Impact of Adjuvanted H1N1 Vaccine on Cell-Mediated Rejection in Heart Transplant Recipients

During the H1N1 influenza virus pandemic, vaccination of high risk groups including solid-organ transplant recipients was advised. A retrospective case control study of 60 heart transplant patients, 15 having received the H1N1 virus antigen and ASO3 adjuvant vaccine (GlaxoSmithKline, Mississauga, ON, Canada) within 21 days and 45 having not been vaccinated, all undergoing routine surveillance endmyocardial biopsies, was performed. The overall rate of cellular rejection (all grades) was not statistically different between groups; however, acute cellular rejection, ≥grade 2 (1990 ISHLT criteria), was more frequent among those having recently vaccinated (control: 1/45 vs. 6/15, p = 0.001). On multivariate analysis, the only risk factor found to be associated with acute cellular rejection was recent H1N1 viral antigen and adjuvant vaccination (OR 26.5: 95% CI 02.59–270.5). Vaccine adjuvants increase host response to vaccine antigens by immune upregulation potentially increasing risk of rejection in solid-organ transplant recipients. The potential hazard of vaccination this study raises must be weighed with the clear benefit vaccination has proven to be. During the H1N1 influenza virus pandemic, vaccination of high risk groups including solid-organ transplant recipients was advised. A retrospective case control study of 60 heart transplant patients, 15 having received the H1N1 virus antigen and ASO3 adjuvant vaccine (GlaxoSmithKline, Mississauga, ON, Canada) within 21 days and 45 having not been vaccinated, all undergoing routine surveillance endmyocardial biopsies, was performed. The overall rate of cellular rejection (all grades) was not statistically different between groups; however, acute cellular rejection, ≥grade 2 (1990 ISHLT criteria), was more frequent among those having recently vaccinated (control: 1/45 vs. 6/15, p = 0.001). On multivariate analysis, the only risk factor found to be associated with acute cellular rejection was recent H1N1 viral antigen and adjuvant vaccination (OR 26.5: 95% CI 02.59–270.5). Vaccine adjuvants increase host response to vaccine antigens by immune upregulation potentially increasing risk of rejection in solid-organ transplant recipients. The potential hazard of vaccination this study raises must be weighed with the clear benefit vaccination has proven to be. During the 2009 influenza A (H1N1) virus pandemic, groups at highest risk were prioritized for vaccination. Influenza infections in solid-organ transplant (SOT) recipients are associated with high morbidity, mortality and graft dysfunction (1Vilchez RA McCurry K Dauber J et al.Influenza virus infection in adult solid organ transplant recipients.Am J Transplant. 2002; 2: 287-291Crossref PubMed Scopus (202) Google Scholar,2Wendt CH Community respiratory viruses: Organ transplant recipients.Am J Med. 1997; 102 (Discussion 42–43.): 31-36Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In medically stable SOT recipients, routine immunizations (trivalent, nonadjuvanted) for seasonal influenza are encouraged by the Advisory Committee on Immunization Practices (3Fiore AE Shay DK Broder K et al.Prevention and control of seasonal influenza with vaccines: Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2009.MMWR Recomm Rep. 2009; 58: 1-52PubMed Google Scholar). Following the announcement by the World Health Organization (WHO) of pandemic status there was a push for the rapid development of an effective vaccine (4WHONew influenza A (H1N1) virus: Global epidemiological situation, June 2009.Wkly Epidemiol Rec. 2009; 84: 249-257PubMed Google Scholar). Use of adjuvants, potent immunopotentiators without intrinsic antigenicity, was encouraged by the WHO with the goal of minimizing antigen requirements, while increasing/maintaining efficacy (5WHOStrategic advisory group of experts on immunization—Report of the extraordinary meeting on the influenza A (H1N1) 2009 pandemic, 7 July 2009.Wkly Epidemiol Rec. 2009; 84: 301-304PubMed Google Scholar,6WHO. Summary of: WHO virtual consultation on the safety of adjuvanted influenza vaccines. 2009 [updated June 3, 2009; cited 2011 May 1, 2011]; Available from: http://www.who.int/vaccine_research/documents/Report_on_consultation_on_adjuvant_safety_2.pdf.Google Scholar). It was during this period that Health Canada, Canada’s regulatory agency, authorized the interim use of Arepanrix (GlaxoSmithKline, Mississauga, ON, Canada), a two-component vaccine consisting of inactivated H1N1 virus antigen and AS03 adjuvant (7HealthCanada. Notice of decision for AREPANRIX™ H1N1. Ottawa, ON, Canada 2009 [updated November 30, 2009]; 30: 685–690. Available from: http://www.hc-sc.gc.ca/dhp-mps/alt_formats/pdf/prodpharma/sbd-smd/phase1-decision/drug-med/nd_ad_2009_arepanrix_h1n1_132070-eng.pdf.Google Scholar). AS03 a squalene and DL-α-tocopherol based, oil-in-water emulsion adjuvant system is currently one of two oil-in-water emulsion adjuvant systems currently approved for use with influenza vaccines (6WHO. Summary of: WHO virtual consultation on the safety of adjuvanted influenza vaccines. 2009 [updated June 3, 2009; cited 2011 May 1, 2011]; Available from: http://www.who.int/vaccine_research/documents/Report_on_consultation_on_adjuvant_safety_2.pdf.Google Scholar). This adjuvant has been previously studied with few serious adverse events in up to 6 months of follow-up with only local irritation reported and no documented increase in systemic immune-mediated events (6WHO. Summary of: WHO virtual consultation on the safety of adjuvanted influenza vaccines. 2009 [updated June 3, 2009; cited 2011 May 1, 2011]; Available from: http://www.who.int/vaccine_research/documents/Report_on_consultation_on_adjuvant_safety_2.pdf.Google Scholar). In SOT patients, efficacy of ASO3-adjuvanted influenza vaccines has been demonstrated in a case series of lung transplant recipients with reduced clinical infections (symptoms, or drop in FEV1 with positive nasopharygeal swab) and no increase in bronchiolitis obliterans syndrome at 6 months (8Schuurmans MM Tini GM Dalar L Fretz G Benden C Boehler A Pandemic 2009 H1N1 influenza virus vaccination in lung transplant recipients: Coverage, safety and clinical effectiveness in the Zurich cohort.J Heart Lung Transplant. 2011; 30: 685-690Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The capabilities of adjuvants to increase both antigen-specific immune responses as well as increase immunological memory are of concern for the SOT patient. This study was designed to evaluate whether using an adjuvanted vaccine system in heart transplant recipients increases rates of allograft rejection. This retrospective case cohort study evaluated cardiac transplant patients undergoing routine post-transplant endomyocardial biopsies within 21 days of inactivated H1N1 virus/AS03 vaccine intramuscular injection. Control patients were those who had not received the vaccination but underwent biopsy within 1 business day to study patient and were randomly selected in a 3:1 fashion (3 control:1 study patient). The protocol of this study was reviewed and approved by the Research Ethics Board at the University Health Network. All cardiac allograft biopsies were reviewed and reported by a single cardiac pathology laboratory experienced in reading and reporting post-transplant samples and blinded to patient vaccination status. By local practice, biopsies are reported simultaneously using both the 1990 ISHLT grading system nomenclature and 2004 revision (9Billingham ME Cary NR Hammond ME et al.Heart Rejection Study Group. The International Society for Heart TransplantationA working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection..J Heart Transplant. 1990; 9: 587-593PubMed Google Scholar,10Stewart S Winters GL Fishbein MC et al.Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection.J Heart Lung Transplant. 2005; 24: 1710-1720Abstract Full Text Full Text PDF PubMed Scopus (1384) Google Scholar). Patient variables including demographics, pretransplant diagnosis, current and past immunotherapy, and rejection history were examined. The primary endpoint was acute cellular rejection, ≥ISHLT grade 2 (1990 criteria). Baseline characteristics among the vaccinated and nonvaccinated control groups were compared by the Student t-test (continuous variables) and the chi-square analysis (categorical variables). Logistic regression models were used to calculate odds ratios and confidence intervals. Multivariate analysis was performed on factors predicting rejection. Exploratory analysis was done with both forward and backward stepwise regression model and validated with logistic regression. In the multivariate model, a cut off p-value of <0.2 from the univariate analysis was used. The level of significance for multiple comparisons was adjusted with the Bonferonni correction. For all remaining analyses, p-values <0.05 were considered to be significant. Clinical and demographic characteristics for both patients recently receiving inactivated H1N1 virus/AS03 vaccine and the control group are listed in Table 1. No variable reached statistical significance between groups. The overall mean age at time of biopsy was 52.5 ± 12.0 years. Patients in the control group were a mean of 50.1 ± 53.1 months post-transplant ranging between a minimum of 1.8 and a maximum 222.3 months. In the recently vaccinated group, subjects were a mean of 59.4 ± 44.0 months post-transplant with a range of 5.7 to 140.0 months.Table 1:Baseline characteristics of heart transplant population undergoing a routine post-transplant endomyocardial biopsyControl N = 45Recent H1N1 virus antigen/AS03 vaccine N = 15p-ValueMale32 (71.1%)10 (66.7%)0.745Age (years)51.4 ± 12.955.9 ± 8.00.210Time posttransplant (months)50.1 ± 53.159.4 ± 44.00.543Immune therapyNumber of immunosuppressant agents2.9 ± 0.63.1 ± 0.70.385Prednisone40 (88.9%)11 (73.3%)0.144Calcineurin inhibitor39 (86.7%)14 (93.3%)0.486Mycophenolate27 (60.0%)12 (80.0%)0.160mTOR inhibitor20 (44.4%)8 (53.3%)0.550Thymoglobin (prior 6 months)1 (2.2%)0 (0.0%)0.560Rejection ≥ grade 2 past 3 months8 (17.8%)4 (26.7%)0.456 Open table in a new tab Immunosuppressive regimens were similar in both groups with a trend toward increased mycophenolate use among recently vaccinated patients (p = 0.16) and prednisone use in the control arm (p = 0.14). The average total number of immunosuppressive medications per patient was similar with an average of 3.1 ± 0.7 agents in recently vaccinated patients and 2.9 ± 0.6 in control patients. Diagnosis of acute cellular rejection in the 3 months prior to the study biopsy was also similar in both groups (control: 8/45 vs. vaccinated: 4/15 p = 0.456). The primary endpoint of acute cellular rejection ≥ grade 2 occurred more frequently in patients recently receiving the H1N1 vaccine in comparison to the control group (control: 1/45 vs. 6/15 p = 0.001) (Figure 1). The rate of cellular rejection (all grades) did not significantly differ between the nonvaccinated and vaccinated groups (control: 15/45 vs. vaccinated 8/15 p = 0.168). On multivariate analysis of cellular rejection ≥grade 2, only recent H1N1 vaccination remained statistically significant (OR 26.5: 95%CI 2.59–270.5) (Table 2; Figure 2). This finding was consistent when results were reanalyzed with 2-factor modeling, independently comparing recent H1N1 virus/AS03 vaccination and potential confounding variables with their association to acute cellular rejection, ≥grade 2 (Table 3).Table 2:Multivariate analysis of predictors of acute cellular rejection ≥grade 2 (ISHLT 1990 criteria) in the 21 days following H1N1 virus/AS03 vaccineOdds ratioSEp-Value[95% Confidence interval]Gender1.501.730.7240.16–14.40Age at biopsy1.030.060.6410.92–1.15Months postcardiac transplant1.000.010.9050.98–1.02≥Grade 2 cellular rejection in past 6 months1.392.110.8270.07–27.27≥Grade 2 cellular rejection in past 3 months1.381.920.8160.09–21.06Recent H1N1 virus/AS03 vaccine26.5131.430.0062.59–270.75 Open table in a new tab Figure 2:Odds ratio from multivariate analysis and SE of predictors of ≥ grade 2 acute cellular rejection (ISHLT 1990 criteria) in the 21 days following H1N1 virus/AS03 vaccine.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 3:Two factor models of predictors of acute cellular rejection ≥grade 2 (ISHLT 1990 criteria) in the 21 days following H1N1 virus/AS03 vaccine. Level of significance p-value <0.01 (adjusted for multiple comparisons with Bonferroni correction 0.05/number of comparisons)Odds ratio95% Confidence intervalp-ValueVaccination29.903.17–281.720.003Gender1.340.18–10.000.773Vaccination27.672.92–262.020.004Age at biopsy1.020.92–1.130.740Vaccination29.943.15–284.790.003Months postcardiac transplant1.000.98–1.020.864Vaccination28.162.78–284.990.005≥Grade 2 cellular rejection in past 6 months1.130.17–7.610.897Vaccination28.783.07–270.170.003≥Grade 2 cellular rejection in past 3 months1.290.16–10.420.809 Open table in a new tab Given the potential morbidity and mortality associated with influenza infections in SOT patients, primary prevention is important. In the immune-competent population, the influenza vaccination has been shown to provide protective immunity, reducing the incidence of infection and societal costs (11Campbell DS Rumley MH Cost-effectiveness of the influenza vaccine in a healthy, working-age population.J Occup Environ Med. 1997; 39: 408-414Crossref PubMed Scopus (166) Google Scholar). The immunosuppression required to maintain graft function in the transplant population may diminish the immunogenicity of vaccinations. Fraund et al. reported reduced antibody titers among heart transplant patients following immunization with a trivalent influenza virus (without adjuvant) in comparison to healthy controls (12Fraund S Wagner D Pethig K Drescher J Girgsdies OE Haverich A Influenza vaccination in heart transplant recipients.J Heart Lung Transplant. 1999; 18: 220-225Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In this study nine heart transplant patients underwent 23 endomyocardial biopsies within 3 months of immunization with no reported cases of cellular rejection ≥grade 2 (ISHLT 1990 criteria) (grade 0 = 17, grade 1 = 6) (12Fraund S Wagner D Pethig K Drescher J Girgsdies OE Haverich A Influenza vaccination in heart transplant recipients.J Heart Lung Transplant. 1999; 18: 220-225Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Blumberg et al. raised concerns of a potential increase in histologic rejection (grade 2 and 3A) in the study of heart transplant recipients randomized to influenza vaccination compared to unvaccinated controls (13Blumberg EA Fitzpatrick J Stutman PC Hayden FG Brozena SC Safety of influenza vaccine in heart transplant recipients.J Heart Lung Transplant. 1998; 17: 1075-1080PubMed Google Scholar). Despite a trend toward increased rejection (4 of 14 vs. 1 of 14, p = 0.326) they concluded that vaccination was likely safe in stable heart transplant recipients (13Blumberg EA Fitzpatrick J Stutman PC Hayden FG Brozena SC Safety of influenza vaccine in heart transplant recipients.J Heart Lung Transplant. 1998; 17: 1075-1080PubMed Google Scholar). The addition of an adjuvant may maximize the immunogenicity of a single-dose vaccine however it may be at the expense of increased rejection. Multidose vaccination with repeated exposure to an antigen is an alternative option to enhance immune response in transplant patients obviating the need for adjuvant exposure (8Schuurmans MM Tini GM Dalar L Fretz G Benden C Boehler A Pandemic 2009 H1N1 influenza virus vaccination in lung transplant recipients: Coverage, safety and clinical effectiveness in the Zurich cohort.J Heart Lung Transplant. 2011; 30: 685-690Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). In a small open-label randomized trial, Magnani et al. reported similar antibody responses in heart transplant recipients immunized against seasonal influenza irrespective of the addition of an adjuvant emulsion. No increases in episodes of clinical rejection were noted; however the mean time from vaccination to biopsy was 44 ± 26 days (14Magnani G Falchetti E Pollini G et al.Safety and efficacy of two types of influenza vaccination in heart transplant recipients: A prospective randomised controlled study.J Heart Lung Transplant. 2005; 24: 588-592Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Our study did not examine the efficacy of the H1N1 immunization. In fact no patient vaccinated against H1N1 went on to develop the clinical disease. However, in this small case-control study the rate of cellular rejection of at least ISHLT (1990) grade 2 was increased in the short-time period following H1N1 immunization. No patient progressed to resistant rejection, and only 1 was found to have an elevation in filling pressures on right heart catheterization without an associated reduction in ventricular function. We did not see an increase in de novo donor-specific antibody (DSA) production or antibody-mediated rejection in our population. The clinical significance of mild cellular rejection including grade 2 is controversial as the majority of cases will improve without intervention with a minority progressing to more severe forms (15Brunner-La Rocca HP Sutsch G Schneider J Follath F Kiowski W Natural course of moderate cardiac allograft rejection (International Society for Heart Transplantation grade 2) early and late after transplantation.Circulation. 1996; 94: 1334-1338Crossref PubMed Google Scholar). The only other study evaluating the safety of adjuvanted vaccines in thoracic organ transplant recipients did not specifically report the rate of acute cellular rejection in their cohort of lung transplant recipients (8Schuurmans MM Tini GM Dalar L Fretz G Benden C Boehler A Pandemic 2009 H1N1 influenza virus vaccination in lung transplant recipients: Coverage, safety and clinical effectiveness in the Zurich cohort.J Heart Lung Transplant. 2011; 30: 685-690Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Intuitively, this association of rejection and adjuvanted vaccines is logical. The upregulation of the immune system following adjuvant exposure presumably can augment a low level of inflammation within the transplanted graft. This is demonstrated in our study as grade 2 rejection. As the overall rate of cellular rejection (all grades) was not increased it can be hypothesized that we saw a shift of rejection from the mild grade 1 to moderate grade 2. The small size and limited sampling period in our study prevents any determination of duration and peak of this effect. Influenza vaccination has been associated with increased humoral and cellular alloreactivity in SOT and healthy patients (16Danziger-Isakov L Cherkassky L Siegel H et al.Effects of influenza immunization on humoral and cellular alloreactivity in humans.Transplantation. 2010; 89: 838-844Crossref PubMed Scopus (54) Google Scholar). This response peaking 2–4 weeks postvaccine exposure has not been associated with changes in response to self-B cell, suggesting the altered alloimmunity may not result in increased self-reactivity or graft rejection. Our data regarding the association between rejection grade and use of this adjuvant vaccine should be interpreted with caution. We believe that studies evaluating the safety of both adjuvanted and nonadjuvanted vaccines in SOT patients are necessary to adequately address this safety issue. Until then, our program will continue to offer medically stable patients routine nonlive vaccines including the annual influenza vaccine as clinically indicated. H. Ross is on the DSMB for the Wyeth SRL study and is a principal investigator of a Novartis sponsored clinical trial. The remaining authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

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