Comparison of DNA delivery systems for vaccination against intracellular bacteria [original]

Comparison of DNA delivery systems for

vaccination against intracellular bacteria

vorgelegt von

M. Sc.

Nayoung Kim

aus Seoul, Korea

von der Fakultät III – Prozesswissenschaften

der Technischen Universität Berlin

zur Erlangung des akademischen Grades

Doktor der Naturwissenschaften

Dr. rer. Nat

Genehmigte Dissertation

Promotionsausschuss

Vorsitzender: Prof. Dr. Ulf Stahl

Berichter: Prof. Dr. Roland Lauster

Berichter: Prof. Dr. Stefan H. E. Kaufmann

Tag der wissenschaftliche Aussprache: 2. Sep.2004

Berlin 2004

D 83

Contents

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Introduction................................................................... 1

1.1. DNA vaccine development against tuberculosis............................1

1.1.1 Vaccines....................................................................................1

1.1.2 Tuberculosis as a global health problem.......................................3

1.1.3 Novel TB vaccine development ...................................................5

1.1.4 DNA vaccine development ..........................................................6

1.1.5 DNA vaccine carrier systems..................................................... 11

1.2. Pathology of and immunity to tuberculosis..................................13

1.2.1 Mycobacterium tuberculosis ......................................................13

1.2.2 Pathology of and immunity to tuberculosis in human...................14

1.2.3 Pathology of and immunity to tuberculosis in animal models........18

1.3. Experimental listeriosis .............................................................21

1.3.1 Listeria monocytogenes ............................................................21

1.3.2 Pathology of and immunity to listeriosis......................................23

1.4. Selection of the target genes for TB DNA vaccine.......................27

1.5. Specific aims of the study..........................................................29

Materials and Methods.................................................30

2.1. Materials..................................................................................30

2.1.1 Mice ........................................................................................30

2.1.2 Cell lines..................................................................................30

2.1.3 Bacteria and virus.....................................................................31

2.1.4 Plasmid DNAs..........................................................................31

2.1.5 Peptides ..................................................................................35

2.1.6 DNA carrier systems.................................................................35

2.1.7 Antibodies and tetramers ..........................................................36

2.1.8 Buffers.....................................................................................38

2.2. Methods...................................................................................39

2.2.1 Vaccination ..............................................................................39

2.2.2 Protection assay.......................................................................39

2.2.3 Cytokine ELISpot assay............................................................40

2.2.4 Flow cytometry.........................................................................42

2.2.5 Measurement of CTL activity.....................................................43

2.2.6 ELISA......................................................................................44

Results..........................................................................45

3.1. DNA vaccination in the listeriosis model .....................................45

Contents

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3.1.1 Verification of plasmid DNAs for vaccination...............................45

3.1.2 Protection against L. monocytogenes by DNA vaccination and

comparison of DNA vaccine delivery systems.........................................46

3.1.3 Antigen-specific CD8

T cells induced by DNA vaccination against

L. monocytogenes ................................................................................51

3.1.4 IFN-γ secretion induced by DNA vaccination against L.

monocytogenes ....................................................................................55

3.1.5 Th 1/Th 2 immune response......................................................59

3.1.6 Failure to detect CTL activity .....................................................60

3.2. DNA vaccination against M. tuberculosis....................................62

3.2.1 Protection against M. tuberculosis by naked DNA or by DNA with

PLG 62

3.2.2 Antigen-specific CD8

T cells induced by DNA vaccination against

M. tuberculosis .....................................................................................65

3.2.3 IFN-γ secretion induced by DNA vaccination against M.

tuberculosis..........................................................................................66

Discussion....................................................................71

4.1. Selection of DNA vaccine candidates.........................................72

4.2. Comparison of DNA delivery systems for vaccination..................74

4.3. Immune responses induced by DNA vaccination.........................78

4.4. Conclusion...............................................................................82

Summary.......................................................................86

Zusammenfassung.......................................................87

References....................................................................88

Abbreviations.............................................................110

Acknowledgements....................................................113

10.

Curriculum Vitae.........................................................114

Introduction

1. Introduction

1.1. DNA vaccine development against tuberculosis

1.1.1 Vaccines

Vaccines are amongst the most powerful developments in modern medical

science and a cost-effective way for disease prevention. The incidences of

diseases such as smallpox, diphtheria, measles, mumps, pertussis, rubella,

poliomyelitis, tetanus, and hepatitis B have declined dramatically as

vaccinations have become common. Smallpox was eradicated in 1979, and

efforts are currently under way to eradicate or eliminate three vaccine-

preventable diseases – polio, measles, and maternal and neonatal tetanus

(State of the world’s vaccines and immunization. WHO ISBN92/4/154623/9.

World Health Organization. Geneva. 2003). However, no vaccine is 100%

effective and live attenuated vaccines can be detrimental in

immunocompromised individuals. Many of current common vaccines are live

attenuated, for instance, tuberculosis, typhoid, measles, and mumps

(Zinkernagel, 2003; Welsh et al., 2004). Furthermore, there is a critical need

for vaccines against diseases which cause over 5 millions of deaths

throughout the world every year, for example, malaria, tuberculosis (TB), and

acquired immune deficiency syndrome (AIDS). There is no vaccine against

malaria or AIDS. Vaccines againnst pneumococcal disease, meningococcal

disease, and rotavirus diarrhea are also required urgently in developing

countries (State of the world’s vaccines and immunization. WHO

ISBN92/4/154623/9. World Health Organization. Geneva. 2003). It should be

noted that most unsuccessful vaccines require T cell-mediated immunity,

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which is difficult to achieve with conventional vaccines. Vaccines that do not

work satisfactorily and do not induce long-term protection include vaccination

against TB, leprosy, and most classical parasitic infections, such as malaria,

leishmaniasis and schistosomiasis, but also against some viral infections,

including herpes, papilloma, and human immunodeficiency viruses (HIV). The

efficient control of virtually all these agents requires T cell-mediated effector

mechanisms in addition to protective antibodies (Zinkernagel, 2003).

The concept of vaccination originated in the prevention from infectious

disease by E. Jenner in the late 18

century. In modern medicine, vaccinology

has been enlarging its scope to cover not only prophylactic but also

therapeutic vaccines against cancer, allergy, autoimmune diseases as well as

infectious diseases including bacterial and viral diseases by novel

achievements in science and technology (Moingeon et al., 2003). There are

two main categories of vaccines: whole organism vaccines and subunit

vaccines. Whole organism vaccines include inactivated/killed, live attenuated,

and recombinant vaccines. Genetic engineering technology provides a new

way to produce deletion or other mutant strains and recombinant strains as

vaccine candidates. Subunit vaccines include proteins, peptides, DNAs,

polysccharides, and toxoids, and the development of subunit vaccine are

accelerated by genetic engineering technology, enlarged knowledge of

immunology, genomics, and proteomics. There are several features that

vaccines should accomplish: safety, sustained pathogen-specific protection,

induction of neutralizing antibodies/protective T cell responses, and practical

considerations (Duclos, 2004; Bonhoeffer et al., 2004) (Table 1).

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Table 1 Features of effective vaccines

Safe Vaccine must not itself cause illness or death

Protective Vaccine must protect against illness resulting from

exposure to live pathogen

Sustained protection Protection against illness must last for several years

Induces neutralizing

antibody

Some pathogens (such as poliovirus) infect cells that

cannot be replaced (e.g. neurons). Neutralizing

antibody is essential to prevent infection of such

cells.

Induces protective T

cells

Some pathogens, particularly intracellular, are more

effectively dealt with by cell-mediated responses.

Practical considerations Low cost per dose, biological stability, ease of

administration, few side-effect.

1.1.2 Tuberculosis as a global health problem

Tuberculosis (TB) is a major global health problem with two million deaths and

8.8 million new cases in 2002. The global incidence rate of TB was growing at

approximately 1.1% per year, and the number of cases at 2.4% year (Global

tuberculosis control: WHO report 2004. WHO/HTM/TB/2004.331. World

Health Organization, Geneva, 2004). In many cases, TB is a curable disease

with a complex, and long-term regimen of drug treatment. Drug-resistant

strains of Mycobacterium tuberculosis, which is the major causative agent of

TB, develop as a consequence of inconsistent or partial treatment. Rapid

global dissemination of the W-Beijing family strains, notable multi-drug-

resistant strains, is an emerging public health threat (Bifani et al., 2002). More

than 50 million people are already infected with multi-drug resistant strains. TB

and HIV/AIDS form a lethal combination, each speeding the other pathogen’s

progress. Fifteen millions are already coinfected, and it results in extra 0.5

(adapted from C. Janeway. Immunobiology. 5

ed.)

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million deaths. The emergence of multi-drug-resistant strains of M.

tuberculosis and the susceptibility of patients infected with HIV to TB have

fueled the spread of the disease.

Currently only one vaccine is available. Mycobacterium bovis Bacille

Calmette-Guérin (BCG) is an attenuated strain derived from M. bovis and

developed by Calmette and Guérin in the early 19

century (Kaufmann, 2001).

Although BCG prevents disseminated TB in newborns, it fails to protect

against the most common form of the disease, pulmonary tuberculosis in

adults (Kaufmann, 2001). Protective efficacy is variable (ranging from 1-80%)

against adult pulmonary disease and wanes with time (Colditz et al., 1994).

Furthermore, different BCG strains showed different protection rates and

different levels of immune responses in mice (Lagranderie et al., 1996).

Strikingly, some strains, such as the Prague and Japanese strains, were

unable to protect mice against a secondary mycobacterial challenge

(Lagranderie et al., 1996). Protection from TB is associated with the

maintenance of a strong cell-mediated response to infection involving both

cluster of differentiation (CD) 4

and CD8

T cells and the ability to respond

with T helper type 1 (Th 1) type cytokines, particularly Interferon-γ (IFN-γ)

(Dupuis et al., 2000; Flynn and Chan, 2001). BCG vaccination induces IFN-γ-

secreting T cells, predominately of the CD4

T cell phenotype (Lalvani et al.,

1998; Goonetilleke et al., 2003). Recent studies suggest that BCG delivered

parenterally may fail to induce T cell immune responses in the lung mucosa,

which are considered critical for protection against pulmonary disease

(Gallichan and Rosenthal, 1996; Belyakov et al., 1999). However, the basis of

the variability is still uncertain. Therefore, the development of a novel, more

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effective vaccine is urgently required.

1.1.3 Novel TB vaccine development

Three broad approaches in vaccine development against TB are being

pursued: whole bacterial vaccines, subunit vaccines, and combination

vaccines (Britton and Palendira, 2003; Kaufmann, 2000). The first approach

includes either attenuated strains of M. tuberculosis produced by random

mutagenesis or targeted deletion of putative virulence factors, or by genetic

manipulation of BCG to express new antigens or cytokines. Live bacterial

vaccines have the advantage that many antigens can act together to induce

maximum protection, but in the case of TB pathogens, there are serious safety

concerns. A modified BCG overexpressing the 30 kDa protein Ag85A, a major

secreting protein of M. tuberculosis is in phase I trial since Jan. 2004 (Hoag,

2004; Horwitz and Harth, 2003). This is the first TB vaccine candidate showing

evidence of higher potency than the current BCG vaccine in preclinical trials.

Recombinant BCG into which the region of deletion-1 (RD1) locus was

reintroduced also showed enhanced protection against M. tuberculosis (Pym

et al., 2003). In an effort to improve access to the major histocompatibility

complex (MHC) pathway of antigen processing, recombinant BCG strains

were generated which secrete a hemolytic fusion protein containing

listeriolysin O (LLO) of Listeria monocytogenes (Hess et al., 1998) and proved

enhanced vaccine potency in vivo (Grode et al., manuscript in preparation).

The second approach utilizes non-viable subunit vaccines to deliver

immunodominant mycobacterial antigens. Both protein and DNA vaccines can

induce partial protection against experimental tuberculosis infection in mice

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(Britton and Palendira, 2003). Protein subunit vaccines mostly induce humoral

immunity, which is less significant for protection against intracellular bacteria.

In contrast, DNA vaccines can be expressed, processed in the cell, and

presented by MHC molecules to the cell surface. This mechanism enables to

mimic the antigen presentation process of intracellular bacteria and to induce

cell-mediated immune responses including both CD4

and CD8

T cell

immune responses. The genes encoding Antigen 85A/B, ESAT-6, HSP65, and

many others have been exploited as subunit vaccine candidates (Huygen et

al., 1996; Tascon et al., 1996; Lowrie et al., 1997) but still protection levels

with DNA vaccination against tuberculosis has been generally less effective

than BCG vaccination alone (Britton and Palendira, 2003). DNA vaccine

encoding Hsp 65 also showed therapeutic effect in mice (Lowrie et al., 1999)

but prophylactic vaccination is considered to be the most cost-effective way to

control TB.

The third approach includes tests of immunomodulatory adjuvants and prime-

boost protocols. For example, a subunit vaccine can be used as a boost

vaccine. Several prime-boost strategies have been tested: DNA-protein, DNA-

recombinant virus expressing the same respective antigens, DNA-BCG, and

BCG-DNA (Tanghe et al., 2001; McShane et al., 2001; Feng et al., 2001;

Goonetilleke et al., 2003; Mollenkopf et al., submitted).

1.1.4 DNA vaccine development

During the past decade, DNA vaccination has been increasingly employed in

an attempt to achieve simpler, safer, and more effective vaccination protocols.

DNA vaccination involves inoculation with an expression vector that encodes

Introduction

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an antigenic protein. The encoded antigen is then produced in situ and elicits

an immune response. Since the idea of DNA vaccines was proposed, studies

have shown immunogenecity or protective efficacy of DNA vaccines for a

variety of disease targets, including cancer, allergy, autoimmune diseases,

bacterial diseases, and viral diseases. DNA vaccines against intracellular

organisms that require cell-mediated immunity, such as the agent of TB,

malaria, leishmaniasis, hepatitis, and AIDS, would be highly desirable as well

as those against cancer. Immunization of various species (ranging from mice

to human) with unique plasmid DNA constructs encoding foreign proteins has

resulted in immune responses to antigens derived from a variety of infectious

agents, including influenza (Ulmer et al., 1993; Fynan et al., 1993), HIV (Wang

et al., 1994), rabies (Xiang et al., 1994), hepatitis B and C (Major et al., 1995;

Sallberg et al., 1997), malaria (Wang, 1998), and mycobacteria (Tascon et al.,

1996; Huygen et al., 1996).

Exogenous antigens provided by killed/inactivated pathogens, recombinant

protein, or protein derived from live vaccines are taken up by antigen

presenting cells by phagocytosis or endocytosis and are presented by MHC

class II molecules to stimulate CD4

T cells, which can help to generate

effective antibody responses. In contrast, MHC class I molecules associate

with antigenic peptides synthesized within the cytoplasm of the cells and are

elicited by live or DNA vaccines (Gurunathan et al., 2000). DNA vaccination

favors a Th 1 response. The predominant immunoglobulin (Ig) isotype

detected after DNA vaccination is IgG2a (Roman et al., 1997). It was also

shown that the frequency of cytolytic T lymphocyte (CTL) precursors in mice

that were vaccinated with plasmid DNA encoding a Sendai virus nucleoprotein

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were comparable to those elicited by live Sendai virus infection (Chen et al.,

1998). Until now, Th 1 responses and CTLs induced by DNA vaccination have

been shown in various bacterial and viral systems (Fu et al., 1997; Seaman et

al., 2004; Cho et al., 2001).

Fig. 1 Mode of action of DNA vaccines. Following injection of an antigen-

encoding plasmid, transfected muscle or skin cells act as antigen depots.

Transfer to antigen-presenting cells occurs via cross-presentation. Direct

transfection of antigen-presenting cells can also occur. CpG sequences in the

plasmid DNA stimulate the innate immune system. The outcome is induction

of all arms of the immune response. (Adapted from Stevenson, 2004. DNA

vaccines and adjuvants. Immunol. Rev. 199:5-8)

There are at least 3 mechanisms by which the antigen encoded by plasmid

DNA is processed and presented to elicit an immune response (Fig. 1): direct

transfection of bone-marrow derived APCs (Iwasaki et al., 1997; Doe et al.,

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1996), direct transfection of somatic cells (Agadjanyan et al., 1999), and

cross-priming (Ulmer et al., 1996). Some evidence suggests that bone

marrow-derived APCs, but not somatic cells, directly induce immune

responses after DNA vaccination (Iwasaki et al., 1997; Doe et al., 1996).

However, because somatic cells such as myocytes or keratinocytes constitute

the predominant cell populations transfected after DNA inoculation via muscle

or skin injection, respectively, these cells may serve as a reservoir for antigen.

Thus, somatic cells can be important in the induction of immune responses via

cross-priming and may play a role in augmenting and/or maintaining the

response (Gurunathan et al., 2000).

In 1990, it was shown that direct intramuscular inoculation of plasmid DNA,

so-called ‘naked DNA’ encoding several different reporter genes, could induce

protein expression in muscle cells (Wolff et al., 1990). The first demonstration

of protective efficacy of a DNA vaccine in an animal model was reported in the

influenza model in 1993 (Ulmer et al., 1993) and the induction of an antigen

specific immune response was shown for the first time in humans by a malaria

DNA vaccine in 1998 (Wang et al., 1998).

Ideally, DNA vaccine candidates should be pathogen-specific and

immunogenic and should not induce any detrimental response to the host.

The vectors for DNA vaccines usually contain viral promoters for transcription

in mammalian cells and bacterial unmethylated cytidine-phosphate-guanosine

(CpG) motifs in bacterial plasmid backbone, which activate B cells and

dendritic cells (DCs) through Toll-like receptor 9 (TLR9) in human to produce

Th 1 cytokines and promote priming and differectiation of Th 1 T cells (Sato et

al., 1996; Ahmad-Nejad et al., 2002; Tascon et al., 2000). The TLRs are

Introduction

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pattern recognition receptors like MMR, that enable macrophages and

dendritic cells to recognize bacteria, thus ensuring that an appropriate

immune response is generated to defend against the particular pathogen

causing infection (Medzhitov and Janeway, Jr., 1998).

There have been tremendous efforts to improve the efficacy of DNA vaccines,

for example, developing new viral or non-viral vectors, insertion of gene

regulatory elements in the plasmid backbone, and exchanging codon-usages

to frequently used ones in mammals (Doria-Rose and Haigwood, 2003). Other

approaches are construction of plasmids for co-expression of cytokines or

costimulatory molecules, co-immunization of plasmids containing genes

encoding cytokines or costimulatory molecules, and application of cytokines or

CpG motifs as immunoadjuvants. These molecules can influence not only the

magnitude but also the type of immune response induced by DNA vaccines

(Scheerlinck, 2001). The usage of cytokines, costimulatory molecules, their

genes, and other immunomodulatory agents may increase these safety

concerns and has to be dealt with very carefully since all cytokines exhibit

dose-dependent toxicity.

Several major safety concerns were identified by the Food and Drug

Administration, U. S. A. (Smith and Klinman, 2001): the possibility that DNA

vaccination could stimulate the production of autoantibodies against plasmid

DNA, potentially inducing or accelerating the development of systemic

autoimmune diseases; the possible induction of a local inflammatory response

against organ-specific autoimmunity; the development of tolerance rather than

immunity to the encoded antigen, putting vaccine recipients at increased risk

from infection; polarization of the hosts cytokine response profile owing to

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CpG motifs present in the plasmid backbone; and/or the potential for

integration of the plasmid DNA vaccine into the genome of host cells. So

these need to be controlled and avoided.

1.1.5 DNA vaccine carrier systems

DNA vaccines have enormous advantages: they are economical, relatively

safe, easy to handle, and stable at room temperature. These characteristics

are particularly beneficial to use in less developed countries where the

majority of infectious disease incidences are reported. However, low efficiency

of DNA delivery is one of the disadvantages of naked DNA. Thus effective

DNA delivery systems can enhance cellular uptake of DNA, facilitate

intracellular targeting of DNA to cytoplasm or nucleus, and reduce the amount

of DNA required. The reduction of the amount of DNA also reduces the

possible risk of integration and any potentially harmful side effects.

Several methods have been reported to improve DNA delivery: gene-gun,

liposomes and lipids, proteins, biodegradable polymers, and attenuated

bacteria. Gene-gun is a gas-driven biolistic bombardment device that propels

gold particles coated with plasmid DNAs directly in an intradermal way. Initially,

the DNA delivered by gene-gun was known to induce predominantly B cell

immune responses and Th 2 immune responses (Fynan et al., 1993) but

recently there are a few reports that claimed that gene-gun methods could

also induce Th 1 immune responses and CTLs (Trimble et al., 2003) though

inconsistently (Bartholdy et al., 2003).

Liposomes are bilayered membranes consisting of amphiphathic molecules

such as phospholipids, forming unilayered or multilayered vesicles. Cationic

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liposomes have been used for in vitro transfection for a long time, and showed

improved TB DNA vaccine efficacy by formulation in cationic lipids (D'Souza et

al., 2002). Although many lipid particles and oil-in-water emulsions proved too

toxic for widespread use in humans, a squalene oil-in-water emulsion, MF59,

was developed without the presence of additional immunostimulatory

adjuvants, which proved to be a potent adjuvant with an acceptable safety

profile (De Donato et al., 1999).

Virus-like particles (VLPs) from various viruses, and histone-like protein

(TmHU) from hyperthermostable eubacterium Thermotoga maritima have

shown the potential of effective DNA delivery in vitro and/or in vivo. Envelope

proteins (VP1) from murine polyoma virus can self-assemble into particles.

Virus-like particles have also been used as a gene delivery vehicle for long-

term expression of a reporter gene with small amounts (5 µg) of DNA in mice

(Krauzewicz et al., 2000). In many studies, VLPs have been designed to

express pathogen-specific antigens. For example, a candidate vaccine

against human papilloma virus (HPV) based on VLP composed of the L1

capsid protein is likely to be available in the near future (Koutsky et al., 2002).

Histone-like protein showed a capability as an efficient mediator for

transfection of eukaryotic cells in vitro and in vivo (Esser et al., 2000). In that

report, 5 µg of DNA with TmHU protein successfully expressed a reporter

gene in mice. Recombinant hepatitis B surface antigen (HBsAg) is used as a

hepatitis B vaccine and suggested as a subunit vaccine carrier (Singh and

O'Hagan, 2002).

Microparticles or biodegradable polymers, such as poly(lactide-co-glycolide)s

(PLGs) have been used in humans for many years as suture material and as

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controlled-release drug delivery systems for proteins, peptides,

oligonucleotides, and drugs. Biodegradable particles also appear to have

significant potential as a vehicle for DNA vaccines (Hedley et al., 1998); Jung

et al., 2000; Singh et al., 2000). PLG is biocompatible, biodegradable, and

non-immunogenic, thus PLG particles can be repeatedly administered.

Absorption of these particles into cells depends on the size of particles, and

DNA loading capacity depends on the positive charge and the pH. There are

two kinds of particles: One is a surface-loading type, and the other is an

encapsulating type. The particles can be applied by intramuscular,

subcutaneous, oral, intranasal, or intravaginal route.

Attenuated strains of invasive bacteria Shigella flexneri, Salmonella

typhimurium, and Listeria monocytogenes have been used for the delivery of

plasmid DNA (Sizemore et al., 1997; Darji et al., 1997; Dietrich et al., 1998).

Attenuated bacteria also can deliver DNA into host cells but express

heterologous proteins at higher levels than conventional DNA vaccines.

1.2. Pathology of and immunity to tuberculosis

1.2.1 Mycobacterium tuberculosis

Mycobacteria are rod-shaped, aerobic, non-spore forming, non motile bacteria

and called acid-fast bacilli because they do not stain readily by Gram staining,

but once stained they resist decolorization by acid or alcohol despite being

categorized as gram-positive bacteria. Because mycobacteria grow 20 to 100

times slower than other bacteria, it takes 4-6 weeks to obtain a colony of M.

tuberculosis for drug sensitivity studies. The Mycobacterium genus has a cell

wall of unique composition due to the dominant presence of mycolic acids that

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make up more than 50% of its dry weight. The genome of M. tuberculosis has

been sequenced and shown to be 4.41 Mb in size and to contain about 4000

protein-coding genes of which 52% can be assigned a function (Cole et al.,

1998). Only 376 putative proteins share no homology with known proteins and

presumably are unique to M. tuberculosis (Camus et al., 2002).

1.2.2 Pathology of and immunity to tuberculosis in human

The main route of infection for the tubercle bacillus is the respiratory tract. The

bacteria are inhaled in airborne droplets that proceed distally to the lung to

establish an infection (Kaufmann, 2001). After entering the lung, the first cell

type encountered by the bacteria is the alveolar macrophage, which has the

microbicidal armory to destroy most potential invaders. The immune response

is initiated when M. tuberculosis arrives in the alveolar space, where it

encounters alveolar macrophages. However, the tubercle bacillus has the

extraordinary ability to persist and even to replicate in this extremely hostile

environment, where most other pathogens perish. M. tuberculosis resides in

phagosomes, which are not acidified into lysosomes (Clemens, 1996).

Inhibition of acidification has been associated with urease secreted by

mycobacteria and with uptake of mycobacteria by complement- or mannose-

binding receptors rather than Fc receptors (Schlesinger, 1993). The inhibition

of phagosomal acidification occurs by accumolation of a proton-ATPase

(Schaible et al., 1998). Residing in the early recycling endosome, M.

tuberculosis has ready to access to iron, which is essential for intracellular

survival (Schaible et al., 2002). The pathogenicity of M. tuberculosis has been

attributed to several cell wall components, for example, cord factor, a surface

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glycolipid and lipoarabinomannan (LAM). Interestingly, the macrophage

mannose receptor (MMR) binds the virulent M. tuberculosis strains H37Rv

and Erdman but not the avirulent strain H37Ra, although both strains contain

the same amount of terminal dimannosyl residues (Schlesinger et al., 1996;

Schlesinger et al., 1994). A major heteropolysaccharide, LAM inhibits

macrophage activation by IFN-γ, and induces macrophages to secret tumor

necrosis factor-α (TNF-α) and interleukin-10 (IL-10) (Barnes et al., 1992).

Lectin DC-specific intercellular adhesion molecule-3 grabbing nonintegrin

(DC-SIGN) is also known as a M. tuberculosis receptor on human DCs

(Tailleux et al., 2003). The tubercle bacillus and its cell wall glycolipid

lipoarabinomannan seem to bind to and to induce, via DC-SIGN, an

intracellular signal leading to IL-10 production, which in turn could impair

activation of protective T cell responses directed against M. tuberculosis

(Kaufmann and Schaible, 2003). Ingestion of M. tuberculosis by macrophages

is also believed to depend on the engagement of TLRs, TLR2 and TLR4.

However, the role of TLRs are still controversial since there are contradictory

results from TLR2- or 4-deficient mice (Abel et al., 2002; Shim et al., 2003;

Reiling et al., 2002).

The bacteria enter the parenchyma and can replicate within the alveolar

macrophages or in resident lung macrophages. The signals induced result in

migration of monocyte-derived macrophages and resident DCs to the focal

site of infection in the lungs. Immunohistochemical, electron microscopic, and

flow cytometric analyses showed that M. bovis BCG purified protein derivative

(PPD) beads mobilized CD11c

DCs of comparable maturation. Transfer of

DCs from PPD antigen-challenged lungs conferred a Th 1 anamnestic

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cytokine response in recipients (Chiu et al., 2004). Once there, CD4

and

CD8

T cells are primed against mycobacterial antigens. Primed T cells

expand and migrate back to the lungs and then through the lung tissue to the

focus of infection, presumably in response to signals such as chemokines

produced by or in response to infected cells (Gonzalez-Juarrero and Orme,

2001).

Formation of compact granulomas that contain the pathogen at these sites

begins with the accumulation of macrophages at sites of bacterial implantation

and multiplication (Dannenberg, Jr., 1989). The migration of macrophages and

T cells, as well as B cells, to the site of infection culminates in the formation of

a granuloma, a characteristic feature of tuberculosis. In addition to T cells and

macrophages, the granuloma consists of other host cells including B cells,

dendritic cells, endothelial cells, and fibroblasts (Gonzalez-Juarrero and Orme,

2001). The granuloma can later show central caseous necrosis and give rise

to cavities, although this does not occur in all cases of disease. A key aspect

of granuloma formation is the development of fibrosis within the granuloma

and in surrounding parenchyma, which produces macroscopic nodules

(tubercles). The massive activation of macrophages that occurs within

tubercles often results in the concentrated release of lytic enzymes (Converse

et al., 1996; Chandrasekhar and Mukherjee, 1990). These enzymes destroy

nearby healthy cells, resulting in circular regions of necrotic tissue which

eventually form a lesion with caseous consistency. As these caseous lesions

heal, they become calcified and are readily visible on X-rays, where they are

called Ghon complexes. In adults, the disease advances as a necrotizing

pneumonic process that can involve bronchioles and result in the spread of

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infection to other areas of the lungs (North and Jung, 2004).

Tuberculosis immunity relies mainly on cell-mediated immunity rather than

humoral immunity. The acquired cellular immune response to M. tuberculosis

is complex. CD4

and CD8

T cells, as well as unconventional T cells such as

γδ T cells and CD1-restricted CD4

CD8

or CD4

/CD8

single positive αβ T cell

subsets, and natural killer (NK) cells are involved (Shen et al., 2002; Schaible

et al., 2000; Suzuki et al., 1986) but generally CD4

T cells play a central role

in protection. Interferon-γ is a key cytokine in the immune response against M.

tuberculosis (Flynn et al., 1993). This is demonstrated by the considerably

increased risk of TB patients with reduced cell-mediated immunity, such as

those infected with HIV or individuals undergoing immunosuppressive therapy,

compared with patients with defective humoral immunity, such as those with

multiple myeloma, who show no increased predisposition to TB (Cohen et al.,

1987). The patients who were deficient IL12Rc signaling and IFN-γ production

suffered from severe mycobacterial and Samonella infections (de Jong et al.,

1998).

The macrophage has multiple functions in TB, including antigen processing

and presentation, and effector cell functions. Ingestion of M. tuberculosis by

macrophages triggers, via NF-κB activation, transcription of numerous

macrophage genes including those that code for proinflammatory cytokines

and chemokines. The infected macrophage releases IL-12 and IL-18, which

stimulate T lymphocytes, predominantly CD4

T cells to release IFN-γ (Wang

et al., 1999). However, at least in the mouse model of infection, M.

tuberculosis has the ability to evade the onslaught of innate immunity, as

Introduction

______________________________________________________________

virulent bacilli replicate exponentially within mouse lung during the first few

weeks after infection, yet after the onset of acquired immunity, the growth of

the bacilli plateaus (Hingley-Wilson et al., 2003).

Protective acquired immunity to M. tuberculosis is dominated by CD4

and

CD8

T cells with the Th 1 cytokine profile (Flynn and Chan, 2001). The

importance of the Th 1 cytokines IFN-γ and IL-12 is supported by the high

susceptibility to mycobacterial infection of individuals with defects in IL-12, its

receptor, and the IFN-γ receptor (Doffinger et al., 2002; Fieschi et al., 2003;

Lichtenauer-Kaligis et al., 2003). The primary producers of IFN-γ are CD4

and CD8

T cells and NK cells. It is also noteworthy that humans with TB can

generate a Th 1 response to M. tuberculosis, as evidenced by the presence in

their blood and lungs of CD4

and CD8

T cells capable of responding

specifically to M. tuberculosis antigens by replicating and synthesizing IFN-γ

and other Th 1 cytokines, such as IL-12 and IL-18 in vitro (Arend et al., 2000;

Ulrichs et al., 2000; Lalvani et al., 2001). At least some of the CD8

T cells, γδ

T cells, and CD1 restricted T cells secrete perforin and granulysin in human

which apparently kills mycobacteria within macrophages directly (Stenger et

al., 1997; Ernst et al., 2000). T cells with specificity for mycobacterial

glycolipids presented by CD1 (CD1a, b, and c) molecules seem to have a

unique role in human tuberculosis. CD1 molecules are abundantly expressed

on DCs but not on macrophages. Generally CD1-glycolipid-specific T cells

produce IFN-γ and express cytolytic activity (Schaible et al., 2000).

1.2.3 Pathology of and immunity to tuberculosis in animal models

Animal models are essential for investigating the immune response in vivo

Introduction

______________________________________________________________

where the ability to control infection can be assessed in a physiological setting,

after the selective removal of one or more components of the host response

suspected of being involved. Much of what is known about the immunology of

TB has come from studies of immunity to TB in mice, although the

histopathology of TB in the rabbit and guinea pig is more human-like than in

the mouse during early stages of infection. Additionally, the costs are relatively

low, and there are more experimental tools and strains available including

transgenic mice and gene knock-out mice.

Tuberculosis in mice is also mostly a lung disease, progressive and lethal, in

spite of the generation of Th 1-mediated immunity (North and Jung, 2004).

Mice deficient in CD4 T cells showed impaired ability to control infection and

died of tuberculosis (Caruso et al., 1999). These mice showed deficiency in

early production of IFN-γ in the lung and macrophage activation (Caruso et al.,

1999). The development of and bacterial containment in granulomatous

lesions was markedly impaired in TcR-β-/-, and less severely affected in TcR-

δ-/- mutants. Mycobacteria-induced IFN-γ production by spleen cells in vitro

was almost abolished in TcR-β-/- and virtually unaffected in TcR-δ-/- mice.

(Ladel et al., 1995). A recent comparative study of targeted gene-deleted mice

incapable of making αβ T cells, MHC class I (β2m), MHC class II, or γδ T cells

showed that both CD4

and CD8

αβ T cells contribute to the ability of mice to

inhibit M. tuberculosis growth from about day 20 post infection initiated with

100 colony-forming unit (CFU) of M. tuberculosis via the respiratory route. In

contrast, mice incapable of generating γδ T cells were identical to wild-type

mice in their ability to control infection and survival (Mogues et al., 2001).

Whereas in the absence of MHC class I-dependent immunity, lung infection

Introduction

______________________________________________________________

progressed to a 1 log higher level than in wild-type mice and was controlled at

a stationary level for a long period of time, in the absence of MHC class II

dependent immunity, infection remained progressive and was lethal (Mogues

et al., 2001). However, the data obtained in mice that are genetically deficient

for β2m or transporter associated with antigen processing 1 (TAP1) gene

clearly demonstrate an important role for CD8

T cells in protection (Behar et

al., 1999; Flynn et al., 1992). The increased susceptibility of β2m-KO mice

over MHC class I (K

)-KO mice was due to defective iron metabolism, and

iron overload represented an exacerbating cofactor for TB (Schaible et al.,

2002). CD8

T cells are apparently required to control TB in the latent phase

(van Pinxteren et al., 2000) and require IFN-γ (Tascon et al., 1998). CD8

cells also participate in the memory immune response to M. tuberculosis in

mice (Serbina and Flynn, 2001). CD1d - restricted T cells, responding to

mycobacterial glycolopids such as glucosemonomycolate, LAM, and

isoprenoids, also participate in optimal protection through DCs as antigen

presenting cells expressing CD1 molecules (Schaible et al., 2000).

There are some recent supportive results on macrophages as the effectors of

Th 1 immunity after activation by IFN-γ. Nitric oxide is one of the major

antimicrobial defense molecules of macrophages. It is generated from L-

arginine by action of the inducible isoform of nitric oxide synthase 2 (NOS2).

The other antimicrobial defense mechanism is based on reactive oxygen,

which is generated by the transfer of an electron from NADPH to molecular

oxygen by NADPH-oxydase. The NOS2-deficient mice showed impaired

protection from M. tuberculosis infection but NADPH-oxidase-deficient mice

were only slightly susceptible and only virulent strains of M. tuberculosis could

Introduction

______________________________________________________________

overcome the growth inhibitory action of a Th1-dependent, NOS2-independent

mechanism of defense (Macmicking et al., 1997; Jung et al., 2002; Cooper et

al., 2000). Protective immune response dominantly depends on Th 1

cytokines as revealed in IFN-γ or IL-12 deficient mice (Cooper et al., 1993).

IFN-γ works synergistically with TNF-α in activating macrophages. Gene

knock-out mice deficient in TNFR1 showed exacerbated TB, and granuloma

formation was impaired (Flynn et al., 1995). CD4

T cells also produce

lymphotoxin α, which participates in protection against M. tuberculosis (Roach

et al., 2001). Therefore the whole range of acquired immunity is required for

optimal protection from M. tuberculosis infection.

1.3. Experimental listeriosis

1.3.1 Listeria monocytogenes

Intracellular bacteria do not only include M. tuberculosis but also Listeria

monocytogenes, Salmonella species, Legionella pneumophila, other

Mycobacterium species, and many others (Fig. 2). Many of them are

pathogenic (Schaible et al., 1999). L. monocytogenes is a gram-positive,

motile facultative intracellular bacterium that causes severe food-borne

infections, i. e. listeriosis. Pregnant women, their neonates, and

immunosuppressed individuals are particularly susceptible to severe Listeria

infection. The bacteria tolerate high concentrations of salt, and relatively low

pH, and are able to multiply at refrigeration temperatures.

Introduction

______________________________________________________________

Class I

MIIC

CD8

T cell

CD4

T cell

phagolysosome

late

phagosome

(

pH 4.5)

early phagosome

(pH 6.5)

Class II

lysosome

Mycobacterium blocks

phagosome maturation

Listeria escapes into cytoplasm

Salmonella modifies

in phagosome

listeriolysin, phospholipase C

Fig. 2 Antigen presentation and intracellular bacteria (Adapted from L.

Grode).

Virulence factors of L. monocytogenes are very well known. Upon

phagocytosis by macrophages, L. monocytogenes escapes the vacuole by

secreting listeriolysin O (LLO), an essential virulence factor (Mengaud et al.,

1987; Bielecki et al., 1990). The protein LLO is a sulfhydril-activated, pore-

forming cytolysin, which is active at the low pH existing in the phagosome and

promotes evasion from the phagosome into the cytoplasmic compartment.

Cell culture studies of the effects of hly gene, encoding LLO, showed that LLO

is required for the survival and proliferation of L. monocytogenes within

macrophages and non-professional phagocytes (Portnoy et al., 1988; Kuhn et

al., 1988). The locus, including the hly (lisA) gene is a 9 kb virulence gene

cluster that is involved in functions essential to intracellular survival (Bieleki et

al., 1990). A PEST sequence (P, Pro; E, Glu; S, Ser; T, Thr) close to N-

terminus of LLO is essential for the virulence and intracellular

Introduction

______________________________________________________________

compartmentalization of this pathogen (Decatur and Portnoy, 2000). In the

cytosol, L. monocytogenes expresses ActA, which polymerizes actin, enabling

bacterial mobility and cell-to-cell spread (Kocks et al., 1992) While both ActA

and LLO-deficient bacteria are avirulent upon inoculation of mice, ActA-

deficient bacteria induce long-term, CD8 T cell-mediated protective immunity

while LLO-deficient bacteria do not (Goossens and Milon, 1992; Bouwer et al.,

1994). Two different phospholipase C molecules (PlcA, and PlcB) contribute to

the escape of L. monocytogenes into cytoplasm and to cell-to-cell spread

(Marquis et al., 1995). Internalins (InlA, and InlB) and a secreted 60 kDa

protein (p60) encoded by iap gene are involved in invasion of non-phagocytic

host cells (Dramsi et al., 1995; Gaillard et al., 1994; Kuhn and Goebel, 1989).

The internalin locus encodes the first invasin described in a gram-positive

bacterium, implicated in internalization by cells that are not usually phagocytic,

such as epithelial and endothelial cells and hepatocytes (Gaillard et al., 1996).

Deletion of the gene encoding p60 in L. monocytogenes led to abnormal cell

division and loss of actin-based mobility (Pilgrim et al., 2003).

1.3.2 Pathology of and immunity to listeriosis

Perinatal listeriosis mainly results from invasion of the fetus via the placenta

and develops as chorioamnionitis. Its consequence is abortion, usually from 5

months of gestation onwards, or the birth of a baby or stillborn fetus with

generalized infection, a clinical syndrome known as granulomatosis

infantiseptica and characterized by the presence of pyogranulomatous

microabscesses disseminated over the body and a high mortality (Klatt et al.,

1986). L. monocytogenes is one of the three principal causes of bacterial

Introduction

______________________________________________________________

meningitis in neonates.

The gastrointestinal tract is thought to be the primary site of entry of L.

monocytogenes into the adult host. In the initial stages, the bacteria were

detected mostly in the absorptive epithelial cells of the apical area of the villi,

whereas in later phases most were inside macrophages of the stroma of the

villi (Racz et al., 1972). The bacteria that cross the intestinal barrier are carried

by the lymph or blood to the mesenteric lymph nodes, the spleen, and the liver.

Experimental infections of mice via the intravenous route have shown that L.

monocytogenes bacteria are rapidly cleared from the bloodstream by

neutrophils and resident macrophages in the spleen and liver but not all

bacteria are destroyed by tissue macrophages, and the surviving bacteria

start to grow for 2 to 5 days in mouse organs (Conlan and North, 1991). The

principal sites of bacterial multiplication in the liver are the hepatocytes. During

the early steps of liver colonization, polymorphonuclear neutrophils are

recruited at the sites of infection, forming discrete microabscesses. Two to

four days after infection, neutrophils are gradually replaced by blood-derived

mononuclear cells together with lymphocytes to form the characteristic

granulomas. Between days 5 and 7 post infection, L. monocytogenes bacteria

start to disappear from mouse organs until their sterile clearance as a result of

IFN-γ-mediated macrophage activation and the induction of an acquired

immune response primarily mediated by CD8

lymphocytes, which together

destroy infected cells (Harty et al., 1992; (Kaufmann, 1993). These CD8 T

cells are focused on listerial epitopes present in secreted virulence-associated

proteins, such as lysteriolysin O (LLO), metalloprotease, and 60 kb protein,

p60 (Vazquez-Boland et al., 2001).

Introduction

______________________________________________________________

Acquired immunity against L. monocytogenes is entirely cell-mediated and

largely dependent on CD8

T cells, but antibodies play little or no role in

immunity. Protection against L. monocytogenes also involves a vigorous Th 1-

biased CD4

T cell response and innate immunity such as the activation of

IFN-γ-producing NK cells in response to IL-12 and TNF-α secretion by

infected macrophages.

Innate immunity to L. monocytogenes plays a crucial role in controlling the

initial bacterial burden, allowing time for acquired immune responses to

develop and confer sterilizing immunity (Unanue, 1997). Several lines of

investigation have implicated monocyte recruitment in early defense against

infection. Antibody-mediated blockade of CD11b or deficiency of the CCR2

chemokine receptor markedly enhances susceptibility to L. monocytogenes

infection by preventing monocyte recruitment to infected tissues (Rosen et al.,

1989; Kurihara et al., 1997). The proinflammatory response induced by L.

monocytogenes in the liver may be initiated by the interaction of the bacteria

with receptors, such as TLRs. Human TLR2 promotes monocyte activation by

L. monocytogenes (Flo et al., 2000). However, MyD88-deficient mice, which

are largely defective in TLR signaling, are fully proficient to mount a protective

acquired immune response to L. monocytogenes despite their increased

susceptibility to primary infection (Seki et al., 2002; Edelson and Unanue,

2002; Way et al., 2003). TLR2

-/-

mice did not show increased susceptibility to

L. monocytogenes, either (Edelson and Unanue, 2002).

Invasion of host cell cytosol by LLO and phosphilipases provides an essential

stimulus that promotes the development of protective adaptive immunity

Introduction

______________________________________________________________

against L. monocytogenes (Serbina et al., 2003). T cell-mediated immune

responses become operative 4-5 days following bacterial inoculation and are

essential for complete clearance of L. monocytogenes from infected mice

(Busch and Pamer, 1999; Bhardwaj et al., 1998). Experiments using MHC

class I (β2m)

and MHC class II

deficient mice demonstrated that CD8

T cells

are the principle T cell effectors for host defense to L. monocytogenes (Ladel

et al., 1994). Mice lacking IFN-γ, TNF, or their specific receptors have

inadequate innate immune responses and are highly susceptible to infection

with virulent L. monocytogenes (Huang et al., 1993; Pfeffer et al., 1993; Rothe

et al., 1993). Adoptive transfer experiments using CD8

T cells derived from

IFN-γ deficient mice, however, demonstrated that IFN-γ production by CD8

cells is not the sole effector mechanism (Harty et al., 1992; Harty and Bevan,

1995). It is noteworthy that IFN-γ is also produced by NK cells, especially in

the early phase of infection (Nishibori et al., 1996). There are 2 major cytolytic

mechanisms of CD8

T cells; perforin-dependent and Fas/FasL-dependent.

Both perforin deficient mice and Fas-decifient mice showed higher

susceptibility to L. monocytogenes but perforin-mediated mechanisms are

more significant early during primary infection, whereas Fas/FasL-mediated

mechanisms appear more significant late during primary infection (Kagi et al.,

1994; Jensen et al., 1998).

Experimental murine listeriosis has been studied for the past 4 decades to

examine basic aspects of innate and acquired cellular immunity to intracellular

bacteria. Critical features of the murine model are that it yields rapid and

quantitative results, either by enumeration of colony forming units (CFU) in the

Introduction

______________________________________________________________

liver and spleen or by determination of a lethal infection dose (Portnoy et al.,

2002). Median survival times of mice are about 250 days for resistant mice

(C57BL/6 and BALB/c) and about 100 days for susceptible mice (DBA/2, C3H,

CBA, and 129Sv) after infection of 100 CFU of M. tuberculosis by aerosol, but

survival results can be assessed with a lethal dose of L. monocytogenes in

less than 10 days. Well-characterized antigens and epitopes also provide

defined scopes to investigate antigen-specific immune responses to

intracellular bacteria. For instance, large numbers of CTL responding to L.

monocytogenes infection in BALB/c mice are specific for immunodominant

epitope LLO

91-99

presented by the H2-K

MHC class I molecule (Pamer et al.,

1991). Two other epitopes, mpl

84-92

and p60

449-457

, elicit subdominant T cell

responses, while another epitope, p60

217-225

, elicits an intermediate response

(Pamer, 1994; Busch et al., 1997). Less information is available concerning

MHC class II-restricted epitopes. Only the H2-E

-restricted peptide LLO

215-234

and the H2-A

-restricted peptide p60

301-312

have been elucidated in some

detail (Geginat et al., 1998).

1.4. Selection of the target genes for TB DNA vaccine

The state-of-the-art of genomics and proteomics has brought changes in

vaccinology. Particularly, it has greatly benefited the rational selection of

effective DNA vaccine candidates. The current vaccine against TB, M. bovis

BCG, is closely related to M. tuberculosis (>90% DNA homology) but a large

number of deletion mutations in many open reading frames (ORFs) has been

discovered, and 16 regions of deletion (RDs) encoding 129 ORFs were

reported so far, for example, a gene encoding the early secretory antigenic

Introduction

______________________________________________________________

target of 6 kDa (ESAT-6) (Behr et al., 1999). Out of them, 39 ORFs are

missing in all BCG strains. There are 376 putative, unique proteins in M.

tuberculosis that share no homology with known proteins (Camus et al., 2002).

By comparative proteome analysis based on two-dimensional electrophoresis

of M. tuberculosis H37Rv and M. bovis BCG, 39 M. tuberculosis-specific

secreting proteins were identified (Mattow et al., 2001). Out of these genes,

effective DNA vaccine candidates can be selected.

The genes encoding Antigen 85A/B, ESAT-6, HSP65, and many others have

been exploited as subunit vaccine candidates (Huygen et al., 1996; Lowrie et

al., 1997; Tascon et al., 1996) but still protection levels with DNA vaccination

against challenge with M. tuberculosis has been generally less effective than

BCG vaccination alone (Britton and Palendira, 2003). Ergo, heterologous

prime-boost strategies would be worth adopting.

Three novel DNA vaccine candidates were selected by Mollenkopf et al.

(Mollenkopf et al., submitted), based on 2D-electrophoresis analysis to select

M. tuberculosis-specific secreting protein (Mattow et al., 2001). The proteoms

of M. tuberculosis H37Rv was compared with those of M. bovis BCG Chicago.

These are Rv3407, Rv2520c, and Rv1511. Rv3407 is a 300 bp non-essential

gene (Sassetti et al., 2003), and encodes a 99 a. a. conserved hypothetical

protein of unknown function. Rv2520c is a 228 bp non-essential gene

(Sassetti et al., 2003), and encodes a 72 a. a. possible conserved membrane

protein of unknown function. Rv1511 or gmdA is a 1023 bp non-essential

gene (Lamichhane et al., 2003), and encodes a 340 a. a. GDP-mannose 4, 6

dehydratase, which is probably involved in nucleotide-sugar metabolism.

Introduction

______________________________________________________________

1.5. Specific aims of the study

The goal of this study was to compare DNA delivery systems for vaccination

against intracellular bacteria. Specifically, this study was performed to select

the most potent DNA vaccine candidates against L. monocytogenes as an

experimental model system and against M. tuberculosis, to detect antigen-

specific immune response thereby, to compare the different DNA vaccine

carrier systems in listeriosis model, to find the most effective one, and to apply

the results to TB.

To select the most potent DNA vaccine candidate, plasmid DNAs encoding

wild-type LLO, p60, or mutant LLO against L. monocytogenes and plasmid

DNA encoding Rv1511, Rv2520, and Rv3407 against M. tuberculosis were

tested by protection assays. To compare the efficiency of DNA vaccine

delivery, PLG stabilized with PVA, PLG stabilized with CTAB, a novel

encapsulating particle, VLP, and TmHU were tested because these DNA

carriers showed improved DNA transfer of reporter genes in vitro and/or in

vivo, and were regarded relatively safe compared with lipid DNA carriers. The

antigen specific immune responses were determined by flow cytometry with

MHC class I/peptide tetramers, IFN-γ intracellular flow cytometry, IFN-γ

ELISpot, IgG1/IgG2a ELISA, and CTL assays.

Materials and Methods

_______________________________________________________________

2. Materials and Methods

2.1. Materials

2.1.1 Mice

Female BALB/c mice (6-8 week-old) were purchased from the Federal

Institute for Risk Assessment, Berlin, Germany and maintained under specific-

pathogen-free conditions in the animal facilities at the Federal Institute for

Risk Assessment, Berlin, Germany, or in the animal facilities of the Max-

Planck-Institute for Infection Biology, Berlin, Germany. All animal experiments

were performed in accordance with German and institutional animal care

guidelines.

2.1.2 Cell lines

Mastocytoma cell line, P815 was obtained from the American Type Culture

Collection (ATCC, Manassas, VA, U.S.A.) and cultured in RPMI 1640

(Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (FCS)

(Biochrom, Berlin, Germany), 100 U/ml penicillin (Biochrom, Berlin, Germany),

100 U/ml streptomycin (Biochrom, Berlin, Germany), 4 mM L-Glutamine

(Gibco, Karlsruhe, Germany), and 5 µM 2-Mercaptoethanol (ME) (Gibco,

Karlsruhe, Germany), named complete RPMI 1640, in a fully humidified

incubator in 5% CO

at 37°C. Macrophage/monocyte cell line, J774A.1 was

obtained from ATCC and cultured in Dulbeco’s modified Eagle’s medium

(DMEM) (Biochrom, Berlin, Germany) supplemented with 10% FCS, 100 U/ml

penicillin, 100 U/ml streptomycin, and 4 mM L-Glutamine (Gibco, Karlsruhe,

Materials and Methods

______________________________________________________________

Germany) in a humidified incubator in 10% CO

at 37°C.

2.1.3 Bacteria and virus

Listeria monocytogenes EGD strain Sv

1/2a was originally obtained

from G. B.

Mackaness. The bacteria were grown in Luria-Bertani (LB) broth (Difco,

Heidelberg, Germany) without any antibiotics to an OD

600

of 0.6, harvested by

centrifugation, and stored as stock in final 10% glycerol in LB at –80ºC. The

next day, one stock was thawed, plated onto LB agar plates, and colony-

forming units (CFU) were assessed.

Mycobacterium tuberculosis H37Rv strain was grown in Middlebrook 7H9

broth (Difco, Heidelberg, Germany) containing 0.05% Tween 80,

supplemented with albumin-dextrose complex, and stored in aliquots at –70°C.

Mycobacterium bovis BCG Danish 1331 (Statens Serum Institute,

Copenhagen, Denmark) was cultured in Dubos broth base (Difco, Heidelberg,

Germany) supplemented with Dubos medium albumin (Difco, Heidelberg,

Germany) at 37°C. A mid-logarithmic culture was aliquoted and stored at -

80°C before use. Mycobacterial cell stocks were plated onto Middlebrook

7H11 agar plates supplemented with oleic acid albumin-dextrose complex

(Difco, Heidelberg, Germany) and CFU were assessed.

Lymphocytic choriomeningitis virus (LCMV) WE strain was originally obtained

from Dr. F. Lehmann-Grube (Schwenk et al., 1971) and grown in L929

fibroblast cells.

2.1.4 Plasmid DNAs

Plasmid DNAs encoding p60 named pCiap, listeriolysin O (LLO) named

Materials and Methods

______________________________________________________________

pClisA, and non-hemolytic, mutant LLO named pChly492 were constructed by

Fensterle et al. (Fensterle et al., 1999) and J. Hess et al. (Hess et al., 2000)

(Fig. 3). Briefly, wild-type LLO gene and p60 gene of L. monocytogenes

without the bacterial signal sequence were amplified by polymerase chain

reaction (PCR) and inserted into EcoRI/XbaI site and XhoI/XbaI site of pCI

mammalian expression vector (Promega, Madison, WI, U.S.A.), respectively.

L. monocytogenes strain BUG337 encoding an LLO version with a single

amino acid (a. a.) exchange at the a. a. position 492 (Trp-492-Ala) was kindly

provided by Dr. P. Cossart (Michel et al., 1990). The mutant LLO gene was

amplified from genomic DNA of L. monocytogenes strain BUG337 by PCR,

and integrated into XhoI/XbaI site of pCI vector, which is named pChly492.

The gene map of pChly492 is the same as pClisA.

Plasmid DNAs encoding M. tuberculosis specifically expressed genes were

constructed by Mollenkopf et al. (Mollenkopf et al, submitted) (Fig. 4). Briefly,

M. tuberculosis specific genes were determined by comparative proteomics

comparing M. tuberculosis strain H37Rv and strain Erdmann with M. bovis

BCG strain Chicago and strain Copenhagen using two-dimensional gel

electrophoresis, mass spectrometry, and N-terminal sequencing techniques

(Mollenkopf et al., 2002). Three TB DNA vaccine candidate genes, Rv1511,

Rv2520, and Rv3407 were selected by preliminary DNA vaccination

experiments and amplified from M. tuberculosis strain H37Rv genomic DNA

by PCR and cloned into BamHI sites of pCMVtpa4 mammalian expression

vector containing an ER-targeting leader sequence, tpa4, which is originally

the human tissue plasminigen activator signal sequence (Weiss et al., 1999).

This sequence improves induction of immune responses and protection with

Materials and Methods

______________________________________________________________

DNA vaccine against M. tuberculosis (Delogu et al., 2002; Li et al., 1999). This

pCMVtpa4 vector was a kind gift from Dr. J. Ulmer, Chiron, U.S.A..

All molecular biological techniques followed standard techniques and

enzymes were purchased from New England Biolabs (Frankfurt am Main,

Germany). All plasmid DNAs for DNA vaccination were prepared using Qiagen

Endotoxin-Free Plasmid Purification kit (Qiagen, Hilden, Germany) following

manufacturer’s instruction. After purification, the DNAs were digested by

proper restriction enzymes to confirm by 1 % agarose gel electrophoresis in 1

X TAE buffer. For example, pCI vector was digested by NdeI, pCiap was

digested by HpaI, and pClisA and pChly492 were digested by NheI to yield

fragments of 1542 b. p. and 2466 b. p. bands from pCI, 1211 b.p. and 4165

b.p. bands from pCiap, 1320 b.p. and 4191 b.p. from pCliaA and pChly492.

pClisA

5511 bps

1000

2000

3000

4000

5000

BsrGI

SpeI

SnaBI

Ecl136II

SacI

PstI

BspMI

BbsI

XhoI

NsiI

Ppu10I

Bst1107I

EcoRI

Eco47III

XbaI

SalI

SmaI

XmaI

NotI

XmaIII

MunI

BsaBI

BamHI

NaeI

NgoMIV

DraIII

NspI

EcoO109I

AhdI

AlwNI

BglII

CMV-IE promotor

Intron

Hly

SV40-poly(A)

f1 ori

Amp

ori

pCiap

5376 bps

1000

2000

3000

4000

5000

BsrGI

SpeI

SnaBI

NcoI

Ecl136II

SacI

BbsI

XhoI

EcoRI

PvuII

BstXI

XbaI

AccI

SalI

SmaI

XmaI

NotI

XmaIII

MunI

ClaI

BsaBI

BamHI

NaeI

NgoMIV

DraIII

NspI

EcoO109I

XmnI

BglII

CMV-I.E. promotor/enhancer

Intron

iap

SV40 poly(A)

f1-ori

Amp-r

ori

A B

Fig. 3 Plasmid DNAs encoding L. monocytogenes genes.

Immunodominant

antigens of L. monocytogenes

were inserted into pCI mammalian expression

vector

under a CMV promoter. A. Plasmid pClisA encodes listeriolysin A antigen.

Plasmid pChly492 encoding mutant LLO has the same gene map. B. Plasmid

pCiap encodes p60 antigen.

Materials and Methods

______________________________________________________________

pCMV-TPA3407

4728 bps

1000

2000

3000

4000

HindIII

BlnI

StuI

BseRI

SfiI MscI

BsrGI

SpeI

SnaBI

NsiI

Ppu10I

Bpu1102I

XcmI

Van91I

AccIII

AflII

PvuII

HpaI

PstI

EcoRI

EcoNI

Eco47III

NheI

BamHI

NaeI

NgoMIV

BsgI

BamHI

MluI

BclI

PciI

AhdI

BsaI

FspI

PvuI

XmnI

SspI

tpa'

ORF-3

pCMV-TPAgmdA

5454 bps

1000

2000

3000

4000

5000

HindIII

BlnI

StuI

BseRI

SfiI

SpeI

SnaBI

NsiI

Ppu10I

Van91I

AccIII

AflII

PvuII

EcoRI

EcoNI

NheI

SmaI

XmaI

NotI

ClaI

BsgI

PmlI

SgrAI

AgeI

BamHI

NruI

BsiWI

RsrII

NaeI

NgoMIV

MluI

BclI

PciI

AhdI

BsaI

FspI

PvuI

SspI

tpa'

ORF-1

pCMVtpa2520c

4659 bps

1000

2000

3000

4000

HindIII

BlnI

StuI

BseRI

SfiI MscI

BsrGI

SpeI

SnaBI

SacII

NsiI

Ppu10I

Bpu1102I

XcmI

Van91I

AccIII

AflII

PvuII

HpaI

PstI

EcoRI

Eco47III

NheI

SmaI

XmaI

NotI

BamHI

ClaI

Bsu36I

Tth111I

BamHI

MluI

BclI

PciI

AhdI

BsaI

Cfr10I

FspI

PvuI

XmnI

SspI

tpa'

ORF-3

'tpa'

''tpa

A B

Fig. 4 Plasmid DNA encoding M. tuberculosis s

pecifically expressed

genes. M. tuberculosis-

specifically expressed genes were inserted into

mammalian expression vector under a CMV promoter to be fused to

downstream of tpa4, the ER-targeting sequence. A. Plasmid pCMV-

TPA3407

encodes Rv3407 gene. B. Plasmid pCMV-

TPAgmdA encodes Rv1511 gene.

C. Plasmid pCMV

TPA2520c encodes Rv2520 gene.

Materials and Methods

______________________________________________________________

2.1.5 Peptides

The LLO-derived peptide corresponding to a. a. 91-99 (single-letter code,

GYKDGNEY), immunodominant epitope restricted by MHC class I molecule,

H2-K

(Pamer et al., 1991) and the p60-derived peptide corresponding to a. a.

217-225 (KYGVSQDI), immunodominant epitope restricted by H2-K

(Pamer,

1994) were synthesized by Gerini Biotools (Berlin, Germany).

The Rv1511-derived peptide (GYVKFDQRYL) corresponding to a putative

epitope restricted by H2-K

and the Rv3407-derived peptides (IPARRPQNL,

RPQNLLDVT) corresponding to putative epitopes restricted by H2-L

were

synthesized as described above. The putative epitopes and affinities to class I

MHC molecules were predicted by computer programs, MAPPP (MHC-I

Antigenic Peptide Processing Prediction) (http://www.mpiib-

berlin.mpg.de/MAPPP/) (Hakenberg et al., 2003; Mollenkopf et al., submitted)

and FragPredict, provided by Max-Planck-Institute for Infection Biology, Berlin,

Germany (Nussbaum et al., 2003).

The LCMV NP

118-126

(RPQASGVYM) peptide, representing a H-2

–restricted T

cell epitope, was kindly provided by Dr. P. Aichele (Aichele et al., 1990).

All peptides were synthesized by Gerini Biotools (Berlin, Germany) and stored

at –20ºC as stocks resolved in sterile distilled water at 1mg/ml.

2.1.6 DNA carrier systems

Three different kinds of DNA vaccine delivery systems were used in this study:

cationic poly(lactic-co-glycolic acid) (PLG) microparticles, histone-like protein

from hyperthermostable eubacterium Thermotoga maritima (TmHU), and

Materials and Methods

______________________________________________________________

virus-like particles, VP1 protein from mouse polyoma virus (VLP).

Cationic PLG particles were produced by a reaction with RG 502H (Boeringer

Ingelheim, Ingelheim, Germany) and polyethylenimin (BASF, Ludwigshafen,

Germany), stabilized with hexadecyltrimethyl ammonium bromide (CTAB)

(Fluka, Steinheim, Germany) or with polyvinylalcohol (PVA) (Hoechst,

Frankfurt am Main, Germany) (Singh et al., 2000a). The size of particles was

830.63 nm ± 36.2. Particles were resolved in sterile endotoxin-free distilled

water (DW), sonicated for 30 seconds, and added to a DNA solution at a ratio

of 1:100 = DNA: PLG (weight/weight) in 100µl of sterile, endotoxin-free DW

per injection. The mixture of DNA and PLG was incubated at 4ºC overnight

and then injected intramuscularly into mice. For encapsulating particles, novel

amine-modified polyesters were made to encapsulate DNA at a ratio of 5:1

(w/w) in 20 µl/application, and applied intranasally. All particles were kindly

provided by Prof. Dr. T. Kissel, University of Marburg, Marburg, Germany.

Histone-like protein from Thermotoga maritima (TmHU) and VP1 protein from

mouse polyoma virus (VLP) were purified and kindly supplied by Dr. J. Hess,

November AG, Erlangen, Germany. TmHU protein was mixed with DNA at a

ratio of 1:1.2=DNA: protein (w/w) in 100µl of sterile, endotoxin-free distilled

water (DW) per injection and injected subcutaneously (s. c.) into mice. The

VP1 protein was mixed with DNA at a ratio of 1:1 (w/w) in 100µl of sterile,

endotoxin-free DW per injection, incubated for 15 min at room temperature,

and then injected s. c. into mice.

2.1.7 Antibodies and tetramers

Rat immunoglobulin (Ig), anti-mouse CD8α monoclonal antibody (mAb) (clone

Materials and Methods

______________________________________________________________

YTS169), anti-mouseCD4 mAb (clone YTS191.1), anti-mouse CD62L mAb

(clone Mel14), anti-mouse IFN-γ (clone XMG1.2 and clone R4), and anti-

mouse IgG Fc Rc (clone 2.4G2) (Karpovsky et al., 1984) were purified from

rat serum or hybridoma supernatants with protein G sepharose or purchased

from Becton Dickinson (B & D, Heidelberg, Germany). Antibodies were

cyanine 5 (Cy5)-, phycoerythrin (PE)-, or fluorescein-5-isothiocyanate (FITC)-

conjugated according to standard protocols.

A modified form of the full-length cDNA of the H2-K

H chain and human β2m

were kindly provided by Dr. E. Pamer (Altman et al., 1996; Busch and Pamer,

1998). Tetrameric H2-K

/peptide complexes were generated following the

protocols described in Busch and Pamer, 1998. In brief, a specific biotinylation

site was inserted to the COOH terminus of truncated H2-K

heavy chain (no

transmembrane region, truncation after a. a. 284). This fusion protein and β2m

were expressed in large amounts as recombinant proteins in Escherichia coli

using the isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible pET3a vector

system (Novagen, Madison, U.S.A.) and Escherichia coli strain BL21 (DE3)

(Novagen, Madison, U.S.A.) as an expression host. Purified heavy chain and

β2m were dissolved in 8 M urea and diluted into refolding buffer containing

high concentration of synthetic peptides (60 µM) to generate monomeric,

soluble H2-K

-peptide complexes. MHC-peptide complexes were purified by

gel filtration over a Superdex 200HR column (Pharmacia Biotech AB,

Piscataway, NJ., U.S.A.), and in vitro biotinylated for 12 hrs at 20°C in the

presence of 15 µg biotin operon repressor protein A (BirA) (Avidity, Boulder,

CO., U.S.A.), 80 µM biotin, 10 mM ATP, 10 mM MgOAc, 20 mM bicine, and 10

mM Tris-HCL (pH 8.3). To remove free biotin, MHC complexes were again

Materials and Methods

______________________________________________________________

purified by gel filtration, and then tetramerized by addition of PE-conjugated

streptavidin (Molecular Probes, Eugene, OR., U.S.A.) at a molar ratio of 4:1.

Tetramers were purified by gel filtration over a Superdex 200HR column and

stored at 3-5 mg/ml at 4°C in 1X PBS containing 0.02% sodium azide, 1 µg/ml

leupeptin, and 0.5 mM EDTA.

2.1.8 Buffers

Carbonate buffer, pH9.7

15mM Na2CO3

35mMNaHCO3

TAE buffer

36mM Tris-HCl

30mMNa2HPO4/NaH2PO4

Phosphate buffered saline (PBS), pH 7. 4

8 g/l NaCl

0. 2 g/l KCl

0. 2 g/l KH

1. 3 g/l Na

HPO

Refolding buffer pH8.0

100mM Tris-HCl

400mM L-arginine-HCl

1mM NaEDTA

Materials and Methods

______________________________________________________________

5mM red. gluthione

2.2. Methods

2.2.1 Vaccination

Sex- and age-matched BALB/c mice were immunized with 10 or 100 µg of

naked DNA or with 10 µg of DNA with each carrier in 100 µl of volume

intramuscularly (i.m.), subcutaneously (s.c.), or intranasally 3 times at 3 weeks

intervals (Fig. 3). As positive control, sublethal dose (1 X 10

or 5 X 10

) of L.

monocytogenes EGD strain or 1 X 10

M. bovis BCG Danish 1331 was

injected intravenously (i.v.) into mice at the same time as the prime

vaccination.

2.2.2 Protection assay

Mice vaccinated with DNA encoding L. monocytogenes genes were

Fig. 5 Experimental scheme Mice were vaccinated with 10 or 100 µ

g of

naked DNA or with 10 µg of DNA with each carrier in 100 µ

l i. m., s. c., or i. n.

3 times at 3 weeks intervals. Immunoassays from mice vaccinated with

monocytogenes genes were performed from day –

14, one week after the last

boost and those from mice vaccinated with M. tuberculosis

genes were

performed from day 7, 4 weeks after the last boost.

Materials and Methods

______________________________________________________________

challenged i.v. with a lethal dose (1x10

or 5x10

) of L. monocytogenes strain

EGD in 100µl of sterile PBS, at day 0, 3 weeks after the last boost. Survival

was checked daily until day 10 post infection.

For protection assays against M. tuberculosis, mice were challenged with an

aerosol generated from a 10 ml, single-cell suspension containing a total of

1×10

CFU of M. tuberculosis strain H37Rv, using a Middlebrook Airborne

Infection Apparatus (Middlebrook, Terre Haute, IN, U.S.A.), at day 0 (Grode et

al. submitted). These aerosol doses delivered 100-200 live bacilli to the lungs

of each animal. At days 30, 60, and 90 post infection, lungs, livers, and

spleens were aseptically removed from infected mice to assess CFU. Serial

dilutions from organ homogenates were plated onto Middlebrook 7H11 agar

plates supplemented with oleic acid albumin-dextrose complex (Difco,

Heidelberg, Germany) and were incubated at 37°C for 3-4 weeks.

Naïve mice as negative controls and mice of positive control groups as

described above were also challenged in the same way as experimental

groups. Statistics were assessed by t-test or logrank test using Graph Pad

Prism software.

2.2.3 Cytokine ELISpot assay

Enzyme-linked immunospot assays (ELISpot) were performed to determine

the number of antigen specific T cells secreting IFN-γ (Miyahira et al., 1995).

Spleens were removed from mice aseptically and prepared as single cell

suspension by passing through a stainless-steel mesh. Red blood cells were

removed by low osmotic pressure shock. One day before the assay, 96-well

nitrocellulose plates (Millititer HA; Millipore, Bedford, MA, U. S. A.) were

Materials and Methods

______________________________________________________________

coated with 5 µg/ml of the anti-mouse IFNγ mAb (clone R4) in 100 µl of

carbonate buffer, pH 9.6, per well. After overnight incubation at 4°C, the plates

were washed twice with PBS and blocked for 2 hrs at 37°C with 100 µl of 1%

bovine serum albumin (BSA) (Sigma-Aldrich, Munich, Germany) in PBS per

well. Splenocytes (1x10

or 5x10

cells/well) were added in 100 µl complete

RPMI 1640 media per well. P815 cells were coated with 10

-6

M of designated

peptides at 37°C for 2 hrs and then washed twice with complete RPMI 1640.

On the other hand, J774A.1 cells were incubated with 10ug/ml of L.

monocytogenes crude extracts or M. tuberculosis crude extracts at 37°C for 2

hrs. Otherwise, autolougous splenocytes were incubated with peptides or

bacterial crude extract. P815 cells, J774A.1 cells, or autologous splenocytes

(10

cells/well) with or without antigen were added into wells containing

responder-splenocytes in 100 µl of complete RPMI 1640. After 20-24 hrs

incubation at 37°C, 5% CO

the plates were washed 10 times with 0.05%

Tween-20 in PBS (washing buffer). To detect IFNγ secreting cells, 0.25 µg/ml

biotinylated anti-mouse IFNγ mAb (clone XMG1.2) in 100 µl washing buffer

per well was added and incubated at 37°C for 2 hrs. The plates were washed

10 times in washing buffer, and incubated for 1 h at 37°C in 0.05 µg/ml

alkaline phosphatase-coupled streptavidin (Dinova, Hamburg, Germany) in

100 µl of 1 X PBS per well. After 5 washes, spots of IFNγ-secreting cells were

visualized by adding 50 µl of the ready-to-use substrate 5-Bromo-4-Chloro-3-

Indolyl Phosphate/Nitro Blue Tetrazolium (BCIP/NBT) (Sigma-Aldrich, Munich,

Germany) dissolved in water. The reaction was stopped after 15 min at 37°C

by washing several times with distilled water. After drying, spots were counted

under a dissecting microscope at 3-fold magnification. The frequency of

Materials and Methods

______________________________________________________________

antigen-specific T cells is expressed as the number of spots of IFNγ-secreting

cells per 1x10

or 5x10

splenocytes.

2.2.4 Flow cytometry

Single cell suspensions from spleens were prepared as described above and

stained for fluorescence-activated cell sorting (FACS). Before intracellular IFN-

γ staining, 2x10

splenocytes per reaction were incubated with 10

-6

M of

designated peptides at 37°C for 5 hrs and washed once with 1X PBS. During

the last 4 hrs of incubation, 10 µg/ml Brefeldin A (Sigma-Aldrich, Munich,

Germany) was added. Thereafter, the cells were resuspended, incubated on

ice with 10 µg/ml anti-mouse IgG Fc receptor (Rc) (clone 2.4G2), 10 µg/ml rat

IgG, and 50 µg/ml unconjugated strepavidin (Molecular Probes, Eugene, OR,

U.S.A.) in 100 µl of 1X PBS for 5 min, and extracellularly stained with Cy5-

conjugated anti-mouse CD8α mAb (clone YTS169) and PE-conjugated anti-

mouse CD4 mAb (clone YTS191.1) for 20 min. The cells were washed with 1X

PBS and fixed with 1% paraformaldehyde (PFA) (Sigma-Aldrich, Munich,

Germany) for 20 min at room temperature. After fixation, the cells were

washed with 0.1% BSA in 1X PBS. The cells were perforated with 0.5%

saponin, 0.1% BSA, blocked with 10 µg/ml anti-mouse IgG Fc Rc (clone

2.4G2), 10 µg/ml Rat IgG (Dianova, Hamburg, Germany), and 50 µg/ml

unconjugated strepavidin (Molecular Probes, Eugene, OR, U.S.A.) in 100 µl of

1X PBS for 10min and intracellularly stained with FITC-conjugated anti-mouse

IFN-γ mAb (clone XMG1.2).

For tetramer analyses, 1x10

splenocytes were blocked as described above,

stained on ice with Cy5-conjugated anti-mouse CD8α mAb (clone 169), FITC-

Materials and Methods

______________________________________________________________

conjugated anti-mouse CD62L mAb (clone Mel. 14), and PE-conjugated H2-

/peptide tetramers for 60min, and washed with 1X PBS.

Cells were analyzed using a FACSCalibur and the CELLQuest software (B

& D, Mountain View, CA, U.S.A.).

2.2.5 Measurement of CTL activity

The activity of antigen specific CTL was detected by peptide-loaded target

cells stained with 5- (and 6-) carboxyfluorescein diacetate succinimidylester

(CFSE), which is a highly stable amine-reactive reagent readily incorporated

into cells. When membrane damage occurs, the dye is almost instantaneously

lost and the cells are no longer able to take up or retain the charged dye.

Several reports have shown that CFSE-prelabeled target cells can be

successfully evaluated to detect cytolysis (Sheehy et al., 2001). Splenocytes

from naïve mice were prepared as described above and washed twice with

ice-cold PBS. The cells were resuspended at a concentration of 2x10

cells/ml,

and stained with a low concentration (0.25 µM) of CFSE (Molecular Probes,

Eugene, OR, U.S.A.) or with a high concentration (5 µM) of CFSE for 4 min.

After labeling, complete RPMI 1640 medium was added to stop the reaction,

and splenocytes labeled with a high concentration of CFSE were loaded with

designated peptides at 37°C for 1-2 hrs. The two populations of different

CFSE intensities were mixed at a 1:1 ratio, and 6x10

cells adoptively

transferred into each recipient mouse (Aichele et al., 1990). The recipient mice

were primed with 200 plaque-forming units (pfu) of LCMV intraperitoneally or

5x10

L. monocytogenes i.v. at designated days before analysis. After 24 hrs,

recipient mice were sacrificed, and splenocytes were analyzed by FACS

Materials and Methods

______________________________________________________________

(FACScalibur, B & D, Mountain View, CA, U.S.A.).

2.2.6 ELISA

Enzyme-linked immunoabsorbent assay (ELISA) was performed to determine

whether Th 1 or Th 2 immune responses are dominantly induced by DNA

vaccination. IgG1 antibody responses indicate Th 2 immune response and

IgG2a antibody responses indicate Th 1 immune response. Briefly, 96 well

microtiter plates (Nunc) were coated with 10 µg/ml of L. monocytogenes crude

extract in 0.1M bicarbonate buffer overnight, washed five times in washing

buffer, blocked with PBS, 0.05% Tween 20, 1% BSA at 37°C for 2 hrs, and

washed again. Blood sera were collected from mice, pooled, and serially

diluted in PBS, and added into ELISA plates as triplicates. As positive control,

mouse IgG1 Ab (clone 25D1) and mouse IgG2a Ab (clone F23.1) was used

and as negative control, BSA was used at the concentration of 100 µg/ml in

PBS. The plates were incubated at 37°C for 1 hr, washed, and 2

Abs added,

anti-mouse IgG1 Ab (clone X56) and anti-mouse IgG2a (clone R19-15)

(PharMingen) at the concentration of 0.5µg/ml in 100µl/well of PBS. After

incubation at 37°C for 1 hr, the plates were washed, and 50 µl/well of 1mg/ml

p-nitrophenyl phosphate solution (Sigma) added. After incubation at room

temperature for 20 min, 50 µl/well of 0.5M EDTA, pH 8 was added to stop the

coloring reaction. The intensity of the reaction was measured at OD405 by

SpectraMax250 (Molecular Devices) and SoftmaxPro software.

Results

_______________________________________________________________

3. Results

3.1. DNA vaccination in the listeriosis model

3.1.1 Verification of plasmid DNAs for vaccination.

The plasmid DNAs encoding p60 named pCiap, wild-type LLO named pClisA,

and non-hemolytic, mutant LLO named pChly492 for DNA vaccination were

purified and confirmed by the sizes of the DNA fragments after restriction

enzyme digestion in 1 % agarose gel electrophoresis (Fig. 6). The gene maps

of the DNAs are displayed in Fig. 3. The pCI vector was digested with NdeI,

pCiap was digested with HpaI, and pClisa and pChly492 were digested with

NheI. The enzymes were chosen to distinguish each vector by different sizes

of DNA fragments. The pCI vector (Lane 1) revealed a 1542 b. p. band and a

2466 b. p. fragment after digestion with NdeI, pCiap (Lane 2) revealed a 1211

b.p. and a 4165 b.p. fragment with HpaI digestion, and pClisA and pChly492

(Lane 3 and 4) revealed a 1320 b.p. and a 4191 b.p. fragment with NheI

digestion. All fragments’ sizes were correct and the sequence of pChly492

was confirmed by DNA sequencing. The result of sequencing displayed 3

additional unexpected mutations regardless of the mutation of a. a. 492 (Trp-

Ala). These mutations are located in a.a. 64 (Tyr-Cys), a. a. 160 (Gly-Ser),

and a. a. 197 (no a. a. change) and not located in any known functional

domains, such as PEST sequence (a.a. 32-50), and immunodominant

epitopes.

Results

______________________________________________________________

1 2 3 4

2 Kb →

→→

→

1.5Kb →

→→

→

Fig. 6 The DNAs encoding L. monocytogenes genes. The pCI vector was

digested with NdeI, pCiap was digested with HpaI, and pClisa and pChly492

were digested with NheI. The pCI vector (Lane 1) revealed a 1542 b.p. and a

2466 b.p. fragments, pCiap (Lane 2) revealed a 1211 b.p. and a 4165 b.p.

fragments, and pClisA and pChly492 (Lane 3 and 4) revealed a 1320 b.p. and

a 4191 b.p. fragments.

3.1.2 Protection against L. monocytogenes by DNA vaccination and

comparison of DNA vaccine delivery systems

In order to determine which plasmid DNA shows the best protection and which

infection dose would be appropriate to reveal the improvement of DNA

vaccine efficacy by DNA vaccine carriers, preliminary protection assays were

performed with 100 µg of naked DNA upon infection with 1xLD50 of L.

monocytogenes. As described in Materials and Methods, sex- and age-

matched BALB/c mice were injected with 100 µg naked DNA i. m. 3 times at 3

weeks intervals. Three weeks after the last boost, mice were infected with

1xLD50 of L. monocytogenes. Groups of 6 mice were vaccinated with vector

control as mock, with plasmid DNA encoding p60, with plasmid DNA encoding

wild-type LLO, or with plasmid DNA encoding non hemolytic, less virulent

mutant LLO. As positive control, a group of 6 mice was immunized with a

sublethal dose of L. monocytogenes at the time point of priming. The plasmid

Results

______________________________________________________________

DNA encoding mutant LLO induced the best protection against L.

monocytogenes infection. The protection was as efficient as after a primary L.

monocytogenes infection of sublethal dose. All mice survived and protection

was statistically significant compared to mock treated mice by logrank test

using Prism software. The summary of logrank test was * (P=0.0190). The

difference of the survival curves between naïve and mutant LLO was also

statistically significant (**, P=0.0043). The second best plasmid DNA as a

vaccine candidate was the one encoding p60. The summary of the logrank

test was * (P=0.0345), compared with naïve, but not significant compared with

mock treated mice (P=0.1151) (Fig. 7). Mice injected with vector control also

showed slightly increased survival, but not significant. The infection dose for

challenge was increased for further experiments for the test of DNA carriers

because the improvement of efficacy by DNA carrier systems could not be

detected with the infection dose of 1xLD50.

First, the efficacy of 10 µg of naked DNA and DNA with PLG stabilized with

0 2 4 6 8 10

100

naive

mock

p60

LLO

mutant LLO

L. monocytogenes

Days after LD

Listeria monocytogenes

challenge

Survival (%)

Fig. 7 Survival curves of BALB/c mice immunized with 100µ

µµ

g of naked

DNA from 1xLD

L. monocytogenes challenge.

Difference of survival of

mice vaccinated upon 100 µg of naked DNA encoding mutant LLO or wit

sublethal dose of L. monocytogenes

from that of mock treated mice is

statictically significant (*, P=0.0190). Statistics were assessed by logrank test

using Graph Pad Prism software.

Results

______________________________________________________________

CTAB were compared. The vaccination procotols were the same as before but

the infection dose for challenge was 5 times LD

. Difference of survival of

mice immunized with 10 µg of naked DNA encoding mutant LLO from that of

mock treated mice was not statistically significant (P=0.5143). Difference of

survival of mice immunized with sublethal dose of L. monocytogenes from that

of mock treated mice was significant (*, P=0.0185) (Fig. 8, A). Difference of

survival of mice immunized with mutant LLO/PLG from that of mock treated

mice was not significant (P=0.2029) but the difference of survival curves

between mutant LLO and naïve was significant (**, P=0.0015). Difference of

survival of mice immunized with sublethal dose of L. monocytogenes from that

of mock treated mice was significant (*, P=0.0178), and the difference

between L. monocytogenes immunization and naive was also significant (***,

P=0.0006) (Fig. 8, B). Difference of survival of mice immunized with mutant

LLO/PLG from that of mutant LLO was not significant (P=0.4441) (Fig. 8, A

and B). The PLG stabilized with CTAB could not significantly improve the

efficacy of DNA vaccine compared with same amount of naked DNA. Four

mice out of 60 mice died after vaccination with DNA and particles. In contrast,

no mouse died after vaccination with naked DNA. From this experiment, the

DNA encoding mutant LLO was also confirmed as the best DNA vaccine

candidate against L. monocytogenes.

The purpose of this study was not only to improve protection and immune

response against intracellular bacteria but also to reduce the amount of DNA

required for vaccination. Thus, 100 µg of naked DNA was compared with 10

µg of DNA and carriers. To clarify the improvement, the infection dose for

challenge was increased up to 10xLD

. The vaccination protocols were the

Results

______________________________________________________________

same as before. Survival tests were performed with mice vaccinated with 10

µg of naked DNA i. m., with 100 µg of naked DNA i. m., with 10 µg of DNA and

PLG stabilized with PVA i. m., with 10 µg of DNA and VLP s. c., with 10 µg of

DNA and TmHU s. c., or with 10 µg of DNA and newly developed

encapsulating particles intranasally. As a result, 100 µg of naked DNA

encoding mutant LLO showed 50 % protection from L. monocytogenes

infection (*, P=0.0498) but no particle improved the efficacy of DNA vaccine

significantly (Fig. 9).

0246810

100

naive

mock

p60

LLO

mutant LLO

L. monocytogenes

Days after 5XLD

Listeria

monocytogenes challenge

Survival (%)

0246810

100

naive

mock

p60/PLG

LLO/PLG

mutant LLO/PLG

L. monocytogenes

Days after 5XLD

Listeria

monocytogenes challenge

Survival (%)

Fig.8 Survival curves of BALB/c mice immunized with 10 µ

µµ

g of naked DNA

or with 10 µ

µµ

µg of DNA and PLG. A. Intramuscular immunization with 10 µ

g of

naked DNA. Difference of survival of mice immunized with mutant LLO from that

of mock treated mice was not statistically significant (P=0.2029). Difference o

survival of mice immunized with sublethal dose of L. monocytogenes

from that

of mock was significant (*, P=0.0178). B. Intramuscular immunization with 10 µ

DNA absorbed onto 1 mg of PLG stabilized with CTAB. Difference of survival of

mice immunized with

mutant LLO/PLG from that of mock treated mice was not

significant (P=0.5143). Difference of survival of mice immunized with sublethal

dose of L. monocytogenes

from that of mock treated mice was significant (*,

P=0.0185). Difference of survival of mice imm

unized with mutant LLO/PLG from

that of mutant LLO was not significant (P=0.4441). Statistics were assessed by

logrank test using Graph Pad Prism software.

Results

______________________________________________________________

0 2 4 6 8 10

100

naive

mock

mutant LLO

L. monocytogenes

Days after 10XLD50 Listeria

monocytogenes challenge

Survival (%)

0 2 4 6 8 10

100

naive

mock

mutant LLO

L. monocytogenes

Days after 10XLD

Listeria

monocytogenes challenge

Survival (%)

0246810

100

naive

mock

mutant LLO/TmHU

L. monocytogenes

Days after 10XLD

Listeria

monocytogenes challenge

Survival (%)

0 2 4 6 8 10

100

naive

mock

mutant LLO/VLP

L. monocytogenes

Days after 10XLD

Listeria

monocytogenes challenge

Survival (%)

0 2 4 6 8 10

100

naive

mock

mutant LLO/PLG

L. monocytogenes

Days after 10XLD

Listeria

monocytogenes challenge

Survival (%)

0 2 4 6 8 10

100

naive

mock

mutant LLO/encap

L. monocytogenes

Days after 5XLD

Listeria

monocytogenes challenge

Survival (%)

A B

C D

E F

Fig. 9 Survival curves of mice immunized with 10 µ

µµ

g of naked DNA (A),

100 µ

µµ

µg of naked DNA (B), 10 µ

µµ

µg of DNA and TmHU (C), 10 µ

µµ

g of DNA and

VLP (D), 10 µ

µµ

µg of DNA and PLG stabilized with PVA (E), and 10 µ

µµ

g of DNA

and encapsulating particle (F).

Difference of survival of mice vaccinated with

100 µg of

naked DNA encoding mutant LLO from that of mock treated mice

was statistically significant (*, P=0.0498) and that of

L. monocytogenes

was

also significant (***, P=0.0009) (B). Statistics were assessed by logrank test

using Graph Pad Prism software.

Results

______________________________________________________________

3.1.3 Antigen-specific CD8

T cells induced by DNA vaccination against L.

monocytogenes

In order to determine antigen-specific immune response induced by DNA

vaccination, flow cytometric analysis with MHC class I / LLO

91-99

peptide

tetramer was performed. The preparation and staining protocol is described in

Materials and Methods. The splenocytes were gated by FSC/SSC scatters

Fig. 10 Gating for flow cytometric analysis with MHC/pe

ptide

tetramers. The splenocytes were gated by FSC/SSC scatters (A), PI

to exclude dead cells (B), and CD8

cells (C). Finally, LLO

tetramer/CD62L dot plot was displayed (D).

A B

C D

Results

______________________________________________________________

(Fig. 10, A), PI- cells to exclude dead cells (Fig. 10, B), and CD8

cells (Fig.

10, C). Finally, LLO tetramer/CD62L dot plots were displayed (Fig. 10, D). The

cell surface marker, CD62L is known as an effector/memory cell marker.

When the T cell is activated, CD62L expression level decreases and

afterwards, the expression level is restored again during memory phase

(Busch and Pamer, 1999).

Results

______________________________________________________________

4.99 0.74 0.05 0.12

0.84 0.18

0.02 0.07

A B C

D F

0.77 0.75 0.03 0.11

Fig. 11 Antigen-specific CD8

T cells induced by DNA vaccination.

Splenocytes were prepared from a mouse infected secondary with

monocytogenes 5 days before analysis (A), from mice injected with 100 µ

g of

naked DNA of empty vector (B), from mice injected with 10 µ

g of DNA of empty

vector and PLG stabilized with PVA (C), from a mouse immunized with sublethal

dose of L. monocytogenes 7 weeks before analysis (D),

from mice vaccinated

with 100

g of naked DNA encoding mutant LLO (E), and from mice vaccinated

with 10

g of DNA and PLG stabilized with PVA (F). Splenocytes were prepared

5 days after the last boost. The cells were gated as in Fig. 10. The results were

ummarized in a gragh (G). Values represent averages from 2 mice except that

of one L. monocytogenes

infected mouse. Error bars represent standard

deviation.

0.2

0.4

0.6

0.8

1.2

1st L. m.

naked pCI

naked pChly492

PLG pCI

PLG pChly492

LLOtet(+)CD62L(-)

LLOtet(+)CD62L(+)

Results

______________________________________________________________

Groups of 2 mice were vaccinated with 100 µg of naked DNA or with 10 µg of

DNA and PLG stabilized with PVA as described above. Mice were sacrificed

and were analyzed 5 days after the last boost. As positive control, two mice

were immunized with a sublethal dose 5x10

CFU of L. monocytogenes 7

weeks before analysis, and one of them was infected secondarily with 1x10

monocytogenes 5 days before analysis, while the other one was regarded as

long-term immunity positive control. As expected, splenocytes from secondary

infection showed markedly expanded tetramer positive cell population (Fig. 11,

A) and those of primary infection as long-term immunity positive control also

showed significant tetramer positive cell population (Fig. 11, D). Both of vector

control showed background level of tetramer positive cell populations (Fig. 11,

B, C) and mice vaccinated with 10 µg of DNA encoding mutant LLO and PLG

also showed no significant tetramer positive cell population (Fig. 11, F). On the

other hand, vaccination of 100 µg of DNA encoding mutant LLO induced

increased tetramer positive cell population (Fig. 11, E). The tetramer positive

and CD62L negative cell population was 0.49% on average and it was 8.9 fold

increased (Fig. 11, G). Interestingly, one out of 2 mice showed dramatically

increased tetramer positive cell population (0.84%) but the other one showed

less (0.14%). Tetramer

CD62L

cell populations were 87% out of total

tetramer

cell population of secondary infection at day 5, 82% out of that of

DNA vaccination 5 days after the last boost, and 51% out of that of long-term

immunity. Thus, 5 days after the last boost, many antigen-specific T cells

induced by DNA vaccination were activated. There exist tetramer positive cell

populations after 7 days and 10 days after the last boost (Table 2). As

conclusion of this experiment, DNA vaccination could induce antigen-specific

Results

______________________________________________________________

immune response in the listeriosis model.

LLO tetramer positive cell population (%)

01450.1850.9650.1451.22510

0.911.7454.232.0816.5*2.8*7

0.1450.1450.660.1855.73*1.52*5Days

after

the

last

boost/

infecti

10 µg

DNA/PLG

mutant

LLO

10 µg

DNA/PLG

vector

100 µg

naked

Mutant

LLO

100 µg

naked

vector

2nd

infection

1st

infection

(6 wks

before

the last

boost)

3.1.4 IFN-γ secretion induced by DNA vaccination against L. monocytogenes

Since IFN-γ is known as a key cytokine for protection against intracellular

bacteria, IFN-γ secreting splenocytes were detected by ELISpot assay as

described above. Groups of 3 mice were sacrificed 7 days after the last boost

and splenocytes were prepared and incubated with P815 cells (Fig. 12) or

autologous splenocytes (Fig. 13) loaded with/without LLO

91-99

peptides or

p60

217-225

peptides onto ELISpot plates for 24 hrs. These peptides are

immunodominant MHC class I H2-K

-restricted epitopes. Splenocytes from

mice vaccinated with 100 µg of naked DNA encoding mutant LLO showed

IFN-γ secreting cells after restimulation with LLO

91-99

peptides, but those of

p60 and wild-type LLO did not show increased levels of IFN-γ secreting

Table 2. Kinetics of LLO

91-99

tetramer positive populations from mice

vaccinated with DNA

. * data from one mouse. All other values represent

averages from 2 mice.

Results

______________________________________________________________

splenocytes after restimulation with p60

217-225

peptides or LLO

91-99

peptides,

respectively. The LLO

91-99

peptide-specific IFN-γ secreting cell population

induced by vaccination with DNA encoding mutant LLO was 17 IFN-γ

secreting splenocytes/10

cells, while that of vector control was 10.6 IFN-γ

secreting splenocytes/10

cells, The increase of IFN-γ secreting cells by DNA

encoding mutant LLO was up to 70% (Fig. 12). However, the LLO

91-99

peptide-

specific IFN-γ secreting splenocyte population was not detected by

vaccination with 10 µg of DNA and PLG stabilized with PVA (Fig. 13). The IFN-

γ secreting cell population from mice vaccinated with 100 µg of naked DNA

encoding mutant LLO was 26.5/10

cells, while that of vector control was

13/10

cells. The increase was 50%. As positive control, a mouse was

immunized with sublethal dose of L. monocytogenes 7 weeks before analysis,

and the mouse exhibited high levels of IFN-γ-secreting splenocytes (67.5/10

This result is similar to Fig. 12 and consistent with FACS data in Fig. 11.

Splenocytes from mice vaccinated with 10 µg of DNA and TmHU or VLP were

also tested by ELISpot assay, but failed to show significant level of antigen-

specific IFN-γ secreting cell population (data not shown). These results

suggest plasmid DNA mutant LLO could induce antigen-specific MHC class I-

restricted CD8

T cell immune response, but plasmid DNA encoding p60 or

wild-type LLO failed to induce detectable amounts of antigen-specific MHC

class I-restricted CD8

T cell immune responses. Additionally, no DNA carrier

improved DNA vaccination to induce detectable level of antigen-specific

immune responses. These results are in line with the protection results.

Results

______________________________________________________________

naïve

pCI

pCiap

pClisA

pChly492

IFN-g secreting cells/1X10

Media

P815

LLOpep

p60pep

Fig. 12. Frequencies of antigen-specific IFN-γ

γγ

secreting splenocytes

by DNA vaccination.

Groups of 3 mice were vaccinated with pCI as

vehicle control, with pCiap encoding p60, with pClisA encoding wild-

type

LLO, or with pChly492 encoding mutant LLO. All vaccination doses were

100 µg of naked DNA. Splenocytes were prepared 7 days after t

he last

boost and MHC class I-restricted LLO

91-99

or p60

217-225

peptide-

specific

IFN-γ

secretion determined by ELISpot assay. Values represent means

from 3, and error bars represent standard deviation.

Results

______________________________________________________________

100

120

140

160

naïve

naked pCI

naked pClhy492

PLG pCI

PLG pChly492

L.m.

IFN-g secreting cells/2X10

Media

LLOpep

Spl

Fig. 13 Frequencies of antigen-specific IFN-γ

γγ

secreting

splenocytes by DNA vaccination. Groups o

f 2 mice were vaccinated

with 100

µg of pCI as vehicle control, with 100 µ

g of pChly492 encoding

mutant LLO, with 10 µg of pCI and PLG stabilized with PVA, or 10 µ

g of

pChly492 and PLG stabilized with PVA. Splenocytes were prepared 7

days after the last boost and MHC class I-restricted LLO

91-99

peptide

specific IFN-γ

secretion determined by ELISpot assay. As positive

control, one mouse was immunized with a sublethal dose of

monocytogenes

. Values represent means from 2 mice except for

positive control, and error bars represent standard deviations.

Results

______________________________________________________________

3.1.5 Th 1/Th 2 immune response

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

naïve pCI pCiap pClisA pChly492

IgG1 OD405

0.2

0.4

0.6

0.8

1.2

1.4

naïve pCI pCiap pClisA pChly492

IgG2a OD405

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

naïve pCI pCiap pClisA pChly492

IgG1 OD405

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

naïve pCI pCiap pClisA pChly492

IgG2a OD405

0.2

0.4

0.6

0.8

1.2

1.4

1.6

1.8

naïve pCI pCiap pClisA pChly492

IgG1 OD405

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

naïve pCI pCiap pClisA pChly492

IgG2a OD405

0.2

0.4

0.6

0.8

1.2

1.4

naïve pCI pCiap pClisA pChly492 L.m.

IgG2a OD405

A B

C D

E F

G H

Fig. 14 Comparison of antibody subclasses induced by DNA vaccination

Blood sera were colleted at day 0 from mice vaccinated with 100 µ

g of naked

DNA (A, B), from mice vaccinated with 10 µ

g of DNA and TmHU (C, D), from

mice vaccinated with 10 µ

g of DNA and VLP (E, F), from mice vaccinated with

10 µ

g of DNA and PLG stabilized with PVA (G, H). Sera were pooled from

roups of three mice and put into plates as triplicates. To detect IgG1 and

IgG2a antibody titer, ELISA assay was performed. All dilution factors were 64

except in G and H (128). Left panels (A, C, E, G) show IgG1 titers, and right

panels (B, D, F, H) show

IgG2a titers. Abscissas indicate OD405. Values

represent means from triplicates, and error bars represent standard

deviations.

Results

______________________________________________________________

For protection and immunity against intracellular bacteria, the Th 1 T cell

immune response plays a pivotal role. To determine the balance between Th

1/Th 2 immune responses, ELISA assays were performed to detect serum

IgG1 and IgG2a antibody titers as described above. IgG1 antibody response

indicates Th 2 immune response, and IgG2a antibody response indicates Th 1

immune response. Blood sera were colleted from mice vaccinated with 100 µg

of naked DNA, from mice vaccinated with 10 µg of DNA and TmHU, from mice

vaccinated with 10 µg of DNA and VLP, and from mice vaccinated with 10 µg

of DNA and PLG stabilized with PVA at day 0, 3 weeks after the last boost. As

positive control, sera were collected from mice immunized with a sublethal

dose of L. monocytogenes 9 weeks before analysis. As expected, mice

vaccinated with 100 µg of naked DNA encoding mutant LLO exhibited high

titer of IgG2a and interestingly, DNA encoding p60 also induced high IgG2a

production (Fig. 14, B). DNA encoding p60 protected mice the second best

from L. monocytogenes infection. On the IgG1/IgG2a antibody titer from mice

vaccinated with 10 µg of DNA and PLG, PLG is not protein and simply 10 µg

of DNA may not be sufficient to induce significant Th 1 immune response.

3.1.6 Failure to detect CTL activity

In vivo killing assay using CFSE labeled target cells was adopted to detect

CTL activity. This method is established to give quantitative and qualitative

results of actual in vivo killing and little spontaneous leakage (Aichele et al.,

1997; Sheehy et al., 2001).

Results

______________________________________________________________

No peptide LCMVpep

1.59 1.32

No peptide LCMVpep

0.70 0.01

No peptide L.m.pep

1.39 0.66

No peptide L.m.pep

0.77 0.02

LCMVpep L.m.pep

1.35 0.78

LCMVpep L.m.pep

0.07 0.27

LCMVpep L.m.pep

1.01 0.66

A B

C D

Mice were primed with 200 pfu of LCMV or 5x10

L. monocytogenes and 9

days or 24 days after the priming, splenocytes from naïve mice were loaded

with LCMV-derived MHC class I-restricted peptide or with L. monocytogenes

derived MHC class I-restricted peptide, labeled with CFSE, and transferred

into primed mice i.v. respectively, as described above. At day 9 post infection,

splenocytes loaded with antigen-specific peptides were killed in both LCMV-

primed mice and L. monocytogenes-primed mice (Fig. 15, C, D), but at day 24,

splenocytes loaded with antigen-specific peptides were killed only in LCMV-

Fig. 15 CTL activities in LCMV-primed or L. monocytogenes-

primed

mice Mice were primed with LCMV (C, F) or with L. monocytogenes

(D,

G), and in vivo killing assay was performed at day 9 (A, B, C, D) and day

24 (E, F, G) post infection. Naïve

mice were used as negative control (A,

B, E).

Results

______________________________________________________________

primed mice (Fig. 15, F), while L. monocytogenes-primed mice failed to kill

splenocytes loaded with antigen-specific peptides (Fig. 15, G). Naïve mice did

not show cytotoxic activity (Fig. 15, A, B, E).

3.2. DNA vaccination against M. tuberculosis

3.2.1 Protection against M. tuberculosis by naked DNA or by DNA with PLG

Sex- and age- matched BALB/c mice were vaccinated with 10 µg of DNA and

PLG stabilized PVA 3 times at 3 weeks intervals. As positive control, 1x10

CFU M. bovis BCG Danish strain were injected i.v.. Mice were challenge-

infected with 100-200 M. tuberculosis H37Rv by aerosol 3 weeks after the last

boost. At day 30 and 60 post infection, lungs and spleens were removed from

groups of five mice, and bacterial loads were assessed. At day 30, mice

vaccinated with Rv3407 DNA and PLG showed about 0.3 log

decreased

CFU, and at day 60, about 0.5 log

CFU decreased, but the difference was

statistically not significant (Fig. 16). At day 30, P value between vector control

and Rv3407 was 0.1152, and at day 60, P=0.1490. This result also suggested

Rv3407 DNA is the better DNA vaccine candidate than Rv2520 and Rv1511,

but in a previously report with 100 µg of naked DNA, Rv1511 also showed

significant protection against M. tuberculosis (Mollenkopf et al., submitted).

The DNA vaccine efficacy of 10 µg of DNA and PLG was compared with that

of 100 µg of naked DNA (Fig. 17). At day 30, CFU from mice vaccinated with

100 µg of naked Rv3407 DNA was approximately 5.5 log

10,

that of vector

control (tpa) was approximately 5.9 log

10,

but the difference was statistically

not significant (P=0.0952). Mice vaccinated with 10 µg of DNA and PLG also

showed similar level of decreased CFU. However, this effect did not last until

Results

______________________________________________________________

day 90 post infection.

Results

______________________________________________________________

Day 30

Naive

BCG

Vector/PLG

Rv1511/PLG

Rv2520/PLG

Rv3407/PLG

3.0

3.5

4.0

4.5

5.0

5.5

6.0

H37Rv CFU (log) Lung

Day 60

Naive

BCG

Vector/PLG

Rv1511/PLG

Rv2520/PLG

Rv3407/PLG

3.0

3.5

4.0

4.5

5.0

5.5

6.0

H37Rv CFU (log) Lung

Fig. 16 Protection of BALB/c mice immunized with 10 µ

µµ

g of DNA and

PLG stabilized with PVA from M. tuberculosis infection.

Data

represent the mean number of CFU/lung in log

values for groups of five,

and error bars represent standard deviation. Statistics were assessed by

t test (Mann-Whitney test) using Prism program.

Day 30

Naive

BCG

tpa

3407

tpa/PLG

3407/PLG

4.0

4.5

5.0

5.5

6.0

6.5

H37Rv CFU (log) per lung

Day 90

Naive

BCG

tpa

3407

tpa/PLG

3407/PLG

H37Rv CFU (log) per lung

Fig. 17 Protection of BALB/c mice immunized with 100 µ

µµ

g of naked

DNA or with 10 µ

µµ

µg of DNA/PLG from M. tuberculosis infection.

Data

represent the mean number of CFU/lung in log

values and error bars

represent standard deviation. At day 30, the difference of CF

U from mice

vaccinated with vector control (tpa) and Rv3407 was not significant

(P=0.0952) by t test (Mann-Whitney test) using Prism program.

Results

______________________________________________________________

3.2.2 Antigen-specific CD8

T cells induced by DNA vaccination against M.

tuberculosis

0.65

0.85

0.02



 

 

 

 



 

tet(+)CD62(-) tet(+)CD62(+)

% gated

Rv1511

Mock

0.85

0.28

In order to detect antigen-specific immune response induced by DNA

vaccination, flow cytometric analysis with MHC class I Hd-K

/ Rv1511-derived

putative epitope tetramer was performed. Mice were vaccinated with 100 µg of

naked Rv1511 DNA as described above. The preparation and staining

Fig. 18 Antigen-specific CD8

T cells induced by DNA vaccination

Splenocytes from mice vaccinated with 100 µ

g of naked Rv1511 DNA, B.

Splenocytes from mice vaccinated with 100 µ

g of naked empty vector

DNA (Mock). The dot plots showed CD8+ living cells. C. Summary of

tetramer

analysis. Values represent averages from 3 mice and error bars

represent standard deviations.

Results

______________________________________________________________

protocol was described in Materials and Methods. The splenocytes were

gated by FSC/SSC scatters (Fig. 10, A), PI negative cells to exclude dead

cells (Fig. 10, B), and CD8

cells (Fig. 10, C). Finally, LLO tetramer/CD62L dot

plots were displayed (Fig. 10, D). At day 7, 4 weeks after the last boost, the

tetramer positive cell population from mice vaccinated with 100 µg of naked

Rv1511 DNA was 1.36% and that of vehicle control (mock) 0.34%. The

tetramer positive cell population was 4 fold increased by DNA vaccination (Fig.

18). Therefore, antigen-specific immune responses could be induced by DNA

vaccination against M. tuberculosis.

3.2.3 IFN-γ secretion induced by DNA vaccination against M. tuberculosis

In order to detect antigen-specific IFN-γ secreting cells induced by DNA

vaccination, ELISpot assays were performed. Groups of three mice were

vaccinated with 100 µg of naked tpa DNA as vehicle control, with 100 µg of

naked Rv3407 DNA, with 10 µg of tpa DNA and PLG stabilized with PVA, and

with 10 µg of Rv3407 DNA and PLG. Splenocytes were prepared at day –15, 1

week after the last boost (Fig. 19 A, C, E) and at day 30, 7.5 weeks after the

last boost (Fig. 19 B, D, F), stimulated with media only (Fig. 19 A, B), with M.

tuberculosis crude extract (Fig. 19 C, D), or with the mixture of 2 different

Rv3407-derived putative MHC class I epitope peptides (Fig. 19 E, F) for 3

days, and antigen-specific IFN-γ secretion was determined by ELISpot assay

as described above. At day –15, mice vaccinated with 10 µg of Rv3407 DNA

and PLG showed 21.1 IFN-γ secreting splenocytes/1x10

cells responding to

Rv3407-derived epitopes when the splenocytes were incubated in media

without antigen, while mice vaccinated with vector control showed 2.2 IFN-γ

Results

______________________________________________________________

secreting splenocytes/1x10

. However, average background level of spots

was 5/1x10

cells (Fig. 19, A). At day –15, mice vaccinated with 10 µg of

Rv3407 DNA and PLG showed 22.2 IFN-γ secreting splenocytes/1x10

cells

responding to M. tuberculosis crude extract when the splenocytes were

incubated in media containing M. tuberculosis crude extract, while mice

vaccinated with vector control showed 8.9 IFN-γ secreting splenocytes/1x10

cells (Fig. 19, C). However, Rv3407-derived peptide specific IFN-γ secreting

cells were not increased (Fig. 19, E) and IFN-γ secreting splenocytes by 100

µg of naked Rv3407 DNA were not increased, either.

At day 30, mice vaccinated with 100 µg of naked Rv3407 DNA showed 25.8

IFN-γ secreting splenocytes/1x10

cells responding to M. tuberculosis crude

extract, while mice vaccinated with vector control showed 14.4 IFN-γ secreting

splenocytes/1x10

, which is 79% increased. IFN-γ secreting cells induced by

100 µg of naked Rv3407 DNA also were 85% increased responding to

Rv3407-derived peptides, when the splenocytes were incubated in media only

(Fig. 19, B). Mice vaccinated with 100 µg of naked Rv3407 DNA showed 36.7

IFN-γ secreting splenocytes/1x10

cells responding to M. tuberculosis crude

extract when the splenocytes were incubated in media containing M.

tuberculosis crude extract, while mice vaccinated with vector control showed

22 IFN-γ secreting splenocytes/1x10

(Fig. 19, D). Mice vaccinated with 100

µg of naked Rv3407 DNA showed 38.3 IFN-γ secreting splenocytes/1x10

cells responding to Rv3407-derived peptides when the splenocytes were

incubated in media containing M. tuberculosis crude extract, while mice

vaccinated with vector control showed 5.6 IFN-γ secreting splenocytes/1x10

(Fig. 19, F). However, 10 µg of DNA and PLG failed to induce significant IFN-γ

Results

______________________________________________________________

secreting cells at day 30. Ergo, these results suggested DNA vaccination

could induce antigen-specific IFN-γ secretion and PLG might improve the

induction of immune response at early time point but it did not last longer.

Results

______________________________________________________________

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Media

Mtb lysate

Peptide mix

A B

C D

E F

Fig.19 Frequencies of antigen-specific IFN-γ

γγ

secreting splenocytes by

DNA vaccination. Groups of 3 mice were vaccinated with 100 µ

g of naked

tpa DNA as vehicle control, with 100 µg of naked Rv3407 DNA, with 10 µ

g of

tpa DNA and PLG, and with 10 µ

g of Rv3407 DNA and PLG. Splenocytes

were prepared at day

–

15, 1 weeks after the last boost (A, C, E) and at day

30, 7.5 weeks after the last boost (B, D, F), stimulated with media only (A,

B), with M. tuberculosis crude extract (C, D), or with Rv3407-deri

ved putative

epitope peptide (E, F) for 3 days, and antigen-specific IFN-γ

secretion was

determined by ELISpot assay. Values represent means from 3, and error

bars represent standard deviation.

Results

______________________________________________________________

Discussion

_______________________________________________________________

4. Discussion

Tuberculosis (TB) which is mainly caused by M. tuberculosis, is still a major

health problem with two million deaths and 8.8 million new cases annually

(Global tuberculosis control: WHO report 2004. WHO/HTM/TB/2004.331.

World Health Organization, Geneva, 2004). Although M. bovis BCG, the

current vaccine against TB, prevents disseminated TB in newborns, it fails to

protect against the most common form of the disease, pulmonary TB in adults.

Therfore, a novel vaccine against TB is urgently needed. DNA vaccines can

induce antigen-specific Th 1 CD4

and CD8

T cell responses required to

protect mammals from intracellular bacterial infection, and they have several

advantages: they are economical, relatively safe, easy to handle, and stable at

room temperature. However, one of the disadvantages of DNA vaccine is the

low efficiency of DNA delivery. Ergo, effective DNA delivery systems can

reduce the amount of DNA required, reduce safety concerns, and at the same

time improve protection and immune responses.

This study was performed to select the most potent DNA vaccine candidates

against L. monocytogenes as an experimental model system and against M.

tuberculosis, to analyze antigen-specific immune responses to compare the

different DNA vaccine carrier systems in the listeriosis model, to identify the

most effective one, and to apply the results to M. tuberculosis infection. The

plasmid DNAs encoding mutant LLO (Trp492Ala) of L. monocytogenes and

encoding Rv3407 of M. tuberculosis were selected as DNA vaccine

candidates because they induced protection in mice most effectively from L.

monocytogenes and M. tuberculosis, respectively, and also induced antigen-

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specific CD8

T cell immune responses and IFN-γ production, which play a

pivotal role in the control of intracellular bacteria. Three different kinds of

biodegrabable particles and two different proteins were evaluated to select the

most effective DNA delivery system to reduce the amount of DNA required for

DNA vaccination, but no DNA carrier with 10µg of DNA could exceed the

potency of 100 µg of naked DNA alone.

4.1. Selection of DNA vaccine candidates

In order to select the most effective DNA vaccine candidates against L.

monocytogenes, the plasmid DNAs encoding p60, wild-type LLO, and mutant

LLO (Trp492Ala) were compared. The proteins, p60 and LLO are well known

virulence factors and dominant antigens of L. monocytogenes (Mengaud et al.,

1987; Kuhn and Goebel, 1989) and mutant LLO (Trp492Ala) is non hemolytic

and less virulent (Michel et al., 1990). Plasmid DNA encoding p60, LLO

(Fensterle et al., 1999), or mutant LLO (Cornell et al., 1999) under CMV

promoter proved to provide protective immunity in mice against L.

monocytogenes.

Sex- and age-matched BALB/c mice were vaccinated with naked DNA or with

DNA and carrier 3 times at 3 weeks interval. The vaccination protocol was

optimized in previous reports (Fensterle et al., 1999; Mollenkopf et al.,

submitted). The potency of three plasmid DNAs encoding L. monocytogenes

genes were compared by protection assay to check survival, and the plasmid

DNA encoding mutant LLO showed the best protection against L.

monocytogenes infection at different infection doses with or without DNA

carrier. The second best one was the plasmid DNA encoding p60. The

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plasmid DNA encoding wild-type LLO did not show significant protection at

high infection doses. This result is consistent with a previous report (Cornell et

al., 1999). Interestingly, vector control itself improved protection slightly. This

seems to be due to the CpG motif effects (Elkins et al., 1999). For further

experiments, the plasmid DNA encoding mutant LLO was selected as the best

DNA vaccine candidate.

To develop a novel DNA vaccine against M. tuberculosis, three genes were

selected by Mollenkopf et al. (Mollenkopf et al., submitted), based on 2D-

electrophoresis analysis to select M. tuberculosis-specific secreting protein

(Mattow et al., 2001). They are Rv3407, Rv2520c, and Rv1511. Rv3407 is a

300 bp non essential gene (Sassetti et al., 2003), and encodes a 99 a. a.

conserved hypothetical protein with unknown function. Rv2520c is a 228 bp

non essential gene (Sassetti et al., 2003), and encodes a 72 a. a. putative

conserved membrane protein with unknown function. Rv1511 or gmdA is a

1023 bp non essential gene (Lamichhane et al., 2003), and encodes a 340 a.

a. GDP-mannose 4, 6 dehydratase, which is probably involved in nucleotide-

sugar metabolism.

The 3 plasmid DNAs encoding M. tuberculosis fused with tpa4 under the CMV

promoter were compared by protection assays to assess the CFUs, since TB

is a chronic disease. The ER-targeting leader sequence, tpa4, representing

the human tissue plasminogen activator signal sequence, has been

demonstrated to improve induction of immune response and protection with

DNA vaccines against M. tuberculosis (Delogu et al., 2002; Li et al., 1999).

Mice were vaccinated with 10 µg of DNA loaded onto PLG stabilized with PVA

and infected with 100-200 M. tuberculosis H37Rv by aerosol as decribed

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above. The Rv3407 DNA induced protection at day 30 and 60 post infection,

but Rv2520 and Rv1511 failed to protect mice from M. tuberculosis infection.

Surprisingly, the protection by 10 µg of Rv3407 DNA loaded onto PLG was

comparable to that of 100 µg of naked DNA at day 30, but DNA vaccination of

Rv3407 with or without PLG did not induce protection against M. tuberculosis

infection significantly at day 90. In contrast to that of L. monocytogenes

infection, CpG motifs in empty vector did not improve protection against M.

tuberculosis. Bacterial CpG motifs stimulate protection against L.

monocytogenes (Elkins et al., 1999) but they fail to enhance the protective

efficacy of subunit vaccine against M. tuberculosis (Hsieh et al., 2004). The

results in this dissertation support these reports.

4.2. Comparison of DNA delivery systems for vaccination

To compare the efficiency of DNA vaccine delivery, PLG stabilized with PVA,

PLG stabilized with CTAB, a novel encapsulating particle, VLP, and TmHU

were tested because these DNA carriers showed improved DNA transfer of

reporter genes in vitro and/or in vivo, and were regarded relatively safe

compared with lipid DNA carriers.

The efficiency of DNA vaccine delivery systems was evaluated by protection

assay with 10 µg of DNA at high infection dose of L. monocytogenes to verify

the results. One biodegradable particle, PLG stabilized with CTAB displayed

slightly increased protection compared with 10 µg of naked DNA, but was not

statistically significant. All other DNA carriers, i. e. PLG stabilized with PVA,

encapsulating biodegradable particle, VLP, and TmHU failed to improve

protection at all compared with 100 µg of naked DNA.

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The amount of DNA used in prevous reports varies (ranging 10 ng-150 µg)

(O'Hagan et al., 2001; Cui and Mumper, 2002; Briones et al., 2001; Singh et

al., 2000b). Most reports described that DNA delivery systems improved

protection or immune responses. In these studies, DNAs and carriers were

compared with the same amount of naked DNA, not with an optimized

vaccination protocol established. They focused on the improvement of

protection or immune response, not on the reduction of the amount of DNA

required for DNA vaccination, but the reduction of the amount of DNA is

important to reduce safety concerns and costs. Numerous factors might affect

optimal dose for DNA vaccination in addition to the delivery efficiency of DNA

carriers: expression efficiency of vector, which is dependent on promoter,

stability of mRNA and protein produced by DNA vaccine, immunogenicity of

antigen, prime-boost strategies (how many times and how often to administer),

the way of administration (i.m., s.c., intradermal, intranasal, or oral), and

infection system (Gurunathan et al., 2000; Colosimo et al., 2000). Furthermore,

there are many different kinds of biodegradable particles, for example, PLG

stabilized with PVA and PLG stabilized with CTAB, and many different

methods of production of biodegrabable particles. Therefore, the results in

previous reports are hardly comparable to the results in this study.

In a recent interesting report, 150 µg of M. leprae Hsp65-encoding plasmid

DNA was loaded onto PLG particles in two different ways: addition of DNA

after polymerization of PLG and addition of DNA at the beginning of reaction

(Johansen et al., 2003). These products were administered s.c. once while

150 µg of naked DNA was administered i.m. three times. As a result, neither

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DNA loaded onto PLG particles improved protection, nor immune response

compared with 3 times vaccination of naked DNA. Interestingly, when DNA

was added at the beginning of reaction, the vaccine efficacy was higher than

when DNA was added after polymerization although both of them were lower

than that of 3 times administration of naked DNA (Johansen et al., 2003). The

PLG particles used in this dissertation were mixed with DNA after

polymerization.

Additionally, the DNA loading capacity of PLG is quite low (20-50%) (Briones

et al., 2001) and PLG stabilized PVA prodeuced thick precipitates so much

that some of the precipitates could not pass through the needle. So the actual

amount of DNA loaded onto PLG to inject might be less than expected.

In many reports, reporter genes, such as β-galatosidase and green

fluorescent protein (GFP), were used to assess the potential of DNA delivery

but the actual pathogen for DNA vaccine was not tested (Cui et al., 2003; Cui

and Mumper, 2003; McKeever et al., 2002; Stern et al., 2003). For example,

TnHU proved the potential of DNA delivery in vitro and in vivo using lacZ gene

as a reporter gene (Esser et al., 2000) but no more papers have come out of

this.

Many methods to improve DNA vaccine delivery have been proposed,

including new biodegradable particles, PLG bound immunogenic lipid or other

immunomodulator, for instance, cholera toxin and lipid A, and antigen-

expressing VLP. Many trials did not prove to be efficient and safe enough for

use in humans. In another recent report, vaccine efficacy of 30 µg of

mycobacterial Hsp65-encoding DNA loaded onto PLG particles with or without

trehalose dimicolate (TDM) once was compared with that of 100 µg of naked

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DNA three times (Lima et al., 2003). Without TDM, DNA loaded PLG could not

improve vaccine efficacy significantly (Lima et al., 2003). TDM did not prove

safe enough. Cationic PLG particles formulated with CTAB was one of the

attempts to improve the DNA delivery efficacy, and the particles enhanced

transfection efficiency up to 100 fold in vitro (C. Oster et al., personal

comminication), but 4 out of 60 mice died after administration of the particle

while no mice died from naked DNA vaccination or DNA and other carriers.

The reason of the death is unclear, but CTAB is a strong detergent and safety

of PLG with CTAB in vivo is not proven yet. However, a recent paper showed

enhanced protective efficacy of DNA vaccine encoding Ag85A by absorption

onto cationic PLG particles (Mollenkopf et al., 2004)

The results from ELISA assay implicated another problem with the use of

proteins as DNA vaccine delivery system. The IgG1/IgG2a antibody titers in

blood sera from mice vaccinated with DNA and VLP or with DNA and TmHU

were seriously biased. Since VLP and TmHU themselves are proteins, they

elicit Th 2 and B cell immune responses after repeated administrations and

might induce non-specific immune responses or cross-priming, too (Skoberne

et al., 2002). Finally, many of the DNA delivery systems are not practically

easy to handle, nor stable at room temperature, and some of them are more

expensive than DNA. For example, usually, proteins should be purified and

stored at low temperature and the purification process takes time, money, and

expertise.

In conclusion, there are many kinds of DNA delivery systems, methods of

production of DNA delivery systems, and DNA vaccination protocols, so it is

very difficult to identify the best DNA vaccine delivery system. They should be

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______________________________________________________________

compared using same plasmid DNA and specific infection system. Above all,

development of more efficient and safer DNA delivery system for DNA

vaccination is still required and on the other hand, the improvement of DNA

vaccine itself is also worth taking into consideration.

4.3. Immune responses induced by DNA vaccination

Vaccines should elicit pathogen-specific immune responses. To determine

antigen-specific CD8

T cells induced by DNA vaccination against L.

monocytogenes, tetramer analysis was performed using MHC class I H2-

/LLO

91-99

tetramers. These tetramers stain antigen-specific CD8

T cell

populations in a quantitative way (Busch and Pamer, 1998; Busch and Pamer,

1999). As expected, mice at day 5 post secondary infection showed greatly

expanded CD8

tetramer

cell polulation and mice 7 weeks after the primary

infection also showed significant CD8

tetramer

cell population. Mice

vaccinated with 100 µg of naked DNA encoding mutant LLO showed

significant level of CD8+ tetramer+ cells comparable to long term immune T

cells after L. monocytogenes infection. These data imply that 5 days after the

last boost, many antigen-specific T cells induced by DNA vaccination were still

activated. However, mice vaccinated with 10 µg of DNA and PLG did not elicit

measurable CD8

tetramer

cells. There exist tetramer positive cell

populations after 7 days and 10 days after the last boost. These results

suggested DNA vaccination elicited antigen-specific CD8+ T cells.

In order to determine antigen-specific CD8+ T cells induced by DNA

vaccination against M. tuberculosis, a putative epitope from Rv1511

(GYVKFDQRYL) and one from Rv3407 (IPARRPQNL) and their affinity to

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MHC class I molecule were predicted by computer programs, MAPPP (MHC-I

Antigenic Peptide Processing Prediction) (Hakenberg et al., 2003) and

FragPredict, provided by Max-Planck-Institute for Infection Biology, Berlin,

Germany (http://www.mpiib-berlin.mpg.de/MAPPP/) (Mollenkopf et al.,

submitted; Nussbaum et al., 2003) and two MHC class I/peptide tetramers

were produced as described above. At day 7, 4 weeks after the last boost,

mice vaccinated with 100 µg of naked Rv1511 DNA showed increased CD8

tetramer

cell population, but at day 10, 20, 30, and 60, there was no

detectable tetramer+ cell population any more (data not shown). Mice

vaccinated with 100 µg of naked Rv3407 DNA were also tested to detect the

tetramer

cell population by MHC class I/putative epitope of Rv3407, but there

was no detectable tetramer+ cell population at day 14, 20, 30, and 60 (data

not shown). There are two possibilities to explain this onservation: one is that

the tetramer

cell population might be so rapidly retracted that it is not

detectable at day 10 any more, and the other and more likely one is that the

epitope prediction or affinity prediction might not be acrurate enough. The

affinity of MHC class I/peptide was predicted only by computer program, and

no more information is available. Thus, the half-life of MHC class I/peptide

might be too short to be used after several days, the putative epitope from

Rv3407 might not be immunodominant, or the affinity of peptide from Rv3407

to MHC class I might be much weaker than predicted.

It is generally believed that IFN-γ is a key cytokine in the immune response

against intracellular bacteria. Therefore, ELISpot assay was performed to

detect IFN-γ secreting cells. The results showed that DNA vaccination with the

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plasmid DNA encoding mutant LLO and the Rv3407 DNA induced IFN-γ

secreting cells responding to MHC class I-restricted peptides and to bacterial

proteins. The DNA vaccination with 100 µg of naked DNA encoding mutant

LLO successfully induced IFN-γ, but 10 µg of DNA and PLG did not.

Remarkably, DNA vaccination with 10 µg of Rv3407 DNA and PLG induced

IFN-γ secreting splenocytes at the early phase, one week after the last boost,

but not at the late phase, 7 weeks after the last boost. In contrast, DNA

vaccination with 100 µg of naked Rv3407 DNA did not induce IFN-γ secreting

splenocytes at the early phase, but did so at the late phase. Generally, these

results are consistent with protection data. Vector control also induced slightly

increased levels of IFN-γ secreting splenocytes, which implied innate immunity

was elicited by CpG motifs.

On the other hand, IFN-γ intracellular staining for FACS analysis was

performed, too, but no significant population of CD8+ IFN-γ+ cells was

detectable although mice infected with L. monocytogenes 7 weeks before

analysis showed increased CD8+ IFN-γ+ cell populations (data not shown).

There are many kinds of cell types producing IFN-γ, including NK cells, NK T

cells, and macrophages as well as Th 1 CD4+ cells. In addition, several recent

reports suggested DNA vaccination induced protective immune responses

against L. monocytogenes without IFN-γ (Barry et al., 2003; Yoshida et al.,

2001) and antigen presentation was efficient without IFN-γ (Skoberne and

Geginat, 2002). Taken together, the source of IFN-γ secreted after intracellular

bacterial infection and the role of IFN-γ in immunity against intracellular

bacteria should be investigated in more detail.

DIscussion

______________________________________________________________

To protect mice from intracellular bacteria, CD8

T cells play an important role.

The protection against L. monocytogenes is dependent mostly on CD8

cells. BCG vaccination induces predominantly CD4

T cell immune response

but optimal protection against M. tuberculosis requires both CD4

and CD8

cell immune responses (Kaufmann, 2001; Goonetilleke et al., 2003).

Therefore, CTL assays were performed. Firstly, conventional

Cr-release

assays were performed but failed to detect significant CTL activity. One of the

reasons was high spontaneous release (data not shown). Secondly, an in vivo

killing assay using CFSE labeled target cells was adopted to detect CTL

activity. This method is established to give quantitative and qualitative results

of actual in vivo killing and less spontaneous leakage (Aichele et al., 1997;

Sheehy et al., 2001). The activity of antigen specific CTL was detected by

peptide-loaded target cells stained with 5- (and 6-) carboxyfluorescein

diacetate succinimidylester (CFSE), which is a highly stable amine-reactive

reagent readily incorporated into cells. When membrane damage occurs, the

dye is almost instantaneously lost and the cells are no longer able to take up

or retain the charged dye. Several reports have shown that CFSE-prelabeled

target cells can be successfully evaluated to detect cytolysis (Sheehy et al.,

2001). The antigen-specific CTL activity was successfully detected 9 days

after both primary infection of L. monocytogenes and LCMV. However, 24

days after primary infection, only LCMV-primed mice showed significant CTL

activity. Similar results showed that rapid protection in vivo is long-lived after

immunization with LCMV but short-lived after vaccination with L.

monocytogenes (Ochsenbein et al., 1999). Therefore, the results imply CTL

activity induced by intracellular bacteria is rapidly retracted and the detection

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of CTL activity of long term immunity against intracellular bacteria requires

much more sensitive methods.

4.4. Conclusion

Several studies have demonstrated that DNA vaccines elicit CD8

T cell as

well as CD4

T cell responses, but effective DNA vaccine delivery systems

can enhance cellular uptake of DNA vaccines and/or facilitate intracellular

targeting of DNA to the cytoplasm and nucleus, and also reduce the amount of

DNA required. BALB/c mice were vaccinated with 100 µg of naked DNA

encoding p60, wild-type LLO, or non-hemolytic mutant LLO under the control

of a CMV promoter or 10 µg of DNA with 10 µg of VLP, 12 µg of TmHU, or 1

mg of PLG i. m. 3 times at 3 weeks intervals. Vaccination with naked DNA

encoding mutant LLO protected mice efficiently against L. monocytogenes but

no delivery system significantly improved the efficacy of DNA vaccine. IFN-γ

secreting CD8+ splenocytes responding to LLO

91-99

peptide were determined

by ELISpot assay and MHC class I-restricted, LLO

91-99

peptide specific CD8

splenocytes were assessed by the tetramer technique.

These results suggest that DNA encoding mutant LLO is a potent vaccine

candidate against L. monocytogenes and that DNA vaccination can induce

antigen-specific immune responses. More sensitive techniques are required to

detect memory/effector immune responses elicited by intracellular bacterial

antigens. Since memory/effector cells from intracellular bacterial pathogens

were short lived, and DNA vaccines express just one antigen or a few

antigens at relatively low levels, there might be natural limitations to detect

memory/effector cells induced by DNA vaccination against intracellular

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bacteria. In addition, critical reviews and optimized comparisons are required

to use DNA vaccine carrier systems.

As previously reported, DNA vaccine candidates were identified by comparing

the proteomes of M. tuberculosis H37Rv and M. bovis BCG Chicago, and

DNA vaccination with Rv3407 DNA provided protection against M.

tuberculosis H37Rv by aerosol at post-infection day 30 and 60. DNA

vaccination with Rv3407 DNA induced IFN-γ secreting CD8

splenocytes

responding to peptides of putative epitopes derived from Rv3407 as

determined by ELISpot. The DNA vaccination also induced MHC class I-

restricted, putative epitope-specific CD8

splenocytes determined by FACS

analysis with Rv1511 peptide/H-2K

tetramers. Vaccination with 10 µg of DNA

loaded onto PLG also showed protection but antigen-specific immune

response could not be detected.

These results suggest that Rv3407 is the best DNA vaccine candidate against

M. tuberculosis, and DNA vaccine encoding a M. tuberculosis-specifically

expressed gene can induce protection against M. tuberculosis and antigen-

specific immune response, but awaits improvement.

The improvement of DNA vaccine itself is worth taking into consideration.

There have been several efforts to enhance the efficacy of DNA vaccines:

developing new viral or non-viral vectors, insertion of gene regulatory

elements in plasmid backbone, and exchanging codon usages to frequently

used in mammals (Doria-Rose and Haigwood, 2003). There are reports on

enhanced immunogenicity against M. tuberculosis by new vectors, for

example, alphavirus plasmid replicon (Kirman et al., 2003) and recombinant

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modified vaccinia virus Ankara (Goonetilleke et al., 2003). Fusion to tpa4

signal sequence or ubiquitin intracellular targeting sequence also improved

vaccine efficacy against M. tuberculosis (Delogu et al., 2002; Li et al., 1999).

Optimization of codon usage of DNA vaccine was reported to enhance

effective immune response against L. monocytogenes (Uchijima et al., 1998).

A linear minilalistic vecotor (MIDGE) is also available to reduce any side-effect

owing to bacterial plasmid backbones, including CpG motif effect (Lopez-

Fuertes et al., 2002). Construction of DNA encoding fusion protein of different

antigens and combinations of different DNA vaccines, so-called DNA vaccine

cocktail could be considered, too (Delogu et al., 2002).

Protection levels with DNA vaccination against challenge with M. tuberculosis

have been generally less effective than BCG vaccination alone. Thus it would

also be worthwhile taking prime-boost strategies. The prime-boost strategies

are effective at generating high levels of T cell memory (Ramshaw and

Ramsay, 2000). This approach is being tested in humans using a DNA

vaccine against malaria (Moorthy et al., 2004), and has shown promising

results against HIV (Takeda et al., 2003).

Several prime-boost strategies using DNA vaccine have been tested: DNA-

protein, DNA-recombinant virus expressing the same respective antigens,

DNA-BCG, and BCG-DNA against M. tuberculosis (Britton and Palendira,

2003). Recently, several studies have demonstrated the efficacy of prime-

boost vaccination strategies in generating cellular immunity to M. tuberculosis

(McShane et al., 2001; Tanghe et al., 2001). Especially, vaccination of DNA

encoding Ag85A as prime and BCG as boost markedly improved protection

(Feng et al., 2001) and mice that had been intranasally vaccinated with BCG

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and then boosted with a recombinant vacinia virus expressing Ag85A had

remarkable reduction in bacterial load in the lungs following aerosol challenge

with M. tuberculosis (Goonetilleke, et al., 2003). The 3 plasmid DNAs used in

this study was already tested the efficacy as a boost vaccine after BCG prime

(Mollenkopf et al., submitted). Especially, BCG prime-Rv3407 DNA boost

vaccination showed superior protection to BCG alone. Since huge populations

in the world were previously administered with BCG, the prime-boost strategy

of BCG as prime and DNA vaccine as boost is regarded as a more important

and practical approach. In addition, recombinant BCG expressing M.

tuberculosis antigens and deletion mutant M. tuberculosis as TB vaccine

candidates have the dangerous potential to become more virulent, and

repeated administration of DNA vaccines also have potentially harmful side

effects to induce autoimmunity or immunity against the vectors. In comparison,

the prime-boost strategies with BCG and DNA vaccines have the advantage

of less safety concerns. Since BCG elicits mostly CD4

immune responses,

subunit vaccines which can induce CD8

immune responses would be

particularly advantageous. Ergo, the next step to improve TB vaccine should

be to adopt prime-boost strategies.

Summary

_______________________________________________________________

5. Summary

L. monocytogenes induces an acute course of infection in mice and the

immune response is mediated by CD4

Th1 cells and CD8

cytotoxic T cells.

Protection is mostly mediated by CD8

T cells, which are directed against two

immunodominant antigens, LLO and p60. Several studies have demonstrated

that DNA vaccines elicit CD8

as well as CD4

T-cell responses, but low

efficiency of DNA delivery is one of the disadvantages of DNA vaccines. The

purpose of this thesis was to compare different DNA vaccine carrier systems,

to identify the most effective one(s), and to apply the results to TB vaccination.

I vaccinated BALB/c mice with 100 µg of naked DNA encoding p60, wild-type

LLO, or non-hemolytic mutant LLO under the control of a CMV promoter or 10

µg of DNA with 10 µg of VLP, 12 µg of TmHU, or 1 mg of PLG i. m. 3 times at

3 weeks intervals. Vaccination with naked DNA encoding mutant LLO

protected mice efficiently against L. monocytogenes but none of the delivery

systems tested significantly improved the efficacy of 10 µg of DNA vaccine

compared with vaccination with 100 µg of naked DNA. IFN-γ secreting CD8

splenocytes responding to LLO

91-99

peptide were determined by ELISpot

assay and MHC class I-restricted, LLO

91-99

peptide specific CD8

splenocytes

were assessed by the tetramer technique. These results suggest that DNA

encoding mutant LLO is a promising vaccine candidate against L.

monocytogenes and that DNA vaccination can induce antigen-specific

immune responses. However, further optimization of these carrier systems is

required before application to DNA vaccination. DNA vaccination with Rv3407

DNA provided protection against M. tuberculosis H37Rv aerosol infection.

DNA vaccination with antigen Rv1511 or Rv3407 induced IFN-γ secreting

CD8

splenocytes responding to peptides of putative epitopes derived from

Rv1511 or Rv3407, respectively, as determined by ELISpot. The DNA

vaccination also induced MHC class I-restricted, putative epitope-specific

CD8

splenocytes as determined by FACS analysis with Rv1511 peptide/H-

tetramers. Vaccination with 10 µg of Rv3407 DNA loaded onto PLG also

showed partial protection. These results suggest that DNA vaccines encoding

M. tuberculosis-specific genes can induce pathogen-specific protection

against M. tuberculosis, but await further improvement.

Summary

______________________________________________________________

6. Zusammenfassung

Die Listeriose ist eine akute Infektion, und die Immunantwort wird durch CD4

Th1 Zellen und CD8

zytotoxische T Zellen vermittelt. Der Infektionsschutz

wird überwiegend durch CD8

getragen, die mit zwei immundominanten

Antigenen, LLO und p60, reagieren. Verschiedene Studien haben gezeigt,

dass DNA Impfungen sowohl CD8

T Zell- als auch CD4

T-Zell-Antworten

erzeugen, aber die geringe Effizienz der DNA-Aufnahme ist einer der

Nachteile von DNA-Impfungen. Ziel dieser Arbeit ist es, durch den Vergleich

verschiedener DNA Trägersysteme das effektivste System zu identifizieren,

und die erhobenen Resultate auf die Tuberkulose-Impfung zu übertragen. Ich

habe BALB/c Mäuse mit 100 µg reiner DNA, die p60, Wildtyp LLO oder

mutiertes, nicht hämolytisches LLO unter der Kontrolle eines CMV Promotors

kodiert, geimpft, oder mit 10 µg DNA zusammen mit 10 µg VLP, 12 µg TmHU,

oder 1 mg PLG. Die Impfung mit reiner, für mutiertes LLO kodierender DNA

schützte die Mäuse effizient gegen L. monocytogenes, aber keines der

untersuchten Transportsysteme verbesserte die Effizienz der DNA Impfung

(10 µg) verglichen mit der Gabe von 100 µg reiner DNA. IFN-γ sezernierende

CD8

Milzzellen, die auf das LLO

91-99

Peptid reagierten, wurden durch

ELISpot Tests bestimmt, und MHC Klasse I-restringierte, LLO

91-99

Peptid-

spezifische CD8

Milzzellen mittels Tetramer Färbung untersucht. Die

Ergebnisse legen nahe, dass modifiziertes LLO ein wirkungsvoller Impfstoff-

Kandidat gegen L. monocytogenes ist, und dass dieser DNA-Impfstoff

antigen-spezifische Immunantworten induziert. Weitere Optimierung ist jedoch

notwendig, bevor DNA-Impfstoff-Träger-Systeme eingesetzt werden können.

DNA-Impfung mit dem Antigen Rv3407 erzeugte Schutz gegen Aerosol-

Infektion mit M. tuberculosis H37Rv. Die Mäuse wurden mit 100 µg DNA durch

drei i. m. Injektionen in dreiwöchigen Intervallen geimpft. DNA Impfung mit den

Antigenen Rv1511 oder Rv3407 induzierte IFN-γ sekretierende CD8

Milzzellen, die auf die Peptide der putativen dominanten Epitope von Rv1511

und Rv3407 reagierten, wie durch ELISpot Test nachgewiesen. Die DNA

Impfung induzierte auch MHC Klasse I-restringierte, epitopspezifische CD8

Milzzellen, die mittels FACS Analyse mit Rv1511 Peptid/H-2K

Tetrameren

bestimmt wurden. Eine Impfung mit 10 µg Rv3407 DNA, die auf PLG

aufgebracht war, induzierte Protektion. Diese Ergebnisse lassen darauf

schließen, dass ein DNA-Impfstoff, welcher ein M. tuberculosis-spezifisches

Gen kodiert, antigen-spezifischen Schutz gegenüber M. tuberculosis

vermitteln kann, jedoch einer weiteren Optimierung bedarf.

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Abbreviations

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8. Abbreviations

Ab antibody

Ag antigen

AIDS acquired immune deficiency syndrome

ββ

β2m beta 2-microglobulin

BCG bacille Calmette-Guérin

BCIP/NBT 5-Bromo-4-Chloro-3-Indolyl Phosphate/Nitro Blue Tetrazolium

BSA bovine serum albumin

CD cluster of differentiation

CFSE 5- (and 6-) carboxyfluorescein diacetate succinimidylester

CFU colony forming unit

CTAB hexadecyltrimethyl ammonium bromide

CTL cytolytic T lymphocyte

Cy5 cyanine 5

DMEM Dulbeco’s modified Eagle’s medium

DC dendritic cells

DC-SIGN DC-specific intercellular adhesion molecule-3 grabbing nonintegrin

DW distilled water

ELISA enzyme-linked immunoabsorbent assay

ELISpot enzyme-linked immunospot assays

FACS fluorescence-activated cell sorter

FITC fluorescein-5-isothiocyatate

HIV human immunodeficiency virus

HPV human papilloma virus

Abbreviations

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111

Ig immunoglobulin

IFN interferon

IL interleukin

i.m. intramuscular

IPTG isopropyl-β-d-thiogalactopyranoside

i.v. intravenous

LAM lipoarabinomannan

LCMV lymphocytic choriomeningitis virus

LLO lysteriolysin O

mAb monoclonal antibody

ME mercaptoethanol

MHC major histocompatibility complex

MMR macrophage mannose receptor

NK natural killer

PBS phosphate buffered saline

PE phycoerythrin

PFA paraformaldehyde

PFU plaque forming unit

PLG poly(lactide-co-glycolide)

PPD purified protein derivative

PVA polyvinylalcohol

s.c. subcutaneous

TAP-1 transporter associated with antigen processing 1

TB tuberculosis

TDM trehalose dimicolate

Abbreviations

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112

Th 1 T helper type 1

TLR Toll-like receptor

TmHU histone-like protein from Thermotoga maritima

TNF tumor necrosis factor

VLP virus-like particle

w/w weight/weight

Acknowledgements

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9. Acknowledgements

I appreciate Prof. Dr. Stefan H. E. Kaufmann for making everything I have

done here possible and Prof. Dr. Roland Lauster for taking care of the final

steps of the Ph. D. thesis. I also deeply appreciate Dr. Peter Seiler and Dr.

Leander Grode for helpful discussions and encouragement by all means.

I thank Dr. Christine Oster, Prof. Dr. Thomas Kissel, and Dr. Jürgen Hess for

providing DNA carriers and superb collaboration, Dr. Miso Kursar and Dr.

Hans-Willi Mittruecker for invaluable advices, and Dr. Robert Hurwitz for

skillfully producing tetramers. My thanks go to Ellen Heineman, Peggy Mann,

Maik Stein, Tharsana Tharmalingam, and Manuela Stäber for technical

supports. I am grateful to Dr. Ulrich Steinhof, Dr. Peter Aichele, Dr. Helmy

Rachman, Dr. Jong Seok Lee, Nathalie Jänner, Sabine Seibert, Chun Kim,

Ulrike Rapp, Markus Koch, and Marcus Niemeyer for warm encouragement

and conversation. I also thank Mrs. Yvonne Bennett for the help to improve

this dissertation and Nathalie Jänner for German language skills. I am so sorry

that I cannot include all colleagues at Max-Planck-Institute for Infection

Biology, but I appreciate all of them from the bottom of my heart.

My special thanks go to Prof. Dr. Sang Dai Park for giving me a chance to

start graduate study and to my family and my old friends. Bo Young,

Sunkyoung, Dong Im, Sang Hee and I have been struggling to live our own

lives and encouraging each other for a long time. I strongly believe Asian girls

should be much more encouraged and supported to do what they want to do

and someday we will.

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10. Curriculum Vitae

Name: (First Name) Nayoung (Last Name) Kim

Date of Birth: 6, July, 1970

Place of Birth: Seoul, Korea

Nationality: Republic of Korea (South Korea)

Address: (Residence) Ahornallee 40/41 D-14050 Berlin, Germany

(Lab) Max-Planck Institute for Infection Biology, Department of

Immunology, Schumannstr. 21/22, D-10117 Berlin,

Germany

Telephone Number: (Office) +49-(0)30-28460-562

(Lab) +49-(0)30-28460-545

(FAX) +49-(0)30-28460-505

(Mobile) +49-(0)174-5186687

E.mail address: kim@mpiib-berlin.mpg.de

[email protected]

Education

2004. Ph.D. candidate

Feb.1997. Department of Molecular Biology, College of Natural Sciences,

Seoul National University (finished the course work of Ph.D.

program)

Feb.1995. Department of Molecular Biology, College of Natural Sciences,

Seoul National University (M.Sc.)

Feb.1993. Department of Molecular Biology, College of Natural Sciences,

Seoul National University (B.Sc.)

Feb.1989. Sacred Heart Girls’ High School

Work Experience

Aug.2001 – present. Max-Planck Institute for Infection Biology, Department of

Immunology (Ph. D. Student)

Oct.2000 - Jun. 2001. Microarray Center, Biomedlab Institute, Biomedlab Co.

(Senior Researcher)

Nov.1999 - May.2000. Department of Pathology, College of Medicine,

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115

Seoul National University (Researcher)

Mar.1996 - Aug.1996. Department of Molecular Biology, College of Natural

Sciences, Seoul National University (Teaching

Assistant)

Jan.1993 - Oct.1999. Laboratory of Molecular Immunology, Institute of

Molecular Biology and Genetics, Seoul National

University (Graduate Student)