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Impact of Bacille Calmette-Guérin revaccination on serum IgE levels in a randomized controlled trial

Abstract

INTRODUCTION:

Bacille Calmette-Guérin (BCG) downmodulates allergen-specific IgE levels and prevents other atopic responses in experimental models but fails to protect against respiratory allergies. Human responsiveness to BCG is variable and may interfere with protection.

METHODS:

Multivariate models were evaluated to test the possible effect of responsiveness (assessed by IFN-γ production) to BCG revaccination on the modulation of total and allergen-specific serum IgE levels in healthy volunteers participating in a randomized controlled trial.

RESULTS:

Serum total or Derp-specific IgE levels did not change regardless of the increase in IFN-γ levels.

CONCLUSIONS:

BCG responsiveness does not affect protection against atopy.

Keywords:
Allergy; Atopy; Tuberculosis vaccine

Studies in humans show no evidence that immunization with Mycobacterium bovis Bacille Calmette-Guérin (BCG) protects against allergic diseases11. Arnoldussen DL, Linehan M, Sheikh A. BCG vaccination and allergy: a systematic review and meta-analysis. J Allergy Clin Immunol. 2011;127(1):246-53, 253.e1-21.

2. Flohr C, Nagel G, Weinmayr G, Kleiner A, Williams HC, Aït-Khaled N, et al. Tuberculosis, bacillus Calmette-Guérin vaccination, and allergic disease: findings from the International Study of Asthma and Allergies in Childhood Phase Two. Pediatr Allergy Immunol Off Publ Eur Soc Pediatr Allergy Immunol. 2012;23(4):324-31.
-33. Linehan MF, Nurmatov U, Frank TL, Niven RM, Baxter DN, Sheikh A. Does BCG vaccination protect against childhood asthma? Final results from the Manchester Community Asthma Study retrospective cohort study and updated systematic review and meta-analysis. J Allergy Clin Immunol . 2014;133(3):688-95.e14.. Nevertheless, there is increasing evidence that BCG vaccination modulates several aspects of atopy in experimental models of asthma and humans44. Choi IS. Immunomodulating Approach to Asthma Using Mycobacteria. Allergy Asthma Immunol Res. 2014;6(3):187-8.. 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)55. Gouveia ACC, Brugiolo ASS, Alves CCS, Silva FMC, Mesquita FP, Gameiro J, et al. Th2 responses in OVA-sensitized BALB/c mice are down-modulated by Mycobacterium bovis BCG treatment. J Clin Immunol. 2013;33(1):235-45.. Increased levels of allergen-specific serum IgE and eosinophilia are hallmarks of atopic asthma, and the former is correlated with the severity of clinical symptoms66. Sinz H, Renz H, Skevaki C. Cellular and noncellular bloodborne biomarkers in asthma. Ann Allergy Asthma Immunol. 2017;118: 672-9.. 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 humans77. Barreto ML, Pilger D, Pereira SM, Genser B, Cruz AA, Cunha SS, et al. Causes of variation in BCG vaccine efficacy: examining evidence from the BCG REVAC cluster randomized trial to explore the masking and the blocking hypotheses. Vaccine. 2014;32(30):3759-64..

In Salvador, Bahia, Brazil, neonatal BCG has been shown to protect children with respiratory allergies and sneezing against asthma88. da Cunha SS, Cruz AA, Dourado I, Barreto ML, Ferreira LDA, Rodrigues LC. Lower prevalence of reported asthma in adolescents with symptoms of rhinitis that received neonatal BCG. Allergy. 2004;59(8):857-62.. 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 individuals99. Barbosa T, Arruda S, Fernandes BD, Carvalho LP, Cardoso S, Cunha S, et al. BCG (Bacille of Calmette-Guérin) revaccination leads to improved in vitro IFN-gamma response to mycobacterial antigen independent of tuberculin sensitization in Brazilian school-age children. Vaccine. 2003;21(17-18):2152-60..

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 infection1010. Oliveira ES, Marinho JM, Barbosa T, study group. Interferon-gamma production by mononuclear cells in Bacille Calmette-Guérin-revaccinated healthy volunteers predicted long-term antimycobacterial responses in a randomized controlled trial. Vaccine . 2013;31(37):3778-82.. 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.

Ethical considerations

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)1010. Oliveira ES, Marinho JM, Barbosa T, study group. Interferon-gamma production by mononuclear cells in Bacille Calmette-Guérin-revaccinated healthy volunteers predicted long-term antimycobacterial responses in a randomized controlled trial. Vaccine . 2013;31(37):3778-82.. All subjects were healthy undergraduate students with a negative tuberculin skin test upon repeated administration of protein purified derivative (PPD)1010. Oliveira ES, Marinho JM, Barbosa T, study group. Interferon-gamma production by mononuclear cells in Bacille Calmette-Guérin-revaccinated healthy volunteers predicted long-term antimycobacterial responses in a randomized controlled trial. Vaccine . 2013;31(37):3778-82.. 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 elsewhere1010. Oliveira ES, Marinho JM, Barbosa T, study group. Interferon-gamma production by mononuclear cells in Bacille Calmette-Guérin-revaccinated healthy volunteers predicted long-term antimycobacterial responses in a randomized controlled trial. Vaccine . 2013;31(37):3778-82.. 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 group1010. Oliveira ES, Marinho JM, Barbosa T, study group. Interferon-gamma production by mononuclear cells in Bacille Calmette-Guérin-revaccinated healthy volunteers predicted long-term antimycobacterial responses in a randomized controlled trial. Vaccine . 2013;31(37):3778-82.. 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 deviation1010. Oliveira ES, Marinho JM, Barbosa T, study group. Interferon-gamma production by mononuclear cells in Bacille Calmette-Guérin-revaccinated healthy volunteers predicted long-term antimycobacterial responses in a randomized controlled trial. Vaccine . 2013;31(37):3778-82..

TABLE 1:
Sociodemographic and clinical characteristics of the study subjects.

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)1111. Solé D, Yamada E, Vana AT, Costa-Carvalho BT, Naspitz CK. Prevalence of asthma and related symptoms in school-age children in São Paulo, Brazil--International Study of Asthma and Allergies in Children (ISAAC). J Asthma Off J Assoc Care Asthma. 1999;36(2):205-12.questionnaire. 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 studies1212. Ponte JC, Junqueira SB, Veiga RV, Barreto ML, Pontes-de-Carvalho LC, Alcântara-Neves NM. A study on the immunological basis of the dissociation between type I-hypersensitivity skin reactions to Blomia tropicalis antigens and serum anti-B. tropicalis IgE antibodies. BMC Immunol. 2011;12:34.. 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 studies1313. Marks GB, Ng K, Zhou J, Toelle BG, Xuan W, Belousova EG, et al. The effect of neonatal BCG vaccination on atopy and asthma at age 7 to 14 years: an historical cohort study in a community with a very low prevalence of tuberculosis infection and a high prevalence of atopic disease. J Allergy Clin Immunol . 2003;111(3):541-9.. 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 revaccination99. Barbosa T, Arruda S, Fernandes BD, Carvalho LP, Cardoso S, Cunha S, et al. BCG (Bacille of Calmette-Guérin) revaccination leads to improved in vitro IFN-gamma response to mycobacterial antigen independent of tuberculin sensitization in Brazilian school-age children. Vaccine. 2003;21(17-18):2152-60.,1414. Conceição EL, Nascimento-Sampaio FS, Schwingel PA, Oliveira ES, Rocha MS, Vieira I, et al. Revisiting the heterogeneous IFN-γ response of Bacille of Calmette-Guérin (BCG)-revaccinated healthy volunteers in a randomized controlled trial: effect of the body mass index and of the IFNG+874 A/T polymorphism. PloS One. 2016;11(7):e0160149. or the relevance of these variables in previous studies investigating determinants of asthma1515. Grabenhenrich LB, Gough H, Reich A, Eckers N, Zepp F, Nitsche O, et al. Early-life determinants of asthma from birth to age 20 years: a German birth cohort study. J Allergy Clin Immunol . 2014;133(4):979-88..

FIGURE 1:
A. Total serum IgE levels in revaccinated volunteers. B. Anti-Derp serum IgE levels in revaccinated volunteers. C. Total serum IgE levels in control volunteers. D. Anti-Derp serum IgE levels in control volunteers. E. Multivariate linear models of the relationship between total serum IgE levels 2 months after baseline quantification and total IgE levels at baseline. F. Multivariate linear models depicting the relationship between anti-Derp serum IgE levels 2 months after baseline quantification and total IgE levels at baseline. Confidence intervals at 95% are indicated in brackets. The blue line and squares depict the data from high responders; the red line and squares depict the values obtained for low responders, and the green line and squares represent data from controls. IgE: immunoglobulin E; Derp: Dermatophagoides pteronyssinus.

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 above1010. Oliveira ES, Marinho JM, Barbosa T, study group. Interferon-gamma production by mononuclear cells in Bacille Calmette-Guérin-revaccinated healthy volunteers predicted long-term antimycobacterial responses in a randomized controlled trial. Vaccine . 2013;31(37):3778-82.. 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 determined1616. Choi IS, Koh YI. Therapeutic effects of BCG vaccination in adult asthmatic patients: a randomized, controlled trial. Ann Allergy Asthma Immunol . 2002;88(6):584-91..

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 subjects22. Flohr C, Nagel G, Weinmayr G, Kleiner A, Williams HC, Aït-Khaled N, et al. Tuberculosis, bacillus Calmette-Guérin vaccination, and allergic disease: findings from the International Study of Asthma and Allergies in Childhood Phase Two. Pediatr Allergy Immunol Off Publ Eur Soc Pediatr Allergy Immunol. 2012;23(4):324-31.. 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 sensitization55. Gouveia ACC, Brugiolo ASS, Alves CCS, Silva FMC, Mesquita FP, Gameiro J, et al. Th2 responses in OVA-sensitized BALB/c mice are down-modulated by Mycobacterium bovis BCG treatment. J Clin Immunol. 2013;33(1):235-45.. Children with respiratory allergies who received neonatal BCG in Salvador were protected against asthma88. da Cunha SS, Cruz AA, Dourado I, Barreto ML, Ferreira LDA, Rodrigues LC. Lower prevalence of reported asthma in adolescents with symptoms of rhinitis that received neonatal BCG. Allergy. 2004;59(8):857-62.. 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 response1616. Choi IS, Koh YI. Therapeutic effects of BCG vaccination in adult asthmatic patients: a randomized, controlled trial. Ann Allergy Asthma Immunol . 2002;88(6):584-91.,1717. von Hertzen LC, Haahtela T. Could the Risk of Asthma and Atopy Be Reduced by a Vaccine That Induces a Strong T-helper Type 1 Response? Am J Respir Cell Mol Biol. 2000;22(2):139-42.. 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.

Acknowledgements

The authors are grateful to Jorge Lessa Tolentino, Silvana Sousa da Paz, and Cleriston Farias Queiroz for technical assistance, and Andris Walter for editing the manuscript. Universidade Federal da Bahia and Escola Bahiana de Medicina e Saúde Pública kindly provided logistical support. Ataulpho de Paiva Foundation kindly donated the M. bovis BCG Moreau-RJ vaccine. M. tuberculosis H37Rv culture lysate was kindly provided as part of NIH, NIAID Contract No. HHSN266200400091C, titled Tuberculosis Vaccine Testing and Research Materials, which was awarded to Colorado State University.

REFERENCES

  • 1
    Arnoldussen DL, Linehan M, Sheikh A. BCG vaccination and allergy: a systematic review and meta-analysis. J Allergy Clin Immunol. 2011;127(1):246-53, 253.e1-21.
  • 2
    Flohr C, Nagel G, Weinmayr G, Kleiner A, Williams HC, Aït-Khaled N, et al. Tuberculosis, bacillus Calmette-Guérin vaccination, and allergic disease: findings from the International Study of Asthma and Allergies in Childhood Phase Two. Pediatr Allergy Immunol Off Publ Eur Soc Pediatr Allergy Immunol. 2012;23(4):324-31.
  • 3
    Linehan MF, Nurmatov U, Frank TL, Niven RM, Baxter DN, Sheikh A. Does BCG vaccination protect against childhood asthma? Final results from the Manchester Community Asthma Study retrospective cohort study and updated systematic review and meta-analysis. J Allergy Clin Immunol . 2014;133(3):688-95.e14.
  • 4
    Choi IS. Immunomodulating Approach to Asthma Using Mycobacteria. Allergy Asthma Immunol Res. 2014;6(3):187-8.
  • 5
    Gouveia ACC, Brugiolo ASS, Alves CCS, Silva FMC, Mesquita FP, Gameiro J, et al. Th2 responses in OVA-sensitized BALB/c mice are down-modulated by Mycobacterium bovis BCG treatment. J Clin Immunol. 2013;33(1):235-45.
  • 6
    Sinz H, Renz H, Skevaki C. Cellular and noncellular bloodborne biomarkers in asthma. Ann Allergy Asthma Immunol. 2017;118: 672-9.
  • 7
    Barreto ML, Pilger D, Pereira SM, Genser B, Cruz AA, Cunha SS, et al. Causes of variation in BCG vaccine efficacy: examining evidence from the BCG REVAC cluster randomized trial to explore the masking and the blocking hypotheses. Vaccine. 2014;32(30):3759-64.
  • 8
    da Cunha SS, Cruz AA, Dourado I, Barreto ML, Ferreira LDA, Rodrigues LC. Lower prevalence of reported asthma in adolescents with symptoms of rhinitis that received neonatal BCG. Allergy. 2004;59(8):857-62.
  • 9
    Barbosa T, Arruda S, Fernandes BD, Carvalho LP, Cardoso S, Cunha S, et al. BCG (Bacille of Calmette-Guérin) revaccination leads to improved in vitro IFN-gamma response to mycobacterial antigen independent of tuberculin sensitization in Brazilian school-age children. Vaccine. 2003;21(17-18):2152-60.
  • 10
    Oliveira ES, Marinho JM, Barbosa T, study group. Interferon-gamma production by mononuclear cells in Bacille Calmette-Guérin-revaccinated healthy volunteers predicted long-term antimycobacterial responses in a randomized controlled trial. Vaccine . 2013;31(37):3778-82.
  • 11
    Solé D, Yamada E, Vana AT, Costa-Carvalho BT, Naspitz CK. Prevalence of asthma and related symptoms in school-age children in São Paulo, Brazil--International Study of Asthma and Allergies in Children (ISAAC). J Asthma Off J Assoc Care Asthma. 1999;36(2):205-12.
  • 12
    Ponte JC, Junqueira SB, Veiga RV, Barreto ML, Pontes-de-Carvalho LC, Alcântara-Neves NM. A study on the immunological basis of the dissociation between type I-hypersensitivity skin reactions to Blomia tropicalis antigens and serum anti-B. tropicalis IgE antibodies. BMC Immunol. 2011;12:34.
  • 13
    Marks GB, Ng K, Zhou J, Toelle BG, Xuan W, Belousova EG, et al. The effect of neonatal BCG vaccination on atopy and asthma at age 7 to 14 years: an historical cohort study in a community with a very low prevalence of tuberculosis infection and a high prevalence of atopic disease. J Allergy Clin Immunol . 2003;111(3):541-9.
  • 14
    Conceição EL, Nascimento-Sampaio FS, Schwingel PA, Oliveira ES, Rocha MS, Vieira I, et al. Revisiting the heterogeneous IFN-γ response of Bacille of Calmette-Guérin (BCG)-revaccinated healthy volunteers in a randomized controlled trial: effect of the body mass index and of the IFNG+874 A/T polymorphism. PloS One. 2016;11(7):e0160149.
  • 15
    Grabenhenrich LB, Gough H, Reich A, Eckers N, Zepp F, Nitsche O, et al. Early-life determinants of asthma from birth to age 20 years: a German birth cohort study. J Allergy Clin Immunol . 2014;133(4):979-88.
  • 16
    Choi IS, Koh YI. Therapeutic effects of BCG vaccination in adult asthmatic patients: a randomized, controlled trial. Ann Allergy Asthma Immunol . 2002;88(6):584-91.
  • 17
    von Hertzen LC, Haahtela T. Could the Risk of Asthma and Atopy Be Reduced by a Vaccine That Induces a Strong T-helper Type 1 Response? Am J Respir Cell Mol Biol. 2000;22(2):139-42.
  • Financial support: Fundação de Amparo à Pesquisa do Estado da Bahia (Programa Primeiros Projetos Nº 160/2003).

Publication Dates

  • Publication in this collection
    Jan-Feb 2018

History

  • Received
    07 Apr 2017
  • Accepted
    12 Sept 2017
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