Vijaya Lakshmi V., Sunil Kumar, Surekha Rani H.,
Suman Latha G. and
Murthy K.J.R.
From the Immunology Unit, Bhagawan Mahavir Medical
Research Center, 10-1-1, Mahavir Marg, Hyderabad 500 004, India.
Correspondence to: Dr. Vijaya Lakshmi V., Immunology
Unit, Bhagawan Mahavir Medical Research Center, 10-1-1, Mahavir Marg,
Hyderabad 500 004, India,
E-mail:
[email protected]
Manuscript received: February 18, 2003, Initial
review completed: July 21, 2003;
Revision accepted: July 5, 2004.
The effector mechanisms of BCG protection were examined 5-7
years after vaccination. The in vitro lymphoproliferation,
following stimulation with tuberculin, in normal, (A) vaccinated
and (B) unvaccinated children and children with tuberculosis (C),
were assayed. The mean stimulation index (SI) of lymphocyte
transformation in normal subjects were significantly (P<0.05)
higher than those with tuberculosis. The ratio of
tuberculin-specific CD4 to CD8 cells in short-term cultures were
significantly (P<0.05) higher in the vaccinees. In group (A), 70%
had positive ratios as against 20% and 0% in groups (B) and (C),
respectively. Secretion of IL-2 by the cells was significantly
(P<0.05) high in the vaccinated. None of the unvaccinated children
had positive levels of IL-2. The vaccines also had highly
significant (P<0.01) levels of IFN-(g) in the supernatants of
cell-cultures, following tuberculin stimulation. In majority of
the BCG vaccinated children, the stimulation of specific TH1 cells
seem to be considerably high, in short-term in vitro cultures.
While these responses were not so marked in the unvaccinated, they
were almost totally absent in the patients.
The incidence of tuberculosis is on the rise. The
rate of increase cannot decrease by chemotherapy alone.
Immunoprophylaxis has a pivotal role in the control of the
disease(1). BCG, a vaccine currently in use in several countries
including India, was developed nearly 80 years ago, when many of the
aspects of cellular immunity were still unknown. The efficacy of BCG
is in doubt. There is a need therefore, to elucidate whether BCG
specifically stimulates those subsets of T cells, which are
beneficial to the host.
Subjects and Methods
This study was conducted in normal children in
the age-group of 5-7 years and categorized into the following
groups: (Group A) normal and vaccinated with BCG during the first
year (vaccination was confirmed by presence of BCG scar and
interrogation of the parents), n = 45; (Group B) normal and without
a BCG scar and with no evident history of vaccination, n = 31; and
(Group C) children with active tuberculosis (meningitis, miliary and
lymphadenitis forms), n = 31. Peripheral venous blood was collected
follow-ing an informed consent from the parents.
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-IFN-(g) levels and
interleukin-2 (IL-2) were measured. Also, specific CD4 and CD8 Cells
were measured by ELISA (1,2).
Results
The results indicated that the stimulation index
(SI) in lymphocytes stimulated by concanavalin-A was positive in all
the normal children. With tuberculin, the mean value of the patients
was significantly (P <0.05) lower than that of the normal children,
irrespective of the vaccination status. When SI value of 2 and above
were considered to be positive, 34/34 (100%) of the vaccinated,
18/21 (85.7%) of the unvaccinated and 15/30 (50%) of children with
tuberculosis had positive values. The individual values are shown in
(Fig. 1).
 |
 |
|
Fig. 1. Stimulation indices against tuberculin in
lymphocyte transformation test of vaccinated (group A),
unvaccinated (group B) and tuberculous children (group C). |
Fig. 2. Ratios of CD4 to CD8 cells after stimulation
with tuberculin in short-term cultures of BCG vaccinated(group
A), unvaccinated (group B)and tuberculous children (group C). |
The levels of specific CD4 and CD8 cells in the
vaccinated group (group A) were significantly (P <0.05) higher than
the levels in the other groups, i.e. unvaccinated (group B) and
those with tuberculosis (group C). When 1.5 was considered as a
cut-off value, 70%, 20% and 0% of the children in the three groups
respectively, had positive ratios (Fig. 2).
The difference in IL-2 levels between groups A
and B was significant (P <0.05). This assay was not done in group C.
While 20/30 (66.7%) of the children in group A had positive values,
none in group B had a SI of 2 and above (Fig. 3). Mean values
of IFN-(g) levels were statistically different in the three groups
(P <0.01). When 100 pg/ml was considered as the cut-off value, 55.0
%, 47.6% and 3.1% of the children in groups A, B and C respectively,
had positive values (Fig. 4).
 |
 |
| Fig. 3.
Stimulation indices in cultures of IL-2- dependent cells
supplemented with supernatants of short-term cultures of
lymphocytes from BCG vaccinated (group A) and unvaccinated
children (group B). |
Fig. 4.
IFN-g levels in supernatants of short-term cultures of
lymphocytes from BCG vaccinated (group A), unvaccinated (group
B) and tuberculous (group C) children. |
Discussion
The results of a large-scale trial conducted in
India on BCG, indicated that the efficacy of the vaccine was 0%(3).
Ever since, neither the level of protection that the vaccine
provides in children, nor the duration of its effect have been
firmly established in children who receive the vaccination. In a
previous study, it was shown that 33% of the unvaccinated and 70% of
the vaccinated children had positive in vitro cell mediated
immune responses to PPD; the vaccinated children had higher levels
which started to decline after about four years (4). In another
study from India, the overall vaccine effectiveness estimated was
60%(5).
In the present study, conducted in children aged
5-7 years, the results suggested that the in vitro
lymphoproliferation of tuberculin-specific cells were more or less
similar in both the vaccines and those who did not receive the
vaccination. The information gained from both the above tests was
that tuberculin-sensitized cells were present in children
irrespective of the vaccination status; the magnitude of the
response, was albeit, higher in the vaccinated group. However,
whether these cells are of the beneficial type or not was the issue
addressed in this study.
Within the complex immunoregulatory response to
mycobacterial infection, it is established that T cells provide
protection(6). It is also known that CD4 subset of T cell is the
primary cell responsible for regulating immune responses to M.
tuberculosis(7). Bulk CD4 cell populations and CD4 clones from M.
tuberculosis-infected individuals have been found to be directly
cytotoxic for monocytes pulsed with mycobacterial antigens(6). CD4
cells cross inflamed endothelial surfaces to reach the sites of
mycobacterial infection. Infections by M. tuberculosis were
subs-tantially enhanced by CD4 depletion in mice(7). In this study
majority (70%) of the BCG vaccinees had elevated levels of specific
CD4/CD8 cell ratios, as against a minor (20%) proportion of the
unvaccinated, suggesting that BCG vaccine specifically stimulates
CD4 T cells in children. Whether the efficacy wanes in the remaining
30% of the vaccinated children or whether the vaccine provided any
protection in the first place, needs to be clarified.
IFN-g, an essential component of the host defence
against mycobacteria, is responsible for the activation of
macrophages, stimulation of anti-mycobacterial properties(8,9) and
secretion of IL-1, tumor necrosis factor, granulocyte
macrophage-colony stimulating factor and platelet derived growth
factor(10) by the macrophages(11) . That BCG vaccine stimulates the
secretion of IFN-g by lymphocytes from vaccinated children has been
shown in this study, wherein the levels of the cytokines were
elevated after in vitro stimulation. However, about 40% of
them still had low levels of the cytokine. The levels of the
cytokine present soon after vaccination may throw light on whether
there has been a decline in the immunity. Majority (66.7%) of the
vaccinated children in this study had significantly elevated
(positive) levels of IL-2, while all of the unvaccinated children
had low (negative) levels, suggesting that BCG selectively induces
human TH1 cells.
Tuberculin-specific in vitro
lympho-proliferative responses in patients with tuberculosis (group
C) were low, as also the secretion of IFN-g. It was reported earlier
that patients with newly diagnosed, pulmonary tuberculosis had a
tuberculin-specific defect in IL-2 production(12). Poor CD4 T cell
responses, both in vivo (skin test anergy) and in vitro (with PBMC),
were reported in patients with advanced disease(13). A reciprocal
relationship between T cell responsiveness and the extent of disease
in patients has been demonstrated several times. According to
Orme(9), when mice or guinea pigs are immunized with BCG and later
challenged in the lungs with virulent M. tuberculosis, the
progression of the infection is slowed and there is an accelerated
development of granulomatous response, probably a result of rapid
recognition of the primary lesion by memory T-cells.
The results of this 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. Future research
needs to be directed towards augmentation of the magnitude and
incidence of the beneficial effects of BCG.
Acknowledgements
The authors thank Prof. Indira Nath, All India
Institute of Medical Sciences, New Delhi; Dr. K. Sridhar Rao, Center
for Cellular & Molecular Biology, Hyderabad; Director, State TB
Center; General Secretary, TB Association of Andhra Pradesh; and
Superintendent, Nilofer Hospital for Women and Children, Hyderabad.
Contributors: VLV, SK, SRH, SLG were involved
in the conception, design, acquisition and analysis of data. VLV and
MKJR drafted and reviewed the manuscript. VLV will be the guarantor
of the study.
Funding: Department of Science and
Technology, Government of India.
Competing interests: None declared.
