Hepatitis B virus (HBV) infection is an important worldwide public health issue, and this infection is responsible for significant morbidity and mortality. The HBV vaccine was introduced in the early 1980s for use in individuals at high risk for acquiring HBV infection. In the United States it is currently administered to all infants, previously unvaccinated children, and unvaccinated adults at high risk in an attempt to achieve universal protection against HBV infection (1). Between 90% and 95% of the adult population responds to HBV vaccination (2,3). Nonresponders are often found to carry specific human leukocyte antigen (HLA) haplotypes, including B8, DR3, and DQ2 (4,5).
Celiac disease (CD) is a common autoimmune disorder with an estimated prevalence of 1 in 120 to 300 in Europe and North America (6–9). It is strongly associated with the DQ2 HLA haplotype, and individuals lacking the DQ2 haplotype are generally positive for DQ8 (10–13). In fact, studies suggest that as many as 99% of patients with CD can be expected to carry ≥1 of these HLA alleles (14,15).
Because these same haplotypes are overrepresented in patients in the general population who do not show a response to HBV vaccination, it seems reasonable to hypothesize that patients with CD are, as a group, less able to respond to HBV vaccine than the general population, whose frequency of HLA haplotypes B8, DR3, and DQ2 are much lower. In this study we therefore sought to determine whether children with CD fail to respond to HBV vaccine more frequently than children without CD. To assess whether a lack of response to HBV is specific to HBV vaccine or a reflection of a more generalized inadequate response to childhood vaccination, we also evaluated patients with CD and control subjects' responses to tetanus, rubella, and Haemophilus influenzae type b (Hib) vaccines. The HBV vaccine is a DNA-recombinant vaccine, tetanus vaccine is a toxoid vaccine, rubella vaccine is a live attenuated vaccine, and Hib vaccine is a polysaccharide vaccine.
PATIENTS AND METHODS
Children previously diagnosed with CD based on standard clinical criteria including abnormal serological markers (anti-gliadin immunoglobulin [Ig] A antibody, tissue transglutaminase IgA antibody, anti-endomysial IgA antibody, anti-gliadin IgG antibody) and typical small-bowel biopsy histologic findings such as villous atrophy with crypt hyperplasia and increased intraepithelial lymphocytes, who were studied by members of the Division of Pediatric Gastroenterology and Nutrition at Schneider Children's Hospital, North Shore-Long Island Jewish Health System, were approached to participate in this study by a letter or by their primary gastroenterologist during an office visit. Inclusion criteria required that subjects must have completed 3 full doses of recombinant HBV vaccination at least 6 months before enrolling in the study. Immunization to tetanus, rubella, and Hib also had to have been completed or updated. The childhood and adolescent immunization schedule approved by the Advisory Committee on Immunization Practices (www.cdc.gov/nip/acip), American Academy of Pediatrics (www.aap.org), and American Academy of Family Physicians (www.aafp.org) for 2002 was applied to evaluate the completeness or update of immunizations. Vaccination records were obtained from parents or from the primary pediatrician with written permission of the parents and reviewed in every case to ensure the completeness of childhood vaccination.
Healthy age- and sex-matched controls were recruited from the children evaluated or treated by the same group of physicians for functional bowel disorders. Control subjects needed to have a negative celiac serological panel or unremarkable duodenal histology. The celiac serological panel was interpreted as abnormal if the anti-gliadin IgA antibody, tissue transglutaminase IgA antibody, or anti-endomysial IgA antibody was higher than the normal range in a subject with a normal level of total serum IgA. In children with serum IgA deficiency, the celiac panel was considered abnormal if there was an increased anti-gliadin IgG antibody. All subjects with abnormal celiac panels not already known to have biopsy-proven CD were recruited to undergo upper endoscopy for small-bowel biopsy to determine whether they had CD. Control subjects should have received immunizations as required in the patients with CD. Exclusion criteria for control subjects also included the presence of an underlying immune disorder, receipt of medication currently or in the past known to modulate or suppress the immune system (ie, corticosteroids, azathioprine, 6-mercaptopurine), and family history of CD in any first-degree relative. Subjects who had an increased erythrocyte sedimentation rate (>20 mm/h) or low serum albumin level (<3.5 g/dL), or whose history and physical examination suggested possible inflammatory bowel disease, were also excluded from participation in the study.
Informed consent was obtained from a parent or legal guardian. Assent was obtained from subjects if they were 9 years of age or older. All aspects of the study were approved by the North Shore-Long Island Jewish Health System's institutional review board.
Clinical data were compiled for each subject from a review of previous medical and vaccination records. These data included current age, sex, immunization status (HBV, tetanus, rubella, and Hib) including timing of immunizations, age of diagnosis for CD, results of celiac panel if it was done within 2 months before the antibody testing (as a reflection of adherence to gluten-free diet in CD group), medication (current and past), coexisting medical condition, and family history.
The results of a subject's immune status to the vaccines studied were conveyed to the subject's parent or guardian. If a subject was found not to have developed HBsAb, then the primary care physician was notified with the permission of the parent/guardian. Repeat immunization was not performed as a part of this study, but was left to the discretion of the child's primary care provider.
Approximately 1 tablespoon of blood was collected by phlebotomy and was processed at the clinical laboratory of North Shore-Long Island Jewish Health System. HBsAb was measured using enhanced chemiluminescence (Ortho Clinical Diagnostics, Raritan, NJ). Samples with results ≥12 IU/mL were flagged as antibody positive. Samples with results between 5 and 12 IU/mL were retested, and those <12 IU/mL for 2 of 3 tests were flagged as antibody-negative. An enzyme-linked immunosorbent assay was used for antibodies to rubella (Biomeriux, Durham, NC), tetanus (The Binding Site, Birmingham, UK), and Hib (The Binding Site, Birmingham, UK). The cutoff values were 0.75 index, 1.0 IU/mL, and 1.0 μg/mL, respectively.
The primary outcome variable of the study was the response to HBV vaccine (positive or negative for the antibody). A negative response was considered a vaccine failure. Secondary outcomes included response to tetanus, rubella, and Hib vaccines.
Demographic variables such as age, sex, family history, age when vaccination was completed, number of years since last vaccination, and titer level for HBsAb in HBV responders were also collected. The Fisher exact test was used to compare the proportions of “failed response” between CD and control groups, male and female patients among the CD group, and CD subjects who completed vaccinations before or at/after 1 year of age. The Mann-Whitney test was used to compare age, age when HBV vaccination was completed, years since immunizations, and titer level of HBsAb between groups. Continuous variables are expressed as mean ± SD. Multiple logistic regression was used to investigate independent effect of CD on failed response to HBV vaccine after the adjustment for confounders (sex, age when HBV vaccination was completed, and years since vaccination). Differences were considered significant at the P < 0.05 level. All statistical analyses were carried out using SAS software (SAS, Cary, NC).
The study was designed to enroll 25 subjects per group, which would have yielded approximately 82% power to detect a 35% difference in the primary outcome variable between the CD and control groups (assuming 40% vs 5% negative response) using the Fisher exact test at the 0.05 significance level.
In total, the parents or guardians of 48 children or adolescents signed the study consent. However, 4 subjects were ultimately excluded per protocol. One subject had a family history of CD and another had inflammatory bowel disease. Another 2 subjects had abnormal duodenal histological findings, 1 with nonspecific duodenitis and the other with eosinophilic duodenitis. Although both CD and control groups should have 25 subjects as initially designed because of logistical constraints, 26 patients with CD and only 18 control subjects were accrued in the study. All of the patients with CD were white, but the control group included 5 subjects of Asian descent. Two patients with CD had comorbidity with insulin-dependent diabetes mellitus. The mean age of the patients with CD at the time of the study was 9.2 ± 4.6 years. Fifty-two percent were female. Ten of the 26 patients with CD (38%) had a family history of CD. There were 3 pairs of siblings included in the CD group. None of the control subjects had a positive family history for CD. The mean age of subjects at the time of CD diagnosis was 7.1 ± 5.2 years. A tabulation of age, sex, age when HBV vaccine was completed, and years since last vaccination according to group (CD vs controls) is summarized in Table 1. There were no significant differences between CD and controls with respect to age, age when HBV vaccination was completed, or years since last vaccinations.
The rates of failure to each immunization are summarized in Table 2.
Hepatitis B Vaccine
All of the subjects had received 3 doses of HBV vaccine at least 9 months before the HBsAb testing. A significantly higher proportion of subjects in the CD group (14 of 26) failed to respond to HBV vaccine compared with controls (2 of 18) (53.9% vs 11.1%; P < 0.05). Multiple logistic regression showed an independent effect of CD on failed response to HBV vaccine adjusted for confounders (sex, age when HBV vaccination was completed, and years since vaccination; Table 3). Subjects with CD were 8.33 times more likely to have negative results for HBsAb than controls after adjusting these confounders (95% CI, 1.5–46.5). There was no statistically significant difference in mean titers of HBsAb in vaccine responders between CD and control groups (293.1 ± 420.9 IU/mL vs 653.1 ± 1777.6 IU/mL; P = 0.43).
Nine of 14 female patients with CD were negative for HBsAb, as were 5 of 12 male patients with CD (64.3% vs 41.7%; P = 0.11). Eleven of 16 patients with CD who completed HBV vaccination before 1 year of age did not develop HBsAb; conversely, 3 of 10 patients with CD who completed it at or after 1 year of age failed to develop HBsAb (68.8% vs 30.0%; P = 0.11). Two HBsAb-negative control subjects were male. One completed HBV vaccine before 1 year of age and the other one did it at 1 year of age. Two CD subjects with insulin-dependent diabetes mellitus successfully developed HBsAb. Within the patients with CD, the mean time between the last dose of HBV vaccine and HBsAb test was not significantly different in responders versus nonresponders (6.1 ± 3.6 years vs 6.6 ± 3.0 years; P = 0.13). A celiac serological panel was tested in 22 patients with CD at the time of or within 2 months before HBsAb testing to evaluate adherence to a gluten-free diet. The panel findings were abnormal in 36% of subjects in the HBsAb-positive (4 of 11) and HBsAb-negative groups (4 of 11). There was a positive family history of CD in 25% of those who had positive HBsAb findings, compared with 50% of those who tested negative for HBsAb. Concordant results were found among the 3 pairs of siblings included in the CD group. Two pairs were HBV vaccine nonresponders and the other pair had positive HBsAb findings.
All of the subjects in both the CD and control groups tested positive for rubella antibodies.
There was only 1 subject in the CD group who tested negative for tetanus antibody, and there were 0 in the control group (3.9% vs 0%; P = 1.0).
Haemophilus Influenzae Type B Vaccine
Haemophilus influenzae type b antibody was not analyzed in 2 subjects because of an error in specimen processing at the laboratory. Although there were fewer subjects in the CD group with a negative response to Hib than in the control group, the difference was not significant (33.3% vs 44.4%; P = 0.53). Mean durations between the last dose of Hib vaccine and Hib antibody testing were 6.4 ± 4.1 years for Hib vaccine responders and 7.6 ± 2.7 years for Hib nonresponders. There was no statistically significant difference between these 2 groups. None of the nonresponders were younger than 5 years of age, and repeat immunization was not indicated.
We have demonstrated that children with CD have a significantly higher prevalence of failed response to HBV vaccine than does a healthy control population. By contrast, their response to other childhood vaccines is not impaired. Patients with CD who completed HBV vaccination before 1 year of age had a higher frequency of failure to develop HBsAb than did the group of patients with CD who completed it at or after the first year of life. None of the patients with CD were diagnosed before 1 year of age, and it is unknown whether ongoing exposure to gluten, possible nutritional deficiencies, or age may have contributed to the failure to develop HBsAb.
In a similar study in an adult cohort, Noh et al determined the HBsAb status of 23 adults with CD who had received a full series of HBV vaccine (16). Of the 19 subjects who had testing for HBsAb, 13 did not show long-term immunity. Human leukocyte antigen class II typing was performed on 15 of these subjects (13 nonresponders and 2 responders). All tested subjects were heterozygous or homozygous for DQ2. Noh et al postulated that HBV vaccine nonresponders fail to induce the Th2 response needed for B cell differentiation and formation of memory B cells (16). A defect in the production of memory B cells has been demonstrated, associated with splenic hypofunction, in adults with CD complicated by refractory disease, enteropathy-associated T cell lymphoma, and ulcerative jejunoileitis (17). This defect was found to be present irrespective of the duration of a gluten-free diet.
The ability to respond to recombinant HBV vaccine is immunogenetically conditioned by multiple candidate genes (4,5,18,19). Certain HLA haplotypes appear to be particularly important. Alper et al (4) prospectively vaccinated 5 homozygotes and 9 heterozygotes for extended MHC haplotypes (HLA-B8, SC01, DR3). Four of the 5 homozygotes produced low levels of HBsAb 2 months after their third HBV vaccination, whereas all 9 heterozygotes had significantly higher titers of HBsAb. Stachowski et al (5) studied 153 patients with end-stage renal disease immunized with a recombinant HBV vaccine. Thirty-four patients were considered nonresponders. Homozygotes for HLAs A1, B8, DR3, and DQ2 were found almost exclusively in the nonresponder group, and significantly more heterozygotes for these alleles were found in the nonresponder group compared with the responders. Martinetti et al performed an HLA study in 9 absolute nonresponder and 8 hyporesponder infants who underwent HBV vaccination in the neonatal period (18). Many of the subjects carried HLA haplotypes classically involved in autoimmune disease, including DQ2 and DQ8.
Belloni et al assessed the frequency of autoantibody production after HBV vaccination in 210 six-year-old children who were vaccinated at birth (20). Only 10 children (4.7%) did not respond to HBV vaccine. Overall, HBV vaccination did not increase autoantibody production compared with a nonvaccinated control population, and only 1 child (a responder to HBV vaccine) was shown to have a detectable anti-endomysial antibody. However, HBV vaccine nonresponders had a significantly higher frequency of autoantibodies, particularly smooth muscle antibody, compared with responders (30% vs 2%; P = 0.0025). The SMA-positive children carried the HLA-C4AQ0, DRB1*0301, DQB1*02 haplotype, a widely known predisposing factor for autoimmune disorder. This finding further supports the important role of HLA haplotypes for response to HBV vaccine and the development of autoimmune disease.
Although other factors may contribute to successful HBV immunization, these observations support the hypothesis that HLA haplotypes play a role in responding to HBV vaccine. The concordant responses in the sibling pairs with CD in our study also support the probable genetic contribution to HBV vaccine response. In the present study, we did not perform HLA typing because it is widely known that virtually all children with CD are positive for HLA-DQ2 or DQ8, whereas only approximately 30% of the general population in North America is DQ2 positive (21). Our study was not designed to determine whether healthy children who are HLA DQ2 positive are at greater risk for nonresponse to HBV vaccine. Evaluating the HLA haplotypes for CD and control groups in future studies may characterize the role of HLA typing in response to HBV vaccine more strongly.
A high nonresponse rate to HBV vaccine puts children with CD at significant risk of acquiring HBV infection. This can be particularly true in individuals who do not take appropriate precautions because of a history of having undergone a recommended vaccination schedule. Given the high prevalence of CD in the population, a lack of response to HBV vaccine can also hinder efforts to develop “herd” immunity through universal vaccination. Evaluating the response to HBV vaccine should be considered a routine assessment in children with CD. Whether repeat immunization should be considered to increase the chance of response to HBV vaccine in children with CD remains to be determined. New modalities to enhance vaccine response may also need to be investigated. However, our findings suggest that there may be a significant public health problem that needs to be addressed. Given current vaccination strategies and the worldwide frequency of CD, it appears that a large reservoir of HBV-susceptible people will persist even if universal vaccination becomes a reality.
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