Influenza infection carries serious morbidity and mortality implications in vulnerable patient populations such as organ transplant recipients (1, 2). Because of immunosuppression- induced T- and B-cell immunodeficiency, these patients are considered to be at higher risk than healthy individuals for serious progression of influenza, possibly culminating in pneumonia (3). In the past decades, a considerable body of evidence has been acquired attesting to the safety and efficacy of vaccination against seasonal influenza in heart transplant recipients (4–11). This is reflected in guideline recommendations (12). With the emergence of the novel influenza A (H1N1) virus strain in April 2009 (1, 13, 14) and its rapid spread to a pandemic (15), an urgent need for new vaccines had arisen (16). It was suspected that the H1N1 strain would be the most dominant and dangerous strain of the 2009/2010 influenza season. Thus, public health authorities and medical societies advocated the immediate administration of an A/H1N1 pandemic influenza vaccine (17, 18). However, different pandemic vaccines, both adjuvanted and nonadjuvanted, have been developed (16, 19–21).
There is an unmet clinical need to specify vaccination regimens against the novel influenza A (H1N1) in high-risk groups such as immunosuppressed patients (18). To date, no data on the antibody response, adverse-effect profiles, and optimal doses of the new pandemic vaccines are available for organ transplant recipients. We sought to assess the serologic efficacy and general tolerability of a novel AS03-adjuvanted A/H1N1 vaccine in heart transplant recipients.
Prevaccination Antibody Levels
Of the 47 patients enrolled in the study, no patient exhibited a titer of greater than or equal to 1:40 on hemagglutination-inhibition (HI) testing at baseline. In contrast, eight patients (17%; 95% confidence interval [CI], 8%–31%) were found to have a baseline IgG concentration of more than 11 Virotech units (VU) on enzyme-linked immunosorbent assay (ELISA) testing.
Antibody Response to A/California/7/2009 (H1N1) Vaccination
At 20±2 days, vaccination had caused an HI antibody titer increase from less than 1:10 to greater than or equal to 1:40 in 13 patients and a greater than fourfold increase from baseline titers greater than or equal to 1:10 in 2 patients, for an overall seroconversion rate of 32% (15 of 47 patients; 95% CI, 19%–47%). As all 15 patients had a postvaccination titer greater than or equal to 1:40, the seroprotection rate was also 32%.
ELISA testing revealed a statistically significant increase of IgG concentration from 8.2±2.9 VU at baseline to 12.0±4.7 VU postvaccination (P<0.001) in the 47 patients. A total of 22 patients had an IgG concentration more than 11 VU.
The ELISA results in relation to the HI antibody test results are shown in Figure 1. Regarding HI antibody testing as the diagnostic reference, sensitivity, specificity, positive predictive value, and negative predictive value for the ELISA test were 80.0%, 68.8%, 54.5%, and 88.0%, respectively.
Variables Affecting the Primary Endpoint
Dichotomizing patients by age revealed seroconversion rates of 31% (8 of 26; 95% CI, 14%–52%) in patients younger than 60 years and 33% (7 of 21; 95% CI, 15%–57%; P>0.9999) in patients aged 60 years or older. With regard to time posttransplantation, seroconversion was observed in 9 of 22 patients on immunosuppression for less than 3 years (41%; 95% CI, 21%–64%) and in 6 of 25 patients on immunosuppression for 3 years or longer (24%; 95% CI, 9%–45%; P=0.347).
Univariate logistic regression analyses showed that age, time posttransplantation, or the immunosuppressive regimen (calcineurin inhibitor vs. everolimus based) did not significantly affect the primary endpoint (Table 1).
Testing all three variables simultaneously by multivariate regression analysis also revealed no independent effect of either variable on the primary endpoint (Table 1).
At 4 months of follow-up, no patient reported any influenza-like symptoms. No influenza-related hospitalizations and deaths were observed.
Adverse Effects of Vaccination
The AS03-adjuvanted vaccine emulsion was generally well tolerated. At 4 months, a median of 2 (range, 1–9) minor adverse effects had been reported by a total of 32 patients (68%; 95% CI, 63%–81%). Most frequently reported effects were fatigue (15 patients [32%]), tiredness (13 patients [28%]) and myalgia (14 patients [30%]). Serious adverse effects such as seizures, palsy, vasculitis, or allergies were not reported. Relevant (International Society for Heart and Lung Transplantation [ISHLT] 2R) rejections occurred in two patients at 1 and 3 months, respectively.
The results of our study demonstrate that a single 3.75 μg hemagglutinin (HA) dose of the AS03-uvanted pandemic influenza A (H1N1) vaccine Pandemrix elicits seroprotection— defined by a postvaccination antibody titer greater than or equal to 1:40—in only 32% of heart transplant recipients, with the 95% CI ranging from 19% to 47%. Thus, according to the current requirements for efficacy of influenza vaccines, which demand a seroprotection rate of more than 70% (22, 23), a single-dose vaccination using Pandemrix must be considered as ineffective.
The immunogenicity of Pandemrix observed in our study must be evaluated in relation to the European and U.S. acceptance criteria for influenza vaccines (24, 25). Both demand a seroprotection rate more than 70% and a seroconversion rate more than 40%, with the U.S. acceptance criteria also demanding the lower limit of the 95% CI for seroconversion to be greater than or equal to 40%. Both requirements were not met in our patients. Hence, the immunogenicity of a single dose of Pandemrix against novel influenza A (H1N1) virus in heart transplant recipients is relatively poor. It turned out to be markedly less effective than in healthy subjects, in whom single-dose vaccination with Pandemrix has been demonstrated to achieve a seroconversion rate of 98.2% in 56 healthy vaccinees (20). Our study implies that, with the future emergence of a novel, more virulent and dangerous than the existing H1N1 strain, a second-dose vaccination should be considered in heart transplant recipients. However, sufficient enhancement of immunogenicity by second-dose vaccination against novel influenza A (H1N1) virus still needs to be confirmed in immunosuppressed patients.
Given the lack of long-term safety data on AS03-adjuvanted vaccines and following current local public health advisories, we did not recommend second-dose vaccination.
HI Antibody Response to Vaccination in Comparison With Other Studies
Our results are in accordance with previous studies in heart transplant recipients, indicating for different influenza strains a slower and impaired antibody response to vaccination than in healthy controls (3, 8, 22). However, protective antibody levels have been reported after single-dose vaccination against seasonal influenza (4, 22).
The 32% seroprotection rate observed in our patients is substantially less than the 56% reported for the A/Singapore (H1N1) strain by Dengler et al. (8) and far less than the 100% reported by Fraund et al. (22). This variation in antibody response might be caused by the different strains analyzed and the diverse vaccine preparations used. The utilization of adjuvants is an established concept in vaccination (23). Although a superiority of adjuvanted vaccines could not be demonstrated for seasonal influenza in a previous trial with heart transplant recipients (7), adjuvanted vaccines have proven effective for the novel influenza A (H1N1) virus in healthy adults (3, 16, 20, 21). In most studies, MF59-adjuvanted vaccines were used, whereas in our study, AS03 served as the adjuvant. To our knowledge, there have been no studies on the use of AS03-adjuvanted vaccines in heart transplant recipients.
With respect to second-dose vaccination, current data are inconclusive. Multiple-dose vaccinations have proven effective in heart transplant recipients and significant titer increases have been shown after first-dose vaccination for the A/H1N1/Singapore virus strain (3). Data from other solid-organ transplant recipients are contradictory: some showed effectiveness of booster immunization (26) and others did not demonstrate titer increases (5, 27). However, the optimal vaccination regimen regarding the novel influenza A (H1N1) virus still remains to be elucidated.
Comparison of IgG Antibody Response on ELISA With HI Antibody Titers
When compared with the diagnostic reference of HI antibody testing, the new pandemic influenza A IgG ELISA test kit proved to be of limited clinical use. Although 12 of the 15 responders to vaccination were correctly identified on ELISA (corresponding to a sensitivity of 80%), the incidence of false-positive ELISA results was unacceptably high. All our patients were seronegative prevaccination, yet ELISA falsely identified 8 patients (17%) as seropositive. Postvaccination, the positive predictive value of ELISA testing was only 54.5%, which essentially translates into a 50:50 chance of correctly identifying a responder to vaccination. False-positive results also affect the specificity, that is, the “ability” of the test to correctly identify nonresponders; it was only 69% in our patient cohort.
Variables Affecting Antibody Response to Vaccination
The physiologic age-related slowed immune response of elderly subjects (23) has translated into lower seroconversion rates after second-dose vaccination against influenza A/California/7/2009 (H1N1) in healthy adults older than 60 years (28). Our study included a substantial proportion of elderly immunosuppressed patients. Yet, we could not prove age to be a variable significantly affecting vaccination success after single-dose vaccination. Also, the time posttransplantation and the immunosuppressive regimen (calcineurin inhibitor-based vs. everolimus-based) did not show an effect on the antibody response to vaccination. Previous studies have reported different effects of immunosuppressants on influenza-vaccine-induced antibody response in transplanted patients, with a dampening effect of cyclosporine A (29) and mycophenolate mofetil (27), and a neutral (30) or even enhancing (31) effect of proliferation signal or mammalian target-of-rapamycin inhibitors. There is encouraging preclinical evidence that the latter agents have enhancing effects on memory T cells (32), which are considered essential for acquiring immunity to infections (33).
We did not observe any serious adverse effects such as myalgia or allergies. The allograft rejections observed in two patients were hemodynamically not relevant (ISHLT 2R). These observations are consistent with recent studies (4, 7, 8, 22).
We realize that the number of patients studied is fairly small. This may have affected in particular our analyses of variables affecting the primary endpoint. However, former studies assessing seasonal influenza vaccination enrolled similarly small numbers of heart transplant recipients (3, 7, 8, 22). A further limitation of our study is the observational nonrandomized design. Because immunosuppressed patients are known to have a slowed immunization response, it might be speculated that a repeat serologic assessment 6 weeks after vaccination might have shown a greater increase in IgG concentration. Finally, female patients are underrepresented in our study.
This study provides first data on the HI antibody response and adverse-effect profile of a single dose of an AS03-adjuvanted split-virion influenza A (H1N1) vaccine in heart transplant recipients. It showed that a substantial proportion of these patients did not exhibit seroprotective antibody concentrations after single-dose vaccination. The new pandemic influenza A IgG ELISA test kit proved to be an imprecise tool for the assessment of antibody response.
MATERIALS AND METHODS
Data acquisition was performed in a single-center, observational, nonrandomized and noninterventional fashion. From November 1 to December 31, 2009, all patients of our outpatient clinic in routine posttransplantation care were informed about the vaccination campaign against the ongoing influenza A (H1N1), which advised vaccination according to the latest recommendations of local German public health authorities and the ISHLT advisory (18).
During the 2-month enrollment phase, 101 adult heart transplant recipients presented for routine care at our institution. Of these, 47 patients (44 men; 56±13 years) consented to participate in the study. They were all in stable clinical condition, beyond 6 months after transplantation (with 47% of patients for <3 years, and the remainder for at least 3 years, on an immunosuppressive regimen at the time of vaccination), free from infection, and lacked echocardiographic signs or biopsy proof of allograft rejection. No patient had received prior pandemic influenza vaccination or reported a history of signs and symptoms suggestive of the influenza syndrome (i.e., fever, sore throat, cough, malaise, myalgia). Pertinent patient characteristics are listed in Table 2. Of note, the study comprised patients with different regimens of maintenance immunosuppression, including 24 patients (51%) with everolimus-based immunosuppression.
The 47 patients underwent a baseline visit at our outpatient clinic. We performed routine clinical assessment including echocardiography and blood sampling for clinical chemistry and immunosuppressant trough blood levels. A separate serum sample was stored at −80°C to determine baseline antibody titers. Thereafter, the patients were referred to public vaccination sites to get vaccinated. All patients underwent vaccination using the pandemic influenza vaccine (H1N1) (split-virion, inactivated, adjuvanted) A/California/7/2009 (H1N1)v like strain (X-179A) (Pandemrix, GlaxoSmithKline Biologicals S.A., Rixensart, Belgium) (34). The single intramuscular dose of 0.5 mL vaccine consisted of 3.75 μg HA inactivated split influenza virus plus the adjuvant AS03 (11.86 μg of dl-α-tocopherol, 10.69 μg of squalene, and 4.86 μg of polysorbate). This was the only vaccine preparation available in Germany at that time.
Patients were asked to return for a repeat assessment of antibody response 21 days after vaccination; a postvaccination serum sample was stored −80°C to determine antibody titers. Clinical follow-ups up to 4 months were scheduled on an individual basis as routine practice. Screening for acute rejection episodes comprised echocardiography, immunosuppressant trough blood levels, cytomegalovirus infection status, and clinical assessment. After vaccination, special emphasis was placed on the assessment of symptoms such as fever, sore throat, cough, malaise, and myalgia as correlates for an influenza syndrome. Adverse effects of vaccination were addressed by means of a custom-made questionnaire asking for incidence of flu-like illness, proven diagnosis of H1N1 infection, irritation at the injection site, fever of more than 38°C, fatigue, swollen lymph nodes, tiredness, headache, paresthesia, dizziness, insomnia, abdominal discomfort, arthralgia, myalgia, seizures, palsy, vasculitis, allergy, or unforeseen hospitalizations from any cause.
Both baseline and postvaccination serum samples were analyzed by a validated microtiter HI test as previously described (35), with the A/California/7/2009 strain used as antigen. Before testing, each serum was treated with receptor-destroying enzyme to inactivate non-specific inhibitors, achieving a final serum dilution of 1:10. Sera were then diluted serially twofold into microtiter plates. The live virus was adjusted to 4 HA units/25 μL, which was verified by back titration; 25 μL of this virus suspension was added to each of the 96 wells. After incubation at room temperature for 30 min, freshly prepared 0.5% turkey red blood cells were added; the plates were mixed for agitation followed by a further incubation at room temperature for 30 min. Human sera serving as positive controls, an international pandemic H1N1 serum standard received from the National Institute for Biological Standards and Control, London, UK, and negative controls were included on each plate. The determination of the HI titer was performed by identification of the reciprocal of the last serum dilution that contained nonagglutinated red blood cells.
Samples were also examined using a novel pandemic new influenza A IgG ELISA test kit (Genzyme Virotech, Rüsselsheim, Germany) that became available in Germany in January 2010. It provides a semiquantitative measurement of specific immunoglobulin G (IgG) antibodies against the influenza A/California/7/2009 (H1N1) virus. Based on a spectrophotometric analysis of absorbance values at 450 nm, with a reference value of 620 nm, the test provides optical densities that reflect concentrations of IgG antibodies in so-called Virotech units (VU), with VU less than 9 representing no relevant antibody concentrations and VU greater than 11 representing significantly increased antibody concentrations (VU between 9 and 11 represent “borderline” [inconclusive] antibody concentrations). The processing of samples and the performance of test procedures were performed according to the manufacturer's instructions.
HI antibody testing and ELISAs were performed at the Robert Koch Institute, Berlin, Germany, and the Department of Medical Microbiology, Virology and Hygiene of the University Medical Center Hamburg-Eppendorf, Hamburg, Germany, respectively.
The primary endpoint was seroprotection on HI testing, defined by a postvaccination titer greater than or equal to 1:40.
Secondary endpoints comprised:
- Seroconversion on HI testing, defined by a postvaccination titer greater than or equal to 1:40 in patients with a prevaccination titer less than 1:10 or by an at least fourfold postvaccination titer increase in patients with a prevaccination titer greater than or equal to 1:10;
- A relevant antibody response as defined by VU more than 11 on ELISA testing;
- Variables affecting the primary endpoint, specifically, patient age, time posttransplantation (i.e., time on immunosuppression), and immunosuppressive regimen;
- And a comparison of ELISA and HI test results.
Continuous variables are presented as mean±SD or median plus range, where appropriate. Nominal variables are presented as counts and percentages. Exact 95% CIs based on a binomial distribution were calculated for proportions. Comparisons of continuous variables were performed by Student's t test for paired samples. The effect of patient age, time posttransplantation, and dichotomized immunosuppressive regimen on the primary endpoint was assessed by univariate and multivariate logistic regression analysis. All statistical tests were performed using SPSS software (version 17.0). A two-sided P value less than 0.05 was considered statistically significant.
The study was approved by the local Ethics Committee and was conducted according to the Declaration of Helsinki. Patients provided written informed consent before enrollment.
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