The first task of the innate immune system is sensing a potential infectious agent by identifying pathogen-associated molecular patterns (PAMPs) through innate cells’ pathogen recognition receptors (PRRs) ^[1][7]. Once pathogens are identified, the innate immune system stimulates the production of chemokines and cytokines responsible for the inflammatory response and upregulation of the stimulatory molecules for T cell responses ^[2][3][8,9]. In the context of oral vaccines and other mucosal vaccines, the innate immune system presents a challenge of tolerance. The mucosal barriers are constantly presented with antigens and stimuli from commensal or foreign microbes; the mingling of ‘self’ and ‘non-self’ is so commonplace that inducing an immune response for each instance would lead to severe inflammation of the mucosal barriers and negative impacts on the animal’s health. Therefore, the innate immune system must operate with some tolerance towards these antigens. Meanwhile, mucosal vaccines, including oral vaccines, must find a way to break through this tolerance to provoke a robust immune response that will provide protection against a specific pathogen ^[4][10]. The tolerance of gut mucosal immune systems will be discussed later in this rpapesearchr.

Immunology is an important field of vaccinology, with a special focus on the activation of the adaptive immune response. However, it is important to note that, without the innate immune response’s ability to recognize and signal potential infectious bodies within a host, the adaptive response would be ineffective. The adaptive response is characterized by a slow response that can take days or weeks to manifest and is highly dependent on water temperature, with lower temperatures being associated with a slower response ^[5][6][11,12]. The adaptive immune response can be broadly split into the following two categories: humoral and cell-mediated responses ^[7][13]. The cell lineage vital to this process is the lymphocyte, divided into B (humoral) and T (cell-mediated) cells ^[8][14]. In teleost fish, both T and B cells originate in the head kidney; B cells also mature here, while T cells migrate to the thymus for maturation ^[9][10][15,16].

B cells are responsible for the humoral, or antibody-mediated, immune response. To express and secrete antibodies, B cells are first exposed to antigens, with the help of the innate immune system and antigen-presenting cells (APCs). B cells then develop into plasmablasts and eventually mature plasma cells secreting specific antibodies ^[11][17]. B cells and their antibodies are important defenders and can combat pathogens through direct neutralization as well as through indirect mechanisms such as opsonization, complement activation, and antigen presentation to other immune cells ^[12][18].

Three distinct types of immunoglobulins have been described in teleost fish, each following the basic structure of two heavy chains and two light chains: IgM, IgD, and IgT/IgZ ^[13][19]. The most abundant immunoglobulin in fish, IgM, was the first type described in fish and can be found in almost all vertebrates ^[14][20]. IgM can be found on B cell membranes (membrane-bound) and in its secreted tetrameric form (secretory) in blood circulation and mucus. Some amount of IgM can be detected in circulation without antigen stimulation, but its levels are increased after antigen exposure and subsequent immune system activation ^[13][19]. Studies have also shown that the secreted tetrameric form of IgM can be transported into mucosal tissues, including skin mucus and other mucosal tissues discussed later in this resviearchw ^[15][21]. This antibody class assists with a wide range of pathogen defenses, including complement activation, agglutination, and neutralization ^[12][13][16][18,19,22]. As the most abundant immunoglobulin, it is a popular parameter measured in fish vaccination studies to evaluate immune stimulation and predict efficacy ^[17][18][19][23,24,25].

Other immunoglobulin classes are not nearly as well-described in function or origin relative to IgM. IgD was first discovered in channel catfish (Ictalurus punctatus) in 1997 ^[20][26], and its functions remain somewhat of a mystery, even in mammalian species ^[13][19]. There is evidence of B cells expressing both IgM and IgD, as well as solely IgD ^[21][27]. IgD is found in several other fish species ^[22][23][24][28,29,30], and the highest levels are generally found within the head kidney tissue ^[25][31]. A monoclonal antibody specific to rainbow trout (O. mykiss), IgD was used to measure levels within sera, when researchers found that its levels were up to 400 times lower relative to the IgM in the sera ^[26][32]. IgT (known as IgZ in zebrafish (Danio rerio)) was the focus of several recent studies aimed at characterizing the mucosal immunity of fish ^[21][27]. It was first discovered in rainbow trout and zebrafish in 2005, and no ortholog has been found in mammals or birds to date ^[27][28][33,34]. One study found that specific IgT antibodies were increased in the mucosal barriers of the gastrointestinal tract, but sera antibodies were dominated by IgM during a parasitic infection ^[21][27]. Bath and oral vaccinations that act on the mucosal barriers of the skin and intestinal tract have been shown to induce greater levels of IgT expression in tissues for both bacterial and viral antigens ^[29][30][35,36]. To date, no B cells have been identified that jointly express IgT and IgM, indicating a separate development and lineage for each isotype ^[12][18]. The hypothesis of separate lineages is further supported by the lack of evidence demonstrating class-switching capabilities of Igs in teleost fish ^[31][37]. The results from these studies are only possible because monoclonal antibodies (mAbs) have been developed that are specific to various Ig forms in teleosts.

Cell-mediated immunity is another important component of the immune response, particularly when combatting intracellular pathogens which have escaped antibody-mediated defense mechanisms. The cell-mediated immune response is governed primarily by T lymphocytes. There are two broad types of T cells: γδ and αβ T cells. The γδ T cells behave similarly to innate pathogen recognition cells in that they do not require antigen processing and presentation through the normal signaling proteins. The function of γδ T cells in fish is not fully understood, but it is hypothesized that they are involved in antigen recognition and may play an important role in mucosal immunity ^[32][38]. This is supported by an increased prevalence of γδ T cells in mucosal and epithelial tissues, the barriers at which pathogens are first met with resistance from the host ^[1][7]. However, studies identifying and characterizing the function of this type of T cell are lacking. To date, the characterization of γδ T cells has largely used genomic and molecular techniques to identify genes and characterize their expression ^[33][39]; antibody tools have only been used for zebrafish ^[34][35][40,41].

The other form of T cells, αβ T cells, are better characterized in fish immunology and vaccinology, particularly for viral vaccines. The αβ T cells are divided into cytotoxic T lymphocytes (CTLs) or T helper cells (T_H). CTLs are directly responsible for killing infected or abnormal cells within the host, based on the antigens presented on the surface of the targeted cells. Viral pathogens typically provoke antigen presentation on the outside of host cells with major histocompatibility complex (MHC) molecules, which CTLs target. Thus, for many anti-viral vaccines, CTL activity is measured to characterize the immune response. Several anti-viral oral vaccines have characterized CTL responses, including inactivated preparations ^[36][42], live-attenuated ^[37][43], and DNA vaccines ^[38][39][44,45]. A recent review of teleost CTL responses to infection and vaccination by Yamaguchi et al. provides more detailed information for studies on viral infection and vaccination ^[40][46]. CTLs are often identified through the CD8 marker on their outer membrane, which assists with cell-to-cell interactions and signaling with MHC-1 proteins; T_H cells are identified by their presence of CD4 on their outer membrane, which mediates interactions with MHC-II proteins ^[1][7]. Genetic expression of these proteins, CD8 and CD4, is the most common way of identifying and characterizing these populations of T cells; antibody tools targeting the cellular characterization of T cells have yet to be developed for many teleost species ^[10][16]. T_H cells assist the cell-mediated and humoral immune response by producing cytokines that act as signalers to other immune cells ^[41][42][47,48]. These cytokines can play important roles in stimulating the humoral immune system and provoking stronger antibody responses to viral antigens, demonstrating the cooperation between the cell-mediated and humoral arms of the immune system ^[43][49]. The use of antibodies specific to teleost CD4 have helped characterize T_H cell responses to viral antigens in rainbow trout, zebrafish, and olive flounder (Paralichthys olivaceus), according to a recent review of T_H cell responses ^[44][50], but continued development of these tools is necessary for a deeper understanding of T_H cell function and response to various stimuli.

Fundamentally, the mechanisms of vaccines stimulating a systemic immune response are similar across injection and mucosal delivery methods. APCs present the vaccine antigen through their MHC-II class molecules, a step which activates T cells and stimulates B cell proliferation and antibody secretion ^[45][51]. The major differences in the response to the vaccine between the two delivery methods are the following: where the antigens are introduced, and, subsequently, where the immune response radiates from. For injection vaccination, the antigen bypasses all mucosal defenses of the host fish; this results in a robust immune response that causes IgM antibodies to circulate throughout the serum of the fish ^[46][52]. Unlike mucosal vaccines, there is no commensal microbe population to account for in an injected vaccine, so antigens are directly exposed to the host cells. Oral and immersion vaccines aim to stimulate an immune response in specific tissues that are most likely to encounter target pathogens, with the added benefit of stimulating IgT in addition to IgM. These tissues are characterized by a complex equilibrium of host and commensal microbe communities that is still not fully understood ^[47][53]. Immersion vaccines can stimulate the production of mucosal antibodies in skin and gill tissue, which ideally protects the fish from pathogens crossing these barriers and causing disease. The stimulation of mucosal antibodies was demonstrated for several antigen targets, though protection against pathogen exposure was variable ^[46][52]. Oral vaccines stimulate immune responses local to the intestinal tract, but have a more complex antigen uptake route compared to the injection or immersion methods because of the intestinal transit of the vaccine ^[46][52].