Case Report: Molecular autopsy underlie COVID-19-associated sudden, unexplained child mortality.

Herein, we report a child with COVID-19 and seemingly no underlying disease, who died suddenly. The autopsy revealed severe anemia and thrombocytopenia, splenomegaly, hypercytokinemia, and a rare ectopic congenital coronary origin. Immunohistochemical analysis demonstrated that the patient had acute lymphoblastic leukemia of the B-cell precursor phenotype (BCP-ALL). The complex cardiac and hematological abnormalities suggested the presence of an underlying disease; therefore, we performed whole-exome sequencing (WES). WES revealed a leucine-zipper-like transcription regulator 1 (LZTR1) variant, indicating Noonan syndrome (NS). Therefore, we concluded that the patient had underlying NS along with coronary artery malformation and that COVID-19 infection may have triggered the sudden cardiac death due to increased cardiac load caused by high fever and dehydration. In addition, multiple organ failure due to hypercytokinemia probably contributed to the patient's death. This case would be of interest to pathologists and pediatricians because of the limited number of NS patients with LZTR1 variants; the complex combination of an LZTR1 variant, BCP-ALL, and COVID-19; and a rare pattern of the anomalous origin of the coronary artery. Thus, we highlight the significance of molecular autopsy and the application of WES with conventional diagnostic methods.

Development of a Peptide Sensor Derived from Human ACE2 for Fluorescence Polarization Assays of the SARS-CoV-2 Receptor Binding Domain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the continuing emergence of infectious variants have caused a serious pandemic and a global economic slump since 2019. To overcome the situation and prepare for future pandemic-prone diseases, there is a need to establish a convenient diagnostic test that is quickly adaptable to unexpected emergence of virus variants. Here we report a fluorescent peptide sensor 26-Dan and its application to the fluorescence polarization (FP) assay for the highly sensitive and convenient detection of SARS-CoV-2. The 26-Dan sensor was developed by fluorescent labeling of the 26th amino acid of a peptide derived from the N-terminal α-helix of human angiotensin-converting enzyme 2 (hACE2) receptor. The 26-Dan sensor maintained the α-helical structure and showed FP changes in a concentration-dependent manner of the receptor binding domain (RBD) of the virus. The half maximal effective concentrations (EC50's) for RBD of Wuhan-Hu-1 strain, Delta (B.1.617.2), and Omicron (BA.5) variants were 51, 5.2, and 2.2 nM, respectively, demonstrating that the 26-Dan-based FP assay can be adaptable to virus variants that evade standard diagnostic tests. The 26-Dan-based FP assay could also be applied to model screening of a small molecule that inhibits RBD binding to hACE2 and identified glycyrrhizin as a potential inhibitor. The combination of the sensor with a portable microfluidic fluorescence polarization analyzer allowed for the detection of RBD in a femtomolar range within 3 min, demonstrating the assay could be a promising step toward a rapid and convenient test for SARS-CoV-2 and other possible future pandemic-prone diseases.

Structural basis of spike RBM-specific human antibodies counteracting broad SARS-CoV-2 variants.

The decrease of antibody efficacy to mutated SARS-CoV-2 spike RBD explains the breakthrough infections and reinfections by Omicron variants. Here, we analyzed broadly neutralizing antibodies isolated from long-term hospitalized convalescent patients of early SARS-CoV-2 strains. One of the antibodies named NCV2SG48 is highly potent to broad SARS-CoV-2 variants including Omicron BA.1, BA.2, and BA.4/5. To reveal the mode of action, we determined the sequence and crystal structure of the Fab fragment of NCV2SG48 in a complex with spike RBD from the original, Delta, and Omicron BA.1. NCV2SG48 is from a minor VH but the multiple somatic hypermutations contribute to a markedly extended binding interface and hydrogen bonds to interact with conserved residues at the core receptor-binding motif of RBD, which efficiently neutralizes a broad spectrum of variants. Thus, eliciting the RBD-specific B cells to the longitudinal germinal center reaction confers potent immunity to broad SARS-CoV-2 variants emerging one after another.

Neutralizing Type I Interferon Autoantibodies in Japanese Patients with Severe COVID-19.

PURPOSE: Autoantibodies (aAbs) to type I interferons (IFNs) have been found in less than 1% of individuals under the age of 60 in the general population, with the prevalence increasing among those over 65. Neutralizing autoantibodies (naAbs) to type I IFNs have been found in at least 15% of patients with life-threatening COVID-19 pneumonia in several cohorts of primarily European descent. We aimed to evaluate the prevalence of aAbs and naAbs to IFN-α2 or IFN-ω in Japanese patients who suffered from COVID-19 as well as in the general population. METHODS: Patients who suffered from COVID-19 (n = 622, aged 0-104) and an uninfected healthy control population (n = 3,456, aged 20-91) were enrolled in this study. The severities of the COVID-19 patients were as follows: critical (n = 170), severe (n = 235), moderate (n = 112), and mild (n = 105). ELISA and ISRE reporter assays were used to detect aAbs and naAbs to IFN-α2 and IFN-ω using E. coli-produced IFNs. RESULTS: In an uninfected general Japanese population aged 20-91, aAbs to IFNs were detected in 0.087% of individuals. By contrast, naAbs to type I IFNs (IFN-α2 and/or IFN-ω, 100 pg/mL) were detected in 10.6% of patients with critical infections, 2.6% of patients with severe infections, and 1% of patients with mild infections. The presence of naAbs to IFNs was significantly associated with critical disease (P = 0.0012), age over 50 (P = 0.0002), and male sex (P = 0.137). A significant but not strong correlation between aAbs and naAbs to IFN-α2 existed (r = - 0.307, p value < 0.0001) reinforced the importance of measuring naAbs in COVID-19 patients, including those of Japanese ancestry. CONCLUSION: In this study, we revealed that patients with pre-existing naAbs have a much higher risk of life-threatening COVID-19 pneumonia in Japanese population.