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Alemtuzumab recipients trended toward lower seroconversion rates (25% vs. 51%; p= 0.11). No ... or alemtuzumab or interleukin 2 (IL-2) receptor antagonist.
Cell Transplantation, Vol. 22, pp. 469–476, 2013 Printed in the USA. All rights reserved. Copyright  2013 Cognizant Comm. Corp.

0963-6897/13 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368912X656135 E-ISSN 1555-3892 www.cognizantcommunication.com

Humoral Immune Response Following Seasonal Influenza Vaccine in Islet Transplant Recipients Moacyr Silva, Jr.,* Atul Humar,* A. M. James Shapiro,* Peter Senior,* Katja Hoschler,† Aliyah Baluch,* Leticia E. Wilson,* and Deepali Kumar* *Alberta Transplant Institute, University of Alberta, Edmonton, Alberta, Canada †Health Protection Agency, Center for Infections, London, UK

Annual influenza vaccine is recommended for organ transplant recipients, but immunogenicity is known to be suboptimal. Islet transplant recipients receive immunosuppressive therapy, but there are no data on the immunogenicity of influenza vaccine in this population. In this prospective cohort study, adult islet transplant recipients at least 3 months posttransplant were enrolled. All patients received the 2010–2011 seasonal influenza vaccine. Serum was obtained pre- and postvaccination to determine humoral response to each of the three influenza strains included in the vaccine. Adverse effects of vaccine were also noted. A total of 61 islet transplant recipients were enrolled and completed the study protocol. The median time from last transplant was 1.9 years (range 0.26–11.4 years), and most patients had undergone multiple prior islet transplant procedures (90.2%). Overall immunogenicity of the vaccine was poor. Seroconversion rates to H1N1, H3N2, and B antigens were 34.4%, 29.5%, and 9.8%, respectively. In the subset not seroprotected at baseline, a protective antibody titer postvaccination was achieved in 58.6%, 41.9%, and 34.5% of patients, respectively. Patients within the first year of transplant were significantly less likely to seroconvert to at least one antigen (23.5% vs. 54.5%; p = 0.029). Alemtuzumab recipients trended toward lower seroconversion rates (25% vs. 51%; p = 0.11). No vaccine-related safety concerns were identified. Seasonal influenza vaccine had suboptimal immunogenicity in islet transplant recipients especially those who were less than 1 year posttransplant or had received alemtuzumab induction. Novel strategies for protection in this group of patients need further study. Key words: Immunogenicity; Immunosuppression; Infection

INTRODUCTION Seasonal influenza infections cause significant mor­ bidity and mortality in transplant recipients (14). Influ­enza is an RNA virus that is classified into three subtypes: A, B, and C. Subtypes A and B cause the majority of human disease. Although most infections remain in the upper respiratory tract, up to one third of transplant patients can develop pneumonia requiring hospitalization (10,14). In addition, transplant recipients have high tissue viral loads and shed virus for prolonged periods (13). Current guidelines recommend annual influenza vaccination with a trivalent inactivated vaccine for all transplant recipients (4,15). The vaccine consists of two influenza A strains based on the surface glycoproteins, hemagglutinin (H) and neuraminidase (N), as well as a B strain. Seroresponses (i.e., seroprotection or seroconversion) to seasonal influenza vaccine in organ transplant recipients have been quite variable and

range from 15% to 93% for at least one vaccine virus strain, but in the majority of studies is suboptimal (12). This variability is likely due to type of transplant studied, time from transplant, and various immunosuppressive regimens. Allogeneic pancreatic islet transplantation has been used successfully as a therapy for patients with poorly controlled type 1 diabetes mellitus for over a decade. With current immunosuppressive protocols, insulin independence rates at 5 years posttransplant are more than 50% (20). Nevertheless, given the intense immuno­suppression required to control both auto- and allo-immunity, patients remain at risk of complications of immunosuppression including viral infections such as influenza. There are no published data with regard to influenza vaccine immunogenicity in patients who have undergone islet transplantation. Islet transplant patients are a unique population to study the immune response to vaccination, since they receive a cellular rather than organ

Received September 15, 2011; final acceptance April 5, 2012. Online prepub date: September 21, 2011. Address correspondence to Deepali Kumar, M.D., M.Sc., FRCPC, Alberta Institute for Transplant Sciences, University of Alberta, 6-030 Katz Center for Health Research, Edmonton, Alberta, Canada T6G 2E1. Tel: +1 (780) 492-3885; Fax: +1 (780) 492-4805; E-mail: [email protected]

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transplant, undergo induction using various immunosuppressive regimens, and can often undergo multiple transplant procedures. Commonly used immunosuppressant agents used for induction include antithymocyte globulin (ATG) or alemtuzumab or interleukin 2 (IL-2) receptor antagonist (daclizumab or basiliximab). In addition to one of these, patients may also receive an anti-tumor necrosis factor (TNF) agent such as etanercept or infliximab. Maintenance immunosuppressive strategies usually include tacrolimus and mycophenolate mofetil (MMF) or sirolimus. There are little comparative data in the literature evaluating vaccine responses in patients that receive these induction regimens or combinations of induction regimens. The aim of the present prospective cohort study was to assess seasonal influenza vaccine immunogenicity and safety in a cohort of islet transplant recipients receiving tacrolimus and MMF. materials and METHODS Patient Population This was an observational cohort study conducted at the University of Alberta Hospital. The study was approved by the Health Research Ethics Board, and all study participants gave written informed consent. Adult islet transplant recipients were identified and recruited from outpatient clinics in August 2010. Patients currently enrolled in NIH trials of islet transplantation were excluded. To be eligible, patients were at least 3 months from their most recent transplant and receiving at least one immunosuppressive medication. For transplant, all patients received induction immunosuppression that consisted of either an IL-2 receptor antagonist (daclizumab 2 mg/kg at days 0 and 5 or basiliximab 20 mg on days 0 and 4), antithymocyte globulin (ATG), or alemtuzumab. These were given with or without a TNF-a antagonist (etanercept 50 mg weekly or infliximab 10 mg/kg given at time of transplant). Patients also received maintenance immunosuppression with a combination of tacrolimus (to maintain a trough of 8–10 ng/ml) and MMF. Patients had to be willing to receive the influenza vaccine and have no contraindications to vaccination such as egg allergy or history of vaccine-related Guillain–Barre syndrome. The annual influenza vaccine Fluviral® (GlaxoSmithKline, Canada) was available to all transplant patients in October 2010, and study participants received vaccine at their local public health unit. The 2010–2011 influenza vaccine contained 15 mg each of the following virus strains: A/California/7/2009 (H1N1)-like strain, A/Perth/16/2009 (H3N2)-like strain, and B/Brisbane/60/2008. The vaccine dose was administered as a 0.5-ml intramuscular injection in the deltoid muscle. Patients were vaccinated between October 2010 and January 2011. Venous blood samples were collected prevaccination and at 4–6 weeks postvaccination.

Baseline demographic and transplant data were collected for each patient. For adverse event reporting, patients were contacted at 48 h postvaccination, and followed until 6 months postimmunization for the development of microbiologically documented influenza infection. Laboratory Methods Sera collected pre- and 4–6 weeks postvaccination were stored at –80°C until testing. All serum samples were tested for antibodies against the three influenza strains present in the vaccine using the hemagglutination inhibition assay (HAI). The test was performed at the Microbiology Services, Colindale, Health Protection Agency, UK, using methods previously described (6). Briefly, serum was incubated with a receptor-destroying enzyme to remove any nonspecific inhibitors of hemagglutination. Serially diluted sera were then incubated with influenza virus (containing 4 hemagglutination units of virus) followed by addition of 0.5% turkey red blood cells. Titers were determined by doubling dilutions of antibody. All sera were tested in duplicate. The lower limit of detection of the assay was a dilution of 1:10. Definitions The primary endpoints were based on criteria defined by the EMEA (European Agency for Evaluation of Medicinal Products) and the US Food and Drug Admi­ nistration for use in trials assessing influenza vaccine immunogenicity (7,22). Primary endpoints included seroprotection defined as a HAI titer of ≥1:40 postvaccina­ tion and seroconversion, defined as a fourfold increase in HAI titers from pre- to postvaccination and achieving a titer of ≥1:40. For statistical analysis, a titer reported as less than 10 was assigned a value of 5. Statistical Analysis Geometric mean titers (GMTs) of strain-specific antibody and mean seroconversion factors were compared between baseline and postvaccination using a Wilcoxon rank-sum test for paired data. Seroconversion and seroprotection rates were compared between baseline and postvaccination using the McNemar test. The seroconversion factor or the fold increase in titer was calculated by dividing the postvaccination titer by the pre­vaccination titer. A geometric mean of the seroconversion factor for each influenza strain was then calculated, termed the geometric mean fold rise (GMFR). Multivariate analysis using a forward conditional method was done for factors affecting seroconversion to at least one antigen. Variables were included that had a value of p 60 Gender (male/female) Number of islet transplants 1 2 3 4 Time from most recent transplant to ­vaccination (years; median and range) Induction therapy at most recent transplant IL2RA-based Antithymocyte globulin-based Alemtuzumab-based Belatacept TNF-a antagonist (in combination with above) Maintenance immunosuppression Tacrolimus MMF Sirolimus Insulin use

N = 61 (%) 53.8 (32.6–72.5) 18 (29.5) 34/27 6 (9.8%) 36 (59%) 13 (21.3%) 6 (9.8%) 1.9 (0.26–11.4)

31 (50.8%) 17 (27.9%) 12 (19.7%) 1 (1.6%) 30 (49.2%)

58 (95.1%) 56 (91.8%) 1 (1.6%) 37 (60.7%)

IL2RA, interleukin 2 receptor antagonist; TNF, tumor necrosis factor; MMF, mycophenolate mofetil; SD, standard deviation; IQR, interquartile range.

a TNF-a antagonist, either etanercept or infliximab, in addition to the induction therapy. For long-term immuno­ suppression, most patients were prescribed a combination of tacrolimus and MMF. Only one patient received sirolimus. This patient was transplanted several years ago when the immunosuppression protocol for islet transplantation was sirolimus based. The sole kidney-islet recipient in the cohort was also receiving prednisone 5 mg daily. Immunosuppressant use remained stable from vaccination to postimmunization sample collection. Supplementary exogenous insulin was being used to ensure excellent glycemic control in 37 (60.7%) recipients. Almost all patients (60/61, 98.4%) had previously received influenza vaccine. No patient had developed microbiologically documented influenza infection by the 6 months of follow-up. Vaccine Response Seroprotection and seroconversion rates for each of three influenza strain contained in the vaccine are shown in Table 2. GMTs for all strains increased significantly after vaccination. Postvaccination seroprotection rates ranged from 67.2% to 78.7%. Seroconversion rates were lower and ranged from 9.8% to 34.4%. Seroconversion to at least one antigen was seen in 28/61 (45.9%) of patients. Of these, 14 (23%) patients seroconverted to one antigen only, 11 (18%) patients seroconverted to two antigens, and only 3 (4.9%) patients seroconverted to all three vaccine antigens. Older age defined as >60 years also negatively affected some vaccine responses; for example, patients of age >60 were less likely to be seroprotected with H1N1 strain (61.1% vs. 86.0%; p = 0.043) and B strain (44.4% vs. 76.7%; p = 0.014) and also less likely to seroconvert with H3N2 (11.1% vs. 37.2%; p = 0.042). Absolute pre- and postimmunization titers and seroprotection and seroconversion rates by induction immunosuppressive are shown in Figures 1 and 2. For statistical purposes, the induction agent assigned to a patient was the last one they received. The single patient who received belatacept was excluded from this analysis. There were no demographic differences in the three immunosuppressive groups. The patients classified as receiving IL-2 receptor antagonist had no previous exposure to alemtuzumab. Overall postimmunization seroprotection rates did not significantly differ between the three groups; however, the seroconversion rate to at least one antigen tended to be lower in those that received alemtuzumab versus other induction [3/12 (25%) with alemtuzumab vs. 25/49 (51%) with other induction; p = 0.11]. The geometric mean fold rise (GMFR or seroconversion factor) was significantly lower for H1N1 in those that had received alemtuzumab versus other induction therapy (1.33 vs. 3.10; p = 0.025) (Fig. 3). No significant differences in seroresponse were found in those who also received a TNF-a antagonist.

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Table 2.  Geometric Mean Titers and Seroprotection/Seroconversion Rates to Vaccine Strains (N = 61)

GMT preimmunization GMT postimmunization Seroprotection preimmunization Seroprotection rate postimmunization Seroconversion rate Seroconversion factor (geometric mean fold rise; GMFR)

A/H1N1

A/H3N2

B

30.4 80.0* 32 (52.5%) 48 (78.7%)** 21 (34.4%) 2.63

21.7 41.4* 30 (49.2%) 41 (67.2%)** 18 (29.5%) 1.91

24.3 35.3* 32 (52.5%) 41 (67.2%)** 6 (9.8%) 1.46

*For postimmunization GMTs, p 60 [OR 1.74 (1.12–29.2); p = 0.036]. No variable was predictive of seroconversion to influenza B.

Figure 3.  Seroconversion factors for influenza A/H1N1 in individual patients grouped by most recent induction immuno­ suppression. Horizontal lines represent geometric mean fold rise (GMFR) for each group. One patient was excluded from the alemtuzumab group to increase clarity. The GMFR for this patient was 0.06. IL2RA, interleukin 2 receptor antagonist; ATG, antithymocyte globulin. *GMFR for alemtuzumab, 1.33 versus 3.10 for other induction (p = 0.025)

within 1 year of transplant, alemtuzumab induction, TNF-a antagonist therapy, MMF >2 g daily, and age >60 years, time from transplant was the only independently significant variable (p = 0.035) (Table 3). There were only nine patients that were within 6 months from last trans­ plant. Of these, only 2/9 (22.2%) seroconverted to at least one antigen. Univariate and multivariate analyses of factors that affect seroconversion to each vaccine antigen were done and included the above factors plus baseline seroprotection for each vaccine strain. In this subanalysis of each serotype, the following variables were significant on multivariate analysis: for H1N1, baseline seroprotection [odds ratio (OR) 1.20 (1.10–10.1); p = 0.03]; for H3N2, time from transplant [OR 1.66 (1.02–27); p = 0.048] and

Baseline Seroprotection Baseline rates of seroprotection (i.e., a strain-specific titer of ≥1:40) ranged from 49.2% to 52.5%, depending on the influenza strain. For the subset of patients that were not seroprotected at baseline, GMTs to each strain increased significantly from baseline to post­ vaccination, although they remained low overall (Table 4). In this cohort, seroprotection rates after immunization ranged from 34.5% to 58.6%, whereas seroconversion rates ranged from 20.7% to 51.7%. The lowest responses were seen for the B strain. Patients who were not seroprotected at baseline had a greater GMFR in antibody titer. This was true for both influenza A strains, H1N1 (1.68 vs. 4.30; p = 0.004) and H3N2 (1.29 vs. 2.80; p = 0.02), as well as influenza B (1.14 vs. 1.91; p = 0.012). Patients who were not protected at baseline were more likely to seroconvert to vaccine antigens. For H1N1, 7/32 (21.9%) with baseline seroprotection converted, whereas 14/29 (48.3%) without baseline seroprotection had a fourfold rise, p = 0.03. For H3N2, 6/30 (20%) with baseline protection seroconverted, whereas 12/31 (38.7%) naive at baseline seroconverted, p = 0.11. We also analyzed whether any variables affected seroprotection and seroconversion rates to each strain for patients not previously seroprotected. These variables included age ≥60 years, time from transplant (2 g daily Baseline seroprotection NS, not significant.

Univariate (Odds Ratio; 95% CI); p Value

Multivariate (Odds Ratio; 95% CI); p Value

0.26 (0.07–0.91); 0.029 0.48 (0.15–1.50); 0.20 0.32 (0.077–1.33); 0.10 1.42 (0.51–3.91); 0.50 0.49 (0.18–1.36); 0.17 0.73 (0.22–2.38); 0.60 N/A

1.36 (1.10–13.9); p = 0.035 NS NS NS NS NS N/A

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Table 4.  Humoral Responses in Subgroup of Patients Who Were Not Seroprotected Prior to Immunization

GMT preimmunization GMT postimmunization Seroprotection rate postimmunization Seroconversion rate Seroconversion factor (GMFR)

A/H1N1 (n = 29)

A/H3N2 (n = 31)

B (n = 29)

7.16 30.8* 17 (58.6%) 14 (48.3%) 3.83

8.74 24.5* 13 (41.9%) 12 (38.7%) 2.80

7.87 15.0* 10 (34.5%) 4 (13.8%) 1.37

*p 70%, seroconversion rate of >40%, and seroconversion factor >2.5 (7). Each antigen must meet at least one of these criteria. For Fluviral, pooled immunogenicity data

in the healthy 18–60 years population over three seasons (2004–2007) show a seroprotection rate of 71–97% and a seroconversion rate of 64–79% (Fluviral product monograph) (9). In contrast, seroconversion rates in this study were 9.8%, 29.5%, and 34.4% for influenza B, H3N2, and H1N1, respectively. In studies on the healthy population, the geometric mean fold rise ranged from 6.8–13.8 compared to 1.46–2.63 in our study. In those with no prior seroprotection, the seroconversion rates ranged from only 13.8% to 48.3%, and seroprotection after vaccination ranged from 34.5% to 58.6%. As noted above, there are no previously published data on vaccine immunogenicity in islet transplant recipients. Previous data in solid organ transplant recipients have indicated response rates of 15–93%, depending on several factors including the organ studied, time from transplant, and use of certain immunosuppressives (12). Previous studies have shown diminished responses in patients receiving mycophenolate mofetil (MMF) (11,18,21). One study found a 2.6- to 5-fold reduction in seroconversion in those receiving more than 2 g daily of MMF versus no MMF (19). Although most of our patients were receiving MMF, we did not find a difference in seroprotection or seroconversion rates for those receiving more than 1 g daily versus less than 2 g. There are little data on the role of different induction regimens and their effect on vaccine response. We previously conducted immunogenicity studies of influenza vaccine in two cohorts of lung transplant recipients (16,17). Responses to one dose of influenza vaccine in the 2007 cohort were significantly better than those in the 2011 cohort. This may have been partly due to the fact that only 30% of patients received any induction therapy in the 2007 cohort whereas 89% received induction in the 2011 cohort. However, maintenance immunosuppression was also different in the two studies (primarily calcineurin-inhibitor/azathioprine in the 2007 cohort and MMF/tacrolimus in the 2011 cohort), making a post hoc comparison difficult. The current study is novel in that provides a direct comparison of vaccine responses using three main induction strategies: IL-2 receptor antagonist, antithymocyte globulin, and alemtuzumab. Our study found significantly lower GMFR for

INFLUENZA VACCINE IN ISLET TRANSPLANT

H1N1 strain in those that received alemtuzumab and a trend to lower seroconversion for this strain. We also addressed timing of influenza vaccine. Current guidelines by the American Society of Transplantation recommend that influenza vaccine be given starting at 3–6 months posttransplant; however, data on vaccine immunogenicity in the early posttransplant period are limited. Studies in organ transplant recipients have been conflicting with one study not demonstrating any difference in response based on time from transplant (3); however, more recently, Birdwell et al. showed diminished responses in kidney transplant recipients who received vaccine at less than 6 months posttransplant (2). Consistent with this observation, our study also showed a diminished seroconversion to at least one vaccine antigen when vaccine was administered within the first year post-transplant (23.5% vs. 54.5%, p = 0.029). We believe that the reason time from transplant is significant is in fact due to the degree of immunosuppression. Specifically, induction therapy likely has a prolonged immunological effect. For example, in a study of islet transplant patients, this effect was recently shown with CMV reactivation in patients receiving induction therapy (8). Preimmunization vaccine titer was also an important predictor of the response to vaccination. Almost half the cohort had a strain-specific titer ≥1:40 prior to vaccination, and this group was less likely to have a significant response to vaccination. High baseline seroprotection rates lead to difficulty in analysis of vaccine response and have led to the use of the additional criteria of seroconversion rate and seroconversion factor (1,23). Therefore, we performed a separate analysis of the subgroup that was not protected prior to vaccination. Other studies of influenza vaccination in the organ transplant population have also found high baseline seroprotection rates, where up to 78% of subjects are already seroprotected prior to vaccination (19). Baseline seroprotection was also found to be a significant factor for vaccine response in another study of organ transplant recipients, where patients whose preimmunization titer was ≥1:40 had significantly less seroconversion (p