The cellular characterisation of SARS-CoV-2 spike protein in virus-infected cells using Receptor Binding Domain-binding specific human monoclonal antibodies.

A human monoclonal antibody panel (PD4, PD5, PD7, SC23 and SC29) was isolated from the B cells of convalescent patients and used to examine the S protein in SARS-CoV-2-infected cells. While all five antibodies bound conformational-specific epitopes within SARS-CoV-2 Spike (S) protein, only PD5, PD7, and SC23 were able to bind to the Receptor Binding Domain (RBD). Immunofluorescence microscopy was used to examine the S protein RBD in cells infected with the Singapore isolates SARS-CoV-2/0334 and SARS-CoV-2/1302. The RBD-binders exhibited a distinct cytoplasmic staining pattern that was primarily localised within the Golgi complex and was distinct from the diffuse cytoplasmic staining pattern exhibited by the non-RBD binders (PD4 and SC29). These data indicated that the S protein adopted a conformation in the Golgi complex that enabled the RBD recognition by the RBD-binders. The RBD-binders also recognised the uncleaved S protein indicating that S protein cleavage was not required for RBD recognition. Electron microscopy indicated high levels of cell-associated virus particles, and multiple cycle virus infection using RBD-binder staining provided evidence for direct cell-to-cell transmission for both isolates. Although similar levels of RBD-binder staining was demonstrated for each isolate, the SARS-CoV-2/1302 exhibited slower rates of cell-to-cell transmission. These data suggest that a conformational change in the S protein occurs during its transit through the Golgi complex that enables RBD recognition by the RBD-binders, and suggests that these antibodies can be used to monitor S protein RBD formation during the early stages of infection.

A Case of Diffuse Alveolar Hemorrhage Associated with High-Titer of MPO-ANCA Demonstrating Cytoplasmic Staining Pattern

Diffuse alveolar hemorrhage (DAH) is a life-threatening complication of ANCA-associated vasculitis (AAV) that requires urgent recognition and treatment. A presumptive diagnosis is often rendered without histopathology if concordant positivity of ANCA by indirect immunofluorescence (IIF) and ELISA assays, i.e., P-ANCA+/myeloperoxidase (MPO) Ab+ or C-ANCA+/proteinase-3 (PR3) Ab+, is documented in the context of pulmonary-renal syndrome or rapidly progressive glomerulonephritis. In this respect, the discordance between IIF and ELISA assays poses a diagnostic challenge in the absence of convincing histopathology and involves the risks of delaying the implementation of timely immunosuppressive therapy. Here, we report a 74-year-old woman who developed DAH and was found to have a high titer of MPO-ANCA exhibiting cytoplasmic staining on IIF, i.e., MPO-C-ANCA. The literature suggests that the availability of distinct epitopes on the MPO molecule dictates the perinuclear versus cytoplasmic staining pattern, which potentially explains the discordance between ELISA and IIF assays. Her DAH was controlled in association with seven sessions of plasmapheresis, methylprednisolone 1 gram daily for 3 days followed by 1 mg/kg/day, and rituximab. This case exemplifies the importance of consideration of pretest probability of suspected diagnosis that would realize a plausible interpretation of seemingly inconsistent serological profile and its effective incorporation into the diagnostic reasoning.

Langerin (CD207) Staining in Normal Pediatric Tissues, Reactive LymphNodes, and Childhood Histiocytic Disorders

Langerin is a recently identified lectin for which antibodies can be used as immunohistochemical markers of Langerhans cells (LCs). We describe the distribution of staining in autopsy pediatric tissues, dermatopathic and other reactive lymph nodes, and childhood histiocytic lesions using the 12D6 antibody (Novocastra). We also correlate CD1a (antibody O1O) staining to these factors. Langerin on epidermal LCs has a coarsely granular cell membrane and a cytoplasmic staining pattern that is always associated with CD1a expression. All 6 skin samples had Langerin+/CD1a+ LCs within the epidermis. Six of 8 thymuses showed single scattered dendritic-shaped cells in the medulla and rare cells within Hassall corpuscles that coexpressed Langerin and CD1a. Cortical thymocytes were CD1a+/Langerin-. Four of 8 livers examined showed a sinusoidal lining pattern of Langerin+/CD1a-. All 15 autopsy lymph nodes showed a similarly strong Langerin+/CD1a- sinus pattern of staining on fixed tissue elements, mostly in medullary sinuses. All 12 dermatopathic lymph nodes showed accumulation of Langerin+/CD1a+ cells in the pale paracortical nodules. All 24 instances of LC histiocytosis (LCH) were Langerin+/CD1a+. All 12 non-LCH histiocytic disorders are negative for Langerin in the histiocytes of interest. We conclude that Langerin is coexpressed with CD1a on LCs and LCH. Lymph node sinuses and hepatic sinusoids show Langerin+/CD1a-cells, indicating that, when used alone to confirm LCH infiltration, the 12D6 antibody should be used with caution. At other sites, its diagnostic accuracy is similar to that of CD1a.