Fig 1: Histological features of kidney biopsy showing diffuse active tubulointerstitial nephritis. (A) H&E slide. Marked lymphocytic infiltration in the interstitium with an admixture of neutrophils and plasma cells. Focal features of acute tubulitis are also present (arrowheads). Glomeruli reveal no apparent histological changes. (B) Electron microscopy sample is taken from interstitium between renal tubules. Few virus-like particles in size of ~120 nm with barely visible spikes are present intracellularly. Cross-sections of the particles contain interior electron-dense black dots which could be interpreted as helical nucleocapsid (lower magnification is available in Supplementary Figure 1). (C,D) Immunohistochemistry (IHC) for Anti-SARS-CoV-2 spike glycoprotein highlighted positive scattered immune cells in the interstitium of renal parenchyma (arrows). Rabbit polyclonal antibody against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (ab272504, Abcam) was applied. Lung tissue of a deceased COVID-19-infected patient was used as a positive tissue control and a negative reagent control was performed on a consecutive biopsy tissue section to ensure sensitivity and specificity of the IHC test.
Fig 2: Immunohistochemical/immunofluorescence expression of Sars-Cov-2 antigens in relation to histopathological changes in thyroid gland obtained from patient who deceased from COVID-19. Sars-Cov-2 spike (S) protein was found notably expressed throughout the thyroid tissue and was localized diffusely in the cytoplasm of follicular cells (A, B, C, D, E, F and J, K, L, M, N, O). Concomitantly, the thyroid gland harboured copious mononuclear infiltrate (A, B, C, D, E, F, G and H) forming granuloma-like structures (B, C, D, E and F) with giant multinuclear cells (arrows on B, D, E, F), epithelial desquamation, colloid depletion and extensive follicular disruption (A, B, C, D, E, F, G, H and I, corresponding to findings consistent with subacute thyroiditis. Spike protein positivity was also found in epithelioid cells within the granulomatous tissue (F). Sars-Cov-2 nucleocapsid protein immunopositivity was noticeably sparser, arranged in punctiform inclusions and localized predominantly in the perinuclear region of follicular cells (arrows on G and H; J, K, L, M, N, O). The majority of remaining follicular cells showed positivity for cleaved caspase-3 (I). Staining was performed using rabbit anti-SARS-CoV-2 spike glycoprotein antibody (Abcam, ab272504; dilution 1:4000), mouse anti-SARS-CoV-2 nucleocapsid protein antibody (Cell Signaling Technology, clone E8R1L; dilution 1:200) and rabbit anti-caspase-3 antibody (Abcam, ab13847; diluted 1:50). Immunoreactions were visualized by DAKO EnVision+System (DAKO Cytomation) for immunohistochemistry or by Alexa Fluor 488 donkey anti-rabbit IgG (Thermo Fisher Scientific; diluted 1:300) and Alexa Fluor 555 goat anti-mouse IgG (Thermo Fisher Scientific; diluted 1:500) secondary antibodies for immunofluorescence studies. Magnifications: A × 100; B, C, D, E, I × 400; F, J, K, L × 600; G, H, M, N, O × 1000.
Fig 3: Western blot assay to examine SARS-CoV-2 proteins production.The hCoV-19/Turkey/ERAGEM-001/2020 strain was used to infect the cell lines at an MOI of 0.5. The whole cell lysate samples were collected at 24 h post-infection. The Western blot assay was performed to examine the production of viral proteins using a rabbit polyclonal antibody to the SARS-CoV-2 spike glycoprotein (S) (1/1000) (Abcam; ab272504) and a human antibody to the SARS-CoV-2 nucleocapsid protein (NP) (1:2500) (GenScript; HC2003). The membrane was reacted with the ECL substrate solution (Pierce ECL, USA). The membrane was exposed to an autoradiograph film (KODAK X-OMAT, Sigma Germany), and was developed using a Kodak developer (X-OMAT 1000A, Sigma Germany). The arrows indicate that the bands at approximately 180 kDa (Fig 5A) and 48 kDa (Fig 5B) represent S and NP, respectively.
Fig 4: The pPAL-Sfs + pPAL-N vaccine candidate. (A) pPAL-Sfs map. Sfs is a codon-optimized modified version of the SARS-CoV-2 Wuhan reference sequence that contains a modification to avoid furin cleavage of the protein product. Large-scale laboratory preparation of the pPAL-Sfs plasmid containing supercoiled (CCC), open-circular (OC), CCC-dimer, and CCC-trimer conformers. Western blot of whole protein extracts from HEK293 cells transfected with pPAL, pPAL-S, and pPAL-Sfs. The primary antibody was 1:800-diluted goat anti-S polyclonal antibody (Abcam #ab272504, Cambridge, United Kingdom), which only recognizes the S2 subunit. The secondary antibody was 1:2,000-diluted HRP-conjugated goat polyclonal anti-rabbit Ig. Furin cleaves the S protein obtained from pPAL-S but not Sfs from pPAL-Sfs. (B) Large-scale laboratory preparation of the pPAL-N plasmid containing CCC, OC, CCC-dimer, and CCC-trimer conformers. Western blot of whole protein extracts from HEK293 cells transfected with pPAL and pPAL-N. The primary antibody was 1:500-diluted rabbit anti-N polyclonal antibody. The secondary antibody was 1:2,000-diluted HRP-conjugated goat polyclonal anti-rabbit Ig. (C, D) immunofluorescence of cultured HEK293 cells transfected with pPAL, pPAL-Sfs, and pPAL-N plasmids. The anti-S and anti-N antibody dilutions were 1:50. The pPAL control was incubated with both antibodies. The secondary antibody was Alexa Fluor® 488-conjugated goat anti-rabbit IgG diluted to 1:200. Sfs and N expression is observed.
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