Non-Invasive Antibody Production in the Chicken

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List of Papers

This thesis is based on the following publications. The papers will be re- ferred to in the text by their Roman numerals.

I. Mayo, S.L., Persdotter-Hedlund, G. Tufvesson, M., Hau, J. 2003. Systemic immune response of young chickens orally immunized with bovine serum albumin. In Vivo 17: 261-268.

II. Mayo, S.L., Tufvesson, M., Carlsson, H-E., Royo, F., Gizurar- son, S., Hau, J. 2005. RhinoVax® is an efficient adjuvant in oral immunization of young chickens and cholera toxin B is an effective oral primer in traditional subcutaneous immunization with Freund’s incomplete adjuvant. In Vivo 19: 375-382.

III. Mayo, S., Royo, F., Hau, J. 2005. Correlation between adjunctivity and immunogenicity of cholera toxin B subunit in orally immunized young chickens. APMIS 113: 284-287.

IV. Mayo, S., Royo, F., Tufvesson, M., Carlsson, H-E. Hau, J. 2006. Immunospecific antibody concentration in egg yolk of chickens orally immunized with varying doses of bovine serum albumin and the mucosal adjuvant, RhinoVax®, using different immunization regimes. Scand J. Lab Anim. Sci. 33(3): 163-169.

V. Mayo, S., Carlsson, H-E., Zagon, A., Royo, F., Hau, J.2009. Enhancement of anamnestic immunospecific antibody response in orally immunized chickens. J. Immunol. Methods 342: 58-63.

Reprints were made with permission from the publishers:

Contents

INTRODUCTION...... 9 Importance of the Study ...... 9 Aim of the Study ...... 11 The Avian Immune System...... 11 Function...... 11 Anatomy ...... 11 Types of Immune Response ...... 13 Innate (non-specific) immune response...... 13 Adaptive (specific) immune response...... 13 Antigen-Antibody Interaction ...... 14 Immunoglobulin Classes in Chickens (IgA, IgM, IgY) ...... 15 IgM ...... 15 IgA...... 16 IgG and IgY...... 16 Transport of IgG from Maternal Serum to Egg Yolk...... 17 Antibody Production from Egg Yolk (IgY) ...... 17 Advantages of IgY Antibody Production...... 18 Immunization Routes ...... 19 Classical/Subcutaneous immunization ...... 19 Oral immunization...... 19 Adjuvants ...... 20 Freund’s Adjuvant (FCA and FIA)...... 20 Cholera toxin B-subunit (CTB) ...... 21 RhinoVax® (RV)...... 22 MATERIALS AND METHODS ...... 23 Animals and Husbandry ...... 23 Age of Chickens...... 24 Treatment Groups and Immunization Schemes ...... 24 Paper I...... 24 Paper II...... 25 Paper III...... 26 Paper IV...... 27 Paper V...... 28 Collection of serum samples (Paper I, II, III)...... 28 Collection of eggs (Paper IV and V)...... 29

Antibody extraction from egg yolk (Paper IV and V)...... 29 ELISA quantification of immunospecific antibodies...... 29 Paper I and II ...... 30 Paper III ...... 31 Paper IV and Paper V ...... 32 Statistical analyses...... 32 Ethics committee license...... 33 RESULTS ...... 34 Paper I ...... 34 Paper II ...... 35 Paper III...... 36 Paper IV ...... 37 Paper V...... 39 DISCUSSION ...... 41 SUMMARY ...... 48 REFERENCES...... 50 ACKNOWLEDGEMENTS ...... 61 ERRATA...... 63

Abbreviations

AU arbitrary unit BSA bovine serum albumin CTB cholera toxin b subunit DPI days post immunization DPSI day post subcutaneous immunization ELISA enzyme linked immunosorbent assay FCA Freund’s complete adjuvant FIA Freund’s incomplete adjuvant IgA immunoglobulin A IgG immunoglobulin G IgM immunoglobulin M IgY immunoglobulin from egg yolk RV RhinoVax® SC subcutaneous immunization SF Softigen®

Introduction

Importance of the Study The majority of laboratory animals are used as model for man in the development of drugs, toxicity studies and studies of diseases. A large proportion, however, are used for the production of polyclonal antibodies which are used for laboratory and therapeutic purposes. Mice take up approximately 75% of the total animal species used for polyclonal antibody production followed by rabbits (10%), birds (5%) and other ungulates (2.5%) Domestic fowl or chickens accounted for 87% of all birds used (UK Statistics, 2008). In EU countries, Canada, the United States and Queensland Australia, birds, domestic foul and/or poultry make up 4% of the total animal species used in scientific research related to animal welfare or non-invasive procedures (EBRA 2002, USDA 2008; CACC, 2008, Queensland, 2004). This production is mainly carried out not only by commercial companies but also by academic and government research institutes. The reason why rabbits, birds and farm animals are classified as laboratory animals is that the procedures used are associated with some discomfort for the animals. In antibody production, animals are injected on several oc- casions with antigens in combination with so-called adjuvant, i.e. the classic Freund’s Complete Adjuvant, which stimulate the immune system but often also result in unwanted side effects in the animals such as development of lesions including granulomas on the injection site (Alving, 2002; Gupta and Siber, 1995; Stills, 1994; Gupta et al., 1993; Claassen et al., 1992; Stills and Bailey, 1991; Weidemann et al., 1991; Broderson, 1989 and Toth et al., 1989); necrotic dermatitis (Leenars, et al 1998) and spinal cord compression from an injected site granuloma (Kleinman et al., 1993). For review see Stills, 2005; Alving, 2002. Once the animals have produced satisfactory concentrations of antibodies in the blood they are subjected to routine sampling of quite large amounts of blood from which the antibodies are purified. Polyclonal antibody production in chickens has proven to be a worthwhile alternative to conventional antibody production in mammals (Hau and Hen- dricksen, 2006). Previous studies have demonstrated that chickens are just as good antibody producers as mammals (Svendsen et al., 1994; Bollen et al., 1995; Bollen and Hau, 1996) and that antibody, IgY, can be purified from the egg yolk (Jensenius et al., 1981; Svendsen et al., 1995; for review see Schade, et al, 2005) making blood sampling obsolete. This procedure elimi-

9 nates the discomfort for the animals associated with being restrained and omits pain caused by invasive blood sampling. Immunity can also be induced through antigenic stimulation of mucus membrane e.g. in the alimentary tract (Zhang, et al, 2007; Yang, et al., 2007; Muir, et al, 1999; Holmgren et al, 1994, 1992). Therefore, the development of an effective oral immunization method for chickens is the remaining step to eliminate all animal distress associated with polyclonal antibody production. Oral immunization is accepted to have a great potential in vaccine development in mammals because of its easy and safe administration. Increas- ing evidence indicates that mucosal vaccination can induce both systemic and local mucosal immunity, while systemic immunization generally fails to elicit strong mucosal immunity (Zhang et al, 2007; Valosky et al, 2005; Zhang et al, 2002). However, large and repeated antigen doses are often required to achieve a protective immune response because of degradation of the antigen by gastric acid and proteases present in the gastrointestinal tract. Hence, there is a great need to overcome this problem by developing strategies for enhancing delivery of antigen to the mucosal immune system and identifying mucosa-active immunostimulating agents, i.e. adjuvants (Holm- gren et al, 1992). Although there are still hurdles to overcome before oral immunization and IgY production in chickens can be perfected, this study contributes to providing the foundation and additional information that could eventually lead a very useful tool for a humane way of producing and har- vesting polyclonal antibodies. The experiments discussed in this thesis focused on developing efficient oral immunization regimes for chickens using non-aggressive adjuvants such as cholera toxin B-subunit (CTB) and RhinoVax® (RV)*, a pegylated mono/diglyceride. Both CTB and RV have been demonstrated to result in egg yolk immunospecific antibody concentration above that of classical subcutaneous immunization with Freund’s incomplete adjuvant (Hedlund and Hau, 2001). The researches conducted in this thesis were specifically aimed to identify the optimal age of the chickens to be immunized, as well as determine the preparation and dosage of a model antigen (bovine serum albumin), which could produce the optimum serum immune response and development of IgY in chicken egg. Bovine serum albumin (BSA) and human immunoglobulin G (hIgG) are two common antigens used in polyclonal antibody production and these are the antigens previously tested for production of antibodies in egg yolk (Bollen and Hau, 1996; 1997; 1998). Furthermore, the efficacy of commercially known oral non-aggressive adjuvants such as CTB and RV were also evaluated.

* RhinoVax® was previously termed Softigen®

10 Aim of the Study The aim of this study was to develop an efficient method for non-invasive antibody production in the chicken. In particular, the objective was to optimize an oral immunization regime using a non-aggressive adjuvant, to establish the optimal age of the chickens to be immunized and to determine the optimal dose of immunogen as well as the sequencing of the doses for optimal yield of antibodies.

The Avian Immune System Function The immune system defends the host against intruding foreign (non-self) substances such as pathogens or foreign organisms. The body of the host has developed sophisticated defense systems, in which reactions depend specifically on the kind of antigen and the route of entrance. Table 1 summa- rizes the avian immune system.

Table 1. The Avian Immune System Organs/Tissues Cellular Elements Humoral Elements

Primary Lymphoid Lymphocytes,T-cells, Immunoglobulins (IgY, Organs: B-cells,Macrophages, IgA, IgM), complement, Bursa of Fabricius NK cells cytokines and thymus Secondary Lymphoid Organs: Spleen, bone marrow, Harderian gland, pineal gland, mucosa-associated lymphoid tissue (MALT), lymphoid nodules

Anatomy The bird’s immune system includes lymphatic vessels and lymphoid tissue. Primary lymphoid organs are the thymus, located in the neck along the jugu- lar vein, and the Bursa of Fabricius (BF), (Fig.1) a blind sac located adja- cent, extending from the dorsal side of the cloaca, the common portal of the reproductive, urinary, and digestive systems (Glick, 1964). The Bursa of Fabricius is the primary lymphoid organ in birds but is not found in mammals. The main function of the Bursa of Fabricius is the immunological maturation of the pre-bursal stem cells to form immunoglobulin-producing

11 cells or antibodies (B-lymphocytes or B-cells) (Fig3) (Glick et al., 1956; Cooper et al., 1980; Nieminen, 2001; Kohonen et al, 2007). These cells are produced in the embryonic liver, yolk sac and bone marrow. The cells move to the bursa of Fabricius (BF) after 15 days incubation through 10 weeks of age. The BF programs these cells which then move to the blood, spleen, cecal tonsils, bone marrow, Harderian gland (Fig 2), and thymus.

Fig 1. Fig 2. Fig 1.Bursa of Fabricius Birds (and other animals with a cloaca) have a bursa, or outpouching of the cloaca, in which b-cells mitose, mature, and then migrate out into the general body tissues, i.e. blood, spleen, cecal tonsils, bone marrow, thymus and the Harderian gland Fig 2.

Fig 3. In birds, most of the Ig diversification occurs by gene conversion in the bursa of Fabricius. However, further Ig diversification is achieved by somatic hypermuta- tion in secondary lymphoid organs Figures adapted from: Kohonen et al. 2007. Source: http://people.eku.edu/ritchisong/birdcirculatory.html

Although birds lack lymph nodes, the secondary lymphoid system is well developed and lymphoid tissue is scattered throughout the body (Fletcher and Barnes, 1998; Sharma, 1999). Secondary lymphatic organs and tissues are located in the spleen, bone marrow, mural lymph node and lymph nodes.

12 There is also a lymphatic circulatory system of vessels and capillaries that transport lymph fluid through the bird’s body and communicate with the blood circulation.

Types of Immune Response The immune systems of chickens and mammals are quite similar, although there are some differences. Like in mammals, the chicken’s immune system is divided into two major components, one being non-specific or innate and the other is specific or acquired.

Innate (non-specific) immune response The non-specific or innate immune response responds to all intruding organisms and molecules. The first line of defence against foreign/infectious organisms is the physical barrier of the skin, cilia and mucous membranes. If this fails, the non-specific immune response is activated through two mecha- nisms, a humoral response including complement, interferon, lyzozyme and a cellular response including macrophages, neutrophils, thrombocytes, and Natural Killer (NK) cells which converge on the invader, killing or slowing it down until the components of the specific immune response have been generated and can join in.

Adaptive (specific) immune response The specific or adaptive immune response is characterized by specificity, heterogeneity, and memory. It is divided into the cell-mediated (cellular) and the humoral (non-cellular) response. These responses together provide an excellent defense against foreign invaders. Both are adaptive and respond specifically to most foreign substances, although depending on the antigen one response generally dominates over the other. Cell-mediated immunity is particularly effective against fungi, parasites, intracellular viral infections, cancer cells, and foreign tissue. The humoral immune response defends primarily against extracellular bacteria and viral infections. The cells involved in both immune systems are the lymphocytes which in mammals originate in the bone marrow and migrate to different lymphoid organs. There are two types of lymphocytes which are known as T cells and B cells. All lymphocytes are derived from bone marrow but those which mature in the thymus are called T-type cells (T lymphocytes). The T cells are responsible for cell- mediated immunity. Since the immunity involving T cells is associated with the T cells themselves, this type of immunity is called cell-mediated immunity. B-cells destroy extracellular pathogens and their products by releasing antibody, a molecule which specifically recognizes and binds to a particular

13 target molecule, called the antigen (for review see, Sharma, 1999). B-cells are produced in the embryonic liver, yolk sac and bone marrow. In birds, lymphocytes pass through the Bursa of Fabricius and differentiate to become B cells. The cells move to the Bursa of Fabricius (BF) after 15 days incubation through 10 weeks of age. The bursa-derived lymphocytes (B cells) produce antibodies which can react specifically with antigen. Be- cause B cells produce antibodies that circulate, the immunity is called humoral immunity. During the embryonic stage in chickens, the bursa is colonized by the prebursal stem cells derived from extrabursal regions (Cooper et al., 1980; Nieminen et al., 2001; Kohonen et al, 2007). After colonization of the bursa by pre-destined prebursal stem cells, diversification of rearranged immunoglobulin (Ig) genes within the bursal micro-environment follows (Sayegh et al., 2000). B cells in the bursa mature and differentiate in order to provide the diversity needed to protect against the great variety of potential pathogens (Paramithiotis and Ratcliffe, 1994). These differentiated B cells then leave the bursa to seed other organs of the immune system (Schmidt, 1997). The diversity achieved by the bursa during the first six weeks of life is what the bird will have for its lifetime. The bursa continues to produce B cells until it involutes at puberty and the only source of B-cell renewal in adult birds is the pool of postbursal B cells in the peripheral lymphoid organs (Nieminen et al., 2001). The development of B lymphocytes in avian species is reviewed in detail by Ratcliffe and colleagues (1996) and Nieminen and co- workers (2001).

Antigen-Antibody Interaction The acquired immune response is highly specific for a particular antigen. For the specific immune response to be activated, the invading antigen must be processed by an antigen presenting cell (APC) such as a macrophage, ingest- ing the antigen and fragmenting it into antigenic peptides. Pieces of these peptides are joined to the major histocompatibility complex (MHC) molecules and are displayed on the surface of the cell (peptide-MHC complex) (Ryan et al., 2001).The T lymphocytes, have receptor molecules that enable each of them to recognize a different peptide-MHC combination. T cells activated by that recognition divide and secrete lymphokines that mobilize other components of the immune system. One set of cells that responds to those signals are the B cells which also have receptor molecules of a single specificity on their surface. Unlike the receptors on the T cells, those on the B cells can recognize parts of antigens free in solution, without MHC molecules. Upon antigen stimulation, B-cells are activated which takes place in the germinal centers of the spleen (Arakawa et al., 1996). When activated, the B cells divide and differentiate into plasma cells that secrete immu-

14 noglobulins, which are soluble forms of their receptors. These plasma cells are abundantly present in a unique terminal stage B-cell organ called the Harderian gland (Mansikka et al., 1989). By binding to the antigens that they find, the antibodies can neutralize them or precipitate their destruction by complement enzymes or by scavenging cells and the antigenic material is then removed from the body through the spleen and liver. Some T and B cells become memory cells that persist in the circulation and boost the immune system's readiness to eliminate the same antigen if it presents itself in the future. With successive exposure to the same antigen (boosters), antibody isotype switching or class switching takes place, which enables antibodies of given specificity to acquire a different constant region, change their effector function and produce a higher magnitude of antibody response (Zouali, 2001).

Immunoglobulin Classes in Chickens* (IgA, IgM, IgY) Antibody molecules are glycoproteins called immunoglobulins with two or more binding sites which attach to a specific site (epitope) on an antigen. They are constructed from paired heavy and light polypeptide chains. The three major immunoglobulin classes in chickens analogous to mammalian Igs are IgA, which is secreted at the mucosa, IgM and IgY(IgG), which circulate in blood and lymph (Benedict and Berestecky, 1987). Evidence for IgE and IgD in chickens, homologous to those in mammals has also been proposed (Burns and Maxwell, 1981; Chen et al, 1982). The different classes or isotypes of antibodies are distinguished by the structure of their heavy chain, varying in chemical structure and numbers of attachment sites per molecule.

IgM The chicken IgM molecule is similar to mammalian IgM with respect to immunoelectrophoretic mobility and morphology (Tureen et al., 1966). The molecular weight is 890 kD, comparable to mammalian IgM, 900 kD (Hig- gins, 1975). Chicken IgM has the same function as mammals’ IgM and de- livers the first immune response. IgM is detectable in chicken serum at levels around 0.7 mg/ ml. In the chicken egg, IgM is present in the white but not in the yolk (Pattersson et al., 1962; Rose et al., 1974).

* By tradition the term used for adult hens as well as chickens is “chicken” although this is semantically odd.

15 IgA The physico-chemical structure and function of chicken IgA is also similar to mammalian IgA, although the molecular weight of the secretory chicken IgA is 350-360 kD in comparison to 320 kD for secretory mammalian IgA (Leslie and Martin, 1973). Like mammalian IgA, chicken IgA is found in fluids of the gall bladder and in secretions) and the detectable normal level in chicken serum is 0.6 mg/ml. Like IgM, IgA is not detectable in egg yolk but present in small quantities in the egg white.

IgG and IgY The main antibody from chicken egg yolk is called IgY. Initially, IgY was thought of as IgG-like immunoglobulin, whereas IgY is now considered to be an evolutionary ancestor of mammalian IgG and IgE antibodies (Warr et al., 1995). As early as 1969, Leslie and Clem, suggested that the chicken’s main serum immunoglobulin be called IgY rather that IgG to distinguish it from the mammalian IgG. In the present work the term IgG is favored when the molecule is present in the chicken and IgY when it has been transferred into the egg yolk. Despite similarities between IgY and mammalian IgG antibodies, there are also some profound differences in their structure. In terms of structure, the molecular mass of IgY was reported to be 167 250 Da, slightly larger that IgG (~160 kDa). Furthermore, the IgY heavy chain is 67-70 kDa, whereas the molecular mass of the mammalian IgG heavy chain is approximately 50 kDa (Sun et al., 2001). The greater molecular mass of the IgY is due to an increased number of heavy chain constant domains and carbohydrate chains. The IgY heavy chain constant region has four domains and no hinge, whereas that of mammalian IgG has three domains and a hinge region (Fig 4). In addition, the hinge region of IgY is much less flexible compared to mammalian IgG. It has been suggested that IgY is a more hydrophobic molecule than mammalian IgG (Davalos-Pantoja et al., 2000).

Figure adapted from Cova, 2005 Fig.4.Schematic structural difference between mammalian IgG and chicken IgY. The IgY molecule consists of two heavy (H) and two light (L) chains. The heavy (H) chain of the full length chicken IgY molecule has one variable (VH) and four constant region domains (Cv1, Cv2, Cv3, Cv4) The truncated IgY, lacking the two terminal domains of the constant region, Cu3 and Cu4, of the H chain, resembles a structural equivalent F(ab')2 fragment of IgY, thus named as IgY(Fc) or IgY(-Fc) for the intact molecule and u(Fc) for its H chain.

Transport of IgG from Maternal Serum to Egg Yolk Antibody is transported to the egg yolk through receptors on the membranes of the chicken oocyte. As the chicken oocyte develops, receptors on its membrane sequester large amount of chicken antibodies which are stored within the sterile confines of the yolk (Kowalczyk et al., 1985). By the time the egg is oviposited, as much as 200 mg of chicken antibodies (IgY) are present in the yolk (Lösch et al., 1986). Several regions within the antibody molecule are important for its transport into the chicken egg yolk such as the intact Fc and hinge regions (Morrison et al., 2001). At any given point in time during egg maturation, the concentrations of IgY in egg yolk are similar to those of IgG in the circulation of the hen (Bollen and Hau, 1997).

Antibody Production from Egg Yolk (IgY) Production of polyclonal antibodies through parenteral immunization of laboratory animals is a routine procedure worldwide. The species traditionally used for immunization have been mammals such as rabbits, sheep, goats and horses. However, production of antibodies from chicken egg yolk may repre- sent an attractive alternative to antibodies from serum. As described more than 100 years ago, avian maternal antibodies are transferred from serum to

17 egg yolk to confer a passive immunity to embryos and neonates (Klemperer, 1893). Immunization of chickens results in abundant supply of immunoglobulin Y (IgY) in the egg yolk, because IgY antibodies from chicken blood serum are efficiently transferred across the follicular epithelium of the ovary and accumulated in the yolk during oogenesis up to concentrations of 100 to 250 mg IgY per egg yolk (Rose et al.,1974; Kowalczyk et al., 1985; Lösch et al., 1986; Akita and Nakai, 1992). To date, perhaps the most humane way to produce polyclonal antibodies is from chicken egg yolk.

Advantages of IgY Antibody Production Using the chicken as the immunization host brings several advantages to antibody production, the most apparent being the non-invasive collection of antibodies by using egg yolk as a source of antibodies. In terms of animal welfare, there is no need to bleed the chicken as is done when using mammals for antibody production. A single chicken can produce large amounts of IgY, up to 3 grams/month which is 10-20 times the amount obtainable from a rabbit. The yield in IgY antibodies can be compared to that of IgG antibodies obtained by conventional immunization methods, 200 mg IgG can be obtained monthly, with approximately 5% constituting the specific antibody. In the case of the chicken, approximately 1500 mg of IgY can be harvested each month, and between 2% and 10% are specific antibodies (Schade et al., 1994). Taken together, chicken antibody collection and isolation can be described as non-invasive, rapid and economical. Compared to antibody production in rabbits, the IgY technology offers several advantages (Fischer et al., 1996). No blood sampling, only egg collection is required upon immunization and low quantities of antigen are required to obtain high and long- lasting IgY titres in the yolk from immunized hens (Gassmann et al., 1990; Larsson et al., 1998) Another advantage is that IgY antibodies tend to recognize analogous proteins in a number of mammalian species making IgY antibody production of antibodies more applicable and successful in chicken than in other animals. (Hau et al., 1980; 1981; Gassmann et al., 1990; Tini et al., 2002). In practice this means that chicken antibodies against a protein in one species can often be used for analyses of the analogous protein in another species (Hau et al., 1990). With regard to function, three important differences between IgY and mammalian IgG need to be considered. Firstly, IgY does not bind to proteins A or G (Jensenius et al., 1981; Guss et al., 1986), an important feature of IgG that allows simple IgG isolation. Secondly, IgY does not bind rheumatoid factors (autoantibodies, RFs) (Larsson et al., 1991). IgG molecules often cause false positive results by interaction with RFs in immunoassays

18 (Bollen and Hau, 1996). Thirdly, chicken egg yolk immunoglobulin does not interfere with mammalian IgG and does not activate the mammalian complement system (Jensenius et al., 1981; Carlander and Larsson, 2001).

Immunization Routes Various routes of immunization (subcutaneous, intramuscular, intradermal) are routinely used to deliver an antigen with the aim to induce a humoral and/or a cellular immune response. Certain immunization procedures are more effective than others depending on the nature of the antigen and the type of immune response that is desired for a particular antigen. The classical or parenteral route by injection is the most efficient route to induce a humoral or systemic response. On the other hand, several studies have demonstrated that oral immunization through delivery of antigens with carrier systems can induce a simultaneous peripheral and mucosal response (Huang et al., 2008, Cox et al, 2006; Rask, et al, 2000). This may be an attractive alternative to subcutaneous or intramuscular immunization. For animal welfare and practical reasons, an optimized oral immunization procedure would be a more preferable way to deliver an antigen, as it would les- sen the animals’ discomfort and stress. There are various ways to administer an antigen and there is no specific route that is universally superior over another. Hence, the choice of immunization route is usually based upon choices of species and adjuvant. In this thesis, discussion will be limited to subcutaneous and oral immunization.

Classical/Subcutaneous immunization Subcutaneous immunization generally provides a slow absorption of antigen, which occurs primarily through the lymphatic vessels to the local lymphoid tissues. This route should be used whenever possible instead of e.g. intramuscular and intradermal injection as it offers the advantage of easy access for visualization and palpitation to monitor for post immunization complica- tions such as abscess formation and is easy to use in most species.

Oral immunization The induction of serum or mucosal antibody responses to orally administered antigens is often difficult and generally requires the administration of relatively large quantities of antigen, as the amount of antigen actually ab- sorbed and capable of eliciting an immune response is usually low. How- ever, oral administration of large quantities of antigen required to produce an immune response often leads to simultaneous induction of systemic tolerance (Tomasi, 1980; Miller et al., 1992).

19 In general, the mechanism by which antigen is taken up by the small intestine is primarily via non-specific sampling of the contents of the lumen by microfold cells (M cells) which are special epithelial cells that overlie the Peyer’s patches and other lymphoid clusters of the gut-associated lymphoid tissue (Bland and Britton, 1984; Muir et al., 2000). Antigen molecules that posses lectin or lectin-like binding activities are able to bind to glycolipids and glycoproteins on the intestinal mucosa and to stimulate these cells to transport the proteins into the systemic circulation, thereby eliciting a systemic immune response (De Aizpurua and Russell-Jones, 1988).

Adjuvants The use of adjuvants to stimulate the immune system and to enhance the immune response is routine in antibody production. The word adjuvant is derived form the Latin word “adjuvare” which means to help, aid or to enhance (Vogel, 1998). In oral immunization, adjuvants are considered important for induction of a systemic immune response. Most protein antigens, especially small polypeptides (<10kDa) and non-protein antigens, usually need to be conjugated to a large immunogenic carrier molecule to become good immunogens. These antigens, especially if administered in small quantity, need to be administered with an adjuvant to assure a high quality/high quantity antibody response by the immunized animal. Several mucosal adjuvants have been reported to work well in mammals (Lycke and Holmgren, 1986; Clements et al., 1988; Butler, et al., 1990; Van der Heijden et al., 1991; Wilson et al., 1993; Wikingsson and Sjöholm, 2002.). However, there are few reports of effective mucosal adjuvants in chickens (Hoshi et al., 1998; 1999a; Takada and Kida, 1996; Vervelde et al, 1998; Girard et al., 1999; Hedlund and Hau, 2001). Although there are several adjuvants reported (for review see Stills, 2005), this thesis will be limited to the discussion of Freund’s adjuvant, Cholera toxin B-subunit and RhinoVax®.

Freund’s Adjuvant (FCA and FIA) Freund’s Complete Adjuvant (FCA) and Freund’s Incomplete Adjuvant (FIA) are the most common adjuvants used for parenteral immunization. These adjuvants are a mixture of mineral oil, a surfactant and heat-killed Mycobacterium tuberculosis or M. butyricum (FCA) or without mycobacteria (FIA) (Freund et al., 1937). The water-in-oil emulsion is prepared by mixing one volume of the adjuvant (FCA or FIA) to one volume aqueous antigen solution. In the emulsion, the antigen is distributed in oil droplets which after injection disperse widely in the body thereby increasing the potential for interaction with relevant cells. Antibody production is enhanced

20 by Freund’s adjuvant primarily because of the depot effect, a depot of antigen forms at the injection site resulting in the sustained release of small quantities of the antigen over a long period of time and non-specific im- munopotentiation of macrophages by surfactants and the mycobacteria (for review see Stills, 2005). FIA is frequently used to boost animals that received a primary antigen injection with FCA but it can be used as adjuvant for primary injection as well. It has adjuvant properties that favor humoral immunity without cell mediated-immunity, although less potently than FCA. FCA is less frequently used because of its severe reactions i.e. severe inflammation causing necrosis and ulceration of the tissue (Wanke et al., 1996; Weidemann et al, 1991; Broderson, 1989 and Toth et al., 1989); including the risk of anaphy- lactic shock if injected a second time (CCAC, 1991; VPHI, 1993). The dis- cussions of FCA to induce pain and distress have resulted in the preparation of numerous guidelines in Canada and several European countries. The use of FCA is currently limited in experimental animals which prompted the process of developing alternative adjuvant products (Hendriksen, 2005). In chickens, the use of FCA is discouraged because it results in reduction in egg laying frequency and consequently the reduction in IgY productivity (Bollen and Hau, 1999b).

Cholera toxin B-subunit (CTB) CTB is one of the most often used adjuvants as a means to enhance a mucosal and systemic immune response via orally administered antigen mixed with cholera toxin (CT), or conjugated to its B-subunit (CTB). Cholera toxin (MW approx. 84kDa) is the primary enterotoxin produced by Vibrio cholera, consisting of two components (Gill, 1976): the A subunit which is toxic (MW 27.2 kDa), and the non-toxic pentamer CTB (MW 11.6 kDa) (Mekalanos, 1988) which mediates the binding of CT to the surface of nu- cleated cells through interaction with the GM1 gangliosides (Cuatrecasas, 1973). CTB is the non-toxic binding moiety of cholera toxin and consists of a ring of 5 identical subunits (ring-shaped pentamer) which can be purified as a separate molecular species called “choleragenoid”. CTB functions as a mucosal adjuvant by targeting to the cell surface GM1 ganglioside (Holm- gren et al., 1993). Aside from this, several studies proposed other mecha- nisms of CT adjuvanticity, which include increasing permeability of the intestinal mucosal membranes to antigens (Lycke et al., 1991) by enhancing the transepithelial flux of antigens from the lumen to the lamina propia (Gi- zurarson et al., 1992); enhancing the antigen-presenting cell function (MHC class II expression and IL-1 production) (Lycke et al., 1989); induction of isotype switching of Peyer’s patches B cells from IgM to IgG and IgA (Lebman et al., 1988; Lycke and Strober, 1989; Wilson et al., 1993) and inhibition of suppressor T-cells (Dertzbaugh and Elson,1991).

21 Cholera toxin (CT) has been reported to abrogate oral tolerance as well as enhance IgA response in rodents (Elson and Ealding, 1984b; Lycke and Holmgren, 1986). In chickens, oral immunization of CTB resulted in an egg yolk immunospecific antibody response (Hedlund and Hau, 2001). Immu- nostimulation of the humoral immune response was likewise observed in chickens given CT/CTB orally (Girard et al., 1999) as well as in chickens administered by the intranasal (Takada and Kida, 1996), intra-intestinal (Vervelde et al., 1998) and intravenous (Quéré and Girard, 1999) routes. By contrast, CT was not found to exert satisfactory effects in orally immunized chickens by Meinersmann and Porter (1993).

RhinoVax® (RV) RhinoVax® (RV) is a non-ionic bioadhesive surfactant containing glycerides (Gizurarson and Heron, 1994). It was previously termed Softigen®. This adjuvant formulation is based on caprylic-capric glycerides dissolved in polysorbate 20 and water. RV was proven to have no adverse biological effects (Gizurarson et al., 1996a) and to be safe for intranasal and oral administration to man. Various bacterial and viral protein antigens administered with RV intranasally induced significant mucosal IgA, as well as systemic IgG responses both in experimental animals (Hrafnkelsdottir, et al., 2005, Gizurarson et al., 1995; 1996b; 1998a; Jakobsen et al., 1999; Hraf- nkelsdottir, K., et al 2005) and in humans (Gizurarson et al., 1998b). The biological mechanism of RV is still under investigation. However, an early study by Gizurarson and co workers (1996a) proposed that an observed absorption enhancing effect after intranasal immunization may be due to alteration of the mucus layer, alteration of tight junctions, and extraction of membrane components by co-micellisation or prolonged contact at the absorption site, accompanied by specific stimulation of the local lymphoid tissue.

22 Materials and Methods

Animals and Husbandry A total of 32 outbred (inbreeding coefficient <3%) (Paper I); 45 approximately 50% in-bred (40%-60% homozygosity) (Paper II); 13 chickens from lines selected for more than 20 years for large comb size (4 outbred, Line 36: inbreeding coefficient <3%; and 9 Line 33: 50% inbred, 40–60% homozygosity) (Paper III), one-day old White Leghorn female chickens, (Gallus domesticus) from Lövsta Poultry Facility of the Swedish Agricultural Uni- versity were acclimatized for two weeks. All batches of chickens used in Paper I, II and III were group housed in pens with aspen wood chips (Tap- vei, Oy, Kaavi, Finland) as bedding in a temperature controlled room with a heating lamp providing temperatures of 20oC to 35oC in the pen (30oC for the first week after hatching and 25oC for later rearing), with 8 hours dark and 16 hours light cycle. Food granules (batch no. 55102, Odal Lantmän, Uppsala, Sweden) free of animal protein and tap water were available ad libitum. The average body weight of the chickens, at 15 days of age, was 129.4 g ± 10.2 (Paper I); 128.3g ± 4.56 (Papers II and III) at the start of the experiment. Body weight was recorded at every immunization and blood-sampling event. For Paper IV a total of 45* approximately 50% in-bred (40%-60% homozygosity) White Leghorn (Line 43) layers (Gallus domesticus) selected from male comb related traits for 15 generations were used in this study. For Pa- per V, a total of 20** White Leghorn (Line 44) layers were used. The hens used in papers IV and V were from GAL AB breeding stock housed at Söderby gård, Uppsala, Sweden. They were kept in single cages with the dimension of height 65 cm, length 48 cm and depth 41 cm. The ambient temperature varied from 18 to 21 ºC and the light cycle was 8 hours dark and 16 hours light. Food was crushed pellets containing 15% protein (Johan Hansson, Uppsala Sweden) and water was available ad libitum.

*One chicken from treatment 3 (HP 5 days) died spontaneously during the study. **One chicken died spontaneously during the study.

23 Age of Chickens The chickens were 15 days old when first immunized (Papers I, II, III), except for one treatment group in Paper I in which 22-day-old chickens were immunized with BSA without adjuvant. In Paper IV and V, chickens were 35 and 20 weeks old, respectively when first immunized.

Treatment Groups and Immunization Schemes Paper I. The study consisted of 8 treatment groups with 4 chickens in each group. Each chicken was individually marked with a numbered plastic leg ring (Palms Zoo, Uppsala, Sweden). The immunogen mixture was administered by gavage to the pharynx, 200 l/chicken. After every administration of the adjuvant/antigen mixture, 1 ml of saline was administered by gavage. The treatment groups were: Group A: 5% RhinoVax® and 600 )g BSA. (Softigen® from Sasol Gmbh, Witten, Germany) was added to a concentration of 1:20 (v:v) in a BSA (Sigma, Sweden, Cat. No. A-4503) solution (3 mg/ml distilled water). The chickens were immunized on days 1, 14 and 28. Group B: 10% RhinoVax® and 600 )g BSA. RhinoVax® was added to a concentration of 1:10 (v:v) in a BSA solution (3 mg/ml distilled water). The mixture was administered on days 1, 14 and 28. Group C: 20 % RhinoVax® and 600 )g BSA. RhinoVax® was added to a concentration of 1:5 (v:v) in a BSA solution (3 mg/ml distilled water). The mixture was administered on days 1, 14 and 28. Group D: CTB and BSA glutaraldehyde-conjugated. CTB (Sigma, Swe- den, Cat. No. C9930) and BSA were conjugated (10:1 molar ratio) with glutaraldehyde. The protocol was based on Van der Heijden and co-workers (1991). Briefly, CTB (containing 0.5% Cholera toxin A-subunit) and BSA dissolved in PBS (pH 8.0) were mixed, after which, glutaraldehyde was slowly added to a concentration of 15 mM. The mixture was gently stirred for one hour at room temperature. To stop the reaction, glycine was added at a concentration wherein there is at least 60mM glycine in the solution. The mixture was dialyzed against phosphate buffered saline (PBS) overnight at room temperature and administered on days 1, 14 and 28. Group E: 6 mg BSA in H2O. BSA was dissolved in distilled water at a concentration of 30 mg/ml. Two hundred l of 6 mg BSA solution was fed daily by gavage for 5 consecutive days. A booster, 6 mg BSA was given 30 days after the first administration of antigen. Group F: 6 mg BSA in H2O. The protocol was identical to Group E except that the chickens were 22 days old when first immunized. Group G: 120 ug BSA with FIA (Positive control). A solution of 600 μg BSA dissolved in 1 ml PBS was emulsified in equal volume of FIA (Sigma,

24 Sweden). Chickens were immunized subcutaneously with 200l on days 1, 14 and 28. Group H: Saline (Negative control).Two hundred l of saline was given to each chicken by gavage on days 1, 14 and 28.

Table 1. Summary of Immunization Scheme for Paper I Treatment Group (n) Age Route Dosage(vol/ Immuni- Sampling (days) chick/day) zation (dpi) (day) A) 5%RV+600μg BSA 4 15 gavage 200l 1, 14 & 28 7,21 & 35 B) 10%RV+600μg BSA 4 15 gavage 200l 1, 14 & 28 7,21 & 35 C) 20%RV+600μg BSA 4 15 gavage 200l 1, 14 & 28 7,21 & 35 D) CTB+BSA(conj.1:10) 4 15 gavage 200l 1, 14 & 28 7,21 & 35 E) 6mg BSA in H2O 4 15 gavage 200l 1 to 5, & 30 12 & 37 F) 6mg BSA in H2O 4 22 gavage 200l 1 to 5, & 30 12 & 37 G) 120μg BSA + FIA 4 15 s.c. 200l 1, 14 & 28 7,21 & 35 H) saline 4 15 gavage 200l 1, 14 & 28 7,21& 35

Paper II. The chickens originated from 11 hens (A, B, C, D, E, F, G, H, I, J and K). Five chickens from each hen (A to E) were distributed among the 5 treatments groups so that there was one sister in each treatment group. Chickens from hens, which had less than five offspring (F to K) were distributed evenly among the treatment groups to make a total of 9 chickens per treatment group from 9 different hens (9 blocks). The immunization protocol consisted of two phases, 1 and 2. Phase 1 involved administration of an antigen, BSA, in combination with various adjuvant preparations, either CTB and/or RV and given by oral route (gavage), except for the treatment of the positive control group which received BSA emulsified in FIA by subcutaneous injections. There were five treatment groups with 9 chickens each in group 1 (BSA+20% RV), group 2 (BSA+CTB), group 3 (BSA+CTB+RV) and group 5 (100%RV); and 8 chickens in group 4 (BSA+FIA). The immunogen mixture was administered by gavage to the pharynx at 200 l/chicken. All chickens were immunized on days 0, 14 and 28. They were 15 days old when first immunized (day 0). The treatment groups were: Group 1: 2 mg BSA and 20% RhinoVax®. (Softigen from Sasol Gmbh, Witten, Germany) was added to a concentration of 1:5 (v:v) in a BSA (Sig- ma, Sweden, Cat. No. A-4503) solution (10mg/ml phosphate buffered saline (PBS), pH 8.0). Group 2: BSA and CTB glutaraldehyde-conjugated. BSA and CTB (Sigma, Sweden, Cat. No. C9930) were conjugated (20:1 molar ratio) with glutaraldehyde. The protocol was based on Van der Heijden and co-workers (1991). Briefly, CTB (containing 0.5% Cholera toxin A-subunit) and BSA dissolved in PBS (pH 8.0) were mixed, after which glutaraldehyde was slow-

25 ly added to a concentration of 15 mM. The mixture was gently stirred for one hour at room temperature. To stop the reaction, glycine was added, at a concentration at which glycine reached at least 60 mM in the solution. The mixture was dialyzed against PBS overnight at room temperature. Group 3: BSA and CTB and 20% RhinoVax® glutaraldehyde-conjugated. BSA and CTB (20:1 molar ratio) and 20% RV were conjugated with glutaraldehyde as described in Group 2. Group 4: 1 mg BSA with FIA (Positive control). A solution of 10 mg BSA dissolved in 1 ml PBS was emulsified in 1 ml of FIA (Sigma, Sweden). Chickens were immunized subcutaneously with 200 l on days 0, 14 and 28. Group 5: 2 mg BSA and 100% RhinoVax®. Softigen from Sasol Gmbh, Witten, Germany) was added to a concentration of 1:1 (v:v) in a BSA (Sig- ma, Sweden, Cat. No. A-4503) solution (10mg/ml phosphate buffered saline (PBS), pH 8.0).

Table 2a. Summary of Immunization Scheme for Paper II (Phase I) Treatment Group* (n) Age Route Dosage(vol/ Immuni- Sampling (days) chick/day) zation (day) (dpi) 1) 20%RV+2mg BSA 9 15 gavage 200l 0, 14, 28 14, 28, 42 2) CTB+BSA(conj.1:20) 9 15 gavage 200l 0, 14, 28 14, 28, 42 3) 20%RV+CTB+BSA 9 15 gavage 200l 0, 14, 28 14, 28, 42 4) 1mg BSA+FIA 9 15 s.c. 200l 0, 14, 28 14, 28, 42 5) 100%RV+2mg BSA 9 15 gavage 200l 0, 14, 28 14, 28, 42

In Phase 2, another antigen, human IgG emulsified with FIA was administered by subcutaneous injection to all chickens in all treatment groups of Phase 1, 14 days after the last oral immunization, i.e. 42 days after the first immunization was given (42 dpi). On the last blood sampling in Phase 1, at 42 dpi, Phase 2 was initiated. Each chicken in every treatment group was immunized subcutaneously (4 sites on the breast with 200ul of 200ug human IgG emulsified in FIA). Injections were given on days 0 and 14.

Table 2b. Summary of Immunization Scheme for Paper II (Phase II) Treatment Group* (n) Age Route Dosage(vol/ Immuni- Sampling (days) chick/day) zation (day) (dpi) All Phase I group 200μg human IgG+FIA 9/grp 57 s.c. 200l 0,14 14,28 * 9 families of 5 sisters

Paper III. When chickens were 15 days old, they were immunized with bovine serum albumin (BSA) conjugated to cholera toxin B subunit (CTB). The immunogen mixture was administered by gavage to the larynx at a dose of 200 l/chicken. After every administration of the adjuvant/antigen mix-

26 ture, 1 ml of saline was administered by gavage. BSA-CTB glutaraldehyde- conjugated mixture was prepared by conjugating BSA (Sigma, Sweden, Cat. no. A-4503) and cholera toxin B (CTB) subunit (Sigma, Sweden, Cat. no. C9930).The protocol was based on Van der Heijden, 1991 and described in Hedlund and Hau, 2001.

Table 3. Summary of Immunization Scheme for Paper III (BSA+CTB Conjugated) Chicken Line (n) Age Route Dosage(vol/ Immunization Sampling (days) chick/day) (day) (dpi) 1) Inbred 4 15 gavage 200l 1, 14, 28 7, 21, 35 2) Outbred 9 15 gavage 200l 0, 14, 28 14, 28, 42

Paper IV. In all the groups but the control, the animals were fed with a solution of bovine serum albumin (BSA) (Sigma-Aldrich Chemie GmbH, Stein- heim, Germany) in bi-distilled water. The control was given bi-distilled water, and in those groups with adjuvant, 20% of RhinoVax® (formerly Softi- gen® from Sasol Gmbh, Witten, Germany) where used. Five individual chickens were grouped in each of the nine treatments. Amounts, concentration and days of immunization were described in the Table 4.

Table 4. Summary of Immunization Scheme for Paper IV Treatment RhinoVax Days x volume Conc. BSA Total BSA/ Group treatment LP 5 days No 5 x 250 l 20 mg/ml 25 mg LP 3 days No 3 x 500 l 20 mg/ml 30 mg HP 5 days No 5 x 250 l 200 mg/ml 250 mg HP 3 days No 3 x 500 l 200 mg/ml 300 mg LP 5d + RV Yes 5 x 250 l 20 mg/ml 25 mg LP 3d + RV Yes 3 x 500 l 20 mg/ml 30 mg HP 5d + RV Yes 5 x 250 l 200 mg/ml 250 mg HP 3d + RV Yes 3 x 500 l 200 mg/ml 300 mg Control No 5 x 250 l No No LP: Low protein, 20 mg/ml HP: High protein, 200 mg/ml 5days: 5 consecutive days of 250μl 3 days: 3 consecutive days of 500μl + RV: with RhinoVax®

The first booster was performed 1 month after first immunization with ex- actly the same protocol as described in Table 1. The second booster was performed 1 month after first booster, with just a single dose, but the volume and concentration were maintained for the different groups as what they previously received.

27 Paper V. Five chickens were randomly allocated to each of the four study groups. Groups 1 and 2 received oral immunization with BSA in one, two, three, or four treatment periods (Table 5). A treatment period consisted of three consecutive days of oral immunization. The immunogen mixture, which consisted of 200 mg BSA (Sigma-Aldrich)/ml dissolved in MilliQ H2O with 20% RhinoVax®, was administered by gavage, the dose volume was 0.5ml per day. Group 3 chickens received subcutaneous injections with a mixture of 20mg BSA/ml in 50% Freund’s Incomplete Adjuvant (FIA) (Sigma-Aldrich) total volume 0.2 ml, and served as positive controls. In week 18, group 3 chickens received BSA+RV as an oral booster. Group 4 was used as a negative control group and the chickens in this group received water by gavage at relevant time points (Table 5). Week 1-17 was designated Phase 1, and week 18-26 was designated Phase 2.

Table 5. Summary of Immunization Scheme for Paper V Grp # Week 1 Week 4 Week 7 Week 18 Chickens/grp 1 p.o.BSA+RV p.o.BSA+RV p.o.BSA+RV p.o.BSA+RV 5 2 p.o.BSA+RV p.o H2O p.o.BSA+RV p.o.BSA+RV 5 3 s.c. BSA+FIA s.c. BSA+FIA s.c.BSA+FIA p.o.BSA+RV 41 4 p.o H2O p.o H2O p.o H2O ------5 BSA=Bovine Serum Albumin, RV=RhinoVax®, FIA=Freund’s Incomplete Adjuvant, p.o.=oral administration, s.c.=subcutaneous administration. 1 One chicken died during phase 2.

Collection of serum samples (Paper I, II, III) All chickens were blood sampled for 1-2 ml using a 23 G needle on a 2.5 ml syringe from the wing/brachial vein. In the Paper I study, all chickens were bled seven days after every treatment. For Groups E and F, blood samples were obtained seven days after the last day of antigen administration and seven days after boosting. In Paper II, blood sampling was done before every oral or sc immunization, every fortnight, at the same time when immunization treatments were given, i.e. for Phase 1 on days 14, 28, and 42, and for Phase 2, on days 14 and 28 after the initial subcutaneous injection (dpsi). In Paper III, all chickens were bled 7 days after each immunization for the outbred group (on days 7, 21 and 35) and the inbred chickens were bled every fortnight, at the same time as immunizations were given (on days 14, 28 and 42). On the final day of sampling, the chickens were exsanguinated by cardiac puncture during anaesthesia with 200-400 l Ketamine (Ketalar®, Park-Davis/Warner Lambert, Sweden) (50mg/ml) and 100-200 l Xylazine

28 (Rompun® Vet, Bayer, Sweden) (20mg/ml), injected intramuscularly. Serum samples were collected from centrifuged clotted blood and stored at –20oC until analysis.

Collection of eggs (Paper IV and V) In Paper IV, eggs from each chicken were collected every week starting at week 1 (about 3 days after last immunization) and every 4 weeks thereafter (i.e. week 4, 9 and 13). In Paper V, eggs were collected twice a week.Each animal was kept in individual cages. Each collected egg were numbered and kept in 4 0C until analysis. All eggs were weighed before separation of the egg yolks from the whites.

Antibody extraction from egg yolk (Paper IV and V) To extract the antibodies from yolk, a slight modification of the method described by Persdotter-Hedlund and Hau (2001) and Svendsen et al (1995) was performed. Briefly, the egg yolk was separated from the white, and washed with bi-distilled water. The vittelline membrane was punctured and the yolk was collected in 50 ml tubes. The total weight of yolk was measured, ho- mogenised mechanically, and just a sample of 10 g was retained (when the yolks had little volume 6.5 g was retained, Paper V). After which, 40 ml of bi-distilled water was added (1:5 dilution for Paper V), shaken for 20 minutes to 1 hour and frozen at –20ºC until further purification. The mixture was thawed out at 4°C overnight or briefly at room temperature, 1 to 2 ml of di- choloromethane was added to extract the lipid content of the yolk. After a couple of minutes of manual vigorous shaking the mixture was placed in a mechanical shaker for 20 to 30 minutes and centrifuged for 45 minutes at 4°C, at 3000xg or 400 rpm. The supernatant was collected and concentrated in a dialysis tube (MWCO 12000-14000) in Polyethylenglycol (PEG) 6000 (Merck Schucardt CHG, Hohenbrunn, Germany) to less than 4 ml. Then volume was adjusted to 4 ml with 4°C PBS pH 7.4 and homogenized. Samples were added with 0.1% Sodium Azide, vortexed and kept at 4ºC.

ELISA quantification of immunospecific antibodies Indirect ELISAs were used to quantify the immunospecific IgM, IgA and IgG antibodies in chicken serum for Papers I, II and anti-human IgG in Pa- per III and anti-BSA IgY in Papers IV and V.

29 Paper I and II ELISA quantification of immunospecific chicken anti-BSA IgA, IgM, IgG antibodies Microtiter plates (Nunc, Roskilde, Denmark) were coated with 100 g/ml BSA protein standard (Sigma Cat. No. P-0834) in carbonate buffer (pH 9.6, 100 l/well) and incubated overnight at 4oC in a moist chamber. The plates were washed four times in PBS-Tween-20 (pH 7.4) in between each step. After coating and washing, the plates were incubated with the chicken serum samples diluted 1:100. As standard series, a pool of serum (n=4) from the positive control (FIA-treated group) was diluted 1.5-fold starting at 1:2,000 to 1:512,000, for IgG; and 2-fold dilutions at 1:2 to 1:4096 for IgM and 1:2 or 1:64 to 1: 2048 or 1:65536 for IgA in Paper I and Paper II, respectively. The standard serum pool was defined to contain 100 arbitrary units (AU)/ml for IgG, IgM and IgA. The chicken antibodies were detected with horseradish peroxidase (HRP) conjugated rabbit anti-chicken IgG (1:10,000, Sigma, Swe- den); with HRP conjugated goat anti-chicken IgM (1:10,000, Nordic Immu- nology Lab, Tilburg, The Netherlands); or with HRP conjugated goat anti- chicken IgA (1:1,000, Nordic Immunology Lab, Tilburg, The Netherlands), respectively. An OPD-substrate (100 l/well, Kem-En-Tec, Copenhagen, Denmark) was used for colour development. Treated plates were incubated for 15 min in the dark at room temperature to react after which the reaction was stopped with 1 M H2SO4 (150 l/well). The plates were read in an ELISA reader (Multiskan RC, Labsystems, Sweden) at 492 nm. For Paper I, the inter assay coefficient of variation for IgG was 3.0%, 5.0% for IgM and 5.3% for IgA. The intra assay coefficient for IgG, IgM and IgA was 2.3%, 3.6% and 3.9%, respectively. For Paper II (Phase 1), the inter assay coefficient of variation for IgG was 3.4%; 4.7%; for IgM and 5.8% for IgA. The intra assay coefficient variation for IgG, IgM and IgA was 2.5%, 4.5% and 5.1%, respectively.

To optimize the assay and minimize high background staining in sera from chickens in the negative control group(H), various techniques were used (Paper I): 1. Blocking with 1% tryptophan (200 l/well) for 2 hrs after coating plates with 100 g/ml BSA. 2. Same as 1) plus add sample and block again with 10% normal rabbit serum. 3. Blocking with 10% milk solution (commercial semi-skimmed, 3% milk) after coating plate with 100 g/ml BSA. 4. No blocking, but increasing the ionic strength of wash buffer from 0.15M to 0.5M. 5. Use of Capture ELISA using 1:1,200 dilution of rabbit anti-bovine albumin (100 l/well) (Nordic, Immunology Lab, Tilburg, The Netherlands) as the primary catcher antibody.

30 Since unspecific binding to BSA cannot be eliminated by the techniques mentioned above, only values with an absorbance at 492 nm larger than the mean of serum from saline treated control chickens plus one standard devia- tion were considered positive. For quantification of anti-human IgG in chicken serum (Paper II, Phase 2), microtitre plates were coated with 1 mg/ml human IgG (Sigma, Sweden) in carbonate buffer (pH 9.6, 100 l/well) and incubated overnight at 4oC in a moist chamber. The plates were washed four times in PBS-Tween-20 (pH 7.4) in between each step. The plates were incubated for 1 hour at room temperature (RT) with 1% L-tryptophan (Sigma, Sweden), washed and incubated for 1 hour with the chicken serum samples (diluted to fix the standard curve). After washing, the plates were blocked with 1:500 rabbit anti-human IgG (Sigma, Sweden) for one hour at RT. IgY purified from egg yolk of chickens subcutaneously immunized with human IgG emulsified with FIA was used as standard (Hedlund and Hau, 2001). The IgY standard preparation was diluted 2-fold starting at 1:1040 to 1: 104000. The standard serum pool was defined to contain 100 AU/ml. The IgG antibody was detected with horseradish peroxidase (HRP) conjugated rabbit anti-chicken IgG (1:10,000, Sigma, Swe- den). An OPD-substrate (100 l/well, Kem-En-Tec, Copenhagen, Denmark) was used for colour development. Treated plates were incubated for 15 min in the dark at room temperature to react after which the reaction was stopped with 1 M H2SO4 (150 l/well). The absorbance at 492 nm was read in an ELISA reader (Multiskan RC, Labsystems, Sweden). The inter and intra assay coefficients of variation were 1.35% and 1.51% respectively.

Paper III ELISA quantification of immunospecific chicken anti-CTB IgG antibodies Microtitre plates (Nunc, Roskilde, Denmark) were coated with 1 g/ml CTB (Sigma, Sweden, Cat. no. C9930) in carbonate buffer (pH 9.6, 100 l/well) and incubated overnight at 4 °C in a moist chamber. The plates were washed four times in PBS-Tween-20 (pH 7.4) in between each step. After coating and washing, the plates were incubated with the chicken serum samples diluted 1:100. As standard series, a pool of serum (n=4) from CTB+BSA immunized chickens was diluted 2-fold starting at 1:100. The standard serum pool was defined to contain 100 AU/ml. The chicken antibodies were detected with horseradish peroxidase-conjugated rabbit anti-chicken IgG (1:10,000, Sigma, Sweden). An OPD-substrate (100 l/well, Kem-En-Tec, Copenhagen, Denmark) was used for colour development. Treated plates were incubated for 15 min in the dark at room temperature to react, after which the reaction was stopped with 1 M H2SO4 (150 l/well). The absorbance at 492 nm was recorded. Intra-assay coefficient variation was 1.8% and inter-assay coefficient variation was 5.4%.

31 Paper IV and Paper V ELISA quantification of immunospecific chicken anti-BSA IgY antibodies Indirect ELISA was used to quantify the immunospecific anti-BSA IgY antibodies in egg yolk, as described elsewhere (Persdotter-Hedlund and Hau 2001). Microtiter plates (Nunc, Roskilde, Denmark) were coated with 100 g/ml BSA protein standard (Sigma-Aldrich) in carbonate buffer pH 9.6 over night (100 ml/well). The following day the remaining binding sites were blocked using 10 mg/ml L-tryptophane (Sigma-Aldrich) in PBS-Tween 20 pH 7.4 (200 l/well, 1 h with shake). The plates were washed five times with PBS-Tween 20 pH 7.4 in between each step. After washing, the plates were incubated with different egg yolk samples for 1 h with shake (100 l/well). All samples were added in duplicates. The plates were again blocked; this time using rabbit serum diluted 1:10 in PBS-Tween 20 (100 l/well, 1 h with shake). ). The chicken antibodies were detected with horseradish peroxidase (HRP) conjugated rabbit anti-chicken IgY (1:10,000, Sig- ma-Aldrich) diluted in PBS-Tween 20. These antibodies were added to the plates (100 l/well) and incubated 50 minutes in 37°C. An O- phenylenediamine dihydrochloride-substrate (OPD) (Kem-En-Tec, Copen- hagen, Denmark) was used for color development (100 l/well). Treated plates were incubated in the dark 15 minutes at room temperature to react, after which the reaction was stopped using 1 M H2SO4 (150 l/well). The absorbance at 492 nm was read in an ELISA reader (Multiskan RC, Labsys- tems, Sweden. In Papers IV and V, the concentration of each sample was described using arbitrary units as 100AU/ml defined from a pool of serum from classically immunized chickens used as standard series in Papers I, II and III. A standard curve was prepared from a pool of 3 eggs which gave the highest anti- BSA IgY titres by preparing a twofold dilution series from 1/20 to 1/5160. Since the amount of IgY in yolk is dependent of maternal serum concentration, and independent of egg size (Woolley and Landon,1995), the total amount IgY in every egg could be calculated The inter assay coefficient of variation was 3.4% and the intra assay coefficient of variation was 2.5%.

Statistical analyses Data for Papers I, II and III were analyzed by ANOVA and correlations with Excel (Microsoft) software programme. P values <0.05 were considered significant. For Paper IV, non-parametric statistics was used due to the non- normal distribution of the data. To compare between groups, these were collapsed as “Low protein without RhinoVax® (LP)”, “High protein without RhinoVax® (HP)”, “Low protein with RhinoVax® (LP+RV)”, “High protein with RhinoVax® (HP+RV)” and control. Five groups were analyzed using

32 Kruskal-Wallis test, and the results exposed week by week. To compare between 3 and 5 days groups of high protein with RhinoVax®, Mann- Whitney test was used. The entire test was performed with SPSS v. 12.1.01. To analyze data of Paper V, non-parametric Mann-Whitney tests based on ranks were performed with SPSS 147 v. 14.0. Values were considered significant when p<0.05.

Ethics committee license All experimental protocols involving animals were approved by the Regional Ethics Committee on Animal Research at Tierp, Sweden

33 Results

Paper I Mayo, S.L., Persdotter-Hedlund, G. Tufvesson, M., Hau, J. 2003. Sys- temic immune response of young chickens orally immunized with bovine serum albumin. In Vivo 17:261-268. This study investigated the efficacy of oral immunization of an antigen, BSA with and without commercial adjuvants, in terms of mounting a systemic immune response in 15-day old chickens (age of the chicken at the time of the first oral administration of antigen-adjuvant mixture). CTB of varying concentrations of RV(formerly Softigen®) were used as adjuvants. Chickens orally fed by gavage with 600μg of BSA mixed with 5, 10 or 20% RV or with 11 mg BSA conjugated to 0.2 mg CTB, produced circulating immunospecific anti-BSA IgG, IgM and IgA antibodies. On the other hand, chickens first immunized at 15-days old and fed daily with 6 mg BSA in 0.2 ml H2O for 5 days with a total of 30 mg BSA without adjuvant, produced IgM and IgA antibodies in their circulation but not IgG (Fig 4a, Paper I) How- ever, all three antibody classes were observed in the serum of 22 days old chickens (Fig 4b Paper I) IgG and IgM serum concentrations were 4 to 10 times higher in CTB-and RV-treated groups compared to groups given BSA alone. The IgA serum levels in groups fed with BSA alone were about the same as those of adjuvant-treated groups. There was no significant difference in immunospecific titres between oral-treatment groups. Chickens subcutaneously immunized with 120g BSA emulsified in FIA exhibited a stronger immune response, approximately 10 times higher IgG, IgM and IgA concentrations compared with chickens orally immunized with CTB and RV.

34 Paper II Mayo, S.L., Tufvesson, M., Carlsson, H-E., Royo, F., Gizurarson, S., Hau, J. 2005. RhinoVax® is an efficient adjuvant in oral immunization of young chickens and cholera toxin B is an effective oral primer in traditional subcutaneous immunization with Freund’s incomplete adjuvant. In Vivo 19(2):375-382. No significant differences were found among chickens orally treated with RV of different concentrations in the previous study (Paper I). Therefore, a higher concentration of RV, 20% and 100%, was used for this study, to determine the most efficient dose for oral immunization of young chickens. Fifteen-day old White Leghorn female chickens approximately 50% in-bred, originating from 11 hens were divided into 5 treatments groups so that there was one sister in each treatment group. Phase 1 involved oral administration of an antigen, BSA, in combination with various adjuvant preparations, either CTB and/or RV. A positive control group received BSA emulsified in FIA by subcutaneous injections. All chickens responded with immunospecific titres of IgA, IgM and IgG. Classical parenteral immunization with FIA was generally the most potent. The highest IgG titres recorded in the orally immunized chickens were in the group immunized with 20% RV as the adjuvant. The titres in this group were higher than those recorded in the other orally immunized chickens and approximately 5 times lower than those recorded in the FIA group. In Phase 2, all chickens were subjected to traditional subcutaneous immunization with another antigen, human IgG. The two groups of chickens that received CTB orally during Phase 1 responded with significantly higher titres than did the other chickens indicating that oral administration of CTB prior to traditional parenteral immunization may have a priming effect on the humoral immune system. The immunospecific antibody response varied between the 11 families of chickens. There was no correlation between familial responsiveness to oral and subcutaneous immunizations. Families that were high responders to oral immunization were not high responders to parenteral immunization and vice versa.

35 Paper III Mayo S., Royo F., Hau J. 2005. Correlation between adjuvanticity and immunogenicity of cholera toxin B subunit in orally immunised young chickens. Brief report. APMIS 113:284–287. The observations in Paper II indicated that variation in immune response may be correlated with genetic variation. However, the results here showed that there was no significant difference between the immune responses of the outbred and the partly inbred chickens. As expected, the titres against BSA and CTB increased throughout the 42-day study period. Furthermore, There was a positive correlation (r=0.66, n=39, p<0.001) between the immunospecific antibody concentrations against BSA and the adjuvant CTB in the individual samples obtained throughout the study period, demonstrating the correlation between adjunctivity of CTB to it’s immunogenicity.

36 Paper IV Mayo S., F. Royo, M. Tufvesson, H-E Carlsson and J. Hau. 2006. Im- munospecific antibody concentration in egg yolk of chickens orally immunised with varying doses of BSA and a mucosal adjuvant, Rhino- Vax® using different immunization regimes. Scand. J. Lab Animal Sci. 33(3): 163-169. In Paper II we showed that 20% Rhinovax® (RV) was the efficient concentration of oral adjuvant in producing a peripheral antibody response. Paper IV described the effect of varying doses of antigen (BSA) i.e., between 25 and 300 mg total bovine serum albumin (BSA) with and without 20% Rhi- noVax® and; days of administration, i.e., 3 or 5 consecutive days, as a way to develop immunization scheme that would boost IgY concentration after oral booster immunizations. Furthermore, Paper IV analyzed anti-BSA immune response in the egg yolk (IgY) instead of the circulation or blood serum (IgG) as used in Papers I, II and III. Hens fed with BSA in combination with 20% RhinoVax®, contained significantly higher concentrations of immunospecific IgY than did egg yolks of chickens fed with BSA without adjuvant. The most efficient dose in the RhinoVax®-treated groups was 250 to 300 mg total BSA regardless of whether the chickens were initially immunized daily for three or five days (Fig 1, Paper IV). Continuous feeding of high doses (200mg/ml) BSA w/ 20% RV of either 3 days (HP3d+RV) or five days (HP5d+RV) showed high IgY titre compared to other treatment groups, i.e. LP+RV or BSA, on a week by week analyses. Only the high dose groups developed IgY titre at the 9th and 13th week while other groups did not. No differences among groups were observed in the 1st and 4th week. Furthermore, analysis of individual chicken eggs sampled every 4 weeks showed that all of the 5 chickens in the HP+ RV (3 day or 5 day) groups responded to the treatment as compared to the LP+RV treated and the BSA alone-treated groups, wherein at most 2 of 5 chickens responded (data not shown). Groups given low protein doses of BSA (25-30 mg total BSA) with Rhi- noVax® (LP 3d or 5d +RV) produced anti-BSA IgY titres but was within almost the same range as treatment groups fed with BSA alone or control. An IgY response in BSA alone-treated groups were observed whereby groups fed for 3 days of either low or high protein had one out of five chickens responding as early as week 4. However, compared with the RV group the IgY titre of the BSA fed group are still lower, even almost the same titre as the control. Data of Fig 2 (Paper IV) where the average of 5 eggs from 5 individual chickens in each treatment group taken every week for the entire duration of the experiment showed that highest titre was often after the 2nd immunization. Although Fig. 2 shows a peak on week 3, this peak correspond to the

37 highest titre one single egg sample from one chicken (see Table 1, chicken # 6329), which responded to the first immunization and did not recover in the succeeding weeks. Unlike the 2nd immunization (given by continuous 3 or 5 day feeding), the 3rd immunization did not give a high titre, equal or more than the observed peak at week 7 or week 8. Neither was there a significant difference in the highest IgY titre from 5 egg samples between HP5d+RV versus HP3d+RV groups in weeks 13.

38 Paper V Mayo, S.L., H-E. Carlsson, A. Zagon, F. Royo and J. Hau. 2009. En- hancement of anamnestic immunospecific antibody response in orally immunized chickens. Journal of Immunological Methods 342:58-63 Having observed from the Paper IV study that memory can be developed after daily oral immunization for 3-5 consecutive days but was not able to sustain a traditional booster effect; this study aimed to evaluate immunization schemes in which immunospecific IgY concentration would increase and be sustained following oral booster immunizations. Four treatment groups with five chickens per treatment group were subjected to various immunization regimes. Two groups, Groups 1 and 2 of egg laying hens (5 in each group) were immunized orally (each immunization event consisted on dosing on three consecutive days) with BSA in combination with RV using different immunization schemes. Group 3 was given parenteral immunization with FIA followed by an oral booster of BSA with RV. Lastly, Group 4 served as a negative control given water throughout the treatment period. In Group 1, given 3 oral immunizations weeks 1, 4 & 7 followed by an oral dose 17 weeks after the first immunization, two chickens (# 1143 and #1220) responded to all four immunization periods, another 2 chickens (#1343 and #1151) responded to the last three while one chicken (# 1212) showed a low response after the third immunization period. The chickens in Group 1 reached a significantly higher ‘plateau’ (weeks 12-18) between the last two immunization periods compared to the period between the first two immunizations. After the fourth immunization period (Phase 2) all chickens exhibited a significant (p<0.018) increase in immunospecific anti- BSA antibodies in the egg yolks (Fig 1, PaperV). In Group 2, given 2 oral immunizations weeks 1 and 7 followed by an oral dose 17 weeks after the first dose four out of five chickens responded to every immunization period. One chicken (#2306) maintained an IgY egg yolk contents around 100-300 AU between the second and third immunization period (Figure 2, Paper V). This chicken exhibited a relatively strong reaction giving the highest contents: 582 AU at day 57 after the second immunization. Anti-BSA IgY stayed at a high level (starting around 200 AU) after the second immunization, further increasing in titre beyond the 3rd immunization, week 18 (Phase 2). The IgY concentration was maintained at a high level and did not decrease to a level similar to that between the first and second first immunization. Likewise, chicken #2261, a weak reaction after the second immunization compared to the first reaction, showed a significantly higher response after the third immunization and onwards of Phase 2. Group 2 chickens had a significantly higher IgY response in Phase 2 (after week 18, the last immunization period) than group 1 chickens (p<0.05). In group 2, peak 3 at 46 dpi, was significantly higher than both peak 1 at 14 dpi (p<0.002) and peak 2 at 56 dpi (p<0.005).

39 Comparison of the means of group 1 and 2 for the whole period showed that group 2 chickens had a significantly higher IgY-concentration than group 1 (p<0.002). The total yield of anti-BSA IgY of eggs harvested from chickens in Group 1 was a significantly lower (6%) than Group 2, (12%) in comparison to parenterally immunized positive control Group 3. Group 3 chickens had clear signs of booster effects after each subcutaneous immunization period. The eggs of Group 3 chickens, had significantly higher immunospecific anti BSA IgY-concentration than did any of the eggs from the orally immunized chickens (p<0.001). In Phase 2, one chicken (#2331) died spontaneously and chicken #2314 laid only seven eggs. Chick- ens #2131 and #2138 responded to the oral immunization, as did chicken #2232, however only slightly (Figure 3, Paper V). The peak after the oral booster immunization in Group 3 was significantly higher than any of the earlier minor peaks (p<0.003). A fairly steady IgY production could be seen in all chickens up until the last immunization. None of the chickens in Group 4 that received only water by gavage developed anti-BSA antibodies.

40 Discussion

Oral immunization is preferable as an easy, safe and less invasive route for delivery of antigen than the invasive classical immunization which involves injection either intramuscular, subcutaneous or peritoneal routes. The development of an oral immunization technique for subsequent use in non- invasive polyclonal antibody production is a strategy to totally eliminate distress associated with antigen administration in the animal used for antibody production. Researchers have conducted studies comparing oral immunization strategies aimed at inducing a simultaneous peripheral and mucosal immune response with the aid of adjuvants. However, the usual response of the mammalian gastro-intestinal tract is tolerance rather than immunity. On- ly certain antigens seem to stimulate a mucosal, peripheral or combined immune response and usually only when used in combination with an adjuvant (for review see Mann, et al, 2009). Use of adjuvants has been made in the development of new vaccine delivery systems in which antigens have been administered orally or intranasally in biodegradable biospheres or associated with carrier proteins derived from cholera toxin (CT) (Holmgren et al., 1993;1994; 2003; 2005; Lebens and Holmgren, 1994; Sanchez and Holmgren, 2008) or Escherichia coli heat labile enterotoxin (Rask et al, 2000; Elson and Ealding, 1984a; 1984b; El- dridge et al., 1991; Dertzbaugh and Elson 1991; Challacombe et al., 1992; Elson and Dertzbaugh, 1994). Similar positive results have been obtained in animals using glyceride based adjuvants and nasal administration (Gizurar- son et al., 1998a, Jakobsen et al., 1999, Hrafnkelsdottir et al, 2005). In hens, Hedlund and Hau (2001) used oral immunization to test a number of adjuvants including Poly (lactide-co-glycolide) (PLG) microspheres mi- croencapsulation of the antigen (Aguado and Lambert 1992; Alonso et al., 1994; Hoshi et al., 1999b); Cholera toxin B-subunit (CTB) which results in a strong peripheral and mucosal immune response in mice (Elson and Dertz- baugh, 1994); PLG combined with CTB, CTB conjugated with glutaraldehyde, Dimethyl dioctadecyl ammonium bromide (DDA), and RhinoVax® (formerly Softigen®). They observed that both CTB and RhinoVax® resulted in egg yolk immunospecific antibody concentrations above the positive controls (classical s.c. immunization with FIA). CTB and RV may both be alter- natives to Freund’s Adjuvant (FA) which is the adjuvant most commonly used in antibody production.

41 Very few studies have investigated if young chickens, less than one month old, could mount a systemic immune response when fed with an antigen alone or with antigen conjugated to an adjuvant (Klipper at al., 2000; 2001). In Paper 1, very young chickens (> 15 days old) produced a peripheral immune response following an oral immunization with RV or CTB as adjuvants, or even with the antigen alone. Chickens fed continuously for 5 days with the antigen alone (BSA), when they were 15-20 days old or when they 22-27 days old, both produced immunospecific IgM and IgA responses, but an IgG response could only be detected in the older age group. It seems likely that the age when the first immunization is given is an important factor. Klipper and co-workers (2000) reported that chickens initially fed with BSA solution as early as 10 days old produced serum antibody responses. These responses were, however, lower compared to the immune response of chickens fed with antigen and adjuvants. Our study in Paper I confirmed that the IgG response was low in chickens fed without adjuvant. The IgG level was even below the detection level in the group initially fed at 15 days old with BSA alone. CTB and RV caused a stimulating effect on the humoral response even in these very young chickens. The IgG and IgM levels were more than 4 and 10 times, respectively, and higher in the CTB and RV groups compared to chickens not given adjuvants with the antigen. On the other hand, none of these adjuvants had an effect on the IgA response as the concentrations in both groups fed with antigen alone or with adjuvants were similar. These responses, however, were not significantly different between treatment groups. This may be due to the large variation in antibody concentrations between animals within the individual groups, specifically because about half of the chickens in the study did not produce sufficient immunospecific antibody to result in concentrations above the sensitivity of the assays. The use of out-bred chickens may account for the large variations as it is known that antibody response to most antigens is under polygenic control (Hendrik- sen and Hau, 2002). Further test therefore calls for larger group sizes and inbred animals. To follow through the results from Paper I, another study with higher RV concentrations (20% and 100%) were tested using families of partly inbred chickens and bigger group sizes (Paper II, Phase 1). Upon comparison among the orally-treated groups, 20% RV was the most efficient adjuvant in inducing an IgG antibody response in chickens. Oral immunization with 20% RV was more potent than 100% RV, perhaps because water may be a necessary factor for keeping the correct three-dimensional structure when antigen is presented for the antigen presenting cells Gizurarson et al, 1995 noted that the relationship of hydrophile-lipophile balance value (HLB) which is the balance between size and strength of hydrophilic and lipophilic groups to the immunological response of various organs and biological fluids after an intranasal vaccination of diphtheria toxoid suspended in non-

42 inonic surfactants polysorbate 20 or Span 85. Formulation having HLB= 9.0 (with 12.5 being the highest HLB value) gave the best result of IgG, IgA and IgM concentration in the serum, spleen, lungs and the GI tract. Likewise, Verheul and Snippe (1992) described that some non-ionic block polymers having HLB values between 1.0 and 2.9 were the best to stimulate both humoral and cellular responses. Earlier studies of Hunter et al, 1981, 984 showed that large insoluble polymers adjuvants with hydrophilic moieties flanking hydrophobic moieties favored chemotaxis, complement activation and antibody formation. These studies demonstrate that the host response to an antigen depends upon its local presentation in addition to its concentration, and such response raises the possibility of manipulating antigen presentation for therapeutic advantage. Their use is dependent upon the ability to alter an antigen presentation on the surface of the oil droplets. The effect of type and concentration of a cosurfactant and oil on the ability of nonionic surfactant PEG-8 caprylic/capric glycerides (Labrasol®) to solubi- lize both oil and water phases plays a factor in the adjuvants efficacy. Djekic and coworkers (2008) evaluated. the amount of surfactant required to completely homogenize equal masses of oil and water to form a single phase microemulsion (termed as balanced microemulsion) (S min, %w/w), the mini- mal concentration of the surfactant/cosurfactant blend required to produce a balanced microemulsion (SCoS min, %w/w) as well as the maximum concentration of water solubilized in investigated surfactant/oil and surfactant/cosurfactant/oil mixtures [W(max), %w/w]. Results indicated that Labra- sol® showed a good efficiency in the presence of lower molecular volume fatty acid esters with a preferred chemical structure such as isopropyl myris- tate [S min 56.14% (w/w); W(max) 12.28% (w/w)]. Oils with high molecular volume (olive oil and mineral oil) do not result in microemulsion formation). The chemical structure of RV is very similar to the asylglycerides that are found as breakdown (apoptosis) products in inflamed tissue. These com- pounds stimulate the macrophages through the Fc receptor and affect the production of arachidonic acid. They are found to augment the antigen presentation cells and dendritic cells and stimulate the cytokine production between macrophages and B- and T-cells (Gizurarson, pers. comm.). Although the IgG concentrations induced by 20% RV were 5 times lower than in the FIA immunized group, this level of response by an oral regime seems for animal welfare reasons an attractive practical alternative for egg yolk antibody production as compared to the more invasive technique of injecting antigen with FIA emulsions. The IgM and IgA responses were found to be significantly higher in the chicken group orally immunized with CTB+RV combined than in the other groups (oral). Moreover, both mean IgM and IgA concentrations of the CTB+RV treated groups were similar to those of chickens immunized with FIA. The combination of RV and CTB thus seems to have a synergistic effect on the immunospecific IgM and IgA response.

43 When the chickens in the same treatment groups were parenterally immunized with another antigen, human IgG (Paper II, Phase 2), the chickens in the groups that had received CTB orally in the Phase 1 study produced significantly higher IgG antibodies against human IgG as compared to the other groups in Phase 2. This indicates that CTB seems to act as a primer in animals subsequently immunized by the classical parenteral route with FIA. The mammalian antibody response to most antigens seems to be geneti- cally determined and under polygenic control involving histocompatibility genes (Long, 1957; Lifschitz et al., 1980). In order to determine if genetic variation was a factor contributing to animal variation in the immune response and to test if chicken responsiveness to oral immunization is correlated with responsiveness to traditional (subcutaneous) immunization, sisters from hens were allocated to the various treatment groups in the study reported in Paper II, Phase 2. The study showed a clear and significant difference between families in their humoral immune response. This result is consistent with other reports on mammals. Outbred stocks of rabbits, although selected through generations for high antibody response, have been docu- mented to exhibit a remarkable inter-individual variation in their immune response (Harboe and Ingild, 1983). Carlander and colleagues (2003) reported that IgY concentration varies between chicken genotypes and individual chickens of the same line. The immune response in chickens has also been demonstrated to be subject to genetic variation (Gehad et al., 1999; Al- Murrani et al., 2002; Carlander et al., 2003). In the present study, it was found that chicken lines that produce a good humoral response to parenteral immunization may not necessarily respond equally well to oral immunization and vise versa. The present study suggests that the humoral response appears to be under genetic control and that the response is dependent on the mode of antigen administration. It is known that the efficacy of cholera toxin subunit B (CTB) as an adjuvant in inducing an immune response also plays a role in oral immunization and subsequent production of immunospecific antibody (Holmgren et al, 2005; 1994; 1993). The Paper III study re-enforced the concept that the adjuvant effect of CTB is associated with its immunogenicity. We showed a positive correlation between the concentrations of antibodies against BSA and antibodies against the adjuvant CTB suggesting that the adjuvant effect of CTB is associated with its immunogenicity. Other studies have likewise elucidated a correlation of CTB’s adjunctivity with its immunogenicity. To- chikubo and co-workers (1998) showed that by using recombinant CTB (rCTB) produced by Bacillus bevis carrying pNU212-CTB, CTB alone was able to enhance both mucosal IgA and serum IgG immune responses to BSA given by intranasal and oral immunization in mice. Similar results were found by Park and colleagues (2003) where CTB and rCTB enhanced BSA- specific antibody titers of IgM, IgG1 and IgA, both in sera and in the intestinal and peritoneal fluids of intraperitoneally immunized mice. Also, in a

44 study of a vaccine, Peru-15pCTB, a plasmid construct engineered by site- directed mutagenesis of CTB to Peru-15, a live attenuated Vibrio cholera strain demonstrated 30-fold higher anti-CTB serum IgG titers in mouse and rabbit models than in Peru-15 vaccine alone (Roland et al., 2007). Based on the principle that that the reactivity of CTA subunit could only be detected if CTA was associated with CTB (Holmgren,1982). Sanchez and colleagues (2002) explored to generate detoxified cholera toxin without loosing its im- munoadjuvant properties by making CTA constructs and reacting with CTB by GM1-ELISA. They elicited significant anti-CTB antibody serum responses after immunization of mice via nasal route. There were no significant differences in immune response between the inbred and outbred chickens against BSA or CTB in Paper III, perhaps because all chickens originated from the same lines. The significant relationship shown between concentrations of antibodies against BSA and CTB may provide a tool which can be used to compare the usefulness of different stock or strains of chickens for antibody production by correlating the concentration of the antibodies of any antigen with specificities against CTB. Optimization of the dose of an antigen-adjuvant complex is important for the efficacy of the immunogen. Paper IV showed that the most efficient dose in the 20% RhinoVax®-treated groups was 200 mg/ml BSA regardless of whether the chickens were initially immunized daily for three or five days. Moreover, the same dose treatment administered to hens for three continuous days the first week, and every 3rd week thereafter for three months followed by a booster, showed an increasing IgY titre in the chicken eggs (Paper V). Both results in Papers IV and V provided some evidence of immunological memory in chickens based on the increase of IgY concentration starting on the 2nd immunization treatment (Paper IV). Also, a booster effect was clear- ly demonstrated in the last single dose immunization in week 18 in Paper V, thereby showing the presence of memory cells following the two previous oral immunizations, i.e. weeks 4 and 7. In addition, the consistent results of significant immune response in the above studies confirmed that RhinoVax® is an efficient oral adjuvant In Paper IV, immunization dynamics of HP+RV groups based on weekly analyses of 5 eggs from individual hens revealed that the highest titres were observed on weeks 7 or 8, between the 2nd and 3rd immunization in 8 out of 10 chickens (Figure 2). The difference between 3 or 5 days feeding was not significant upon comparison of IgY peak values for both treatments. A constant peak for all the 5 eggs collected every week was not observed as each chicken responded at different times but the majority of the highest titres was observed on weeks 7 or 8 for either 4 of 5 chickens in the HP3d+RV group or 5 of 5 chickens in HP5d+RV group (Table 1). The chickens received oral booster immunizations one and two months after the initial immunization. No real effect could be recorded after the second and third immunization. Although the IgY responses to the booster

45 treatment did not exhibit a traditional booster effect whereby high IgY concentrations are maintained and increase towards a new plateau, this study provided some evidence of memory based on an optimum IgY concentration recorded for each individual chicken in the later period of the treatment. The studies reported in Paper V demonstrated a clear manifestation of a booster effect of the immunization in week 18 or Phase 2 in Paper V. In Group 2, all chickens exhibited higher immunospecific IgY response in all three immunizations starting at week 1, 7 and continued beyond week 18, although at varying levels among the chickens. Group 1 showed a similar response whereby 4 of 5 chickens produce increased IgY to all 4 immunizations from weeks 1, 4, 7 to week 18. Group 2 had 12% of the amount of IgY produced by the parenterally immunized positive control Group 3. While Group 1 had 6% compared to Group 3. This suggested that intervals longer than 3 weeks between immunization events are beneficial to obtain a sus- tainable high antibody concentration in egg yolk. Other studies suggested a similar result whereby a strong anamnestic response lasting 4 weeks after booster immunization was observed in mice given multiple oral and nasal immunization liposomal Strep. mutants coupled to a lipid mucosal adjuvant (Childers et al, 2000). The vaccine given over a 2 hour period for 2 days with a booster administered 14 weeks after initial immunization showed significant IgG anti-S.mutants response given by nasal route. This anamnestic response was observed starting at 2 weeks post booster and lasted for another 2 weeks. Del Rio and co-workers (2008) likewise showed that 4 weeks interval between a total of three oral (intragastric) immunizations of a recombinant antigen in mice produced IgG antibodies 4 weeks post immunization and increased considerably for another 5 weeks thereafter. On the contrary, a single injection of E. coli lysate containing a recombinant clone from a tick strain failed to elicit a protective anamnestic response in mice (Gilmore, et al, 2003). The reason for this failure in immune response might be that memory cells were not activated by single priming and that a longer lasting immune response is dependent on the circulating antibodies provided by booster immunizations subsequently producing an expansive anamnestic response. In both orally immunized groups, there were large inter-individual variations in the amounts of anti-BSA IgY produced and in the responses to booster immunizations. This implies that selection and breeding of animals with a strong immune response could be a possible way to increase the efficacy of this method. However, it is not certain that a strong response to BSA guarantees strong response to other antigens of interests. In conclusion, this is the first demonstration of a clear anamnestic immune response in orally immunized chickens and the results suggest that by modifying the interval between booster immunizations it may well be possible to increase the concentrations of immunospecific IgY thereby stimulating antibody producers to make use of this more lenient alternative to parenteral

46 immunization. Although oral immunization results in approximately ten times lower concentrations of immunospecific antibodies in the egg yolk, compared to traditional subcutaneous immunization schemes, the oral immunization routines, when further refined, may eventually compete with parenteral immunization protocols in terms of practicality and efficacy.

47 Summary

The results presented here are considered the first phase toward the ultimate goal to develop a non-invasive route for antigen administration. The delivery system of antigen for the experiments presented here was by gavage, which albeit not an invasive technique, may be considered not entirely stress-free for the animal. However, this is an initial stage of the research project to provide basic reliable information of the efficacy of a pegylated mono/diglyceride (RhinoVax®/Softigen®) as adjuvant for antigen administration in young chickens. Once the dosage formulation has been optimized, the next step will be to develop a practical delivery system of the antigen whereby the antigens will be voluntarily ingested by the chickens through e.g. incorporation of the antigen-adjuvant mixture into an attractive food item for the chicken. The antigen may be incorporated into a food item, either as a solid or as a liquid solution. As a model, we prepared an antigen/adjuvant mixture and fed each chicken with the mixture through ga- vages. Using the basic knowledge of the efficacy and dosing of an antigen- RhinoVax® mixture gained in this thesis, this should provide avenues for future researches to test different ways to feed an antigen-adjuvant mixture to the chicken. The amounts of immunospecific IgY produced in these studies were lower than those harvested from traditional parenteral immunizations, and may seem disappointing. However, considering that this antibody production method can be accomplished with the use of unregulated procedures, not requiring a license from the authorities, and that the amounts of antibody produced per egg-laying hen is probably more than what can be achieved from a parenterally immunized rabbit (Bollen, 1997), it may still be an attractive alterative in large scale production systems. The potential of oral immunization of chickens as a non-invasive alternative to traditional antigen administration has been demonstrated. Combined with harvest of antibodies from chicken eggs, oral immunization through voluntary intake of a camouflaged antigen-adjuvant mixture, will completely eliminate all discomfort from animals used for polyclonal antibody production. The long-range objective of this study was to fully implement the concept of the Three Rs (Replacement, Reduction, and Refinement) according to Russell and Burch (1959) to polyclonal antibody production:

48 Replacement: Because traditional immunization regimes involve injections and blood sampling, animals used for immunization are currently cate- gorized as laboratory animals, and their use is regulated like any other use of laboratory animals. Ultimately, this project will result in procedures, which are not stressful for the animals, and the production can be carried out at normal egg producing farms. Reduction: The antibody productivity of chickens is far greater than that of mammals of similar size because egg-laying chickens produce eggs with an antibody concentration similar to that of blood. Consequently, the number of animals can be greatly reduced from switching from small mammals like rabbits to chickens. Refinement: The oral immunization implies that antigen can be administered camouflaged in food eliminating the stress associated with capture, restraint and antigen administration.

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60 Acknowledgements

My deepest gratitude to the following people who meant so much to me in helping complete this thesis….

To my supervisors, Professor Hans-Erik Carlsson and Professor Jann Hau, for your patience, unceasing encouragement and confidence in my capability as a scientist. Your positive view of situations, especially in stressful mo- ments, has given me the inspiration to reach my dream. Thank you so much for not giving up on me!

Professor Hans-Erik Carlsson, to whom I so owe much for offering me to join the Department of Comparative Medicine that gave me the best years of working with a wonderful group of scientists. Thank you for helping me finalize my thesis.

To Professor Sveinbjörn Gizurarson for your collaboration and guidance in understanding the mechanism of RhinoVax®.

Dr.Måns Tufvesson for your constant help and support in this study and for allowing us to use your animals.

My former colleagues at the Department, Gabriella, Klas Mahinda, Felix, Emma and Lena, a million thanks for sharing your time, knowledge and friendship that will be forever be part of my cherished memories at BMC. Thank you for discussing “science”, patience in all my questions, teaching me Swedish and of course sharing those wonderful picnics and dinners! Tack så mycket!

To Professor Kenneth Söderhäll and Dr. Irene Söderhäll at the Department of Comparative Physiology who opened the opportunity for me to study in Sweden. I’m forever grateful for all your support during my early years in Uppsala.

To my current supervisor at the University of California San Diego, Dr. Pa- mela Mellon, for allowing me to take off from work to go to Sweden for the PhD defense and to all my lab colleagues, for all your constant encouragement to complete my PhD studies. My sincerest thanks to you all!

61 My Filipino, Swedish and other international friends, Jopac, Josie, Malu and Sepideh, Veron, Ramon, Eliza,Gloria, Jealy,Merose, Niklas, Keith and Inger, Doris, Berit and Leif , Wilhelm and Marie-Louise Ahlgren, Pikul, So Young and Felix for your prayers, constant support, friendship and enjoyable get- togethers. To my mom, Amy and stepfather, Karl, and my family in San Diego, Detz, Kuya Zaldy, Sazah, Jillian, Tintin and Ms. Rosey for your un- conditional love and faith in me.

My husband, Alex and my “babies” Blessie and Jamie, for understanding and loving me…always. You are my purpose for being. I love you so much!

Finally, I give praise to the Lord Almighty for being the source of strength and faith for me and my family and for blessing me with the presence of all these people!

The projects conducted in this thesis received generous funding from the Swedish National Board for Laboratory Animal Science and the Swedish Research Council for Environment, Agricultural Sciences and Spatial Plan- ning.

My sincere appreciation to the International Foundation for Ethical Research (IFER) that kindly provided financial support through the “Graduate Student Fellowship” grant.

62 Errata

Paper I: page 262, right hand column, 1st paragraph Group G: 120 mg should be μg

Paper II: pages 337- 339, Figures 1-4 labels (x-axis) SF should be RV

Paper IV: page 165, Table 1, under “Days x volume” ml should be μl

Paper IV: pages 166- 167, Figures 1 and 2 Missing labels and legends. See corrected Figures 1 and 2 below

4 1 wk 4 wk

AU/ml 3 9 wk 2 13 wk

0 control LP 5 daysLP 3 daysLP HP 3 daysHP HP 5 days 5 HP LP 5d + RV + 5d LP RV + 3d LP HP 5d + RV 5d + HP RV 3d + HP Treatment Groups

Fig 1. Immunospecific anti-BSA IgY concentrations of chickens in all treatments groups. Data represents average the average of 5 eggs from individual hen in each treatment group (5 animals each group) analyzed each month. Error bars ± SEM

Legend: LP: Low protein , 20 mg/ml (5 days: 5 consecutive days of 250 ul) HP: High protein, 200 mg/ml (3 days: 3 consecutive days of 500 ul + RV: with adjuvant RhinoVax® (RV) 5d: 5 consecutive days ; 3d: 3 consecutive days Weeks: 1 : 1st immunization (multiple dose) 4 : 2nd immunization (multiple dose) 9 : 3rd immunization (single dose) 13: end of experiment

63 Anti-BSA IgY of HP 5d and 3d+ RV treated groups 20 18 16 14 12 10 HP5d+RV

Au/ml 8 HP3d+RV 6 4 2 0 12345678910111213 weeks

Fig 2. Average anti-BSA IgY titres from 5 eggs of 5 individual chickens analyzed weekly. Each value is the average IgY titre of 5 eggs. Arrows indicate time of immunization. Weeks: 1 : 1st immunization (multiple dose) 4 : 2nd immunization (multiple dose) 9: 3rd immunization (single dose)

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