Fig. 2: Viral pneumonia in the lungs of cynomolgus macaques challenged 5 weeks or 5 years after vaccination.

Cynomolgus macaques were subcutaneously vaccinated and infected as described in Fig. 1a. Lung tissues were collected 7 days after challenge infection. For each macaque, at least one section was made from the proximal part of the lobe vertical to the bronchus: left upper, left middle, left lower, right upper, right middle, and right lower lobes. a H & E staining and lung histological score (right graph). Histological scores were evaluated in each section. Each dot represents the average score of 6–8 sections for each macaque. b–f Immunohistochemical analysis of infiltrating cells. A section that showed the closest histological score to the average was selected. Left panels: b myeloid cells (monocytes and granulocytes), c CD163-positive inhibitory macrophages, d granzyme B-positive killer cells, e CD3-positive T cells, and f NKG2A-positive natural killer cells. Brown: positive cells; blue: nucleus. Right graphs: quantification of positive cells detected by immunohistochemistry. Each dot represents the average number of more than ten fields for each macaque. g Fibrin clots were visualized by Carstairs staining, and the number of clots was counted manually under the light microscope for each section. Left: Carstairs staining. Fibrin, red blood cells, collagen and platelets are stained in orange-red, yellow, blue, and gray, respectively. The asterisk indicates a fibrin clot in blood vessels. Right graph: the number of fibrin clots per pixel (area). Each dot represents the average number of 6–8 sections for each macaque. The area of each slide was measured by Image J, and the number of clots per area was calculated. a–g Left panel, representative photos are shown with macaque identification numbers in photos. Right panel, black: unvaccinated macaques; red: macaques challenged 5 years after vaccination; blue: macaques challenged 5 weeks after vaccination. **: p < 0.01, statistical analysis was performed with the two-tailed Student’s t-test. There is no significant difference by the analysis with the two-tailed Mann-Whitney U-test.