1. INTRODUCTION

1.1. Mycobacteria and tuberculosis

Mycobacterium tuberculosis (Mtb ), the agent of human tuberculosis (TB), was

discovered in, 1882 by Robert Koch and for a long time called after his name (the Koch

bacillus). Mtb is an obligate aerobe, non-motile and rod-shaped with 2·A~-tm in length and

0.2-0.5~-tm in width. It is a facultative intracellular pathogen that can survive and multiply

inside macrophages and inside other mammalian cells. Mtb is not classified as either Gram­

positive or Gram-negative because it does not have the chemical characteristics of either,

although the bacteria do contain peptidoglycan (murein) in their cell wall (Prescott et al.,

1996). Phylogenetic studies among mycobacteria by 16S rRNA sequencing (Rogall et

al., 1990) showed that Mtb belongs to a group of 'slow growers', also known as 'Mtb

complex' requiring 3-4 weeks to form colonies, with generation time qftypically ~24 hours

in solid mediua. The Mtb complex include six members: Mtb the causative agent in the vast

majority of human tuberculosis cases; M africanum, an agent of human TB in sub-Saharan

Africa; M microti, the agent of TB in voles; M bovis, which infects a very wide variety of

mammalian species including humans; BCG, an attenuated variant of M bovis; and M

canetti, a smooth variant that is very rarely encountered but causes human disease. The

important features shared by all members of Mtb complex include a cell wall of unique

composition composed by a complex ou~er cell wall consisting of large amount of cell wall

lipid. It consists of several unique components such as lipoarabinomannan (LAM),

lipomannan (LM), pthiocerol dimycocerostate (PDIM), mycocerostate, mycolic acid,

trehalose dimycolate (TDM)and sulpholipids (Beman et al., 1990; Bersa and Chatterjee,

1994). These components are suggested to be responsible for mycobacterial hydrophobicity,

ability to form clumps or cords, ability to survive intracellularly and it is the cell wall that

gives acid-fastness, enabling it to retain basic dyes in the presence of acid alcohol. The

metabolic activity of mycobacteria, including assimilation of nutrients, energy production,

metabolism and biosynthesis of macromolecules are similar to those of other bacteria

(Ratledge, 1982; Wheelar and Ratledge, 1994). Many non pathogenic mycobacteria are

components of the normal flora of humans, found most often in dry and oily locales.

TB is a common and deadly infectious disease caused by the Mtb or M bovis. Over

one-third of the world's population now has the TB bacterium in their bodies and new

Introduction Page 1

infections are occurring at a rate of one per second (WHO, 2006). In 2004, 14.6 million

people had active TB and there were 8.9 million new cases and 1.7 million deaths, mostly in

developing countries (WHO, 2006). A rising number of people in the developed world

contain TB because their immune systems are compromised by immunosuppressive drugs,

substance abuse, or HIV/AIDS. The rise in HIV infection levels and the neglect of TB

control programs have caused a resurgence of TB. Drug-resistant strains of TB have

emerged and are spreading (2006).

In the past, TB was called consumption, because it seemed to consume people from

within, with a bloody cough, fever, pallor, and long relentless wasting. Other names

included phthisis (Greek for consumption) and phthisis pulmonalis; scrdfula, (in adults),

affecting the lymphatic system and resulting in swollen neck glands; tabes mesenterica, TB

of the abdomen and lupus vulgaris, TB of the skin; wasting disease; white plague, because

sufferers appear markedly pale; king's evil, because it was believed that a king's touch

would heal scrofula; and Pott's disease, or Gibbus of the spine and joints. Miliary TB is an

archaic term that is still occasionally used, and is when the infection invades the circulatory

system- resulting in X-ray lesions with the appearance of millet seeds (Tuberculosis 1911).

This form of TB is now more commonly named disseminated TB. In the patients where TB

becomes an active disease, 75% of these cases affect the lungs, where the disease is called

pulmonary TB. Symptoms include a productive, prolonged cough of more than three weeks

duration, chest pain, and coughing up blood. Systemic symptoms include fever, chills, night

sweats, appetite loss, weight loss, paling, and . those affected are often easily fatigued

(WHO, 2006). When the infection spreads out of the lungs, extra pulmonary sites include

the pleura, central nervous system in meningitis, lymphatic system in' scrofula of the neck,

genitourinary system in urinogenital TB, and bones and joints in Pott's disease of the spine.

An especially serious form is disseminated or miliary TB. Extrapulmonary forms are more

common in immunosuppressed persons and in young children. Infectious pulmonary TB

may co-exist with extrapulmonary TB, which is not contagious (CDC 2000).

1.2. The Macrophage

The word 'macrophage' is Greek for "big eater". Macrophages differentiate into their

terminal phenotype from monocytes. Monocytes and macrophages are both phagocytic,

with roles in both antigen-specific and non-specific (innate) defense responses.

Macrophages engulf pathogens, and being classical antigen-presenting cells, process and

Introduction Page2

present pathogen-derived peptides to T lymphocytes. Several biochemical and cell

biological processes relate to the defense responses of macrophages. Some of these include:

(a) Acidification of the pathogen-containing phagosome by inserting proton pumps

into the membrane, and assembling proteins on the surface to facilitate docking

and fusion oflysosomes (phagosome maturation),

(b) Fusion of the mature phagosome with lysosomes to enable enzymatic lysis of the

phagocytosed pathogen (antigen processing),

(c) Loading pathogen-derived peptides on major histocompatibility complex II

molecules for presentation toT lymphocytes (antigen presentation),

(d) Secretion of chemokines and cytokines to attract T lymphocytes to the vicinity,

and influence their differentiation to appropriate cytotoxic/helper phenotype,

(e) Generation of free radicals such as reactive oxygen species (ROS) and reactive

nitrogen intermediates (RNI) to cause oxidative stress to the pathogen,

(f) Execution of an apoptotic programme designed to deny sanctuary to intracellular

pathogens, and package these pathogens in apoptotic bodies for uptake by antigen

presenting cells (APC), etc.

During infection with TB-causing bacteria, all of the above are invoked in

immunocompetent individuals, whereby most people infected with these pathogens manage

to clear the infection without drug therapy. In susceptible individuals, one ·or more of the

above responses is compromised, since the pathogen has evolved several biochemical

mechanisms to subvert the host responses. Such interplay between the host and the

pathogen biochemistry and cellular biology is increasingly being addressed by research into

host-pathogen interactions.

1.3. Immunology of tuberculosis

Mtb is equipped with numerous immune evasion strategies, including modulation of

antigen presentation to avoid elimination by T cells. Mtb infected macrophages appear to be

diminished in their ability to present antigens to CD4+ T cells, which leads to persistent

infection (Hmama et al., 1998). Another mechanism by which antigen presenting cells

(APCs) contribute to defective T cell proliferation and function is by the production of

cytokines, including TGF-B, IL-10 (Rojas et al., 1999) or IL-6 (vanHeyningen et al., 1997).

Since Mtb is an intracellular pathogen, the serum components may not get access

· and may not play any protective role. Although many researchers have dismissed a role for

Introduction Page 3

B cells or antibody in protection against TB (Johnson eta!., 1997), recent studies suggest

that these may contribute to the response to TB (Bosio eta!., 2000).

1.3.1. Cells involved in immune response

Mtb is a classic example of a pathogen for which the protective response relies on

cell mediated immune response (CMI). In the mouse model, within 1 week of infection with

virulent Mtb, the number of activated CD4+ and CD8+ T cells in the lung draining lymph

nodes increases (Feng eta!., 1999). Between 2 and 4 week post-infection, both CD4+ and

CD8+ T cells migrate to the lungs and demonstrate an effector/memory phenotype

(CD44hiCD451°CD62L-); approximately 50 per cent of these cells are CD69+. This indicates

that activated T cells migrate to the site of infection and are interacting with APCs. The

tuberculous granulomas contain both CD4+ and CD8+ T cells (Randhawa et a!., 1990) that

contains the infection within the granuloma and prevent reactivation.

Mtb resides primarily in a vacuole within the macrophage, and thus, maJor

histocompatibility complex (MHC) class II presentation of mycobacterial antigens to

CD4+ T cells is an obvious outcome of infection. These cells are most important in the

. protective response against Mtb. Murine studies with antibody depletion of CD4+ T cells

(Maller et al., 1987), adoptive transfer (Orme eta!., 1984), or the use of gene-disrupted

mice (Caruso eta!., 1999) have shown that the CD4+ T cell subset is required for control

of infection. In humans, the pathogenesis of HIV infection has demonstrated that the loss

of CD4+ T cells greatly increases susceptibility to both acute and reactivation TB (Selwyn

eta!., 1989). The primary effector function ofCD4+ T cells is the production ofiFN-y and

possibly other cytokines, sufficient to activate macrophages. In MHC class II-/- or CD4-/­

mice, levels of IFN-y were severely diminished very early in infection (Muller et a!.,

1987). NOS2 expression by macrophages was also delayed in the CD4+ T cell deficient

mice, but returned to wild type levels in conjunction with IFN-y expression (Caruso eta!.,

1999). In a murine model of chronic persistent Mtb infection, CD4+ T cell depletion

caused rapid re-activation of the infection (Scanga eta!., 2000). IFN-y levels overall were

similar in the lungs of CD4+ T cell depleted and control mice, due to IFN .. y production by

CD8+ T cells. Moreover, there was no apparent change in macrophage NOS2 production

or activity iri the CD4+ T cell-depleted mice. This indicated that there are IFN-y and NOS2

independent, CD4+ T cell dependent mechanisms for control of TB. Apoptosis or lysis of

infected cells by CD4+ T cells may also play a role in controlling infection (Keane et a!.,

1997). Therefore, other functions of CD4+ T cells are likely to be important in the

Introduction Page4

protective response and must be understood as correlates of immunity and as targets for

vaccine design.

CD8+ cells are also capable of secreting cytokines such as IFN-y and IL-4 and thus

may play a role in regulating the balance ofThl and Th2 cells in the lungs of patients with

pulmonary TB. The mechanism by which mycobacterial proteins gain access to the MHC

class 1 molecules is not fully understood. Bacilli in macrophages have been found outside

the phagosome after 4-5 days of infection (McDonough et al., 1993), but presentation of

mycobacterial antigen by infected macrophages to CD8+ T cells can occur as early as 12h

after infection. Reports provide evidence for a mycobacteria-induced pore or break in the

vesicular membrane surrounding the bacilli that might allow mycobacterial antigen to

enter the cytoplasm of the infected cell (Teitelbaum et al., 1999). Yu et al (1995) analyzed

CD4+ and CD8+ populations from patients with rapid, slow, or intermediate regression of

disease while receiving therapy and found that slow regression was associated with an

increase in CD8+ cells in the BAL. Taha et al (1997) found increased CD8+ T cells in the

broncho alveolar lavage (BAL) of patients with active TB, along with striking increases in

the number of BAL cells expressing IFN-y and IL-12 mRNA. These studies point to a

potential role for CD8+ T cells in the immune response to TB. Lysis of infected human

dendritic cells and macrophages by MHC class 1 restricted CD8+ T cells specific for Mtb

antigens reduced intracellular bacterial numbers (Stenger et al., 1997). The killing of

intracellular bacteria was dependent on perforin/granulysin (Stenger et al., 1998). Lysis

through the Fas/Fas L pathway did not reproduce this effect (Stenger et al., 1997). At high·

effector-to-target ratio (50: 1 ), this lysis reduced bacterial numbers (Silva et al., 2000). It

was shown that IFN-y production in the lungs by the. CD8+ T cell subset was increased at

least four-fold in the perforin deficient (P-/-) mice, suggesting that a compensatory effect

protects P-1- mice from acute infection (Matloubian et al., 1999). Studies defining antigens

recognized by CD8+ T cells from infected hosts without active TB provide attractive

vaccine candidates and support the notion that CD8+ T cell responses, as well as CD4+ T

cell responses must be stimulated to provide protective immunity.

y/8 T-cells are large granular lymphocytes that can develop a dendri#c morphology , in lymphoid tissues; some y/8 T cells may be CD8+. In general, y/8 T cells are felt to be

non-MHC restricted and they function largely as cytotoxic T cells. Animal data suggest

that y/8 T cells play a significant role in the host response to TB in mice and in other

species (Izzo et al., 1992), including humans. Mtb reactive y/8 T cells can be found in the

peripheral blood of tuberculin positive healthy subjects and these cells are cytotoxic for

Introduction PageS

monocytes pulsed with mycobacterial antigens and secrete cytokines that may be involved

in granuloma formation (Munk eta!., 1990). Studies (Ueta et al., 1994; Tazi e't al., 1992)

demonstrated that y/8 cells were relatively more common (25 to 30% of the total) in

patients with protective immunity as compared to patients with ineffective immunity.

Studies in childhood TB patients showed that the proportion ofT cells expressing the y/8

T cell receptor was similar in TB patients and controls (Swaminathan et al., 2000). Thus

y/8 cells may indeed play a role in early immune response against TB and is an important

part of the protective immunity in patients with latent infection.(Ladel et al., 1995).

Increased accumulation of neutrophils in the granuloma and increased chemotaxis

has suggested a role for neutrophils (Fleischmann et al., 1986). At the site of

multiplication of bacilli, neutrophils are the first cells to arrive followed by NK cells, y/8

cells and alp- cells. There is evidence to show that granulocyte macrophage-colony

stimulating factor (GM-CSF) enhances phagocytosis of bacteria by neutrophils. Majeed et

al., (1998) have shown that neutrophils can bring about killing of Mtb in the presence of

calcium under in vivo conditions.

Natural killer (NK) cells are also the effector cells of innate immunity. These cells

may directly lyse the pathogens or can lyse infected monocytes. Culture of NK cells with

live Mtb causes expansion of NK cells implicating that they may be important responders

to Mtb infection in vivo (Esin eta!., 1996). During early infection, NK cells are capable of

activating phagocytic cells at the site of infection. A significant reduction in NK activity is

associated with multi drug resistant TB (MDR-TB). NK activity in BAL has revealed that

different types of pulmonary TB are accompanied by varying degrees of depression

(Ratcliffe et al., 1994). IL-2 activated NK cells can bring about mycobactericidal activity

in macrophages infected with M avium complex (MAC) as a non specific response

(Bermudez eta!., 1991). Apoptosis is a likely mechanism of NK cytotoxicity. NK cells

produce IFN-y and can lyse mycobacterium pulsed target cells (Molloy et al., 1993).

Augmentation of NK activity with cytokines implicates them as potential adjuncts to TB

chemotherapy (Nirmala eta!., 2001).

Dendritic cells (DCs) are among the most potent antigen presenting cells (APCs)

which are indispensable for the activation of naive T lymphocytes during primary immune

response (Banchereau et al., 1998). Differentiated from bone marrow leukocyte precursors,

immature DCs are programmed for antigen capture and display very low levels ofMHC and

T cell-costimulatory molecules. After contact with various stimuli, including some

microbial products such as LPS, DCs undergo a process of maturation, during which they

Introduction Page 6

upregulate their MHC (class I and II) and costimulatory molecules (CD80, CD86, CD40,

and CD54) and become very efficient T cell stimulators. Secretion of MTSAs from the

phagosomal complex of infected macrophages into the extracellular matrix is likely to be

followed up by their uptake by DCs and their precursors, which are recruited almost

immediately to the site of the infection. Therefore, the outcome of the interactions of

MTSAs with the DCs may well constitute the driving force for the nature of immune

responses to Mtb that are subsequently generated and can eventually determine the course

of an infection.

1.3.2. Role of cytokines and chemokines

Recognition of Mtb by phagocytic cells leads to cell activation and production of

cytokines, which in itself induces further activation and cytokine production in a complex

process of autoregulation and cross-regulation. This cytokine network plays a crucial role

in the inflammatory response and the outcome of the mycobacterial infections.

Interferon-y is produced by both CD4+ and CD8+ T cells, as well as by NK cells in TB and

is important in macrophage activation and perhaps other functions (Lalvani et al., 1998;

Serbina et al., 1999). IFN-y might augment antigen presentation, leading to recruitment of

CD4+ T lymphocytes and/or cytotoxic T lymphocytes which might participate in

mycobacterial killing. The protective role of IFN-y in tuberculosis is well established,

primarily in the context of antigen-specific T cell immunity (Andersen et al., 1997). IFN-y

is produced by T cells from healthy PPD+ subjects as well as those with active TB.

Although some studies suggest that IFN-y levels are depressed in patients with active TB

(Zhang et al., 1995; Lin et a!., 1996), this cytokine may not be ideal as an immune correlate

of protection. The recent report that Mtb has developed mechanisms to limit the activation

ofmacrophages by IFN-y (Ting et al., 1999) suggests that the amount ofiFN-y produced bY.

T cells may be less predictive of outcome than the ability of the cells to respond to this

cytokine. IFN-y is the major activator of macrophages and it causes mouse, but not human,

macrophages to inhibit the growth ofMtb in vitro (Cooper et al., 1993). IL-4, IL-6 and GM­

CSF could bring about in vitro killing of mycobacteria by macrophages either alone or in

synergy with IFN-y in the murine system (Blanchard et a!., 1991). Although IFN-y

production may vary among subjects, some studies suggest that IFN-y levels are depressed

in pati~nts with active TB (Lin et a!., 1996; Zhang et a!., 1995). Another study

demonstrated that Mtb could prevent macrophages from responding adequately to IFN-y

(Ting et al., 1999). Mycobacterial antigen-specific IFN-y production in vitro can be used as

Introduction Page 7

a surrogate marker of infection with Mtb (van Crevel et al., 1999). Individuals defective in

genes for IFN-y or the IFN-y receptor are susceptible to serious mycobacterial infections; . including Mtb (reviewed in Ottenhoff et al., 1998). IFN-y knockout (GKO) mice are the

most susceptible mouse strain to virulent Mtb (Cooper et al., 1993). Macrophage activation

is defective in GKO mice and NOS2 expression is low (Dalton et al., 1993). These factors

likely contribute to the extreme susceptibility of and unchecked bacterial growth in GKO

mice. However, the mean survival time for Mtb-infected NOS2-/- mice is at least twice that

of GKO mice, suggesting that there is IFN-y-dependent, NOS2-independent mechanisms of

protection against tuberculosis (MacMicking et al., 1997; Flynn eta!., 1993).

Tumor necrosis factor (TNF-a) may have multiple roles in immune and pathologic

responses in TB, and is required for the control of the infection. Mtb induces TNF-a

secretion by macrophages, dendritic cells and T cells (Henderson et al., 1997; Serbina and

Flynn, 1999). In mice deficientin TNF-a or 55kDa TNF receptor, Mtb infection resulted in

rapid death of the mice, with substantially higher bacterial burdens compared to control

mice (Bean et al., 1999). TNF-a in synergy with IFN-y induces NOS2 expression (Liew and

Millott, 1990). TNF-a is important for walling off infection and preventing dissemination.

Convincing data on the importance of this cytokine in granuloma formation in TB and other

mycobacterial diseases have been reported (Flynn et al., 1995; Flesch et al., 1990).TNF-a

affects cell migration and localization within tissues in Mtb infection. During chronic

infection, NOS2 expression in the lungs was reduced following TNF-a neutralization

(Mohan et al., 2001) TNF-a influence expression of adhesion molecules as well as

chemokines and chemokine receptors, and this is certain to affect the formation of

functional granuloma in infected tissues. TNF -a has also been implicated in

immunopathologic response and is often a major factor in host-mediated destruction of hmg

tissue (Moreira et a!., 1997). Increased level of TNF -a was found at the site of lesion

(pleural fluid), as compared to systemic response (blood) showing that the

compartmentalized immune response must be containing the infection (Prabha et al., 2003).

In response to Mtb infection, NOS2 expression in the granulomas of 1NFRp55-/- mice was

delayed (Flynn et al., 1995), although a similar delay was not observed in TNF-a -1- mice

(Bean et al., 1999). The requirement for TNF -a in control of Mtb infection is complex, but

it clearly is an important component for macrophage activation.

Interleukin-1 p is a second proinflammatory cytokine involved in the host response

to Mtb. Like TNF -a, IL-l p is mainly produced by monocytes, macro phages, and dendritic

cells. In TB patients, IL-l p is expressed in excess at the site of disease (Law et al., 1996).

Introduction Page 8

Studies with mice suggest an important role of IL-l~ in TB: IL-l a and -1 ~ double-KO mice

(Yamada et al., 2000) and IL-lR type !-deficient mice (which do not respond to IL-l~)

display an increased mycobacterial outgrowth and also defective granuloma formation after

infection with Mtb (Juffermans et al., 2000).

Interleukin-2 has a pivotal role in generating an immune response by inducing an

expansion of the pool of lymphocytes specific for an antigen. Therefore, IL-2 secretion by '

the protective CD4+ Th1 cells is an important parameter to be measured. Several studies

have demonstrated that IL-2 can influence the course of mycobacterial infections, either

alone or in combination with other cytokines (Blanchard et al., 1989).

IL-4 and Th2 responses in TB are subjects of some controversy. In human studies, a

depressed Th1 response, but not an enhanced Th2 response was observed in PBMC from TB

patients (Lin et a!.; 1996, Robinson et al.; 1994, Ottenhof et a!.; 1998, Zhang et al.; 1995).

Elevated IFN-y expression was detected in granuloma within lymph nodes of patients with

tuberculous lymphadenitis, but little IL-4 mRNA was detected (Lin et al., 1996). These

results indicated that in humans a. strong Th2 response is not associated with TB. In mice,

studies (Cooper et al., 1993) suggest that the absence of a Th1 response to Mtb does not

necessarily promote a Th2 response and an IFN-y deficiency, rather than the presence ofiL-

4 or other Th2 cytokines, prevent control of infection. In a study of cytokine gene

expression in the granuloma of patients with advanced TB by in situ hybridization, IL-4 was

detected in 3 of 5 patients, but never in the absence of IFN-y expression (Fenhalls et al.,

2000). The presence or absence of IL-4 did not correlate with improved clinical outcome or

differences in granuloma stages or pathology. The deleterious effects of IL-4 in intracellular

infections (including TB) have been described to this cytokine's suppression of IFN-y

production (Powrie and Coffman, 1993) and macrophage activation (Appel berg et al.,

1992). In mice infected with Mtb, progressive disease and reactivation of latent infection

are both associated with increased production of IL-4. Similarly, over-expression of IL-4

intensified tissue damage in experimental infection (Lukacs et al., 1997). Conversely,

inhibition of IL-4 production did not seem to promote cellular immunity. IL-4-1:- mice

displayed normal instead of increased susceptibility to mycobacteria in two studies,

suggesting that IL-4 may be a consequence rather than the cause of TB development (Erb et

al., 1998; North et al., 1998). In contrast, a recent study on IL-4 KO mice showed increased

granuloma size and mycobacterial outgrowth after airborne infection (Sugawara et al.,

2000). Compared with control mice, production of proinflammatory cytokines was

increased in these animals and accompanied by excessive tissue damage.

Introduction Page 9

Interleukin-6 has multiple roles in the immune response, including inflammation,

hematopoiesis and differentiation of T cells. Pro- and anti-inflammatory properties

(vanHeyningen et al., 1997) of IL-6 are produced early during mycobacterial infection and

at the site of infection (Law et al., 1996). A potential role for IL-6 in suppression ofT cell

responses was reported (vanHeyningen et al., 1997). IL-6 may be harmful in mycobacterial

infections, as it inhibits the production of TNF -a and IL-l p and promotes in vitro growth of

M avium (Shiratsuchi et al., 1991). Other reports support a protective role for IL-6. IL-6-

deficient mice display increased susceptibility to infection with Mtb (Ladel et a!., 1997), '

which seems related to a deficient production of IFN-y early in the infection, before

adaptive T cell immunity has fully developed (Saunders et al., 2000).

Interleukin-8 is an important chemokine in the mycobacterial host-pathogen

interaction. It recruits neutrophils, T lymphocytes, and basophils in response to a variety of

stimuli. It is released primarily by monocytes/macrophages, but it can also be expressed by

fibroblasts, keratinocytes, and lymphocytes (Munk et al., 1995). IL-8 is the neutrophil

activating factor. Elevated levels of IL-8 in BAL fluid and supernatants from alveolar

macrophages were seen in patients (Law et al., 1996). IL-8 gene expression was also

increased in the macrophages as compared with those in normal control subjects. In a series

of in vitro experiments it was also demonstrated that intact Mtb or LAM, but not deacylated

LAM, could stimulate IL-8 release from macrophages (Zhang et al., 1995). Friedland et al.,

(1996) studied a group of mainly HIV positive patients, and reported that both plasma IL-8

and secretion of IL-8 after ex vivo stimulation of peripheral blood leukocytes with

lipopolysaccharide remained elevated throughout therapy for TB. Other investigators had

previously shown that IL-8 was also present at other sites of disease, such as the pleural

space in patients with TB pleurisy (Ceyhan et al., 1996).

IL-l 0, an anti-inflammatory cytokine is produced by macrophages after

phagocytosis of Mtb (Shaw et al., 2000) and after binding of mycobacterial LAM (Dahl et

al., 1996). T lymphocytes, including Mtb-reactive T cells, are also capable of producing

IL-l 0. In patients with tuberculosis, expression of IL-l 0 mRNA has been demonstrated in

circulating mononuclear cells, at the site of disease in pleural fluid, and in alveolar lavage "".

fluid (Gerosa et al., 1999). IL-l 0 directly inhibits CD4+ T cell responses, as well as by

inhibiting APC functions of cells infected with mycobacteria (Rojas et al., 1999). IL-l 0

antagonizes the proinflammatory cytokine response by down regulation of production of

IFN-y, TNF-a, and IL-12 (Fulton et al., 1998; Hirsch et al., 1999). Indeed, IL-10

transgenic mice with mycobacterial infection develop a larger bacterial burden (Murray et

Introduction Page 10

al., 1997). In line with this, IL-l 0-deficient mice showed a lower bacterial burden early

after infection in one report (Murray and young, 1999), albeit normal resistance in two

other reports (Erb et al., 1998; North, 1998). In human TB, IL-l 0 production was higher in

anergic patients, both before and after successful treatment, suggesting that Mtb-induced

IL-l 0 production suppresses an effective immune response (Boussiotis et al., 2000).

IL-12 is a key player in host defense against Mtb. IL-12 is produced mainly by

phagocytic cells, and phagocytosis of Mtb seems necessary for its production (Fulton et

al., 1996). IL-12 has a crucial role in the induction of IFN-y production (0' Neill and

Greene, 1998). In TB, IL-12 has been detected in lung infiltrates, in pleurisy, in

granulomas, and in lymphadenitis. The expression of IL-12 receptors is also increased at

the site of disease (Zhang et al., 1999). The exogenous administration of IL-12 to BALB/c

mice can improve survival (Flynn et al., 1995). The protective role of IL-12 can be

inferred from the observation that IL-12 KO mice are highly susceptible to mycobacterial

infections (Cooper et al., 1997; Wakeham et al., 1998). Apparently, IL-12 is a regulatory

cytokine which connects the innate and adaptive host response to mycobacteria (Sieling et

al., 1994; Trinchieri et al., 1995) and which exerts its protective effects mainly through the

induction of IFN-y (Cooper et al., 1997). An intriguing study indicated that the

administration of IL-12 DNA could substantially reduce bacterial numbers in mice with

chronic Mtb infection, (Lowrie et al., 1999) suggesting that the induction of this cytokine

is an important factor in the design of a TB vaccine.

TGF -~ also seems to counteract protective immunity in TB. Mycobacterial

products induce production of TGF-~ by monocytes and dendritic cells (Toossi et al.,

1995). TGF-~ is present in the granulomatous lesions of TB patients and is produced by

human monocytes after stimulation with Mtb (Toossi et al., 1995) or lipoarabinomannan

(Dahl et al., 1996). TGF-~ has important anti-inflammatory effects, including deactivation

of macrophage production of ROI and RNI (Ding et al., 1990), inhibition of T cell

proliferation (Rojas et al., 1999), interference with NK and CTL function and

downregulation of IFN-y, TNF-a and IL-l release (Ruscetti et al., 1993). Toossi et al

(1995) have shown that when TGF-~ is added to co-cultures of mononuclear phagocytes

and Mtb, both phagocytosis and growth inhibition were inhibited in a dose dependent

manner. Part of the ability of macro phages to inhibit mycobacterial growth may depend on

the relative influence ofiFN-y and TGF-~ in any given focus of infection.

Chemotactic cytokines (chemokines) are largely responsible for recruitment of

inflammatory cells to the site of infection. About 40 chemokines and 16 chemokine

Introduction Page 11

receptors have now been identified (Zlotnik and Y oshie, 2000). A number of chemokines

have been investigated in TB. First, several reports have addressed the role of IL-8, which

attracts neutrophils, T lymphocytes, and possibly monocytes. Upon phagocytosis of Mtb

or stimulation with LAM, macrophages produce IL-8 (Juffermans et al., 1999; Zhang et

al., 1995). This production is substantially blocked by neutralization of TNF -a and IL-l~'

indicating that IL-8 .Production is largely under the control of these cytokines (Zhang et

al., 1995). A second major chemokine is monocyte chemo-attractant protein 1 (MCP-1),

which is produced by and acts on monocytes and macrophages. Mtb preferentially induces

production ofMCP-1 by monocytes (Kasahara et al., 1994). In murine models, deficiency

of MCP-1 inhibited granuloma formation (Lu et al., 1998). A third chemokine is

RANTES, which is produced by a wide variety of cells and which shows promiscuous

binding to multiple chemokine receptors. In murine models, expression of RANTES was

associated with development of M bovis induced pulmonary granulomas (Chensue et al.,

1999). Apart from IL-8, MCP-1, and RANTES, other chemokines may be involved in cell

trafficking in TB (Ragno et al., 2001). Inhibition of chemokine production may lead to an

insufficient local tissue response.

1.3.3. Th1 and Th2 response

Two broad (possibly overlapping) categories ofT cells have been described: Th1

type and Th2 type, based on the pattern of cytokines they secrete, upon antigen

stimulation. Th1 cells secrete IL-2, IFN-y and TNF-a and Th2 type cells secrete IL-4, IL-5

and IL-l 0. The balance between the two types of response is reflected in the resultant host

resistance against infection. The differentiation of Th1 and Th2 from these precursor cells . \

may be under the control of cytokines such as IL-12.

In mice infected with virulent strain of Mtb, initially Th1 like and later Th2 like response

has been demonstrated (Orme et al., 1993). There are inconsistent reports in literature on

preponderance ofTh1 type ofcytokines, ofTh2 type, increase ofboth, decrease ofTh1, but

not increase of Th2 etc. Moreover, the response seems to vary between peripheral blood

and site of lesion; among the different stages of the disease depending on the severity. It

has been reported that PBMC from TB patients, when stimulated in vitro with PPD,

release lower levels ofiFN-y and IL-2, as compared to tuberculin positive healthy subjects

(Huygen et al., 1988). Other studies have also reported reduced IFN-y (Vilcek et al., 1986)

increased IL-4 secretion (Sanchez et al., 1994) or increased number of IL-4 secreting cells

(Surcel et al., 1994). These studies concluded that patients with TB had a Th2-type

Introduction Page 12

response in their peripheral blood, whereas tuberculin positive patients had a Th1-type

response. Recently, cellular response at the actual sites of disease has been examined.

Zhang eta! (1994) studied cytokine production in pleural fluid and found high levels of

IL-12 after stimulation of pleural fluid cells with Mtb. IL-12 is known to induce a Th1-

type response in undifferentiated CD4+ cells and hence there is a Tht response at the

actual site of disease. Lin eta!., (1996) observed that TB patients showed evidence of high

IFN-y production and no IL-4 secretion by the lymphocytes in the lymph nodes. There

was no enhancement of Th2 responses at the site of disease in human TB. Robinson eta!.,

(1994) found increased levels of IFN-y mRNA in situ in BAL cells from patients with

active pulmonary TB.

In addition, reports suggest that in humans with TB, the strength of the Th1-type

immune response relate directly to the clinical manifestations of the disease. Sodhi et a!.,

(1997) have demonstrated that low levels of circulating IFN-y in peripheral blood were

associated with severe clinical TB. Patients with limited TB have an alveolar

lymphocytosis in infected regions of the lung and these lymphocytes produce high levels

of IFN-y (Nirmala eta!., 2001). In patients with far advanced or cavitary disease, no Tht­

type lymphocytosis is present.

Infected macrophages in the lung, through their production of chemokines, attract

inactivated monocytes, lymphocytes, and neutrophils (van Crevel et a!., 2002), none of

which kill the bacteria very efficiently (Fenton et al., 1996). Then, granulomatous focal

lesions composed of macrophage-derived giant cells and lymphocytes begin to form

(Dannenberg and Rook, 1994). It has been demonstrated that TNF-a (Chensue et a!.,

1994) and IFN-y are involved in granuloma formation (Enelow eta!., 1992). This process

is generally an effective means of containing the spread· of bacteria. As cellular immunity

develops, macrophages loaded with bacilli are killed, and this results in the formation of

the caseous center of the granuloma, surrounded by a cellular zone of fibroblasts,

lymphocytes and blood-derived monocytes (Dannenberg and Rook., 1994). Although Mtb

bacilli are postulated to be unable to multiply within these caseous tissues due to its acidic

pH, the low availability of oxygen, and the presence of toxic fatty acids, some organisms

may remain dormant but alive for decades. The strength of the host cellular immune

response determines whether an infection is arrested here or progress to the next stages.

This enclosed infection is referred to as latent or persistent TB and cali persist throughout

the person's life in an asymptomatic and non-transmissible state. In persons with efficient

cell mediated immunity, the infection may be arrested permanently at this point. Live

Introduction Page 13

bacilli have been isolated from granulomas or tubercles in the lun~s of persons with

clinically inactive TB, indicating that the organism can persist in a granulomatous lesion

for many years (Opie an~ Aronson, 1927).

The lymph node. biopsy specimens showing histological evidence of TB could be

classified into four groups based on the organization of the granuloma, the type and

numbers of participating cells and the nature of necrosis (Ramanathan et al., 1999). These

were (i) hyperplastic (22.4%) - a well-formed epithelioid cell granuloma with very little

necrosis; (ii) reactive (54.3%) - a well-formed granuloma consisting of epithelioid cells,

macrophages, lymphocytes and plasma cells with fine, eosinophilic caseation necrosis;

(iii) hyporeactive (17.7%) - a poorly organized granuloma with macrophages, immature

epithelioid cells, lymphocytes and plasma cells and coarse, predominantly basophilic

caseation necrosis; and (iv) nonreactive (3.6%) unorganized granuloma with macrophages,

lymphocytes, plasma cells and polymorphs with non caseating necrosis. It is likely that the

spectrum of histological responses seen in tuberculous lymphadenitis is the end result of

different pathogenic mechanisms underlying the disease (Ramanathan et al., 1999).

Different animal models have been employed to address the question of how the

complex spatiotemporal processes underlying granuloma formation ensue during primary

infection and prevent mycobacterial spread (Dannenberg et al., 1990; Dannenberg et al.,

2001; Orme and Roberts., 1998; Saunders et al., 1999). Different human models were

recently established taking advantage of human cells and tissue. The common model of a

human tuberculous granuloma describes an area of central necrosis, which provides the

nutritional source for persisting mycobacteria, surrounded by a d.ense leukocyte wall ·

preventing mycobacterial spread. Measurement of the size of granulomas and calculation of

the ratio between the surrounding leukocyte coat and the necrotic core revealed that the

more the granuloma enlarges, the smaller this ratio becomes, suggesting that necrosis

expands at the expense of the surrounding cell layers, rather than because of a general

proportional growth of the granuloma by increased leukocyte recruitment from the

circulation. Segovia-Juarez et al., (2004) developed an agent based in vitro system in a

cellular environment, suggesting that chemokine diffusion and prevention of macrophage

'overcrowding' in the system as well as T cell recruitment are crucial prerequisites for

granuloma formation. Leukocyte infiltration also contributes to massive impairment of the

affected tissue. Classical granuloma structure with a necrotic core and a surrounding cell

layer does not serve as the focus of host-pathogen interactions (Ulrichscet al., 2004).

Introduction Page 14

The measurement of cellular infiltration in subsequent aspirates serves as a model in

patients for the early infiltration to the region in primary infection. Neutrophils were the first

cell type observed in the mouse model (Seiler et a!., 2003), followed by macrophages.

Lymphocytes formed the predominant cell type at later time points (Ulrichs eta!., 2004). In

addition, proliferative activity and cytokine production are mainly observed outside the classical

granuloma structure rather than at the interface between the cellular layer and necrosis (Ulrichs

eta!., 2004; Fenhalls eta!., 2000), supporting the notion that the classical granuloma represents

an abandoned battlefield, surrounded by lymphocyte infiltration, where the direct cross-talk

between (latent or active) Mtb and the host immune response takes place.

Formation of central necrosis within the developing granulomatous tissue,. however,

required strong activation of matrix metalloproteinases. Further characterization of

mycobacterial presence in the affected lung revealed that some granulomas, indeed, harbour

mycobacterial material within their necrotic core, but that many necrotic areas are devoid of

mycobacteria, as measured by immunohistological staining and PCR of microdissected

material. Mycobacterial material was detected in the leukocyte infiltrates, indicating that antigen

presentation takes place within specialized regions at the periphery of the classical granuloma

structure (Ulrichs eta!., 2004). These distribution patterns confirm earlier fmdings (Fenhalls et

al., 2002),. who employed in situ hybridization techniques for the detection of mycobacterial

spread. Immunohistological surface marker staining revealed that the inner cell layer of

tuberculous granulomas does not harbour CD8+ T cells (Ulrichs et al., 2004). This finding

argues against the notion that cytolytic CD8+ T cells play a major role in augmentation of the

necrotic core by killing infected antigen-presenting cells (APCs) in their neighbourhood

(Ulrichs eta!., 2004). Rather, CD8+ T cells as well as CD4+ T cells are found in abundance in

the peripheral leukocyte infiltration, mainly surrounding APC and B cell containing follicular

aggregates with high proliferative activity. This architecture suggests the formation of lymphoid

tissue resembling that of secondary lymphoid organs or lymphoid follicles. Lung tissue is

particularly amenable to infiltration and organization of leukocytes, which ensures that an

efficacious local immune response is orchestrated in these lymph node-like structures

surrounding the site of mycobacterial infection. Fine dissection of the local structures and

immunological functions by a combination of laser-microdissection and analysis of gene

expression profiles in distinct cells of these regions of interest for specific gene expression will

provide insight into the precise organization of the immune response operative in this

battlefield.

Introduction Page 15

The granulomas subsequently heal, leaving small fibrous and calcified lesions.

However, if an infected person cannot control the initial infection in the lung or if a latently

infected person's immune system becomes weakened by immunosuppressive drugs, HIV

infection, malnutrition, ageing, or other factors, the granuloma center can become liquefied

by an unknown process and then serves as a rich medium in which the new revived bacteria

can replicate in an uncontrolled manner. At this point, viable Mtb can escape from the

granuloma and spread within the lungs (active pulmonary TB) and even to other tissues via

lymphatic system and the blood (miliary or extrapulmonary TB). When this happen, the

person becomes infectious and requires antibiotic therapy to survive (Dannenberg and

Rook, 1994).

1.3.4. Pathogenesis of TB

Mtb is the most common cause of mycobacterial disease in humans. TB can be

experimentally modeled in mice, guinea pigs, rabbits and rats depending on the

requirements of study. It is primarily a pulmonary disease and is initiated by the deposition

of Mtb, contained in aerosol droplets, onto lung alveolar surfaces. This disease has many

manifestations, affecting bone, the central nervous system, and other organ systems

(Wiegeshaus et al., 1989; Smith, 2003). Based on Lurie's fundamental studies in rabbits

(Lurie and Dannenberg., 1965), four stages of pulmonary tuberculosis have been

d~stinguished (Dannenberg et al., 1994). The first stage begins with inhalation of tubercle

bacilli. Alveolar macrophages ingest the bacilli and often destroy them. At this stage, the

destruction of mycobacteria depends on the intrinsic microbicidal capacity of host

phagocytes and virulence factors of the ingested mycobacteria. Mtb which escapes the

initial intracellular destruction will multiply, and this will lead to disruption of the

macrophage. When this happens, blood monocytes and other inflammatory cells are

attracted to the lung (second stage). These monocytes will differentiate into macrophages

which again readily ingest but do not destroy the mycobacteria. In this symbiotic stage,

mycobacteria grow logarithmically, and blood-derived macrophages accumulate, but little

tissue damage occurs. Two to three weeks after infection, T cell immunity develops, with

antigen-specific T lymphocytes that arrive, proliferate within the early lesions or tubercles,

and then activate macrophages to kill the intracellular mycobacteria. Subsequent to this

phase the early logarithmic bacillary growth stops (third stage). The free bacteria or their

components are thought to interact with sensitized CD4+ T lymphocytes that are attracted

and then proliferate and release inflammatory cytokines (Karnholz, 1996). Central solid

Introduction Page 16

necrosis in these primary lesions inhibits extracellular growth of mycobacteria. As a result,

infection may become stationary or dormant. In fourth stage, disease may progress, and

hematogenous dissemination may take place after primary infection, as well as months or

years afterwards (post primary TB), under conditions of failing iinmune surveillance.·

Liquefied caseous foci provide excellent conditions for extracellular growth of Mtb. Cavity

formation may lead to rupture of nearby bronchi, allowing the bacilli to spread through the

airways to other parts of the lung and the outside environment. The final outcome of

infection with Mtb depends on the balance between outgrowth, killing of Mtb and the extent

of tissue necrosis, fibrosis, and regeneration.

1.4. Management of TB

Proper management and treatment of TB is necessary. TB can be prevented by

vaccination with considerable success. Advances have also been made in the effective

treatment of TB, in particular with the adoption of directly observed therapy short course

(DOTS), in national TB control programs, but in spite of this the currently available

regimens are suboptimal.

1.4.1. TB Vaccines

First vaccine against TB was developed by Albert Calmette and Camille Guerin in

1921, using a live attenuated strain of M bovis, bacillus Calmette-Guerin (BCG). To date,

some three billion people have been vaccinated with BCG worldwide. There have been

numerous controlled clinical trials of the BCG vaccine, yielding diverse and often

contradictory results due to the fact that, although BCG protects against severe forms of

childhood TB, especially meningeal TB, its protective efficacy progressively wanes during

adolescence and the vaccine does not protect against pulmonary TB in adults. BCG is at

best credited with a 50% overall protective efficacy (Brewer et al., 1995, Fine et al., 1995).

This has prompted the search for new, improved TB vaccines. A heap of promising new

approaches has been developed during the last two decades. Advances in gene and antigen

identification, availability of genome sequences, a greater understanding of immune

mechanisms possibly able to control mycobacterial disease, the development of adjuvants

and delivery systems to stimulate T-cell immunity, and increased funding from the public as

well as the private sectors are some of the reasons for progress in this area (Kaufmann and

McMichael, 2005 and Reed et al., 2003). Dozens of vaccine candidates have been tested in

recent years in animal models, including subunit protein/peptide vaccines in adjuvants,

Introduction Page 17

DNA vaccines, rationally attenuated strains of Mtb, recombinant mycobacteria and live

vectors expressing genes coding for immunodominant mycobacterial antigens· or

mycobacterial lipids (Dietrich eta!., 2003; Doherty et al., 2004; Glyn et al., 2005; Kumar et

al., 2003; McMurray et al., 2003; Nor et al., 2004). By culturing M bovis isolate from a . cow for a period of 13 years and a total of 231 passages, Calmette, a physician, and Guerin,

a veterinarian, created an attenuated variant of M bovis, bacillus Calmette-Guerin (BCG).

BCG was first tested in infants in 1921 as an oral vaccine. New methods of administration

were later introduced, such as intradermal, multiple puncture, and scarification. Since 1974,

BCG vaccination has been included in the WHO Expanded Program on Immunization,

resulting in more than three billion doses injected worldwide (approximately 100 million

immunizations in children each year). As recently shown by sequencing, the original BCG

strain lost the RD1 region ofthe Mtb genome in the course of the selection process (Cole et

al., 1998). Major BCG vaccine strains in use today differ even further from the original

BCG strain and from each other, with "stronger" strains (Pasteur 1173 P2, Danish 1331)

being more reactogenic and, presumably, more immunogenic, than "weaker" strains (Glaxo

1077, Tokyo 172) (Brewer et al., 1995). No other widely used vaccine is as controversial as

BCG. Its effects in large randomized, controlled, and case-control studies have been widely

disparate, from excellent protection against TB to no protection (Fine et al., 1995). Most

studies have demonstrated that BCG vaccines afford a higher degree of protection against

severe forms of TB, such as meningitis and disseminated TB, than against moderate form~\ of the disease. The efficacy of neonatal BCG vaccination also wanes with age, dropping in

one study from 82% in children less than 15 years of age to 67% in the 15-24-year-old

group, and to 20% only in persons over 25. Studies that evaluated meningitis or miliary TB

demonstrated that BCG can. provide good protection against these serious forms of TB in

young children, with reported efficacy ranges from 46-1 00%. In contrast, efficacy against

pulmonary TB, which is more prevalent in adolescents and adults, has ranged from 0-80%.

In addition, BCG vaccination may only provide protection against primary infection and be

of little help in already infected individuals or in cases of reactivation TB (Smith et al.,

2004; Young et al., 1995). Efficacy of BCG vaccination also appears to vary with

geographic latitude - the farther from the equator, the more efficacious the vaccine.

Presumably, exposure to nonpathogenic mycobacteria, which is more intense in warm

climates, induces a degree of protective immunity in exposed populations, interfering with

BCG take and therefore masking potential protection from BCG. BCG growth was inhibited

in mice sensitized with M avium, and protection by BCG against subsequent infection with

Introduction Page 18

Mtb was decreased in these mice as compared with unsensitized control mice (Brandt et al.,

2002). Vaccination with BCG still however remains the standard for TB prevention in most

countries because of its efficacy in preventing life-threatening forms of TB in infants and

young children, and also because it is the only vaccine available, is inexpensive, requires

only one encounter with the baby, and side effects such as BCG adenitis are relatively

minor. Nevertheless, BCG has failed to control the increase of new TB cases worldwide.

There is, therefore, an urgent need to develop better TB vaccines as an alternative or a

complement to BCG (Nor et al., 2004). More than 120 vaccine candidates have now been

tested in the low-dose aerosol mouse and guinea pig models (Izzo et al., 2005), including

DNA, attenuated Mtb, recombinant BCG, and subunit vaccines (Andersen et al., 2001;

Brandt et a/2000).

The first recombinant BCG vaccine reported to induce greater protective immunity

to TB than the standard BCG vaccine in animal models was BCG30, a BCG Tice strain

engineered by Horwitz and coworkers to express the 30 kDa major secretory protein Ag85B

(Horwitz et al., 2000). This vaccine is in Phase I trial in the USA. Another recombinant

BCG that expresses two epitopes from ESAT-6 has more recently been constructed (Nor et

al., 2004). A BCG::RD1 recombinant, in which the RD1 genomic segment of the Mtb

genome has been reintroduced, resulting in the expression of ESAT -6 and Ag85A proteins,

has been developed at the Pasteur Institute, Paris. BCG: :RD 1 shows increased persistence

and improved protection against challenge with virulent Mtb in animal models, as compared

with standard BCG. Another improved BCG, rBCG:ureC-Hly, was engineered at the Max

Planck Institute for Infection Biology in Berlin (Germany) to express listeriolysin 0, which

increases MHC class I presentation (Hess et al., 1998) and its urease gene was deleted in

order to prevent neutralization of the acidic pH in phagosomes. This recombinant BCG was

found to be devoid of pathogenicity for SCID mice and provided greatly improved

protection against aerosol TB in the mouse model. A different live vaccine approach

consists in developing attenuated auxotrophic Mtb mutants (Guleria et al., 1996; Jackson et

a/1999; Smith et al., 2001). These include a PhoP mutant ofMtb, developed at the Pasteur

Institute in Paris, and two double auxotrophic mutants developed at the Albert Einstein

College of Medicine in New-York (Collins et al., 2000; Sambandamurthy et al., 2002).

These vaccines have been shown to be safe in animals but their evaluation in humans still is

met with the problem of safety and stability.

Due to safety concerns, in particular in immuno-compromised persons, as well as to

technical challenges regarding manufacture and reproducibility, live mycobacterial vaccines

Introduction Page 19

are not the product of choice of most vaccine manufacturers. Many new TB vaccines

approaches are therefore focused on recombinant subunit vaccines, DNA vaccines (Kamath

et al., 1999; Lowrie et al., 1997), or attenuated Salmonella vector- (Hess et al., 1998) or

virus vector- based vaccines (Zhu et al., 1997) that express mycobacterial antigens. A

variety of antigens obtained from whole bacteria, or isolated from bacterial short-term

culture filtrates (Andersen et a1., 2001; Roberts et al., 1995) such as Ag85A and. 85B,

MTP64, ESAT-6, hsp60, the R8307 protein, a 36 kDa proline-rich mycobacterial antigen,

or the 19 kDa and 45 kDa proteins, have been found to provide protection levels in mice

similar to that obtained with BCG, especially when combining them with a strong Th1-

inducing adjuvant (Andersen et al., 2001). Several Mtb antigens delivered as DNA vaccines •

were effective in reducing bacterial counts in mice following aerosol challenge (Kamath et

al., 1999). The first of the genuinely new candidates, a recombinant attenuated vaccinia

virus MV A strain construct carrying the Mtb secretory Ag85A, has been developed by

McShane and colleagues at Oxford University (McShane et al., 2005). The vaccine has

completed phase I safety evaluation in humans in the United Kingdom without major

adverse events and is now being evaluated in The Gambia (McShane et al., 2004). Another

live recombinant vaccine based on a nonreplicative adenovirus vector expressing Ag85A is

developed by the Aeras Global TB Vaccine Foundation and Crucell NV. Several non-living

TB vaccine candidates also have entered or will soon be entering human clinical trials,

including two recombinant protein subunit vaccines, one based on an Mtb32/Mtb39 ('Mtb

72F') fusion protein produced by Corixa Inc and adjuvanted by a MPL-based adjuvant

formulation from GSK (Skeiky et al., 2004) and the other based on an ESAT-6/Ag85B

fusion protein, developed by the Statens Serum Institute in Copenhagen (Langermans et al.,

2005). In addition, multi-epitope polypeptides, as well as nonproteinic antigens such as

mycolic acids and carbohydrate moieties, are being developed as candidate subunit

vaccines. The search for a new and improved vaccine against TB is a very active field of

research, which in the last 10 years has benefited tremendously from the progress in

molecular biology, genomics, proteomics and transcriptomics, resulting in the identification

of a large number of antigens with vaccine potential (Andersen et al., 2005).

1.4.2. Treatment of TB

The era of chemotherapy TB began with the discovery of streptomycin in 1943.

Para-aminosalicylic acid was discovered in 1946. Isoniazid (INH) and pyrazinamide (PZA)

were added to the list of medicines active against TB in 1952 and 1954, respectively. PZA

Introduction Page 20

is a bactericidal drug highly active against intracellular bacteria and those within the acidic

microenvironment of caseous material. Rifampin (RIF), first used in 1966, was shown to

have excellent activity against populations of rapidly dividing and inactive bacilli. Existing

and newer medications and the primary activity against Mtb are listed in Table below:

Table. Anti-TB drugs and characteristics (Elizabeth et al., 2008).

Name

First-line drugs lsoniazid

Rifampin PZA EMB

Second-line drugs Ethionamide p-Aminosalicylic acid Capreomycin

Aminoglycosides

Fluoroquinolones. Cycloserine

Level of activity

Bactericidal against actively dividing bacteria Bacteriostatic against nonreplicating bacteria Bactericidal Bactericidallactive at acidic pHI Bacter:iotatic

Bacteriostatic Bacteriostatic Bactericidal (active against nonreplicating bacterial Bactericidal against actively dividing extracellular organisms Bactericidal Bacteriostatic

Approved drugs with anti- TB activity Metronidazole Bactericidal Li nezolid Ba cter icida l Clolazimine Bacteriostatic Ampicillin-sulbactam Bactericidal

Promising drugs in clinical trials PA-824 Bactericidal OPC-6 7683 Bactericidal TMC-207 Bactericidal SQ-109 Bactericidal

Mechanism of action

lnhibits mycolic acid synthesis lnhibit catalase-peroxidase enzyme lnhibits DNA dependent RNA polymerase Unknown lnhibits arabinosyl tansferase enzymes

lnhibits mycolic acid synthesis Interferes with bacterial folic acid synthesis Inhibits protein synthesis

Disrupt bacterial protein synthesis

lnhibit DNA gyrase lnhibits cell wall synthesis

Forms reactive radicals that damage DNA Inhibits ribosomal protein synthesis Binds to mycobacterial DNA 1nhibits cell wall mucopeptide synthe.sis; Inhibits be.ta-lactamases

Inhibits cell wall protein and lipid synthesis 'lnhibits mycotic acid synthesis Inhibits proton pump for ATP synthesis Inhibits tell wall synthesis

It was quickly recognized that single drug therapy of TB rapidly resulted in the emergence

of drug resistance while combination chemotherapy prevented it (Elizabeth et al., 2008).

This paradigm of treatment, long the standard in TB, is similarly used in cancer and HIV

therapy. Multiple randomized controlled trials conducted by the British Medical Research

Council demonstrated the synergistic sterilizing activity of RIF and PZA in a multidrug

regimen.(Mitchison, 2005; Fox et al., 1999). As a result, TB therapy could be shortened I

from 12-18 months to 6-9 months with relapse rates of 5% or less. This is the regimen that

has been adopted worldwide for the treatment of drug-susceptible pulmonary TB. The

short-course six-month regimen for the treatment of TB consists of an initial two month

intensive phase using four drugs: INH (also abbreviated H), RIF (R), PZA (Z), and

ethambutol (EMB, E). Ethambutol can be discontinued if the organism is found to be

pansensitive. This intensive phase is followed by a continuation phase consisting of four

months of INH and RIF. Directly observed therapy is recommended for all patients. Patients

with cavitary pulmonary disease who remain culture positive after two months of therapy

(delayed conversion) are at higher risk of relapse with six months of therapy, and the

Introduction Page 21

guidelines recommend extending the continuation phase of therapy to seven months (total

of nine months of treatment) (Blumberg et al., 2003).

It has been.recognized for a long time that the completion rate for such long courses

of treatment for TB is lower than expected. Patients with TB stop treatment for various

reasons. These factors likely interact in a complex, dynamic way that ultimately influence

treatment outcomes. Patient-centered treatment programs need to be developed and further

research is needed to understand how the patient experiences his/her TB treatment. In 1994,

the WHO put forward the STOP TB initiative as a global platform to combat TB built on·a

strategy of directly observed therapy short course (DOTS). One major element of this

strategy calls for supervised administration of standardized treatment doses with the goal of

increasing completion rate to 85%. Relapse of TB most commonly occurs within the first

two years after treatment. Some patient-related factors associated with relapse include

presence of cavities on chest X-ray, sputum culture positivity after eight weeks of intensive

phase treatment, being underweight > 10% ideal body weight (Benator et al., 2002), and

failure to gain >5% weight during the intensive phase treatment period (Khan et al., 2006).

In addition, HIV co-infected patients were noted to have higher risk of relapse in a cohort

from San Francisco compared with HIV -negative patients (Nahid et al., 2007). Treatment­

related risk factors for relapse also have been reported. Chang et al. (2004) in Hong Kong

found that patients who received daily treatment were less likely to relapse and that

prolongation of treatment protected against relapse. In a systematic review of relapse rate

associated with the six-month treatment regimen, the same authors reported similar

relationship between relapse and dosing schedule: they found an increasing odds ratio for

relapse as the dosin~ frequency decreased from daily to once weekly; for example,

rifapentine-. INH in the continuation phase (Chang et al., 2006). Nahid and colleagues

reported that HIV infected patients treated with a six'-month regimen were more likely to

relapse than those treated for longer (Nahid et al., 2007). They also noted that daily

treatment was associated with lower odds of relapse in the HIV -infected population (Nahid

et al., 2007). The above-mentioned studies support the CDC recommendation that if sputum

culture remains positive at eight weeks and there was cavitary disease on chest-X-ray; the

continuation phase should be extended to nine months total therapy. Other situations where

nine months treatment of pan-susceptible pulmonary TB is recommended are when PZA is

not used during the intensive phase and at the discretion of the treating physician when

other aforementioned risk factors for relapse are present. Development of drug resistance

during recurrence or relapse of TB is also a major concern. When treatment is completed

Introduction Page 22

according to standard guidelines without interruptions, it is most likely that recrudescent

Mtb is still susceptible to first-line medications. However, incomplete, erratic and

inadequate therapy with subsequent failure or recrudescence is the most important risk

factor for development of drug resistance. Worldwide, there is an increasing number of

multidrug- resistant TB (MDR-TB) cases, estimated to be 490, 000 new cases per year

(2008a). The standard definition of MDR-TB is resistance to both INH and RIF.

Extensively drug resistant tuberculosis (XDR-TB), recently redefined as resistance to INH,

RIF any fluoroquinolone, and any second-line injectable drug (i.e. amikacin, kanamycin or

capreomycin) has been reported in 45 countries that performed drug~susceptibility testing

(2006). Reports of high fatality rates of XDR-TB in South Africa (Pillay and Sturm, 2007)

and poorer treatment outcome compared to MDR-TB (Kim eta/., 2007) have brought into

sharp focus the urgent need to develop new drugs for treating TB. TB is the leading cause of

death ofH,IV co-infected patients in the developing world. The WHO estimated 231, 000

patients died of HIV -related TB in 2006 (2008a). The current rifampin-based therapy has

significant drug interactions with anti-retroviral agents, including protease inhibitors and

non-nucleoside reverse transcriptase inhibitors, rendering the treatment of this co-infection

complicated and requiring close patient monitoring (Mcllleron et al., 2007). The need for

new regimens without these potential drug interactions is another major impetus driving

new drug development in the TB arena.

There are several important considerations in the development of new TB drugs.

These drugs should be developed with an aim to shorten the present treatment to four

months or possibly shorter, be effective against susceptible and resistant strains, be

compatible with antiretroviral therapies for those HIV-TB patients currently on such

therapies, and improve treatment of latent infection. The pipeline for new TB drugs looks

more promising now compared to the past 40 years. There are newer congeners in the

rifamycin group that are Food and Drug Administration (FDA) approved, and classes of

drugs approved for other indications that have significant antimycobacterial properties and

are undergoing clinical testing for MDR-TB treatment. Multiple new compounds are also in

various stages of preclinical and clinical development. In the rifamycin class of

medications, rifabutin is approved for treatment of Mycobacterium. avium-intracellulare

complex (MAC) and can be used in HIV-TB co-infected patients to reduce the drug

interactions with antiretroviral agents (as compared with rifampin). Rifapentine is a long

acting rifamycin approved in 1998 for treatment of TB with the attractive feature of once­

weekly dosing. However, its use is limited to the continuation phase of treatment for

Introduction Page 23

noncavitary, HIV negative pulmonary TB when the sputum is smear negative at eight weeks

(Blumberg eta!., 2003). Rifapentine is also under investigation for treatment of latent TB

infection (L TBI). In a mouse model of TB, Rosenthal et al. (2007) demonstrated that a

combination of rifapentine, moxifloxacin and PZA cured mice with TB disease after three

months of treatment. This increased sterilizing activity was attributed to the longer half-life

of r!fapentine. Serum drug levels of rifapentine exceeded the minimum inhibitory

concentration (MIC) for much longer than for rifampin (Rosenthal et al., 2007). Various

drugs that are FDA approved for other indications and have known anti-tuberculous activity

are currently in phase II and III clinical trials. The fluoroquinolone class of antibiotics has

broad-spectrum activity against gram-positive and gram-negative Rathogens as well as

against Mtb. The fluoroquinolones inhibit DNA gyrase causing failure of the bacterial DNA

to uncoil, killing the pathogen. The respiratory quinolones moxifloxacin, gatifloxacin and

levofloxacin have the greatest activity against Mtb. Moxifloxacin was studied by the TBTC

in two randomized trials. It was found that two month culture conversion rate, the primary

endpoint, was no different when moxifloxacin replaced ethambutol or INH in the intensive

phase of therapy as compared with the standard regimen (2008d; Burman et al., 2006). The

Oflotub trial tested the sterilizing activity of gatifloxacin, moxifloxacin and ofloxacin in

smear positive pulmonary TB and found that gatifloxacin and moxifloxacin but not

ofloxacin are associated with shorter time to sputum conversion (Rustomjee et al., 2008).

The ReMox trial is a phase III trial that will evaluate whether using moxifloxacin in the

intensive and continuation phases can shorten treatment to four months (2008c). The

oxazolidinones are a new class of antibiotics that inhibit protein synthesis by blocking

translation at the initiation step (Sood et a!., 2006). Linezolid, the only currently available

agent in this class, was approved by the FDA in 2000 for treatment of resistant gram­

positive infections. It has significant in vitro activity against multiple strains of susceptible

and MDR-TB (Sood et al., 2006; Alcala et al., 2003). Clinical experiences with the use of

linezolid for the treatment of MDR-TB indicate positive microbiological and clinical

responses in a significant proportion of patients, although the numbers reported to date are

small (Park eta/., 2006; von der Lippe et al., 2006; Fortun et al., 2005). Linezolid has been

used for variable periods ranging from six weeks to 18 months, and dose reduction was

employed in some cases in an effort to mitigate the adverse effects (Park et a!., 2006).

However, serious adverse reactions including reversible bone marrow suppression and

peripheral and optic neuropathy were seen in a substantial number of patients. Although

early studies show linezolid to have a clinically important role in MDR and XDR-TB, its

Introduction Page 24

use will have to be weighed against its toxicity profile as well as its high cost. Several

additional compounds with potent activity against Mtb are in preclinical and early clinical

phases of drug development. Among the nitroimidazole compounds, three are currently in

trials for MDR-TB treatment. Metronidazole has antimycobacterial activit~ but only under

anaerobic conditions and is active against dormant organisms (Brooks et al., 1999;

Paramasivan et al., 1998; Desai et al., 1989). It is currently being studied in a phase II trial

for MDR-TB in South Korea (2008b). Nitroimidazopyran PA-824 has bactericidal activity

against actively dividing and nonreplicating populations of Mtb (Tyagi et al., 2005). It

inhibits biosynthesis of cell wall lipid components, and has in vitro activity against sensitive

and drug-resistant strains with a low MIC in the 0.015-0.25 flg/ ml range (Tomioka, 2006).

In a murine. model of TB, Nuermberger et al. (2006) found that when combined with

standard anti-TB drugs, PA-824 was no better than standard therapy, but its combination

with moxifloxacin and ·pzA led to greater sterilizing activity compared to standard therapy

(Nuermberger et al., 2008). A third nitroimidazole compound, OPC-67683, also has potent

activity against Mtb with very low MICs (Matsumoto et al., 2006; Tomioka, 2006). It

inhibits synthesis of mycolic acids, a cell wall component. Bactericidal activity, both in

vitro and in vivo against susceptible and drug-resistant strains, has been reported. Under

development by Otsuka Pharmaceuticals, OPC-67683 is also in phase II trials at multiple

clinical sites worldwide (2008e ). Another particularly promising compound in the

antituberculous drug development pipeline is the diarylquinoline R207910, now designated

TMC207. The mechanism of action of this drug involves inhibition of a subunit of

mycobacterial ATP synthase, thus blocking ATP production (Koul et al., 2007). In vitro

studies have shown TMC207 to be highly active against multiple mycobacterial species

including Mtb, MAC, M kansasii, M fortuitum, and M abscessus (Tomioka, 2006).

Importantly, TMC207 was highly effective in vitro against clinical isolates ofMtb that were

resistant to many of the currently available anti-TB therapies including INH, RIF, SM,

EMB, PZA, and moxifloxacin indicating a lack of cross-resistance which makes this an

especially promising agent for the future treatment of MDR-TB (Tomioka 2006). Early

studies in murine TB treatment have shown that use of TMC207 in combination regimens

that also included PZA (e.g., combined with INH+PZA or with RIF+PZA) led to more rapid

conversion to culture negativity (Ibrahim et al., 2007). Phase II clinical trials of TMC207-

containing regimens in treatment of MDR-TB are underway (2008£). SQ 109 is a diamine

compound selected for further development because of its potent activity against drug

sensitive and drug resistant Mtb in culture studies. Although it is· a derivative of ethambutol,

Introduction Page 25

SQ 109 is considered a new compound in that it has a much lower MIC (0.16- 0.64

mg/liter) and regulates different genes compared with ethambutol (Protopopova et al.,

2005). It has synergistic activity against multiple strains of Mtb when combined with INH

or rifampin (Chen et al., 2006). When combined with rifampin, SQ109 also demonstrated

significant activity against ridampin-resistant Mtb (Chen et al., 2006). These important

characteristics have led to its designation as an orphan drug by the US FDA as well as fast­

track clinical development under the auspices of the TB Alliance. In conclusion, there is

reason for optimism regarding the prospects for new drug therapies for TB in the next

decade.

1.5. Current research problems and objectives

Mycobacteria enter into macrophages through various receptors on the surface of

macrophages. Arabinosylated-lipoarabinomannan (Ara-Lam) preferably binds to CD-14

receptor while mannosylated- Lipoarabinomannan (Man-Lam) binds to mannose receptor of

the macrophages. Man-Lam of pathogenic mycobacteria activates PI-3 kinase and results in

activation of PKCs and MAP kinases (Chen et al., 1999; Roach et al., 2002). There is a

possibility that attachment of macrophage receptors with ligands on the surface of

mycobacteria could result in the phosphorylation/dephosphorylation of PKC isoforms

further resulting in the cellular response to infection. Infected macrophage secretes

cytokines and present pathogen derived antigens in association with MHC molecules which

are recognized by receptors on the T cells resulting in the generation of specific immune

response. Cytokines secretes by T cells further activate macrophages resulting in the

enhanced bactericidal activity of macrophages. Resident macrophages and macrophages

activated by cytokines released in response to infection show different bactericidal activity.

Prtoein kinases C (PKCs) are involved in the regulation of expression and secretion of

cytokines by macrophages and T cells. Alternatively, cytokines can also modulate the

activity of PKCs. The interdependence of cytokines and protein kinases tends to suggest a

cytokine mediated regulation of protein kinases and vice versa and that such mechanisms

play a leading role in clearing intracellular mycobacteria. Considering the role of PKC in

the regulation of immune response evaluation of the entire repertoire of PKCs during

infection of macrophages with pathogenic and non-pathogenic mycobacteria would be of

fundamental importance in understanding the role of PKC in pathogenesis of tuberculosis.

How host PKCs behave during infection? If mycobacteria induce any change in the host

PKCs, whether this alteration is mediated by direct host-bacilli interaction or is mediated by

Introduction Page 26

cytokines released during process of infection? Ser/Thr kinases of Mtb have been shown to

regulate various processes like regulation of cell shape and "morphology, signal

transduction, regulation of stress response and survival of mycobacteria within

macrophages. PknG have been reported to be essential for the survival of mycobacteria

within macrophages. Mammalian PKC-a has the similarity with PknG. Whether there is any

interlink between host PKCs and S/TPKs of mycobacteria during the infection and if any,

how these interactions are linked with killing or survival of pathogen? The localization of

PknG in the macrophage cytosol as well as the homology to eukaryotic protein kinases

rather suggest that PknG phosphorylates a host substrate involved in the modulation of

phagosome-lysosome fusion, although the identity of such a substrate remains elusive. It is,

however, interesting to note that mammalian PKC-a, besides being associated with

phagosomal membranes itself, modulates the association of p57, th~ human homolog of

coronin-1, to phagosomes (Itoh, et al., 2002; Yan Hing et al., 204). PKC-a has important

role in the uptake and killing of intracellular pathogens by macrophages. Considering the

importance of PKC-a in membrane trafficking events and its possible involvement in

phagosome maturation, PknG might modulate signaling pathways involved in

phagolysosome biogenesis by competing with host PKC-a for substrate binding. The

downstream targets of PknG in host cell are currently unknown.

To address the above stated issues present study was conducted with the following

objectives:

1. Study of expression and phosphorylation of different PKC isoforms in macrophages

during infection with pathogenic and non-pathogenic mycobacteria.

2. Study of distribution of Mtb specifics S/TPKs in pathogenic"' and non-pathogenic

mycobacteria.

3. Cloning, over-expression and 'purification of PknG of Rv and study the expression

of this protein in different mycobacterial species by immunoblotting.

4. Study the effect of PknG on expression and phosphorylation of PKC-a of

macrophage during infection.

5. To study the role ofPKC-a in survival or killing of mycobacteria.

Introduction Page 27