Of the 58 KTRs who were enrolled in a study that evaluated the immunogenicity of two doses of SARS-CoV-2 mRNA in KTRs at our department, 54 KTRs were enrolled in this study (16). All participants completed three doses of the SARS-CoV-2 mRNA-1273 vaccine (Moderna) or BNT162b2 SARS-CoV-2 mRNA vaccine (Pfizer-BioNTech) between January and June 2022. This study was conducted in accordance with the principles outlined in the Declaration of Helsinki. All the participants provided written informed consent. The ethics committee of Yamagata University Faculty of Medicine approved the protocol for this research project (approval no. 2021-329).

Blood samples were obtained within 2 weeks before the first dose, 2–4 weeks after the second dose, and 1–7 weeks after the third dose of the vaccine. Serum creatinine levels recorded on the day of blood sample collection were retrieved from the patient records.

The blood samples were tested using an anti-SARS-CoV-2 S enzyme immunoassay (Elecsys anti-SARS-CoV-2 S RUO; Roche Diagnostics, Mannheim, Germany), which detects antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein, according to the manufacturer’s instructions. Values below 0.8 U/mL were considered negative.

To analyze cellular responses, an ELISpot assay measuring interferon-gamma produced by specific SARS-CoV-2 T cells was performed as previously described (16). Briefly, PBMCs were isolated by specific gravity centrifugation using Ficall-Paque Premium (Cytiva, Tokyo, Japan), and cryopreserved until analysis. Stimulation was conducted with individual sequences containing 11 amino acids overlapping a 15-mer peptide pool derived from a peptide scan of the full-length sequence of the vaccine (BNT162b2), which encoded the receptor-binding domain of the SARS-CoV-2 spike glycoprotein (2 µg/mL/peptide; JPT Peptide Technologies, Berlin, Germany). All tests were performed in duplicate. Negative control wells lacked peptides, and positive control wells contained anti-CD3 monoclonal antibodies (1:1000; Mabtech, Stockholm, Sweden). Then, 2×10⁵ PBMCs per well were stimulated, placed in a plate pre-coated with anti-IFN-γ (Human INF-γ ELISpotPro kit, Mabtech, Stockholm, Sweden) at 37°C in a humidified incubator with 5% CO₂, and incubated for 48 h. The cells were removed, and the plates were washed five times with phosphate-buffered saline. Next, 100 μL of a 200-fold diluted secondary anti-INF-γ antibody conjugated with horseradish peroxidase (ELISpotPro kit, Mabtech, Stockholm, Sweden) was added to each well and incubated at room temperature for 2 h. After five washes with phosphate-buffered saline, a tetramethylbenzidine substrate was added. ELISpot analysis was performed using an ELISpot Reader (Autoimmun Diagnostika; Strasberg, Germany). Cytokine activity was calculated from spot size and intensity values, as previously described (16, 17). The rate of change in cytokine activity in each test was calculated using the formula below, and the mean value of the two measurements was used as the measured value.

The rate of change in cytokine activity = 100 × (cytokine activity in peptide-stimulated wells − cytokine activity in negative control wells)/cytokine activity in negative control wells.

The cut-off value was determined by calculating the mean ± 2 standard deviations in a group of healthcare workers obtained prior to the first vaccination in our previous study, and was determined as the rate of change in cytokine activity greater than 164 (16).

Adverse reactions after each dose of vaccine were documented using a specific questionnaire that included local reactions (pain, redness, and swelling at the injection site) and systemic reactions (fever, fatigue, headache, chills, myalgia, arthralgia, vomiting, and diarrhea). Participants were also asked to rate their symptoms on an ordinal scale (none, mild, moderate, or severe). Mild symptoms were defined as those that did not interfere with daily activities, moderate symptoms were defined as those that caused some interference with daily activities, and severe symptoms were defined as those that prevented daily activities.

Clinical episodes of acute rejection, including acute elevation in eGFR after the third vaccination, were also evaluated. eGFR was calculated using a formula modified for Japanese patients, as regulated by the Japanese Society of Nephrology (eGFR = 194 serum creatinine mg/dL^1.094 × age^0.287 × 0.739 [if female]).

All clinical data were collected from patient records and analyzed retrospectively. Statistical analysis of various parameters was performed for each group using Fisher’s exact test for categorical variables and Mann–Whitney U or Wilcoxon signed-rank tests for continuous variables. The significance level was set at p < 0.05. All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface that is a modified version of R commander designed to add statistical functions frequently used in biostatistics (The R Foundation for Statistical Computing, Vienna, Austria) (18).

Of the 58 KTRs who were enrolled in the study that evaluated the immunogenicity of two doses of SARS-CoV-2 mRNA in KTRs at our department, 1 patient did not receive the third vaccine, 2 patients did not want to participate in the study, and 1 patient was infected with SARS-CoV-2 before the third vaccination. Finally, 54 KTRs who did not have a clinical episode of COVID-19 before the third vaccine were enrolled in this study. The clinical characteristics of the participants are shown in Table 1 .

In 53 of 54 (98%) KTRs who received the BNT162b2 vaccine as the first and second vaccine, 28 (53%) received the BNT162b2 vaccine and 25 (47%) received the mRNA-1273 vaccine as the third vaccine. Only 1 of 54 (2%) KTRs who received mRNA-1273 as the first and second vaccine received the mRNA-1273 vaccine as the third vaccine. The duration between the second and third vaccinations was 7 ± 1 month. No significant difference between KTRs with BNT162b2 as third vaccine and mRNA-1273 as third vaccine was observed in the positivity rate of the anti-SARS-CoV-2 antibody (KTRs with BNT162b2, 32%; KTRs with mRNA-1273, 31%; p=1.000), anti-SARS-CoV-2 antibody titer (KTRs with BNT162b2, 83 ± 296 U/mL; KTRs with mRNA-1273, 108 ± 442 U/mL; p=0.749), the positivity rate of ELISpot activity (KTRs with BNT162b2, 50%; KTRs with mRNA-1273, 58%; p=0.597), and the rate of change in ELISpot cytokine activity (KTRs with BNT162b2, 386 ± 572; KTRs with mRNA-1273, 240 ± 238; p=0.545) after the second vaccination. Forty-nine of 54 (91%) were living donor KTRs, and 16 of 54 (30%) KTRs received ABO-incompatible kidney transplants (KT). All KTRs were treated with calcineurin inhibitors, and 48 of 54 (89%) KTRs were treated with steroids. Seventeen KTRs were converted from mycophenolate mofetil (MMF) to everolimus (EVL) (0.75 mg bid) only one week before and after the third vaccination in 46 of 54 KTRs who had been treated with MMF. All 21 KTRs with rituximab received rituximab only for the induction of ABO-incompatible and/or sensitized KT. There were 6 patients who received rituximab within 24 months before the first vaccination. The duration between the rituximab induction and the first vaccinations was 61 ± 41 month. Only 17 of 54 KTRs (31%) were SARS-CoV-2 anti-spike antibody-positive, and 29 of 54 (54%) were ELISpot activity-positive after the second vaccination.

After the third vaccination, all 17 (100%) KTRs with anti-SARS-CoV-2 antibody positivity after the second vaccination and 27 of 37 (73%) KTRs without anti-SARS-CoV-2 antibodies after the second vaccination were positive for anti-SARS-CoV-2 antibodies ( Figure 1A ). The anti-SARS-CoV-2 antibody seroconversion rate after the third vaccination was 81.4%, which was higher than that reported in previous studies (6.4-69.2%) ( Table 2 ) (8, 10–15). Moreover, anti-SARS-CoV-2 antibody titers were significantly higher than those after the second vaccination not only in all KTRs (2^nd, 95 ± 370 U/mL; 3^rd, 6673 ± 10676 U/mL; p<0.001) but also in KTRs with anti-SARS-CoV-2 antibody positivity after the second vaccination (2^nd, 301 ± 622 U/mL; 3^rd, 15385 ± 14173 U/mL; p<0.001) ( Figure 1B ). Both neutrophil counts and IgG titers before the third vaccination did not differ between KTRs with anti-SARS-CoV-2 antibody and those without it after the third vaccination; however, lymphocyte count was significantly lower in KTRs without anti-SARS-CoV-2 antibody after the third vaccination (p=0.006). In the univariate model, age ≥ 60 years, first vaccination within 2 years after kidney transplantation, and lymphocyte count < 1150/mm³ were associated with a lack of humoral response to the third vaccination ( Table 3 ). Multivariate regression analysis, accounting for age ≥ 60 years and lymphocyte count < 1150/mm³, confirmed these associations. Rituximab did not affect anti-SARS-CoV-2 antibody positivity; however, anti-SARS-CoV-2 antibody was not detected in 4 of 6 KTRs who received rituximab within 24 months before the first vaccination. Short-term peri-vaccination conversion from MMF to EVL did not improve anti-SARS-CoV-2 antibody positivity. MMF, which was one of the risk factors for a lack of humoral response to the second vaccination, did not significantly affect the anti-SARS-CoV-2 antibody positivity rate after the third vaccination. In contrast, the anti-SARS-CoV-2 antibody titer was significantly lower in KTRs with MMF (KTRs with MMF, 4853 ± 8407 U/mL; KTRs without MMF, 17141 ± 16174 U/mL; p=0.016).

After the third vaccination, 26 of 29 (90%) KTRs with positive ELISpot cytokine activity after the second vaccination, and 12 of 24 (50%) KTRs without ELISpot cytokine activity after the second vaccination were positive for ELISpot cytokine activity ( Figure 2A ). All three KTRs whose ELISpot cytokine activity turned negative after the third vaccination received triple immunosuppressants consisting of steroids, MMF, and tacrolimus and the doses of these immunosuppressants did not change during the study period. Their lymphocyte counts did not change significantly. Anti-SARS-CoV-2 antibody test was negative after the second vaccination in all three KTRs; however, the anti-SARS-CoV-2 antibody test was positive after the third vaccination (22, 7913, and 11167 U/mL). The rate of change in cytokine activity after the third vaccination was significantly higher than that after the second vaccination, not only in all KTRs (3^rd, 34690 ± 133690; 2^nd, 316 ± 445; p<0.001) but also in KTRs without ELISpot cytokine activity after the second vaccination (2^nd,554 ± 496; 3^rd, 58932 ± 178090; p<0.001) ( Figure 2B ). Neither neutrophil counts, lymphocyte counts, nor IgG titers differed between KTRs with anti-SARS-CoV-2 antibody positivity and those without it after the third vaccination. In univariate analysis, hypertension and lymphocyte count < 1150/mm³ were associated with a lack of positive ELISpot activity after the third vaccination ( Table 4 ). Multivariate regression analysis, accounting for lymphocytes less than 1150/mm³, confirmed this association.

In 44 KTRs who were anti-SARS-CoV-2 antibody-positive after the third vaccination, 28 (64%) KTRs were also ELISpot cytokine activity-positive. Neither humoral nor cellular responses after the third vaccination were observed in 9 of 54 (17%) KTRs. Only one KTR was ELISpot cytokine activity-positive but anti-SARS-CoV-2 antibody negative. In 44 KTRs with anti-SARS-CoV-2 antibody positivity after the third vaccination, the anti-SARS-CoV-2 antibody titer in KTRs with positive ELISpot cytokine activity was significantly higher than that in KTRs without ELISpot cytokine activity (9547 ± 12179 U/mL vs. 2909 ± 4127 U/mL, p=0.047).

Adverse events at the injection site after the third vaccination (72%) were significantly lower than those after both the first (96%, p<0.001) and second vaccinations (96%, p<0.001) ( Figure 3A ). Systemic adverse events after the third vaccination were not significantly higher (57%) than those after both the first (37%, p=0.053) and second vaccinations (48%, p=0.441) ( Figure 3B ). Systemic adverse events classified as greater than moderate did not differ between the first (15%), second (17%), and third vaccinations (17%) (p=1.000). Systemic adverse events classified as greater than moderate after the third vaccination were not correlated with anti-SARS-CoV-2 antibody positivity after the third vaccination (positive, 16%; negative, 20%; p=1.000), ELISpot cytokine activity positivity after the third vaccination (positive, 16%; negative, 20%; p=0.701), or third vaccine type (BNT162b2 vaccine, 20%; mRNA-1732 vaccine, 14%; p=0.719). There was no significant deterioration of the eGFR after the third vaccination (2^nd, 52.1 ± 18.1 mL/min/1.73 m²; 3^rd, 52.6 ± 18.5 mL/min/1.73 m²; p=0.926). None of the patients showed clinical symptoms of acute rejection.