Identification of Immunohistochemical Reagents for In Situ Protein Expression Analysis of Coronavirus-associated Changes in Human Tissues.

We studied the suitability of commercially available monoclonal antibodies (mAbs) for the immunohistochemical (IHC) detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) in standard archival specimens. Antibodies were screened on HEK293 cells transfected with viral nucleoprotein, S1 subunit and S2 subunit of spike protein and on untransfected cells, as well as a panel of normal tissue. Lung tissue with presence of SARS-CoV2 confirmed by in situ hybridization (ISH) was also used. A total of 7 mAbs were tested: (1) mAb 001 (Sino Biological, 40143-R001), (2) mAb 007 (Sino Biological, 40150-R007), (3) mAb 019 (Sino Biological, 40143-R019), (4) mAb 1A9 (GeneTex, GTX632604), (5) mAb ABM19C9 (Abeomics, 10-10007), (6) FIPV3-70 (Santa Cruz, SC-65653), and (7) mAb 6F10 (BioVision, A2060). Only 2 mAbs, clone 001 to the nucleoprotein and clone 1A9 to the S2 subunit spike protein displayed specific immunoreactivity. Both clones showed strong staining in the acute phase of COVID-19 pneumonia, mostly in areas of acute diffuse alveolar damage, but were not completely congruent. Viral protein was also found in kidney tubules, endothelia of multiple organs and a nasal swab of a patient with persistent SARS-CoV2 infection. The other tested reagents were either poorly reactive or demonstrated nonspecific staining in tissues and lesions not infected by SARS-CoV2. Our study demonstrates that rigid specificity testing is mandatory for the evaluation of mAbs to SARS-CoV2 and that clones 001 to nucleoprotein and 1A9 to S2 subunit spike protein are useful for the in situ detection of SARS-CoV2.

Insights into pathogenesis of fatal COVID-19 pneumonia from histopathology with immunohistochemical and viral RNA studies.

INTRODUCTION: We describe post-mortem pulmonary histopathologic findings of COVID-19 pneumonia in patients with a spectrum of disease course, from rapid demise to prolonged hospitalisation. METHODS AND RESULTS: Histopathologic findings in post-mortem lung tissue from eight patients who died from COVID-19 pneumonia were reviewed. Immunohistochemistry (IHC) and next-generation sequencing (NGS) were performed to detect virus. Diffuse alveolar damage (DAD) was seen in all cases with a spectrum of acute phase and/or organising phase. IHC with monoclonal antibodies against SARS-CoV-2 viral nucleoprotein and spike protein detected virus in areas of acute but not organising DAD, with intracellular viral antigen and RNA expression seen predominantly in patients with duration of illness less than 10 days. Major vascular findings included thrombi in medium- and large-calibre vessels, platelet microthrombi detected by CD61 IHC and fibrin microthrombi. CONCLUSIONS: Presence of SARS-CoV-2 viral RNA by NGS early in the disease course and expression of viral antigen by IHC exclusively in the acute, but not in the organising phase of DAD, suggests that the virus may play a major role in initiating the acute lung injury of DAD, but when DAD progresses to the organising phase the virus may have been cleared from the lung by the patient's immune response. These findings suggest the possibility of a major change during the disease course of COVID-19 pneumonia that may have therapeutic implications. Frequent thrombi and microthrombi may also present potential targets for therapeutic intervention.