Studies in humans show no evidence that immunization with Mycobacterium bovis Bacille Calmette-Guérin (BCG) protects against allergic diseases1–3. Nevertheless, there is increasing evidence that BCG vaccination modulates several aspects of atopy in experimental models of asthma and humans4. Mice immunized with BCG and sensitized intranasally with ovalbumin (OVA) had a lower percentage of eosinophils in the bronchoalveolar lavage, reduced levels of total and allergen-specific serum immunoglobulin E (IgE), decreased levels of T-helper 2 (Th2) cytokine, decreased lung inflammation, as well as increased levels of interferon gamma (IFN-γ), tumor necrosis factor beta (TGF-β), and interleukin-10 (IL-10)5. Increased levels of allergen-specific serum IgE and eosinophilia are hallmarks of atopic asthma, and the former is correlated with the severity of clinical symptoms6. A limited modulation of the immune response by the BCG vaccine in humans may partially explain the discrepancies in the findings of studies using experimental models and humans7.
In Salvador, Bahia, Brazil, neonatal BCG has been shown to protect children with respiratory allergies and sneezing against asthma8. However, children’s responses to BCG revaccination are variable, and the levels of IFN-γ and IL-10 are increased in response to mycobacterial antigens in vitro in only a few individuals9.
Here, we investigate the immune modulation elicited by revaccination with BCG in a previously conducted clinical trial that evaluated the in vitro responses to mycobacterial challenge in a sample of healthy BCG-revaccinated volunteers negative for TB infection10. We hypothesized that BCG revaccination differentially modulated serum IgE levels in individuals with the high levels of IFN-γ compared with those in which the effects of the vaccine were modest. This hypothesis was tested as a secondary objective of the clinical trial.
This study was approved by the Institutional Review Board of Instituto Gonçalo Moniz (CAAE-0010.0.225.069-09) and was registered in the Brazilian Clinical Trials Registry (primary ID No. RBR-2gv984). We analyzed the serum samples from 75 volunteers who participated in a previous randomized, open, controlled (2-arms) clinical trial, of which 46 subjects received BCG revaccination and 29 received no intervention (Table 1)10. All subjects were healthy undergraduate students with a negative tuberculin skin test upon repeated administration of protein purified derivative (PPD)10. The serum samples of two volunteers (one revaccinated and one control) were not analyzed. The Consolidated Standards of Reporting Trials (CONSORT) 2010 statement guidelines were followed, and the study’s enrollment and follow-up flowcharts have been published elsewhere10. All individuals had been vaccinated with BCG in early childhood and tested negative for latent tuberculosis (in the tuberculin skin test) and human immunodeficiency virus (HIV) before inclusion in the study. Whole blood cultures (blood diluted 1:10 in Roswell Park Memorial Institute (RPMI) 1640 stimulated with 10µg/ml Mycobacterium tuberculosis H37Rv culture lysate) from 46% of BCG-revaccinated individuals in this trial showed consistently higher levels of IFN-γ at 2 and 12 months after the intervention compared with baseline levels and the control group10. The fold-increase in the IFN-γ levels at 2 months after revaccination relative to baseline levels (T2/T0) was predictive of the IFN-γ responses measured at 12 months, which exceeded the median values of the control group by at least 2-fold the standard deviation10.
|Revaccinated (n=46) a||Controls (n=29) b|
|atopic (n =29)||non-atopic (n =16)||(atopic n =17)||non-atopic (n =11)|
|Age in years (95% CI)c||20 (19-21)||20 (19-21)||20 (17-23)||20 (18-22)|
|male||9 (31.0)||2 (12.0)||5 (29.0)||4 (36.0)|
|female||20 (69.0)||14 (88.0)||12 (71.0)||7 (64.0)|
|Family income > 5 MWd (%)||22 (76.0)||11 (69.0)||13 (76.0)||8 (73.0)|
|n/a||1 (3.0)||0 (0.0)0||1 (6.0)||0 (0.0)|
|Smoking (%) yes||2 (7.0)||0 (0.0)||0 (0.0)||0 (0.0)|
|n/a||0 (0.0)||0 (0.0)||0 (0.0)||0 (0.0)|
|Drug use (%)|
|alcohole||8 (28.0)||6 (38.0)||1 (6.0)||3 (27.0)|
|otherf||1 (3.0)||0 (0.0)||0 (0.0)||0 (0.0)|
|n/a||3 (10.0)||0 (0.0)||0 (0.0)||0 (0.0)|
|yes||3 (10.0)||1 (6.0)||2 (12.0)||0 (0.0)|
|no||13 (45.0)||8 (50.0)||8 (47.0)||5 (45.0)|
|n/a||13 (45.0)||7 (44.0)||7 (41.0)||6 (55.0)|
|BMIg (95% CI)c||21.0 (20.1-22.0)||20.3 (19.2-21.3)||21.3 (19.6-23)||20.4 (18.8-22.0)|
|BCGh scar size from revaccination in mm (95% CI)c||8.3 (6.3-10.2)||7.4 (4.1-10.8)||–||–|
|Current medication use (%)|
|antihistamine||0 (0.0)||1 (6.0)||0 (0.0)||0 (0.0)|
|corticosteroid||0 (0.0)||0 (0.0)||1 (6.0)||0 (0.0)|
|other non-related||6 (21.0)||3 (19.0)||3 (18.0)||2 (18.0)|
|none||23 (79.0)||12 (75.0)||13 (76.0)||9 (82.0)|
|asthma/wheezing||6 (21.0)||2 (12.0)||4 (24.0)||2 (18.0)|
|rhinitis/sneezing||16 (55.0)||4 (25.0)||16 (94.0)||2 (18.0)|
|rhinitis/sneezing combined with asthma/wheezing||2 (7.0)||1 (3.0)||1 (6.0)||1 (9.0)|
|dermatitis/eczema||5 (17.0)||2 (12.0)||1 (6.0)||0 (0.0)|
|not allergic||8 (28.0)||11 (69.0)||1 (6.0)||8 (73.0)|
|n/a||1 (3.0)||0 (0.0)||0 (0.0)||0 (0.0)|
|Allergy symptoms in the past 12 months (%)i||13 (45.0)||5 (31.0)||13 (76.0)||3 (27.0)|
|n/a||0 (0.0)||0 (0.0)||0 (0.0)||0 (0.0)|
|to any allergen in the SPTj||17 (59.0)||–||8 (47.0)||–|
|to Derpk in the SPT||24 (83.0)||–||14 (82.0)||–|
|SPT conversion 2 months after intervention*||–||1 (6.0)||–||1 (9.0)|
|Total IgE (ng/ml)c|
|baseline (T0)||90.0 (45.4-134.5)||19.4 (3.2-35.7)||23.6 (10.8-36.5)||21.4 (2.2-40.6)|
|two months after intervention (T2)||66.7 (36.0-97.4)||21.9 (7.9-36.0)||34.4 (8.9-60.0)||25.5 (1.8-49.2)|
|Anti-Derp IgE (kU/ml)c|
|baseline (T0)||29.40 (13.18-45.63)||0.00 (0.00-0.00)||23.70 (6.54-40.85)||0.00 (0.00-0.00)|
|two months after intervention (T2)||29.3 (10.5-48.0)||0.12 (-0.10 to -0.33)||8.09 (-2.69 to -18.87)||0.22 (-0.47 to -0.90)|
95% CI: 95% confidence intervals; MW: minimum wage; n/a: information not available; BMI: body mass index; BCG: Bacille Calmette-Guérin; ISAAC: International Study of Asthma and Allergies in Childhood; SPT: skin prick test; Derp: Dermatophagoides pteronyssinus; IgE: immunoglobulin E; T0: time zero (baseline); T2:two months after the intervention. a One revaccinated individual not tested. b One control individual not tested. cResults are expressed as means (95% CI). d MW (>5 MW indicates an upper-class or class “A” consumer)3. eAlcohol use was defined as alcohol consumption at least once per week. f Marijuana use. g BMI (weight in kg divided by the square of the height in m). h BCG. i Data obtained from the ISAAC questionnaire. j SPT. k Derp whole extract. *Percentage of individuals non-reactive to Derp at baseline that became reactive 2 months after revaccination.
The clinical trial assessed skin reactivity against respiratory aeroallergens, including Dermatophagoides pteronyssinus (Derp), Blomia tropicalis, Blattella germanica, Periplaneta americana, dog epithelium, cat epithelium, pollen mixture IV (Dactylis glomerata, Festuca arundinacea, Lolium perenne, Phleum pratense, and Poa pratensis), and fungal mixture V [Aspergillus (A.) Aspergillus amstelodami, Aspergillus fumigatus, Aspergillus niger, Aspergillus terrus, Penicillium (P.) Penicillium brevicompactum, Penicillium expansum, Penicillium notatum, Penicillium roqueforti, Cladosporium fulvum, and Cladosporium herbarum] using skin prick tests [(SPT); ALK-Abelló, São Paulo, Brazil]. Sociodemographic and clinical characteristics were obtained using a standard questionnaire and the International Study of Asthma and Allergies in Childhood (ISAAC)11questionnaire. Total serum IgE levels were measured using the Human IgE Flex Set assay and a FACSArray flow cytometer (Becton Dickinson, Palo Alto, CA, USA). Anti-Derp serum IgE levels were measured using an ImmunoCAP kit (ImmunoCAP Phadia AB, Uppsala, Sweden). Multivariate linear models were evaluated, adjusting for a combination of the following variables: gender, age, body mass index (BMI), income, revaccination scar size, occurrence of allergies in the previous 12 months, atopy (defined as a positive SPT and/or anti-Derp IgE level >0.35kU/L at baseline), and the IFN-γ T2/T0 ratio.
The percentage of atopic and non-atopic individuals was not significantly different between the revaccinated and control group (Table 1). Demographic characteristics, smoking, drug use habits, and allergy symptoms did not differ between revaccinated and control individuals, either atopic or non-atopic. Atopic individuals were usually sensitized against aeroallergens from the house dust mite D. pteronyssinus, as observed in previous studies12. Baseline total IgE levels were increased in atopic individuals of the revaccinated group compared with atopic controls. The levels of total and anti-Derp IgE were not significantly different between T2 and T0 in both groups.
Total IgE levels (Figure 1A and Figure 1C) and anti-Derp IgE levels (Figure 1B and Figure 1D) remained unchanged up to 12 months after the intervention in the revaccinated (Figure 1Aand Figure 1B) and control (Figure 1C and Figure 1D) groups. In BCG-revaccinated individuals, total IgE levels at T2 were positively correlated with total IgE levels (p < 0.0001; Spearman r=0.88) and anti-Derp IgE levels (p < 0.0001; Spearman r=0.54) at T0, suggesting a lack of IgE modulation from BCG revaccination, which agrees with the results of previous studies13. Although the results of bivariate analysis indicated that there was no association between total or anti-Derp IgE levels and the parameters used in the multivariate linear models, these variables were included in the models because of a previously reported correlation with IFN-γ levels upon mycobacterial antigen stimulation after BCG revaccination9,14 or the relevance of these variables in previous studies investigating determinants of asthma15.
The modulation of serum IgE (total and anti-Derp-specific) at 2 months after BCG revaccination was evaluated using multivariate linear models (Figure 1). Vaccine responsiveness was taken into account by stratifying the BCG-revaccinated group according to the IFN-γ T2/T0 ratio into a high-responder group (IFN-γ T2/T0 ratio >3.262) and a low-responder group (IFN-γ T2/T0 ratio ≤3.262) because this cut-off was predictive of the IFN-γ response to M. tuberculosis antigens after 12 months of revaccination, as discussed above10. Combinations of the following variables were tested: gender, age, body mass index (BMI), income, revaccination scar size, occurrence of allergies in the previous 12 months, atopy (defined as a positive SPT and/or anti-Derp IgE level >0.35kU/L). The results indicated that these variables did not affect the regression coefficients compared with the bivariate analysis of serum IgE levels (total or anti-Derp) between T2 and T0. Therefore, the results of the two bivariate regression models were analyzed considering the vaccine responsiveness in a stratified analysis (Figure 1). There was no significant difference between the regression coefficients of the models describing the relationship between total IgE levels at T2 and T0 in the control and BCG-revaccinated individuals of the low- and high-responder groups (Figure 1E). The regression coefficients of the models describing the relationship between anti-Derp IgE levels at T2 and T0 were also close to 1 in all groups (Figure 1F), suggesting that BCG did not affect the serum IgE response, regardless of the capacity of responding to the vaccine evaluated by the IFN-γ levels.
The individuals included in the clinical trial reported mild respiratory allergy symptoms, and most had not used medications to treat allergic symptoms in the previous 12 months (Table 1). A minority (15%) reported dry cough in the absence of respiratory infection in the previous 12 months; only one individual reported having symptoms after physical exercise that impaired activities of daily living and speech. However, in this study, no clinical evaluations were performed to assess the regression of allergic symptoms after revaccination with BCG. Therefore, we could not determine the possible effects of BCG revaccination on allergic responses in the volunteers of this trial. Notwithstanding, a previous study reported that respiratory function was improved and medication use was reduced after repeated BCG administration in patients with moderate to severe perennial asthma, although the effect on eosinophil counts and sputum cytokine levels was null, and IgE levels were not determined16.
It has been suggested that the inability of BCG revaccination to modulate atopic responses may be related to an individual’s age at the time of antigen exposure and/or the genetic background of the study subjects2. In animal models, the effect of the modulation of the Th2 response was protective and improved parameters related to allergic conditions in animals exposed to microbial antigens before allergen sensitization5. Children with respiratory allergies who received neonatal BCG in Salvador were protected against asthma8. Age at the time of vaccination may have played a protective role in these cases, possibly modulating the children’s response before allergic sensitization. It is also possible that the route of administration and antigen load affect the ability of BCG to modulate the atopic response16,17. Alternatively, an overlooked effect of non-atopic responses may explain this apparent protection by vaccination with BCG.
In conclusion, BCG revaccination did not modulate total IgE levels in vaccine-responsive individuals and allergen-specific IgE levels in the studied population, regardless of the magnitude of the increase in the IFN-γ levels.