|Year : 2006 | Volume
| Issue : 2 | Page : 59-63
Invitro immune responses in children following BCG vaccination
V Vijayalakshmi, Sunil Kumar, H Surekha Rani, G Suman Latha, KJR Murthy
Immunology Unit, Bhagawan Mahavir Medical ResearchCentre, 10-1-1, Mahavir Marg, Hyderabad 500 004., India
Immunology Unit, Bhagawan Mahavir Medical ResearchCentre, 10-1-1, Mahavir Marg, Hyderabad 500 004.
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: There is still no consensus on the efficacy of BCG vaccine in the prevention of tuberculosis. This study therefore addressed the question of the magnitude of immunity afforded by BCG, by studying the effector mechanisms of protection in children. The main objectives were to assess the degree of immunity conferred by BCG vaccine in children and to identify the most immunogenic antigen(s) of BCG by conducting in-vitro studies.
Materials and methods: Children in the age-group of 1 to 10 years, were categorized: (A) normal, and vaccinated with BCG during the first year, n=45, (B) normal, without scar and with no evident history of vaccination, n=31: and (C) children admitted in the hospital with a confirmed diagnosis of tuberculosis, n=31. Fractions of BCG were obtained by lysis, sonication, separation by gel chromatography, HPLC and confirmed by SDS-PAGE. In lymphoproliferative assays PBMC were cultured and stimulated with either Concanavalin-A or Tuberculin or the fractions of BCG. Stimulation indices (SI) in lymphoproliferation, CD4/CD8 cells, levels of Interferon-γ (IFN- γ) in the culture supernatants were measured by ELISA.
Results: The vaccinated children displayed significantly high (P<0.05) mean values of SI in LTT, CD4/CD8 cell ratio against the unfractionated, 67kDa fraction and BCG-CF Ags. While 100% of the vaccinated children had positive lymphoproliferation indices to BCG-CF, only 8.3% of the unvaccinated children were positive.
Conclusion: Some of the components of BCG induced a strong Thl cell response in children. These immunogenic antigens were present in the whole cell lysate. The use of BCG vaccine for tuberculosis is worthwhile till a new vaccine is developed.
Keywords: BCG vaccine, Children, Immune responses, T cells, IFN- γ
|How to cite this article:|
Vijayalakshmi V, Kumar S, Rani H S, Latha G S, Murthy K. Invitro immune responses in children following BCG vaccination. Lung India 2006;23:59-63
|How to cite this URL:|
Vijayalakshmi V, Kumar S, Rani H S, Latha G S, Murthy K. Invitro immune responses in children following BCG vaccination. Lung India [serial online] 2006 [cited 2018 Mar 21];23:59-63. Available from: http://www.lungindia.com/text.asp?2006/23/2/59/44410
| Introduction|| |
BCG vaccine was developed nearly 75 years ago, when most of the aspects of cellular immunity were unknown. Despite several studies, there is still no consensus on the efficacy of the vaccine in various parts of the world. In South India the efficacy was reported to be 0%. The subjects in majority of the studies on the efficacy of BCG, were either animals or human adults. This study therefore addressed the question of the magnitude of immunity afforded by BCG, by studying the effector mechanisms of protection in children. Also, an attempt was made to identify the most immunogenic component of BCG. The main objectives therefore were to assess the degree of immunity conferred by BCG vaccine in children and to identify the most immunogenic antigen(s) of BCG by conducting in vitro studies on the effector mechanisms of protection.
| Materials and Methods|| |
Children: Clearance from the ethical committee of the institute and informed consent from the parents were obtained. Children in the age-group of 1 to 10 years, visiting Immunization Centers were examined clinically and categorized accordingly into the following groups: (A) normal, asymptomatic and vaccinated with BCG during the first year (vaccination was confirmed by presence of scar and interrogation of the parents), n=45, (B) normal, asymptomatic without a BCG-scar and with no evident history of vaccination (confirmed by interrogation of the parents), n=31: and (C) children admitted in the hospital with a confirmed diagnosis of tuberculosis, n=31. TB meningitis was confirmed by CSF analysis; miliary TB by skin test negativity and X-ray and TB lymphadenitis by biopsy results. Peripheral venous blood was collected from the children. The identity of the samples was blinded when further assays were carried out.
Antigens (Ags): BCG was lysed, sonicated and separated by gel chromatography, HPLC and confirmed by SDS-PAGE, in the laboratory of Prof. P. J. Brennan, CO, USA. The unfractionated as well as the fractionated components were used as antigens in the assay. BCG was cultured in this laboratory and short-term filtrates were used in the assays. Tuberculin was a kind gift from Prof. J.L.Stanford, London, UK.
Lymphoproliferative assays: Briefly, the assays were carried out by culturing lymphocytes in complete RPMI 1640 medium and stimulated with either Concanavalin-A or Tuberculin. Stimulation index (SI) in lymphoproliferation and levels of Interferon - γ (IFN- γ) in the supernatant were measured by ELISA using commercially available kit(Quantakine, USA). Also, specific CD4 and CD8 cells were measured by ELISA , .
| Results|| |
LTT: The mean values with 1 standard deviation of the SI in the three groups of children are mentioned in [Table 1]. The values in group (a) were significantly high (P<0.05) against the unfractionated, 67kDa fraction and BCG-CF Ags, compared to that against the same Ags in the groups (b) and (c). On the contrary, the means values of SI of fractions 35kDa and lOkDa were significantly low (P<0.05) in the first group. [Table 2] shows the percent number of children with positive SI s of the Ags. Majority of the children had a positive SI; however, least number of the unvaccinated children (8.3%) were positive as against 100% of the vaccinated.
Specific CD4 and CD8 cells: The results of only three antigens are mentioned in [Table 3]. Group (a) had significantly higher (P<0.05) mean values against Tuberculin and 67kDa fraction, compared to the values in other groups. The table also depicts the percent number of children with a positive ratio ( > 1.5) of CD 4/CD8 cells. Majority of the vaccinated children had positive ratios to Tuberculin and 67kDa fraction.
Interferon-γ (IFN-γ) assay : The mean values of IFN-g levels [Table 4] in group (a) were significantly high (P<0.05) against Tuberculin, 67kDa and l8kDa fractions. A large number of the vaccinated children had positive IFN-γ levels after in-vitro stimulation with tuberculin, 67kDa, l8kDa, lOkDa fractions, none of the children had positive levels of IFN-γ, when the cut-off was considered to be 100pg/ml. Also, none of the tuberculous children had positive levels against any of the fractions of BCG.
| Discussion|| |
Despite the fact that there is no consensus on the efficacy of BCG, it is widely accepted that the vaccine reduces disseminated tuberculosis in children, thereby preventing the severe forms of the disease , BCG therefore assumes great importance in the quest for a new vaccine for tuberculosis. Identification of both, correlates of protective immunity and the antigens that can induce them, in the context of BCG vaccination may lead to the development of the new subunit vaccine for tuberculosis. CD4+ Thl cell response with the release of IFN-γ, a cytokine which activates macrophages, can presumably be a good marker of protective immunity for tuberculosis. The identification of antigens can probably be achieved by the demonstration of a strong Th 1 cellular response induced by the antigen in `protected' individuals, in contrast to the lack of a similar response in immunocompromised individuals.
According to Ginsberg, a number of lessons have been learned from screening potential vaccine candidates in small animal models. One of the points that the studies highlighted is the need to develop animal models that better mimic the conditions of the host - i.e. hosts that have already been immunized with BCG, exposed to environmental mycobacteria, and/or are persistently infected with M. tuberculosis  . This study comprised of children who were either immunized with BCG, or exposed to environmental mycobacteria and not vaccinated, or have the disease.
When various components of BCG were tested for their capacity to stimulate PBMC from children, HSP antigens, 67kDa, and l8kDa fractions, 35kDa and 1OkDa fractions, unfractionated crude sonicate and BCG -culture filtrate were found to be immunostimulatory in at least one group of children. It was demonstrated in an earlier study indicate that BCG vaccination in children entails positive TH1 immune responses in the majority of them. These responses were not marked in the unvaccinated and absent in the tuberculous children .
BCG vaccination contributed to elevated in vitro response of the vaccinated children, suggesting the presence of highly responsive mycobacteria-specific T cells  .
The responses of 67kDa fraction were consistently high in the vaccinees, when all the parameters studied were considered. The differences in lymphoproliferative responses to 18kDa fraction between the different groups of children were only subtle with all of them having positive values, while the levels of IFN-γ were markedly high only in the vaccinated children. None of the unvaccinated or tuberculous children had positive IFN-γ levels.
There were several reports on the immunogenicity of HSP-65, -70, -71kDa and -18kDa, of M.tuberculosis, M.bovis, and M.leprae , . When T cell reactivity to the 70 and 65 KD protein antigens derived from Mycobacterium bovis BCG strain was studied by measuring the proliferative responses of PBMC from members of a community resident in the Torres Straits islands, the pattern of responsiveness to the purified antigens paralleled that to whole sonicates from M.leprae and BCG  . However, it was demonstrated in the present study that the in vitro responses towards the HSP antigens were greater than that against the whole crude sonicate. This suggested that there could be certain suppressive antigens in BCG. Ab et al  reported that HSP-65 stimulated- effector cells produced interferon. Lysates containing an epitope of a M.leprae protein of relative molecular mass 18,000 (18K) were shown to stimulate nearly half of the M. leprae specific T cell clones  . It was reported that PBMC proliferated in response to mycobacterial HSP, irrespective of their HLA types  . In an ex vivo transfection approach with a retroviral vector even a single antigen, hsp65 could evoke strong protection when expressed as a transgene  .
There is some sceptism regarding the use of HSPs in the vaccine, as there is a homology in the amino acid sequences between the mycobacterial HSP and the human protein, responsible for causing arthritis. Worthington et a1  reported a lack of any association between HSP 65 and RA. A recombinant ® 65kDa protein from M. leprae did not induce arthritis .
The lymphoproliferative response to lOkDa fraction was least in the vaccinated children, with only 40% of them having positive responses as against 60% and 83% in the unvaccinated and diseased children. Surprisingly, the IFN-γ levels were positive in 75% of the vaccinated, while none of the unvaccinated and diseased children had positive levels. Whether the stimulated cells in tuberculous children have any role in suppressing the Thl responses needs to be elucidated. It was reported earlier that in patients with tuberculous pleuritis, proliferative responses to lOkDa antigen of M. tuberculosis were higher in pleural fluid mononuclear cells than in PBMC, indicating that T cell reactivity to this antigen is enhanced at the site of the disease  .
In the present study it was demonstrated that the 35kDa subunit present in the cytoplasm of BCG stimulated a normal proportion of CD4/CD8 cells (not shown in the table), despite low lymphoproliferative responses and IFN-γ in the vaccinees. This observation and significantly high lymphoproliferative responses in patients and unvaccinated children probably indicate that the stimulated cells are of the Th2 type. Th2 type activity was reported in tuberculosis patients combined with raised levels of the IgE and sometimes eosinophilia  . IFN-g nonproducing Th2 cells do not have the cytolytic activity against antigen-pulsed APCs  . It was earlier reported that this fraction stimulated antibody production in leprosy patients and also proliferative and suppressive to T cells responses , .
The proliferative responses induced by Tuberculin and 67KDa HSP present in BCG cell lysate were more or less similar, but IFN-γ levels were high for Tuberculin, in the vaccinated children. The lymphoproliferative responses to BCG culture filtrate were high in the vaccinated, but lower than that of 67KDa HSP. In conclusion, this study demonstrated that some of the components of BCG were capable of inducing a strong Th1 cell response in children. The most immunogenic antigens were present in the whole cell lysate. The use of BCG vaccine for tuberculosis is worthwhile till a new vaccine is developed.
| Acknowledgements|| |
The authors thank Prof. Indira Nath, AIIMS, New Delhi, India; Prof. P.J. Brennan, Colorado State University, USA; Dr. K.Sridahar Rao, Centre for Cellular and Molecular Biology, Hyderabad; Director, State TB Center and General Secretary, TB Association of AP, India, for their guidance and cooperation.
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[Table 1], [Table 2], [Table 3], [Table 4]