IMMUNOGENIC POTENTIAL OF EGG SHELL MEMBRANE IN COMBINATIONAL ADJUVANT SYSTEM OF RECOMBINANT HEPATITIS B VACCINE IN MICE

ABSTRACT

Hepatitis B caused by the Hepatitis B virus (HBV) is one of the most common infectious diseases in the world and a major health problem. Its preventive measure is seen in hepatitis B vaccine. The inability of alum adjuvanted hepatitis B vaccine to trigger Th1 response and the need for reduced booster doses necessitate the quest for novel adjuvant. The present study is  aimed  at  evaluating  the  immunogenicity  of  recombinant  hepatitis  B  vaccine  when combined with egg shell membrane (ESM) as a possible adjuvant for the prevention of hepatitis B infection. There was no difference in the trend with which the mice gained weight in their different groups. There was non significant (p>0.05) decrease in the liver weight of the treated mice. There was non significant (p>0.05) difference in the activity of the liver marker enzyme aspartate aminotransferase (AST) when compared across the groups. The alanine aminotransferase (ALT) levels indicated a non significant (p>0.05) decrease when HBV-ESM  immunised  mice  were  compared  to  HBV  (2  doses)  and  HBV  (3  doses) immunised mice. The cellular component of the immune system showed relative differences in the differential percentage count when compared across and within the various groups. The neutrophil count showed non significant (p> 0.05) difference across the groups on day 0 and day 14 after first immunization. But on day 21 there was significant (p< 0.05) decrease across the groups. The lymphocyte counts showed a significant (p<0.05) increase within the groups on days 14 to 21. Across the groups the lymphocytes showed a significant (p<0.05) increase when HBV- ESM immunized mice were compared to HBV (2 doses) and a non significant (p>0.05) decrease when compared to HBV (3 doses) immunised mice. The eosinophil counts showed non significant (p> 0.05) increase within the groups across the days and a non significant (p>0.05) decrease across the groups. There was no basophil recorded. The monocyte counts showed significant (p< 0.05) increase when HBV- ESM immunized mice were compared to HBV (3 doses) immunized mice and HBV (2 doses) immunised mice on days 21 and 28. The IgG titre showed a significant (p< 0.05) increase on days 21 and 28 when HBV- ESM immunized mice were compared to HBV (2 doses) immunised mice but non significant (p> 0.05) difference compared to HBV (3 doses) immunised mice. A significant (p< 0.05) increase was observed in IgG titre within HBV (3 doses), HBV (2 doses) and HBV- ESM immunized mice on days 21 and 28.  The IgG1 titre across the groups on day 14 showed that HBV-ESM immunized mice increased significantly (p< 0.05) when compared to mice immunized with 3 doses of HBV. On day 21 and 28 a significant (p< 0.05) increase was observed in the IgG1 titre of HBV- ESM immunized mice when compared to HBV (2 doses) immunised mice. IgG2a showed a significant (p< 0.05) increase in HBV- ESM immunized mice when compared to both HBV (3 doses) and HBV (2 doses) immunised mice. Based on these findings, the present study showed that Hepatitis –B vaccine mixed with egg shell membrane at 2 doses elicited  more cellular and humoral response when compared to the recombinant hepatitis B vaccine at 2 doses and non significantly different when compared to the recombinant hepatitis B vaccine administered at 3 doses.  Hepatitis –B vaccine mixed with egg shell membrane at 2 doses shows no toxicity and as such it is not lethal.  Summarily, it could also be inferred from the results that the ESM adjuvant could be acting through cellular recruitment evident in the increasing number of cellular components during the course of the study and also through formation of depot sites seen by the slow and continued release of vaccine antigen resulting in progressive increase in antibody titre.

CHAPTER ONE

INTRODUCTION

Hepatitis B is one of the most common infectious diseases in the world and a major health problem. It is caused by the Hepatitis B virus (HBV) which is a DNA virus of the family Hepadnaviridae (Bertoletti and Gehring, 2006). World Health Organization estimates that about 2 billion people worldwide have serologic evidence of past or present HBV infection, and 350 million are chronically infected and at risk for HBV-related liver disease (WHO,

2015). The large infected populace and the hyper virulence of the causative agent makes it pertinent to provide highly efficient curative and preventive measures hence the advent of hepatitis B vaccine in the management of hepatitis B infection (Gerlich, 2013).

Most recent immunization against HBV is active vaccination with hepatitis B vaccine (HBsAg) which is an antigen used in the formulation of the hepatitis B vaccine produced from yeast through recombinant DNA technology (WGO, 2015).   The currently available vaccine is a recombinant vaccine devoid of live microorganism and as a result needs an adjuvant to boost the immunogenicity of the vaccine.

Adjuvants are substances that can enhance and modulate the immunogenicity of the vaccine antigen  and  are  particularly  important  when  the  vaccine  antigens  are  purified  and  lack intrinsic  innate  and/or  adaptive  immune  triggers  (Garçon  et  al.,  2012).  The  currently available recombinant HBV vaccines contain hepatitis B virus surface antigen (HBsAg) as the antigen and aluminum based compounds as an adjuvant.  Aluminum based adjuvants is the most commonly licensed adjuvant in vaccines for human use and   has been shown to predominantly activate Th2-type humoral immune responses, but is poorly effective in inducing  Th1-type  cellular  responses  essential  for  the  treatment  of  viral  infections (Apostólico et al., 2016). Irrespective of tremendous positive results in preventing the infection, it remains a huge challenge to induce protective immunity in the groups that are poorly responsive and in those that do not respond at all to recombinant vaccines (Kundi,

2007). Leroux‑Roels (2015) opined that if hepatitis B vaccines are improved, it may also

induce rapid and longer-lasting protective immune responses in all vaccine recipients as well as in poor responders. Therefore, there is a pertinent need for an improved and effective adjuvant for Hepatitis B vaccine. Novel adjuvants such as MF59 an oil in water emulsion, ASO4 and some ISS have elicited stronger response than alum type adjuvant but they also have some disadvantages such as reactogenicity and cost effectiveness (Sivakumar et al.,

2016). Egg shell membrane is a readily available resource from eggs. Studies have shown that this otherwise waste product gotten from eggs has immunomodulatory properties, affordable and natural with minimal side effects (Masuda and Hiramatsu, 2008). This present study is steered towards evaluating the immunogenicity of recombinant hepatitis B vaccine when combined with egg shell membrane (ESM) as a possible adjuvant for possible therapeutics use in the prevention of hepatitis B infection.

1.1       Overview of Immune System

Normal human homeostasis is constantly challenged by pathogenic microbes which contain a vast array of toxic or allergenic substances (Chaplin, 2010). Immunity is the capability of the body to fight against pathogenic microbes in order to prevent us from infectious diseases. It is capable of generating a wide variety of cell networks and molecules capable of specifically recognizing and removing an endless spread of foreign invaders (Kimbrell, 2001).

1.1.1      Historical Orientation

Immunity is derived from the Latin word immunitas, meaning “to be exempt from.”   This concept dates back to 1500s, before the causes of disease were understood (Mak et al., 2014). People  who  lived  during  a  first  exposure  to  a  devastating  disease  and  didn’t  get  sick following subsequent exposure were said to have become “exempt from” the disease, or “immune” (Kindt, 2007). The concept of immunity from disease dates back at least to Greece in the 5th century BC. Thucydides wrote of individuals who recovered from the plague, which was raging in Athens at the time (Mak et al., 2014). An early milestone in the 10th century  in  China  was  intentionally  inducing  immunity  to  an  infectious  disease  where smallpox was endemic. The process of “variolation” involved exposing healthy people to material from the lesions caused by the disease, either by putting it under the skin, or, more often, inserting powdered scabs from smallpox pustules into the nose (Cavaillon, 2011).

Over 100 years prior to Koch’s postulates in 1890, which definitively identified microbes as the causative agent of disease, Edward Jenner had made a crude vaccine from the pus of cow pox lesions to successfully immunize people against small pox. (Janeway and Travers 2001). Eli  Metchnikoff  observed  in  1882,  that  white blood  cells  were  destroying  the  engulfed pathogen. The word phagocyte, from the Greek words “phagein,” to eat and “cyte” cell, was used to describe this cellular action (McComb et al., 2013). In addition to Metchnikoff’s discovery of cellular immunity, other researchers were examining the ability of bodily fluids to provide protection against disease. In 1890,  antibodies were discovered by  Emil von

Behring and Shibasaburō Kitasato when they identified acellular components of the blood that conferred immunity when transferred from one animal to another (kantha, 1991). The discovery of antibodies raised different school of thought about the importance each type of immunity played in overall host immunity. The gap was bridged in 1903 when scientists Almroth Wright and Steward Douglas proved that humoral responses helped the cellular immune response indicating that both cellular and humoral immune responses played important roles. They observed that antibodies and complement enhanced the phagocytosis of bacteria by binding to the bacteria, an event termed opsonization (Turk, 1994).

1.2       Types of Immunity

In ensuring that there is swift and effective response to pathogens, the body employs the use of two broad complementary systems: The innate immunity and the adaptive or acquired immunity (Spiering, 2015).

Innate immune is described Delves and Reott (2000) described the system to include all aspects of the host’s immune defence mechanisms that are encoded in their mature functional forms by the germ-line genes of the host. The term innate immunity could be used to include microbiological, chemical and physical barriers and likewise include the elements of the immune system (neutrophils, monocytes, macrophages, complement, cytokines, and acute phase proteins) that provides first line defence to the immediate host. The innate immune system is non-specific and lacks immunological memory (Prescott et al., 2002).

Adaptive immune system also called acquired immune system or rarely known as specific immune system. It is a subsystem of overall immune system because it composed of systemic cells and processes that inhibits pathogen growth. The Adaptive immunity is distinctive from other type of immunity because of its ability to distinguish self and non-self-components inside the body (Tomar, 2016). Antigen-specific receptors expressed on the surfaces of T- and  B-lymphocytes  are  the  primary  basis  of  adaptive  responses.  The  antigen  specific receptors of the adaptive response are encoded by genes that are assembled by somatic rearrangement of germ-line gene elements to form intact T cell receptor (TCR) and immunoglobulin (B cell antigen receptor; Ig) genes (Iwasaki & Medzhitov, 2010).

Adaptive immunity has four intrinsic features;

•   Antigenic  specificity  which  permits  it  to  distinguish  subtle  differences  among antigens.

•   Diversity wherein the immune system is capable of generating tremendous diversity in its recognition molecules, allowing it to recognize billions of unique structures on foreign antigens.

•   Immunologic memory that enables the immune system to recognize, respond and keep a memory of an antigen that causes a heightened state of immune reactivity when next it is encountered.

•   Self/non-self recognition this aids the immune system in responding only to foreign antigens (Mak et al., 2014).

Adaptive immunity is categorized into two types: Passive immunity and Active immunity.

Passive Immunity

Passive immunity involves the transfer of immunity from one individual to another in the form of prefabricated antibodies. Passive immunity could be naturally or artificially transmitted. In passive immunity, the patient is at risk of epidemic by the same microbe later because body can’t develop memory cells (Tomar, 2016).

Naturally acquired passive immunity: Maternal passive immunity is an example of naturally acquired passive immunity. This illustrates antibody-mediated immunity (mostly IgG) which is transfered to a fetus from its mother at the time of pregnancy through the placenta cells. (Yang, 2014).

Artificially acquired passive immunity: Artificially acquired passive immunity is a type of short-term immunity which can be conferred by the transfer of antibodies. These antibodies are administered into the body in various forms such as monoclonal antibodies from animal blood serum (Tomar, 2016).

Active Immunity

The entry of pathogen inside the body leads to the stimulation of B cells and T cells which develops memory B-cells and T- cells (Spiering, 2015). These memory cells produce secondary response when they encounter the same pathogen again. Active immunity may also be natural or artificial.

Naturally acquired active immunity: This type of immunity develops when an individual is exposed to a live pathogen which produces a primary immune response and then leads to immunological memory. This immunity is natal because it is not conferred by deliberate exposure.

Artificially acquired active immunity: Artificially acquired active immunity is stimulated by the administration of vaccine. This is due to the presence of antigens in the vaccine. They are aimed at inducing a primary response against the antigen without causing any lethal side effects (Tomar, 2016).

1.3       Mediators of Innate Immunity

The term innate immunity could be used to include microbiological, chemical and physical barriers and also include the elements of the immune system such as neutrophils, monocytes, macrophages,  complement,  cytokines,  and  acute phase proteins.  These provide  first  line defence to the immediate host. The innate immune system is non-specific and lacks immunological memory (Prescott et al., 2002).

1.3.1 Barrier Defence

The first line of innate defence is interceded by a pre-existing collection of physical, chemical and molecular barriers that exclude foreign material in a way that is totally non-specific and requires no induction. These elements include anatomical barriers and physiological barrier (Kindt, et al., 2007).

The anatomical barriers include the skin and the surfaces of the mucus membrane. The intact skin  and  mucous  membranes  of  the  body  afford  a  high  degree  of  protection  against pathogens. The skin is a resistant barrier because of its outer horny layer consisting mainly of keratin, which is indigestible by most micro-organisms, and thus shields the living cells of the epidermis from micro-organisms and their toxins (Mak et al., 2014).

The relatively dry condition of the skin and the high concentration of salt in drying sweat are inhibitory or lethal to many microorganisms. The sebaceous secretions and sweat of the skin contain bactericidal and fungicidal fatty acids, which constitute an effective protective mechanism against many potential pathogens (Prescott et al., 2002). The respiratory tract has sticky mucus covering which acts as a trapping mechanism for inhaled particles. The action of cilia sweeps the secretions containing the foreign material; towards the pharynx so that when  swallowed,  acidic  secretions  in  the stomach  destroy  most  of the micro-organisms present (Mak et al., 2014). Mucopolysaccharides contained in nasal secretions and saliva is capable of blocking some viruses.   The washing action of tears and flushing of urine are effective in stopping invasion by micro-organisms. The commensal micro-organisms that make up the natural bacterial flora covering epithelial surfaces offers protection by using up a

niche that cannot be used by a pathogen, competing for nutrients, producing byproducts that can inhibit the growth of other organisms (Kindt et al., 2007)

1.3.2           Physiologic Barriers

The physiologic barriers that contribute to innate immunity include temperature, pH, and various soluble and cell associated molecules. A variety of soluble factors such as lysozyme, interferon and complement also contribute to innate immunity (Kindt et al., 2007).

Lysozyme is a hydrolytic enzyme found in mucous secretions and in tears, is able to cleave the peptidoglycan layer of the bacterial cell wall (Khan, 2009). Interferon comprises a group of proteins produced by virus-infected cells. Interferons bind to nearby cells and induce a generalized antiviral state (Pestka, 2007).

Compliment is a group of serum proteins that circulate in an inactive state. Compliments are capable of causing  damage  pathogenic organisms by  either destroying the pathogens  or facilitating their clearance (Stewart, 2012). These serum proteins are activated to their active forms  by  variety  of  specific  and  nonspecific  immunologic  mechanisms  (Janeway  and Travers, 2001).

Pattern Recognition: When the first line defence (the anatomical and physiological barriers) have been circumvented by invaders, innate leukocytes start to take action as a result of pattern recognition mediated by the binding of pattern recognition molecules (PRMs) to pathogen–assisted  molecular  pattern  (PAMPs)  furnished  by  pathogens  and  to  damage- assisted molecular pattern (DAMP) emanating from damaged host cells (Turvey and Broide,

2010).   PRMs come in several different forms, some of which are membrane-bound and others of which are soluble. PRMs that are expressed by innate leukocytes are called pattern recognition receptors (PRRs) (Mak et al., 2014).  PRRs are located in the plasma membrane of an innate leukocyte, or are soluble molecules free in the leukocyte’s cytoplasm, or are fixed in the membranes of intracellular vesicles called endosomes (Stewart, 2012). Other PRMs are made by non-leukocytes and are present as soluble molecules free in the extracellular milieu. When these latter PRMs bind to a given PAMP or DAMP, they must then bind to another receptor fixed in a leukocyte membrane in order to trigger the appropri- ate clearance response. (Matzinger, 2007)

1.3.3    Inflammation

The activation of leukocyte PRRs by their ligands results in the induction of new gene transcription and the synthesis of various “pro-inflammatory” cytokines. These cytokines propagates the influx of adaptive leukocytes into the site of injury or infection. This influx is an integral part of inflammation or an inflammatory response (Stewart, 2012). The resulting swelling and redness are the outward physical signs associate with inflammation. Innate leukocytes secrete powerful molecules that take part in inflammatory response and can cause tissue damage and hamper immune system function if left to operate unchecked (Kindt et al.,

2007).   Therefore it is of great importance that inflammation is properly controlled as excessive or prolonged inflammation is pathologic and unbalances homeostasis (Moser and Leo 2010).

1.3.4    Phagocytosis

Phagocytosis (“eating of cells”) is a means used by innate leukocytes in engulfing pathogens such as bacteria. Phagocytosis is carried out primarily by three types of PRR-expressing cells: neutrophils, macrophages and dendritic cells (DCs); these cell types are consequently known as phagocytes (Moser and Leo 2010).

1.3.5    Cells Involved in Innate Immunity

White blood cells also known as leukocytes are key cells involved in innate immunity. These cells differ from other cells of the body in that they are not associated with a specific organ therefore their function is autonomous (Staros, 2005). These cells are the products of hematopoietic stem cells. They move freely inside the plasma and capture infectious particles and invading microorganisms (Turvey and Broide, 2010). These leukocytes cells include: Natural  killer  cells,  eosinophils,  mast  cells,  basophils,  neutrophils,  macrophages,  and dendritic cells. These cells work along with the immune system by eliminating pathogenic agents which causes infection (Abad and VarTan, 2016).

Natural Killer Cells

Natural killer cells have the morphology of lymphocytes but do not bear a specific antigen receptor (Janeway and Travers, 2001). They recognize abnormal cells through the use of immunoglobulin receptors (FcR) with which they bind antibody coated targets leading to antibody-dependent cellular cytotoxicity (Bettelli, 2010). Furthermore, they have receptors on their surface for MHC class I (Stewart, 2012). If on interaction with a cell, this receptor is not bound, the natural killer cell is programmed to lyse the target by secretion of perforins onto

the cell surface to which the natural killer cell has adhered. Perforins make holes in the cell membrane and granzymes are injected through the pores (Wang, 2013). These cells are called natural killer because they do not require any activation. They kill the microbes as soon as they produced inside the body (Tomar, 2016).

Eosinophils

Eosinophils are employed in the protection of the host from parasitic (particularly nematode) infections (Prescott et al., 2002). Eosinophils are not phagocytic, but have large granules containing major basic protein, eosinophilic cationic protein, eosinophil peroxidase, and eosinophil-derived neurotoxin, which are highly cytotoxic when released onto the surface of organisms (Abbas et al., 2007). They are present in low numbers in a healthy individual about 1–3% of leucocytes (Zschaler et al., 2014), but their numbers rise in certain allergic conditions (Stewart, 2012). Eosinophils in murine sera as reported by O’Connell (2015) have characteristic orange to red coloured round cytoplasmic granules and takes up about 0-7% of the leucocyte count in the peripheral blood of mice.

Mast Cells and Basophils

Irrespective of the relatively fewer numbers of the basophils and mast cells compared with the other white cells, they are involved in some of the most severe immunological reactions, such as anaphylaxis and angioedema (Spínola et al., 2014). Mast cells can be differentiated on the basis of the enzymes they contain and their tissue location. T mast cells (mucosal mast cells) contain only trypsin, while connective tissue mast cells contain both chymotrypsin and trypsin (Crivellato and Ribatti, 2013).  Basophils are morphologically similar cells found in the blood. Mast cells and basophils have high affinity receptors for IgE Fc_RI (CD23) which rapidly absorbs any local IgE (Prescott et al., 2002). Binding of antigen to IgE results in cross linking of these receptors which in turn leads to degranulation and release of preformed mediators, such histamine and serotonin which are vasoactive amines (Spínola et al., 2014). Basophils circulate in human blood in low numbers of about 0.2 % (Stewart, 2012). In mice basophils are rare and are characterized by large round and segmented nuclei (O’Connell,

2015).

Neutrophils

Neutrophils  comprising  of  approximately  70%  of  total  white  blood  cells  are  the  most abundant leukocytes in human blood. Neutrophils are indispensible in defence against pyogenic (pus forming) bacteria (Weber, 2015).  They are not present in normal tissues, but

are rapidly recruited to sites of acute inflammation. Neutrophils are highly phagocytic. They constitutively possess a wide variety of antimicrobial killing mechanisms (Parkin and Cohen,

2001). The neutrophil has a multilobed nucleus and a granulated cytoplasm that stains with both acid and basic dyes; it is often called a polymorphonuclear leukocyte (PMN) for its multilobed nucleus which constitute 50%–70% of the circulating white blood cells (Kindt et al., 2007). In mice the leucocytes contain about 10-25% neutrophils (Zschaler et al., 2014) with nuclei in the shape of ‘8’ (Leliefeld et al., 2015)

Macrophages and Monocytes

Macrophage means large eaters. Macrophages are one of the three main types of specialized phagocyte that can engulf and internalize (phagocytose), and subsequently kill microbes such as bacteria (Moser and Leo 2010). Macrophages reside in almost every tissue of the body and are important components of both innate and adaptive immune responses (Hirayama et al.,

2017).  They can either be Resident macrophages which are present in steady-state tissues (i.e. before infection occurs) or Recruited (or elicited) macrophages are not tissue-resident cells, but they develop from circulating precursors called monocytes (Shepherd and Hoidal

1990). Circulating precursors of macrophages, which are produced in the bone marrow, are called monocytes. The macrophages are larger than the monocytes (Kindt et al., 2007). The monocytes are mononuclear with kidney-shaped or ovoid nucleus which adopt grayish blue colour when stained (O’Connell, 2015). Murine leukocytes contain about 2% monocytes (Zschaler et al., 2014).

Dendritic Cells

Dendritic cells are present in tissues that comes in contact with the external environment, mainly with the skin (i.e. Langerhans cells), and the inner mucosal lining of the stomach, nose and lungs (Pugholm, 2016). Dendritic cells play an important role in the mechanism of antigen presentation, and provide a network between the innate and adaptive immune system (Bikash and Mahesh, 2012).

Cytokines

Cytokines are small molecular weight messengers secreted by virtually all cells to alter the behaviour of itself or another cell (Mak et al., 2014). Cytokines send intracellular signals by binding to specific cell-surface receptors (Precott, 2002). The biological effect depends on the cytokine and  the cell  involved,  but  typically  these molecules  will  affect  cell  activation, division, apoptosis, or movement. They act as autocrine, paracrine, or endocrine messengers.

Examples  include  interleukins,  chemokines,  colony-stimulating  factors  and  interferons

(Vaillant, 2015).

1.4       Mediators of Adaptive Immunity

The key components of the adaptive immune system are the lymphocytes. The recognition of molecules from infectious agents by lymphocytes is mediated by their specialized antigen receptors, which are not present on cells of innate immunity (Willey et al., 2014). Lymphocytes are produced in the bone marrow by the process of hematopoiesis (Kindt et al.,

2007). In addition, lymphocytes are normally small, relatively inactive cells. Thus, adaptive immune responses require the activation and proliferation of specific clones of B cells or T cells in order to reach a critical mass that can deal with the infectious agents (Mak et al.,

2014). An antigen can be defined as a molecular structure against which a specific adaptive immune response can be made. Collectively, lymphocytes express a vast range or repertoire of antigen receptors of different specificities, but each lymphocyte expresses multiple copies of a receptor of only a single given specificity (Kindt et al., 2007). In humans lymphocytes constitute about 30%-50% of the leukocytes in peripherial blood and about 70-80% of peripherial blood of mice (Zschaler et al., 2014).

1.4.1    T Lymphocytes

T lymphocytes though produced in the bone marrow migrates to the thymus gland to mature. During its maturation within the thymus, the T cell comes to express a unique antigen- binding molecule, called the T-cell receptor, on its membrane which can recognize only antigen that is bound to cell-membrane proteins called major histocompatibility complex (MHC) molecules  (Kindt  et  al.,  2007).  T  cells  are generally  classified  into  two  groups expressing either cell surface CD4 or CD8 receptors. CD8 T cells are most commonly known as cytotoxic T lymphocytes (CTL) while the CD4 T cells are commonly referred to as helper T cells (McComb et al., 2013).

CD4 Helper and Regulatory T Cells

CD4 T cells, which when activated become conventional helper T (Th) cells or regulatory T (Treg) cells, are one of the two main types of T cells. They are so-called because they possess a molecule called CD4 that is involved in recognition of the cells with which they interact (Nauta, 2011). As for all naive T-lymphocytes, CD4 T cells circulate in the bloodstream and migrate through secondary lymphoid tissues and to function they need to be activated. In secondary lymphoid  tissues  DCs  dendritic cells  activate CD4 T  cells and  regulate their

differentiation into cells capable of mediating different functions (Lanzavecchia and Sallusto,

2000). These effector T cells then either interact with other cells locally within the secondary lymphoid  tissues  or  they  migrate  to  peripheral  sites  of  inflammation  and  infection  and interact with different cell types. There are at least four main ways in which CD4 T cells can be instructed to function, and the respective T cell subsets are termed Th1, Th2, Th17 and T reg cells (MacPherson and Austyn, 2012).

•   Th1 cells are formed when CD4 T cells are activated in the presence of IL-12 and IFNγ. They stimulate anti-microbicidal and cytotoxic effector functions of immunity by activating macrophages, recruiting and activating cytotoxic cells, both NK cells and CD8 T lymphocytes (McComb et al., 2013).

•   Th2 secretes IL-4, IL-5, IL-6, and IL-10. This subset functions more effectively as a helper for B-cell activation and immune responses that depend upon antibodies. Th2 cells appear to block development of autoimmune disease and also block the progression of the disease once it is established (Orsbone, 2000).

•   Th17 cells are particularly efficient at recruiting neutrophils to the site of infection; hence, neutrophils can  be recruited in both the innate and adaptive responses of immunity. This type of response may be particularly important for defence against other types of bacteria, particularly bacteria that cause acute inflammation such as Staphylococcus and Streptococcus, and perhaps some fungi (MacPherson and Austyn,

2012).

•   Treg  cells  suppress  the  responses  of  other  cells,  including  DCs  and/or  other lymphocytes. This type of response may be particularly important in switching off immune responses when an infectious agent has been eliminated and in ensuring that harmful responses are not made against innocuous agents (including components of the body itself) (Willy et al., 2014).

CD8 T cells

These T cells possess multiple copies of clusters of differential molecules called CD8 that is involved in recognition of the cells with which they interact. Following activation, these T cells can become cytotoxic T cells capable of inducing apoptosis in cells they recognize. CD8

T  cells  like  CD4  T  cells  circulate  in  the  bloodstream  and  migrate  through  secondary lymphoid tissues. (VonAndrian and Mackay 2000). These cells leave the secondary lymphoid tissues, and enter peripheral sites of inflammation and infection. Here they can kill virally

infected cells. They do so in two main ways –through perforin and granzymes, and through Fas-ligand-Fas interactions (MacPherson and Austyn, 2012). As cytotoxic cells, CD8 T cells are  a  central  component  of  adaptive  immunity  to  viruses  such  as  the  influenza  virus. However, they can also produce cytokines that are directly toxic, such as tumour necrosis factor (TNF)-a, which induces apoptosis by binding to death-inducing receptors on other cells, or can modulate or enhance the functions of innate cells (Parkin and Cohen, 2001).

1.4.2    B Lymphocytes

B lymphocytes are also small, resting cells, morphologically indistinguishable from T cells. They  are  produced  and  also  mature  in  the  bone  marrow.  When  they  are  appropriately activated their primary function is to develop into plasma cells, which can be regarded as antibody-synthesizing factories, or to become memory cells (Mak et al., 2014). In many cases B cells need help from T cells to become activated and to develop into plasma cells and memory cells while the T cells control the type of antibody that they make (Stewart, 2012). This type of response, which is typically made in response to protein antigens by B cells in specialized sites of secondary lymphoid tissues (termed follicles), is therefore termed a T- dependent (TD) response (MacPherson and Austyn, 2012). Another subset of B cells, located in a specialized site of the spleen (called the marginal zone) can produce antibodies to other types of antigen, such as polymeric carbohydrates, without needing any help from T cells and this is an example of a T-independent (TI) response (MacPherson and Austyn, 2012). Finally, a different type of non-conventional B cell (called B-1 cells in the mouse) can produce natural antibodies in the apparent absence of any antigenic stimulation (Fagarasan and Honjo,

2000). A central feature of adaptive immunity is the phenomenon of immunological memory. This is a crucial property of lymphocytes and a function of adaptive immune responses in general (Mak, 2014). Immunity in these cases may last for many years and is due to the development of populations of memory lymphocytes. Once an infectious agent has been cleared, most of the expanded populations of effector T cells, or plasma cells, die. However, populations of antigen-specific memory T cells, both CD4 and/or CD8 T cells, and memory B cells persist (Tangye and Tarlinton 2009).

1.4.3    Major Histocompatibility Complex (MHC) Molecules

B cells can recognize extracellular antigens directly with the aid of their B cell receptors (BCRs). However in T cells, antigen recognition cannot occur directly, because the antigens for which TCR are specific are inside other cells. It then becomes necessary for any cell

containing  antigen  to  show  it,  or  present  it  to  the  T  cells  (Chaplin,  2010).  The  MHC molecules which are expressed by almost every cell of the body binds representative samples (usually peptides) of molecules (usually proteins) that are synthesized within a cell, or which have been internalized by the cell and transports them to the cell surface (Galson et al.,

2015). The major histocompatibility complex (MHC) MHC molecules are also called the human leukocyte-associated (HLA) antigens in human and histocompactibility-2 in mice is a large genetic complex with multiple loci (Zabriskie, 2012). The MHC loci encode two major classes of membrane-bound glycoproteins: class I and class II MHC molecules (Willey et al.,

2014) During their biosynthesis MHC class I and II molecules are routed through different cellular compartments.  Hence, they can bind peptides derived from different sites. As a general rule MHC class I molecules, which are widely expressed on different cell types, bind peptides that have been produced within the cytoplasm. In contrast MHC class II molecules, which are expressed on a more limited number of cell types, bind peptides that have been produced within endosomes or phagosomes (Galson et al., 2015). Helper T cells (CD4) recognize MHC class II antigens while suppressor cytotoxic T cells (CD8) recognize MHC class I antigens.

Thymus-independent Antigens

A number of antigens will stimulate specific immunoglobulin production directly. These T- independent antigens are of two types: mitogens and certain large molecules. Mitogens are substances that cause cells, particularly lymphocytes, to undergo cell division. Certain glycoproteins, called lectins, have mitogenic activity (Stewart, 2012). These molecules have specificity for sugars; they bind to the cell surface and activate all responsive cells. The response to the mitogens is therefore polyclonal, as lymphocytes of much different specificity are activated. However, at low concentrations these mitogens do not cause polyclonal activation but can lead to the stimulation of specific B cells. (Zabriskie, 2012)

Thymus–dependent Antigens

Many antigens do not stimulate antibody production without the help of T lymphocytes. These antigens first bind to the B cell, which must then be exposed to T cell-derived lymphokines (helper factors) before antibody can be produced. For the second activation signal to be targeted effectively at the B cell, the T and B cells must be in direct contact. For this to happen, the B and T cell epitopes must be linked physically (Stewart, 2012). However, T cells only recognize antigen that has been processed and presented in association with products of the major histocompatibility complex (MHC), so it is impossible for native

antigen to form a bridge between surface immunoglobulin and the T cell receptor. The B cell binds to its epitope on free antigen, but there is no site on this molecule to which the T cell can bind, because it requires antigen associated with MHC products (Zabriskie, 2012).

1.4.4    Antigen Processing and Presentation

T dependent antigen requires that the antigen becomes associated with MHC class II molecules and expressed on the cell surface in a form that helper T cells can recognize for an antibody response to occur (Zabriskie, 2012). All cells express MHC class I molecules, but class II molecules are confined to cells of the immune system – the antigen-presenting cells (APCs). These cells present antigen to MHC class II-restricted T cells (the CD4+) and therefore play a key role in the induction and development of immune responses (Prescott et al., 2002). There are a large number of antigen-presenting cells in the body, most of which constitutively express MHC class II molecules. Other cells, such as T lymphocytes and endothelium, can be induced to express MHC class II molecules by suitable stimuli such as lymphokines.  The  relative  importance  of  each  type  depends  on  whether  a  primary  or secondary response is being stimulated and on the location (Stewart, 2012).

The most studied antigen-presenting cells are the macrophages and dendritic cells. However, it is now apparent that in certain situations B cells may be important antigen presenting cells. The relative importance of B cells becomes greatest during secondary responses, especially when the antigen concentration is low Here the B cells can specifically engulf antigen via their surface immunoglobulin (Mak et al., 2014). In a primary response, specific B cells are at a low frequency and their receptors are of low affinity; in this situation macrophages and dendritic cells are probably most important (Kindt  et al., 2007). The key feature of all antigen-presenting cells is that they can ingest antigen, degrade it and present it,  in the context  of  MHC  class  II  molecules,  to  T  cells.  The  antigen  is  taken  into  the  antigen- presenting cells and enters the endocytic pathway (Zabriskie, 2012). Before it is destroyed completely, peptide fragments are taken to a structure called the compartment for peptide loading. MHC class II molecules are synthesized within the endoplasmic reticulum and are also transported to the compartment for peptide loading, where they associate with the processed antigen. The MHC class II molecule with the bound peptide is then transported to the cell surface (Willey et al., 2014).

1.5       Antibody (Immunoglobulins)

Janeway and Travers (2001) defines an antibody, also known as an immunoglobulin as a large Y-shaped protein produced by B-cells that is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen. Each tip of the “Y” of an antibody contains a paratope (a structure analogous to a lock) that is specific for one particular epitope (similarly analogous to a key) on  an antigen, allowing  these two structures to  bind together with precision. Antibodies are produced by a type of white blood cell called a plasma cell (Zabriskie, 2012). Antibodies can occur in two physical forms, a soluble form that is secreted from the cell, and a membrane-bound form that is attached to the surface of a B cell and is referred to as the B cell receptor (BCR). The BCR is only found on the surface of B cells and facilitates the activation of these cells and their subsequent differentiation into either antibody factories called plasma cells, or memory B cells that will survive in the body and remember that same antigen so the B cells can respond faster upon future exposure (Borghesi and Milcarek, 2006).

Antibodies are typically made of basic structural units each with two large heavy chains and two small light chains. Heavy chains are structurally distinct for each immunoglobulin class or subclass. Both light and heavy chains contain two different regions. Constant regions (CL and CH) have amino acid sequences that do not vary significantly between antibodies of the same class (Pier et al., 2004). The variable regions (VL and VH) from different antibodies do have different sequences. It is the variable regions (VL, VH) that when folded together form the antigen binding sites. The four chains are arranged in the form of a flexible Y with a hinge region. This hinge region allows the antibody molecule to assume a T shape (Borghesi and Milcarek, 2006). The stalk of the Y is termed the  crystallizable fragment (Fc) and contains the site at which the antibody molecule can bind to a cell. The top of the Y consists of two antigen-binding fragments (Fab) that bind with compatible epitopes (or antigenic determinant sites). The Fc fragments are composed only of constant regions, whereas the Fab fragments have both constant and variable regions (Prescott et al., 2002).

More specifically the light chain may be either of two distinct forms called kappa (κ) and lambda (λ). These can be distinguished by the amino acid sequence of the carboxyl portion of the chain. In human immunoglobulins, the carboxyl terminal portion of all chains is identical; thus this region is termed the constant (CL) domain. With respect to the lambda chains, there are  four  very  similar  sequences  that  define  the  subtypes  λ1,  λ2,  λ3,  and  λ4   with  their

corresponding constant regions C λ1, C λ2, Cλ3, and Cλ4 (Wintrobe and Meyer, 2004). In humans there are five classes of heavy chains designated by lowercase Greek letters: gamma (γ), alpha (α), mu (µ), delta (Δ), and  epsilon (ε). The properties of these heavy  chains determine, respectively, the five immunoglobulin classes—IgG, IgA, IgM, IgD, and IgE. Each immunoglobulin class differs in its general properties, half-life, distribution in the body, and interaction with other components of the host’s defensive systems. (Kindt et al., 2007)

1.5.1    Immunoglobulin Classes

IgM molecule is the oldest class of immunoglobulins, and it is a large molecule consisting of five basic units held together by a J chain (Mak et al., 2007). The major role IgM plays is the intravascular neutralization of organisms, especially viruses. The reason for this important physiological role is that it contains five complement-binding sites, resulting in excellent complement activation (Willey et al., 2014). This activation permits the segment removal of antigen–antibody complement complexes via complement receptors on phagocytic cells or complement-mediated lysis of the organism. It has relatively low affinity binding to the antigen in question. Second, because of its size, it does not usually penetrate into tissues (Zabriskie, 2012). IgM accounts for about 10% of the immunoglobulin pool. IgM is the first immunoglobulin made during B-cell maturation and is expressed as membrane-bound antibody  on  B  cells.  IgM  is  secreted  into  serum  during  a  primary  antibody  response. Although most IgM appears to be pentameric, around 5% or less of human serum IgM also exists in a hexameric form. Hexameric IgM activates complement up to twentyfold more effectively than does the normal pentameric form (Prescott et al., 2002). In both mice and humans only one isotype of IgM exists but appears to be lesser in mice as result of difference in B cell development in mice (Mestas and Hughes, 2004).

IgG accounts for 80% of the immunoglobulin pool and hence is the major immunoglobulin in human serum IgG is a smaller molecule that penetrates easily into tissues and is present in blood plasma and tissue fluids (Mak et al., 2014). The IgG class acts against bacteria and viruses by opsonizing the invaders and neutralizing toxins. It is also one of the two immunoglobulin classes that activate complement by the classical pathway. IgG is the only immunoglobulin molecule able to cross the placenta and provide natural immunity in uterus and to the neonate at birth (Keller and Stiehm, 2000). There are four major classes of IgG: IgG1 and IgG3 activate complement efficiently and clear most protein antigens, including the removal of microorganisms by phagocytic cells. In contrast, IgG2 and IgG4 react mostly with carbohydrate antigens and are relatively poor opsonins. This is the only molecule that crosses the placenta to provide immune protection to the neonate (Prescott et al., 2002). In contrast to human IgG, which can be divided into four subclasses (IgG1–4), murine IgG only knows three subclasses (IgG1–3). In addition, murine IgG2 can be split in the isotypes IgG2a, 2b and 2c, of which IgG2a and 2c are allelic variants and further sequence variants are known for IgG1 and IgG2b (Haan et al., 2017).

IgA The major mucosal immunoglobulin, consists of two basic units joined by a J chain. The addition of a secretion molecule prevents its digestion by enzymes present in mucosal and intestinal secretions (Mak et al., 2014). There are two isotypes of IgA in humans IgA1 and IgA2 and just IgA in mice (Mestas and Hughes, 2004). IgA2 is the major IgA molecule in secretions and is quite effective in neutralizing antigens that enter via these mucosal routes. IgA1, the main IgA molecule in serum, is, however, susceptible to inactivation by serum proteases and is thus less active for defence. Its function is unclear at present (Zabriskie,

2012).  IgA accounts for about 10- 15% of the immunoglobulin pool. Secretory IgA (sIgA), as the modified molecule is now called, is the primary immunoglobulin of mucosal associated lymphoid tissue. Secretory IgA is also found in saliva, tears, and breast milk. In these fluids and related body areas, sIgA plays a major role in protecting surface tissues against infectious microorganisms by the formation of an immune barrier (Kindt et al., 2007) IgD is an immunoglobulin found in trace amounts in the blood serum constituting about 0.2% of the total  immunoglobulin  in  serum.  It  has  a monomer structure similar to  IgG.  IgD antibodies do not fix complement and cannot cross the placenta, but they are abundant in combination with IgM on the surface of B cells and bind antigens, thus signaling the B cell to start antibody production. (Chen et al., 2009) IgE makes up only a small percent of the total immunoglobulin pool. The classic skin- sensitizing and anaphylactic antibodies belong to this class. When two IgE molecules on the surface of these cells are cross-linked by binding to the same antigen, the cells degranulate. This degranulation releases histamine and other pharmacological mediators of anaphylaxis. It also stimulates eosinophilia and gut hypermotility (increased rate of movement of the intestinal contents) that aid in the elimination of helminthic parasites. Thus, though IgE is present in small amounts, this class of antibodies has very potent biological capabilities. (Woof and Burton, 2004).

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