Sensitive and specic immunohistochemistry protocols for detection of SARS-CoV-2 nucleocapsid and spike proteins in formalin-xed, paran-embedded COVID-19 patient tissues

Human coronavirus disease 2019 (COVID-19) is a life-threatening and highly contagious disease caused by coronavirus SARS-CoV-2. Sensitive and specic detection of SARS-CoV-2 virus in tissues and cells of COVID-19 patients will support investigations of the biologic behavior and tissue and cell tropism of this virus. We identied two commercially available anity-puried polyclonal antibodies raised against Nucleocapsid and Spike proteins of SARS-CoV-2 that provide sensitive and specic detection of the virus by immunohistochemistry in formalin-xed, paran-embedded tissue. Protocols are presented that are mutually validated by matched detection patterns of virus-infected cells in autopsy lung tissue of COVID-19 deceased patients by the two distinctly different antibodies. Negative controls include autopsy lung tissue from patient who died from non-COVID-19 respiratory disease and control rabbit immunoglobulin. SARS-CoV-2 detection in human tissues will provide insights into viral tissue and cell distribution and load in patients with active infection as well as provide insight into clearance of virus in late COVID-19 disease stages.


Introduction
In December of 2019 an outbreak of pneumonia cases of unknown aetiology occurred in Wuhan, China, leading to the identi cation of a novel beta-coronavirus named SARS-CoV-2 as the causative agent 1-3 .
The infectious disease was termed COVID-19 in January of 2020 by the World Health Organization 4 . COVID-19 patients with lower respiratory tract infection often develop acute respiratory disease syndrome (ARDS) that is also observed in patients with severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), other corona-viral pneumonias. However, COVID-19 is much more transmissible between people than SARS and MERS and was declared a pandemic in February of 2020. Extensive efforts to identify treatments and develop vaccines are ongoing.
The SARS-CoV-2 virus causes a wide spectrum of clinical manifestations in patients with COVID-19 5 . For rational development of treatments for COVID-19 there is a great need to understand the pathogenesis and pathology of COVID-19, not only in the respiratory tract but also in numerous other organs that become directly infected through vascular or neuronal spread. Commonly affected non-respiratory organs include GI-tract, kidney, heart, skin and central nervous system [6][7][8] . In addition, numerous pathological changes appear secondary to, or indirectly from, viral infection, including aberrant immune cell activation, vascular changes and coagulopathies, in turn affecting numerous organs [9][10][11] . Sensitive tools are required to determine the virus distribution in organs and cells 12,13 and to identify associated adaptations in the proximal microenvironment, including immune cell recruitment and local in ammatory changes. Immunohistochemistry for viral protein provides a rapid means to identify virally infected cells within histological sections of formalin-xed, para n-embedded (FFPE) tissues. Initial IHC assays with ability to detect SARS-CoV-2 were based on antibodies originally raised against SARS-CoV protein epitopes with su cient cross-reactivity to SARS-CoV-2 epitopes 14, 15 . Here we present IHC protocols for two commercially available antibodies raised directly against epitopes of SARS-CoV-2 Nucleocapsid protein or SARS-CoV-2 Spike protein.

Anticipated Results
We present IHC protocols for two antibodies raised directly against epitopes of SARS-CoV-2 Nucleocapsid protein or SARS-CoV-2 Spike protein (S1-domain; Figure 1). Speci city of the antibodies have been independently validated in western blotting and ELISA. The clearance rates of SARS-CoV-2 virus within infected human tissues have yet to be determined. We expect that many COVID-19 patients die from disease complications after virus has been cleared. In the absence of human tissue with known positive virus presence at the time of autopsy, we mutually validated the IHC protocols for the two antibodies by documenting matched patterns of virus-infected cells in adjacent sections of autopsy lung tissue of COVID-19 deceased patients. Negative controls include autopsy lung tissue from patient who died from non-COVID-19 respiratory disease or control rabbit immunoglobulin.
Sensitive and speci c detection of SARS-CoV-2 in human tissues will provide insights into viral organ and cell distribution and load in patients with active infection, as well as provide insights into viral tissue loads during early and late stages of COVID-19. Figure 1 Immunohistochemistry (IHC) of SARS-CoV-2 antigens in FFPE lung tissue. Detection of SARS-CoV-2 nucleocapsid protein (A,B; brown staining, red arrows) or SARS-CoV-2 Spike protein (D,E; brown staining, red arrows) in adjacent sections of autopsy lung tissue from COVID-19 deceased patient. Negative