Preventive and therapeutic benefits of nelfinavir in rhesus macaques and human beings infected with SARS-CoV-2.

Effective drugs with broad spectrum safety profile to all people are highly expected to combat COVID-19 caused by SARS-CoV-2. Here we report that nelfinavir, an FDA approved drug for the treatment of HIV infection, is effective against SARS-CoV-2 and COVID-19. Preincubation of nelfinavir could inhibit the activity of the main protease of the SARS-CoV-2 (IC50 = 8.26 µM), while its antiviral activity in Vero E6 cells against a clinical isolate of SARS-CoV-2 was determined to be 2.93 µM (EC50). In comparison with vehicle-treated animals, rhesus macaque prophylactically treated with nelfinavir had significantly lower temperature and significantly reduced virus loads in the nasal and anal swabs of the animals. At necropsy, nelfinavir-treated animals had a significant reduction of the viral replication in the lungs by nearly three orders of magnitude. A prospective clinic study with 37 enrolled treatment-naive patients at Shanghai Public Health Clinical Center, which were randomized (1:1) to nelfinavir and control groups, showed that the nelfinavir treatment could shorten the duration of viral shedding by 5.5 days (9.0 vs. 14.5 days, P = 0.055) and the duration of fever time by 3.8 days (2.8 vs. 6.6 days, P = 0.014) in mild/moderate COVID-19 patients. The antiviral efficiency and clinical benefits in rhesus macaque model and in COVID-19 patients, together with its well-established good safety profile in almost all ages and during pregnancy, indicated that nelfinavir is a highly promising medication with the potential of preventative effect for the treatment of COVID-19.

D3AI-Spike: A deep learning platform for predicting binding affinity between SARS-CoV-2 spike receptor binding domain with multiple amino acid mutations and human angiotensin-converting enzyme 2.

The number of SARS-CoV-2 spike Receptor Binding Domain (RBD) with multiple amino acid mutations is huge due to random mutations and combinatorial explosions, making it almost impossible to experimentally determine their binding affinities to human angiotensin-converting enzyme 2 (hACE2). Although computational prediction is an alternative way, there is still no online platform to predict the mutation effect of RBD on the hACE2 binding affinity until now. In this study, we developed a free online platform based on deep learning models, namely D3AI-Spike, for quickly predicting binding affinity between spike RBD mutants and hACE2. The models based on CNN and CNN-RNN methods have the concordance index of around 0.8. Overall, the test results of the models are in agreement with the experimental data. To further evaluate the prediction power of D3AI-Spike, we predicted and experimentally determined the binding affinity of a VUM (variants under monitoring) variant IHU (B.1.640.2), which has fourteen amino acid substitutions, including N501Y and E484K, and 9 deletions located in the spike protein. The predicted average affinity score for wild-type RBD and IHU to hACE2 are 0.483 and 0.438, while the determined Kaff values are 5.39 ± 0.38 × 107 L/mol and 1.02 ± 0.47 × 107 L/mol, respectively, demonstrating the strong predictive power of D3AI-Spike. We think D3AI-Spike will be helpful to the viral transmission prediction for the new emerging SARS-CoV-2 variants. D3AI-Spike is now available free of charge at https://www.d3pharma.com/D3Targets-2019-nCoV/D3AI-Spike/index.php.

Exploring the Binding Affinity and Mechanism between ACE2 and the Trimers of Delta and Omicron Spike Proteins by Molecular Dynamics Simulation and Bioassay.

Five major variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged and posed challenges in controlling the pandemic. Among them, the current dominant variant, viz., Omicron, has raised serious concerns about its infectiousness and antibody neutralization. However, few studies pay attention to the effect of the mutations on the dynamic interaction network of Omicron S protein trimers binding to the host angiotensin-converting enzyme 2 (ACE2). In this study, we conducted molecular dynamics (MD) simulations and enzyme linked immunosorbent assay (ELISA) to explore the binding strength and mechanism of wild type (WT), Delta, and Omicron S protein trimers to ACE2. The results showed that the binding capacities of both the two variants' S protein trimers to ACE2 are enhanced in varying degrees, indicating possibly higher cell infectiousness. Energy decomposition and protein-protein interaction network analysis suggested that both the mutational and conserved sites make effects on the increase in the overall affinity through a variety of interactions. The experimentally determined KD values by biolayer interferometry (BLI) and the predicted binding free energies of the RBDs of Delta and Omicron to mAb HLX70 revealed that the two variants may have the high risk of immune evasion from the mAb. These results are not only helpful in understanding the binding strength and mechanism of S protein trimer-ACE2 but also beneficial for drug, especially for antibody development.

New laboratory evidence for the association between endothelial dysfunction and COVID-19 disease progression.

There is growing evidence that angiotensin-converting enzyme 2 is highly expressed on endothelial cells, endothelial dysfunction plays a critical role in coronavirus disease 2019 (COVID-19) progression, but laboratory evidence is still lacking. This study established a multicenter retrospective cohort of 966 COVID-19 patients from three hospitals in Wuhan, China. We found that male (62.8% vs. 46.5%), old age [72 (17) vs. 60.5 (21)], and coexisting chronic diseases (88.5% vs. 60.0%) were associated with poor clinical prognosis in COVID-19. Furthermore, the deteriorated patients exhibited more severe multiorgan damage, coagulation dysfunction, and extensive inflammation. Additionally, a cross-sectional study including 41 non-COVID-19 controls and 39 COVID-19 patients assayed endothelial function parameters in plasma and showed that COVID-19 patients exhibited elevated vascular cell adhesion molecule-1 (VCAM-1) (median [IQR]: 0.32 [0.27] vs. 0.17 [0.11] µg/ml, p < 0.001), E-selectin (21.06 [12.60] vs. 11.01 [4.63] ng/ml, p < 0.001), tissue-type plasminogen activator (tPA) (0.22 [0.12] vs. 0.09 [0.04] ng/ml, p < 0.001), and decreased plasminogen activator inhibitor-1 (0.75 [1.31] vs 6.20 [5.34] ng/ml, p < 0.001), as compared to normal controls. Moreover, VCAM-1 was positively correlated with d-dimer (R = 0.544, p < 0.001); tPA was positively correlated with d-dimer (R = 0.800, p < 0.001) and blood urea nitrogen (R = 0.638, p < 0.001). Our findings further confirm the strong association between endothelial dysfunction and poor prognosis of COVID-19, which offers a rationale for targeting endothelial dysfunction as a therapeutic strategy for COVID-19.

CoVac501, a self-adjuvanting peptide vaccine conjugated with TLR7 agonists, against SARS-CoV-2 induces protective immunity.

Safe, effective, and economical vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to achieve adequate herd immunity and end the pandemic. We constructed a novel SARS-CoV-2 vaccine, CoVac501, which is a self-adjuvanting peptide vaccine conjugated with Toll-like receptor 7 (TLR7) agonists. The vaccine contains immunodominant peptides screened from the receptor-binding domain (RBD) and is fully chemically synthesized. It has been formulated in an optimized nanoemulsion formulation and is stable at 40 °C for 1 month. In non-human primates (NHPs), CoVac501 elicited high and persistent titers of protective neutralizing antibodies against multiple RBD mutations, SARS-CoV-2 original strain, and variants (B.1.1.7 and B.1.617.2). Specific peptides booster immunization against the B.1.351 variant has also been shown to be effective in improving protection against B.1.351. Meanwhile, CoVac501 elicited the increase of memory T cells, antigen-specific CD8+ T-cell responses, and Th1-biased CD4+ T-cell immune responses in NHPs. Notably, at an extremely high SARS-CoV-2 challenge dose of 1 × 107 TCID50, CoVac501 provided near-complete protection for the upper and lower respiratory tracts of cynomolgus macaques.

Predicting spike protein NTD mutations of SARS-CoV-2 causing immune evasion by molecular dynamics simulations.

The coronavirus disease 2019 (COVID-19) pandemic was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Among all the potential targets studied for developing drugs and antibodies, the spike (S) protein is the most striking one, which is on the surface of the virus. In contrast with the intensively investigated immunodominant receptor-binding domain (RBD) of the protein, little is known about the neutralizing antibody binding mechanisms of the N-terminal domain (NTD), let alone the effects of NTD mutations on antibody binding and thereby the risk of immune evasion. Based on 400 ns molecular dynamics simulation for 11 NTD-antibody complexes together with other computational approaches in this study, we investigated critical residues for NTD-antibody binding and their detailed mechanisms. The results show that 36 residues on the NTD including R246, Y144, K147, Y248, L249 and P251 are critically involved in the direct interaction of the NTD with many monoclonal antibodies (mAbs), indicating that the viruses harboring these residue mutations may have a high risk of immune evasion. Binding free energy calculations and an interaction mechanism study reveal that R246I, which is present in the Beta (B.1.351/501Y.V2) variant, may have various impacts on current NTD antibodies through abolishing the hydrogen bonds and electrostatic interaction with the antibodies or affecting other interface residues. Therefore, special attention should be paid to the mutations of these key residues in future antibody and vaccine design and development.