Recognition of CpG oligodeoxynucleotides by human Toll-like receptor 9 and subsequent cytokine induction.

Toll-like receptor 9 (TLR9) recognizes a synthetic ligand, oligodeoxynucleotide (ODN) containing cytosine-phosphate-guanine (CpG). Activation of TLR9 by CpG ODN induces a signal transduction cascade that plays a pivotal role in first-line immune defense in the human body. The three-dimensional structure of TLR9 has not yet been reported, and the ligand-binding mechanism of TLR9 is still poorly understood; therefore, the mechanism of human TLR9 (hTLR9) ligand binding needs to be elucidated. In this study, we constructed several hTLR9 mutants, including truncated mutants and single mutants in the predicted CpG ODN-binding site. We used these mutants to analyze the role of potential important regions of hTLR9 in receptor signaling induced by phosphorothioate (PTO)-modified CpG ODN and CpG ODNs only consist entirely of a phosphodiester (PD) backbone, CpG ODN2006x3-PD that we developed. We found truncated mutants of hTLR9 lost the signaling activity, indicating that both the C- and N-termini of the extracellular domain (ECD) are necessary for the function of hTLR9. We identified residues, His505, Gln510, His530, and Tyr554, in the C-terminal of hTLR9-ECD that are essential for hTLR9 activation. These residues might form positive charged clusters with which negatively charged CpG ODN could interact. Furthermore, we observed ODN-PD induced interleukin-6 (IL-6) through TLR9 in a CpG-sequence-dependent manner in human peripheral blood mononuclear cells and B cells, whereas ODN-PTO induced IL-6 in a CpG-sequence-independent manner. These finding are relevant for the mechanism of hTLR9 activation by CpG ODNs.

Evaluating Saliva Sampling with Reverse Transcription Loop-mediated Isothermal Amplification to Improve Access to SARS-CoV-2 Diagnosis in Low-Resource Settings.

Standard diagnosis of SARS-CoV-2 by nasopharyngeal swab (NPS) and real-time reverse transcriptase-polymerase chain reaction (PCR) requires a sophisticated laboratory, skilled staff, and expensive reagents that are difficult to establish and maintain in isolated, low-resource settings. In the remote setting of tropical Sumba Island, eastern Indonesia, we evaluated alternative sampling with fresh saliva (FS) and testing with colorimetric loop-medicated isothermal amplification (LAMP). Between August 2020 and May 2021, we enrolled 159 patients with suspected SARS-CoV-2 infection, of whom 75 (47%) had a positive PCR on NPS (median cycle threshold [Ct] value: 27.6, interquartile range: 12.5-37.6). PCR on FS had a sensitivity of 72.5% (50/69, 95% confidence interval [CI]: 60.4-82.5) and a specificity of 85.7% (66/77, 95% CI: 75.9-92.6), and positive (PPV) and negative (NPV) predictive values of 82.0% (95% CI: 0.0-90.6) and 77.6% (95% CI: 67.3-86.0), respectively. LAMP on NPS had a sensitivity of 68.0% (51/75, 95% CI: 56.2-78.3) and a specificity of 70.8% (63/84, 95% CI: 58.9-81.0), with PPV 70.8% (95% CI: 58.9-81.0) and NPV 72.4% (95% CI: 61.8-81.5%). LAMP on FS had a sensitivity of 62.3% (43/69, 95% CI: 49.8-73.7%) and a specificity of 72.7% (56/77, 95% CI: 61.4-82.3%), with PPV 67.2% (95% CI: 54.3-78.4) and NPV 68.3% (95% CI: 57.1-78.1%). LAMP sensitivity was higher for NPS and FS specimens with high viral loads (87.1% and 75.0% for Ct value < 26, respectively). Dried saliva on filter paper was stable for 4 days at room temperature. LAMP on either NPS or FS could offer an accessible alternative for SARS-CoV-2 diagnosis in low-resource settings, with potential for optimizing sample collection and processing, and selection of gene targets.