key: cord-0770803-jipmmanx
authors: Davidov, Yana; Indenbaum, Victoria; Tsaraf, Keren; Cohen-Ezra, Oranit; Likhter, Mariya; Ben Yakov, Gil; Halperin, Rebecca; Levy, Itzchak; Mor, Orna; Agmon-Levin, Nancy; Afek, Arnon; Rahav, Galia; Lustig, Yaniv; Ben Ari, Ziv
title: A third dose of the BNT162b2 mRNA vaccine significantly improves immune responses among liver transplant recipients
date: 2022-04-19
journal: J Hepatol
DOI: 10.1016/j.jhep.2022.03.042
sha: 1c7a83e52cc89773ab741381b5f315fea9f70520
doc_id: 770803
cord_uid: jipmmanx

BACKGROUND: Immune responses of solid organ transplant recipients to two doses of the BNT162b2 mRNA anti-SARS-CoV-2 vaccine is impaired. The immunogenicity and safety of a third dose among liver transplant (LT) recipients are unknown. This work aimed to evaluate the immune response of LT recipients to a third dose of the BNT162b2 mRNA vaccine. METHODS: Consecutive LT recipients (n=61) in follow-up at Sheba Medical Center were included. Receptor-binding domain (RBD) IgG, neutralizing antibody (NA) titers, and T cells levels before and 21-28 days a third vaccine dose were determined. Adverse effects after the third dose were monitored. RESULTS: The LT recipients were of a median age of 65 years; 57.4% were male. The humoral immune response rate improved significantly, with 56% of patients showing a response before the third vaccine to 98% after the third vaccine. The cellular response in 12 evaluated patients improved significantly (p=0.008). The geometric mean of anti-RBD IgG levels, NA levels, and T cell count also increased significantly after the third dose. NA titers after the third dose negatively correlated with age (p=0.03), mycophenolate mofetil treatment (p=0.005), and combined immunosuppression as compared to calcineurin inhibitor monotherapy (p=0.001). After the third dose, adverse effects were reported by 37% of recipients and were mostly mild (local pain and fatigue). CONCLUSION: After a third BNT162b2 mRNA vaccine, the immune response improved significantly among LT recipients, without serious adverse effects. Further studies are needed to evaluate immune response durability and determine the optimal number and schedule of boost vaccines. LAY SUMMARY: The Pfizer-Biotech BNT162b2SARS-CoV-2 vaccine induced significant immunity among liver transplant recipients. The majority of the patients developed sufficient levels of both humoral and cellular immune responses. Factors that predict non-response were older age and immunosuppressive medications.

The BNT162b2 mRNA vaccine against SARS-CoV-2 has been shown to be safe and effective in immunocompetent subjects ,1,2,3 . In contrast, solid organ transplant (SOT) recipients were reported to have impaired immune responses after two doses of the vaccine 4-8 , with liver transplant (LT) recipients showing a better immune response to the mRNA vaccine than other SOT recipients 4, 9, 10 . The reduced immune response was observed more in patients who received combined immunosuppression treatments (combination of calcineurin inhibitor (CNI) with mycophenolate mofetil (MMF), or sirolimus, or everolimus, and/or low-dose steroids) and patients with renal impairment [4] [5] [6] [7] . We have reported a 72% positive immune response rate among 76 LT recipients after the second dose of the BNT162b2 mRNA vaccine 10 . Kamar et al. reported improved immune response after the third BNT162b2 mRNA dose, administered to SOT recipients (78 kidney-transplant recipients, 12 LT recipients, eight lung-trans-plant or heart-transplant recipients, and three pancreas transplant recipients) 61 days after the second dose 11 . Benotmane et al. 12 We aimed to prospectively assess, for the first time, the safety as well as the efficacy of the third Pfizer-BioNTech BNT162b2 mRNA vaccine dose among LT recipients by evaluating both humoral and cellular immune responses.

This study was conducted at the Sheba Medical Center between 13 July 2021 and 5 September 2021 and was approved by the institutional review board. All participants agreed to participate in the study and signed a written informed consent before any study-related procedures were conducted. The study population included 61 adult (age >18 years old) LT recipients routinely followed at the Liver Diseases Center. The median time between the second vaccine dose,   serology after the second vaccine, and the third vaccine dose were 174 days (IQR, 168-182 days) and 135 (119-149 days), respectively.

Demographic, clinical, and laboratory data were extracted from electronic patient records. Blood tacrolimus or everolimus trough levels were determined, and routine blood tests were performed between the time the third vaccine was administered and before the serology test. Renal function was calculated using the chronic kidney disease epidemiology collaboration (CKD-EPI) creatinine equation. Chronic kidney disease was defined as eGFR <60 ml/min/1.73 m 2 for a duration of >3 months 14 .

The study was approved by our institutional review board (8314-21-SMC). Written informed consent was obtained from each patient included in the study.

Antibody detection testing Samples were evaluated with an "in-house" enzyme-linked immunosorbent assay (ELISA) that detects IgG antibodies against the receptor-binding domain (RBD) of SARS-CoV-2 15, 16 . A SARS-CoV-2 pseudo-virus (psSARS-2) neutralization assay was performed, as previously described 17 , to detect SARS-CoV-2 neutralizing antibodies using a green fluorescent protein (GFP) reporter-based pseudotyped virus with a vesicular stomatitis virus (VSV) backbone coated with the SARS-CoV-2 spike (S) protein, which was generously provided by Dr. Gert Zimmer (Institute of Virology and Immunology (IVI), Mittelhäusern, Switzerland). Sera not capable of reducing viral replication by 50% at 1 to 8 dilution or below were considered non-neutralizing.

IgG antibody titers above 1.1 Sample/Cut-off (S/CO) were defined as positive (responders), J o u r n a l P r e -p r o o f 8 while anti-RBD IgG <1.1 S/CO was defined as negative (non-responders). Patients who were still considered responders before receiving the third vaccine dose were defined as having a maintained immune response, while responders whose anti-RBD IgG levels dropped to below 1.1 S/CO prior to the third dose were defined as patients who failed to maintain an immune response.

Peripheral blood mononuclear cells were isolated by density gradient centrifugation using UNI-SEP+ (Novamed). Plasma was collected and spun at 1000 × g for 20 min to remove platelets before collection of PBMC. Following one wash with PBS and one wash with 4 Cell Nutri-T-Medium (Sartorius), cells were resuspended in 4Cell Nutri-T-Medium and counted using the Countess II Cell counter (Invitrogen).

Fresh PBMC were used in all ELISpot assays using the Elispot IFN-γ kit (AID). Briefly, fresh PBMC were added in duplicate wells at 2 × 10 5 cells in 50 μl per well and stimulated with 50 μl of SARS-CoV-2 peptide pools (S-complete, Miltenyi Biotech) (2 μg/ml per peptide). 4Cell

Nutri-T-Medium was used as negative control and Phytohaemagglutinin (PHA) was used as positive controls. After 16-20 h at 37 °C, 5% CO2, 95% humidity, cells were removed and secreted IFN-γ was detected by adding alkaline phosphatase conjugated secondary antibody for 2h. The plates were developed using BCIP/NBT substrate according to the manufacturer's instructions. ELISpot plates were scanned on an AID ELISpot Reader. The unspecific background (mean SFU from negative control wells) was subtracted from experimental readings.

J o u r n a l P r e -p r o o f Laboratory provided only two days for the T-cell evaluation test. Only the patients who agreed to participate during these days received a T-cell evaluation.

Inclusion criteria for participating in the current study of the third vaccine were: all LT recipients who received the second dose at least one months prior to the third dose.

LT patients who had recovered from SARS-CoV-2 or had an active SARS-CoV-2 infection up to seven days after the third vaccine dose, were excluded from the study. 

Baseline demographics and clinical and laboratory characteristics of the 61 LT recipients vaccinated with the third dose are presented in Table 1 patients; CNI and prednisone -6 patients). Triple immunosuppression therapy was administered to 6 (9.8%) patients (combination of CNI, MMF, and prednisone) (see Table 1 ). Fig. 1A and Fig. 1B) . Anti-RBD IgG titers positively correlated with NA titers both before (r=0.6, p<0.0001) and after the third vaccine (r=0.7, p<0.0001).

Due to the small sample size and the highly significant response to the third dose among the LT recipients (98.4%), predictors of negative immune response after the third dose could not be evaluated.

NA titers after the third dose negatively correlated with age (r= -0.32, p>0.03), MMF treatment (r=-0.4, p>0.005), and combined immunosuppression (double and triple therapy) as compared to CNI monotherapy (r=-0.5, p>0.007). NA titers after the third dose also positively correlated with hemoglobin levels (r=0.6, p<0.0001) and alanine aminotransferase levels (r=0.5, p<0.0014). No correlation was found between NA titers following the third dose and sex, comorbidities (DM, HTN, dyslipidemia, impaired renal function), or treatment with mTOR inhibitors or prednisone.

Monotherapy with CNI predicted response with an area ratio under the curve (ARUC) of 0.75, and a cut-off value of IgG anti-RBD 3.9 S/CO with a sensitivity 87% and specificity 58%, or J o u r n a l P r e -p r o o f cut-off value of the 5.8 S/CO with a sensitivity 50% and specificity 84%, p=0.001. Additionally, CNI monotherapy also predicted higher levels of NA with ARUC of 0.76, for a cut-off value of 1536; the sensitivity was 77% and the specificity 56% (p=0.003; Supplementary Figure 1 ).

During the preparation of this manuscript, two of the 61 LT recipients became infected with SARS-CoV-2 two months after receiving the third dose and suffered from mild symptoms Both patients were responders after the third vaccine, as shown by IgG anti-RBD levels of 2.1 S/CO for the first and 5.8 S/CO for the second. The first patient had undetectable NA levels, while the NA titer in the second patient was 4096. However, the first patient was a non-responder prior to the third dose, with IgG anti-RBD levels of 0.2 S/CO and the second patient was a responder, with IgG anti-RBD levels of 3.2 S/CO. .1-2.3, p=0.016 ). Age at vaccination, sex, time after transplantation, comorbidities, renal failure, and combined immunosuppression had no influence (Fig. 2, Supplementary Table 3 ).

The humoral immune response was evaluated in 47 of 61 patients after the second vaccine Table 4 ).

Adverse effects (mostly mild) were reported by 50% of the patients after the second dose of BNT162b2 mRNA vaccine 10 . Following the third dose, adverse effects were mild and reported by 37% of patients. Among these patients, 29% had local adverse reactions (mostly localized post-injection pain) and 22% reported systemic adverse reaction (mostly fatigue).

Age, gender, comorbidities, and level of immune response (levels of anti-RBD, NA and T cells) did not correlate with development of adverse effects.

The third dose of the BNT162b2 mRNA vaccine significantly improved humoral and cellular immune response among LT recipients. The low immune response rate measured among LT recipients 38 days after the second vaccine (68%) further declined 5 months after the second dose (57%), but significantly improved, 3 weeks after the third dose (98%). The vast majority (96%) of LT recipients who had a negative immune response before the third dose, achieved a positive immune response after the third dose. Our findings are in line with a recent report by 20 . Taken together, similarly to the higher immune response rate noted after the second vaccine dose in our LT recipients 10 , the third vaccine dose induced a substantially better immune response compared to heart 20 and renal transplanted recipients 11, 19 .

While the immune response after the second vaccine dose among all SOT recipients was relatively low 4-8 , our results demonstrated a remarkably higher response rate (72%) among LT recipients 10 . Rabinowich et al. 4 , Rueter at al 21 and Rashidi-Alavijeh 9 et al. reported 47.5%, 74% and 79% response rates, respectively, to two doses of the BNT162b2 mRNA vaccine among LT recipients (n=80, 141, 43 patients, respectively).

After the second dose of the vaccine, negative immune responses among cohorts of SOT recipients that included also LT recipients, correlated with older age, impaired renal function, MMF treatment or combined (double or triple) immunosuppression [4] [5] [6] 8 . Due to the very high response rate and relatively small sample size in our study, we were unable to identify immune response predictors. However, we found a significant negative correlation between anti-RBD IgG levels and MMF treatment, combined immunosuppression, older age and impaired renal function.

CNIs are the principal immunosuppression agents prescribed after LT. Among the available CNIs, tacrolimus is the drug of choice in almost 90% of LT recipients 22 . Due to the known nephrotoxicity that is associated with administration of CNI, patients are commonly presceibed reduced dosages, with or without renal-sparing agents such as sirolimus, or everolimus, low-dose steroids, or MMF 22 . likely to decline, similarly to the pattern observed 5-6 months after the second dose. Assessment of the durability of the immune response after the third dose is our next research goal; samples will soon be collected and analyzed and data hopefully published.

The presented study was limited by its small sample size. In addition, although our findings revealed significantly improved humoral and cellular immune responses following the third vaccine dose among LT recipients, no comparative data was provided regarding such responses among healthy individuals. While two patients developed SARS-COV-2 infection, and suffered from mild disease that did not require hospitalization, conclusion regarding protection against severe infection cannot be made at this time point.

In summary, the third BNT162b2 mRNA vaccine significantly improved humoral and cellular immune response among LT recipients, particularly among non-responders to the second vaccine, and enhanced the immune response among LT recipients who showed a waning immune response after the second vaccine dose. The third vaccine dose was not associated with severe adverse effects. Further studies will be necessary to determine whether a 5-month period between booster doses is necessary or if scheduling vaccination in immunocompromised patients requires optimization, with particular emphasis on SOT recipients treated with combined immunosuppression therapies. 

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