Mucosal Vaccine Delivery Research Articles

Enhanced Mucosal Immune Responses Against Tetanus Toxoid Using Novel Delivery System Comprised of Chitosan‐Functionalized Gold Nanoparticles and Botanical Adjuvant: Characterization, Immunogenicity, and Stability Assessment

Approaches based on combined use of delivery systems and adjuvants are being favored to maximize efficient mucosal delivery of antigens. Here, we describe a novel delivery system comprised of chitosan-functionalized gold nanoparticles (CsAuNPs) and saponin-containing botanical adjuvant; Asparagus racemosus extract (ARE) for oral delivery of tetanus toxoid (TT). A significant increase in TT-specific IgG (34.53-fold) and IgA (43.75-fold) was observed when TT-CsAuNPs were formulated with ARE (TT-ARE-CsAuNPs). The local IgA immune responses for TT also showed a significant increase (106.5-fold in intestine washes and 99.74-fold in feces) with ARE-based formulations as compared with plain TT group. No effect of ARE was observed on size, charge, and loading properties of CsAuNPs. Additionally, no effect of ARE and CsAuNPs was observed on antigenicity and secondary structure of TT as determined by fluorescence, circular dichroism, and Fourier transform infrared spectroscopy. The stability studies demonstrated excellent stability profile of formulation at recommended storage conditions. The study establishes the possible role of immunomodulatory adjuvants in particulate delivery systems for mucosal delivery of vaccines.

Mucosal vaccine delivery by non-recombinant spores of Bacillus subtilis.

Development of mucosal vaccines strongly relies on an efficient delivery system and, over the years, a variety of approaches based on phages, bacteria or synthetic nanoparticles have been proposed to display and deliver antigens. The spore of Bacillus subtilis displaying heterologous antigens has also been considered as a mucosal vaccine vehicle, and shown able to conjugate some advantages of live microrganisms with some of synthetic nanoparticles. Here we review the use of non-recombinant spores of B. subtilis as a delivery system for mucosal immunizations. The non-recombinant display is based on the adsorption of heterologous molecules on the spore surface without the need of genetic manipulations, thus avoiding all concerns about the use and environmental release of genetically modified microorganisms. In addition, adsorbed molecules are stabilized and protected by the interaction with the spore, suggesting that this system could reduce the rapid degradation of the antigen, often observed with other delivery systems and identified as a major drawback of mucosal vaccines.

Novel transgenic rice-based vaccines.

Oral vaccination can induce both systemic and mucosal antigen-specific immune responses. To control rampant mucosal infectious diseases, the development of new effective oral vaccines is needed. Plant-based vaccines are new candidates for oral vaccines, and have some advantages over the traditional vaccines in cost, safety, and scalability. Rice seeds are attractive for vaccine production because of their stability and resistance to digestion in the stomach. The efficacy of some rice-based vaccines for infectious, autoimmune, and other diseases has been already demonstrated in animal models. We reported the efficacy in mice, safety, and stability of a rice-based cholera toxin B subunit vaccine called MucoRice-CTB. To advance MucoRice-CTB for use in humans, we also examined its efficacy and safety in primates. The potential of transgenic rice production as a new mucosal vaccine delivery system is reviewed from the perspective of future development of effective oral vaccines.

Superior protection and distinct phenotype of pulmonary CD8+ T cells elicited by mucosal vaccination (MUC5P.863)

Abstract CD8-positive T cells (CTLs) confer protective immunity against many infectious diseases, and thus the development of safe and efficient CTL-targeted vaccination is of high clinical significance. Although many respiratory pathogens invade the host through the lung mucosa, most licensed human vaccines elicit limited and systemic T cell responses, rather than strong local pulmonary responses. We therefore wondered whether elicitation of pulmonary CTL response with microbe-free CTL-targeted subunit vaccine might benefit the host and protect against respiratory infections. We discovered superior protection of humanized mice from lethal respiratory challenge with vaccinia virus with intranasal, but not intra-peritoneal, vaccination. Critically, this superior protection did not correlate with the magnitude of the systemic CTL response but was dependent on in situ elicitation of robust pulmonary epitope-specific CTLs, which were able to limit viral replication and prevent disease. Global transcriptome analysis revealed that pulmonary CTLs elicited by vaccination possessed distinct phenotypic and functional properties when compared to CTLs that homed to the spleen. To extend this finding, we developed an approach for eliciting protective CTL responses that utilizes nanoparticle-based mucosal delivery of epitope-targeted vaccine formulated with an optimal adjuvant.

Antigen targeting to M cells for enhancing the efficacy of mucosal vaccines

Vaccination is one of the most successful applications of immunology and for a long time has depended on parenteral administration protocols. However, recent studies have pointed to the promise of mucosal vaccination because of its ease, economy and efficiency in inducing an immune response not only systemically, but also in the mucosal compartment where many pathogenic infections are initiated. However, successful mucosal vaccination requires the help of an adjuvant for the efficient delivery of vaccine material into the mucosa and the breaking of the tolerogenic environment, especially in oral mucosal immunization. Given that M cells are the main gateway to take up luminal antigens and initiate antigen-specific immune responses, understanding the role and characteristics of M cells is crucial for the development of successful mucosal vaccines. Especially, particular interest has been focused on the regulation of the tolerogenic mucosal microenvironment and the introduction of the luminal antigen into the lymphoid organ by exploiting the molecules of M cells. Here, we review the characteristics of M cells and the immune regulatory factors in mucosa that can be exploited for mucosal vaccine delivery and mucosal immune regulation.

Mucosal vaccine delivery by non-recombinant spores of

Development of mucosal vaccines strongly relies on an efficient delivery system and, over the years, a variety of approaches based on phages, bacteria or synthetic nanoparticles have been proposed to display and deliver antigens. The spore of Bacillus subtilis displaying heterologous antigens has also been considered as a mucosal vaccine vehicle, and shown able to conjugate some advantages of live microrganisms with some of synthetic nanoparticles. Here we review the use of non-recombinant spores of B. subtilis as a delivery system for mucosal immunizations. The non-recombinant display is based on the adsorption of heterologous molecules on the spore surface without the need of genetic manipulations, thus avoiding all concerns about the use and environmental release of genetically modified microorganisms. In addition, adsorbed molecules are stabilized and protected by the interaction with the spore, suggesting that this system could reduce the rapid degradation of the antigen, often observed with other delivery systems and identified as a major drawback of mucosal vaccines.

Non-recombinant display of the B subunit of the heat labile toxin of Escherichia coli on wild type and mutant spores of Bacillus subtilis

BackgroundMucosal infections are a major global health problem and it is generally accepted that mucosal vaccination strategies, able to block infection at their entry site, would be preferable with respect to other prevention approaches. However, there are still relatively few mucosal vaccines available, mainly because of the lack of efficient delivery systems and of mucosal adjuvants. Recombinant bacterial spores displaying a heterologous antigen have been shown to induce protective immune responses and, therefore, proposed as a mucosal delivery system. A non-recombinant approach has been recently developed and tested to display antigens and enzymes.ResultsWe report that the binding subunit of the heat-labile toxin (LTB) of Escherichia coli efficiently adsorbed on the surface of Bacillus subtilis spores. When nasally administered to groups of mice, spore-adsorbed LTB was able to induce a specific immune response with the production of serum IgG, fecal sIgA and of IFN-γ in spleen and mesenteric lymph nodes (MLN) of the immunized animals. Dot blotting experiments showed that the non-recombinant approach was more efficient than the recombinant system in displaying LTB and that the efficiency of display could be further increased by using mutant spores with an altered surface. In addition, immunofluorescence microscopy experiments showed that only when displayed on the spore surface by the non-recombinant approach LTB was found in its native, pentameric form.ConclusionOur results indicate that non-recombinant spores displaying LTB pentamers can be administered by the nasal route to induce a Th1-biased, specific immune response. Mutant spores with an altered coat are more efficient than wild type spores in adsorbing the antigen, allowing the use of a reduced number of spores in immunization procedures. Efficiency of display, ability to display the native form of the antigen and to induce a specific immune response propose this non-recombinant delivery system as a powerful mucosal vaccine delivery approach.

Enhanced Nasal Mucosal Delivery and Immunogenicity of Anti-Caries DNA Vaccine through Incorporation of Anionic Liposomes in Chitosan/DNA Complexes

The design of optimized nanoparticles offers a promising strategy to enable DNA vaccines to cross various physiological barriers for eliciting a specific and protective mucosal immunity via intranasal administration. Here, we reported a new designed nanoparticle system through incorporating anionic liposomes (AL) into chitosan/DNA (CS/DNA) complexes. With enhanced cellular uptake, the constructed AL/CS/DNA nanoparticles can deliver the anti-caries DNA vaccine pGJA-P/VAX into nasal mucosa. TEM results showed the AL/CS/DNA had a spherical structure. High DNA loading ability and effective DNA protection against nuclease were proved by gel electrophoresis. The surface charge of the AL/CS/DNA depended strongly on pH environment, enabling the intracellular release of loaded DNA via a pH-mediated manner. In comparison to the traditional CS/DNA system, our new design rendered a higher transfection efficiency and longer residence time of the AL/CS/DNA at nasal mucosal surface. These outstanding features enable the AL/CS/DNA to induce a significantly (p<0.01) higher level of secretory IgA (SIgA) than the CS/DNA in animal study, and a longer-term mucosal immunity. On the other hand, the AL/CS/DNA exhibited minimal cytotoxicity. These results suggest that the developed nanoparticles offer a potential platform for DNA vaccine packaging and delivery for more efficient elicitation of mucosal immunity.

Evaluation of Mucosal and Systemic Immune Responses Elicited by GPI-0100- Adjuvanted Influenza Vaccine Delivered by Different Immunization Strategies

Vaccines for protection against respiratory infections should optimally induce a mucosal immune response in the respiratory tract in addition to a systemic immune response. However, current parenteral immunization modalities generally fail to induce mucosal immunity, while mucosal vaccine delivery often results in poor systemic immunity. In order to find an immunization strategy which satisfies the need for induction of both mucosal and systemic immunity, we compared local and systemic immune responses elicited by two mucosal immunizations, given either by the intranasal (IN) or the intrapulmonary (IPL) route, with responses elicited by a mucosal prime followed by a systemic boost immunization. The study was conducted in BALB/c mice and the vaccine formulation was an influenza subunit vaccine supplemented with GPI-0100, a saponin-derived adjuvant. While optimal mucosal antibody titers were obtained after two intrapulmonary vaccinations, optimal systemic antibody responses were achieved by intranasal prime followed by intramuscular boost. The latter strategy also resulted in the best T cell response, yet, it was ineffective in inducing nose or lung IgA. Successful induction of secretory IgA, IgG and T cell responses was only achieved with prime-boost strategies involving intrapulmonary immunization and was optimal when both immunizations were given via the intrapulmonary route. Our results underline that immunization via the lungs is particularly effective for priming as well as boosting of local and systemic immune responses.

Stabilized lipid nanocapsules eliciting potent mucosal immune response (P4509)

Abstract We investigated the use of a novel lipid nanocapsule, Interbilayer-Crosslinked Multilamellar Vesicles (ICMV), for mucosal vaccine delivery. ICMV co-encapsulated with MPLA and/or polyI:C as adjuvants, was used to vaccinate mice using model antigen and SIV-gag epitopes. When delivered via the lungs, we found that ~60-fold more antigen transported to the draining lymph nodes than equivalent subcutaneous vaccination, mediated by the vast number of antigen presenting cells in the lungs. Potent CD8 T-cell response was elicited by ICMV pulmonary vaccine and established a substantially greater memory population than a soluble vaccine, with 5.2-fold and 4.8-fold higher frequencies of antigen-specific cells in lungs and spleen, at 11 weeks post-priming. T-cells primed by ICMV pulmonary vaccination were also found to be strongly biased toward an effector memory phenotype, and a 5-7-fold higher number of effector memory cells were found in both the local and distal mucosa compared to soluble vaccines. Protection against a viral challenge was observed in animals given ICMV pulmonary vaccine while animals that received subcutaneous ICMV or pulmonary soluble vaccines showed steady weight loss that led to 100% mortality by day 5 post challenge. These particles can be prepared using aqueous solution processing techniques currently used for production of commercial liposomal products. They can also be freeze-dried for long term storage and have shown to retain immunogenicity after 1 month.

Enhanced protection against pulmonary mycobacterial challenge by chitosan‐formulated polyepitope gene vaccine is associated with increased pulmonary secretory IgA and gamma‐interferon+ T cell responses

Induction of local (pulmonary) immunity plays a critical role in preventing dissemination of Mycobacterium tuberculosis (M. tb) during the early infection stage. To induce specific mucosal immunity, chitosan, a natural cationic polysaccharide, was employed as a mucosal gene carrier and complexed with pHSP65pep, our previously constructed multi-epitope gene vaccine, which induces splenic gamma-interferon (IFN-γ)(+) T helper cell 1 responses. The resultant chitosan-pHSP65pep was administered intranasally to BALB/c mice with four doses of 50 μg DNA followed by mycobacterial challenge 4 weeks after the final immunization. It was found that the chitosan formulation significantly induced production of secretory immunoglobulin A (P < 0.05) as determined by measuring its concentrations in lung lavage fluid and enhanced pulmonary CD4(+) and CD8(+) IFN-γ(+) T cell responses (P < 0.001) compared with naked gene vaccine. Improved protection against Mycobacterium bovis bacillus Calmette-Guérin (BCG) challenge was consistently achieved by the chitosan-DNA formulation both as the vaccine alone or in a BCG prime-vaccine boost immunization scenario. Our study shows that mucosal delivery of gene vaccine in a chitosan formulation remarkably enhances specific SIgA concentrations and mucosal IFN-γ(+) T cell response, which correlated positively with immunological protection.

Enhanced protection against pulmonary mycobacterial challenge by chitosan-formulated polyepitope gene vaccine was associated with elevated pulmonary SIgA and IFN-γ+T cell response

Induction of local (pulmonary) immunity plays a critical role in preventing dissemination of Mycobacterium tuberculosis (M. tb) during the early infection stage. To induce specific mucosal immunity, chitosan, a natural cationic polysaccharide, was employed as a mucosal gene carrier and complexed with pHSP65pep, our previously constructed multi-epitope gene vaccine, which induces splenic gamma-interferon (IFN-γ)+ T helper cell 1 responses. The resultant chitosan–pHSP65pep was administered intranasally to BALB/c mice with four doses of 50 μg DNA followed by mycobacterial challenge 4 weeks after the final immunization. It was found that the chitosan formulation significantly induced production of secretory immunoglobulin A (P < 0.05) as determined by measuring its concentrations in lung lavage fluid and enhanced pulmonary CD4+ and CD8+IFN-γ+ T cell responses (P < 0.001) compared with naked gene vaccine. Improved protection against Mycobacterium bovis bacillus Calmette–Guérin (BCG) challenge was consistently achieved by the chitosan–DNA formulation both as the vaccine alone or in a BCG prime-vaccine boost immunization scenario. Our study shows that mucosal delivery of gene vaccine in a chitosan formulation remarkably enhances specific SIgA concentrations and mucosal IFN-γ+ T cell response, which correlated positively with immunological protection.

TARGETING CELLULAR RECEPTORS AS A STRATEGY FOR THE DEVELOPMENT OF NEW GENERATION MUCOSAL VACCINES

Mucosal pathogens are a primary source of infection that require the development of vaccines to induce a specific, protective and long lasting immune respon se. Despite an urgent need, development of effectiv e mucosal immunization strategies has proven difficul t. The literature was reviewed to elucidate cell specific targets that may be utilized to increase t he immunogenicity of protective antigens through mucosal vaccination. Vaccine vehicles such as lipos omes, outer membrane vesicles and Poly Lactic Acid (PLA) nanoparticles serve as antigen delivery systems as well as immuno-stimulatory agents. Even with the advances in mucosal vaccine delivery mechanisms many systems lack specificity and fail to generate a protective immune response. Henc e, targeting extracellular receptors by harnessing antibody specificities, bacterial molecules, or tox ins in conjunction with protective antigens provide s an attractive alternative to conventional vaccine r egiments that may circumvent the need for delivery systems and adjuvants. Fusion proteins targeting M cells, dendritic cells, or lymphocytes, are promising candidates for enhancing antigenic traffi cking across the mucosal membrane and subsequent uptake by antigen presenting cells. Directing anti gens to extracellular receptors of epithelial and immune cells via mucosal immunization provides an attractive model for generating a protective memory immune response that is essential to a succe ssful vaccine regiment.

Quillaja saponaria extract as mucosal adjuvant with chitosan functionalized gold nanoparticles for mucosal vaccine delivery: Stability and immunoefficiency studies

Carrier mediated delivery of vaccines along with adjuvants can possibly address the issue related to oral vaccines like inadequate immune potentiation. In this study, chitosan functionalized gold nanoparticles (CsAuNPs) were used as a carrier for the model antigen tetanus toxoid (TT) along with immunostimulant Quillaja saponaria extract (QS). Physicochemical properties (size, zeta potential, pH value) of formulation were investigated as stability indicating parameters. The synthesized CsAuNPs were spherical in shape, around 40 nm in size, positively charged (around +35 mV) and had TT and QS payload of 65% and 0.01%, respectively. Formulation parameters did not alter the secondary structure of TT, as determined by FTIR, fluorescence and CD spectroscopy. Antigen specificity, determined by an ELISA, was also not compromised. The CsAuNPs conferred protection to TT against gastric hydrolysis as studied in vitro. TT-QS-CsAuNPs induced up to 28-fold immune responses compared to control formulations (TT, TT-QS) after oral administration of formulations in BALB/c mice. The immune responses were quantified by measuring the TT-specific IgG and IgA titers using ELISA. Findings herein demonstrate that co-delivery of TT and QS with functionalized CsAuNPs promotes better systemic and local immune responses and hence can be considered as a sound approach for oral vaccine delivery.

Cell-Penetrating Peptide-Linked Polymers as Carriers for Mucosal Vaccine Delivery

We evaluated the potential of poly(N-vinylacetamide-co-acrylic acid) modified with d-octaarginine, which is a typical cell-penetrating peptide, as a carrier for mucosal vaccine delivery. Mice were nasally inoculated four times every seventh day with PBS containing ovalbumin with or without the d-octaarginine-linked polymer. The polymer enhanced the production of ovalbumin-specific immunoglobulin G (IgG) and secreted immunoglobulin A (IgA) in the serum and the nasal cavity, respectively. Ovalbumin internalized into nasal epithelial cells appeared to stimulate IgA production. Ovalbumin transferred to systemic circulation possibly enhanced IgG production. An equivalent dose of the cholera toxin B subunit (CTB), which was used as a positive control, was superior to the polymer in enhancing antibody production; however, dose escalation of the polymer overcame this disadvantage. A similar immunization profile was also observed when ovalbumin was replaced with influenza virus HA vaccines. The polymer induced a vaccine-specific immune response identical to that induced by CTB, irrespective of the antibody type, when its dose was 10 times that of CTB. Our cell-penetrating peptide-linked polymer is a potential candidate for antigen carriers that induce humoral immunity on the mucosal surface and in systemic circulation when nasally coadministered with antigens.

Adsorption of β-galactosidase of Alicyclobacillus acidocaldarius on wild type and mutants spores of Bacillus subtilis

BackgroundThe Bacillus subtilis spore has long been used as a surface display system with potential applications in a variety of fields ranging from mucosal vaccine delivery, bioremediation and biocatalyst development. More recently, a non-recombinant approach of spore display has been proposed and heterologous proteins adsorbed on the spore surface. We used the well-characterized β-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius as a model to study enzyme adsorption, to analyze whether and how spore-adsorption affects the properties of the enzyme and to improve the efficiency of the process.ResultsWe report that purified β-galactosidase molecules were adsorbed to purified spores of a wild type strain of B. subtilis retaining ca. 50% of their enzymatic activity. Optimal pH and temperature of the enzyme were not altered by the presence of the spore, that protected the adsorbed β-galactosidase from exposure to acidic pH conditions. A collection of mutant strains of B. subtilis lacking a single or several spore coat proteins was compared to the isogenic parental strain for the adsorption efficiency. Mutants with an altered outermost spore layer (crust) were able to adsorb 60-80% of the enzyme, while mutants with a severely altered or totally lacking outer coat adsorbed 100% of the β-galactosidase molecules present in the adsorption reaction.ConclusionOur results indicate that the spore surface structures, the crust and the outer coat layer, have an negative effect on the adhesion of the β-galactosidase. Electrostatic forces, previously suggested as main determinants of spore adsorption, do not seem to play an essential role in the spore-β-galactosidase interaction. The analysis of mutants with altered spore surface has shown that the process of spore adsorption can be improved and has suggested that such improvement has to be based on a better understanding of the spore surface structure. Although the molecular details of spore adsorption have not been fully elucidated, the efficiency of the process and the pH-stability of the adsorbed molecules, together with the well documented robustness and safety of spores of B. subtilis, propose the spore as a novel, non-recombinant system for enzyme display.

Microneedle and mucosal delivery of influenza vaccines

In recent years with the threat of pandemic influenza and other public health needs, alternative vaccination methods other than intramuscular immunization have received great attention. The skin and mucosal surfaces are attractive sites probably because of both noninvasive access to the vaccine delivery and unique immunological responses. Intradermal vaccines using a microinjection system (BD SoluviaTM) and intranasal vaccines (FluMist®) are licensed. As a new vaccination method, solid microneedles have been developed using a simple device that may be suitable for self-administration. Because coated microneedle influenza vaccines are administered in the solid state, developing formulations maintaining the stability of influenza vaccines is an important issue to be considered. Marketable microneedle devices and clinical trials remain to be developed. Other alternative mucosal routes such as oral and intranasal delivery systems are also attractive for inducing cross-protective mucosal immunity, but effective non-live mucosal vaccines remain to be developed.

Mucosal Vaccine Design and Delivery

Mucosal surfaces are a major portal of entry for many human pathogens that are the cause of infectious diseases worldwide. Vaccines capable of eliciting mucosal immune responses can fortify defenses at mucosal front lines and protect against infection. However, most licensed vaccines are administered parenterally and fail to elicit protective mucosal immunity. Immunization by mucosal routes may be more effective at inducing protective immunity against mucosal pathogens at their sites of entry. Recent advances in our understanding of mucosal immunity and identification of correlates of protective immunity against specific mucosal pathogens have renewed interest in the development of mucosal vaccines. Efforts have focused on efficient delivery of vaccine antigens to mucosal sites that facilitate uptake by local antigen-presenting cells to generate protective mucosal immune responses. Discovery of safe and effective mucosal adjuvants are also being sought to enhance the magnitude and quality of the protective immune response.

Efficacy of intranasal and spray delivery of adjuvanted live vaccine against infectious bronchitis virus in experimentally infected poultry

Live vaccines are widely used in the avian industry. Such vaccines can be either injected or delivered on animal mucosa and are usually not adjuvanted. In this study we show that live vaccines efficacy can be improved by formulation with adjuvants in a model of mucosal delivery of live infectious bronchitis vaccine in chicken. Three adjuvant technologies have been tested using intranasal and spray delivery methods to poultry. Those technologies are water in oil in water emulsion, nanoparticles and polymer adjuvants. Intranasal delivery of polymer and nanoparticles adjuvanted live vaccines improved significantly the antibody titer and protection to challenge observed compared to a commercial non-adjuvanted reference. Moreover, spray delivery of the polymer adjuvanted vaccine showed a significantly higher protection compared to the non-adjuvanted reference. Our data demonstrates that the use of MontanideTM adjuvants in the formulation of live poultry vaccines for mucosal delivery can confer to vaccinated animals a significantly improved protection against pathogens.

Mucosal Immune System and M Cell-targeting Strategies for Oral Mucosal Vaccination

Vaccination is one of the most effective methods available to prevent infectious diseases. Mucosa, which are exposed to heavy loads of commensal and pathogenic microorganisms, are one of the first areas where infections are established, and therefore have frontline status in immunity, making mucosa ideal sites for vaccine application. Moreover, vaccination through the mucosal immune system could induce effective systemic immune responses together with mucosal immunity in contrast to parenteral vaccination, which is a poor inducer of effective immunity at mucosal surfaces. Among mucosal vaccines, oral mucosal vaccines have the advantages of ease and low cost of vaccine administration. The oral mucosal immune system, however, is generally recognized as poorly immunogenic due to the frequent induction of tolerance against orally-introduced antigens. Consequently, a prerequisite for successful mucosal vaccination is that the orally introduced antigen should be transported across the mucosal surface into the mucosa-associated lymphoid tissue (MALT). In particular, M cells are responsible for antigen uptake into MALT, and the rapid and effective transcytotic activity of M cells makes them an attractive target for mucosal vaccine delivery, although simple transport of the antigen into M cells does not guarantee the induction of specific immune responses. Consequently, development of mucosal vaccine adjuvants based on an understanding of the biology of M cells has attracted much research interest. Here, we review the characteristics of the oral mucosal immune system and delineate strategies to design effective oral mucosal vaccines with an emphasis on mucosal vaccine adjuvants.