INVESTIGATION AND CHARACTERIZATION OF THE ENHANCED HUMORAL RESPONSE FOLLOWING IMMUNIZATION WITH THE LETHAL AND EDEMA OF ANTHRACIS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of

Philosophy in the Graduate School of The Ohio State University

By

Karen E. Brenneman, B. S.

*****

The Ohio State University

2007

Dissertation Committee: Approved by: Dr. Darrell R. Galloway, Adviser

Dr. Brian Ahmer ______Dr. Paula W. Bryant Adviser Dr. Richard F. Mortensen Graduate Program in Microbiology

ABSTRACT

Bacillus anthracis is the causative agent of the disease anthrax. Although the organism produces multiple virulence factors during an infection, a large portion of the disease pathology can be attributed the effects of two A-B toxins: lethal and edema .

These toxins selectively target innate immune effector cells, preventing containment and clearance of the developing infection. Additionally, lethal and edema toxin have been shown to impair the priming and proliferation of adaptive immune cells. Despite these immunosuppressive effects, lethal toxin has been shown to stimulate a strong immune response the context of an immunization. Co-immunization with the two components of lethal toxin (lethal factor and protective ) increases response to each component.

In this work, the functional activities of lethal toxin were investigated in order to identify the toxin actions responsible for the enhanced immune response. The effects of each step of the cellular intoxication process were examined, as the toxin specifically target immunological cells and shuttle through different antigen processing compartments during the course of intoxication. The catalytic activity of lethal toxin was also examined, since the downstream consequences of toxicity on a developing immune response have not been characterized. To study this, single amino acid changes were

ii made in each of the lethal toxin components in order to remove specific functional activities of the toxin complex. The immune response generated by toxin combinations lacking a single functional activity were then compared to the response stimulated by wild type lethal toxin.

The work from this project revealed a complicated relationship between lethal toxin and the . The enhanced antibody response depended on the catalytic activity of lethal toxin and was observed only when wild type lethal toxin was present in an immunization. Although the inactive forms of lethal toxin did not produce an enhanced immune response, altering the functional abilities of the toxin complex illuminated a fundamental immunological difference between the two components of lethal toxin. A strong antibody response to lethal factor depended on the production of

IFN- and the cytosolic localization of this antigen. In contrast, the antibody response to protective antigen required endosomal processing and the concurrent production of IL-4.

These two immune responses antagonized each other, resulting in a competition between the antibody responses against the lethal toxin components that was observed under all circumstances tested.

This project also determined that the combination of edema toxin components

(protective antigen and edema factor) enhanced antibody production, and subsequently investigated the functional mechanism by which this response was provoked. Although edema toxin forms a protein complex analogous to lethal toxin, edema toxin interacted

iii with the immune system in a manner quite different from lethal toxin. The combination of the edema toxin components was able to act as an adjuvant on the immune system. An enhanced immune response to edema toxin did not depend on either the catalytic activity of the toxin or the cellular intoxication process. Competition between the antibody responses against the edema toxin components was observed to a lesser degree than for the lethal toxin components. A robust immune response to edema factor was accompanied by the production of both IL-4 and IFN-, and thus competition between the antibody responses to the edema toxin components was observed only when edema factor was localized to the cytosol and separated from protective antigen. Co-localization of edema factor and protective antigen in the endosome resulted in an enhanced immune response to both proteins.

iv Dedicated to my husband Rob, who still isn’t sure what do,

but who has always supported and encouraged me

v ACKNOWLEDGMENTS

This document would not be possible without the help of numerous individuals.

First, I would like to thank my adviser, Dr. Darrell Galloway, for his commitment to me and to this project. My committee also deserves recognition for their flexibility in allowing this project to be completed under unusual circumstances. Thanks go to Dr. Les

Baillie and CAPT Al Mateczun for providing a temporary home with the NMRC/BDRD group. Without the help of CDR Gail Chapman, LT Matthew Weiner, Stephanie Gray,

Tatiana Pervaia and Gordon Heissler, it would not have been possible to undertake such comprehensive animal studies. I am grateful to the scientists of the BDRD Vaccine group – Drs. Arya Akmal, Mark Albrecht, Christian Darko, Olga Pomerantseva, John

Charles Rodenberry and Ramjay Vatsan – for all their insights and suggestions. Special thanks go to CDR Joe Morris, who taught me everything I needed to know about protein purification and endotoxin. Special thanks also go to Dr. Stan Goldman, who has been invaluable in challenging me and providing focus to my usually scattered ideas. I am extremely grateful to Dr. Bart Legutki for teaching me the importance of planning ahead, and to Matthew Bell for teaching me the importance of having all the controls. Both of them have been more than fellow graduate students over the past six years and I am privileged to have had their support, encouragement and friendship.

vi VITA

10 September 1978 ……………………………Born – Raleigh, North Carolina, USA

2000 …………………………………………..B. S. Biochemistry and Molecular Biology, Pennsylvania State University

2000 – 2005 …………………………………...Graduate Teaching and Research Associate, The Ohio State University

2005 – present …………………………………Research Associate, Naval Medical Research Center, Biological Defense Research Directorate

PUBLICATIONS

Hermanson, G., V. Whitlow, S. Parker, K. Tonsky, D. Rusalov, P. Lalor, M. Komai, R. Mere, M. Bell, K. Brenneman, A. Mateczun, T. Evans, D. Kaslow, D. Galloway and P. Hobart. A cationic -formulated plasmid DNA vaccine confers sustained antibody-mediated protection against aerosolized anthrax spores. PNAS. 2004. 101(37):13601-13606.

FIELDS OF STUDY

Major Field: Microbiology

Minor Field: and Molecular Biology

vii TABLE OF CONTENTS

Page Abstract ……………………………………………………………………………..….ii

Dedication ………………………………………………………………………...... v

Acknowledgments ………………………………………………………………..…...vi

Vita ………………………………………………………………………………...... vii

List of Tables ……………………………………………………………………….....xi

List of Figures …………………………………………………………………….…..xii

List of Abbreviations ………………………………………………………………....xv

Chapters

1. Introduction ………………………………………………………….…...... 1

1.1 Bacillus anthracis .………………………….…………………………..1

1.1.1 Microbiology …………………………………………………….....1 1.1.2 Virulence factors and the regulation of virulence ………………….4

1.2 The bipartite anthrax toxins ……...………………………………...…..8

1.2.1 Protective antigen ………………………………………………….9 1.2.2 Lethal factor ………………………………………………………14 1.2.3 Edema factor ……………………………………………………...20

1.3 Anthrax: disease, prevention and treatment ………………………….23

1.3.1 Pathogenesis and disease ……....…………………………………23 1.3.2 Anthrax vaccination ………………………………………………28

1.4 Statement of the problem ……………………………………………..31 viii 2. Materials and Methods ………………………………………………………..33

2.1 Toxin mutagenesis and plasmid construction ………………………..33

2.2 Protein purification ……………………………………………………38

2.3 Characterization of protein activity ………………………………….46

2.4 Animal manipulations and immunological analyses …………………56

3. The humoral immune response to lethal toxin is altered by cellular intoxication as well as MEK proteolysis ……………………………………...68

3.1 Introduction …………………………………………………………...68

3.2 Results ………………………………………………………………...72

3.2.1 Characterization of lethal toxin mutants ……………………...72 3.2.2 Immunization with non-proteolytic mutants of lethal toxin ….78 3.2.3 Immunization with lethal toxin mutants deficient in cellular intoxication …………………………………………...82

3.3 Discussion …………………………………………………………….85

4. Edema toxin is an adjuvant whose activity does not depend on either cAMP production or cellular intoxication …………………………………………..115

4.1 Introduction ………………………………………………………….115

4.2 Results ……………………………………………………………….118

4.2.1 Characterization of edema toxin mutants …………………...118 4.2.2 Immunization with edema toxin mutants lacking adenylyl cyclase activity ………………………………………………123 4.2.3 Immunization with edema toxin mutants deficient in cellular intoxication …………………………………………126

4.3 Discussion …………………………………………………………...131

ix 5. The antibody titers to lethal and edema factor are increased by co-administration of bacterial products with inactive toxin …………………………………….154

5.1 Introduction ………………………………………………………….154

5.2 Results ……………………………………………………………….157

5.2.1 Immunogenicity of inactive lethal and edema toxin components prepared without endotoxin removal …………...... 157 5.2.2 Immunization with truncated lethal toxin in the presence of purified lipopolysaccharide and monophosphoryl lipid A ....159

5.3 Discussion …………………………………………………………...161

Conclusions …………………………………….……………………………………172

References …………………………………………………………………………...177

x LIST OF TABLES

Table Page

2.1 Functional mutants of the B. anthracis toxin proteins …………………..……63

2.2 Plasmid vectors used in this study ……………………………………………64

2.3 Bacterial strains used in this study ……………………………………………64

2.4 Point mutations introduced into the B. anthracis toxin components …………65

2.5 Expression conditions for recombinant B. anthracis proteins …...…..……….66

3.1 responses to lethal toxin components deficient in complex formation …………………………………………………………………….110

3.2 Cytokine responses to lethal toxin components deficient in cellular receptor binding ………………………………………………………………………112

3.3 Cytokine responses to lethal toxin components deficient in translocation ….114

4.1 Cytokine responses to edema toxin components deficient in complex formation …………………………………………………………………….149

4.2 Cytokine responses to edema toxin components deficient in cellular receptor binding ………………………………………………………………………151

4.3 Cytokine responses to edema toxin components deficient in translocation ...153

5.1 Effect of endotoxin on the lethal and edema factor titers in the presence of protective antigen ………………………………………………………...168

xi LIST OF FIGURES

Figure Page

2.1 SDS-PAGE visualization of purified PA, LF and EF proteins ……………….67

3.1 Structure of lethal factor bound to the amino terminal portion of Mek1 ……..94

3.2 Immunoblot of Mek1 proteolysis by LF, LF687 and LFn ……………………95

3.3 Structure of activated protective antigen ……………………………………..96

3.4 Gel mobility shift assay for complex formation by PA and LF binding mutants ……………………………………………………………….97

3.5 Sandwich ELISA to measure complex formation by PA and LF binding mutants ………………………………………………………………..98

3.6 Comparison of cellular binding by PA83 and PA682 on J774A.1 ……………………………………………………………….....99

3.7 Immunofluorescence control reactions in J774A.1 macrophages …………..100

3.8 Intracellular localization of wild type lethal toxin components ……………..101

3.9 Intracellular localization of lethal toxin mutants deficient in translocation ....102

3.10 Survival of J774A.1 macrophages following intoxication with lethal toxin mutants ………………………………………………………….103

3.11 Serum IgG anti-PA responses to lethal toxin components deficient in MEK proteolysis …………………………………………………………………...104

3.12 Serum IgG anti-LF responses to lethal toxin components deficient in MEK proteolysis …………………………………………………………………...105

xii 3.13 Neutralizing antibody response to lethal toxin components deficient in MEK proteolysis ………………………………………………………….107

3.14 Serum IgG responses to lethal toxin components deficient in complex formation …………………………………………………………………….109

3.15 Serum IgG responses to lethal toxin components deficient in cellular receptor binding ……………………………………………………………..111

3.16 Serum IgG responses to lethal toxin components deficient in translocation …………………………………………………………………113

4.1 Structure of edema factor bound to calmodulin and ATP …………………..139

4.2 Intracellular cAMP levels following intoxication with edema factor mutants lacking adenylyl cyclase activity …………………………………...140

4.3 Structure of activated protective antigen ……………………………………141

4.4 Gel mobility shift assay for complex formation by PA and EF binding mutants ……………………………………………………………...142

4.5 Sandwich ELISA measuring complex formation by PA and EF binding mutants ………………………………………………………………143

4.6 Intracellular cAMP levels following intoxication with protective antigen mutants deficient in cellular intoxication ……………………………………144

4.7 PA-specific serum IgG responses to edema toxin components deficient in adenylyl cyclase activity ...…………………………………………………..145

4.8 EF-specific serum IgG responses to edema toxin components deficient in adenylyl cyclase activity ...…………………………………………………..146

4.9 Serum IgG responses to edema toxin components deficient in complex formation …………………………………………………………………….148

4.10 Serum IgG responses to edema toxin components deficient in cellular receptor binding ……………………………………………………………..150

xiii 4.11 Serum IgG responses to edema toxin components deficient in translocation …………………………………………………………………152

5.1 Endpoint titers of mice immunized with inactive lethal toxin ………………164

5.2 Endpoint titers of mice immunized with truncated edema toxin ……………165

5.3 Serum IgG responses of mice immunized with inactive lethal toxin ……...166

5.4 Serum IgG responses of mice immunized with truncated edema toxin ……..167

5.5 Serum IgG responses of mice immunized with an endotoxin-free, truncated form of lethal toxin ………………………………………………………….169

5.6 Serum IgG responses of mice immunized with a truncated form of lethal toxin in the presence of lipopolysaccharide ……………………………………….170

5.7 Serum IgG responses of mice immunized with a truncated form of lethal toxin in the presence of monophosphoryl lipid A …………………………………171

xiv LIST OF ABBREVIATIONS

ABTS – 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) ATP – adenosine triphosphate BSA – bovine serum albumin cAMP – adenosine 3’,5’-cyclic monophosphate CTL – cytotoxic T DMEM – Dulbecco’s modified Eagle’s medium EDTA – ethylenediaminetetraacetic acid ELISA – enzyme-linked immunosorbent assay ELISPOT – enzyme-linked immunosorbent spot assay EU – endotoxin unit (equivalent to 0.1 ng/ml) FITC – fluorescein isothiocyanate HEPES – N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) IL – interleukin IFN – interferon gamma IPTG – isopropyl -D-1-thiogalactopyranoside kDa – kilodalton LB – Luria Bertani LPS – lipopolysaccharide MHC (I or II) – major histocompatibility complex (class I or II) MWCO – molecular weight cutoff NP-40 – nonylphenyl-polyethylene glycol N-terminal/terminus – amino terminal/terminus PAGE – polyacrylamide gel electrophoresis PBS – phosphate buffered saline PBST – phosphate buffered saline with 0.1% Tween 20 PCR – polymerase chain reaction SDS – sodium dodecyl sulfate TAE – Tris acetate EDTA TEMED – N,N,N’,N’- tetramethylethylenediamine TLR – Toll-like receptor TMB – 3,3’,5,5’-tetramethylbenzidine dihydrochloride TNF – tumor necrosis factor alpha TRITC – tetramethylrhodamine isothiocyanate

xv CHAPTER 1

INTRODUCTION

1.1 Bacillus anthracis

1.1.1 Microbiology

Bacillus anthracis is a gram positive, facultatively aerobic soil microbe that belongs to Bacillus subgroup 1 [1]. Unlike many Bacillus species, B. anthracis is nonmotile. B. anthracis is also non-hemolytic on sheep blood agar. This is an important diagnostic characteristic that distinguishes it from many closely related species. Like all bacilli, B. anthracis is a spore-forming organism and thus has two phases of life: vegetative and metabolically dormant endospore. Under nutrient-rich conditions, vegetative cells proliferate. Once nutrients are depleted or conditions become unfavorable for growth, the cells sporulate. The spores are resistant to a wide variety of harsh circumstances [2, 3] and can persist in the soil for long periods of time [4-6].

B. anthracis is most closely related to Bacillus cereus and Bacillus thuringiensis, although it also has sequence homology to Bacillus mycoides [1, 7-10]. Genetically, this is a very diverse group of organisms [11]. Although all members of this group are normally considered saprophytes, many strains have the ability to cause infection in

1 mammalian hosts. B. anthracis is the causative agent of the disease anthrax. Anthrax is primarily a disease of herbivores, but can develop in humans. B. cereus is a ubiquitous soil microbe, but is also an opportunistic in humans. It most commonly causes food poisoning [12], but has also been associated with soft tissue infections [13]. B. thuringiensis is another common soil microbe that is well known as an insect pathogen

[14], but is also able to cause opportunistic infections in humans [15]. B. anthracis is most closely related to the pathogenic strains of B. cereus and B. thuringiensis, even though the types of infection caused by each species are quite different [16, 17].

B. anthracis is unusual among bacterial species, in that the species itself shows very little diversity [18, 19]. Analysis of the Bacillus cereus group (which includes B. cereus, B. thuringiensis and B. anthracis) by conventional methods groups all B. anthracis isolates together as a phylogenetically monomorphic species [8, 17, 20, 21].

Distinguishing separate B. anthracis strains has only become possible in recent years by increasing the amount of genome analyzed in each strain [1, 22, 23]. This lack of diversity across the species indicates a clonal population structure [22, 24, 25] indicative of either a very recent origin or population size bottlenecks associated with the spread of this disease.

In addition to its chromosome, B. anthracis carries two large plasmids. Both plasmids are essential for virulence [26] and differences in the virulence of various B. anthracis strains have been linked to the plasmid copy number as well as the strain lineage [27]. The larger of the two plasmids, pXO1, is approximately 180 kb and contains the anthrax toxin genes pagA, lef and cya [28] as well as the virulence regulator atxA [29] and a germination operon [30]. The virulence factors contained on pXO1 are

2 part of a pathogenicity island, which is flanked by inverted IS1627 elements and contains multiple elements involved in horizontal transfer, including insertion elements, integrases and transposases [31]. The smaller plasmid, pXO2, is approximately 95 kb and contains the capsule genes capBCAD [28]. Both plasmids appear to be subject to internal rearrangements. Inversion of the pathogenicity island on pXO1 has been reported [24], as has the spontaneous loss of the capsulated phenotype in pXO2+ strains [32].

Additionally, the horizontal transfer of one or both plasmids to related Bacillus species seems highly likely. Recent studies have shown anthrax-like disease cases linked to B. cereus strains carrying pXO1-like plasmids [25, 33, 34].

Although B. anthracis was identified and characterized in the late 19th century, only the part of its life cycle pertaining to infection and disease is currently understood.

Anthrax spores, either from soil or contaminated animal parts, are the infectious agent of anthrax [35]. Once inside a mammalian host, the high nutrient concentrations trigger germination, although there may be host-specific germination factors as well [30]. As infection progresses, fatal bacteremia develops and the vegetative bacilli can reach concentrations of 109 cells/mL in the blood [36]. Sporulation does not appear to occur inside the host [37], perhaps because once the available nutrients are depleted in the dead or dying host, the oxygen tension is too low for sporulation [38] or possibly due to the repression of sporulation by the virulence gene regulator AtxA [39]. Once the host dies, the vegetative bacilli are released back into the environment [40] to either sporulate or continue in a non-pathogenic lifestyle. The efficiency of sporulation following environmental release appears to be low [40]. However, the vegetative cells do not seem to do much better, as B. anthracis requires high levels of nutrients in order to maintain

3 vegetative cell growth [41]. Although related Bacillus species routinely undergo life cycles of germination, vegetative growth and sporulation in the soil as environmental conditions dictate, it remains unclear how well B. anthracis can survive outside of a mammalian host. It may be that the few bacilli that sporulate following infection simply persist until environmental conditions are permissible and then undergo a limited amount of growth, leading to a “hot spot” of soil contaminated by anthrax [42]. It may also be that B. anthracis is able to survive by exploiting the high nutrient concentrations available in the rhizosphere of plants [43], thus allowing multiple rounds of vegetative growth following re-entry into the environment.

1.1.2 Virulence factors and the regulation of virulence

As a pathogen, B. anthracis has several virulence factors that allow it to establish infection in a host and then to evade the host defenses. The major virulence factors are the two bipartite toxins, lethal and edema toxin, and the poly-D-glutamic acid capsule, all of which are encoded by the plasmids. There are also chromosomally encoded virulence factors whose roles are just now beginning to be investigated. The virulence factors of B. anthracis focus on impairing the innate immune system long enough to establish a successful infection. Thus, the major targets are the effector functions of and macrophages.

The most studied virulence factors of B. anthracis are the two bipartite toxins: lethal and edema toxin. The genes coding for the toxins are contained on the larger of the two virulence plasmids, pXO1 [31]. Toxin gene expression begins shortly after germination and can be detected while the vegetative bacilli are still contained within the [44]. The two toxins are essential for virulence, as strains missing the toxin

4 genes are severely attenuated [26, 45]. However, questions about the roles of the toxins in infection remain. Although lethal and edema toxin are both toxic to mice [46, 47], and removal of the infecting bacilli in the late stages of anthrax does not prevent death [48], there is no clear connection between the actions of the toxins and the cause of death in anthrax. For many years, the similarity between the secondary shock occurring in the final stages of anthrax infection [49] and the cytokine-like shock death that resulted from lethal toxin intoxication [46, 50] was interpreted as lethal toxin being the ultimate cause of death [46]. However, research in recent years has shown that both lethal and edema toxin act primarily as immune suppressors. Lethal toxin causes apoptosis specifically in macrophages [51] as well as prevents the production of pro-inflammatory [52].

Edema toxin attacks neutrophils and , preventing the respiratory burst [53], pro-inflammatory cytokine production [54] and phagocytosis [55]. Thus, toxin action appears to silence the innate arm of the immune system, preventing an effective response from being mounted against the invading bacilli. The specifics of each toxin will be reviewed in greater detail in a later section.

The other major virulence factor of B. anthracis is its poly-D-glutamic acid capsule. The capsule synthesis genes (capBCAD) are carried on the smaller of the two virulence plasmids, pXO2 [32]. Although the exact enzymatic activities of the CapB,

CapC and CapA proteins have not been defined, the proteins are presumed to be the synthesis genes for the capsule, based on their similarity to the described pgsBCA operon in B. subtilis [56] and the ability of the capBCAD operon to confer the capsulated phenotype in transformed E. coli cells [57]. The capsule of B. anthracis is unusual in that it is a homopolymer of glutamic acid residues [58] linked together in chains of

5 approximately 215 kDa [59]. Peptide capsules are uncommon in , although some

Bacillus species do produce glutamic acid polymers [60, 61]. Of these species, only

Bacillus licheniformis produces a capsule similar to B. anthracis.

However unusual, the function of the peptide capsule is the same as those of the more common polysaccharide variety; vegetative bacilli are protected from macrophage phagocytosis if capsule is present [57, 62]. In addition to the polymerized capsule associated with the cells, recent studies indicate that the small fragments of glutamic acid released by CapD from the capsule polymers may protect vegetative cells from complement [63]. Finally, the capsule behaves as a thymus-independent type 2 antigen

[64], inducing proliferation in the absence of help. This allows the capsule to provide two more important immunological advantages to the infecting bacilli – obscuring cell surface from the immune system and preventing a robust humoral response to the only surface antigen available. Capsule production thus allows the bacilli to escape from the initial site of infection and spread systemically [65]. The advantage conferred by simply avoiding the immune system is impressive, as capsulated, non- toxigenic strains of B. anthracis are highly toxic in mice [66, 67].

Expression of the capsule and toxin genes is regulated at the level of transcription by the presence of bicarbonate [68]. Toxin gene expression also depends on temperature

o [69]. Optimal expression occurs at 37 C in 5% atmospheric CO2, which is roughly equivalent to the concentration of bicarbonate in the bloodstream [70-72]. Transcription is regulated by the global virulence regulator AtxA found on pXO1 [73, 74]. In the presence of physiological levels of bicarbonate, the protein activates expression of the bicarbonate regulon, which includes the toxin genes on pXO1, the capsule synthesis

6 genes on pXO2, and multiple chromosomal genes [75]. Although it controls the bicarbonate regulon, transcription of the atxA gene is unaffected by the presence of CO2

[76], but is upregulated at 37oC [77]. The regulatory network in which AtxA participates is poorly understood. The upstream components, including the bicarbonate sensor, any signaling proteins associated with it, and proteins which directly influence atxA expression, are currently unknown. atxA may fall under the control of the transition state regulator AbrB, although this seems to be more related to the timing of expression than the host environment [78].

The downstream components are better understood, although many gaps remain.

AtxA positively regulates both the capsule operon and the three toxin genes. It is not a direct activator of transcription, as it has no homology to any known DNA or RNA binding protein [73] and no DNA or RNA binding activity has ever been associated with it. AtxA increases expression of the acpA and acpB capsule regulatory genes by initiating transcription from an alternative promoter [79]. AcpA and AcpB directly increase the transcription levels of the capBCAD operon [79], again, by initiating transcription from an alternative promoter [80]. As soon as the capsule synthesis genes are expressed, capsule production increases [81]. AcpA and AcpB have no effect on the toxin genes (pagA, lef and cya). No other gene products have been linked to the positive control of the toxin genes, but at least one is presumed to exist. A negative regulator,

PagR, has been found. It appears to fine tune expression levels of pagA and perhaps cya

[82] via a feedback loop [83].

The bicarbonate regulon dependent on AtxA is unique to B. anthracis. The pathogenic strains of B. cereus and B. thuringiensis have multiple chromosomally

7 encoded virulence factors under the control of the PlcR regulon [13, 84, 85]. B. anthracis has homologs to many of these proteins, which include phospholipases, enterotoxins, and [7]. These genes are regulated by PlcR in B. anthracis as well [86], but were thought to be unexpressed due to a nonsense mutation in plcR that truncated the gene product [86]. However, recent work has shown that the hemolytic genes under the control of PlcR are expressed in B. anthracis under anaerobic conditions [87]. It is unclear at this time if the other genes in the PlcR regulon are also expressed and if so, what contribution they make to the virulence of B. anthracis.

One of the hemolysins under the control of PlcR is the -dependent cytolysin, anthrolysin O. Anthrolysin O is produced under anaerobic conditions [87, 88], which may include the period immediately following spore germination inside the macrophage as well as the terminal phase of infection. Anthrolysin O is able to cause cellular of erythrocytes [88], macrophages, and neutrophils [89] at high concentrations. At lower concentrations, many gram positive cytolysins impair phagocytic cell functions, such as chemotaxis, the respiratory burst or phagocytosis [90-

92]. Thus, anthrolysin may be able to cooperate with lethal and edema toxin to impair the innate immune system. There is evidence that anthrolysin O may be able to assist in the escape of the newly germinated bacilli from the macrophage [89]. Additionally, anthrolysin O may be able to cooperate with lethal toxin to cause macrophage apoptosis

[93].

1.2 The bipartite anthrax toxins

Three toxin genes are contained on pXO1: pagA, lef and cya [31]. These encode the toxin proteins protective antigen (PA), lethal factor (LF) and edema factor (EF),

8 respectively [94-96]. The toxin proteins can be combined to form two bipartite toxins.

Lethal toxin results from the combination of lethal factor and protective antigen [97-99].

Edema toxin is the combination of edema factor and protective antigen [99-101].Lethal and edema toxin represent a variation on the A-B channel-forming toxins [102]. In A-B toxins, one protein carries the toxic activity, while the other functions as a means to recognize target cells and deliver the toxic protein to the correct cellular compartment.

The anthrax toxins are interesting because although the toxins target different cell types with different effects, both toxins use protective antigen as their delivery molecule.

1.2.1 Protective antigen

The delivery mechanism for both lethal and edema toxins is protective antigen

(PA). PA is an 83 kDa protein composed of 4 domains of anti-parallel -sheets [103].

PA has sequence homology to another Gram positive toxin, the iota-b toxin of

Clostridium perfringens [104]. It is structurally similar to Staphylococcus aureus - [105], and functionally similar to the B part of diphtheria toxin [106, 107].PA is responsible for the translocation of LF and EF from the extracellular milieu to the cytoplasm of the target cell. The cellular intoxication process by PA can be divided into three essential steps. First, PA must form the toxin complex by binding to LF or EF as well as to itself. Second, PA must recognize the target cell via its receptor. Third, PA must transport LF or EF to the cytoplasm from the endocytic compartment.

Formation of the toxin complex is one of the earliest events in the intoxication process. The initial step is the proteolytic processing of full-length PA (PA83) to its active form. Activation reveals the LF and EF binding sites [108] and allows for the heptamerization of PA [109]. PA is cleaved near the end of domain 1 at the site 9 164RKKR167, resulting in the release of a 20 kDa and a 63 kDa fragment (PA20 and PA63, respectively) [110]. PA20 quickly separates from PA63 [111] and is thought to play no further role in infection, while PA63 is the molecule responsible for the delivery of LF and EF. Classically, conversion of full-length PA83 to active PA63 is carried out by a furin-like cell-surface protease, necessitating that PA83 bind to its cellular receptor prior to complex formation [110, 112]. However, PA has been shown to be activated by numerous proteases [113, 114], including extracellular proteases [115, 116]. Toxin complexes formed in the absence of cells exhibit similar activity to those formed on the cell surface [116, 117], indicating that in vivo, toxin complex formation can occur at any point after from B. anthracis and prior to cellular uptake.

The toxin complex forms after PA has been activated. PA63 heptamerizes and binds to LF and EF in a multi-step process. Initially, two PA63 molecules dimerize via interactions between amino acids 510-518 and 483-486 of domain 3 [118]. The formation of the PA dimer creates a single binding site for either LF or EF on the surface of domain 1 that spans the interface of the two PA proteins [119, 120]. The LF/EF binding site requires amino acids 178, 207, 210 and 214 on one PA molecule and residues 197 and 200 of its neighbor [121]. LF and EF bind to this site via their N- terminal domains [122, 123]. The N-terminal domains of LF and EF share 55% sequence similarity, and their PA-binding sites are structurally similar [124]. LF and EF bind PA via a single patch of residues, consisting of amino acids 182-188 and 223-236 on LF and

169-175 and 214-227 of EF [120]. The interaction between PA and LF or EF is a net electrostatic attraction between the positively charged PA residues and the negatively

10 charged LF and EF residues [125]. Binding between PA and LF or EF is stabilized by the presence of two Ca2+ ions immediately beneath the binding surface on PA [126].

The PA2LF or PA2EF heterotrimer is the basic unit that drives toxin complex formation [127]. The full complex consists of seven molecules of PA bound to three molecules of LF or EF [128] with very high affinity [129]. The complex forms by the polymerization of three heterotrimers and is capped by the binding of a PA63 monomer

[127]. In the final complex, LF and EF are positioned on PA such that only three LF or

EF molecules can bind (the remaining LF/EF binding sites are partially obscured) [121,

130]. LF and EF are oriented on the heptamer with their amino termini centered over the pore in the center of the PA heptamer [125]. Although lethal and edema toxin are usually referred to as separate entities, the toxin complex can consist of both PA2LF and PA2EF heterotrimers [131]; thus, hybrids of lethal and edema toxin are possible in vivo.

PA must recognize the target cell by means of a cellular receptor, either before or after complex formation. Thus far, two separate anthrax toxin receptors have been described – tumor endothelial marker 8 (TEM8) [132] and human capillary morphogenesis protein 2 (CMG2) [133]. Both receptors are expressed at moderately high levels (approximately 104 receptors/cell) on a variety of cell types, including epithelial cells, heart muscle, lung tissue and leukocytes [132-137]. The extracellular domains of TEM8 and CMG2 contain an integrin-like inserted (I) domain (also described as a von Willebrand factor type A domain). The I domain contains a metal ion-dependent adhesion site (MIDAS) motif essential for regular integrin binding [138, 139]. Although the natural ligands of the receptors are unknown, both are suspected to be involved in cellular adhesion [136, 137, 140].

11 PA recognizes the MIDAS motif and binds to the divalent metal ion within [141] by means of an exposed loop on domain 4, consisting of amino acids 681-688 [142-144].

An additional contact is made between amino acids 340-348 in domain 2 of PA and

CMG2 [145, 146]. Binding to the cellular receptor occurs within minutes [147] due to the high affinity of PA for its receptors. PA has an affinity 1000 times that of a regular integrin for its ligand (10-6 vs. 10-3) [148], implying that PA competes with the natural ligands of TEM8 and CMG2 for binding [141].

Multiple forms of TEM8 and CMG2 exist due to splicing variations in the cytoplasmic portions of the proteins [133, 147]. All three variants of TEM8 and two of the variants of CMG2 are able to function as anthrax toxin receptors [133, 147], most likely because the cytoplasmic tail of the receptor plays no role in either uptake or subsequent vesicular routing [147]. The physical binding of PA and complex formation results in the clustering of the anthrax toxin receptors and sequestration of the toxin complex-bound receptors within a lipid raft [149]. The lipid raft promotes rapid internalization [149] by receptor-mediated endocytosis [150] via clathrin-coated pits

[149].

After internalization into the endosome, the only step left in the intoxication process is the transport of LF and EF from the endosome to the cytoplasm. This is accomplished by the pH-dependent translocation of LF and EF through a transmembrane channel created by PA. The structural rearrangements necessary to form the transmembrane pore require that PA be released from its receptor [130, 151]. Receptor release is pH-dependent, and appears to differ between TEM8 and CMG2 [151].

Complexes bound to TEM8 are released from the receptor at mildly acidic pH (pH 6-6.5)

12 in the early endosomes [151-153]. Complexes bound to CMG2 must traffic to the more acidic compartment of the late endosomes (pH 5.2-5.5) in order to be released from the receptor [151]. The difference in TEM8 and CMG2 may be due to differences in the receptor’s affinity for PA or the amino acid interactions with PA.

As PA releases its receptor, pore formation begins simultaneously [154]. The channel-forming pore is predicted to be an extended -barrel (similar to Staphylococcus aureus -hemolysin [105]) composed of at least amino acids 285-340 in domain 2 of PA

[155-157]. The exact sequence of molecular events required to convert PA from a receptor-bound heptamer to a transmembrane channel is currently unknown. However, it is clear that major structural rearrangements must occur in domains 2 and 3 of PA in order to form the pore [154, 155]. The rearrangements are not limited to the pore-forming amino acids, as residues 397, 425 and 427, while not physically part of the pore, make an essential contribution to the structural conversion from heptamer to membrane-spanning channel [158]. The pore is predicted to be 12-30 angstroms in diameter [130, 159, 160], although it may be able to stretch to as much as 75 angstroms [130].

Once the pore is formed, translocation of LF and EF begins. Unless the very largest estimates of the channel diameter are correct, the transmembrane pore is too small to allow LF or EF to pass through without at least partially unfolding [103, 130, 161,

162]. Indeed, experiments indicate that some degree of structural flexibility in LF and EF is necessary for efficient translocation [163]. The unfolding of LF and EF is a pH- dependent process that occurs at pH 5-6 [164]. Under these acidic conditions, LF and EF unfold to an intermediate state between a molten globule and simple secondary structures

[164], which reduces their size enough to pass through the lumen of the pore. LF and EF 13 appear to be threaded through the pore amino terminus first [165]. The membrane spanning channel of PA appears to function not only as a portal to the cytoplasm, but also as a chaperone to actively facilitate the translocation of LF and EF [166]. LF and EF may also actively contribute to the translocation process, as both proteins have been shown to interact with lipid membranes at pH 5-6 [122, 167].

Most translocation occurs from late endosomes [152], probably due to the pH requirements of LF and EF unfolding. Translocation is an independent event for each of the three LF and/or EF molecules bound to the complex [168]. The LF and EF molecules do not cooperate during translocation, with the result that complexes containing a single ligand translocate LF or EF as efficiently as “fully loaded” complexes [168]. After translocation, LF separates completely from the transmembrane complex and is free to diffuse through the cytoplasm to reach its targets [153, 167]. EF, however, permanently associates with the vesicle membrane during translocation [122, 153]. The nature of

EF’s interaction with the membrane is unknown, but similar to many adenylyl cyclases, the structure of EF is predicted to contain multiple transmembrane helices [153]. After

LF and EF are translocated, vesicles containing the transmembrane PA channel continue to the lysosome where PA is degraded [149].

1.2.3 Lethal factor

Lethal factor, the A portion of lethal toxin, is a 90 kDa zinc metalloprotease [169,

170]. The protein is composed of 4 domains that are largely alpha-helical [161]. The first domain binds to PA and plays no role in substrate recognition or enzyme catalysis

[120, 123, 161]. Domains 2-4 comprise the enzymatic portion of the toxin [161].

Domain 2 provides substrate recognition and binding sites [161, 171]. Domain 2 shares

14 sequence homology with the VIP2 toxin of B. cereus, but lacks the catalytic residues to carry out the NAD binding and ADP-ribosylation activities of VIP2 [161]. The positioning of domain 3 provides substrate specificity by sterically blocking all but the exposed termini of proteins from the substrate binding site [161]. The protease active site is located on domain 4. A zinc ion is coordinated by amino acids 686, 690 and 735 in a structure similar to that of thermolysin [161]. Additional contacts with the C-terminal domain of the substrate are required for substrate recognition and binding, but it is unclear which residues of LF are involved in these interactions [172].

LF is a metalloprotease that has a very specific and limited set of substrates. LF acts on members of the mitogen-activated protein kinase kinase (MEK) family [173,

174]. LF is able to proteolytically inactivate Mek1, Mek2 [173, 174], MKK3 [175],

MKK4, MKK6 and MKK7, but not MKK5 [176]. LF binds to the amino terminal tail of a MEK protein and cleaves between the first 7-10 amino acids (depending on the MEK involved) [173, 176]. The amino terminus of a MEK protein defines both its substrate specificity and cellular location, and thus the sequences vary widely although they tend to be rich in basic residues [177-179]. LF is able to cleave these varying sequences and structures by recognizing the homologous stretch of similar basic residues [176]. Amino terminal proteolysis destroys the docking site for the substrate MAPK [180]. The loss of the docking site reduces the affinity of MEK for its MAPK substrate [172]. It also decreases the rate of MEK kinase activity [172]. The effects of proteolysis range in severity from total loss of activity to a modification of activity, depending on the MEK

15 proteolyzed [175, 179]. LF cleavage may also destabilize MEK, leading to its premature destruction [173]. The inactivation of MEK proteins leads to a loss in cell signaling through the MAPK pathways.

MAPK signaling plays an important role in host defense, by mediating such effects as TLR signaling [181, 182] and the response to inflammatory cytokines [183].

These pathways are critical in the struggle between host and infecting pathogen, and thus

MAPK signaling is a target for several bacterial virulence factors. Two such effectors are the YopP and YopJ proteins produced by Yersinia enterocolitica and Yersinia pseudotuberculosis, respectively. Both YopJ and YopP bind to MEK proteins, preventing their activation and that of the downstream MAPKs [184, 185]. The downstream effect of YopJ/P action results in the loss of TNF- production in response to LPS [185-187]. YopJ and YopP have also been associated with the apoptosis of host macrophages [188, 189]. These effects are very similar to those produced by lethal toxin.

LF is highly specific not only in its protein substrates, but also in the cell types that it affects. Although the anthrax toxin receptors are present in high concentrations on numerous cell types, only a small subset of cells are noticeably affected, and it is the macrophage that experiences the full range of lethal toxin action [190, 191].

Macrophages play a central role for LF, as mice without macrophages are immune to lethal toxin challenge [46]. Curiously, macrophages from different strains of mice are not equally susceptible to LF-induced cell death [192, 193], even though LF impairs

MAPK signaling in all macrophages [175, 193]. Sensitivity to lethal toxin action depends on the particular allele of Nalp1b present in the mouse genome [194]. At this

16 time, it is unknown whether Nalp1b is a substrate for LF or if particular alleles of Nalp1b are simply more sensitive to changes in MAPK signaling than the other Nalp1b alleles.

The hallmark of lethal toxin action in macrophages is LF-induced cell death. At low concentrations of lethal toxin, human and mouse macrophages undergo apoptosis

[51, 195], silently removing effector cells from the immune population. Apoptosis appears to proceed via two different mechanisms, depending on whether the cell line is resistant or sensitive to lethal toxin. Sensitive macrophages have allele 1 of Nalp1b

[194]. In these macrophages, apoptosis occurs through a caspase-dependent mechanism

[51] mediated by the inflammosome [196]. Upon LF action, Nalp1b effects the activation of caspase 1 [194] leading to a pathway involving caspases 4, 6 and 8 [51]. In sensitive cells, apoptosis occurs as a direct result of LF action.

Human and resistant mouse macrophages have a more complex interaction with

LF. At low concentrations of LF, MKK3 and MKK6 are cleaved [197]. The loss of activity from these two kinases prevents activation of p38 [197]. Normally, p38 combines with NF-B to promote expression of gene products that prevent activation- dependent macrophage apoptosis [197]. Thus, a resistant macrophage intoxicated with

LF that subsequently receives an activation signal (LPS, lipoteichoic acid or TNF-)

[197, 198], will undergo apoptosis instead of becoming activated [197]. The loss of p38 activation alone may not be sufficient for this effect in all cell lines, and the additional loss of signals from ERK1/2 and/or JNK may be necessary [198].

At sub-lethal concentrations of LF, the innate immune response to bacterial infection is impaired. Macrophages lose the ability to produce cytokines in response to bacterial products. The lack of signal through p38 leads to the inactivation of the 17 transcription factor IFN-regulatory factor 3 (IRF3) [199]. IRF3 controls cytokine expression dependent on bacterial stimulation [199], and its inactivation prevents expression of the pro-inflammatory cytokines TNF- and IL-1 [52]. The bactericidal activity of macrophages also suffers after intoxication [51]. Nitric oxide is produced less efficiently, resulting in less bacterial killing [175]. A similar effect occurs in neutrophils.

Here, the lack of signal from p38 leads to a loss of activity in NADPH oxidase, causing a decrease in superoxide production [200].

High concentrations of LF are able to cause the rapid necrotic death of macrophages [46, 201]. Necrosis may be the result of intracellular damage from overproduction of reactive oxygen species [50]. Macrophages also produce increased levels of the pro-inflammatory cytokine IL-1 both by upregulating expression and by triggering the release of pre-formed IL-1 through a caspase 1-dependent pathway [202].

TNF-, IL1 and IL-6 expression remains repressed [202].

High concentrations of lethal toxin also affect adaptive immune cells. B cell proliferation and IgM production in response to antigen are reduced following lethal toxin intoxication [203]. T cell activation levels also decline [204], leading to decreased

T cell proliferation and IL-2 expression [205] and the repression of IL-6 and TNF-

[204]. Some of these defects may be due to impaired function. High concentrations of lethal toxin decrease the levels of costimulatory molecules expressed on the cell surface, and reduce the ability of dendritic cells to prime naïve T cells, both in vitro and in vivo [206]. Additionally, dendritic cells produce less TNF-, IL-1, IL-6 and

IL-12 after intoxication [206].

18 At high concentrations (50 g/mouse), lethal toxin causes mortality in mice [207].

Lethal toxin challenge does not result in the over-production of pro-inflammatory cytokines in either rats or mice [207, 208]. The continuous perfusion of lethal toxin has no effect on IL-1, IL-6 or TNF- production [208], while a direct challenge results in only a transient increase in IL-1 and IL-6 [207]. Likewise, inflammatory tissue damage from the overproduction of nitric oxide and other reactive oxygen species does not occur

[208]. Death from lethal toxin intoxication appears to be the result of circulatory shock leading to hypoxia [207, 208], although how lethal toxin action results in this pathology is unknown. Lethal toxin action is unlikely to be the sole cause of death in an anthrax infection. However, the loss in vascular integrity leading to fluid accumulation and the overall state of circulatory shock may be direct contributions of lethal toxin to anthrax pathology [208].

In vitro and in vivo, the effects of lethal toxin are dose dependent. At low concentrations, LF acts to impair cytokine production and other innate immune functions.

Moderate doses of LF are sufficient for macrophage apoptosis, and high doses result in macrophage necrosis and damage to the adaptive immune cells. Although MEK family members are cleaved whenever LF is present, the range of effects that LF produces hinges on the severity of the cellular insult [175, 193, 209, 210].

Because lethal toxin’s effects are dose dependent, it is very difficult to ascertain the contribution of lethal toxin to anthrax pathogenesis. Although lethal toxin is expressed throughout infection [211, 212] and is present in very high concentrations in the blood in the final hours of infection [213], the local concentration of toxin in the early stage of disease (presumably when immunological responses are critical) is unknown. 19 This makes the specific effects of lethal toxin on the host at a given stage of disease a matter of speculation. However, in vivo evidence indicates that some of the dramatic results produced by high concentrations of lethal toxin in vitro may not occur at all in an infection. Lethal toxin challenge does not result in the production of a pro-inflammatory cytokine storm [207, 208]. Similarly, apoptotic, not necrotic, macrophages are found in anthrax patients [214]. Thus, care must be taken in extrapolating the in vitro attributes of lethal toxin to an infection scenario.

1.2.4 Edema factor

Edema factor, the A portion of edema toxin, is an 89 kDa adenylate cyclase [215].

Introduction of EF into eukaryotic cells results in a rapid increase in the intracellular levels of cAMP [215], due to its high specific activity – 1000-fold higher than eukaryotic adenylyl cyclases [216, 217]. Enzymatic activity depends on the presence of the eukaryotic protein calmodulin (CaM) [218]. The requirement for CaM serves as a regulatory mechanism to ensure that EF exerts its effects only on host target cells [219].

Like LF, EF is composed of four largely alpha-helical domains [162]. The amino terminal domain of EF has a sequence and structure similar to that of LF [120, 124]. The

N-terminal domain of EF binds to PA [120, 216] and has no role in the catalytic function of EF [216, 220]. The ATP-binding pocket and active site of the enzyme is located at the interface between domains 2 and 3 [162, 220]. When CaM is not bound, the active site of EF is structurally disordered and the enzyme is inactive. The binding of CaM is a multi-step process that effects large structural alterations in EF. Except for a connecting linker to domain 3, domain 4 separates completely from domains 2 and 3 and three additional loops rearrange substantially to form the CaM binding site. The result of these

20 alterations is a tightly bound CaM molecule and an active site capable of binding ATP

[162]. The conversion of ATP to cAMP is thought to occur by a two metal ion mechanism, similar to that found in polymerases [221].

Intracellular cAMP levels regulate numerous innate effector cell functions, including cytokine production, chemotaxis, phagocytosis and superoxide production

[222-225]. As a result, many produce toxins that alter the concentration of cAMP in order to disable these functions [226-230]. Many of these adenylyl cyclase toxins have effects similar to EF on neutrophils and monocytes. Cholera toxin prevents production of the pro-inflammatory cytokines TNF- and IL-12 [231, 232]. Bordetella pertussis actually produces two cAMP modulators. Pertussis toxin prevents chemotaxis and degranulation [233] while adenylyl cyclase toxin inhibits superoxide production and phagocytosis as well as chemotaxis in neutrophils [234, 235].

Neutrophils are responsible for the majority of microbial clearance and killing during the initial response to an infection. Edema toxin counters these actions by impairing both phagocytosis and the respiratory burst in neutrophils. Within 60 minutes of intoxication, neutrophils experience a six-fold decrease in their level of phagocytosis

[55]. Phagocytosis impairment persists as long as EF is active [55]. The respiratory burst is also impaired within 60 minutes of intoxication [53]. Superoxide production drops to negligible levels in intoxicated neutrophils, even in the presence of bacterial products [53]. The increase in cAMP prevents the cAMP-dependent protein kinase

(PKA) from activating NADPH oxidase [200]. Unlike phagocytosis, the effect of EF on superoxide production depends on the activation state of the neutrophil. Neutrophils which have been stimulated with bacterial products prior to intoxication can maintain

21 effective levels of superoxide production, while unprimed neutrophils intoxicated with

EF lose the ability to respond to bacterial products even after EF is no longer active [53].

EF is also able to act on monocytes in a cAMP-dependent manner. In both macrophages and monocytes, EF activity upregulates the expression of both anthrax toxin receptors (TEM8 and CMG2) [236]. The increased level of receptor on the cell surface increases the susceptibility of the intoxicated cell to the effects of LF [236]. EF is also able to alter the cytokine profile of monocytes. The presence of EF prevents expression of TNF-, even when the monocytes are stimulated with TNF--eliciting signals [54] In contrast, EF increases expression of IL-6 by monocytes to levels equivalent to those provoked by LPS [54]. The production of IL-6 is directly attributable to increased levels of cAMP [237], but the increase in IL-6 makes a striking contrast to the other immunosuppressive effects of EF.

New research indicates that EF is able to affect the adaptive immune response in addition to innate effector cells. T lymphocyte activation and proliferation are both impaired by edema toxin intoxication [238]. T cell expression of IFN-, IL-2, IL-5 and

TNF- also decrease [238]. These effects are due to the disruption of the signaling pathway from the T cell receptor by the increased levels of cAMP [238]. EF also prevents cytokine expression by dendritic cells, reducing the levels of IL-12 and TNF- produced [239]. EF is able to cooperate with LF action to further repress TNF- production [239].

At high concentrations (37.5 g/mouse), intravenous injection of edema toxin causes rapid lethality in mice [47]. Edema toxin targets the GI tract, causing extensive fluid accumulation; similar to cholera toxin [240]. Tissue lesions, especially on the 22 adrenal glands, occur, along with lymphocyte depletion. High levels of the pro- inflammatory cytokines IL-1, IL-1 and IL-6 are expressed shortly before death. Death appears to be the result of multiple organ failure [47]. Edema toxin action is probably not the cause of death in an anthrax infection. However, some of the disease pathology, namely the widespread tissue lesions and alterations in serum chemistry, may be attributable to the actions of this toxin.

Although EF and LF have different cytoplasmic targets and alter different signaling pathways, there is some redundancy in their downstream effects. EF and LF are both able to decrease the production of superoxide, reduce T cell activation and prevent cytokine expression. The duplicity of these effects is likely due to the intersection of the cAMP-dependent pathways with MAPK signaling [238]. The high concentrations of intracellular cAMP produced by EF activate the cAMP-dependent protein kinase (PKA). Activated PKA phosphorylates Raf, leading to Raf’s inactivation.

The inhibition of Raf leads to the loss of signaling through the MAPK pathways [241].

1.3 Anthrax: disease, prevention and treatment

1.3.1 Pathogenesis and disease

B. anthracis is the etiological agent of the disease anthrax. Anthrax can infect all mammals, including humans, but is primarily a disease of herbivores. In all cases of anthrax, the spores are the infectious agent [35]. No reliable reports exist of vegetative bacilli causing infection, or either animal to animal or animal to human transmission.

Anthrax is common in animal populations, usually infecting cattle, goats or sheep, although horses, deer and other game animals are also frequently infected [242]. B.

23 anthracis and anthrax are found throughout the world [6, 42]. In several countries, including the United States, the disease is controlled by widespread animal vaccination programs [243]. However, outbreaks continue to occur in parts of the world where anthrax is endemic, namely the Middle East (Turkey, Iran and Pakistan) and Africa [244].

In humans, as in animals, the first step in infection is the introduction of spores into the body. The spores are phagocytosed by the local macrophage population and transported to the draining lymph nodes [245]. Although B. anthracis is an extracellular pathogen, spore uptake by the macrophages appears to be required for the establishment of infection [30, 246, 247]. Once inside the phagolysosome, the spores germinate [44].

The nutrient-rich environment of the macrophage may be sufficient to stimulate germination through one or more of the multiple germination pathways available to B. anthracis [247-250]. Alternatively, a host-specific signal may be required [30, 247].

Immediately following germination, expression of the anthrax toxin genes pagA, lef and cya and the global virulence regulator atxA is upregulated [44]. The toxins are not required for phagolysosome escape, but are necessary to survive inside the macrophage [251, 252]. The toxins may promote escape from the macrophage, or may initiate useful downstream immunological effects. Anthrolysin O may also be involved in the escape of the bacilli from both the phagosome and the macrophage [89]. Although the exact escape mechanism is undescribed, the bacilli reach the extracellular milieu 4-6 hours after germination [251]. Bacterial replication within the macrophage is limited if it occurs at all [251, 252]. The extracellular bacteria then spread through the lymph node and if not contained by the immune system, will spread to neighboring lymph nodes. As successive lymph nodes are overwhelmed, the vegetative bacilli go on to spread

24 systemically through the bloodstream. Here, in the late stage of the disease, the bacilli replicate to extremely high numbers [36], toxin and capsule production reach their maximum levels [78], and an overwhelming bacteremia and toxemia result [213]. The combination of bacteremia and toxemia leads to secondary shock and the death of the host [253].

In humans, anthrax takes one of three forms, and is categorized by the route of infection. Introduction of spores into the body through cuts or abrasions in the skin during the handling of contaminated animal parts or soil results in the cutaneous form of the disease [242]. The infecting spores germinate approximately five days after exposure, resulting in a localized infection [254]. Initially, the infection is characterized by the development of an ulcer or vesicles and localized edema. The vesicles eventually merge into a painless black eschar [242]. Resolution of the infection usually occurs within 2-3 weeks [254]. Antibiotic therapy has little effect on either the development or healing of the eschar, but it does decrease the likelihood of systemic complications [242].

Cutaneous anthrax is the mildest form of the disease; without treatment, mortality approaches 20% of patients. Most of these deaths are due to the development of systemic complications [255].

Ingestion of spores from under-cooked contaminated meat leads to gastrointestinal anthrax. This form of the disease is infrequently reported, which has led to the view that this form of the disease rarely occurs. Instead, the range of symptoms and difficulty of diagnosis may lead to under-reporting [256]. Symptoms vary from asymptomatic to mild gastroenteritis to hemorrhagic shock and death [256]. After ingestion, the spores germinate somewhere along the gastrointestinal (GI) tract. The

25 initial ulcerative lesions, possibly similar to cutaneous lesions, develop at the location of spore germination. Depending on the location of the lesions, different symptoms develop. Spores in the upper GI tract frequently cause ulcerative lesions in the esophagus, stomach and jejunum, and can progress to massive hemorrhage [256-258].

Spores in the lower GI tract cause intestinal lesions, and can progress to complications such as hemorrhage [256], bowel obstruction [259, 260] and bowel perforation (sepsis)

[260, 261]. Abdominal ascites can also occur with this form of the disease [256, 262,

263]. Gastrointestinal anthrax is thought to have a mortality rate between 25-60% [264], but without accurate case reporting, it is difficult to determine either the frequency or the severity of this form of anthrax [256].

The inhalation of spores leads to the most serious form of the disease. In inhalational anthrax, spores are inhaled into the alveolar ducts and alveoli. The spores are taken up by the alveolar macrophages and transported back to the mediastinal and hilar lymph nodes [254]. Germination of the spores usually occurs within a week of exposure, but may take as long as six weeks [265]. Once the spores have germinated, inhalational anthrax rapidly develops. The initial stage lasts approximately four days and is marked by malaise, a non-productive cough, fatigue and fever [254, 266]. The initial stage is followed by a fulminant stage in which the patient experiences severe respiratory distress and shock [266]. Death occurs 24 to 36 hours after respiratory distress begins

[266]. Treatment during the initial stage of infection may extend the length of the initial phase and decrease the risk of death in some patients, however many patients progress into the fatal fulminant stage even with treatment [266]. Treatment is largely ineffective in the final stage of inhalational anthrax and mortality approaches 100% [266, 267].

26 Anthrax is not a common disease of humans, partly because of the low incidence of exposure and partly because humans are not as susceptible as herbivores [268, 269].

Approximately 2,000 cases of human anthrax occur worldwide each year [242]. The majority of reported cases (95%) are cutaneous infections [242], although there is some uncertainty about the prevalence of gastrointestinal anthrax [256]. Most cases of anthrax occur after agricultural [270] or occupational exposure (meat packing, leather tanning, wool/hair sorting) to spore-contaminated animal products [271]. Human anthrax is mainly a problem in developing countries with infrastructure disrupted by war or other political instability [264]. The industrial world is largely unaffected, due to the vaccination of at-risk personnel and the practice of industrial hygiene [264].

Since anthrax is uncommon in humans, there is little if any in the population at large. Because of its rarity, inhalational anthrax is almost impossible to diagnose in time for effective treatment without a high index of suspicion. The spores are infectious, easy to produce and stable for decades if not longer [272, 273]. Aerosolized spores are able to penetrate closed buildings with almost the same infectivity as outdoor areas [242]. Thus, the deliberate release of aerosolized B. anthracis spores could result in a very large number of casualties with a high mortality rate, and could potentially overwhelm local health care facilities and local infrastructure as well as cause widespread panic. These characteristics make B. anthracis an ideal biological weapon.

Currently, 17 nations are thought to have biological weapons capabilities [255], and it is unknown how many additional nations or groups may have access to these weapons. Although the generation of a large-scale lethal anthrax aerosol is difficult without advanced technology, small-scale releases are within the means of numerous

27 well-funded autonomous groups [242]. In October 2001, the mailing of anthrax spores in the United States resulted in 22 cases of anthrax (11 cutaneous, 11 inhalational) with 5 deaths (all from inhalational anthrax) [274]. The Japanese terrorist cult Aum Shinrikyo dispersed aerosolized spores in Tokyo multiple times in the 1990’s, fortunately without incident [275]. The closest approximation of a large-scale biological attack occurred in

Sverdlovsk in the former Soviet Union in 1979. Aerosolized spores from multiple strains of B. anthracis [276] were accidentally released from a military microbiology facility and contaminated the southern portion of the city [265]. As a result, 17 cases of cutaneous anthrax and 79 cases of inhalational anthrax developed, with 68 fatalities (all from inhalational anthrax) [265, 277].

1.3.2 Anthrax vaccination

Since inhalational and gastrointestinal anthrax infections are difficult to diagnose and rapidly develop life-threatening symptoms, vaccination prior to infection is the best defense against anthrax. With the notable exception of Louis Pasteur’s capsulated, non- toxigenic attenuated vaccine strain [278], most anthrax vaccines have focused on the bipartite toxins as opposed to the vegetative bacilli. Although various cell surface proteins provoke an immune response in an infection, the responses to these proteins are unable to prevent either the systemic spread of the bacilli or the damaging effects of lethal and edema toxin [45, 279, 280]. An immune response directed against the toxins attenuates the pathology of anthrax sufficiently to allow the host immune response to control and eradicate the vegetative bacilli [281, 282].

In order to protect against the toxins, the host immune system must be able to block the cellular intoxication process and prevent lethal and edema factor from reaching

28 the cytoplasm of target cells. Therefore, a successful toxin-based vaccine will stimulate an extracellular or TH2 response that produces a high level of toxin neutralizing antibodies (i.e. able to interfere with the cellular intoxication process) [283, 284]. The presence of antibodies able to neutralize lethal toxin action is one of the only characteristics shown to correlate directly with survival in a challenge [285, 286]. Most vaccines achieve the production of a protective response by immunization with PA, since as the common component of lethal and edema toxins, it is able to block the effects of both toxins [282].

There are two general categories of anthrax vaccines: non-capsulated, toxigenic attenuated strains and purified toxin components. Although both kinds of vaccines include PA and provide protection, the protective immune responses generated may be quite different. The former Soviet Union developed a live spore vaccine for humans using the STI-1 strain of B. anthracis. The STI-1 vaccine was reported to have low immunogenicity [287], but this may be due to the induction of a CTL-based response instead of antibody production (K. Brenneman and A. Cross, unpublished data).

Immunization with STI-1 was reported to provide 75-85% protection [288]. A similar strain, Sterne 34F2, is used as an animal vaccine and has been responsible for the worldwide reduction of anthrax epidemics [289-291].

Despite the effectiveness of the STI-1 and Sterne spore vaccines, both strains retained a residual amount of virulence [287] that the Western world deemed unsafe for use in humans. This led to the development of the purified toxin vaccines, based on the observation that PA alone is able to stimulate protective immunity in a range of animal models including primates [282, 292, 293]. The current U.S. licensed vaccine (anthrax

29 vaccine adsorbed – AVA) is produced from a cell-free culture filtrate of anaerobically grown B. anthracis strain V770-NP1-R. This strain is a non-encapsulated, non- proteolytic variant of a bovine strain isolated in Florida in 1951 [282]. This cell-free material, which consists largely of PA, is adsorbed onto aluminum hydroxide [294]. The immune response is primed by vaccination at 0, 2 and 4 weeks and 6, 12 and 18 months and then boosted yearly [295, 296]. The U.K. vaccine (anthrax vaccine precipitated –

AVP), while similar in principle to AVA, is produced from an alum precipitate of the cell-free culture filtrate of a static, aerobic culture of the B. anthracis Sterne strain 34F2

[297]. The immunization schedule is also slightly different, comprising vaccinations at

0, 3 and 6 weeks and 6 months followed by annual boosts [298]. In addition to large amounts of PA, AVP also contains trace amounts of LF, EF and other bacterially-derived , which have been shown to stimulate antibody responses in recipients and may contribute to protection [280, 299].

Both AVA and AVP provide protection from anthrax challenges in animals [300,

301], and the limited amount of data available indicate that humans are protected as well

[302]. However, multiple doses are necessary to maintain a protective level of PA- specific antibodies [303]. Although both vaccines are considered safe due to the low incidence of serious side effects [304, 305], 3-20% of vaccine recipients develop mild, local reactions following immunization [304], most likely in response to the additional bacterial products in the vaccine [299, 304]. Thus, there is a desire for a cleaner and more effective vaccine. To address this, a highly purified, recombinant form of PA adsorbed to Alhydrogel is currently being developed as a replacement for AVA [292,

306, 307].

30 However, except in the presence of potent adjuvants containing bacterial cell wall components, purified PA is unable to stimulate as strong or as protective of an immune response as live spore vaccines [307-309]. While PA remains the primary protective antigen in a spore vaccine [45], the immune response is supplemented by additional spore and vegetative cell antigens. The addition of spore antigens significantly improves the level of protection over that provided by PA alone [310, 311]. Likewise, directing an antibody response against the capsule improves the opsonization and phagocytosis of infecting microbes [312]. Thus, although the immune response to PA plays a central role in mediating protection in an anthrax infection, the additional spore, vegetative cell and toxin antigens in the attenuated vaccines supplement that protective immune response.

1.4 Statement of the problem

The goal of this project was to determine the mechanism by which the humoral response to lethal toxin is enhanced. Previous work has shown that the combined presence of PA and LF in an immunization increases the antibody titer to both antigens

[313]. While some work has implied that lethal toxin activity is required for this effect

[314], other studies have indicated that the increase in antibody titer is predicated on the formation of the toxin complex [116, 313]. This project sought to resolve that discrepancy by clarifying the effects of LF activity and PA function on the murine immune response. To that end, single amino acid changes removing specific protein activities were introduced into PA and LF and the resulting immune responses to these toxin mutants were compared. This project also investigated the murine immune response to edema toxin. Since edema toxin forms a protein complex analogous to lethal

31 toxin, this project examined whether the combination of PA and EF also enhanced antibody production, and the mechanism by which edema toxin provoked this response.

32 CHAPTER 2

MATERIALS AND METHODS

2.1 Toxin mutagenesis and plasmid construction

Toxin gene characteristics: The genes encoding PA83, LFn, LF687 and EF were kindly provided by Dr. Steve Leppla at NIH. PA83 and EF are the wild type toxin genes from

Bacillus anthracis [94, 96]. LF687 is an inactivated form of the full-length LF gene containing the point mutation E687C. This mutation is in the active site of the metalloprotease and results in the complete loss of protease activity [169, 170]. LFn is a truncated form of LF consisting of amino acids 1-254. The truncation removes the substrate recognition and enzyme activity domains [161, 171], preventing enzyme activity, but retaining the N-terminal domain responsible for binding to PA. Thus, LFn can be translocated to the cytoplasm of target cells [123]. The EFn gene was created by truncating full-length EF at amino acid 303. This removes the ATP-binding site as well as the calmodulin binding site, preventing enzymatic activity [162]. Truncation at amino acid 303 allows EFn to bind to PA and enter the target cell [122], similar to LFn. The functional abilities of each protein are summarized in Table 2.1.

33 All of the native anthrax toxin genes contain a signal peptide of approximately 30 amino acids that promotes secretion from B. anthracis. Primers were designed to exclude this signal sequence from the recombinant expression constructs, and thus all expressed proteins begin with the first amino acid after the signal peptide cleavage site [94-96].

The genes encoding PA83, LF687 and LFn were cloned into the eukaryotic expression vector pVR1020 by Matthew Bell. The gene encoding EF was amplified by PCR from pSE42 using the forward primer: 5’ CCCGGATCCATGAATGAACATTACACTGAG 3’, and reverse primer: 5’ GGGGATCCTTTTTCATCAATAATTTTTTGG 3’. EFn was amplified from the pSE42 template using the forward primer shown above and the reverse primer: 5’

GGGGGAATTCTTATTTAAGTGCTTTTTC 3’. All PCR reactions with the EF and EFn genes were conducted with the Taq polymerase (Invitrogen, Carlsbad, CA) using an annealing temperature of 50oC and 4 minute, 60oC extension steps, as the GC content of the primers precluded extension at 72oC. PCR products for EF and EFn were cloned into the expression vector pVR1020 using BamHI sites at the 5’ and 3’ ends of the gene. The sequence of each construct was confirmed by BigDye Terminator Cycle sequencing on the 3730 DNA Analyzer (Applied Biosystems, Foster City, CA) at the OSU Plant-

Microbe Genomics Facility. See Table 2.2 for plasmid descriptions. pQE-30 protein expression constructs: For recombinant protein expression, the genes were cloned into the pQE-30 protein expression vector (Qiagen, Valencia, CA). The pQE-30 vector places the cloned gene under the control of the T5 phage promoter, which utilizes host E. coli RNA polymerase (Table 2.2). The promoter is regulated by the lac repressor protein. In order to control expression, the cells must constitutively over express lacI, and thus either the chromosomal mutation lacIq or the repressor plasmid

34 pREP4 must be present. Cloning into pQE-30 results in a hexahistidine tag fused in- frame to the N-terminus of the protein of interest, allowing the protein to be purified by metal affinity chromatography. The genes for LF687, LFn, EF, EFn and PA83 were subcloned from the parent pVR1020 vector. The genes were excised from pVR1020 with

BamHI and KpnI, generating a fragment with a 5’ BamHI end and a 3’ KpnI end. LFn was excised solely with BamHI. After digestion, the reaction was run on a 1% agarose gel and the gene-containing fragment was purified. The restriction fragment was ligated into appropriately digested pQE-30 by T4 ligase (Invitrogen) for 4 hours at room temperature. The ligation reaction was then transformed into chemically competent XL-1

Blue cells (which contain the lacIq mutation), and the cells were grown overnight at 37oC.

The resulting colonies were screened by restriction digest – plasmids resulting in the correct restriction pattern following digestion with two different enzymes had their sequences confirmed by BigDye Terminator Cycle sequencing on the 3730 DNA

Analyzer (Applied Biosystems) at the OSU Plant-Microbe Genomics Facility.

Agarose gel electrophoresis and purification of DNA: DNA fragments were separated for analysis and purification by agarose gel electrophoresis. Electrophoresis grade agarose (1.0% (w/v)) (Fisher, Atlanta, GA) was melted in TAE buffer (0.01 M Tris base,

0.04 acetic acid, 0.001 M EDTA), then 0.8 g/ml ethidium bromide was added and the gel was cast in a horizontal electrophoresis system (BioRad, Hercules, CA). DNA was mixed with 2x sample buffer (50% w/v sucrose, 40 mM Tris base, 0.24% w/v each of bromophenol blue, xylene cyanole and orange G) and electrophoresed at 120 V until resolved. For size comparison, a minimum of one lane was reserved for the 1 Kb Plus

DNA ladder (Invitrogen). The DNA in the gel was visualized by UV transillumination at

35 302 nm. Gels for DNA analysis were photographed using the BioRad ChemiDoc gel documentation system. Gels that served as a means of purification were first photographed, and then the DNA fragment of the desired size was excised from the gel with a clean razor blade and purified with the Gel Extraction Kit (Qiagen) according to the manufacturer’s instructions.

Transformation of chemically competent E. coli: Chemically competent E. coli cells from the DH5,XL-1 Blue, M15 or SG13009 strain were prepared as follows [315].

Cells were grown in 50 mL of SOB (20 g/L tryptone, 5 g/L yeast extract, 0.5 g/L NaCl,

1.86 g/L KCl with 10 mM MgCl2) to an OD600 of 0.5. Cells were collected by centrifugation at 4,000 x g at 4oC for 5 minutes. Pelleted cells were gently resuspended in cold 0.1 M CaCl2, incubated on ice for 20 minutes and then pelleted as before. The competent cells were resuspended in 3 mL cold 0.1 M CaCl2 containing 15% glycerol

(v/v) and divided into 50 L aliquots. Cells were snap frozen in liquid nitrogen and stored at -70oC until use.

Competent cells were thawed on ice immediately prior to transformation.

Approximately 100 ng of DNA were added to the competent E. coli cells and mixed gently. The cells were allowed to take up DNA for 30 minutes before a heat shock at

42oC for one minute. Following heat shock, the cells were rested on ice for five minutes.

To each transformation, 450 L of SOC media (20 g/L tryptone, 5 g/L yeast extract, 0.5 g/L NaCl, 1.86 g/L KCl with 10 mM MgCl2 and 20 mM glucose) was added and the transformation incubated at 37oC for 45 minutes to allow expression of the antibiotic resistance gene. Transformed cells were then pelleted by centrifugation in a microcentrifuge and resuspended in 100 L of LB broth. Cells were plated on LB agar 36 supplemented with 100 g/mL ampicillin (and 25 g/mL kanamycin for M15 and

SG13009) and incubated at 37oC overnight. See Table 2.3 for a description of the strains used in this study.

Site-directed mutagenesis of the PA, LF and EF genes: Single amino acid point mutations were introduced into the genes encoding PA83, LF, LF687 and EF with the

QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Mutations were made in order to knock out specific functional activities of each protein (Table 2.1).

Point mutations were introduced directly into the pQE-30 expression constructs as follows. A pair of mutagenic primers (one to complement each strand of the template plasmid) was designed for each mutation (see Table 2.4 for codon changes). The template plasmid (50 ng) was mutated by the PCR extension of the mutagenic primer pair

(125 ng of each) with PfuTurbo polymerase. Primers were annealed to the template at

55oC, and then extended at 68oC for 2 minutes per each kilobase of plasmid length for a total of 12 cycles. This resulted in double-stranded full-length copies of the template plasmid that contained the desired mutation. The parental template DNA was selectively destroyed by DpnI digestion at 37oC for 1 hour, and the remaining mutated DNA was transformed into competent XL-1 Blue cells as described above. Cells from the transformation were plated on LB agar containing 100 g/mL ampicillin and incubated overnight at 37oC. Colonies were screened by BigDye Terminator Cycle sequencing on the 3730 DNA Analyzer (Applied Biosystems) at the OSU Plant-Microbe Genomics

Facility in order to identify clones containing the desired mutation.

37 2.2 Protein purification

Growth and recombinant protein expression: Optimal expression conditions – defined as those yielding the highest level of protein expression and lowest level of protein degradation – were determined for each pQE-30 expression construct. Each construct was transformed into the XL-1 Blue and M15 strains and 5 ml cultures were grown to an

OD600 of 0.6. Protein expression was induced with 1 mM IPTG and continued for 4, 6 or

16 hours at 37oC. Expression was monitored by SDS-PAGE and western blot of culture samples. Constructs that had no detectable expression from either XL-1 Blue or M15 were transformed into SG13009 for further optimization.

The effect of expression time, expression temperature, induction OD, growth medium and IPTG concentration were examined to determine optimal conditions for the

SG13009 constructs. Cultures were grown in either LB or 2xYT (16 g/L tryptone, 10 g/L yeast extract and 5 g/L NaCl) broth to an OD600 of 0.4, 0.5, 0.55, 0.6, 0.65 or 0.7, then induced with 0.5, 1.0 or 2.0 mM IPTG, and expressed for 4, 6, 8, 14, 16 or 20 hours at either 37oC, 30oC or room temperature. Protein expression was monitored by SDS-

PAGE electrophoresis. Alterations in the growth medium or IPTG concentration had no noticeable effects on the level of protein expression. A significant increase in protein expression was observed for several constructs when the expression temperature was decreased and the length of expression was increased (Table 2.5). The pQE-30 construct containing LF did not produce detectable amounts of protein under any conditions tested.

Another construct containing LF in the pET15b vector was generously provided by Dr.

Susan Wimer-Mackin at Ligocyte Pharmaceuticals. This construct was expressed from the protein expression strain BL21, as pET15b requires the presence of the T7

38 polymerase for protein expression. Expression conditions for the pET15b/LF construct were determined by the same methods as for the SG13009 constructs (Table 2.5).

Once expression conditions were determined, one liter cultures were grown in LB broth containing 100 g/mL ampicillin. Strains containing the repressor plasmid pREP4 had an additional 25 g/mL kanamycin added to the growth media. Strains were grown

o at 37 C with 250 rpm rotation until the cultures reached on OD600 of 0.55-0.6. Protein expression was induced by the addition of 1 mM IPTG. Cultures were then incubated for the time and at the temperature shown in Table 2.3. Following expression, cells were pelleted at 10,000 x g for 15 minutes at 4oC. Pellets from cultures expressed for 4-6 hours were stored overnight at 4oC, but cultures expressed 16-20 hours were purified immediately over Ni-NTA or TALON resins.

Protein purification over Ni-NTA resin: The cell pellet from an expression culture was resuspended in 2x the wet weight of the pellet of lysis buffer (300 mM NaCl, 50 mM

NaH2PO4, 10 mM imidazole, pH 8.0). The protease inhibitors Pefabloc SC (10 mg), leupeptin (5 mg) and aprotinin (5 mg), and DNase I (5 mg) were added to the lysis buffer following resuspension. Cells were lysed by French Press at 14,000 psi twice and then centrifuged at 10,000 x g for 20 minutes to separate the soluble and insoluble fractions.

The soluble fraction of the cells was saved and added to 3 ml of 50% Ni-NTA slurry

(Qiagen) and batch bound for four hours at 4oC with shaking. After the batch bind, the entire slurry was poured into a gravity column. After the resin had settled, the non- binding fraction was allowed to flow through. The column was washed with eight column volumes of wash buffer (300 mM NaCl, 50 mM NaH2PO4, 20 mM imidazole, pH

8.0) to remove contaminating proteins. Purified protein was eluted in 1 mL fractions 39 over three column volumes of elution buffer (300 mM NaCl, 50 mM NaH2PO4, 250 mM imidazole, pH 8.0). Protein-containing fractions were identified by SDS-PAGE.

Fractions were pooled and dialyzed into PBS using a MWCO of 3000 daltons. The dialysis process occurred over 24 hours and included two buffer changes.

Endotoxin-free protein purification over TALON resin: In order to reduce the number of contaminating cellular proteins, an alternative purification strategy was developed using TALON resin (Clontech, Mountain View, CA). TALON contains a Co2+ metal ion instead of Ni2+, resulting in a lower affinity for background contaminants and also decreasing the amount of metal ion contaminating the purified protein. This procedure was also developed to remove contaminating E. coli endotoxin so that the purified proteins could be used in cell culture and animal manipulations. Endotoxin-free buffers were made by dissolving the appropriate amounts of each component in certified endotoxin-free water (Invitrogen). The pH was adjusted by removing a 2.5 ml aliquot of buffer for measurement, then adding 0.5 M NaOH dropwise to the buffer and removing another 2.5 ml aliquot for measurement.

The cell pellet from an expression culture was resuspended in 2x the wet weight of the pellet with lysis buffer (300 mM NaCl, 50 mM NaH2PO4, pH 7.0). The protease inhibitors Pefabloc SC (10 mg) and leupeptin (5 mg), and DNase I (10 mg) were added to the lysis buffer following pellet resuspension. Cells were lysed two times with the

French Press at 16,000 psi and then centrifuged at 45,000 x g for 20 minutes to separate the soluble and insoluble cellular fractions. During the centrifugation, 5 mL of 50%

TALON slurry was equilibrated with 50 mL of lysis buffer. Following centrifugation, the soluble fraction of the cells was saved and added to the equilibrated TALON resin in

40 a Falcon tube. Endotoxin removal (ER) buffer (Qiagen) was added to make up 10% of the final volume. The proteins were batch bound to the TALON resin for 16-20 hours at

4oC with shaking.

After the batch bind, the entire slurry was poured into a gravity column and the non-binding fraction was allowed to flow through. The column was washed with ten column volumes of wash buffer (300 mM NaCl, 50 mM NaH2PO4, 20 mM imidazole, pH

7.0) with 10% ER buffer to remove the majority of endotoxin. Contaminating proteins were removed by washing with an additional ten column volumes of endotoxin-free wash buffer. Purified protein was eluted in 1 mL fractions of five column volumes of endotoxin-free elution buffer (300 mM NaCl, 50 mM NaH2PO4, 150 mM imidazole, pH

7.0). Protein-containing fractions were identified by either SDS-PAGE electrophoresis or absorbance at 280 nm. Fractions were pooled and dialyzed into endotoxin-free HEPES buffer (10 mM HEPES, 50 mM NaCl, pH 7.5) using a MWCO of 3000 daltons. The dialysis process occurred over 24 hours and included three buffer changes. This purification process was able to lower endotoxin concentrations to approximately 100

EU/mL. The remaining endotoxin was removed by purification over an EndoTrap column.

Endotoxin removal over an EndoTrap Blue column: Residual E. coli endotoxin contaminating purified protein preparations was removed with an EndoTrap Blue column

(Cambrex, Walkersville, MD). The column was charged with 6 column volumes of regeneration buffer (provided by the manufacturer), then equilibrated with 6 column volumes of equilibration buffer (provided by the manufacturer). Following equilibration, the purified protein sample was loaded onto the column and allowed to pass through by

41 gravity flow. Fractions were collected immediately after the sample was loaded.

Protein-containing samples were identified by absorbance at 280 nm and pooled.

Proteins eluted from the EndoTrap column were in HEPES buffer and were endotoxin- free (less than 1.0 endotoxin units/mL). The quality of the purified protein was assessed by SDS-PAGE (Figure 2.1).

SDS-PAGE electrophoresis of protein samples: Proteins were separated for analysis using the polyacrylamide gel electrophoresis method of Laemmli with the following modifications. Each discontinuous gel consisted of an upper 4% stacking portion and a lower 10% resolving portion. The resolving portion of the gel was prepared first.

Acrylamide and bis-acrylamide in a ratio of 29:1 (National Diagnostics, Atlanta, GA) were diluted to a final concentration of 10% in buffer containing 0.1% SDS and 380 mM

Tris HCl, pH 8.8. Ammonium persulfate (0.5% w/v) and TEMED (0.111% v/v) were added as polymerization agents and the gel was cast in the BioRad Protean II system at a thickness of 0.75 mm. The acrylamide mixture was overlaid with a thin layer of n- butanol in order to facilitate polymerization. Polymerization was allowed to occur for

30-45 minutes at room temperature.

Once the resolving portion of the gel was polymerized, the n-butanol was wicked off and the stacking portion of the gel was prepared. Acrylamide and bis-acrylamide in a ratio of 29:1 (National Diagnostics) were diluted to a final concentration of 4% in buffer containing 0.1% SDS and 125 mM Tris HCl, pH 6.8. Ammonium persulfate (0.5% w/v) and TEMED (0.111% v/v) were then added as polymerization agents. A comb containing the appropriate number of wells was inserted into the top of the gel apparatus.

42 The exposed corners of the gel were sealed with petroleum jelly to aid polymerization and the mixture was incubated for an additional 30-45 minutes at room temperature.

Protein samples were combined with an equal volume of 2x sample loading buffer

(125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 2% -mercaptoethanol, 0.01% bromophenol blue) and heated for 5 minutes at 95oC. The comb was removed from the stacking gel and the wells were thoroughly flushed with distilled water. Samples were loaded into the wells of the gel, along with a minimum of one lane of the Precision Plus protein standards (BioRad). Electrophoresis was conducted at 200 V for approximately

45 minutes in SDS-PAGE tank buffer (30 mM Tris base, 192 mM glycine, 0.1% SDS).

After electrophoresis, the gels were removed from the apparatus and washed three times in distilled water for 5 minutes each to remove the SDS. The protein was stained in

BioSafe Coomassie (BioRad) overnight. Background was removed by washing twice for five minutes in distilled water. Gels were photographed using the BioRad ChemiDoc

XRS gel documentation system.

Protein quantification by bicinchoninic acid assay: The amount of total protein in samples was quantified by bicinchoninic acid (BCA) assay (Pierce, Rockford, IL).

Protein-containing samples were diluted in duplicate two-fold dilutions ranging from 1:2 to 1:16 on an Immulon 4 microtiter plate (Thermo Scientific, Waltham, MA). A seven- point standard curve of bovine serum albumin (BSA) was also prepared in concentrations ranging from 1500 to 25 g/mL, according to the manufacturer’s instructions. The total volume of each sample or standard for assay was 25 L. Working reagent (sodium carbonate/bicarbonate buffer containing bicinchoninic acid, sodium tartrate and cupric sulfate) was added to each well of the microtiter plate and incubated for 30 minutes at 43 37oC. The absorbance of each sample was read at 562 nm on a PowerWave 340 microplate spectrophotometer (BioTek Instruments, Winooski, VT). The average absorbance values for the standard curve were plotted and the linear regression equation for the best fit line through the points was calculated by Microsoft Excel. Average sample absorbance values in the range of the standard curve were interpolated to determine the protein concentration. Protein samples with more than one dilution in range of the standard curve used the average concentration of the dilutions as the total protein concentration.

Limulus amebocyte lysate assay: The limulus amebocyte lysate (LAL) assay was used to quantitate the amount of endotoxin present in protein samples following purification.

The QCL-1000 assay was performed (Cambrex) according to the manufacturer’s instructions. All kit reagents were reconstituted immediately before use. A standard curve of endotoxin was created ranging from 0.2 – 1.0 EU/mL. The purified stock of endotoxin was vortexed vigorously for 15 minutes prior to dilution, and each standard dilution was vortexed an additional two minutes before transferring to the next tube or microtiter plate. Samples were diluted directly in the assay plate in duplicate 10-fold dilutions ranging from pure sample to 1:1000. Each sample and standard was pre- warmed to 37oC before an equivalent amount of limulus amebocyte lysate was added and incubated 10 minutes at 37oC. Chromogenic substrate solution was then added to each well on the plate and incubated an additional six minutes at 37oC. The assay was stopped by the addition of 50 l of 10% SDS. The absorbance of each sample at 405 nm was read on the PowerWave 340 microplate spectrophotometer (BioTek Instruments) and the values were compared to the standard curve of known endotoxin concentrations. The

44 linear regression equation of the best fit line through the standard curve points was calculated by Microsoft Excel, and then used to determine the endotoxin concentration of the purified protein samples. Protein samples with more than one dilution in range of the standard curve used the average concentration of the dilutions as the total endotoxin concentration.

Purification of E. coli lipopolysaccharide: Lipopolysaccharide (LPS) was purified from the protein expression strain XL-1 Blue according to the method of Hancock and

Darveau [316]. Two 1 L cultures of XL-1 Blue were grown to an OD600 of 0.6, and then grown an additional 4 hours in order to mimic protein expression conditions. The cells were harvested by centrifugation at 10,000 x g for 15 minutes at 4oC. The pellets were resuspended in 10 mM Tris, pH 8.0, combined, and then washed twice more with 10 mM

Tris, pH 8.0. After the final wash, the cell pellet was weighed and resuspended in 30 ml of 10 mM Tris, pH 8.0, 2 mM MgCl2, 100 g/mL DNase I and 25 g/mL RNase I for each 5 g wet weight of cells. Cells were lysed at 16,000 psi by French Press two times.

DNase I and RNase I were added again at 100 g/mL and 25 g/mL, respectively. The lysate was then incubated for 2 hours at 37oC with shaking.

Following incubation, tetrasodium EDTA and SDS were added to a final concentration of 0.1 M tetrasodium EDTA and 2% SDS in order to disrupt the cell wall.

The solution was mixed thoroughly and then centrifuged at 50,000 x g for 30 minutes at

20oC to pellet the peptidoglycan. The supernatant was saved and Pronase E was added to a concentration of 200 g/mL. The solution was incubated overnight at 37oC with shaking to degrade cellular protein.

45 LPS was precipitated by the addition of two volumes of 0.375 M MgCl2 in 95% ethanol; the solution was mixed well and cooled to 0oC. The sample was then centrifuged at 12,000 x g for 15 minutes at 0oC. The supernatant was discarded and the pellet was resuspended in 2% SDS, 0.1 M tetrasodium EDTA and 10 mM Tris, pH 8.0.

This solution was then heated to 85oC and incubated for 30 minutes to denature membrane associated proteins. Following denaturation, the sample was cooled to room temperature and 25 g/mL of Pronase E was added and incubated overnight at 37oC with shaking.

Finally, the LPS was precipitated with two volumes of 0.375 M MgCl2 in 95% ethanol; the solution was mixed well and cooled to 0oC. The sample was then centrifuged at 12,000 x g for 15 minutes at 0oC. The supernatant was discarded and the pellet was resuspended in 10 mM Tris, pH 8.0. This solution was centrifuged at 200,000

o x g for two hours at 20 C in the presence of 25 mM MgCl2. The supernatant was discarded and the pellet resuspended in distilled water. The LPS was then dialyzed in distilled water using a MWCO of 10,000 Da for 24 hours with two changes of water to remove salts and SDS. The dialyzed LPS was lyophilized and stored at room temperature. The activity of the purified LPS was quantified by Limulus amebocyte lysate assay.

2.3 Characterization of protein activity

Lethal toxin cell viability assay: The ability of the lethal toxin mutants to cause cell death was assessed on the mouse cell line J774A.1 (ATCC, Manassas, VA).

J774A.1 cells were grown in DMEM (Invitrogen) supplemented with 10% fetal calf

46 serum (Invitrogen), 2 mM L-glutamine (Invitrogen), 100 U/mL penicillin (Invitrogen)

o and 100 U/mL streptomycin (Invitrogen) at 37 C in a 5% CO2 humidified atmosphere.

Cells were seeded at a concentration of 3x105 cells/mL onto a 96-well microtiter plate

(Corning, Acton, MA) 18-20 hours before the assay. The day of the assay, serial two- fold dilutions of toxin (beginning at 10 g/mL PA and 10 g/mL of LF) were made in quadruplicate across the plate. PA proteins containing a mutation were combined with wild type LF and vice versa. Wild type lethal toxin was also incubated with cells as a

o control. Following a four-hour incubation at 37 C in 5% CO2, the toxin-containing media was removed and replaced with DMEM containing XTT (sodium 3’- all circumstances tested, and it was conclueded that edema toxin and inactive edema toxin are able to act as adjuvants -bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate))

(Roche, Indianapolis, IN) for 16 hours to determine cell viability [317]. The assay was read at 480 nm on a PowerWave 340 microplate spectrophotometer (BioTek

Instruments). The dilution series data (absorbance at 480 nm versus toxin concentration) for toxin combinations that decreased cell viability were modeled with 4-parameter logistic (4PL) curves of the form:

OD480(y) = B1 + (B2-B1)/ [1 + exp {B3 (B4-x)}]

The data were fit by DataFit 8.1 software (Oakdale Engineering, Oakdale, PA) via a nonlinear least-squares analysis, yielding the parameters (B1,B2,B3,B4) of the best fit.

Toxin combinations that had no effect on cell viability were not modeled.

Intracellular cAMP capture ELISA: The ability of the edema toxin mutants to cause an increase in the amount of intracellular cAMP in CHO-K1 cells (ATCC) was measured by direct enzyme immunoassay (Assay Designs, Ann Arbor, MI). CHO-K1 cells were

47 grown in F-12 media (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen), 2 mM L-glutamine (Invitrogen), 100 U/mL penicillin (Invitrogen) and 100 U/mL

o streptomycin (Invitrogen) at 37 C in a 5% CO2 humidified atmosphere. Cells were seeded onto a 96-well microtiter plate (Corning) 18-20 hours before the assay. The day of the assay, serial 2-fold dilutions of toxin (beginning at 3 g/mL PA and 3 g/mL of

EF) were made in triplicate across the plate. PA proteins containing a mutation were combined with wild type EF and vice versa. Wild type edema toxin was also incubated

o with cells as a control. The cells were intoxicated for 45 minutes at 37 C in 5% CO2.

Toxin-containing media was removed and the cells were washed with sterile PBS.

Intracellular cAMP was extracted by the addition of 0.1 M HCl with 0.1% Triton X-100 for 10 minutes. Lysed cells were centrifuged at 1,000 x g for ten minutes. The supernatants were removed and diluted 1:10 in 0.1 M HCl containing 0.1% Triton X-100.

The cAMP capture ELISA was performed according to the manufacturer’s instructions. Diluted samples were mixed with neutralizing reagent (provided by the manufacturer) and added to a microtiter plate coated with goat anti-rabbit IgG. A five- point standard curve of cAMP ranging from 200 – 0.78 pmol/mL was made in 0.1 M HCl containing 0.1% Triton X-100 according to the manufacturer’s instructions. The standard curve was mixed with neutralizing reagent and loaded onto the assay plate. Equivalent amounts of cAMP conjugated to alkaline phosphatase and polyclonal rabbit anti-cAMP were added to each sample and standard. The assay was incubated for two hours at room temperature with shaking. The plate was then washed three times with Tris-buffered saline and developed by the addition of p-nitrophenyl phosphate substrate for one hour at

48 room temperature. The reaction was stopped with trisodium phosphate and the plate was read at 405 nm on the PowerWave 340 microplate spectrophotometer (BioTek

Instruments).

For data analysis, the mean absorbance of each sample or standard was converted to the percent of maximum binding by the equation:

% Bound = Average A405/ (average A405 of 0 pmol/mL cAMP standard) x 100

The standard curve was graphed as the percent of maximum binding vs. the cAMP concentration and the equation of the logarithmic fit through the points was calculated by

Microsoft Excel. The cAMP concentrations of the samples were determined by interpolating the percent bound value for the sample into the equation of the standard curve.

Detection of Mek1 proteolysis by immunoblot: The ability of LF, LF687 and LFn to remove the N-terminal residues of Mek1 in the presence of PA was assessed on J774A.1 mouse macrophages (ATCC). J774A.1 cells were grown in DMEM (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen), 2 mM L-glutamine (Invitrogen),

100 U/mL penicillin (Invitrogen) and 100 U/mL streptomycin (Invitrogen) at 37oC in a

5% CO2 humidified atmosphere. Cells were seeded at a concentration of 3x105 cells/mL

o onto a 12-well microtiter plate (Corning) and incubated at 37 C in a 5% CO2 humidified atmosphere until the cells had formed a monolayer (18-20 hours). The day of the assay, spent media was removed and replaced with 100 ng/mL of PA combined with 100 ng/mL of LFn, LF687 or LF. Cells exposed to each protein individually were used as controls.

o The cells were exposed to toxin for 2.25 hours at 37 C in 5% CO2 – sufficient for complete MEK proteolysis [175]. Following incubation, the toxin media was removed

49 and the cells were washed with DMEM to remove residual extracellular toxin then lysed with 100 L of 2x SDS-PAGE sample buffer. Samples were heated at 95oC for 10 minutes to denature cellular proteins, then equivalent amounts of sample were loaded onto a 10% denaturing acrylamide gel and electrophoresed at 200 V for 50 minutes.

Following SDS-PAGE electrophoresis, samples were immobilized on PVDF membrane (Invitrogen) by the wet electophoretic transfer method of Towbin [318]. Gels were removed from the apparatus and aligned with a PVDF membrane, pre-wet in methanol and then rinsed in transfer buffer (30 mM Tris base, 192 mM glycine, 0.1%

SDS and 20% methanol). The gel and membrane were sandwiched between sheets of filter paper and loaded into a transfer apparatus. Enough transfer buffer was added to cover the sandwich. The gel was transferred at 30 V for 60 minutes. After the transfer, the location of the pre-stained standards was marked in pencil and the membrane was blocked in 3% nonfat milk PBST for one hour at room temperature.

The membrane was washed three times in wash buffer over ten minutes before the addition of primary antibody. Cellular lysates were probed with two different primary antibodies in order to assess Mek1 proteolysis. Anti-MEK specific for amino acids 2-18 of Mek1 (Upstate Cell Signaling Solutions, Lake Placid, NY), was used at 0.5 g/mL as a marker for proteolysis. Anti-MEK specific for the carboxyl terminus of Mek1 (Santa

Cruz Biotechnology, Santa Cruz, CA) was used at 1 g/mL as a control for the total amount of MEK present. Primary antibody was diluted in 3% nonfat milk in PBST and incubated on the membrane for 1 hour at room temperature with shaking. Following three washes with PBST to remove unbound antibody, bound primary antibody was detected using species-specific secondary antibody conjugated to horseradish peroxidase 50 (KPL, Gaithersburg, MD). Secondary antibody was diluted 1:7000 in 3% nonfat milk in

PBST and incubated on the membrane for one hour at room temperature. The membrane was then washed three times with PBST, and then developed with TMB membrane peroxidase substrate (KPL). Once sufficiently developed, the reaction was quenched with distilled water, and the membrane was photographed with the ChemiDoc XRS gel documentation system.

Gel mobility shift of the toxin complex: The ability of PA197, LF236 and EF227 to form a complex with wild type PA, LF or EF was visualized by the shift in mobility that occurs when the toxin proteins complex from monomers to a heterodecamer [120, 121].

A 4% native acrylamide gel was prepared by diluting acrylamide and bis-acrylamide in a ratio of 29:1 (National Diagnostics, Atlanta, GA) to a final concentration of 4% in buffer containing 0.1% SDS and 125 mM Tris HCl, pH 6.8. Ammonium persulfate (0.5% w/v) and TEMED (0.111% v/v) were then added as polymerization agents. The gel was cast in the BioRad Protean II system at a thickness of 0.75 mm. A comb containing the appropriate number of wells was inserted into the top of the gel apparatus. The exposed corners of the gel were sealed with petroleum jelly to aid polymerization and the mixture was incubated 30-45 minutes at room temperature.

PA83 and PA197 were activated by partial trypsin digestion. Trypsin was added at 1/1000 the PA concentration for 30 minutes at 37oC. The digestion was stopped by the addition of trypsin soybean inhibitor at a 10 M excess. To form the complexes, 20 g of activated PA or PA197 were combined with 10 g of either LF, LF236, EF or EF227 for

30 minutes at 37oC. Samples containing each individual protein were used as controls.

51 Each sample was mixed with an equivalent amount of native PAGE sample buffer

(BioRad) and loaded onto the 4% native gel. Native electrophoresis buffer (30 mM Tris base, 192 mM glycine) was added and the gel was run at 50 V for 20 minutes, then 80 V for an additional hour. After electrophoresis, the gels were removed from the apparatus and washed three times in distilled water for five minutes each. The protein was stained in BioSafe Coomassie (BioRad) overnight. Background was removed by washing twice in distilled water. Gels were photographed using the BioRad ChemiDoc XRS gel documentation system.

Sandwich ELISA of the toxin complex: The binding of PA to LF or EF was also assessed by a modified sandwich ELISA. Immulon 4 microtiter plates (Thermo

Scientific) were coated overnight at 4oC with the mouse anti-PA

3E2-071100-02 at 10 g/mL. This monoclonal was the kind gift of Drs. Robert Bull and

Jill Czarnecki at the Naval Medical Research Center, Biological Defense Research

Directorate. Following coating, the plates were washed four times with PBST and then blocked with PBST containing 3% nonfat dry milk for 90 minutes at 37oC. While blocking, PA83 and PA197 were activated by the addition of trypsin (trypsin was diluted to 1/1000 the PA concentration) for 30 minutes at 37oC. Further activation was stopped by the addition of trypsin soybean inhibitor at a 10 M excess. The plates were then washed four times with PBST and 20 ng of activated PA83 or PA197 was added to each well and incubated for 90 minutes at 37oC. Unbound PA was removed by four washes with PBST. A serial two-fold dilution series starting at 200 ng/mL of LF, LF236, EF or

EF227 was made across the PA-containing wells in order to determine the interaction between the molecules. The LF or EF was incubated with PA for 30 minutes at 37oC.

52 The plates were washed four times and the bound LF or EF was detected by the addition of polyclonal rabbit anti-LF or anti-EF. The polyclonal antibody was diluted to

5 g/mL in PBST containing 3% nonfat milk and incubated for 90 minutes at 37oC.

After washing, the bound rabbit antibody was detected by the addition of anti-rabbit IgG conjugated to horseradish peroxidase (KPL) diluted 1:7000 in PBST with 3% nonfat milk. Secondary antibody was incubated for 60 minutes at 37oC before unbound antibody was washed off. The plates were developed in the presence of ABTS substrate

(KPL) for 30 minutes at 37oC. The reaction was stopped by the addition of 1% SDS and the absorbance of each sample was read at 405 nm on the PowerWave 340 microplate spectrophotometer (BioTek Instruments). The extent of LF and EF binding to PA was presented by graphing the average absorbance at 405 nm against the LF or EF concentration.

Fluorescent ELISA of PA binding the cellular receptors: Binding of PA83 and PA682 to the surface of J774A.1 mouse monocyte cells was compared using a fluorescent

ELISA. J774A.1 cells were seeded into 96-well black microtiter plates (Corning) at a concentration of 3x105 cells/mL and grown for 16 hours in DMEM (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen), 2 mM L-glutamine (Invitrogen),

100 U/mL penicillin (Invitrogen) and 100 U/mL streptomycin (Invitrogen) at 37oC in a

5% CO2 humidified atmosphere. The overnight growth media was removed and replaced with triplicate two-fold serial dilutions of PA83 or PA682 starting at 200 g/mL. The PA

o was incubated with the cells for eight minutes at 37 C in 5% CO2, then the PA-containing media was removed and the cells were washed three times over five minutes with sterile

PBS. The cells were fixed with 4% paraformaldehyde for ten minutes at room 53 temperature, then the fixative was removed and the cells were washed three times over five minutes with PBS. The plates were blocked with PBS containing 3% BSA for 30 minutes at room temperature, then washed three times over five minutes with PBS.

Primary antibody (mouse anti-PA monoclonal 3E2-071100-02 – kindly provided by Drs.

Robert Bull and Jill Czarnecki) was diluted to 2.5 g/mL in PBS containing 3% BSA and added to the cells for 30 minutes at room temperature. Unbound antibody was removed by washing three times over five minutes. Primary antibody was detected by the addition of anti-mouse IgG conjugated to FITC (KPL). Secondary antibody was diluted 1:250 in

PBS containing 3% BSA and incubated on cells for 30 minutes at room temperature in the dark. Plates were washed three times with PBS over five minutes, then 100 l of PBS was added to each well and the plates were read on the Tecan Ultra Evolution microplate reader (Tecan, Research Triangle Park, NC) at 494 nm. The values for PA-containing wells were normalized against the background fluorescence readings of unlabeled cells.

The amount of PA bound to the cells was presented as the average fluorescence intensity plotted against the PA concentration.

Intracellular localization of PA, LF and EF by immunofluorescence: J774A.1 mouse monocyte cells were seeded into Lab-Tek II 4-well chamber slides (Nunc, Rochester,

NY) at a concentration of 3 x 105 cells/well and grown for 20 hours in DMEM

(Invitrogen) supplemented with 10% fetal calf serum (Invitrogen), 2 mM L-glutamine

(Invitrogen), 100 U/mL penicillin (Invitrogen) and 100 U/mL streptomycin (Invitrogen)

o at 37 C in a 5% CO2 humidified atmosphere. The overnight growth media was removed and replaced with DMEM containing 50 g/mL of each toxin. Cells were intoxicated with LF in the presence of either PA83 or PA425. Cells were also incubated with each 54 protein alone as a control. The cells were intoxicated for 45 minutes at 37oC in the presence of 5% CO2. Extracellular toxin was removed by washing the cells three times over five minutes with sterile phosphate buffered saline. The cells were fixed by the addition of 4% paraformaldehyde for ten minutes at room temperature. Fixative was then removed and the cells were washed three times over five minutes with permeabilization buffer (PBS containing 0.2% NP-40). The slides were blocked with blocking buffer

(PBS containing 0.2% NP-40 and 3% BSA) for 30 minutes at room temperature.

After washing, primary antibody was diluted in blocking buffer and incubated on the cells for 30 minutes at room temperature. PA was detected by the mouse monoclonal

3E2-071100-02 (kindly provided by Drs. Robert Bull and Jill Czarnecki) at a concentration of 2.5 g/mL; LF was detected by rabbit polyclonal antibody at a concentration of 1 g/mL. Primary antibodies (anti-PA and anti-LF) were combined and added to all toxin-containing wells. Control antibodies against Rab5b and Mek1 (Santa

Cruz Biotechnology, Santa Cruz, CA) were diluted to 10 g/mL in blocking buffer and incubated on control wells for 30 minutes at room temperature. Excess antibody was removed by washing three times over five minutes with permeabilization buffer.

Secondary antibody (anti-mouse IgG conjugated to FITC or anti-rabbit IgG conjugated to TRITC) (KPL) was diluted 1:250 in blocking buffer and incubated on the cells for 30 minutes at room temperature protected from light. Unbound antibody was removed by washing three times over five minutes. The slide chambers were removed and the slides allowed to air dry protected from light. Slides were mounted with ProLong mounting medium (Invitrogen) and allowed to set overnight at room temperature in the

55 dark. The following morning, the slides were sealed with clear nail polish and cells were visualized on a Nikon Eclipse TS100 microscope and documented with the Nikon

DXM1200 camera.

2.4 Animal manipulations and immunological analyses

Vaccine formulation and immunization: During vaccine formulation, proteins were administered in a 1:1 molar ratio resulting in the following amounts: 30 g of PA83, 32.5

g of LF (and all full-length forms of LF), 32.1 g of EF (and all forms of full-length

EF), 10.6 g of LFn and 12.4 g of EFn. Reagents were prepared for an entire group of mice at a time so that all mice in a group received identical formulations. Purified protein (containing no more than 10 EU/mg protein) was added to an endotoxin-free microfuge tube and diluted to a final volume of 60 L/mouse using sterile endotoxin-free

HEPES buffer (10 mM HEPES, 50 mM NaCl, pH 7.5). Protein samples for the first and second immunizations were prepared simultaneously. Once prepared, all reagents were stored at -20oC until the morning of the immunization. On the day of the immunization, the protein samples were thawed and warmed to room temperature. An equivalent volume of Imject Alum (Pierce) was added dropwise to the protein solution while vortexing. Absorption to the Alum was allowed to continue for an additional 30 minutes while vortexing. For immunizations given with monophosphoryl lipid A (MPL) as the adjuvant, lyophilized MPL (Lot 220A-25) (Avanti, Alabaster, AL) was resuspended in sterile water and mixed with prepared protein immediately before immunization, according to the manufacturer’s instructions.

56 Groups of ten female Balb/cAnNCr mice (NCI Frederick, Frederick, MD) were immunized intraperitoneally with 120 L of protein adsorbed to Alum. In one experiment (noted in the text), groups of five female Balb/c mice (Jackson Laboratory,

Bar Harbor, ME) were immunized according to the same schedule. Mice received immunizations on days 1 and 15 of the study. Mice were restrained and the abdomen was disinfected with 70% ethanol. Injections were administered intraperitoneally with a

26 gauge needle on the left side of the abdomen. The injection site was aspirated prior to injection to ensure the vaccine was given in the correct location. After injection, mice were monitored for 24 hours to check for any adverse reactions or complications due to the immunization.

Venous blood collection for serum analysis: Venous blood was collected at 14-day intervals during the course of each experiment beginning on the day prior to the first immunization. To promote dilation of the tail veins, mice were placed under a heat lamp for 5-10 minutes. Mice were monitored during this procedure to prevent overheating.

Once vasodilation had occurred, individual mice were restrained, and the tails were disinfected with alcohol. The tail vein was nicked with a disinfected razor blade. Up to

100 L of blood was collected from each mouse in a Microvette Z-gel serum separator tube (Sarstedt Inc., Newton, NC). After phlebotomy, pressure was applied to the incision with sterile gauze and the mouse returned to its cage when bleeding had ceased. Within four hours of collection, blood was separated by centrifugation at 6,000 x g for 10 minutes. The serum-containing fraction was removed and stored at -20oC, with a maximum of one freeze-thaw cycle during the course of analysis.

57 Quantification of serum IgG by indirect ELISA: Immulon 4 microtiter plates (Thermo

Scientific) were coated with the antigen of interest at a concentration of 1 g/mL in

o ELISA coating buffer (0.06 M Na2CO3, 0.14 M NaHCO3 at pH 9.5) overnight at 4 C.

Plates were washed four times with ELISA wash buffer (PBS, pH 7.4 (Cambrex) with

0.1% Tween 20) then blocked with wash buffer containing 3% nonfat powdered milk for

1 hour at 37oC. After washing, serum samples were diluted in triplicate serial two-fold dilutions across the plate for an eleven-point dilution series and incubated 1 hour at 37oC.

Serum samples expected to have an endpoint titer of 3200 or less were diluted in a seven- point dilution series. After washing, secondary antibody was used to detect bound anti- toxin IgG. Horseradish peroxidase-conjugated goat anti-mouse IgG (KPL) was diluted

1:12,000 in ELISA wash buffer and incubated on the plate for 1 hour at 37oC. The plate was washed a final four times with ELISA wash buffer. Reactions were developed in the presence of ABTS substrate (KPL) for 30 minutes at 37oC, stopped with 1% SDS and read at 405 nm on the PowerWave 340 microplate spectrophotometer (BioTek

Instruments).

A standard curve was generated by diluting purified mouse IgG (Sigma) in serial two-fold dilutions from 1000 ng/mL in 0.1 M carbonate/bicarbonate buffer. Diluted IgG standards were used to coat one row of wells on each antigen plate with 100 L of each dilution. Except for the incubation with diluted sera, these wells were treated exactly the same as the wells containing antigen. Instead of being incubated with diluted sera, these wells were incubated with 100 L of ELISA wash buffer. The average absorbance at 405 nm for each point of the standard curve was plotted against IgG concentration and the equation of the best fit line through the linear portion of the curve was determined by 58 Microsoft Excel. The average absorbance from the diluted serum samples in the linear portion of the ELISA graph and within the range of the standard curve was interpolated to determine the antigen-specific IgG concentration of the serum sample. For each group, the titer from each mouse was determined. The individual titers were then averaged (geometric mean) and the standard error of the mean was calculated. Groups were compared by Student’s T test with a p value < 0.05 considered significant.

Lethal toxin neutralization assay: The ability of serum to protect macrophages from the apoptotic effects of lethal toxin was assessed on the mouse monocyte cell line J774A.1

(ATCC). J774A.1 cells were grown in DMEM (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen), 2 mM L-glutamine (Invitrogen), 100 U/mL penicillin

o (Invitrogen) and 100 U/mL streptomycin (Invitrogen) at 37 C in a 5% CO2 humidified atmosphere. Prior to neutralization, the amount of toxin necessary for 99.9% cell killing was determined. This concentration was re-calculated for each experiment, as the sensitivity of macrophages to lethal toxin varies, depending on the number of cell passes and overall cell health. Cells were seeded at a concentration of 3x105 cells/mL onto a 96- well microtiter plate (Corning) 18-20 hours before the assay. The next day, serial two- fold dilutions of lethal toxin (beginning at 1000 ng/mL PA and 800 ng/mL of LF) were

o made in quadruplicate across the plate and incubated at 37 C in 5% CO2. Following a four-hour incubation, the toxin-containing media was removed and replaced with DMEM containing XTT (sodium 3’-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-

6-nitro) benzene sulfonic acid hydrate)) (Roche) for 16 hours to determine cell viability

59 [317]. The assay was read at 480 nm on the PowerWave 340 microplate spectrophotometer (BioTek Instruments). The dilution series data were modeled with 4- parameter logistic curves of the form:

OD480(y) = B1 + (B2-B1)/ [1 + exp {B3 (B4-x)}]

The data were fit by DataFit 8.1 software (Oakdale Engineering) via a nonlinear least- squares analysis, yielding the parameters (B1,B2,B3,B4) of the best fit. The y axis was then converted to % cell viability (ranging from 0-100%), and the toxin concentration at which 0.1% of the target cells remained viable was calculated. This concentration was the amount the test sera were challenged with.

For the neutralization assay, cells were seeded onto a 96-well microtiter plate

(Costar) at 3x105 cells/mL 18-20 hours before the assay. The following day, serum samples were diluted in quadruplicate two-fold serial dilutions and then mixed with lethal toxin (at the concentration calculated to cause 99.9% cell death) for 1 hour at 37oC.

Spent media was removed from the cells and replaced with the serum-toxin mixture.

o Following four hour incubation at 37 C in 5% CO2, the toxin-containing media was removed and replaced with DMEM containing XTT for 16 hours. The absorbances were then read at 480 nm. The dilution series data (absorbance at 480 nm vs. serum dilution) were modeled with 4-parameter logistic (4PL) curves via a nonlinear least-squares analysis as described above. The inflection point of the fit (B4 parameter) corresponds to the serum dilution able to protect 50% of the cells in the assay from lethal toxin action

(ED50) [319]. Data reported here represent the geometric mean of the individual ED50 values of the mice in each group and are shown with the standard error for the group.

Groups were compared by Student’s T test with a p value < 0.05 considered significant.

60 IFN and IL-4 ELISpot of mouse splenocytes: MultiScreen HTS plates (Millipore,

Bedford, MA) were pre-wet with 35% ethanol, and then washed six times with sterile distilled water. Capture antibody (either anti-mouse IFN or anti-mouse IL-4 (Mabtech,

Cincinnati, OH) was diluted to 10 g /mL in PBS and incubated on the plates overnight at 4oC. The plates were then washed six times with RPMI (Invitrogen) and blocked for two hours at 37oC with R10 (RPMI 1640 containing 10% fetal calf serum, 2 mM L- glutamine, 100 U/mL penicillin and 100 U/mL streptomycin).

Mice from the desired vaccine regimen were euthanized by CO2 asphyxiation and their spleens were removed. The spleens were disrupted with the sterile flat end of a 10 ml syringe in R2 (RPMI 1640 containing 2% fetal calf serum, 2 mM L-glutamine, 100

U/mL penicillin and 100 U/mL streptomycin) and allowed to settle for 3-4 minutes on ice to separate large clumps of connective tissue from the single cell suspension. The suspended cells were removed and centrifuged at 400 x g for 10 minutes at 4oC. The cell pellet was washed twice with R2 then resuspended in R10. The splenocytes were counted on a Z1 Coulter particle counter (Beckman Coulter, Fullerton, CA) using an upper threshold of 6.125 m and a lower threshold of 4.000 m. Blocking solution was discarded from the plates and replaced with stimulant. Splenocytes were stimulated with

R10, 5 g/mL Concavalin A, or 1 g of target antigen. Cells were added to the stimulant at a concentration of 2.5 x 105 cells/well and incubated for 48 hours at 37oC.

After stimulation, the cells were removed and the plates were washed six times with ELISPOT wash buffer (PBS with 0.05% Tween 20). Secreted cytokines were detected with anti-mouse IFN- or IL-4 conjugated to biotin (Mabtech). Primary antibody and capture antibody consisted of a matched pair of mouse monoclonal 61 antibodies specific for the cytokine of interest. Primary antibody was diluted to 1 g/mL in PBS containing 0.5% fetal calf serum and incubated on the plates for two hours at room temperature. The plates were washed six times again with ELISPOT wash buffer, and then captured cytokines were detected with streptavidin conjugated to alkaline phosphatase. Streptavidin was diluted 1:1000 in PBS with 0.5% fetal calf serum and incubated on the plates for 1 hour at room temperature. The plates were washed six times with ELISPOT wash buffer, and then three times with PBS. The spots were developed with NBT/BCIP for approximately 15 minutes at room temperature. Once the plate was fully developed, the reaction was stopped by flushing the plate with water. Quenched plates were dried and read on an AID EliSpot 04 reader (Autoimmun Diagnostika GmbH,

Strassberg, Germany). Data reported here represent the geometric mean of the individual spot numbers for the mice in each group and are shown with the standard error for the group. Groups were compared by Student’s T test with a p value < 0.05 considered significant.

62 Protein Mutation Functional Effect Reference

PA83 none wild type protein [94]

PA197 K197A cannot bind to LF or EF [121]

PA425 D425A cannot translocate LF or EF to cytoplasm [158]

PA682 N682S cannot bind cellular receptor [132]

LF none wild type protein [95]

LFn aa 1-254a truncated – binds PA, but enzymatically [123] inactive LF687 E687C catalytic residue replaced - inactive [169, 170]

LF236 b Y236A cannot bind to PA [120]

EF none wild type protein [96]

EFn aa 30-303a truncated – binds PA, but enzymatically [122] inactive EF346 K346R ATP-binding site disrupted - inactive [320]

EF227 b Y227A cannot bind to PA [120]

a The amino acids in LF and EF are numbered differently. Both LFn and EFn begin with the first amino acid after the signal peptide cleavage site b LF236 and EF227 have unaltered substrate binding sites and are enzymatically active

Table 2.1: Functional mutants of the Bacillus anthracis toxin proteins

63 Plasmid Description Gene Expression Source or Reference pSE42 Cloned pXO1 fragment None [96] containing EF cya gene pVR1020 Vector for protein expression CMV promoter, N-terminal Vical from mammalian cells fusion to TPA signal [321] pQE-30 Vector for protein expression T5 promoter, N-terminal Qiagen from E. coli fusion to 6x His tag pET15b Vector for protein expression T7 promoter, N-terminal Novagen from E. coli fusion to 6x His tag

Table 2.2: Plasmid vectors used in this study

Strain Species Use Vendor

DH5 Maintenance and plasmid propogation Stratagene of pVR1020 XL-1 Blue Escherichia coli Maintenance and plasmid propogation Stratagene of pQE-30; limited protein expression M15 Escherichia coli Protein expression from pQE-30 Qiagen constructs SG13009 Escherichia coli Protein expression from pQE-30 Qiagen constructs BL21 Escherichia coli Protein expression from pET15b Novagen construct

Table 2.3: Bacterial strains used in this study

64 b Protein Template Codon Primer Change a

PAK197A pQE-30/PA83 AAA - GCA GGATATACGGTTGATGTCGCAAATAAAAGAAC

PAD425A pQE-30/PA83 GAC - GCC CATTAAATGCACAAGCCGATTTCAGTTCTAC

PAN682S pQE-30/PA83 AAT - AGT GATTTTAAAAAATATAGTGATAAATTACC

LF pQE-30/LF687 TGC - GAA GAGGGTTTTATACACGAATTTGGACATGCTG

c LFY236A pET15b/LF TAT - GCC GATGTTTTACAGCTTGCCGCACCGGAAGC

EFK346R pQE-30/EF AAG -AGG TGTGGCTACAAGGGGATTGAATGTTC

EFY227A pQE-30/EF TAT - GCC GGTATTAGAGTTAGCCGCCCCCGACATG

a Codons were altered in order to meet two criteria: (1) new codons were chosen to be one of the two most commonly used codons for that amino acid in E. coli, and (2) as few nucleotides as possible were changed b Bases that are a mismatch to the native sequence are underlined c pQE-30/LF did not express protein under any circumstances tested, and thus the LFY236A mutation was made in the pET15b/LF construct

Table 2.4: Point mutations introduced into the Bacillus anthracis toxin components

65 Expression Expression Expression Gene Producta Induction ODc Strainb Time Temperature PA83 M15 0.6 4-6 hrs 37oC LF BL21 0.55 16-20 hrs 22-25oC d LF687 SG13009 0.55 16-20 hrs 22-25oC d LFn XL-1 Blue 0.6 4-6 hrs 37oC EF SG13009 0.55 16-20 hrs 22-25oC d EFn XL-1 Blue 0.6 4-6 hrs 37oC a Constructs containing a single amino acid change in the toxin protein were expressed under the same conditions as the parent construct b The expression strains M15 and SG13009 contain the repressor plasmid pREP4 and require the presence of 25 g/ml kanamycin for growth c Optical density readings were taken at 600 nm d Protein expression was conducted at ambient room temperature, which varied between 22 and 25oC

Table 2.5: Expression conditions for recombinant Bacillus anthracis proteins

66 Figure 2.1: SDS-PAGE visualization of purified PA, LF and EF proteins. Proteins were purified over TALON resin under endotoxin-removing conditions, then passed over an EndoTrap Blue column. Equivalent amounts of purified protein were loaded onto a 10% acrylamide denaturing gel. The proteins were electrophoresed at 200 V for 45 minutes, then stained with BioSafe Coomassie for visualization. Shown are the (A)PA mutants, (B) LF mutants and (C) EF mutants. Proteins are compared to the Precision Plus molecular weight standards.

67 CHAPTER 3

THE HUMORAL IMMUNE RESPONSE TO LETHAL TOXIN IS ALTERED BY CELLULAR INTOXICATION AS WELL AS MEK PROTEOLYSIS

3.1 Introduction

Lethal toxin, comprised of PA and LF, is one of the key virulence factors in anthrax. In the early stage of disease, lethal toxin is responsible for the pro-inflammatory cytokine suppression and macrophage apoptosis assisting the systemic spread of bacilli

[322, 323]. In the late stage of disease, lethal toxin may be responsible for circulatory shock contributing to death in anthrax [208]. The loss of LF abrogates the ability of B. anthracis to establish a lethal infection, thus, strains lacking LF are severely attenuated

[281]. Additionally, the neutralization of lethal toxin by antibodies directed against either

PA or LF protects macrophages in vitro [284, 324-326]. In vivo, the presence of PA and

LF neutralizing antibodies correlates directly with protection following challenge [285,

317, 324]. The importance of lethal toxin in both the early and late stages of disease makes elicitng a robust immune response to it highly desirable. However, very few studies have investigated the immune response to LF either alone or in combination with

PA.

68 The British anthrax vaccine (AVP) consists of PA and LF precipitated onto aluminum hydroxide. Individuals immunized with the vaccine produce antibody responses to both PA and LF as well as neutralizing antibodies to lethal toxin

[298](Matthew Hepburn, personal communication). Similarly, mice immunized with a strain of B. anthracis attenuated by the removal of the capsule and EF genes respond with high PA and LF antibody titers [327]. Interestingly, comparison of the immune responses generated by a strain producing only LF and a strain producing lethal toxin revealed that the antibody titer to LF was greatly improved by the presence of PA [327].

The authors of this study attributed the increase in antibody titer to the general impact of lethal toxin activity on the immune system. However, the ability of lethal toxin to suppress immune function during infection is well established and it is not immediately apparent how lethal toxin could enhance the development of the humoral response. and subsequent T cell priming is impaired in dendritic cells and B cells [203, 206]. Macrophages either undergo apoptosis or lose the ability to be activated [51, 52]. B and T cells have reduced levels of proliferation and antibody or cytokine production [203, 205]. However, these inhibitory effects of lethal toxin appear to be transient, as a strong humoral response to both PA and LF rapidly develops following infection [328](K. Brenneman, manuscript in preparation).

In order to understand the effect of lethal toxin on the antibody response to LF, all of the activities of lethal toxin must be considered. The proteolytic inactivation of MEK family members by LF has been examined in depth; however, investigations have focused on the immediate consequences of MEK cleavage in specific cell types. The immunological events triggered by lethal toxin in a mixed population of cells in the days

69 and weeks following intoxication are largely unknown. One report found that lethal toxin acted as a T cell mitogen 4-6 days after intoxication. In this study, T cell proliferation depended on the metalloprotease activity of LF as well as the presence of intoxicated monocytes [314], implying that the interaction between lethal toxin and the immune system may be much more complicated than in vitro studies indicate.

The cellular intoxication process, carried out largely by PA, also affects the antibody response to LF, as immunization studies with lethal toxin defective in MEK proteolysis have revealing results. Immunization of mice with DNA constructs encoding

PA and the N-terminal domain of LF showed an improvement in LF titer that was dependent on the presence of PA, but not on lethal toxin activity [313]. A more comprehensive investigation examined the immune response to strains of B. anthracis producing functionally inactivated PA [116]. The introduction of deletions into the various functional regions of PA indicated that the increase in LF titer could be attributed to the formation of a soluble toxin complex [116]. Both of these studies indicate that

MEK proteolysis by LF plays a limited role in the enhancement of the immune response to lethal toxin.

Although both studies implied that an increaseed response to LF depends on the activity of PA, not MEK proteolysis by LF, a more detailed investigation is necessary.

Protein expression from attenuated strains was quantified only by western blot and no attempt was made to examine the stability of the deleted proteins. More recent studies on the toxin complex have shown that some of the deletions were in functionally irrelevant locations while others may have had multiple functional effects. The study also failed to include the immune response to wild type lethal toxin. The DNA immunization study did

70 not account for the effect of DNA on the immune response. the addition of a second plasmid in the PA+LF immunization may have increased the amount of CpG motifs present. Changing the number of CpG motifs present could have altered TLR signaling and subsequent immune activation, regardless of the protein encoded on the vector. Also, protein expression from the DNA constructs resulted in the cytoplasmic localization of both PA and LF. The normal cellular intoxication process of lethal toxin results in the cytosolic localization of only LF. Altering the subcellular distribution of PA in the DNA immunization may have altered the resulting immune response due to differences in antigen processing and presentation.

PA contributes three functions towards lethal toxin activity. Each of these functions has immunological relevance and can be hypothesized to affect the development of the anti-toxin immune response. First, the high affinity of the lethal toxin components for each other and the slow rate of complex dissociation [127, 129] may stabilize the lethal toxin complex in vivo, thus retarding extracellular degradation and providing a higher concentration of PA and LF available for an immune response.

Second, the binding of lethal toxin to the cellular receptors may enhance the development of an immune response to LF by increasing the amount of LF taken up by antigen presenting cells. The low dissociation rate of the toxin complex [127] and the high affinity of PA for its cellular receptor [148] ensures that both LF and PA will concentrate at the surface of cells expressing the anthrax toxin receptors. Third and finally, the translocation of LF to the cytosol from the endosome by PA may alter antigen processing and presentation in a way that enhances the anti-LF response. A significant increase in

71 the amount of LF in the cytoplasm of immune effector cells may result in improved MHC

I presentation. Alternatively, if the efficiency of LF translocation is poor [168, 329], the increased concentration of LF at the cell surface may also result in an increased amount of LF present in the late endosomes and lysosomes and thus improve MHC II presentation.

In order to determine the effects of MEK proteolysis by LF and the cellular intoxication activities of PA on the immune response to lethal toxin, detailed information about the lethal toxin complex was used to produce single amino acid mutations in both

PA and LF. Each of these mutations removed a specific functional activity without grossly affecting the physical properties of either PA or LF. Mice were then immunized with lethal toxin combinations defective in a single functional activity and the resulting antibody responses were compared to the response generated by each component alone as well as wild type toxin. Although many other animal species are susceptible to the effects of lethal toxin, mice were chosen as a model for two reasons. First, the effects of lethal on the immune system are best described in mice. Second, the increase in antibody titers following co-immunization thus far has been observed exclusively in mice.

3.2 Results

3.2.1 Characterization of lethal toxin mutants

In order to investigate the effects of specific lethal toxin functions on the downstream immune response, each of lethal toxin’s described activities was knocked out. To accomplish this, a variety of single amino acid mutations were introduced in PA and LF. The mutations removed the following functional activities: MEK proteolysis,

72 complex formation, cellular receptor binding and cytoplasmic translocation. Each mutation was introduced by site directed mutagenesis into the protein expression construct. Proteins were then expressed from E. coli and purified over a metal affinity column via an N-terminal 6X his-tag. All proteins were purified under endotoxin- removing conditions.

MEK proteolysis by lethal factor: In order to remove the proteolytic activity of LF on

MEK family members, the mutation E687C was made in LF [169]. This removes the general base from the active site [161] and reduces enzyme activity to negligible levels

[170]. Although LF687 is unable to inactivate MEK family members and has no toxicity in macrophages, the MEK binding site on LF687 is intact. Since the N-terminal domain of MEK serves as a means to localize MAPK in the cytoplasm for efficient phosphorylation and signal transduction [178, 180], binding of LF687 at the amino terminus of MEK may displace MAPK and alter cell signaling without adverse effects on cell viability. In order to examine the immunological effect of cytoplasmic LF without any MEK interactions, an additional construct was made that contained only the amino terminal domain of LF (LFn). LFn is able to bind PA and be translocated to the cytoplasm of target cells [123], but lacks the substrate binding domains and the active site

[161]. See Figure 3.1 for the structural location of these mutations.

The ability of LF687 and LFn to cleave the N-terminus of Mek1 in J774A.1 macrophages was compared to the function of wild type LF. Following intoxication with

PA and either LF, LF687 or LFn, cell lysates were probed for the presence of the N- terminal and C-terminal epitopes of Mek1 (Figure 3.2). Exposure to PA, LF, LF687 or

LFn alone had no effect on the N-terminal of Mek1. Intoxication with PA+LF

73 resulted in the loss of the N-terminal Mek1 epitope. No decrease in the amount of Mek1 present was observed with the C-terminal specific antibody, although a slight increase in the electrophoretic mobility of Mek1 was detected. In contrast, no effect on the N- terminal or C-terminal Mek1 epitope was observed for cells intoxicated with either

PA+LF687 or PA+LFn, demonstrating the lack of enzymatic activity for these two mutants of LF.

Lethal toxin complex formation: The formation of the toxin complex was averted by two mutations. The binding of PA to LF was prevented by the point mutation K197A in

PA. Substitution of this residue removes a critical positive charge from the LF binding site [121, 125] and results in the loss of more than 95% of wild type activity [121]. The binding of LF to PA was disrupted by the point mutation Y236A in LF. This mutation results in the loss of any detectable PA binding [120] presumably due to the removal of a stabilizing hydrophobic interaction at the center of the binding site [125]. See Figures 3.1 and 3.3 for the structural locations of these mutations.

The binding of PA197 to wild type LF and LF236 to wild type PA was assessed using two different methods. First, the shift in electrophoretic mobility between monomeric proteins and the heterodecameric complex was visualized on a 4% native acrylamide gel (Figure 3.4). The combination of wild type PA and LF results in a discrete band of low electrophoretic mobility that corresponds to the full-size toxin complex [121]. The combination of PA and LF236, PA197 and LF, or PA197 and LF236 does not produce a similar low mobility band. The ability of PA197 to bind wild type LF and LF236 to bind wild type PA was quantified using a modified sandwich ELISA

(Figure 3.5). PA and PA197 were captured with equal efficiency using a non-

74 neutralizing monoclonal antibody (data not shown). The amount of LF or LF236 bound to PA was detected using a non-neutralizing polyclonal antibody. Negligible amounts of

LF and LF236 were captured by the anti-PA monoclonal in the absence of PA (data not shown). When compared to wild type lethal toxin, the single mutation combinations of

PA197 with LF and PA with LF236 showed a 10-15 fold decrease in complex formation.

The combination of PA197 and LF236 resulted in a more than 50-fold decrease in complex formation.

Cellular receptor binding: Binding of the toxin complex to the cellular receptors TEM8 and CMG2 was inhibited by introducing the mutation N682S into PA (Figure 3.3). The residue is located on an exposed loop of domain 4 of PA that is responsible for interacting with the metal ion contained in the metal ion-dependent adhesion site of the receptor [141]. Although it is the neighboring aspartate 683 that makes the essential contact with the metal ion, mutation of residue 682 prevents receptor binding [132, 330] most likely by destabilizing either the D683-metal ion interaction or the structure of the entire exposed loop [141, 145, 146].

Binding of PA682 to cell surface receptors was measured on J774A.1 macrophages by fluorescent ELISA (Figure 3.6). Cells were exposed to either PA or

PA682 for 8 minutes – sufficient for significant binding, but minimal internalization

[147] – before the cells were fixed and stained for the presence of PA. Wild type PA exhibits dose-dependent binding consistent with a high number of cellular receptors.

Only minimal amounts of PA682 were detected bound to the macrophages.

Cytoplasmic translocation: The translocation activity of lethal toxin was precluded by the mutation D425A in PA (Figure 3.3). The mutation prevents the formation of the

75 transmembrane channel and results in a 10-fold decrease in LF translocation to the cytoplasm [158]. Although residue 425 is not predicted to be part of the transmembrane pore [155, 156], the aspartate is thought to make a specific contribution to the conformational changes that occur during the conversion from receptor-bound heptamer to membrane-spanning pore [158]. The PA425 protein was more difficult to purify than the other mutants due to protein degradation prior to purification. Although proteolytic digest experiments predict that this mutant to be folded similarly to wild type PA [158], the exact contribution of this residue in translocation is currently unknown and alteration at this site may have resulted in the local destabilization of domain 2.

In order to assess the functional activity of PA425, the subcellular location of LF in the presence and absence of PA was examined by indirect immunofluorescence.

J774A.1 macrophages were intoxicated with PA, PA425, LF, PA+LF or PA425+LF, then fixed and stained for PA and LF. The resulting patterns were compared to those of endosomal and cytoplasmic markers (Figure 3.7). In cells that received either LF or PA alone, both proteins stained with the punctate pattern characteristic of endosomal localization (Figure 3.8). Cells that received active lethal toxin (Figure 3.8) showed a punctate pattern of PA localization unaffected by the addition of LF. However, in the presence of PA, LF no longer produced punctate staining, but was detected throughout the cell in a pattern consistent with cytoplasmic localization. In contrast, cells intoxicated with PA425+LF show punctuate staining for LF in both the presence and absence of PA

(Figure 3.9). These data imply that LF is unable to separate its cellular location from

PA425 and are consistent with the more extensive studies done on this mutant by

Sellman, et. al. in 2001 [158].

76 Cellular toxicity of lethal toxin mutants: Although each mutation was found to impair the specific functional activity it corresponded to, the ability of each mutant to cause death in J774A.1 macrophages was also assessed (Figure 3.10). No single form of either

PA or LF alone affected cell viability (data not shown). The toxin combinations

PA+LF687, PA+LFn and PA682+LF did not exhibit toxicity at any concentration tested.

At extremely high concentrations (> 5 g/mL), PA425+LF and PA+LF236 were able to cause death in 15-20% of cells. While the data cannot be accurately analyzed by 4- parameter logistic fit, an estimation of the toxin concentration necessary for 50% cell death is approximately 15 g/mL of PA425+LF and 20 g/mL of PA+LF236. In comparison with wild type lethal toxin, this represents a 25-fold and 40-fold decrease in macrophage lethality, respectively. The combination of PA197+LF was able to kill macrophages; however, the toxin concentration necessary for 50% cell death was substantially more than that for wild type toxin (4 g/mL PA197+LF vs. 0.5 g/mL lethal toxin).

To summarize, all of the single point mutations significantly impaired the specific function targeted. The loss of specific functional activity translated into a loss of macrophage lethality for all mutants except PA197. While PA197 showed severe defects in binding to LF, this defect was partially overcome on live cells, resulting in a quantifiable amount of cell death. However, PA197 was included in the set of functionally inactivated lethal toxin mutants, as the activity of this PA197+LF was 10- fold less than that of wild type lethal toxin.

77 3.2.2 Immunization with non-proteolytic mutants of lethal toxin

The immune response to the non-proteolytic mutants of LF – LF687 and LFn – in combination with PA was compared to the response generated by immunization with active lethal toxin. Proteins were purified under endotoxin-removing conditions and administered to mice in a 1:1 molar ratio. The 1:1 molar ratio was used in order to keep the proportion of protein involved in a toxin complex constant across all groups of mice.

Mice received the molar equivalent of 30 g of PA. The proteins were mixed prior to absorption onto alum. Groups of 10 Balb/cANnCr mice were immunized intraperitoneally on days 1 and 15. Although wild type lethal toxin retained a significant amount of activity following absorption to alum, the immunizing dose was less than the published LD50 [207], and no toxicity was observed during the immunization regimen.

PA-specific antibody response: Following immunization, the serum IgG titers to PA were analyzed at two week intervals for 70 days. No mouse had a detectable anti-PA response prior to the immunization series. Serum IgG specific for PA was detectable at day 14, although the levels of antibody present were extremely low (<1 g/mL). The antibody response did not begin to fully develop until after the second immunization

(Figure 3.11). The PA antibody response peaked at 1.33 mg/mL on day 56, one month after the final immunization. Addition of LF, LF687 or LFn to PA had no effect on the kinetics of the humoral response to PA. The addition of LF, LF687 or LFn also had no effect on the production of PA-specific antibodies at the early timepoints of the study.

No significant difference was observed between PA alone and PA combined with LF,

LF687 or LFn at either day 14 or 28. However, the addition of any of the three forms of

LF to PA significantly reduced the PA antibody response after day 42. The PA antibody 78 titer at day 42 dropped from 0.920 mg/mL to 0.322 mg/mL with LF687 (p = 0.004). At day 56, titer were further reduced from 1.33 mg/mL to 0.797 mg/mL with LFn (p =

0.041), to 0.466 mg/mL with LF687 (p = 0.008), and to 0.713 mg/mL with LF (p =

0.030). By day 70, the PA-specific titer had been reduced from 1.15 mg/mL to 0.756 mg/mL with LFn (p = 0.046), to 0.460 mg/mL with LF687 (p = 0.001) and to 0.432 mg/mL with LF (p = 3.2x10-4).

LF-specific antibody response: The serum IgG titers to LF were also measured at two week intervals for 70 days during the course of the experiment. Prior to immunization, no mouse had a detectable anti-LF response. Following immunization, the humoral response to LF developed rapidly (Figure 3.12). After only one immunization, the LF- specific titers ranged between 43 and 110 g/mL. This was significantly higher than the corresponding anti-PA response at day 14. The antibody response to LF also developed more rapidly than the antibody response to PA. Slight differences occurred between the full-length and truncated forms of LF. Serum IgG titers to LFn peaked on day 28 and declined steadily thereafter. The titers to LF687 and LF peaked between days 28 and 42, although the peak appeared to be closer to day 42 than day 28. Although a smaller g amount of LFn was used in the immunization compared to full-length LF, the antibody response to LFn was comparable to that of LF and LF687 at every point except day 42.

The addition of PA to LFn had no significant effect on the LF-specific antibody response (Figure 3.12). Neither the amount of LF-specific antibody produced nor the kinetics of the response differed from LFn alone. Likewise, the combination of PA with

LF687 did not alter either the kinetics of the response or the amount of antibody produced against LF687. Although the LF titer for PA+LF687 appeared higher than for 79 LF687 at days 56 and 70, the differences were statistically insignificant (p = 0.268 and

0.467 for day 56 and 70, respectively) and can be attributed to variation among group members in the rate of antibody reduction. In contrast, the addition of PA to wild type

LF changed both the kinetics of the LF response and the amount of antibody produced.

The antibody response to LF peaked on day 28 for lethal toxin instead of between day 28 and 42 for LF alone. Higher antibody levels against LF were present on day 14 and 28 in mice immunized with lethal toxin compared with those immunized with LF alone. At day 14, the antibody titers were 44 g/mL and 107 g/mL for LF and lethal toxin, respectively (p = 0.0002). On day 28, the LF-specific titers were 4.03 mg/mL and 6.32 mg/mL for LF and lethal toxin, respectively (p = 0.0148). The alteration of the LF- specific humoral response by lethal toxin only occurred in the early stage of the immune response, as after day 42, the levels of antibody produced in response to LF and lethal toxin were equivalent.

Lethal toxin neutralization: In addition to the general antibody titers produced against

PA and LF, the ability of antibodies to neutralize lethal toxin was also measured at two week intervals for 70 days. Since the amount of lethal toxin neutralization corresponds directly to survival in anthrax infection [285], this activity was assessed as a measure of the effectiveness of the antibody response. Despite the presence of PA and LF-specific antibodies in the serum on day 14, neutralizing activity was not detected in any serum samples prior to day 28 (data not shown). Likewise, neutralizing activity was not detected on any day for mice immunized with adjuvant alone (data not shown).

Significant levels of lethal toxin neutralization occurred in mice that received PA, LF and

LF687 at all points after day 28 (Figure 3.13). The kinetics of the PA neutralizing

80 response paralleled those of the overall PA antibody response, with the peak response on day 56. In contrast, the neutralizing response to all forms of LF differed from the kinetics of the original antibody response. LF-specific antibody titers peaked between day 28 and

42 for all forms of LF. The level of neutralizing activity generated by LF, LF687 and

LFn remained relatively constant throughout the experiment. Interestingly, although LFn contains the PA binding site expected to be a key neutralizing epitope, very low levels of neutralizing antibody were produced in response to LFn.

Immunization with PA and LFn resulted in a significantly greater amount of neutralizing activity than immunization with LFn alone at all time points (Figure 3.13).

However, the lethal toxin neutralizing ability of serum from mice immunized with PA in addition to LFn was not significantly different from mice that received PA alone except on day 70. On day 70, the neutralizing titer for PA was 2,170, while PA+LFn had a neutralizing titer of 5,380 (p = 0.0002). In contrast, the amount of lethal toxin neutralizing antibodies produced in response to PA was significantly enhanced by the addition of LF687 at days 28, 42 and 70 (p = 0.0018, 0.0023 and 5.851x10-5, respectively). A significant change in the neutralizing titers between LF687 alone and

PA+LF687 was observed only on day 70 (titers increased from 2,170 to 6,650; p =

0.0046). Immunization with lethal toxin increased the neutralizing titer over that observed with PA alone at days 28, 42 and 70 (p = 0.0058, 0.0323 and 0.0017, respectively). Similarly, immunization with lethal toxin significantly increased the neutralizing titer that observed for LF alone at all time points (p = 0.0167, 0.0134, 0.0054 and 0.0145 for days 28, 42, 56 and 70, respectively)

81 3.2.3 Immunization with lethal toxin mutants deficient in cellular intoxication

Although the enhancement of the LF antibody response depended on the activity of LF, the decrease in the PA antibody response after day 42 did not. Therefore, the immune response to mutants deficient in the cellular intoxication process – PA197,

PA682 and PA425 – was compared to the response generated by active lethal toxin.

Endotoxin-free protein was combined in a 1:1 molar ratio and absorbed to alum. Groups of 10 Balb/cANnCr mice received 30 g of PA and/or the molar equivalent of LF on days 1 and 15 in an intraperitoneal immunization. Mice that received active lethal toxin did not exhibit any symptoms of toxicity. The serum IgG titers against PA and LF were measured at two-week intervals for 56 days after the first immunization. None of the mice had detectable PA or LF-specific antibody responses prior to immunization.

Immunization with toxin complex formation mutants: PA197, although unable to bind to LF and form the toxin complex, produced an immune response similar to that of wild type PA. The kinetics and strength of the antibody response and the T cell cytokine production were equivalent at all time points for these two molecules. However, combination with LF produced quite different results for PA and PA197. The addition of

LF to PA had no effect on the antibody response to PA. Adding LF to PA197 resulted in a substantial increase in PA-specific antibody production at day 28 (Figure 3.14). The

PA titer increased from 45 g/ml to 750 g/ml (p = 0.016). Combining PA197 with LF did not affect the kinetics of the humoral response – antibody titers peaked on day 28 in both cases. However, the amount of LF-specific antibody produced decreased significantly at days 14 and 28 (p = 2.02x10-6 and 0.044, respectively) in the presence of

PA197. 82 Cytokine production during the T cell response also differed between lethal toxin and PA197+LF. The number of IL-4 and IFN- producing T cells specific for LF and PA was measured at the conclusion of the experiment in order to understand the general features of the T lymphocyte response. With lethal toxin, both the IFN- response and the IL-4 response to PA were unaffected by the addition of LF (Table 3.1). Likewise, the IFN- response to LF was unchanged by the addition of PA. Only the IL-4 response to LF altered in the presence of PA. In this case, the number of IL-4 producing cells grew from 67.6 to 154 (p = 7.74x10-6). When the association of the two proteins was prevented, the T lymphocyte response to LF in the presence of PA197 was equivalent to the response seen with LF alone. In contrast, the response to PA197 in the presence of

LF changed significantly when compared to PA197 alone. The number of IL-4 producing T lymphocytes increased from 5.52 to 56.4 (p = 8.2x10-4) with the addition of

LF. Similarly, the number of IFN- producing cells changed from 2.74 to 18.3 (p =

8.2x10-4). This difference between lethal toxin and PA197+LF was striking. Active toxin affected cytokine production in the LF-specific response. Removing the ability to form the toxin complex not only failed to improve LF-specific cytokine production, but also changed the amount of IL-4 and IFN- produced in response to PA.

Immunization with cellular receptor binding mutants: When the toxin complex was allowed to form, but not localize at the cell surface on receptors, a slightly different effect was observed. PA682 produced a humoral response equivalent to PA, but behaved differently from both PA and PA197 when in the presence of LF. Similar to PA197, the antibody titer to PA682 was unaffected by the addition of LF (Figure 3.15). The kinetics of this antibody response were the same as those for PA682 alone. The amount of 83 antibody produced against LF was significantly decreased by the presence of PA682, dropping from 8.35 mg/mL to 3.26 mg/mL on day 28 (p = 0.006), from 4.67 mg/mL to

2.61 mg/mL on day 42 (p = 0.018), and from 4.63 mg/mL to 2.66 mg/mL on day 56 (p =

0.040). Thus, the humoral response to PA682+LF appeared to be an intermediate between active lethal toxin and protein deficient in complex formation.

A similar hybridization was observed with the T lymphocyte cytokine profiles

(Table 3.2). The number of cells producing IFN- in response to PA682 increased in the presence of LF from 3.54 to 10.8 (p = 0.023), as did the number of cells producing IL-4

(from 11.8 to 44.8; p = 0.001). This was also observed for toxin deficient in complex formation. In contrast, the response to LF in the presence of PA682 resembled that produced by lethal toxin. The amount of T lymphocytes secreting IFN- in response to

LF was unaffected by the addition of PA682, but the quantity of T cells producing IL-4 in response to LF doubled from 67.5 to 141 (p = 4.85x10-5) when PA682 was added.

Immunization with cytoplasmic translocation mutants: A dramatic change in both the antibody and the T cell responses occured when the proteins were localized in the endosome and prevented from translocating. The antibody response to PA425 was not equivalent to the antibody response produced by PA (Figure 3.16). The amount of antibody produced against PA425 dwarfed the amount specific for PA at days 28, 42 and

56. The kinetics of the response to PA425 were also significantly altered. The levels of antibody specific for PA425 climbed throughout the 56 days of the experiment, while the amount of antibody specific for PA leveled off by day 42. The reason for the difference between PA and PA425 is unknown. When LF was added to PA425, the PA antibody response decreased significantly at days 42 and 56 (p = 0.009 and 0.004, respectively). 84 However, the amount of PA-specific antibody produced for PA425+LF was comparable to the amount of PA-specific antibody observed for wild type lethal toxin. Antibody production in response to LF decreased from 8.35 mg/mL to 5.48 mg/mL at day 28 (p =

0.045) when PA425 was added, but was equivalent to LF alone at all other time points.

Although the antibody responses to PA and PA425 were quite different, the IL-4 and IFN- T lymphocyte responses to PA and PA425 alone were equivalent (Table 3.3).

However, the combination of PA425 and LF had a negative impact on the T lymphocyte response. The number of cells producing IFN- upon stimulation with PA425 was unaffected by co-immunization with LF, but the number of IL-4 producing cells decreased significantly from 38.0 to 11.6 (p = 4.1x10-4) when LF was present. The opposite effect occurred for LF. The number of lymphocytes producing IL-4 in response to LF did not change significantly with the addition of PA425. The amount of cells producing IFN- in response to LF was diminished (from 154 to 88.0) by the presence of

PA425 (p = 0.013).

3.3 Discussion

Active lethal toxin exerts two separate effects on the adaptive immune response.

The first is the early increase in LF antibody titers and the increase in the number of IL-4 producing T lymphocytes. This effect of lethal toxin depends on the enzymatic activity, including proteolysis of MEK family members, and will be referred to as the active toxin effect. The second effect depends not on MEK proteolysis, but on the intoxication ability of PA and the combined presence of PA and LF. This will be referred to as the inactive

85 toxin effect, for although PA and the toxin complex still interact with the immune system in important ways, the metalloprotease activity of lethal toxin has been removed.

The active toxin effect results from the downstream consequences of MEK proteolysis and the resultant toxicity. One possible explanation for the active toxin effect is the necrotic death of macrophages. Necrotic cell death has been associated with the release of alarmins – self molecules capable of activating the innate immune cells [331,

332]. These molecules include heat shock proteins [333-338], nuclear proteins [339] and free nucleotides [340, 341], reactive oxygen intermediates [342] and extracellular matrix components [343]. Necrotic cell death is a sufficient stimulus to result in the maturation of dendritic cells and the priming of CD4+ T lymphocytes [344, 345]. High concentrations of lethal toxin have been observed to produce the necrotic death of macrophages in vitro [346]. This toxin-induced cell death has been reported to result in the release of reactive oxygen intermediates [50] as well as production of the pro- inflammatory cytokines TNFIL-1 and IFN- [50, 204], all of which may contribute to a danger response by the immune system leading to the increase in LF titer.

While the necrotic death of macrophages at the immunization site could result in the activation and maturation of dendritic cells and thus improved T cell priming, it is unclear to what degree macrophage necrosis occurs in vivo. Minimal amounts of necrotic macrophages are observed in mice challenged with a lethal dose of lethal toxin [207].

Similarly, a lethal toxin challenge does not result in the release of reactive oxygen intermediates or IL-1 [207, 208]. Thus, macrophage necrosis may only be an in vitro phenomenon, and another explanation for the active toxin effect may be necessary.

86 The hallmark of lethal toxin action is the apoptosis of intoxicated macrophages.

Apoptosis is often considered an immunologically silent form of cell death that does not promote the maturation and activation of innate immune cells [344, 347-349]. However, a growing number of studies have indicated that the uptake of apoptotic bodies by dendritic cells can induce maturation and efficient T cell priming [350, 351]. The generation of a strong immune response appears to depend on the degree of cellular apoptosis [352] and possibly infection-related signals [353-355]. Phagocytosis of apoptotic bodies can serve as an important source of antigens for dendritic cells [356,

357]. These antigens are displayed on MHC I molecules via cross-presentation [350,

356, 358] and presentation results in efficient CTL priming [357, 359, 360]. Although the processing of apoptotic antigens is best described for the priming of CTL responses, antibody production and MHC II presentation can also be enhanced [353, 361].

The specifics of an immune response primed by apoptosis fit with the details of lethal toxin intoxication. Increased levels of T cell proliferation occur only in a mixed lymphocyte population that has been exposed to intoxicated macrophages [314].

Macrophages readily undergo apoptosis when exposed to lethal toxin [51]. Normally, macrophages are responsible for the majority of apoptotic cell clearance [362], but uptake results in the prevention of pro-inflammatory cytokine expression and antigen presentation [363]. Efficient antigen presentation and T cell priming results when the amount of apoptosis exceeds the phagocytic capacity of the local macrophage population and immature dendritic cells are recruited [352]. During lethal toxin-induced apoptosis, the phagocytic capability of macrophages is impaired [51]. This could easily result in a bolus of apoptotic bodies requiring increased dendritic cell involvement.

87 Dendritic cells recruited to the immunization site may be intoxicated by free lethal toxin, but they cannot be intoxicated from the phagocytosis of cells damaged by lethal toxin, due to the nature of the A-B toxicity. This limits the degree of dendritic cell intoxication. The intoxication of dendritic cells results in the loss of cytokine production and costimulatory molecule upregulation in response to TLR stimuli, and therefore a decrease in T cell priming efficiency [206]. However, the maturation of dendritic cells in response to apoptosis appears to proceed through a TLR-independent pathway [364], circumventing some of the effects of lethal toxin. The intoxication and apoptosis of macrophages may remove a large number of innate immune effector cells, but the resulting apoptotic debris could be more than enough to stimulate an adaptive response.

In vivo, a sub-lethal challenge of toxin could be expected to enhance the adaptive immune response to lethal toxin due to additional dendritic cell maturation and T cell priming. However, the adaptive responses to both components of lethal toxin are not enhanced – it is only the response to LF that improves. This is the result of the inactive toxin effect, which depends on the activity of PA and the toxin complex’s contribution to the immunogenicity of LF. Immunization with PA results in moderate antibody titers that peak around day 56. The T cell response to PA is composed almost entirely of IL-4- producing lymphocytes, which agrees with studies showing that PA is processed exclusively in the endosomal and lysosomal compartments for subsequent MHC II presentation [365]. In contrast, immunization with LF results in high antibody titers that peak between day 28 and 42. The majority of T lymphocytes accompanying the LF antibody response produce IFN-, although IL-4-producing T cells are a large minority.

The immune response to LF also results in a significant amount of cytotoxic T

88 lymphocytes [366]. Although LF in the absence of PA is usually considered to be an extracellular protein, LF may be able to reach the cytoplasm of cells in a PA-independent manner [367], explaining the strong TH1 character of the LF immune response.

The differences in the T cell responses (TH1-like for LF versus TH2 for PA) imply that the responses to PA and LF will antagonize each other when both antigens are present. Translocation of LF to the cytoplasm by active PA would be expected to enhance the TH1-like character of the LF response, further exacerbating the antagonism between the responses. This is exactly what happens when active PA is combined with

LF. The LF antibody response develops at the expense of the PA antibody response.

The antibody titers to PA decrease in the presence of both active and inactive LF, while the antibody response to inactive LF is unaffected. The antibody response to active LF in the presence of PA is significantly improved, due to the immunological contribution of intoxicated macrophage cell death. The antagonism between the LF and PA responses extends beyond these two proteins – the development of a TH2 response to ovalbumin (aa

323-329) is enhanced by the presence of PA, but impaired when PA and LF are both present [206]. Interestingly, the T lymphocyte response to each antigen appears to be affected by the immune response to the other protein. The number of T cells producing

IFN- in response to PA increases in the presence of LF; likewise, the number of LF- specific cells producing IL-4 increases in the presence of PA.

The immunodominance of the LF antibody response depends on the activity of

PA. Mutations in PA that disable the cellular intoxication process and thus prevent the cytoplasmic localization of LF enhance the TH2 characteristics of the adaptive immune response and change the predominant antigen from LF to PA. There is a general decrease

89 in the level of LF-specific antibodies and an increase in the number of IL-4 producing T lymphocytes to PA. The extent to which the PA response is improved and the LF response harmed depends on the specific PA function removed.

The simplest competition between the TH1-like LF response and the TH2 PA response occurs when complex formation is prevented. In this situation, the PA and LF are physically separated and interact with the immune system much as they do when the other protein is absent. LF no longer has a direct route into the cytoplasm via PA, and an antigen presenting cell displaying PA peptides may or may not display LF peptides. The resulting LF response when PA197 is present is very similar to that of LF alone. The number of IL-4 and IFN- producing T cells remains the same. The only noticeable effect of PA197 on LF is a small decrease in the LF-specific antibody titer at day 28.

However, there is a dramatic improvement in the PA response. Antibody titers increase, as do the number of IL-4 and IFN- producing T cells. When LF is forced to reach the cytosol on its own via PA-independent uptake, only a fraction of the LF present is internalized [367]. All of the PA molecules are able to traffic to the endosome by receptor-mediated uptake [148-150]. Thus, the resulting TH1 response to LF is overshadowed by the strong TH2 response generated by the endosomal PA.

The prevention of receptor binding by PA complicates the immune response to both proteins. Under these circumstances, LF is prevented from interacting with cells in a PA-independent fashion, as a significant portion is sequestered in toxin complexes

[127, 129]. Thus, the immune response to LF in the presence of PA682 is substantially altered. Antibody titers to LF drop to their lowest levels with this form of PA. IL-4 production by T lymphocytes increases to its highest levels for both PA and LF. The 90 IFN- response to PA is almost non-existent. All of these conditions indicate the generation of a strong TH2 response that should favor PA, however, there is no increase in the amount of PA-specific antibody produced. There are two probable reasons for this.

First, the endosomal localization of PA682 is less efficient than wild type PA, as PA682 molecules can no longer concentrate at the cell surface [148]. Second, PA must be processed in a sequential fashion by different proteases for efficient processing, with peptides from the LF binding site being released first in the early endosome [365].

However, LF does not dissociate from the LF binding site until the complex reaches the late endosome [152, 164]. Thus, the binding of LF likely interferes with the endosomal processing of PA.

The most dramatic effect on the PA and LF immune responses occurs when PA is prevented from translocating LF out of the endosome. This strongly biases the ensuing response in the TH2 direction by ensuring the endosomal and lysosomal processing of both proteins as well as subsequent display on MHC II molecules. Endosomal localization of LF results in a significant decrease in both LF-specific antibodies and

IFN--producing T lymphocytes, emphasizing the importance of cytosolic processing for this antigen. Surprisingly, the endosomal localization of LF also had a negative effect on the PA immune response, decreasing both antibody titers and IL-4 production by T cells.

Why this occurred is unclear. It is unlikely that cytosolic processing is important for PA, as several studies have reported endosomal processing for PA and the subsequent development of a TH2 response [206, 283, 365]. Also, both the antibody response and the IL-4 producing T cell response to PA425 alone were significantly better than the response to any other form of PA. The reason for the decreased PA425 response in the 91 presence of LF may be related to the stability of PA425. PA425 was much more prone to degradation during purification, which may have enhanced endosomal digestion and antigen presentation. The addition of LF and complex formation may stabilize PA425 and provide resistance to protease digestion that the PA monomer lacks [127].

Regardless of the competition between the LF and PA-specific immune responses, the ability of serum to neutralize lethal toxin benefited from the presence of both antigens in an immunization. Data from the toxin neutralizing experiments imply that although the antibody responses from PA+LFn and PA+LF687 appeared to be similar, the antibodies generated in the response were quite different. The neutralizing antibodies of mice immunized with PA+LFn were primarily directed against PA. The early increase in neutralizing activity in PA+LF687 sera was most likely due to the presence of LF-specific neutralizing antibodies, as the addition of PA to LF687 had no effect on the neutralizing ability of serum at either day 28 or 42. The neutralizing response to lethal toxin appeared to be more balanced, incorporating neutralizing epitopes from both PA and LF.

For all combinations of PA and LF, co-immunization resulted in an increase in the length of time that a high level of circulating toxin neutralizing antibodies were present.

The addition of LF to PA results in high levels of lethal toxin neutralization shortly after immunization, most likely due to the early production of LF antibodies. Interestingly, the

PA binding site on LF did not contribute significantly to neutralization, implying that LF- based toxin neutralization occurs at a later step in the intoxication process than complex formation. The combination of PA and LF also results in higher levels of toxin neutralization than either protein alone at the conclusion of the immune response. The

92 enhancement of lethal toxin neutralization resulting from co-immunization with PA and

LF make a very strong case for the inclusion of LF in any future toxin-based anthrax vaccines.

In conclusion, combining PA and LF in an immunization has numerous effects on the adaptive immune response. PA and LF are presented differently to the immune system. These two responses compete with each other, resulting in the predominance of one antigen over the other. Further enhancement of the immune response occurs through the cytotoxic effects of lethal toxin, most likely due to the maturation of dendritic cells exposed to apoptotic bodies carrying the lethal toxin components. Interestingly, while the competition between the TH1-like LF response and the TH2-like PA response has a negative effect on either PA or LF-specific antibody production (depending on whether

PA is active or inactive), the T cell responses to both PA and LF are improved by the addition of the other protein. The improvement occurs in the T cells producing the cytokine associated with the other protein’s concurrent immune response. This highlights the fact that the immune response neither antigen can be classified as fully TH1 or TH2 in this case. It also emphasizes the complexity of cytokine cross-talk between the TH1 and

TH2 pathways and the resulting immune responses.

93 Figure 3.1: Structure of lethal factor bound to the amino terminal portion of Mek1. The coordinates of the structure for PA were obtained from Swiss-Prot (accession # P15917) and modeled using the SwissPDBViewer software. Residues targeted by site directed mutagenesis are shown in red. Domain 1 (blue) spans amino acids 1-254. This domain binds to PA and contains the tyrosine at position 236 important in that interaction. The catalytic portion of the enzyme is comprised of domains 2, 3 and 4. Domains 2 and 3 (green and purple, respectively) provide substrate specificity. Domain 4 contains the active site, which includes glutamate 687 as the catalytic base. The first 16 amino acids of Mek1 (represented as a white space-filling model) are shown bound to the active site.

94 Figure 3.2: Immunoblot of Mek1 proteolysis by LF, LF687 and LFn. J774A.1 cells were incubated with 100 ng/mL of LF, LF687 and LFn in the presence and absence of PA for 2.25 hours. Cells were lysed and run on a 10% acrylamide denaturing gel. The gels were transferred to PVDF membrane and probed for the presence of the (A) amino terminus of Mek1 and (B) carboxyl terminus of Mek1. The absence of the N-terminal epitope in a sample containing the carboxyl terminus of Mek1 (46 kDa) was considered a positive reaction for MEK proteolysis.

95 Figure 3.3: Structure of activated protective antigen. The coordinates of the structure for PA were obtained from Swiss-Prot (accession # P13423) and modeled using the SwissPDBViewer software. The structure represents the activated (PA63) molecule. The PA20 fragment is not shown. Residues targeted by site directed mutagenesis are shown in red. Domain 1’ (blue) is responsible for LF and EF binding and contains the lysine 197 residue necessary for that interaction. Translocation and pore formation is carried out by domain 2 (purple). The aspartate residue at position 425 is located at the center of the heptameric pore. PA heptamerizes via interactions between domain 3 (green) with neighboring PA molecules. Domain 4 (gold) interacts with the cellular receptor via the loop containing asparagine 682.

96 Figure 3.4: Gel mobility shift assay for complex formation by PA and LF binding mutants. PA83 and PA197 were activated by trypsin (1:1000) for 30 minutes at 37oC. To form the complexes, 20 g of activated PA or PA197 was combined with 10 g of LF or LF236 for 30 minutes at 37oC. Samples containing each protein singly were used as controls. The samples were mixed with 2x native PAGE sample buffer and run on a 4% native acrylamide gel at 50 V for 20 minutes, then 80 V for an additional 60 minutes. Following electrophoresis, protein was visualized with BioSafe Coomassie.

97 Figure 3.5: Sandwich ELISA to measure complex formation by PA and LF binding mutants. Immulon 4 plates were coated with mouse anti-PA monoclonal antibody 3E2- 071100-02. PA83 and PA197 were activated with trypsin (1:1000), and then bound to the monoclonal antibody. Unbound PA was removed, and LF or LF236 was allowed to bind to PA or PA197 for 30 minutes at 37oC to determine the degree of interaction between the two molecules. Polyclonal rabbit anti-LF was added to detect the bound LF. The bound rabbit antibody was detected by the addition of anti-rabbit IgG conjugated to horseradish peroxidase. The plates were developed with ABTS substrate and the absorbance at 405 nm was read. Values shown are the average absorbance for each series +/- the standard deviation.

98 Figure 3.6: Comparison of cellular binding by PA83 and PA682 on J774A.1 macrophages. J774A.1 cells were incubated with either PA or PA682 in triplicate for 8 o minutes at 37 C in 5% CO2 before unbound protein was washed away. Cells were fixed with paraformaldehyde and bound protein was detected by indirect immunofluorescence. The average fluorescence intensity for each series was normalized to the fluorescence of unbound cells. Values shown are the normalized average fluorescence +/- the standard deviation.

99 Figure 3.7: Immunofluorescence control reactions in J774A.1 macrophages. J774A.1 macrophages were fixed with 4% paraformaldehyde. Cells were permeabilized, and organelles were detected with either rabbit anti-Rab5b or rabbit anti-MEK1 antibodies. Unintoxicated macrophages were incubated with mouse anti-PA (3E2) and rabbit anti-LF (polyclonal). Bound antibodies were visualized by the addition of anti- mouse-FITC and anti-rabbit-TRITC. Slides shown are (A) the early endosome marker Rab5b (B) the cytoplasmic marker MEK1 (C) unintoxicated J774A.1 cells (TRITC filter) and (D) unintoxicated J774A.1 cells (FITC filter). Photographs of panes A and B were taken with 2 second exposures, pane C was taken with a 5 second exposure, and pane D was taken with a 7 second exposure.

100 Figure 3.8: Intracellular localization of wild type lethal toxin components. J774A.1 o macrophages were exposed to PA, LF or PA+LF for 45 minutes at 37 C in 5% CO2, then fixed with 4% paraformaldehyde. Cells were permeabilized, and toxin proteins were detected with mouse anti-PA (3E2) and rabbit anti-LF (polyclonal). Antibody-toxin complexes were visualized by the addition of anti-mouse-FITC and anti-rabbit-TRITC. Slides shown are (A) PA83 (B) LF (C) PA83+LF (FITC filter) and (D) PA83+LF (TRITC filter). Photographs were taken with 5 second (TRITC) or 7 second (FITC) exposures.

101 Figure 3.9: Intracellular localization of lethal toxin mutants defective in translocation. J774A.1 macrophages were exposed to PA425, LF or PA425+LF for 45 o minutes at 37 C in 5% CO2, then fixed with 4% paraformaldehyde. Cells were permeabilized, and toxin proteins were detected with mouse anti-PA (3E2) and rabbit anti-LF (polyclonal). Antibody-toxin complexes were visualized by the addition of anti- mouse-FITC and anti-rabbit-TRITC. Slides shown are (A) PA425 (B) LF (C) PA425+LF (FITC filter) and (D) PA425+LF (TRITC filter). Photographs were taken with 5 second (TRITC) or 7 second (FITC) exposures.

102 Figure 3.10: Survival of J774A.1 macrophages following intoxication with lethal toxin mutants. Lethal toxin combinations were generated by combining equivalent amounts of PA protein with LF protein (50 ng toxin = 50 ng PA+50 ng LF). The toxin combinations were incubated in triplicate on J774A.1 macrophages for 4 hours at 37oC in 5% CO2, then cell viability was assessed by XTT assay. Wild type lethal toxin was compared to mutants deficient in (A) MEK proteolysis and (B) cellular intoxication. Y values were converted from A480 to % survival, and graphed as the average % survival +/- the standard deviation. 103 Figure 3.11: Serum IgG anti-PA responses to lethal toxin components deficient in MEK proteolysis. Groups of 10 Balb/cAnNCr mice were immunized on days 1 and 15 with equivalent molar amounts of protein (30 g PA, 32 g of LF, 32 g LF687 and 10.5 g LFn) bound to Alum and the antigen specific titers were tracked over 70 days. PA- specific titers were determined by indirect quantitative ELISA against the protein of interest. The graph is broken over 5-15 g/mL in order to better observe the values at day 14. Values shown are the geometric mean titer of the group plus/minus the standard error of the mean. Significant changes in PA titer (p < 0.05) are marked with an *.

104 Figure 3.12: Serum IgG anti-LF responses to lethal toxin components deficient in MEK proteolysis. Groups of 10 Balb/cAnNCr mice were immunized on days 1 and 15 with equivalent molar amounts of protein (30 g PA, 32 g of LF, 32 g LF687 and 10.5 g LFn) bound to Alum and the antigen specific titers were tracked over 70 days. LF- specific titers for groups of mice immunized with (A) LFn, (B) LF687 or (C) LF were determined by indirect quantitative ELISA against the protein of interest. The graph is broken over 75-200 g/mL in order to better observe the values at day 14. Values shown are the geometric mean titer of the group plus/minus the standard error of the mean. Significant changes in LF titer (p < 0.05) are marked with an *.

105 106 Figure 3.13: Neutralizing antibody responses to lethal toxin components deficient in MEK proteolysis. Groups of 10 Balb/cAnNCr mice were immunized on days 1 and 15 with equivalent molar amounts of protein bound to Alum and the lethal toxin neutralizing titers were tracked over 70 days. Serum was incubated with 85.1 ng/mL of PA and 68.1 ng/mL of LF (sufficient for 99.5% cell death) and then added to J774A.1 macrophages. Cell viability was assessed after 4 hours. Data were fit by 4-parameter logistic fit to determine the serum dilution at which 50% of toxin was neutralized (ED50). Comparisons are shown between PA and (A) LFn-containing groups (B) LF687- containing groups and (C) LF-containing groups. Data represent the geometric mean titer of the group plus/minus the standard error of the mean. Significant changes in meutralizing titer (p < 0.05) are marked with an *.

107 108 Figure 3.14: Serum IgG responses to lethal toxin components deficient in complex formation. Groups of 10 Balb/cAnNCr mice were immunized on days 1 and 15 with equivalent molar amounts of protein bound to Alum and the antigen specific titers were tracked over 70 days. (A) PA83 and (B) LF -specific titers were determined by indirect quantitative ELISA against the protein of interest. In order to better observe the data at day 14, the graphs are broken at 5-15 g/mL (PA) and 50-200 g/mL (LF). Values shown are the geometric mean titer of the group plus/minus the standard error of the mean. Significant changes in titer (p < 0.05) are marked with an *. 109 Cytokinea PA PA197 LF PA+LF PA197+LF

PA IL-4 20.5 (9.4)b 5.5 (2.8) --- 46.1 (9.0) 56.4 (20.8)c

LF IL-4 ------67.5 (7.7) 154.7 (11.6) 68.4 (13.9)

PA IFN 2.2 (3.2) 2.7 (0.6) --- 17.6 (6.1) 18.3 (3.8)

LF IFN ------154.1 (17.7) 195.4 (15.9) 198.0 (19.1)

a splenocytes from mice immunized with lethal toxin components were stimulated with either PA or LF and assayed for the production of either IL-4 or IFN b values shown are the geometric mean (+/- the standard error) of the group c underlined values are statistically different (p < 0.05) from the value obtained for mice immunized with that toxin component alone

Table 3.1: Cytokine responses to lethal toxin components deficient in complex formation

110 Figure 3.15: Serum IgG responses to lethal toxin components deficient in cellular receptor binding. Groups of 10 Balb/cAnNCr mice were immunized on days 1 and 15 with equivalent molar amounts of protein bound to Alum and the antigen specific titers were tracked over 70 days. (A) PA83 and (B) LF -specific titers were determined by indirect quantitative ELISA against the protein of interest. In order to better observe the data at day 14, the graphs are broken at 5-15 g/mL (PA) and 50-200 g/mL (LF). Values shown are the geometric mean titer of the group plus/minus the standard error of the mean. Significant changes in titer (p < 0.05) are marked with an *.

111 Cytokinea PA PA682 LF PA+LF PA682+LF

PA IL-4 20.5 (9.4)b 11.8 (3.3) --- 46.1 (9.0) 44.8 (8.7)c

LF IL-4 ------67.5 (7.7) 154.7 (11.6) 141.5 (12.0)

PA IFN 2.2 (3.2) 3.5 (1.2) --- 17.6 (6.1) 10.8 (2.7)

LF IFN ------154.1 (17.7) 195.4 (15.9) 181.1 (20.5)

a splenocytes from mice immunized with lethal toxin components were stimulated with either PA or LF and assayed for the production of either IL-4 or IFN b values shown are the geometric mean (+/- the standard error) of the group c underlined values are statistically different (p < 0.05) from the value obtained for mice immunized with that toxin component alone

Table 3.2: Cytokine responses to lethal toxin components deficient in cellular receptor binding

112 Figure 3.16: Serum IgG responses to lethal toxin components deficient in translocation. Groups of 10 Balb/cAnNCr mice were immunized on days 1 and 15 with equivalent molar amounts of protein bound to Alum and the antigen specific titers were tracked over 70 days. (A) PA83 and (B) LF -specific titers were determined by indirect quantitative ELISA against the protein of interest. In order to better observe the data at day 14, the graphs are broken at 5-15 g/mL (PA) and 50-200 g/mL (LF). Values shown are the geometric mean titer of the group plus/minus the standard error of the mean. Significant changes in titer (p < 0.05) are marked with an *. 113 Cytokinea PA PA425 LF PA+LF PA425+LF

PA IL-4 20.5 (9.4)b 38.0 (5.7) --- 46.1 (9.0) 11.6 (3.0)c

LF IL-4 ------67.5 (7.7) 154.7 (11.6) 67.5 (6.9)

PA IFN 2.2 (3.2) 6.0 (5.6) --- 17.6 (6.1) 6.1 (1.1)

LF IFN ------154.1 (17.7) 195.4 (15.9) 88.0 (14.8)

a splenocytes from mice immunized with lethal toxin components were stimulated with either PA or LF and assayed for the production of either IL-4 or IFN b values shown are the geometric mean (+/- the standard error) of the group c underlined values are statistically different (p < 0.05) from the value obtained for mice immunized with that toxin component alone

Table 3.3: Cytokine responses to lethal toxin components deficient in translocation

114 CHAPTER 4

EDEMA TOXIN IS AN ADJUVANT WHOSE ACTIVITY DOES NOT DEPEND ON EITHER cAMP PRODUCTION OR CELLULAR INTOXICATION

4.1 Introduction

The immune response resulting from exposure to edema toxin (PA+EF) was investigated for two reasons. First, a growing amount of evidence indicates that edema toxin plays a significant role in the pathology of anthrax infection. B. anthracis strains lacking LF are attenuated, but not avirulent [281]. Likewise, LF-specific lethal toxin neutralizing antibodies provide partial protection in an anthrax challenge, while PA- specific neutralizing antibodies provide full protection [321]. The slow-healing edema at the infection site of all forms of anthrax is attributable to the action of edema toxin [242,

368, 369]. Although the localized edema of cutaneous anthrax is rarely a serious problem

[242], patient survival may depend on the mitigation of pleural edema in inhalational anthrax [266]. Systemic exposure to edema toxin may result in a cytokine storm, as the levels of the pro-inflammatory cytokines IL-6, MCP-1 IL-1 and IL-1 sharply increase following toxin challenge [47]. Thus, the damaging effects of edema toxin make an immune response to this toxin desirable.

115 Second, characterizing the immune response to edema toxin allows comparisons to be made with the immune response to lethal toxin. Following immunization with inactive lethal toxin, the localization of PA and LF to different intracellular compartments is sufficient to result in competition between the PA and LF antibody responses. When cellular intoxication proceeds normally, cytoplasmic LF is the dominant B cell antigen and the LF antibody response develops at the expense of the PA antibody response. This effect is the result of the translocation activity of PA. Since edema toxin also uses PA as a way to recognize and enter target cells, the immune response to inactive edema toxin may be similar to that produced by inactive lethal toxin.

LF and EF compete for binding to PA at the same site [121, 131]. Both binding of PA to cellular receptors and receptor-mediated endocytosis occur independently of EF and LF.

The translocation of LF and EF from the endosome is thought to occur by the same molecular mechanism [125], although there is one important difference. The translocation of EF is predicted to result in a transmembrane protein [122, 153] instead of a soluble cytoplasmic antigen.

As a membrane protein, EF is present in both the cytoplasmic and endosomal compartments, and could presumably be processed for both MHC class I and II presentation. Immunization with EFn in the presence of PA has been shown to result in

EF-specific antibodies, but no attempt was made to assess the production of EF-specific

CTL’s [370]. Membrane proteins have been shown to be processed by a variety of mechanisms that give rise to MHC-I associated peptides and subsequent CTL responses.

However, these mechanisms depend on the localization of the membrane protein to either the endoplasmic reticulum or the Golgi compartments [371-374]. These locations are

116 inconsistent with the intoxication process of edema toxin. As it contains no cellular localization signals, edema factor may eventually reach the membranes of these compartments through vesicle trafficking. However, there is no indication that cytosolic processing will be as important for edema factor as it was for lethal factor.

There are two important differences between active lethal and edema toxin that are expected to influence the immune response generated by active edema toxin. First, it is the lethal toxin-induced cell death that enhances the antibody response to LF. Edema toxin has never been associated with either necrosis or apoptosis in any cell type. In fact, edema toxin prevents lethal toxin-mediated apoptosis [368]. Thus, edema toxin action is unlikely to result in a bolus of apoptotic bodies scavenged by dendritic cells. Second, toxins that alter the level of intracellular cAMP, namely cholera toxin, pertussis toxin and labile toxin, have been shown to be potent adjuvants [375-377]. Two recent reports link active edema toxin with adjuvant activity as well [378, 379]. Both studies report an increase in the PA antibody titer following immunization with active edema toxin. No mention was made of the EF antibody titer. Edema toxin was also able to improve the serum IgG1 and IgG2b titers to an unrelated antigen administered simultaneously [378], implying that the immune response produced to active edema toxin has TH2-like characteristics. This differs from the TH1-like immune response observed following immunization with active lethal toxin.

Despite the differences between LF and EF, edema toxin is expected to share some similarities with lethal toxin in an immunization. Although the immune response to edema toxin has been shown to have TH2 characteristics [378], active edema toxin may be able to stimulate a simultaneous TH1 response. This is suspected for two reasons.

117 First, even though EF is localized to the endosomal membrane following translocation, a substantial portion of the translocated protein is accessible to cytoplasmic enzymes for subsequent cytosolic processing. Second, many of the cAMP-elevating toxins have been associated with the production of mixed TH1/TH2 immune responses [380, 381]. Both the cytoplasmic localization of EF and the adjuvant activity associated with cAMP production depend on the functionality of PA in the cellular intoxication process.

Removing the cellular intoxication functions of PA will remove the potential TH1 biases of active edema toxin. Therefore, edema toxin mutants defective in the cellular intoxication process are expected to behave similarly to the corresponding lethal toxin mutants. In order to investigate this hypothesis, single amino acid mutations were introduced into both PA and EF. Each of these mutations removed a specific functional activity without grossly affecting the physical properties of either PA or EF. Mice were then immunized with edema toxin combinations defective in a single functional activity and the resulting antibody responses were compared to the response generated by each component alone as well as wild type toxin.

4.2 Results

4.2.1 Characterization of edema toxin mutants

Each of the published activities of edema toxin was knocked out by site-directed mutagenesis in order to investigate the effect of specific edema toxin functions on the downstream immune response. To facilitate the comparison between lethal and edema toxin, the mutations introduced into PA and EF were comparable to those used when assessing the functions of lethal toxin. For each mutation, a single amino acid change

118 was introduced that prevented a specific functional activity. The functional activities removed were ATP binding, complex formation, cellular receptor binding and cytoplasmic translocation. Each mutation was introduced directly into the protein expression construct. Proteins were then expressed from E. coli and purified over a metal affinity column via an N-terminal 6X his-tag. All proteins were purified under endotoxin-removing conditions. cAMP production by edema factor: To prevent the production of cAMP, the mutation

K346R was made in EF [320]. This mutation removes the active site residue responsible for coordinating the three phosphate groups of ATP and thus EF346 is unable to effectively interact with ATP (Figure 4.1) [162]. The lack of substrate binding reduces enzyme activity to negligible levels [320]. The calmodulin (CaM) binding site on EF346 remained intact, making it probable that EF346 interacts with CaM. Calmodulin is an intracellular calcium sensor that responds to transient increases in the calcium concentration. The calcium-bound form of CaM is able to interact with numerous cellular proteins and modulate downstream cellular events [382]. Although the affinity of EF for CaM is not high enough to effectively sequester CaM from cellular proteins (Kd for EF = 10-8, eukaryotic proteins range from 10-7 to 10-9) [221, 383-385], the possibility of altered CaM-dependent signaling was addressed by the deletion of the catalytic domains of EF. A truncated form of EF containing only the N-terminal domain (EFn) was made in order to specifically examine the effect of cytosolic localization on EF

(Figure 4.1). Similar to LFn, EFn is able to bind PA and be translocated to the cytoplasm of target cells [122], but lacks the substrate binding domains and the active site [122,

162].

119 The ability of EF346 and EFn to increase intracellular cAMP concentrations in

CHO-K1 cells was compared to wild type EF (Figure 4.2). Following intoxication with

PA and either EF, EF346 or EFn, the intracellular cAMP was extracted and quantified.

PA, EF, EF346 and EFn had no effect on the level of cAMP in cells when administered separately. Similarly, the intracellular cAMP concentration of cells exposed to PA+EFn or PA+EF346 was not significantly different from that of unintoxicated cells.

Intoxication with wild type edema toxin raised the cAMP concentration from 1.42 to

1920 pmol/mL (a 1000-fold increase).

Toxin complex formation: Two mutations prevented the formation of the toxin complex. The PA197 mutant from the lethal toxin study was used to interrupt the binding of PA to EF (Figure 4.3). The loss of the arginine residue at position 197 removes a critical positive charge from the EF binding site [121, 125] and reduces binding by more than 95% [121]. The binding of EF to PA was disrupted by the point mutation Y227A (Figure 4.1). No detectable PA binding occurs with this mutation [120], presumably due to the loss of a hydrophobic interaction that stabilizes the binding of EF to PA [125].

The binding of PA197 to wild type EF and EF227 to wild type PA was assessed by two different methods. To qualitatively visualize the formation of the toxin complex, the toxin proteins were combined and electrophoresed on a 4% native acrylamide gel to detect the shift in electrophoretic mobility between unassociated monomers and the heterodecameric complex (Figure 4.4). The combination of wild type PA and EF resulted in a discrete band of low mobility. This corresponds to the full-size toxin complex [121]. PA197 was unable to produce a similar low mobility band in the

120 presence of either EF or EF227, indicating a lack of toxin complex formation for this mutant. However, a limited amount of complex was observed with the combination of

PA and EF227.

The ability of PA197 and EF227 to bind wild type PA and EF was quantified by a modified sandwich ELISA (Figure 4.5). PA and PA197 were captured with equal efficiency by a non-neutralizing monoclonal antibody (data not shown). The amount of

EF or EF227 bound to PA was determined by detection with non-neutralizing polyclonal antibody. Negligible levels of EF and EF227 in the absence of PA were captured by the anti-PA monoclonal (data not shown). When compared to wild type edema toxin, the single mutation combination of PA with EF227 showed approximately a ten-fold decrease in complex formation. However, the combination of PA197 with EF resulted in wild type levels of binding. This was surprising, since Chapter 3 demonstrated that binding of PA197 to LF was significantly impaired. The difference in LF and EF binding to PA197 indicates that although LF and EF bind to the same site on PA, the PA residues that interact with LF are not necessarily those that interact with EF. The combination of

PA197 and EF227 resulted in binding levels similar to those observed for PA+EF227.

The amount of EF227 bound to PA197 was approximately 10-fold less than the amount of EF bound to wild-type PA.

Cell surface binding and translocation: Binding of edema toxin to the cellular receptors

TEM8 and CMG2 was inhibited with the PA682 mutant introduced in the lethal toxin study. As described previously, the mutation N682S prevents receptor binding [132,

330] most likely by destabilizing either the entire receptor-binding loop of PA or the specific interaction with the MIDAS motif of the receptor [141, 145, 146]. Likewise,

121 translocation of EF from the endosome was precluded by the mutation D425A in PA.

The mutation prevents the formation of the transmembrane channel and results in a 10- fold decrease in translocation [158]. The functional activity of both the PA682 and

PA425 mutant was characterized in chapter 3.

Cellular toxicity of edema toxin mutants: Although the targeted function of each mutation was shown to be impaired, the ability of the mutated forms of edema toxin to raise the intracellular cAMP concentration was assessed in CHO-K1 cells (Figure 4.6).

No PA or EF mutant by itself was able to significantly alter the amount of cellular cAMP from that observed with unintoxicated cells (data not shown). At concentrations greater than 0.15 g/mL, wild type edema toxin significantly increased the amount of intracellular cAMP. The toxin combinations PA682+LF and PA425+EF had no effect on cAMP levels at any concentration tested, confirming their defects in the cellular intoxication process. Although PA+EF227 produced a slight increase in cAMP amounts at the highest toxin concentration, the increase was not enough to be statistically different from the cAMP levels of unintoxicated cells. However, intoxication with PA197+EF resulted in an increase in cAMP levels. A significant increase was observed at concentrations greater than 0.31 g/mL of PA197+EF. The amount of intracellular cAMP produced by PA197+EF was 35-65% of that produced by wild type edema toxin, confirming the results of the modified sandwich ELISA for complex formation.

Although the PA197 mutant showed significant binding defects with LF, the same was not true for EF. For this reason, the PA197 protein was excluded from the set of edema toxin mutants deficient in cellular intoxication. Although the -helical structures of the PA binding site are very similar for LF and EF, there are significant structural 122 differences in the loops at the ends of the helices [221]. Subtle differences in the interactions between these loop residues and PA may account for the different interactions of LF and EF with PA197. The other toxin complex formation mutant,

EF227, was able to form a low mobility band in the presence of PA during the gel shift assay. This activity correlated with a small but insignificant increase in cAMP production in live cells. Since this activity was substantially less than that observed for wild type edema toxin, EF227 was included in the set of edema toxin mutants deficient in cellular intoxication. The single point mutations addressing cellular receptor binding and cytosolic translocation significantly impaired the specific function targeted. This loss of specific functional activity translated into a loss of cAMP production for both PA682 and

PA425.

4.2.2 Immunization with edema toxin mutants lacking adenylyl cyclase activity

The immune response produced by immunization with the non-catalytic mutants of EF in combination with PA was compared to the response generated by active edema toxin. Proteins were purified under endotoxin-removing conditions and administered to mice in a 1:1 molar ratio. As with the lethal toxin study, the 1:1 molar ratio was used in order to keep the proportion of protein involved in a toxin complex constant across all groups of mice. Mice received the molar equivalent of 30 g of PA. Proteins were absorbed to alum and groups of 10 Balb/cANnCr mice were immunized intraperitoneally on days 1 and 15. This study was conducted prior to the LD50 determination for edema toxin. As it turned out, the immunizing dose in this study was slightly less than the published LD50 (37.5 g/mouse) [47], and thus significant toxicity was observed in the group that received active toxin. All the mice in that group developed peritoneal edema 123 and respiratory distress and one mouse in the group died as a result of toxin effects. The survivors exhibited no signs of toxicity following the second immunization at day 15.

Following immunization, the serum IgG titers to PA were analyzed at two week intervals for 70 days. No mouse had a detectable anti-PA response prior to the immunization series. Very low levels of PA-specific IgG were detected at day 14

(<1 g/mL). Similar to the results obtained with lethal toxin, the antibody response to

PA did not begin to develop fully until after the second immunization (Figure 4.7). The

PA antibody response peaked at 1.33 mg/mL on day 56, one month after the final immunization. The addition of wild type EF to PA had no effect on the kinetics of the

PA antibody response, but strongly inhibited antibody production. In the presence of wild type EF, production of PA-specific antibodies was significantly decreased after day

42. The PA titer was reduced from 918 g/mL to 239 g/mL at day 42 (p = 0.005), from

1330 g/mL to 356 g/mL at day 56 (p = 0.005) and 1150 g/mL to 263 g/mL at day

70 (p = 3.12x10-4). Removal of the catalytic activity of EF reduced the impact on the anti-PA response. EF346 significantly reduced the PA-specific titer on days 56 and 70 (p

= 0.040 and 0.001, respectively), but the decrease was not as much as was observed for wild type toxin. The presence of EF346 had no effect on the kinetics of the humoral response to PA. Similar results were obtained when EFn was combined with PA. The

PA-specific antibody titer decreased on days 56 and 70 (p = 0.046 and 0.028, respectively). The addition of EFn appeared to shift the peak PA antibody titer from day

56 to day 42; however it is unclear if this is due to a change in the kinetics of antibody production or to the decrease in titer at day 56.

124 The serum IgG titers to EF were also measured at two week intervals for 70 days during the course of the experiment. Prior to immunization, no mouse had a detectable anti-EF response. The humoral response to full-length EF developed rapidly following immunization (Figure 4.8). The kinetics of the antibody response to EF resembled those observed for LF and differed substantially from PA. Serum IgG titers to EFn peaked on day 42 and declined steadily thereafter, as did the titers to EF346 and EF. EFn did not appear to be as immunogenic as either EF or EF346, although this effect may be due to the different g amounts of full-length and truncated protein present in the immunization.

The addition of PA to EFn had no significant effect on the antibody response to

EF (Figure 4.8). Neither the amount of EF-specific antibody produced nor the kinetics of the response differed from EFn alone. The difference in antibody titer at day 42 (871

g/mL for EFn vs. 471 g/mL for PA+EFn) was not significant (p = 0.174). Likewise, the combination of PA with EF346 did not alter the kinetics of response to EF. Adding

PA to EF346 did change the amount of EF-specific antibody produced at day 28 from

2.09 mg/mL to 2.97 mg/mL (p = 0.046). The difference in EF titers between EF346 and

PA+EF346 at day 70 was not significant (p = 0.223) and was attributable to variation among group members in the rate of antibody reduction. The most dramatic effect on the

EF response was observed with the addition of PA to wild type EF. Wild type edema toxin shifted the peak EF-specific titer to day 28 as opposed to day 42 for EF alone.

Higher antibody levels against EF were present on day 14 and 28 in mice immunized with edema toxin compared with those immunized with EF alone. At day 14, the antibody titers were 26 g/mL and 74 g/mL for EF and edema toxin, respectively (p =

0.014). On day 28, the EF-specific titers were 4.33 mg/mLand 7.54 mg/mL for EF and 125 edema toxin, respectively (p = 0.010). At day 42 and all timepoints thereafter, the levels of antibody produced in response to EF and edema toxin were statistically equivalent.

To summarize, the combination of PA with active or inactive EF altered the humoral response. The levels of PA-specific antibody produced decreased after day 56 when catalytically inactive forms of EF were present. This decrease in titer is cAMP- independent and is most likely related to the cellular intoxication process by PA. Active

EF substantially impaired the production of antibody in response to PA. The cellular intoxication process may also have contributed to this decrease, although the action of edema toxin exacerbated this effect. In contrast, the presence of PA enhances the early antibody response to both EF and EF346, by increasing the levels of EF-specific antibodies produced. This effect on EF titers depends on the presence of the catalytic domains of EF, as the truncated EFn was unable to produce the change in titer. However, toxin activity further increased the EF-specific titers and also decreased the amount of time necessary to produce the peak response.

4.2.3 Immunization with edema toxin mutants deficient in cellular intoxication

Immunization with active edema toxin increased the EF antibody titer and decreased the PA titer. Removing the catalytic activity mitigated these effects only partially, implying that toxin activity and cellular intoxication combine to produce this pattern. To ascertain the role of intoxication in the altered immune responses to PA and

EF, mice were immunized with the intoxication mutants EF227, PA682 and PA425 in order to compare the resulting responses to that generated by active edema toxin.

Endotoxin-free protein was combined in a 1:1 molar ratio and absorbed to alum.

126 Groups of 10 Balb/cANnCr mice received 30 g of PA and/or the molar equivalent of EF on days 1 and 15 in an intraperitoneal immunization. The group that received active edema toxin was increased to 15 mice to account for toxicity. However, immunization with edema toxin in this study resulted in the death of all group members within 72 hours. The reason for the increased toxicity in this study is unclear, as immunization with active toxin in the previous study did not result in effects this severe.

The increased toxicity was unlikely to be due to the presence of a contaminant in the purified PA or EF, as mice that received either PA or EF alone did not exhibit any symptoms of toxicity.

The remaining groups in the study (each protein alone and EF227, PA682 and

PA425 in combination) were assayed for PA and EF-specific antibody production. The serum IgG titers were measured at two-week intervals for 56 days after the first immunization. None of the mice had a detectable PA or EF-specific response prior to immunization. At the conclusion of the experiment (70 days after the first immunization), the splenocytes from the mice were harvested and assayed for IL-4 and

IFN- production in response to both PA and EF.

Immunization with toxin complex formation mutants: EF227 is unable to form the edema toxin complex with PA and is functionally similar to the PA197 mutant used in the lethal toxin study. Both PA and EF227 are present in the immunization but they are unable to physically interact. The kinetics of the EF227 immune response were similar to that of EF (Figure 4.9). Both responses peaked on day 42. However, the amount of antibody produced against the two proteins differed at days 42 and 56. The peak EF titer

(4.62 mg/mL) was nearly twice that of the peak EF227 titer (2.47 mg/mL) on day 42 127 (p = 0.013). Similarly, the EF titer at day 56 (3.23 mg/mL) significantly exceeded that of

EF227 (1.87 mg/mL; p = 0.030). The reason for the immunological difference between these two proteins is unknown.

Combining EF227 with PA resulted in a very different immune response from the one obtained with PA+EF in the previous study. There, the EF antibody titer increased significantly at days 14 and 28, while the PA antibody titer failed to increase after day 28.

The addition of EF227 to PA significantly increased the PA antibody response at days 14 and 28 (p = 0.044 and 0.034, respectively) (Figure 4.9). The difference between the PA and PA+EF227 responses at day 42 was not significant (p = 0.099). Likewise, the antibody response to EF227 was significantly enhanced by the addition of PA at days 14 and 28 (p = 0.039 and 0.005, respectively). The combination of PA and EF227 also impacted the kinetics of both the PA and EF227 responses. Peak antibody titers to both proteins occurred on day 28, representing a shift of two and four weeks for EF227 and

PA, respectively.

The number of IL-4 and IFN- producing T cells specific for PA and EF was determined at the conclusion of the experiment in order to understand the general features of the T lymphocyte response. T lymphocyte cytokine production also differed when PA and EF227 were combined (Table 4.1). When the proteins were administered separately, the majority of T lymphocytes specific for PA and EF227 produced IL-4 (20.5 and 94.6, respectively), although EF227 stimulated a large minority of cells (27.6) to produce IFN-

. The combination of the two proteins in an immunization altered the T cell responses considerably. The addition of PA to EF227 significantly increased the number of EF- specific T lymphocytes producing IFN- to 188.4 (p = 7.7x10-4). The difference in the 128 number of IL-4-producing lymphocytes was not significant. Thus, the combination of

PA+EF227 changed the nature of the T cell response from predominantly IL-4 to predominantly IFN-. The changes were even greater for PA. In the presence of EF227, the number of T lymphocytes producing both IL-4 and IFN- in response to PA increased significantly. IL-4-producing cells increased to 59.3 (p = 0.048), while the number of

IFN--producing cells jumped to 65.3 (p = 4.15x10-4). Thus, the addition of EF227 to PA changed the T lymphocyte response from one composed entirely of IL-4 to one evenly split between IL-4 and IFN-.

Immunization with the cellular receptor binding mutant: Preventing cell surface localization by disrupting receptor binding had less of an effect on the antibody response than preventing toxin complex formation (Figure 4.10). The antibody response to EF in the presence of PA682 was significantly increased at day 28 from 1.86 mg/mL to 5.2 mg/mL (p = 0.004). The difference between EF and PA682+EF at days 42 and 56 was not significant. A similar pattern was observed for the PA682 titers. The PA-specific titer increased from 122 g/mL to 463 g/mL in the presence of EF at day 28 (p = 0.011).

A significant difference was not observed at any other timepoint. The combination of

PA682+EF had no effect on the kinetics of the EF antibody response, but the peak PA682 titer shifted to day 28 in the presence of EF.

The combination of PA682 with EF also had less of an effect on the T lymphocyte cytokine profiles than the mutants deficient in complex formation. Both the

PA682 and EF-specific responses were dominated by cells producing IL-4 (Table 4.2).

When the two proteins were combined, the number of cells producing IFN- in response to PA682 increased from 3.5 to 25.3 (p = 0.007), as did the number of cells producing 129 IL-4 (11.8 to 71.2; p = 2.4x10-4). Although both the IFN- and IL-4 responses were enhanced by EF, the T lymphocyte response to PA remained one that is predominantly

IL-4. In contrast, only the IFN- response to EF increased significantly in the presence of

PA682 (from 10.0 to 177.6; p = 7.12x10-5). The increase in EF-specific IL-4 production in the presence of PA682 was not significant (80.8 vs. 140.0, p = 0.079). Although the majority of EF-specific T lymphocytes produced IFN-in the presence of PA682 (177.7), a substantial number of IL-4-producing cells were also present (140.0).

Immunization with the cytosolic translocation mutant: The antibody responses to both

EF and PA were significantly altered by localization in the endosome. As previously discussed, the antibody response to PA425 was not equivalent to the antibody response produced by PA (Figure 4.11). The amount of antibody produced against PA425 is significantly higher than the amount of antibody to PA at all timepoints. When EF was added to PA425, the PA antibody response decreased significantly at days 42 and 56 (p =

0.001 and 0.002, respectively). The difference at day 28 was not significant due to the variation among group members that received PA425. In contrast, the EF-specific antibody titer was significantly enhanced by the addition of PA425. Antibody production increased from 1.86 mg/mL to 4.64 mg/mL at day 28 (p = 0.019), from 4.61 mg/mL to

7.91 mg/mL at day 42 (p = 0.022), and from 3.20 mg/mL to 5.48 mg/mL at day 56 (p =

0.023). No difference was observed at day 14. Unlike the combination of the other intoxication mutants, the combination of PA425 and EF had no effect on the kinetics of either the EF-specific or the PA-specific antibody responses.

Although the antibody responses to PA and PA425 were quite different, the IL-4 and IFN- T lymphocyte responses to PA and PA425 alone were equivalent 130 (Table 4.3). The addition of EF to PA425 had a positive impact on the IL-4 T lymphocyte response. The number of cells producing IFN- upon stimulation with

PA425 was unaffected by co-immunization with EF, but the number of IL-4 producing cells increased significantly from 38.0 to 57.1 (p = 0.034) when EF was present. In contrast, PA425 had a strongly negative effect on cytokine production in response to EF.

The number of lymphocytes producing IL-4 in response to EF dropped from 80.8 to 8.1 with the addition of PA425 (p = 0.012). The amount of IFN- producing cells was unchanged by the presence of PA425: IFN- production remained slightly above background levels.

4.3 Discussion

Although the immune responses to lethal and edema toxin are quite different, there are two important similarities between the toxins. First, the catalytic activity of both toxins is able to enhance the early anti-toxin immune response. Second, the cellular intoxication process of lethal and edema toxin affects the cellular location of LF and EF, respectively. This PA-dependent change in antigen location has a large impact on the characteristics of the subsequent immune response to both lethal and edema toxin components.

Cellular intoxication results in the translocation of LF and EF from the endosomal compartment to the cytosol [158]. The intoxication process also separates LF and EF from PA and results in different pathways of antigen processing and presentation for the toxin components. Under these circumstances, the immune responses to the endosomal

PA and the cytosolic LF or EF compete with one another. Similar to the results observed 131 with lethal toxin, when PA and EF were separated into different cellular compartments, the EF antibody response increased while the PA-specific titer decreased. Competition between the EF and PA antibody responses was observed for active and inactive forms of

EF and depended on the presence of wild type PA. Although the cytokine profile of T cells associated with the EF-specific response to edema toxin is unknown, the competition with the TH2-based anti-PA response [283, 284] and the similarity to the immune response against lethal toxin imply that the antibody response to cytosolic EF is associated with the generation of a TH1-like response. The production of the TH1-like response to EF then supercedes the TH2 response to PA.

When PA is rendered non-functional by the introduction of a mutation blocking any of the cellular intoxication steps, both EF and PA are essentially confined to the endosome. EF227 and EF bound to PA682 are both soluble antigens unable to interact with target cells and are presumably endocytosed by fluid phase uptake. Since EF227 has no contact with functional PA, it cannot be translocated to the cytoplasm [120], while the lack of contact with the cellular receptor abrogates cytosolic translocation with

PA682 [145, 146]. EF bound to PA425 is actively targeted to the cell surface and the endosomal pathway, but is unable to be translocated to the cytosol. Under these circumstances, both PA and EF are processed in the endosomal compartments and generate a robust antibody response accompanied by IL-4 producing T cells. This represents a significant divergence from the immune responses observed for PA and LF under similar conditions. The reason for this divergence rests with a fundamental difference in the immunological properties of LF and EF.

132 Immunization with LF individually results in an antibody response dependent on cytosolic processing and IFN- production by LF-specific T cells. PA is processed exclusively in the endosomal compartments and a robust antibody response depends on the secretion of TH2 cytokines by T lymphocytes [365]. Thus, co-immunization with PA and LF results in competition between the two antibody responses under all circumstances. Conditions which favor the development of a TH2 response to endosomal

PA hinder the development of a robust TH1 response to LF and vice versa.

In contrast, the antibody response to EF given alone in an immunization was accompanied by T lymphocytes producing IL-4. Only a small number of IFN- producing T cells were detected. Concentrating EF in the endosome with PA425 significantly enhanced the antibody response to EF. This implies that EF is associated with the production of a TH2 immune response, and is in agreement with studies showing that EF is unable to reach the cytoplasm of target cells in the absence of PA [122, 153].

Thus, combining EF with PA puts two TH2 antigens in the same immunization. The functional inactivation of PA and subsequent endosomal confinement of both proteins results in a robust antibody response to EF, since both proteins are able to generate similar TH2 responses. However, the data indicate that EF is not exclusively a TH2 antigen. Cytosolic localization does not hinder the development of an EF antibody response. The development of a robust antibody response to EF following either endosomal or cytosolic processing implies that the immune response to EF can proceed through either the TH1 or TH2 pathway.

The cellular intoxication process for edema toxin is sufficient to predict the interaction between the PA and EF antibody responses. However, the cellular 133 intoxication process is not responsible for the increase in antibody titers when PA and EF are combined. The enhanced immune response to PA and EF results from edema toxin’s ability to act as an adjuvant in an immunization. The cAMP-elevating toxins, namely cholera toxin, heat-labile toxin and pertussis toxin, have long been known to be potent mucosal adjuvants. Studies with these toxins have demonstrated that a large portion of this adjuvant activity depends on the alteration of intracellular cAMP levels [386, 387].

The increase in intracellular cAMP triggers dendritic cell maturation resulting in enhanced naïve T cell priming due to the upregulation of costimulatory molecules and

TH2 cytokine production by the dendritic cells [388-390]. A recent report linked edema toxin with mucosal adjuvanticity, and although the details of its adjuvant effects are largely uncharacterized, edema toxin has been shown to upregulate TH2 cytokine production and costimulatory molecule expression by macrophages [378].

Consistent with its reported adjuvant ability, the greatest effect on the EF-specific response was observed with catalytically active edema toxin. Immunization with active edema toxin resulted in a dramatic increase in the EF-specific antibody response and the acceleration of EF-specific antibody production. Antibody production to EF peaked 14 days sooner under the influence of toxin activity. However, the catalytically inactivated form of edema toxin was able to duplicate the increase in EF-specific antibodies, as were forms of edema toxin that carried mutations preventing cellular intoxication. No edema toxin mutant was able to alter the kinetics of the resulting antibody response following immunization. This mirrors the abilities of cholera, labile and pertussis toxins, for although cAMP production contributes a large portion of the adjuvanticity of cholera, labile and pertussis toxins, catalytically inactivated toxins have been shown to retain

134 adjuvant activity [381, 391-393]. The results observed for active and inactive forms of edema toxin are similar to those published for heat-labile toxin from E. coli. Active labile toxin has been shown to not only increase antibody titers, but also to accelerate the kinetics of the resulting antibody response. Inactive labile toxin is able to increase titers to bystander antigens, but is unable to affect the kinetics of the developing response [391,

394].

There was an important difference between the published results for cAMP- elevating toxins and those observed for edema toxin. Even though inactive forms of toxin can act as mucosal adjuvants, the degree of adjuvant activity is significantly reduced in these mutants. The use of inactive toxin as an adjuvant frequently results in antibody titers that are at least ten-fold less than the titers obtained using wild type toxin

[392, 394, 395]. While the inactive edema toxin mutants were unable to increase EF titers as much as active edema toxin, the difference was approximately two-fold. The discrepancy may have resulted from differences in the route of immunization, as this can have a significant effect on the adjuvanticity of the toxin. Inactive mutants may exhibit wild-type levels of adjuvanticity when administered intraperitoneally, but have much less of an effect in a mucosal immunization [392, 396].

The reason for inactive edema toxin’s adjuvanticity is unclear. Co-immunization with PA and EF resulted in an increase in the EF antibody titers for all forms of PA and

EF except EFn. The adjuvant activity of edema toxin did not depend on either the adenylyl cyclase activity of EF or the intoxication ability of PA. Although cholera, labile and pertussis toxin can be inactivated and still act as adjuvants, these mutants must retain the ability to bind their cellular receptors [381, 397, 398]. The ability of edema toxin to

135 act as an adjuvant without contacting its target cell is without precedent among the cAMP-elevating toxins. One possible explanation is that the adjuvanticity of inactive edema toxin resulted from residual toxin activity. Attenuated labile toxin mutants that possess low levels of activity are almost as potent of adjuvants as wild type toxin [380,

399]. The combination of PA+EF227 resulted in a very small but detectable increase in the level of intracellular cAMP, indicating that the toxin was attenuated, not inactive.

Although no increase in intracellular cAMP levels was detected for PA+EF346,

PA425+EF or PA682+EF, it is possible that at the high concentrations administered in the immunization, enough cAMP was produced to stimulate an enhanced immune response. Another possible explanation is that the adjuvant activity resulted from dose effects. Increasing the dose of inactive toxin in an immunization increases the strength and scope of the downstream immunological effects [381, 391, 393]. A third possibility is that edema toxin interacts with the immune system in a different way than the other cAMP-elevating toxins. The requirement for receptor binding for cholera and labile toxin adjuvanticity may actually be a requirement for receptor-mediated signals upon toxin binding [397, 398]. There is no evidence that either of the anthrax toxins trigger receptor-mediated signaling events upon binding. Lethal and edema toxin are able to intoxicate cells using surface receptors lacking the entire cytoplasmic domain [147], and

PA has never been associated with any of the immunomodulatory effects described for the B subunits of cholera and labile toxin [398]. Edema toxin may be able to forgo the requirement for receptor-mediated signals by interacting with immunological target cells in a novel manner.

136 Regardless of the underlying mechanism, active and inactive forms of edema are able to act as their own adjuvants in an immunization. Similar to cholera, labile and pertussis toxins, the adjuvanticity of edema toxin appears to depend on contributions from both enzymatic activity and the cellular intoxication process [381, 391-393]. These two contributions can have quite different effects on the resulting immune response. The increase in intracellular cAMP drives the proliferation of TH2 cells and the production of

TH2 cytokines [400-403], and thus, all of these toxins are able to act as TH2 adjuvants.

Despite the TH2-enhancing effects common to all cAMP-elevating toxins, each toxin has its own adjuvant characteristics. Cholera toxin functions almost entirely as a TH2 adjuvant [401, 402, 404]. Pertussis toxin, while able to promote TH2 responses, is a very strong TH1 adjuvant [381, 405, 406]. Labile toxin is able to stimulate both TH1 and TH2 responses [380, 393, 407]. This effect is mediated by the nature of the interaction between the toxin complex and the immune system [380, 381, 392]. Immunization with inactive edema toxin resulted in an increase in endosomal EF and PA antibody titers, as well as an increase in the number of IL-4 producing T lymphocytes. Immunization with an attenuated form of edema toxin was also reported to enhance the TH2 response to bystander antigens [378]. Edema toxin is clearly able to promote the production of a strong TH2 response. However, edema toxin may also be able to promote a robust TH1 response. The antibody titers to cytosolic EF were significantly enhanced to the detriment of the TH2-based PA response. Also, even forms of inactive edema toxin that promoted a strong TH2 response to PA and EF resulted in the production of very high numbers of IFN- producing T lymphocytes specific to each antigen. The production of

IFN- is modest following immunization with cholera toxin, but is a salient feature of the

137 immune responses generated by labile and pertussis toxin [393, 401, 405]. Thus, edema toxin may be able to stimulate both T cell pathways and function as a TH1/TH2 adjuvant.

138 Figure 4.1: Structure of edema factor bound to calmodulin and ATP. The coordinates of the structure for EF were obtained from Swiss-Prot (accession # P40136) and modeled using the SwissPDBViewer software. Residues targeted by site directed mutagenesis are shown in red. Domain 1 (blue) spans amino acids 34-303. This domain binds to PA and contains the tyrosine at position 227 important in that interaction. The catalytic portion of the enzyme is comprised of domains 2 and 3 (purple and green, respectively). An ATP molecule (represented as a yellow ball and stick model) is shown bound to the active site, along with the arginine residue at position 346 that facilitates ATP binding. Domain 4 (gold) is shown bound to a molecule of calmodulin (pink).

139 Figure 4.2: Intracellular cAMP levels following intoxication with edema factor mutants lacking adenylyl cyclase activity. EF, EF346 and EFn (5 g/mL) were mixed with an equivalent amount of PA and incubated on CHO-K1 cells for 45 minutes at 37oC in 5% CO2. CHO-K1 cells were also incubated with each protein singly as controls. All incubations were performed in triplicate. Following incubation, the cells were lysed and intracellular cAMP was extracted by the addition of 0.1 M HCl with 0.1% Triton X-100. The concentration of cAMP in each sample was quantitated by indirect capture ELISA. Shown above are the average cAMP concentrations +/- the standard deviation of the mean.

140 Figure 4.3: Structure of activated protective antigen. The coordinates of the structure for PA were obtained from Swiss-Prot (accession # P13423) and modeled using the SwissPDBViewer software. The structure represents the activated (PA63) molecule. The PA20 fragment is not shown. Residues targeted by site directed mutagenesis are shown in red. Domain 1’ (blue) is responsible for LF and EF binding and contains the lysine 197 residue necessary for that interaction. Translocation and pore formation is carried out by domain 2 (purple). The aspartate residue at position 425 is located at the center of the heptameric pore. PA heptamerizes via domain 3 (green) interactions between neighboring PA molecules. Domain 4 (gold) interacts with the cellular receptor via the loop containing asparagine 682.

141 Figure 4.4: Gel mobility shift assay for complex formation by PA and EF binding mutants. PA83 and PA197 were activated by trypsin (1:1000) for 30 minutes at 37oC. To form the complexes, 20 g of activated PA or PA197 was combined with 10 g of EF or EF227 for 30 minutes at 37oC. Samples containing each protein singly were used as controls. The samples were mixed with 2x native PAGE sample buffer and run on a 4% native acrylamide gel at 50 V for 20 minutes, then 80 V for an additional 60 minutes. Following electrophoresis, protein was visualized with BioSafe Coomassie.

142 Figure 4.5: Sandwich ELISA measuring complex formation by PA and EF binding mutants. Immulon 4 plates were coated with mouse anti-PA monoclonal antibody 3E2- 071100-02. PA83 and PA197 were activated with trypsin (1:1000), and then bound to the monoclonal antibody. Unbound PA was removed, and EF or EF227 was allowed to bind to PA or PA197 for 30 minutes at 37oC to determine the degree of interaction between the two molecules. Polyclonal rabbit anti-EF was added to detect the bound EF. The bound rabbit antibody was detected by the addition of anti-rabbit IgG conjugated to horseradish peroxidase. The plates were developed with ABTS substrate and the absorbance at 405 nm was read. Values shown are the average absorbance for each series +/- the standard deviation.

143 Figure 4.6: Intracellular cAMP levels following intoxication with protective antigen mutants deficient in cellular intoxication. Edema toxin combinations were generated by combining equivalent amounts of PA protein with EF protein (3 g toxin = 3 g PA + 3 g EF). The toxin combinations were incubated in triplicate on CHO-K1 cells for 45 o minutes at 37 C in 5% CO2. Following incubation, the cells were lysed and intracellular cAMP was extracted by the addition of 0.1 M HCl with 0.1% Triton X-100. The concentration of cAMP in each sample was quantitated by indirect capture ELISA. Shown above are the average cAMP concentrations +/- the standard deviation of the mean.

144 Figure 4.7: PA-specific serum IgG responses to edema toxin components deficient in adenylyl cyclase activity. Groups of 10 Balb/cAnNCr mice were immunized on days 1 and 15 with equivalent molar amounts of protein (30 g PA, 32 g of EF, 32 g EF346 and 12.4 g EFn) bound to Alum and the antigen specific titers were tracked over 70 days. PA83-specific titers were determined by indirect quantitative ELISA against PA. The graph is broken over 5-50 g/mL in order to better observe the values at day 14. Values shown are the geometric mean titer of the group plus/minus the standard error of the mean. Significant changes in PA titer (p < 0.05) are marked with an *.

145 Figure 4.8: EF-specific serum IgG responses to edema toxin components deficient in adenylyl cyclase activity. Groups of 10 Balb/cAnNCr mice were immunized on days 1 and 15 with equivalent molar amounts of protein (30 g PA, 32 g of EF, 32 g EF346 and 12.4 g EFn) bound to Alum and the antigen specific titers were tracked over 70 days. EF-specific titers from groups immunized with (A) EFn, (B) EF346 and (C) EF were determined by indirect quantitative ELISA against EF. The graph is broken over 50-200 g/mL in order to better observe the values at day 14. Values shown are the geometric mean titer of the group plus/minus the standard error of the mean. Significant changes in EF titer (p < 0.05) are marked with an *.

146 147 Figure 4.9: Serum IgG responses to edema toxin components deficient in complex formation. Groups of 10 Balb/cAnNCr mice were immunized on days 1 and 15 with equivalent molar amounts of protein bound to alum and the antigen specific titers were tracked over 56 days. (A) PA83 and (B)EF-specific titers were determined by indirect quantitative ELISA against the protein of interest. To better observe the data at day 14, the graphs are broken between 5-15 g/mL (PA) and 25-100 g/mL (EF). Values shown are the geometric mean titer of the group plus/minus the standard error of the mean. Significant changes in antibody titer (p < 0.05) are marked with an *.

148 Cytokinea PA EF EF227 PA+EF227

PA IL-4 20.5 (9.4)b ------59.4 (15.8)c

EF IL-4 --- 80.8 (22.0) 94.6 (15.3) 119.0 (16.6)

PA IFN 2.2 (3.2) ------65.4 (15.6)

EF IFN --- 10.0 (16.8) 27.6 (26.3) 188.4 (22.6)

a splenocytes from mice immunized with lethal toxin components were stimulated with either PA or LF and assayed for the production of either IL-4 or IFN b values shown are the geometric mean (+/- the standard error) of the group c underlined values are statistically different (p < 0.05) from the value obtained for mice immunized with that toxin component alone

Table 4.1: Cytokine responses to edema toxin components deficient in complex formation

149 Figure 4.10: Serum IgG responses to edema toxin components deficient in cellular receptor binding. Groups of 10 Balb/cAnNCr mice were immunized on days 1 and 15 with equivalent molar amounts of protein bound to alum and the antigen specific titers were tracked over 56 days. (A) PA83 and (B)EF-specific titers were determined by indirect quantitative ELISA against the protein of interest. To better observe the data at day 14, the graphs are broken between 5-15 g/mL (PA) and 25-100 g/mL (EF). Values shown are the geometric mean titer of the group plus/minus the standard error of the mean. Significant changes in antibody titer (p < 0.05) are marked with an *. 150 Cytokinea PA PA682 EF PA682+EF

PA IL-4 20.5 (9.4)b 11.8 (3.3) --- 71.2 (15.0)c

EF IL-4 ------80.8 (22.0) 140.0 (12.8)

PA IFN 2.2 (3.2) 3.5 (1.2) --- 25.3 (8.7)

EF IFN ------10.5 (16.8) 177.7 (28.9)

a splenocytes from mice immunized with lethal toxin components were stimulated with either PA or LF and assayed for the production of either IL-4 or IFN b values shown are the geometric mean (+/- the standard error) of the group c underlined values are statistically different (p < 0.05) from the value obtained for mice immunized with that toxin component alone

Table 4.2: Cytokine responses to edema toxin components deficient in cellular receptor binding

151 Figure 4.11: Serum IgG responses to edema toxin components deficient in translocation. Groups of 10 Balb/cAnNCr mice were immunized on days 1 and 15 with equivalent molar amounts of protein bound to alum and the antigen specific titers were tracked over 56 days. (A) PA83 and (B)EF-specific titers were determined by indirect quantitative ELISA against the protein of interest. To better observe the data at day 14, the graphs are broken between 5-15 g/mL (PA) and 25-100 g/mL (EF). Values shown are the geometric mean titer of the group plus/minus the standard error of the mean. Significant changes in antibody titer (p < 0.05) are marked with an *.

152 Cytokinea PA PA425 EF PA425+EF

PA IL-4 20.5 (9.4)b 38.0 (5.7) --- 57.1 (7.7)c

EF IL-4 ------80.8 (22.0) 8.1 (8.6)

PA IFN 2.2 (3.2) 6.0 (5.6) --- 3.19 (1.0)

EF IFN ------10.5 (16.8) 9.6 (3.3)

a splenocytes from mice immunized with lethal toxin components were stimulated with either PA or LF and assayed for the production of either IL-4 or IFN b values shown are the geometric mean (+/- the standard error) of the group c underlined values are statistically different (p < 0.05) from the value obtained for mice immunized with that toxin component alone

Table 4.3: Cytokine responses to edema toxin components deficient in translocation

153 CHAPTER 5

THE ANTIBODY TITERS TO LETHAL AND EDEMA FACTOR ARE INCREASED BY CO-ADMINISTRATION OF BACTERIAL PRODUCTS WITH INACTIVE TOXIN

5.1 Introduction

The previous two chapters have dicussed the effects of active lethal and edema toxin on the immune response and described the circumstances necessary to produce and increase in the lethal or edema factor antibody titer. For lethal toxin, the increase in antibody titer depends on the the activity of LF. For edema toxin, an increase in EF- specific titer depends not on toxin activity, but on the adjuvant properties of the toxin.

The downstream consequences of these two stimuli result in the alteration of the T lymphocyte cytokine profile and a titer increase in the developing antibody response.

Removing either the MEK proteolysis activity of LF or the catalytic domains of EF results in an LF or EF-specific antibody response equivalent to that obtained with LF or

EF by itself in an immunization. Although inactivated forms of lethal and edema toxin have significant effects on the development of an anti-toxin response, this chapter will explore the production of an enhanced antibody response in the absence of toxin activity.

154 The DNA immunization study published by Price, et. al. in 2001 demonstrated that the combination of plasmids encoding PA and LFn in an immunization resulted in increased antibody titers to both PA and LFn compared to each plasmid given alone

[313]. Two discrepancies may be noted between the results of this study and those reported in the previous chapters – the simultaneous increase in PA and LF antibody titer and the increase in antibody titer in the absence of toxin activity. The simultaneous increase in PA and LF titer may be due to differences between DNA and protein immunizations. In a protein immunization, the toxin interacts with cells through the defined intoxication process. Immunization with plasmid DNA results in the transfection of target cells that subsequently express the PA and LFn antigens. Following transfection, the majority of expressed protein is retained within the cytosol (Matthew

Bell, personal communication). The importance of cytoplasmic localization in the immune response to LF was demonstrated in chapter 3. It is possible that the retention of

PA with LFn in the cytoplasm would result in the production of a TH1-like response to both proteins and any improvement in the immune response would affect both proteins.

However, chapter 3 indicated that the mere cytoplasmic localization of LF was insufficient to trigger an increase in LF-specific antibody production. The results from the DNA immunization study imply that an immune stimulus separate from the toxin components can produce the same immunological effects as lethal toxin activity.

One possible immune stimulus in a DNA immunization is the plasmid DNA itself. Bacterial DNA falls into the category of immunological signals known as pathogen-associated molecular patterns (PAMPs). These are evolutionarily conserved molecules found in a wide variety of pathogens, but are foreign to the host [408, 409].

155 The presence of PAMPs is sufficient not only to generate an immediate inflammatory response by innate effectors, but also to stimulate efficient antigen presentation and T cell priming [410, 411]. Bacterial DNA has a higher frequency of unmethylated CpG motifs than eukaryotic DNA [412]. That difference is sufficient for recognition of unmethylated bacterial CpG motifs by TLR9 and subsequent activation of macrophages, dendritic cells and B cells [413-417]. Doubling the number of plasmids present in the DNA immunization may have resulted in an improved response to inactive toxin simply because the number of CpG motifs present increased, leading to a stronger immune stimulus.

Another possibility is that a TH1 determinant such as CpG motifs [418] is necessary for the improvement in antibody titers. Antibody titers to inactive toxin absorbed to alum may be unaffected not because of toxin inactivity, but because of the release of TH2 cytokines [419]. This is an interesting idea, as unmethylated CpG DNA is not the only PAMP capable of promoting a TH1 response. Several currently recognized

PAMPs are able to stimulate the release of TH1 cytokines, including bacterial lipopolysaccharide (LPS) and peptidoglycan [182, 420].

In order to test the ability of bacterial PAMPs to mimic the effects of active lethal toxin, crude preparations of inactive lethal toxin contaminated with LPS and other non- protein cell wall components were used in a small study. The resulting immune responses to both PA and LFn were compared with those obtained from mice immunized with highly purified protein preparations. The results from these investigations demonstrate that toxin activity is not the only immunological stimulus capable of improving the antibody response to the toxin proteins.

156 5.2 Results

5.2.1 Immunogenicity of inactive lethal and edema toxin components prepared without endotoxin removal

The truncated forms of LF and EF (LFn and EFn, respectively) were combined with PA, as was the proteolytically inactivated LF687. No attempt was made to compare the inactive toxins with wild type toxin. Proteins were purified over Ni-NTA resin and administered to mice in a 1:1 molar ratio. Mice received the molar equivalent of 50 g of

PA. The protein was absorbed to alum and groups of five female Balb/c mice were immunized intraperitoneally on days 1 and 15. Mice were monitored for adverse effects following immunization. The endpoint serum titers against PA, LF and EF were measured at two-week intervals for 28 days after the first immunization. None of the mice had detectable PA, LF or EF-specific antibody responses prior to immunization.

The results show that the addition of PA to both LFn and LF687 increased the antibody titer to LF at both day 14 and 28 (Figure 5.1). At day 14, the LFn endpoint titer increased from 3,200 to 8,445 with the addition of PA (p = 0.0126). Similarly, at day 14 the LF687 titer rose from 264 to 2,425 in the presence of PA (p = 2.388x10-4). The increase at day 28 was even more pronounced. Combining PA with LFn raised the LF titer from 36,234 to 399,544 (p = 1.312x10-7). The LF687 titer increased from 26,787 to

365,430 with the addition of PA (p = 2.482x10-4). In contrast, the addition of either LFn or LF687 had no effect on PA-specific antibody production at any time. A similar pattern was observed with the combination of EFn and PA. The EF-specific antibody titers increased significantly in the presence of PA (Figure 5.2). At day 14, the EFn titer increased from 459 to 1270 with the addition of PA (p = 0.0029). At day 28, the

157 presence of PA raised EFn titers from 15,222 to 45,614 (p = 0.0032). The PA-specific antibody titer was not significantly affected by the addition of EFn at either day 14 or 28.

The results from the preliminary study stand in stark contrast to the results obtained when an effort to remove endotoxin was made. Although these data have been previously presented (see Figures 3.12 and 4.8), they are shown here again in a simplified form for clarity and comparison. Mice immunized with truncated lethal or edema factor

(LFn or EFn, respectively) or with inactive lethal factor (LF687) alone and in conjunction with PA showed no significant difference in the LF or EF-specific antibody titer at any time during the experiment (Figures 5.3 and 5.4). Similarly, the PA-specific antibody titer in these groups remained unchanged by the presence of LF or EF through day 28, and then declined in the later half of the study.

The largest difference between the crude protein preparations used in this study and the highly purified protein preparations used in chapters 3 and 4 was the amount of

E. coli LPS present. The highly purified preparations of protein underwent an exhaustive endotoxin removal procedure specifically targeting this contaminant. The correlation between the amount of endotoxin present and the inactive toxin titers is shown in Table

5.1. As the endotoxin levels were reduced in the protein samples, the effect of PA on the antibody titers to inactive forms of LF or EF decreased to nothing. Although E. coli LPS is the most likely candidate for this alteration of the immune response, the possibility remains that some other non-protein bacterial product was also present and was responsible for this effect.

158 5.2.2 Immunization with truncated lethal toxin in the presence of purified lipopolysaccharide and monophosphoryl lipid A

The results from the crude protein preparations imply that the presence of non- protein bacterial products in a protein immunization significantly alters the resulting immune response. To examine this more closely, a study was conducted to determine the effect of purified LPS on the antibody titers raised against inactive lethal toxin. PA+LFn was chosen as the inactive form of toxin, as it had the most dramatic increase in titer when endotoxin was present. PA83 and LFn were purified over a TALON affinity column under endotoxin-removing conditions. The amount of endotoxin remaining was determined to be 0.37 and 6.0 EU/ml for PA and LFn, respectively. This amounted to

0.012, 0.087 or 0.099 EU/mouse for PA, LFn and PA+LFn groups, respectively.

The immune response to endotoxin-free protein was compared to an equivalent protein immunization to which 10,000 EU of purified endotoxin was added. Since the structure and immunogenicity of endotoxin varies widely among gram negative bacterial strains [421, 422], LPS was purified from the E. coli strain XL-1 Blue in order to most closely replicate the conditions of the crude protein study. As a positive control, a third set of mice were immunized with protein complexed with 5 g monophosphoryl lipid A

(MPL). MPL is a detoxified form of LPS purified from Salmonella minnesota R595.

The immunological potency and low toxicity of MPL has resulted in its use as a vaccine adjuvant [423, 424]. Mice received protein in a 1:1 molar ratio (30 g of PA and/or 10.5

g of LFn) on days 1 and 15. The antibody response to both proteins was measured at two-week intervals for 42 days after the first immunization. Mice had no detectable PA or LFn-specific antibody titers prior to immunization. 159 Proteins purified under endotoxin-removing conditions were unable to produce an increase in antibody titer when the proteins were combined (Figure 5.5). Mice responded in an identical manner to mice previously immunized with endotoxin-free protein. The

LFn-specific antibody response did not change significantly when PA was added, nor was the PA-specific antibody response altered by the presence of LFn. Interestingly, although the pattern of inactive toxin titers was the same as in the previous study, the overall titer levels were not. LFn provoked an extremely strong antibody response (in excess of 16 mg/mL), while the PA response was less than usual. Immunization with 30

g of PA historically produces a maximum antibody titer of 1 mg/mL; in this study antibody titers peaked at 0.75 mg/mL.

When purified LPS was reintroduced into the purified protein, the response was not appreciably different from the response to endotoxin-free protein (Figure 5.6). The

LFn-specific titers did not increase in the presence of PA, nor did the PA-specific titers change significantly in the presence of LFn. These results indicate that the presence of

LPS in the crude protein preparations was not responsible for the significant increase in

LFn antibody titers. The total amounts of antibody are similar to those in the endotoxin- free protein groups, with very high LFn titers and lower-than-usual PA titers.

When MPL was added as an adjuvant, the immune response was strikingly different (Figure 5.7). First, the amounts of antibody produced against both proteins were much lower than when alum was present. Neither PA nor LFn was able to stimulate a peak antibody response in excess of 1 mg/mL. This is most likely due to differences in adjuvanticity between 60 l of alum and 5 g of MPL. The second difference between immunization with MPL and endotoxin-free protein was that with MPL, the antibody 160 response to LFn was significantly improved by the presence of PA at days 28 and 42. On day 28, the LFn-specific titer increased from 37.9 to 199.1 g/mL (p = 8.93x10-4) when

PA was added. Likewise, on day 42, the antibody titer to LFn increased from 37.3 to

205.3 g/mL with the addition of PA (p = 0.0026). The PA-specific antibody titers were unaffected by the addition of LFn at all time points. The presence of MPL was sufficient to replicate the pattern of inactive toxin titers observed in the preliminary study.

5.3 Discussion

Contrary to expectations, purified E. coli lipopolysaccharide, a known immunomodulator, was unable to affect the inactive toxin antibody titers. This was unexpected for two reasons. First, the presence of LPS has long been linked with the release of IFN- and a strong bias towards a TH1 adaptive immune response [425]. The importance of a TH1 component in the improved antibody response to the cytosolic LF proteins has already been discussed in chapter 3. Second, MPL, another gram negative endotoxin expected to behave similarly to E. coli LPS, was able to stimulate an increase in the LFn titer when PA was present.

The different inactive toxin antibody responses may be due to the level of purification of the TLR agonists present. In the initial study, while LPS may have been present in the greatest amounts, it is highly unlikely that LPS was the only bacterial

PAMP present in the protein preparation. While attempting to assess the effect of LPS on the inactive toxin response, highly purified LPS was used in conjunction with highly purified protein. The stringent purification procedures for both LPS and protein probably removed some other PAMP(s) which was responsible for the titer increase. Although 161 CpG motifs were unlikely to be present in the crude protein preparations, trace amounts of cell wall and lipoproteins could easily have been present. The presence of even small amounts of these molecules is sufficient to activate innate immunity via the Toll-like receptors and alter TLR signaling [413, 426-429].

The presence of a second contaminant explains why highly purified LPS was unable to replicate the results produced by crude protein preparations, but it does not explain the increase in titer induced by MPL. One possibility is that the MPL used in the second study was not highly purified. Highly purified preparations of E. coli LPS and

MPL stimulate innate cells through interaction with only TLR4 [430-432]. However, many commercial preparations of MPL are not purified to this degree, and as a result, stimulate both TLR2 and TLR4 [431]. This is not an insignificant difference, as TLR4 ligands promote the release of IFN-, IFN- and IL-12 and bias the immune response towards TH1, while TLR2 ligands promote IL-8 and IL-23 production and result in a TH2 bias [182, 433-435]. The combination of TLR2 and TLR4 signals together may result in the mixed TH1/TH2 response frequently associated with MPL [424, 436].

Few conclusions can be drawn about the either the TLR pathways stimulated or the resulting cytokine profile during immunization with MPL, due to the limited nature of the LPS study. The goal of the study was to determine if purified LPS could produce the increase in antibody titers observed with crude protein preparations. The data indicate that LPS could not stimulate increased antibody production to LFn in the presence of PA, and imply that a second gram negative cell component was present in the preliminary study (and MPL) that was able to affect antibody production. Suspicion immediately

162 falls on lipoproteins and endotoxin binding peptides, as these are the most frequent contaminants of LPS and MPL preparations and they have been implicated in the mixed

TLR response in impure preparations [431, 432, 437-440].

Although the cytokines produced by LFn-specific T cells were not measured following co-administration of LPS with inactive toxin, LPS is classically associated with the production of IFN- and a TH1 response [182, 433-435]. It is interesting to note that the presence of a strong TH1 adjuvant did not improve the antibody titers to the cytoplasmic LFn. Cytosolic processing is essential for the antibody response to LF, and this has led to the hypothesis that it is the induction of a TH1-like response when PA and

LF are both present that drives the increase in LF-specific antibodies. However, a “pure”

TH1 response results in the generation of antigen-specific CTL’s, not antibodies. In order to increase antibody production during a TH1-like response, TH2 cytokines may be required. The data in chapter 3 show that the number of LF-specific IL-4 producing T lymphocytes present increases whenever the LF-specific antibody titer increases.

The lack of effect on the LFn titer when LPS is present implies that while TH1 characteristics may be important, the antibody response to LF may depend on some degree of hybridization between the TH1 and TH2 pathways.

163 Figure 5.1: Endpoint titers of mice immunized with inactive lethal toxin. Groups of 5 Balb/c mice were immunized with 50 g of PA83 and either LF687 or LFn absorbed to Alum on days 1 and 15. PA83 (A) and LF (B)-specific endpoint titers were determined by indirect ELISA against the protein of interest. Endpoint titers were defined as the inverse of the serum dilution providing an absorbance twice that of naïve mice. Values shown are the geometric mean titer of the group plus/minus the standard error of the mean. 164 Figure 5.2: Endpoint titers of mice immunized with truncated edema toxin. Groups of 5 Balb/c mice were immunized with 50 g of PA83 and EFn absorbed to Alum on days 1 and 15. PA83 (A) and EF (B)-specific endpoint titers were determined by indirect ELISA against the protein of interest. Endpoint titers were defined as the inverse of the serum dilution providing an absorbance twice that of naïve mice. Values shown are the geometric mean titer of the group plus/minus the standard error of the mean.

165 Figure 5.3: Serum IgG responses of mice immunized with inactive lethal toxin. Groups of 10 Balb/cAnNCr mice were immunized on days 1 and 15 with equivalent molar amounts of endotoxin-free protein (30 g PA, 32 g LF687 and 10.5 g LFn) bound to Alum and the antigen specific titers were tracked over 70 days. PA83 (A) and LF (B)-specific titers were determined by indirect quantitative ELISA against the protein of interest. Values shown are the geometric mean titer of the group plus/minus the standard error of the mean.

166 Figure 5.4: Serum IgG responses of mice immunized with truncated edema toxin. Groups of 10 Balb/cAnNCr mice were immunized on days 1 and 15 with equivalent molar amounts of endotoxin-free protein (30 g PA and 12.5 g EFn) bound to Alum and the antigen specific titers were tracked over 70 days. PA83 (A) and EF (B)-specific titers were determined by indirect quantitative ELISA against the protein of interest. Values shown are the geometric mean titer of the group plus/minus the standard error of the mean.

167 Endotoxin LF/EF LF/EF+PA Protein Net Effectd p valuea (EU/mouse) Titera Titera LFn 3125 36,230b 399,500 b 10-fold increase 1.31E-07 LF687 5434 26,790 b 276,900 b 10-fold increase 0.0002 EFn 9008 15,220 b 45,610 b 3-fold increase 0.0032 LFn 0.1402 2514c 3165 c No change 0.2261 LF687 0.6050 2664 c 3397 c No change 0.2370 EFn 0.1452 421 c 392 c No change 0.9054

a Values are from day 28 b Values shown are endpoint titers c Values are quantitative titers and have units of g/mL d Net effect is defined as the change in LF/EF titer when PA is present

Table 5.1: Effect of endotoxin on the lethal and edema factor titers in the presence of protective antigen

168 Figure 5.5: Serum IgG responses of mice immunized with an endotoxin-free, truncated form of lethal toxin. Groups of 10 Balb/cAnNCr mice were immunized on days 1 and 15 with equivalent molar amounts of protein bound to Alum. Proteins were purified under endotoxin-removing conditions, such that no mouse received more than 0.5 units of endotoxin in an immunization. PA83 (A) and LFn (B)-specific titers were determined by indirect quantitative ELISA against the protein of interest. Values shown are the geometric mean titer of the group plus/minus the standard error of the mean. 169 Figure 5.6: Serum IgG responses of mice immunized with a truncated form of lethal toxin in the presence of lipopolysaccharide. Groups of 10 Balb/cAnNCr mice were immunized on days 1 and 15 with equivalent molar amounts of protein bound to Alum. Protein samples were spiked with purified E. coli endotoxin immediately prior to alum absorption, such that each mouse received 10,000 EU in each immunization. PA83 (A) and LFn (B)-specific titers were determined by indirect quantitative ELISA against the protein of interest. Values shown are the geometric mean titer of the group plus/minus the standard error of the mean. 170 Figure 5.7: Serum IgG responses of mice immunized with a truncated form of lethal toxin in the presence of monophosphoryl lipid A. Groups of 10 Balb/cAnNCr mice were immunized on days 1 and 15 with equivalent molar amounts of protein (30 g PA and 10.5 g LFn) complexed with 5 g of MPL. PA83 (A) and LFn (B)-specific titers were determined by indirect quantitative ELISA against the protein of interest. Values shown are the geometric mean titer of the group plus/minus the standard error of the mean.

171 CONCLUSIONS

The focus of this project was to investigate the immune responses to lethal and edema toxins from B. anthracis. Both toxins use the same B subunit to target cells, and thus have almost identical intoxication processes. Both toxins are trophic for innate immune effector cells, and both toxins serve to suppress the immune response during infection. Because of these similarities, the two toxins were expected to interact with the immune system in similar ways and generate similar immune responses. However, the adaptive immune response perceives lethal and edema toxin quite differently, and immunization with these two similar toxins produces very different results.

Lethal and edema factor form homologous complexes with protective antigen and intoxicate cells through the same mechanism. The cellular intoxication process, from complex formation to cytoplasmic translocation, affects the immune response to both toxins. However, due to differences between lethal and edema factor, the effect of cellular intoxication is different for each toxin. The immune response to lethal factor depends on cytosolic processing and IFN- production by LF-specific T lymphocytes.

Protective antigen requires an immune response based on processing in the endosomal compartments and subsequent IL-4 production. The combination of a TH1 antigen (LF) and a TH2 antigen (PA) in the same immunization sets up a situation with conflicting

172 immune responses. The TH1 and TH2 pathways negatively regulate each other, and thus, the antibody response to either PA or LF suffered in every combination tested. Inactive lethal toxin mutants possessing active PA molecules resulted in the cytoplasmic localization of LF. The immunogenicity of LF under these circumstances drove the production of a TH1-like response, resulting in a decrease in the PA antibody titers.

Inactive lethal toxin mutants possessing inactive PA molecules resulted in the endosomal localization of LF. Under these conditions, the resulting immune response was biased in the TH2 direction, which resulted in a decrease in LF titer.

Edema toxin is composed of PA (a TH2 antigen) and EF. The immune response to EF by itself results in the endosomal processing of antigen and subsequent production of IL-4. Under these circumstances, EF is a TH2 antigen. However, the immune response to EF also allows cytosolic processing and IFN- production. Thus, a robust antibody response to EF can be generated by either the TH1 or TH2 pathway. This sets up an entirely different scenario for the edema toxin complex. When inactive PA mutants are included in the edema toxin combination, both antigens are contained in the endosomal pathway and the resulting TH2 response results in increased antibody titers to

PA and EF. Only when functionally active PA translocates EF to the cytoplasm does EF generate a TH1 response and compete with the development of the PA response. Under these circumstances, the EF antibody response is enhanced, while the PA antibody titers are reduced.

The formation of a toxin complex and the subsequent cellular intoxication process has a considerable effect on the development of the immune response to both lethal and edema toxin. Similarly, the downstream immunological consequences of toxin action

173 make a significant contribution to the early stage of the anti-toxin response. Lethal toxin is able to cause the death of macrophages, through toxin-mediated apoptosis or possibly necrosis. The presence of apoptotic bodies or other cellular debris has a strong stimulatory effect on dendritic cells and improves the ability of dendritic cells to prime naïve T cells. The action of the toxin results in an enhanced anti-LF antibody response in the first month after immunization. If the ability of lethal toxin to proteolyze MEK proteins is removed, toxin-mediated cell death no longer occurs following immunization and subsequently, no enhancement of the LF-specific immune response is observed.

Active edema toxin also affects the developing immune response. Similar to several other cAMP-elevating toxins, active edema toxin is able to act as an adjuvant.

Following immunization, the antibody response to EF is significantly increased and the kinetics of the response are altered. Edema toxin differs from lethal toxin, in that the inactive forms of edema toxin retain many of the adjuvant properties of the wild type toxin in an intraperitoneal immunization. Thus, inactive forms of edema toxin are able to increase EF and PA-specific antibody responses, although the kinetics of the enhanced responses do not differ from those observed when the toxin components are given alone.

One important conclusion from the LPS study is that an increase in LF and EF antibody titers can be produced in the absence of toxin activity. A separate immune stimulus (MPL) was also able to increase antibody titers to LF. Although there is no obvious connection between MEK proteolysis, cAMP production and TLR signaling, the activities of lethal and edema toxins (and MPL) intersect at the innate immune system and antigen presenting cells. Lethal toxin mediated cell death results in dendritic cell maturation through either danger signals or apoptotic body processing. Edema toxin

174 stimulates antigen presenting cell maturation through a cAMP-dependent pathway. MPL interacts with antigen presenting cells through the toll-like receptors. Although the interactions of each toxin with the immune system are different, the downstream consequences produce the same immunological effects. This implies that the enhancement of the lethal and edema factor titers results from increased stimulation of the innate immune response, which then drives an increase in antigen-specific T and B cell activity.

Although the goal of this project was not to develop an anthrax vaccine, two observations from this work suggest improvements to existing immunization strategies.

First, the ability of an anthrax immunization to generate lethal toxin neutralizing antibodies is crucial. The neutralization of lethal toxin action in an infection is a key predictor of survival, and neutralizing antibodies are currently the only known correlate of protection. Although the mechanism of cellular intoxication dictates that the PA and

LF antibody responses compete with each other, the overall effect of co-immunization on lethal toxin neutralization was positive. The addition of active, inactive and truncated forms of LF to PA resulted in a significant increase in toxin neutralization following immunization. In particular, the presence of full length LF raised the neutralizing titer to very high levels within 28 days of immunization. These LF-specific antibodies provide neutralization before the immune response to PA has had a chance to develop. This is of benefit in situations where rapid protection from anthrax infection is necessary. Also, the combination of LF with PA resulted in high neutralizing titers after the peak immune

175 response. PA-specific neutralizing antibodies rapidly decline to negligible levels when

PA is administered alone. The addition of LF appears to lengthen the time that high levels of circulating neutralizing antibody are present.

Second, edema toxin does not require catalytic activity in order to increase the toxin-specific antibody response. Edema toxin makes a significant contribution to the pathology of anthrax. Additionally, edema toxin antibodies directed against PA are also able to react with lethal toxin, the importance of which has already been discussed. Since the two toxins share PA, and since the PA binding sites on LF and EF are homologous, neutralizing antibodies against one toxin have a high level of activity against the other toxin. Immunization with an inactive form of edema toxin provides anthrax toxin- specific antibodies as well as lethal toxin neutralizing antibodies. Also, the adjuvant properties of edema toxin make it an ideal molecule for a combination vaccine including other anthrax epitopes or antigens from another pathogen entirely.

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