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Vanderbilt University School of Medicine, Departments of Medicine, Preventive Medicine, Pediatrics
Microbiology and Immunology
Pediatric Clinical Research Office, Vanderbilt University School of Medicine, Nashville, Tennessee.
Background.
During the recent smallpox vaccination campaigns, ischemic cardiac complications were observed after vaccination. To examine a possible association between the smallpox vaccine and postvaccination ischemic events, we investigated alterations in levels of prothrombotic proteins (plasminogen activator inhibitor type 1 [PAI-1] and soluble CD40 ligand [sCD40L]) in recently vaccinated individuals.
Methods.
Vaccinia-naive (cohort N; aged 1832 years) and vaccinia-experienced (cohort E; aged 3349 years) healthy adults were vaccinated with a 1 : 5 dilution of the Aventis Pasteur smallpox vaccine. Plasma levels of PAI-1 and sCD40L were measured in 30 subjects (cohort N, n = 15; cohort E, n = 15) at baseline and twice after vaccination (between days 7 and 9 and between days 26 and 30).
Results.
Baseline mean PAI-1 levels significantly differed between cohorts N and E (P = .04). Within each exposure cohort, mean PAI-1 levels did not significantly change after vaccination. Baseline sCD40L levels did not differ between cohorts N and E. In cohort N, sCD40L levels significantly decreased after vaccination but returned to baseline levels within 1 month. Vaccination did not significantly alter levels of sCD40L in cohort E.
Conclusions.
Levels of PAI-1 and sCD40L did not significantly increase after smallpox vaccination. Vaccine-induced alterations in levels of these prothrombotic proteins do not appear to play a role in ischemic events observed after smallpox vaccination.
During the recent smallpox vaccination campaigns, 18 cases of ischemic heart disease were identified in recently vaccinated individuals, including 2 cases of fatal myocardial infarction [1, 2]. Although a causal role of smallpox vaccination in these ischemic events has not been established, the potential for such an association generated significant concern [35]. After these events, the Centers for Disease Control and Prevention (CDC) recommended that future candidates for smallpox vaccination be excluded if they are at increased risk for ischemic cardiac disease [6].
One potential pathophysiologic mechanism that may explain the observed ischemic events relates to the vigorous inflammatory response that develops after smallpox vaccination, which theoretically could trigger coronary thrombosis. Over the past few years, atherosclerotic lesion progression and thrombosis have been implicated as inflammation-mediated processes involving cytokine production and complex interactions between inflammatory cells and platelets within the vasculature [7]. In addition, alterations in the levels of serum prothrombotic proteinsspecifically, plasminogen activator inhibitor type 1 (PAI-1) and soluble CD40 ligand (sCD40L)have been linked both to ischemic cardiovascular events [810] and to vigorous systemic inflammatory responses [1113]. To examine a possible pathophysiologic association between the smallpox vaccine and postvaccination ischemic events, we investigated the effect of smallpox vaccination on levels of these prothrombotic proteins in a subset of both vaccinia-naive and vaccinia-experienced individuals participating in a larger clinical trial.
SUBJECTS, MATERIALS, AND METHODS
Study participants.
Participants for this substudy were recruited from among healthy adult volunteers enrolled in a larger clinical trial comparing various types of bandages for use over smallpox vaccination sites. Approval for the trial and substudy was granted by the Vanderbilt University Institutional Review Board, and all volunteers provided written informed consent. Volunteers were classified as vaccinia naive (cohort N; aged 1832 years) or vaccinia experienced (cohort E; aged 3349 years) on the basis of prior history of vaccine exposure. Exclusion criteria for vaccination are noted in the Appendix.
To exclude volunteers with potential risk factors for ischemic events, those with a history of the following conditions were also excluded from participation: myocardial infarction or other ischemic heart disease, angina, congestive heart failure, cardiomyopathy, stroke or transient ischemic attack, or other heart condition being treated by a doctor. Potential participants with an immediate family member (father, mother, brother, or sister) with a history of ischemic heart disease before age 50 years, as well as subjects who demonstrated a 10% risk of developing a myocardial infarction or coronary death within the next 10 years (assessed using the National Cholesterol Education Program's Risk Assessment Tool [14]), were also excluded.
All volunteers underwent baseline electrocardiograph (ECG) analysis, to allow ascertainment of the presence of prior ischemic heart disease as well as to provide a baseline study in the event that the volunteer developed chest pain, dyspnea, or other potential cardiac-related symptoms after vaccination. The study cardiologist (J.B.) reviewed all ECGs prior to enrolling a subject in the main clinical trial.
Vaccination methods and follow-up.
Eligible volunteers were vaccinated with a 1 : 5 dilution of the Aventis Pasteur smallpox vaccine (APSV), a liquid formulation of calf lymphorigin smallpox vaccine derived from the New York Board of Health vaccinia strain and maintained frozen at -20°C. The frozen vaccine was reconstituted with sterile water diluent containing 50% glycerin and 0.25% phenol (Chesapeake Biological Laboratories). Vaccine was administered to the deltoid area via scarification by 15 punctures with a bifurcated needle [15]. The inoculation site was covered with either gauze or 2 different types of occlusive dressings and was changed regularly by the study investigators until the site was epithelialized.
Volunteers were seen every 35 days for scheduled dressing changes, assessment of vaccine response, and evaluation of adverse events. Vaccine success was indicated by the development of a vaccination site "take," defined as the presence of a vesicle or pustule at the injection site 611 days after vaccination [15]. At each follow-up visit, study staff inspected and measured the vaccination lesion, the surrounding erythema and induration, and any regional (axillary or cervical) lymphadenopathy. At each follow-up visit, volunteers were questioned about the presence of any vaccine-related adverse events and were instructed to report both these symptoms and daily oral temperatures on a diary card. Fever was defined as an oral temperature of 37.8°C.
At each visit, volunteers were actively screened for the development of chest pain or pressure, dyspnea, and peripheral edema, and all positive responses were further evaluated by symptom assessment and physical examination. Volunteers exhibiting clinically significant symptoms were also evaluated by means of cardiac enzyme measurement (serum creatine kinase, creatine kinase MB fractionation, and troponin T) and ECG analysis, if indicated after the clinical assessment.
Prothrombotic protein specimen collection and measurement.
To assess alterations in PAI-1 and sCD40L activity after vaccination, serum samples were obtained from each volunteer at baseline (prior to vaccination), between days 7 and 9 after vaccination, and between days 26 and 30 after vaccination. Because of circadian variability in the levels of PAI-1, all samples were collected between 8:00 and 10:00 A.M. [10].
Because of transient activation of platelets, which may subsequently release PAI-1 after the initial venipuncture, specimens for PAI-1 analysis were obtained at the end of the phlebotomy. Blood samples were collected on ice and were centrifuged immediately at 0°C for 20 min. All plasma or serum was separated and stored at -70°C until the time of assay. Blood for measurement of PAI-1 levels was collected in tubes containing 0.105 mmol/L acidified sodium citrate (Biopool AB), and antigen levels were determined using a 2-site ELISA (Biopool AB), which measures active and latent PAI-1 in plasma. Normal PAI-1 levels range from 4 to 43 ng/mL (mean ± SD, 18 ± 10 ng/mL) [16].
Serum for sCD40L analysis was collected into serum separator tubes and frozen at -70°C until analysis. Serum sCD40L levels were determined using a commercial quantitative sandwich enzyme immunoassay technique (Quantikine; R&D Systems). Briefly, sCD40L in serum diluted 1 : 2 in assay diluent or purified standard was captured on plates coated with polyclonal antibody specific for sCD40L, washed, and detected with an enzyme-linked polyclonal antibody to sCD40L and a colorimetric substrate. Plates were read in a SpectraMAX plate reader (Molecular Devices), per the manufacturer's instructions. The sCD40L standard was tested at a starting concentration of 8000 pg/mL and then in 2-fold dilutions, to generate a standard curve. Results were expressed as nanograms per milliliter, and all serum sample results were within the dynamic range of the standard curve.
Statistical analysis.
A sample size of at least 13 volunteers per exposure cohort (N and E) was required in order to achieve 90% power to detect a mean difference of 10 ng/mL before and after vaccination (SD of assays, 10 ng/mL). To account for potential loss of volunteers for follow-up and lack of clinical response to vaccination, the target sample size was set at 15 volunteers per cohort (total n = 30). Comparisons of demographics and prothrombotic marker activity at each time point (at baseline, days 79, and days 2630 after vaccination) between cohorts N and E were performed using Student's t test. Comparisons of prothrombotic marker activity between each time point within each cohort were performed using paired t tests. Initial analyses were conducted using data from the entire N and E cohorts, regardless of vaccination site status. Secondary analyses excluding any volunteers who did not develop a clinical vaccination site take were also performed.
RESULTS
Fifteen volunteers were enrolled in each of the cohorts, N and E, for a total of 30 participants. In terms of sex, race, and ethnicity, volunteers enrolled in the cardiac substudy did not significantly differ from the participants enrolled in the larger clinical trial. However, when compared with the volunteers enrolled in the larger trial yet not enrolled in the cardiac substudy, the cohort E volunteers enrolled in the substudy were significantly older (mean age, 45.4 vs. 41.7 years; P = .01). The mean ages of volunteers in cohorts N and E were 24.0 years (range, 19.630.9 years) and 45.4 years (range, 33.448.9 years), respectively (table 1). In cohort N, 53.3% of volunteers were men, whereas 46.7% of volunteers in cohort E were men. Samples were collected before vaccination and then at a mean of 7.3 days (day 79 sample) and 28.2 days (day 2630 sample) after vaccination. Baseline ECGs did not show significant abnormalities or evidence of prior ischemia in any volunteer.
All cohort N volunteers exhibited evidence of a clinical take at the vaccination site; however, 2 members of cohort E did not have evidence of a take at their vaccination site. The median peak sizes of the vaccination lesions were 20 mm and 15 mm for the cohort N and E volunteers, respectively, and lesion size peaked at 12 days after vaccination in cohort N volunteers and at 9.5 days in cohort E volunteers (table 1). Regional lymphadenopathy was detected in 46.7% and 26.7% of cohort N and E volunteers, respectively. Local reactogenicity symptoms are noted in table 1.
None of the participants experienced clinically significant chest pain, chest pressure, or lower-extremity swelling during the duration of the trial. Of note, 1 participant underwent additional ECG analysis 14 days after vaccination, after experiencing an isolated episode of shortness of breath that lasted a few minutes. The volunteer denied having any other symptoms, including chest pain, pressure, or discomfort. Physical examination revealed no abnormalities, the repeat ECG showed no signs of ischemia or pericarditis, and the episode resolved with no further symptoms. Cardiac enzyme analysis was not performed on any volunteers in the substudy, because of the lack of persistent clinically significant cardiac-related symptoms.
PAI-1 analysis.
There was a significant difference in mean plasma PAI-1 levels at baseline between cohort N and E volunteers (P = .04) (figure 1). However, no difference was detected in mean plasma PAI-1 levels between cohort N and E volunteers at days 79 or days 2630 after vaccination. In addition, there was no significant difference in the change in PAI-1 levels from baseline to days 79, from days 79 to days 2630, or from baseline to days 2630, within either cohort or between the 2 cohorts (P > .05, for each comparison). When the 2 cohort E volunteers without a clinical take were excluded from the PAI-1 analyses, the difference between baseline PAI-1 levels between cohorts N and E was no longer significant (P = .06).
DISCUSSION
On the basis of our investigation, vaccination with APSV does not appear to increase levels of either PAI-1 or sCD40L, prothrombotic proteins related to ischemic cardiac disease. This finding is important, given the recent emphasis on the adverse cardiac events that can potentially occur after smallpox vaccination and that have been highlighted in recent campaigns. Over the past several years, the safety and efficacy of stockpiled smallpox vaccines have been evaluated in carefully designed clinical trials [17, 18]. Each of these studies convincingly demonstrated a high rate of systemic and local adverse events occurring after vaccinia vaccination. In contrast, during these trials, no evidence of ischemic cardiac disease was detected in vaccine recipients, although these studies were conducted before the increased awareness of potential postvaccination cardiac complications. When the vaccine was administered more broadly to the military and civilian populations, however, several cases of postvaccination ischemic cardiac events were reported. In total, 13 cases of angina and 5 cases of myocardial infarction have been described to date. Two people with myocardial infarction died [1, 2, 19]. Interestingly, reports of ischemic cardiac events occurring after smallpox vaccination are not new, as highlighted by a report of an acute "stenotic" cardiac event that occurred after vaccination of a previously vaccinated man in 1979 [20].
All vaccine recipients with myocardial infarctions in the recent campaigns had clearly defined risk factors for ischemic heart diseasespecifically, hypertension, hypercholesterolemia, a previous history of transient ischemic attacks, diabetes, tobacco use, and prior atherosclerotic coronary disease [1, 2, 19]. An analysis using rates of death from cardiac-associated conditions among a population matched for age and sex found that the number of deaths due to myocardial infarction within 3 weeks of smallpox vaccination was not increased above the expected baseline of cardiac events in this population [21]. Although a relationship between the smallpox vaccine and ischemic complications was not definitively determined, the CDC appropriately recommended that potential vaccine recipients be screened for risk factors for cardiac disease and excluded from vaccination if they meet these criteria [6]. In contrast, the multiple reports of myocarditis and pericarditis during the vaccination campaigns were subsequently shown to occur at a statistically increased rate after vaccination [22]. Although the underlying pathophysiologic causes of vaccinia-associated myopericarditis is still unclear, it does not seem to be related to coronary thrombosis or ischemic cardiac disease [22].
To better understand how vaccinia might trigger postvaccination ischemic cardiac events, it is important to review some fundamental concepts. First, the importance of platelet aggregation and subsequent thrombosis in ischemic cardiovascular disease is well established. Elevations of prothrombotic proteins, such as coagulation factors and fibrinolysis inhibitors, have been associated with an increased risk for ischemic cardiac disease [9, 10]. PAI-1, in particular, has been implicated as an important mediator of coronary artery disease and myocardial infarction [10, 23, 24]. A linear glycoprotein released from platelets, adipose tissue, and activated endothelial cells, PAI-1 modulates tissue plasminogen activator (t-PA) and regulates fibrinolysis. Activated PAI-1 resides on the platelet surface and protects the blood clot from premature lysis by inhibiting the fibrinolytic activity of t-PA. Increased plasma concentrations of PAI-1 are associated with various thrombotic disorders and are an independent risk factor for reinfarction in patients who have had a first myocardial infarction before the age of 45 years [10].
Second, inflammation of the cardiac vasculature has also been implicated in the pathogenesis of coronary atherosclerosis and thrombosis [7]. CD40 ligand, a protein abundant in platelets, plays an important role in the inflammatory aspects of atherosclerotic lesion progression and thrombosis [25]. Its soluble form, sCD40L, is released from stimulated lymphocytes and activated platelets and promotes coagulation by inducing cellular expression of tissue factor and facilitating platelet aggregation. Likely because of its role in the pathogenesis of arterial thrombosis, sCD40L has recently been associated with acute coronary syndromes and appears to be an indicator of increased risk of both fatal and nonfatal myocardial infarction [9].
Exhibition of both of these factors may also be influenced by inflammation. PAI-1 is a known acute-phase reactant whose release is influenced by certain cytokines, including IL-6, IL-1, tumor necrosis factor (TNF), and tissue growth factor (TGF) [12, 13]. In vitro, PAI-1 RNA expression increases in the presence of interferon (INF) stimulation of astrocytoma cells [11]. The exact effect of cytokine release on PAI-1 in vivo, however, remains unclear. The role of inflammation and elevations in cytokines in the increase and activation of prothrombotic proteins may be particularly relevant when attempts are made to correlate smallpox vaccination with ischemic cardiac events. We have recently shown that levels of INF-, IL-10, and TNF- are significantly increased in vaccinia-naive subjects 1 week after vaccination with APSV, providing a potential prostimulatory environment for PAI-1 and sCD40L [26].
Recent evidence has shown a potential correlation between the incidence of coronary atherosclerosis and 2 types of infectious microorganisms, herpes simplex virus (HSV) and Chlamydia pneumoniae [27]. Although there is no direct evidence that these microorganisms initiate atheromatous plaque formation, both have been identified in atheromatous lesions of coronary arteries at autopsy, and increased titers of antibody to these organisms have been used to predict risk of further complications after myocardial infarction [27]. Interestingly, in vitro studies investigating the effect of infection with these microorganisms on PAI-1 levels have had variable results in different tissues. Infection of human vascular endothelial cells by C. pneumoniae has been shown to lead to the overexpression of tissue factor, PAI-1, and IL-6 [28], whereas infection of human umbilical vein endothelial cells by HSV leads to a decrease in PAI-1 levels [29]. The relationship between these infections and PAI-1 levels remains unclear in vivo; however, the variable response in PAI-1 levels may be due to differing cytokine responses in different tissues.
In our investigation, after smallpox vaccination in healthy adults with and without prior vaccinia exposure, elevations in levels of PAI-1 and sCD40 ligand were not detected. At baseline, mean plasma PAI-1 levels were significantly greater in cohort E than in cohort N. However, this finding was not unexpected, since levels of PAI-1 are known to increase with age [21]. Thus, although vaccinia-induced activation of coagulation factors and fibrinolysis inhibitors may prove to be a factor in the pathogenesis of cardiac ischemic events noted after smallpox vaccination, vaccine-induced elevations of PAI-1 and sCD40L do not appear to play a role. Since most persons who experienced cardiac complications after vaccination had clearly defined cardiac risk factors, the development of ischemic cardiac events may have been an unfortunate coincidence secondary to vaccination of an older population with multiple comorbidities and not directly related to the vaccine.
The present study has several potential limitations. Our relatively small sample size may not have allowed us to detect the significance of small alterations in PAI-1 or sCD40L activity after vaccination. As a result of specifics regarding the timing of sample collection for PAI-1 analysis, our sample selection may have been biased to include those who were able to attend follow-up visits in the morning. However, comparisons of baseline characteristics between those who participated in the cardiac substudy and those who remained in the larger clinical trial did not reveal significant differences. Although participants in cohort E in the cardiac substudy were significantly older than participants in the same cohort in the larger clinical trial, it is unlikely that this disparity is of any clinical significance.
In addition, persons with significant risk factors for ischemic events and cardiovascular disease were appropriately excluded from study participation and vaccination. Activity of prothrombotic proteins after vaccination, as well as vascular biological factors in these persons, might differ from that of a healthy adult, and, as a result, our findings might have been biased by the exclusion of those at higher risk for cardiac disease. Finally, subjects in our study also received a smallpox vaccine, APSV, that differed from the lyophilized vaccine (Dryvax; Wyeth-Ayerst) used in the military and civilian campaigns in which adverse cardiac events were recently noted. Nonetheless, both vaccines are derived from the same New York Board of Health vaccinia strain, and reactogenicity after vaccination with APSV appears to be slightly greater when compared with Dryvax [18], suggesting that adverse events related to vaccine-induced inflammation should have been comparable between the 2 vaccines.
The occurrence of adverse cardiac events in persons recently vaccinated with smallpox vaccine has caused concern regarding the safety of vaccinia inoculation. Additional studies are now needed to examine other potential interactions between smallpox vaccination and the physiologic mechanisms involved in intravascular thrombosis and atherosclerotic plaque rupture.
Acknowledgments
We thank the members of the Vanderbilt Pediatric Clinical Research Office, especially Debbie Hunter, Miriam Swihart, Roberta Cornell, Adam Michel, Christina Powell, Jennifer Hicks, and Jennifer Kissner; William Schaffner; the Vanderbilt General Clinical Research Center; Aventis Pasteur; the EMMES Corporation, especially Heather Hill; and colleagues at the Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, particularly Stephen Heyse, Mamodikoe Makhene, Walla Dempsey, and Holli Hamilton, for their support for and guidance with this project.
APPENDIX
Exclusion criteria for vaccinia vaccination
Exclusion criteria for both cohorts (N and E):
History of autoimmune disease
Use of immunosuppressive medications
History of HIV infection
History of solid organ or bone marrow transplantation
History of malignancy
History of or current illegal injection drug use
Eczema (active or quiescent)
Current exfoliative skin disorders
Prior vaccination with any vaccinia-vectored or other pox-vectored experimental vaccine
Presence of medical or psychiatric conditions or occupational responsibilities that precluded subject compliance with the protocol
Acute febrile illness (fever of 38.1°C) on the day of vaccination
Allergies to components of the vaccine
Pregnancy or lactation
Household contact with children <12 months of age or household or sexual contacts with anyone with the following conditions: history of or concurrent eczema, a history of exfoliative skin disorders, history of the immunosuppressive conditions noted above, ongoing pregnancy
Additional exclusion criterion for cohort N:
Presence of a typical vaccinia scar or history of smallpox vaccination.
Additional exclusion criterion for cohort E:
Lack of confirmation of prior smallpox vaccination.
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