Fig 2: Histopathological analysis of XBB.1.5 infection in K18-hACE2 mice. (A) Representative images of dual immunofluorescence-stained lung tissues. Lung samples were collected from mock-infected control and at days 3 and 5 post XBB.1.5 infection and labeled with the SARS-CoV-2 nucleocapsid protein (green), DsRNA (red), and nuclei (blue). Scale bars for mock-infected control lung: 100 μm and 50 μm on the original and high-magnification images, respectively. Scale bars for XBB.1.5-infected lungs: 200 μm and 50 μm on the original and high-magnification images, respectively (A representative image is shown for each group). (B) Images of whole lungs collected from mock-infected control (n = 1) and at 5 dpi with XBB.1.5 (n = 3). (C) Representative images of hematoxylin and eosin (H&E)-stained lung sections collected from mock-infected control and at 3 or 5 dpi with XBB.1.5 (A representative image is shown for mock and 3 dpi; 2 representative images from 2 individual mice are shown for 5 dpi; Scale bars: 2,000 μm and 200 μm on the original and high-magnification images, respectively). (D) Representative images of dual immunofluorescence-stained lung tissues collected from mock-infected control and at days 3 and 5 post XBB.1.5 infection. Lung samples were labeled with CD45-Alexa Fluor® 488 (green), Anti-Actin α-Smooth Muscle-Cy3™ (red), and DAPI (blue). Scale bars for mock-infected control lung: 50 μm and 20 μm on the original and high-magnification images, respectively. Scale bars for XBB.1.5-infected lungs: 200 μm and 50 μm on the original and high-magnification images, respectively (A representative image is shown for each group).

Fig 3: Cellular immune response to XBB.1.5 infection. Flow cytometric analysis of lung tissues in K18-hACE2 mice following intranasal inoculation with XBB.1.5 or mock infection. (A) CD45+ cell numbers in mock- and XBB.1.5-infected lungs. (B) CD45+ CD3+ cell numbers. (C) CD8+ T-cell numbers. (D) CD4+ T-cell numbers. (E) Representative expression of CD4 and CD8 visualized by surface staining on CD45+ CD3+ cells in mock- and XBB.1.5-infected lungs. (F) Frequency of CD45+, CD3+, CD8+ T, and CD4+ T cells in the lungs. (G) Representative expression of CD11c+ and CD11b+ visualized by surface staining on CD45+ cells in mock- and XBB.1.5-infected lungs. (H) CD45+ CD11b+ cell numbers. (I) CD45+ CD11c+ cell numbers. (J) CD45+ CD11c+ CD11b+ cell numbers. Each point represents an individual mouse. The bars indicate the mean ± SEM. n = 3 both for mock- and 5 dpi XBB.1.5-infected animals; two-tailed unpaired t-test (*p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001).

Fig 4: Histopathological analysis of XBB.1.5 infection in K18-hACE2 mice. (A) Representative images of dual immunofluorescence-stained lung tissues. Lung samples were collected from mock-infected control and at days 3 and 5 post XBB.1.5 infection and labeled with the SARS-CoV-2 nucleocapsid protein (green), DsRNA (red), and nuclei (blue). Scale bars for mock-infected control lung: 100 μm and 50 μm on the original and high-magnification images, respectively. Scale bars for XBB.1.5-infected lungs: 200 μm and 50 μm on the original and high-magnification images, respectively (A representative image is shown for each group). (B) Images of whole lungs collected from mock-infected control (n = 1) and at 5 dpi with XBB.1.5 (n = 3). (C) Representative images of hematoxylin and eosin (H&E)-stained lung sections collected from mock-infected control and at 3 or 5 dpi with XBB.1.5 (A representative image is shown for mock and 3 dpi; 2 representative images from 2 individual mice are shown for 5 dpi; Scale bars: 2,000 μm and 200 μm on the original and high-magnification images, respectively). (D) Representative images of dual immunofluorescence-stained lung tissues collected from mock-infected control and at days 3 and 5 post XBB.1.5 infection. Lung samples were labeled with CD45-Alexa Fluor® 488 (green), Anti-Actin α-Smooth Muscle-Cy3™ (red), and DAPI (blue). Scale bars for mock-infected control lung: 50 μm and 20 μm on the original and high-magnification images, respectively. Scale bars for XBB.1.5-infected lungs: 200 μm and 50 μm on the original and high-magnification images, respectively (A representative image is shown for each group).

Fig 5: Immunopathology in the lungs of C57BL/6 mice following SARS-CoV-2 XBB.1.5 infection. (A) Representative immunofluorescence images of lung sections from mock- and XBB.1.5-infected mice stained with CD45-Alexa Fluor® 488 (green, leukocytes), Anti-Actin α-Smooth Muscle-Cy3™ (red, smooth muscle), and DAPI (blue, nuclei). Scale bars: 150 μm (low magnification) and 75 μm (high magnification). (B) Representative immunofluorescence images of lung sections stained with CD68-Alexa Fluor® 488 (green, macrophages), Anti-Actin α-Smooth Muscle-Cy3™ (red, smooth muscle), and DAPI (blue, nuclei). Scale bars: 250 μm (low magnification) and 75 μm (high magnification). n = 4 animals per group.