Clin Exp Vaccine Res.  2016 Jan;5(1):31-40. 10.7774/cevr.2016.5.1.31.

Assessment of immunological markers and booster effects of Ag85B peptides, Ag85B, and BCG in blood of BCG vaccinated children: a preliminary report

• Husain AA
• Daginawla
• Singh L
• Kashyap RS

• KMID: 2152695
• DOI: http://doi.org/10.7774/cevr.2016.5.1.31

Abstract

PURPOSE
In the present study, the protective immunological markers in serum and peripheral blood mononuclear cells (PBMCs) of bacillus Calmette-Guerin (BCG) vaccinated and unvaccinated children were evaluated after vaccination. Further, PBMCs of children with low protective levels were boosted with BCG, Ag85B, and Ag85B peptides to study their booster effects to increase waning BCG induced immunity.
MATERIALS AND METHODS
Fifty children from 1 month to 18 years of age were randomized for the study. Blood samples were collected from 27 participants with/without BCG vaccination. Immunological markers (anti-BCG, interferon gamma [IFN-gamma], and adenosine deaminase activity) were assessed in both serum and PBMCs of children. Children with low levels of protective immunological markers were further recruited and their PBMCs were boosted with BCG, Ag85B, and Ag85B peptides.
RESULTS
Children in age group of 4-6 years were associated with significantly (p<0.05) higher BCG-specific IgG and IFN-gamma levels compared to those in age group greater than 10 years. Vaccinated children had greater repertoire of immunological memory which on in vitro stimulation with BCG showed increase in BCG-specific response compared to unvaccinated controls. Assessment of booster effects of BCG, Ag85B, and Ag85B peptides in PBMCs of children revealed greater potential of peptides to boost BCG induced immunity compared to BCG and Ag85B.
CONCLUSION
To conclude, children within age 4-6 years are associated with high immunological markers which eventually diminish with age thereby suggesting need for booster dose in later years. Mycobacterium tuberculosis peptides along with BCG may be used as attractive candidates to boost such waning BCG induced immunity in children.

Keyword

BCG vaccine; PBMC; Interferon-gamma; Child; Peptides; Ag85B

MeSH Terms

Adenosine Deaminase
Bacillus
BCG Vaccine
Child*
Humans
Immunoglobulin G
Immunologic Memory
Interferon-gamma
Interferons
Mycobacterium bovis*
Mycobacterium tuberculosis
Peptides*
Vaccination
Adenosine Deaminase
BCG Vaccine
Immunoglobulin G
Interferon-gamma
Interferons
Peptides

Figure

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  Fig. 1 Study participation diagram. The figure represents the inclusion/exclusion criteria adopted for recruitment of 27 children.

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  Fig. 2 Box plots of immunological markers (anti–bacillus Calmette–Guérin [BCG], adenosine deaminase [ADA], and interferon γ [IFN-γ]) in serum (A) and cell supernatants (B) of peripheral blood mononuclear cells (PBMCs) of children of different age groups. Isolated PBMCs were stimulated with BCG vaccine (100 µL). Data is representative of 48 hours after BCG stimulation. Data are shown as SEM. ANOVA (one way) was used for multiple comparisons. *,**Significant (p < 0.05, p < 0.001), ***Highly significant (p < 0.001).

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  Fig. 3 Immunological markers (anti–bacillus Calmette–Guérin [BCG], adenosine deaminase [ADA], and interferon γ [IFN-γ]) in serum (A) and cell supernatants (B) of peripheral blood mononuclear cells (PBMCs) of BCG vaccinated children with/without BCG scar post-vaccination. Data is representative of 48 hours after BCG stimulation (for PBMCs). Data are shown as ±SD. Paired t-test was used for obtaining statistical significance. Significant (*p<0.05, **p<0.001), ***Highly significant (p<0.0001).

• 

  Fig. 4 Immunological markers (anti–bacillus Calmette–Guérin [BCG], adenosine deaminase [ADA], and interferon γ [IFN-γ]) in blood (A) and cell supernatants (B) of peripheral blood mononuclear cells (PBMCs) of BCG vaccinated children and unvaccinated children. Data is representative of 48 hours after BCG stimulation (for PBMCs). Data are shown as ±SD. Paired t-test was used for obtaining statistical signifcance. Significant (*p<0.05, **p<0.001), ***Highly significant (p<0.0001).

• 

  Fig. 5 Anti–bacillus Calmette–Guérin (BCG) (A) and interferon γ (IFN-γ) (B) levels in 72-hour culture supernatants of children boosted with BCG, Ag85B, and Ag85B peptides. Peripheral blood mononuclear cells of children were isolated and stimulated with BCG (100 µL/105 CFU). Twelve hours after incubation, cells were stimulated with BCG vaccine (50 µL/102 CFU), 0.1 mL of Ag85B (25 µg/mL in Freund's adjuvant) and 0.1 mL cocktail of Ag85B peptides (25 µg/mL p1 and p3 in Freund's adjuvant). Paired t-test was used for obtaining statistical signifcance. **Significant (p<0.001), ***Highly significant (p<0.0001).

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  Fig. 6 Comparative assessment of immunological markers: anti–bacillus Calmette–Guérin (BCG) (A) and interferon γ (IFN-γ) (B) in 72-hour cell culture supernatants of children boosted with BCG, Ag85B, and Ag85B peptides. Titres of both immunological markers were compared on Log2 for percentage fold rise from initial response after primary stimulation with BCG vaccine. **Significant (p<0.001) values.

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