Download Licentiate thesis from the Department of Immunology,

Licentiate thesis from the Department of Immunology,
Wenner-Gren Institute, Stockholm University, Sweden
Diagnostic biomarkers and improved vaccination
against mycobacterial infection
Muhammad Jubayer Rahman
Stockholm 2008
"It is a good morning exercise for a research scientist to discard a pet hypothesis every day
before breakfast - it keeps him/her young."
Konrad Lorenz (1903 - 1989)
1973 Nobel Laureate in Medicine
2
Summary
Tuberculosis (TB) remains one of the world’s most serious infectious diseases. It is
estimated that a third of the world’s population is latently infected and 8 million new
cases are recorded each year. Although BCG vaccination triggers protective immune
responses in the neonates, it confers protection against only certain forms of childhood
TB. Protection mediated by BCG, against pulmonary TB, is controversial as reported
with variable efficacy ranging from 0-80%. In addition to the problems associated with
the BCG vaccine, diagnosis of TB cannot be performed readily with the available tools.
At present, an effective control of TB is highly dependent on the development of a new
TB-vaccine as well as proper identification and treatment of individuals with active
disease. Therefore, we particularly focused on identification of biomarker (s) of infection
and the development of better vaccines, with special emphasis on the immune responses
in the respiratory tract.
In the first study, we aimed to identify immune biomarker (s) of infection for better
diagnosis of TB. Mice were infected with BCG administered i.n. or i.v., and the bacterial
burden in the lungs, spleen and liver was examined. We measured IL-12, IFN-γ, TNF,
soluble TNF receptors (sTNFR) and mycobacteria-specific antibodies in the bronchoalveolar lavage (BAL) and in serum in order to find immune correlates of infection.
Results showed that sTNFR and mycobacteria-specific antibodies in BAL, but not in
serum, might be useful in distinguishing active from latent infection or exposure to
mycobacterial antigens.
In the second study, we investigated whether we could improve the currently used BCG
vaccine. For this purpose, we tested a combination of neonatal vaccination protocol using
BCG and posterior boosting with the protein heparin-binding hemagglutinin adhesion
(HBHA). It has been described that immunization with native (n) HBHA but not
recombinant (r) HBHA conferred protection against M. tuberculosis challenge in mice.
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This protection was comparable to that afforded by the BCG vaccine. In order to improve
the protective efficacy of the nHBHA vaccine we followed heterologous prime-boost
strategy, comprising BCG vaccination at the neonatal age, followed by nHBHA boosting
at the infant and adult ages. We also examined whether the rHBHA protein could boost
BCG-mediated protective immunity. Cellular immune responses and protection as
measured by control of bacterial growth in the lungs of the treated animals were
followed. Our results showed an improved effect of BCG-priming on HBHAimmunization. The BCG/HBHA immunization protocol was more effective in induction
of HBHA-specific immune responses, as well as in protection than when the animals
received only BCG or HBHA alone. Importantly, our study revealed that nHBHA does
not require co-administration with adjuvant provided that mice were primed with live
BCG before boosting.
Finally, we hypothesized that in utero sensitization of the fetal immune system with
nHBHA may improve nHBHA-specific immune responses after birth. The pregnant
mother was immunized with nHBHA 1 week before delivery. After birth, the offspring
received two doses (week 1 and week 4) of nHBHA formulated with cholera toxin. We
examined HBHA-specific recall responses and protection after challenge with a high
dose of BCG. We found that immune responses were improved by priming the pregnant
mother, and that this also provided better protection than when the offspring received
only BCG or HBHA neonatal vaccinations.
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LIST OF PAPERS
This thesis is based on the following original papers (manuscripts), which will be referred
to by their Roman numerals:
I. Arko-Mensah J1*, Rahman MJ1*, M. Singh2, Fernández C1. Immunodiagnosis
of mycobacterial infection: Increased levels of immunological markers in the
respiratory tract but not in serum correlate with active pulmonary infection in
mice (Submitted).
*
Authors contributed equally to this work
II. Rahman MJ1, Locht C2 and
Fernández C1. Improvement of HBHA
vaccination by BCG priming in a neonatal mouse model (Manuscript).
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LIST OF ABBREVIATIONS
AFB
APC
BAL
BALT
BCG
BCG-BMM
CFP-10
CR
CT
DC
ESAT-6
HIV
i.n.
i.v.
IFN-γ
IL-12
iNOS
kDa
LALT
LAM
MALT
MHC
NALT
nHBHA
NO
PCR
PknG
PPD
rBCG
rHBHA
sTNFR
TB
TCR
TLRs
TNF
XDR
Acid fast bacilli
Antigen presenting cell
Broncho-alveolar lavage
Bronchus-associated lymphoid tissue
Bacillus Calmette-Guérin
BCG infected bone-marrow derived macrophages
10-kDa culture filtrate protein
Complement receptor
Cholera toxin
Dendritic cell
6-kDa early secretory antigenic target
Human immunodeficiency virus
Intranasal
Intravenous
Interferon gamma
Interleukin 12
Inducible nitric oxide synthase
kiloDalton
Larynx-associated lymphoid tissue
Lipoarabinomannan
Mucosa associated lymphoid tissue
Major histocompatibility complex
Nose associated lymphoid tissue
Heparin-binding hemagglutinin native
Nitric oxide
Polymerase chain reaction
protein kinase G
Purified protein derivative
Recombinant Bacillus Calmette-Guérin
Heparin-binding hemagglutinin recombinant
Soluble tumor necrosis factor receptor
Tuberculosis
T cell receptor
Toll-like receptor
Tumor necrosis factor
Extensively drug resistant
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CONTENTS.................................................................................Page
INTRODUCTION ---------------------------------------------------------------- 8
TUBERCULOSIS (TB) IS A GLOBAL BURDEN ------------------ 8
TUBERCULOSIS IS AN INFECTIOUS DISEASE ----------------------- 8
Mycobacterium tuberculosis --------------------------------------------- 8
Infection and outcomes --------------------------------------------------- 9
M. tuberculosis survival program --------------------------------------- 10
IMMUNE RESPONSES TO M. tuberculosis ------------------------------- 12
Innate immunity ----------------------------------------------------------- 12
Humoral immunity -------------------------------------------------------- 13
Cellular immunity --------------------------------------------------------- 14
Host’s protective kits------------------------------------------------------ 17
Mucosal immunity in the lung ------------------------------------------- 18
VACCINES FOR TB------------------------------------------------------------- 20
The BCG vaccine---------------------------------------------------------- 20
New TB vaccines ---------------------------------------------------------- 21
DIAGNOSIS OF TB-------------------------------------------------------------- 23
Conventional methods ---------------------------------------------------- 24
Newer methods ------------------------------------------------------------ 25
Immune biomarker (s) of infection-------------------------------------- 27
PRESENT STUDY --------------------------------------------------------------- 28
AIMS--------------------------------------------------------------------------------- 28
Results and discussions ---------------------------------------------------------- 29
Paper I-------------------------------------------------------------------------------- 29
Paper II------------------------------------------------------------------------------- 31
Preliminary results ----------------------------------------------------------------- 34
CONCLUDING REMARKS --------------------------------------------------- 38
FUTURE STUDIES -------------------------------------------------------------- 39
ACKNOWLEDGEMENTS------------------------------------------------------- 41
REFERENCES --------------------------------------------------------------------- 42
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INTRODUCTION
TUBERCULOSIS (TB) IS A GLOBAL BURDEN
‘TB anywhere is TB everywhere’-remains a serious global burden due to the contagious
nature of the infection. The World Health Organization (1) recorded 1.6 million deaths
resulted from TB in 2005. The highest number of deaths and mortality per capita are in
Africa, where estimated incidences are nearly 350 cases per 100 000 individuals. The
most important weapon to keep the disease under control is a vaccine. The BCG (live
attenuated Mycobacterium bovis BCG) was discovered in early 1900s. Unfortunately,
very poor efficacy of the BCG vaccine has been reviewed over time. BCG has been
shown to protect against disseminated TB in children but not against pulmonary TB.
With the current context of human immunodeficiency virus (HIV) prevalence and the
continued increased number of virulent strains, the TB epidemic is exacerbated.
TUBERCULOSIS IS AN INFECTIOUS DISEASE
Mycobacterium tuberculosis causes TB primarily in the lungs. The bacteria are
transmitted from person to person through the air by droplet nuclei, usually 1-5 µm in
diameter, having only 1-5 tubercle bacilli. Organisms in the droplet nuclei survive for
long periods and remain infective. Bacilli reach the alveoli within the lungs and multiply.
Air droplets are formed by sneezing, coughing or speaking, as well as in the hospital or
laboratory during sputum collection, bronchoscopy or tissue processing.
M. tuberculosis
M. tuberculosis was first described on March 24, 1882 by Robert Koch, who
subsequently received the Nobel Prize in 1905. The M. tuberculosis belongs to the genus
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Mycobacterium, which comprises a number of gram-positive, acid-fast, rod-shaped
aerobic bacteria and is the only member of the family Mycobacteriaceae within the order
Actinomycetales. Mycobacteria are classified into two categories, the fast-growing and
the slow-growing strains. The special characteristics of the cell wall of mycobacteria are
the peptidoglycan layer, free lipids and waxes that provide hydrophobic characters, acid
fast properties and intracellular survival (2). Most mycobacteria live in habitats such as
water or soil. A few are intracellular pathogens in animals and humans causing serious
diseases include TB and leprosy. M. tuberculosis, M. bovis, M. africanum, and M. microti
are members of the tuberculosis species complex, and all cause a disease known as TB.
Usually M. tuberculosis is pathogenic for humans while M. bovis is pathogenic for
animals.
Infection and outcomes
Infection is characterized by the presence of bacilli in the host, without any clinical
symptoms. As shown in Fig. 1, after inhalation, mycobacteria are taken up by the alveolar
macrophages and may be killed immediately. Alternatively, bacteria may start replication
in the macrophages, and afterwards may localize to the other parts of lungs or regional
lymph nodes (3-5). Interactions between the pathogen and the host terminate by killing of
the bacilli or result in lesions in the lungs. The lesions can be detected by X-ray but in
some cases lesions can not be seen in chest radiographs. The clinical manifestations of
TB after infection in the lungs are known as pulmonary TB. Bacilli continue their
replication and form tubercle which can undergo so-called caseation necrosis showing
semi-solid or "cheesy" consistency. A significant proportion of the patients at this
caseation stage can be diagnosed by chest radiograh. At this point the patients are highly
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contagious. However, failure in early detection of infection and therefore lack of
treatment in a minority of cases lead disease progression or hematogenous dissemination
to other parts of the body such as, bones and joints (6), kidneys and genital tract (7) or the
central nervous system resulting in TB meningitis (8).
Inhalation of M.tuberculosis
PPD+
PPD-
Immediate killing
of M. tuberculosis
Stabilization
(latency)
Primary complex
Localized disease
(primary TB)
Dissemination of M.
tuberculosis
Stabilization
(latency)
Acute disease
(meningitis, miliary TB)
Fig. 1: Chronological events after inhalation
of M. tuberculosis.
Clin Microbiol Rev. 2002;15:294-309
Reactivation
(post-primary TB)
M. tuberculosis survival program
Under natural circumstances, alveolar macrophages take up mycobacteria from the
alveolar mucosa and enclose them into the phagosomes. Subsequently, activation of
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macrophages leads to the fusion of the phagosomes with lysosomes, which results in
killing of bacteria. M. tuberculosis escapes this destruction by altering the maturation of
phagolysosome fusion in some way, and extends its length of survival in the host, which
is called a state of clinical latency. Bacteria set up their dormant life by the rapid
upregulation of ‘dormancy regulon’--contains atleast 48 genes. At the dormant state,
bacteria survive under low oxygen level and become drug-resistant. Under such a
situation bacteria shut off protein synthesis and stop replication in the host.
Three possible mechanisms have been suggested to be involved for the intracellular
survival of bacteria (9-12). First, Voskuil et al demonstrated that hypoxia and low
nontoxic levels of nitric oxide (NO) induced the expression of a 48-gene regulon, which
limits the M. tuberculosis replication by inhibiting their metabolic process. Second,
Walburger et al proposed that the phagosome-lysosome fusion can be interrupted by the
active production of protein kinase G (PknG), which is one of several eukaryotic-like
serine/threonine protein kinases found in M. tuberculosis and related organisms. PknG
blocks the lysosomal delivery and ensures a friendly environment to the bacterium (Fig.
2).
Fig. 2: PknG affects the
intracellular traffic of M.
tuberculosis in macrophages.
Nat Med. 2007;13:282-4
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Third, Rengarajan et al proposed that phosphate transport proteins may be crucial for the
survival of bacteria. Probably, lung macrophages have limited levels of phosphates
therefore, transport of inorganic phosphate by phosphate transport proteins is critical for
the bacterial growth in macrophages.
IMMUNE RESPONSES TO M. tuberculosis
As other microorganisms, an infection with M. tuberculosis stimulates both arms of the
immune system, innate and adaptive. After intracellular infection, the protective immune
response to M. tuberculosis is mainly cell mediated. Collaborations between lymphocytes
and macrophages are basically important for the successful eradication of M.
tuberculosis.
Innate immunity
It is generally believed that alveolar macrophages are the primary cells that initially
takeup M. tuberculosis. Ingestion of M. tuberculosis by macrophages signals NF-κB
mediated expression of several genes that code for cytokines and chemokines.
Chemokines signal leukocytes to travel to the site of infection and this process further
activates macrophages.
Many receptors have been identified for endocytosis of M. tuberculosis by macrophages
and dendritic cells (DCs) (reviewed in ref. 13). Complement receptor 1 (CR1), CR3, CR4
(14-16) recognize and bind to the cleavage products of complement component C3
deposited on the surface of M. tuberculosis (opsonization) following complement
activation. Mannose receptors bind to the nonopsonized M. tuberculosis (16). Outcomes
of the interactions depend on the type of receptors. Fc receptors increase the production
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of reactive oxygen intermediates that favours phagosome-lysosome fusion (17) and CR3
prevents the respiratory burst and inhibits the maturation of phagosomes (18).
Toll-like receptors (TLRs) are pattern recognition receptors that play an important role in
the recognition of mycobacterial components. Thirteen TLRs (TLR1 to TLR13) have
been identified in humans and mice. TRL2 recognizes the 19-kDa lipoprotein (19) and
the major mycobacterial cell wall component lipoarabinomanann (LAM) (20). In animal
models, defective functions of TLR2 and TRL4 have been associated with susceptibility,
at an early stage of infection (21). TLR-9 has also been proposed to be involved in the
recognition of CpG-motifs in mycobacteria (22). Usually, stimulation of TLRs by
mycobacterial components triggers a proinflammatory response by the production of IL12 and inducible nitric oxide synthase (iNOS) (23), which can promote mycobacterial
killing (24). TLR signalling is also important in the generation of cellular populations
such as differentiation of monocytes into macrophages and DCs (25).
Humoral immunity
Mycobacterial infection is basically intracellular and therefore, the humoral immune
response is considered to be non-protective. However, the benefits from antibodies have
been explored in the control of intracellular infections especially chlamydial respiratory
infection, and chronic infections induced by actinomycetes (26, 27). While the role of
antibody in protection against TB is uncertain, several mechanisms of action mediated by
antibody are considered, which could modify the outcome of infection. Antibody may
interfere with the adhesion of mycobacteria (28) or bind and neutralize the function of
mycobacterial products (29). Recent studies have also demonstrated the protective
function of antibody in experimental mouse models of TB (30-33). Recently,
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mycobacterium-specific antibodies have been found to be capable of enhancing cellmediated immune responses to mycobacteria (34). Antimycobacterial antibodies
increased the proliferation of CD4+ and CD8+ T cells after in vitro stimulation with BCG
in the presence of antigen presenting cells (APC) (34). Disregarding the role of antibody
in protection, specific antibodies to mycobacterial components can be valuable in
diagnosis of TB (35).
Cellular immunity
CD4+ T cells
CD4+ T cells are central in driving cellular immunity to TB. Cytokines produced by
CD4+ T cells are of critical importance for the outcome of immune responses, for
example Th1 type or Th2 type. Th1 cells secreting interleukin-2 (IL-2) and interferon-γ
(IFN-γ) are associated with cell-mediated immunity, whereas Th2 cells that produce
typically IL-4, IL-5, IL-10 and IL-13, are responsible for humoral immunity. After
phagocytosis of mycobacteria, antigens are processed and presented to the CD4+ T cells
by macrophages or DCs in association with class II MHC molecules, encoded by the
major histocompatibility gene complex (MHC). Activated CD4+ T cells express CD40L
on the surface that interacts with its receptor, CD40, on antigen presenting cells. The
activated CD4+ T cells secrete cytokines such as IFN-γ and tumor necrosis factor (TNF),
which in turn activate macrophages to kill the ingested bacteria (36). The importance of
CD4+ T cells in the control of TB has been demonstrated in mice and humans. Studies
with CD4+ T cell-deficient mice showed a defect in IFN-γ production, which led to the
increase of bacterial numbers in various organs (37). In a murine model of chronic
persistent M. tuberculosis infection, depletion of CD4+ T cells resulted in a steady
14
increase of bacterial growth in all organs (38). In humans, the importance of CD4+ T cells
is clear because humans infected with the HIV are more susceptible to develop TB (39).
Fig-3: Antigen presentation and activation of CD4+ and CD8+ T cells. Nat Rev Microbiol. 2006;4:469-76
CD8+ T cells
CD8+ T cells are also important in protection against TB although to a lesser extent than
CD4+ T cells. Antigens are processed and presented to CD8+ T cells in association with
class I MHC molecules. Mice, lacking CD8+ T cells or MHC class I molecules are more
susceptible to infection (40). Although CD4+ T cells are the major source for IFN-γ
production, CD8+ T cells are also able to produce IFN-γ (41). CD8+ T cells are efficient
in lysing infected cells and reducing bacterial load (42). The effector mechanisms
mediated by CD8+ T cells are largely dependent on the production of perforin/granulysin
(43).
Despite the fact that CD8+ T cells are important in protection, it is still unclear how the
presentation of processed antigens to CD8+ T cells is done. As shown in Fig. 4, three
15
possible mechanisms are proposed to be involved: first, the classical pathway of antigen
processing and presentation by macrophages; second, M. tuberculosis infected cells may
produce exosomes containing mycobacterial antigens, which can be presented by
bystander APC for MHC class I cross processing; third, apoptosis of M. tuberculosis
infected cells releases apoptotic vesicles containing mycobacterial antigens, which are
taken up by bystander APC. The mechanisms are likely to be operated in the presence of
the specific type of APC.
M. tuberculosis
Fig. 4: Cross processing of M. tuberculosis (MTB) antigens for CD8+ T cells. J. Clin. Invest. 2007;117:2092–2094
Gamma/delta (γδ) T cells
γδ T cells are large granular lymphocytes, which comprise 10% of circulating Tlymphocytes in healthy individuals (44). γδ T cells display T cell receptor 1 (TCR-1)
receptors whereas αβ T cells (CD4+ and CD8+ T cells) display TCR-2 receptors. The γδ
TCRs and αβ TCRs are similar in both sequence and structure. However, γδ T cells may
16
directly recognize in the form of intact proteins (45) or non-protein phosphoantigens (44,
46), and αβ T cells recognize small peptides together with MHC molecules. The exact
role of γδ T cells in protection against TB remains to be elucidated. It has been
demonstrated that γδ T cells may produce IFN-γ during early infection, and that they can
function as APC and give proliferation signals to αβ T cells (47). It has also been
suggested that γδ T cells probably limit the migration of inflammatory cells to the site of
infection that may cause tissue damage (48, 49). Mice infected with M. tuberculosis/BCG
intranasally showed a quick localization of γδ T cells in the lungs (50, 51). It has been
shown that upon in vitro stimulation with BCG-infected macrophages, γδ T cells showed
cytotoxic activity (52), and blocking of γδ T cells’ functions reduced cytotoxic activity
and IFN-γ production by lung CD8+ T cells (52). Thus γδ T cells have been proposed to
be important for the control of infection in the period between the innate and adaptive
immune responses (52). γδ T cells can also produce IL-17 during early in the infection
that might help in the movement of immune cells to the site of the infection (53).
Host’s protective kits
Experiments in animal models showed that immune responses against TB are Th1
dominated (54-56). Macrophages ingest bacteria and produce the IL-12 cytokine, which
is important for the induction and generation of Th1 immunity. Fig. 3 displays the
sequence of events driven by the host after phagocytosis of bacilli. Activated CD4+- and
CD8+ T- cells play a crucial role by producing IFN-γ and TNF, which together drive the
immune system towards a Th1-type of immune response. In humans, defective in the
receptors for IFN-γ and IL-12 reiterate the importance of these cytokines in protection
(57, 58). Mice deficient in the IFN-γ-gene expression are very susceptible to fatal TB (59,
17
60). TNF is also an important cytokine in the control of TB. In mice, it was demonstrated
that in absence of TNF, effective granuloma formation is impeded and bacterial growth is
rapidly increased, which reduced the survival time of the mice (61, 62). However, high
levels of TNF are detrimental for the host (63) and may cause severe pathology.
Therefore, the persistence and the levels of TNF determine whether TNF activity is
helpful or harmful. TNF functions by interacting with one of two receptors: TNFR1 (55
kDa) or TNFR2 (75 kDa) expressed on almost all nucleated cells. The extracellular
domain of both receptors can be cleaved by metalloproteases and then the soluble form
(sTNFR) can bind to TNF and neutralize TNF-mediated activities. It has been described
that complete neutralization of TNF activity by sTNFR prevents the induction of cellmediated immunity in mice and therefore succumb to BCG infection (64).
Both TNF and IFN-γ-signaling activate macrophages and activation leads to the synthesis
of iNOS, which regulates the production of NO. NO is an important mediator in the
killing of intracellular M. tuberculosis.
Mucosal immunity in the lung
The immune response in the respiratory tract has been studied during the last decade,
with the intention to induce high level of protection again TB. The hypothesis is that,
since TB is primarily a lung disease, the immune responses in the lungs might play a
significant role in protection. Mucous membrane lines up the respiratory tract and
initially prevents invasion of the pathogens. The defensive mechanism of the mucosal
lining predominantly involves mucosa associated lymphoid tissue (MALT), where
pathogens are sampled, processed and presented to the immune cells. Lymphoid tissues
in the respiratory tract can be divided into three groups (65): nose associated lymphoid
18
tissue (NALT), larynx-associated lymphoid tissue (LALT), and bronchus-associated
lymphoid tissue (BALT). Luminal antigens are taken up by a specific type of cells called
microfold (M cells), that are present in the epithelium overlying NALT. A shown in Fig.5, DCs process and present antigens to the T lymphocytes. Activated T cells help B cells
to produce antigen-specific antibodies especially of the IgA isotype. Antigen-specific T
cells and IgA-producing B cells migrate to the effector sites via thoracic duct and blood
circulation. After proteolysis of the polymeric Ig receptor, the dimeric IgA binds with a
parts of the polymeric Ig receptor and form secretory-IgA. Secretory-IgA antibodies are
then released into the mucosal secretions. Secretory IgA binds to the pathogens and block
their replication and colonization (66, 67).
Fig-5: The mucosal immune system. Nature Rev. Imm 2004;4:699-710
19
VACCINES FOR TB
The BCG vaccine
The BCG (bacille Calmette-Guèrin) vaccine was developed in the early 1900s by Albert
Calmette and Camille Guèrin. BCG is made of a live, attenuated strain of M. bovis. BCG
was first given to a newborn in 1921, whose mother had died of TB and who was looked
after by a grandmother, was also suffering from the disease. This vaccinated individual
remained free of TB throughout his life (68). In 1928 the League of Nations suggested
widespread vaccination with BCG. In the first half of the 20th Century, BCG vaccines
were prepared and preserved under varying conditions by different laboratories and
therefore both genotypical and phenotypical changes occurred. As shown in Fig. 6,
Japanese and Danish strains were evolved from the original Pasteur BCG in 1921 and
Glaxo strain from the Danish in 1331.
Fig. 6: Documented evolution of the four M. bovis BCG strains. DR indicates the deleted regions and the
number in the arrows is the in vitro passages of bacteria which were occurred during the evolution of
BCG Pasteur and daughter strains since 1921 to the freeze dried of BCG in 60th.
Ann Clin Microbiol Antimicrob. 2004,3:10
20
The BCG vaccine is effective against disseminated and meningeal TB in infants and
young children. However, the protective efficacy of BCG vaccination against pulmonary
TB is highly questioned. Results from field studies showed unsatisfactory efficacy
varying between 0% and 80% (69). There could be many reasons for this such as
methodological differences among the clinical trials, genetic differences within and
among host populations, endogenous reactivation of persistent infection, exogenous reinfection, and effects of environmental mycobacteria on the host immune response to
BCG. Furthermore, there have been reports about the influence of helminth infections
that divert Th1 immunity towards Th2, resulting in reduced protection (70). Today, the
BCG vaccine is not considered as a secured vaccine against pulmonary TB.
New TB vaccines
Live attenuated vaccines: Several strategies have been taken into consideration in order
to improve the BCG vaccine. Recent studies with comparative genomic analysis
identified that some genes were missing in the BCG (71, 72). Therefore it was presumed
that BCG was not a complete vaccine against M. tuberculosis. Accordingly, it was
hypothesized that the introduction of the deleted genes to the BCG vaccine could
improve the vaccination. Subsequently, vaccination of mice with a recombinant BCG
(rBCG) reconstituted with the missing genes, 6-kiloDalton early secretory antigenic
target (kDa ESAT-6) and CFP-10 (10-kDa culture filtrate protein) showed better
protection than in the control animals immunized with BCG alone (73). rBCG overexpressing mycobacterial antigen, Ag85B, was also reported to possess a better efficacy
than wild-type BCG, in guinea pigs (74), as well as, in macaques (75). Although the
modified live BCG vaccines have been found to be attractive, the major concern for the
21
clinical use of this kind of vaccine is safety. Introduction of missing genes to the BCG
vaccine might include the possibility of reactivation of virulence.
Subunit vaccines (single or fusion proteins): Subunit vaccines contain purified antigens
rather than whole organisms. The development of subunit vaccines has been accelerated
due to the sequencing of the M. tuberculosis genome (76). This led to the identification of
many protective antigens including antigen 85 complex proteins (Ag85, 30-32 kDa),
ESAT-6, CFP-10 and TB 10.4 (belong to the esat-6 gene family), 19 and 38 kDa
lipoproteins, heparin-binding hemagglutinin adhesion (HBHA) etc. However, almost all
of the single subunit proteins with adjuvants used for vaccination showed a certain level
of protection that is even lower than the BCG vaccination. To our knowledge, only the
HBHA has been proposed to be a potent vaccine candidate against TB, based on the
observation that vaccination with HBHA, in mice, could reduce bacterial counts as
efficiently as vaccination with wild-type BCG (77). Another important characteristic of
the HBHA protein is that lymphocytes isolated from the infected healthy subjects
produce more IFN-γ in response to HBHA than patients with active disease (78).
Recently, it has been described that vaccination with fusion proteins is highly protective
against M. tuberculosis infection in animals. The fusion protein Ag85B-ESAT-6 has been
tested in mice (79) and found a level of protection that was better than that with either
Ag85B or ESAT-6. Exchange of ESAT-6 with TB10.4 in the Ag85B-ESAT-6 construct
has been found to induce a level of protection comparable to that provided by BCG
vaccination (80).
However, a major challenge in developing a subunit vaccine is the requirement of a safe
and effective adjuvant.
22
DNA vaccines: DNA vaccine is injected into muscle cells usually with a "gene gun" that
uses compressed gas to blow the DNA. Some muscle cells express the inoculated DNA
and are able to stimulate the immune system. Over the last few years, DNA vaccinations
have become a strategic way to deliver mycobacterial antigens that stimulate the cellular
immune system. In mice, DNA-vaccines expressing several immunodominant antigens
have been found to be protective against M. tuberculosis (81-85). DNA vaccines
stimulate both CD4+ and CD8+ T cells (reviewed in ref. 88) and induce mixed Th1/Th2
type of immune responses (87). The efficacy of DNA vaccines can be further improved
by co-delivery of multiple DNA plasmids or chimeric DNA vaccines (88, 89). Although
DNA vaccines are probably a good alternative, they have been disappointing so far when
tested in larger animals like guinea pigs and non-human primates (90).
With the knowledge from different types of vaccines and the context of current global
BCG vaccination, it has increasingly been realized that a combination of BCG or rBCG,
DNA vaccines, and subunit proteins may provide greater levels of protection.
Heterologous prime-boost strategies involving BCG as a priming vaccine and either
DNA-based vaccine or subunit protein as a booster have been found very attractive (91,
92).
DIAGNOSIS OF TB
A timely and accurate diagnosis of TB is essential for better treatment, prevention and
control of the disease. Currently there are a number of methods used to diagnose
mycobacterial infection but none of them are optimal. Identification of M. tuberculosis in
conventional laboratory tests is difficult.
23
Conventional methods
Microscopy and culture
Diagnosis of active pulmonary TB by microscopic or culture techniques is based on the
detection of bacilli in specimens from the respiratory tract -- the most common type is
sputum. Acid fast bacilli (AFB) smear microscopy and culture on Löwenstein-Jensen
medium are the “gold standard” methods for the diagnosis of active TB. AFB smear
microscopy is most commonly used to identify highly contagious patients. AFB smear
microscopy is a quick and inexpensive technique. However, an estimation of 500010,000 organisms per ml of sputum is needed to visualize the bacilli under microscope.
This situation makes mycobacteriologic identification by smear difficult, especially in
children who rarely produce adequate sputum. Culture based methods are more sensitive
and can detect as few as 10 bacteria/ml of specimen. Culture techniques are also used to
monitor drug sensitivity and resistance pattern. A real disadvantage of the culture method
is that it takes 4-6 weeks for confirmation. However, a positive culture for M.
tuberculosis confirms the diagnosis of active disease.
Tuberculin skin test
“Koch´s Old Tuberculin” is a heat-inactivated filtrate from cultures of M. tuberculosis
that was first proposed for the treatment of TB. However, TB patients who received
tuberculin suffered from systemic reactions, including fever, muscle aches, and
abdominal discomfort with nausea and vomiting. These observations were the basis of
using tuberculin, also known as purified protein derivative (PPD), for the diagnosis of
TB. Tuberculin skin test was described by Mantoux, and his method became widespread.
A positive Mantoux tuberculin skin test appears between 24 to 72 hours after application
24
of tuberculin, is used to detect infection. But PPD skin test is not reliable to detect
infection, since people exposed or vaccinated with BCG also respond. In addition, in a
positive skin test, major confounding factors are exposure with mycobacteria other than
M. tuberculosis.
Radiology
Radiographic screening is another way of monitoring the progression of TB disease.
Although radiography is often useful to detect advanced stages of disease, it is also an
initial screening method used together with PPD skin test. In active pulmonary TB, chest
radiograph views cavities often in the upper lung. Chest radiographs may be suggestive
of, but are never specific to TB.
Newer methods
Nucleic acid amplification methods
Polymerase Chain Reaction (PCR) is an efficient technique that allows rapid
amplification of mycobacterial species. Among many genomic targets for diagnostic PCR
there are, the insertion element IS6110 (present as multiple copies, 4-20, in M.
tuberculosis), the 65 kDa heat-shock protein gene, the 126 kDa fusion protein gene, and
the gene encoding the β-subunit of RNA polymerase. All of them have been found to
provide accurate determination of the presence of M. tuberculosis. However,
implementation of the PCR technique remains technically challenging because it requires
strong laboratory capacities and good quality control procedures that are frequently not
available in affected countries.
25
Serological diagnosis
In the case of patients who do not produce adequate sputum or sputum smears are
negative or patients with extrapulmonary TB, serological diagnosis is an attractive
option. Serological diagnosis is based on the detection of TB-specific antibodies in the
sera of patients. There have been many studies showing TB-specific antibody responses
in patients suffering from active disease (93, 94). However, the general scenario of
serological diagnosis is very poor in terms of sensitivity and specificity (95, 96). Crossreactions also occur in healthy individuals exposed to environmental mycobacteria or
BCG vaccination, and therefore difficulties arise in the interpretation of the results.
A possible explanation for the low success of serological studies may be that TB is
primarily a disease in the lungs and therefore measurements of specific antibodies locally
in the respiratory tract would be more accurate as markers of lung infection than
serological studies that would reflect a systemic response.
Cell mediated immunity and diagnosis
The only widely used method based on cell-mediated immune response is the PPD.
However, a major drawback of PPD is that it gives false positive results in people
exposed to the environmental mycobacteria or vaccinated with BCG. Thus, efforts have
been made to identify antigens that are not cross-reactive with BCG. Antigens, such as
ESAT-6 and CFP-10 have been proposed for TB diagnosis (97, 98). This diagnosis
method is based on the assumption that T cells from sensitized individuals produce IFN-γ
when they re-encounter antigens of M. tuberculosis (99). One of the recently developed
assays is QuantiFERON-TB Gold, which is the advanced version of QuantiFERON-TB.
A positive test indicates that the M. tuberculosis infection is likely and a negative test
26
indicates infection is unlikely. However, one of the shortcomings of using ESAT-6 and
CFP-10 is that orthologues of these antigens in other mycobacteria (M. smegmatis) might
influence the test results (100). Also, QuantiFERON-TB Gold test alone is not sufficient
to differentiate between active and latent infection because QuantiFERON-TB Gold test
can be positive for both active and latent cases. Moreover, some patients with advanced
TB have been found to be not responsive to the antigenic stimulation, which also limits
the use of QuantiFERON-TB Gold test.
Immune biomarker (s) of infection
Biomarkers are biological features or substances that can be used as indicators of
infection. Recently, it has been described that cytokine levels in broncho-alveolar lavage
(BAL) could be good markers of infection. TNF, IFN-γ and IL-2 levels in BAL of
patients with smear-negative pulmonary TB are increased significantly compared to the
other pulmonary disease (101). Moreover, other candidate biomarkers for TB infection
such as lactoferrin, CD64, and the Rab33A (member of the Ras-associated GTPase) have
recently been suggested (102). Expression of CD64 on monocytes has been found higher
in TB patients than M. tuberculosis-infected healthy subjects. Similarly, a higher gene
expression of lactoferrin and Rab33A was reported in TB patients. However, these
molecules whether or not specific for TB are yet to be defined.
27
PRESENT STUDY
Aims
TB is primarily a lung disease and the respiratory tract is the natural route of M.
tuberculosis infection. In order to improve diagnostic methods and protection against TB,
in the present study we have aimed at investigating the immune responses at the entry
port of mycobacteria and downstream along the respiratory tract.
Our specific objectives were:
ƒ
To investigate the induction of immune responses in mice to mycobacterial
infection and to identify immunological parameters or biomarkers associated
with infection (Paper I, Submitted)
ƒ
To increase the protective efficacy of the HBHA vaccine based on the
induction of HBHA-specific immune responses in the lungs (Paper II,
Manuscript).
ƒ
To examine the effect of maternal immunization with HBHA on postnatal
immunity in the offspring (Preliminary results)
Materials and Methods
The materials and methods for these studies are described in the separate papers.
28
Results and discussion
Identification of immune biomarker (s) of mycobacterial infection for better diagnosis
of TB (Paper I)
Diagnosis of TB based on the immune responses to mycobacterial antigens (103-107) has
been extensively studied over the past years. However, methods based on the detection of
marker (s) in serum are limited by low sensitivity and specificity. Diagnosis based on the
local but not peripheral immune responses might be highly sensitive and specific. In this
regard, we modeled natural infection in mice by using BCG administered intranasally
(i.n.). We examined levels of several immune parameters both in the lungs and in serum
and analysed immune correlates of active infection.
The major findings of this study are:
a) Active mycobacterial infection, but not exposure to non-replicating mycobacteria
resulted in elevation of IL-12, IFN-γ and sTNFR in BAL independently of the route of
infection. Serum levels were not equally conclusive.
b) Reactivation of controlled BCG infection resulted in increased bacteria growth in the
lungs and in increased levels of sTNFR in BAL.
c) Active infection, but not exposure to non-replicating mycobacteria resulted in
production of antigen-specific IgG or IgA in BAL.
In recent times, attention has been focused on the detection of immune parameters locally
in the lungs which might be of good correlates of infection. Expression of IL-12 and IFNγ mRNA in the BAL (108) and elevated levels of cytokines such as IFN-γ, IL-12, IL-1β,
IL-8 and TNF produced by broncho-alveolar cells were described to be associated with
29
active pulmonary TB (109). In addition to the cytokine levels, cytokine receptors, such as
sTNFR, have been reported to be sensitive marker of infection. Increased shedding of
sTNFR1 into serum is predominantly associated with TB rather than HIV infection. The
levels of sTNFR1 are reduced even after anti-tuberculosis treatment (110). Transgenic
mice expressing high serum levels of sTNFR1 exhibited reduced bactericidal activity,
undifferentiated granulomas and succumbed to BCG infection (64). In the present study,
we, however, measured cytokines TNF, IL-12 and IFN-γ or sTNFR levels in both BAL
and in serum after active infection as well as treatment with heat-killed BCG or BCGlysate and compared with the bacterial burden in the lungs. In contrast to the previous
reports, we found that increased serum levels of sTNFR were not restricted to active
infection but induced after treatment of mice with killed-BCG. However, increased levels
of sTNFR, IL-12 and IFN-γ in BAL correlated better with the bacteria counts. This
suggests that levels of cytokines or soluble TNF receptors in BAL, but not in serum, are
more proper to detect infection in the lungs. We also confirmed our findings in the mouse
model of active infection induced by the systemic route. After intravenous route of
infection with live BCG, we observed a similar correlation between the bacterial burden
and the sTNFR levels in the lungs. The undetectable TNF might indicate that TNF and
sTNFR levels are inversely correlated.
sTNFR levels are not only increased in mycobacterial infections, but also in many other
inflammatory diseases (111-113). Therefore, we reasoned that detection of BCG-specific
antibodies, together with cytokines or soluble cytokine receptors, could be used as
biomarkers for distinguishing an active infection from exposure to killed bacteria. We
detected higher levels of antibodies in both BAL and serum at week-9 after infection,
30
compared to the early time points. We detected antigen-specific IgA only in the BAL but
not in the serum. The reason for this could be that our maximum observation time was
week-9 after infection, which might limit the detection of systemic IgA. In contrast to the
results with live BCG, treatment with killed-BCG or BCG-lysate induced only detectable
levels of antibodies in serum. Live BCG is a very strong stimulant and therefore,
probably, continuous stimulation of immune cells by the secreted antigens resulted in
significant production of antibodies over time. Specific antibodies in BAL but not in
serum probably reflect better with the results of sTNFR in BAL.
In conclusion, our observation suggested a new strategy of detecting mycobacterial
infection based on the determination of more than one immune marker in the lung
microenvironment.
Improved immunogenicity and protective efficacy of HBHA vaccine (Paper II)
BCG vaccine is the most widely used immunization in the world (144). The failure of
BCG vaccine in life-long protection against TB is one of the important factors for the
current threat of TB. This suggests that a new vaccine against TB is urgently needed in
the control of the disease. In recent times, there have been significant advances in the
development of a new TB vaccine particularly in improving BCG vaccination. The best
model for the improvement of BCG vaccination is probably the heterologous prime-boost
strategy (115). In this protocol, priming with BCG and boosting with either DNA-based
vaccine or fusion protein vaccine have been reported to be very efficient (92, 116). In this
study, we evaluated a single protein, HBHA as a BCG boost vaccine. HBHA is one of the
most potent mycobacterial antigens that has been shown to induce protection against M.
tuberculosis challenge in mice similar to the protection provided by the BCG (117). We
31
tested immunogenicity and protective efficacy of HBHA in a neonatal BCG-vaccinated
mouse model.
The major findings of this study are:
a) Neonatal BCG vaccination primed the immune system for native (n) HBHA and
therefore upon nHBHA boosting, recall immune responses to HBHA were improved.
b) BCG/nHBHA combination significantly improved protection against BCG infection
independently of the route of nHBHA immunization and the coadministration of
adjuvant.
c) Boosting with rHBHA induced protection in the BCG-primed mice.
The neonatal immune system is not fully matured and biased to Th2 type response. The
challenge of neonatal vaccination arises due to the poor response of neonates to most
vaccines. Neonatal BCG vaccination is one of the few examples that can shift the
immune system from a Th2 to Th1 type response (118). Although the BCG-promoted
immunity wanes with time, BCG vaccination can induce even longer protective immunity
than subunit vaccines (119).
In this study, following vaccination with BCG/HBHA combination we observed that
BCG vaccination not only primed the immune system but also BCG itself acted as an
immune adjuvant. We found that introduction of cholera toxin (CT) in nHBHA vaccine
did not show a better protection than the nHBHA formulated without CT. Interestingly,
priming with BCG improved protective immune responses even for recombinant (r)
HBHA, which was previously reported to be non-protective (117). We observed that lung
lymphocytes from the BCG-primed and rHBHA-boosted mice produced large amounts of
32
IFN-γ upon stimulation with BCG-infected bone-marrow derived macrophages (BCGBMM), a methodological approach suggested to correlate with the level of protection.
IFN-γ levels upon stimulation with HBHA were also induced but were unable to correlate
with the level of protection. It is possible that upon BCG infection, BMM present mostly
the protective epitope to the lymphocytes from HBHA-immunized mice, which is not the
case with HBHA stimulation. In this regard, it has been reported that some mycobacterial
antigens may induce higher levels of IFN-γ but do not offer protection (120). Presently,
we cannot explain the mechanism of protection induced after rHBHA boosting. Most
probably rHBHA acted as a carrier molecule for the protective epitopes generated after
BCG vaccination.
We examined the effect of route of nHBHA administration on the immune responses and
protection under the BCG/nHBHA vaccination protocol. We found that, provided the
animals were primed with BCG, the level of protection was comparable between the i.n.
and subcutaneous (s.c.) routes of immunization. In vitro stimulation of lung cells with
BCG-BMM showed comparable levels of IFN-γ between the i.n. and s.c. groups, which
correlated with the levels of protection. Νeonatal BCG vaccination predominantly
induced IFN-γ+CD4+ T cells as measured by the expression of CD40L on the CD4+ T
cells. Consistent with this, in human neonates, it has also been found that BCG
vaccination induced higher frequency of IFN-γ+CD4+ T cells than IFN-γ+CD8+ T cells.
Thus, the general consensus is that BCG vaccination is highly important for the induction
of Th1 dominated immune response in the Th2 biased neonates.
33
Collectively, our study showed an improvement of HBHA vaccination, when combined
with the neonatal BCG priming, which would have a significant impact on future TBvaccine development.
Effect of maternal priming with HBHA during the gestation period on postnatal
cellular immunity in the offspring (Preliminary results)
Previously, it was described that vaccination of adult mice with nHBHA could induce a
level of protection similar to that provided by the BCG vaccination (117). However, in
our neonatal model, we have experienced that the protection, offered by the nHBHA
vaccination alone, was lower than that offered by the BCG vaccination. In order to
improve the nHBHA vaccination in neonates, we hypothesized that in utero sensitization
of the fetal immune system with nHBHA may improve nHBHA-specific immune
responses after birth.
It has been reported previously that not only antibodies but also antigens can be
transferred from the mother to the fetus through the placenta, and to the neonates via milk
(121-123). Recently, increasing numbers of evidence suggest that transplacental
sensitization of the fetal immune system could improve postnatal immune responses
(123, 124). In this context, we were especially interested to improve postnatal T-cell
immunity by immunizing the pregnant mother at 2 weeks of gestation. Immunization of
pregnant mice at 1-week ahead of delivery might exclude the possibility of HBHAspecific antibody transportation through the placenta from the mother to the fetus. Within
this short period, IgM, but not IgG can be produced. However, IgG is the only class of
immunoglobulin that can cross the placental barrier. Therefore, our hypothesis was that
immunization of pregnant mother at 2th week of gestation will prime the fetal cellular
34
immune system and will exclude HBHA-specific antibody-mediated effect on the
offspring.
In this study, we vaccinated the pregnant mice with n- or rHBHA at 2 weeks of gestation.
Following delivery, mice were vaccinated with either BCG s.c. or n- or rHBHA i.n. at 1week and later boosted with n- or rHBHA i.n. at 4 weeks of age. Mice vaccinated with
only BCG, at 1-week, were used as a control. One week after the last immunization, lung
lymphocytes were isolated and restimulated in vitro with rHBHA in order to assess the
recall immune responses. As shown in Fig. 7, offspring from the immunized mother had
significantly higher recall immune responses to rHBHA than the offspring from the
unimmunized mother. Maternal priming also improved recall immune responses in the
BCG-primed-rHBHA-boosted neonates although the effect was not statistically
significant.
We next examined whether the improved recall immune responses, due to maternal
immunization, could impact on better protection. To examine this, we used nHBHA
instead of rHBHA and followed the similar immunization protocol as mentioned above.
Four weeks after the last immunization, mice were challenged with a high dose of BCG
i.n., and 3 weeks postchallenge the bacterial burden in the lungs was determined. As
shown in Table 1, the offspring from the immunized mother controlled infection better
than the offspring from the unimmunized mother, which was even higher than the BCG
control group. The effect of maternal priming was less clear in the group that received
BCG instead of nHBHA at 1-week and boosted later with nHBHA.
35
Vaccination
Recall IFN-γ response
Fig. 7: Maternal immunization primes
Neonates
for
improved
postnatal
cellular Pregnant
immunity. Pregnant mice at 2th week of Mother
W1
W4
gestation were primed with rHBHA s.c.
rHBHA
BCG
rHBHA
Following delivery, neonates were
immunized with BCG s.c or rHBHA+CT
BCG
rHBHA
i.n. at one week of age. Second booster
dose of rHBHA+CT was applied i.n. at
BCG
4-week of age. One week after the last
immunization, lung cells from the
rHBHA
rHBHA rHBHA
rHBHA-immunized animals were rerHBHA rHBHA
stimulated in vitro with rHBHA to
*
measure recall IFN-γ responses. Data for
IFN-γ shows mean stimulation index
with s.d. calculated as a ratio of HBHA2
3
4
5
6
stimulated IFN-γ levels to Con A0
1
S.I.×10
stimulated IFN-γ levels multiplied by a
factor 10. Data for IFN-γ represents mean±sd of triplicate wells. Cells were pooled from four animals per group.
Results were analyzed from two independent experiments. p value (s) was calculated by comparing two groups
using t test. * significant when p<0.05.
Taken together, these results suggest that maternal
ternal immunization with HBHA could
prime the fetal immune system, and therefore subsequent immunization of the offspring
with HBHA induces better protection.
The results from this study were encouraging because when compared with the BCG
vaccinated control group, the HBHA vaccinated group (maternal and postnatal
immunization) had higher levels of recall responses and protection. This indicates that a
combination of maternal and postnatal immunization with only nHBHA might be
beneficial for the group of children who are at risk of developing BCGosis due to the
BCG vaccination. The effect of maternal priming in the BCG vaccinated group was not
so prominent and this could be due the fact that BCG itself is a very strong
immunostimulant and therefore BCG/HBHA vaccination of the naive offspring was
sufficient to induce protection. Under the natural circumstances, heterologous primeboost strategy with BCG/HBHA vaccination is a better approach as we have seen in our
previous study.
36
Table 1. Bacterial burden in the lungs of offspring mice born from the mother primed with or without nHBHA
Vaccination1
Pregnant Mother
Protection, nHBHA2
Neonates
W1
W4
CFU in the lungs
mean±sd
×104
% of reduction3
nHBHA+CT
BCG
nHBHA+CT
2.25±5.9
55**
-
BCG
nHBHA+CT
2.40±2.3
52**
-
BCG
-
3.95±6.6
21
nHBHA+CT
nHBHA+CT
nHBHA+CT
3.15±7.0
37*
-
nHBHA+CT
nHBHA+CT
4.40±5.8
12
-
-
5.00±6.7
1
Vaccination was done with nHBHA in the pregnant mother at 2th week of gestation which followed offspring immunizations with
BCG or nHBHA+CT at 1 week and nHBHA+CT at 4 weeks of age.
2
Mice were challenged with BCG i.n. at a dose of 10 million CFU/animal four weeks after the last immunization. Three weeks
postchallenge bacterial burden in the lungs was determined.
3
Percent of reduction was calculated with respect to the unimmunized group. Results were analyzed from two independent
experiments. p value(s) was calculated by comparing two groups using t test.
**p<0.001 and *p <0.05, when compared with BCG group.
37
Concluding remarks
An effective control of TB is highly depended on the development of a new TB-vaccine,
as well as proper identification and treatment of individuals with active disease. From our
study with the identification of immune markers for better diagnosis of mycoabcaterial
infection, we can conclude that sTNFR and mycobacteria-specific antibodies in BAL, but
not in serum might be useful in distinguishing active from latent infection or exposure to
mycobacterial antigens. Cytokines such as IL-12 and IFN-γ can also be used as markers of
infection. Although we could not detect TNF in our particular model of infection, BAL
TNF has been reported to be a poor marker of detecting smear-negative pulmonary TB
(101).
The experience with the BCG and HBHA subunit vaccines in a neonatal mouse model
indicates that BCG vaccination primes the immune system for nHBHA. Priming with
BCG and boosting with nHBHA could substantially improve BCG-derived immune
responses and induce improved protection. Boosting with a non-protective antigens,
rHBHA, could also induce protection, however, only when the mice were primed with
BCG. Finally, upon BCG priming, HBHA immunization induces higher levels of
protection irrespective of the route of administration and the co-administration of adjuvant.
An alternative strategy of improving neonatal nHBHA vaccination is the priming of the
fetal immune system by vaccinating the pregnant mother at 2th week of gestation. This
strategy might only be useful for the children who are at risk of developing BCGosis.
Otherwise, BCG/HBHA combinations seem to be the best effective vaccine in our model
against mycobacterial infection.
38
Future studies
1. How long-lasting is the memory induced after BCG/HBHA vaccination?
In the previous study we have assessed the recall immune responses and protection 1week and 4-week, respectively, after the last immunization with HBHA. Therefore, we
will investigate the presence of protective memory, at 2 months and 4 months, after the
last immunization in a longitudinal experiment.
2. How long does the effect of BCG priming last?
In the previous study we considered only 3 weeks interval between prime and boost
vaccination. We will therefore examine the kinetics of BCG-primed immunity in order to
choose the best time point for boosting BCG-mediated immune response with HBHA.
3. Does BCG vaccination prime for only BCG-related antigens or has it a more
general adjuvant effect?
We have seen that BCG primes the immune system for HBHA, which is a BCG antigen.
In the future study we will ask whether the effect of BCG priming is specific to BCGrelated or unrelated antigens. A comparative study between HBHA and other
immunodominant antigens (BCG-related or unrelated) will be done by following
heterologous prime-boost approach.
4. Do mycobacteria other than BCG improve HBHA vaccination?
M. vaccae is a non-pathogenic mycobacterium, has been reported to exert adjuvant
effects (125). We will examine whether vaccination with M. vaccae prior to the HBHA
immunization could induce HBHA-specific immune responses and protection.
5. Is the effect of maternal priming with HBHA antibody mediated or cell mediated
or both?
The preliminary experiment with the maternal immunization focused exclusively on the
cellular immunity. Transferable maternal antibodies are crucial during the period when
the fetal T-cell immunity is not fully developed. In the future experiments we will
39
examine whether the milk of HBHA-primed mother has any influence on protection in
the offspring. We will also investigate the effect of maternal antibody in protection by
using IFN-γ-gene knockout mice, since IFN-γ is critical for the cell-mediated immune
response and protection against mycobacterial infection.
40
Acknowledgements
This study was carried out at the Department of Immunology, Stockholm University. I
am grateful to all who were involved in these studies, in particular my supervisor Prof.
Carmen Fernández for her continuous support from the first day in Sweden. I thank
Carmen for creating a PhD position for me and teach me how to approach a research
problem and to keep the integrity in expression of ideas. I am really grateful to Prof.
Marita Troye-Blomberg for giving me her valuable times in correcting thesis.
I thank Prof. Klavs Berzins, Prof. Eva Severinson and Associate Prof. Eva
Sverrenmark for all the important suggestions towards improving the quality of the
work.
My special thanks to John Arko-Mensah who is working in the TB project, for his
relentless cooperation in the laboratory and also for important suggestions.
I appreciate Eva Nygren and Solveig for their important support in the animal house.
I thank Manijeh Avedi for helping me with a FACS antibody.
I thank Magaretha Hagstedt, Ann Sjörlund, Gelana Yadeta, Anna Leena and
Elizabeth Bergner for their invaluable assistance.
Thanks to all past and present colleagues at the department especially Shanie, Yvonne
and Salah for sharing the knowledge of Flow cytometry. Thanks also to Stefania, Anna,
Petra, Halima, Manijeh, Lisa, Nora, Ylva, Nancy, Khosro, Jacqueline, Pablo, Amre,
Nnaemeka, Jacob, Esther, Gudrun, Ebba, Sandra, Christian, Ulrika, Magdi, and
Camilla for the nice discussion at the department.
I appreciate beyond words the families who over the years support me for everything. A
special thanks to my wife for her patience, and constant encouragement. Thank you Ilma,
my little daughter with a smile always, a special gift from the Almighty.
My special thanks to Qazi Khaleda Rahman who actually paved the way for us in
Sweden for higher study. I thank Dr. Atikul Islam and Dr. Zarina Kabir for helping my
family on several occasions.
I owe a debt of gratitude to my parents and sisters for their continuing support, grounding
and love.
41
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52
Immunodiagnosis of mycobacterial infection: Increased levels of immunological
markers in the respiratory tract but not in serum correlate with active pulmonary
infection in mice.
Arko-Mensah J1*, Rahman MJ1*, M. Singh2, Fernández C1
1
Department of Immunology, Wenner-Gren Institute, Stockholm University, S-10691,
Stockholm, Sweden
2
Lionex Diagnostics and Therapeutics GmbH, Braunschweig, Germany
*Both authors contributed equally to the manuscript
Corresponding author: John Arko-Mensah
Department of Immunology, The Wenner-Gren Institute, Stockholm University,
S-10691 Stockholm, Sweden
Tel: 00468164174; Fax: 004686129542
Email: [email protected]
1
Abstract
In developing countries where the vast majority of Mycobacterium tuberculosis (M.
tuberculosis) infections occur, diagnosis relies mainly on identification of acid-fast bacilli
in unprocessed sputum smears, which fails to identify approximately 50% of active
pulmonary disease cases, or mycobacteria culture which is usually slow and could take
several weeks. On the other hand, immunological tests for the diagnosis of TB have
relied mostly on detection of immune markers in serum or release of cytokines by
mononuclear cells in vitro. Here, we have modelled the natural route of mycobacterial
infection by infecting mice intranasally (i.n), but also systemically with Mycobacterium
bovis bacillus Calmette-Guérin (BCG) and assessed the immune response generated at
the site of infection and in serum. We demonstrate that active infection of mice with
BCG, but not exposure to non-replicating mycobacteria, induced production of IL-12,
IFN-γ and sTNFR locally in the lungs as detected in the broncho-alveolar lavage (BAL).
There was a positive relationship between bacteria growth in the lungs and levels of
sTNFR or IL-12, and to some extent IFN-γ in BAL. Moreover, sTNFR levels increased
significantly in BAL after reactivation of controlled infection with dexamethasone, which
resulted in increased bacteria growth in the lungs. Finally, infection but not exposure to
non-replicating mycobacteria induced specific IgG and IgA antibodies in BAL. Taken
together, the detection of sTNFR and mycobacteria specific antibodies, especially IgA
locally in the lungs to elected antigens expressed at different stages of disease
manifestation may be used as immunological markers for the diagnosis of TB.
2
1. Introduction
The global epidemic of tuberculosis (TB) results in eight to ten million new cases of
disease per year (1), with an annual projected increased rate of 3%. It is estimated that
between 5 and 10% of immunocompetent individuals are susceptible to TB, and of these,
85% develop pulmonary disease (2) in the first 2 years after exposure. The chronicity or
latency of Mycobacterium tuberculosis infection has made eradication of TB a very
difficult goal.
Immunity against TB depends on several factors, including cytokines, chemokines,
antibodies, macrophages, neutrophils, several T-cell subsets and specific pattern of T-cell
migration (3, 4). The infected host generates a T helper (Th)1 type of immune response in
which mycobacterial antigen-specific T lymphocytes are recruited to the lungs, and play
a significant role in protection against M. tuberculosis infection (5, 6). Interferon gamma
(IFN-γ) is the central mediator in protection against M. tuberculosis infection and
synergises with tumour necrosis factor (TNF) in activating macrophages (7). Interleukin
(IL)-12, together with TNF and IFN-γ, plays a critical role in the development of
protective granulomas, which contain activated macrophages producing specific
enzymes, such as inducible nitric oxide synthase (iNOS) or NOS2, responsible for the
elimination of bacteria. These mechanisms form the basis for protection against
mycobacterial spreading (8, 9). Even if less clear, specific antibodies particularly IgA in
mucosal secretions have been related to protective immunity against pulmonary
infections (10-12).
3
To effectively control the global TB epidemic, an improvement in the currently available
diagnostic tools is of utmost importance. In the vast majority of low- and middle-income
countries, where TB is prevalent, diagnosis primarily relies on identification of acid-fast
bacilli in unprocessed sputum smears using microscopy. Although acid-fast staining is
relatively quick, sensitivity is variable, ranging from 20-80% (13), because greater than
104 bacilli/ml of sputum are required for reliable detection. Subsequently, approximately
50% of active pulmonary TB is sputum smear negative, the failure rate being greater for
paediatric and HIV-associated TB (14, 15). While mycobacteria culture is the ultimate
proof of M. tuberculosis infection and often used as a reference method for diagnosis due
to its high sensitivity and specificity (16, 17), it involves direct handling of live bacteria
and also takes 4-6 weeks to grow on solid culture medium. The Mantoux or Tuberculin
skin test is used widely in several countries as the standard method for the diagnosis of
latent TB. The interpretation of this test, which is based on the detection of delayed type
hypersensitivity to purified protein derivative (PPD) after intradermal injection, could
however be affected by factors such as age, exposure to environmental mycobacteria or
BCG vaccination (18).
In recent times, the diagnostic potential of immune-based tests for the identification of M.
tuberculosis infection has been assessed and thought to offer the potential to improve
case detection in a general population. In this regard, a number of alternative testing
strategies, some based on IFN-γ release have been developed to address some of the
problems presented by the Tuberculin skin test in the detection of latent M. tuberculosis
infection. IFN-γ release assays involve stimulation of blood lymphocytes with the early
secreted antigenic target 6 (ESAT-6) or culture filtrate protein (CFP-10), followed by the
4
measurement of IFN-γ by enzyme-linked immunosorbent assay (ELISA) or the detection
of IFN-γ-producing cells by ELISPOT (19, 20). However, the infrastructure required to
collect specimen, deliver them to the proper laboratory and perform the test make it
difficult to establish in resource-limited countries (21). The development of
serodiagnostic tests for the detection of antibodies, antigens and immune complexes has
been attempted for decades. Serological methods are usually simple, rapid, inexpensive
and relatively non-invasive. To this end, different mycobacterial antigens have been
evaluated, culture filtrate proteins (22), mycobacterial sonicates (23), and purified
proteins (24, 25). Until recently, researchers have looked mainly in serum for
identification of specific mycobacterial antibodies as markers of infection. With regard to
the use of cytokines or cytokine receptors as surrogate markers of mycobacterial
infection, levels have been assessed mainly in serum of TB patients (26-30). Serological
methods have been used with limited success. One possible explanation is that serum
may not be the best biologic material to be analyzed.
Since TB is a disease of the lungs, antibodies and cytokines detected locally in the lungs
and associated fluids will more likely reflect infection status compared to serum. In this
study, we aimed to identify both cellular markers and specific mycobacterial antibodies
locally in broncho-alveolar lavage (BAL) and serum which in combination or separately
may be indicative of active mycobacterial infection. We demonstrate that active infection
of mice but not exposure to non-replicating mycobacteria resulted in increased levels of
IL-12, IFN-γ and soluble TNF receptors (sTNFR) in the BAL. Moreover, infection but
not exposure to non-replicating mycobacteria induced production of specific IgA
antibodies in BAL, which were undetectable in serum.
5
2. Materials and Methods
2.1. Mice
The studies were performed using 8-12 weeks old female BALB/c mice purchased from
Taconic Europe, Denmark and housed in pathogen free conditions. All animals were kept
at the Animal Department of the Arrhenius Laboratories, Stockholm University, Sweden.
All experiments were done in accordance with the guideline of the animal research ethics
board at Stockholm University. Mice were supervised daily and sentinel mice were used
to assess and ensure pathogen free conditions in the facility.
2.2. Bacteria cultivation
M. bovis BCG (Pasteur strain) obtained from Dr. Ann Williams, HPA, Salisbury, UK,
was grown in Middlebrook 7H9 broth with glycerol supplemented with albumindextrose-catalase (ADC) at 37oC, and aliquots frozen in PBS at -70oC. Three vials picked
randomly from the stock were thawed, serially diluted in plating buffer (PBS with 0.05%
Tween-80 [vol/vol]) and colony forming units (CFU) counted at 2-3 weeks after plating
on Middlebrook 7H11 agar (Difco, Sparks, MD, USA), with glycerol and oleic-acidalbumin-dextrose-catalase (OADC) enrichment.
2.3. Preparation of heat killed (hk)- and soluble BCG lysate
Bacteria were grown until they reached approximately 5-10 × 107 CFU/ml. To prepare
hk-BCG, 107 CFU/ml of BCG were autoclaved at 121˚C for 15 min. Killed bacteria were
washed once and re-suspended in sterile PBS before use. For the preparation of BCG
lysate, 107 CFU/ml bacteria were pelleted by spinning at 8,000 × g, resuspended in 0.05%
Tween 80 in PBS and washed two more times in this solution. The bacteria were then
6
resuspended in 5 ml of ice-cold PBS and sonicated on ice for 14 cycles of 1 minute each
as described by Power CA et al (31). The sonicated suspension was spun at 20,000 rpm
for 30 minutes at 4˚C to remove particulate matter and the supernatant containing soluble
antigens (referred to as BCG lysate) was collected. Protein concentration of the lysate
was determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, CA,
USA) according to the manufacturers instructions. Soluble Bovine serum albumin
fraction V (BSA) was used as protein standard, and the supernatant was stored at –20˚C.
2.4. Single mycobacteria antigens
The antigen 85 complex (Ag85c) was obtained from the Colorado State University.
Ag85c is a major secreted protein belonging to the mycolyl transferase family of the M.
tuberculosis complex (32). Purified 38-kDa, 19-kDa and 16-kDa antigens were all
obtained from LIONEX Diagnostics & Therapeutics GmbH, Germany. These antigens,
also secreted, have been commonly used in the serodiagnosis of TB (33-35).
2.5. Experimental infection or treatment with non-replicating BCG, determination of
CFU and reactivation of controlled infection
Mice were infected i.n. with 107 CFU of live BCG or treated with 107 CFU of nonreplicating BCG (BCG lysate or hk-BCG) or PBS as control in 30 µl PBS. Before
infection or treatment, mice were anaesthetised with isofluorane (Baxter Medical AB,
Kista, Sweden). I.n. administration was carried out by inoculation of live or nonreplicating BCG to the nostrils (15 µl per nostril given in two doses) and mice allowed to
breathe the suspension into the lung naturally (36). At day 3, weeks 1, 3, 5, and 9 after
infection, mice were sacrificed by cervical dislocation. Briefly, lungs, spleen and liver
7
were removed aseptically and placed in 2 ml PBS with 0.05% Tween-80 and
homogenized in glass homogenizers. Serial dilutions of the lung homogenates were
plated on Middlebrook 7H11 agar plates with OADC enrichment and incubated at 37˚C.
The number of CFU was determined 2-3 weeks after plating. Reactivation of controlled
infection was done as described by Lowrie et al. (37) with some modifications. Briefly,
each mouse was injected intraperitoneally (i.p.) at week 10 after infection (day 0) with
180 µg dexamethasone in 100µl PBS. I.p. injection of dexamethasone was repeated each
other day (days 0, 2, 4, and 6) for a total of 4 doses.
2.6. Sample collection
Serum and BAL were collected from each group of mice at the different time points
indicated above. To obtain sera, mice were bled from the tail vein and serum collected
after centrifugation. BAL was obtained by flushing 1.5 ml of PBS into the lungs of
sacrificed mice. All samples were kept at -20oC until use.
2.7. Cytokine and cytokine receptor detection assays
TNF and sTNFR enzyme-linked immunosorbent assay (ELISA) was performed using the
commercially available DuoSet ELISA Development Systems (R&D Systems Europe,
Abingdon, UK) according to the manufacturer’s recommendations, with slight
modifications. Streptavidin conjugated to alkaline phosphatase (MABTECH, Nacka,
Sweden) was used instead of horseradish peroxidase at 1:1000 dilution. IL-12 and IFN-γ
ELISA was performed using commercially available kits (MABTECH, Nacka, Sweden).
The enzyme-substrate reaction was developed using p-nitrophenyl phosphate (SIGMA,
St. Louis, MO, USA). Optical density was read in a multiscan plate reader (Anthos
8
Labtech Instruments, Salzburg, Austria) at 405 nm and concentrations were obtained by
comparison with calibration curves established with recombinant TNF, TNFR1 & 2, IL12 or IFN-γ standards.
2.8. Detection of antibodies in serum and BAL
Antibodies in serum and BAL were analyzed by ELISA. ELISA plates (Costar, high
binding, NY, USA) were coated with either BCG lysate (20 µg/ml) or Ag85c, 38-kDa,
19-kDa or 16-kDa (2 µg/ml) in carbonate-bicarbonate buffer pH 9.6, overnight (ON) at
room temperature (RT). Plates were washed four times with washing buffer (0.9% NaCl0.05% Tween-20 [vol/vol]). After washing, pools of samples (4 mice per treatment)
serially diluted were incubated in the antigen coated plates, starting as follows 1:100
(sera) and 1:5 (BAL) and plates were then incubated ON at RT. Following sample
incubation, plates were washed and incubated for 2 h at RT with alkaline-phosphatase
(ALP) labelled goat anti-mouse IgG or IgA (Southern Biotech, Birmingham, USA) and
the enzyme substrate reaction developed using p-nitrophenyl phosphate as substrate.
Absorbance was measured in a multiscan reader at 405 nm. Non-specific antibodies were
determined in parallel using BSA as unrelated antigens, and these values were deducted
from those obtained for specific anti-BCG antibodies.
2.9. Statistical analysis
Data are presented as the mean value ± SEM. Student’s t test was used to determine
statistical significance between treatment points. Differences were considered significant
when (*, p<0.05, (**, p<0.01).
9
3. Results
3.1. Intranasal infection but not treatment of mice with non-replicating BCG induces
elevated levels of IL-12, IFN-γ and sTNFR in the lungs
TNF, IL-12 and IFN-γ are essential components of the protective immune response
against mycobacterial infection, and elevated levels of these cytokines in the lungs, the
principal organ involved during M. tuberculosis infection, could be indicative of disease
activity. To assess this, BAL from mice infected i.n. with BCG or treated with nonreplicating BCG (lysate or hk-BCG) was collected at different time points and analyzed
for TNF, IL-12 and IFN-γ amounts. To quantify bacterial load in lungs, spleen and liver,
serial dilutions of organ homogenates were plated on Middlebrook 7H11 agar, and CFU
counted 2-3 weeks after plating.
Live, but not non-replicating BCG induced levels of IL-12 and IFN-γ in the lungs (Figure
1a-b). The amount of IL-12 detected in BAL progressively increased during infection to
week 3, and declined significantly (*P< 0.05) by week 9. There was a direct relationship
between IL-12 levels in BAL and bacteria growth in the lungs (Figure 1c). The highest
level of IFN-γ in BAL, unlike IL-12, did not exactly coincide with the peak bacteria
growth in the lungs, but was indicative of active infection. In contrast to IL-12 or IFN-γ,
minimal amounts of TNF were detected at all time points (data not shown). Since TNF
was undetectable in BAL we reasoned that elevated levels of sTNFR could result in TNF
neutralization (38). To assess this, we measured the levels of sTNFR1 and 2. Similarly to
IL-12 and IFN-γ, live, but not non-replicating BCG induced levels of sTNFR in the lungs
as detected in BAL (Figure 1d-e). The concentrations of sTNFRs, like IL-12 and to some
10
extent IFN-γ, seem to have a direct relationship with bacteria growth in the lungs.
Compared to the lungs, there was significantly lower infectivity of the spleen or liver
after i.n. infection of mice with BCG.
3.2. Elevated levels of sTNFR and IL-12 in serum are more indicative of exposure to
mycobacterial antigens than to active infection
Generally, peripheral blood is used for assessing the state of infection in several diseases.
To determine whether serum levels of sTNFR or IL-12 could be used similarly as BAL
levels to predict an ongoing mycobacterial infection, sera collected from the tail-vein at
the time points mentioned above were analyzed for presence of cytokines. Increased
levels of sTNFR and IL-12 were measured in serum not only after infection, but also after
treatment of mice with non-replicating BCG (Figure 2a-c). There was no direct
relationship between sTNFR or IL-12 levels and bacterial growth (Figure 1c) in the
lungs, spleen or liver, indicating that presence of sTNFR or IL-12 in serum may be more
indicative of antigen exposure rather than active infection. In these experiments, TNF and
IFN-γ were undetectable in serum. These results suggest that the detection of cytokines or
soluble receptors in serum may not be reflective of bacteria growth in the lungs, and
therefore not reliable for prediction of an ongoing mycobacterial infection.
3.3. Intravenous infection of mice with BCG results in elevated levels of sTNFR in BAL
which correlate with bacterial growth in the lungs
Since i.n. infection did not result in significant bacterial growth in the spleen and liver,
we administered BCG systemically via the i.v. route, as infection via the i.v. route results
in generalized infection of the lungs, spleen and liver. From the results obtained with i.n.
11
infection, levels of sTNFR were measured since concentrations seemed to have the
strongest correlation with bacteria growth. Intravenous administration of BCG resulted in
a generalized infection, with bacteria growth in all three organs (Figure 3a). Similarly to
the observation in BAL after i.n. infection, elevated levels of sTNFR (Figure 3b-c),
followed increased bacteria growth in the lungs. Overall, higher amounts of sTNFR were
detected in the serum of i.v., compared to i.n. infected mice. There was however no direct
relationship between levels of sTNFR in serum and bacterial load in the lungs, spleen or
liver, perhaps because many organs are infected and their contributions are unclear.
These results corroborate the observation that the local immune response in the lung
microenvironment but not the generalized or systemic may reflect an ongoing bacterial
growth in the lungs.
3.4. Reactivation of mycobacterial infection results in elevated levels of sTNFR in BAL
In a lifetime, 5-10% of people latently infected with M. tuberculosis reactivate their
infection as a consequence of immunosuppression, which results in increased bacterial
growth in the lungs and other organs. In animal experiments, reactivation of controlled
mycobacterial infection has been achieved by the administration of dexamethasone (34,
35), a corticosteroid that suppresses the effector functions of T cells central to the control
of infection. To determine whether increased bacteria growth in the lungs will result in
elevated levels of sTNFR in BAL, mice infected, but with controlled infection (week 10
after i.n. infection) were treated with dexamethasone as described above and BAL and
sera analyzed for presence of sTNFR.
12
Administration of dexamethasone resulted in increased bacterial growth in the lungs
(Figure 4a) by week 2 after treatment. There was also a slight but not significant increase
in bacteria load in the spleen and liver (data not shown). sTNFR levels increased
significantly in the BAL 2 weeks after dexamethasone treatment (Figure 4b-c), which
correlated with the bacteria growth in the lungs. In contrast, sTNFR levels in serum after
dexamethasone treatment were comparable to that of untreated mice (data not shown).
These results confirm our observation that sTNFR levels in BAL correlate with bacteria
growth in the lungs, and higher sTNFR amounts may be indicative of an ongoing but not
controlled chronic mycobacterial infection.
3.5. Infection but not treatment of mice with non-replicating BCG results in production of
BCG or antigen specific antibodies in BAL and serum
Thus far, our results have demonstrated a correlation between bacterial load in the lungs
and sTNFR levels in the lung microenvironment. However, several infections, especially
upper respiratory tract infections may induce inflammation and increase cytokine
production in the respiratory tract including the lungs. To confirm that infection was
caused by mycobacteria, we analyzed BAL and sera for the production of specific IgG
and IgA antibodies using BCG lysate as antigen. Only infected mice produced detectable
levels of antibodies in BAL and serum (Figure 5a-b), which increased over time. BCG
specific IgA was detected in BAL, but not serum, and had a similar kinetics as IgG
(Figure 5a). Next, we determined whether detection of antibodies to single mycobacterial
antigens in BAL and serum could be used to predict an ongoing infection compared to
total mycobacterial antigens. Similarly, to the results obtained with BCG lysate, only
active infection resulted in production of antigen specific IgG or IgA in BAL (Fig. 5c, d).
13
In contrast to BCG lysate, moderate to high amounts of IgG to single antigens were
detectable in serum (Fig. 5e). Overall, these data suggest that detection of antibodies to
multiple or single mycobacterial antigens in BAL, especially IgA may be predictive of an
ongoing infection in the lungs.
14
4. Discussion
A critical element of TB control is early diagnosis of infection and treatment. The
obtention of a microbiologically confirmed diagnosis of TB by the traditional methods
are disadvantaged by low sensitivity in sputum smear microscopy or slow growth in the
case of mycobacteria culture. These shortcomings, which could lead to diagnostic delay,
underscore the need for alternative diagnostic tests that are simple, rapid and sensitive. In
recent years, there has been renewed interest in the development of immune-based assays
for the diagnosis of M. tuberculosis infection. In this regard, IFN-γ release (19, 20) or
antibody based diagnostic assays (reviewed in ref. 39) have been developed.
Notwithstanding, the biological complexity of M. tuberculosis infection means that
election of a single immunological marker as predictive of infection or disease activity
would probably have limited diagnostic value, and analysis of several biomarkers would
offer the possibility of enhanced diagnosis. The importance of our study therefore lies in
the fact that we have assessed elements of the Th1 immune profile, known to be
important for the control of M. tuberculosis infection (5, 6), and specific mycobacterial
antibodies in serum and at the site of infection.
In the present study, we have demonstrated that: 1) active mycobacterial infection, but
not exposure to non-replicating mycobacteria resulted in concomitant elevation of IL-12,
IFN-γ and sTNFR in BAL; 2) levels of IL-12, sTNFR and IFN-γ in BAL depend on
bacteria growth in the lungs; 3) serum levels of IL-12 and sTNFR are probably more
related to exposure to mycobacterial antigens since live and non-replicating BGC induced
similar levels of IL-12 and sTNFR; 4) independently of the route of infection, sTNFR
levels in BAL correlate with bacteria growth in the lungs, but bacteria growth in the
15
spleen or liver do not seem to directly influence sTNFR levels in BAL; 5) reactivation of
controlled BCG infection with dexamethasone results in increased bacteria growth in the
lungs, which correlated with sTNFR levels in BAL; 6) active infection, but not exposure
to non-replicating mycobacteria resulted in production of antigen specific IgG or IgA in
BAL.
Over the years, the majority of studies that have investigated into cytokines or their
soluble receptors as markers of active disease in TB patients have looked mainly in the
serum (26-30). However, M. tuberculosis infection occurs via the respiratory tract, and
currently, there is the belief that in TB, as well as in other lung diseases, the markers of
disease activity need to be measured locally in the lungs, and not in peripheral blood. In
the present study, elevated levels of IL-12 or sTNFR in serum were associated with
exposure to mycobacterial antigens in general and not exclusively to active infection. On
the other hand, elevated levels of IL-12, IFN-γ in BAL, or the shedding of sTNFR
depended on the infection status. In TB, several studies have directly demonstrated a
relationship between cytokine production in the lungs and active tuberculosis based on
cytokine release by mononuclear cells at the site of infection (41-42). In these studies,
there was significant increase in release of IFN-γ, IL-12, IL-1β, IL-8 and TNF by
broncho-alveolar cells lavaged from patients with pulmonary TB.
In contrast, few studies have systematically assessed cytokines or their soluble receptors
locally in the lungs of TB patients. For example, Condos and co-workers (42)
demonstrated elevated levels of TNF and IL-1β in BAL from patients with active
pulmonary TB compared with that from healthy subjects. However, in their study, a TB
16
patient was defined as any patient with chest radiograph suggestive of TB regardless of
HIV status. In this regard, a possible contribution by some other co-infections cannot be
entirely excluded. In their work, Kupeli and co workers (43) demonstrated elevated levels
of TNF in BAL of smear-negative pulmonary TB patients compared to healthy controls.
However, BAL IL-2 and IFN-γ levels were not significantly different from that obtained
from patients with other pulmonary diseases. In our study, IFN-γ level in BAL was
lower, Compared to IL-12, and peaked later at week 5. IL-12 produced by activated
antigen presenting cells acts as a pro-inflammatory cytokine that bridges the innate and
adaptive immune responses and skews T-cell reactivity toward a Th1 cytokine pattern
(44).
The two forms of TNF, soluble TNF and transmembrane TNF function physiologically
by interacting with TNFR1 or TNFR2 expressed on a diverse range of cell types (45).
Elevated levels of sTNFR were measured in BAL after i.n. or i.v. infection of mice with
BCG. It is notable that mycobacteria have a predilection for the lungs even when
inoculated systemically. The bioavailability of TNF is modulated by binding to TNFR
Although TNF neutralization is an effective therapy in some debilitating conditions like
rheumatoid arthritis, it could increase the risk of reactivation of latent TB (46). For
example, transgenic mice expressing high serum levels of TNFR1 exhibited reduced
bactericidal activity, had undifferentiated granulomas and succumbed to BCG infection
(47). TNF neutralization may explain our inability to detect TNF and account for the
relatively lower levels of TNFR1, known to account mainly for binding to TNF. While
elevated levels of TNF were found in the BAL of TB patients (42, 48), its been suggested
that low TNF levels relative to sTNFR are found in chronic infection like TB, compared
17
to high TNF levels in acute infections which could overcome the potential neutralizing
activity of sTNFR in the circulation (49). Overall, our findings strengthen the notion that
TNF, IFN-γ, IL-12 and sTNFR are important components of the anti-mycobacterial
protection systems in humans, even if it is still unclear which levels could be considered
adequate, and elevated levels may be used as surrogate markers of disease activity.
In the first part of our study, we identified cellular immune markers associated with
mycobacterial infection. Later, we analyzed mycobacteria-specific antibodies in BAL and
serum after active infection of mice or treatment with non-replicating antigens by using
BCG lysate or selected mycobacterial antigens commonly used in the serodiagnosis of
TB. The use of crude antigens like whole-culture filtrate (22) or lysate (23) composed of
a mixture of several antigens has had the limitation of lack of sensitivity and/or
specificity. In the last decade however, studies of new assays that use various purified
and well-characterised proteins (24, 25) and lipid antigens (50, 51) for measurement of
serum antibodies in TB patients have been reported. While antibody production during a
primary immune response is usually low, the chronicity of mycobacterial infection means
that there is continuous stimulation of immune cells by surface exposed as well as
secreted antigens, resulting in significant production of antibodies over time. The higher
levels of antibodies measured in both BAL and serum at week 9, compared to the early
time points is therefore expected. In contrast, non-replicating antigens would need
continuous priming as well as help from relevant adjuvants to be optimally immunogenic.
This may explain the inability of the non-replicating antigens administered as single
doses to induce detectable antibodies in BAL. Nevertheless, detectable levels of specific
IgG were measured in serum but still not in BAL of mice treated with non-replicating
18
antigens. One explanation could be that the single antigens used, which were purified
antigens, had better affinity and higher specificity compared to BCG lysate antigens. In
this regard, it has been suggested that antibody-based diagnostics that utilize purified or
multiple antigens would achieve high levels of sensitivity and specificity (52).
In this study, IgA was detectable only in BAL. IgA is the major immunoglobulin in
mucosal secretions and our group and others have demonstrated the induction of IgA in
mucosal secretions after i.n. immunization with mycobacterial antigens (10-12) or
infection with mycobacteria (53). Nevertheless, mycobacteria specific IgA was detected
in the serum of adult TB patients (54, 55). This is not entirely surprising, considering the
protracted nature of TB in humans, which could result in constant release of secretory
mycobacterial antigens into the general circulation resulting in continuous priming of IgA
secreting B-cells. The four antigens selected have been commonly used for the
serodiagnosis of TB (32-35). In contrast to Ag85c, 38-kDa or 19-kDa, lower amounts of
anti-16-kDa antibodies were detectable in BAL and serum. The 16-kDa antigen is a
cytosolic regulatory protein specific to the M. tuberculosis complex and expressed during
latency (56). A systematic analysis of humoral immune responses of TB patients has
shown that the profile of antigenic proteins of M. tuberculosis recognized by antibodies
differs at different stages of infection and disease progression (57). This suggests that an
accurate diagnostic test for TB will need to be based on a combination of antigens. At
this point, it is conceivable to propose the detection of mycobacterial antigens in BAL,
saliva or serum as an alternative to soluble immune markers. However, serological tests
based on detection of mycobacterial antigens, like the skin test, could be confounded by
cross-reactivity with non-pathogenic mycobacteria or immunization.
19
Its noteworthy to mention that measurement of immune markers directly in saliva would
be rapid, simple, inexpensive and non-invasive compared to obtention of BAL.
Moreover, the use of BAL may be technically demanding in resource-limited settings. So
far, we have been unsuccessful in detecting cytokines or antibodies to single
mycobacterial antigens in saliva from infected mice. Notwithstanding, we are working to
improve our detection assays in saliva. In conclusion, detection of a tailored combination
of mycobacteria specific antibodies and cellular markers in the respiratory tract would be
useful for the immunodiagnosis of TB, and may help prediction of the clinical course of
disease.
20
Acknowledgements
We are grateful to Esther Julián and Gudrun Horner for their initial contribution to this
work and fruitful discussions. We are also indebted to Irene García and María Olleros for
sharing with us their vast knowledge about TNF and sTNFRs. The study was supported
by grants from the Swedish Institute and Hjärt-Lungfonden.
21
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26
Figure Legends
Figure 1
Intranasal infection but not treatment of mice with non-replicating BCG induces elevated
levels of sTNFR, IL-12 and IFN-γ in the lungs. Mice were infected i.n. with 107 CFU of
BCG or treated with corresponding amounts of BCG lysate or hk-BCG. Control mice
were treated with PBS. BAL was collected at day 3, weeks 1, 3, 5 and 9 after infection or
treatment. Levels of IL-12 (a), IFN-γ (b) and sTNFR (d and e) were measured with
standard ELISA kits and mean concentrations expressed as pg/ml. Serial dilutions of
lungs, spleen and liver were plated on Middlebrook 7H11 agar and bacterial loads were
determined 2-3 weeks after plating (c). Results are expressed as mean concentration (a,b),
(d,e) or CFU × 103 (c) ± SEM from 4 mice per group. A representative of three different
experiments is shown. * p<0.05, ** p<0.01 versus D3.
Figure 2
Elevated levels of sTNFR and IL-12 in serum are more indicative of exposure to
mycobacterial antigens than to active infection. Sera from mice infected with live or nonreplicating BCG as described in Figure 1 were analyzed for presence of sTNFR (a-b) and
IL-12 (c) with standard ELISA kits and mean concentrations expressed as pg/ml. Results
are expressed as mean concentration (a-c) ± SEM from 4 mice per group. A
representative of three different experiments is shown.
27
Figure 3
Intravenous infection of mice with BCG results in elevated levels of sTNFR in BAL,
which correlate with bacterial growth in the lungs. Mice were infected i.v. with 107 CFU
of BCG and BAL and sera collected at day 3, weeks 1, 3, 5 and 9 after infection. Levels
of sTNFR in BAL (b-c) and sera (d-e) were assayed as described above and mean
concentrations expressed as pg/ml. Serial dilutions of lungs, spleen and liver were plated
on Middlebrook 7H11 agar and bacterial loads determined 2-3 weeks after plating (a).
Results are expressed as mean concentration (b-e) or CFU × 103 (a) ± SEM from 4 mice
per group. A representative of three different experiments is shown. * p<0.05, ** p<0.01
versus D3.
Figure 4
Reactivation of mycobacterial infection results in elevated levels of sTNFR in BAL.
Mice perceived to have controlled BCG growth in the lungs at week 10 after i.n. infection
with 107 CFU of BCG were injected intraperitoneally with 180ug dexamethasone per
mouse every other day for a total of 4 injections. BAL collected at weeks 1, 2 and 3 after
dexamethasone treatment was assayed as described above for the presence of sTNFR,
and mean concentrations expressed as pg/ml (b-c). Serial dilutions of lungs were plated
on Middlebrook 7H11 agar and bacterial loads determined 2-3 weeks after plating (a).
Results are expressed as mean concentration (b-c) or CFU × 103 (a) ± SEM from 4 mice
per group. A representative of three different experiments is shown. * p<0.05, ** p<0.01
versus D3.
28
Figure 5
Infection but not treatment of mice with non-replicating BCG results in production of
BCG or antigen specific antibodies in BAL and serum. Mice were infected i.n. with 107
CFU of BCG or treated with 107 CFU non-replicating BCG, and BAL and sera collected
as above. Micro-titre plates were coated with 25 µg/ml of BCG lysate as antigen
overnight in bicarbonate buffer, pH 9.6, and the amounts of BCG-specific antibodies
assayed in BAL (a) and serum (b) serially diluted in PBS. 2 µg/ml of purified Ag85c, 38kDa, 19-kDa or 16-kDa was coated as described and antigen specific antibodies assayed
in BAL (c, d) and in serum (e). OD was read at 405nm and was in the linearity phase.
Results are expressed as mean OD from 4 mice per group, and a representative of three
different experiments is shown.
29
a)
d)
2000
2500
**
**
2000
(pg/ml)
**
1500
1500
*
1000
1000
500
500
0
0
D3
w1
w3
w5
w9
D3
BCG
BCG lysate
hk-BCG
PBS
b)
120
IFNγ
**
w5
w9
e)
sTNFR2
14000
(pg/ml)
(pg/ml)
w3
**
10000
80
60
w1
12000
100
**
**
40
*
8000
6000
4000
20
2000
0
0
D3
w1
w3
w5
w9
D3
w1
w3
w5
w9
c)
Bacteria load
180
Lung
Spleen
Liver
160
**
140
120
CFU x 103
(pg/ml)
sTNFR1
2500
IL-12
3000
100
*
*
80
60
40
20
0
D3
w1
w3
w5
Fig. 1
w9
30
b)
a)
sTNFR2
sTNFR1
25000
7000
20000
6000
(pg/ml)
4000
3000
15000
10000
2000
5000
1000
0
0
D3
w1
w3
w5
D3
w9
w1
w3
w5
BCG
BCG lysate
hk-BCG
PBS
c)
IL-12
6000
5000
4000
(pg/ml)
(pg/ml)
5000
3000
2000
1000
0
D3
w1
w3
w5
w9
Fig. 2
31
w9
a)
Bacteria load
Lung
Spleen
Liver
90
80
70
*
CFU x 103
60
50
40
30
20
10
0
D3
w1
w3
w5
w9
BAL
SERUM
d)
b)
12000
sTNFR1
sTNFR1
10000
1600
1400
(pg/ml)
(pg/ml)
8000
**
1200
1000
800
*
6000
4000
600
2000
400
200
0
0
D3
D3
w1
w3
w5
w1
w3
w5
w9
w9
c)
e)
sTNFR2
sTNFR2
10000
45000
**
35000
6000
(pg/ml)
(pg/ml)
8000
4000
*
25000
15000
2000
5000
0
0
D3
w1
w3
w5
w9
D3
w1
w3
w5
Fig. 3
32
w9
Bacteria load
30
a)
25
*
CFU x 103
20
15
10
5
0
w9
w11
w12
w13
DXM treatment
b)
BAL
1200
sTNFR1
(pg/ml)
1000
*
800
600
BCG(-DXM)
BCG(+DXM)
400
w9
w11
w12
w13
DXM treatment
c)
8000
sTNFR2
7000
(pg/ml)
6000
*
5000
4000
3000
2000
1000
w9
w11
w12
DXM treatment
w13
Fig. 4
33
OD (405nm)
a)
BAL
2
1,8
1,6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
w1
w3
w5
w9
w1
w3
IgG
b)
w5
Coating antigen: BCG lysate
w9
IgA
Serum
2.5
BCG
lysate
OD (405nm)
2
hk-BCG
1.5
1
0.5
0
w1
w3
w5
w9
w1
w3
IgG
c)
w9
IgA
2.5
d)
BAL
1.6
IgG
1.5
1
0.5
0
1
0.8
0.6
0.4
0.2
0
w1 w3 w5 w9
BCG
w1 w3 w5
w1 w3 w5 w9
BCG lysate
w1 w3 w5 w9
hk-BCG
BCG
BCG lysate
w1 w3 w5 w9
hk-BCG
Ag85c
38-kDa
19-kDa
16-kDa
e)
2.5
w1 w3 w5
Groups
Groups
IgG
2
OD (405nm)
IgA
1.4
1.2
OD (405nm)
2
OD (405nm)
w5
1.5
SERUM
1
0.5
0
w1 w3 w5 w9
BCG
w1 w3 w5
BCG lysate
w1 w3 w5 w9
hk-BCG
Groups
34
Fig. 5
Improvement of HBHA vaccination by BCG priming in a neonatal mouse model
Rahman MJ1, Locht C2 and Fernández C1
1
Department of Immunology, Wenner-Gren Institute, Stockholm University, S-10691,
Stockholm, Sweden
2
INSERM U629, Institut Pasteur de Lille, 1, rue du Prof. Calmette, F-59019 Lille Cedex,
France.
Corresponding author: Muhammad Jubayer Rahman
Department of Immunology, The Wenner-Gren Institute, S-10691, Stockholm University,
Stockholm, Sweden
Tel: 00468164174; Fax: 004686129542
Email: [email protected]
1
Abstract
The neonatal immune system is not fully developed which results in suboptimal
responses to many vaccines. The Mycobacterium bovis BCG vaccine is one of the few
examples that can actively induce Th1 cellular immunity in the neonates and confers
protection against severe forms of disseminated but not pulmonary tuberculosis in
children. Moreover, protection mediated by the BCG vaccine wanes over time. There is a
need to develop an effective booster vaccine, which can improve the BCG-stimulated
protective immunity. Since tuberculosis (TB) is primarily a pulmonary disease we
addressed whether the route of administration and the election of a good antigen could be
of importance in the improvement of neonatal BCG vaccination. For this purpose, we
have tested the mycobacterial antigen heparin-binding hemagglutinin (HBHA), a potent
new vaccine candidate. Our results showed that priming with BCG at the neonatal period
followed by boosting with native (n) HBHA, improved HBHA-specific immune
responses in the lungs, including the frequency of IFN-γ producing CD4+ and CD8+ T
cells. Interestingly, priming with BCG biased the immune response towards a Th1 type
since the vast majority of CD4/CD40L expressing cells was also found to be IFN-γ
positive. A comparison of intranasal (i.n.) and subcutaneous (s.c) immunization with
nHBHA showed that i.n. immunization induced higher levels of IFN-γ as observed upon
in vitro stimulation of lung cells with HBHA. However, IFN-γ levels upon stimulation
with BCG-infected macrophages and protection against high-dose of BCG infection were
comparable between the i.n. and s.c. groups. Also, the use of adjuvant in the boosting
with nHBHA was not necessary provided that the animals were previously primed with
BCG. Immunological responses correlated with the levels of protection measured by
control of bacterial growth in the lungs upon i.n. infection. Finally, priming with BCG
improved the immunogenicity and protection promoted by the recombinant form of
HBHA regarded previously as non-protective. We discuss the improvement of HBHA
vaccination for better protection against TB.
2
1. Introduction
Tuberculosis (TB) is a disease of global concern caused by the pathogen Mycobacterium
tuberculosis. The World Health Organization (WHO) reported that there were 8.8 million
new cases of TB in 2005 and a total of 1.6 million people died of TB, including patients
infected with HIV (1). Nearly 80% of the world’s population is vaccinated with
Mycobacterium bovis bacille Calmette-Guérin (BCG) (2), which has been the only
licensed vaccine against TB. Despite this large coverage, TB remains one of the leading
causes of human death (3). In a number of field studies, it has been reported that the
protective efficacy of BCG vaccination against pulmonary TB varies from 0-80% (4-5).
Only well-nourished children vaccinated with BCG at the neonatal stage have protection
against TB with an average of 50% (5-6), which has been reported as a highly cost
effective intervention against severe childhood TB such as meningitis or miliary TB (7).
Therefore, vaccination with BCG is still recommended in many countries as the standard
for TB prevention in infants and young children. Unfortunately, BCG-mediated
protection lasts only during the first years (5) of life and revaccination with BCG does
not provide additional protection (8). With the increasing appearance of multi-drug
resistant TB and the co-infection with human immune deficiency virus (HIV), incidence
of TB is now escalating. All together, there is an urgent need to develop an effective
vaccine.
BCG is a live attenuated strain of M. bovis, which was altered with many undefined
genetic changes during the process of attenuation. The reasons for the variable efficacy of
BCG in protection against M. tuberculosis are not well understood. Possible explanations
have been suggested (reviewed in refs 9, 10) including influence of environmental
3
mycobacteria on the immune response to BCG, deletion of protective antigens, route of
administration, and genetic differences among vaccines. In order to improve the BCG
vaccine or to develop a new TB vaccine, several approaches have already been addressed
in different experimental models with recombinant BCG (rBCG), DNA vaccines or
subunit proteins. It has been shown that rBCG over-expressing Ag85B provided better
protection than the BCG vaccine (11, 12). Another strategy is the use of subunit vaccines
but the majority of them failed to impart better protection than the BCG vaccine (13, 14).
DNA vaccines expressing mycobacterial antigens have shown protection in mice but they
have been disappointing in the non-human primate model of TB (15). Heterologous
prime-boost strategies using DNA and subunit proteins have been demonstrated
promising in mice, although protection was not significantly better than BCG when tested
in the larger animals like guinea pigs (16). Subsequently, it was found that BCG primeDNA boost and BCG prime-fusion protein boost regimes were efficient for enhancing
protective immunity in mouse models of TB (17, 18).
The mycobacterial heparin-binding hemagglutinin adhesion (HBHA) has been reported
to be a potent mycobacterial antigen that displayed a level of protection similar to the
BCG vaccine in a mouse model of TB (19, 20). HBHA is a surface associated protein
involved in adherence to epithelial cells (21), and has also been shown to be important for
extrapulmonary dissemination of M. tuberculosis (22). In mice, protection mediated by
HBHA was found to be dependent on the methylation of the c-terminal site of native (n)
HBHA but not to the recombinant (r) HBHA without methylation (20). In humans,
methylation-dependent T-cell immunity to nHBHA was proposed to be protective against
TB, since T lymphocytes from healthy subjects infected with M. tuberculosis produce
4
significant amounts of HBHA-specific interferon-γ (IFN-γ), but T cells from patients
with active disease do not (23).
One of the requirements for an effective vaccine against pulmonary TB is that it should
be able to induce long-lasting protective immunity in the lungs, where the initial infection
takes place. Many investigations have focused on the examination of the mucosal
immune responses at the entry port of the mycobacteria and downstream along the
respiratory tract, which have profiled the significant strength of vaccine-induced lung
immunity against TB (24, 25). Generally, the mucosal route of immunization enhances
immune responses on the mucosal sites, provided that antigen is delivered with a strong
adjuvant (26). In this context, cholera toxin (CT) and E. coli heat-labile enterotoxin (LT)
have been described as successful mucosal adjuvants (reviewed in ref. 27), although both
are toxic for human use. However, progress has been made to modify CT and LT in a
way that could reduce the toxicity but retain the adjuvanticity (28, 29).
Protection against pulmonary TB concerns the development of a new vaccine with
improved immunogenicity and protection, which is especially difficult to achieve by
vaccinating children after birth. Studies in newborn mice demonstrated that they are
biased to a Th2-type of immune response and basically unable to induce a Th1-type of
immune response (30, 31). Importantly, BCG is an example of one of the few vaccines
that can induce Th1-type immune responses at birth (32), and this might be one of the
reasons why BCG is effective in reducing the incidence of childhood TB. Since BCGmediated protection wanes over time and repeated vaccination with BCG is not effective,
strategy for future vaccines could be to focus on the boosting of BCG-induced protective
5
immune responses and this may be best accomplished by a subunit booster vaccine. Due
to the protective capacity of HBHA, we evaluated a heterologous prime-boost protocol
by priming with BCG at the neonatal stage and boosting with n- or rHBHA at the
systemic or mucosal routes during the infant and adult ages.
We show here that priming with BCG at the neonatal age and boosting with nHBHA later
improved protection, which was significantly better than the protection provided by BCG
alone. Protection correlated with the enhanced levels of IFN-γ in the respiratory tract.
6
2. Materials and Methods
2.1. Animals
The studies were performed in neonatal (1-week-old), infant (4 to 6-week-old) and adult
(more than 8-week-old) BALB/c mice. Adult mice were purchased from Taconic Europe,
Denmark and housed in pathogen free conditions. Neonates were obtained from
laboratory breeding facilities followed by timed (hand) mating protocol using adult males
and females manually placing them together over night. Three weeks after birth mice
were weaned and separated from their mothers. All animals were kept at the Animal
Department of the Arrhenius Laboratories, Stockholm University, Sweden. All
experiments were done in accordance with the relevant guideline of the animal research
ethics board at Stockholm University. Mice were supervised daily and sentinel mice were
used to assess and ensure pathogen free conditions in the facility.
2.2. Antigens and adjuvants
Both n- and rHBHA were kindly provided by Dr. Camille Locht, Pasteur de Lille, France.
Briefly, nHBHA was extracted from M. bovis or M. tuberculosis H37Ra and purified by
heparin-sepharose
chromatography
(21),
followed
by
high-performance
liquid
chromatography (HPLC) as described previously by Masungi et al. (23). rHBHA was
expressed in E. coli and purified by nickel chromatography as previously described (33).
Cholera toxin was obtained from Quadratech Ltd, Surrey, UK.
2.3. Mycobacteria
M. bovis BCG (Pasteur strain) obtained from Dr. Ann Williams, UK was grown in
Middlebrook 7H9 (DIFCO, Sparks, MD, USA) broth supplemented with albumin-
7
dextrose-catalase (ADC) enrichment, 0.5% glycerol and 0.05% Tween 80 (vol/vol) for
10-15 days at 37oC. Aliquots frozen in phosphate buffer saline (PBS) with 10% glycerol
were kept at -70oC. Three vials picked randomly from the stock were thawed, serially
diluted in plating buffer (PBS with 0.05% Tween-80 [vol/vol]) and colony forming units
(CFU) counted 2-3 weeks after plating on Middlebrook 7H11 agar (Difco, Sparks, MD,
USA), with glycerol and oleic acid-albumin-dextrose-catalase (OADC) enrichment.
2.4. Immunization and challenge
As shown in Fig. 1, neonates at 1-week of age were primed with either BCG or nHBHA
formulated with CT administered subcutaneously (s.c.). A total of 105 CFU of BCG or
nHBHA (1 µg) formulated with CT (0.2 µg) in 50 µl of phosphate buffer saline (PBS)
was administered s.c. at the dorsal neck region. For boosting studies, mice were given nor rHBHA intranasally (i.n.) or s.c. three times at 2-week interval from the age of 4
weeks. For i.n.-boosting, mice were anesthetized with isofluorane (Baxter Medical AB,
Kista, Sweden) and given 5 µg of HBHA together with 1 µg of CT in 20 µl total volume
(5 µl per nostril given in two doses). Mice were allowed to breathe the suspension into
the lung naturally. For s.c.-boosting, 5 µg of nHBHA was formulated with 1 µg of CT in
100 µl of PBS and administered into the dorsal neck region. Adult mice were vaccinated
similarly as mentioned above with five times the neonatal doses. Age-matched control
mice received PBS (i.n. or s.c.) or BCG s.c. Some mice were sacrificed one week after
the last immunization for in vitro cellular responses. The rest of the mice were infected
with BCG i.n. (107 CFU per animal) four weeks after the last immunization. Three weeks
after the challenge, mice were sacrificed for the observation of bacterial burden in the
lungs as described previously (34).
8
2.5. Mononuclear cell isolation
One week after the last immunization with HBHA, lungs and spleens were removed
aseptically and placed in sterile PBS. Lungs were cut into small pieces (2-3mm) and
incubated for one hour at 37˚C with collagenase type I (1 mg/ml, Roche, Mannheim,
Germany) and DNase (30 U/ml, Sigma, St. Louis, MO, USA). Lung pieces were then
homogenised using a glass homogenizer and the cell suspension was filtered through a 70
µm nylon membrane (BD, Franklin Lakes, NJ USA) and subjected to gradient separation
using Lympholyte M (CEDARLANE laboratories, Ontario, Canada). After centrifugation
at 1500 g for 20 minutes, the interface was collected and washed twice with PBS. Viable
cells were enumerated using trypan blue. Thereafter, cells were cultured in complete
DMEM medium containing 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml
penicillin, 100 µg/ml streptomycin and 2 mM sodium pyruvate (all from Invitrogen,
Paisley, UK).
2.6. Generation of bone marrow derived macrophages
Bone marrow derived macrophages (BMM) were generated as previously described (35).
Briefly, after sacrifice, the femur and tibia of the hind legs were taken. Bone marrow
cavities were flushed with cold, sterile PBS followed by washings and resuspension of
the cells in complete DMEM supplemented with 20% L929 cell conditioned medium (as
a source of M-CSF). Bone marrow cells were plated in 6-well plates and incubated for 7
days at 37˚C, 5% CO2, with replacement of medium every second day. Before use, BMM
were washed vigorously to remove non-adherent cells.
9
2.7. In vitro stimulation of lymphocytes
Ag stimulation: Mononuclear cells isolated from the lungs were plated at a concentration
of 106 cells/ml in 96-well flat bottom plates (Costar, NY, USA) and stimulated with
HBHA (5 µg/ml) or Con A (2 µg/ml) for 72 h at 37˚C, 5% CO2. Cell culture supernatant
was collected and stored at -20˚C until tested for cytokines by Enzyme-linked immuno
sorbent assay (ELISA) assay.
Stimulation with BCG-infected BMM (BCG-BMM): It has been described that IFN-γ
levels upon in vitro stimulation of lymphocytes from HBHA-immunized animals with
BCG- or M. tuberculosis pulsed macrophages correlate better to protection against M.
tuberculosis (19, 20). Macrophages were infected with BCG for 4 h at a multiplicity of
infection of 5:1. Extracellular bacteria were removed by vigorous washings and cultures
incubated again for 24 h at 37˚C. Lung cells were cultured with BCG-BMM for 72 h at a
ratio of 10:1. Culture supernatants were collected and analyzed for IFN-γ by captured
ELISA.
2.8. IFN-γ ELISA
A commercially available kit for IFN-γ (MABTECH, Stockholm, Sweden) was used to
determine the cytokine levels in the culture supernatants according to the manufacturer’s
recommendations, with slight modifications. Streptavidin conjugated to alkaline
phosphatase (MABTECH, Stockholm, Sweden) was used instead of horseradish
peroxidase at 1:1000 dilution. The enzyme-substrate reaction was developed using pnitrophenyl phosphate (Sigma). Optical density was measured in a multiscan ELISA
reader at 405 nm and concentrations were calculated from the standard curves established
10
with corresponding purified recombinant mouse IFN-γ. Data were represented as a
stimulation index calculated as a ratio of HBHA or BCG-BMM-stimulated IFN-γ levels
to Con A-stimulated IFN-γ levels multiplied by a factor 10.
2.9. Intracellular IFN-γ staining
Lymphocytes were isolated from the lungs as described above. Lymphocytes at 106
cells/ml were stimulated with HBHA (5 µg/ml), BCG-BMM (105 cells/ml) or Con A as a
positive control (2 µg/ml, Sigma) for 6 h at 37˚C. GolgiStop (BD), containing monensin
was added for the last 4 h of stimulation. Thereafter, cells were washed and incubated
with mouse FcBlock (FcγII/III, BD). Following washings, cells were stained for surface
markers: allophycocyanin conjugated anti-CD3 (eBioscience, San Diego, USA),
fluorescein isothiocyanate (FITC) conjugated anti-CD4 (BD) and phycoerythrin (PE)
conjugated anti-CD8 (BD). At this stage, cells were permeabilized using the
Cytofix/Cytoperm kit (BD Pharmingen), and stained for intracellular R-PE-coupled
cyanine dye (PE-cy7) conjugated anti-IFN-γ antibody (BD). After washing, cells were
resuspended in PBS containing 0.1% sodium azide and analyzed by FACS Calibur.
Detection of CD40L on the cell surface was done by stimulation of cells according to the
protocol described previously (36). Briefly, cells were cocultured with PE-conjugated
anti-CD40L antibody (BD) in the presence of CD28-specific antibody (BD) during the
stimulation with HBHA or BCG-BMM as described above. After stimulation, cells were
washed and blocked with Fc receptors (FcγII/III, BD) and stained for the surface markers
CD3 and CD4 and subsequently permeabilized for intracellular IFN-γ staining as
described above. Following washings, cells were resuspended in PBS containing 0.1%
sodium azide and analyzed by FACS Calibar.
11
2.10. Statistics
Comparison between two experimental groups was done by Student’s t test. A p value of
<0.05 was considered significant.
12
3. Results
3.1. Improvement of nHBHA-immunization by neonatal BCG priming
3.1.1. BCG priming improves immune responses to nHBHA boostings
To examine whether BCG vaccination could prime the immune system for the induction
of HBHA-specific immune responses, we vaccinated mice at 1-week of age with BCG
s.c. and later boosted them s.c. with nHBHA three times at 2-week intervals (Fig. 1). One
week after the last immunization, lung lymphocytes were prepared and stimulated in vitro
with n- or rHBHA. IFN-γ levels were measured in the culture supernatants as a sign of
cell activation. Both the native and recombinant proteins were equally efficient in the in
vitro restimulation of lung cells (data not shown). We here present only data obtained
with recombinant form of HBHA. Since rHBHA is easier to obtain, and in the following
restimulation experiments only rHBHA was used. As shown in Fig. 2A, significantly
higher levels of IFN-γ were produced by cells from BCG-primed-nHBHA-immunized
animals (BCG/nHBHA) (Gr. 5) compared to the control groups receiving only with PBS
(Gr. 1), BCG (Gr. 2) or nHBHA (Gr. 3) alone. Similar results were observed when lung
cells were cultured with BCG-BMM (Fig. 2B: Gr. 5). Collectively, these results showed
an improved effect of BCG priming on recall immune responses to HBHA.
3.1.2. Effect of BCG as adjuvant
It has been shown in adult mice that nHBHA, contrary to other mycobacterial antigens,
induced a degree of protection similar to that provided by BCG. In these experiments
HBHA was administered with adjuvant (19, 20). Generally, adjuvants make proteins
immunogenic but unfortunately, there are not good adjuvants for human use. The current
13
ones are either too toxic or not so efficient. Therefore, it was of interest to test the need of
adjuvant in our immunization protocol. nHBHA was administered with or without CT to
mice primed neonatally with BCG. Lung lymphocytes from the immunized mice were
isolated and restimulated in vitro as described above. IFN-γ levels in culture supernatants
were determined. Data showed that in the BCG-primed mice, vaccination with nHBHA
given together with CT did not result in a significantly higher IFN-γ response compared
to nHBHA administered without CT (Fig. 2A, B: Gr. 4 and 5). Our observations
suggested that adjuvants might not be necessary provided that mice were primed with
live BCG before boosting.
3.1.3. The effect of route of immunization with nHBHA
The i.n. route of immunization preferentially induces better immune responses in the
lungs compared to the parenteral immunization (37). In order to examine this issue in our
BCG/nHBHA model, we compared the recall immune responses induced after
immunization following i.n.- or s.c.-routes. One week after the last immunization with
nHBHA, cells were isolated from the lungs and restimulated with rHBHA or BCG-BMM
and IFN-γ levels were measured in the culture supernatants. Restimulation with rHBHA
induced significantly higher levels of IFN-γ in the i.n. group compared to the s.c. group
(Fig. 2A: Gr. 5 and 6). In contrast, IFN-γ responses after BCG-BMM stimulation were
comparable in both groups (Fig. 2B: Gr. 5 and 6). Thus, both i.n. and s.c. immunizations
may be similar in the stimulation of lung cells at least in the model described here using
BCG as a priming agent.
14
3.1.4. Boosting with nHBHA significantly enhances protection promoted by BCG
vaccination
We next examined whether the higher levels of IFN-γ observed in the animals primed
with live BCG and boosted with nHBHA under the various conditions mentioned above
correlated with higher protection against BCG infection. Four weeks after the last
immunization, mice were challenged with a high dose of BCG given i.n. and three weeks
later, bacterial numbers in the lungs were enumerated. Our results showed that in all
groups, independently of the route of immunization or the co-administration with
adjuvant, there was a better protection (Fig. 2C: Gr.4, 5, 6) when the mice were
neonatally primed with BCG.
3.2. Identification of cells producing IFN-γ following prime-boost immunization
Priming with BCG followed by boostings with nHBHA i.n. or s.c. significantly improved
IFN-γ responses and protection. We next examined the frequency of IFN-γ producing
CD4+ and CD8+ T cells. Mice were immunized as described above, and 1-week after the
last boost, lymphocytes were isolated from the lungs and stimulated with rHBHA or
BCG-BMM for 6 h. Cells were permeabilized and stained for intracellular IFN-γ. As
shown in Table 1, upregulation of CD4+IFN-γ+ and CD8+IFN-γ+ cells was observed in
both the i.n.- and s.c.-boosted groups. Restimulation with BCG-BMM induced higher
percentages IFN-γ producing cells than restimulation with rHBHA. Compared with the
BCG-vaccinated control group, i.n. and s.c. groups had higher percentages of CD4+IFNγ+ and CD8+IFN-γ+ cells. Compared to each other, higher percentages of IFN-γ+ cells
were observed in the i.n. immunized mice.
15
CD40L/CD154 is an activation marker that is expressed transiently mostly by activated
CD4+ T cells (36). To closely assess the proportion of antigen-specific CD4+ T cells and
IFN-γ production in our experimental conditions, we further investigated the proportion
of activated CD4+ T cells in presence of HBHA or BCG-BMM by cell surface staining
with anti-CD40L antibody. The proportion of CD4+ T cells expressing CD40L (33% and
38% respectively upon stimulation with HBHA or BCG-BMM) was higher in the i.n.
group compared to the s.c. and BCG-vaccinated groups (Table 2). Intracellular staining
for IFN-γ showed that the majority of the CD40L+CD4+ T cells were IFN-γ expressing
cells (74% and 80% respectively upon stimulation with HBHA or BCG-BMM,
respectively) suggesting that after BCG priming, T cells had a tendency to produce a Th1
type of immune response.
Using a cell depletion assay we next examined the contribution of CD4+ T cells on IFN-γ
production. Lung cells were depleted of CD4+ T cells using anti-CD4 antibody tagged
magnetic beads and cultured with BCG-BMM for 72 hours. Undepleted cells as well as
isolated CD4+ T cells were cultured with BCG-BMM. Levels of IFN-γ in the culture
supernatants were detected by ELISA. We observed more than 70% reduction following
depletion of CD4+ T cells from the lung cell suspensions (Fig. 3). Reconstitution of lung
cell suspensions by adding CD4+ T back to the culture resumed IFN-γ levels.
3.3. Priming with BCG improves also protection promoted by rHBHA
It has been described previously that both n- and rHBHA were immunogenic in terms of
cellular and humoral immune responses but that a protective immune response was only
generated by nHBHA (20). The only difference was observed when splenocytes from
16
immunized animals were restimulated with mycobacteria-pulsed BMM. Under these
conditions, significantly more IFN-γ was secreted by the lymphocytes from nHBHAimmunized mice than by those from rHBHA-immunized mice (20). In order to see if
neonatal vaccination with BCG could impact on rHBHA-boosters in the generation of
protective immune responses, we tested i.n.-rHBHA-immunization in the BCG-primed
neonates and compared with the BCG/nHBHA (Fig. 2: Gr. 6, i.n.) and BCG-vaccinated
(Fig. 2: Gr. 2) groups. Results showed that restimulation of lung lymphocytes with
rHBHA induced similar levels of IFN-γ production in both n- or rHBHA vaccinated
groups (Table 3). More interesting, restimulation with BCG-BMM showed increased
levels of IFN-γ production in both groups particularly in the rHBHA-boosted group
(Table 3), which was previously reported to be an indication of protective immune
responses. To test whether this immune response was protective, mice were challenged
with BCG i.n. Results showed that there was a remarkable decrease of the bacterial load
(51%) in the rHBHA-boosted animals, which was higher, even if not statistically
significant than the reduction observed in the BCG-vaccinated (37%) group (Table 3).
Taken together, these observations indicate a possible role of protection by rHBHA when
given to the BCG-primed animals.
17
4. Discussion
In this study, we have aimed to improve the protection provided by neonatal vaccination
with BCG by additional boosting with the mycobacterial antigen HBHA later in life
(infant and adult mice). We chose HBHA because it has been found to be one of the most
potent antigens able to provide a protection against M. tuberculosis challenge in mice
similar to the protection provided by BCG (20). This type of immunization protocol has
been successfully used before but with relatively less protective mycobacterial antigens
(17, 18). Therefore, we expected to be able to induce even higher levels of protection by
using HBHA. Also of interest was to optimise protective immunization in neonates more
prone to Th2 type of responses (30, 31). BCG is an immunomodulator that can induce
better Th1 type of immune responses than the conventional adjuvant mono phosphoryl
lipid A (38). Upon vaccination with BCG, human neonatal immune response to BCG has
been found to be Th1 dominated which is characterized by the higher frequencies IFN-γ+CD4+ T lymphocytes (32).
Induction of protective immune responses in the respiratory compartment is probably the
most effective way to prevent pulmonary TB since the respiratory tract is the natural
route of M. tuberculosis infection (24, 25). Thus we have tested the efficacy of HBHA on
the induction of protection against mycobacterial infection based on the lung immunity.
Based on previous studies, our assumption was that for the induction of strong and
protective immune responses in the respiratory tract, i.n. route of immunization would be
better than s.c. route. We show here that in fact, vaccination of neonatal mice with BCG
drives the immune system to Th1 responses, possibly more protective regarding a
mycobacterial infection. We also show that the BCG/nHBHA combination, significantly
18
improved protection against lung infection compared to vaccination with BCG or
nHBHA administered separately and that, under these conditions, adjuvant was not
required. Moreover, priming with BCG improved protective immune responses even for
rHBHA, which was previously reported to be non-protective (20). In contrast to our
expectations we found that, provided the animals were primed with BCG, the level of
protection was comparable between the i.n. and s.c. routes of immunization.
Increasing attention has recently been paid on BCG prime-boost vaccination strategies
not only towards the development of vaccines against TB (17, 18) but also against other
diseases (39-42). It is known that live BCG, but not killed, is a good immunostimulant
(43). It has been proposed that the viability of BCG may be crucial for the induction of
immune responses because live BCG but not killed could migrate to the draining lymph
nodes, which results in an increase in mononuclear cells in the lymph nodes and
expression of inducible nitric oxide synthase (iNOS) both in the lymph nodes and at the
site of infection (43). Consistent with the notion that BCG can act as an adjuvant, we
observed an immunostimulatory and/or adjuvant role of BCG in the mice vaccinated with
BCG/nHBHA, where addition of CT did not show any effect on the immune response
and protection. Apart from the adjuvant effect, we show here that BCG vaccination
primes the immune system for nHBHA. There are two possible explanations for this:
first, immunization of the BCG-primed mice with nHBHA may amplify HBHA-specific
immunity because BCG itself contains nHBHA protein; second, live-BCG probably acts
as an immune adjuvant, enhancing the immunogenicity of HBHA.
19
Since BCG priming shows immunostimulatory or adjuvant effects regarding nHBHA, we
asked if this immunization protocol could also have an effect on rHBHA, not only
enhancing immune responses but also providing higher levels of protection. We found
that IFN-γ levels upon stimulation of lung cells with BCG-BMM were increased and that
protection was also higher in the BCG/rHBHA compared to the BCG group. The
mechanisms behind the improved immune responses and protection in the rHBHAboosted mice are not clear to us. In this context, it has been shown in an in vitro
experiment that stimulation of lymphocytes from M. tuberculosis-infected healthy
individuals with methylated peptide together with rHBHA increased IFN-γ production
(20). This was assumed to be a carrier protein effect on immunogenicity. In our in vivo
situation, we speculated that rHBHA probably acted as a carrier molecule for the
methylated peptides generated after BCG priming and therefore it could enhance
protective immune responses specific to the methylated epitope.
Generally i.n. immunization with protein antigens induces higher immune responses and
better protection in the lungs than systemic immunization (25). In contrast to this general
experience, we have observed in the present study that administration of BCG improved
immune responses and protection regardless of the route of administration of HBHA.
Recently, a comparative study between i.n. and intramuscular (i.m.) boosting of BCGprimed mice showed that both routes induced similar frequencies of IFN-γ+CD4+ T cells
in the lungs (44). However, the i.n. route of vaccination showed better protection than the
i.m. route. In contrast, we observed that i.n. boosting with HBHA induced higher
frequencies of IFN-γ+CD4+- and CD8+-T cells than the s.c. boosting in response to BCGBMM, as observed by the flow cytometric analysis of intracellular staining for IFN-γ.
20
However, IFN-γ levels measured by ELISA were similar in both groups. Thus, these
results might indicate that the capacity of the cells to produce IFN-γ is more important
than the higher proportion of cells, which probably correlates with the protection.
The importance of CD4+ and CD8+ T cells in protection has been evaluated in several
studies (45-47). In fact, IFN-γ producing CD4+ T cells were found important in the early
phases of the acquired immune response (48) and in absence of CD4+ T cells, M.
tuberculosis infection caused severe pathology and reduced survival time (47). Consistent
with this notion, we show in this study that after priming with BCG, the majority of the
activated CD4+ T cells were driven to the IFN-γ production as demonstrated by the
detection of CD40L expression. We speculate that the primary vaccination with BCG is
critical, and favors the establishment of a strong Th1 type of immune response.
In conclusion, this study has shown the importance of BCG priming in the development
of protective vaccines against mycobacterial infection. Neonatal BCG vaccination is
fairly effective against severe forms of TB but not consistent with the level of protection
against pulmonary TB and unable to provide sufficiently long memory. Therefore, if the
HBHA boosting can improve the BCG derived protection, this would have a significant
impact on the future TB vaccine development.
21
Acknowledgments
This study has been supported by grants from the Swedish Institute and the European
Commission, Sixth Framework Program, LSHP-CT- 2005-018736.
22
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27
Table 1. IFN-γ producing CD4/CD8 lung cells
Vaccination
Prime
IFN-γ producing cells (%)
1
Boost
1
rHBHA
BCG-BMM
CD4
CD8
CD4
CD8
BCG
-
5.12
3.03
6.11
2.24
BCG
nHBHA+CT (i.n.)
7.98(56)
3.58(18)
9.64(58)
3.74(67)
BCG
nHBHA+CT (s.c.)
5.66(11)
3.00(0)
7.39(21)
2.99(25)
1
In vitro restimulation was done for 6 h in presence of GolgiStop followed by surface staining of CD3, CD4 and CD8 markers. After
the restimulation, cells were permeabilized and stained for intracellular IFN-γ expression. Data represent pooled samples from 3
animals per group. Cells producing IFN-γ were expressed as percentage of total lymphocytes.
Values shown in the parentheses are increased cell population by HBHA immunization over BCG control group.
(HBHA-immunized – BCG-immunized)×100/ BCG-immunized
28
Table 2. IFN-γ+CD40L+ cells within CD4+ T cells
Prime
1
Vaccination
Boost
1
rHBHA
BCG-BMM
CD40LCD4
IFN-γCD40LCD4
CD40LCD4
IFN-γCD40LCD4
BCG
-
22
60
31
67
BCG
nHBHA+CT (i.n.)
33
74
38
80
BCG
nHBHA+CT (s.c.)
24
64
31
70
1
In vitro restimulation was done for 6 h in presence CD40L antibody and GolgiStop followed by surface staining of CD3 and
CD4 markers. After the restimulation, cells were permeabilized and stained for intracellular IFN-γ expression. Data represents
pooled samples from 3 animals per group. Cell expresses CD40L was represented as percentage of total CD4+ T lymphocytes
and cell expresses IFN-γ was represented as percentage of total CD40L+CD4+ T lymphocytes.
29
Table 3. Immune response and protection induced by rHBHA immunization in the BCG-primed animals
IFN-γ indexa
Vaccination
Prime
CFU countsb
Boost
rHBHA
BCG-BMM
×104
% of
reduction2
1
BCG
-
0.74±0.47
0.96±0.22
3.08±0.98
37
1
BCG
nHBHA+CT (i.n.)
4.71±1.41**
3.03±0.84 **
1.56±0.49
68 **
BCG
rHBHA+CT (i.n.)
4.33±1.04**
1.79±0.39**
2.42±0.88
51*
a
Priming with BCG and boosting with rHBHA improve protective immunity. One week after the last immunization, cells from the
lungs were isolated and stimulated in vitro with rHBHA or BCG-BMM for 72 h. IFN-γ levels were determined in the culture
supernatants. IFN-γ index is a ratio of HBHA-stimulated IFN-γ levels to Con A-stimulated IFN-γ levels multiplied by a factor 10.
b
Four weeks after the last immunization mice were challenged with 107 CFU of BCG given i.n. Three weeks postchallenge, bacterial
load in the lungs was enumerated.
Data represents mean ±sd.
1
Data were taken from Fig. 2 A and B
2
Percent of reduction of CFU was calculated with respect to the unimmunized group in Fig. 2C.
**p<0.05 and *p=0.18, when compared with BCG group.
30
Figure legends
Figure 1. Schematic representation of vaccination schedule. Neonatal mice were primed
with BCG or nHBHA s.c. and boosted later with n- or rHBHA (A). Adult mice were
primed with BCG and boosted later with nHBHA (B). One week after booster
vaccination, some of mice were sacrificed and lung-derived cells were tested for
immunogenicity. The rest of the mice were challenged with BCG administered i.n. 4week after the last immunization. Challenged mice were killed after 3-week and bacterial
counts were determined in the lungs by colony forming unit (CFU) assay.
Figure 2. Strong immune response and protection by nHBHA-immunization in the BCGprimed animals. IFN-γ levels after in vitro restimulation of lymphocytes with HBHA (A)
and BCG-BMM (B). Mice at 1-week were immunized with BCG s.c. nHBHA boosters
were formulated with or without CT and administered via i.n./s.c route thrice at two
weeks interval after weaning at 3 weeks of age. Lung lymphocytes from immunized or
unimmunized animals were cultured with HBHA or BCG-BMM for 72 h and IFN-γ
levels in the culture supernatants were measured. Data for IFN-γ shows mean stimulation
index with s.d. calculated as a ratio of HBHA or BCG-BMM-stimulated IFN-γ levels to
Con A-stimulated IFN-γ levels multiplied by a factor 10. Cells were pooled from four
animals per group. Data represents mean±sd of triplicate wells. C, protection against i.n.BCG challenge compared with unimmunized and BCG controls. Four weeks after the last
immunization, mice were challenged with BCG i.n. Three weeks postchallenge, bacterial
load in the lungs was determined. Graph shows bacterial counts from individual animal
31
with mean of ten animals per group. Results were analyzed from two independent
experiments. p value(s) was calculated by comparing two groups using t test. *, p<0.05.
Figure 3. CD4+ T cells are the major source of IFN-γ production. Adult mice were
primed with BCG (5×105 CFU) s.c. at dorsal region of the neck and were rested for 3
weeks. Afterward, mice were immunized three times i.n. at 2 weeks interval with
nHBHA (5 µg/animal) formulated with CT (1 µg/animal). Animals primed with only
BCG were considered as a control. One week after the last immunization, lung
lymphocytes from the immunized mice were collected and allowed for magnetic
depletion of >95% CD4+ T cells. Coculture experiment of lymphocytes and BCG-BMM
(10:1) showed removal of the CD4+ T cells are major IFN-γ-producers as detected in
ELISA. Depletion of CD4+ T cells affected IFN-γ production. IFN-γ levels were resumed
in the coculture of the lymphocytes reconstituted with CD4+ T cells and BCG-BMM.
Data represents mean±s.d. of triplicate wells prepared from the pooled samples of 4
animals per group. p value(s) was calculated by comparing two groups using t test. *,
p<0.05.
32
Immunogenicity
Neonates
W0
BCG or
nHBHA
(s.c.)
W3
W7
W5
W8
Challenge
W11
Lung CFU count
W14
(A)
n- or rHBHA (i.n/s.c..)
Immunogenicity
Adults
W0
BCG (s.c.)
W3
W5
W7
W8
(B)
nHBHA (i.n.)
Fig. 1
33
A)
B)
HBHA
7.0
*
6.0
s.c.
*
4.0
i.n.
S.I.×10
5.0
S.I.×10
BCG-BMM
*
5.0
4.0
3.0
3.0
2.0
2.0
1.0
1.0
0.0
0.0
W1
PBS
BCG
nHBHA
BCG
BCG
BCG
PBS
-
nHBHA
nHBHA
nHBHA
nHBHA
CT
-
-
+
-
Gr.
1
2
3
4
W4,6,8
+
5
W1
W4,6,8
PBS
BCG
nHBHA
BCG
PBS
-
nHBHA
nHBHA
+
CT
-
-
+
-
6
Gr.
1
2
3
4
BCG
BCG
nHBHA
nHBHA
+
+
5
Protection
C)
*
70000
60000
50000
s.c.
40000
i.n.
30000
20000
10000
0
W1
W4,6,8
PBS
BCG
nHBHA
BCG
PBS
-
nHBHA
nHBHA nHBHA
CT
-
Gr.
1
-
+
2
3
4
BCG
BCG
nHBHA
+
+
5
6
Fig. 2
34
6
BCG
*
BCG/nHBHA+CT
2500
IF N -γ (p g /m l)
2000
1500
1000
500
0
Undepleted
CD4+T-depleted
re-constituted with CD4+T
Fig. 3
35