Licentiate thesis from the Department of Immunology, Wenner-Gren Institute, Stockholm University, Sweden

Diagnostic biomarkers and improved against mycobacterial

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 , 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 , 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 broncho- alveolar 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 with native (n) HBHA but not

recombinant (r) HBHA conferred protection against M. challenge in mice.

3 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 HBHA- immunization. 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 .

4 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).

5 LIST OF ABBREVIATIONS

AFB Acid fast bacilli APC Antigen presenting cell BAL Broncho-alveolar lavage BALT Bronchus-associated lymphoid tissue BCG Bacillus Calmette-Guérin BCG-BMM BCG infected bone-marrow derived macrophages CFP-10 10-kDa culture filtrate protein CR Complement receptor CT Cholera toxin DC Dendritic cell ESAT-6 6-kDa early secretory antigenic target HIV Human immunodeficiency virus i.n. Intranasal i.v. Intravenous IFN-γ Interferon gamma IL-12 Interleukin 12 iNOS Inducible nitric oxide synthase kDa kiloDalton LALT Larynx-associated lymphoid tissue LAM Lipoarabinomannan MALT Mucosa associated lymphoid tissue MHC Major histocompatibility complex NALT Nose associated lymphoid tissue nHBHA Heparin-binding hemagglutinin native NO Nitric oxide PCR Polymerase chain reaction PknG protein kinase G PPD Purified protein derivative rBCG Recombinant Bacillus Calmette-Guérin rHBHA Heparin-binding hemagglutinin recombinant sTNFR Soluble tumor necrosis factor receptor TB Tuberculosis TCR T cell receptor TLRs Toll-like receptor TNF Tumor necrosis factor XDR Extensively drug resistant

6 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

7 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 , who subsequently received the Nobel Prize in 1905. The M. tuberculosis belongs to the genus

8 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

9 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 Primary complex of M. tuberculosis

Stabilization Localized disease Dissemination of M. (latency) (primary TB) tuberculosis

Stabilization Acute disease (latency) (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

10 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

11 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

12 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 IL-

12 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 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,

13 mycobacterium-specific antibodies have been found to be capable of enhancing cell-

mediated 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).

Fi g-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 T-

lymphocytes 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 cell-

mediated 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 re- infection, 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 over- expressing 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 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 5000-

10,000 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 ” 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). Cross- reactions 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 IFN-

γ 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 BCG-

lysate 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 (BCG-

BMM), 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 TB- vaccine 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 HBHA- specific 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 1-

week 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 for improved postnatal cellular Pregnant Neonates th Mother immunity. Pregnant mice at 2 week of W1 W4 gestation were primed with rHBHA s.c. Following delivery, neonates were rHBHA BCG rHBHA 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-w eek of age. One week after the last immunization, lung cells from the rHBHA rHBHA rHBHA rHBHA-immunized animals were re- stim ulated in vitro with rHBHA to - rHBHA rHBHA * measure recall IFN-γ responses. Data for - - IFN -γ shows mean stimulation index with s.d. calculated as a ratio of HBHA- stimulated IFN-γ levels to Con A- 0 1 2 3 4 5 6 stim ulated IFN-γ levels multiplied by a S.I.×10 factor 10. Data for IFN-γ represents mean±sd of triplicate wells. Cells were pooled from four animals per group. Re sults 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 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 prime-

boost 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 Protection, nHBHA2

Pregnant Mother Neonates CFU in the lungs % of reduction3 mean±sd W1 W4 ×104

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 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 1- week 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 BCG- related 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.

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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

1Department of Immunology, Wenner-Gren Institute, Stockholm University, S-10691,

Stockholm, Sweden

2Lionex 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 with glycerol supplemented with albumin-

dextrose-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-acid-

albumin-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 non- replicating 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 of live or non- replicating 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, IL-

12 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% NaCl-

0.05% 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 non- replicating 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.

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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 non- replicating 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, 38- kDa, 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) sTNFR1 IL-12 2500 3000

2500 2000 ** ** 2000

** 1500 * 1500

(pg/ml) (pg/ml) 1000 1000

500 500

0 0 D3 w1 w3 w5 w9 D3 w1 w3 w5 w9

e) BCG BCG lysate b) sTNFR2 hk-BCG 14000 PBS 120 IFNγ 12000 ** 100 ** 10000

80 * 8000

60 ** ** (pg/ml) 6000 (pg/ml) 40 4000 20 2000

0 0 D3 w1 w3 w5 w9 D3 w1 w3 w5 w9

c) Bacteria load 180 Lung 160 Spleen 140 ** Liver 120

3 100 * * 80 CFU x 10 60 40

20

0 D3 w1 w3 w5 w9 Fig. 1

30

b) a) sTNFR2 sTNFR1 25000 7000

6000 20000

5000 15000

4000 (pg/ml) 10000 (pg/ml) 3000

2000 5000 1000

0 0 D3 w1 w3 w5 w9 D3 w1 w3 w5 w9

BCG BCG lysate hk-BCG PBS

c)

IL-12 6000

5000 4000

3000 (pg/ml) 2000

1000

0 D3 w1 w3 w5 w9

Fig. 2

31

a)

Bacteria load Lung 90 Spleen 80 Liver 70 * 60 3 50 10 40 30 CFU x 20 10 0 D3 w1 w3 w5 w9

SERUM BAL d)

b) 12000 sTNFR1 sTNFR1

10000 1600 1400 8000 * 1200 ** 6000

1000 (pg/ml) 800 4000 (pg/ml) 600 400 2000 200 0 0 D3 w1 w3 w5 w9 D3 w1 w3 w5 w9

c) e)

sTNFR2 sTNFR2 10000 45000

8000 ** 35000 *

6000

(pg/ml) 25000

4000 (pg/ml) 15000 2000 5000 0 0 D3 w1 w3 w5 w9 D3 w1 w3 w5 w9

Fig. 3

32

Bacteria load

30 a) 25 * 20 3

10 15

10 CFU x 5

0 w9 w11 w12 w13 DXM treatment

b) 1200 BAL sTNFR1

1000 *

800

(pg/ml) 600 BCG(-DXM) BCG(+DXM) 400 w9 w11 w12 w13 DXM treatment

c) 8000 sTNFR2 7000

6000 *

5000

4000 (pg/ml) 3000 2000

1000 w9 w11 w12 w13 DXM treatment Fig. 4

33 2 BAL a) 1,8

1,6 ) 1,4 m 1,2 1

405n 0,8 ( 0,6 0,4 OD 0,2 0 Coating antigen: BCG lysate w1 w3 w5 w9 w1 w3 w5 w9

IgG IgA

b) Serum 2.5 BCG lysate 2

hk-BCG m) 1.5

1 0.5 OD (405n 0 w1 w3 w5 w9 w1 w3 w5 w9 IgG IgA

c) 2.5 d) BAL 1.6 IgG IgA 2 1.4 1.2

1.5 1 405nm) 0.8 1 OD ( 0.6

0.5 OD (405nm) 0.4 0.2 0 0 w1 w3 w5 w9 w1 w3 w5 w1 w3 w5 w9 w1 w3 w5 w9 w1 w3 w5 w1 w3 w5 w9

BCG BCG lysate hk-BCG BCG BCG lysate hk-BCG Groups Groups

Ag85c e) 38-kDa 2.5 IgG 19-kDa 2 16-kDa

1.5 1 SERUM 405nm)

OD ( 0.5 0 w1 w3 w5 w9 w1 w3 w5 w1 w3 w5 w9 Fig. 5 BCG BCG lysate hk-BCG Groups

34 Improvement of HBHA vaccination by BCG priming in a neonatal mouse model

Rahman MJ1, Locht C2 and Fernández C1

1Department of Immunology, Wenner-Gren Institute, Stockholm University, S-10691,

Stockholm, Sweden

2INSERM 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 prime-

DNA 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 BCG- mediated 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 n- or 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 p-

nitrophenyl 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+IFN-

γ+ 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 nHBHA- immunized 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 rHBHA-

boosted 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 BCG-

primed 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 BCG-

BMM, 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 References

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27

Table 1. IFN-γ producing CD4/CD8 lung cells

Vaccination IFN-γ producing cells (%)

Prime Boost 1rHBHA 1BCG-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 anim als 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-i mmunized – BCG-immunized)×100/ BCG-immunized

28

+ + + Table 2. IFN-γ CD40L cells within CD4 T cells

Vaccination 1rHBHA 1BCG-BMM Prime Boost 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

1In 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

Vaccination IFN-γ indexa CFU countsb % of reduction2 Prime Boost rHBHA BCG-BMM ×104

1BCG - 0.74±0.47 0.96±0.22 3.08±0.98 37

1BCG 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 7 Four weeks after the last immunization mice were challenged with 10 CFU of BCG given i.n. Three weeks postchallenge, bacterial load in the lungs was enumerated.

Data represents mean ±sd. 1Data 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 . 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. 4- week 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 BCG- primed 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

Lung CFU count Immunogenicity Challenge Neonates W0 W3 W5 W7 W8 W11 W14 (A)

BCG or nHBHA n- or rHBHA (i.n/s.c..) (s.c.)

Immunogenicity

Adults W0 W3 W5 W7 W8 (B)

BCG (s.c.) nHBHA (i.n.)

Fig. 1

33

A) HBHA B) BCG-BMM 7. 0 * 5.0 * s.c. 6. 0 * 4.0 5. 0 i.n. 3.0

4.0 10 .I.×10 I.×

S 3. 0 S. 2.0 2. 0 1.0 1.0

0.0 0.0 W1 PBS BCG nHBHA BCG BCG BCG W1 PBS BCG nHBHA BCG BCG BCG W4,6,8 PBS - nHBHA nHBHA nHBHA nHBHA W4,6,8 PBS - nHBHA nHBHA nHBHA nHBHA CT - - + - + + CT - - + - + + Gr. 1 2 3 4 5 6 Gr. 1 2 3 4 5 6

Protection 70000 * C) 60000 50000 s.c. 40000 i.n. 30000

20000 10000 0 W1 PBS BCG nHBHA BCG BCG BCG

W4,6,8 PBS - nHBHA nHBHA nHBHA nHBHA

CT - - + - + +

Gr. 1 2 3 4 5 6

Fig. 2

34

BCG * BCG/nHBHA+CT

2500

2000

) l m / g 1500 (p

γ - N 1000 IF

500 0

Undepleted CD4+T-depleted re-constituted with CD4+T

Fig. 3

35