key: cord-0937539-uyn1hcqs
authors: Toniutto, Pierluigi; Falleti, Edmondo; Cmet, Sara; Cussigh, Annarosa; Veneto, Laura; Bitetto, Davide; Fornasiere, Ezio; Fumolo, Elisa; Fabris, Carlo; Sartor, Assunta; Peressutti, Roberto; Curcio, Francesco; Regattin, Laura; Grillone, Lucrezia
title: Past COVID-19 and immunosuppressive regimens affect the long-term response to anti-SARS-CoV-2 vaccination in liver transplant recipients
date: 2022-03-10
journal: J Hepatol
DOI: 10.1016/j.jhep.2022.02.015
sha: 332ab1200ff49df34c0f67f471abc0e3ec6efd71
doc_id: 937539
cord_uid: uyn1hcqs

BACKGROUND & AIMS: The long-term immunogenicity of anti-SARS-CoV-2 vaccines in liver transplant patients (LT) is unknown. We aimed to assess the long-term antibody response of the Pfizer-BioNTech® BNT162b2 vaccine in LT compared to controls. METHODS: LT underwent anti-SARS-CoV-2 anti-receptor binding domain protein IgG (anti-RBD) and anti-nucleocapsid protein IgG antibody (anti-N) measurements at the first and one, four and six months after the second vaccination dose. RESULTS: One hundred forty-three LT and 58 controls were enrolled. At baseline, 131/143 (91.6%) LT tested anti-N negative (COVID-19 naïve), and 12/143 (8.4%) tested positive (COVID-19 recovered) compared to negative controls. Among COVID-19 naïve, 22.1% were anti-RBD positives one month after the first vaccine dose, while 66.4%, 77%, and 78.8% one, four and six months following the second vaccine dose. In contrast, 100% of controls were positive at 4 months (p<0.001). The median anti-RBD titer four months after the second vaccine dose was significantly lower (32 U/ml) in COVID-19 naïve than in controls (852 U/ml, p<0.0001). Higher mycophenolate (MMF) daily dose (p<0.001), higher frequency of ascites (p=0.012), and lower serum leukocyte count (p=0.016) were independent predictors of anti-RBD negativity at six months. All COVID-19 recovered tested positive for anti-RBD at each time point. The median antibody titer was similar in those taking MMF (9400 U/ml, 11925 UI/ml, 13305 UI/ml, and 10095 UI/ml) or not taking MMF (13950 UI/ml, 9575 UI/ml, 3500 UI/ml, 2835 UI/ml, p=NS) three weeks after the first and 1, 4 and 6 months after the second vaccine dose, respectively. CONCLUSIONS: In COVID-19-naïve, the immunogenicity of anti-SARS-CoV-2 vaccination was significantly lower than that in controls. MMF was the main determinant of vaccination failure in SARS-CoV-2 naïve patients. LAY SUMMARY: The long-term (up to six months) anti-SARS-CoV-2 mRNA Pfizer-BioNTech® BNT162b2 vaccine-induced humoral response in liver-transplanted patients (LT) is unknown. We performed a prospective study to evaluate the long-term vaccine-induced humoral response (positivity of anti-SARS-CoV-2 s-RBD IgG antibodies) in 131 COVID-19-naïve and 12 COVID-19-recovered LT compared to a group of 58 healthy COVID-19 naïve controls. We found that the COVID-19-naïve LT that tested anti-RBD positive three weeks after the first vaccine dose were 22.1%, and 66.4%, 77%, 78.8% after one, four and six months following the second vaccine dose, compared to 100% controls evaluated at four months. An increasing daily dose of mycophenolate mofetil (MMF), in addition to the presence of severe graft dysfunction leading to cirrhosis with ascites and a lower serum leukocyte count, were selected as independent predictors of a negative vaccine-induced long-term immune response. In contrast, all COVID-19-recovered LT presented a full response to vaccination, which was detectable after the first vaccine dose and was maintained until the sixth month, regardless of the use of MMF.

The new coronavirus pathogen, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has been identified as the cause of coronavirus disease 2019 (COVID-19) 1 . Preliminary reports indicated that in liver transplant (LT) recipients, the clinical outcome following COVID-19 was better compared to other solid organ transplant recipients 2 and not per se worse compared to the general population 3 . However, more recent reports indicate that mortality in LT recipients remains particularly elevated 4, 5 . Two anti-SARS-CoV-2 vaccines based on mRNA technology (Pfizer-BioNTech® BNT162b2 and Moderna® mRNA-1273) 6, 7 have been approved. After the administration of two doses of these vaccines in immunocompetent patients, nearly all of them developed neutralizing antibodies against SARS-CoV-2 s-receptor binding domain (RBD) protein 8 .

The development of neutralizing antibodies seems to reduce the risk of symptomatic severe SARS-CoV-2-related disease in immunocompetent patients 9 . However, in LT recipients, the short-term (up to three months) humoral immune response induced by SARS-CoV-2 mRNA vaccines seems to be lower than that in immunocompetent patients [10] [11] [12] . At present, no data are available regarding the rate and duration of the immune response after vaccination in the long term (up to six months) in this population. Despite this, all scientific societies recommend that LT patients should undergo two anti-SARS-CoV-2 doses with mRNA vaccines 3-6 months after LT, when immunosuppression can be reduced [13] [14] [15] , with the possibility of a third booster dose 16 . The aim of this prospective study was to assess the safety and the long-term (up to six months) humoral immune response induced by two doses of the Pfizer-BioNTech® BNT162b2 vaccine in a cohort of LT patients compared to healthy controls.

Study protocol. The staff at the academic hospital in our Italian region launched the anti-SARS-CoV-2 vaccination program for all LT patients, adopting two doses of the Pfizer-BioNTech® BNT162b2 vaccine, in March 2021. Both vaccine doses were administered directly in the hospital for all LT patients who were in long-term follow-up at the hospital hepatology and liver transplantation unit.

Patients fulfilling this condition along with their demographic and clinical characteristics were extracted from the electronic database. The exclusion criteria were age at transplant <18 years, pregnancy, past known SARS-CoV-2 infection, and LT performed less than three months before vaccination. A group of physicians and nurses without known past SARS-CoV-2 infection who followed patients in the clinic served as controls. All patients and controls provided written informed consent to the vaccination protocol and to participate in this study, which was approved by the regional Ethical Committee and conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution's human research committee.

Before vaccination, both patients and controls completed a detailed interview reporting the presence of signs and/or symptoms (i.e., fever, cough, anosmia, and diarrhea), suggesting recent or past SARS-CoV-2 exposure. In addition, control subjects were periodically tested for SARS-CoV-2 infection via real-time reverse transcription (RT-PCR) on nasopharyngeal swabs. A vaccination self-reported side effects questionnaire was administered to participants within 30 days of receipt of the second vaccination dose.

In all patients, a blood sample was collected at the following time points: at the first and the second vaccine doses (performed 19 days after the first) and at one (31±2 days), 4 (125±5 days), and 6 months (165±4 days) thereafter. One blood sample was collected 4 months (134±15 days) after the second vaccine dose in controls ( Figure 1 ). Anti-SARS-CoV-2-N protein IgM and IgG antibodies (iFlash® -Shenzhen Yhlo Biotech Co. Ltd) and anti-spike glycoprotein-specific immunoglobulin G receptorbinding domain (s-RBD) antibodies (Roche Elecsys®, F. Hoffmann-La Roche Ltd) were measured in blood samples collected at every time point in both LT patients and controls. In accordance with the manufacturer's inserts, cutoff values used to identify positive patients were >10.0 kAU/L and ≥0.8 U/ml for the anti-SARS-CoV-2 N and s-RBD protein antibodies, respectively. Whiney) test was used, and data are presented as medians and interquartile (IQR) ranges. The comparison of categorical variables was carried out using the Pearson chi-square test, and data are presented as frequencies (%). Stepwise logistic regression analysis with a forward approach was used to select independent predictors for the development of a positive anti-SARS-CoV-2 vaccine-induced humoral response. All variables showing a p value ≤0.10 in the univariate analysis were included.

Pseudo R 2 , the area under the ROC curve, and the percentage of correct classification are presented as quality estimations of the regression model. Multivariate linear regression analysis with a stepwise forward approach was used to discriminate the best fitting variables in predicting the antibody response after vaccination, considering antibody titer as a continuous variable. All variables significantly associated with antibody response post vaccination in the univariate regression test were selected to run in the multivariate linear model.

Patients. One hundred sixty-four LT patients were selected for enrollment in the study. Among them, 19 (11.6%) declined to participate in the vaccination program, and two did not receive the second vaccine dose: one died due to the progression of hepatocellular carcinoma recurrence, and the other was lost to follow-up. Thus, 143 LT patients and 58 healthy controls were ultimately evaluated. LT patients were more frequently male (71.7% vs. 32.9%, p<0.001) and older (67.7 vs. 47.6 years, p<0.001) than controls.

None of the LT patients and controls reported in the interview as having a current or recovered SARS- and six (28.5 U/ml) months remained stable in LT, but it was significantly lower (32 U/ml) than that in the controls (852 U/ml, p<0.0001) at four months ( Figure 2 ).

COVID-naïve patients. In the multivariate analysis, independent predictors of immune response failure (anti-SARS-CoV-2 s-RBD IgG antibody titer <0.8 U/ml) three weeks after the first dose of vaccination were alcohol consumption >40 gr/day (p<0.001), taking a higher daily dose of mycophenolate mofetil (MMF) (p=0.002), and having a lower estimated glomerular filtration rate J o u r n a l P r e -p r o o f (eGFR) (p=0.016) ( Table 2 ). In addition to taking a higher daily dose of MMF, immunosuppression employing >2 drugs, having lower serum leucocytes and having older age at LT were selected as independent predictors of unsuccessful antibody response one and four months after the second vaccine dose, respectively (Supplementary CTAT Tables 1S and 2S) . A higher daily MMF dose assumption (p<0.001), a more frequent presence of ascites (p=0.012) and having a lower number of leukocytes (p=0.016) were selected as independent predictors of the negative antibody response six months after vaccination (Table 3) Table 4S ).

To identify patients who developed a strong antibody response after the full course of vaccination, the anti-SARS-CoV-2 s-RBD IgG antibody cutoff titer was selected at 100 U/ml. This was derived from the observation that adoptive transfer of purified polyclonal IgG from convalescent macaques robustly protected naïve recipient rhesus macaques against challenge with SARS-CoV-2 when the antibody titer was at least 100 U/ml 17 . Considering this cutoff level, the number of patients who tested positive for anti-SARS-CoV-2 s-RBD IgG antibodies at 1, 4 and 6 months after the second vaccine dose was 51 (38.9%), 45 (35.7%) and 36 (29.3%), respectively. In the multivariate analysis, independent predictors of the achievement of a strong antibody response (>100 U/ml) were a younger age at LT (p=0.0013), alcohol consumption <40 gr/day (p<0.001) and taking a lower daily dose of MMF (p<0.001) at 1 month and no alcohol consumption at 4 months after the second vaccine dose J o u r n a l P r e -p r o o f (Supplementary CTAT Tables 5S and 6S , respectively). A lower daily dose of MMF (p=0.006) in addition to a younger age at LT (p=0.012), alcohol consumption <40 gr/day (p<0.001) and higher hemoglobin serum levels (p=0.047) were selected as independent predictors of a strong antibody response 6 months after vaccination (Supplementary CTAT Table 7S ).

Anti-SARS-CoV-2 s-RBD antibody response after BNT162b2 vaccination in LT COVID-19-recovered patients. All 12 recovered COVID-19 patients presented a positive anti-SARS-CoV-2 s-RBD antibody response before vaccination. Furthermore, all increased their antibody titers following vaccination, which were significantly higher than those reported in COVID-19-naïve patients ( Figure   4 ). Interestingly, the median antibody titers developed three weeks after the first vaccine dose were significantly higher than those present before vaccination but not significantly different from those developed one month after the second vaccine dose. As reported for COVID-19 naïve patients, the mean antibody titers remained stable four and six months after vaccination, and none of the patients became antibody-negative. Unlike COVID-19-naïve patients, in COVID-19-recovered patients, the positive antibody response rate was the same in those receiving MMF (N=6) as in those who did not 

The long-term antibody response to the full course of BNT162b2 vaccination was recorded in 78.8% of COVID-naïve patients compared to 100% of the controls. Moreover, the peak of responder patients J o u r n a l P r e -p r o o f was reached four months after the second vaccine dose and remained stable up to six months. Recent reports indicated that the rate of antibody response to anti-SARS-CoV-2 vaccination in LT patients ranged from 45.5% to 82% 11, 12, 16, [18] [19] [20] [21] [22] , which is comparable to what we observed. However, all these studies evaluated the early (up to 3 months) immune response to vaccination. Our study presents, for the first time to our knowledge, data regarding the persistence of the antibody response to vaccination in the long term (up to 6 months). Our findings could be considered unexpected, since the effect of immunosuppressive therapies could reduce antibody response duration over time more rapidly compared to immunocompetent patients. In a recent report conducted in healthcare professionals, six months after a full course of BNT162b2 vaccine, antibody titer decline was observed in approximately 89.6% of cases, and approximately 45% of them became seronegative 23 . However, the peak response in these patients was reached 1 month after the second vaccine dose, which was earlier than what we observed in our series. One possible explanation could be that the vaccine-induced antibody response in immunosuppressed patients might be delayed and therefore detected to last longer. Whether this kinetic antibody response might be used to plan the vaccine booster dose would require appropriate clinical studies. In any case, to maintain the immunological response to vaccination for a long time, and in the hope of increasing the number of patient responders, it is desirable that booster doses are carried out in this category of patients. This strategy appears to be further justified by the recent emergence of the Omicron viral variant, the clinical impact of which in LT is not yet known.

No sex differences in the rate of long-term vaccine antibody response were detected. This agrees with previous reports 11, 18, 22 but is in contrast to what was reported in LT recipients by Herrera et al. 19 , who documented a significantly lower response rate in females. However, this difference may be due to the different vaccine types (mRNA1273) adopted in these patients compared to BNT162b2 adopted in our patients.

A detrimental effect on the long-term antibody response to vaccination was exerted by increasing the daily dose of MMF rather than adopting double or triple immunosuppressive drug combinations. This J o u r n a l P r e -p r o o f confirms data reported both in liver and in other solid organ transplants 24 . Rabinowich et al. 18 showed that the use of MMF, in addition to a triple immunosuppression regimen, higher doses of steroids and lower eGFR were selected as negative predictors of vaccination response. In our series, only 13/131 (9.9%) patients were taking prednisone at a dosage >5 mg compared to 24/80 (30%) of those reported in the aforementioned study. Thus, the impact of steroids may be influenced by the different number of treated patients between the two studies. Furthermore, only 4 patients adopting triple immunosuppression were enrolled in our study. The influence of eGFR in conditioning the antibody vaccine response has seldom been evaluated in studies 11, 12, 19, 22 . In our series, a lower eGFR was selected as a negative predictor of vaccine response only three weeks after the first dose and not thereafter, which may be considered in agreement with what has been demonstrated in other series 22 .

In addition to the use of MMF, the presence of severe graft dysfunction leading to ascites formation and a lower serum leukocyte count were associated with a poor antibody response. Patients with advanced liver disease frequently show a suboptimal response to the anti-SARS-CoV-2 vaccine 12 , which is also observable when employing other types of vaccines, such as that for hepatitis B 25 . The detrimental combination of immunosuppressive treatment and the presence of graft cirrhosis after liver transplantation may justify our results. This may also explain the negative impact of a lower leukocyte count, which could be considered a surrogate marker of drug-induced immunosuppression and severe portal hypertension 26 .

The strong vaccination response was evaluated adopting an antibody cutoff value of 100 U/ml 17 . The percentage of responder patients six months after vaccination decreased from 78.8% if evaluated by an antibody cutoff of 0.8 U/ml to 29.3%. Interestingly, alcohol consumption >40 gr/day showed a negative impact of a strong vaccination response, in addition to previously reported negative predictors. This finding agrees with studies indicating that alcohol consumption decreases the humoral response to some vaccines, such as those against streptococcal pneumonia 27 . Since alcohol relapse after LT is described in up to 5% of recipients 28 , the effectiveness of anti-SARS-CoV-2 vaccination in this category of patients may be further reduced.

In COVID-19-recovered patients in our series, higher BMI, diabetes mellitus, and recurrent cirrhosis with portal hypertension were significantly more frequent than in COVID-19-naïve patients. In contrast, no significant differences were recorded regarding which immunosuppression treatment was adopted, particularly regarding the use of MMF. This may be expected, since the presence of metabolic comorbidities has been associated with a worse clinical outcome for COVID-19 in LT but not as a factor leading to increased susceptibility to infection 29, 30 . Similar consideration may be made with regard to the use of MMF, since it has been associated only with a more severe form of COVID-19 31 , whereas the use of tacrolimus has been associated with a more benign course 32 . Although in solid organ transplant recipients, a robust antibody and T cell response can be elicited regardless of COVID-19 severity 33, 34 , to our knowledge, no data are available on the impact of MMF use on developing natural immunity after SARS-CoV-2 infection in LT. The novelty of our findings, although derived from a small number of patients, is that among the COVID-19-recovered patients (50% treated with MMF), prevaccination anti-s-RBD protein antibody titers were detectable in all of them, independent of the use of MMF. Moreover, after the first vaccine dose, the antibody titer increased significantly and was comparable to that obtained 1 month after the second vaccine dose and remained positive up to six months, regardless of the use of MMF. This finding, if confirmed in a larger series, seems to support the observation that every immunosuppressive regimen adopted after LT has no meaningful impact on the ability to mount natural antibody response after SARS-CoV-2 infection 34 .

Regarding vaccine safety in our study, no severe adverse events were reported in either patients or controls, nor were any liver biochemical abnormalities found during routine postvaccination patient follow-up. These observations agree with previous studies performed in solid organ transplant recipients 10, 18, 19 .

Our study has some limitations. First, we did not evaluate the cellular immune response to vaccination. The correlation between humoral and cellular immune responses to anti-SARS-CoV-2 vaccination remains unclear. In a recent report evaluating 138 LT recipients, there was no evidence J o u r n a l P r e -p r o o f of a spike-specific T cell response in the majority of those without any detectable antibody response 22 , suggesting that, in some cases, humoral and cellular immune responses could overlap. Second, we did not adopt systematic surveillance of patients to assess the efficacy of vaccination in preventing SARS-CoV-2 infection. Although this was not the aim of our study, we did not observe symptomatic SARS-CoV-2 infections in vaccinated patients during the follow-up period. Third, we enrolled patients with a long interval between transplant and vaccination; thus, our results may not be comparable to those obtainable when vaccination has been performed close to transplant.

In conclusion, in COVID-19-naïve LT patients, the anti-SARS-CoV-2 vaccination antibody response rate, although significantly lower than that in controls, was maintained for at least six months. The J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f 

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LT: liver transplantation; BMI: body mass index; HBV: hepatitis B virus; HCV: hepatitis C virus; AH: alcoholic hepatitis; AI: autoimmune; HCC: hepatocellular carcinoma; DM: diabetes mellitus; HTN: arterial hypertension; MMF: mycophenolate mofetil

P: prednisone; # reference blood levels evaluated within 1 month before vaccination for each IS drug were calculated in accordance with Cillo et al. 35 ; eGFR: estimated glomerular filtration rate; AST: aspartate aminotransferase; ALT: alanine aminotransferase; INR international normalized ratio

The authors thank Cristina Minissale, Federica Sandri, Marcello Ferro, Deborah Di Giusto, Annalisa Sostero for their logistic support and all patients and their families for permitting the realization of the study.J o u r n a l P r e -p r o o f