Protein sequence alignment, phylogenetic analysis and data analysis of the 3th dose of the WHO-approved inactivated SARS-CoV-2 vaccine
Protein sequence alignment was performed using the MUSCLE algorithm by MEGA-X software (v.10.1.8) or ClustalW (https://www.genome.jp/tools-bin/clustalw). For phylogenetic analysis, nucleotide or protein sequences of the viruses were first aligned using the ClustalW and the MUSCLE algorithm, respectively. Phylogenetic trees were subsequently generated using the maximal-likelihood method in MEGA-X (1,000 bootstraps). The model and the other parameters used for phylogenetic analysis were applied following the recommendations after finding the best DNA/protein models using the software. The nucleotide similarity of coronaviruses was analysed using SimPlot (v.3.5.1) with a sliding windows size of 1,000 nucleotides and a step size of 100 nucleotides using gap-stripped alignments and the Kimura (two-parameter) distance model. The prediction of the effect of some genes on others was conducted by the mCSM-PPI2.
All of the vaccinated sera were collected from volunteers at about 21 days after the third dose of the WHO-approved inactivated SARS-CoV-2 vaccine (CoronaVac, Sinovac). The median age of volunteers was older than 37. There were a total of male and female participants. All of the volunteers were recruited by Sinovac. None of the participants had a history of previous infections and there were no serious adverse events after they were given the vaccine. All of the volunteers were provided informed written consent forms, and the whole study was conducted according to the requirements of Good Clinical Practice of China. The procedures about human participants were approved by the Ethics Committee (seal) of Beijing Youan Hospital, Capital Medical University with an approval number of LL-2021-042-K.
Cells washed with PBS were lysed with 2% TritonX-100/PBS containing 1 mM freshly prepared PMSF (Beyotime, ST506) on ice for 10 min. Cell lysates were centrifugationd at 12,000g at 4 C for 5 min and then mixed with a small amount of SDS-elution buffer and cultured at 95 C for 5 min. The supernatant was centrifugationd to 7,000g at 4 C and resuspended with 50 to detect the HA tag or VSV-M on pseudotyped viruses. In the case of the block, 5% milk in PBS containing 0.1% Tween-20 was supplemented with 0.05% Tween-20 at room temperature. [23H12] (Kerafast, EB0011) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (AntGene, ANT325) were added at a 1:10,000 dilution in PBST with 1% milk, or 1:250 dilution in TBST with 1% milk in the case of the stem-helix targeting monoclonal antibody B6, and incubated on a shaker at 4 °C. After three washes in PBST or TBST, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody AffiniPure goat anti-mouse IgG (H+L) (Jackson Immuno Research, 115-035-003), AffiniPure goat anti-rabbit IgG (H+L) (Jackson Immuno Research, 111-035-003, 1:10,000 dilution) or Alexa Fluor 680-conjugated goat anti-human secondary antibody (1:50,000 dilution, Jackson ImmunoResearch, 109-625-098) for 1 hour. The blots were subsequently washed three times before visualization using the LI-COR Odyssey CLx or the Omni-ECL Femto Light Chemiluminescence Kit (EpiZyme, SQ201) and a ChemiDoc MP Imaging System (Bio-Rad). Supplementary table 3 contains information about the antibodies. Uncropped and unprocessed full scans of gel source data are provided in Supplementary Figs. 1 and 2.
Stable cell lines overexpressing different receptors were generated by lentivirus transduction and antibiotic selection. Specifically, the lentiviruses carrying the target genes were produced by co-transfection of lentiviral transfer (pLVX-EF1a-Puro,) and packaging plasmids pMD2G (Addgene, 12259) and psPAX2 (Addgene, 12260) into HEK293T cells using Lip2000 transfection reagent (Biosharp, BL623B). The lentivirus-containing supernatant was collected and pooled at 24 and 48 h after transfection. There were cells transduced by the lentivirus with 8 g polybrene. Stable cells were selected and maintained in the growth medium with puromycin (1 μg ml−1). Cells selected for at least 10 days were considered to be stable cell lines and were used in various experiments.
The 96-well immuno-plates with the ACE2 ectodomains were coated overnight at a maximum concentration of 100 l per well. After three washes with PBST, wells were blocked by 3% skimmed milk/PBS at 37 °C for 2 h. The wells were added to and incubated for 1 hour at a temperature of 37 C. After extensive washes, wells were put into the 3% skimmed milk/PBS for 1 hour at 37C. Finally, the substrate solution (Solarbio, PR1210) was added to the plates, and the absorbance at 450 nm after reaction termination was measured using the SpectraMax iD3 Multi-well Luminometer (Molecular Devices).
The complex structures were manually built using the refined maps. The atomic models were further refined by positional and B-factor refinement in real space using Phenix (v.1.19)59. For building the PDF-2018 RBD–Bat37ACE2 complex model, the refinement NeoCoV RBD–Bat37ACE2 complex structures were manually docked into the refined maps using UCSF Chimera (v.1.15) and further corrected manually by real-space refinement in Coot. The atomic models were further refined by using both the Phenix and Rosetta models. For the PDF-2180 S model building, UCSF ChimeraX (v.1.1)71 and Coot were used to fit a MERS-CoV spike atomic model (PDB: 5W9J) into the PDF-2180 cryo-EM map. The model was rebuilt with a tool called Coot. N-linked glycans were hand built into the density where visible, and the models were rebuilt and refined using Rosetta (v.1.2.5)69,70. All of the models were validated and analysed using MolProbity72 and Privateer73. The figures were generated using ChimeraX. The datasets and refinement statistics are shown in Supplementary Table 1.
The total number of micrographs were recorded for the complex. The PDF-2018 RBD–Bat37ACE2 complex had a total of over 3000 micrographs recorded. Both datasets were similarly processed. MotionCor2 is used to process raw data. The raw data were aligned and averaged into motion-corrected summed images, after which, defocus values for each micrograph were determined using Gctf. Particles were picked and analyzed to get a two-dimensional alignment. Well-defined partial particles were selected for initial model reconstruction in Relion57. The initial model was used as a reference for 3D classification. After refinement and post-processing, the overall resolution of the PDF-2018 RBD–Bat37ACE2 complex was up to 3.8 Å based on the gold-standard Fourier shell correlation (FSC; threshold = 0.143)58. C2 symmetry was expanded prior to 3D refinement for the NeoCoV RBD–Bat37ACE2 complex. Finally, the resolution of the NeoCoV RBD–Bat37ACE2 complex reached 3.5 Å. The quality of the local resolution was evaluated using ResMap (v.1.95)59. For the PDF-2180 spike trimer, a total of 1,746 micrographs were collected. Video frame alignment, estimation of the microscope contrast-transfer function parameters, particle picking and extraction were performed using Warp. Particles were put in a box of 800 binned to 400 square feet. Reference-free 2D classification was performed using Relion (v.3.0) to select well-defined particles images before 3D classification without symmetry applied using a MERS-CoV cryo-EM map61 as an initial model in Relion. 3D refinements and CTF refinement (to refine per-particle defocus values) were performed in Relion (v.3.0)62. Particle images were processed using the Bayesian polishing procedure implemented in Relion (v.3.0)63 before performing another round of 3D refinement and per-particle defocus refinement. Subsequently, 3D classification without alignment was performed using a mask focused on the N-terminal domain to improve its resolution, and the selected particles were processed for 3D refinement imposing C3 symmetry using non-uniform refinement in cryoSPARC (v.3.3.1)64, which yielded a final reconstruction of the PDF-2180 S at a resolution of 2.5 Å. Local-resolution estimation, filtering and sharpening were performed using CryoSPARC (v.3.3.1)65. The gold-standard FSC of 0.143 criterion is the basis of reported resolutions and the curves were corrected for the effects of soft masking when noise substitution was used.
Source: https://www.nature.com/articles/s41586-022-05513-3
Immunofluorescence assay for the characterization of 3Flag receptors in HEK293 cell lines and to determine the intracellular luciferase activity
Expression levels of receptors were evaluated by immunofluorescence assay detecting the C-terminal 3×Flag tags. Cells expressing receptors were seeded in the 96-well plate (poly-lysine-pretreated plates for HEK293 cell lines) at a cell density of about 1–5 × 104 cells per well and cultured for 24 h. Cells were fixed with 100% methanol at room temperature for 10 min and then incubated with a mouse monoclonal antibody (M2) targeting the Flag-tag (Sigma-Aldrich, F1804) diluted in 1% BSA/PBS at 37 °C for 1 h. After one wash with PBS, the cells were submerged in a mixture of 1%BSA/PBS at 37 C for 1 h. The nuclei were stained with Hoechst 33342 (1:5,000 dilution in PBS). Images were captured with a fluorescence microscope (Mshot, MI52-N).
Cells obtained by transfection were trypsinized and held in a 96-well plate for 48 hours to allow attachment and entry simultaneously. For viruses without known susceptible cells, infections were performed using the same genome copies of a reference virus with a calculated TCID50 titre (commonly 1 × 105 TCID50 per 100 μl). For TPCK-treated trypsin (Sigma-Aldrich, T8802) treatment, pseudotyped viruses in serum-free DMEM were incubated with 100 μg ml−1 TPCK-treated trypsin for 10 min at 25 °C. The reactions were stopped by adding a 100 g liter of soybean trypsin inhibitor to DMEM. After 16–20 h, GFP images were acquired using a fluorescence microscope (Mshot, MI52-N), and intracellular luciferase activity was determined using the Bright-Glo Luciferase Assay Kit (Promega, E2620) and measured using the SpectraMax iD3 Multi-well Luminometer (Molecular Devices) or a GloMax 20/20 Luminometer (Promega).
The cells were transfected with plasmids that were full-length hACE2 or Bat40, for pseudotyped virus neutralization. During the night, 50,000 cells per well were used to seed the 96-well plates at the 37 C that were used for transfection. For neutralizations, twofold serial dilutions of B6, S2P6 or S2H14 IgGs were prepared in DMEM. After 5 l of the corresponding pseudo typed viruses were mixed with 20 l of DMEM and 25 l of each IgG dilution, they were incubated for 45 min at 37 C. The HEK293T cells were washed 3 times before adding 40 l of the mixture. Then, 1 h later, 40 μl DMEM was added to the cells. After 17–20 h, 70 μl of One-Glo-EX substrate (Promega) was added to each well and incubated on a plate shaker in the dark. The plates were read using a plate reader. There were copies of the measurements taken. Relative light units were plotted and normalized in Prism (GraphPad, v.8). Cells alone without pseudotyped virus was defined as 0% infection, and cells with virus only was defined as 100% infection.
The coronaviruses were put in the cells at 37C after they were added to DMEM at indicated concentrations. After the cells were washed with DMEM, they were put into a Hanks’ balanced salt solution and incubated with 2 g ml1 of baby goat anti-human IgG. Cells were washed twice with PBS and then put in a petri dish for nucleus staining. Images were captured using a fluorescence microscope (MI52-N). For flow cytometry analysis, cells were detached by 5 mM of EDTA/PBS and analysed using the CytoFLEX Flow Cytometer (Beckman). The dead cells, as indicated by SSC/FSC, were excluded by gating. There were 10,000 events analysed for the gated live-cell population. The cells were gated when compared with control cells without expression. The data was analysed using FlowJo. The gating strategy for flow cytometry analysis can be found in supplementary fig. 3
Octet RED 96 instrument was used to determine the target binding affinities of the proteins. The biosensors were dipped into the buffer after the 20 g ml1 is loaded onto them. bioSensors were dipped into the buffer with a range of 0 to 500nM for 120 s and then dropped into the buffer for 300 s to record dissociation and association kinetics. Kinetic buffer without ACE2 was used to define the background. The corresponding affinities were calculated with the Octet Data Analysis software (v.12.2.0.20) using curve-fitting kinetic analysis or steady-state analysis with global fitting. KD,app values were reported because of the use of dimeric ACE2.
A cell-cell fusion test was done on HEK293T cells that were stably expressing different genes. Group A cells were transfected with spike and rLucN(1–155)-sfGFP1–7(1–157) expressing plasmids. Group B cells were transfected with spike and sfGFP8–11(158–231)-rLuc(156–311) expressing plasmids. After 12 h after transfection, cells from both groups were trypsinized and mixed into a 96-well plate. After 24 h, cells were washed and then trypsin was taken out for 10 min at room temperature. The nuclei were stained with Hoechst 33342 for 30 min at 37 C. The fluorescent images were captured using a microscope. 20 M of EnduRen live-cell subtitlature was added to the cells in DMEM and then taken for a count using the Varioskan LUX multi-well luminometer.
Source: https://www.nature.com/articles/s41586-022-05513-3
Infection-related experiments and statistical analysis of a non-pathogenic virus with gain-of-function mutations to expand tropism for human cells
The State Key Laboratory of Verology, with the permission of the University, conducted the infections-related experiments. Indeed, a VSV-based pseudotype virus system was used for all entry and neutralization assays, including new mutations in NeoCoV or PDF-2180 spikes that expand tropism for human cells. There are non-pathogenic pseudotyped viruses that are useful in studies of coronaviruses entry and host range determination. The gain-of-function change, NeoCov-T510F, was generated when we discovered that a Phe residue was present in the same location as PDF-2180 S and that it has an effect on binding affinity. We cautiously avoided extensively testing and showing the gain of function mutations on NeoCoV to expand its tropism on human cells.
Most infection and live-cell binding-related experiments were repeated between 2 and 5 times with around 3–4 biological repeats. Technical repeat experiments were conducted with in petri dishes. Similar results were obtained in all of the experiments, and representative data are shown. Data can either be presented as mean or mean s.e.m. as specified in the figure legends. Most statistical analyses were conducted using GraphPad Prism (v.8) using unpaired two-tailed Student’s t-tests, unless otherwise specified. P < 0.05 was considered to be significant; P < 0.05, P < 0.01, P < 0.005, **P < 0.001.
Antigen retrieval and sequencing of whole-genome sequences from FFPE tissue sections collected from the next of kin at the National Cancer Institute
The legal next of kin consented to the collection of the tissues in the laboratory at the National Cancer Institute. The patients in this cohort were notvaccinated against the disease. Tissues preserved for histopathologic analysis and special staining were dissected fresh at the time of autopsy, placed into tissue cassettes, fixed for 24 h in neutral-buffered formalin, and then transferred to 70% ethanol for 48 h before impregnation with paraffin.
The correlation between the target and control samples was demonstrated in the case of apandemic control cases. 5 m FFPE tissue sections were hydrated in serial alcohol solutions of distilled water after being deparaffinized. Heat antigen retrieval was carried out using a pressure cooker (DAKO) by submerging slides in 1× pH 6 citrate buffer for 20 min. Endogenous enzyme activity was quenched with 3% hydrogen peroxide containing sodium azide for 10 min with additional 10% non-fat dry milk (Bio-Rad) for 20 min to prevent nonspecific binding. Tissue sections were then incubated with polyclonal SARS or SARS-CoV-2 N antibody (1:500, custom made, GenScript U864YFA140-4/CB2093, 0.447 mg ml−1)43,44,45 for 1 h at room temperature. There was congruently stained negative controls on subsequent sections, following the same protocol as replacing the primary antibody with a rabbit IgG control antibody. I-1000-5 is from Vector Laboratories. The reaction was visualized with a microscope using the Dako and Envision+Rb systems. Sections were lightly counterstained with haematoxylin, dehydrated in graded alcohols, cleared in xylene, mounted and coverslipped.
Supernatant from the virus isolation plates of thalamus of P38 were sequenced using short-read, whole-genome sequencing. The manufacturer used Ribo-Zero+ to deplete rRNA following their protocol. The water was eluted in order to prepare the libraries that were prepared after the kit was put into use. The 10 l of depletedRNA was used as a template for first and second-step synthesis. KAPA Uniquedual-Indexed adapters, as well as samples enriched with KAPA Hi fi HotStart Ready mix and a range of 9 to 19PCR amplification cycles, were used for the multiplexing. The manufacturer’s manual has a range of 12 to 18 cycles of post-capture amplification for myBaits Expert Viruses, which can be found at arborbiosciences.org. Purified, enriched libraries were quantified on a CFX96 Real-Time System (Bio-Rad) using Kapa Library Quantification kit (Roche Sequencing Solutions). Libraries were pooled together as needed in equimolar concentrations to obtain 2 150-bp reads on the MiSeq. Raw sequence reads were trimmed of Illumina adapter sequence using Cutadapt version 1.12 (ref. 40) and then trimmed and filtered for quality using the fastq_quality_trimmer and fastq_quality_filter tools from the FASTX-Toolkit 0.0.14 (Hannon Lab, CSHL). Reads were then mapped to the SARS-CoV-2 2019-nCoV/USA-WA1/2020 genome (MN985325.1) using Bowtie2 version 2.2.9 (ref. 41) with parameters -local -no-mixed -X 1500. PCR duplicates were removed using picard MarkDuplicates, version 2.26.10 (Broad Institute).
To prove the presence of infectious virus, tissues with high viral RNA levels through dd and subgenomic RNA RT-qPCR measuring across a broad range of 16 to 35 Cq were isolated. Virus isolation was carried out on tissues by homogenizing the tissue in 1 ml DMEM and inoculating Vero E6 cells in a 24-well plate with 250 µl of cleared homogenate and a 1:10 dilution thereof. Plates were centrifuged for 30 min at 1,000 r.p.m. and incubated for 30 min at 37 °C and 5% CO2. The inoculum was replaced by 500 l DMEM containing penicillin, streptomycin, and a couple of other drugs. Six days after inoculation, the cytopathic effect was scored. The same method was used for a blind passage of samples in which cytopathic effect was not present. There was additional isolation of the virus from P38 thalamus and hypothalamus. The cells were grown with 50 U mil, penicillin, 50 g and strechicomycin and 10 g. There was no selection antibiotic used in the isolation of the virus on Vero E6 cells. The cytopathic effect was ruled out using the results of the analysed tissue homogenate from flash-frozen specimens and plates that had been analysed using the expression of the SARS-coV-2 E genes. Cell lines were confirmed to be free of mycoplasma in the house.
Chromogenic ISH detection was carried out using the manual RNAScope 2.5 HD assay (catalogue no. 322310, Advanced Cell Diagnostics) with a modified pretreatment protocol. Briefly, formalin-fixed and paraffin-embedded (FFPE) tissue sections were cut at 7 μm, air dried overnight, and baked for 1–2 h at 60 °C. The tissues were deparaffinized and dehydrated before being treated for 15 minutes at room temperature. The slides were boiled with pretreatment reagent for 15 min, digested with protease at 40 °C for 20 min, and then hybridized for 2 h at 40 °C with probe-V-nCov2019-S (catalogue no. 848561, Advanced Cell Diagnostics) or probe-V-nCoV-N (catalogue no. 846081, Advanced Cell Diagnostics)42. In addition, probe-Hs-PPIB (peptidylprolyl isomerase B, catalogue no. 313901, Advanced Cell Diagnostics) and probe-dapB (catalogue no. 310043, Advanced Cell Diagnostics) were used as a positive and negative control, respectively. Subsequent amplification was carried out according to the original protocol. The detection of specific probe-binding sites was visualized by using the Advanced Cell Diagnostics kit. The slides were covered in a substance.
Source: https://www.nature.com/articles/s41586-022-05542-y
Statistical methods for multiple comparisons of blood ddenum chromosome levels between two continuous variables using stochastic regression models with log-transformations
Correlations between two continuous variables were assessed using Spearman’s rank correlation coefficient (ρ). Fisher’s z-transformation was used for the calculations of 95% CI and P values. To compare ddPCR levels between tissue types (respiratory versus non-respiratory), we used linear mixed models with compound symmetry correlation structure to account for repeated measures within each subject. Residual diagnoses are used to check assumptions. Log-transformations were used when needed. A small positive random number is used to replace 0 values with log10-transform ddPCR values. Logistic regression models were used to generate ROC curves. Optimal cutoff values were selected by treating sensitivity and specificity as equally important. SAS version 9.2 was used for the analyses. All P values are two-sided and reported without adjustment for multiple comparisons.