research participants
This was a prospective cross-sectional study of patients with CKD. We recruited patients from the outpatient department or dialysis center of Queen Mary Hospital between August 11 and October 5, 2022. Patients were eligible if they were 18 years of age or older and had one of the following kidney diseases: (1) CKD stage 3 or above (defined as estimated glomerular filtration rate (eGFR) < 60 ml/min 持续 > 3 months); (2) Receiving peritoneal dialysis (PD) for more than 1 month; (3) Obtaining HD for more than 1 month; (4) KTR receiving maintenance immunosuppression; (5) Receiving maintenance immunosuppression of immune-mediated glomerulonephritis. Patients were excluded if they had acute kidney injury, refused to provide written informed consent, were mentally incapacitated to provide written informed consent, or were unable to contribute sufficient blood. At our center, we use a regimented immunosuppressive regimen for patients with KTR and immune-mediated glomerulonephritis. KTR received triple maintenance immunosuppression, including corticosteroids, a calcineurin inhibitor (CNI), and an antimetabolite (mycophenolate mofetil) as first-line therapy, which was replaced with an mTOR inhibitor in patients who developed post-transplant malignancy. ). Patients with immune-mediated glomerulonephritis are given maintenance immunosuppression based on renal pathology (corticosteroids and antimetabolites are used in patients with lupus nephritis, ANCA vasculitis, and membranoproliferative glomerulonephritis; patients with minimal change nephropathy and membranous nephropathy are Corticosteroids with or without CNI). Blood specimens were collected using coagulated vessels and lithium heparin vessels. This study was approved by the Institutional Review Board of the University of Hong Kong/Hong Kong Western Hospital Authority (HKU/HA HKW IRB) (reference numbers UW 22-555 and UW 21-313). The study was conducted in accordance with the 1964 Declaration of Helsinki. Written informed consent was obtained from all study participants.
definition
As defined by the World Health Organization, patients are considered to have completed the primary series of COVID-19 vaccines if they have received at least 2 doses of BNT162b2 or CoronaVac28. Patients were classified as previously infected with SARS-CoV-2 if they had a known positive reverse transcription polymerase chain reaction (RT-PCR) or rapid antigen test result; or in this study, if they had not been previously vaccinated Live whole-virion COVID-19 vaccines tested positive for IgG against the SARS-CoV-2 nucleocapsid (N) protein in this study.
Virus culture and live virus neutralizing antibody assay
Viral culture was performed with TMPRSS2-expressing VeroE6 (VeroE6/TMPRSS2) cells in a biosafety level 3 facility as we described previously, with minor modifications11,26,29. Briefly, TMPRSS2-expressing VeroE6 (VeroE6/TMPRSS2) cells (JCRB Cat#JCRB1819) were inoculated in 1 mL minimal essential medium (MEM) (Gibco®, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS ) and 1 mg/mL G418 (Gibco®, Thermo Fisher Scientific), 1 × 105 Cells in shell bottles (Diagnostic Hybrids, Inc).Shell vial incubated at 37°C, 5% CO2 Inoculation was carried out until confluence. Each vial was inoculated with 100 μL of clinical specimen. After one hour of incubation, clinical samples were removed and the cells were supplemented with 1 mL of 1% FBS, 100 IU/ml penicillin, 100 μg/mL streptomycin, 20 U/ml nystatin, and 25 mM HEPES (Gibco®, MoFisher Technology).Cells were incubated at 37°C, 5% CO22 Virus-induced cytopathic effects (CPE) were observed daily. Cultures with more than 50% CPE expanded to large volumes in VeroE6/TMPRSS2 cells under the same culture conditions. 50% tissue culture infective dose (TCIDFifty) were measured in VeroE6/TMPRSS2 cells. The SARS-CoV-2 lineage of viral culture isolates was confirmed using whole-genome sequencing.
Live viral nAb assays were performed as we previously described11,26,29. The SARS-CoV-2 ancestral strain (GISAID accession number EPI_ISL_17668119) and four Omicron variant sublines (BA.2.2 (GISAID accession number EPI_ISL_17668120), BA.2.3.20 (GISAID accession number EPI_ISL_16342299), BA.5.2 (GISAID accession number EPI_ISL_16342299) No. EPI_ISL_1) 37776 58), XBB.1 (GISAID accession number EPI_ISL_15602393)) are included. Briefly, serum samples were heat-inactivated at 56°C for 30 min and serially diluted 2-fold in MEM containing 1% FBS.Mix copies of each diluted serum with the SARS-CoV-2 virus isolate to achieve a final concentration of 100 TCIDFifty and incubate at 37°C for 1 hour. After incubation, 100 μL of serum-virus mixture was added to VeroE6/TMPRSS2 cells seeded into 96-well plates 48 h before infection. The cells were incubated with the mixture at 37°C. After 3 days of incubation, the CPE of each well was visually scored by two independent observers. 50% Neutralization Titer (NTFifty) was determined by using the log(inhibitor) versus normalized response variable slope in GraphPad PRISM version 9.4.0. For statistical analysis, if the live virus nAb titer was <10, a value of 5 was assigned.
Interferon-gamma release assay
Following the manufacturer’s instructions (Wantai SARS-CoV-2 IGRA, Wantai Biopharm, Beijing, China). Lithium heparin blood collected from each patient via VACUETTE® LH lithium heparin tubes (Greiner Bio-One, Kremsmünster, Austria) was processed on the day of collection and stored at 37 °C in the three tubes provided Incubate for 24 hours under: i) Background control culture tube for individual IFN-γ background (N tube); (ii) Positive control culture tube (P tube) containing SARS-CoV-2 non-specific antigen for non-specific IFN-γ secretion was used as a control; (iii) culture tubes (T-tubes) were tested for specific IFN-γ secretion using SARS-CoV-2 spike protein antigen. The concentration of IFN-γ released in the plasma fractions obtained after centrifugation of the three tubes was then measured using an enzyme-linked immunosorbent assay (ELISA) provided in the same assay kit. IFN-gamma responses were measured by a SkanIt microplate reader at a wavelength of 620–450 nm and analyzed using SkanIt software for microplate reader RE version. 6.0.2.3, (Thermofisher Scientific, Waltham, MA, USA). Results are interpreted according to manufacturer’s recommendations.
IgG detection against SARS-CoV-2 nucleocapsid (N) protein
IgG levels against the SARS-CoV-2 N protein were determined using the SARS-CoV-2 IgG test (Alinity, Abbott).
Whole-genome sequencing and genomic data analysis
Randomly selected SARS-CoV-2 RT-PCR-positive archival clinical specimens were retrieved for whole-genome sequencing to determine viral lineage using our previously described Oxford Nanopore MinION device (Oxford Nanopore Technologies)29 (Supplementary Table S1). Nanopore sequencing was performed following the Nanopore protocol – PCR tiling for COVID-19 (version: PTC_9096_v109_revH_06Feb2020) according to the manufacturer’s instructions (Oxford Nanopore Technologies). Briefly, extracted RNA was first reverse transcribed into cDNA using SuperScript™ IV reverse transcriptase (ThermoFisher Scientific, Waltham, MA, USA). PCR was then performed using the hCoV-2019/nCoV-2019 version 3 amplicon kit (Integrated DNA Technologies, Coralville, IA, USA) and the Q5® Hot Start High-Fidelity 2X Master Mix Kit (New England Biolabs, Ipswich, Massachusetts) Amplification, USA) according to the Nanopore protocol. PCR products were purified using 1× AMPure XP beads (Beckman Coulter, Brea, CA, USA) and quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The purified DNA was then subjected to standardization of end preparation and native barcoding ligation reactions according to PCR tiling of the COVID-19 virus protocol using Native Barcoding Expansion 96 (EXP-NBD196, Oxford Nanopore Technologies). Barcoded libraries were then pooled, purified with 0.4× AMPure XP beads, and quantified using the Qubit dsDNA HS Assay Kit. Purified pooled libraries were ligated to sequencing adapters and sequenced for 24–48 hours using an Oxford Nanopore MinION device using an R9.4.1 flow cell.
For bioinformatics analysis, use the recommended ARTIC bioinformatics workflow (version 1.2.1) with minor modifications as previously described29. These modifications include reducing the minimum length of the guppyplex step to 350 to allow detection of potentially small deletions and increasing the “-normalize” value to 999.999 to merge all sequencing reads and use high-precision mode for base calling Out and enhance QC pass score from 7 to 10. The sequence NC_045512.2 obtained from NCBI was used as a reference and the alignment files generated by Medaka were checked using Integrative Genomics Viewer (IGV) (2.8.0) to verify the mutations called by the ARTIC pipeline.SARS-CoV-2 lineages were assigned using the online Nextclade tool (version 2023-03-28)30. All sequences were deposited into the GISAID database (Supplementary Table S2).
Statistical Analysis
Statistical analysis was performed using PRISM 9.4.0 or SPSS 26.0.0. Categorical and continuous variables were compared using Fisher’s exact test and Mann Whitney U test, respectively. One ANOVA with Dunn’s multiple comparison test was used to compare >2 groups. Subgroup analyzes were performed to exclude the influence of potential confounding effects of previous infection. Sample size was based on feasibility. Multivariable regression analysis was performed to control for confounders including KTR, use of immunosuppressants, number of immunosuppressants, and use of antimetabolite immunosuppressants.arrive ask Values less than 0.05 were judged to be statistically significant.