M-cell Targeting Research Articles

Quality by design (QbD) approach-based development of optimized nanocarrier to achieve quality target product profile (QTPP)-targeted lymphatic delivery

Background. Insulin, commonly used for diabetes treatment, needs better ways to improve its effectiveness and safety due to its challenges with poor permeability and stability. Various system has been developed for oral peptide delivery. The non-targeted system can prevent gastric and enzymatic degradation of peptides but cannot increase the bulk transport of peptides across the membrane. However, the non-selectivity is the limitation of the existing system. Numerous carbohydrate-binding receptors overexpressed on intestinal macrophage cells (M-cells) of gut-associated lymphoid tissue. It is the most desirable site for receptor-mediated endocytosis and lymphatic drug delivery of peptides. Objective. The prime objective of the study was to fabricate mannose ligand conjugated nanoparticles (MNPs) employing a quality-by-design approach to address permeability challenges after oral administration. Herein, the study’s secondary objective of this study is to identify the influencing factor for producing quality products. Considering this objective, the Lymphatic uptake of NPs was selected as a quality target product profile (QTPP), and a systematic study was conducted to identify the critical formulation attributes (CFAs) and critical process parameters (CPP) influencing critical quality attributes (CQAs). Mannosylated Chitosan concentrations (MCs) and TPP concentrations were identified as CFAs, and stirring speed was identified as CPP. Methods. MNPs were prepared by the inotropic gelation method and filled into the enteric-coated capsule to protect from acidic environments. The effect of CFAs and CPP on responses like particle size (X) and entrapment (Y) was observed by Box-Behnken design (BBD). ANOVA statistically evaluated the result to confirm a significant level (p < 0.05). The optimal conditions of NPs were obtained by constructing an overlay plot and determining the desirability value. HPLC and zeta-seizer analysis characterized the lyophilized NPs. Cell-line studies were performed to confirm the safety and M-cell targeting of NPs to enhance Insulin oral bioavailability. Results. The morphology of NPs was revealed by SEM. The developed NPs showed a nearly oval shape with the average size, surface potential, and % drug entrapment were 245.52 ± 3.37 nm, 22.12 ± 2.13 mV, and 76.15 ± 1.3%, respectively. MTT assay result exhibited that MNPs safe and Confocal imaging inference that NPs selectively uptake by the M-cell. Conclusion. BBD experimental design enables the effective formulation of optimized NPs. The statistical analysis estimated a clear assessment of the significance of the process and formulation variable. Cell line study confirms that NPs are safe and effectively uptake by the cell.

Application of M-cell targeting ligand Co4B enhances Ag-specific mucosal and systemic immunity and protection against MERS-CoV infection after intranasal immunization

Abstract Middle East respiratory syndrome is associated with severe pneumonia and poses a global public health threat. Authorized vaccines against the causative agent of MERS-CoV are urgently needed. In order to optimize the efficacy of recombinant vaccines, adjuvants should be required. Therefore, we previously identified a ligand Co4B as an adjuvant to enhance Ag delivery to the nasal mucosa immune compartment and promote Ag-specific mucosal and systemic immune responses via intranasal administration against A. pleuropneumoniae infection in a murine model. Based on the pathogenesis of MERS-CoV which infects host following respiratory route; thus, we generated a recombinant protein conjugated with Co4B to the MERS-CoV spike protein receptor-binding domain (S-RBD) to develop mucosal subunit vaccine candidate. Ag-specific mucosal IgA and systemic IgG antibody responses, together with virus-neutralizing capacities, were highly elicited in C57BL/6 and human dipeptidyl peptidase 4-transgenic (hDPP4-Tg) mice intranasally immunized with Co4B-conjugated S-RBD compared to those immunized with originally S-RBD. Ag-specific T cell-mediated immunity was also induced in the spleen and lungs of mice intranasally immunized with Co4B-conjugated S-RBD. Intranasal immunization of hDPP4-Tg mice with Co4B-conjugated S-RBD prominently reduced immune cell infiltration into the tissues of virus-challenged mice. Finally, Co4B-conjugated S-RBD-immunized mice demonstrated were improved protection against infection, more likely to survive, and exhibited less body weight loss. Collectively, our results suggest that Co4B-conjugated S-RBD could be used as an improved subunit vaccine candidate against MERS-CoV infection. This work was supported by the Ministry of Science and ICT No. 2020K1A4A7A02095058 and the NRF funded by the Ministry of Education No. 2019R1I1A3A01062224. Ms. Y. L. Yang and Ms. B.-H. Cho were supported by the BK21 FOUR program of the Department of Bioactive Material Sciences.

Engineered Nanoparticles inside a Microparticle Oral System for Enhanced Mucosal and Systemic Immunity.

Antigen delivery through an oral route requires overcoming multiple challenges, including gastrointestinal enzymes, mucus, and epithelial tight junctions. Although each barrier has a crucial role in determining the final efficiency of the oral vaccination, transcytosis of antigens through follicle-associated epithelium (FAE) represents a major challenge. Most of the research is focused on delivering an antigen to the M-cell for FAE transcytosis because M-cells can easily transport the antigen from the luminal site. However, the fact is that the M-cell population is less than 1% of the total gastrointestinal cells, and most of the oral vaccines have failed to show any effect in clinical trials. To challenge the current dogma of M-cell targeting, in this study, we designed a novel tandem peptide with a FAE-targeting peptide at the front position and a cell-penetrating peptide at the back position. The tandem peptide was attached to a smart delivery system, which overcomes the enzymatic barrier and the mucosal barrier. The result showed that the engineered system could target the FAE (enterocytes and M-cells) and successfully penetrate the enterocytes to reach the dendritic cells located at the subepithelium dome. There was successful maturation and activation of dendritic cells in vitro confirmed by a significant increase in maturation markers such as CD40, CD86, presentation marker MHC I, and proinflammatory cytokines (TNF-α, IL-6, and IL-10). The in vivo results showed a high production of CD4+ T-lymphocytes (helper T-cell) and a significantly higher production of CD8+ T-lymphocytes (killer T-cell). Finally, the production of mucosal immunity (IgA) in the trachea, intestine, and fecal extracts and systemic immunity (IgG, IgG1, and IgG2a) was successfully confirmed. To the best of our knowledge, this is the first study that designed a novel tandem peptide to target the FAE, which includes M-cells and enterocytes rather than M-cell targeting and showed that a significant induction of both the mucosal and systemic immune response was achieved compared to M-cell targeting.

Intranasal immunization with a Middle East respiratory syndrome-coronavirus antigen conjugated to the M-cell targeting ligand Co4B enhances antigen-specific mucosal and systemic immunity and protects against infection

Middle East respiratory syndrome (MERS) is a threat to public health worldwide. A vaccine against the causative agent of MERS, MERS-coronavirus (MERS-CoV), is urgently needed. We previously identified a peptide ligand, Co4B, which can enhance antigen (Ag) delivery to the nasal mucosa and promote Ag-specific mucosal and systemic immune responses following intranasal immunization. MERS-CoV infects via the respiratory route; thus, we conjugated the Co4B ligand to the MERS-CoV spike protein receptor-binding domain (S-RBD), and used this to intranasally immunize C57BL/6 and human dipeptidyl peptidase 4-transgenic (hDPP4-Tg) mice. Ag-specific mucosal immunoglobulin (Ig) A and systemic IgG, together with virus-neutralizing activities, were highly induced in mice immunized with Co4B-conjugated S-RBD (S-RBD-Co4B) compared to those immunized with unconjugated S-RBD. Ag-specific T cell-mediated immunity was also induced in the spleen and lungs of mice intranasally immunized with S-RBD-Co4B. Intranasal immunization of hDPP4-Tg mice with S-RBD-Co4B reduced immune cell infiltration into the tissues of virus-challenged mice. Finally, S-RBD-Co4B-immunized mice exhibited were better protected against infection, more likely to survive, and exhibited less body weight loss. Collectively, our results suggest that S-RBD-Co4B could be used as an intranasal vaccine candidate against MERS-CoV infection.

Development of an M cell targeted nanocomposite system for effective oral protein delivery: preparation, in vitro and in vivo characterization

BackgroundThere is a strong need for non-invasive and patient-friendly delivery systems of protein drugs for long-term therapy. However, oral delivery of protein drugs is a big challenge due to many barriers including instability in the gastrointestinal (GI) tract and low permeability. To overcome the absorption barriers in GI tract and improve the patient compliance, this study aimed to develop an M cell targeted-nanocomposite delivery system of protein drugs.ResultsAn aminoclay-protein core complex (AC-Ins) was prepared by using insulin as a model protein and then sequentially coated with Ulex europaeus agglutinin 1 (UEA-1) for M-cell targeting and the pH sensitive polymer, Eudragit® L100 (EUAC-Ins). All nanoparticles were obtained with a high entrapment efficiency (> 90%) and their structural characteristics were confirmed by Fourier transform-infrared spectroscopy, energy dispersive X-ray spectroscopy, and circular dichroism. Among the developed nanoparticles, EUAC-Ins effectively suppressed drug release at pH 1.2, while rapidly released drugs at pH 6.8 due to dissolution of the outer coating layer. The conformational stability of insulin entrapped in EUAC-Ins was well maintained in the presence of proteolytic enzymes. Compared to free insulin, EUAC-Ins increased the membrane transport of insulin by 4.4-fold in M cells. In parallel, oral administration of EUAC-Ins in mice enhanced insulin uptake by 4.1-fold in the intestinal Peyer’s patches and 2.6-fold in intestinal epithelium tissues with normal villi, compared to free insulin. Orally administered EUAC-Ins decreased significantly the blood glucose level in diabetic mice, while the effect of oral insulin solution was negligible.ConclusionAn M cell targeted-ternary nanocomposite system obtained by dual coating of the aminoclay-protein core complex with UEA-1 and a pH dependent polymer is promising as an effective oral protein delivery carrier.

Present Scenario of M-Cell Targeting Ligands for Oral Mucosal Immunization.

The immune system plays an important role in the prevention of infection and forms the first line of defense against pathogen attack. Delivering of antigen through mucosal route may elicit mucosal immune system as the mucosal surface is the most common site of pathogen entry. Mucosal immune system will be capable to counter pathogen at mucosal surface. Oral mucosal immunization opens the ways to deliver antigens at gut-associated lymphoid tissue. This can elicit both local and systemic immune response. Mucosal vaccines are economical, highly accessible, non parenteral delivery and capacity to produce mass immunization at the time of pandemics. To deliver antigens on the mucosal surface of the gastrointestinal tract, the immune system relies on specialized epithelial cell i.e. Microfold (M)-cell. An approach to exploit the targeting specific receptors on M-cell for entry of antigens has made a breakthrough in vaccine development. In this review, various strategies have been discussed for the possible entry of antigens through M-cells and an approach to increase the uptake and efficacy of vaccines for oral mucosal immunization.

Oral Gavage Delivery of PR8 Antigen with β-Glucan-Conjugated GRGDS Carrier to Enhance M-Cell Targeting Ability and Induce Immunity.

Oral gavage is known as one of most convenient routes for therapeutic administration in comparison with other available routes such as intravenous, intra muscular, suppository, etc. An oral vaccine delivery system has additional potential as it may provide a convenient way to prevent infectious diseases by introducing optimum immunization in mucus. Although oral vaccine delivery has attracted tremendous interest in vaccine delivery research, various limitations have prevented its rate of progress up to the level that was initially expected. However, the major problems of oral vaccine delivery are vaccine instability and lack of absorbability, resulting from degradation of the sophisticated antigens in the acidic medium in the stomach. In order to obtain adequate microfold-cell (M-cell) targeting and uptake, the therapeutic material is required to pass through the stomach and reach the small intestine without degradation. In this project, we have introduced a conjugate of β-glucan and Glycine-Arginine-Glycine-Aspartic acid-Serine (GRGDS) that is effective for simultaneous protection of the antigen (PR8) and M-cell targeting. According to the experimental results, the cationic β-glucan-GRGDS conjugate can encapsulate a certain amount of anionic PR8 through electrostatic interaction, which forms nanoparticles with a range of diameter of 200-250 nm. Also, the PR8 incorporated nanoparticles showed high cell viability and stability in diverse environments. Finally, excellent M-cell targeting ability was verified in an in vitro M-cell model. Most importantly, the in vivo test obviously demonstrated the superiority of this system, which significantly increases antibody concentration in serum, intestine, and mucus as measured 21 days after immunization.

Evaluation of microparticulate ovarian cancer vaccine via transdermal route of delivery

Ovarian cancer is the fifth most commonly occurring malignancy in women, with the highest mortality rate among all the gynecological tumors. Microparticulate vaccine can serve as an immunotherapeutic approach with a promising antigenic delivery system without a need for conventional adjuvants. In this study, a microparticulate vaccine using whole cell lysate of a murine ovarian cancer cell line, ID8 was prepared by spray drying. Further, the effect of interleukins (ILs) such as IL-2 and IL-12 was evaluated in a separate study group by administering them with vaccine particles to enhance the immune response. The vaccine microparticles were administered to C57BL/6 female mice via transdermal alone and in combination with the oral route. The transdermal vaccine was delivered using a metallic microneedle device, AdminPen™. Orally administered microparticles also included an M-cell targeting ligand, Aleuria aurantia lectin, to enhance the targeted uptake from microfold cells (M-cells) in Peyer's patches of small intestine. In case of combination of routes, mice were given 5 transdermal doses and 5 oral doses administered alternatively, beginning with transdermal dose. At the end of vaccination, mice were challenged with live tumor cells. Vaccine alone resulted in around 1.5 times tumor suppression in case of transdermal and combination of routes at the end of 15th week when compared to controls. Inclusion of interleukins resulted in 3 times tumor suppression when administered with transdermal vaccine and around 9 times tumor suppression for the combination route of delivery in comparison to controls. These results were further potentiated by serum IgG, IgG1 and IgG2a titers. Moreover, CD8+ T-cell, CD4+ T-cell and NK (natural killer) cell populations in splenocytes were elevated in case of vaccinated mice. Thus, vaccine microparticles could trigger humoral as well as cellular immune response when administered transdermally and via combination of route of delivery. However overall, vaccine administered with interleukins, via combination of route, was found to be the most efficacious to suppress the tumor growth and lead to a protective immune response.

Formulation and evaluation of oral microparticulate ovarian cancer vaccines

Ovarian cancer is the fifth most leading cause of cancer related deaths in women in the US. Customized immunotherapeutic strategies may serve as an alternative method to control the recurrence or progression of ovarian cancer and to avoid severe adverse effects of chemotherapy. In this study, a microparticulate vaccine using whole cell lysate of a murine ovarian cancer cell line, ID8 was prepared with the use of a spray dryer. These particles were designed for oral delivery using enteric polymers such as methacrylic copolymer, Eudragit® FS30D and hydroxyl propyl methyl cellulose acetate succinate. These particles were targeted for uptake via microfold cell (M-cell) in Peyer's patches of small intestine using M-cell targeting ligand, Aleuria aurantia lectin. The interleukins (ILs) such as IL-2 and IL-12 were added to the vaccine formulation to further enhance the immune response. The particles obtained were of 1.58±0.62μm size with a charge of 12.48±2.32mV. The vaccine efficacy was evaluated by administering the particles via oral route to C57BL/6 female mice. At the end of vaccination, mice were challenged with live tumor cells. Vaccinated mice showed significant (around six-fold) retardation of tumor volume in comparison to non-vaccinated animals for 3 weeks after the tumor challenge (p<0.001). The serum IgG antibody levels were found to be elevated in case of vaccinated animals in comparison to non-vaccinated group (p<0.05). Analysis of IgG1 titers (indicative of Th2 response) and IgG2a titers (indicative of Th1 response) showed a mixed Th1 and Th2 immune response in case vaccine alone and Th2 response in case of vaccine with interleukins group. Moreover, CD8+ T-cell, CD4+ T-cell and B-cell populations in different lymphatic organs were elevated in case of vaccinated mice. Thus, whole cell lysate vaccine microparticles formulated by spray drying could trigger humoral as well as cellular immune response when administered orally. Such vaccine could potentially be an effective treatment for patients with residual tumor or high tumor-relapse probability.

Avian reovirus sigma C enhances the mucosal and systemic immune responses elicited by antigen-conjugated lactic acid bacteria

Mucosal surfaces are common sites of pathogen colonization/entry. Effective mucosal immunity by vaccination should provide protection at this primary infection site. Our aim was to develop a new vaccination strategy that elicits a mucosal immune response. A new strain of Enterococcus faecium, a non pathogenic lactic acid bacteria (LAB) with strong cell adhesion ability, was identified and used as a vaccine vector to deliver two model antigens. Specifically, sigma (σ) C protein of avian reovirus (ARV), a functional homolog of mammalian reovirus σ1 protein and responsible for M-cell targeting, was administered together with a subfragment of the spike protein of infectious bronchitis virus (IBV). Next, the effect of immunization route on the immune response was assessed by delivering the antigens via the LAB strain. Intranasal (IN) immunization induced stronger humoral responses than intragastic (IG) immunization. IN immunization produced antigen specific IgA both systemically and in the lungs. A higher IgA titer was induced by the LAB with ARV σC protein attached. Moreover, the serum of mice immunized with LAB displaying divalent antigens had much stronger immune reactivity against ARV σC protein compared to IBV-S1. Our results indicate that ARV σC protein delivered by LAB via the IN route elicits strong mucosal immunity. A needle-free delivery approach is a convenient and cost effective method of vaccine administration, especially for respiratory infections in economic animals. Furthermore, ARV σC, a strong immunogen of ARV, may be able to serve as an immunoenhancer for other vaccines, especially avian vaccines.

105 M-Cell Targeting by Neuraminidase Functionalized Microparticles for Future Application in Oral Immunotherapy

BackgroundRecently, we demonstrated in an experimental mouse study that mucosal M-cell targeting with Aleuria aurantia lectin (AAL) coated Poly(D,L-lactide-co-glycolide) (PLGA) microspheres represents a promising oral treatment approach in IgE mediated allergy. Due to its structural similarities with AAL we aimed to assess Neuraminidase (NA) from Vibrio cholerae as a novel M-cell specific targeters and compared its properties to AAL and wheat germ agglutinin (WGA) representing 2 plant lectins, which target either M-cells or epithelial cells, respectively.MethodsThe resistance against gastric digestion of NA, AAL and WGA was analyzed in simulated gastric fluid (SGF) experiments. Intestinal epithelial binding was determined using the colon carcinoma cell line Caco2, which represents a well established model for the human intestinal epithelium. Binding specificity was evaluated by inhibition experiments by incubating Caco2 cells with Biotin-labeled NA, AAL or WGA, after preincubation with a-L fucose, monoganglioside (GM1) or N,N′,N″-triacetyl-chitotriose (TCT). The stimulatory effects of the targeting substances on the intestinal microenvironment were investigated by cytokine read-out experiments in real-time PCR. Further, the transeptihelial uptake of NA-, AAL- or WGA-functionalized fluospheres was evaluated in a human M-cell co-culture model.ResultsAll 3 targeters were stable up to 180 minutes in SGF, indicating their suitability for oral application. The binding partners were a-L fucose for AAL and TCT for WGA, whereas NA interacts with intestinal epithelial cells via a-L fucose and additionally GM1. NA skewed the cytokine production by inducing a 2-fold increase of the Th1 cytokine IFNg after 60 minutes, whereas AAL decreased the overall cytokine expression. In a human M-cell co-culture model, a higher transepithelial transport rate of fluospheres coated with NA and AAL was observed as compared to WGA and plain particles.ConclusionsNA specifically targets M-cells via a-L fucose and additionally GM1 and, thus, increases the transepithelial transport of NA coated particles. Due to the immunomodulatory capacity on intestinal epithelial cells, NA functionalized microspheres may represent a promising M-cell specific targeting approach for oral immunotherapy.

Oral Mucosal Immunization: Recent Advancement and Future Prospects

To induce immunity against infectious diseases, vaccination at mucosal sites represents one of the major inventions in the field of healthcare. Since decades, various mucosal routes including nasal, oral and rectal have been exploited but the oral route is more patient compliance better than others due to its astonishing advantages as mucosal immunity generated by oral vaccines serves as a strong first line of defense. Proper stimulation of the cellular and humoral immune response is vital for the effective protection of mucosal surfaces against invasion of infectious pathogens. Recently, colloidal carriers like liposomes, niosomes, bilosomes, virosomes, ISCOMS, archaeosomes, chochleates, micronanoparticles and dendrimers have shown great attention for delivery of antigenic materials via gastrointestinal route. Moreover, delivery of mucosal adjuvant in combination with these carriers has also been proved as a successful strategy to enhance the stimulation of mucosal immune system. Further, selective and specific delivery of antigen to Peyers patch or any other antigen presenting cells are appropriate for efficient uptake of antigen to and induce enhanced immune responses. In this paper, we have reviewed various carrier systems, immuno-adjuvants and targeting ligands to deliver vaccines efficiently through oral route providing non invasive method of immunization. Some of these have also been evaluated in the pre clinical and clinical trials. In future, these studies could help in developing vaccines at commercial scale having potential of eradicating life threatening infectious diseases. Keywords: Oral immunization, delivery systems, antigen, M-cell targeting, adjuvant

Microencapsulation of Vaccine Antigens and Adjuvants for Mucosal Targeting

Delivery of vaccine antigens that can trigger potent mucosal immune response is one of the effective strategies to overcome a wide array of infectious diseases. Microencapsulation of vaccine antigens with Poly(lactide-co-glycolic acids) (PLGA), an FDA approved biodegradable polymer, has been investigated for targeted M-cell uptake. While PLGA possesses many attractive properties, a successful PLGA based mucosal-targeted vaccine has yet to be introduced. This review focuses on the physiochemical properties important in the preparation of antigen-loaded PLGA microparticles, properties that influence M-cell specific uptake, and the induction of effective immune responses. In addition, a possible role of microparticle properties in immune adjuvant activity is discussed. A careful consideration of these factors may yet lead to the development of an effective needle-free mucosal vaccine using polymer microparticles. Keywords: M-cell targeting, PLGA, microparticles

Targeting of whole killed bacteria to gastrointestinal M-cells induces humoral immunity in the female reproductive tract

Recently, we demonstrated that oral delivery of whole killed bacteria, when agglutinated by an M-cell targeting lectin, resulted in an enhanced systemic and mucosal antibody response, as well as a protective immunity, against the gut pathogens Helicobacter pylori and Campylobacter jejuni. Importantly, this protection was achieved without the addition of exogenous adjuvant. Here, in this addendum, we extend this initial study by reporting on the vaginal antibody response induced by these vaccinations. These data show that the targeting of M-cells within the gastrointestinal tract also induces the secretion of antigen-specific antibodies (IgG and IgA) at a distal mucosal site, namely the vaginal mucosa. This observation raises the possibility that oral delivery of a whole, killed bacteria vaccine that target intestinal M-cells could potentially provide a strategy for inducing protective immunity against pathogenic bacteria that infect mucosal sites outside the gastrointestinal tract.

M-Cell Targeting of Whole Killed Bacteria Induces Protective Immunity against Gastrointestinal Pathogens

As the majority of human pathogens infect via a mucosal surface, delivery of killed vaccines by mucosal routes could potentially improve protection against many such organisms. Our ability to develop effective killed mucosal vaccines is inhibited by a lack of adjuvants that are safe and effective in humans. The Ulex europaeus agglutinin I (UEA-I) lectin specifically binds M cells lining the murine gastrointestinal tract. We explored the potential for M-cell-targeted vaccination of whole, killed Helicobacter pylori, the main causative agent of peptic ulcer disease and gastric cancer, and Campylobacter jejuni, the most common cause of diarrhea. Oral delivery of UEA-I-agglutinated H. pylori or C. jejuni induced a significant increase in both serum and intestinal antibody levels. This elevated response (i) required the use of whole bacteria, as it did not occur with lysate; (ii) was not mediated by formation of particulate clumps, as agglutination with a lectin with a different glycan specificity had no effect; and (iii) was not due to lectin-mediated, nonspecific immunostimulatory activity, as UEA-I codelivery with nonagglutinated bacteria did not enhance the response. Vaccination with UEA-I-agglutinated, killed whole H. pylori induced a protective response against subsequent live challenge that was as effective as that induced by cholera toxin adjuvant. Moreover, vaccination against C. jejuni by this approach resulted in complete protection against challenge in almost all animals. We believe that this is the first demonstration that targeting of whole killed bacteria to mucosal M cells can induce protective immunity without the addition of an immunostimulatory adjuvant.

Ovalbumin-Protein σ1 M-Cell Targeting Facilitates Oral Tolerance With Reduction of Antigen-Specific CD4+ T Cells

The follicle-associated epithelium (FAE) plays key roles in antigen uptake and subsequent induction of mucosal immunity. In this study, we examined whether M-cell targeting using a protein antigen (Ag) delivery system would induce oral tolerance instead of enhancement of Ag-specific mucosal antibody (Ab) responses. Mice were fed different doses of a recombinant protein sigma 1 of reovirus genetically conjugated to ovalbumin (OVA-psigma1), psigma1 only, or phosphate-buffered saline (PBS) before oral challenge with OVA plus cholera toxin as mucosal adjuvant. OVA-specific Ab and CD4-positive (CD4(+)) T-cell responses were determined. A low dose of OVA-psigma1 reduced anti-OVA Ab and CD4(+) T-cell responses in both mucosal and systemic lymphoid tissues. OVA/MHC I-A(d) tetramer staining showed that the numbers of OVA-specific CD4(+) T cells were significantly reduced in lamina propria of mice fed OVA-psigma1 than those fed psigma1 only or PBS only. In fact, Foxp3 expressing CD25(+) CD4(+) T cells were markedly increased in this tissue. Nonetheless, CD25(+) CD4(+) T cells from the spleen, mesenteric lymph nodes, and Peyer's patches of orally tolerized mice showed increased transforming growth factor beta1 (TGF-beta1) and interleukin-10 (IL-10) production compared with nontolerized mice. These results show that an FAE M-cell targeting protein Ag delivery system facilitates oral tolerance induction because of a reduction in Ag-specific CD4(+) T cells and increased levels of TGF-beta1 and IL-10 producing, CD25(+) CD4(+) regulatory T cells in both systemic and mucosal lymphoid tissues.

Implication of nanoparticles/microparticles in mucosal vaccine delivery

Although polymeric nanoparticles/microparticles are well established for the mucosal administration of conventional drugs, they have not yet been developed commercially for vaccine delivery. The limitation of the mucosal (particularly oral) route of delivery, including low pH, gastric enzymes, rapid transit and poor absorption of large molecules, has made mucosal vaccine delivery challenging. Nevertheless, several polymeric delivery systems for mucosal vaccine delivery are currently being evaluated. The polymer-based approaches are designed to protect the antigen in the gut, to target the antigen to the gut-associated lymphoid tissue or to increase the residence time of the antigen in the gut through bioadhesion. M-cell targeting is a potential approach for mucosal vaccine delivery, which can be achieved using M-cell-specific lectins, microbial adhesins or immunoglobulins. While many hurdles must be overcome before targeted mucosal vaccine delivery becomes a practical reality, this is a potential area of research that has important implications for future vaccine development. This review comprises various aspects that could be decisive in the development of polymer based mucosal vaccine delivery systems.

M-cell surface beta1 integrin expression and invasin-mediated targeting of Yersinia pseudotuberculosis to mouse Peyer's patch M cells.

Quantitative analysis of Yersinia pseudotuberculosis infection of murine gut loops revealed that significantly more wild-type bacteria associated with Peyer's patch M cells than with dome enterocytes or goblet cells. An invasin-deficient mutant was significantly attenuated for M-cell invasion, while beta1 integrin expression was demonstrated in the apical membranes of M cells but not enterocytes. M-cell targeting by Yersinia pseudotuberculosis in vivo may, therefore, be mediated primarily by the interaction of invasin with cell surface beta1 integrins.

Antigen transport by intestinal M cells

M cells play a critical role in the generation of protective secretory immunity against intestinal pathogens. For this reason, they have become an important focus for studies on oral vaccine development. In this review, we outline the work on M cells reported in the past year, with particular emphasis on findings that have relevance to vaccine development. The most important findings in this regard are the demonstration of antibody-enhanced M-cell targeting and the analysis of increased M-cell numbers in response to Salmonella infection.