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By Rosa Moreno, PhD.
As the race for developing a vaccine to tackle the ongoing coronavirus disease (COVID-19) pandemic evolves, scientists at NIH, CDC and Harvard Medical School continue to make strides in understanding the often-unpredictable pathology and immune response associated with SARS-CoV-2 infections. Several recent studies shed light on the organ and tissue distribution of the virus following infection, the predominant tissue damage induced by the virus, and the immune responses triggered by infection. These findings, the product of studies with animal models and tissue derived from human COVID fatalities, provide critical base knowledge to support the development of strategies aimed at curving disease progression.
In a recent Nature publication , which reiterates findings from an earlier pre-print , Munster and colleagues at the NIH studied SARS-CoV-2 pathogenesis in a rhesus macaque model. Animal model systems such as this primate model, are critical for understanding the progression and outcomes of infectious diseases. Pre-clinical animal models help provide the fundamental knowledge needed for the development of strategies aimed at treatment (e.g., vaccine development), and curving infectivity (e.g., through the implementation of public health recommendations).
Similar to humans, SARS-CoV-2 infection in macaques leads to upper airway colonization, demonstrated by the presence of viral shedding from the nose and throat. Additionally, the virus makes its way to the lungs where it’s recovered from broncho-alveolar tissue, leading to tissue damage and pneumonia. Overall, the findings by Munster et al. demonstrated that SARS-CoV-2 infection in macaques parallels many of the clinical findings in humans with mild to moderate symptoms.
Detection of SARS-CoV-2 nucleocapsid protein in Rhesus macaque tissues. SARS Nucleocapsid Protein Antibody [NB100-56576] - Pathological changes in rhesus macaques infected with SARS-CoV-2. (g) SARSCoV-2 antigen is detected by immunohistochemistry in type I pneumocytes. Magnification 400x. (j) SARS-CoV-2 antigen is detected by immunohistochemistry in type I pneumocytes (asterisk) and type II pneumocytes (arrow) as well as alveolar macrophages (arrowheads). Magnification 400x. (k) SARS-CoV-2 antigen is detected by immunohistochemistry in mediastinal lymph node. Magnification 400x. (l) SARSCoV-2 antigen is detected by immunohistochemistry in macrophages and lymphocytes in the lamina propria of the cecum. Magnification 400x. bioRxiv March 21, 2020 //doi.org/10.1101/2020.03.21.001628
Further demonstrating the usefulness of primate model systems, in a new
Science publication , Chandrashekar and colleagues at Harvard Medical School developed a rhesus macaque model of SARS-CoV-2 infection and re-challenge. This model addresses a critical question: Is there protective immunity following COVID-19 infection? The answer is yes, but much remains to be elucidated in terms of the type of immune response and its durability. Briefly, following initial SARS-CoV-2 infection, investigators demonstrated the presence of binding (spike and nucleocapsid protein) and neutralizing antibodies. Re-challenge with SARS-CoV-2, by re-infection of animals 35 days following initial exposure, triggered a fast-immune response as indicated by increased virus specific neutralizing antibody levels and improved viral load clearance.
Similar to findings by the group at NIH, Chandrashekar et al. demonstrated the presence of SARS-CoV-2 throughout the upper-respiratory tract and lungs, and to a lesser extent in other tissues such as the gastrointestinal tract. Their model recapitulated many of the pathological findings seen by Munster et al. regarding lung tissue damage and pneumonia. Using the same Novus rabbit polyclonal antibody (NB100-56576) to SARS-CoV, COV-2 nucleocapsid was detected in pneumocytes and in alveolar macrophages. This group also leveraged the sensitivity and specificity of SARS-CoV-2 RNA probes [RNAscope: SARS-CoV2 anti-sense specific probe v-nCoV2019-S (848561) and SARS-CoV2 sense specific probe vnCoV2019-orf1ab-sense (859151) ] to confirm the presence of viral RNA within lung tissue and infiltrated immune cells.
Detection of SARS-CoV-2 nucleocapsid protein in human lung tissue. Tissue sections were obtained from fatal coronavirus disease cases which were confirmed SARS-CoV-2 positive by PCR (provided by Dr. Gordon Love, MD, LSUHSC Department of Pathology, Medical Director of Laboratories, University Medical Center, New Orleans, LA). Sections were analyzed for SARS-CoV-2 RNA by RNAscope (by Dr. Alexander Kalyuzny, PhD, R&D Systems, Minneapolis, MN). Briefly, formalin-fixed paraffin-embedded tissue sections of human CoVID-19-infected lung were probed for SARS-CoV-2 RNA with ACD anti-sense specific probe v-nCoV2019-S (848561) . Left-Fast Red chromogen particles marked by magenta pseudo color indicate positive detection of SARS-CoV-2 RNA. Arrow and circle indicate area shown at higher magnification on Right image, where the red chromogen particles indicating viral RNA detection are visible.
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To understand the tissue pathology and distribution of SASR-CoV-2 in humans with chronic COVID-19 disease, Martines et al. at the Centers for Disease Control and Prevention (CDC) analyzed autopsy tissues from patients who died from COVID-19. All patients had underlying conditions known to increase susceptibility to disease progression such as cardiovascular disease , diabetes , and chronic kidney disease. Evidence of lung tissue damage, particularly diffuse alveolar damage (DAD), was present in most patients. Histopathological findings included desquamated pneumocytes, hyaline membranes, alveolar edema, and alveolar macrophage infiltration.
By immunostaining with the same antibody to SARS-CoV nucleocapsid protein (NB100-56576), Martines et al. identified the virus within the upper respiratory tract and pulmonary tissue (e.g., bronchial epithelium, type I and type II pneumocytes, and alveolar macrophages). Interestingly, the viral nucleocapsid protein was not detected in other organs such as heart, liver, kidney, spleen, or intestine. Given that ACE-2, the receptor for SARS-CoV-2 entry, is expressed in cardiac, kidney, and gastrointestinal tract tissues, Martine et al. conceded that SARS-CoV-2 associated extrapulmonary pathologies require further investigation.
Detection of SARS-CoV-2 nucleocapsid protein in human tissues. Detection of SARS-CoV-2 nucleocapsid antigen in tracheal epithelial ciliated cells by immunohistochemistry with a rabbit polyclonal antibody to SARS-CoV nucleocapsid protein (NB100-56576). Source: modified from figure 3 in "Pathology and Pathogenesis of SARS-CoV-2 Associated with Fatal Coronavirus Disease, United States" .
Investigators at the CDC confirmed that the rabbit polyclonal antibody to SARS-CoV nucleocapsid protein cross-reacts and detects SARS-CoV-2 by staining infected Vero cells. Moreover, they showed that the antibody has no cross-reactivity towards a variety of respiratory viruses including influenza A(H1N1), influenza B, respiratory syncytial virus, parainfluenza virus type 3, human coronavirus (HCoV) 229E, or MERS-CoV.
Lastly, in a recent publication in Modern Pathology , Dr. Lynette Sholl's group reported on the detection of SARS CoV-2 in autopsied respiratory tissues from confirmed COVID-19 cases. Importantly, their findings positively correlated the presence of the virus in lung tissues with the acute-phase of DAD. In contrast, SARS-CoV-2 nucleocapsid protein was undetectable in patients at an organizing (proliferative)-phase of DAD. About the use of the SARS-CoV nucleocapsid antibody (NB100-56576), Dr. Sholl of Harvard Medical School said: "We were able to quickly optimize and validate the antibody for use in clinical diagnostics on formalin fixed paraffin embedded tissues and have found that it is a robust tool for evaluation of SARS-CoV-2 in human autopsy tissue".
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Rosa Moreno, PhD.
Product Marketing Specialist at Novus Biologicals LLC
