Supplemental Digital Content (SDC) Supplementary Methods
1. Detection of SARS-CoV-2 IgG antibodies and common cold coronavirus antibodies using commercially available tests
2. Detection of neutralizing antibodies using in-vitro assays 3. Assessment of T-cellular response
4. Assessment of donor-specific anti-HLA antibodies (DSA) and donor-derived cell- free DNA (dd-cfDNA) in patients with MPA withdrawal
5. Statistics Supplementary Table S1 Supplementary References
Supplementary Methods
1. Detection of SARS-CoV-2 IgG antibodies and common cold coronavirus antibodies using commercially available tests
IgG response against the spike S1 and the nucleocapsid protein were determined by using the SARS-CoV-2 Total Assay (Siemens, Eschborn, Germany) and the Elecsys anti-SARS-CoV-2 Assay (Roche, Mannheim, Germany), respectively. In line with manufacturers’ instructions, the cut-off index ≥ 1.0 indicated positive (reactive) results and was established by 2-point calibration of the assays with low and high concentrations of the spike protein. The cut-off value for seroconversion was defined as an anti-S1 IgG index ≥ 10.0.
Surrogate neutralizing antibodies were determined using plate-based assays (Medac, Wedel, Germany). The test mimics the virus-host interaction, as the serum samples were conjugated with soluble SARS-CoV-2 receptor binding domain-horseradish peroxidase (RBD- HRP), and subsequently added to the ELISA plates, which are coated with angiotensin- converting-enzyme-2 (ACE2) receptorsS1. RBD-HRP bound by non-neutralizing antibodies were able to bind the ACE2 receptors, while RBD-HRP bound by neutralizing antibodies were washed out. Subsequently, a chromogenic substrate for HRP (3,3′,5,5′-Tetramethylbenzidine (TMB) / hydrogen peroxide (H2O2)) was added. The percent inhibition was calculated based upon the optical density (OD) measured at 450nm. A cut-off ≥ 30% inhibition was applied according to manufacturer’s instructions to define positivity for this assay.
IgG response against five SARS-CoV-2 epitopes (full spike, the spike S1 and S2 subunits, RBD and nucleocapsid protein) as well as antibody reactivity against the spike S1 of four common cold coronaviruses (HCoV-229E, HCoV-HKU1, HCoV-NL63, HCoV-OC43) were determined by using a multiplex bead-based assay (LabScreen Covid Plus, One Lambda, West Hill, CA, USA)S2. Each of the above-mentioned proteins were conjugated to individual beads. The beads were read using the Luminex 200 analyzer (Luminex, Austin, Texas, USA).
The MFI cut-off values for each target epitope were adopted as recommended by the manufacturer. Individual cutoff values for each target antigen are as follows:
Target Cut-off (MFI)
SARS-CoV-2 Spike 6800
SARS-CoV-2 Spike S1 2700
SARS-CoV-2 Spike RBD 5800
SARS-CoV-2 Spike S2 3200
SARS-CoV-2 Nucleocapsid Protein 5900
HCoV-229E Spike S1 8012
HCoV-HKU1 Spike S1 4235
HCoV-NL63 Spike S1 4407
HCoV-OC43 Spike S1 3599
MFI, mean fluorescence intensity; RBD, receptor-binding domain; HCoV, human corona virus
2. Detection of neutralizing antibodies using in-vitro assays
Titers of neutralizing antibodies were determined using a SARS-CoV-2 live virus in titration experiments. The assay has a similar workflow as a focus reduction neutralization test (FRNT).
SARS-CoV-2 wild-type and viral variants were gained from naso- and oro-pharyngeal swabs of RT-PCR-confirmed SARS-CoV-2-positive patients. The variants were amplified in SARS- CoV-2 susceptible cells, specifically the delta-variant and wild-type in Vero E6 cells, and the omicron-variant in Calu-3 cells. The virus titers of stocks produced were measured by plaque assay and stored at -80°C before use. 2-fold serial dilutions were performed for each patient serum, and each dilution was incubated with an equal volume of the designated variant. After one hour incubation at 37°C, the mixtures were added to the corresponding SARS-CoV-2 susceptible cells and were fixed in plates with 5% formaldehyde at 24 hours post infection.
Virus replication was determined after immunostaining for the viral nucleocapsid protein with an in-cell ELISA. In contrast to FRNT, the staining signal of the assay was visualized with colorimetric methods using an HRP-conjugated secondary antibody and read with a plate reader. By extension, results were expressed as OD values as opposed to virus infected foci counts. The described visualization method has been validated with FRNT assay
data with Sotrovimab (S309). Values were normalized to infected control without serum (100%
infection) and the non-infected control (0% infection).
3. Assessment of T-cellular response
T-cellular response was assessed by quantitative detection of plasmatic IFN-γ following in- vitro stimulation of T-cells after contact with SARS-CoV-2 spike antigen using the QuanT-Cell SARS-CoV-2 (IGRA) assay (EUROIMMUN, Lübeck, Germany). 1ml of heparinized whole blood sample was added to a stimulator tube coated with SARS-CoV-2 spike antigen as well as to a negative and positive control tube, all of which were incubated overnight at 37°C.
Following centrifugation, the supernatant plasma was extracted and analyzed by an IFN-γ ELISA (EUROIMMUN, Lübeck, Germany). In accordance with the manufacturer’s recommendations, results > 200IU/ml were considered positive, 100-200IU/ml as borderline positive and < 100IU/ml as negative. Values from samples with inadequate responses to positive or negative controls were exempted from analysis.
4. Assessment of donor-specific anti-HLA antibodies (DSA) and donor-derived cell- free DNA (dd-cfDNA) in patients with MPA withdrawal
The presence of donor-specific anti-human leukocyte antigen antibodies (HLA) of IgG isotype was tested in the serum samples, and DSA were identified as de novo or resurgence of previously detected DSA by drawing comparisons to pre-vaccination sera. DSA were analyzed using the commercially available LABScreen Single Antigen Beads Assay Kit (One Lambda, West Hills, CA, USA) for the Luminex platform.
Levels of circulating donor-derived cell-free DNA (dd-cfDNA) were measured using the AlloSeq cfDNA test (CareDx, Inc., Brisbane, CA). Duplicate 10ml samples of venous blood were drawn into Streck cell-free DNA BCT tubes (Streck, Omaha, NE). Following centrifugation, cfDNA was extracted using the Circulating Nucleic Acid kit (Qiagen, Redwood City, CA) and quantified using the AlloSeq cfDNA PCR assay (CareDx, Brisbane, CA). The
next generation sequencing (NGS)-based assay identifies the fraction of donor-specific cfDNA by analyzing 202 targeted single-nucleotide polymorphisms (SNPs). Sequencing was performed using the MiniSeq system (Illumina, San Diego, CA) and the results were generated by the AlloSeq cfDNA software (CareDx).
5. Statistics
Data were expressed either as median with interquartile range (IQR) or as counts with percentage. Two-group comparisons of continuous variables were performed by applying the Mann-Whitney U test for unpaired data and the Wilcoxon matched-pairs test for paired data.
Spearman’s rho was employed as a nonparametric measure of rank correlation to test the relationship between anti-S1 IgG as determined by commercially available assay and neutralization against the delta- and omicron-variants using a live virus assay, respectively. All statistical tests were two-tailed, conducted with a 95% confidence interval and a p-value < 0.05.
The statistical analysis was performed using GraphPad Prism version 9.3.1 (GraphPad Software, San Diego, CA, USA).
Supplementary Table S1 Baseline Characteristics of KTR undergoing MPA withdrawal with and without seroconversion at t1 and t2
Characteristics of KTR undergoing MPA withdrawal N=28
t1 seropositive N=15
t1 seronegative N=13
P-Value t2 seropositive N=14
t2 seronegative N=14
P-Value
Basic patient characteristics Female, N (%)
Age at t0, median (IQR) BMI (kg/m2), median (IQR)
4 (27) 58 (52–62) 25 (22–29)
6 (46) 61 (53–66) 25 (22–28)
0.43 0.32 0.86
4 (29) 56.5 (50.3–61.3) 24.9 (21.8–27.5)
6 (43) 61.5 (52.8–66.3) 24.8 (22.4–28.9)
0.70 0.12 0.91 Vaccine administered
Homologous mRNA vaccination, N (%) 3x BNT162b2
4x BNT162b2
Heterologous mRNA vaccination, N (%) 2xBNT162b2 + 1xmRNA-1273 2xmRNA-1273 + 1xBNT162b2 3xBNT162b2 + 1xmRNA-1273
Heterologous vaccination incl. viral vector, N (%) 2xChAdOx1 + 1xBNT162b2
2xChAdOx1 + 1xmRNA-1273 1xChAdOx1 + 2xBNT162b2
1xChAdOx1 + 1xmRNA-1273 + 1xBNT162b2 3xBNT162b2 + 1Janssen + 1xmRNA-1273 More than three previous vaccine doses, N (%)
7 (47) 6 (40) 1 (7) 4 (27) 2 (13) 2 (13) 0 (0) 4 (27)
1 (7) 0 (0) 2 (13)
0 (0) 1 (7) 2 (13)
8 (62) 8 (62) 0 (0) 2 (15)
0 (0) 1 (8) 1 (8) 3 (23)
1 (8) 1 (8) 0 (0) 1 (8) 0 (0) 1 (8)
0.48 / / 0.66
/ / /
>0.99 / / / / /
>0.99
7 (50) 6 (43) 1 (7) 3 (21) 2 (14) 1 (7) 0 (0) 4 (29)
1 (7) 0 (0) 2 (14)
0 (0) 1 (7) 2 (14)
8 (57) 8 (57) 0 (0) 3 (21)
0 (0) 2 (14)
1 (7) 3 (21)
1 (7) 1 (7) 0 (0) 1 (7) 0 (0) 1 (7)
>0.99 / /
>0.99 / / /
>0.99 / / / / /
>0.99 Transplant data
Time since plantation (years), median (IQR) Donor characteristics
Deceased, N (%) Living, N (%)
7.2 (3.2–11.4)
10 (67) 5 (33)
2.3 (1.6–8.8)
9 (69) 4 (31)
0.045
>0.99
>0.99
8.1 (3.4–12.2)
9 (64) 5 (36)
2.8 (1.6–8.7)
10 (71) 4 (29)
0.03
>0.99
>0.99
Rejection during the past 12 months, N (%) S-Creatinine at t1 (mg/dl), median (IQR) S-Creatinine at t2 (mg/dl), median (IQR)
Tacrolimus trough level at t0 (µg/l), median (IQR)*
Methylprednisolone dose at t0 (mg), median (IQR)
0 (0) 1.56 (1.32–1.61) 1.47 (1.36–1.64)
6.1 (5.89.3) 4 (4–4)
0 (0) 1.38 (1.17–1.55) 1.36 (1.11–1.59) 7.9 (5.4–12.9)
4 (4–4)
/ 0.17 0.25 0.62 0.23
0 (0) 1.6 (1.4–1.7) 1.5 (1.4–1.6) 6.1 (5.89.3)
4 (3.75–4)
0 (0) 1.3 (1.1–1.5) 1.3 (1.1–1.6) 7.9 (5.4–12.9)
4 (4–4)
/ 0.06 0.09 0.62 0.22 Cause of nephropathy
Glomerular disease, N (%) PKD, N (%)
Reflux/chronic pyelonephritis, N (%) Vascular, N (%)
Systemic, N (%) Diabetes, N (%) Other/Unknown, N (%) Comorbidities
Arterial hypertension, N (%) Diabetes, N (%)
CAD, N (%)
Chronic lung disease, N (%) Chronic liver disease, N (%) Malignancy, N (%)
7 (47) 2 (13) 2 (13) 1 (7) 0 (0) 0 (0) 3 (20)
14 (93) 1 (7) 3 (20) 2 (13) 0 (0) 4 (27)
9 (69) 1 (8) 0 (0) 0 (0) 1 (8) 0 (0) 2 (15)
10 (77) 4 (31) 5 (38) 4 (31) 0 (0) 4 (31)
0.28
>0.99 / / / /
>0.99
0.31 0.15 0.41 0.37 /
>0.99
7 (50) 2 (14) 2 (14) 0 (0) 0 (0) 0 (0) 3 (21)
13 (93) 1 (7) 3 (21) 2 (14) 0 (0) 3 (21)
9 (64) 1 (7) 0 (0) 1 (7) 1 (7) 0 (0) 2 (14)
11 (79) 4 (29) 5 (36) 4 (29) 0 (0) 5 (36)
0.70
>0.99 / / / /
>0.99
0.60 0.33 0.68 0.65 / 0.68
BMI, body-mass index; CAD, coronary artery disease; IQR, interquartile range; KTR, kidney transplant recipients; MPA, mycophenolic acid; N, number; PKD, polycystic kidney disease; Seronegative:
Anti-spike S1 IgG index < 10; Seropositive: Anti-spike S1 IgG index ≥ 10
*Analyses based on a reduced study cohort consisting of patients medicated with Tacrolimus (10 seropositive KTR at t1 and t2, 12 seronegative KTR at t1 and t2)
Supplementary References
S1. Tan CW, Chia WN, Qin X, et al. A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2–spike protein–protein interaction. Nat Biotechnol.
2020;38(9):1073-1078. doi:10.1038/s41587-020-0631-z
S2. Bray RA, Lee JH, Brescia P, et al. Development and Validation of a Multiplex, Bead- based Assay to Detect Antibodies Directed Against SARS-CoV-2 Proteins. Transplantation.
2020;105(1):79-89. doi:10.1097/tp.0000000000003524
