Fig 2: Antihistamine drugs prevented SARS-CoV-2 infection in transgenic hACE2 mice. (A) Acrivastine at serially diluted concentrations was premixed with authentic SARS-CoV-2 D614G viruses, followed by incubation with HEK293T-hACE2 cells. After 48 h, the viral RNA in the supernatants was extracted and quantified via RT-PCR-based detection. The IC50 value was calculated based on the relative number of viral RNA copies within each concentration. (B) The IC50 of triprolidine against the authentic SARS-CoV-2 D614G strain was measured and calculated as described in panel A. (C) Schematic of the animal infection assay. Four transgenic hACE2 mice (C57BL/6) were intravenously administrated with acrivastine at a dosage of 10 mg/kg of body weight. Mice in the control group were administrated with an equal volume of saline. Six hours later, all mice were intranasally challenged with 1 × 105 focus-forming units of the SARS-CoV-2 D614G virus. On Day 5 post-infection, the mice were euthanized. Lung tissues were harvested and subjected to HE, immunohistochemistry (IHC), and viral quantification assays. (D) Lung tissues from both saline- and acrivastine-treated mice (challenged with authentic SARS-CoV-2) were analyzed by HE staining as well as IHC with antibodies against the SARS-CoV-2 N protein. (E) SARS-CoV-2 RNA copies in lung tissues were quantified via RT-PCR and are presented as log10 copies per milliliter (mL). (F) Schematic of ACE2- or HRH1-dependent SARS-CoV-2 entry. In ACE2high/HRHlow cells, SARS-CoV-2 viruses enter susceptible cells mainly by binding to ACE2 receptors through S proteins, while in cells with low ACE2 expression and high HRH1 expression (ACE2low/HRH1high cells), SARS-CoV-2 viruses can utilize HRH1 as alternative receptors to enter target cells. In cells expressing both ACE2 and HRH1 (ACE2medium/HRH1medium cells), SARS-CoV-2 can use both receptors to enter target cells. In addition, the presence of HRH1 could enhance ACE2-dependent viral entry. The data in panels A and B are presented as the means ± SEMs of biological triplicates. The data in panel E represent the mean ± SEM of biological quadruplicate samples. The P value in panel E was calculated by Student’s t-test. ***P < 0.001. The scale bars in panel D represent 100 µm.

Fig 3: Antiviral activity of VHH60.a VHH60 inhibits pseudovirus carrying wildtype spike protein infection on Caco-2 cell line. b VHH60 inhibits ancestral SARS-CoV-2 infection on Vero-E6. c Survival curve of mice infected by SARS-CoV-2. Vehicle group all died at 4 d.p.i (5/5), one in VHH60 group (1/5) died. d Body weight change of mice infected by SARS-CoV-2. Data is represented as ratio of body weights at indicated timepoint versus day 0 (n = 10 at 0 and 3 d.p.i, n = 4 at 4 d.p.i, n = 5 at 5.d.p.i). e Virus load in lung after 3 days of infection (n = 5). f Representative image of immunofluorescence from lung. Blue: Nuclei, Red: ACE2, Green: nucleocapsid protein (NP). g Representative image of hematoxylin-eosin (H&E) staining from the lung. Scar bar, 2000 μm in the up row, 150 μm in the down row. One-way ANOVA with Tukey’s multiple comparison test was used. *P < 0.05. Error bars represent the means with SDs.

Fig 4: Screening of high-impact mutant RBD antigen. (A) SimPlot analysis of nucleotide sequence similarity between SARS-CoV-2 Omicron BA.2 and other variant strains. The upper portion indicates the proteins encoded by SARS-CoV-2, with The orange box highlighting the RBD region. (B) Analysis of the Correlation between Antibody Immune Escape and S Protein Mutation Sites. (C) Normalization of data on RBD binding to ACE2(X-axis) and the neutralization of antibody escape(Y-axis) using Z-scores.The black circle delineates the region of mutation sites classified as low-impact. (D) Process flow for RBD vaccine mutation sites.

Fig 5: ACE2/MAPK mediates the 1 potentiating effects of SARS-CoV-2 on platelet activation. A SARS-CoV-2 virus and its Spike protein phosphorylate ACE2, Erk, p38, and JNK in human platelets. Platelets from healthy donors were pretreated with SARS-CoV-2 (1 × 105 PFU) or its Spike protein (2 μg/mL) for various times, as 5 indicated. For ACE2 phosphorylation detection, cell lysates were prepared and subjected to immunoprecipitation (IP) with anti-phospho-Ser/Thr antibody, followed by immunoblotting analysis with anti-ACE2 antibody. Cell lysates without the process of immunoprecipitation (10% of input) were analyzed in parallel as loading controls. For p-Erk, p-p38, and p-JNK detection, cell lysates were prepared and directly subjected to immunoblotting. Representative results are presented from 4 experiments using platelets from different healthy donors and summary data is presented in the Additional file 1: Online Figure 7. B Enhanced platelet aggregation by SARS-CoV- 2 is abolished by MAPK inhibitors. The healthy human platelets were pretreated with 10 μM PD98059 (ERK1/2 inhibitor), 10 μM SB203580 (p38 inhibitor), or 10 μM SP600125 (JNK inhibitor) for 10 min, then treated with SARS-CoV-2 (1 × 105 PFU, 30 min) before stimulation by 0.025 U/mL thrombin or 0.6 μg/mL collagen. Representative results are presented from 3 experiments using platelets from different donors. C Enhanced platelet aggregation by Spike protein is abolished by MAPK inhibitors. Human platelets were pretreated with 10 μM PD98059, 10 μM SB203580, or 10 μM SP600125 for 10 min, then treated with Spike protein (2 μg/mL, 5 min) or vehicle as control before stimulation by 0.025 U/mL thrombin or 0.6 μg/mL collagen. Representative results are presented from 3 experiments using platelets from different healthy donors. D Accelerated clot retraction by SARS-CoV-2 (D1) or its Spike protein (D2) is abolished by MAPK inhibitors. Platelets were pretreated with 10 μM PD98059, 10 μM SB203580, or 10 μM SP600125 for 10 min, and then incubated with SARS26 CoV-2 (1 × 105 PFU, 30 min) or Spike protein (2 μg/mL, 5 min) as in B and C. Clot retraction was initiated as in Fig. 3d. Representative images and summary data of 3 experiments are presented using platelets from different healthy donors. Representative images and summary data are presented from 3 experiments using platelets from different donors. E Increased phosphorylation of Erk, p38, and JNK in platelets from COVID-19 patients, compared with healthy donors. Representative results are presented using platelets from 6 individuals from different COVID-19 patients (n = 3) and healthy donors (n = 3). Statistical analyses were performed using unpaired two34 tailed Student’s t test in (D1) and (D2). *P < 0.05; **P < 0.01. MAPK indicates mitogen activated protein kinase; PD, PD98059; SB, SB203580; SP, SP600125