Seroprevalence of SARS-CoV-2 IgG Antibodies After the Second BNT162b2 mRNA Vaccine in Japanese Kidney Transplant Recipients


 We aimed to evaluate the rate of anti–SARS-CoV-2 IgG seropositivity and investigated factors associated with seropositivity after the second SARS-CoV-2 mRNA vaccination in kidney transplant (KT) recipients. This retrospective study conducted between June 2021 and November 2021 included 106 KT recipients and 127 healthy controls who received the second dose of the BNT162b2 mRNA vaccine at least seven days before the measurement of antibody titers. The titers of immunoglobulin G (IgG) antibodies against the receptor-binding domain of SARS-CoV-2 spike (S) protein were determined. Seropositivity was defined as an anti–SARS-CoV-2 IgG level of ≥15 units/mL, which was considered as the presence of sufficient neutralizing antibodies. The median ages and the seroprevalence rates of the healthy controls and KT recipients were 68 and 56 years and 98% and 22%, respectively. Univariate logistic regression analysis revealed that age >53 years, rituximab use, mycophenolate mofetil use, and KT vintage <7 years were negatively associated with anti–SARS-CoV-2 IgG seropositivity in KT recipients. Humoral response after the second BNT162b2 mRNA vaccine was greatly hindered by immunosuppression therapy in KT recipients. Older age, rituximab use, mycophenolate mofetil use, and KT vintage may play key roles in seroconversion.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a critical disease associated with high mortality rate in kidney transplant (KT) recipients with immunosuppression 1 , for whom SARS-CoV-2 vaccination is recommended for infection prevention. However, several studies reported that KT recipients exhibited a signi cantly impaired response to standard dose of SARS-CoV-2 mRNA-based vaccination compared to the general population [2][3][4][5][6][7] . Su cient data are not available for KT recipients, who were not included in SARS-CoV-2 vaccine clinical trials 8 . Additionally, most studies evaluating immunoglobulin G (IgG) antibody levels against SARS-CoV-2 mRNA vaccines (P zer/BioNTech BNT162b2 or Moderna mRNA-1273) were from Western countries. As KT protocols vary across countries and regions, the vaccine e cacy has not been fully validated in KT recipients in Japan. In Japan, ABO blood-type incompatible (ABOi) KT protocols with strong immunosuppression strategies are necessary due to the absence of donor exchange programs and the serious donor shortage [9][10][11][12][13] . Currently, one-third of the recipients undergo ABOi KT with rituximab desensitization 13 . However, the anti-SARS-CoV-2 IgG seropositivity rate after the second SARS-CoV-2 mRNA-based vaccination in patients who undergo ABOi KT with contemporary immunosuppressive strategies remains unknown. Therefore, we measured the titers of immunoglobulin G (IgG) antibodies against the receptor-binding domain of SARS-CoV-2 spike (S) protein and investigated risk factors for inadequate humoral response after the second dose of the P zer/BioNTech BNT162b2 mRNA vaccine in KT recipients, including those who underwent ABOi KT.

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The background characteristics of the study cohort are summarized in Table 1. Brie y, the median ages were 68 (IQR: 38-77) and 56 (IQR: 44-65) years in the controls and KT recipients, respectively. Rituximab was administrated in 43 (41%) KT recipients, including 24 (23%) ABOi KT recipients and 19 (18%) ABOc KT recipients. Biopsy-proven rejection and viral infections before enrollment in the current study were observed in 10 (9%) and 11 (10%) patients, respectively. Steroids were used in most of all recipients (n=97, 92%), with a median prednisone dose of 5.0 mg. All recipients received combined immunosuppressive therapy including a median of three agents. Everolimus was used in 12 recipients. The median period after KT was 6.3 years. No recipient experienced biopsy-proven rejection or viral events during the current study period.
The current study ndings suggest that humoral response after SARS-CoV-2 mRNA vaccination is strongly inhibited in KT recipients >53 years of age, those treated with rituximab or MMF, and those with a KT vintage of < 7 years. Interestingly, similar factors were reported to be associated with weaker humoral response in a recent study that investigated the SARS-CoV-2 S IgG antibody in 142 KT recipients using the LIAISON® assay and showed age ≥54 years, KT vintage ≤8 years, and treatment with ≥2 immunosuppressants were signi cantly associated with seroconversion 16 . These results suggest that combination immunosuppressive therapy may induce strong immunosuppression which might interfere with antiviral antibody production for 7−8 years. Factors that impact antibody response in KT recipients should be further investigated.
Among the immunosuppressive agents, rituximab and MMF exhibited a signi cant impact on humoral response in not only KT recipients but also patients with other chronic clinical conditions. Several studies reported the negative effects of rituximab and MMF use on anti-SARS-CoV-2 IgG seropositivity in KT recipients 17,18 and in patients with autoimmune in ammatory rheumatic diseases 19 . Kantauskaite et al.
showed that MMF-free immunosuppressive regimens were signi cantly associated with seroconversion (OR 13.25, 95% CI 3.22−54.6, P < 0.001) 17 . The authors suggested that MMF had a dose-dependent unfavorable effect on antibody titers, such as MMF levels of >1000 mg/day. We also examined the association between anti-SARS-CoV-2 IgG seropositivity and MMF dose and found that the seropositivity was lower in those treated with MMF doses > 1000 mg (6.3%) compared to those who were not treated with MMF (59%) and those treated with 500-1000 mg MMF (16%), which indicated that MMF dose modi cation might improve immune response to the SARS-CoV-2 vaccine. However, further investigation is warranted to address the balance between rejection and immune acquisition.
Rituximab use was also signi cantly associated with impaired humoral response in the current study. A multicenter observational study examining SARS-CoV-2 seropositivity in adult patients with autoimmune in ammatory rheumatic diseases (n = 686) reported that rituximab use was signi cantly associated with impaired humoral response to the BNT162b2 mRNA vaccine 19 . As B cell depletion is associated with a lack of serological response, those ndings are reasonable regarding the negative impact of rituximab on humoral response to various vaccines 20 . Therefore, these results emphasize the importance of SARS-CoV-2 vaccination before the administration of MMF and /or rituximab in KT recipients.
The impact of ABOi KT on humoral response to SARS-CoV-2 vaccines should be addressed. Albeit uncommon across the globe, ABOi KT is a common alternative for donor exchange programs in Japan.
As it requires extensive immunosuppression including rituximab and therapeutic apheresis, we hypothesized that ABOi KT might also have a great impact on anti-SARS-CoV-2 IgG seropositivity. However, we found that ABOi KT had a limited impact on seropositivity in the present study; this result might be associated with the lower statistical power due to the limited sample size, which should be addressed in future studies.
The major limitations of the present study include the limited sample size and retrospective study design. The IgG antibody titers were determined during the early phase of mass immunization in Japan. ABOi KT and rituximab use in immunologically high-risk recipients might not be common worldwide. Additionally, measurement of antibody titers is one of the several methods to assess immunologic response to vaccination. Despite those limitations, this is the rst study evaluated the seroprevalence of SARS-CoV-2 IgG antibodies after the second BNT162b2 mRNA vaccine in Japanese KT recipients.
In conclusion, we con rmed that the rate of anti-SARS-CoV-2 IgG seroconversion was low in KT recipients after the second BNT162b2 mRNA vaccine. However, several outstanding questions remain and further investigation is warranted to determine the duration of immunity under immunosuppressive therapy, the effect of reduced titers on the protective activity of vaccines against breakthrough infections, and the e cacy of third vaccination in KT recipients.

Methods
This retrospective study was approved by the Ethics Committee of Hirosaki University (2021-089). All participants had previously provided written informed consent for other biomarker studies. Additional informed consent for the current study was waived with approval by the Ethics Committee of Hirosaki University. The clinical and research activities being reported are consistent with the Principles of the Declaration of Istanbul and Helsinki.

Participants
The current study conducted between June 21, 2021 and November 1, 2021 included 106 KT recipients and 127 healthy controls who received the second BNT162b2 dose at least seven days before the measurement of anti-SARS-CoV-2 antibody titers. The control group included members of the medical staff, medical students, and posttreatment patients with localized cancers who were not actively receiving any treatment. Those with previous SARS-CoV-2 infection and who provided blood samples for titer measurement within the rst seven days after the second BNT162b2 dose were excluded. Clinical parameters of age, gender, primary kidney disease, KT vintage (years), dialysis vintage (years), ABO blood type compatibility, immunosuppressant agents, past history of rejection events, past history of viral events, and renal function were obtained from the medical records.

Immunosuppression
Flow cytometry and Luminex-based single-antigen bead assay were used to select the immunosuppression protocol 21 . Low-dose rituximab (100 mg/m 2 or 100 mg/body) and donor-speci c human leukocyte antigen antibodies were administered in recipients of ABOi and ABO blood-type compatible (ABOc) KT, respectively. For ABOc KT recipients, basic immunosuppression included calcineurin inhibitors (CNIs), mycophenolate mofetil (MMF), and steroids. Most KT recipients, i.e., those who underwent KT after February 2002, were treated with induction therapy using the anti-CD25 monoclonal antibody basiliximab on the day of operation and postoperative day 4. Intravenous immunoglobulin was not given in all KT patients due to the lack of insurance coverage ABOi KT recipients received basic immunosuppressive agents, rituximab and therapeutic apheresis. Low-dose rituximab was administered three weeks before transplantation. Basic immunosuppressive agents (CNIs, MMF, and steroids) were administered seven days before transplantation in all ABOi KT recipients. Several sessions of double-ltration plasmapheresis and one session of plasma exchange were performed on the day before surgery to remove anti-A/B antibodies until the anti-A/B antibody titers decreased to a level of <1:32-1:64. Those with viral infection or malignancies were switched from CNIs or MMF to everolimus.

Measurement of anti-SARS-CoV-2 IgG antibody titers
Cross-sectional blood samples collected for regular evaluation were used to measure the titers of IgG antibodies against the SARS-CoV-2 S receptor-binding domain, and the Elecsys Anti-SARS-CoV-2 S RUO assay (Covas 8000/e 801; Roche Diagnostics, Mélan, France) was used. According to the manufacturer's data, seropositivity was de ned as an anti-SARS-CoV-2 IgG level of ≥15 U/mL, which was shown to be su cient for the presence of neutralizing antibodies. Antibody titers between 0.80 and 15 U/mL were de ned as marginal values.

Outcomes
In the current study, we compared the rates of anti-SARS-CoV-2 IgG seropositivity as well as the rates of marginal anti-SARS-CoV-2 IgG seropositivity between the controls and KT recipients. We also determined the anti-SARS-CoV-2 IgG seropositivity rate in ABOi KT recipients, and investigated the factors associated with anti-SARS-CoV-2 IgG seropositivity after the second SARS-CoV-2 mRNA vaccination in KT recipients.

Statistical analysis
Qualitative and quantitative variables were described as numbers with percentages and medians with interquartile ranges (IQRs), respectively. The chi-squared, Fisher's exact, Mann-Whitney U, and Student's t tests were used for the statistical comparison between the healthy controls and KT recipients. Univariable logistic regression analysis was performed to identify factors associated with anti-SARS-CoV-2 IgG seropositivity after the second SARS-CoV-2 mRNA vaccination, and odds ratio (OR) with 95% con dence interval (CI) were calculated. Predictive accuracy and optimal cutoff values for anti-SARS-CoV-2 IgG levels were evaluated by area under the receiver operating characteristic (ROC) curve analysis. All statistical analyses were performed using BellCurve for Excel 3.10 (Social Survey Research Information, Tokyo, Japan) and GraphPad Prism 7.00 (GraphPad Software, San Diego, CA, USA).

Declarations
Author contributions: Shingo Hatakeyama had full access to all data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.  VisualabstractSciRep.pdf