Stimulation of local antibody production: parenteral or mucosal vaccination?

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Stimulation of local antibody production: parenteral or mucosal vaccination? Jean-Pierre Bouvet, Nipa Decroix and Perayot Pamonsinlapatham Mucosal vaccines aim to prevent the penetration of pathogens, including HIV, into the body. Their cost can be low compared with curative therapies and thus, they are well suited to the fight against pandemic diseases in developing countries. Efficacy has been demonstrated for the oral poliovirus vaccine, but only very few other vaccines administered by the mucosal route are available commercially at present. Tremendous research efforts have now improved significantly the classical approach of these vaccines, and alternative methods of immunization, based on new concepts of mucosal immunity, are being developed.

The induction of an immune response at the mucosal surface is of major interest because of its ability to modulate colonization by commensals, as well as increase defenses against the penetration of pathogens through the epithelium, reducing their concentration to a harmless level. Secretory IgA (SIgA) is the main Ig isotype mediating humoral immunity in human secretions, but recent studies have demonstrated the presence of locally produced IgG antibodies also [1,2]. The oral poliovirus vaccine is highly efficient at preventing poliomyelitis but, unfortunately, many attempts to induce the same level of protection by oral administration of other vaccines have failed in humans. Although the mucosally administered vaccines are being improved progressively, recent studies have demonstrated that the secretion of mucosal antibodies (IgG and IgA) can be induced by parenteral routes. One important application of this discovery could be the development of a mucosal anti-HIV vaccine to prevent the sexual transmission of AIDS. Humoral immunity of mucosae

Jean-Pierre Bouvet* Nipa Decroix Perayot Pamonsinlapatham Unité d’Immunopathologie Humaine, INSERM UR430, Hôpital Broussais, 75014 Paris, France. *e-mail: jean-pierre.bouvet@ brs.ap-hop-paris.fr

SIgA reaches the epithelial and glandular lumens by active transcytosis mediated by the polymeric Ig receptor (pIgR), which is formed by the membrane secretory component. SIgA is involved in two protection mechanisms, described as ‘immune exclusion’, which prevents the entry of pathogens into the body, and ‘immune elimination’, which eliminates pathogens from epithelial cells and subepithelial stroma [3,4]. SIgA is resistant to endogenous proteases, but can be degraded in the colonic lumen by microbial IgA-proteases [5], leading to the production of Fab-α. These fragments maintain some activity by combining with the protein Fv, a hexavalent glycoprotein that binds the http://immunology.trends.com

VH3 variable domain of the SIgA heavy chain to form macromolecular complexes [6]. The production of SIgA antibodies is triggered after translocation of the eliciting antigen from the lumen to inductive areas across M cells [7]. The inductive areas comprise the mucosa-associated lymphoid tissue (MALT) (e.g. Peyer’s patches, solitary lymphoid follicles and tonsils). Primed B cells from these areas mature in the lymph and blood before homing to the subepithelial stroma. This system is compartmentalized [8,9], with most cells from each inductive site migrating to defined areas. The association between lymphocytes from different MALT sites and different effector areas influences the choice of route of administration of a vaccine. SIgA contains high levels of natural antibodies [10]. These polyreactive antibodies, which have been described in polyclonal [11] and monoclonal human sera [12], are present irrespective of any immunization procedure, and probably represent the modern form of an ancestral immunity [13]. In secretions, as well as in the serum [14,15], they are involved in the preimmune barrier against pathogens. Their reactivity with microbial components might interfere with measurements of the response to a vaccine. The IgG present in secretions can be derived from the serum during swelling of the digestive lumen, local inflammation or bleeding. This leakage of IgG can be particularly high in the airways [16]. Under normal conditions, the clearance of serum IgG in the liver releases this isotype into the biliary tract and thereafter, into the gut [17]. A mucosal origin for IgG antibodies in secretions has been demonstrated by their specificity, which differs clearly from that of autologous serum antibodies in healthy subjects [1]. Moreover, a comparison of the IgG antibodies from different autologous secretions has shown striking variation in their specificities, which indicates the compartmentalization of IgG production in the mucosae [1]. Although IgG is degraded by proteases in the gastrointestinal tract (but not in genital secretions or milk), its Fab fragments can neutralize toxins and viruses [18]. The mechanism of active transport of IgG through epithelial cells has been investigated recently in an intestinal cell line [19].

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Unlike pIgR, the receptor for IgG is recycled after dissociation of the ligand–receptor complex at the apical pole of the cell. Live vaccines administered by the mucosal route

The Sabin oral poliovirus vaccine is presently the only human mucosal vaccine to be universally commercialized. This live vaccine is efficient and relatively safe, despite the occurrence of revertants. To mimic this vaccine, genetically attenuated pathogens have been constructed and administered by the mucosal route. Some of them have been enriched with genes encoding mucosal adjuvants, cytokines or other molecules favoring the mucosal response, as reviewed by Czerkinsky et al. [20]. A large number of microorganisms has been investigated as vectors for genes encoding ‘protective’ molecules. Computer-assisted bibliography shows that, to date, as many as ten different species of attenuated bacteria and seven different species of virus have been used as vectors for experimental mucosal immunization against various components of 18 species of bacteria, 13 species of virus and three species of parasite. These genetically modified microorganisms were selected for experimentation according to their local persistence, which allows a long duration of the immune response. However, investigators have encountered severe problems: (1) the extent of the immune response is associated with the degree of colonization by the vaccine microorganism, often depending on a background of pathogenicity; (2) this residual pathogenicity varies according to individuals, but is unacceptable in infants; (3) spontaneous genetic recombinations can occur between the local flora and the vaccine, which might become pathogenic; and (4) severe infections, not observed during the trials, can sometimes occur during mass campaigns or industrial production. These risks are important because the vaccines are administered to healthy individuals and public opinion is sensitive to the use of recombinant organisms. Inactivated vaccines and mucosal adjuvants

In contrast to live vaccines, inactivated microorganisms are not pathogenic usually, but their short-term persistence at mucosal surfaces impairs the level and duration of the immune response. To bypass these limitations, inactivated microorganisms or purified antigens have been enclosed in microcapsules [21], liposomes [22] or immunostimulating complexes (ISCOMs) [23]. Recently, the use of free virus-like particles has improved the immunogenicity of inactivated mucosal vaccines and allowed their study in humans [24]. Associated with these long-lasting vaccines, the most commonly used adjuvants are cholera toxin and the heat-labile enterotoxin of Escherichia coli [25]. Cholera toxin is delivered as its atoxic http://immunology.trends.com

B subunit, alone or with a harmless level of the A subunit. Nontoxic derivatives of these toxins have been produced by mutagenesis, permitting their increased use as mucosal adjuvants in humans [26]. A recombinant B subunit combined with inactivated bacteria has been used recently in a new anti-cholera vaccine and gave significant protection in adults [27]. It is commercially available in some countries, and its low cost is of particular importance [28]. Other molecules, such as oligodeoxynucleotides containing immunostimulatory CPG motifs [29], as well as interleukin-12 given by the nasal route in mice [30], have been shown to be promising mucosal adjuvants. The main problem of mucosal administration is local degradation and possible expelling of the vaccine, both of which impair control of the dose delivered. Human mucosal vaccines by parenteral administration

Most investigations of the secretory immune response after parenteral administration of a dead or constitutive human vaccine have been carried out with the tetanus vaccine, which is known to selectively induce the production of IgG antitoxins. The antibodies detected in secretions after this vaccination are mainly IgG and thus, were considered to be mainly serum-derived by means of diffusion across the mucosal surface. As early as 1973, the presence of IgG antibodies in the vaginal fluid of a subject vaccinated by intramuscular injections was reported [31]. It was observed later that the saliva of 77 of 151 infants vaccinated against tetanus contained specific IgA antibodies [32]. Tarkowski et al. [33] analyzed the IgA antibody response to Neisseria meningitidis after systemic immunization and found an increased titer of specific IgA in the saliva. In 1994, our group described a dramatic increase in the level of IgG antitoxins in the vaginal fluid after tetanus vaccination [34]. Moldoveanu [35] and colleagues observed a salivary IgA response to systemic immunization with a subvirion of influenza virus A. Similarly, Brokstad et al. [36] observed specific SIgA antibodies in the oral fluid after parenteral administration of an inactivated influenza virus A vaccine. Mucosal responses to chemically defined, injected antigens

Two groups, including ours, have investigated the possibility of parenteral administration of different soluble, mucosal vaccines in the murine model. Enioutina et al. have shown that subcutaneous injections of the capsular polysaccharide of Haemophilus influenzae induce the secretion of neutralizing IgA and IgG antibodies into nasal and vaginal fluids [37]. This vaccine was conjugated with both diphtheria toxoid and 1α,25-dihydroxyvitamin D3, and associated with alum. The mucosal

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Moreover, IgA and IgG antibody-producing cells were observed in the Peyer’s patches and intestinal lamina propria. In our study, antibodies in the cytoplasm of mucosal plasma cells were analyzed by the perfusion–extraction method [38]. After intramuscular immunization in the absence of adjuvant, the mice produced mucosal IgG and, to a lesser extent, IgA antibodies specific for the tetanus toxoid. The mucosal origin of these antibodies was confirmed by their higher specific activities in the mucosal organs compared with the spleen and serum. After the last booster injection, the mucosal response persisted for as long as eight months, whereas the serum response decreased progressively. This response was observed against carrier-coupled haptens also, particularly against an important epitope of the gp41 protein of the HIV-1 virus containing the protective consensual neutralizing sequence ELDKWA. Moreover, preliminary results with this peptide, synthesized in-line with the pan-DR epitope PADRE, indicate a preferential mucosal response in mice. In addition to intramuscular and/or subcutaneous injections, the intradermal immunization of mice with diphtheria toxoid in the presence of alum and different adjuvants – such as dihydroxide vitamin D3, cholera toxin, heat labile lymphotoxin of E. coli, pertussis toxin or forskolin – induced high levels of IgA antibodies in secretions [39]. Variants of parenteral immunization

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Fig. 1. Possible mechanisms of triggering humoral immunity in mucosal sites. (a) The classical means of antigen (Ag) penetration by the mucosal route. The Ag translocates from the lumen to the Peyer’s patches (PPs) across M cells (M), where it encounters Ag-presenting cells (APCs), CD4+ T cells and B cells. Activated B cells then migrate to the mesenteric lymph nodes (MLNs), before maturing in the lymph and blood. They home to the lamina propria (LP), where they differentiate into plasma cells in the presence of CD4+ T cells. Locally synthesized antibodies (Abs) are transported actively across epithelial cells (ECs) into the lumen by the polymeric Ig receptor (pIgR) and/or FcRn. (b) An intramuscular Ag migrates to the draining peripheral lymph node (DPLN), where it activates APCs, such as B cells, macrophages and dendritic cells. A mucosal response is triggered when these APCs reach the mucosa-associated lymphoid tissue (MLNs or PPs). (c) Alternatively, an intramuscular Ag can diffuse directly to MLNs or PPs, where it is presented by APCs to CD4+ T cells, which activate B cells. (d) A transcutaneous Ag is processed by skin APCs, which migrate to MLNs and PPs.

antibodies were not serum-derived, because they were not detected after the passive transfer of large doses of immune serum IgG into control animals. http://immunology.trends.com

Numerous studies have demonstrated that various routes of parenteral immunization can induce the production of mucosal antibodies. Specific examples include intramuscular vaccination with a live retrovirus [40,41] or papilloma virus-like particles [42], and an attenuated equine virus administered by the subcutaneous route [43]. The injection of DNA vaccines by the ‘gene-gun’ method is of interest; this method has been shown to be more efficient at inducing protection against experimental influenza than administration of the same vaccine in the nostrils or trachea [44]. However, a major problem of DNA vaccination is the risk of genetic inclusions favoring cancer or other genetic dysregulations. Nonetheless, this principle is fascinating and would allow mass vaccinations at a reasonable cost. Transcutaneous immunization by the topical application of large amounts of antigen, together with cholera toxin or E. coli enterotoxin, induces the production of specific antibodies in secretions and antibody-secreting cells in the mucosa [45–47]. The application of these toxins is harmless and might, thus, be of interest in future vaccines [48]. This method has been investigated already in humans by the simple application of patches [49]. The direct injection of regional lymph nodes has been shown to induce a targeted secretory immune response also [50], but this interesting procedure is technically difficult and probably

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Acknowledgements We thank Sylvio Iscaki for helpful discussion and critical review of the manuscript. This work was supported by Agence Nationale de Recherches sur le SIDA (ANRS) and Institut National de la Santé et de la Recherche Médicale (INSERM). N.D. is the recipient of a fellowship from ANRS. She is on leave from the National Science and Technology Development Agency, Division of Biotech (Bangkok, Thailand). P.P. is working on a collaborative project between INSERM UR430 (France) and the Faculty of Pharmacy, Silpakorn University, Thailand.

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dependent on the pathological status of the injected lymph node. Indeed, inguinal and iliac lymph nodes might become sclerotic and can be latently infected by parasites. Dendritic-cell vaccination using antigen-pulsed bone-marrowderived cells administered intravenously was shown recently to induce protective immunity against a genital infection in mice [51]. These results are of biological interest, but the cost of the procedure will probably be too high for application to human vaccines.

serum albumin (68 kDa), which is detected in all secretions. In another hypothesis (Fig. 1d), soluble antigen administered by an intradermal or transcutaneous route activates antigen-presenting cells of the skin [53]. These cells migrate to the MALT, where they present the antigen to lymphocytes. Cutaneous microenvironmental factors (e.g. those provided by adjuvants such as vitamin D3), increase the efficiency of the local antigen-presenting cells [39]. Finally, antibody secretion by plasma cells depends on the help of CD4+ T cells in the lamina propria.

Mechanisms of parenterally induced mucosal immunity

Several different, nonexclusive mechanisms (Fig. 1) have been proposed to explain the production of secretory antibodies after parenteral administration of the antigen. Evidence from adoptive-transfer experiments [52] showed that an antigen diffuses from an intramuscular inoculation site to draining peripheral lymph nodes (Fig. 1b). Here, it is picked up by local antigen-presenting B cells, as well as macrophages and dendritic cells, which migrate to the MALT, where they activate CD4+ T cells and B cells. These data are in agreement with Lehner’s results involving antiretroviral vaccines injected into iliac lymph nodes [50]. Alternatively, soluble or phagocytosed antigens might reach the MALT directly (Fig. 1c). The possibility of diffusion of large molecules from serum to mucosa is demonstrated by

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Conclusion and future applications

The major aim of immunization of the mucosal immune system is to provide protection against pathogens that invade an organism across the epithelial barrier. The extension of our basic knowledge has allowed investigators to improve the classical methods of mucosal vaccination and investigate alternative, and maybe complementary, routes of administration. The relative risks of live vaccines and the increasing strength of opinion against the use of genetic recombinants have prompted the development of safe, chemically defined vaccines, allowing the administered dose to be controlled. These parenterally administered antigens might be good candidates for future mucosal vaccines.

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Antifertility vaccines Peter J. Delves, Torben Lund and Ivan M. Roitt The possibility of using immunization as a method of birth control has been explored actively since the 1930s, with several different sperm, egg or hormonal antigens having been studied as suitable targets for intervention. However, it is only in the past decade that the efficacy of vaccination against fertility has become established firmly in both humans and free-roaming animal populations. We will review recent progress in the continuing development of antifertility vaccines, with an emphasis on vaccines intended ultimately for use in humans, whilst highlighting also some of the notable successes achieved with vaccines produced for use in other species.

Peter J. Delves* Torben Lund Ivan M. Roitt Dept of Immunology and Molecular Pathology, Windeyer Institute of Medical Sciences, University College London, 46 Cleveland Street, London, UK W1T 4JF. *e-mail: p.delves@ ucl.ac.uk

Although fertilization of the oocyte by a spermatozoon is clearly a key event in the reproductive process, egg and sperm antigens are not the only potential targets for immunological antifertility vaccines. For example, the development of the gametes is under hormonal regulation, and these hormones can be inhibited immunologically (Fig. 1). Neutralizing antibodies could operate by steric hindrance (for example, by blocking the binding of a hormone to its receptor or spermatozoon to an oocyte) or by facilitating the rapid clearance of the target antigen through the formation of immune complexes. http://immunology.trends.com

Most antifertility vaccines would need to elicit neutralizing antibodies secreted into the reproductive tract. In the female, the cervix might be the main site of such antibody production (Fig. 2). The lamina propria of the endocervix contains plasma cells and the cervical mucus has been shown to contain IgG, IgA (at lower concentrations than IgG) [2] and complement components [3]. Both IgA and IgG are present in the oviductal fluid, derived perhaps from the transudation of circulating antibodies [4]. The antibodies that are present in the uterine fluid are thought to be washed down from the oviducts. Optimal vaccination schedules for genital-tract immunity are under intensive investigation [5], not least because of the importance of such studies to the prevention of sexually transmitted diseases [6]. Intranasal immunization appears to be a particularly effective method to generate the production of antibodies in the female reproductive tract, with the B subunit of cholera toxin being an especially potent stimulator of such responses [2].

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