FEMS Microbiology Immunology 89 (1991) 1-10 © 1991 Federation of European Microbiological Societies 0920-8534/91/$03.50 Published by Elsevier

FEMSIM 00189

Review

Mucosal immunity and vaccination Jan H o l m g r e n Department of Medical Microbiology and Immunology, University of GiSteborg, GiJteborg, Sweden Received 7 August 1991 Accepted 2 September 1991

Key words: Mucosal immunity; Vaccination; Oral vaccine; Enteric infection; Cholera vaccine; Secretory IgA

1. SUMMARY The gut mucosal immune system is a critical component of the body's defense against pathogenic organisms, especially those responsible for enteric infections associated with diarrhoeal disease. Attempts to vaccinate against infections of mucosal tissues have been less successful than vaccination against systemic infections, to a large extent reflecting a still incomplete knowledge about the most efficient means for inducing protective local immune responses at these sites. Secretory IgA (SIgA) is the predominating immunoglobulin along mucosal surfaces, and SIgA antibodies generated in gastrointestinal, respiratory or genito-urinary mucosal tissues can confer protection against infections affecting or originating in these sites. An efficacious intestinal SIgA

Correspondence to: J. Holmgren, Department of Medical Microbiology and Immunology, University of G6teborg, Guldhedsgatan 10, G6teborg $413-46, Sweden.

immunity-inducing oral vaccine against cholera has been developed recently, and development of oral vaccines against other enteric infections such as those caused by enterotoxigenic Escherichia coli, Shigella and rotaviruses is in progress as well. Based on the concept of a common mucosal immune system through which activated lymphocytes from the gut can disseminate immunity to other mucosal and glandular tissues, there is currently also much interest in the possibility of developing oral vaccines against infections in the respiratory and urogenital tracts. However, the large and repeated antigen doses often required to achieve a protective immune response still makes this vaccination approach impractical for many purified antigens. There is, therefore, a great need to develop strategies for enhancing delivery of antigen to the mucosal immune system as well as to identify mucosa-active immunostimulating agents (adjuvants). These and other aspects of mucosal immunity in relation to immunization and vaccine development are discussed in this short review article.

2. INTRODUCTION It is now widely recognized that in order to be efficacious, vaccines against enteric infections, at least those against infections with non-invasive organisms such as Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC), must be able to preferentially stimulate the local gut mucosal immune system, and that this goal is usually better achieved by administering the vaccines by the oral route rather than parenterally. Indeed, based on the concept of a common mucosal immune system through which activated lymphocytes from the gut can disseminate immunity to other mucosal and glandular tissues, there is currently also much interest in the possibility to develop oral vaccines against, for instance, infections in the respiratory and urogenital tracts. Oral vaccines are, in general, easier to produce and to control quality than parenteral vaccines. They are also easier (and safer) to administer to large population groups since they do not require any medically trained personnel or sterile supplies. There are, however, many factors that can influence the expression of mucosal immunity and therefore also the outcome of vaccination attempts against enteric and other mucosal infections. In this review, following a condensed presentation of the organization and function of, especially, the gut mucosal immune system, I will discuss some of these factors and also describe some novel approaches now being tested to optimize the stimulation of mucosal immunity by oral vaccines.

3. T H E GUT MUCOSAL IMMUNE SYSTEM The intestine is the largest immunological organ in the body. It comprises 70-80% of all immunoglobulin-producing cells and produces more secretory IgA (SIgA) (50-100 mg/kg body weight/day) than the total production of IgG in the body (approx. 30 m g / k g / d a y ) [1]. The local immune system of the gut has two main functions: to protect against enteric infections, and to protect against uptake of a n d / o r

harmful immune response to undegraded food antigens [2]. An important basis for local immunity is the migration of specific, antigen-activated B and T cells from the Peyer's patches (PP) to the intestinal lamina propria and epithelium. Antigen administered orally is taken up in the PP by the modified epithelial ceils known as M cells, and it is then transported to the lymphoid tissue of the PP where the initial mucosal immune response occurs. PP contain B and T cells as well as antigen-presenting cells (APC). After antigen-induced proliferation and partial differentiation, both B and T ceils enter the regional mesenteric lymph nodes, and then after further differentiation they are transported through the thoracic duct into the circulation. As these cells have surface determinants, so-called adressins, which are specific for lymphocyte homing receptors on endothelial cells in mucosal and glandular tissues, they will eventually return to and extravasate into these tissues. Most of the B and T cells activated in the intestine migrate to the lamina propria of the intestine and another population of T cells to the intestinal epithelium. However, a substantial proportion, perhaps 10-20%, end up in mucosal tissues outside the intestine [3].

3.1. Secretory IgA The best known entity providing specific immune protection for the gut is the SIgA system. The SIgA molecule is synthesized as an IgA dimer by B ceils in the lamina propria. The dimeric IgA is then transported into the gut lumen by a 'lock-and-key' interaction between the J-chain on the lgA dimer and secretory component (SC) receptor present on the basolateral membrane of the enterocyte. At the apical surface, the SIgA molecule is exocytosed, and the SC is cleaved to deliver the classical SIgA molecule with a small piece of SC remaining in the membrane [1]. IgA synthesis is highly T cell-dependent. A T switch cell causes IgM-bearing B cells in the Peyer's patches to instead express surface IgA. Another T cell causes the surface IgA-expressing B cells to differentiate into an IgA secreting plasma cell. A third, less well-defined regulatory

Table 1 Protective functions of secretory IgA 1. Immune exclusion comprising inhibition of: (a) Bacterial adherence, colonization and penetration; (b) Toxin binding and action; (c) Viral attachment and infection; (d) Food antigen uptake (including allergens, carcinogens and toxicants). 2. Antibody-dependent T cell-mediated cytotoxicity (ADCC). 3. Interference with microbial growth, e.g. iron utilization. 4. Stimulation of immune response through: (a) facilitation of antigen uptake by M cells into Peyer's patches; (b) lymphocyte stimulation by anti-idiotypic Ab activity (role in breast-milk immunity?)

mechanism is attributed to a so-called contrasuppressor T cell, which specifically suppresses other T cell-mediated suppression of IgA. Several cytokines, produced both by T cells and other cells, are also known to influence different steps in the IgA differentiation: in the early, 'switch' stage probably mainly IL-4 and transforming growth factor beta (TGF-beta), and in later stages mainly IL-5 and IL-6 but also TGF-beta, IL-4 and possibly interferon-gamma (IFN-y) [4]. The resistance of SIgA against normal intestinal proteases makes antibodies of this isotype uniquely well-suited to protect the intestinal mucosal surface [2]. The main protective function of SIgA antibodies is the 'immune exclusion' of bacterial and viral pathogens, bacterial toxins and other potentially harmful molecules (Table 1). SIgA has also been described to mediate antibody-dependent T cell-mediated cytotoxicity (ADCC), and to interfere with the utilization of necessary growth factors for bacterial pathogens in the intestinal environment, such as iron. Finally, SIgA antibodies might also exert positive influences on the inductive phases of mucosal immunity by facilitating antigen uptake in PP by complexing with antigen and at the same time being able to attach to the PP M cells. 3.2. CMI and other immune mechanisms Cell-mediated immune reactions including MHC-restricted cellular cytotoxicity, natural killer

cell activity and ADCC have also been proposed to play a role in intestinal immunity against invasive enteric infections, caused by, for example, Salmonella and Shigella bacteria and rotaviruses. Gut mucosal gamma interferon (IFN-y) producing T cells might be especially important in the intestinal immune defense. We have recently found that these cells are present in unusually large numbers in the human intestinal mucosa as compared with other lymphoid tissues, and also that they undergo significant expansion following intestinal antigenic exposure [5]. IFN-y can influence both the afferent and efferent steps of mucosal immunity. It enhances IgA production and increases MHC antigen expression and thereby probably antigen presentation by different mucosal cells. It also increases the expression of SC receptors on the enterocyte surface which facilitates SIgA transport [6]. Furthermore, IFN-y has been found to interfere with both tight-junction permeability between intestinal epithelial cells and with active electrolyte secretion by enterocytes in vitro [7,8]. On top of this, IFN-y may interfere with the receptivity of epithelial cells for invasive enteric pathogens such as Shigella bacteria. 3.3. Immunologic memory Contrary to initial beliefs that local immunity was incapable of anamnestic immune responses it has become abunduntly clear during the last decade that memory is indeed an important component of mucosal immunity [9]. The intestinal SIgA response to antigen exposure is of relatively short duration, lasting for a few weeks (mice) to a few months (humans). However, as illustrated in Fig. 1, the SIgA system exhibits potent immunologic memory and can be repeatedly stimulated by renewed contact with antigen. Immunological memory, that could rapidly and efficiently be triggered by repeated antigen exposure, may be the main explanation for the long-lasting immunity that has been observed in both animals and humans after oral immunizations with cholera antigens in the absence of any detectable persisting SIgA antibody response. Indeed, studies from our laboratory have demonstrated a long-lasting, perhaps life-long local immunologic memory with

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Fig. 1. Intestinal immune responses and immunologic memory in Swedish volunteers after oral immunizations with cholera vaccine (adapted from ref. 5). Data are expressed as geometric mean n u m b e r s of specific antibody-secreting cells (ASC) to the B subunit component (CTB) of the vaccine per one million of mononuclear lymphoid cells isolated from duodenal biopsies from the volunteers at the indicated times in relation to oral cholera vaccinations. The ASC were determined with the E L I S P O T method.

properties consistent with such a function (e.g. a rapid anamnestic response to even minute amounts of renewal antigen); specific memory B cells have also been isolated and characterized [9].

4. DISSEMINATION OF GUT IMMUNITY TO DISTANT MUCOSAE The notion of a 'common mucosal immunological system' that provides immune reactivity, not only at the site of antigen deposition but also at remote mucosal sites, is especially important when considering strategies of vaccination against mucosal pathogens. Thus, in addition to the emerging use of the oral route of vaccination against various enteric infections, there is currently also much interest in the possiblity to develop oral vaccines against infections in the respiratory and urogenital tracts. The results of animal experiments have supported the notion of the potential to induce IgA antibody formation and immune protection also in 'distant' extra-intestinal mucosal sites after oral vaccination. Interestingly, these studies have also

highlighted the possibility to convert normally weak mucosal immunogens into strong ones by linking them to a potent mucosal immunogen carrier such as cholera toxin (CT) or its non-toxic binding B subunit (CTB) [10,11]. In this respect we could demonstrate that oral administration of small amounts of a streptococcal protein antigen (a protective antigen against dental caries) covalently linked to CTB induced both mucosal and extramucosal IgA and IgG anti-streptococcal antibody responses in mice [11]. Thus, mice fed low doses (15 /xg) of CTB-linked streptococcal antigen together with free CT as adjuvant, displayed high frequencies of specific antibody-secreting cells in submandibular salivary glands as well as in mesenteric lymph nodes, and spleen (Table 2). In contrast, equivalent or even 20- to 30-fold higher doses of streptococcal antigen given alone or conjugated to a non-intestinal binding protein (bovine serum albumin) were relatively ineffective in eliciting antibody responses to the streptococcal antigen in either salivary glands, mesenteric lymph nodes, or spleen (Table 2), and also in serum [11]. Analogous results were reported by Liang et al. [10] using covalently linked Sendai virus-cholera toxin conjugates. Indeed, as judged by the even higher IgA antibody responses in respiratory secretions after combined oral-inTable 2 Specific antibody-forming cells in mucosal and extramucosal tissues after enteric immunization with a streptococcal antigen conjugated to cholera B subunit (data adapted from ref. 16) Immunogen * strep ag-CTB (15/xg) strep ag-BSA (15/xg) strep ag (150/xg) nil §

MLN

Spleen

SG

1170_+310

280+55

150_+48

< 5

< 5

60_+ 15

Mucosal immunity and vaccination.

The gut mucosal immune system is a critical component of the body's defense against pathogenic organisms, especially those responsible for enteric inf...
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