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The outcome of any serious research can only be to make two questions grow where only one grew before.

Thorstein Veblen

Cover image provided and copyright Dennis Kunkel Microscopy, Inc. (www.denniskunkel.com)

List of papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Nilsson E, Larsson A. (2007) Stability of chicken antibodies

freeze-dried in the presence of lactose, sucrose and threalose. J Poult Sci, 44:58–62.

II Nilsson E, Hanrieder J, Bergquist J, Larsson A. (2008) Proteo-

mic characterization of IgY preparations purified with a water dilution method. J Agric Food Chem, 56(24):11638–42.

III Nilsson E, Amini A, Wretlind B, Larsson A. (2007) Pseudo-

monas aeruginosa infections are prevented in cystic fibrosis pa-tients by avian antibodies binding Pseudomonas aeruginosa flagellin. J Chromatogr B Analyt Technol Biomed Life Sci, 856(1-2):75–80.

IV Nilsson E, Larsson A, Olesen HV, Wejåker PE, Kollberg H.

(2008) Good effect of IgY against Pseudomonas aeruginosa in-fections in cystic fibrosis patients. Pediatr Pulmonol, 43(9):892–9.

Reprints were made with kind permission from the respective publishers.

List of papers not included in the thesis

Nilsson E, Larsson A. (2005) Chicken anti-protein L for the detection of small amounts of protein L in the presence of IgG. Hybridoma (Larchmt.). 24(2):112–4. Nilsson E, Kollberg H, Johannesson M, Wejåker PE, Carlander D, Larsson A. (2007) More than 10 years' continuous oral treatment with specific im-munoglobulin Y for the prevention of Pseudomonas aeruginosa infections: a case report. J Med Food, 10(2):375–8. Amini A, Nilsson E. (2008) Quantitative analysis of polypeptide pharmaceu-ticals by matrix-assisted laser desorption/ionization time-of-flight mass spec-trometry. J Pharm Biomed Anal, 46(3):411–7.

Contents

Introduction................................................................................................... 11�

IgY ........................................................................................................... 11�

Avian immunoglobulins ...................................................................... 11�

Transfer of IgY from hen to egg ......................................................... 12�

Stability ............................................................................................... 13�

Antibody production............................................................................ 13�

Purification .......................................................................................... 14�

Applications of avian vs. mammalian antibodies................................ 14�

IgY in immunological assays and diagnostics .................................... 15�

IgY for oral immunotherapy................................................................ 15�

Studies of IgY for immunotherapy...................................................... 16�

Antimicrobial effects of IgY ............................................................... 17�

Antibiotic resistance and other side effects.............................................. 18�

Cystic fibrosis .......................................................................................... 19�

Genetics and basic defect .................................................................... 19�

Clinical manifestations ........................................................................ 20�

Respiratory infections.......................................................................... 21�

Pseudomonas aeruginosa in CF.......................................................... 22�

Treatment and survival ........................................................................ 24�

Anti-Pseudomonas IgY as prophylaxis to CF patients........................ 25�

Proteomics................................................................................................ 26�

Aims.............................................................................................................. 28�

Materials and Methods ................................................................................. 29�

Immunization ........................................................................................... 29�

Antibody extraction.................................................................................. 29�

Freeze-drying and forced stability test (paper I) ...................................... 29�

IgY-activity ELISA (paper I) ................................................................... 30�

Gel electrophoresis and immunoblotting (paper II & III)........................ 30�

NanoLC-MALDI-TOF/TOF MS (paper II) ............................................. 31�

Cholesterol and triglyceride (paper II)..................................................... 31�

MALDI-TOF MS (paper II & III)............................................................ 31�

Peptide sequencing (paper III) ................................................................. 32�

Study subjects (paper IV)......................................................................... 32�

Control group (paper IV) ......................................................................... 33�

Anti-Pseudomonas IgY treatment (paper IV) .......................................... 33�

Statistics (paper IV) ................................................................................. 34�

Results and Discussion ................................................................................. 35�

Paper I. Stability of chicken antibodies freeze-dried in the presence of lactose, sucrose and threalose .................................................................. 35�

Paper II. Proteomic characterization of IgY preparations purified with a water dilution method .............................................................................. 36�

Paper III. Pseudomonas aeruginosa infections are prevented in cystic fibrosis patients by avian antibodies binding Pseudomonas aeruginosa flagellin .................................................................................................... 39�

Paper IV. Good effect of IgY against Pseudomonas aeruginosa infections in cystic fibrosis patients.......................................................................... 40�

Conclusions................................................................................................... 43�

General discussion and future plans ............................................................. 45�

Svensk sammanfattning ................................................................................ 48�

Bakgrund.................................................................................................. 48�

Analyser av IgY ....................................................................................... 48�

IgY-behandling av patienter med cystisk fibros ...................................... 49�

Pågående studier ...................................................................................... 50�

Slutsats ..................................................................................................... 51�

Acknowledgements....................................................................................... 52�

References..................................................................................................... 55�

Abbreviations

1D One-dimensional 2D Two-dimensional 2DGE Two- dimesional gel electrophoresis ABC Adenine nucleotide-binding cassette ACN Acetonitrile BMI Body mass index BSA Bovine serum albumin CAF Chemically-assisted fragmentation cAMP Cyclic adenosine monophosphate CF Cystic fibrosis CFTR Cystic fibrosis transmembrane con-

ductance regulator Da Dalton ELISA Enzyme-Linked ImmunoSorbent

Assay EMEA European Medicines Agency ESBL Extended spectrum ß-lactamases FEV1 Forced expiratory volume in

one second GMP Good medical practice HRP Horseradish peroxidase Ig Immunoglobulin LC Liquid chromatography Lys-tag 2-methoxy-4,5-dihydro-1H-

imidazole MALDI Matrix-assisted laser desorp-

tion/ionization MARPA Multiple-antibiotic-resistant Pseudo-

monas aeruginosa MPA Medical Products Agency MRSA Methicillin-resistant Staphylococcus

aureus MS Mass spectrometry nanoLC Nanoflow liquid chromatography NBT/BCIP Nitro blue tetrazolium/5-bromo-

4-chloro-3-indolyl-phosphate

RP-HPLC Reversed Phase High Performance Liquid Chromatography

PA Pseudomonas aeruginosa PBS Phosphate buffered saline PBS-T PBS containing 0.5% Tween-20 PEG Polyethylene glycol PMNs Polymorphonuclear leukocytes PSD Post source decay QS Quorum sensing SDS-PAGE Sodium dodecyl sulfate

polyacrylamide gel electrophoresis RT Room temperature TFA Trifluoroacetic acid TMB 3.3’, 5.5’-tetramethylbenzidine TNF Tumor necrosis factor TOF Time-of-flight VRE Vancomycin-resistant enterococci

11

Introduction

The aim of this thesis was to characterize an IgY preparation to prevent Pseudomonas aeruginosa infections in cystic fibrosis patients. Antibiotics have saved many lives, but in order to keep them potent and reduce the de-velopment and spread of antibiotic resistance we need to use antibiotics more carefully and develop new treatment strategies. The use of antibodies from egg yolk, for oral immunotherapy and passive immunization is dis-cussed in the thesis.

IgY

Avian immunoglobulins

Antibodies play an important role in the immune system, which serves to protect the body from foreign substances. The chicken has three immuno-globulin (Ig) classes: IgA, IgM and IgY. Avian IgA and IgM are present in the egg white and have properties similar to their mammalian counterparts.1 IgM confers the first protection and IgA is a secretory antibody found in gall fluids, urine, feces and tears. IgY is the major immunoglobulin in serum. IgY is also the dominant immunoglobulin class present in the egg yolk, and was given the name IgY (from egg yolk IgG) by Leslie and Clem in 1969 to distinguish the antibody from mammalian IgG.2 IgY was at first considered to be the equivalent of mammalian IgG since it is the major immunoglobulin in serum, but it is now believed to be related to both mammalian IgG and IgE.3 Like IgE in mammals, IgY mediates anaphylactic reactions. IgY is structurally similar to IgG (Fig. 1). They both consist of two heavy and two light chains bound together with disulphide bounds, but they differ in that IgY has a shorter and less flexible hinge region.3 Another difference is that IgY has a molecular weight of 167 250 Da, which is higher than mammalian IgG (160 kDa) due to a heavy chain with a higher number of heavy chain constant domains and carbohydrate chains.3,4 IgY is also more hydrophobic than IgG and has a lower isoelectric point (5.7–7.6).5 Whereas the diversity of mammalian immunoglobulin is mainly a result of rearrangements, the diversity of chicken immunoglobulins is due to gene conversion and somatic hypermutation.6,7 Gene conversion mainly takes place in the Bursa of Fabri-cus, an important organ for B cell development, which is found only in birds

12

and is active in the beginning of life. As the chicken grows older, gene con-version occurs in germinal centers in the spleen, which are formed after chal-lenge with an antigen. Somatic hypermutation replaces gene conversion as the dominant form of diversification as the germinal centers matures.7

Figure 1. Mammalian IgG and avian IgY. IgG has a more flexible hinge region. IgY is heavier due to a larger heavy chain.

Transfer of IgY from hen to egg

Passive immunity conferred by antibody transfer from hen to chick was dis-covered already in 1893 by Klemperer.8 The transfer of IgY from hen serum to the embryo is comparable to the cross-placental transfer of IgG in mam-mals and aims to protect the newly hatched offspring until it has developed its own humoral immunity. It is an active two-step transport. First, IgY is transferred into the egg yolk and then transported across yolk sac membranes into the blood stream of the embryo primarily during the last days before hatching.9 IgY is bound by the yolk sac via IgY receptor, FcRY, on the yolk sac membrane in a pH dependent manner.10 The receptor binds IgY at pH 6, but not at pH 8. Conformational changes due to pH are suggested to alter the receptors binding site. Moreover, FcRY-expressing cells transcytosed the receptor at pH 6, but not at pH 8.11 The egg yolk is slightly acidic (pH 6) which should favor binding of the FcRY to IgY, and when the complex reaches the blood with a more basic pH (pH 7.4) IgY is released. An intact Fc region is required for uptake into the egg yolk, and IgY has several amino acid sequences that are also of importance.12 IgY concentration during matu-ration of the egg is steady, and the amount of immunospecific IgY corre-sponds linearly to the hen’s serum concentration.13 All subpopulations of maternal IgY are transferred into the chicken oocyte.14

IgG

IgY

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Stability

There are different reports on how much IgY that is active in stool.15 In new-borns, there is more IgY present in the stool since an infant’s stomach pH is higher than the normal pH for an adult. This should also be true for elderly individuals with an increased stomach pH due to reduced acid production. IgY is quite resistant against trypsin and chymotrypsin inactivation, but the anti-bodies are degraded by pepsin.16

Several strategies to protect IgY from hydrolysis by gastric enzymes and acidic condition have been investigated. The stability of IgY at pH 3 was increased in the presence of sorbitol, but not in xylitol.17 IgY is also more stable in sucrose against low pH, heat and pressure.18,19 IgY encapsulated into liposomes were more resistant both to pepsin and acidic conditions,20 and freeze-dried IgY coated with gum arabic was protected against hydroly-sis by trypsin, chymotrypsin and pepsin.19 Chitosan-alginate coated IgY was found to cure enteric colibacillosis in pigs more rapidly than non-coated IgY, but the uncoated IgY still had a better effect than the commonly used antibi-otic.21 IgY has also been dissolved in sodium carbonate buffer to prevent degradation in the stomach.22

IgY is not degraded during pasteurization at 60ºC.23 IgY was stable after 30 minutes in 60–65ºC, but was no longer active after 20 min in 80ºC.24

Antibody production

Specific IgY antibodies are obtained by immunizing the hen with the antigen of interest (Fig. 2). To avoid handling large amounts of highly virulent pathogens Motoi and colleagues immunized hens with a rabies antigen ex-pressed in Escherichia coli instead of using the rabies virus itself. However, the antibodies were not as effective as IgY against the whole rabies virus.25 Usually the antigen is given together with an adjuvant to increase the anti-body response. Hens tolerate Freund’s adjuvant relatively well,26 it stimu-lates a high immune response27 and it is the most commonly used adjuvant in published papers. Intramuscular immunization gives higher yield of IgY than subcutaneous immunization.19 After an initial immunization, booster doses are often given to assure high antibody titers over time. Typically, after the first immunization one or two booster doses are given every fourth week and thereafter every other month. It takes about 6–7 days after detection in serum until specific antibodies are found in the egg yolk.13 A small amount of anti-gen elicits a strong immune response, but the response depends on the type of antigen and the hen.28-30 IgY antibodies have high avidity31,32 and the anti-body titers are persistent over time. One laying hen produces approximately 20 eggs per month, and each egg contains a high concentration of IgY, about 100 mg.33 Thus, over 2000 mg IgY per month can be isolated whereof up to 10% is specific for the antigen used for immunization.34

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Figure 2. A hen will produce specific IgY antibodies in response to immunization with an antigen. The antibodies are transferred from the hen serum to the egg yolk. The eggs are collected, and IgY is purified from the egg yolk.

Purification

The egg yolk contains mainly proteins and lipids and can be divided into a granular and plasma fraction.35 The plasma fraction can be further divided into low-density lipoproteins and a water-soluble part. The water-soluble fraction contains livetins, among them IgY (�-livetin). There are several methods to purify IgY from egg yolk, e.g. salt, dextran sulphate, xanthan, gum, ethanol or polyethylene glycol (PEG) precipitation, thiophilic chro-matography or water dilution.33,36,37 Depending on the type of application and demand, more than one method can be combined. We have applied the water dilution method because the method does not include any toxic addi-tives and it is simple, rapid, suitable for large-scale production, inexpensive, and efficient for obtaining IgY with high activity.33,36 Moreover, the process contains no additives except water. There are methods that give somewhat higher yield, but with the disadvantage of additives. With the water dilution method, the pH can be altered to obtain a higher yield of IgY.33 In our purifi-cation process, the pH was left unchanged because a time consuming ultra-centrifugation could then be replaced by sedimentation. Besides IgY, the water dilution method yields other water-soluble proteins from the egg yolk and some lipids.36 The constituents were examined in paper II.

Applications of avian vs. mammalian antibodies

There are many advantages of IgY compared to mammalian antibodies. For example there is a great phylogenetic distance between birds and mammals, and hence, IgY has affinity to more epitopes of mammalian proteins than a corresponding mammalian antibody.38 IgY will thus display a higher activ-ity.26 IgY also cross-reacts with conserved proteins from different mammal-

15

ian species.22,32 There is no cross-reactivity between mammalian antibodies and IgY.39 IgY has a more stable molecular structure than mammalian IgG.3 It is possible to obtain 5–10 times more antibodies from a hen than from a rabbit under the same period of time, making the production of avian anti-bodies cheaper than the mammalian equivalent. IgY antibodies are prefer-able since the number of animals is reduced and there is no need to bleed them. It is more practical to collect eggs than blood and much less harmful to the animals. Moreover, it is more practical to purify IgY from the egg yolk, since it does not contain any other immuoglobulins than IgY, compared to purification of IgG from sera, which contains other immunoglobulin classes as well as many other constituents. IgY purification is mostly about removal of egg yolk lipids. There are also other properties that make IgY highly in-teresting for oral immunotherapy.

IgY in immunological assays and diagnostics

Due to the evolutionary distance between mammalian and avian antibodies, IgY is very well suited for immunological assays of mammalian proteins, especially since the risk for unspecific cross-reactivity and background can be minimized. The comparison of IgY to IgG has shown that IgY is as good as or even better than IgG in many immunoassays.40 IgY can be labeled, like IgG, with for example biotin with good activity and stability.41 IgY can also be used for diagnostic purposes. An example is anti-cystatin C IgY for detec-tion of cystatin C, a marker of kidney function.42 IgY against recombinant rabies proteins has also been suggested as suitable for diagnosis.43

IgY for oral immunotherapy

IgY has several properties that make the antibodies attractive for oral immuno-therapy. The antibodies neither activate the human complement system, nor react with rheumatoid factors, human anti-mouse IgG antibodies, or human Fc-receptors, all of which are well-known cell activators and mediators of inflam-mation.15,39 This is in contrast to antigen-antibody complexes with mammalian IgG antibodies, which will activate the human complement system and produce potent inflammatory mediators. This is also an advantage of IgY when used in immunological assays and diagnostics. Orally administered immunoglobulins do not pass from the intestines to the blood,44 and thus there will be no systemic effects. In contrast, IgY given intravenously to mice elicited an anti-IgY re-sponse.45 IgY is well tolerated for oral immunotherapy, but less so for intrave-nous injections. There is essentially no risk that bacteria or other pathogens should develop resistance against oral treatment with IgY. It is comparable with eating raw egg yolk and IgY is an ingredient in our regular diet. Thus, it is completely safe as long as the subject is not allergic to eggs. Allergic reactions may occur to egg-derived antibodies and residual egg proteins.46

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Studies of IgY for immunotherapy

Animal studies

Several animal studies have proven IgY against different pathogens to be effective for the treatment and prevention of infections. Oral treatment with IgY against enteric diseases has been studied to the largest extent. For exam-ple, IgY against rotavirus was protective against bovine rotavirus both in calves and mice.15,16 Moreover, salmonellosis has been prevented both in neonatal calves47 and in a mouse model.15 With the highest dose tested all calves treated with IgY survived, whereas all calves in the control group died.47 Enteric colibacillosis causing diarrhea is a problem among newborn piglets post-weaning. Therefore, antibiotics have been given in the feed as prophylaxis, a praxis still employed in some countries. The downside is an increased risk of selection for resistance. IgY against various fimbrial pro-teins of Escherichia coli has shown efficacy in prevention and treatment of enteric diseases in piglets and reduction in mortality.15,48-50

Clostridium difficile is a nosocomial bacterium that causes antibiotic-associated diarrhea and colitis, producing toxin A and B.51 Treatment with IgY against these toxins was effective in a dose-dependent manner. The highest concentration that was tested completely inhibited mortality in a hamster model, whereas 9/10 in the control group died.52 Another interesting study was conducted on experimental rats with induced colitis, which were treated with anti-tumor necrosis factor (TNF) IgY.22 TNF is a proinflamma-tory cytokine that plays a major role in inflammatory bowel disease and is increased in colitis. Large amounts of anti-TNF IgY were found at sites of inflammation. IgY treatment reduced the symptoms of both chronic and acute stages of colitis compared to controls treated with unspecific IgY, even if the inflammation was not reversed.

IgY against Cryptosporidium parvum oocytes in SCID mice is an exam-ple of a treatment against parasites. The infection was not totally cleared but oocyte shedding was reduced.53 The potential against many types of mi-crobes is further exemplified by reduced Candida albicans in the mouth of immunosuppressed mice. In addition, the mice treated with anti-candida IgY also had less C. albicans colonization of other organs.54

Besides oral treatment, IgY was effective given as an infusion in bovine mammary glands to prevent Staphylococcus aureus mastitis.55 This makes IgY a promising and needed alternative to antibiotics, since antibiotic resis-tance is an increasing problem among mastitis-causing bacteria.56 Moreover, IgY given intravenously as antivenom reduced the effect of snake venom, and the mortality in mice was reduced as well as edema.57 Animal hyperim-mune sera is commonly used, but there is a risk of anaphylactic reactions and serum sickness.58 These reactions were not seen with the IgY an-tivenom.57

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Human studies

In humans, IgY against Streptococcus mutans decreased the establishment of these bacteria in dental plaques when used as mouth rinse for seven days.59 The percentage of S. mutans per total Streptococci in vivo was decreased compared to the controls, but there was a large variation within the groups. In a small study, a gel containing IgY antibodies against Porphyromonas gingivalis was given to periodontitis patients. The treatment delayed the time to recolonization compared to controls.60

IgY against urease of Helicobacter pylori reduced the urea test values in volunteers tested positive for H. pylori with the test in two studies.24,61 In one study the antibodies were given as a supplement in drinking yoghurt,24 which illustrates the possibility of using IgY in functional food.

In a double-blind placebo-controlled study, 79 children with rotavirus were randomized to treatment with anti-rotavirus IgY or placebo during four days. The patients treated with IgY had significantly less stool frequency and amount, but the duration of diarrhea was not significantly reduced.62

Our group has performed the longest study of oral IgY treatment in hu-mans by administering Anti-Pseudomonas IgY to cystic fibrosis patients in order to prevent lung infections with Pseudomonas aeruginosa,63 which is further described later on. A small feasibility study performed by our group indicated that IgY might prevent Candida albicans infections in the mouth of children with lymphatic leukemia. None of the children receiving IgY treatment (0/4) had signs of C. albicans in their mouth, compared to 7/13 in the untreated group.64

Antimicrobial effects of IgY

There are several proposed mechanisms of action for IgY therapies, with a common one being IgY’s ability to prevent adhesion of the microbe to cells.48,59,65,66 Adhesion of bacteria to host cells is an important virulence factor. Flagella and fimbriae are protrusions that the bacteria use as adhesins. IgY against a fimbriae of E. coli reduced bacterial adherence to villi of the intestinal lumen in vitro and prevented shedding of the bacteria in weaning pigs, which were treated with these antibodies followed by challenge with E. coli.48 IgY binding to Salmonella alters the surface of the bacteria, which could explain the reduced ability to adhere.67 Porphyromonas gingivalis causes periodontitis by aggregating with other bacteria. IgY against an outer membrane protein of this bacterium inhibits coaggregation68 and IgY against a hemagglutinin inhibited P. gingivalis hemagglutination.69 In addition, IgY against gingipains, i.e. P. gingivalis proteases has been shown to reduce the proteolytic effect of the gingipains in vitro.70

Another suggested mechanism is increased phagocytosis. Specific IgY bound to S. aureus induces phagocytosis by milk macrophages.71 Specific

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IgY has also been shown to reduce, but not totally inhibit, the growth of bacterial species.66,71 The urease activity of H. pylori and bacterially induced injury of the gastric mucosa measured as the number of infiltrated lympho-cytes and neutrophils were reduced by specific anti-urease IgY.66 As men-tioned above, the toxic effect of venom was also reduced with specific IgY. IgY against recombinant mammalian TNF reduced the TNF-induced killing of cells from a murine cell line in vitro.22

The studies of IgY treatment in animals and humans have in general been short-term studies with no follow up. A few of them have shown total cure or prevention of the infection, but most of them have only shown reduced infection or symptoms. However, optimization of treatment strategies, such as more frequent and higher doses, enteric coating etc., could improve the results. Not many studies have been followed up and no IgY treatments exist on the market yet. IgY does not necessarily need to be used for total cure. It could also be used to reduce an infection to make antibiotic treatment more potent and successful.

Antibiotic resistance and other side effects Antibiotics have saved many lives. However, overuse and misuse have led to antibiotic resistance, which is an increasing problem worldwide.72,73 One problem is self-medication with leftover antibiotics and antibiotics sold over the counter, i.e. without a prescription, in some countries.73 This means a higher risk of taking the wrong medication, dose, or duration and, there is a threat for a selection towards resistance. There may be overconfidence in antibiotics, therefore, it is important to state the risks as well as the benefits, and realize that antibiotic treatment is not always required.73 Due to the globalization, people are traveling more and food and other products are distributed all over the world, and together with them resistant bacteria will follow. Not only the use of antibiotics in humans is a risk factor for resis-tance development, but also the high use of antibiotics in animals.74 Most antibiotics to animals in agriculture are not given for therapy, but for promo-tion of growth and prevention of disease. These antibiotics are often the same as, or similar, to antibiotics in human medicine. Antibiotics given as prophylaxis in aquaculture increases the amount of antibiotic-resistant bacte-ria in water.75 Pharmaceutical waste in the water is another factor causing resistance.

Bacteria under antibiotic pressure acquire resistance through mutations or by transfer of resistance genes from other bacteria or viruses. Many bacteria are also intrinsically resistant.72 There are several mechanisms of resistance, e.g. antibiotic-degrading enzymes and enzymes that modify the structure of the antibiotic molecules, whereby the site of antimicrobial effect can be al-tered.74 Multi-drug efflux pumps actively transport antibiotics out of the

19

bacteria. These pumps can be upregulated and porins downregulated to re-duce penetration of antibiotics.72 Examples of highly resistant bacteria that have emerged are MRSA,76 MARPA77, ESBL78, VRE74 and Clostridium difficile.51 The spread of these bacteria are not only caused by antibiotic us-age, but also by poor hygiene, primarily at care settings. Thus, infectious control is also important in minimizing the spread of antibiotic resistance.72

Other problems with antibiotics, besides resistance, are disturbance of the normal flora, which predisposes for infections by other opportunistic and pathogenic bacteria and fungi, and the risk for allergenic79 and toxic side effects of antibiotics. Vaginal candidosis is associated with antibiotic use.80 Treatments with amino glycoside antibiotics have been linked to both neph-rotoxicity81 and hearing loss.82 When there are fewer antibiotics to choose from more toxic alternatives sometimes become the only choice and due to treatment failure the cost is increased.72

Antibiotics are not only important to cure infections, but also to minimize the otherwise large risk for infections during surgery, transplantation or can-cer chemotherapy.73 Although resistance is increasing, development of new antibiotics is decreasing.73 Complements to antibiotics are urgently needed and passive immunization with IgY has the potential to be such a comple-ment.

Cystic fibrosis

Genetics and basic defect

Cystic fibrosis (CF) is the most common autosomal recessive disorder among Caucasians with an incidence of 1/2500–4000 newborns.83 There are about 600 persons affected in Sweden (personal information from the Swed-ish association for cystic fibrosis). CF is caused by a single mutation in each pair of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) genes on chromosome seven coding for the CFTR protein.84-86 This protein is primarily a cAMP-activated chloride-channel belonging to the adenine nu-cleotide-binding cassette (ABC) transporter family (Fig. 3). The channel also transports other ions and influences other ion transporters.87 Defective or absence of CFTR leads to an incorrect transport of chloride, and secondarily, also of sodium and water, which causes abnormally thick mucus.

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Figure 3. Schematic illustration of the CFTR chloride channel. ©Roberto Osti Illus-trations.

There are about 1600 known mutations today (see the CFTR Mutation Data-base: http://www.genet.sickkids.on.ca/cftr/app). The most common is the 508delF, which accounts for approximately 70%.88 The mutations can be divided into five major classes. In class I mutations truncated mRNA leads to no synthesis of the CFTR protein. In class II mutations, including 508delF, CFTR is synthesized, but never matures due to misfolding leading to degradation in the endoplasmatic reticulum. Class III–IV mutated CFTR all reach the cell membrane, but class III mutations cause blocked regulation and thus, CFTR is not activated. Class IV includes nonfunctional CFTR caused by decreased conductance. Class V mutations means functional pro-tein that is expressed in reduced amount.88 The degree of disease depends on class of mutation combined with modifying genes and environmental factors including infections, nutrition and treatment. 89-92

Clinical manifestations

The clinical manifestations seen in CF are mainly a result of defective fluid secretions. Normal mucus produced by exocrine glands lubricates the respi-ratory, digestive, and reproductive systems, prevents tissues from drying, and protects them from infection. In CF patients the mucus is thick and ac-

21

cumulates in the airways and in the pancreas, which primarily results in res-piratory infections, malnutrition and reduced growth. Impairment and de-struction of the exocrine pancreas causes reduced production of pancreatic enzymes followed my malabsorption of fats. With time, this leads to an in-creased risk for diabetes.93 In addition there is a large spectrum of other symptoms in CF, which are of considerable relevance. These include chronic sinusitis, nasal polyps, clubbing of finger and toes, rectal prolapse, impaired liver function, pancreatitis, biliary cirrhosis, and reduced fertility in women and infertility in men.88,94

Respiratory infections

Frequent respiratory infections lead to breathing impairment and eventually permanent lung damage in CF patients. CF is characterized by excessive, persistent inflammation in the airways in response to bacterial infections that causes lung destruction and decline in pulmonary function.95 Inflammation also predisposes for new infections. Relatively often lung transplantation becomes unavoidable.94

In a normal lung there is an airway surface liquid layer above the respira-tory epithelium. First there is one layer with periciliary liquid and above that a layer with mucus. The periciliary layer has low viscosity and allows movement of the cilia and thus mucociliary clearance. This is in contrast to the CF lung with thick and highly viscous mucus, which results in airway obstruction and defective mucociliary transport, entrapment of bacteria and elevated risk for bacterial infections.88

In young CF patients respiratory infections with Staphylococcus aureus and Hemophilus influenzae are most common, but with time Pseudomonas aeruginosa becomes predominant.93 Methicillin-resistant Staphylococcus aureus (MRSA) is especially problematic and is increasing among CF pa-tients. MRSA lung infection has been associated with a more rapid decline in lung function and need for hospitalization and antibiotic treatment.96,97

During the last years other opportunistic and pathological bacteria and fungi, not earlier seen among CF patients, have emerged.93,98,99 The explana-tion is probably a longer life, but also frequent antibiotic treatment,99,100 e.g. Burkholderia cepacia, Stenotrophomonas maltophilia, Achromobacter axy-losoxidans and Aspergillus fumigatus. Infections with bacteria from the Burkholderia cepacia complex have the worst outcome, while the clinical relevance of the other pathogens is more uncertain. A large concern with most of these bacteria is that they are resistant against multiple antibiotics and can transfer the resistance genes to other organisms. An example is mul-tiple-antibiotic-resistant Pseudomonas aeruginosa (MARPA), which is sug-gested to acquire its resistance by gene transfer from other bacteria.99

22

Pseudomonas aeruginosa in CF

Pseudomonas aeruginosa (PA) is a Gram-negative bacterium found in wa-ter, soil and many other environments, which can grow both in anaerobic and aerobic conditions and metabolize various sources.101 PA causes oppor-tunistic and nosocomial infections in immuno-compromised patients and is the most clinically detrimental bacterium for respiratory infections in CF patients.102 Usually, the first acute infections are possible to cure, but after recurrent infections the patient typically becomes chronically colonized in-cluding exacerbations with worse clinical condition. A chronic PA infection leads to a decline in lung function and is one of the main causes of morbidity and mortality for CF patients.88 During the chronic infection the bacteria usually turns mucoid by over-producing alginate and are thereby almost impossible to eradicate. Mucoid PA is associated with worse pulmonary function.92

There is a window of opportunity when PA can be treated and therefore, the strategy has become to give early high dose antibiotic treatment.100,103-108 This has lowered the frequency of chronically colonized patients. In Copen-hagen this strategy has been followed for a long time and they found that early aggressive antibiotic treatment could protect 80% of the patients for up to 15 years from chronic colonization.104 The prevalence of PA among CF patients in Scandinavia was recently compared. The patients at the CF centre in Copenhagen received most antibiotics and only 14.5% of the CF patients >19 years were chronically colonized compared to 30.9% in the Swedish centres.109 The introduction of antibiotic treatment with inhaled tobramycin has been successful105,107 and is now commonly prescribed for eradication of PA colonizations. At a Belgian CF centre, where inhaled antibiotics are given intermittently as prophylaxis, only, 2.8% of the CF patients under 18 years of age were chronically colonized.108 Although there is a clear benefit of early high dose antibiotics to prevent chronic colonization with PA the optimal treatment strategy has not been defined. There is no effective vac-cine against PA on the market, despite several studies in the field.110,111 It is expensive to treat PA infections112 and antibiotic toxicity81,82, allergy79 and resistance77 are other concerns.

PA has many virulence factors and several of them confer resistance. Pili are important for adhesion and PA has flagella, which it uses for twitching motility. Additionally, flagella initiate inflammation. PA also secrets several virulence factors, which cause tissue destruction and inflammatory response and, which are important for colonization and invasion.102 PA is intrinsically resistant against many antibiotics and easily develops resistance under anti-biotic pressure. 77 For example, PA has several efflux pump systems that can pump antibiotics out of the bacterium and mutated porins decreases perme-ability of antibiotics.77 PA also expresses ß-lactamases. There are hypermu-tator PA strains, which are more prone to develop resistance due to muta-

23

tions in mismatch repair.113 Hypermutators are common among CF patients and in addition, MARPA bacteria are increasing among CF patients and are associated with worse prognosis.114,115 There was also an association with antibiotic use. During the last decade highly transmissible and epidemic strains have appeared, which have lead to a segregation policy for CF pa-tients.116,117 Chronic infection with one of these strains is linked to worse disease.116 PA in biofilm is more resistant to antibiotics in a quorum sensing- (QS) dependent way.118

It is not fully understand why CF patients suffer more from PA than from other bacteria. There are several theories to this phenomenon; a) PA are less phagocytosed by cells with defective CFTR119,120 b) adheres better to respira-tory epithelial cells from CF patients121,122 and c) PA has the ability to adapt to the condition in the CF lung environment with low oxygen levels, which favors initiation of growth in biofilm.102 Biofilms are communities of bacte-ria enmeshed in an extracellular matrix consisting of secreted proteins, poly-saccharides, nucleic acids and cellular debris, attached to abiotic or biotic surfaces.123 Conversion to growth in biofilm and to mucoidy has been recog-nized as mechanisms for PA to cope with the CF lung. Mucoid PA produces alginate and the expression of alginate is upregulated under anaerobic condi-tions.124 PA growing in biofilm occurs after infection has been established and once biofilm is formed many other virulence factors are repressed102 (Fig. 4). For example, the alternative sigma factor which induces expression of alginate represses expression of fliC,125 the gene coding for flagellin, which is the main component of the flagellum.126 PA exposed to saliva from CF patients decreased the expression of flagellin of PA.127 Biofilm formation is thus an approach for the bacteria to evade the immune system, i.e. a way to persist after invasion of the host and also to resist the effect of antibiotics. It has been implied that bacteria in biofilm can be 1000 times more resistant to antibiotics than planktonic bacteria.123 Resistance can partly be explained by the fact that bacteria in biofilm are in stationary phase and antibiotics mostly target cell proliferation. In addition biofilm may prevent penetration of antibiotics.101

QS regulates biofilm formation, which is a way of cell-to-cell communi-cation dependent on cell density.101 Bjarnsholt et al., showed that QS defi-cient mutants are more susceptible to tobramycin and polymorphonuclear leukocytes (PMNs) killing, while QS expressing PA growing in biofilm paralyzed PMNs.118 PMNs release toxic oxygen radicals and PA exposed for these radicals adapts by producing alginate.128 QS regulates many other viru-lence factors besides biofilm formation and thus, is important for the patho-genesis. Therefore, ways to prevent this mechanism has attracted focus as possible treatment of PA infections.101,123

24

Figure 4. An important virulence factor for Pseudomonas aeruginosa is the flagel-lum, which confers twitching motility. After initiation of an infection in the airways of CF patients, P. aeruginosa usually stops expressing the flagellum to escape the immune system. To adapt and persist P. aeruginosa grows in biofilm and becomes mucoid due to alginate overexpression.

During chronic colonization of CF lungs with PA the bacteria goes through many genetic changes to adapt to the environment in the lungs.129 LasR is a PA transcription factor regulating many factors including QS, which often becomes inactivated in chronically infected CF patients. Inactivation of lasR confers growth advantage under certain conditions and increases ß-lactamase activity.130 It was recently found that CF patients colonized with PA mutated in lasR, had a worse disease than those with functional LasR. The decline in lung function was similar to what is seen with mucoid PA, but appeared earlier than mucoidy. LasR mutations could therefore be an early marker for progression of PA lung disease.131

Treatment and survival

All respiratory infections – acute, intermittent and exacerbations of chronic infections - are treated with high doses of antibiotics. CF patients are also given vitamin supplements, pancreatic enzymes and a high caloric diet, often with added polyunsaturated fatty acids, which has improved nutritional status. Physiotherapy is also an important part in CF treatment.

When the CFTR gene was discovered large hope was set to find a way to cure the basic defect of CF. However, gene therapy has so far been unsuc-cessful.132 In CF research today, there is a large focus on new therapies to cure downstream defects of CFTR dysfunction, e.g. activation of an inactive

Non-mucoid

Flagellated

Mucoid

Non-�agellated

Bio�lm

Flagellum Alginate

25

CFTR. Another approach is attempts to activate other chloride channels than CFTR to correct the salt balance and compensate for defective CFTR.83,87

Thanks to improved care and treatment strategies the survival of CF pa-tients has increased extensively during the last decades. In Sweden the me-dian life expectancy has increased from 7 � years in 1968133 to over 40 years in 2008 (personal information from the Swedish association for cystic fibro-sis). Still, CF patients die prematurely showing that the disease is life threat-ening and the need for new and further improved treatments.

Anti-Pseudomonas IgY as prophylaxis to CF patients

We have studied the prophylactic effect of oral treatment with Anti-Pseudomonas IgY against PA infections in CF patients for more than four-teen years. The exact mechanism of action for orally given Anti-Pseudomonas IgY is not known. The hypothesis is that IgY forms a barrier in the oropharynx. This barrier of antibodies prevents PA, which enters the lungs via the nose and oropharynx, from binding to the mucosal epithelial surface. Thus, oromucosal treatment with Anti-Pseudomonas IgY neutralizes any PA present in the oral cavity and nasopharyngeal tract, thereby prevent-ing invasion of the bacteria into the lower respiratory tract. PA is believed to enter the lungs primarily at sleep during the night. Therefore, Anti-Pseudomonas IgY should be taken in the evening. CF patients may be colo-nized with PA in their sinuses without colonization of the lungs, which could reflect a state before the bacteria enters the lower respiratory tract. Our group has shown that there are active concentrations of Anti-Pseudomonas IgY in the saliva and in the oropharyngeal mucosa in the morning when gargling with IgY after teeth-brushing in the evening.134

Adhesion is an important virulence mechanism and in fact the adhesion of PA to nasal epithelial cells of CF patients is stronger than to nasal epithelium of healthy persons in vitro.121,122 In the lungs however, PA is rather trapped within the mucus than attached to the respiratory epithelium.124 Anti-Pseudomonas IgY prevents adhesion of PA to dermal epithelial cells in vi-tro,135 but does not affect growth of the bacteria (unpublished data). Passive immunization with IgY could have an effect on multiple virulence mecha-nisms since the treatment contains polyclonal antibodies.

In a first feasibility study of prophylactic treatment with Anti-Pseudomonas IgY to CF patients there were only 2.4 PA positive cul-tures/100 treatment months in the IgY treated group compared to 13.7 PA positive cultures/100 treatment months in the control group.63 There were no adverse events. Due to these promising results the Swedish Medical Products Agency (MPA) has granted treatment on special named patient basis since 2003 and the Swedish government reimburses the cost of treatment.

The intention is that Anti-Pseudomonas IgY should be used as an “add-on” treatment to antibiotics, reducing the needed amount of antibiotics.

26

Acute or intermittent infections with PA have to be cured with antibiotics and daily treatment with Anti-Pseudomonas IgY should follow.

Proteomics Proteomics are studies of a proteome at a given time point. A proteome is all proteins of a cell, an organ or an organism. Proteomics enables large-scale studies of proteins and thousands of proteins can be studied at the same time. Moreover, only a low amount of sample is required thanks to the high sensi-tivity of proteomic approaches. Proteomics can both be used to define the properties of a known protein or for identification of unknown proteins. Comparative expression analysis can be used to find biomarkers expressed during disease or due to drug treatment. Other applications are proteome profiling, identification of posttranslational modifications and protein-protein interactions.136,137

Two-dimensional gel electrophoresis (2DGE) was the first proteomic technology and it requires good protein purification to avoid interference. The first step is isoelectric focusing whereby proteins are separated accord-ing to their isoelectric point. In the second dimension, proteins are separated according to size by SDS-PAGE.136 Advantages of 2DGE are the high reso-lution and separation. Thousands of proteins can be separated in one gel. Limitations are relative low throughput and reduced sensitivity due to sev-eral processing steps with associated loss of material.137 In addition a higher protein concentration is required for visualization on the gel than for detec-tion with mass spectrometry (MS). The proteins of interest in a 2D gel are excised from the gel, enzymatically in-gel digested into peptides and thereaf-ter analyzed by MS. 136

An alternative to gel electrophoresis is based on liquid chromatography (LC). The whole protein sample is digested and thereafter the sample is frac-tionated with LC before MS analysis. Often reversed phase (RP) LC is ap-plied, which has a relatively high resolution and is fully compatible with MS. If the sample is too complex it can be necessary to pre-fractionate the sample before digestion or to separate the sample in more than one LC-dimension in order to get a higher resolution. In this thesis nanoflow LC (nanoLC) was performed, which has an increased sensitivity due to down scaling of flow rates and columns resulting in a higher analyte concentration hitting the MS-detector as well as enhanced ion desorption.137

Mass spectrometers consist of three basic components: an ion source, a mass analyzer and an ion detector.138 For matrix-assisted laser desorption ionization (MALDI) the analyte is mixed with a matrix and added onto a MALDI target. A pulsed laser radiation is applied and thereby the matrix absorbs the radiation leading to desorption and ionization of the analyte. An electric field is applied and the ions travel dependent on their m/z ratio in a

27

flight tube. The time it takes until the ion reaches the MS-detector, time-of-flight (TOF) is measured and a mass spectrum is generated.137,138

The obtained peptide mass spectra can be used for peptide mass finger-printing for protein identification. This approach is based on matching of detected mass peaks with theoretical peptide masses in protein databases.139 Protein quantification can be performed with MALDI-TOF MS by isotope-tagging.140

Antibody based protein micro-arrays are good for high-throughput appli-cations and requires less amount of sample than MS. Protein arrays enables detection of expression of a protein circuit instead of just looking at a single biomarker.141 IgY could be used as antibodies in these arrays.

28

Aims

Antibiotic resistance is a growing concern worldwide and there is an increas-ing need to reduce the use of antibiotics to prevent the development of resis-tance. The overall aim was to study the potential of IgY for oral immuno-therapy with focus on Anti-Pseudomonas IgY as an alternative to antibiotics to prevent or cure infections. More specific aims were: Paper 1 IgY in aqueous solutions must be stored frozen to remain intact. The aims of this study were to investigate the activity of IgY after freeze-drying, if it is maintained over time after storage at 37°C and if it is better maintained in the presence of a sugar as a stabilizing agent. The aim was further to investi-gate this possibility as a way to simplify storage and transport requirements of antibodies for diagnostic and therapeutic applications.

Paper II Characterization of IgY preparations purified with a water dilution method regarding protein content and identification of other proteins than IgY pre-sent in the antibody preparations. Other aims were to investigate the repro-ducibility of the water dilution method and determine triglyceride and cho-lesterol levels. Paper III Identify Pseudomonas aeruginosa recognized by Anti-Pseudomonas IgY to further characterize the prophylactic effect of these antibodies and gain in-creased understanding about the immunological mechanisms behind this immunotherapy. Paper IV Evaluate the effect of prophylactic treatment with Anti-Pseudomonas IgY to CF patients. Primarily, study if the treatment prolongs the time between P. aeruginosa recolonization over time and if the effect earlier seen was sus-tained over a long time and with a new antibody preparation including anti-bodies against four additional strains. Secondarily, study if secondary end-points such as lung function, nutritional status and colonization of the lung with other bacteria or fungi and use of antibiotics are affected compared to a control group.

29

Materials and Methods

Immunization White leghorn hens were immunized intramuscularly in the breast muscle with the emulsified antigen (formalin killed P. aeruginosa bacterial strains). After the initial immunization (immunogen with Freund’s incomplete adju-vant), the animals received booster injections with four-week intervals (im-munogen with Freund’s incomplete adjuvant), and thereafter with two months interval. Eggs were collected continuously after the second booster injection at eight weeks.

Antibody extraction The antibodies were purified by the water dilution method earlier described.33,36 Briefly, the egg yolk was separated from the egg white. One part of egg yolk was mixed with nine parts of water and allowed to settle for at least 6 h at 4ºC. (Egg yolks were pooled together to avoid different yield of specific IgY). Then the lipids were removed as sediment, and left was the supernatant, containing the IgY and other water-soluble organic matters. The supernatant was filtered through a 22-�m-pore filter. The IgY containing supernatant is stored at -20ºC. For research purposes the IgY preparations were sometimes concentrated by PEG or ammonium sulphate precipitation.

Freeze-drying and forced stability test (paper I) IgY preparations were frozen in the presence or absence of a sugar. Lactose, sucrose or threalose was added at the concentrations 0, 0.012, 0.06 and 0.3 M. Frozen samples were freeze-dried in a HETOTRAP CT-60 (Heto Lab equipments A/S Birkerod, Denmark) for around 24 h. Freeze-dried IgY was then incubated at 37ºC (forced stability test) for 0, 1, 2, 4 and 8 weeks or stored in room temperature (RT) for 3 months. After freeze-drying and sub-sequent incubation, samples were reconstituted in MQ-H2O to the original volume and stored frozen until further analysis to reduce the assay-to-assay variations. The antibody activity was measured with ELISA and compared to samples frozen prior to freeze-drying.

30

IgY-activity ELISA (paper I) ELISA was used to measure the IgY activity after freeze-drying. 96-well microtitre plates (F96 Polysorp, Nunc, Roskilde, Denmark) were coated with antigen (Pseudomonas aeruginosa) diluted 1:1000 in 0.1 M NaHCO3, pH 9.5. Thereafter, unbound sites were blocked with 125 �l/well of 3 mg bovine serum albumin (BSA)/ml in 0.1 M NaHCO3, pH 9.5. Then freeze-dried Anti-Pseudomonas IgY dissolved in MQ-H2O and diluted in phosphate buffered saline (PBS) containing 0.05% Tween 20 (PBS-T) was added to the wells in different concentrations. Each sample was added in duplicates. This was followed by addition of HRP-conjugated rabbit anti-chicken IgY (Zymed, San Francisco, CA, USA) diluted 1/2000 in PBS-T. Finally the wells were incubated with TMB substrate (Zymed) for ten minutes. The reaction was stopped with addition of H2SO4 (50 �l/well). From all solutions 100 �l was added per well if nothing else is noted. Between each step the plates were washed three times with 250 �l PBS-T/well. The absorbance was read at 450 nm in a microplate reader (SpectraMax, Molecular Devices, Sunnyvale, CA, USA). For analysis of freeze-dried IgY the activity of samples frozen immedi-ately after freeze-drying was set to 100% and compared to the other samples.

Gel electrophoresis and immunoblotting (paper II & III) Invitrogen’s (Carlsbad, CA, USA) system was used for 1D SDS-PAGE analysis according to their instructions. For 2DGE Bio-Rad’s (Hercules, CA, USA) system and reagents were used. Bacterial proteins were purified with ReadyPrep Sequential Kit, while proteins in IgY preparations were acetone precipitated. The final sample was dissolved in ReadyPrep 2-D starter kit rehydration/sample buffer. Thereafter, iso-electric focusing strips with dif-ferent pH ranges were passively rehydrated with the proteins over night at RT. The following day the first dimension, iso-electric focusing, was per-formed for a total of 40 kVh. Next, the strip was equilibrated and attached to 8–16% SDS gels (Tris-HCl, 193 x 183 x 1 mm3) (Protean II Ready Gel). The second dimension was performed for about 5h (16 mA/gel for 30 min and thereafter the current was increased to 24 mA/gel). The gels were either stained with Colloidal Blue Kit (Invitrogen) to visualize protein spots or immunoblotting was performed with IgY as primary antibody and alkaline phosphatase labeled rabbit anti-chicken IgY (Zymed) as secondary antibody. Unspecific IgY served as negative control. The blots were developed with NBT/BCIP substrate (Roche Diagnostics, Mannheim, Germany) until spots appeared, rinsed in water, and air-dried in the dark.

31

NanoLC-MALDI-TOF/TOF MS (paper II) For shotgun proteomics immediate protein digestion was performed. An aliquot of antibody solution was dried, reduced and alkylated before diges-tion. After the digestion procedure the sample was desalted on ZipTip col-umn. Nano-RP-HPLC was performed with an 1100 nanoLC-System (Agilent Technologies, Waldbronn, Germany), equipped with a fraction collector capable for direct fractionation onto a MALDI Target plate. A volume of 8 �L, corresponding to approx. 400 ng digestion products, was injected into a 10 �L sample loop. A 15 cm x 180 �m, C18 column (Thermo Electron, Waltham, MA, USA) with 5 �m particle size and an H2O:ACN:TFA solvent system (H2O, 0.1% TFA [A] ; ACN, 0.1% TFA [B]) for separating the en-zymatic cleavage products was used. A flow rate of 2 �l/min starting with isocratic elution at 2% B for 20 min, then gradient elution from 2% to 8% B in 5 min, then from 8% to 32% B within 86 min, then from 32% to 40% B in 5 min and finally from 40% to 80% B in 1 min was applied. The peptide elution was followed by on-line fractionation onto a MALDI target with a collection rate of 4 fractions a minute for 96 minutes within the elution-period from 20 min (2% B) and 116 min (40% B) resulting in 384 fractions. For optimal MS results disposable pre-spotted anchorchip targets (PAC-targets, Bruker Daltonics, Bremen, Germany) were chosen. Mass data were acquired with an Ultraflex II MALDI-TOF/TOF (Bruker Daltonics, Bremen, Germany) in reflector mode. The acquisition was assisted by applying the WarpLC Software (Bruker Daltonics) for automatic TOF-MS spectra acquisi-tion followed by optimized precursor selection for subsequent MS/MS ex-periments. For final protein identification all collected MS/MS data were run in a combined Mascot database search in SwissProt and Mascot database.

Cholesterol and triglyceride (paper II) The concentrations of cholesterol and triglycerides of IgY batches were de-termined by cholesterol (reagent: 7D62-20) and triglyceride (reagent: 7D74-20) measurements performed on an Architect Ci8200 analyzer (Abbott Laboratories, Abbott Park, IL, USA) according to the recommendations of the manufacturer and reported using S.I. units.

MALDI-TOF MS (paper II & III) In paper II proteins were excised from the gels and then trypsinated and identified by MALDI-TOF MS analysis at the WCN Expression Proteomics Facility (Department of Medical Biochemistry and Microbiology, Uppsala University).142 In paper III, colloidal blue stained 2D gels and immunoblots

32

were visually matched. Spots on gels corresponding to spots on immunoblots were excised and an in-gel tryptic digestion was performed. MALDI-TOF MS experiments were performed in an Autoflex (Bruker Daltonics, Bremen, Germany) of reflector type time-of-flight mass spectrometer, equipped with a pulsed nitrogen laser working at 337 nm. Before analyses, the instrument was externally calibrated with Bruker standard peptide mixture. One �l tryp-tic digest was mixed with 1 �l matrix, �-cyano-4-hydroxy-trans-cinnamic acid (Agilent Technologies, Palo Alto, CA, USA). Next, 1 �l of the mixture was applied onto the MALDI sample plate (Bruker Scout 384/400 Anchor-Chip) and allowed to air dry (dried-droplet method) before being placed in the mass spectrometer. For identification of proteins, peptide masses as well as their partial amino acid sequence in given mass-spectra were matched to proteins in ProFound sequence database:

http://prowl.rockefeller.edu/profound_bin/WebProFound.exe

Peptide sequencing (paper III) To confirm the identity of the proteins, peptide fragments were sequenced by MALDI-TOF MS working in the post source decay (PSD) mode. In order to improve fragmentation of the peptides as well as to simplify interpretation of the PSD-spectra, the tryptic peptides were modified at the N-terminal with chemically-assisted fragmentation (CAF) using Ettan CAF–MALDI Se-quencing Kit (GE Healthcare Amersham Biosciences AB, Uppsala, Swe-den).143 In some cases the lysine containing peptides were also modified at lysine residues at the C-terminal with Lys-tag (2-methoxy-4,5-dihydro-1H-imidazole) (QMX laboratories Ltd., Thaxed Essex UK). 144

Study subjects (paper IV) Seventeen CF patients received IgY treatment for up to twelve years. The inclusion criteria were that the patient had had one or more PA infections that had been cured, but were not chronically infected and that they were not egg allergic. The rationale to use patients, who have had a first PA infection, was to be able to evaluate the study within reasonable time. Anti-Pseudomonas IgY can also be used for primary prevention to delay the onset of PA infections in CF patients, but the time to the first infection might be long and hard to predict. The patients were instructed to gargle IgY every night for two minutes after teeth brushing and then to swallow the solution. They were also instructed to keep a regular diary with annotations of IgY intake and during the last three years to note sick days and intake of antibiot-ics. All other treatments besides IgY continued as usual and were modified

33

on demand for optimal treatment. During the study several patients had con-tinuous use of Azitromycin introduced in their treatment.

Colonization of the lungs with PA or other bacteria was monitored about four times per year by sputum cultures. Infections with PA were also checked by measuring precipitins against PA once a year.145 Chronic infection was defined as at least three PA positive cultures within six months and/or abnor-mal serology. Medical condition was examined four times yearly, including spirometry of forced expiratory volume in one second (FEV1) to follow lung function. Body mass index (BMI) was determined at the end of the study and recalculated as ISO-BMI for children to regulate for age.

The Swedish MPA and the Ethics Committee at Uppsala University Hos-pital approved the study.

Control group (paper IV) The control group consisted of 23 CF patients, who were treated in Aarhus, Denmark. Since the prevalence of CF is low in Sweden, there were not enough patients in Sweden to find a control group within the country. The best group for comparison was found in Aarhus because treatment strategies are similar to Sweden. However, Azitromycin was not introduced in this group. Results of the control group were recorded retrospectively by the same parameters as the IgY treated patients.

Anti-Pseudomonas IgY treatment (paper IV) Hens were immunized as described above. In the first part of the study the patients were treated with IgY against two PA strains (O1 and O5) and in the second part (November 2003 to December 2006) antibodies against four additional strains were added (O3, O6, O9 an O11), thus the formula con-tained IgY against six strains in total. The antibody preparation is purified with the water dilution method (see above), manufactured by Fresenius-Kabi AB (Brunna, Sweden) and approved according to good manufacturing prac-tice (GMP) by the Swedish MPA. Each dose consists of approximately 50 mg IgY in a total volume of 70 ml, corresponding to half an egg. The activ-ity of IgY is controlled with ELISA.

34

Statistics (paper IV) Kaplan-Meier and log-Rank test were used for analysis of time to event. For comparison of yearly change of pulmonary status linear regression was cho-sen. Mann-Whitney test was used to compare the number of positive cul-tures/100 treatment months and paired t-test for comparison of FEV1 at start and end of the study in the IgY treated group. The children’s BMI was trans-formed to ISO-BMI, which means the prognostic BMI-value at the age of 18 years given that the child follows the general development in the population. P-values <0.05 were regarded as statistically significant throughout the study.

35

Results and Discussion

Paper I. Stability of chicken antibodies freeze-dried in the presence of lactose, sucrose and threalose Freeze-drying is a well-known method to preserve proteins and the tech-nique has frequently been used for pharmaceutical applications, but the process itself might cause degradation. This can be prevented by addition of a stabilizing agent. Here, sugar was added since it is known to protect pro-teins during and after the freeze-drying process146,147 and it is tolerable for oral use. Lyophilized IgY has been used in studies of IgY treatment,48,59 but the activity over time had not been investigated. The IgY activity immedi-ately after freeze-drying was the same as before, but decreased slowly during eight weeks at 37ºC in the absence of a stabilizing agent. Addition of disac-charides resulted in preserved activity after eight weeks in 37ºC. The varia-tions between disaccharides (sucrose (Fig. 5), lactose or threalose) and con-centrations (0.012–0.3 M) used were small. Lactose was, however, slightly less effective to preserve IgY and may be less suited for oral use due to re-duced lactose intolerance, which frequently occurs. Sucrose is less expensive then threalose and thus, the firsthand choice as a stabilizing agent. It was difficult to reconstitute samples with the highest concentration tested, 0.3 M and because the effect was not clearly better than with a lower concentration it is recommended to use the lower sugar concentration. We therefore con-clude that freeze-drying of IgY in the presence of a low concentration of sucrose is preferable.

One week storage at 37ºC is comparable to one year at 4ºC or eight weeks at RT.148 This means that IgY freeze-dried with a sugar in this study is very stable for more than a year at RT and for at least three months in the absence of a sugar.

Today, patients on Anti-Pseudomonas IgY store their IgY in the freezer until the day of use and therefore the possibility to store the antibodies in RT, and in a smaller volume, would simplify for the users. At time of use they would simply just reconstitute an IgY powder in water. It also opens the possibility to make a lozenge rather than a gargling-solution. In addition, the possibility to keep IgY in RT makes transport and storage of IgY easier and cheaper.

36

Figure 5. The activity of freeze-dried IgY in the presence of sucrose was well pre-served, independent of concentration (0.012–0.3M), after storage at 37°C for up to eight weeks or at RT for 3 months. Activity is expressed relative to the activity im-mediately after freeze-drying. The activity before freeze-drying served as control.

Paper II. Proteomic characterization of IgY preparations purified with a water dilution method The water dilution method has been shown earlier to be effective in com-parison with other methods for IgY purification.36 The content of IgY prepa-rations purified with the water dilution method was determined in paper II. The protein pattern on colloidal blue stained 1D and 2D gels were very simi-lar between batches. The protein concentration was also very similar, around 1.8 mg protein/ml (coefficient of variation 7%). This shows a high repro-ducibility of the water dilution method.

When proteins in the IgY preparations were separated with 2DGE many spots appeared on the gel, but streaking disturbed the picture (Fig. 6). There is a large dynamic range of protein abundances in the antibody preparation, which in part can explain the problem with streaking. It can also be a result of lipid and salt contamination in the sample. Five proteins could be identi-fied by MS analyses of excised and trypsinated gelspots (Fig. 6), but identi-fication was hampered due to the huge dynamic range in protein content. Therefore, we applied another proteomic bottom-up approach based on nanoLC MALDI TOF/TOF MS. In this technique the whole sample is di-

0%

20%

40%

60%

80%

100%

120%

140%

Control Freeze-

dried

1 week 2 weeks 4 weeks 8 weeks 3

months

Rela

tive I

gY

acti

vit

y

Stability of IgY freeze-dried with sucrose

0.012 M

0.06 M

0.3 M

37

gested before separation compared to 2DGE where proteins are digested after separation. In total, 21 additional proteins were identified (Table 1). Combined with the results from 2DGE and MS analysis totally 26 proteins were identified in this study. Mann and Mann could identify 100 proteins in the egg-yolk water-soluble fraction, including most of the proteins identified in this work.149 They adjusted the pH to 5, which increases the protein yield, which partly explains why more proteins were detected than in this work. Sedimentation at pH 5 requires centrifugation, which is time-consuming and impractical when large quantities are purified. Therefore, we did not adjust the pH, which means a pH around 6, since that enables decanting of the su-pernatant.

Figure 6. 2D gel of proteins in an IgY preparation extracted with the water dilution method. Due to high dynamic range of the proteins it was hard to find an optimal concentration. Thus, some proteins are overrepresented, causing streaking. Five proteins were identified from the gel: PIT-54 (1): hemopexin (2); Ig � chain (3); similar to Niemann-Pick disease (type 2) ( 4); and transthyretin (chain A) (5).

Intact IgY could not be detected with MS analysis since the sample was re-duced before analysis, which leads to breaking of IgY into heavy and light chains.4 However, Ig � chain regions were identified with nanoLC-MALDI TOF/TOF MS and Ig � chain as a spot on 2D gels. Interestingly, a number of the proteins identified have been found to have antimicrobial and immu-nostimulatory effects,35 which implies an additive effect in IgY treatment

pH 4 pH 7

1 2

3

4

5

38

besides the primary effect of the antibodies. Moreover, one of the identified proteins, chicken ovomucoid, protects to some extent against proteolytic degradation by digestive enzymes and thus,150 might protect degradation of IgY given as oral treatment against gastrointestinal infections.

Table 1. Proteins identified with nanoLC MALDI-TOF/TOF MS

No. Protein name Database-entrya

Mascot

scoreb MWc

No. of

Peptidesd

1 Serum albumin ALBU_CHICK 2034 71867.69 49

2 Ovotransferrin TRFE_CHICK 1117 79551.09 20

3 Ovalbumin OVAL_CHICK 1061 43195.63 17

4 Vitellogenin-1 VIT1_CHICK 783 212608.02 13

5 Vitellogenin-2 VIT2_CHICK 769 206731.51 16

6 Ovalbumin related protein Y OVALY_CHICK 415 44028.85 10

7 Apolipoprotein B (fragment) APOB_CHICK 362 50872.65 9

8 Ovoinhibitor IOV7_CHICK 293 54394.10 6

9 Apovitellenin-1 APOV1_CHICK 259 12015.55 9

10 Ovomucoid IOVO_CHICK 180 23659.68 2

11 Riboflavin-binding protein RBP_CHICK 130 28276.62 3

12 Apolipoprotein A-I APOA1_CHICK 122 30640.25 4

13 Ig lambda chain C region LAC_CHICK 114 11524.60 2

14 Ovalbumin-related protein X OVALX_CHICK 79 26331.26 2

15 Fibrinogen alpha chain FIBA_CHICK 62 83242.38 1

16 Coagulation factor IX FA9_CHICK 77 53082.68 1

17 Complement factor B-like

protease

CFBL_CHICK 77 27719.08 1

18 Ig lambda chain V-1 region LV1_CHICK 41 11801.72 1

19 Vitelline membrane outer

layer protein 1

VMO1_CHICK 32 20677.28 1

20 Filensin BFSP1_CHICK 30 76457.74 1

21 Cystatin CYT_CHICK 29 15561.90 1

a Uniprot knowledgebase entry. b Mascot protein score revealed by MudPIT scoring. Proteins were found and identified by integrated mascot database batch search of all MS/MS in Swissprot v 51.6. All matches are identified significantly. Identified proteins are considered as positive match on at least a 95 % significance level (p < 0.05) corresponding to a significance threshold ionscore of 29. c Molecular weight in Da. d Number of tryptically peptides that match the identified protein. At least one matching pep-tide for each identified protein must fulfil criteria of significance (p < 0.01) and uniqueness.

39

In the 16 batches investigated none contained >0.6 mM of cholesterol, and all but two contained <0.5 mM. Trigyceride levels were in the range of 0.43–2.68 mM (mean= 1.39 mM ± 0.68). The risk of elevated cholesterol levels due to daily intake of IgY could be rejected since the levels found were well below normal human serum levels. Oral intake of IgY is comparable to eat-ing eggs and others have shown that an egg rich diet does not increase the risk for elevated blood pressure or cardiac disease.151,152 In fact, some even states that egg eating can decrease serum cholesterol levels.153

Paper III. Pseudomonas aeruginosa infections are prevented in cystic fibrosis patients by avian antibodies binding Pseudomonas aeruginosa flagellin Earlier studies of Anti-Pseudomonas IgY had shown that the antibodies bind PA, but the antigens were not known. In this work PA proteins recognized by Anti-Pseudomonas IgY were identified by 2DGE, immunoblots and MALDI-TOF MS. Protein patterns of the individual PA strains were very similar both in gel-to-gel reproducibility and between strains on colloidal-blue stained 1D and 2D gels. Although colloidal blue stained 2D gels were similar (Fig. 7A and B), immunoblots could be divided into two groups, group A and B, according to pattern (Fig. 7C and D). No spots appeared when unspecific IgY was tested as negative control.

Antigenic proteins were identified with MALDI-TOF MS analysis, which generated excellent spectra and most spots matched well with proteins in the database. A number of protein spots were all identified as flagellin. PA has two types of flagellin, a- and b-type 154 and the proteins in group A were identified as flagellin type a and in group B as type b. Thus, IgY has affinity to both types of flagellin. The heterogeneity resulting in several spots is due to variation in size and amino acid sequence154 and probably also post-translational modifications.155-157

To confirm the identity of the proteins, the tryptic peptides were analyzed by MALDI-TOF MS in PSD mode to accurately measure their mono-isotopic masses as well as determine their amino acid sequences. The obtained PSD spectra were not of optimal quality due to low analyte concentration and thus low peak resolution and poor mass accuracy. However, the partial amino acid sequence information was useful for confirmation of the results, but not for de novo sequencing. After our results had been published Stephen Lory at Harvard University confirmed our results in a protein chip assay (personal communication).

The fact that flagellin was identified as the major antigen is of great inter-est since flagellin is very antigenic and elicits a strong inflammatory re-sponse via toll-like receptor 5.158 Moreover, it is the main component of the

40

PA flagellum, which is required for motility, adherence and chemotaxis, and is also involved in the invasion and the establishment of infection in the host.126 Mutant strains lacking the gene coding for flagellin, fliC, are less capable of inducing infection and mortality,159 and monoclonal antibodies against flagellin reduce mortality and morbidity in murine models.160,161 Therefore, flagella-based vaccines, used as an early treatment in CF, have been investigated.110 It is also important to note that Anti-Pseudomonas IgY bound all PA strains tested even those that had not been used for immuniza-tion, which strengthens its prophylactic effect.

Figure 7. Colloidal blues stained 2D SDS-PAGE gels of Pseudomonas aeruginosa proteins (A and B) and corresponding immunoblots (C and D) incubated with Anti-Pseudomonas IgY. Immunogenic proteins (circled) were identified with MALDI-TOF MS and database searhes as flagellin types a and b.

Paper IV. Good effect of IgY against Pseudomonas aeruginosa infections in cystic fibrosis patients A group of 17 Swedish CF patients were treated with Anti-Pseudomonas IgY for up to twelve years to prevent Pseudomonas aeruginosa lung infec-tions. They were compared with a Danish control group. The main endpoint was the status of PA infection, intermittent or chronic. The most important results are shown in Table 2. The patients treated with IgY were recolonized

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41

less often than the patients in the control group (p=0.028). Patients on IgY had 2.3 positive cultures/100 treatment months compared to 7.0 positive cultures/100 treatment months in the control group. The number of positive cultures is based on sputum samples taken before chronic colonization. There was a significantly longer time from inclusion in the study until the first recolonization with PA in the IgY treated group (p=0.012) (Fig. 8). It has been shown that patients chronically colonized with PA had a shorter time between first colonization ever to the second positive than those not chronically colonized.104

Figure 8. Kaplan-Meier plot of time from inclusion in the study to the first recoloniza-tion with P. aeruginosa in the IgY treated group (solid line) vs. the control group (bro-ken line). The difference between the groups is significant (p=0.012; log-rank test).

Only two siblings (2/17) became chronically colonized with PA (but their PA have continuously been non-mucoid over the years) in the IgY treated group vs. 7/23 in the control group. Due to few events the difference is insignificant but it seems likely that IgY treatment postpones the onset of a chronic infec-

42

tion. These results are comparable to early aggressive antibiotic therapy, which is consensus in many countries.100 Given that IgY prolongs the time between colonization the need for antibiotics is reduced as well as the risk for selection of antibiotic resistance or other side effects of antibiotics.

Table 1. Main results of Anti-Pseudomonas IgY treatment

IgY treated patients

Control group

No. of PA pos cultures/100 treatment months

2.3 7.0

Median time to first re-colonization

25 months 10 months

No. of patients without re-colonization

4/17 (24%) 1/23 (4%)

No. of patients chronically colonized

2/17 (12%) 7/23 (30%)

New pathogens are emerging among CF patients. This is partly due to the fact that the patients live longer but probably also due to antibiotic treat-ment.99,100 The frequency of these bacteria was however low in the IgY treated group. Two patients were chronically infected with S. maltophilia already at inclusion in the study, but no other patients were or have become chronically infected with another pathogen than PA. Malnutrition and de-cline in lung function are common symptoms among CF patients, especially due to PA infections.94 However, the patients in the IgY group only had a small yearly decline in lung function measured as FEV1 (median: -1.0% predicted), the change within the group was not significant (p=0.730) and most patients had a BMI value within normal limits (median ISO-BMI: 21.3, range 18–25). The good nutritional status probably reflects better clinical status due to fewer infections. The patients in the control group had worse BMI (median 19.7; range <16–27). In addition, other pathogens besides PA were more frequently detected and a number of patients were chronically infected with them.

The patient number is rather low, but the study has been going on for a long time, which strengthens the results. Still, it was difficult to find signifi-cant differences between the treated and the control group and a larger ran-domized study is required to confirm these results. Not least, since CF pa-tients are a heterogeneous group of patients with large variations in symp-toms and degree of disease.

43

Conclusions

• The activity of IgY preparations is maintained for long time when IgY is freeze-dried in the presence of a disaccharide and it was sufficient with the lowest concentration tested, 0.012 M. Sucrose was found to be the firsthand choice of the disaccharides used.

• The possibility of freeze-drying IgY would simplify transport, use and storage of IgY and reduce the cost.

• The water dilution method is highly reproducible, regarding both protein content and concentration.

• IgY preparations purified by the water dilution methods contain proteins with antibacterial and immunostimulatory effects that imply an additive effect in IgY treatment in addition to the primary effect of the antibod-ies.

• The cholesterol and triglyceride levels in IgY solutions are so low that they will not affect the serum levels of patients on IgY treatment.

• The major antigen for Anti-Pseudomonas IgY is P. aeruginosa flagellin, the main component of the flagellum important for host invasion and es-tablishment of infection.

• Anti-Pseudomonas IgY may prevent P. aeruginosa infections by hinder-ing host invasion due to reduced adhesion. Anti-Pseudomonas IgY may directly affect adhesion or indirectly via reduced motility.

• Since flagellin initiates inflammation, IgY may also reduce the inflam-matory response in CF patients.

• Anti-Pseudomonas IgY has affinity for other PA strains than those used for immunization, which strengthens its prophylactic potential.

• CF patients treated with Anti-Pseudomonas IgY got fewer infections than the controls and the time from inclusion to first recolonization was significantly longer. Thus, the need for antibiotic treatment is reduced.

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• Only two siblings in the IgY-treated group (12%) became chronically infected compared to 30% in the control group. Thus, treatment with Anti-Pseudomonas IgY might postpone chronic PA infection in CF pa-tients.

• Other opportunistic gram-negative bacteria have not emerged instead of P. aeruginosa in the IgY-treated group.

• Treated patients had good nutritional status at the end of the study. All had BMI values within or close to normal limits. In addition, their respi-ratory function, measured as FEV1, was not significantly changed over time within the IgY-treated group (p=0.730). This probably reflects the low number of lung infections with PA and other bacteria.

• Treatment with Anti-Pseudomonas IgY is very safe, since there have been no adverse events attributable to Anti-Pseudomonas IgY for any patients in the study.

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General discussion and future plans

In order to maintain potent antibiotics, there is a requirement of using them only when needed, which is achieved by a strict prescription policy and im-proved microbiology techniques for identification of pathogens. In addition there is a need to develop new types of antibiotics and other types of phar-maceuticals. These actions should make it possible to use antibiotics when required, which is extremely important. Otherwise bacterial infections can become life threatening due to lack of treatment options. IgY as a comple-ment or alternative to antibiotics offers a possibility to avoid development of antibiotic resistance. One example is shown in this thesis, i.e. that IgY has great potential as a treatment to prevent Pseudomonas aeruginosa infections in CF patients, which partly can be explained by the fact that the antibodies bind to flagellin. We also found that IgY has cross-reactivity to different PA strains. Taken together these findings strengthen the prophylactic effect of Anti-Pseudomonas IgY, which could be of great benefit for CF patients. Prevention of PA lung infections should prolong the time to chronic coloni-zation and, secondarily, reduce inflammation in the lungs, the main reason for mortality and morbidity in CF due to lung destruction.88

There are extensive applications for IgY, not only in many areas of im-munotherapy, but also for other applications in research and medicine. In paper I we found that the antibody activity of freeze-dried IgY is well pre-served over time after subsequent storage at 37°C or RT. This simplifies the handling and storage of IgY. In paper II we examined the content of IgY preparations purified with a water dilution method and found that the method has high reproducibility. This method is also cheap and easy to perform. The toxicity of IgY is extremely small and in paper II it is shown that the choles-terol levels are very low. Therefore IgY intake should be well tolerated for those not allergic to eggs.

Since the antibody activity of IgY was preserved after freeze-drying we have started to develop lozenges. This would be easier to handle, both for the patients and for the pharmacy and hospital due to the possibility to store the antibodies at RT. A gargle requires that the patient is old enough to be able to gargle. A lozenge to suck could be given much earlier in life and would be more convenient for all patients.

The treatment of CF-patients with Anti-Pseudomonas IgY has continued for 2.5 years after the data in paper IV was compiled. No more patients on IgY have become chronically infected and the number of PA positive cul-

46

tures remains at the same low level. These promising results can only be confirmed with a larger randomized placebo controlled study. In Sweden there are not enough patients, thus such a study must be conducted abroad. Currently there is hard ongoing work to start up such a study. Due to the good results, the European Medicines Agency (EMEA) has granted orphan drug designation to Anti-Pseudomonas IgY, i.e. assistance in setting up a phase three study and reduced costs.

It is also of interest to further determine the mechanism of how IgY pre-vents PA infections in CF patients. Therefore, studies have been initiated to investigate if IgY has an effect on biofilm. Biofilm formation is an important virulence factor as earlier stated, because it is a mechanism by which the bacteria protects themselves from antibiotics and from the immune system.123 The flagellum is important for initiation of biofilm formation.162 Given that IgY binds flagellin speaks for a likely effect also on biofilm for-mation and prevention thereof would be an indication of a clinical effect. Preliminary data from an experiment in a flow cell system shows that IgY can bind to biofilm, thus the biofilm does not protect PA from IgY binding. However, the experiments have to be optimized.

Besides IgY treatment against PA, there are many other applications for IgY. We are currently developing antibodies against other bacterial species. Newborn piglets often suffer from E. coli associated diarrhea and therefore are treated with antibiotics. Anti-E. coli IgY in the feed may prevent these infections48-50 and is under development. Another problematic gastrointesti-nal pathogen is Clostridium difficilie, which often occurs after antibiotic treatment.51 Others have shown a positive effect of IgY against C. difficile in a hamster model.52 We aim to develop IgY against C. difficile for human use. An ongoing project is IgY against Extended Spectrum �-lactamase-(ESBL) producing Klebsiella pneumoniae.

ESBL-producing bacteria, mainly K. pneumoniae and E. coli, is one of the most problematic types of resistance and is present worldwide.78,163 In Uppsala the first Scandinavian outbreak occurred in 2005.164 ESBL-producing bacteria are resistant against �-lactam antibiotics and often these bacteria are resistant against many other types of antibiotics as well.78 Resis-tance often arises due to point mutations in existing �-lactamase genes and is usually plasmid borne. It can be spread to other bacteria of the same or other species.78,163 Asymptomatic gastrointestinal colonization often precedes an infection in another localization, primarily the urinary tract or the lungs, which secondarily can lead to bacteraemia.78 Colonization is not only a risk factor for infection, but also problematic because ESBL-producing bacteria are easily spread to others, both in hospitals and in the community.165,166 It is not known whether colonization of the gastrointestinal tract can be sponta-neously cured and, if so, how long time this takes in general. Patients who seem to have been cured often become recolonized if they receive antibiotic treatment, which indicates that the bacteria were not totally eradicated.

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We have developed IgY against two K. penumoniae strains from the same clone as the one in the outbreak in Uppsala.164 So far, we have found with ELISA, flow cytometry analyses and with immunostaining that these anti-bodies bind specifically to K. pneumoniae. Currently, Klebsiella proteins antigenic for IgY are being identified. We are also investigating if IgY spe-cifically can interfere with growth or prevent biofilm formation. Future plans are to investigate if IgY can prevent adhesion to epithelial cells from the intestines. As mentioned before many studies of IgY against various species prevents adhesion.48,59,65,66

We are also setting up a clinical trial. The plan is to include patients at care centers for elderly, who are colonized with ESBL-producing Klebsiella. They will receive IgY as a drinking solution two times a day together with food. There is a risk for degradation of IgY in the stomach before the anti-bodies enter the gastro intestines, but the food taken together with IgY should be buffering. It is also important to point out that elderly often have a slightly higher pH in their stomach due to reduced acid production. The aim of this study is to investigate if anti-ESBL Klebsiella IgY can eradicate fecal carriage of ESBL-producing K. pneumoniae and thereby prevent spread and eventual infections.

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Svensk sammanfattning

Bakgrund Tack vare antibiotika har många liv kunnat räddas. Tyvärr finns det också en baksida, nämligen att en del bakterier utvecklar resistens mot antibiotika. Det är ett stort och allvarligt problem över hela världen och beror till stor del på över- och felanvändning av läkemedlen. Det är viktigt att antibiotika kan användas när det verkligen behövs, exempelvis vid bakterieinfektioner, men också som förebyggande behandling vid situationer då kroppen blir mer mottaglig för en infektion, såsom vid en operation eller cellgiftsbehanding. För att bakterier inte ska bli motståndskraftiga är det därför viktigt att mins-ka användningen av antibiotika. I denna avhandling har äggantikroppen IgY studerats som ett alternativ och komplement till antibiotika, för att förhindra och bota bakterieinfektioner.

Antikroppar är en viktig del av immunförsvaret. De känner igen och bin-der till sådant som normalt inte ska finnas i kroppen, så att det kan förstöras av immunförsvaret. Nya antikroppar bildas när immunsystemet stöter på främmande ämnen, t.ex. en bakterie eller ett virus. Hönan bildar IgY-antikroppar som överförs till ägget för att skydda den nyfödda kycklingen innan den byggt upp ett eget immunförsvar. För att erhålla önskade an-tikroppar kan hönan vaccineras med t.ex. Pseudomonas aeruginosa. Hönan bildar då IgY-antikroppar mot bakterien som sedan överförs till äggulan, varifrån de kan renas fram. För detta har en vattenutspädningsmetod använts, där inget annat än vatten tillsatts i reningsprocessen. Intaget av IgY kan då jämföras med att äta rå äggula och är i sig ofarligt, så länge man inte är al-lergisk mot ägg. Risken för biverkningar är därför minimal. Den stora förde-len med IgY-behandling är att bakterier inte kan bli resistenta mot IgY.

Analyser av IgY I delarbete I och II undersöktes IgY-antikropparnas egenskaper. Möjligheten att frystorka IgY-lösningar, som ett sätt att konservera och förvara antikrop-parna studerades. Själva frystorkningsprocessen kan dock till viss del förstö-ra antikropparna. Därför undersöktes om tillsatt socker kunde skydda mot nedbrytning eftersom socker fungerar som ett stabiliseringsmedel. De frys-torkade antikropparna band lika bra till bakterier som innan de torkades.

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Genom att förvara frystorkat IgY i 37°C undersöktes stabiliteten över tid. Stabiliteten var bättre i de prov som innehöll tillsatt socker. Idag förvaras IgY-lösningar frysta, men möjligheten att frystorka innebär att de skulle kunns förvaras i vanlig rumstemperatur (20°C). Det skulle underlätta för de patienter som behandlas med IgY, men också förenkla förvaring och trans-port av IgY.

Delarbete II syftade främst till att undersöka vilka andra äggule-proteiner, förutom IgY, som finns i antikroppslösningarna. 26 olika proteiner kunde identifieras. Ett intressant resultat var att flera av proteinerna har en känd antibakteriell effekt. Vilket kan betyda ytterligare en positiv effekt av IgY-behandling. Nivåerna av kolesterol och andra fetter var låga. IgY-behandling bör därför inte påverka nivån av blodfetter.

IgY-behandling av patienter med cystisk fibros Cystisk fibros (CF) är en ärftlig sjukdom som drabbar ca ett av 2500-4500 nyfödda barn. I dagsläget finns det ungefär 600 svenskar med CF. Tack vare förbättrad behandling har livslängden förlängts, men fortfarande dör CF-patienter i förtid. Medellivslängd är över 40 år i Sverige idag, vilket kan jämföras med en medellivslängd på bara 7 � år i slutet av 60-talet. Sjukdo-men orsakas av att en jonkanal, som finns i olika sorters celler, saknas eller är felaktig. Eftersom jonkanalen inte fungerar som den ska, leder detta till att salt- och vätskebalansen i cellen rubbas. Symtom på CF är bl.a. leverpro-blem, problem med bukspottkörteln, infertilitet hos män och lunginfektioner. I lungorna hos en frisk person finns det normalt ett vätskelager ovanpå flimmerhåren, som gör att bakterier och annat som inte ska finnas där kan-transporteras bort. I lungorna hos en CF-patient finns det istället för vätska ett segt slem, som gör att bakterierna lättare fastnar och därför också kan infektera patienten lättare. CF-patienter brukar få lunginfektioner från flera olika bakterier, men den som anses vara den värsta är Pseudomonas aerugi-nosa. De första gångerna kan patienter med pseudomonas-bakterien botas med antibiotika, men efter upprepade infektioner blir många kroniskt infek-terade. Detta innebär att det blir omöjligt att få bort bakterierna från lungor-na. Lunginfektioner med Pseudomonas är den vanligaste orsaken till sjuk-dom och dödlighet bland CF-patienter pga. att lungkapaciteten försämras. Till slut kan det till och med bli nödvändigt med lungtransplantation. Försök har utan framgång gjorts för att vaccinera mot Pseudomonas. Vi har studerat effekten av att behandla CF-patienter med IgY mot Pseudomonas (Anti-Pseudomonas IgY) för att undersöka om detta kan förlänga tiden mellan patienternas pseudomonas-infektioner. Hypotesen var att genom att gurgla med IgY-antikroppar som binder till pseudomonas-bakterier i munhålan, så förhindras bakterierna att vandra ned och bilda infektion i lungorna.

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Sedan tidigare var det visat att Anti-Pseudomonas IgY binder till pseu-domonas-bakterier. För att förstå verkningsmekanismen mer kartlades det mer i detalj i delarbete III var på bakterien som IgY binder. Det visade sig att IgY binder till flagellin, som är den huvudsakliga byggstenen i flagellen. Flagellen kan liknas vid en lång svans som Pseudomonas använder för att röra sig. Flagellen är viktig när Pseudomonas ska infektera, men startar även en inflammatorisk process. I och med att Anti-Pseudomonas IgY binder till flagellin, tyder det på att antikropparna därigenom minskar förmågan för pseudomonas att infektera. IgY biter helt enkelt pseudomonas i svansen!

Resultat från IgY-behandling av CF-patienter sammanfattas i delarbete IV. 17 svenska CF-patienter behandlades med IgY under upp till 12 år. Re-sultatet av behandlingen jämfördes med en kontrollgrupp, bestående av 23 danska CF-patienter som inte fick IgY. I övrigt fick de båda grupperna lik-värdig behandling. Resultaten visade att de patienter som behandlats med IgY fick mer sällan en ny infektion, jämfört med kontrollgruppen. Dessutom blev en mindre andel av patienterna kroniskt infekterade i den IgY-behandlade gruppen. Det var också intressant att IgY-patienterna i mindre utsträckning drabbades av andra bakterieinfektioner, eftersom färre infektio-ner också minskar behovet av antibiotika.

Figure 9. Anti-Pseudomonas IgY binder till flagellin som bygger upp flagellen. Flag-ellen kan liknas vid en svans som Pseudomonas aeruginosa använder för att röra.

Pågående studier Behandlingen av CF-patienter fortsätter och har pågått i ytterligare två år, med fortsatt lovande resultat. Vi arbetar även vidare med att förstå hur IgY kan förhindra lunginfektioner med Pseudomonas aeruginosa. Utöver studier med Anti-Pseudomonas IgY, pågår även arbete med att ta fram IgY mot andra bakterier.

Ett av de stora problemen när det gäller antibiotikaresistens är de bakteri-er som producerar ESBL (extended spectrum ß-lactamases). ESBL är ett enzym som bryter ner en grupp av antibiotika, så kallade ß-laktamer, där bland annat penicillin ingår. Klebsiella pneumoniae är en av de mest före-

51

kommande bakterierna som producerar ESBL. Den kan ge upphov till urin-vägs- och luftvägsinfektioner, för att sedan spridas till blodet, där den orsa-kar blodförgiftning. ESBL-producerande Klebsiella koloniserar ofta tarmsy-stemet. Det betyder att den ”lever i tarmen”, men utan att ge några symptom. Smittan kan sedan spridas från bäraren till andra personer eller orsaka infek-tion hos personen själv. I Uppsala orsakade dessa bakterier ett utbrott 2005, då många insjuknade. Nu har man fått bukt på själva utbrottet, men det finns fortfarande många bärare av bakterierna kvar. Man vet inte om bärarskapet någonsin försvinner. Ofta kommer bakterien tillbaka om patienten får antibi-otika mot någon annan sjukdom. Detta tyder på att när andra bakterier slås ut av antibiotika kan motståndskraftiga Klebsiella ta över. Vi blev därför intres-serade av att undersöka om IgY mot ESBL-producerande klebsiella kan för-hindra och/eller bota bärarskap i tarmen. Hittills har försök med olika meto-der visat att anti-ESBL Klebsiella IgY binder Klebsiella bättre än kontroll IgY, dvs. IgY från höns som inte vaccinerats med Klebsiella. Vilket visar att antikropparna har en specifik aktivitet.

Med anledning av dessa fynd ska vi gå vidare med att undersöka om IgY kan förhindra att klebsiella binder till tarmceller. Detta skulle i så fall vara ett tecken på att IgY kan stoppa bakterierna från att bosätta sig i tarmen, och att de istället kommer ut den naturliga vägen. Vi arbetar även med att starta upp en studie med patienter boende på servicehus, vilka är kända bärare, för att undersöka om de med hjälp av IgY-behandling kan bli av med sitt bärars-kap.

Slutsats Denna avhandling visar att Anti-Pseudomonas IgY har potential att förhindra pseudomonas-infektioner hos CF-patienter. Detta kan delvis förklaras med att IgY binder till flagellen på Pseudomonas aeruginosa och därmed stör en egenskap som är viktig för att bakterierna ska kunna infektera. IgY skulle därför kunna vara till stor nytta för CF-patienter, vars sjukdomsbild till stor del beror på om de drabbas av lunginfektioner med Pseudomonas eller inte. Vidare är detta ett exempel på ett komplement till dagens antibiotikabehand-ling, vilket är väldigt viktigt då antibiotikaresistens blir allt vanligare i sam-hället.

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Acknowledgements

Doktorandtiden har varit en lång och mycket lärorik resa. Även om resan sägs vara viktigare än själva målet, känns det rätt bra att ha det sikte. I fram-tiden är det dock resan som jag kommer att minnas mest. Jag vill tacka alla som följt mig på färden och gjort det möjligt att nå ända fram. Till er som bidragit vill jag uttrycka stor uppskattning och tacksamhet. Min handledare, professor Anders Larsson, för att du gett mig ditt förtroende och låtit mig arbeta under stor frihet. Du har hjälpt mig att bli mer självstän-dig. Jag uppskattar din positiva inställning och att du alltid ger mig tid för långa diskussioner (som man aldrig vet var de slutar). Genom att generöst dela med dig av dina kunskaper har jag lärt mig mycket, särskilt om IgY men framför allt har du inspirerat och motiverat mig under hela min resa. Tack! Professor emeritus Hans Kollberg, min bihandledare som varit mycket när-varande under hela doktorandtiden och varit till stor hjälp. Jag imponeras av ditt stora engagemang för forskning och dina stora kunskaper om CF. Tack för allt du lärt mig om CF och för att du introducerat mig till forskarvärlden. Jag har haft förmånen till många samarbeten och även fått möjlighet att arbe-ta på ett antal avdelningar förutom klinisk kemi. Tack till alla för det motta-gande jag har fått. Jag har känt mig mycket välkommen på alla ställen! Ahmad Amini på Läkemedelsverket för allt du lärt mig. Tack för alla intres-santa samtal och för dina historier. Dan Andersson på BMC för pågående samarbete med anti-ESBL Klebsiella IgY-projektet och för möjligheten att få arbeta i ditt lab. Linus Sandegren som handlett mig i mikrobiologilabbet förtjänar många många tack! Du har alltid ställt upp, vare sig det gällt att odla bakteriekulturer eller att tolka re-sultat. Det senare har inte alltid varit så lätt… Thomas Bjarnsholt vid Köpenhamns universitet för hjälp med experiment och för veckan jag tillbringade i ert lab. Tack till Anne för all hjälp i labbet. Tack Morten för tips om vad man kan göra i Köpenhamn och för att du ut-

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förde experiment åt mig (medan jag roade mig). Jag hoppas snart komma tillbaka! Tack alla medförfattare. Tack också till Jonas Bergquist och Jörg Hanrieder på BMC för analyser av IgY-lösningar med nanoLC-MALDI TOF/TOF MS. Bengt Wretlind på KI för Pseudomonasstammar, bilder och råd. Hanne V. Olesen för hjälp med kontrollgruppen. Anders Lannergård för pågående arbete med studieupplägg av en klinisk prövning av anti-ESBL Klesbsiella IgY. Snart kan jag hjälpa till mer igen. Uppsala CF-center, särskilt till Annika Hollsing och Elisabet Näs för samar-bete med Anti-Pseudomonas IgY-studien, forskarluncher och sällskap på kongresser. Stort tack till alla läkare och sjuksköterskor involverade i Anti-Pseudomonas IgY-studien. Ett särskilt tack till alla CF-patienter i studien för ert envisa gurglande och till alla föräldrar som hjälper till. Utan er skulle studien inte kunna genomföras! Tack till Riksförbundet Cystisk Fibros för stipendier. Per-Erik Wejåker, Per Omstedt, Jan Gabrielsson och alla andra på Immun-system AB för stöd och samarbete. Dennis Kunkel Microscopy, Inc. (www.denniskunkel.com) tackas för omslagsbilden och Roberto Osti för bilden på CFTR. Jättestort tack till alla kollegor på klinisk kemi som bidragit till att jag trivts så bra och särskilt till: Göran, Lena C och Karin som jag delat lab med. Tack också till Lena C för att du lärde mig 2D gel elektrofores. Mindre ordning, men inte mindre roligt har det varit i ägglabbet med sällskap av Carina, Anders N (det är ännu roli-gare att lyssna till ditt skratt än till MNK) och Johan (nu vet jag att man kan ha sms-avisering från glassbilen). Jättestort tack till Johan för bra diskussio-ner och för att du läste min avhandling. Mirja, min rumskompis för samtal om allt från livet som doktorand till dansk film och för att jag kunnat vända mig till dig när en fråga dykt upp. Det blev mycket roligare sen du flyttade in! Tjarlått, för tågsällskap, sms, väskdrömmar och för råd från journalisten till ”Norrlänningen”. Välj glädjen! Tack Jenny för att du svarat på en miljon frågor angående avhandlingen och för alla tips. Det är dags för bubbel och bling! Doktorander och andra kollegor: Kristin, Micke, Mia, Matilda, Anders M, Teet, Cilla, Marie B, Anna W och Oskar för fikaraster, after works,

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filminspelningar, julpyssel och syjuntor. Ni har lyst upp doktorandtiden (både bildligt och bokstavligt)! Tack till alla involverade i Biomedicinarprogrammet och sommarprojekt vid Umeå universitet samt Stockholms Biovetenskapliga Forskarskola med pro-jekt på KI och vid Stockholms universitet. Där fick jag en insyn i vad det in-nebär att vara doktorand och en vilja att bli det. Lina, som förgyllde studietiden i Umeå. Du är favoriten i godispåsen! Tack till Sara L för tiden på forskarskolan och numera för sällskap i löparspåret. Alla mina vänner i Umeå, Uppsala och nu på slutet i Stockholm vill jag tacka för allt roligt vi haft och har ihop på middagar, fester med animaliskt tema m.m. Med er kan jag vara Ekan, ta det med ro, och släppa allt som har med forskning att göra. Sara F, Lotta, Terese, Matilda och Karin för lång vänskap och en underbar tjejhelg som gav energi till skrivandet. Fina Anna A. och Maja, nu har jag all tid i världen att ses! Anna L för luncher som pig-gat upp i vardagen. Ett alldeles speciellt tack till dig Karin, min kära vän. Efter att ha bott i olika städer och länder är det fantastiskt att ha dig som granne. Det betyder så mycket att ha en vän som dig, Kakan! Till alla mina släktingar. Jag är väldigt glad för den nära sammanhållning som vi har och för ert intresse för vad jag gör. Gunnel för din omtänksamhet och för att du tar hand om mig och Per. Min syster Frida, min bror Mattias och mina föräldrar, Anita och Leif, ni betyder väldigt mycket. Tack för ert intresse, er uppmuntran och er omtanke. Det är underbart att få komma hem till Tavelsjö och bara vara. Per för kärlek och uppmuntran. Tack för all hjälp och för att du påmint mig om att fokusera på rätt saker under skrivandet. Du inspirerar mig och gör mig mycket, mycket lycklig. Min kärlek till dig växer hela tiden. Nu är det min tur att ta hand om dig! �

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