Role of secretory antibodies in the defence against infections

Int. J. Med. Microbiol. 293, 3 ± 15 (2003) ¹ Urban & Fischer Verlag http: // www.urbanfischer.de/journals/ijmm

Role of secretory antibodies in the defence against infections Per Brandtzaeg Laboratory for Immunohistochemistry and Immunopathology (LIIPAT), Institute of Pathology, University of Oslo, Rikshospitalet, N-0027 Oslo, Norway

Abstract Adaptive immunity mediated by secretory antibodies is important in the defence against mucosal infections. Specific secretory immunoglobulin A (SIgA) can inhibit initial pathogen colonization by performing immune exclusion both on the mucosal surface and within virusinfected secretory epithelial cells without causing tissue damage. Resistance against toxinproducing bacteria such as Vibrio cholerae appears to be particularly dependent on SIgA antibodies. Like natural infections, live topical vaccines or adequate combinations of inactivated vaccines and mucosal adjuvants give rise not only to SIgA antibodies, but also to longstanding serum IgG and IgA responses. The intranasal route of vaccine application could be particularly attractive to achieve this result, but only if successful stimulation is obtained without the use of toxic adjuvants. The degree of protection after vaccination may range from complete inhibition of reinfection to reduction of symptoms. In this scenario it is generally difficult to determine unequivocally the relative importance of SIgA versus serum antibodies. However, infection models in knockout mice strongly support the notion that SIgA exerts a decisive role in protection and cross-protection against a variety of infectious agents. Key words: Immunoglobulin A (IgA) ± secretory IgA ± secretory component ± secretory IgM ± IgG ± mucosal immunity ± polymeric Ig receptor

Introduction More than 90% of all infections involve the mucosae with regard to microbial colonization or entry into the body. Immune exclusion is a term coined for non-inflammatory adaptive mucosal surface protection mediated by immunoglobulin A (IgA) antibodies in co-operation with innate nonspecific defence factors and thus refers to the `first line' of microbial defence. This mechanism has a formidable task because the mucosal surface area is approximately 400 m2 in an adult individual, and is mostly covered by a vulnerable monolayered epithelium.

The cellular basis for immune exclusion is the fact that secretory mucosae and exocrine glands contain most of the body's activated B cells ± terminally differentiated to Ig-producing blasts and plasma cells (collectively called immunocytes). The majority of these local immunocytes (70 ± 90%) produce dimers and some larger polymers of IgA (collectively called pIgA) which, together with pentameric IgM, can be transported through secretory epithelia to provide secretory IgA (SIgA) and SIgM antibodies (Brandtzaeg et al., 1999a). In the first postnatal period, only traces of SIgA and SIgM occur in human external secretions,

Corresponding author: Per Brandtzaeg, LIIPAT, Institute of Pathology, Rikshospitalet, N-0027 Oslo, Norway. Phone: ‡ 47 2307 2743, Fax: ‡ 47 2307 1511, E-mail: [email protected]

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whereas some IgG is often present as a result of `leakage' from the mucosal lamina propria, which ± because of placental transfer ± contains readily detectable maternal IgG as early as at 34 weeks of gestation (Brandtzaeg et al., 1991). Therefore, an adequate mucosal barrier function of neonates depends on a supply of SIgA antibodies from breast milk (Anonymous, 1994). In the developing countries, mucosal infections are a major killer of children below the age of 5 years, being responsible for more than 14 million deaths annually. In such areas infants are particularly dependent on breast milk antibodies to protect their mucosae, and epidemiological data suggest that the risk of dying from diarrhoea is reduced 14 ± 24 times in sucklings (Hanson et al., 1993; Anonymous, 1994). Moreover, experiments in neonatal rabbits strongly suggest that SIgA is the crucial anti-microbial component of breast milk (Dickinson et al., 1998). Although the value of nursing in `westernized' countries is most apparent in preterm infants (Hylander et al., 1998), exclusively breast-fed infants are better protected against a variety of infections (Pisacane et al., 1994; Wold and Hanson, 1994; Newman, 1995; Wright et al., 1998) and ± although still being debated (Oddy et al., 2002; Sears et al., 2002) ± apparently also against allergic disease, asthma (Saarinen et al., 1995; Oddy et al., 1999; Kull et al., 2001) and coeliac disease (Brandtzaeg, 1997). It should be noted, however, that intestinal uptake of SIgA antibodies from breast milk is of no importance for systemic immunity (Ogra et al., 1977; Klemola et al., 1986), except perhaps to some extent in the preterm infant (Weaver et al., 1991). Although gut closure normally occurs in humans mainly before birth, a patent mucosal barrier may not be established until after 2 years of age; the different variables involved in this process are poorly defined (van Elburg et al., 1992). Interestingly, the postnatal colonization of commensal bacteria is important for both establishing (Hooper et al., 2001) and regulating (Neish et al., 2000) an appropriate epithelial barrier.

Induction of mucosal immune responses The various secretory effector sites receive their primed B cells from inductive mucosa-associated lymphoid tissue (MALT) structures, which sample antigens directly from the epithelial surface (Table 1). Although gut-associated lymphoid tissue (GALT), including Peyer's patches in the distal ileum, the appendix and numerous isolated lym-

phoid follicles constitute the major part of MALT, induction of mucosal immune responses apparently takes place also in the palatine tonsils and other lymphoepithelial structures of Waldeyer's pharyngeal ring, including nasopharynx-associated lymphoid tissue (NALT) such as the adenoids in humans, and perhaps bronchus-associated lymphoid tissue (Brandtzaeg et al., 1987; 1999a, b). However, the latter type of organized lymphoid tissue structures are not present in normal lungs of adult humans and only in approximately 40% of adolescents (Hiller et al., 1998). Accumulating evidence suggests that a certain regionalization exists in the mucosal immune system, especially a dichotomy between the gut and the upper aerodigestive tract with regard to differentiation and homing properties of B cells (Brandtzaeg et al., 1999b). This disparity may be explained by microenvironmental differences in the antigenic repertoire as well as adhesion molecules and chemokines (Table 1) involved in selective leucocyte extravasation (Brandtzaeg et al., 1999a, b; Kunkel and Butcher, 2002). It appears that primed immune cells preferentially home to effector sites corresponding to the inductive sites where they initially were triggered by antigens. Such compartmentalization within the `common' or integrated mucosal immune system has to be taken into account in the development of local vaccines. Nevertheless, as reviewed elsewhere (Brandtzaeg et al., 1987), there is communication among various mucosal effector compartments, and the mucosal and systemic lymphoid cell systems are not completely segregated, particularly not in the upper respiratory tract (Brandtzaeg et al., 1999a). MALT structures resemble lymph nodes with Bcell follicles, intervening T-cell areas, and a variety of antigen-presenting cell (APC) subsets, but there are no afferent lymphatics supplying substances for immunological stimulation. Therefore, the exogenous antigens are sampled directly from the mucosal surface, apparently to a large extent by specialized cells of the follicle-associated epithelium (FAE) called membrane (M) cells (Table 1). Mesenteric and cervical lymph nodes may share immune-inductive properties with the respective regional MALT structures, and most likely receive antigens via afferent lymph as well as via antigen-transporting dendritic cells (DCs), which continuously migrate through draining lymph from the mucosal surface (Fig. 1). It has recently been shown in mice that proliferating T cells rapidly obtain gut- or skin-homing properties during antigen priming in mesenteric or peripheral lymph nodes, respectively (Campbell and Butcher, 2002). The responsible factors remain

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Table 1. Characteristics of the systemic versus the mucosal immune system Systemic immunity Inductive sites Antigen uptake and transport

Shared features

Ordinary surface epithelia

Mucosal immunity Epithelia with membrane ( M ) cells

Dendritic cells ( DCs) Draining lymph, peripheral lymph nodes, blood circulation, spleen and bone marrow

Mucosa-associated lymphoid tissue ( MALT ): Peyer's patches, appendix and solitary lymphoid follicles ( GALT ) Tonsils and adenoids Local (regional) lymph nodes

Influx of circulating lymphoid cells: adhesion molecules and chemokines/chemokine receptors Effectors sites Homing of memory and effector T and B cells

Antibody production

Postcapillary high endothelial venules ( HEVs) PNAd/L-selectin ( CD62L ) SLC ( CCL21), ELC ( CCL19)/CCR7 GALT: MAdCAM-1/a4b7 Peripheral (lymphoid) tissues and sites of chronic inflammation: a variety of adhesion molecules and chemokines/chemokine receptors IgG > monomeric IgA > polymeric IgA > IgM

Mucosal lamina propria and exocrine glands: MAdCAM-1/a4b7 (gut), other adhesion molecules (? extraintestinal), TECK ( CCL25)/ CCR9 (small intestine), MEC ( CCL28)/CCR10 (? elsewhere) Tonsils and adenoids

Polymeric IgA > IgM  IgG

Abbreviations: GALT, gut-associated lymphoid tissue; PNAd, peripheral lymph node addressin; SLC, secondary lymphoid-tissue chemokine; ELC, Epstein Barr virus-induced molecule 1 ligand chemokine; CCR, CC chemokine receptor, MAdCAM-1, mucosal addressin cell adhesion molecule 1; TECK, thymus-expressed chemokine; MEC, mucosaassociated epithelial chemokine.

unknown, but similar mediators can probably induce homing capacity for the upper aerodigestive tract when T and B cells are stimulated by antigens in Waldeyer's ring as well as in the cervical lymph nodes. Antigens reaching the regional lymph nodes via draining lymph, or being transported there by DCs, may hence preferentially elicit or reinforce mucosal immunity in the same region (Fig. 1). Importantly, the human nasal mucosa is extremely rich in various DC types, both within and beneath the epithelium (Jahnsen et al., 2003). To be highly successful, an intranasally applied vaccine should therefore probably aim to target both mucosal DCs in the nasal cavity and FAE (including M cells) of MALT structures in Waldeyer's ring. Among the T cells of MALT structures, the CD4‡ helper subset predominates ± the ratio between CD4 and CD8 cells being similar to that of other peripheral T-cell populations (Brandtzaeg et al., 1999a). In addition, primed (memory/effector) B cells aggregate together with T cells in the M-cell pockets, which thus may represent the first contact site between immune cells and luminal antigens (Yamanaka et al., 2001). The development of these B- and T-cell clusters associated with M cells depends on microbial colonization and are absent

in germ-free animals (Yamanaka et al., 2003). The activated B cells probably perform important antigen-presenting functions in this compartment, perhaps promoting antibody diversification and immunological memory. Other types of professional APCs, such as DCs and macrophages, are located below the FAE and between the follicles (Fig. 1). Aided by DCs the CD4‡ T cells will also become activated by microbial and other antigens in MALT, and secrete cytokines such as transforming growth factor b and interleukin 10 (IL-10), thereby inducing antigen-specific B cells to become predominantly IgA-committed plasma blasts (Brandtzaeg et al., 1999a). These primed B cells proliferate and differentiate further on their route through regional lymph nodes before they home via peripheral blood to mucosal effector compartments (Fig. 1). At these sites they complete their terminal differentiation, mainly to become pIgA-producing plasma cells ± again most likely under the influence of T cells and DCs that may pick up antigens from the epithelial surface (Rescigno et al., 2001). A prerequisite for pIgA production is cellular expression of a peptide called the joining (J) chain, apparently signifying a relatively early maturational stage of effector B cells (Brandtzaeg, 1974a; Brandtzaeg et al., 1999c).

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Fig. 1. Schematic depiction of the human mucosal immune system. Inductive sites for mucosal T and B cells are constituted by regional mucosa-associated lymphoid tissue (MALT) with their B-cell follicles and M cell (M)-containing follicle-associated epithelium through which exogenous luminal antigens are actively transported to reach professional antigen-presenting cells (APCs), including dendritic cells (DCs), macrophages, B cells and follicular dendritic cells (FDCs). In addition, intra- or subepithelial DCs may capture antigens and migrate via draining lymph to regional lymph nodes where they become active APCs, which stimulate T cells for productive or down-regulatory (suppressive) immune responses. Naive B and T cells enter MALT (and lymph nodes) via high endothelial venules (HEVs). After being primed to become memory/effector B and T cells, they migrate from MALT and regional lymph nodes via lymph and peripheral blood for subsequent extravasation at mucosal effector sites. This process is directed by the profile of adhesion molecules and chemokines expressed on the microvasculature, the endothelial cells thus exerting a local gatekeeper function for mucosal immunity. The mucosal lamina propria is illustrated with its various immune cells, including B lymphocytes, J chain-expressing IgA and IgM plasma cells, IgG plasma cells with a variable J-chain level (J), and CD4‡ T cells. Additional features are the generation of secretory IgA (SIgA) and secretory IgM (SIgM) via pIgR (SC)-mediated epithelial transport, as well as paracellular leakage of smaller amounts (broken arrow) of both locally produced and serumderived IgG antibodies into the lumen. Note that IgG cannot interact with J chain to form a binding site for pIgR. The distribution of intraepithelial lymphocytes (mainly T-cell receptor a/b‡CD8‡ and some g/d‡ T cells) is schematically depicted. Insert (lower left corner) shows details of an M cell and its `pocket' containing various cell types.

Local production of secretory antibodies Secretory mucosae and exocrine glands of adults contain more than 80% of all antibody-producing cells in the body (Brandtzaeg et al., 1989). Approximately 80 ± 90% are IgA immunocytes, and a larger fraction generally consists of the IgA2 subclass than in peripheral lymphoid tissue ± reaching a dominance over the IgA1 subclass in the large bowel mucosa (Brandtzaeg et al., 1999a). A relative increase of the IgA2 isotype in secretions compared with serum, could be important for the stability of secretory antibodies because SIgA2, in contrast to SIgA1, is resistant to several proteases synthesized by

a variety of potentially pathogenic bacterial species (Kilian et al., 1996). However, production of IgA1 is dominating both in the nasal (93%) and bronchial (75%) mucosa (Brandtzaeg et al., 1999a), and bacteria such as Haemophilus influenzae, Streptococcus pneumoniae and Neisseria meningitidis show frequent synthesis of IgA1-specific proteases; this could contribute to the fact that those three bacterial species are prone to produce invasive disease of the upper respiratory tract. More than 90% of the mucosal IgA immunocytes normally synthesize the J chain (Brandtzaeg, 1974a; Brandtzaeg et al., 1999a) ± a 15-kDa peptide that is essential for correct polymerization of pIgA and pentameric IgM (Johansen et al., 2000; 2001) and

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Fig. 2. Model for local generation of secretory IgA and secretory IgM. J chain-containing dimeric IgA (IgA ‡ J) and pentameric IgM (IgM ‡ J) are produced by local plasma cells (left). Polymeric Ig receptor (pIgR), or membrane secretory component (SC), is synthesized by secretory epithelial cells in the rough endoplasmic reticulum and matures in the Golgi complex by terminal glycosylation (*-). In the trans-Golgi network (TGN), pIgR is sorted for delivery to the basolateral plasma membrane. The receptor becomes phosphorylated (*-) on a serine residue in its cytoplasmic tail. After endocytosis, ligand-complexed and unoccupied pIgR is delivered to basolateral endosomes and sorted for transcytosis to apical endosomes. Some recycling from basolateral endosomes to the basolateral surface may occur for unoccupied pIgR (not shown). Receptor recycling also takes place at the apical cell surface as indicated, although most pIgR is cleaved to allow extrusion of SIgA, SIgM and free SC to the lumen. During epithelial translocation, covalent stabilization of SIgA regularly occurs (disulfide bond between bound SC and one IgA subunit indicated), whereas free SC in secretions stabilizes the noncovalently bound SC in SIgM (dynamic equilibrium indicated). Modified from (Brandtzaeg et al., 1999a).

their subsequent binding to the polymeric Ig receptor (pIgR). This  100-kDa transmembrane multidomain glycoprotein is expressed basolaterally on secretory epithelial cells as so-called membrane secretory component (SC) (Brandtzaeg, 1974b; Brandtzaeg and Prydz, 1984). The elements of the five extracellular Ig-like SC domains that contribute to the interactions with pIgA and pentameric IgM have been extensively characterized (Norderhaug et al., 1999; Johansen et al., 2000). The ligandreceptor complexes are endocytosed, transcytosed through the epithelial cell and then ± after apical pIgR cleavage ± released into the secretion (Fig. 2). The  80-kDa extracellular portion of the receptor remains as bound SC in SIgA and SIgM, thereby stabilizing the secretory antibodies. Particularly the covalent bonding between SC and an a-chain of pIgA makes SIgA the most stable antibody of the immune system (Brandtzaeg et al., 1999a; Norderhaug et al., 1999; Johansen et al., 2000). Cleaved

unoccupied pIgR is released in variable amounts to the secretions as so-called free SC, which may exert certain innate defence functions as discussed below. More pIgA is translocated to the adult gut lumen by the pIgR every day by this mechanism ( 40 mg/ kg body weight) than the total daily IgG production ( 30 mg/kg) (Conley and Delacroix, 1987). Therefore, the intestinal mucosa is quantitatively the most important effector organ of adaptive humoral immunity Although the receptor expression is constitutively regulated, it can be upregulated at the transcriptional level by the immunoregulatory cytokines interferon-g and IL-4, as well as by the proinflammatory cytokines tumour necrosis factor and IL-1 (Brandtzaeg et al., 1992, 1999a; Norderhaug et al., 1999; Schjerven et al., 2000, 2001). Both constitutive and cytokine-enhanced pIgR expression appears to depend on adequate presence of vitamin A (retinoic acid) and the nutritional state of the subject (Ha et al., 1998; Sarkar et al., 1998). Also

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P. Brandtzaeg Table 2. Antimicrobial characteristics of secretory IgA antibodies Immune exclusion (noninflammatory) * Secretory IgA is exceptionally stable; provides prolonged function in secretions * Inhibition of epithelial adherence and penetration * Efficient microbial agglutination and virus neutralization * Secretory IgA operates both extra- and intracellularly * Extensive polyreactive activity (cross-protection) * Enhanced mucophilic properties * May inhibit growth factors, enzymes and plasmids Immune elimination (potentially proinflammatory) * Phagocytosis and cytotoxicity via FcaR ( CD89) and possibly bound SC

Fig. 3. Schematic representation of three levels at which dimeric IgA or secretory IgA (SIgA) may provide mucosal immune protection after being produced with J chain (IgA ‡ J) by plasma cells in the lamina propria. Left: dimeric IgA is transported by the polymeric Ig receptor (pIgR) across epithelial cells and released into the lumen as SIgA antibodies that bind to the mucus layer and perform immune exclusion by interaction with luminal antigens (**). Middle: dimeric IgA antibodies interact with viral antigens within apical epithelial endosomes during pIgR-mediated transcytosis, thereby performing intracellular virus neutralization and removal of viral products. Right: dimeric IgA antibodies interact with penetrating antigens in the lamina propria and shuttle them back to the lumen by pIgR-mediated transport.

steroids can enhance human pIgR expression (Haelens et al., 1999), and it is likely that this receptor is under hormonal control in the lacrimal gland and genital tract. In addition, parasympathetic and sympathetic autonomic nerve stimulation of rat submandibular glands has been reported to increase the output of SIgA significantly (2.6- and 6-fold, respectively), which might reflect an effect of neurotransmitters on secretory epithelial cells (Carpenter et al., 1998).

Effector mechanisms of secretory antibodies and free or bound SC The main purpose of the secretory antibody system is ± in cooperation with innate mucosal defence mechanisms ± to perform immune exclusion (Fig. 3 and Table 2). Most importantly, SIgA can inhibit colonization and invasion by pathogens (Goldblum et al., 1996). Both pIgA and pentameric IgM antibodies internalized by the pIgR may even inactivate viruses (e.g. rotavirus, influenza virus and human immunodeficiency virus (HIV)) inside of epithelial cells and carry the pathogens and their products back to the lumen, thus avoiding cytolytic damage to

the epithelium (Mazanec et al., 1993; Burns et al., 1996; Bomsel et al., 1998; Fujioka et al., 1998). The agglutinating and virus-neutralization antibody effect of pIgA is in fact superior compared with monomeric antibodies (Brandtzaeg et al., 1987; Goldblum et al., 1996; Renegar et al., 1998), and SIgA antibodies can block microbial invasion quite efficiently (Hocini et al., 1997). Thus, individuals negative for HIV who live together with HIVpositive partners for several years, often appear to be protected by specific SIgA antibodies in their genital tract (Mazzoli et al., 1997). Because of the mucophilic properties of its bound SC (Phalipon et al., 2002), specific SIgA can enhance the sticking of bacteria and other antigens to mucus, thereby promoting clearance by respiratory ciliary movement and intestinal peristalsis. Potentially important additional defence functions are the reported ability of SIgA antibodies to induce loss of bacterial plasmids that code for adherence-associated molecules and resistance to antibiotics (Porter and Linggood, 1983), interference with growth factors (e.g. iron) and enzymes necessary for pathogenic bacteria and parasites (Brandtzaeg et al., 1987; Goldblum et al., 1996), and a putative positive influence on the inductive phase of mucosal immunity by promoting relevant antigen uptake in GALT via an IgA-binding receptor on M cells (Mantis et al., 2002). The latter possibility adds to the importance of breast-feeding in providing a supply of relevant SIgA antibodies from the mother to the suckling's gut (Brandtzaeg, 1996). Induction of SIgA responses has, in addition, been shown to interfere significantly with mucosal uptake of macromolecules in experimental animals (Brandtzaeg et al., 1987), and pIgR/SC knockout mice show evidence of increased mucosal leakiness and uptake of Escherichia coli antigens (Johansen et al., 1999). SIgA antibodies are furthermore essential in protection against cholera toxin (CT) as shown in knock-

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out mice lacking expression of J chain (Lycke et al., 1999) or pIgR (Uren et al., 2003). The same is probably true for heat-labile E. coli toxin, which uses the same ganglioside receptor as CT. The epithelial colonization of non-invasive bacterial pathogens is apparently also largely controlled by SIgA antibodies (Uren et al., 2003). Collectively, therefore, the functions of locally produced pIgA would be to inhibit mucosal colonization of microorganisms, neutralize viruses and hamper penetration of soluble antigens (Table 2). Importantly, because of its stability, SIgA can retain its antibody activity for remarkably prolonged periods in a hostile environment such as the gut lumen (Haneberg, 1974) and the oral cavity (Ma et al., 1998). This immune exclusion function is most likely reinforced by the relatively high levels of cross-reacting SIgA antibodies present in external secretions (Quan et al., 1997). These polyreactive `natural' antibodies apparently are designed for urgent protection before an adaptive immune response is elicited; they are therefore reminiscent of innate immunity (Bouvet and Dighiero, 1998), although their site and mechanism of induction remain unclear. Notably, they do not appear to be generated as `natural' IgA antibodies after switching of peritoneal B.1 cells, because humans ± in contrast to mice ± have no detectable traffic of B cells from the peritoneal cavity to the intestinal lamina propria (Brandtzaeg et al., 2001; Boursier et al., 2002). Finally, the immune exclusion effect of SIgA in the gut may be reinforced by interactions with the liverderived superantigen protein Fv (Fv fragment binding protein) ± leading to the formation of large complexes of intact or degraded antibodies with different specificities (Bouvet et al., 1996). Interestingly, free SC can inhibit epithelial adhesion of E. coli (Giugliano et al., 1995) and bind the potent toxin of Clostridium difficile (Dallas et al., 1998). Moreover, a pneumococcal surface protein (SpsA) has recently been shown to interact directly with both free and bound SC (Hammerschmidt et al., 1997). More recently it has been shown that SC can bind and inhibit IL-8, which is a potent chemotactic factor for neutrophilic granulocytes (Marshall et al., 2001). Altogether, these biological properties of SC suggest that it phylogenetically has originated from the innate defence system like many other proteins involved in specific immunity.

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Evaluation of the protective effect of secretory antibodies It is often difficult to evaluate the protective effect of SIgA and SIgM antibodies during natural mucosal infection because there is always a concurrent induction of systemic humoral immunity; this is also the case after local immunization with a live vaccine and when non-proliferating virus-like particles or subunit vaccines are applied together with an appropriate mucosal adjuvant (Velazquez et al., 1996; O'Neal et al., 1998). Particularly in the respiratory tract, a protective effect of serum antibodies ± mainly IgG ± may contribute to immune exclusion, in addition to the general importance of inhibiting further spread of the infectious agent through antibody-dependent neutralization and immune elimination within the lamina propria. A crucial protective role of SIgA has indeed been questioned by observations in IgA knockout (IgA / ) mice (Mbawuike et al., 1999). These mice remain healthy under ordinary laboratory conditions; and when challenged with influenza virus, they show similar pulmonary virus levels and mortality as control wild-type (IgA‡/‡) mice. Leakage of serum IgG antibodies through an irritated mucosal surface lining (Fig. 1), according to the principle of `pathotopic potentiation of local immunity' (Fazekas de St. Groth, 1951), has probably a greater protective role in the respiratory tract than in the gut (Persson et al., 1998). In addition, the IgA / mice usually show a compensatory SIgM response. Interestingly, IgA / mice on exclusive parenteral nutrition have reduced IgA anti-influenza virus titres in the upper respiratory tract and no compensatory SIgM, which most likely explains that they show impaired mucosal immunity (Renegar et al., 2001). The fact that individuals with selective IgA deficiency do not suffer significantly more than others from intestinal virus infections, may largely be ascribed to their consistently enhanced SIgM and IgG1 responses in the gut (Brandtzaeg and Nilssen, 1995). Altogether, a secretory antibody response appears to be essential for optimal mucosal defence. This notion is supported by the finding that systemic IgG antibody production against E. coli is triggered in pIgR knockout mice lacking both SIgA and SIgM (Johansen et al., 1999). Such knockout mice have recently been used as an in vivo model system to test the role of SIgA antibodies in the defence against influenza (Asahi et al., 2002). After intranasal immunization, wild-type mice showed complete protection or partial cross-protection when challenged with live A/PR8 virus. Lack of SIgA in the pIgR /

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mice resulted in reduced degree of protection and cross-protection, despite leakage of IgG and some IgA from serum into nasal secretions. This result emphasizes the importance of SIgA antibodies in mucosal virus defence, and is in keeping with earlier reports demonstrating that such antibodies induced in humans by intranasal vaccination, have a wider spectrum of activity against influenza virus than serum antibodies (Waldman et al., 1970; Shvartsman et al., 1977). Moreover, a correlation between influenza-specific SIgA in nasal lavage and protection has been suggested in vaccinated subjects (Clements and Murphy, 1986).

Exploiting the secretory immune system by mucosal immunization As the vast majority of infections use the mucosae as portals of entry, it appears desirable to induce a protective immune response by topical application of a relevant vaccine. Such mucosal infections include: in the gastrointestinal tract, Helicobacter pylori, Vibrio cholerae, enterotoxigenic E. coli, Salmonella, Shigella spp., Campylobacter jejuni, Clostridium difficile, rotaviruses and calici viruses; in the respiratory tract, Mycoplasma pneumoniae, influenza virus and respiratory syncytial virus; in the urogenital tract, HIV, Chlamydia, Neisseria gonorrhoeae and herpes simplex virus; and in the urinary tract, selected strains of E. coli. All these infections continue to represent a challenge for the development of vaccines that either can prevent the pathogen from colonizing the mucosal epithelium (noninvasive bacteria), penetrating the surface barrier and replicating within the body (invasive bacteria and viruses), and/or block the binding of microbial toxins and neutralize them (Eriksson and Holmgren, 2002). In most of these cases, it would seem desirable to induce specific SIgA antibodies associated with immunological memory. Regrettably, it has often proven to be rather difficult to stimulate strong SIgA responses by peroral administration of vaccines, particularly when using soluble protein antigens. Thus, only few vaccines approved for human use are administered mucosally: the oral (Sabin) polio vaccine, oral killed whole-cell B subunit and live-attenuated cholera vaccines, an oral live-attenuated typhoid vaccine, and an oral adenovirus vaccine (the latter vaccine being restricted to military personnel). Two recent additional mucosal vaccines, an oral liveattenuated vaccine against rotavirus diarrhoea and a nasal enterotoxin-adjuvanted inactivated influenza

vaccine, were withdrawn after a short time on the market because of potential serious adverse reactions, thus illustrating the complexity of mucosal vaccine development (Eriksson and Holmgren, 2002). There is a hope that the problems with peroral vaccination will be overcome by introducing edible plant vaccines (Mason et al., 2002), but the current main focus has shifted to the development of nasal vaccines as illustrated below in relation to influenza.

Mucosal versus parenteral influenza vaccination A substantial number of antibody-producing cells with specificity for influenza virus have been detected in nasal mucosa from unvaccinated adult subjects (Brokstad et al., 2001); it remains unknown whether these B cells have been induced by previous (subclinical?) influenza infection or by cross-reacting antigens. Notably, the SIgA system shows a significant cross-reactive background activity (Quan et al., 1997). It is important in this context that parenteral immunization with an inactivated trivalent virus vaccine did not result in an increase of influenza-specific antibody-producing cells in nasal mucosa (Brokstad et al., 2002). Conversely, parenteral immunization, particularly in adults, elicited an IgA antibody response in tonsils and oral fluids (Brokstad et al., 1995; El-Madhun et al., 1998). Collectively, these results suggest that in order to obtain an enhanced immune response in nasal mucosa, the vaccine should be targeted against topical DCs and/or M cells of MALT, perhaps particularly the adenoids (Fig. 1). Such immune stimulation of regional MALT (including the cervical lymph nodes) will apparently induce homing properties of primed B cells necessary for subsequent efficient extravasation in nasal mucosa. The amount of antigen reaching Waldeyer's ring or the cervical lymph nodes (perhaps via migrating DCs) after parenteral immunization, is clearly insufficient to this end but may nevertheless induce a weak IgA antibody response in the nasopharynx. This response is most likely expressed via SIgA production mainly in the adenoids where epithelial synthesis of pIgR takes place in contrast to the palatine tonsils (Brandtzaeg, 1998). Not unexpectedly, there may be some communication between the systemic and local immune system in the cervical lymph nodes and probably also in the tonsils and adenoids. Early studies of the IgA response to the influenza virus showed directly that the mucosal and systemic immune systems are not completely separated (McCaughan et al., 1984).

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Recent reports have documented the efficacy and effectiveness of the trivalent, live attenuated nasal spray influenza vaccine (CAIV-T) both in healthy children and adults (Glezen, 2002). Although intranasal vaccination also induces systemic immunity, a combination of intranasal and parenteral immunization may be preferable for optimal protection when an inactivated vaccine is used (Keitel et al., 2001). When inactivated vaccines are applied locally, a whole virus preparation not exposed to formalin or betapropiolactone appears to be preferable (Greenbaum et al., 2002), probably because of better targeting to M cells and DCs. Alternatively, the effect of subunit vaccines applied topically can be enhanced by incorporation into liposomes and with addition of the mucosal adjuvant E. coli heat-labile enterotoxin (de Bernardi di Valserra et al., 2002). Other adjuvants such as outer-membrane protein preparations (proteosomes) from N. meningitidis may also be efficient (Plante et al., 2001). CT and E. coli heat-labile toxin are considered the most potent mucosal adjuvants, being well-known from numerous animal studies. However, their toxicity may limit their use in humans. After nasal application in mice, CT has been shown to reach the olfactory bulb and could thus adversely affect the central nervous system (van Ginkel et al., 2000). Although the wild-type E. coli heat-labile holotoxin has been used in humans (Gl¸ck et al., 2000), problems with cases of facial paresis have recently been noted (Eriksson and Holmgren, 2002). Therefore, it is important to develop genetically detoxified mutants of mucosal adjuvants tolerated by humans. Animal experiments have shown that such mutants can retain their capacity to enhance humoral as well as cell-mediated immune responses to a mucosally applied inactivated influenza vaccine (Lu et al., 2002).

Conclusions Secretory immunity appears to be desirable in the defence against mucosal virus infections because it can inhibit initial pathogen colonization by providing blocking SIgA antibody activity both on the mucosal surface and within infected epithelial cells without causing tissue damage. Importantly, in contrast to IgG antibodies, IgA antibodies do not activate complement and are therefore generally considered to be non-inflammatory. Several virological studies have shown that natural infection and mucosal vaccination are more effective in giving rise to SIgA antibodies than parenteral vaccination.

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Also, topical vaccines have been much more efficient when consisting of live viruses than inactivated antigen preparations. However, various types of mucosal adjuvants can enhance the effect of subunit vaccines. Like natural infections, live topical vaccines or adequate combinations of inactivated vaccines and mucosal adjuvants give rise not only to SIgA antibodies, but also to longstanding serum IgG and IgA responses, which is crucial to obtain complete protection. The intranasal route of vaccine application appears particularly attractive to this end. The degree of immunological protection after vaccination may range from complete inhibition of reinfection to reduction of symptoms. In this scenario it is generally difficult to determine unequivocally the relative importance of SIgA versus serum antibodies, despite our advanced mechanistic understanding of the protective effect of mucosal immune responses against virus infections. It is furthermore not clear to what extent cell-mediated mechanisms or antibody-dependent cytotoxicity contributes to immunological protection of the mucosa, although animal experiments suggest that cytotoxic T cells may be important to eliminate influenza virusinfected epithelial cells (Nguyen et al., 1998). Acknowledgements. Studies in the author's laboratory are supported by the Research Council of Norway, the Norwegian Cancer Society and Anders Jahre's Fund.

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