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Send Orders for Reprints to [email protected] Current Topics in Medicinal Chemistry, 2013, 13, 2609-2628 2609 Mucosal Vaccines: Where Do We Stand? Jean-Pierre Kraehenbuhl1,* and Marian R. Neutra2 1 Health Sciences eTraining (HSeT) Foundation, 155 Chemin des Boveresses, 1066 Epalinges, Switzerland; 2GI Cell Biology Research Laboratory, Children’s Hospital and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA Abstract: Mucosal vaccinology is a relatively young but rapidly expanding discipline. At present the vast majority of vaccines are administered by injection, including vaccines that protect against mucosally acquired pathogens such as influenza virus and human papilloma virus. However, mucosal immune responses are most efficiently induced by the administration of vaccines onto mucosal surfaces. The small number of currently licensed mucosal vaccines have reduced the burden of disease and mortality caused by enteric pathogens including rotavirus, V. cholerae and S. typhi, or those that spread to affect distal organs such as poliovirus. Expanding knowledge about the special features of the mucosal immune system promises to accelerate development of mucosal vaccines that could contribute significantly to protection against pathogens that colonize or invade via mucosal surfaces including HIV, Shigella, ETEC, Campylobacter jejuni, Helicobacter pylori and many others. Keywords: Epithelia, mucosae, vaccines, pathogens. 1. INTRODUCTION The majority of human pathogens that are responsible for infectious diseases worldwide invade the host through mucosal surfaces of the digestive, respiratory or urogenital tracts. Vaccines administered mucosally are most effective at eliciting mucosal immune responses and enhancing immune defenses at mucosal surfaces, and can provide more effective protection against infection by mucosal pathogens. Most licensed vaccines, however, are administered by injection and do not efficiently elicit mucosal immunity. Why Mucosal Vaccination ? For protection against mucosally-acquired pathogens, mucosal administration of vaccines offers several advantages over injected vaccines: i/ Mucosal vaccination generally triggers both systemic and mucosal immune responses whereas the response to injected vaccines is largely systemic ii/ Mucosal vaccination is non-invasive and needle-free. This increases vaccine acceptance and safety, avoiding problems of blood borne infections due to needle re-use, especially in developing countries; iii/ Mucosal vaccination avoids adverse effects such as inflammation at the injection site ; iv/ Mucosal vaccines allow frequent boosting ; and v/ Preexisting systemic immunity generally does not interfere with entry of vaccine into mucosal inductive sites, thus increasing the rate of vaccine “take”, for example in infants with maternally acquired serum antibodies. What are the Challenges? Development, testing and approval of mucosal vaccines for human use have been slowed by both technical and regu*Address correspondence to this author at the 155 Chemin des Boveresses, 1066 Epalinges, Switzerland; Tel:/Fax: +4121 692 5856; E-mail: [email protected] 1873-5294/13 $58.00+.00 latory challenges. Mucosally-delivered vaccines, especially those given orally, have to be designed to be stable in a harsh mucosal milieu and they will inevitably be diluted in mucosal secretions, caught in mucus gels and attacked by enzymes. Mucosal vaccines must then cross a well-defended epithelial barrier and be captured by mucosal antigenpresenting cells (APCs). Thus, unlike injected vaccines, the actual dose of mucosally-administered vaccine that enters the body and is “seen” by the immune system can only be estimated. Mucosal vaccine formulations and delivery systems must also be designed to activate innate immune responses in mucosal cells, much as invasive pathogens would do, and this requires the use of live vectors or strong adjuvants. Selection and approval of vaccine candidates requires accurate measurement of immune responses, and this poses additional challenges. Unlike serum antibodies and cells that are readily sampled, secretory antibodies and mucosal effector cells are more difficult to capture and functionally test, and the local variability of mucosal tissues and secretions makes exact quantitation of total body-wide mucosal immune responses impossible. Mucosal immunization is most efficient with live microorganisms, but the use of attenuated pathogens or genetically modified live vectors raises regulatory concerns. For all of the above reasons, and because injected vaccines have long been the norm, mucosal vaccines face skepticism from the scientific community and regulatory agencies. Drawbacks of using Mucosal Vaccines Although mucosal administration of foreign substances is generally safer than injection, some mucosal vaccine candidates have been withdrawn from the market because of unexpected adverse reactions. For example, the rotavirus vaccine RotaShield® increased the risk for intussusception; one © 2013 Bentham Science Publishers 2610 Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 20 or two cases of intussusception occurred among each 10,000 infants vaccinated. The Advisory Committee on Immunization Practices (ACIP) withdrew its recommendation to vaccinate infants with RotaShield® vaccine, and the manufacturer voluntarily withdrew RotaShield® from the market in October 1999. Nasalflu, an experimental inhaled influenza vaccine has been linked with Bell's palsy, an illness that causes temporary facial paralysis. In this case, the adjuvant used (a modified toxin from E. coli) may have been transported retrograde to the brain via olfactory nerves. Switzerland's Berna Biotech ended its development and a trial of some 11,000 patients after reviewing data collected from 4,000 of them[1]. Experimental oral vaccines against enteric diseases consisting of live attenuated bacteria have induced unacceptable gastrointestinal symptoms, apparently due to the host’s innate immune responses to bacterial components. In general the potential toxicity of vaccines administered mucosally has proven difficult to predict because of our limited understanding of events within the mucosa. 2. SPECIAL FEATURES OF THE MUCOSAL IMMUNE SYSTEM Mucosal vaccination has to take into account the fact that the mucosal immune system differs from its systemic counterpart in several important respects. Mucosal tissues have specialized antigen sampling strategies [2], and specialized immune effector mechanisms such as secretory immunoglobulin A (sIgA) [3], that can prevent entry of pathogens into the body. Effector cells induced by mucosal immunization have distinctive homing programs which allow them to circulate and then return to appropriate mucosal sites [4]. In contrast to the systemic immune system whose cells generally function in a sterile environment, the mucosal immune system is constantly exposed to exogenous antigens. Mucosal immune responses are regulated by a variety of suppressive mechanisms [5], that maintain tolerance to environmental antigens such as those present in food and inhaled air and that avoid dysregulated inflammation against innocuous antigens and commensal microorganisms inhabiting mucosal surfaces. Mucosal tissues maintain a delicate balance between these suppressive mechanisms and defensive immune responses through the unique functions of mucosal dendritic cells (DCs) and macrophages, the activities of which are largely conditioned by factors released by epithelial cells and lamina propria stromal cells. Understanding how these special features translate into immunological function is key to the rational design of prophylactic vaccines to protect against mucosal infections. 3. ANTIGEN AND VACCINE SAMPLING BY MUCOSAL TISSUES Mucosal surfaces are vulnerable to infectious agents because they represent vast surface areas lining internal organs that are open to the outside world. Thus it is not surprising that the vast majority of initial antigen encounters occurs at mucosal surfaces such as the gastrointestinal, respiratory and urogenital tracts. As examples, HIV and polio virus gain entry to the body via the gastrointestinal surface, influenza and adenoviruses via the respiratory surface, and HIV and Kraehenbuhl and Neutra herpes simplex virus through the genital mucosae. All mucosal surfaces have an epithelial lining, the organization of which is adapted to its location in the body, and an underlying loose connective tissue (lamina propria) located between the epithelium and the submucosa. The area covered by mucosal surfaces in a human adult is about 400 m2, while the area covered by the skin is only about 2 m2. Antigen sampling strategies are adapted to the diverse epithelial barriers that cover mucosal surfaces throughout the body, but all involve collaboration with dendritic cells (DCs) [6]. Antigen sampling strategies differ among different mucosal tissues depending on whether they are covered by a stratified (oral cavity, esophagus, lower genital tract) or a simple epithelium (airways, gastrointestinal tract, upper genital tract). 3.1. Organ Specific Sampling Continuous antigen sampling at specific mucosal sites is of crucial importance for initiation of mucosal immune responses against pathogens as diverse as HIV, poliovirus and Salmonella. Antigen sampling at these locations is also important for the induction and maintenance of immunological tolerance to harmless foreign antigens that are best "tolerated", such as food antigens in the gut and air-borne particles in the airways. Mucosal antigen sampling is complicated by the fact that antigens are separated from inductive immune sites (organized lymphoid tissue, either within the mucosa or in draining lymph nodes) by epithelial barriers and thus must be transported across the epithelium in order to be sampled by cells of the immune system. Antigen-sampling strategies at diverse mucosal sites are adapted to the cellular organization of the local epithelial barrier (Fig. 1). 3.2. Targeting Mucosal Sites The induction of immune responses against vaccines that are mucosally delivered requires the presence of organized lymphoid tissue, either within the mucosa or in draining lymph nodes [6]. Organized mucosa-associated lymphoid tissues (MALT) are concentrated in areas where pathogens are most likely to enter the body, for example, the palatine & lingual tonsils and the adenoids in the oral and nasopharyx. They also are abundant at sites of high microbial density such as the aggregates of follicles (Peyer’s patches) in the distal ileum, the abundant isolated follicles in the appendix, colon and rectum [3], and the isolated follicles in the bronchi of young children [8]. The follicular epithelium that covers lymphoid follicles contains M cells. M cells form intraepithelial pockets into which lymphocytes and DCs migrate, and these specialized epithelial cells deliver samples of foreign material including vaccines, by vesicular transport from the intestinal lumen directly into the pocket and to underlying DCs. In the absence of organized MALT, antigens and vaccines may also be sampled on mucosal surfaces through another type of epithelial–dendritic cell (DC) collaboration. Throughout epithelia whether stratified or simple, motile DCs can migrate into the narrow spaces between epithelial cells and even to the outer limit of the epithelium where they can obtain samples of foreign material directly from the lu- Mucosal Vaccines: Where Do We Stand? Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 20 A C B D 2611 Fig. (1). A and B. Mucosae covered by simple epithelium contain subepithelial organized lymphoid tissue with follicles, located at specific sites. At such sites, specialized microfold (M) cells in the follicle-associated epithelium (FAE) sample antigens and deliver them across the epithelial barrier directly to subepithelial DCs that then present antigen locally in adjacent mucosal T-cell areas. In the absence of organized lymphoid tissues, DCs located under epithelia or within intraepithelial spaces may sample antigens or microorganisms that breach the barrier, and can even extend their dendrites into the lumen to capture antigens. Upon antigen uptake, such DCs travel to the nearest draining lymph node (LN) to present antigen to T cells. C and D. Mucosae covered by stratified epithelium are generally devoid of organized lymphoid tissues but are drained by regional lymph nodes. In such mucosae, antigens are sampled by DCs present within and beneath the epithelial layer. Upon antigen uptake, these cells migrate to draining lymph nodes for antigen presentation to naive lymphocytes. The tonsils are an exception: their stratified epithelium covers many lymphoid follicles but the epithelium is thinned in spots to accommodate M cells. minal compartment [9]. This might be most immunologically significant at mucosal locations such as the female genital tract, where there are no organized MALT and the epithelium lacks M cells. Intraepithelial and subepithelial DCs that have captured vaccines could potentially interact with local memory lymphocytes to stimulate memory responses [10] or immune tolerance [11]. They also could exit the mucosa through lymphatics to present antigens to naive T cells in organized lymphoid tissues of draining lymph nodes [12]. Different DC subpopulations have distinct roles in determining the nature of immune responses in vivo, including those in the Peyer’s patches [13]. Migration of DCs following immunization from the Peyer’s patches and intestinal mucosa has been studied in some detail but it is still poorly understood. Recently, it has been reported that transcutaneous immunization can result in mucosal immune responses, and DCs from skin have been found to migrate to Peyer’s patches [14]. 3.3. Mimicking Infectious Agents The design of efficient mucosal vaccines may take advantage of what is known about the pathogenesis of infectious microbes that are able to trigger protective mucosal immune responses [12]. Efficient T and B cell responses depend on the structure and localization of the antigen, its dose, and how it is available, but also on the coordinated action of adjuvants and antigen delivery systems: In general, mucosal vaccines are most effective when they mimic successful mucosal pathogens. Ideally, mucosal vaccines should be live mucosa-tropic vectors or microbe-sized particles that adhere to mucosal surfaces (preferably to the FAE), are transported by M cells, are avidly endocytosed by mucosal DCs, and trigger innate immune responses. Some salient observations are: • Particulate vaccines are taken up most efficiently by follicle-associated M cells and underlying dendritic cells when they are in the size range of viruses (30-200 nm 2612 Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 20 diameter) and can adhere to the epithelium [2, 15]. Bacteria-size particles (1m in diameter) are taken up less efficiently, and larger particles are relatively poor delivery vehicles. • Co-administration of antigens and TLR ligands [16], lactosyl cerebroside [17] or co-stimulatory receptor ligands (CD 40-specific antibodies) [18] triggers enhanced CD8 T cell responses. • Bad timing of adjuvant delivery may impair crosspresentation [19]: antigen and adjuvant are best delivered together to the same DC. • Administration of antigens and adjuvants separately or combined in a mixture or conjugated, also affects the immune response. Co-localization in phagosomes may play an important role in presentation by antigenpresenting cells [20]. • How mucosal vaccination schedules and coordination of the delivery of antigens and adjuvants affect mucosal responses and translate into effective mucosal vaccines has been extensively studied in animal models but is not yet firmly established for humans. In general, nonreplicating vaccines require multiple doses to achieve a booster effect, while live vaccines can be effective as a single dose, due to continued production of antigen over time. 3.4. Choosing the Mucosal Immunization Route As mentioned above, lymphocytes activated in organized mucosa-associated lymphoid tissues express specific adhesion molecules and chemokines called “homing receptors” that recognize endothelial counter-receptors in the mucosal vasculature [21,22]. Some of these receptors and chemokine partners are broadly expressed: for example, IgA-secreting B cells induced in mucosal tissues express CCR10, the receptor for CCL28, which is secreted by epithelial cells throughout the small and large intestines, salivary glands, tonsils, respiratory tract and lactating mammary gland. This explains why mucosal immunization at one site can result in secretion of specific IgA antibodies throughout mucosal and glandular tissues: the so-called "common mucosal immune system" [23]. However, the regional nature of mucosal immune responses is now well documented in mice, nonhuman primates and humans. Receptor-mediated recognition systems serve to focus the mucosal immune response at the site where antigen or pathogen was initially encountered. For example, IgA+ B cells generated in the intestine express the “homing receptor” 4/ 7 integrin that interacts strongly with MADCAM1, an "addressin" expressed by venules in the small and large intestine (and lactating mammary gland) but not in other mucosal tissues [21,22]. T cells activated in the small intestine express both 4/ 7 and CCR9 which attracts them to CCL25, a chemokine secreted by epithelia of the small (but not large) intestine [24] Local vaccination, infection or inflammation can upregulate local expression of chemokines, receptors and addressins [25]. Thus, the choice of mucosal vaccination route requires consideration of the nature of the vaccine and the expected site of challenge. Kraehenbuhl and Neutra Mucosal immunization, especially via the intranasal and sublingual routes, also can induce substantial levels of IgA and IgG in serum [26] because mucosal DCs may migrate and carry antigen to systemic inductive sites (lymph nodes and spleen) [27,28] and a fraction of the B cells activated in the mucosa or mucosa-draining lymph nodes express the “peripheral” or systemic homing receptors, 4/ 1 and Lselectin [22]. By contrast, systemic immunization is usually not effective for induction of mucosal responses because in non-mucosal tissues, DCs that capture antigen are not exposed to retinoic acid and don’t induce expression of mucosal homing or chemokine receptors on lymphocytes [29]. An ideal vaccine should provide “front-line” protection at the relevant mucosal surface and also “back-up” protection against systemic spread. For example, to assure protection against mucosal pathogens such as HIV that enter via mucosal surfaces and spread systemically, both mucosal effectors in the rectal or genital mucosa and systemic effectors will be required. In this regard, nasal and sublingual vaccination routes are of great current interest [26]. Intra-nasal administration of vaccines in animals and humans has been shown to induce IgA antibody responses in the salivary glands, upper and lower respiratory tracts, male and female genital tracts, and the small and large intestines as well as CTLs in distant mucosal tissues including the female genital tract [30]. Importantly, either nasal and sublingual immunization produced greater systemic antibody responses than other mucosal immunization routes, sometimes at levels comparable to those induced by systemic vaccination routes [28]. On the other hand, levels of local mucosal protection should be optimized. Rectal immunization is much more effective than nasal or oral routes for inducing strong responses at the rectal mucosa [12]. Nasal immunization is particularly effective for protection against respiratory pathogens, but oral or vaginal vaccines are more effective for protection of the upper gastrointestinal tract or female genital tract, respectively. To activate both humoral and cellmediated immune effector mechanisms distributed both mucosally and systemically, some vaccine strategies currently under investigation are prime-boost combinations involving both mucosal and systemic delivery. While the vast majority of pathogens invade the host through mucosal surfaces, some remain localized to mucosal tissues, while others spread systemically. This may dictate which route of immunization should be chosen as described below. Mucosal Entry of Systemic Pathogens Some pathogens, i.e. Hepatitis B, Hepatitis A, Measles, Polio, Mumps viruses or group B streptococci and Hemophilus influenzae B, enter the host via genital, respiratory or digestive mucosal tissues and infect distant internal organs. Non mucosal vaccine delivery provides protection against these systemic pathogens. The mechanism of protection is probably mediated by systemic IgG antibodies or T cells that inhibit viral replication. Whether mucosal IgG antibodies that are transported across the mucosal tissues mediate protection is yet not established. Mucosal Pathogens and Non Mucosal Vaccine Delivery Non mucosal vaccine delivery, whether subcutaneous or intramuscular, has been shown to mediate protection against Mucosal Vaccines: Where Do We Stand? certain mucosal pathogens, (i.e. Streptococcus pneumoniae, human papilloma virus, influenza virus, Hemophilus influenzae B, Salmonella typhi) that infect mucosal tissues. The mechanism of protection for such pathogens is not fully understood. It is believed that systemic antibody-mediated inhibition of replication and spread is involved, although one cannot rule out a role for IgG antibodies that are transported into secretions by mucosal epithelia. Pathogens for which both Mucosal and Non Mucosal Vaccines are Effective Mucosal and non mucosal vaccines exist for at least three pathogens. For all three vaccines the correlates of protection still have to be identified, although it is likely that protection is mediated by neutralizing and/or non-neutralizing antibodies. Polio The Oral Polio Vaccine (OPV) is a live attenuated virus that generates both systemic IgG antibodies and intestinal sIgA antibodies. The vaccine is easy to administer and it generates herd immunity, probably due to the shedding of inactivated virus in the stools. In developing countries, the oral polio vaccine is less immunogenic than the injected vaccine, which consists of inactivated polio virus. Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 20 2613 the subepithelial lamina propria. Whether vaccines mucosally administered in humans inhibit mucosal replication of viral (influenza, rotavirus, polio) and bacterial (pertussis, typhoid) pathogens through locally secreted IgA or via transcytosed IgG present on mucosal surfaces is not yet known and remains to be established [35]. Studies on IgA-deficient mice and humans show that serum IgG can indeed be sufficient to provide protection against infection in mucosal tissues. In most individuals with IgA deficiency, there is no increased susceptibility to infections. Probably the major role of sIgA is to control the gut flora and prevent entry of potentially harmful commensals [36], but the presence of sIgA has also been correlated with protection against pathogens such as Vibrio cholerae and Salmonella species. Serum IgG can also be actively transported through human epithelial cells in the gut [37,38], the airways [39] and the genital tract [40] via the neonatal Fc IgG receptor (FcRn). FcRn-mediated transport is a mechanism by which IgG can act locally on mucosal surfaces and play a role in immune surveillance and in host defense against mucosally transmitted pathogens. Table 1. Effect of Infection Pressure on Typhoid Vaccine Efficacy [32]. Influenza The intranasal live attenuated vaccine generates herd immunity and its efficacy is marginally higher when compared to the injected vaccine, which consists of a cocktail of viral surface antigens [31] S. typhi. The oral Ty21 and the Vi polysaccharide vaccine are administered mucosally and systemically, respectively. The efficacy of the Ty21a vaccine is dependent on the disease incidence (Table 1). 4. CORRELATES OF PROTECTION The identification of correlates of protection against mucosal pathogens is crucial for the rational design of effective mucosal vaccines and adjuvants [5,15,33] but has proven very difficult to achieve. The lack of reliable and sensitive techniques for measuring humoral and cellular responses in mucosal tissues contributes to the difficulty to identify correlates of mucosal protection and explains in part why few mucosal vaccines have been developed so far. 4.2. Role of T Cells 4.1. Role of Secretory IgA and IgG Antibodies Cellular immunity at mucosal surfaces is critical both for regulating the differentiation and activities of B and T effector cells and providing direct effector responses. The role of T cells as the immunological correlate of protection following vaccination has been documented for a few vaccines. Secretory IgA functions mainly as a preventive antibody, but its special functions in the mucosal milieu are not always reflected in classical in vitro neutralizing antibody assays. It is a poor activator of the complement system due to the lack of C1q binding site. IgA’s main role is to function on mucosal surfaces, preventing uptake of commensals by “immune exclusion”, a mechanism that prevents inflammation [34]. Secretory IgAs can mediate protection by i/ neutralization of pathogens in the intestinal lumen, ii/ neutralization within intestinal epithelial cells as antibodies are transported by the polymeric immunoglobulin receptor through vesicular transport across the epithelial cells, and iii/ neutralization in Mucosal (intranasal) administration of the live attenuated vaccine FluMist triggers cytotoxic CD8+ T cells that are thought to contribute significantly to cross-reactive protection against variant influenza strains [41]. The contribution of systemic and pulmonary T cell effectors to vaccineinduced protection from H5N1 influenza virus infection in mice demonstrates that airway influenza-specific lymphocytes are the main contributors to clearance of challenge virus from the lungs [42], The contribution of serum influenza-specific antibodies in serum and splenic CD8+ T cells is negligible, and the contribution of airway secretory IgA is not established. 2614 Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 20 Kraehenbuhl and Neutra Fig. (2). Under appropriate stimulation, dendritic cells (DC) produce interleukin (IL)-1 and IL-6 that trigger Th17 differentiation in humans. IL-6 signaling is STAT3-dependent. Th17 cells produce IL-21, which contributes to Th17 differentiation. DCs also produce IL-23 that reinforces Th17 T cell development. Signaling by both IL-21 and IL-23 is in part mediated by STAT3. Th17 cell-derived cytokines play an important role in the protection of the host against various bacteria and fungi, particularly at mucosal surfaces. Indeed, Th17 cells are constitutively present in the human and mouse intestinal mucosa. Th17 cells coordinate immune defense against pathogens through their ability to i/ enhance the recruitment and facilitate the activation of neutrophils, and ii/ stimulate the production of defensins by epithelial cells. Fig. (3). The live polio vaccines now in widespread use were developed by Albert Sabin in 1961. The vaccines were developed from circulating strains that had been adapted to laboratory conditions. The viruses were grown using sub-optimal conditions and different host cells, and the resulting progeny viruses were tested for virulence, usually using monkeys. (Adapted from Minor PD, 2004 [49]). Mucosal Vaccines: Where Do We Stand? Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 20 2615 4.3. Role of Th17 T Cells 5.1. Live-Attenuated or Inactivated Vaccines The role of Th17 cells and IL-17 secretion during infection (Fig. 2) provides new insight into host defense at mucosal surfaces and vaccine-induced immunity [43]. Peripherally-induced regulatory T cells and Th17 effector cells arise in a mutually exclusive fashion, Thus CX3CR1+ dendritic cells (DC) promote Th1/Th17 cell differentiation [44], whereas CD103+ DCs induce T regulatory cell differentiation in mouse colonic lamina propria. Many vaccines consist of intact microbes (virus or bacteria) that are either live-attenuated or inactivated (killed). They are administered for prevention, amelioration, or treatment of infectious diseases. IL-17 has been shown to be required for some vaccineinduced protection, including S. pneumoniae, M. tuberculosis, and influenza [45]. IL-17 alone or in combination with B cell-activating factor (BAFF) controls the survival and proliferation of human B cells and their differentiation into IgAsecreting cells [46] and IL-17 triggers sIgA secretion in the airway mucosa following up-regulation of the polymeric immunoglobulin receptor and polymeric IgA transcytosis [47]. 5. DESIGN OF MUCOSAL VACCINES Mucosal immune responses are most efficiently triggered when the vaccine is delivered directly onto mucosal tissues. Mucosal immunization, however, is difficult to achieve for the reasons cited.. In order to enhance efficacy, mucosal vaccines should be designed to mimic physicochemical properties of opportunistic pathogens, specifically charge and size [12]. To achieve this, the following strategies have been proposed [14]: i/ overcoming physiological barriers at mucosal surfaces; ii/ targeting of mucosal antigen presenting cells for appropriate processing of antigens that lead to specific T and B cell activation ; iii/ controlling the kinetics and location of antigen and adjuvant presentation in order to promote longlived, protective adaptive immune memory responses. In order to cope with the increased number of vaccinations that children around the world receive, new needle-free methods of immunization are being developed [48] such as liquid jet injectors, topical or transdermal application to the skin, sublingual application, oral pills, and nasal sprays. Live attenuated vaccines contain weakened forms of the organism that causes the disease. There are many examples of highly successful vaccines that have been developed using live attenuated viruses, .i.e. measles, mumps, rubella, chicken pox, oral polio (Sabin vaccine), yellow fever, nasalspray flu vaccine (including the seasonal flu nasal spray and the 2009 H1N1 flu nasal spray), rabies (now available in two different attenuated forms, one for use in humans, and one for animal usage), or bacteria, i.e. BCG and typhoid vaccine. Several live-attenuated vaccines administered via the mucosal (oral) route are licensed for enteric infections, including cholera, typhoid, and rotavirus. Inactivated microbial vaccines (or killed vaccine) consist of virus particles or bacteria which are grown in culture and then killed using a method such as heat or formaldehyde. These include: viral, i.e. polio vaccine (Salk vaccine) & influenza vaccine, and bacterial vaccines for typhoid fever (Vi capsular polysaccharide (Typhim Vi®, Sanofi Pasteur; Typherix®, GSK) and cholera (Dukoral R, Crucell, Netherlands). Vaccines consisting of live attenuated or intact inactivated microorganisms are the only vaccines approved for mucosal delivery for which protection efficacy has been correlated with measurements of effector mucosal immune responses [4]. The success of live-attenuated and inactivated vaccines is thought to result from the fact that they present multiple immunogens combined with adjuvant activity that triggers strong antibody and cellular responses as well as long-term memory. Attenuation, however, presents the risk of reversion which compromises safety, as documented for the oral polio vaccine (e.g., Sabin 3) [50]. For this reason inactivation of viruses and bacteria has been the preferred approach, since Fig. (4). Virosomes are virus-like particles, consisting of reconstituted influenza virus envelopes, which do not contain the genetic material of the native virus. 2616 Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 20 inactivated vaccines are much safer. Inactivation, however, may reduce immunogenicity and lose adjuvanticity, with a rapid waning of protective immunity. 5.2. Subunit and Conjugate Vaccines Subunit Vaccines Pathogen-specific proteins or protein conjugated polysaccharides represent the second largest category of licensed prophylactic vaccines. The vaccines, i.e. diphtheria and tetanus toxoid vaccines, however, are administered primarily by subcutaneous or intramuscular routes and not mucosally. The cholera vaccine is the only licensed mucosally administered vaccine that contains protein subunits: The cholera vaccine (Dukoral) consists of the cholera toxin B subunit along with inactivated bacteria (Vibrio cholerae O1 strain). Oral, but not parenteral, immunization protects against V. cholera colonization and toxin binding, induces protective mucosal IgA antibodies against the bacterium and its toxin, and provides long-lasting intestinal immunological memory [51]. Conjugate Vaccines No licensed conjugate vaccines for mucosal administration are presently available. Several novel approaches tested in experimental animals highlight the potential of mucosal vaccine development. For instance: Intranasal administration of soluble influenza hemagglutinin protein, linked to a targeting peptide specific for Claudin-4 on M cells, induced both specific serum IgG and mucosal sIgA antibodies [52]. a Herpes simplex virus 2 subunit vaccine (HSV-2 envelope glycoprotein fused to the IgG Fc) given intranasally induced mucosal B and T cell responses and conferred protection from intravaginal challenge with HSV-2 [53]. Repeated intravaginal immunization of recombinant HIV gp140 protein elicited systemic and mucosal neutralizing IgG antibodies [54]. 5.3. Virus-Like Particles (VLP) and Virosomes Virus-like particles and virosomes constitute a category of subunit vaccines in which immunogens are derived from viral components that have the capacity to self-assemble into higher-order three-dimensional structures that maintain the conformational structure of virus immunogens [55]. Virus-like particles (VLP) resemble viruses, but are noninfectious because they do not contain any viral genetic material. The expression of viral structural proteins, such as envelope or capsid, can result in the self-assembly of VLPs. The hepatitis B vaccine, the first VLP to become a commercially viable vaccine. is produced in yeast by expression of the hepatitis B surface antigen (HBsAg) that self-assembles into particles [56]. The human papillomavirus (HPV) vaccine is the only other VLP since to be licensed for human use. The quadrivalent HPV vaccine (Gardasil, from Merck & Co.) is composed of the L1 capsid proteins of HPV-6, -11, 16, and -18 types expressed in yeast [57]. The mechanism of protection mediated by the HPV vaccines is not fully understood, and no immune correlates have been linked definitely to protection. Protection is believed to be due to serum neu- Kraehenbuhl and Neutra tralizing IgG antibodies that are transported across the cervical epithelium where they bind HPV and prevent infection. The licensed VLP vaccines require co-administration with adjuvants to be efficient. In the light of a pandemic threat, influenza VLPs have been recently developed as a new generation of non-egg based, cell culture-derived vaccine candidates against influenza infection [58,59]. A nasal vaccine candidate (NASVAC), comprising hepatitis B virus (HBV) surface (HBsAg) and core antigens (HBcAg), was tested in a phase I trial in healthy adults and shown to be safe, well tolerated and immunogenic. This was the first demonstration of safety and immunogenicity for a nasal vaccine candidate comprising HBV antigens [60]. Virosomes are vaccine delivery systems (Fig. 4) consisting of mono- or bi-layer phospholipid vesicles incorporating virus derived proteins. Virosome fusion with target cells is mediated by functional viral envelope glycoproteins such as influenza virus hemagglutinin (HA) and neuraminidase (NA). Lipids, antigens, adjuvants, or other materials can be added to the dissolved viral membrane components or can be included in the virosome during reconstitution. The vaccines Inflexal® for influenza and Epaxal® for Hepatitis A are approved products on the market, both using virosomes from influenza as a delivery platform. A Phase 1 evaluation of intranasal virosomal influenza vaccine with and without Escherichia coli heat-labile toxin (HLT) was conducted in adult volunteers. Two nasal spray vaccinations with HLT-adjuvanted virosomal influenza vaccine induced a humoral immune response comparable to that induced by a single parenteral vaccination. A significantly higher induction of influenza virus-specific immunoglobulin A was noted in the saliva after two nasal applications. The immune response after a single spray vaccination was significantly lower. HLT as a mucosal adjuvant was necessary to obtain a humoral immune response comparable to that with parenteral vaccination. In this study, all vaccines were well tolerated. Later, however, the formulation with HLT was withdrawn because of toxicity of HTL, but virosomes without HTL remained immunogenic [61]. 5.4. Polymer-Based Carrier Systems Another approach to improve the effectiveness of mucosal vaccines is based on nanotechnologies [62]. These new carrier systems are expected to overcome oral, nasal, intestinal, rectal and genital mucosal barriers through i/ encapsulation of vaccine components to protect them from the harsh mucosal environment and target them to the mucosal immune system; and ii/ incorporation of mucosal adjuvants which enhance immune responses. These new carriers have been made from natural or synthetic polymers, lipids, proteins, or inorganic material, to form particles and capsules of controlled size and structure. Nanocapsules [62,63] (Table 2) constitute a slow release delivery system in which the vaccine can be enclosed within an aqueous (water-in-oil) or oil-based (oil-in-watercore) surrounded by a solid or semisolid material shell. They vary in size from 20 to 200 nm. Three major nanocapsules have been used for mucosal vaccination: i/ Water-in-oil emulsion, ii/ Oil-in-water emulsion, and iii/ liposomes [63]. Mucosal Vaccines: Where Do We Stand? Table 2. 2617 Features of Nanocapsules. Nanoparticles [68, 69] are solid particles in which the vaccine antigens and adjuvants are dispersed within the polymer matrix or adsorbed to the particle surface (Table 3). Specific types of nanoparticles include micelles, dendrimers, and solid matrix nanoparticles composed of biodegradable or bioeliminable synthetic polymers, e.g., polyesters, polyanhydrides, poly(amino acids), natural polymers (chitosan, alginate, albumin) and copolymers [70]. Recently, a large intestine–targeted oral delivery system with pH-dependent microparticles containing vaccine nanoparticles was shown to induce colorectal immune responses in mice at levels comparable to what is obtained with colorectal vaccination using the same vaccine delivery system [71]. This new vaccine delivery system also protected mice against rectal and vaginal viral challenge. Whether this system will protect nonhuman primates and humans remains to be established. Table 3. Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 20 Features of Nanoparticles. Adapted from Chadwick et al [69]. 6. MUCOSAL ADJUVANTS Mucosal vaccine formulations have to overcome the normal mechanisms operating in mucosal tissues that suppress unwanted immune and inflammatory responses to digested food antigens and harmless commensal bacteria, a phenomenon called "mucosal tolerance". This is particularly true for subunit vaccines. Induction of tolerance can be overcome by adding adjuvants to the vaccine formulation that provide "danger" signals that activate innate responses in mucosal epithelial and immune cells (Fig. 5). Adjuvants can be classified into two broad categories: i Immunostimulatory molecules derived from natural immunostimulants including modified bacterial enterotoxins, Toll-like receptor (TLR) ligands (such as CpG oligonucleotide), and cytokines. 2618 Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 20 Kraehenbuhl and Neutra Fig. (5). Mucosal adjuvants Mucosal vaccine efficiency can be enhanced by mucosal adjuvants. They can act on epithelial cells or on innate immune cells, .i.e. dendritic cells, macrophages, present beneath the epithelium. They provide signals that direct and stimulate the immune responses to co-administered soluble antigen. Adapted from Lawson et al. [73]. ii Delivery vehicles with other forms of immunostimulatory activity including saponin-based systems (described below), Montanide ISA-51 & 720, MP59, Adjuvant System 3 used with flu vaccine Pandermix, and Adjuvant System 4 approved for use with injectable vaccines for hepatitis B and HPV. Adjuvants may possess both immunostimulating and antigen delivery properties. There are many classes of adjuvants [72] but only few are effective at promoting mucosal immune responses [73]. 6.1. Bacterial Enterotoxins The bacterially derived ADP-ribosylating enterotoxins, i.e. cholera toxin (CT) [74], heat-labile enterotoxin from E. coli (LT) [75], and their mutants or subunits are the bestcharacterized mucosal adjuvants. These enterotoxins induce antigen-specific IgA antibodies and long-lasting memory to co-administered antigens when administered mucosally or transcutaneously. However, safety issues prevent their use in mucosal vaccination in humans. CT, LT, and some LT mutants stimulate antigen capture by enhancing DC migration from the subepithelial dome to follicular-associated epithelium in mucosa-associated lymphoid tissue (MALT). After oral administration [76], CT and LT induce Th2 responses and mixed Th1/Th2 responses, respectively, These adjuvants also trigger Th17 responses likely to play a role in vaccine-induced protection [77]. 6.2 Toll-Like Receptor (TLR) Ligands TLR ligands activate TLRs, triggering intracellular signaling pathways that lead to cytokine secretion and immune cell activation [78]. This innate immune response in turn promotes an adaptive immune response. Thus, TLR ligands are used to enhance the induction of vaccine-specific responses. In mucosal tissues the following cell types express TLRs and respond to TLR ligands: i/ epithelial cells, ii/ dendritic cells (DC) located in the subepithelial lamina propria and sometimes extending processes between epithelial cells, and iii/ B lymphocytes. TLR-stimulation of these cells induces the following: • On B cells, TLR signaling enhances thymusindependent IgA class switching through the activation of BAFF and APRIL, two members of the TNF family [79]. • TLR2 ligands stimulate IgA production by B cells and gut homing receptor expression [80]. • In gut enterocytes, flagellin, the ligand of TLR5, rapidly induces expression of the chemokine CCL20 which re- Mucosal Vaccines: Where Do We Stand? Table 4. 2619 Mucosal Adjuvants and Delivery Technologies. cruits DCs to the lamina propria [81]. Flagellin also stimulates the mucosal production of IL-17 and IL-22 and the subsequent expression of target genes [82]. • Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 20 In DCs, TLR ligands induce production of cytokines and chemokines that guide the responses of many neighboring cells, resulting in adaptive immunity and/or inflammation. 6.3. Delivery Vehicles with Saponin-Based Immunostimulatory Activity Several lipid-based or lipid-containing mucosal adjuvants are currently being evaluated for mucosal vaccination [83.84]. Their toxicity, stability and manufacturing methods as well as efficacy remains to be established. Quil A is a complex mixture of chemically related triterpenoid saponins extracted from the bark of the Chilean tree Quillaja saponaria Molina. The adjuvant activity of Quil A is mediated through an aldehyde group on the saponins' triterpene aglycone forming a Schiff base with amino groups on costimulatory T cell receptors [84]. Quil A may potentially be able to replace CD80 as a costimulatory signal and preferentially induce Th1 type immune responses. QS-21 is a highly purified triterpene glycoside saponin from Quillaja saponaria that contains two functional groups likely to be involved in the adjuvant mechanism of action through charge or Schiff base interaction with a cellular target. ISCOM (Immune stimulating complex) was described in 1982 by Morein and colleagues [85]. The Iscom-matrix technology is based on Quillaja saponins and the complex formed when saponin fractions are mixed with cholesterol and phospholipids at defined ratios resulting in the assembly of homogeneous cage-like structures ~ 40 nm in diameter. When the antigen is incorporated in the structure, the resulting particle is referred to as an ISCOM. The integrity of antigens co-administered with the ISCOM-matrix adjuvant is thought to be maintained but incorporation of antigen into ISCOMS requires a denaturation step. Saponins are known to interact irreversibly with cholesterol in cell membranes and this can be associated with adverse reactions at the immunization site. However, this toxicity has been considera- bly reduced in current generation ISCOM-matrix formulations. 7. MEASURING MUCOSAL IMMUNE RESPONSES. The assessment of mucosal immune responses to mucosal vaccine candidates remains difficult despite current efforts to develop, standardize and validate methods for measuring mucosal vaccine-specific antibodies or T cells that are suitable for use in large-scale field trials. The major problem is the sampling of mucosal secretions and tissues since respiratory, gut or genital mucosal surfaces are not readily accessible. Mucosal sampling requires cumbersome and invasive procedures. Key aspects are described in more detail below: 8.1. Sampling Secretions and Tissues Mucosal sampling involves collecting secretions and mucosal tissues from i/ the airways including nasal secretions, bronchoalveolar lavage and bronchial biopsies; ii/ the digestive tract including saliva collection, small intestinal biopsies, rectal secretions by sponge and cells by cytobrush, rectal and sigmoidal biopsies; iii/ the genital tract including semen and vaginal and cervical secretions by lavage or sponge and cells by cytobrush. Cytobrushes usually yield cell numbers (mean= 100,000 cells for rectal or cervical sampling) that allow phenotyping, but are too low for functional assays. Sponge collection is usually sufficient to capture enough undiluted secretion to detect and directly measure luminal antigen-specific antibodies. Mucosal lavage collects diluted secretions, so that specific antibody concentrations must be expressed relative to total immunoglobulin levels. 8.2. Assays Currently used in Human Vaccine Trials Most vaccine trials rely on determination of serum antibody levels, which usually do not accurately reflect mucosal secretory antibody levels [86]. Measurements of vaccine specific antibody secreting cells (ASCs) in the bloodstream that bear mucosal homing markers and thus are transiently migrating in the circulation on their way to the mucosa are 2620 Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 20 Table 5. Kraehenbuhl and Neutra Licensed Mucosal Vaccines. known to better reflect local immune responses [87,88]. Standard operating procedures (SOP) have been developed by several laboratories including those of the HIV Vaccine Trial Network to outline the procedures that site staff follow to collect and process mucosal secretions samples for antibody and T cell testing. 8.3. Antibody Assays Three assays are usually used to evaluate vaccine antibody responses: i/ ELISA, ii/ ELISPOT, and iii/ Antibodies in Lymphocyte Supernatant (ALS) assay. ELISA stands for Enzyme-Linked Immunosorbent Assay [89]. ELISA is a sensitive method used in immunology to detect and/or quantitate the presence of a protein (e.g. antigen, antibody, cytokine) in a test solution such as body fluids (serum, plasma, urine) or cell culture supernatants. This method utilizes enzyme-linked antibodies hence the name of the assay. The two most commonly used versions of ELISA are the indirect ELISA which utilizes an unique labeled antibody and the sandwich ELISA which utilizes an unlabeled primary antibody in conjunction with a labeled secondary antibody. ELISPOT. The Enzyme Linked Immunosorbent SPOT assay [90], initially designed to enumerate B cells secreting antigen-specific antibodies, has been adapted to enumerate cytokine-producing cells at the single cell level [91]. Each spot that develops in the assay represents a single reactive cell. Thus, the ELISPOT assay provides both qualitative (type of immune protein) and quantitative (number of responding cells) information. The ELISPOT assay has been adapted to measure serum IgA secreting cells which serve as surrogate markers for mucosal antibody responses. ELISPOT tests traditionally used to evaluate ASC responses are not adapted for large-scale vaccine trials because i/ they are time-consuming; ii/ require relatively large volumes of blood; iii/ cannot be repeated using the same cell samples; iv/ do not properly reflect mucosal booster responses. ALS assay. A simplified ALS assay, in which cells are incubated in the absence of antigen and stored for later analysis of antibody content by ELISA, makes it possible to analyze pre- and post-vaccination samples in the same test. The ALS assay has been used to monitor antibody responses to an oral cholera vaccine, an oral ETEC candidate vaccine, and a novel typhoid vaccine [88]. 8.4. Mucosal T Cell Assays To address the frequency of antigen-specific CD8+ and CD4+ T-cells in mucosal tissues and to determine their cytokine production patterns, several assays are available. The limiting factor, however, is the sampling of mucosal tissues and the collection of enough lymphocytes to perform quantitative assays. To address this limitation, polyclonal T cell amplification procedures have been developed [92]. The most used assays are i/ ELISPOT assays [93], ii/intracellular cellular cytokine staining assays, and iii/ MHC class I tetramer staining assay. 9. LICENSED MUCOSAL VACCINES • The eight vaccines that are currently approved and administered mucosally to humans target three of the main enteric pathogens (i.e. Vibrio cholerae, Salmonella typhi, and rotavirus), the respiratory pathogen, influenza virus, and the mucosally-transmitted neuropathogen poliovirus. • Vaccines have been developed but are not yet approved, and are thus still lacking against other important causes of enteric diseases, i.e. enterotoxigenic Escherichia coli (ETEC) and Shigella. • The main features of the licensed vaccines [94,95] are summarized in Table 5. 10. MUCOSAL VACCINES IN THE PIPELINE Several mucosal vaccines to protect against mucosally acquired pathogens are currently under development and a few have entered clinical trials. These include: Shigella vaccines, Enterotoxicogenic E. coli vaccines, HIV vaccines, and Mycobacterium tuberculosis vaccines( [96,97]. 10.1. Shigella Vaccines Shigellae, the cause of bacterial dysentery, are Gramnegative, nonmotile, facultatively anaerobic, non-spore- Mucosal Vaccines: Where Do We Stand? forming rods. About 165 million cases of shigellosis are reported each year, with a death toll of ~ 1.1 million people. It is an antigenically diverse pathogen with four species (or groups) and 50 serotypes and subserotypes. A Shigella vaccine should be broad enough to protect against multiple serotypes, including S. dysenteriae 1, all 14 S. flexneri types and S. sonnei, which are the most important serotypes worldwide [98]. Immune responses against Shigella infection. Natural Shigella infection protects (~75%) protection against Shigellosis upon subsequent exposure to the homologous Shigella serotype and in some instances against heterologous serotypes. Antibodies (serum or mucosal) directed against the LPS O-antigen appear to play a major role in protection [99]. Orally administered Shigella-specific immunoglobulins prevent shigellosis, indicating that the first line of defense occurs at the gut surface [100]. In healthy adult volunteers, gutderived O-specific IgA ASCs circulating in the bloodstream after oral vaccination reflects intestinal priming that correlates with vaccine efficacy [98]. The immune response to Shigella is a predominantly a Th1-type response, indicating that cell-mediated immunity may also contribute to the defense against this intracellular pathogen [101]. A detailed description of the Shigella vaccines currently in development is provided in a review by Levine and colleagues [98]. The main features of vaccines currently in development are summarized in Table 6. 10.2. ETEC Vaccines Enterotoxigenic Escherichia coli (ETEC) is the most common cause of bacterial diarrhea in children in Africa, Asia and Latin America and in travelers to these regions. Despite this, no effective vaccine for ETEC is available [112]. ETEC causes disease by colonizing the small intestine with colonization factors including fimbriae, and production of heat-labile and/or heat-stable enterotoxins. Antibodies against heat-labile enterotoxin (LT) and the colonization factors (CF) that act synergistically have been shown to be protective in rabbit models [113], and local immunity in the gut seems to be of prime importance for protection. Several inactivated and live candidate ETEC vaccines consisting of toxin antigens, alone or together with colonization factors, have been evaluated in clinical trials (see Table 7). 10.3. HIV Vaccines HIV might be considered a mucosal pathogen, because transmission occurs mainly through exposure of genital and rectal surfaces to HIV and HIV-infected cells [114]. Therefore, an ideal HIV-1 vaccine candidate should induce protective responses in these mucosal tissues and at their surfaces [114]. Systemic delivery of HIV-1 vaccines has been shown in some studies to induce HIV-specific immune responses at the mucosa [115], but in the vast majority of HIV vaccine studies including human trials, mucosal immune responses were not measured. One report indicates that systemic deliv- Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 20 2621 ery of HIV-1 vaccines can compromise the quality or avidity of the HIV-specific immune responses at mucosal sites [116]. Most of the vaccine formulations in preclinical trials in nonhuman primates and in humans were designed to induce antiviral T cell mediated responses rather than antibodies, and of these only a few have been evaluated specifically for their ability to generate CTLs in mucosal tissues. These studies have been extensively reviewed [117] and are summarized in Table 8. 10.4. Mucosal Vaccines for Tuberculosis Mycobacterium tuberculosis is clearly a mucosallytransmitted pathogen but the only licensed TB vaccine, the Bacille Calmette-Guérin (BCG) vaccine, is administered by intradermal injection and is efficient in children but not in adults. In addition, BCG can cause disseminated mycobacterial disease in immuno-compromised individuals including HIV-infected children [127]. Thus, a safe mucosal vaccine to prevent M. tuberculosis infections is urgently needed. . Live Attenuated M. tuberculosis Vaccines A highly attenuated M. tuberculosis strain (aMtb) orally administered at birth to newborn rhesus macaques (Macaca mulatta) induced mucosal and systemic immune responses and protected against TB infection. All vaccinated animals developed M. tuberculosis -specific plasma IgG antibodies to the PSTS1 antigen measured after oral or intradermal vaccination. The vaccine was effective in inducing TB-specific CD4+ and CD8+T cell immune responses systemically and in mucosal tissues. The recombinant aMtb strain mc26435, harboring attenuations in genes critical for replication (panCD and leuCD) and immune evasion (secA2), was safe after oral or intradermal administration in SIV-uninfected and SIVinfected infant macaques [128]. Safety was defined as absence of clinical symptoms, lack of histopathological changes indicative of M. tuberculosis infection, and lack of mycobacterial dissemination. These data represent an important step in the development of novel TB vaccines and suggest that an oral recombinant aMtb vaccine could be a safe alternative to BCG for the pediatric population as a whole, and more importantly for the high risk group of HIVinfected infants. Nanoparticle-Based Vaccine A synthetic vaccine delivery platform with Pluronicstabilized polypropylene sulfide nanoparticles (NPs), which target antigen-presenting cells in lymphoid tissues due to their small size (~30 nm) and which activate the complement cascade by their surface chemistry, has been developed by Ballester and coworkers [129]. The tuberculosis antigen Ag85B was conjugated to the NPs (NP-Ag85B) and the efficacy of the conjugate in eliciting relevant immune responses was assessed in mice after intradermal or pulmonary administration. Pulmonary administration of NP-Ag85B with the adjuvant CpG induced higher levels of antigen-specific polyfunctional Th1 responses in the spleen, the lung and lungdraining lymph nodes as compared to pulmonary 2622 Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 20 Table 6. Shigella Vaccines Under Development. Table 7. ETEC Vaccines Under Development. Kraehenbuhl and Neutra Mucosal Vaccines: Where Do We Stand? Table 8. Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 20 Mucosal and Systemic Immune Responses to HIV Vaccines Administered to non Human Primates and Humans. administration of soluble Ag85B with CpG and to intradermally-delivered formulations. Mucosal and systemic Th17 responses were also observed with this adjuvanted NP formulation and vaccination route, especially in the lung. Following a Mtb aerosol challenge, animals vaccinated with NP-Ag85B and CpG via the pulmonary route showed a substantial reduction of the lung bacterial burden, compared to either soluble Ag85B with CpG or to the corresponding intradermally delivered formulations. These findings highlight the potential of NP-based formulations administered by aerosol for TB vaccination. models reflect the situation in human children is not clear, so comparative studies should be performed. • The role of mucosal and systemic IgG antibodies should be better characterized. In the human gut, for instance, the fenestrated capillaries are likely to allow IgGs to reach neonatal Fc receptors present on epithelial cells and be transported in the lumen by receptor mediated transcytosis. Whether such transport mechanisms also operate in respiratory, genital and urinary mucosal tissues remains to be established. In a murine model of enteropathogenic E. coli (EPEC) disease, it was shown that IgG but not IgA mediated protection [130]. The human papilloma virus (HPV) vaccine is administered systemically and elicits neutralizing antibodies in genital secretions [131]. This suggests that IgG in secretions or mucosal tissues was important but the immunological correlates of protection are still not identified. • We should better define the differences in nature, duration and intensity of humoral and cell-mediated immunity at specific mucosal sites following non-mucosal and mucosal vaccination via different routes of administration in humans. 11. PERSPECTIVES Current licensed mucosal vaccines have reduced the burden of disease and mortality caused by enteric pathogens including rotavirus, V. cholerae and S. Typhi, and those that enter mucosally and spread to affect distal organs such as poliovirus. Recent advances promise to accelerate development of mucosal vaccines that will contribute significantly to protection against pathogens that invade the host via mucosal surfaces, including HIV, Shigella, ETEC, Campylobacter, Helicobacter and probably many others. What Do We Need to Learn More About? • 2623 We must know how to better induce effective longlasting mucosal immunity during the neonatal period and childhood. The extent to which neonatal animal Which Immunization Strategies Need to be Explored And Exploited? In cases where mucosal immunity is essential for protection against mucosal pathogens, the following approaches should be studied and tested: 2624 Current Topics in Medicinal Chemistry, 2013, Vol. 13, No. 20 • • • • • Find new ways to protect antigens from the harsh mucosal environment and to promote their delivery into the inductive sites where immune responses are initiated to achieve the necessary range of immune responses for protection. As examples, the new delivery systems described in this review are designed to facilitate the preservation of antigenicity and appropriate mucosal targeting of antigens. Enhance the recruitment of professional antigen presenting cells to immunization sites, including the airways, the digestive, urinary and genital tracts, and the skin. Define better strategies for induction of appropriate secretory or systemic antibody and/or T cells responses. The mechanisms that underlie the generation of effective immune responses in mucosal tissues following mucosal immunization are complex and probably distinct for the different mucosal surfaces. Kraehenbuhl and Neutra [2] [3] [4] [5] [6] [7] [8] Define more clearly the interactions of vaccine antigens and adjuvants with innate immune cells in mucosal tissues and how these interactions modulate adaptive immunity [9] Develop additional safe yet effective mucosal adjuvants that can be used with mucosal vaccines and more effieient delivery systems. [10] • Better understand the processes of lymphocyte trafficking and homing to specific mucosal effector sites • Develop ways to more effectively induce robust and persistent mucosal immunological memory. • Improve tools for evaluation of antibodies and other immune effectors at mucosal surfaces and in mucosal secretions, particularly in infants and young children. 12. CONCLUSION Mucosal vaccines hold great promise for reducing the burden of disease and mortality caused by mucosally acquired pathogens. Inducing mucosal immunity is complex and challenging. Mucosal delivery of vaccines may be optimal for protection against some diseases but for others, mucosal delivery may not be critical and other routes should be prioritized. Nevertheless, mucosal vaccination has many practical and safety advantages and may also trigger protective systemic responses [123]. Thus, mucosal vaccines may not only prevent initial pathogen entry but may also protect against systemic disease. [11] [12] [13] [14] [15] [16] [17] [18] [19] CONFLICT OF INTEREST The author(s) confirm that this article content has no conflicts of interest. [20] ACKNOWLEDGEMENTS [21] Declared none. REFERENCES [1] Mutsch, M.; Zhou, W.; Rhodes, P.; Bopp, M.; Chen, R. T.; Linder, T.; Spyr, C.; Steffen, R. 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