Antibody titer after administration of mRNA‐based vaccine against severe acute respiratory syndrome coronavirus 2 in liver transplant recipients

Abstract Introduction The mRNA‐based vaccine was released as a COVID‐19 prophylactic; however, its efficacy in organ transplant recipients is unknown. This study aimed to clarify this in liver transplant recipients. Methods Herein, liver transplant recipients from two hospitals who received vaccines were included. Immunoglobulin‐G antibodies against the spike and nucleocapsid proteins were measured chronologically after the second, third, and fourth vaccine doses. Results Antibody levels in 125 liver transplant recipients and 20 healthy volunteers were analyzed. The median age at transplant was 35 (interquartile range 1, 53) years, and the period between transplant and the first dose was 15.2 ± 7.7 years. After the second and third doses, 89.1% and 100% of recipients displayed a positive humoral response, respectively. Anti‐spike antibodies after the second dose were significantly reduced at 3 and 6 months, compared to that at 1 month (26.0 [5.4, 59.5], 14.7 [6.5, 31.4] vs. 59.7 [18.3, 164.0] AU/mL, respectively, p < 0.0001). However, a booster vaccine significantly elevated anti‐spike antibodies in LT recipients (p < 0.0001) as well as in healthy controls (p < 0.0001). Additionally, the decay rate was comparable between the transplant recipients and controls (2.1 [0.8, 4.5] vs. 2.7 [1.1, 4.1] AU/mL/day, p = 0.9359). Only 4.0% of vaccinated transplant recipients were positive for anti‐nucleocapsid antibodies. Conclusion Liver transplant recipients can acquire immunity similar to that of healthy people through vaccination against SARS‐CoV‐2. The antibody decay rate is the same, and booster vaccinations should be administered similarly to that in healthy individuals.


| INTRODUC TI ON
The new coronavirus disease 2019 (COVID-19) caused a pandemic in 2019. An mRNA-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was rapidly released for prophylaxis, and its high efficacy has been reported. 1 The vaccine reportedly had insufficient efficacy in preventing COVID-19 in organ transplant recipients. [2][3][4][5] The Pfizer-BioNTech BNT162b2 mRNAbased vaccine elicited more inferior humoral immunity in liver transplant (LT) recipients than that in healthy volunteers, 3

and induced
lower specific T cell responses in solid organ transplant recipients than that in immunocompetent subjects. 5 Nevertheless, many countries in Europe, the United States, and Japan recommend vaccination in LT recipients. 6,7 However, the vaccine efficacy is still unknown in organ transplant recipients.
In addition to mRNA-based vaccines, several types of vaccines, such as viral vector-based vaccines, have recently become available.
The Pfizer-BioNTech BNT162b2 was initially developed based on a nucleoside-modified mRNA vector vaccine encoding the pre-fusion spike glycoprotein of SARS-CoV-2 and approved worldwide. 1 The mRNA-based vaccines have been reported to have higher efficacy compared to other vaccines 8 and are now more commonly used in Japan.
SARS-CoV-2 has four key structural proteins: the spike (S-), matrix, envelope, and nucleocapsid (N-) proteins. 9 Immunoglobulin-G (IgG), developed against the S-protein, is believed to be a neutralizing antibody (Ab) immune response and is currently the primary target for SARS-CoV-2 vaccine trials. In many previous studies, the humoral response of the vaccine was evaluated by measuring Ab titers against the S-protein, instead of following up on subsequent infection rates. 3,4 On the other hand, IgG developed against the Nprotein is thought to reflect past SARS-CoV-2 infections. 9 Here, we aimed to clarify the efficacy of SARS-CoV-2 mRNAbased vaccines by measuring serum Ab titers against the S-and Nproteins after vaccination in LT recipients.

| PATIENTS AND ME THODS
This study was designed as a prospective cohort from two Japanese hospitals (Shinshu University Hospital in Matsumoto and Matsunami General Hospital in Gifu). The inclusion criteria were LT recipients who had received two or more doses of an mRNA-based vaccine against SARS-CoV-2. A SARS-CoV-2 infection history before vaccination was not considered, and patients who received the vaccine before LT were excluded. Patient data, including age at LT and first vaccination, sex, type of donor and graft, relationship to donor, body mass index, immunosuppression regimen at first vaccination, duration between LT and vaccinations, and history of SARS-CoV-2 infection, were collected from their medical records and a questionnaire.
IgG Ab titers against the S-and N-proteins were measured at 1, 3, 6, 9, and 12 months after the second dose and at 1, 3, and 6 months after the third dose through outpatient visits to the hospitals. Abs levels were also measured 1 month before and 1 and 6 months after the third dose in 20 healthy volunteers (medical staff) who received the vaccine as controls.
Collected blood samples were immediately separated into serum by centrifugation at 3500 rotations per minute for 6 min and stored at −20°C for 1 to 2 weeks. The samples were sent to a laboratory (SRL Inc.) where SARS-CoV-2 antibodies against the S-and Nproteins were measured by chemiluminescent enzyme immunoassay using SARS-CoV-2 S-IgG measurement reagent (FUJIREBIO) and electrochemiluminescence immunoassay using Elecsys Anti-SARS-CoV-2 (Rosche Diagnostics K.K.), respectively. Titers <1.0 arbitrary units (AU)/mL were considered negative according to the manufacturer's instructions.

| Vaccination
The mRNA-based vaccines (Pfizer-BioNTech BNT162b2 or Moderna mRNA-1273) were administered according to Japanese government protocol: 3-week intervals between the first and second doses, followed by the third dose at more than 6 months. We recommended that LT recipients take the first dose of the vaccine more than 6 months after the operation.

| Calculating decay rates
Based on a previous report, 10 the decay rate of Ab titers was calculated by dividing the difference between maximum and minimum titers by interval days in two points after the third and before the fourth vaccine dose.

| Ethics declarations
The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Shinshu University (registration number: 5265). Written informed consent was obtained from all the patients and/or their families before inclusion. Continuous variables were transformed into categorical variables through cutoff values analyzed using receiver operating characteristic curve analysis for patient age at LT.
All statistical tests were two-sided and performed using JMP software, version 16 (SAS Institute); statistical significance was set at p < 0.05.

| RE SULTS
Out of the initial 126 LT recipients enrolled in this study, one was excluded due to having the first vaccination before the transplant.
The reasons for the LT varied, with 64, 49, 11, and 1 being due to biliary, hepatocellular disease, metabolic liver disease, and multiple liver cysts, respectively. The median age at LT was 35 (interquartile range 1, 53) years, with 91.2% receiving the organ from a living donor. The first vaccine dose was taken at 15.2 ± 7.7 years after LT, at a median age of 48 (interquartile range 25, 67) years, with 96% of LT recipients receiving the Pfizer-BioNTech BNT162b2 (Table 1) Among the 258 samples from 116 LT recipients, an inverse correlation was noted between anti-S Ab titers and interval days after the second dose (r = −0.497503, p < 0.001, Figure 1A). Ab titers were plotted for each LT recipient at 1, 3, 6, 9, and 12 months between the second and third doses, and were found to decrease over time in most cases (n = 106, 91.4%, Figure 1B). The median anti-S Abs reached 59.7 (18.3, 164.0) AU/mL 1 month after the second dose, followed by a significant decrease to 26.0 (5.4, 59.5), 14.7 (6.5, 31.5), and 11.7 (7.2, 17.0) AU/mL at 3, 6, and 9 months after the second dose, respectively, compared to those at 1 month after (p < 0.0001).
Abs reached a positive humoral response (≥1.0 Au/mL) in 89.1% of recipients whose anti-S Abs could be measured after the second dose and before the third dose. This anti-S Ab positive response was maintained in 86.5% of recipients just before the third dose.
Factors influencing a positive humoral response to anti-S Abs after the second dose were explored by regression analysis. Age less than 47 years at LT, a duration of more than 2 years between LT and the first vaccination, and a single immunosuppression regimen were significantly associated with the positivity of anti-S Abs by univariate analysis. Among these factors, an age <47 years at LT and a duration >2 years between LT and the first vaccination were independent predictors of anti-S Ab positivity by multivariate analysis ( Table 2).
Anti-S Abs that were reduced six or more months after the second dose significantly increased 1 month after the third dose in the controls (n = 20; p < 0.0001, Figure 2A) as well as in 93 LT recipients (p < 0.0001, Figure 2B). In addition, the median titers were comparable between these recipients and the controls, both before and Anti-S Abs also decreased over time after the second and third doses ( Figure 2B). When the decay rate was calculated by dividing TA B L E 1 Patient characteristics in liver transplant recipients. Anti-S Abs were positive in 100% of the 93 LT recipients after the third dose. The peak Ab titer was widely different in each patient, ranging from a minimum of 1 to a maximum of 8600 AU/mL ( Figure 2B). Peak titers plotted after the second, third, and fourth   Anti-S Abs that were reduced before the third dose, significantly increased one month after the third dose (**p < 0.0001, B) as well as in 20 controls (*p < 0.0001, A), as determined using the paired t-test. Anti-S Abs decreased over time after the third dose in both healthy controls and LT recipients.

F I G U R E 3
The peak anti-spike protein antibody (anti-S Ab) titers after the second, third, and fourth vaccine doses are plotted for 95 out of 125 liver transplant (LT) recipients whose Ab titers were measured at more than two points. The number of cases at each time point is shown below the graph. Abs levels were increased sequentially after each dose.
reagents were administered systemically but were not effective. Case

| DISCUSS ION
The mRNA-based vaccine against SARS-CoV-2 provided immunity to 89.1% and 100% of LT recipients after receiving the second and third doses, respectively. In addition, the titer was comparable to that of the healthy controls after the third dose. The humoral response rate in LT recipients post-vaccination varied from 47.5% to 72%. 3,4,11 Comparing Ab titers with previous reports is meaningless because there are many differences, including in the methods for measuring Ab titers and sample timing. 12 Lower positive serology values and lower Ab titers have been reported in LT and kidney transplant recipients after the second vaccine dose, compared to those in healthy people. 3,13 Here, positive humoral response rates after the second and third doses were higher than those in previous reports, and the Ab titers after the third dose were comparable to those of healthy controls.
Immunosuppression may have a negative impact on vaccine immunization. 14 Although tacrolimus, 15 MMF, 16 and steroids 3 were reported to inhibit the SARS-CoV-2 vaccine immunization of organ recipients, these reagents or multiple immunosuppression regimens that include them were not associated with the humoral response of anti-S Ab after the second vaccine dose in this study. A younger age (<47 years) at LT and longer duration between LT and vaccination (>2 years) were independent predictive factors for positive humoral response. Older age and shorter interval after LT were risk factors associated with non-response to vaccination in LT recipients. 17 The majority of recipients here had a longer duration between LT and F I G U R E 4 Enhanced abdominal computed tomography. Case 1 (A-D) and Case 2 (E,F). The portal vein was patent 1 year before the third vaccine dose in Case 1 (A and B) and before the fourth dose in Case 2 (E). However, the portal and superior mesenteric veins were completely obstructed by thrombosis 1 month after the third dose in Case 1 (C and D, arrows). Partial thrombosis in the portal vein was observed 2 weeks after the fourth dose in Case 2 (F, arrows).
the first vaccination, with an average interval of 15.2 years. They were already on very small doses of immunosuppressants and possibly similar to healthy people in terms of immunity; therefore, the positive humoral response rate might have been higher here than in previous reports.
Here, ~10% of recipients converted from negative-to positive-Ab status after the third dose. It is unclear how much Ab titer is needed to prevent SARS-CoV-2 infection. In addition, the infectionpreventing effects of Abs differ depending on the viral strain, and different cut-off values have been set for judging negatives and positives of between 0.8 and 50 AU/mL. 3,9,12,15 The Ab neutralization level to protect against symptomatic infection is 20.2%, which corresponds to 54 IU/mL of titer. 18 Here, the cut-off value was set at

| CON CLUS ION
LT recipients can acquire immunity similar to that of healthy individuals using the SARS-CoV-2 vaccine. The Ab decay rate is the same as that in healthy people, and booster vaccinations should be performed in the same way. Based on our results, we recommend vaccination against SARS-CoV-2 in LT recipients; however, vaccination-associated portal vein thrombosis warrants caution, especially in patients with a predisposition to thrombosis.

AUTH O R CO NTR I B UTI O N S
AM was a major contributor in analyzing the data, writing the manuscript, and producing the figures and tables. MM conceived this study and YO, AS, KK, YM, TN, and KY provided suggestions. YO and YM also participated in obtaining informed consent from patients. YS was a major contributor in organizing this study and in revising the manuscript. All authors have read and approved the final manuscript.

ACK N OWLED G M ENTS
We would like to thank Tomomi Eguchi, MT, for handling the blood samples at Shinshu University Hospital, and Tomomi Yoshikawa, who summarized the data in Matsunami General Hospital. We would also like to thank Toshihiko Ikegami, MD, PhD, and Yuichi Nakazawa, MD, PhD, who obtained informed consent from the patients at the outpatient clinic. Mika Goto, a recipient coordinator, helped us correct the patient data. We are greatly indebted to our colleagues, who participated in this study as healthy controls.
We would like to thank Editage (www.edita ge.com) for English language editing.

FU N D I N G I N FO R M ATI O N
This research received no specific grants from any funding agency in the public, commercial, or not-for-profit sectors.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare no conflicts of interest for this article.

E TH I C A L A PPROVA L
The study was conducted in accordance with the Declaration of Helsinki and the protocol was approved by the Ethics Committee of Shinshu University (registration number: 5265). Written informed consent was obtained from all the patients and/or their families before inclusion in the study.