Therapy With m-TOR Inhibitors Decreases the Response to the Pandemic Influenza A H1N1 Vaccine in Solid Organ Transplant Recipients

Authors

  • E. Cordero,

    1. Unit of Infectious Disease, Microbiology and Preventive Medicine, Institute of Biomedicine of Sevilla (IBiS), University Hospital Virgen del Rocío/CSIC/University of Sevilla, Sevilla, Spain
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    • These authors contributed equally to this work.

  • A. Perez-Ordoñez,

    1. Unit of Infectious Disease, Microbiology and Preventive Medicine, Institute of Biomedicine of Sevilla (IBiS), University Hospital Virgen del Rocío/CSIC/University of Sevilla, Sevilla, Spain
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    • These authors contributed equally to this work.

  • T. A. Aydillo,

    1. Unit of Infectious Disease, Microbiology and Preventive Medicine, Institute of Biomedicine of Sevilla (IBiS), University Hospital Virgen del Rocío/CSIC/University of Sevilla, Sevilla, Spain
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  • J. Torre-Cisneros,

    1. Clinic Unit of Infectious Diseases (JTC, RL) and Service of Cardiology, Maimonides Institute for Biomedical Research (IMIBIC) Reina Sofia University Hospital, University of Córdoba (UCO), Cordoba, Spain
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  • J. Gavalda,

    1. Vall d'Hebron University Hospital, Barcelona, Spain
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  • R. Lara,

    1. Clinic Unit of Infectious Diseases (JTC, RL) and Service of Cardiology, Maimonides Institute for Biomedical Research (IMIBIC) Reina Sofia University Hospital, University of Córdoba (UCO), Cordoba, Spain
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  • C. Segura,

    1. Clinic Unit of Infectious Diseases (JTC, RL) and Service of Cardiology, Maimonides Institute for Biomedical Research (IMIBIC) Reina Sofia University Hospital, University of Córdoba (UCO), Cordoba, Spain
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  • O. Len,

    1. Vall d'Hebron University Hospital, Barcelona, Spain
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  • E. Cabral,

    1. Vall d'Hebron University Hospital, Barcelona, Spain
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  • A. Gasch,

    1. Unit of Infectious Disease, Microbiology and Preventive Medicine, Institute of Biomedicine of Sevilla (IBiS), University Hospital Virgen del Rocío/CSIC/University of Sevilla, Sevilla, Spain
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  • J. Pachon,

    1. Unit of Infectious Disease, Microbiology and Preventive Medicine, Institute of Biomedicine of Sevilla (IBiS), University Hospital Virgen del Rocío/CSIC/University of Sevilla, Sevilla, Spain
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  • P. Perez-Romero

    Corresponding author
    1. Unit of Infectious Disease, Microbiology and Preventive Medicine, Institute of Biomedicine of Sevilla (IBiS), University Hospital Virgen del Rocío/CSIC/University of Sevilla, Sevilla, Spain
      Perez-Romero, mariap.perez.exts@juntadeandalucia.es
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  • Other authors in the pandemic vaccine in SOTR study group (REIPI): From University Hospital Virgen del Rocío: Martin-Gandul C, Martin-Peña A, Sobrino M, Suárez G, Gentil MA. From the University Hospital Reina Sofia: Salgueiro I, Vidal E, Arizón JM, Vaquero JM, Santos F, Redel J, Mendoza M, Rodríguez A, Agüera ML, de la Mata M, Barranco JL, Ostos C, Castro L. From Val d'hebron Hospital: Román A, Monforte V, Bilbao I, Castells L, Perelló M.

Perez-Romero, mariap.perez.exts@juntadeandalucia.es

Abstract

Concern has been raised regarding the response to vaccination in solid organ transplant recipients (SOTR) undergoing immunosuppressant regimens and the possibility of rejection related to the immune response associated with pandemic influenza H1N1–2009 vaccination. The goal of this study was to assess the immunogenicity, efficacy and safety of the pandemic vaccine in SOTR. We performed a multicenter prospective study in SOTR receiving the pandemic vaccine. Immunological response was determined in serum 5 weeks after vaccination by microneutralization assays, and immunoglobulins were measured by ELISA. Three hundred and forty-six SOTR were included. Preexisting seroprotection was detected in 13.6% of cases and rates of seroconversion and seroprotection after vaccination were 73.1% and 82.9%, respectively. Patients with baseline antibody titers had better geometric mean titers (GMT)-post after pandemic vaccination (339.4 vs. 121.4, p < 0.001). Younger age, liver disease and m-TOR inhibitor therapy were independently associated with lower seroprotection and GMT-post. There were no major adverse effects or rejection episodes. Pandemic vaccine was safe in SOTR and elicited an adequate response, although lower than in healthy individuals. This is the first study describing a decreased response after vaccination in patients receiving mTOR inhibitors who presented lower seroprotection rates and lower GMT-post.

Abbreviations: 
COPD

chronic pulmonary disease

GMT

geometric mean titers

HG

hypogammaglobulinemia

mTOR

mammalian target of rapamycin

mTORC1

mTOR complex 1

mTORC2

mTOR complex 2

SOTR

solid organ transplant recipients

Introduction

In April 2009, a new strain of influenza A H1N1 virus emerged and spread globally (1). Influenza in solid organ transplant recipients (SOTR) causes significant morbidity and mortality (2–4) and a prolonged time to infection clearance (5). Moreover, it has been associated with acute and chronic allograft rejection in SOTR (6). Recently, substantial morbidity in SOTR due to pandemic H1N1 infection has been described (7).

Influenza vaccination, both seasonal and pandemic, was strongly recommended in SOTR during the H1N1–2009 virus pandemic (8). The seasonal influenza vaccine induced an optimal response, although it was diminished in SOTR compared to healthy individuals (9,10). Thus, in SOTR the immune response to vaccination may be influenced by immunosuppression regimens (9,10). Due to its lack of nephro- and neurotoxicity and its antiproliferative effects, rapamycin (sirolimus), which inhibits the activity of the mammalian target of rapamycin (mTOR), is increasingly being used for prophylaxis of organ rejection as an alternative to calcineurin inhibitors (11). The m-TOR is a serine/threonine kinase that regulates growth, proliferation, motility and cellular survival, protein synthesis and transcription (12). The mTOR nucleates two large physically and functionally distinct signaling complexes: mTOR complex 1 (mTORC1) that plays critical roles in cell growth in response to nutrients and mTOR complex 2 (mTORC2) that controls cell proliferation and survival (13). Sirolimus and everolimus act by forming an inhibitory complex with its intracellular receptor FKBP12, which binds to mTORC1, thus inhibiting TOR activity (12). Recent studies have suggested a higher CD8+ memory T-cell response after vaccination in animals receiving m-TOR inhibitors (14–16). However, the impact of this drug in human response to vaccine has not been established.

To date, there are no studies addressing the immune response to the pandemic vaccine in SOTR. Moreover, a concern has been raised about the effect on the response to vaccination in SOTR receiving immunosuppressant regimens and the possibility of rejection related to the immune response associated with influenza vaccination. Therefore, the purpose of this study was to assess the immunogenicity, efficacy and safety of the pandemic H1N1–2009 vaccine in SOTR.

Materials and Methods

Subjects and study design

We performed a multicenter prospective cohort study of SOTR performed at three Spanish University hospitals: Virgen del Rocío in Sevilla, Reina Sofía in Córdoba and Vall d'Hebron in Barcelona, all belonging to the Spanish Network for the Research in Infectious Diseases (REIPI). The SOTR older than 16 years, who received one dose of the pandemic H1N1–2009 vaccine and provided written informed consent, were enrolled in this study, carried out from November 2009 to January 2010. Patients were excluded if they received the transplant less than 2 months ago, in cases of allergy to chicken egg proteins or any vaccine component and pregnancy. Serum samples were collected from each patient at vaccination (baseline) and at 5 weeks after vaccination, and stored at −80°C for further analysis. To collect and record possible adverse effects after receiving the vaccine, patients were followed-up during 90 days or 10 months, if, events of pandemic virus infection were detected. The study was approved by the Ethics Committee for Clinical Research at participating hospitals.

Clinical parameters and definitions

In all vaccinated patients, baseline characteristics, immunological and clinical response and adverse effects, including graft rejections and mortality, were recorded (for details, see the Supporting Information). According to current guidelines, graft rejection was histologically evaluated if it was evident through biochemical or spirometry testing.

Chronic renal insufficiency was defined as serum creatinine levels >1.5 mg/dL. Obesity was defined as BMI ≥ 30 and morbid obesity as BMI ≥ 40. Vaccinated patients included all individuals who had received the seasonal influenza vaccine in the previous year or any vaccine in the previous month. Hypogammaglobulinemia (HG) was defined as IgG levels less than 700 mg/dL. The use of cytolytic induction therapy within the 6 months, previous to the vaccine, was evaluated.

Vaccines

All participants received the pandemic influenza H1N1–2009 vaccine during a 2-week period (November 2009). Patients were given one 0.5-mL dose of the pandemic H1N1–2009 monovalent MF59-adjuvanted vaccine (Novartis, Vaccines and Diagnostics S.r.l., Siena, Italy). Some of the patients at the Vall d'Hebron Hospital received one 0.5-mL dose of the pandemic H1N1–2009 monovalent AS03-adjuvanted vaccine (GlaxoSmithKline Biologicals, S.A., Bélgica) by intramuscular injection. Adverse events were assessed in an identical manner in all the centers that participated in the project according to well-established criteria (17).

Microneutralization assay

The influenza virus microneutralization assay was performed, as previously described (18), with modifications. Briefly, serial dilutions (from 1:5 to 1:5120) of heat inactivated sera was preincubated for 2 h at 37°C in a 5% CO2 atmosphere with 2 × 103 TCID50/mL of the influenza A/California/7/2009 (H1N1)v virus (obtained from Dr. Pérez-Breña, National Centre of Microbiology, Majadahonda, Madrid). One hundred microliters of 1.5 × 105 Madin–Darby canine kidney cells/mL were added and incubated for 24 h at 37°C and 5% CO2. Detection of viral infection was performed by ELISA using a primary antibody against the influenza A virus nucleoprotein, (Antibodyshop-BioPorto Diagnostics, Gentofte, Denmark) diluted 1:5000 in PBS, containing 1% BSA and 0.1% Tween-20, and a HRP-conjugated antimouse immunoglobulin G (IgG; Sigma-Aldrich Quimica SA, Madrid, Spain), diluted 1:10 000. After washing, 100 μL of peroxidase substrate (3, 3', 5, 5'-Tetramethyl-benzidine substrate, for ELISA; Sigma-Aldrich) was added and incubated at room temperature for 15 min. The reaction was stopped with 1 N sulfuric acid. The absorbance was measured at 490 nm with a plate spectrophotometer (AsysHitech UVM 340, Isogen Life Science, Oudenrijn, Netherlands). The serum dilution that reduced more than 50% of the A490 compared to the virus-infected (VC), was considered positive. Titers below the detection limit (<1:5) were assigned a titer of 1:5. Sera were considered positive if titers ≥40 were obtained in two independent assays. The following parameters for efficacy of vaccination based on the international criteria were: geometric mean titers (GMT) defined as mean antibody titer in the group of vaccinated individuals; seroprotection rate defined as the percentage of subjects with antibody titer ≥1:40; seroconversion rate given as the percentage of subjects with fourfold increase in antibody titer from baseline; geometric mean ratio defined as seroconversion factor post to prevaccination.

Gammaglobulinemia determination

Immunoglobulin (IgG, IgM and IgA) levels were measured by ELISA using the Dimension Vista® Intelligent Lab System 1500T (Siemens Healthcare Diagnostics, Deerfield, IL, USA). The reference ranges for the HG categories were those previously described (19).

Statistical analysis

For analysis, patients were grouped by age. A descriptive statistical analysis was performed. Continuous variables were expressed as median and interquartile range or mean ± standard deviation if adjusted to normal distribution and evaluated by Shapiro—Wilk or Kolmogorov–Smirnov tests when appropriate. For bivariate analysis, the chi-square test or the Fisher exact test were used for categorical variables, the Bonferroni correction was applied when appropriate. For quantitative variables, Mann–Whitney test or Student's t-test were used based on their distribution. If the variance was not homogeneous (Levene test) the Welch test was applied (ANOVA). For multivariate analysis, logistic regression and linear regression were used for factors influencing serum response. For immunogenicity analysis, the geometric mean antibody titers at each time point were used. The GMT and 95% confidence intervals were computed by taking the exponent (log10) of the mean and of the lower and upper limits of the 95% confidence intervals of the log10-transformed titers, as previously shown (20). Results were analyzed using PASW Statistic software (v.18.0.1). Statistical significance was established at p < 0.05. All reported p-values are based on two-tailed tests.

Results

Patient characteristics

A total of 368 SOTR received the pandemic H1N1–2009 vaccine, 22 patients were excluded due to the lack of a second serum sample. The SOTR characteristics, comorbidities and background by group of age are described in Table 1. Of the 346 patients evaluated, the second sample was collected at a median of 34 days (range 22.0–71.0) after vaccination.

Table 1.  Characteristics, comorbidities and background of SOTR receiving the pandemic influenza A (H1N1)-2009 vaccine
 Age in groups (years)Total
17–3536–4950–65>65
  1. COPD = chronic obstructive pulmonary disease.

  2. Parameters were compared by multiple comparison chi-square test, p < 0.012 was considered significantly different: 1p < 0.001; 2p = 0.02; 3p = 0.001; 4p = 0.03, 5p = 0.002; 6p<0.001; 7p = 0.03; 8p = 0.04 and 9p = 0.009.

No. (%)44 (12.7)72 (20.8)171 (49.4)59 (17.1)346 (100.0)
Male, no. (%)122 (50.0)32 (44.4)127 (74.3)44 (74.6)225 (65.0)
Smoking, no. (%)1 (2.3)11 (15.3)16 (9.4)3 (5.1)31 (9.0)
Alcohol, no. (%)0 (0.0)3 (4.2)7 (4.1)2 (3.4)12 (3.5)
Contact with children <3 years, no. (%)10 (22.7)14 (20.3)57 (33.5)21 (35.6)102 (29.8)
Influenza vaccine 08/09, no. (%)23 (52.3)38 (52.8)103 (60.2)42 (71.2)206 (59.5)
Influenza vaccine 09/10, no. (%)33 (75.0)53 (73.6)136 (79.5)45 (76.3)267 (77.2)
Pandemic influenza vaccine A (H1N1) v, no. (%)
 MF59-adjuvanted (Focetria®)35 (79.5)59 (81.9)157 (91.8)59 (100.0)310 (89.6)
 AS03-adjuvanted (Pandemrix®)9 (20.5)13 (18.1)14 (8.2)0 (0.0)36 (10.4)
Type of transplant, no. (%)
 Kidney13 (29.5)27 (37.5)46 (26.9)15 (25.4)101 (29.2)
 Liver28 (18.2)21 (29.2)45 (26.3)26 (44.1)100 (28.9)
 Heart7 (15.9)12 (16.7)48 (28.1)16 (27.1)83 (24.0)
 Lung315 (34.1)12 (16.7)28 (16.4)2 (3.4)57 (16.5)
 Combined transplantation1 (2.3)0 (0.0)4 (2.3)0 (0.0)5 (1.4)
Underlying chronic diseases, no. (%)37 (84.1)61 (84.7)156 (91.2)54 (93.1)308 (89.3)
 Chronic pulmonary disease14 (31.8)11 (15.3)37 (21.6)14 (23.7)76 (22.0)
 COPD41 (2.3)3 (4.2)24 (14.0)8 (13.6)36 (10.4)
 Diabetes mellitus56 (13.6)9 (12.5)53 (31.0)20 (33.9)88 (25.4)
 Chronic heart disease66 (13.6)5 (6.9)27 (15.8)23 (39.0)61 (17.6)
 Chronic renal failure7 (15.9)20 (27.8)52 (30.4)23 (39.0)102 (29.5)
 Chronic liver disease1 (2.3)7 (9.7)10 (5.8)6 (10.2)24 (6.9)
Immunosuppressant drugs no. (%)
 Cyclosporine10 (22.7)22 (30.6)42 (24.6)19 (32.2)93 (26.9)
 Tacrolimus733 (75.0)49 (68.1)119 (69.6)30 (50.8)231 (66.8)
 Mycophenolate mofetil831 (70.5)65 (90.3)129 (75.9)46 (78.0)271 (78.6)
 m-TOR inhibitor6 (13.6)7 (9.7)36 (21.1)11 (18.6)60 (17.3)
 Azathioprine1 (2.3)0 (0.0)2 (1.2)1 (1.7)4 (1.2)
 Corticosteroids >20 days930 (68.2)48 (66.7)83 (48.8)27 (45.8)188 (54.4)
Time from transplant to vaccination, no. (%)
 2–12 months6 (13.6)15 (20.8)29 (17.0)8 (13.6)58 (16.8)
 13–24 months5 (11.4)8 (11.1)26 (15.2)1 (1.7)40 (11.6)
 >24 months33 (75.0)49 (68.1)116 (67.8)50 (84.7)248 (71.7)
IgG levels, No (%)    57 (16.4)
 Mild (IgG levels 500–700 mg/dL)4 (9.1)10 (13.9)23 (13.5)8 (13.6)45 (13.0)
 Moderate (IgG levels 350–500 mg/dL)1 (2.3)2 (2.8)7 (4.1)1 (1.7)11 (3.2)
 Severe (IgG <350 mg/dL)0 (0.0)1 (1.4)0 (0.0)0 (0.0)1 (0.3)

Patients, younger than 35 years, received a lung transplant more frequently compared to other groups of age (34.1%, p = 0.001), whereas patients older than 65 years more frequently received liver transplants (44.1%, p = 0.02). Most of the SOTR (89.3%) presented associated underlying conditions, with chronic renal insufficiency the most frequent (29.5%). Sixty patients received m-TOR inhibitors, half of the patients (50.4%) received triple combined immunosuppressive therapy and 41.4% received double immunosuppressive regimen. The most common immunosuppressive combination was tacrolimus, micophenolate mofetil and corticosteroids therapy (31.5%).

The median time after transplantation in patients receiving the pandemic vaccine was 4.7 years (range 0.2–27.8). Of these, most of the patients, 248 (71.7%) were transplanted for longer than 2 years and 24 patients (6.9%) received the pandemic vaccine between 2 and 6 months after transplantation. In 57 patients (16.4%), an IgG deficiency was detected.

The SOTR cohort presented a preexisting baseline seroprotection with titers ≥1:40 in 13.6% of the cases, with lower rates in patients under 35 years of age than in those between 50 and 65 years old and older than 65 (2.3% vs. 15.8% and 20.3% respectively, Table 2).

Table 2.  Antibody responses to pandemic vaccine in the SOTR cohort according to age in groups, type of transplant, time from transplantation to the pandemic vaccine and type of vaccine
 GMTpre CI 95%Baseline seroprotection, no. (%)GMTpost CI 95%Seroconversion, no. (%)GMR CI 95%Seroprotection, no. (%)
  1. Parameters were compared statistically, p ≤ 0.05 was considered significantly different.

  2. GMT, geometric mean titer; GMR, geometric mean ratio.

  3. All the categories within each variable within each time period (GMTpre and GMTpost) were analyzed by ANOVA test: 1mean differences were: 10.24 CI 95% 0.01–1.31, p = 0.04; 50.12 CI 95% 0.10–0.73, p = 0.004; 30.83 CI 95% (–0.34) –1.70, p = 0.06. Seroprotection at baseline and postvaccine and seroconversion rates were analyzed by multiple comparisons by chi-square test, those comparisons that were statistically significant (p ≤ 0.05) were compared in groups two by two, Bonferroni correction was applied: 217–35 years old versus 50–65 years old (2.3% vs. 15.8%, RR 0.14 CI 95% 0.02–1.03, p = 0.02) and 17–35 years old versus >65 years old (2.3% vs. 20.3%, RR 0.11 CI 95% 0.02–0.83, p = 0.006); 62–12 months since transplantation versus >24 months since transplantation (5.2% vs. 16.5%, RR 0.31 CI 95% 0.10–0.98, p = 0.03); 72–12 months since transplantation versus >24 months since transplantation (84.5% vs. 70.6%, RR 1.90 CI 95% 1.00–3.50, p = 0.03). 4Seroprotection in the age group between 17 and 35 years was always lower than the other groups comparisons in pairs, with values statistically significant, in order of age was: 68.2% versus 86.1%, RR 1.20 CI 95% 1.00–1.60, p = 0.02; 68.2% versus 83.6%, RR 1.20 CI 95% 1.00–1.50, p = 0.02; 68.2% versus 88.1%, RR 1.30 CI 95% 1.00–1.60, p = 0.01.

General cohort
 8.1 (7.1–9.2)47 (13.6)139.6 (117.2–166.3)253 (73.1)17.3 (14.2–20.9)287 (82.9)
Age group (years)
 17–355.3 (4.7–6.0)11 (2.3)276.3 (41.4–140.5)329 (65.9)14.3 (7.7–26.5)30 (68.2)4
 36–497.1 (5.5–9.1)7 (9.7)151.0 (104.6–218.1)56 (77.8)21.4 (14.1–32.2)62 (86.1)
 50–658.7 (7.2–10.7)27 (15.8)145.8 (114.1–186.2)125 (73.1)16.7 (12.7–21.9)143 (83.6)
 >65 years10.4 (7.0–15.3)12 (20.3)175.8 (119.0–260.0)43 (72.9)17.0 (10.5–27.4)52 (88.1)
Type of transplant
 Kidney7.2 (5.8–9.1)10 (9.9)150.4 (106.4–202.6)76 (75.2)20.8 (14.4–30)83 (82.2)
 Liver7.6 (6.0–9.6)12 (12.0)150.3 (107.3–210.7)73 (73.0)19.7(13.4–28.7)83 (83.0)
 Heart9.2 (6.8–12.4)14 (16.9)125.6 (90.0–175.2)60 (72.3)13.6 (9.3–19.9)70 (84.3)
 Lung8.4 (6.1–11.7)9 (15.8)116.6 (75.5–180.0)41 (71.9)13.8 (8.8–21.8)46 (80.7)
Time of transplantation to the influenza vaccine (months)
 2–125.9 (4.9–7.2) 53 (5.2)6163.9 (110.5–242.9)49 (84.5)727.7 (18.3–42.1)51 (87.9)
 13–246.6 (4.8–9.1)3 (7.5)107.4 (60.5–190.7)29 (72.5)16.3 (9.2–28.9)31 (77.5)
 >249.0 (7.6–10.6)41 (16.5)140.3 (113.9–172.8)175 (70.6)15.6 (12.3–9.7)205 (82.7)

Immunological response to pandemic H1N1–2009 vaccine

The overall rate of seroconversion and seroprotection was 73.1% and 82.9%, respectively (Table 2). The antibody titer postvaccination was lower in patients receiving immunosuppressive therapy based on m-TOR inhibitor compared to other immunosuppresions, and also in younger patients (group 17–35 years) compared to the rest of the age groups (36–49 years, 50–65 years and >65 years). No differences were found in GMT-post vaccination in patients receiving the vaccine at different times after transplantation (Table 2). Only six patients (2.6%) received cytolytic/induction within the 6 months before vaccination and all of them responded fairly well to the pandemic vaccine.

Factors related to vaccine antibody response are shown in Table 2. Patient age was correlated with immune response to the pandemic vaccine. Thus, a lower seroprotection rate and GMT-post was observed in the group of patients under 35 years of age (Table 2). All patients having antibody titers at baseline were seroprotected (titers ≥1/40) and reached high levels in those patients with seroprotection at baseline (339.4 vs. 121.4, p < 0.001). The rate of seroprotection after vaccination of patients without antibody titers at baseline was lower than in those with previous antibody titers (80.3% vs. 100%, RR 1.3 CI 95% 1.2–1.3, p < 0.001).

The SOTR, with chronic liver disease, had a reduced immune response to the pandemic vaccine with a seroprotection rate of 62.5% versus 84.5% in the others (RR 0.7 CI 95% 0.5–1.0, p = 0.006) and a seroconversion rate of 50.0% versus 74.8% (RR 0.7 CI 95% 0.5–1.0, p = 0.008). Patients with this comorbidity were mostly liver transplant recipients (66.6% vs. 33.3%, p < 0.001). The antibody titers after vaccination were also lower in patients with liver disease (77.7 CI 95% 30.1–200.4 vs. 145.9 CI 95% 122.4–173.8, p = 0.18; Table 3). The use of an m-TOR inhibitor was associated with lower GMT-post (p = 0.01; Table 5).

Table 3.  Factors influencing seroprotection in bivariate analysis and adjusted multivariate analysis
 Bivariate analysis RR (CI 95%)p-ValueMultivariate analysis adjusted OR (CI 95%)p-Value
  1. Parameters were compared statistically in a logistic regression, p ≤ 0.05 was considered significantly different. Number 1 indicates the reference group for each of the variables analyzed.

  2. UD = undefined.

Sex
 Male1   
 Female1.46 (0.82–2.56)0.190.73 (0.38–1.39)0.34
Age (years)1.02 (1.0–1.4) 0.0161.02 (1.0–1.05)0.01
Chronic liver disease
 Yes1   
 No0.70 (0.50–1.00) 0.0060.27 (0.10–0.70)0.007
One year since transplant to influenza vaccine
 Yes1   
 No1.61 (0.68–3.73)0.261.37 (0.54–3.45)0.52
Hypogammaglobulinemia
 Yes1   
 No1.52 (0.65–3.56)0.321.29 (0.51–3.24)0.53
Cyclosporine
 Yes1   
 No0.73 (0.39–1.34)0.310.64 (0.32–1.28)0.21
Mycophenolate mofetil
 Yes1   
 No1.46 (0.77–2.78)0.240.89 (0.40–1.97)0.78
m-TOR inhibitors
 Yes1   
 No0.90 (0.80–1.01)0.070.42 (0.18–1.00)0.049
Corticosteroids longer than 20 days
 Yes1   
 No1.29 (0.73–2.27)0.361.32 (0.66–2.62)0.42
Type of transplant 0.98 0.82
 Liver1   
 Kidney0.94 (0.45–1.95) 0.74 (0.30–1.80) 
 Heart1.10 (0.50–2.42) 1.34 (0.51–3.50) 
 Lung0.83 (0.36–1.94) 0.86 (0.32–2.32) 
 Combined transplantation3.108 (0.00–UD)0.993.108 (0.00–UD)0.99
Table 5.  Factors influencing GMTpost in the bivariate analysis and adjusted multivariate analysis
 Bivariate analysis RR (CI 95%)p-ValueβMultivariate analysis adjusted OR (CI95%)p-Value
Lower limitUpper limit
  1. Parameters were compared statistically in a linear regression, p ≤ 0.05 was considered significantly different. Number 1 indicates the reference group for each of the variables analyzed.

Sex
 Male1     
 Female−0.29 (−0.47–0.27)0.59−0.02−0.470.310.69
Age (years)0.14 (0.005–0.031)0.0070.170.000.030.003
Chronic liver disease
 No1     
 Yes−0.09 (−1.3–0.07)0.08−0.12−1.5−0.100.02
One year since transplant to influenza vaccine
 No1     
 Yes0.04 (−0.26–0.68)0.380.02−0.360.620.61
IgG levels (mg/dL)0.06 (0.00–0.001)0.240.100.000.0010.06
Cyclosporine
 No1     
 Yes−0.05 (−0.59–0.21)0.35−0.03−0.560.290.53
Mycophenolate mofetil
 No1     
 Yes0.06 (−0.18–0.67)0.25−0.00−0.520.460.90
m-TOR inhibitors
 No1     
 Yes−0.12 (−1.02- (−0.98)0.01−0.15−1.20.120.01
Corticosteroids longer than 20 days
 No1     
 Yes0.00 (−0.35–0.35)0.990.00−0.400.410.97
Type of transplant
 Liver1     
 Kidney0.00 (−0.46–0.46)0.990.01−0.450.580.84
 Heart−0.04 (−0.66–0.30)0.460.02−0.450.660.75
 Lung−0.06 (−0.81–0.27)0.35−0.00−0.630.550.90
 Combined transplantation0.05 (−0.73–2.2)0.320.06−0.652.360.26

In the multivariate analysis, age (OR 1.03, 1.00–1.05, p = 0.01) was significantly associated with higher seroprotection whereas liver disease (OR 0.27, 0.10–0.70, p = 0.007) was an independent factor associated with lower seroprotection (Table 3). The odds ratio of seroprotection of patients receiving mTOR inhibitor therapy was 0.42 (CI 95%, 0.18–1.00, p = 0.05). Liver disease was the only independent factor associated with lower seroconversion (OR 0.30, 0.1–0.8, p = 0.02, Table 3).

The results of the analysis of the GMT-post by linear regression correlated with those shown for seroconversion and seroprotection, with higher antibody titers in older patients (p = 0.003) and lower titers in patients with liver disease (p = 0.02), and m-TOR inhibitor therapy (p = 0.01, Tables 4 and 5). The raw proportion for seroconversion and seroprotection of the different m-TOR combinations were as follows: (1) mTOR with tacrolimus, (a) double combination (N = 666.7% and 50%, respectively), (b) triple combination, with mycophenolate (N = 6, 50% and 33.3%, respectively), with corticoids (N = 7, 71.4% and 71.4%, respectively) and with azatioprine (N = 1, 0% and 0%, respectively) and (c) quadruple combination, with mycophenolate and corticoids (N = 1, 0% and 0%, respectively); (2) mTOR with cyclosporine, (a) double combination (N = 14, 85.7% and 85.7, respectively), (b) triple combination, with mycophenolate (N = 2, 50% and 50%, respectively) and with corticoids (N = 6, 66.6% and 50%, respectively) and (3) mTOR with no calcineurine inhibitors: mycophenolate, (a) double combination (N = 8, 87.5% and 87.5%, respectively) and (b) triple combination, with corticoids (N = 9, 100% and 77.8%, respectively).

Table 4.  Factors influencing seroconversion in bivariate analysis and adjusted multivariate analysis
 Bivariate analysis RR (CI 95%)p-ValueMultivariate analysis adjusted OR (CI 95%)p-Value
  1. Parameters were compared statistically in a logistic regression, p ≤ 0.05 was considered significantly different. Number 1 indicates the reference group for each of the variables analyzed.

Sex
 Male1   
 Female0.78 (0.47–1.28)0.330.73 (0.42–1.26)0.26
Age (years)1.00 (0.99–1.02)0.431.01 (0.99–1.02)0.29
Chronic liver disease
 Yes1   
 No0.70 (0.50–1.00) 0.0080.33 (0.13–0.80)0.01
One year since transplant to influenza vaccine
 Yes1   
 No1.20 (1.04–1.40)0.032.07 (0.92–4.66)0.07
Hypogammaglobulinemia
 Yes1   
 No1.03 (0.53–2.00)0.920.86 (0.42–1.75)0.67
Cyclosporine
 Yes1   
 No0.93 (0.54–1.6)0.800.92 (0.50–1.67)0.78
Mycophenolate mofetil
 Yes1   
 No1.03 (0.57–1.84)0.910.70 (0.34–1.41)0.32
m-TOR inhibitors
 Yes1   
 No0.90 (0.70–1.10)0.210.55 (0.26–1.17)0.12
Corticosteroids longer than 20 days
 Yes1   
 No1.44 (0.86–2.27)0.161.26 (0.71–2.23)0.42
Type of transplant 0.94 0.83
 Liver1   
 Kidney1.12 (0.59–2.11) 0.97 (0.46–2.04) 
 Heart0.96 (0.50–1.85) 1.1 (0.50–2.40) 
 Lung1.01 (0.48–2.11) 1.05 (0.45–2.43) 
 Combined transplantation0.55 (0.08–3.5)0.530.38 (0.65–2.2)0.28

Other factors analyzed, such as sex, HG, use of cyclosporine, mycophenolate mofetil or corticosteroids for longer than 20 days, type of transplant, recent induction therapy, comorbidities or previous influenza vaccines were not related with the immune response to vaccination (Tables 3–5). No differences were found in relation to the type of m-TOR inhibitor used.

Clinical response to influenza vaccine

Four of the patients receiving the vaccine (1.1%) were diagnosed with pandemic influenza A H1N1-2009 infection in the follow-up period. All of them were kidney transplant recipients older than 36 years and 3 of them required hospital admission. The median time to the onset of symptoms after receiving the vaccine was 13 days (range 8.0–35.0). Although none of the infected patients had low immunoglobulin levels at the time of vaccination, none of them developed an antibody response 5 weeks after vaccination. All patients exhibited fever but no cases were complicated with pneumonia or bacterial coinfection. Two patients had coinfection with respiratory syncitial virus B. All patients had a favorable outcome, with fever duration of 2 days (range 1.0–5.0).

Adverse effects

No rejection episodes or major type of adverse effects were detected after pandemic vaccination.

Discussion

This study demonstrates an adequate immune response to the pandemic vaccine in this population, although lower than that for the general population (21), in agreement with those with seasonal influenza vaccine (9).

Although it has been documented that elderly have worse response to vaccination (22), in our study SOTR older than 65 years patients receiving the pandemic vaccine responded similarly to the rest of the age groups. It has been proposed that a preexisting cross-reactive antibodies exist in persons over 60 years for the pandemic influenza A H1N1, perhaps due to previous vaccines or influenza infection exposure (23).

A recent clinical trial showed that a single dose of the pandemic vaccine induced a protective immune response in healthy individuals between 12 and 60 years of age (21). In this study, a single dose of the pandemic vaccine was used with an adequate response, although lower than in healthy individuals. Although a possible option for improving the immune response in the SOTR population might be the use of a booster dose, data regarding boosting with the seasonal vaccine in the SOTR population and pandemic vaccine in healthy individuals showed no improvement in the vaccine response (10). However, we observed that patients with baseline antibody titers had better GMT-post after pandemic vaccination suggesting a possible boosting effect.

Pandemic vaccine safety and its stimulation of a protective immune response have been widely demonstrated in healthy individuals (21). However, a controversy concerning efficacy and safety and the theoretical risk of allograft rejection triggered by the immune response to the influenza vaccine has been raised. Most studies involving SOTR, found no link between vaccination with seasonal influenza vaccine and rejection episodes (6). Our results agree with those because no adverse effects or rejection episodes were found in a large series of SOTR recipients receiving the pandemic vaccine. However, episodes of acute allograft rejection have been related with seasonal influenza virus infection (4,24) and pandemic influenza virus infection (25). In these circumstances, the benefits of the vaccine outweigh the potential risk of infection in SOTR and support the administration of the vaccine in this population.

Although antibody production was significantly decreased in SOTR receiving the seasonal influenza vaccine in less than 6 months after transplantation (9), in our study all patients had a similar response, including those within 2–6 months of transplantation. These results may have clinical relevance as influenza infection during the early posttransplant is associated with a worse outcome and therefore an effort should be made to prevent it (6).

Immunosuppression regimens have been associated with lower overall response rates to vaccination in SOTR (9,10). Although chronic kidney disease has been previously associated to a worse vaccination response (9), we found no association in our study. In this study, a decreased immune response was independently associated with chronic liver disease, younger age and mTOR inhibitor therapy.

The group of patients receiving an m-TOR inhibitor had lower seroprotection rates and lower GMT-post, with antibody titers nearly half of those in patients receiving other drugs. This may be explained by the fact that the m-TOR inhibitors act by blocking the proliferation of antigen induced T cells dependent on IL-2 thus blocking the activation of B and T lymphocytes, and interfere in antigenic recognition through the inhibition of antigen presenting cell maturation and therefore, reduce the antibody response (12,13). In a recent study of renal transplant recipients, the use of m-TOR inhibitors induced increased numbers of regulatory T cells (Treg) in peripheral blood (26). Thus, it has been suggested that the accumulation of Treg cells diminishes CD4+ T-cell activation and thus the ability to mount a robust immune response to primary viral infections in aged mice (27). Paradoxically, recent studies performed in mice have shown that the inhibition of m-TOR promotes T-cell immunity by enhancing the generation of CD8+ memory T cells (14–16). Although these results may look contradictory, a possible mechanistic explanation has been suggested by Rao et al. They propose that rapamycin does not inhibit the IFN-γ production during the primary phase (first 72 h) of antigen stimulation (16). Thus, IFN-γ is available for activating T-cell proliferation and differentiation after antigenic stimulation that leads to the generation of effector cells and, ultimately, to the generation of CD8+ memory T cells. In this context, m-TOR may be acting as an integrator of signals on metabolic demand regulating the further effect on memory differentiation (13,16). This observation may have more biological sense and clinical implications, such as the strategic use of m-TOR inhibitor. Further studies will be necessary to respond to this question.

In conclusion, this is the first study describing a decreased immune response after vaccination in patients receiving mTOR inhibitors. New strategies might be evaluated in this population. The m-TOR inhibitor treated patients may be evaluated as candidates for booster vaccination. Further studies are necessary to address this question. Overall, pandemic H1N1–2009 vaccination was not associated with major adverse effects or rejection episodes and therefore, the use of the pandemic H1N1–2009 vaccine could be recommended in SOTR early after the transplant.

Acknowledgments

We thank Michael McConnell for critical reading of the manuscript and Dr. Pilar Pérez Breña and Inmaculada Casas for supplying reagents and Dr. Ana Delgado for their assistance. This work was supported by the Fondo de Investigación Sanitaria (PI050226/2005 and PI060521/2006); Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III (GR09/0041) and Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III – co-financed by European Development Regional Fund “A way to achieve Europe” ERDF, Spanish Network for the Research in Infectious Diseases (REIPI RD06/0008/0000).

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation. The manuscript was not prepared or funded by a commercial organization.

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