Simultaneous measurement of the antibody responses against SARS-CoV-2 and its multiple variants by a phage display mediated immuno-multiplex quantitative PCR-based assay.

To combat the continued pandemic of COVID-19, multiplex serological assays have been developed to comprehensively monitor the humoral immune response and help to design new vaccination protocols to different SARS-CoV-2 variants. However, multiplex beads and stably transfected cell lines require stringent production and storage conditions, and assays based on flow cytometry is time-consuming and its application is therefore restricted. Here, we describe a phage display system to distinguish the differences of immune response to antigenic domains of multiple SARS-CoV-2 variants simultaneously. Compared with linear peptides, the recombinant antigens displayed on the phage surface have shown some function that requires the correct folding to form a stable structure, and the binding efficiency between the recombinant phage and existing antibodies is reduced by mutations on antigens known to be important for antigen-antibody interaction. By using Phage display mediated immuno-multiplex quantitative PCR (Pi-mqPCR), the binding efficiency between the antibody and antigens of different SARS-CoV-2 variants can be measured in one amplification reaction. Overall, these data show that this assay is a valuable tool to evaluate the humoral response to the same antigen of different SARS-CoV-2 variants or antigens of different pathogens. Combined with high-throughput DNA sequencing technology, this phage display system can be further applied in monitoring humoral immune response in a large population before and after vaccination.

Protein Kinase C Inhibitors Reduce SARS-CoV-2 Replication in Cultured Cells.

Infection by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has posed a severe threat to global public health. The current study revealed that several inhibitors of protein kinases C (PKCs) possess protective activity against SARS-CoV-2 infection. Four pan-PKC inhibitors, Go 6983, bisindolylmaleimide I, enzastaurin, and sotrastaurin, reduced the replication of a SARS-CoV-2 replicon in both BHK-21 and Huh7 cells. A PKCδ-specific inhibitor, rottlerin, was also effective in reducing viral infection. The PKC inhibitors acted at an early step of SARS-CoV-2 infection. Finally, PKC inhibitors blocked the replication of wild-type SARS-CoV-2 in ACE2-expressing A549 cells. Our work highlights the importance of the PKC signaling pathway in infection by SARS-CoV-2 and provides evidence that PKC-specific inhibitors are potential therapeutic agents against SARS-CoV-2. IMPORTANCE There is an urgent need for effective therapeutic drugs to control the pandemic caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). We found that several inhibitors of protein kinases C (PKCs) dramatically decrease the replication of SARS-CoV-2 in cultured cells. These PKC inhibitors interfere with an early step of viral infection. Therefore, the rapid and prominent antiviral effect of PKC inhibitors underscores that they are promising antiviral agents and suggests that PKCs are important host factors involved in infection by SARS-CoV-2.

Sustained Release of Co-Amorphous Matrine-Type Alkaloids and Resveratrol with Anti-COVID-19 Potential.

Matrine (MAR), oxymatrine (OMAR), and sophoridine (SPD) are natural alkaloids with varying biological activities; matrine was recently used for the treatment of coronavirus disease 2019 (COVID-19). However, the short half-lives and rapid elimination of these matrine-type alkaloids would lead to low oral bioavailability and serious side effects. Herein, resveratrol (RES) was selected as a co-former to prepare their co-amorphous systems to improve the therapeutic index. The formation of co-amorphous MAR-RES, OMAR-RES, and SPD-RES was established through powder X-ray diffraction and modulated temperature differential scanning calorimetry. Furthermore, Fourier transform infrared spectroscopy and NMR studies revealed the strong molecular interactions between resveratrol and these alkaloids, especially OMAR-RES. Matrine, oxymatrine, and sophoridine in the co-amorphous systems showed sustained release behaviors in the dissolution experiments, due to the recrystallization of resveratrol on the surface of co-amorphous drugs. The three co-amorphous systems exhibited excellent physicochemical stability under high relative humidity conditions. Our study not only showed that minor structural changes of active pharmaceutical ingredients may have distinct molecular interactions with the co-former, but also discovered a new type of sustained release mechanism for co-amorphous drugs. This promising co-amorphous drug approach may present a unique opportunity for repurposing these very promising drugs against COVID-19.

The evaluation of maximum condyle-tragus distance can predict difficult airway management without exposing upper respiratory tract; a prospective observational study.

BACKGROUND: Routine preoperative methods to assess airway such as the interincisor distance (IID), Mallampati classification, and upper lip bite test (ULBT) have a certain risk of upper respiratory tract exposure and virus spread. Condyle-tragus maximal distance(C-TMD) can be used to assess the airway, and does not require the patient to expose the upper respiratory tract, but its value in predicting difficult laryngoscopy compared to other indicators (Mallampati classification, IID, and ULBT) remains unknown. The purpose of this study was to observe the value of C-TMD to predict difficult laryngoscopy and the influence on intubation time and intubation attempts, and provide a new idea for preoperative airway assessment during epidemic. METHODS: Adult patients undergoing general anesthesia and tracheal intubation were enrolled. IID, Mallampati classification, ULBT, and C-TMD of each patient were evaluated before the initiation of anesthesia. The primary outcome was intubation time. The secondary outcomes were difficult laryngoscopy defined as the Cormack-Lehane Level > grade 2 and the number of intubation attempts. RESULTS: Three hundred four patients were successfully enrolled and completed the study, 39 patients were identified as difficult laryngoscopy. The intubation time was shorter with the C-TMD>1 finger group 46.8 ± 7.3 s, compared with the C-TMD<1 finger group 50.8 ± 8.6 s (p<0.01). First attempt success rate was higher in the C-TMD>1 finger group 98.9% than in the C-TMD<1 finger group 87.1% (P<0.01). The correlation between the C-TMD and Cormack-Lehane Level was 0.317 (Spearman correlation coefficient, P<0.001), and the area under the ROC curve was 0.699 (P<0.01). The C-TMD < 1 finger width was the most consistent with difficult laryngoscopy (κ = 0.485;95%CI:0.286-0.612) and its OR value was 10.09 (95%CI: 4.19-24.28), sensitivity was 0.469 (95%CI: 0.325-0.617), specificity was 0.929 (95%CI: 0.877-0.964), positive predictive value was 0.676 (95%CI: 0.484-0.745), negative predictive value was 0.847 (95%CI: 0.825-0.865). CONCLUSION: Compared with the IID, Mallampati classification and ULBT, C-TMD has higher value in predicting difficult laryngoscopy and does not require the exposure of upper respiratory tract. TRIAL REGISTRATION: The study was registered on October 21, 2019 in the Chinese Clinical Trial Registry ( ChiCTR1900026775 ).

Multiplexed Nanomaterial-Based Sensor Array for Detection of COVID-19 in Exhaled Breath.

This article reports on a noninvasive approach in detecting and following-up individuals who are at-risk or have an existing COVID-19 infection, with a potential ability to serve as an epidemic control tool. The proposed method uses a developed breath device composed of a nanomaterial-based hybrid sensor array with multiplexed detection capabilities that can detect disease-specific biomarkers from exhaled breath, thus enabling rapid and accurate diagnosis. An exploratory clinical study with this approach was examined in Wuhan, China, during March 2020. The study cohort included 49 confirmed COVID-19 patients, 58 healthy controls, and 33 non-COVID lung infection controls. When applicable, positive COVID-19 patients were sampled twice: during the active disease and after recovery. Discriminant analysis of the obtained signals from the nanomaterial-based sensors achieved very good test discriminations between the different groups. The training and test set data exhibited respectively 94% and 76% accuracy in differentiating patients from controls as well as 90% and 95% accuracy in differentiating between patients with COVID-19 and patients with other lung infections. While further validation studies are needed, the results may serve as a base for technology that would lead to a reduction in the number of unneeded confirmatory tests and lower the burden on hospitals, while allowing individuals a screening solution that can be performed in PoC facilities. The proposed method can be considered as a platform that could be applied for any other disease infection with proper modifications to the artificial intelligence and would therefore be available to serve as a diagnostic tool in case of a new disease outbreak.