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New Technologies for Producing Systemic and Muscosal Immunity by Oral Immunization: Immunoprophylaxis in Meals, Ready-to-Eat

Arthur O. Anderson1

INTRODUCTION

Immunity, inflammation, and nutrition form an interactive system that impacts on the performance of soldiers in stressful or hazardous environments. A deficiency in any component of this system diminishes the effectiveness of the others (Beisel, 1994). Decrements in performance during the first few weeks of deployment frequently are associated with illness caused by endemic infectious disease agents. This risk is enhanced by the threat of possible biological weapon attacks (Mobley, 1995). To minimize infection-induced performance decrements, troops should be immunized against recognized endemic disease and biological warfare threat agents months prior to deployment, preferably during basic training.

1  

Arthur O. Anderson, Department of Clinical Pathology, Diagnostics Systems Division (formerly of Department of Mucosal Immunology), U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21701-5333



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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability 22 New Technologies for Producing Systemic and Muscosal Immunity by Oral Immunization: Immunoprophylaxis in Meals, Ready-to-Eat Arthur O. Anderson1 INTRODUCTION Immunity, inflammation, and nutrition form an interactive system that impacts on the performance of soldiers in stressful or hazardous environments. A deficiency in any component of this system diminishes the effectiveness of the others (Beisel, 1994). Decrements in performance during the first few weeks of deployment frequently are associated with illness caused by endemic infectious disease agents. This risk is enhanced by the threat of possible biological weapon attacks (Mobley, 1995). To minimize infection-induced performance decrements, troops should be immunized against recognized endemic disease and biological warfare threat agents months prior to deployment, preferably during basic training. 1   Arthur O. Anderson, Department of Clinical Pathology, Diagnostics Systems Division (formerly of Department of Mucosal Immunology), U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21701-5333

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability Promising new technologies from recombinant DNA and biodegradable polymer research will change the way vaccines are produced and will simplify how immunity is induced and maintained (Michalek et al., 1995; Walker, 1994). Two recent papers demonstrate the feasibility of large-scale agricultural production of recombinant vaccine antigens (Ma et al., 1995) and functional recombinant human antibodies (Haq et al., 1995) in quantities that would satisfy virtually any contingency requirement. Engineered human secretory antibodies could be enterically coated and incorporated into foods for passive protection of soldiers from common diarrheal diseases and enterotoxin reactions that affect nutrition and performance (Hyams et al., 1991, 1993; Sarraf et al., 1997). The advantages of passive and active immunization2 via the oral route are multiple. Recombinant vaccines may be encapsulated in biodegradable polymers to prolong shelf life and provide controlled-release, targeted delivery or protection from denaturation by stomach acid and intestinal enzymes until the product is absorbed in the gastrointestinal tract (Michalek et al., 1994; Morris et al., 1994). Combining encapsulated vaccines or antibodies with nutritious foods makes them more convenient and acceptable to use and removes the logistical and anxiety factors associated with the need for periodic inoculations. In addition to enhancing soldier performance and autonomy, systems for oral immunization will save time and money. It will no longer be necessary for soldiers to delay deployment so that they may assemble for vaccination. Because the new vaccines can be self-administered, they can be taken without need of medically trained personnel. Oral vaccination also eliminates dangerous medical waste and the risk of contamination, which are concerns when needles are used. Taken together, it is now feasible to provide complete passive and active protective immunity along with good nutrition in Meals, Ready-to-Eat (MREs). These new technologies should become an exciting and active area of applied research with 2   Passive immunity is an unsustainable state of immunity produced by transfer of antibodies from an immunized donor (after convalescence or complete immunization regimen) to a nonimmune recipient. Protection may be sustained only if regular treatments of antibody are given. In other words, antibody from an immune donor is put into a nonimmune recipient, thus conferring a state of immunity. In this review, passive immunity is used to indicate the prevention or reduction of ''traveler's diarrhea" or infections that affect soldiers within the first few days of deployment. Active immunity is a sustainable state of immunity that results from vaccinations or recovery from an infectious illness. This kind of immunity is antigen specific and can result in logarithmically increasing amounts of antibody and other forms of immunity upon re-exposure to antigen. The most important aspect of active immunity is that immunological memory is induced. Immunological memory results from clonal expansion of lymphocytes specific for the antigen during the primary response. These lymphocytes reside in lymphatic tissues and circulate in the blood. After immunization, there may be many thousands of antigen-specific lymphocytes circulating or residing in tissues. Upon re-exposure to antigen, the rate and magnitude of the specific secondary immune response is orders of magnitude greater than the primary response. Immunological memory resulting from active immunization is more valuable for protecting soldiers than is passive immunity.

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability numerous opportunities for interdisciplinary collaboration and leveraging of the tasks. CURRENT CONCEPTS AND ISSUES IN IMMUNITY It may be useful to digress from the primary objective (i.e., that of describing the exciting new technologies) in order to explain relevant concepts and critical issues in immunology. This explanation will help establish the importance of certain new approaches to immunization and reveal how these approaches could benefit soldiers of the twenty-first century. Concept of Compartmentalization of Immune Responses Immune responses to vaccines are influenced by the route of immunization (injection or oral), the form of the antigen (live, killed, soluble, peptide subunit, or particulate), and the presence in the vaccine of biologically active elements, such as proteins that mediate specific tissue tropisms (components of the pathogen that enable it to attach to and replicate in specific host cells) or materials included as adjuvants (substances added to enhance antigenicity), vectors, or vehicles (Walker, 1994). For example, differences in immune responses to live versus inactivated viral vaccines may be a function of compartmentalization of immune responses, activities requiring vaccine viability like tropism, or molecular strategies for cell entry and replication (Rubin et al., 1986; Spriggs, 1996). In this review to avoid confusion, general comments about effects of route of administration or vehicles on immune responses will be restricted to responses initiated by simple, unmodified, nonreplicating protein antigens. There is now substantial evidence supporting the existence of at least two immune systems, a "peripheral" immune system (involving the spleen, lymph nodes, and other nonepithelial tissues) and a "mucosal" immune system (involving the epithelial tissue lining the respiratory and gastrointestinal tracts) (Ogra et al., 1994). These systems operate separately and simultaneously in most species, including humans. Protective immunity acquired during convalescence usually is referred to as "systemic immunity," but this term is imprecise. Systemic immunity might be a combination of mucosal and peripheral immunity, or it might be dominated by an incomplete form of immunity dictated by a specific pathogen (Mosmann and Sad, 1996; Salgame et al., 1991). For example, if a pathogen stimulates a cytokine cascade that favors antibody production (at the expense of endocytosis and intralysosomal killing), it would continue to prosper within a compartment in which antibodies are ineffective (Finkelman, 1995; Yamamura et al., 1991). Unless a vaccine stimulates the appropriate system or a combination of systems, protective immunity might not be complete.

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability The concept of anatomic compartmentalization of immunity is supported by observations from several disciplines (Kroemer et al., 1993). The anatomy of antigen uptake and the physiology and biochemistry of lymphocyte recirculation within unique tissue microenvironments may influence significantly the quality of humoral and cellular immune responses. The way antigens are acquired by individual lymphatic tissues affects the outcome of an immune response. For example, the same antigen may produce qualitatively different immune responses in lymph nodes, spleen, or Peyer's patches (lymphoid tissue aggregates under intestinal mucosa) (Anderson, 1990). Antigens in lymph are filtered, trapped, processed, and presented at the site where lymph passes over fixed antigen-presenting cells in lymph nodes. Such antigen handling by lymph nodes most often results in peripheral immunity, characterized by the appearance of specific immunoglobulin (Ig)G in the blood. Antigens in blood are filtered, trapped, processed, and presented in strategic blood-tissue interfaces in the spleen, which also result in peripheral immunity. However, the spleen microenvironment is somewhat more complicated because it also accommodates circulating antigen-presenting cells and immunoreactive T-and B-cells from other tissues committed to either peripheral or mucosal immunity. In contrast, antigens in the lumens of enteric organs (i.e., the respiratory and gastrointestinal tracts) are nondestructively endocytosed by specialized epithelial cells called M (membraneous)-cells and transported across the cytoplasm in vacuoles onto lymphoid cells in Peyer's patches, where response to antigen presentation triggers commitment to "mucosal immunity," characterized by release of specific IgA into the secretions3 (McGhee et al., 1992). Lymphocyte traffic patterns, which are regulated by selective expression of adhesion proteins in peripheral or mucosal lymphatic tissues, maintain anatomic segregation of immunologic memory (that enables the immune system to mount a more vigorous and effective response whenever it is restimulated by a specific foreign antigen) by causing antigen-primed cells to return to specific anatomic destinations, where they will encounter conditions that further facilitate expression of peripheral or mucosal immunity (Butcher and Picker, 1996; Ebnet et al., 1996) (Figure 22-1). The multitude of potential conditions includes the prevalence of specific cytokines, adhesion to and costimulation by specific stromal cells, and still, unknown microenvironmental factors intrinsic to those lymphoid compartments that favor commitment of B-cells to specific immunoglobulin isotypes or T-cells to peripheral or mucosal immunity (Anderson, 1990; Rott et al., 1996). Humoral immunity is mediated by euglobulins called antibodies that are produced locally but act at great distances from where they are made and secreted. Cellular immunity is like hand-to-hand combat. T-cells, natural killer (NK) cells, and "armed" macrophages enter into physical combat with the 3   IgG binds and enables the pathogen to be ingested and destroyed by a phagocytic host cell. IgA binds the pathogen and prevents it from binding to host cells so that the luminal fluid or mucous stream will carry it away.

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability FIGURE 22-1 A simplified drawing of the structures and connections of secondary lymphatic tissues where antigen may most efficiently direct immune responses. Multiple known and unknown factors intrinsic to the microenvironments of lymph node, spleen, and mucosa-associated lymphatic tissue (here, M.A.L.T is represented by Peyer's patch) influence whether a peripheral or mucosal type of response occurs. The drawing also indicates that these tissues are integrated with each other and the rest of the individual through vascular and lymphatic connections and a system of lymphocyte recirculation. Microenvironmental structures in the drawing are identified by the symbols below the drawing. pathogen and kill it by punching holes into its membranes or exposing it to enzymes. Cellular immunity also is associated with secretion of hormone-like molecules called cytokines and chemokines. These enable the effector cells to perform their duties actively. Many of the same cytokines also have effects on the humoral immune response by affecting B-cell division, differentiation, and maturation. The distinction between systems that regulate humoral immunity and those that regulate cellular immunity should not be confused with anatomic compartmentalization or with commitment to peripheral or mucosal immunity. There is a division of labor between cellular and humoral immunity that is parallel in both the peripheral and mucosal systems. Some of the same cell interactions and cytokines involved in cellular or humoral immunity are also involved in favoring peripheral over mucosal immunity, and vice versa (Abbas et al., 1996; Mosmann and Sad, 1996; Rocken and Shevach, 1996), as will be discussed later.

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability Compartmentalized Humoral Immunity All immunoglobulins, whether peripheral or mucosal, function by binding antigen in a pocket formed by the complementarity-determining regions (CDRs) encoded by the immunoglobulin heavy (VH)-and light (VL)-chain variable region genes (Carayannopoulos and Capra, 1993). The pocket formed by the VH-VL CDRs spatially conforms to the surface shape of the antigen that binds to the antibody (Figure 22-2). The method by which the genes for antigen-specific CDRs may be obtained quickly so that recombinant protective antibodies may be produced will be discussed later in this review. The antibody class conferred by the heavy-chain constant (C) region genes determines how the antibody will function and where it will act. Antibody C-region genes are expressed after rearrangement of the selected heavy-chain gene and attachment to the already rearranged variable, diversity, and joining region genes.4 Thus IgM, IgD, IgG3, IgG1, IgG2a, IgG2b, IgE, and IgA each have heavy chains that control how they participate in immunity, especially with regard to third-party molecular interactions such as Fc receptor binding,5 activation of the complement system, and endosomal transport across mucosal epithelial cells. Peripheral Immunity and IgG Antibodies of peripheral immunity protect the parenchymal organs and peripheral anatomic sites that are bathed in tissue fluid and supplied by the 4   Immunoglobulin diversity is a function of different choices in heavy and light chain genes that may be combined to make an immunoglobulin, choices of specific variable region genes for light and heavy chains, and somatic mutations incorporated in these variable regions during B-cell development. The diversity produced by the approximately 30 human D-genes involved in V-D-J recombination during early B-cell development produces a minimal effect on overall antibody diversity. The effect of this would be realized as a change in flexibility of the antigen-combining site, which may permit binding of antigens that deviate slightly from that required for ideal fit. The approximately five J-region genes (associated V-D-J genes produced on one chromosome) are located where the recombined V-D-J may join the heavy chain genes (from another chromosome). These joining regions are not the same thing as J-chain, which is an entirely different protein that associates posttranslationally with multimers of IgM or IgA and participates in transepithelial secretion by associating the polymeric immunoglobulin with poly-Ig receptor (secretory component) expressed on epithelial cell. 5   Immunoglobulins usually link the pathogen to a host cell or product. For example, the antigen-combining site binds to an antigen (the first party), the Fc portion (which is at the opposite end of the antibody) binds to a host cell or a product such as mucus (the second party). Other molecules, such as complement or J-chain bind to antibody near the middle of the molecule (the third party) and participate in functions that are related indirectly to that antigen-antibody interaction. Third party interactions are important modifiers of the function of antibodies and enable the antibody to perform more effectively the organ-specific function that they were designed to do.

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability FIGURE 22-2 Diagram of an immunoglobulin molecule. The heavy and light chains of an immunoglobulin molecule are displayed linearly in Panel A, and the antigen-combining site is displayed in native configuration in Panel B. In Panel A, disulfide bonds link the light to the heavy chains, and the heavy chains to each other, to form a dimer. The light-chain constant region 1 is labeled CL 1, and the three heavy-chain constant regions are labeled CH 1–3. Although CH 2 and 3 together form the Fc portion, only the CH 3 domain binds to Fc receptors and controls (defines) the anatomic specialization of the immunoglobulin. The CH 3 region is most important in functions of specific immunoglobulin isotypes. The light-and heavy-chain variable regions are labeled VL and VH, respectively. This linear diagram helps to show the sequential location of the complementarity-determining regions (CDR 1–3) and the framework regions (FR 1–4). Framework regions are relatively conserved portions of the variable region that serve as spacers to position the antigen-binding sites properly when the variable region is folded. Panel B shows how an antigen-binding pocket forms when the VL-VH CDRs are folded into native configuration.

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability blood microvasculature. These antibodies maximize cellular uptake and internalization of antigens. After pathogens have breached the barriers of the skin and/or mucous membranes, antibodies of the IgM and IgG subclasses work in conjunction with the complement system. This collaboration serves to neutralize, injure, aggregate, and opsonize (to coat the pathogen with antigen-specific antibody and complement so that it can be ingested easily and destroyed by a phagocyte that is bearing receptors for Fc and complement) the pathogens so that they may be engulfed and destroyed by phagocytes. Except in rare instances where pentameric IgM (complexed with joiner [J] chain) may be secreted across epithelium, most circulating antibodies of the IgM and IgM subclasses work in blood, lymph, and tissue fluids. They do not normally appear in mucosal secretions (Underdown and Mestecky, 1994). Mucosal Immunity and Secretory IgA Antibodies of mucosal immunity function outside the body at luminal surfaces of the moist epithelium lining conjunctiva; nasopharynx; oropharynx; gastrointestinal, respiratory, and urogenital tracts; and in the ducts or acini of exocrine glands. The principal antibody involved in mucosal immunity is secretory IgA (Underdown and Mestecky, 1994). This class of antibody requires the cooperation of two cell types for optimal activity in vivo. One cell type, the plasma cell, makes the IgA, while an epithelial cell transports it to the gut lumen where it works (Figure 22-3, Panel A). The plasma cell posttranslationally dimerizes the IgA by joining two molecules with another polypeptide, the J-chain. In addition to holding the two IgA molecules together, the J-chain facilitates binding to a poly-Ig receptor synthesized by and displayed on the abluminal side of epithelial cells. The complex is transported in endosomes to the luminal side of the epithelial cell and secreted. The portion of the poly-Ig receptor retained with secreted IgA is called the secretory component. Pathogens adapted to infect mucosa express virulence factors that allow them to adhere, colonize, or invade epithelium. Secretory IgA prevents absorption of these viruses, bacteria, and toxins by blocking their adhesion while they are still on the external side of an epithelial barrier. This activity is opposite to that of antibodies associated with peripheral immunity, which aid the host cells to bind pathogens. By preventing cellular attachment of the antigen, IgA enables it to be flushed away in the stream of secreted fluids and mucous washing over the epithelial membranes. IgA also may facilitate transport of pathogens and toxins out of the body by causing them to be conveyed into bile and other exocrine secretions (Mazanec et al., 1993). At sites where nondegradative endosomal transport delivers a pathogen into the host, such as across M-cells in the dome epithelium of the Peyer's patch (the region of a Peyer's patch where antigen from the gut lumen is transported into the lymphatic tissue) (Neutra and Kraehenbuhl, 1994; Owen

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability FIGURE 22-3 Diagram of small intestinal mucosa. Panel A shows the synthesis of secretory IgA by plasma cells and transport to the intestinal lumen by epithelial cells (dots and interrupted arrows). Panel B shows an intestinal villus. Large black dots represent sites where intraepithelial lymphocytes (IEL) involved in cellular prophylaxis, thymic independent T-cell maturation, or tolerance induction usually are found. and Jones, 1974), antigen-specific IgA recently has been shown to neutralize viral pathogens while they are in the endosome (Mazanec et al., 1992). IgA is the predominant antibody manufactured by the body. This escaped appreciation for many years because blood contains a relatively low concentration of IgA compared with other immunoglobulins. However, 75 percent of the antibody-producing cells in the body are making IgA, and most of this IgA is released on a continuous basis into gastrointestinal fluid, saliva, tears, urine, and other secretions. In humans, up to 40 mg/kg body weight of IgA is manufactured and secreted daily, which is many orders of magnitude greater than that of all other immunoglobulin isotypes (Brandtzaeg, 1994). Compartmentalized Cell-Mediated Immunity T-lymphocytes involved in peripheral and mucosal cell-mediated immunity compromise numerous functional subclasses of increasing diversity (Punt and Singer, 1996). Both T-helper cells (CD4) and cytotoxic T-suppressor cells (CD8) may assume immunoregulatory roles during immune responses, or they may differentiate into effector cells that exhibit segregated traffic patterns and functions (Anderson and Shaw, 1996; Ebnet et al., 1996; Salgame et al., 1991).

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability The profile of cytokines secreted by T-cells dictates immunoreactive cell commitment to either peripheral or mucosal immune functions. Gamma interferon from T-helper 1 (Th1) cells enhances peripheral immunity and suppresses mucosal immunity. IL-4 from T-helper 2 (Th2) cells enhances mucosal immunity and suppresses peripheral immunity. Other cytokines can further influence this divergence or lead to partial or complete compromises. Peripheral T-Cells. T-cells committed to peripheral immunity circulate in the blood and help B-or T-cell precursors differentiate into antibody-secreting cells or cytotoxic effector cells respectively. These T-cells also release a number of cytokines that activate and arm mononuclear phagocytes and natural killer cells to destroy intracellular parasites. Mucosal T-Cells. Mucosally committed T-cells enter Peyer's patches, the lamina propria (the layer of connective tissue underlying the mucosal epithelium), and the intraepithelial compartment (Figure 22-3, Panel B). There is considerable phenotypic diversity among these mucosal T-cells. The subclass of mucosa-homing T-cells known as intraepithelial lymphocytes is believed to play a role in protecting mucosal surfaces from being injured by infectious pathogens or parasites (London et al., 1987). However, some of the diverse cells that populate the intraepithelial compartment include thymic-independent T-cells, which enter the epithelium to complete maturation (Punt and Singer, 1996). Many of these cells display phenotypic markers that reflect intermediate stages of T-cell differentiation. CD8 T-Cell Heterogeneity. Heterogeneity of T-cells of the suppressor-cytotoxic phenotype (CD8) has been described, especially for those cells located within the mucosal epithelium. The CD8 membrane marker is expressed as a heterodimeric complex consisting of an alpha and a beta chain (i.e., CD8ab). Within the intraepithelial compartment, CD8+ cells6 are commonly found expressing two alpha chains (CD8aa). T-cells bearing CD8ab and CD8aa may exhibit separate traffic patterns, as suggested by the observation that they are found in peripheral and mucosal compartments in different ratios. They also may participate in regulating the balance between peripheral and mucosal immunity to specific antigens because of their nominal "suppressor" activity (Salgame et al., 1991). There is some indication from basic studies that these cells exhibit organ-selective commitments. 6   CD8 is a phenotype. CD8+ indicates that a cell bears that phenotype. However, that phenotype is heterogeneous because the presence or absence of CD8b permits identification of two subtypes, one with both chains (CD8ab) and the other with two a-chains (CD8aa). It is possible that these two phenotypes represent peripheral and mucosal CD8+ cells, respectively.

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability CD8 Homing and Cross-Regulation. When mice with severe combined immunodeficiency (SCID) (which lack mature T-or B-cells) are reconstituted with lymphocytes derived from CB17 mice (normal parent strain of SCID), the fate of cells derived from a particular tissue may be traced in vivo (Hilbert et al., 1994). If residence in that tissue imprints functional commitment or organ selectivity, then that will be reflected by the phenotype and homing characteristics of the cells after transfusion. Normal lymphoid cells derived from CB17 lymph nodes or Peyer's patches were transfused into syngeneic SCID mice. Donor CD8ab T-cells, extracted from CB17 lymph nodes, lodged in lymph node para-cortex and in the intestinal lamina propria of SCID recipients. The CD8ab lymph node cells did not enter the intraepithelial compartment but remained near plasma cells in the lamina propria. The preponderant CD8+ phenotype in CB17 Peyer's patch and intraepithelial lymphocytes (IEL) is CD8aa. Infusion of Peyer's patch cells resulted in excellent reconstitution of SCID IEL, especially of CD8aa T-cells. Thus, the homing preference of these putative suppressor cell types is consistent with the possibility of their playing a role in antigen-specific cross-regulation of peripheral versus mucosal immunity as discussed later. Functions of CD8+ Intraepithelial Lymphocytes. The exact functions of IEL subpopulations are not yet known, but animal experiments and ex vivo cellular cytotoxicity assays suggest that some IEL exhibit cellular cytotoxicity directed against viral antigens on infected mucosal epithelial cells (Cuff et al., 1993; London et al., 1987). IEL also are thought to be involved in initiating tolerance (Elson et al., 1995; Gelfanov et al., 1996; Sim, 1995), but it is not clear whether all IEL, or a unique subset of IEL, induce tolerance to food antigens. Presentation of antigen in a manner similar to that of nonclassical major histocompatibility (MHC) antigens7 displayed on basolateral membranes of intestinal epithelial cells in the absence of costimulatory signals8 (Robey and Allison, 1995) may be involved in triggering tolerogenic T-cells (T-cells that induce immunological tolerance; the concept of tolerance will be discussed later) (Matzinger, 1994). There are many basic questions to be answered in the rapidly enlarging field devoted to IEL function. 7   MHC antigens are cell-surface proteins responsible for determination of tissue type and transplant compatibility. Found on antigen-presenting cells, they are divided into two classes: Class 1 MHC antigens form complexes with intracellular peptide fragments after proteosomal digestion; Class II MHC antigens form complexes with extracellular antigens after lysosomal digestion. 8   Costimulatory signals are signals that must be provided simultaneously with the principal signals for an event to progress as expected. The immune system uses these like passwords. Presentation of an antigen to an activated lymphocyte in the absence of a costimulatory signal may cause the cell to die suddenly by apoptosis, instead of starting the process of dividing and differentiating into antibody-producing cells. Specific receptor-ligand interactions are involved.

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability Wong, Y.W., P.H. Kussie, B. Parhami-Seren, and M.N. Margolies 1995 Modulation of antibody affinity by an engineered amino acid substitution. J. Immunol. 154:3351–3358. Wu, H-Y., and M.W. Russell 1994 Comparison of systemic and mucosal priming for mucosal immune responses to a bacterial protein antigen given with or coupled to cholera toxin (CT) B subunit, and effects of pre-existing anti-CT immunity. Vaccine 12(3):215–222. Xu-Amano, J., H. Kiyono, R.J. Jackson, H.F. Staats, K. Fujihashi, P.D. Burrows, C.O. Elson, S. Pillai, and J.R. McGhee 1993 Helper T-cell subsets for immunoglobulin A responses: Oral immunization with tetanus toxoid and cholera toxin as adjuvant selectively induces Th2 cells in mucosa-associated tissues. J. Exp. Med. 178:1309–1320. Yamamura, M., K. Uyemura, R.J. Deans, K. Weinberg, T.H. Rea, B.R. Bloom, and R.L. Modlin 1991 Defining protective response to pathogens: Cytokine profiles in leprosy lesions. Science 254:277–279. Yan, C., J.H. Resau, J. Hewetson, M. West, W.L. Rill, and M. Kende 1994 Characterization and morphological analysis of protein-loaded poly(lactide-coglycolide) microparticles prepared by water-in-oil-in-water emulsion technique. J. Controlled Release 32:231–241. Young, H.A., P. Ghosh, J. Ye, J. Lederer, A. Lichtman, J.R. Gerard, L. Penix, C.B. Wilson, A.J. Melvin, M.E. McGurn, D.B. Lewis, and D.D. Taub 1994 Differentiation of the T-helper phenotypes by analysis of the methylation state of the IFN-ß gene. J. Immunol. 153:3603–3610. Zhang, R.G., M.L. Westbrook, S. Nance, B.D. Spangler, G.G. Shipley, and E.M. Westbrook 1995 The three-dimensional crystal structure of cholera toxin. J. Mol. Biol. 251:563–573. DISCUSSION ROBERT NESHEIM: Are there any questions? I have a question. How stable are these antibodies in the plant materials? ARTHUR ANDERSON: The antibodies are very stable when expressed in plant materials. The Streptococcus mutans-specific IgA that Ma et al. (1995) expressed in transgenic tobacco plants had been secreted into a plant compartment that made it very easy to extract. All they had to do was freeze, grind up the leaves, centrifuge at 15,000g to remove fiber and large plant molecules, and the antibodies were precipitated out of soluble proteins with 40 and 60 percent (NH4)2SO4. ROBERT NESHEIM: The antibody genes can just be put into foods? What happens with heat processing? ARTHUR ANDERSON: That could be a problem with protein antigens or antibodies expressed in plants. In the experiment of Haq et al. (1995), the mice first

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability were given crude tobacco leaf extract. In later experiments, they ate raw potato tubers. If the tubers were cooked, as we like them, the antigen would be denatured and may not induce immunity. This is also a problem with cooking antibodies. Everything I said about use of cholera toxin B-subunit as a carrier for oral vaccines absolutely depends on it retaining its conformational integrity. If the B-subunit pentameric ring is denatured and it cannot bind host cell membrane GM1 gangliosides, then everything I said is incorrect. However, Arntzen has been working on developing foods like transgenic bananas that could be eaten raw. Banana also could be stabilized and dried using sugars and lyophilization. This would enable it to be microencapsulated in an enteric coating to get it past the denaturing effect of stomach acid. These are issues that people who prepare Meals, Ready-to-Eat (MREs) are most ready to test. I have not done all of the components of the vision I presented. However, the literature says that the field is ready to apply this new technology for protection of soldiers. Obviously, I would first like to protect a soldier against the common mucosal pathogens that cause intestinal pathology. I would use recombinant antibodies from transgenic plants incorporated into every MRE to provide passive protection. At the same time, I would periodically give the soldiers "special MREs" or "vaccine candy bars" with the cholera B-subunit linked vaccines to enable development of acquired active immunity. There is no reason to suspect that oral passive antibody would block development of immunity with encapsulated or carrier-linked antigens. GABRIEL VIRELLA: If the ganglioside is not expressed, how does the recombinant cholera toxin B-subunit get to the right cells? ARTHUR ANDERSON: Cholera toxin B-subunit (CTB) binds to GM1 ganglioside on all eukaryotic cells. The sialomucins on absorptive epithelial cells interfere slightly with binding, which causes the carrier to bind to M-cells over mucosal lymphatic tissues or to IEL [intraepithelial lymphocytes] cells, some of which have higher GM1 ganglioside in their membranes. The mucosal M-cells are exactly where we want the antigen to bind because this is where antigens must be taken up for a mucosal immune response to result. The CTB binds more in the proximal small bowel than in the distal bowel because binding depletes the material before it reaches the distal bowel. However, proximal bowel binding has produced the desired results. In sheep, the distal Peyer's patches in the ileum have a different function from those in the proximal bowel. We do not know if having antigens bind to distal bowel lymphatic tissues might change the ratio of antigen priming to tolerance induction, for example.

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability On the other hand, there is a great deal of interest in using enterotoxin-derived carriers in stimulating immunity via intranasal route, which accesses the tonsils and adenoids as antigen-reactive mucosal lymphatic tissues. DOUGLAS WILMORE: There is another route by which antigens can enter the gut, and that is by paracellular channels—gaps that form when the gut is under-perfused and as a result of the effects of acidosis on mucosal integrity. People are now debating how the influence of endotoxin or other products affect antigens coming up those pathways. Do you see other mechanisms to protect the gut in that way? ARTHUR ANDERSON: There has been research about particulate antigens, such as bacteria and yeast, gaining access to the blood circulation by mechanisms you referred to. Griffiss has shown that levels of IgA in the circulating blood appear to dampen the effects of bacteremia on complement activation. There is a blood threshold maintained by IgA levels, which, when exceeded, allows endotoxin to unleash its effects on cytokine cascades, complement activation, and bradykinin activation. Endotoxin and yeast and bacteria normally enter your blood during eating and eliminating and brushing one's teeth. Most people do not show any physiological reactions to this as long as their IgA levels are normal. On the other hand, IgA-deficient people are very susceptible to endotoxin-mediated shock. Endotoxin-containing immunogens actually have been shown to down-modulate mucosal immunity. This has been confirmed by using C3H/HeN and C3H/HeJ mouse substrains. The C3H/HeJ mice lack the gene for cytokine response to endotoxin. These mice are resistant to induction of endotoxin shock. They also mount very potent IgA responses. The endotoxin-susceptible C3H/HeN mouse shows dose-related decrements in IgA production related to endotoxin added to the GI tract. There are a series of publications on this coming from Gerry McGhee's laboratory in Birmingham, Alabama. JOHANNA DWYER: I remember that, a long time ago, one of the formula companies wanted to put antibodies in infant formulas, and I think it foundered. One of the concerns was that some infants might get leaky guts or something, which prevents complete benefit of the antibodies, and they still get sick. Does what you propose have such a danger? Can it be a two-edged sword? ARTHUR ANDERSON: That is a good question. What I have presented about conventional modes of immunization already creates a two-edged sword. In my review, I hope I have narrowed down the alternatives to reduce this risk.

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability What you referred to is a phenomenon which only occurs in infants. It is called the phenomenon of gut closure. New babies do not have all the functional proteases for a while after they are born. The phenomenon relates to the fact that relatively intact proteins can be directly absorbed across the mucosa. After the mucosa matures to a point where most proteases are released, only proteins with mechanisms to resist protease degradation can get across. This, I believe, is what gut closure means. If non-IgA antibodies to pathogens are put in milk, some of those antibodies could actually enable concentration and absorption of viral pathogens in newborn babies. This would result in a paradoxical adverse effect. Normal cow's milk already has secretory IgA against many pathogens, so adding antibodies would not really help. If the antibodies were not dimeric IgA with J-chain and secretory component, they also would not help. The added IgA was too expensive because of its method of preparation; and once you start, you have to keep giving it. I would not have done it with the technology available then, either. I also want to comment on the double-edged sword of using enterotoxin carriers (CT/LT). Both CTB and LT/mLT have been tested in phase I trials in humans. These vaccines have been tolerated well, and dose finding studies have produced benign toxicities. I have enjoyed being a voyeur to the vaccine trials of products developed by Jan Holmgren, John Mekalanos, and John Clements and tested in the Clinical Studies unit of USAMRIID by Dave Taylor and Dan Scott, respectively. In normal unvaccinated subjects, cholera holotoxin or Vibrio cholerae infection is going to cause cramping and diarrhea. When CTB, with or without killed Vibrios, or LTB is given to unvaccinated volunteers, no signs or symptoms result. I have not referred to all the individual studies which show that oral use of CT/LT or CTB with antigen result in both IgA and IgG immune responses. I don't see a double-edged sword there, unless B-subunit loses GM1-binding activity. DAVID SCHNAKENBERG: How often do you have to present antibodies by the oral route? You said IgA could be put in MREs. To get the effect, do you have to administer it daily? ARTHUR ANDERSON: There is pretty wide variability in gut transit time. In the mouse, it is about 6 hours. In human beings, it can be anything from hours to days. If you use enterically coated antibodies in three meals a day, there should be sufficient antibody present all the time. IgA binds to intestinal mucin. The mucin moves more slowly than does the fecal stream, which should allow a buildup of IgA.

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Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability DAVID SCHNAKENBERG: What is the lag time from dosage to effective protection? If you are using this only to a point of deployment, is this something you start 2 weeks ahead of time? ARTHUR ANDERSON: If you are referring to passive antibody protection, it would be adequate to give soldiers the antibodies in their meals en route. By the time they arrive, their intestines would be protected. If you are referring to active protection with vaccines, it is preferrable that the initial priming doses be given as long in advance as possible. I would say it is best to immunize troops during basic training. It is not essential that antibody levels be maintained. Desert Shield/Desert Storm taught us to trust immunological memory. There were people who got only one dose of vaccine and people who got a full series of shots before going overseas; both had sufficient antibody titers to be protective when given a booster 24 to 36 months later. Circulating memory cells will ensure that soldiers ready to deploy will have a return in protective antibody titer within 3 days of the booster. That is what I believe we need to do. HARRIS LIEBERMAN: There is a special category of foods, I believe, of medical foods. It sounds like the kinds of things you are talking about might go into that category, a special sort of deployment ration that is essentially a medical food. I don't think you would want to put antigens in all the MREs, but you would want to have a special category. ARTHUR ANDERSON: I agree. It is not intended that chronic enteric exposure to vaccine antigens be given. That is one of the ways tolerance to food antigens develops. However, your idea of preparing an easily identifyable MRE as a medical food, with instructions on the cover about frequency of use for optimal immunity, would be useful in assuring the proper use by deployed troops. How this would be utilized effectively is something your institute is better suited for determining.