Neutralizing antibody responses and cellular responses against SARS-CoV-2 Omicron subvariants after mRNA SARS-CoV-2 vaccination in kidney transplant recipients

Although the mRNA SARS-CoV-2 vaccine has improved the mortality rate in the general population, its efficacy against rapidly mutating virus strains, especially in kidney transplant recipients, remains unclear. We examined the anti-SARS-CoV-2 spike protein IgG antibody and neutralizing antibody titers and cellular immunity against B.1.1, BA.1, and BA.5 antigens in 73 uninfected kidney recipients and 16 uninfected healthy controls who received three doses of an mRNA SARS-CoV-2 vaccine. The IgG antibody titers were significantly lower in recipients than in healthy controls. Similarly, neutralizing antibody titers against three viral variants were significantly lower in recipients. When the virus was mutated, the neutralizing antibody titers decreased significantly in both groups. In cellular immunity analysis, the number of spike-specific CD8 + non-naïve T cells against three variants significantly decreased in recipients. Conversely, the frequency of spike-specific Th2 CD4 + T-cells in recipients was higher than that in healthy controls. Nineteen recipients and six healthy controls also received a bivalent omicron-containing booster vaccine, leading to increase IgG and neutralizing antibody titers in both groups. After that, eleven recipients and five healthy controls received XBB.1.5 monovalent vaccines, increasing the neutralizing antibody titers against not only XBB.1.5, but also EG.5.1 and BA.2.86 antigens in kidney recipients. Although kidney recipients did not gain sufficient immunity against Omicron BA.5 with the third dose of vaccine, humoral response against mutant SARS-CoV-2 lineages significantly increased after bivalent Omicron-containing booster vaccine and the XBB.1.5 monovalent vaccine. Therefore, it is important for kidney recipients to continue to administer updated vaccines.


Anti-SARS-CoV-2 spike protein IgG antibody production by SARS-CoV-2 vaccination in KTXRs
Anti-SARS-CoV-2 spike protein IgG antibody titers in both groups are shown in Fig. 1.The median antibody titers in the KTXRs and healthy controls after the third dose of vaccine were 7.8 (IQR: < 1-72.7) and 141.0 (IQR: 58.3-332.5)AU/mL respectively, and the antibody titers in the KTXRs were significantly lower than those in Table 1.Baseline characteristics of kidney transplant recipients and healthy controls.Data are presented by frequency (%) or by median (IQR).P values are calculated using the nonparametric Mann-Whitney U test and chi-square test.KTXR kidney transplant recipient, BMI body mass index, eGFR estimated glomerular filtration rate, TAC Tacrolimus, CYA Cyclosporine A.

KTXRs (n = 73) Healthy (n = 16) P value
Female, n (%) 40  healthy controls (p < 0.0001).The antibody positivity rates were 67.1% and 100%.The background factors for the responders and non-responders among KTXRs are summarized in Table 2.The time after kidney transplantation was significantly longer in the responder group than in the non-responder group (p = 0.0012).Furthermore, the mycophenolic acid area under the plasm concentration-time curve was significantly lower in responders than in non-responders (p = 0.0041).No significant differences were observed in the other background factors.

Neutralizing activity of sera against SARS-CoV-2 variants after the third vaccine dose
To measure the neutralizing activity of sera from KTXRs against B.1.1 (WT), BA.1, and BA.5, we generated green fluorescent protein (GFP)-carrying recombinant SARS-CoV-2 with spike protein of WT, BA.1, or BA.5 by reverse genetics (rWT S-GFP, rBA.1 S-GFP, and rBA.5 S-GFP, respectively).Using these chimeric recombinant viruses, we performed a high-throughput neutralization assay evaluated by visual examination of the GFP signal.We previously compared the 50% neutralizing titer (NT 50 ) calculated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and neutralizing activity titer calculated by visual measurement of GFP expression using a fluorescent microscope.The NT 50 calculated from viral RNA levels in the supernatants of infected cells was well correlated with the titer of neutralizing antibodies calculated by GFP fluorescence intensity 10,11 .The neutralizing antibody titers against rWT S-GFP, rBA.1 S-GFP, and rBA.5 S-GFP in KTXRs and healthy controls after the third dose of the mRNA vaccine were shown in Fig. 2a.The neutralizing antibody titers against all types of viruses in KTXRs were significantly lower than those in healthy controls (p < 0.001).Interestingly, when the virus was mutated, the neutralizing antibody titers in both groups were significantly decreased (Fig. 2b and c).
These results indicate that rBA.1 and rBA.5 S-GFP evade neutralizing antibodies in healthy controls and KTXRs.

Induction of spike-specific T-cell responses in healthy donors and KTXRs
Immunosuppressive drugs, including calcineurin inhibitors, generally suppress T cell activity.We performed flow cytometry analysis to investigate whether spike-specific CD4 + T cell responses in KTXRs were induced by mRNA vaccines.Peripheral blood mononuclear cells (PBMCs) obtained from 22 KTXRs and 15 healthy controls were stimulated with overlapping peptides corresponding to the SARS-CoV-2 spike protein.We defined CD154 + CD4 + T cells expressing IFN-g, TNF, or IL-2 as Th1 cells and those expressing IL4 or IL-13 as Th2 cells (Fig. 3a) [12][13][14] .Spike-specific Th1-cell and Th2-cell frequencies were determined in the stimulated samples by subtracting the background signal observed in the unstimulated samples.The frequency of spike-specific Th1 cells against WT spikes in KTXRs was comparable to that in healthy controls (Fig. 3b, p = 0.7314).Furthermore, mRNA vaccine-induced spike-specific Th1 CD4 T-cell responses against BA.1 and BA.5 were at comparable levels to those of Th1 CD4 T-cell responses against WT in both healthy controls and KTXRs.In contrast, the frequency of spike-specific Th2 cells against WT, BA.1, and BA.5 spikes in KTXRs was higher than that in healthy controls (Fig. 3c, WT: p = 0.0312, BA.1: p = 0.0037, BA.5: p = 0.0474).These results indicate that mRNA vaccines can induce spike-specific CD4 + T-cell responses to omicron sublineages in healthy controls and in KTXRs.
Besides, KTXRs are more susceptible to the induction of Th2-biased CD4 + T cell responses.

Neutralizing activity of sera against rBA.5 S-GFP virus after administering the bivalent Omicron-containing booster vaccine
Characteristics of the KTXRs and healthy controls are presented in Table S1.Among the 19 KTXRs who received the bivalent omicron-containing booster vaccine, three received it after the third dose and 16 after the fourth dose.All six healthy controls received the bivalent omicron-containing booster vaccine after the third dose.We measured the anti-SARS-CoV-2 spike protein IgG and anti-N IgG antibody titers.Furthermore, we performed a neutralizing antibody assay using the rBA.5 S-GFP virus.No KTXRs or healthy controls developed anti-N IgG antibodies after the third and bivalent omicron-containing booster vaccination.This result suggests that the specimens used in this study have never been infected with SARS-CoV-2.The anti-SARS-CoV-2 spike protein IgG antibody titers and neutralizing activity of both groups are shown in Fig. 5. Anti-SARS-CoV-2 spike protein IgG antibody titers were significantly elevated in both groups (Fig. 5a, recipients: p = 0.0155, healthy controls: p = 0.0312).However, antibody titers after the bivalent vaccine in healthy controls were significantly higher than those in KTXRs (Fig. 5a, P = 0.0177).Although the neutralizing activity was significantly elevated in KTXRs (Fig. 5b, p = 0.0312), the neutralizing activity was higher in healthy controls than in KTXRs after the bivalent vaccine (Fig. 5b, P = 0.0094).

monovalent vaccination
Characteristics of the KTXRs and healthy controls are presented in Table S2.No KTXRs or healthy controls developed anti-N IgG antibodies before and after XBB.1.5monovalent vaccination.Anti-SARS-CoV-2 spike protein IgG antibody titers increased significantly after XBB.1.5monovalent vaccination in KTXRs (p = 0.0078).The antibody titers increased in all healthy controls after XBB.1.5monovalent vaccination (Fig. 6a).The neutralizing  All healthy controls showed an increase of the neutralizing activity against each variant.

Discussion
To our knowledge, this is the first study to focus on a single cohort of KTXRs and to investigate the neutralizing antibody titers and T-cell responses against SARS-CoV-2 Omicron BA.5 after a third dose of an mRNA SARS-CoV-2 vaccine, a bivalent omicron-containing booster vaccine, and an XBB.1.5monovalent booster vaccine.Although KTXRs did not gain sufficient immunity against Omicron BA.5 with the third dose of the vaccine, humoral response against mutant SARS-CoV-2 lineages was significantly increased by the bivalent Omicroncontaining booster vaccine and XBB.1.5monovalent vaccine.Although similar studies have reported comprehensive analysis in solid organ transplant recipients (kidney, liver, heart, lung, and multiple organs), there are few studies in a single cohort of KTXRs [19][20][21][22][23] .Since the immunosuppression status in solid organ recipients differs depending on their type of graft organ, analysis of immune responses only in KTXRs is useful for future COVID-19 infection control in KTXRs.Recent studies have reported that the COVID-19 severity of the Omicron variants is less than that of the previous lineages in both the general population and KTXRs [24][25][26][27][28] .However, Solera et al. reported that the rate of hospitalization for COVID-19 in solid organ transplant recipients (SORTs) was 9.8% during the XBB.1.5phase, which was higher than that in general population, suggesting that KTXRs might remain susceptible to current Omicron lineages and develop more severe disease 29,30 .Therefore, the development of an effective prophylaxis strategy for COVID-19 infection is essential in KTXRs.
Vol:.( 1234567890 We showed that the neutralizing titers against Omicron BA.5 in KTXRs were significantly lower than those in healthy controls using the live SARS-CoV-2 virus neutralization assay, indicating that recipients are at a higher risk of infection and that the vaccine is less effective in KTXRs.Werbel et al. reported similar results in a cohort of KTXRs (N = 81) 40 .Furthermore, we showed that neutralizing titers also decreased in KTXRs as the virus showed mutations from WT to BA.1 to BA.5.Tuekprakhon et al. reported that as the mutation evolved, neutralizing titers decreased in the general population after three vaccine doses 41 .Pedersen et al. reported the neutralizing titers after a third dose of vaccination in KTXRs decreased as the virus mutated from B.1.617.2 (delta) to BA.1 to BA.5 42 .These results suggests that KTXRs are at a higher risk of infection as the virus evolves.
T-cell responses have been shown to reduce severe illnesses caused by SARS-CoV-2.KTXRs are at a risk of severe SARS-CoV-2 infection because of their reduced T-cell immunity 43 .A previous study indicated that the third mRNA dose induced a T cell response against WT spike peptides in KTXRs, while cellular immunity toward Omicron BA.1 variant immunogenic peptides was low in both KTXRs and healthy controls 9 .However, no studies have evaluated the T-cell responses to Omicron BA.5 spike peptides.The frequency of spike-specific Th1 cells against BA.5 spike in KTXRs was comparable to that in healthy controls; however, Th2 CD4 + T cell responses were significantly higher in KTXRs.This result is unexpectedly interesting, and several studies describe similar phenomenon.Miyaura et al. reported that steroids inhibit the differentiation of CD4 T cells into Th1 while promoting differentiation into Th2 in vitro 44 .Another study examined the effects of MMF on Th1/Th2 differentiation in a Crohn's disease mouse model 45 .The results indicated that MMF significantly suppressed the mRNA expression of the inflammatory cytokines IFN-g and TNF-a, typically associated with Th1 cells, in this model.However, MMF did not inhibit the expression of IL-10 mRNA, a cytokine associated with Th2 responses.Based on these reports, our findings indicate that immunosuppressants affect the differentiation of CD4 T cells in vitro, however, verification in humans in vivo/ex vivo is lacking.The Th1/Th2 balance of CD4 + T cells is crucial in cellular responses, and skewed distribution towards Th2 cells increases the risk of severe infection and vaccine-associated enhanced respiratory disease 46,47 .Further investigation is needed to evaluate the potential risks of increasing Th2 CD4 + T-cell responses in KTXRs.
The number of spike-specific CD8 + T cells producing IFN-g and TNF was significantly lower in KTXRs than in healthy controls.These results suggest that KTXRs did not acquire sufficient immunity against Omicron BA.5 (neither humoral nor in cellular immunity), even after receiving three vaccine doses 17,46,48 .Furthermore, we evaluated the effects of a bivalent Omicron-containing booster vaccine in KTXRs.Although the current bivalent Omicron-containing booster vaccine produced increased anti-SARS-CoV-2 spike protein IgG and neutralizing antibody titers, these effects were lower in KTXRs than in healthy controls.Fernández-Ruiz et al. reported that the neutralizing antibody responses against the BA.4/5 in SORTs increased after a bivalent Omicron-containing booster vaccine.However, booster-induced cell-mediated immunity remained lower when compared with healthy controls 23 .Yau et al. reported that the neutralizing antibody responses in dialysis patients and KTXRs increased significantly against not only BA.5 but also XBB.1.5after a bivalent Omicron-containing booster vaccine; however, the neutralizing antibodies against XBB.1.5 was 5.8-fold lower than against BA.5 due to high immune evasion 49 .Recent studies have reported the effect of XBB.1.5monovalent vaccines in the general population.The XBB.1.5vaccine strongly increased the anti-SARS-CoV-2 spike protein IgG and elicited potent neutralizing responses against previous and contemporary SARS-CoV-2 lineages, including XBB.1.5,EG.5.1, and BA.2.86 50,51 .We investigated a similar effect of XBB.1.5monovalent vaccines among KTXRs.This is the first study to analyze the anti-SARS-CoV-2 spike protein IgG and neutralizing antibody titers against XBB.1.5,EG.5.1, and BA.2.86 in KTXRs after XBB.1.5monovalent vaccination.These data suggested the best prophylaxis strategy against COVID-19 at this time is to continue to administer updated vaccines in KTXRs.
As an alternative prophylaxis strategy, French researchers have reported the efficacy of tixagevimab/cilgavimab in reducing the morbidity and severity of SARS-CoV-2 in KTXRs who failed to develop a protective humoral response after at least three doses of an mRNA vaccine administration during the Omicron BA.1-BA.2epidemic 52,53 .Recent clinical studies suggested that tixagevimab/cilgavimab improved the neutralization of BA.4/5, but this effect was not observed against BQ.1.1 and XBB.1.5 54,55.Therefore, the effect of monoclonal antibody as prophylaxis for COVID-19 infection may be limited.
The strength of our study is the analysis of neutralizing activity using the live SARS-CoV-2 virus neutralization assay.Furthermore, this is the first study investigating T cell responses against SARS-CoV-2 Omicron BA.5 and the effect of bivalent Omicron-containing booster vaccines and XBB.1.5monovalent booster vaccines exclusively in a kidney recipient cohort.Nonetheless, there are some limitations of our study.First, the time between vaccination and blood collection varied because the blood samples were collected during outpatient visits.Although KTXRs had a shorter term than controls, KTXRs had lower neutralizing activity.Therefore, the timing of sample www.nature.com/scientificreports/collection did not significantly affect the results.Second, healthy controls are significantly younger than KTXRs.
Several studies demonstrated that the antibody responses elicited by the mRNA SARS-CoV-2 vaccine declined with increasing vaccine age 56,57 .However, no statistical correlation between age and antibody titer or neutralizing antibody titers was observed in this cohort (Fig. S1).Therefore, the current study provided rational results, though comparing groups by propensity score matching would be ideal.

Conclusion
The adaptive immunity of KTXRs was not significantly developed after a third dose of an mRNA vaccine, suggesting that recipients are at a higher risk for infection and severe disease than healthy controls.However, humoral response against mutant SARS-CoV-2 lineages significantly increased after the bivalent Omicron-containing booster and XBB.1.5monovalent vaccines.Therefore, it is important for kidney recipients to continue to receive updated vaccines.

Study design and patients
We retrospectively reviewed the data of 403 recipients who underwent kidney transplantation at Hokkaido University between 1996 and 2021.Our inclusion criteria comprised patients who were administered up to three mRNA vaccines after kidney transplantation.Conversely, the following criteria were used to exclude patients from the study: (1) loss of graft function, (2) SARS-CoV-2 infection before initiation of the study and (3) development of anti-N IgG antibody before initiation of the study.Therefore, 73 patients were included in the final analysis.As healthy controls, we reviewed the data from seven kidney donors who underwent nephrectomy at Hokkaido University Hospital and 9 clinical staff members at Hokkaido University.Serum was collected from the recipients and controls after the third dose of the SARS-CoV-2 vaccine.The study protocol was approved by the Institutional Review Board of Hokkaido University Hospital (approval number: 022-0217).This study was conducted following the principles of the Declaration of Helsinki, 1996.Written informed consent was obtained from all the patients and healthy volunteers.

SARS-CoV-2 reverse genetics
Recombinant SARS-CoV-2 was generated using a circular polymerase extension reaction (CPER) as previously described 58 .Briefly, nine DNA fragments encoding the partial genome of SARS-CoV-2 (strain 2019-nCoV/ Japan/TY/WK-521/2020, GISAID ID: EPI_ISL_408667) were amplified with polymerase chain reaction (PCR) using PrimeSTAR GXL DNA polymerase (Takara Bio Inc., Shiga, Japan) and primers sets in the Table S1.A linker fragment encompassing the hepatitis delta virus ribozyme, bovine growth hormone poly A signal, and cytomegalovirus promoter was also prepared using PCR.Ten obtained DNA fragments were mixed and used for CPER 58 .To prepare GFP-expressing replication-competent recombinant SARS-CoV-2, we used fragment 9 (F9), where the sfGFP gene was inserted into the ORF7a frame instead of the authentic F9 58 .The rBA.1 S-GFP virus was provided by K. Sato at Tokyo University 59 and the rBA.5 S-GFP virus was previously generated 11 .To generate rXBB.1.5-GFP,rEG.5.1-GFP, and rBA.2.86-GFP, nine DNA fragments encoding the partial genome of SARS-CoV-2 XBB.1.5(strain TKYmbc30523/2022, GISAID ID: EPI_ISL_16697941), EG.5.1 (strain KU2023071028, GISAID ID: EPI_ISL_18072016), BA.2.86 (strain TKYnat15020, GISAID ID: EPI_ISL_18233521) were amplified with polymerase chain reaction (PCR) using PrimeSTAR GXL DNA polymerase (Takara Bio Inc., Shiga, Japan).sfGFP was inserted into the ORF7a frame of the corresponding each F9 fragment by inverse fusion PCR cloning.Then, these nine fragments and a linker fragment were mixed and used for CPER 58 .The CPER product was transfected into VeroE6/TMPRSS2 cells using TransIT-X2 Dynamic Delivery System (Takara) according to the manufacturer's protocol.At 5-8 days post-transfection, the culture medium was harvested and centrifuged, and the supernatants were collected as seed viruses.Nucleotide sequences were determined using a DNA sequencing service (Fasmac, Kanagawa, Japan), and sequence data were analyzed using ApE.We have confirmed that the recombinant SARS-CoV-2 can infect cells and the virus particles are released from the infected cells to the culture supernatant (Fig. S2).

SARS-CoV-2 preparation and titration
The recombinant SARS-CoV-2 was amplified in VeroE6/TMPRSS2 cells, and the culture supernatants were harvested and stored at − 80℃ until use.Infectious titers in the culture supernatants were determined using www.nature.com/scientificreports/50% tissue culture infective doses (TCID50).The TCID50 was calculated using the Reed-Muench method.The culture supernatants of the cells were inoculated onto VeroE6/TMPRSS2 cells in 96-well plates after serial tenfold dilution with low-glucose DMEM containing 2% FBS and 1 mg/mL G418, and the infectious titers were determined 96 h post-infection.All experiments involving SARS-CoV-2 were performed in biosafety level-3 laboratories following standard biosafety protocols approved by Hokkaido University.

Neutralizing antibody titer assay
In each well, 7.5 × 10 3 VeroE6/TMPRSS2 cells were seeded in 96-well plates and maintained in DMEM (high glucose) containing 10% FBS and 1% PS.The cells were then incubated overnight.Each serum sample was serially diluted three-fold in the culture medium the following day, with a first dilution of 1:10 (final dilution range of 1:21,870).The diluted serum was incubated with 700 TCID50 of the chimeric recombinant SARS-CoV-2 at 37 ℃ in 5% CO 2 for 1 h.Then, the mixture of chimeric recombinant SARS-CoV-2 and serum was added to VeroE6/TMPRSS2 cells in a 96-well plate.After 1 h, the cells were washed with DMEM (high glucose) containing 10% FBS and 1% PS.
In the case of using GFP fluorescence assay, after incubating the plates at 37 °C for 34-36 h (rWT S-GFP, rBA.1 S-GFP, rBA.5 S-GFP, rXBB.1.5-GFP,rEG.5.1-GFP) and 82-84 h (rBA.2.86-GFP), GFP fluorescence was detected using ECLIPSE Ts2 (Nikon, Tokyo, Japan).Subsequently, the luminance of GFP was calculated using Image J. A GFP signal with a luminance value > 150 in one field of view was considered positive.The neutralizing antibody titer was defined as the minimum serum dilution at which the GFP signal was positive.The neutralizing antibody titer of each serum sample was defined as the common logarithm (log10) of the average titer of the neutralizing antibody in triplicate assays.Data were plotted using GraphPad Prism 9 software (GraphPad Software, MA, USA).

Statistical analysis
The association between categorical variables was tested using the Chi-square test.Fisher's exact test was used when appropriate.Statistical significance between the independent and dependent nonparametric data groups was evaluated using the Mann-Whitney U and Wilcoxon signed-rank test, respectively.Dunn's multiple-comparison method was used to test for significant differences among the three groups.Pearson's coefficient was used for the correlation analysis.Statistical significance was set at P < 0.05.GraphPad Prism 8.0.0 (GraphPad Software Inc., San Diego, CA, USA) was used for all analyses.

Figure 1 .
Figure 1.Anti-SARS-CoV-2 spike protein IgG antibody titers in healthy controls and KTXRs.Anti-SARS-CoV-2 spike protein IgG antibody titers in 16 healthy controls and 73 KTXRs.The cut-off value was defined as 1.0 AU/mL.*P < 0.0001, P-values were calculated using the nonparametric Mann-Whitney U test.KTXR, kidney transplant recipient.

Figure 2 .
Figure 2. Neutralizing activity against SARS-CoV-2 variants in healthy controls and KTXRs after the third vaccine dose.(a) The neutralizing antibody titer (log10) against rWT S-GFP, rBA.1 S-GFP, and rBA.5 S-GFP in healthy controls and KTX recipients after the third dose of mRNA vaccine.The chimeric recombinant SARS-CoV-2 and diluted serum mixture was inoculated into VeroE6/TMPRSS2 cells.After post-infection, the expression of GFP in VeroE6/TMPRSS2 cells was observed by fluorescent microscopy and the titer of the neutralizing antibody was calculated.***P < 0.0001, P-values were calculated using the nonparametric Mann-Whitney U test.(b)The neutralizing antibody titer (log10) against rWT S-GFP, rBA.1 S-GFP, and rBA.5 S-GFP in healthy controls.This data was extracted from (a). *P < 0.05, Dunn's multiple-comparison method was used to test for significant differences among the three groups.(c) The neutralizing antibody titer (log10) against rWT S-GFP, rBA.1 S-GFP, and rBA.5 S-GFP in KTXRs.This data was extracted from (a). **P < 0.01, ***P < 0.0001, Dunn's multiple-comparison method was used to test for significant differences among the three groups.KTXR, kidney transplant recipient.

Figure 5 .
Figure 5. Anti-SARS-CoV-2 spike protein IgG antibody titers and the neutralizing activity titers against rBA.5 S-GFP virus in healthy controls and KTXRs after a third vaccine dose and bivalent omicron-containing booster vaccine.(a) Anti-SARS-CoV-2 spike protein IgG antibody titers in healthy controls and KTXRs after a third vaccine dose and bivalent omicron-containing booster vaccine.*P < 0.05, P-values were calculated using the Wilcoxon signed-rank test.(b) The neutralizing antibody titer (log10) against rBA.5 S-GFP virus in healthy controls and KTXRs after a third vaccine dose and bivalent omicron-containing booster vaccine.The mixture of rBA.5 S-GFP virus and diluted serum of healthy controls or KTXRs was inoculated into VeroE6/TMPRSS2 cells.After post-infection, the expression of GFP in VeroE6/TMPRSS2 cells was observed by fluorescent microscopy, and the titer of the neutralizing antibody was calculated.*P < 0.05, **P < 0.01, and P-values were calculated using the Wilcoxon signed-rank test.KTXR, kidney transplant recipient.

Table 2 .
Differences in subject characteristics between responders versus non-responders among kidney transplant recipients.Data are presented as frequency (%) or median (IQR).P values are calculated using the nonparametric Mann-Whitney U test and chi-square test.TAC tacrolimus, MPA AUC mycophenolic acid area under the curve.a TAC Trough levels are available in 37 responders and 22 non-responders.b MPA-AUC levels are available in 43 responders and 24 non-responders.