weeks	0.9	41.4	—-	—-	D	F
c	F	24 weeks	52.5	17.5	—-	—-	D	F
d	M	24 weeks	7.4	16.6	—-	—-	D	F
e	M	24 weeks	1.0	22.1	—-	—-	D	F
f	M	24 weeks	1.2	44.7	—-	—-	D	F
g	M	24 weeks	17.6	38.3	—-	—-	D	F
h	F	20 weeks	35.2	5.0	—-	—-	E	M
i	M	26 weeks	4.9	26.1	—-	—-	E	M
j	M	26 weeks	21.9	30.7	—-	—-	E	M

Lungs from SARS-CoV-2 infected mice (M#2–4, F#11–13) that were euthanized at day 4 post-infection to estimate lung viral copies by RT-qPCR. Lungs from uninfected mice a-j were pooled to assess human ACE2 mRNA and protein expression.

^A: A02:01/A24:02/B13:02/B44:02/DR01:01/DR04:01.

^B: A01:01/A02:01/B08:01/B15:01/DR03:01/DR04:01.

^C: A02:01/A24:02/B44:02/B52:01/DR04:01/DR11:04.

^D: A02:01/A02:01/B18:01/B44:02/DR04:01/DR11:04.

^E: 02:01/A02:01/B08:01/B27:05/DR03:01/DR04:01.

Figure 1. Human ACE2 detection in the lungs of HIS-DRAGA mice.

(a) Amplified RT-PCR products using hACE2 primers (upper panel) and mACE2 primers (lower panel) on RNA samples extracted from human lung (lane 1), from lungs of four non-infected HIS-DRAGA mice (lanes 2–5), and from lungs of 2 non-HIS-reconstituted DRAGA mice (lanes 6–7). NC, negative RT-PCR control (lane 8). (b) hACE2 protein expression on alveolar and bronchiolar epithelia as indicated by binding of S1 (RBD)-mFcγ2a recombinant chimeric protein revealed by a goat anti-mouse IgG-FITC conjugate in lung sections from two HIS-DRAGA male mice and one HIS-DRAGA female mouse. Lower panels show S1 (RBD)-FITC staining, and upper panels show S1 (RBD)-FITC and DAPI (nuclei) co-staining. Shown are enlargements of alveolar epithelia (right panels). (c) Lung section from human lung stained as in panel b. (d) Lung sections from two non-HIS reconstituted DRAGA mice stained as in panel b.

Figure 2. Quantification of hACE2 protein in HIS-DRAGA lungs.

Lung lysates were immunoprecipitated using S1 (RBD)-mFcγ2a protein + rat anti-mouse IgG2a-magnetic beads, and eluted hACE2 levels were quantified by ELISA (upper panel), as described in Materials and Methods. Inserted image shows immunoblot analysis of hACE2 immunoprecipitated from human lungs (lane 1), hACE2 immunoprecipitated from HIS-DRAGA mouse lungs (lane 2), and the absence of hACE2 in the lung immunoprecipitate from lungs of a non-HIS-reconstituted DRAGA mice (lane 3). Lower panel provides the data used to calculate the amount of hACE2 immunoprecipitated from human lungs as compared to HIS-DRAGA mouse lungs.

Figure 3. Co-Localization of hACE2 with hCD326 on alveolar human ECs in HIS-DRAGA lungs.

(a&b) Co-localization of hACE2 protein with alveolar hCD326⁺ ECs revealed by co-staining with S1 (RBD)-mFcγ2a + goat anti-mouse IgG-FITC and anti-hCD326-PE in a representative HIS-DRAGA female and male mouse (upper panels). Shown are enlargements of CD326⁺ hECs co-localized with hACE2 on the alveolar epithelia. (c) Low background staining indicating lack of hCD326⁺ ECs and a lack of hACE2 expression lung section from a representative non-HIS-reconstituted DRAGA mouse.

Figure 4. Dynamics of body weight changes following SARS-CoV-2 infection of HIS-DRAGA mice.

(a) Daily monitoring for body weights of HIS-DRAGA male mouse #M1 (dark circles) infected with SARS-CoV-2 virus (10⁴ pfu) and 2 HIS-DRAGA female mice #F1 and #F2 (gray circles) infected with 10⁴ pfu and 2.8x10³ pfu, respectively (pilot infection experiment). (b) Daily monitoring for body weights of 3 HIS-DRAGA female mice (#F11 - #F13, open circles) and three male mice (#M2 - #M4, dark circles) infected with SARS-CoV-2 virus (10³ pfu/mouse). (c) Viral RNA copies in the lungs of mice from panel B measured as described in the Materials and Methods. (d) Daily monitoring of body weights of 2 HIS-DRAGA female mice (#F3 and #F4) that recovered from infection after challenge with SARS-CoV-2 virus (10³ pfu/mouse). (e) Daily monitoring of body weights of six HIS-DRAGA female mice (#F5 - #F10, 6/8 mice, 75%) that did not recover from infection after challenge with SARS-CoV-2 (10³ pfu/mouse).

Figure 5. Lung pathology of SARS-CoV-2 infected HIS-DRAGA mice.

(a) Images of lungs (left panel) and the H&E and Mason’s trichrome stained lung sections (right panels) from a representative non-infected HIS-DRAGA female mouse. (b) Image of lungs (left panel) and the H&E and Mason’s trichrome stained lung sections (right panels) from a representative HIS-DRAGA female mouse (#F5) infected with 10³ pfu of SARS-CoV-2, which did not recover its initial body mass by 25 dpi. Note the discoloration on the left lobe (arrows), parenchymal lymphocyte infiltration and hemorrhagic patches (arrows). Enlargement shows incipient peri-alveolar collagen deposition. (c) Image of lungs (left panel) and the H&E and Mason’s trichrome stained lung sections (right panels) from a representative HIS-DRAGA female mouse (#F6) infected with 10³ pfu of SARS-CoV-2, which had not recovered its initial body mass by 25 dpi. Note the discoloration on the left lobe (arrows) and heavy peri-bronchiolar infiltration with lymphocytes (arrows). Enlargement shows large hemorrhagic patches surrounding a major bronchiole (arrows). (d) Masson’s trichrome staining of lung sections from mouse #F5 in panel b showing intra-alveolar microthrombi (arrows) and collagen deposition (third left panel, blue color). Right panel shows an intra-alveolar blood clot. (e) Masson’s trichrome staining of lung sections (3 left panels) from mouse #F6 in panel c showing microthrombi adherent to the arteriole wall (two left panels, arrows) and an intra-alveolar hemorrhagic patch (panel f, arrow). H&E staining from the lungs of same mouse (#F6) showing a large intra-bronchiolar hemorrhagic patch (panel g, arrow).

Figure 6. Intra-Alveolar microthrombi in SARS-CoV-2 infected DRAGA mice.

(a) Lung sections from two HIS-DRAGA female mice (#F5 and #F6) infected with 10³ pfu that had not recovered from the infection by 25 dpi. Shown are large intra-alveolar, non-nucleated cell clusters staining positive for CD61, indicating the presence of platelet-rich microthrombi. (b) Representative lung section from a non-infected HIS-DRAGA female mouse; the lung parenchyma of this mouse showed no evidence of CD61⁺ -positive clusters indicating platelet-rich thrombi.

Figure 7. Pulmonary sequelae in SARS-Cov-2 infected HIS-DRAGA mice.

Masson’s trichrome staining of lung sections from female mice #F5 (panel a) and #F6 (panel b) which did not recover their initial body weights by 25 days post-infection with SARS-CoV-2 (10³ pfu/mouse). Shown are collagen fibers (blue color) in the peri-alveolar and intra-alveolar infiltrated areas (left panels) and around major bronchioles.

Figure 8. Infiltrating human lymphocytes in the lungs of SARS-CoV-2 infected HIS-DRAGA mice.

Lung sections from HIS-DRAGA female mice #F4 (upper panels in a & b) and #F3 (lower panels in a & b) that had recovered from SARS-CoV-2 infection(10³ pfu/mouse) by 25 dpi stained for hCD45 and hCD3. Of note, infiltrating hCD45⁺ lymphocytes and hCD3⁺ T cells in the infected mice were more abundant and organized in clusters than those in non-infected mice that were dispersed through parenchyma (panels & d).

Figure 9. Human CD8⁺ T cell residency and cytotoxicity in the hCD326⁺ lung epithelial niches of a SARS-CoV-2-infected HIS-DRAGA mouse.

Lung sections from a representative HIS-DRAGA female mouse (#F3) that had recovered its initial body weight 7 days post-infection with SARS-CoV-2 (10³ pfu). (a-d) Co-localization of hCD3⁺ CD4⁺ and CD8⁺ T cell subsets with the hCD326⁺ epithelia marker (mouse #F3). Enlargements show areas of CD4⁺ and CD8⁺ T cell clusters sequestered in the lung CD326⁺ epithelia, with some CD8⁺ T cells egressing into the alveolar air space. (e&f) hCD8⁺ T-resident cell clusters sequestered in lung epithelia of mouse #F3 expressing the hCD103 marker of lung residency. (g&h) hCD8⁺ T cell resident clusters in bronchiolar epithelia of mouse #F3 staining positive for perforin and granzyme B. Similar findings were also present in the lungs of HIS-DRAGA female mouse (#F4) that had recovered its initial body weight by 25 days post-infection.

Figure 10. Human IgM and IgG serum titers to SARS-CoV-2 viral proteins in infected HIS-DRAGA mice.

Shown are the hIgM and hIgG serum titers to SARS-CoV-2 S1 (RBD) protein (a), stable S trimer (b) and total serum antibody titers to Nucleocapsid protein (c) quantified by ELISA at 25 days post-challenge with SARS-CoV-2 (10³ pfu/mouse). OD450_nm values of IgM and IgG antibodies specific for S protein provided in the ELISA kit are indicated (control C+). Control for serum Abs to Nucleocapsid protein (C-) was obtained from a pool of non-infected HIS-DRAGA mice. Shown are the OD450_nm values of anti-viral antibodies corrected by subtracting the OD570_nm values in each well and the signal-to-noise background (ranging from 0.045 to 0.067) from sera of individual mouse prior to infection. Standard deviations (±SD) from two repeated measurements for each serum sample in duplicate wells were determined at 99% interval of confidence using SigmaPlot v.14 software.