Objectives: Although the effectiveness of vaccines in protecting the host from infection has been proven, few surveys have been conducted on changes in antibody levels after vaccination of kidney transplant recipients in Japan.
Materials and Methods: We analyzed serological responses in kidney transplant recipients after BNT162B2 COVID-19 mRNA vaccine with the use of a reagent capable of simultaneously specifying the antibody response to 5 proteins: a full-spike protein (extracellular domain), 3 individual domains of the spike protein (S1, S2, and receptor-binding domain), and nucleocapsid. The analysis involved 111 patients who had follow-up over 1 month after having received the second of 2 coronavirus vaccines after kidney transplant.
Results: Antibodies were detected in 46 of 111 patients (41%). The antibody-positive rate in the kidney transplant group tended to be lower than that in the healthy control group, which showed an antibody-positive rate of 100%. When the antibody-positive rate was analyzed by the type of immunosuppressor used, the rate was 36% (37/100) for patients who used tacrolimus at the time of vaccination and 90% (9/10) for patients who used cyclosporine. Patients administered CD20 antibody (rituximab) before and/or after transplant showed a lower production of antibodies, which was supported by a smaller number of CD19- and CD20-positive cells in the peripheral blood as well as a shorter period between rituximab administration and vaccination. The percentage of responding viral fragments varied greatly among individual patients and showed no uniformity in the kidney transplant group, whereas the mean fluorescence intensity of individual fragments showed a certain tendency in the control group.
Conclusions: The appropriate timing of vaccination should be considered in transplant recipients who use tacrolimus-mycophenolate mofetil combination and rituximab as these drugs are deeply related to a lower antibody response to SARS-CoV-2 BNT162b2 vaccination.
Key words : Antibody titer, BNT162B2, COVID-19, COVID-19 mRNA vaccination, Immunosuppressive medicine
Since the first report on coronavirus disease 2019 (COVID-19) in January 2020, diabetes mellitus, hypertension, and cardiovascular disease have been listed as risk factors for COVID-19. Kidney disease has also been added to the risk factors for this disease, and dialysis, organ transplant, and chronic kidney disease (estimated glomerular filtration rate <30 mL/min/1.73 m2) were considered as factors involving the highest risk of death from COVID-19.1
The mean incubation period for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is about 5 days, and clinical features upon infection with this virus include pyrexia, cough, dyspnea, and malaise. COVID-19 is a systemic disease and has been reported to be often accompanied not only by endothelial cell damage but also by gastrointestinal symptoms, hepatopathy, cardiac lesions, encephalitis, atypical stroke, acute kidney injury, and coagulation disorder.2-7 COVID-19 has been shown to more frequently present with a severe course in patients after organ transplant, along with use of immuno-suppressors, versus other patients in general. That is, the risk for a severe course of COVID-19 is higher in organ transplant recipients. It has also been reported that the antibody-positive rate after vaccination is low in organ transplant recipients.8,9
In Japan, vaccination against COVID-19 was started in February 2021, which also gave priority to organ transplant recipients. However, because 93.5% of the phase 3 clinical trials on this vaccine, reported to date, have excluded patients who use immuno-suppressives, there are no detailed data that have clearly demonstrated efficacy of this vaccine in organ transplant recipients.10 Although the effectiveness of vaccination in protecting the host from infection has been proven, few surveys have been conducted on changes in antibody levels following vaccination of Japanese people. Under such circumstances, we recently conducted simultaneous measurements of antibody responses to the 5 proteins constituting the SARS-CoV-2 in blood before and after vaccination in patients who received 2 doses of the BNT162B2 (mRNA) vaccine, which has been approved and available for use in kidney transplant recipients.
Materials and Methods
The study involved 111 patients who had kidney transplant at the Tokyo Women’s Medical University Hospital and who had subsequently received COVID-19 vaccination twice (61 males and 50 females, mean age of 53.7 ± 14.7 years, mean length of time after transplant of 6.4 ± 3.2 years) (Table 1). Kidney transplant procedures were performed with organs from living sixth-degree relatives living related donors according to Japanese law. Ten health care staff members at our facility (5 males and 5 females) served as healthy controls. The study was carried out retrospectively in accordance with the Declaration of Helsinki after approval by the ethics committee (approval no. 2134) and acquisition of consent from each participant.
To assess the status of immunoglobulin G (IgG) antibody production in transplant recipients, antibodies to the following 5 proteins constituting the SARS-CoV-2 were measured: full-spike protein (extracellular domain [ECD]), spike proteins (S1, S2, and receptor-binding domain [RBD]), and nucleocapsid. Proteins were measured simultaneously in patient sera collected immediately before the first COVID-19 vaccine and 1 month after the second vaccine, with measurements made using LABScreen COVID Plus (One Lambda Inc.) and LABScan3D. With the yielded data, analyses were conducted on antibody-positive rate, percentage of responding viral fragments, and antibody-positive rate by immunosuppressants.
SARS-CoV-2 antibody measurement
Each serum sample was processed in a 96-well enzyme-linked immunosorbent assay tray. The serum (2 μ L) was combined with 1× phosphate-buffered saline (17 μL) and 0.0 2 M EDTA (1 μL), followed by addition of LABScreen COVID Plus beads (5 μL). The mixture was incubated for 30 minutes in the dark at temperature of 20 °C to 25 °C with gentle agitation. The 1× washing buffer (150 μL) was added to each well of the plate, followed by centrifugation at 1300g for 5 minutes. After the washing buffer was removed from each well of the plate, 1× washing buffer (200 μL) was added to each well, followed by 5-minute centrifugation at 1300g (washing done twice). Next, 100× phycoerythrin antihuman IgG (100 μL) was added to each well, followed by 30-minute incubation in the dark at 20 °C to 25 °C, again with gentle agitation. After centrifugation for a further 5 minutes at 1300g, supernatant was removed from each well (washing done twice), 1× phosphate-buffered saline (80 μL) was added to each well, and the LABScan or LABScan3D system was used for measurements. The data in the form of trimmed mean were analyzed with HLA Fusion 4.5 software.
Data are shown as means ± SD or as medians. We used t test for analysis of continuous variables possible to assume a normal distribution. P < .05 (2-tailed) was regarded as statistically significant.
As shown in Table 1, there was no difference between the antibody-positive group and the antibody-negative group in terms of physical characteristics or age. There were significant differences between the 2 groups in terms of days from transplant to vaccination, blood group incompatibility, the presence/absence of preoperative double filtration plasmapheresis (DFPP), preoperative rituximab administration, and the chronic antibody type rejection. The number of days from transplant to vaccination was longer in the antibody-positive group (4160 ± 3362 days) than in the antibody-negative group (2655 ± 2307 days). As shown in Table 2, there were significant differences in terms of estimated glomerular filtration rate and blood trough levels of tacrolimus and mycophenolate mofetil (MMF) at the time of vaccination between the antibody-negative patients and the antibody-positive patients. Blood levels of mizoribine and azathioprine are not presented in this report because their measurement during this study was incomplete. Cyclosporine was not included in statistical analysis because the number of cases was too small to allow valid comparison.
Antibody-positive rate according to viral fragments
All participants in the transplant group and in the healthy control group tested negative for SARS-CoV-2 IgG before vaccination. As shown in Figure 1, the antibody-positive rate after vaccination was 42% (46/111) in the transplant group, which included 30 positive male and 16 positive female patients with a mean age of 53.1 ± 14.2 years. Of all antibody-positive transplant recipients, 65% were male. Antibodies to the following fragments were detected: 11% ECD alone; 16% ECD, S1, RBD, and S2; 8% ECD, S1, and RBD; 3% ECD and S2; 1% ECD and S1; 1% ECD, S1, and S2; 1% ECD and RBD; and 1% S2 alone. Thus, antibody-positive patients tended to have antibodies to 1 or more of all fragments of the virus other than nucleocapsid.
Mean fluorescence intensity of viral fragments in transplant recipients and healthy controls
Figure 2 graphically represents the distribution of responding viral fragments in the 46 transplant recipients who tested positive for antibodies. In analysis of the mean fluorescence intensity (MFI) of each fragment in transplant recipients, ECD recorded the highest MFI (26 176 on average), followed by RBD (13 996), S1 (11 421), and S2 (6825). In the healthy control group, MFI was 27 067 on average for ECD, 28 830 for RBD, 26 089 for S1, and 19 737 for S2. The MFI for ECD differed little between the transplant group and the healthy control group, whereas the MFI for RBD, S1, and S2 was significantly higher in the healthy control group. The distribution of MFI of individual fragments differed little among healthy controls, with each fragment showing MFI near the average level. In the transplant group, however, the MFI varied greatly among individual fragments and among individual patients. The IgG antibody to N protein is not produced after mRNA vaccine injection. Therefore, this antibody was not included in the statistical analysis.
Percentage of viral fragments in transplant recipients and healthy controls
Figure 3 shows the production rate among antibody-positive participants. In analysis of individual fragments in the transplant group, the mean production rate was 47.1% for ECD, 18.2% for S1, 19.9% for RBD, and 11.4% for S2. In the healthy control group, the mean reaction rate was 26.5% for ECD, 25.5% for S1, 28.3% for RBD, and 19.2% for S2. In the transplant group, the production rate of ECD was high and tended to vary greatly among individual patients. In the healthy control group, on the other hand, the antibody-positive rate was uniform for all fragments, with a tendency of small variation among individual subjects.
Of the 111 patients, 100 used oral tacrolimus and 10 used oral cyclosporine. Antibody was positive in 36 of the 100 patients who used tacrolimus (36%) and 9 of the 10 patients who used cyclosporine (90%). One patient who used neither tacrolimus nor cyclosporine was antibody positive.
Of the 111 patients, 89 used oral MMF and 21 used oral mizoribine. Antibody was positive in 28 of the 89 patients who used MMF (31%) and 16 of the 21 patients who used mizoribine (76%). Thus, the antibody-positive rate was higher in patients who used mizoribine than in patients who used MMF. Of the 111 patients, 14 used everolimus and 6 of these 14 patients (43%) were antibody positive. One of these 6 patients had not used any calcineurin inhibitor.
The antibody-positive rate was analyzed in 77 patients who used rituximab before or after transplant. The antibody was positive in 23 of the 77 patients (30%), with the antibody-positive rate tending to be higher among male than among female transplant recipients. There was a significant intergroup difference in the days from rituximab administration to vaccination. The number of days from the start of rituximab administration to vaccination was significantly longer in the antibody-positive group (2419 ± 1440 days) than in the antibody-negative group (2037 ± 1585 days). As shown in Table 3, white blood cell count at the time of vaccination, especially CD19- and CD20-positive cells, which should be targeted by rituximab, was significantly smaller in the antibody-positive group than in the antibody-negative group (CD19: 68.1 ± 69/μL in the antibody-positive group vs 51.0 ± 72/μL in the antibody-negative group; CD20: 72.3 ± 69/μL in the antibody-positive group vs 50.0 ± 67/μL in antibody-negative group). Blood tacrolimus and MMF levels were lower in the antibody-positive group than in the antibody-negative group, although these differences were not statistically significant.
Age, sex, presence/absence of underlying disease (eg, hypertension, diabetes mellitus), and laboratory data (eg, D-dimer, C-reactive protein) are now considered as useful in predicting the severity of COVID-19 in individual cases, but these variables do not always have a sufficient predictive capability. With the expectation that SARS-CoV-2 antibody might be useful in reducing the risk for SARS-CoV-2 infection, we analyzed the data on COVID-19 in relation to the presence and absence of SARS-CoV-2 antibody. According to a report on antibody acquisition following vaccination in healthy individuals, characteristics associated with higher potential of antibody formation after vaccination include past history of COVID-19 infection, female sex, and use of oral dose antiallergy drugs, whereas features associated with lower potential of antibody formation include use of oral dose immunosup-pressors, use of oral dose corticosteroid, advanced age, and frequent alcohol consumption.11
The World Health Organization does not recommend the use of antibody tests independently as a means of making diagnosis. In Japan, multiple reagents for SARS-CoV-2 antibody measurement (for research purposes only) are available, including the immunochromatography kit for simplified quick detection and kits for enzyme-linked immunosorbent assay, electrochemiluminescence immunoassay, and chemiluminescent microparticle immunoassay, although these have shortcomings of large variance in the sensitivity and specificity. Most of these kits are designed to judge qualitatively (either “positive” or “negative”) or semiquantitatively. These kits have not yet been approved as in vitro diagnostics in Japan and hence are currently not covered by the national health insurance. Taking note of the report that the antibody acquisition rate was low in patients who had undergone organ transplant,12 we conducted this analysis of antibody acquisition in patients who received COVID-19 vaccination after organ transplant using a reagent capable of simultaneously identifying and quantifying the responses to 5 proteins (ECD, S1, S2, RBD, and nucleocapsid), constituting the SARS-CoV-2.
Although structural homology has been noted between SAR-CoV-2 and other coronaviruses,13 it remains unknown to which extent there occurs cross-reaction between SARS-CoV and the Middle East respiratory syndrome (MERS) coronavirus. The LABScreen COVID Plus, which was used in the present study, contains the SARS-CoV-2 spike, RBD, nucleocapsid, spike S1, seasonal coronavirus, MERS antigen, and SARS antigen. Because of these features, this reagent has been reported to have a low potential of cross-reaction as one of the striking characteristics. This reagent has been reported to be high in specificity (98.6%) and sensitivity (100%).14 SARS-CoV-2 is an enveloped virus with single-stranded RNA encoding 4 major structural proteins (spike, envelope, membrane, and nucleocapsid) and more than 12 nonstructural proteins. During the initial stage of SARS-CoV-2 invasion into human cells, the spike protein on the viral surface adsorbs, through binding, the cell surface angiotensin-converting enzyme 2, to allow invasion and infection.15
The spike protein, which is included in the test menu, is the most important large-size protein of coronavirus. It is composed of a trimmer and can be roughly divided into S1 (N-terminal domain, RBD) and S2 regions. S1, primarily composed of RBD, plays the role of recognizing the receptor on the cell surface, whereas S2 includes basic elements needed for membrane fusion.16,17
S protein is the target protein whose antibody is produced primarily by vaccines. In the antibody test after vaccination, the IgG antibody to S protein will become positive. The mRNA vaccines currently used in Japan are produced by utilization of the genes capable of artificially producing S protein alone in human cells. It is known that, if the immune system reacts intensely to the artificial S protein formed within human cells, the antibody to SARS-CoV-2 infection can be acquired. Nucleocapsid protein (N protein) is a highly immunogenic phosphoprotein. Many kits for the coronavirus antigen test are designed to detect this protein. However, N protein is a part of the coronavirus not related to mRNA vaccines, and the IgG antibody to N protein is not formed by mRNA vaccine injection. The RBD is a major target of neutralizing antibodies. Inhibition of its binding to angiotensin-converting enzyme 2 (a receptor on human cells) is an important function of neutralizing antibodies in suppression of SARS-CoV-2 infection.18,19 The reagent used in the present study is capable of simultaneously measuring these important proteins.
In the present study, we observed no marked or significant differences in pretransplant renal func-tioning level between the antibody-positive group and the antibody-negative group. On the other hand, significant intergroup differences were noted in the number of days from transplant to vaccination, donor-recipient blood group incompatibility, presence/absence of DFPP, use of rituximab, and chronic antibody-related rejection. We considered that the significant differences observed are attributable to the fact that we make it a rule at our department to perform plasma exchange/DFPP and rituximab treatment preoperatively for the purpose of desensitization. As shown in Table 1, the percentage of ABO-incompatible patients was higher in the antibody-negative group (32%) than in the antibody-positive group (17%).
When the antibody-positive rate was analyzed by preoperative use of rituximab, more patients were preoperatively using rituximab in the antibody-negative group (77%) than in the antibody-positive group (48%). The significantly higher incidence of chronic antibody-related rejection in the antibody-negative group can probably be explained by the use of rituximab for treatment of rejection. When the influence of rituximab on antibody formation was evaluated in 77 patients with history of rituximab use (Table 3), 23 of the 77 patients (30%) were antibody-positive. The number of days from rituximab administration to vaccination differed significantly between the antibody-positive group and the antibody-negative group. Antibody formation was not affected by the rituximab dose, blood tacrolimus trough level, and blood MMF trough level at the time of rituximab administration, but it was affected greatly by the presence or absence of rituximab use. The influence of rituximab on antibody acquisition is unlikely to be ignored, in view of findings that the antibody-positive rate was lower in those who used rituximab than in the general population20 and that those who used rituximab responded poorly to vaccination against influenza.21
Significant differences between the antibody-positive group and the antibody-negative group were also noted in terms of blood tacrolimus and MMF trough levels at the time of vaccination (Table 2). However, among the patients with history of rituximab administration, there was no significant difference in blood tacrolimus or MMF trough level between the antibody-positive group and the antibody-negative group (Table 3). These findings suggest that the B-cell-targeting inhibitory action due to rituximab can overcome the influence of the blood tacrolimus and MMF trough levels on antibody formation after vaccination.
The percentage of patients who acquired SARS-CoV-2 antibody about 1 month after the second vaccination was 41%. This result was close to the finding reported by Kamar and colleagues.9 The antibody acquisition rate was higher according to some reports from other facilities and lower according to other reports.22-24 Thus, the antibody-positive rate may differ among medical facilities. One major factor possibly explaining such variance among medical facilities is the difference in the type or dose level of immunosuppressors used at the time of vaccination or the difference in the combination of immunosuppressors used.
In analysis of the distribution of fragments to which the antibody was formed, there was a trend (common for the posttransplant antibody-positive group and the healthy control group) that S2 had lower immunogenicity than the other fragments. The percentage of each fragment to which antibody was formed ranged from 20% to 28% in the healthy control group, whereas the range was wider (from 11% to 47%) in the posttransplant antibody-positive group. The fragment S1 is a major element of vaccination and has been reported to be specific to SARS-CoV-2.25 According to our data, S1 antibody was positive in 31 of 46 patients (67%), although the MFI varied among individual patients. We observed MFI to be higher for the fragment ECD than for S1 and RBD, and the antibody to ECD was positive in 45 of 46 patients (95%). These differences in MFI seem to reflect (1) the high reactivity of ECD, which extends widely from the N-terminal domain to RBD and, beyond the site of cleavage, to the heat repeat 2 near the membrane-perforating area; and (2) differences in the number of antigen determinants possessed by spike proteins. The antigen determinant plays a significant role in protection against viruses, and it is necessary for us to further understand the specificity of RBD and ECD in this context.
In analyses of the pattern and frequency of responses, there was no difference in the response of each fragment among individual participants from the control group. In the posttransplant antibody-positive group, on the other hand, the response of each fragment varied greatly among individual patients. Most patients manifested strong responses to ECD, whereas there was no uniform trend in the responses to the other fragments. Although the immune responses to viral infection have not been fully clarified, this result suggests that the antibody responses to SARS-CoV-2 are not uniform in all patients because of possible interindividual differences in underlying disease, B-cell repertoire, and T-cell responses due to the various immunosuppressive medications.
A limitation of this study was the lack of observation of T-cell responses. It is necessary to evaluate to which extent tacrolimus, MMF, and other immunosuppressive medications affect T-cell responses, antigen presentation, and, eventually, antibody formation. We cannot say definitely how the data obtained from the testing method that we used will affect protection against infection. To date, however, coronavirus infection has not developed in any of the patients rated as antibody-positive by our testing method. Similar to the findings reported by Khoury and associates,26 it seems highly probable that the capability of protection against infection can be predicted from the intensity of the antibody. For patients rated as antibody-negative, careful measures for protection from possible infection will be needed. In any event, the results of the present study suggested that transplant recipients have less potential of SARS-CoV-2 antibody formation after vaccination. For antibody-positive transplant recipients, we aim to monitor the extent to which the neutralizing antibody continues to manifest the neutralizing activity. In addition, careful follow-up of these patients for rejection is needed, although the possibility for the mRNA vaccine to induce rejection seems to be low.
Serological antibody monitoring is useful not only as a means of checking the presence/absence of antibody formation after vaccination but also as a screening test for identification of symptom-free infected patients. Antibody measurement with the reagent that we used in this study is considered to have sufficient clinical significance in assessing the extent of immune responses in patients who are positive for infection as well as in determining the optimum interval, dose level, frequency, and timing of additional vaccination. The appropriate timing of vaccination needs consideration in transplant recipients who use immunosuppressive medications such as tacrolimus-MMF combination and rituximab because these drugs are deeply related to a lower antibody response to the SARS-CoV-2 BNT162b2 vaccine, as demonstrated in this study.
Volume : 20
Issue : 5
Pages : 463 - 471
DOI : 10.6002/ect.2022.0020
From the 1Department of Urology and the 2Department of Transplant Medicine, Women’s Medical University Hospital; and the 3Yochomachi Clinic Follow-up Center for Kidney Transplant Recipients, Tokyo, Japan
Acknowledgements: The authors have not received any funding or grants in support of the presented research or for the preparation of this work and have no declarations of potential conflicts of interest.
Corresponding author: Hideki Ishida, Department of Transplant Medicine, Tokyo Women’s Medical University Hospital, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
Phone: +81 3 3353-8111
E-mail: firstname.lastname@example.org, email@example.com
Table 1. Background Characteristics
Table 2. Characteristics of Antibody-Positive and Antibody-Negative Groups at Time of Vaccination
Figure 1. Antibody-Positive Rate According to Viral Fragments
Figure 2. Mean Fluorescence Intensity of Viral Fragments in Transplant Recipients (A) and Healthy Controls (B)
Figure 3. Percentage of Viral Fragments in Transplant Recipients (A) and Healthy Controls (B)
Figure 4. Antibody-Positive Rate According to Calcineurin Inhibitors)
Table 3. SARS CoV-2 Antibody Positivity in Patients Administered Rituximab Before and/or After Transplant (N = 77)