High Disease Activity: An Independent Factor for Reduced Immunogenicity of the Pandemic Influenza A Vaccine in Patients With Juvenile Systemic Lupus Erythematosus
ClinicalTrials.gov identifier: NCT01151644.
Faculdade de Medicina da Universidade de São Paulo, Reumatologia, Av Dr Arnaldo, 455, 3° Andar, Sala 3105, Sao Paulo, Brazil, 01246-903. E-mail:
Recent findings demonstrated a reduced immunogenicity of the influenza A H1N1/2009 vaccine in juvenile rheumatic diseases. However, a point of concern is whether the vaccine could induce disease flares. The aim of this study was to assess the disease safety of and the possible influence of disease parameters and therapy on nonadjuvant influenza A H1N1 vaccine response of juvenile systemic lupus erythematosus (SLE) patients.
One hundred eighteen juvenile SLE patients and 102 healthy controls of a comparable age were vaccinated. Seroprotection rate, seroconversion rate, and factor increase in geometric mean titer (GMT) were calculated and effective immune response was defined by the Food and Drug Administration and the European Committee for Proprietary Medicinal Products vaccine immunologic standards. Disease parameters, treatment, and adverse events were evaluated.
Age was comparable in juvenile SLE patients and controls (mean ± SD 16.0 ± 3.5 versus 15.9 ± 4.5 years; P = 0.26). Three weeks after immunization, seroprotection rate (73.7% versus 95.1%; P < 0.001), seroconversion rate (63.6% versus 91.2%; P < 0.001), GMT (90.8 versus 273.3; P < 0.001), and factor increase in GMT (8.1 versus 19.9; P < 0.001) were significantly lower in juvenile SLE patients versus controls. Nonseroconversion was associated with a higher frequency of patients with a Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) score ≥8 (48.8% versus 24%; P = 0.008) and a higher mean ± SD current glucocorticoid dosage (18 ± 21.4 versus 10.5 ± 12.5 mg/day; P = 0.018). Multivariate logistic regression including a SLEDAI-2K score ≥8 revealed that only the SLEDAI-2K remained a significant factor for nonseroconversion (odds ratio 0.42, 95% confidence interval 0.18–0.98; P = 0.045). Disease parameters remained stable throughout the study and no severe vaccine adverse events were observed.
The present study demonstrated adequate disease safety and is the first to discriminate that high disease activity impairs influenza A H1N1/2009 vaccine antibody production in juvenile SLE, in spite of an overall immune response within recommended levels.
Infections are recognized as an important cause of morbidity and mortality in patients with juvenile systemic lupus erythematosus (SLE) and may also induce disease flares (). Immunologic abnormalities related to the disease itself and its treatment seem to be a major contributing factor to this higher susceptibility to infections ().
Children and adolescents were recognized as a risk group for hospitalization and death in the recent influenza pandemic caused by the new influenza A H1N1/2009 virus, particularly in those with preexisting chronic disorders (). Vaccination is considered as the most effective measure to control the spread of the virus and to reduce associated morbidity and mortality ([4, 5]). In fact, the Advisory Committee on Immunization Practices stated that all children and adolescents ages between 6 months and 18 years should receive the trivalent seasonal influenza vaccine containing the A/California/7/2009 (H1N1) strain, and this recommendation is particularly important for those with chronic conditions (). More recently, the European League Against Rheumatism published their recommendations for vaccinations in pediatric patients with rheumatic diseases and reinforced that an annual influenza vaccination should be considered for these patients ().
The efficacy and safety of the seasonal influenza vaccine in children with rheumatic diseases has been reported in previous studies with a limited number of patients ([7, 8]). An appropriate response to the seasonal influenza vaccine in patients with juvenile rheumatic diseases, including 11 patients with juvenile SLE, regardless of their immunosuppressive therapy, was reported by Kanakoudi-Tsakalidou et al (). Likewise, a satisfactory response for the seasonal influenza vaccine independent of treatment was observed in a population of pediatric rheumatic patients, 12 of them with juvenile SLE ().
With regard to the pandemic vaccine, we have recently published a study focusing solely on vaccine immunogenicity and safety in a large cohort of 237 patients with juvenile autoimmune rheumatic diseases and demonstrated an overall reduced immunogenicity, particularly in those receiving glucocorticoid therapy (). The inclusion of a heterogeneous group of illnesses in our cohort hampers the accurate interpretation of the possible influence of a specific disease and/or therapy. Moreover, we have not evaluated disease safety, since another point of concern is whether the vaccine could induce flares ().
Therefore, the aim of the present study was to evaluate disease safety of and the possible influence of disease and therapy on juvenile SLE patients immunized with the nonadjuvant pandemic influenza A H1N1/2009 vaccine.
Box 1. Significance & Innovations
- High disease activity impairs antibody response to the influenza A H1N1/2009 vaccine in juvenile systemic lupus erythematosus (SLE) patients.
- Influenza A/H1N1 2009 vaccine is safe in juvenile SLE patients.
SUBJECTS AND METHODS
Patients and controls
One hundred eighteen juvenile SLE outpatients routinely followed at the Pediatric Rheumatology Unit and the Rheumatology Division of Hospital das Clinicas, São Paulo, Brazil, were included in this study. All patients fulfilled the American College of Rheumatology classification criteria for juvenile SLE (). A total of 102 healthy subjects were concomitantly included in the control group. All participants were ages 9–21 years. Exclusion criteria included previous proven infection by influenza A (H1N1) 2009, anaphylactic response to vaccine components or to eggs, previous vaccination with inactivated vaccines within 2 weeks or live vaccines in the last 4 weeks or even the 2010 seasonal influenza vaccination in the last 6 months before the study entry, acute infection resulting in fever >38°C at the time of vaccination, Guillain-Barré syndrome or demyelinating syndromes, heart failure, blood transfusion within 6 months, and hospitalization.
An interventional, open-label, phase IV study was conducted between March 2010 and April 2010. All juvenile SLE patients were invited by letter to participate in the public health influenza A H1N1/2009 vaccine campaign at the immunization center of the same hospital. Healthy volunteers who came to this center seeking vaccination in response to the Public Health National Campaign were included as the control group. This protocol was approved by the local institutional review board. All participants or their legal guardians signed the informed consent form.
In the period from March 22, 2010 to April 2, 2010, H1N1 A/California/7/2009–like virus vaccine (A/California/7/2009, Butantan Institute/Sanofi Pasteur) was administered to patients and controls as a single intramuscular injection (0.5 ml). All participants were evaluated on the day of vaccination and 3 weeks after that. Blood samples were obtained from each participant immediately before and 21 days after vaccination.
Patient demographic data, treatment, and disease activity
The medical records of all patients were reviewed in terms of demographic data (disease duration) and treatment (glucocorticoid and immunosuppressant use). Disease activity was assessed by clinical and laboratorial parameters at study entry and 21 days and 4 months after vaccination, including articular involvement (arthralgia or nonerosive arthritis), cutaneous lesions (malar or discoid rash, oral ulcers, vasculitis, or photosensitivity), cardiopulmonary disease (serositis, myocarditis, restrictive lung disease, and pulmonary hypertension), renal involvement (proteinuria >0.5 gm/24 hours, cellular casts, persistent hematuria >10 red blood cells/high-power field, or renal failure), neuropsychiatric disease (seizure, psychosis, depression, or peripheral neuropathy), and hematologic abnormalities (hemolytic anemia, leukopenia with a white blood cell count <4,000/mm3, lymphopenia <1,500/mm3, and thrombocytopenia with a platelet count <100,000/mm3). Complement levels (C3 normal range 79–152 mg/dl) were measured by radial immunodiffusion (Siemens Health Care) and anti–double-stranded DNA (anti-dsDNA; normal value <100 IU/ml) was detected by enzyme-linked immunosorbent assay (Inova Diagnostics). The Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) () was calculated at study entry, after 21 days, and after 4 months.
A novel monovalent, nonadjuvant, inactivated, split-virus vaccine was supplied by the Butantan Institute/Sanofi Pasteur. The vaccine contained an inactivated split influenza virus with 15 μg of hemagglutinin antigen equivalent to the A/California/7/2009 (H1N1) virus–like strain (NYMCX-179A), one of the candidate reassortant vaccine viruses recommended by the World Health Organization. Embryonated chicken eggs were employed using the same standard techniques for the production of seasonal, trivalent, inactivated influenza vaccine. The vaccine was presented in 5-ml multidose vials with thimerosal (45 μg per 0.5-ml dose) as a preservative ().
The antibody levels against H1N1 A/California/7/2009–like virus were evaluated using the hemagglutination inhibition assay (HIA) at the Adolfo Lutz Institute. Sera were tested for antibodies to the H1N1 A/California/7/2009 influenza strain supplied by the Butantan Institute. Titers were tested at an initial dilution of 1:10 and at a final dilution of 1:2,560. For the purposes of calculations, negative titers were assigned a value of 1:5, and for titers greater than 1:2,560, a value of 1:2,560 was assigned. Samples were tested in duplicate and geometric mean values were used in the analyses.
Virus concentrations were previously determined by hemagglutinin antigen titration, and the HIA test was performed after removing naturally occurring nonspecific inhibitors from the sera, as previously described ().
The immunogenicity end points after vaccination were the seroprotection rate (titer ≥1:40), seroconversion rate (prevaccination titer <1:10 and postvaccination HIA titer ≥1:40 or prevaccination titer ≥1:10 and postvaccination titer ≥4-fold increase), geometric mean titers (GMTs), and factor increase in GMT (ratio of the GMT after vaccination to the GMT before vaccination).
On the day of vaccination, patients or legal guardians received a 21-day personal diary card containing the following list of predefined adverse events: local reactions (pain, redness, swelling, and itching) and systemic adverse events (arthralgia, fever, headache, myalgia, sore throat, cough, diarrhea, rhinorrhea, and nasal congestion). Participants were asked to give “yes/no” responses to each side effect and to return their diary cards on the second evaluation day (21 days after vaccination). The participants were encouraged to report any other adverse events that were not on the list. All local reactions were considered related to the influenza A H1N1/2009 vaccine, while systemic adverse events were analyzed by the investigators to determine their causality. Severe side effects were defined as those requiring hospitalization or death.
The immunogenicity and safety analyses were descriptive, and the 2-sided 95% confidence intervals (95% CIs) were calculated assuming binomial distributions for dichotomous variables and log-normal distribution for hemagglutination inhibition titers. The GMTs and factor increase in GMT were compared between each subgroup of patients with juvenile SLE and the healthy control group using a 2-sided Student's t-test or the Mann-Whitney U test on the log10-transformed titers. Chi-square or Fisher's exact tests were used for categorical variables. Multivariate logistic regression analysis was performed using the seroconversion rate as the dependent variable and those with P values less than 0.05 in the univariate analyses as independent variables (SLEDAI-2K score ≥8 and current glucocorticoid dose). All tests were 2-sided, and significance was set at a P value less than 0.05.
One hundred eighteen juvenile SLE patients and 102 healthy controls were included in the study. Current age was comparable between the patients and controls (mean ± SD 16 ± 3.5 versus 15.9 ± 4.5 years; P = 0.26), with a predominance of female sex in the juvenile SLE group (77.1% versus 50%; P < 0.001) (Table 1). The mean ± SD disease duration was 5.0 ± 3.6 years and the mean ± SD SLEDAI-2K score was 6.0 ± 5.8. Renal involvement was observed in approximately half (50.8%) of the patients and lymphopenia was observed in 27.1% of the patients at study entry. Ninety-two patients (78%) were receiving antimalarials, 83 (70.3%) were receiving prednisone with a mean ± SD dosage of 18.8 ± 17 mg/day, and 72 (61.0%) were taking immunosuppressive drugs (azathioprine [37.3%], mycophenolate mofetil [12.7%], and methotrexate [11.9%]) (Table 1).
Table 1. Demographic data, disease features, and treatment in juvenile SLE patients and healthy controls at study entry*
|Demographic data|| || || |
|Age, mean ± SD years||16.0 ± 3.5||15.9 ± 4.5||0.26|
|Female sex||91 (77.1)||51 (50)||< 0.001|
|Disease duration, mean ± SD years||5.0 ± 3.6||–||–|
|Disease features|| || || |
|SLEDAI-2K score, mean ± SD||6.0 ± 5.8||–||–|
|Renal involvement||60 (50.8)||–||–|
|Neuropsychiatric involvement||0 (0)||–||–|
|Dosage, mean ± SD mg/day||18.8 ± 17||–||–|
|Dosage ≥20 mg/day||40 (33.9)||–||–|
|Mycophenolate mofetil||15 (12.7)||–||–|
Influenza A H1N1/2009 vaccine immunogenicity
Before influenza A H1N1/2009 immunization, seroprotection rates were comparable in patients and controls (P = 0.736), as was GMT (P = 0.684) (Table 2). Regarding vaccination status prior to the study entry, 6% of juvenile SLE patients had a history of seasonal influenza immunization 1 year earlier, and none of them had pandemic prevaccination seroprotection. Moreover, 11% of healthy controls also had received seasonal influenza immunization during the previous year and only 1% had pandemic prevaccination seroprotection.
Table 2. Serologic data before and after influenza A pandemic (H1N1) 2009 vaccine in juvenile SLE patients and healthy controls*
|Before immunization|| || || |
|Seroprotection, %||18.6 (12.1–26.9)||20.6 (13.2–29.7)||0.736|
|GMT||11.2 (8.9–14.0)||11.9 (9.6–14.9)||0.684|
|After immunization|| || || |
|Seroprotection, %||73.7 (64.8–81.4)||95.1 (88.9–98.4)||< 0.001|
|Seroconversion, %||63.6 (54.2–72.2)||91.2 (83.9–95.9)||< 0.001|
|GMT||90.8 (67.8–121.7)||237.3 (188.8–298.3)||< 0.001|
|Factor increase in GMT||8.1 (6.3–10.5)||19.9 (15.6–25.4)||< 0.001|
Three weeks after vaccination, all parameters were reduced in juvenile SLE patients compared to controls, including seroprotection rates (P < 0.001), seroconversion rates (P < 0.001), GMT (P < 0.001), and factor increase in GMT (P < 0.001) (Table 2).
Comparison of seroconverted and nonseroconverted juvenile SLE patients showed that the groups were similar regarding current age (P = 0.92) and female sex (P = 0.366). The nonseroconverted group showed a higher mean ± SD preimmunization SLEDAI-2K score (7.5 ± 5.8 versus 5.2 ± 5.7; P = 0.035) and higher frequency of a SLEDAI-2K score ≥8 (48.8% versus 24%; P = 0.008) (Table 3).
Table 3. Demographic data, disease activity, and treatment in seroconverted and nonseroconverted juvenile SLE patients at study entry*
|Demographic data|| || || |
|Age, mean ± SD years||16.5 ± 3.9||16.5 ± 3.3||0.92|
|Female sex||31 (72.1)||60 (80)||0.366|
|Disease duration, mean ± SD years||6.1 ± 3.9||5.6 ± 3.4||0.419|
|Disease characteristics|| || || |
|SLEDAI-2K score, mean ± SD||7.5 ± 5.8||5.2 ± 5.7||0.035|
|SLEDAI-2K score ≥8||21 ( 48.8)||18 ( 24.0)||0.008|
|Renal involvement||24 ( 55.8)||36 ( 48.0)||0.413|
|Neuropsychiatric involvement||0 ( 0)||0 ( 0)||1.0|
|Lymphopenia||15 ( 34.9)||17 ( 22.7)||0.197|
|C3, mg/dl ||79 ± 27.7||83 ± 27.7||0.455|
|Anti-dsDNA ||29 ( 67.4)||34 ( 45.3)||0.02|
|Treatment|| || || |
|Antimalarials||34 ( 79.1)||58 ( 77.3)||1.0|
|Prednisone ||32 ( 74.4)||51 ( 68)||0.533|
|Dosage, mean ± SD mg/day||18 ± 21.4||10.5 ± 12.5||0.018|
|Dosage ≥20 mg/day, no./total ( %)||18/32 ( 56.3)||22/51 ( 43.1)||0.268|
|Immunosuppressants||25 ( 58.1)||47 ( 62.7)||0.696|
|Azathioprine||15 ( 34.9)||29 ( 38.6)||0.698|
|Mycophenolate mofetil||7 ( 16.3)||8 ( 10.6)||0.401|
|Methotrexate||4 ( 9.3)||10 ( 13.3)||0.571|
|Cyclophosphamide||1 ( 2.3)||2 ( 2.6)||1.0|
|Cyclosporine||2 ( 4.6)||0 ( 0)||0.131|
Regarding the current treatment at study entry, the mean ± SD prednisone dosage was significantly higher in the nonseroconverted juvenile SLE patients (18 ± 21.4 versus 10.5 ± 12.5 mg/day; P = 0.018). In fact, the mean ± SD prednisone dosage in patients with a SLEDAI-2K score ≥8 was significantly higher compared to patients with low SLEDAI-2K scores (22.4 ± 21.5 versus 8.7 ± 11.2 mg/day; P < 0.001). The frequencies of antimalarial and immunosuppressant agent use were comparable in nonseroconverted and seroconverted patients (Table 3).
Multivariate logistic regression was performed to determine possible deleterious parameters for nonseroconversion, including a high SLEDAI-2K score (≥8) and current glucocorticoid dose, and only a SLEDAI-2K score ≥8 (odds ratio 0.42, 95% CI 0.18–0.98; P = 0.045) remained a significant factor.
No change was observed in the median SLEDAI-2K score before, 21 days after, and 4 months after (4 [interquartile range (IQR) 0–28] versus 4 [IQR 0–25] versus 4 [IQR 0–21]; P = 0.11) the pandemic influenza A H1N1/2009 vaccine. The frequencies of articular involvement (3.4% versus 2.5% versus 3.4%; P > 0.05), neuropsychiatric disease (0% versus 1.7% versus 1.7%; P > 0.05), and hematologic abnormalities (4.2% versus 6.8% versus 4.2%; P > 0.05) were similar before, 21 days after, and 4 months after pandemic influenza A H1N1/2009 vaccination. Whereas the frequency of renal involvement decreased significantly from baseline to the 4-month evaluation (50.8% versus 27.9%; P = 0.0001), mucocutaneous lesions were significantly higher 4 months after vaccination (5.9% versus 16.9%; P = 0.015).
No serious adverse events were reported in both groups. Juvenile SLE patients presented higher frequencies of local redness and itching (11% versus 2.0%; P = 0.007 and 16.9% versus 0.0%; P < 0.0001, respectively), arthralgia (16.9% versus 1.0%; P < 0.0001), and rhinorrhea (12.7% versus 3.9%; P = 0.02) when compared to healthy controls.
The present study is the first to discriminate that disease activity impairs nonadjuvant influenza A H1N1/2009 vaccine antibody production in patients with juvenile SLE, in spite of an overall immune response within recommended levels in these patients.
The analysis of solely lupus patients with a control group of a comparable age was essential, since we have previously demonstrated that vaccine immune response has a diversity related to disease () and age has been recognized as a major factor for this vaccine antibody production ([15-17]). The selection of a sizeable number of patients regardless of disease activity status or immunosuppressive treatments better expresses a real-life situation and allows a more accurate interpretation of the influence of these factors in vaccine humoral response. However, the absence of a concomitant nonvaccinated juvenile SLE control group hinders a definitive conclusion about disease flare. Other limitations of the present study were absence of assessment of the influenza-like illness during the year following immunization, the long-term evaluation of immunogenicity, and the analysis of B cell memory after immunization.
Interestingly, we have identified that an overall high disease activity score at immunization is a relevant factor for the pandemic vaccine nonseroconversion in juvenile SLE, possibly by a direct effect on humoral and cell-mediated immunity () that may ultimately affect the response to antigenic challenge (). Further analysis of SLEDAI-2K parameters has not revealed involvement of a specific major organ underlying this process. On the other hand, lymphopenia did not seem to influence this weaker response in juvenile SLE as also reported for children with cancer () and an adult SLE population (), although Mathian et al (2011) have observed such an association in the latter group (). Additionally, we have identified a higher frequency of low complement levels and anti-dsDNA antibodies unrelated to renal involvement linked to low vaccine response, which appears to reflect the known correlation of immune inflammatory markers with global lupus activity ().
Of note, the large enrollment of a single disease and the multidimensional comparison enabled a more precise definition that glucocorticoid was the only medication associated with lower immunogenicity in juvenile SLE, as also observed in preceding data that identified glucocorticoids as a determinant of a weaker vaccine response in adult SLE ([24, 25]), autoimmune rheumatic diseases (), and juvenile rheumatic diseases (). In contrast, a recent study with the adjuvanted influenza A H1N1/2009 vaccine in adult lupus showed no influence of therapy in immunogenicity (). The limited sample size of the latter study may hamper the interpretation of their finding regarding the possible deleterious effect of therapy on vaccine response. In the present study, none of the patients were receiving B cell depletion therapy, and therefore the previous description of a deleterious effect of this biologic agent in pandemic influenza vaccine response was not assessed herein (). On the other hand, regarding antimalarials, the small representation of juvenile SLE patients without this medication in the present study precludes an accurate interpretation of the absence of a beneficial effect previously reported in adult lupus patients ().
Regarding early and late disease safety of the H1N1 pandemic vaccine, our findings of largely unchanged organ and system involvements confirmed the preceding observation of unchanging lupus biomarkers () and SLEDAI-2K scores ([18, 27, 31]) in spite of the fact that immunization may induce B cell hyperactivity with a possible production of pathogenic autoantibodies and/or disease flare (). The mucocutaneous worsening observed in our juvenile SLE population after a 4-month evaluation may be attributed to the H1N1/2009 immunization; however, other factors such as increased sun exposure were not assessed herein.
Furthermore, the influenza A H1N1/2009 vaccine was well tolerated in juvenile SLE patients without any severe short-term adverse event, as also reported previously by others evaluating a limited number of juvenile SLE ([7, 8]) and by our group analyzing a large group of pediatric patients with autoimmune rheumatic diseases ().
Importantly, the vaccine reached all 3 current immunologic standard parameters for seroprotection (>70%), seroconversion (>40%), and factor increase in GMTs (>2.5) ([32-34]), regardless of the impaired antibody response to the influenza A H1N1/2009 vaccine compared to healthy controls. The use of 2 doses of vaccination could increase these immunologic parameters. In fact, in a previous study of patients with human immunodeficiency virus, a second dose of the pandemic H1N1/2009 influenza vaccine resulted in an additional increase of the seroconversion rate ().
In conclusion, this large prospective study demonstrated an appropriate immune response to the pandemic influenza A/H1N1 2009 virus vaccine with an excellent disease safety profile in patients with juvenile SLE. Lower seroconversion rates were particularly associated with high disease activity scores and these rates were also possibly influenced by glucocorticoid use, suggesting the need of a second boost in this subgroup of patients.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Pereira had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Campos, Silva, Aikawa, Jesus, Moraes, Miraglia, Bueno, Pereira, Bonfa.
Acquisition of data. Campos, Silva, Aikawa, Ishida, Bueno, Pereira, Bonfa.
Analysis and interpretation of data. Silva, Miraglia, Pereira, Bonfa.
The authors would like to thank the staff of the Hospital das Clinicas Faculdade de Medicina da Universidade de São Paulo, the Laboratório de Investigação Médica, the Faculdade de Medicina da Universidade de São Paulo, and the Adolfo Lutz Institute and Butantan Institute for their contribution for the study.