Enhancing Oral Vaccine Potency by Targeting Intestinal M Cells

Download PDF České info

The immune system in the gastrointestinal tract plays a crucial role in the control of infection, as it constitutes the first line of defense against mucosal pathogens. The attractive features of oral immunization have led to the exploration of a variety of oral delivery systems. However, none of these oral delivery systems have been applied to existing commercial vaccines. To overcome this, a new generation of oral vaccine delivery systems that target antigens to gut-associated lymphoid tissue is required. One promising approach is to exploit the potential of microfold (M) cells by mimicking the entry of pathogens into these cells. Targeting specific receptors on the apical surface of M cells might enhance the entry of antigens, initiating the immune response and consequently leading to protection against mucosal pathogens. In this article, we briefly review the challenges associated with current oral vaccine delivery systems and discuss strategies that might potentially target mouse and human intestinal M cells.

Published in the journal: Enhancing Oral Vaccine Potency by Targeting Intestinal M Cells. PLoS Pathog 6(11): e32767. doi:10.1371/journal.ppat.1001147
Category: Review
doi: https://doi.org/10.1371/journal.ppat.1001147

Summary

Advantages and Challenges Surrounding Mucosal Vaccines

The mucosal immune system is a critical line of defense against infectious diseases, as the majority of infections are initiated at mucosal sites [1]–[3]. Therefore, the induction of specific immune responses at mucosal sites may be able to control infections at their point of entry into the body. Over the past few decades, several candidate vaccines have been designed and tested by various mucosal routes in pre-clinical or clinical trials. Although the mucosal immune system comprises several anatomically remote and functionally distinct compartments, it is firmly established that the oral ingestion or intranasal administration of antigens induces humoral and cellular responses not only at the site of antigen exposure but also in other mucosal compartments [4], [5]. This is due to the dissemination of antigen-sensitized precursor B and T lymphocytes from the inductive (e.g., intestinal Peyer's patches) to the effector sites such as the above mentioned glands. However, not all inductive sites display comparable ability to induce equal responses at all effector sites. Despite several advantages, as compared to systemic injections, the delivery of vaccines by mucosal routes, particularly through the genitals or rectum, has not been shown to be very practical in human trials [6]–[8]. In addition, it is hard to administer a mucosal vaccine through the genital tract, as the immunological features of the female reproductive tract, in particular, alter dramatically in response to hormonal fluctuations during the menstrual cycle [9]–[11]. In addition, both male and female genital tracts lack inductive mucosal sites analogous to intestinal Peyer's patches [12]. Furthermore, rectal vaccinations have been shown to induce only modest and localized immune responses, and are not very effective in larger animals and humans [13], [14]. The pitfalls in quantifying effector cells in rectal tissues, combined with the intricacies of the inoculation route, are some other major challenges associated with rectal immunization. Therefore, in order to advance a mucosal vaccine for human use, the routes of administration appear to be limited to oral and nasal administration.

Nasally delivered vaccines are easy to administer and have been shown to be more promising for inducing both mucosal as well as systemic immune responses [15]–[17]. It should be stressed that the immune system of the upper respiratory tract (nasal cavity, oropharynx, trachea, and large bronchi) and lower respiratory tract (bronchioli and alveoli) display marked differences with respect to the dominance of Ig isotypes and induction of humoral immune responses. While the induction of dominant IgA responses in the upper respiratory tract is of importance in the protection at this locale, the lower respiratory tract is the domain of antibodies, of the IgG isotype of circulatory origin. Consequently, systemic immunizations with, for example, pneumococcal polysaccharide vaccines, induce protective immune responses. A nasal spray influenza vaccine (FluMist) containing live attenuated influenza has been approved for human use since 2003 [18], [19]. In an HIV study, macaques that were intranasally vaccinated with SHIV-capturing nanospheres demonstrated elevated levels of IgA and IgG antibodies [20]. Additionally, these vaccinated macaques showed a higher frequency of CD4 +T cells and lower viral loads compared to control macaques after a SHIV challenge. However, two human clinical trials involving nasal administration of HIV-1-derived antigens were recently terminated due to safety concerns. The potential for side effects such as Bell's palsy and damage to the olfactory nerves and the nasal epithelium have been cause for concern; however, these side effects could have occurred due to the use of highly reactogenic adjuvants and not because of the route of administration [21]–[23]. The possibility of such side effects, and the reason that the gastrointestinal (GI) tract is the first line of defense against mucosal pathogens, has led many scientists to pursue oral vaccination. The advantages and disadvantages of each route of mucosal immunization are summarized in Table 1. In this article, the advantages, challenges, and pitfalls with this route of vaccination are addressed. We also briefly review current options for oral delivery systems and approaches that have been explored to improve the uptake of potential vaccines. Oral vaccines have the ability to induce both mucosal and systemic immune responses and are safer, easier to administer, and do not require sterile needles and syringes [24]–[27]. Therefore, oral vaccines could more easily meet the needs of affected people in developing countries, where access to trained medical professionals is frequently limited. Although oral vaccines have several attractive features, the limited numbers of approved oral vaccines attest to the challenges associated with mucosal vaccine design. Studies involving oral vaccine use have been limited due to several challenges, such as difficulties in the collection and processing of external secretions, a lack of standardized assays, the induction of tolerance, the stability of antigens in the harsh conditions of the GI tract, and the antigen–microbial interactions that are continuously occurring in the large intestine [28], [29]. It is for these reasons that only a limited number of oral vaccines are currently licensed, compared to many parenteral vaccines.

**Tab. 1. Advantages and Disadvantages of Each Route of Mucosal Immunization Is Summarized.**

Oral Vaccine Delivery Systems

Recombinant or attenuated strains of various bacteria such as Salmonella, Escherichia coli, Listeria, Shigella, and Lactobacilli have been used as a vectors to deliver antigens into the gut-associated lymphoid tissue (GALT) [30]–[35]. While some interesting results have been reported for these oral delivery systems, immune responses against the vectors eventually predominated over time [36], [37]. In addition, glycosylated antigens cannot be produced in bacteria [38]. Furthermore, over 10¹⁴ microorganisms of >20,000 species reside in the large intestine [43]. Such a large competing population would greatly diminish the chances of colonization and subsequent induction of a vigorous immune response through such vector microorganisms. Oral delivery of live attenuated recombinant viruses such as adenoviruses (Ad), poxviruses, influenza, herpes viruses, and polioviruses encoding specific antigens has been also tested in several oral vaccine studies. While these viral vectors showed promising results, pre-existing immunity to these viruses may prevent their ability to deliver desired antigens.

Oral delivery of DNA vaccines encoding various antigens has also been evaluated in various animal studies [1], [2], [39]–[41]. DNA vaccines contain unmethylated CpG motifs with binding activity to TLR9 receptors. This characteristic assists in activating a variety of cells including dendritic cells (DCs), macrophages, monocytes, and splenocytes [42]. The TLR9 signaling pathway leads to IL-1β and INF-γ secretion, polarizing the immune response to a Th1 type. One of the pitfalls associated with DNA vaccines is the low uptake of DNA from the intestinal tract, which consequently limits B and T cell immune responses [43].

Over the past few years, specific T and B cell epitopes have been characterized in tumor and viral antigens. Synthesis of peptide epitopes for use as a vaccine requires an understanding of T and B cell immunodominant epitopes in the protein structure, and their interaction with major histocompatibility complexes (MHCs) or human leukocyte antigen (HLA) complexes [44]–[48]. The design and development of immunodominant multivalent epitopes representing diverse HLA types is an attractive strategy against hypervariable viruses such as HIV-1 and hepatitis C virus (HCV). One of the pitfalls with this approach is that peptide vaccines are not immunogenic alone, and thus require carriers and potent adjuvants to enhance their immunogenicity. The use of lipidated peptide immunogens is one of several strategies currently being pursued for the improvement of peptide immunogenicity [49]–[51]. Previous studies have demonstrated that the presence of lipid moieties on peptides prolongs the duration of antigen presentation, enhances cytosolic uptake of peptide immunogens, activates innate immunity due to a TLR2 agonist effect, and differentiates non-activated B cells into immunoglobulin-secreting plasma cells [52]–[55]. Although no commercialized peptide vaccine is yet available, this approach has shown promising results in animal studies [56]. Oral delivery of peptide vaccines has been evaluated in pre-clinical and clinical trials. In a phase I study, 33 HIV-seronegative volunteers were primed orally three times with a V3 peptide derived from HIV-1 isolate MN, followed by a systemic boosting [57]. While no broad humoral or cellular immune responses were detected, the results could prove helpful in the further development of orally administered peptide vaccines.

Plant-based oral vaccines are another delivery system that has been tested in recent years [58]–[61]. Seed crops such as rice, maize, and soybean appear to be suitable expression and delivery systems that offer several advantages, such as resistance to intestinal enzymes, rapid scale-up of exogenous antigens, low-cost production, and a decreased risk of contamination by human pathogens [62], [63]. In a mouse study, MucoRice-expressed cholera toxin subunit B (CTB) was administered orally to animals, and specific immune responses and neutralizing activity in both systemic and mucosal compartments were detected [64]. Interestingly, immunized animals with MucoRice-CTB demonstrated protection from an oral challenge with cholera toxin compared to control animals. In a similar study conducted in a non-human primate model, cynomolgus macaques received orally administered MucoRice-CTB. Animals were found to have CT-specific, neutralizing antibodies, and high levels of systemic IgG and intestinal IgA antibodies [65].

Over the past few years, several oral vaccine delivery vehicles such as liposomes, dendrimers, multiple emulsions, immune stimulating complexes (ISCOMs), biodegradable polymers such as poly (lactide-co-glycolide acid), and dendrimers have also been identified [66]–[70]. Antigens, adjuvants, and targeting molecules could be incorporated individually or in combination into these microparticles. These vehicles may thus act as immunostimulants while preventing the degradation of immunogens by enzymes in the GI tract. These particulate formulations might also interact with microfold (M) cells and release immunogens slowly, consequently promoting phagocytosis. Some microparticle studies have shown that the addition of polymers such as chitosan might increase the interaction of antigens with the intestinal mucosal surface [66], [67]. The efficacy of these microparticles has been tested in several animal studies and in a limited number of clinical trials. In a human trial, five volunteers were orally immunized with a surface Enterotoxigenic Escherichia coli (ETEC) polymeric protein (CS6) associated with a biodegradable polymer, poly-lactide-co-glycolide (PLG) [68]. Oral administration of these microparticles was safe, and four out of five volunteers showed IgA responses and a 3.5-fold increase in the levels of serum IgG antibody responses.

In a study by Frey et al. [69], oral administration of CTB was tested as a model for enhancing antigen uptake by intestinal epithelial cells. CTB was chosen as it promotes immune responses when co-administered orally, and its receptor (ganglioside GM1) is present on all intestinal epithelial cell surfaces. In vivo results in rabbits showed that soluble CTB-FITC (diameter of 6.4 nm) was able to bind to apical membranes of both enterocytes and M cells. Whereas CTB coupled to colloidal gold (diameter of 28.8 nm) bound only to M cells and not enterocytes, CTB-coated microparticles (diameter of 1.13 µm) failed to bind to either rabbit enterocytes or rabbit M cells. In a study by Mann et al. [70], two different sizes of a liposome-entrapped influenza antigen were delivered orally in a mouse model. The group of mice that was orally immunized with larger liposomes (60–350 nm and 400–2,500 nm) showed a greater Th1 bias, serum IgG2a production, and antigen-induced splenocyte IFN-γ production, compared to mice having received liposomes 10–100 nm in size. While this study also showed that microparticle size is an important factor associated with particle uptake, the size of the microparticles was quite different from a previous study.

However, sizing is not the only issue with these microparticles. A variety of additional parameters, including the ratio and quantity of chemical components, the amount of encapsulated antigen, hydrophobicity, the ionic surface charge, the type of associated adjuvants, and the dose of administration are also crucial, and should be optimized. In this context, the association of M cell–targeting ligands on the surface of the delivery vehicles might also enhance the binding specificity to intestinal Peyer's patches. In the next section, we briefly describe the M cell surface markers that could be considered in a strategy to enhance capture and uptake of orally administered vaccines.

Targeting the Apical Surface of M Cells

M cells are specialized epithelial cells that predominantly reside in the follicle-associated epithelium (FAE) overlying Peyer's patches. M cells also reside in other sections of the intestinal tract such as the colon and rectum [71]–[74]. M cells are identifiable by their flattened apical surfaces, fewer numbers of cytoplasmic lysosomes, greater numbers of mitochondria, and the absence of glycocalyx covering their surfaces. It also appears that mouse M cells express particular surface markers, compared to enterocytes, such as β1 integrin or α-L-fucose-specific (L-fucose) lectin [75]. In contrast to enterocytes, M cells take up antigens or microorganisms from the intestinal lumen (Figure 1) by phago-, endo-, or pinocytosis and transcytosis, and deliver them to the underlying immune system of the mucosae. This phenomenon also occurs by other mechanisms, for instance in intestinal DCs; however, this will not be discussed here. M cells are not limited to the GALTs, and are also present in other mucosal tissues such as nasopharyngeal-associated lymphoid tissue (NALT) and bronchus-associated lymphoid tissues (BALT) and tonsils [76], [77]. It has been shown that M cells in NALT are a major site of virus entry as well as vaccine delivery; however, limited studies have been reported with regards to the roles of NALT and BALT in the uptake and transport of vaccine-delivered antigens.

**Fig. 1. Schematic diagram of intestinal epithelium showing M cells, Peyer's patches, intestinal epithelial cells, and pathway of Ag transport.**

The ability of M cells in Peyer's patches to take up and transcytose diverse numbers of microorganisms to antigen-presenting cells (APCs) have made M cells an ideal target for vaccine delivery to the mucosal immune system [78]–[80]. It is estimated that only 1 out of 10 million epithelial cells in the intestinal tract is an M cell (approximately 5% in humans and 10% in mice) [81]. Due to these low numbers of M cells, several approaches have been attempted to enhance M cell targeting. It has been indicated that M cell numbers in Peyer's patches are increased after exposure to Streptococcus pneumonia R36a [82]. However, these increased numbers of M cells may uptake all antigens in the intestinal epithelium and not just the antigens of interest, consequently increasing the probability of inducing food allergies and inflammatory diseases. Therefore, it might be more reasonable to target the existing M cells in Peyer's patches than to try to amplify their numbers.

Targeting specific receptors on the apical surface of M cells may have the ability to specifically increase the uptake and presentation of antigens, consequently initiating the immune response and inducing protection against infectious challenge. To date, only limited numbers of M cell receptors and their ligands have been identified, and most of these receptors are not only expressed in M cells but in neighboring enterocytes as well. Some important pathogen recognition receptors (PRRs), such as toll-like receptor-4 (TLR-4), platelet-activating factor receptor (PAFR), and α5β1 integrin, are expressed on the surface of human and mouse M cells [83]–[85]. These innate immune system molecules interact with pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide, lipotechoic acid, peptidoglycan, and bacterial flagellin. This interaction is crucial for the translocation of bacteria across the lumen. Consequently, targeting PRRs might be a suitable strategy for enhancing the uptake of orally administered vaccines by M cells. This interaction activates several signaling pathways that may play important roles in M cell functions. For instance, M cells take up many enteropathogenic microorganisms, such as Yersinia spp., via the α5β1 integrin, and inhibition of this adhesion molecule significantly inhibits transcytosis of M cells [86]–[88]. While PRRs are also expressed on neighboring enterocytes (a challenge in targeting only M cells), the expression patterns of these receptors are varied. For instance, α5β1 integrin is dispersed on the lateral and basolateral surfaces of enterocytes, while in M cells, α5β1 is distributed only on the apical surface.

Lectin-binding studies in experimental animals have shown that M cells express on their surface a particular glycosylation pattern [89], [90]. Several studies showed that Ulex europaeus agglutinin-1 (UEA-1), a lectin specific for α-l-fucose residues, selectively binds to M cells in murine Peyer's patches [91]–[94]. In a study by Manocha et al. [94], the UEA-1 coated on the surface of microparticles encoding HIV genes had the capability to bind to the apical surface of M cells. In another study, by Chionh et al. [95], oral vaccination in a mouse model with killed whole Helicobacter pylori and UEA-1 or Campylobacter jejuni and UEA-1 induced protective responses against live challenge. However, M cell glycosylation patterns are not common to all species, and it remains to be seen whether it can be used to effectively target human M cells [96]. Human M cells have proven to be largely anonymous, as it has been difficult to isolate enough of such cells for further characterization and functional evaluation. Therefore, the specific receptor requirements for human M cells and how to specifically target these receptors remains a challenge. In recent years, a few in vitro human M cell models have been established [97], [98]. One of the most common M cell–like models is comprised of co-cultures of human colon carcinoma cells (Caco-2) along with human lymphoblastoid B cell lines (Raji B cells) [99], [100]. This in vitro model has been used to study the morphology and expression of M cell surface markers and antigen absorption, and to screen oral drug/vaccine delivery systems, as it closely imitates human M cells. While these M cell–like models have been used to attempt to further understand human M cells, one of the concerns of this model pertains to its over-simplification of in vivo events, as well as the lack of signaling factors from other immune cells such as T cells that are required for the formation and optimal function of M cells.

Microarray and three-dimensional imaging of specific molecules associated with M cells has revealed that a surface marker called glycoprotein 2 (GP2) is expressed on both human and mouse M cells [101], [102]. It appears that GP2 plays an important role in molecular mechanisms responsible for antigen uptake by M cells. GP2 serves as a transcytotic apical receptor on the surface of M cells that specifically binds to type I pili on bacterial outer membranes (FimH) [101]. Elimination of GP2 reduced the entry and uptake of bacteria into Peyer's patches and decreased T cell proliferative and antibody responses. Altogether, these results suggest that the GP2 protein might be a promising vaccine target for immunizing against infectious diseases. Several studies have shown that FimH adhesion–based vaccines are able to prevent infection by impeding colonization, enhancing humoral immune responses, and blocking bacterial attachment [103]–[105]. It would be exciting to determine if FimH could direct other antigens to M cells as well.

In an interesting study by Giannasca et al. [106], Peyer's patches were biopsied from volunteers with blood groups type O (two individuals) and type A (one individual). The binding and cellular localizations of 31 lectins and ten anticarbohydrate monoclonal antibodies from biopsy samples were performed by histochemistry and compared to the nearby enterocytes on Peyer's patches. Lectin and antibody results revealed a higher expression of carbohydrates on enterocytes than M cells. Some lectins and antibodies such as OPA and anti-Lewis A also reacted with both M cells and enterocytes. Interestingly, only one (anti-sialyl Lewis A) out of the 41 tested lectins or antibodies largely reacted with human M cells (∼80%) and bound only weakly to the FAE enterocytes (∼20%). While a larger number of human tissue specimens are required to confirm this oligosaccharide repertoire, an anti-sialyl Lewis A–mediated vaccine delivery system might be appropriate approach to enable M cell–targeted mucosal vaccines in humans.

In a study by Misumi et al. [107], the capability of tetragalloyl-D-lysine dendrimer (TGDK) to target M cells was examined in an in vitro human M–like cell culture and a rhesus macaque animal model. The results indicated that TGDK specifically bound to a human intestinal M–cell like model under in vitro conditions and was delivered from the M cell surface to the basolateral area. To examine the in vivo effect of TGDK on M cell targeting, rhesus macaques were orally administered with enteric coated capsules containing TGDK-conjugated multiantigens at weeks 0, 2, and 6. ELISA from feces samples of immunized macaques indicated a high level of IgA antibody responses. Conversely, the control macaques did not induce specific IgA in fecal samples. Furthermore, the immunized macaques with TGDK-conjugated multiantigens also showed neutralizing activity against SIV infection. These results concluded that TGDK transports from the lumen into intestinal M cells, and can consequently be considered for use in mucosal vaccine delivery in humans and non-human primates.

Mucosal Immune Responses and Mucosal Tolerance

Repeated oral administration of large doses of antigen in animal models result in decreased or abrogated T cell–mediated responses to a subsequent systemic immunization with the same antigen [108]. This phenomenon prompts a question concerning the possible induction of mucosal tolerance by mucosally delivered vaccines. Importantly, for vaccine efficacy the dominant target of oral tolerance is the T and not the B cell compartment. As a matter of fact, initial mucosal administration of antigens by the oral or nasal routes primes for B cell responses in parallel with diminished T cell responses in humans as well as in animals [109]. Thus, vaccines whose protective effect is dependent on the induction of antibodies (which is the target of all currently used vaccines in humans) are not likely to diminish their efficacy by mucosal administration of antigens. Furthermore, pre-existing immune responses induced by systemic immunization cannot be attenuated or suppressed by subsequent mucosal administration of the same antigen [109]. However, initial mucosal immunization of immunologically naïve subjects (e.g., with HIV-1 vaccines) might have the undesirable effect of diminishing cell-mediated responses, including cytotoxic T cell–dependent immunity. Thus, the temporal sequence of immunization with initial systemic priming and mucosal boosting as well as the use of certain adjuvants is likely to prevent the induction of mucosal tolerance.

Concluding Remarks

Over the past few decades, oral immunization has been extensively studied due to its many attractive features. The immunological potential, absorption, or limitation in the uptake of antigens, as well as the characteristic distribution of functional cell types in the GI tract, have made it a vital target in the development of oral vaccines. The phenomenon of tolerance is a crucial challenge to overcome in the development of effective oral vaccines. Experimental animal studies have indicated that oral administration of antigens targets the systemic T cell compartment, diminishes cell-mediated immune responses, and induces tolerance. This phenomenon might lead to the induction of cytokines such as TGF-β and IL-10, and consequently enhance antigen-specific antibody responses such as IgA and IgG. While the humoral immune response is critical in the control of some mucosal pathogens, its effect might be questionable on other mucosal pathogens such as HIV and HCV where cell-mediated immune responses may play a larger role. Opponents to this tolerance hypothesis, including the authors of this article, believe that tolerance is not an issue in humans, as it occurs through a completely different mechanism. Furthermore, some clinical studies have showed that a combination of oral priming and systemic boosting might activate both humoral and cellular arms of the immune system. On the other hand, we think that the absence of a potent oral vaccine might be due to other challenges, including antigen degradation by proteolytic enzymes, the low dose of antigen absorbed, a lack of potent mucosal adjuvants, and not actively directing antigens to M cells. To overcome these issues, further work regarding oral vehicle delivery systems that protect antigens and specifically target M cells is required. Targeting M cells by mimicking the entry of mucosal pathogens such as E. coli, Salmonella, and Yersinia may reflect the in vivo binding specificity required by orally administered antigens. Regarding this aspect, a number of studies showed that these pathogens bind to specific lectins expressed on the apical surface of M cells. The binding of orally administered vaccines to M cell lectins was further studied in murine models and indicated that α-L-fucose-lectin (UEA-1) is able to bind specifically to M cells and, to a lesser degree, enterocytes. However, the characterization of murine M cells by this lectin-binding pattern did not reflect the glycosylation patterns present on human M cells. Unfortunately, human M cell features, function, and differentiation from neighboring enterocytes are not well understood.

Based on previous studies, by using tetragalloyl-D-lysine dendrimers, a monoclonal antibody targeting GP2, or using a monoclonal antibody targeting sialyl Lewis A, it might be possible to more specifically direct oral delivery systems to human M cells. However, as these molecules are also expressed on neighboring enterocytes (albeit at lower levels), it will likely be difficult to devise an ideal oral delivery system for targeting human M cells. The understanding of human M cell function, identification of more specific apical surface molecules, and the improvement of intestinal M cell–like models are crucial for the design and further development of M cell–targeted vaccines.

Zdroje

1. McGheeJR

MesteckyJ

DertzbaughMT

EldridgeJH

HirasawaM

1992

The mucosal immune system: from fundamental concepts to vaccine development.

Vaccine

2. McGheeJR

KiyonoH

1992

Mucosal immunity to vaccines: current concepts for vaccine development and immune response analysis.

Adv Exp Med Biol

327

3. Xu-AmanoJ

BeagleyKW

MegaJ

FujihashiK

KiyonoH

1992

Induction of T helper cells and cytokines for mucosal IgA responses.

Adv Exp Med Biol

327

107

117

4. MesteckyJ

1987

The common mucosal immune system and current strategies for induction of immune responses in external secretions.

J Clin Immunol

265

276

5. AziziA

GhunaimH

Diaz-MitomaF

MesteckyJ

2010

Mucosal HIV vaccines: a holy grail or a dud?

Vaccine

4015

4026

6. ParrEL

ParrMB

1990

A comparison of antibody titres in mouse uterine fluid after immunization by several routes, and the effect of the uterus on antibody titres in vaginal fluid.

J Reprod Fertil

619

625

7. HanebergB

KendallD

AmerongenHM

ApterFM

KraehenbuhlJP

1994

Induction of specific immunoglobulin A in the small intestine, colon-rectum, and vagina measured by a new method for collection of secretions from local mucosal surfaces.

Infect Immun

8. KozlowskiPA

Cu-UvinS

NeutraMR

FlaniganTP

1997

Comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women.

Infect Immun

1387

1394

9. SentmanCL

MeadowsSK

WiraCR

ErikssonM

2004

Recruitment of uterine NK cells: induction of CXC chemokine ligands 10 and 11 in human endometrium by estradiol and progesterone.

J Immunol

173

6760

6766

10. WiraCR

RossollRM

2003

Oestradiol regulation of antigen presentation by uterine stromal cells: role of transforming growth factor-beta production by epithelial cells in mediating antigen-presenting cell function.

Immunology

109

398

406

11. LuFX

MaZ

RourkeT

SrinivasanS

McChesneyM

1999

Immunoglobulin concentrations and antigen-specific antibody levels in cervicovaginal lavages of rhesus macaques are influenced by the stage of the menstrual cycle.

Infect Immun

6321

6328

12. MesteckyJ

MoldoveanuZ

RussellMW

2005

Immunologic uniqueness of the genital tract: challenge for vaccine development.

Am J Reprod Immunol

208

214

13. HolmgrenJ

CzerkinskyC

2005

Mucosal immunity and vaccines.

Nat Med

S45

S53

14. LagranderieM

WinterN

BalazucAM

GicquelB

GheorghiuM

1998

A cocktail of Mycobacterium bovis BCG recombinants expressing the SIV Nef, Env, and Gag antigens induces antibody and cytotoxic responses in mice vaccinated by different mucosal routes.

AIDS Res Hum Retroviruses

1625

1633

15. BergquistC

JohanssonEL

LagergardT

HolmgrenJ

RudinA

1997

Intranasal vaccination of humans with recombinant cholera toxin B subunit induces systemic and local antibody responses in the upper respiratory tract and the vagina.

Infect Immun

2676

2684

16. DurraniZ

McInerneyTL

McLainL

JonesT

BellabyT

1998

Intranasal immunization with a plant virus expressing a peptide from HIV-1 gp41 stimulates better mucosal and systemic HIV-1-specific IgA and IgG than oral immunization.

J Immunol Methods

220

103

17. HirabayashiY

KurataH

FunatoH

NagamineT

AizawaC

1990

Comparison of intranasal inoculation of influenza HA vaccine combined with cholera toxin B subunit with oral or parenteral vaccination.

Vaccine

243

248

18. NarayanKM

DelRC

2010

Comparative efficacy of influenza vaccines.

N Engl J Med

362

179

180

19. FioreAE

BridgesCB

CoxNJ

2009

Seasonal influenza vaccines.

Curr Top Microbiol Immunol

333

20. MiyakeA

AkagiT

EnoseY

UenoM

KawamuraM

2004

Induction of HIV-specific antibody response and protection against vaginal SHIV transmission by intranasal immunization with inactivated SHIV-capturing nanospheres in macaques.

J Med Virol

368

377

21. StoweJ

AndrewsN

WiseL

MillerE

2006

Bell's palsy and parenteral inactivated influenza vaccine.

Hum Vaccin

110

112

22. LewisDJ

HuoZ

BarnettS

KromannI

GiemzaR

2009

Transient facial nerve paralysis (Bell's palsy) following intranasal delivery of a genetically detoxified mutant of Escherichia coli heat labile toxin.

PLoS ONE

e6999

doi:10.1371/journal.pone.0006999

23. MutschM

ZhouW

RhodesP

BoppM

ChenRT

2004

Use of the inactivated intranasal influenza vaccine and the risk of Bell's palsy in Switzerland.

N Engl J Med

350

896

903

24. BaumannU

2008

Mucosal vaccination against bacterial respiratory infections.

Expert Rev Vaccines

1257

1276

25. BrangerCG

Torres-EscobarA

SunW

PerryR

FetherstonJ

2009

Oral vaccination with LcrV from Yersinia pestis KIM delivered by live attenuated Salmonellaenterica serovar Typhimurium elicits a protective immune response against challenge with Yersinia pseudotuberculosis and Yersinia enterocolitica.

Vaccine

5363

5370

26. FooksAR

2000

Development of oral vaccines for human use.

Curr Opin Mol Ther

27. KostrzakA

CervantesGM

GuetardD

NagarajuDB

Wain-HobsonS

2009

Oral administration of low doses of plant-based HBsAg induced antigen-specific IgAs and IgGs in mice, without increasing levels of regulatory T cells.

Vaccine

4798

4807

28. GrdicD

SmithR

DonachieA

KjerrulfM

HornquistE

1999

The mucosal adjuvant effects of cholera toxin and immune-stimulating complexes differ in their requirement for IL-12, indicating different pathways of action.

Eur J Immunol

1774

1784

29. CzerkinskyC

HolmgrenJ

2009

Enteric vaccines for the developing world: a challenge for mucosal immunology.

Mucosal Immunol

284

287

30. HallLJ

ClareS

PickardD

ClarkSO

KellyDL

2009

Characterisation of a live Salmonella vaccine stably expressing the Mycobacterium tuberculosis Ag85B-ESAT6 fusion protein.

Vaccine

6894

6904

31. CasiniE

1972

[Bacterial vaccines for oral administration and local mechanisms of immunity].

Ann Sclavo

547

553

32. LiangS

HosurKB

NawarHF

RussellMW

ConnellTD

2009

In vivo and in vitro adjuvant activities of the B subunit of Type IIb heat-labile enterotoxin (LT-IIb-B5) from Escherichia coli.

Vaccine

4302

4308

33. BoyerJD

RobinsonTM

MaciagPC

PengX

JohnsonRS

2005

DNA prime Listeria boost induces a cellular immune response to SIV antigens in the rhesus macaque model that is capable of limited suppression of SIV239 viral replication.

Virology

333

101

34. PerdigonG

AlvarezS

Nader de MaciasME

Pesce de Ruiz HolgadoAA

1988

[Adjuvant activity of lactic bacteria: perspectives for its use in oral vaccines].

Rev Argent Microbiol

141

146

35. PouwelsPH

LeerRJ

ShawM

Heijne den Bak-GlashouwerMJ

TielenFD

1998

Lactic acid bacteria as antigen delivery vehicles for oral immunization purposes.

Int J Food Microbiol

155

167

36. WellsJM

MercenierA

2008

Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria.

Nat Rev Microbiol

349

362

37. TuckerSN

TingleyDW

ScallanCD

2008

Oral adenoviral-based vaccines: historical perspective and future opportunity.

Expert Rev Vaccines

38. MesteckyJ

NguyenH

CzerkinskyC

KiyonoH

2008

Oral immunization: an update.

Curr Opin Gastroenterol

713

719

39. FuJ

BianG

ZhaoB

DongZ

SunX

2009

Enhancing efficacy and mucosa-tropic distribution of an oral HIV-PsV DNA vaccine in animal models.

J Drug Target

803

812

40. NingJF

ZhuW

XuJP

ZhengCY

MengXL

2009

Oral delivery of DNA vaccine encoding VP28 against white spot syndrome virus in crayfish by attenuated Salmonella typhimurium.

Vaccine

1127

1135

41. CazorlaSI

BeckerPD

FrankFM

EbensenT

SartoriMJ

2008

Oral vaccination with Salmonella enterica as a cruzipain-DNA delivery system confers protective immunity against Trypanosoma cruzi.

Infect Immun

324

333

42. KindrachukJ

PotterJ

WilsonHL

GriebelP

BabiukLA

2008

Activation and regulation of toll-like receptor 9: CpGs and beyond.

Mini Rev Med Chem

590

600

43. ForsmanA

UshameckisD

BindraA

YunZ

BlombergJ

2003

Uptake of amplifiable fragments of retrotransposon DNA from the human alimentary tract.

Mol Genet Genomics

270

362

368

44. Van KaerL

Ashton-RickardtPG

EichelbergerM

GaczynskaM

NagashimaK

1994

Altered peptidase and viral-specific T cell response in LMP2 mutant mice.

Immunity

533

541

45. ToesRE

NussbaumAK

DegermannS

SchirleM

EmmerichNP

2001

Discrete cleavage motifs of constitutive and immunoproteasomes revealed by quantitative analysis of cleavage products.

J Exp Med

194

46. YorkIA

GoldbergAL

MoXY

RockKL

1999

Proteolysis and class I major histocompatibility complex antigen presentation.

Immunol Rev

172

47. MoXY

CascioP

LemeriseK

GoldbergAL

RockK

1999

Distinct proteolytic processes generate the C and N termini of MHC class I-binding peptides.

J Immunol

163

5851

5859

48. AziziA

Diaz-MitomaF

2007

Viral peptide immunogens: current challenges and opportunities.

J Pept Sci

776

786

49. DuesbergU

von demBA

KirschningC

MiyakeK

SauerbruchT

2002

Cell activation by synthetic lipopeptides of the hepatitis C virus (HCV) —core protein is mediated by toll like receptors (TLRs) 2 and 4.

Immunol Lett

50. YamashitaY

MaedaY

TakeshitaF

BrennanPJ

MakinoM

2004

Role of the polypeptide region of a 33 kDa mycobacterial lipoprotein for efficient IL-12 production.

Cell Immunol

229

51. SchroderNW

HeineH

AlexanderC

ManukyanM

EckertJ

2004

Lipopolysaccharide binding protein binds to triacylated and diacylated lipopeptides and mediates innate immune responses.

J Immunol

173

2683

2691

52. JacksonDC

LauYF

LeT

SuhrbierA

DeliyannisG

2004

A totally synthetic vaccine of generic structure that targets Toll-like receptor 2 on dendritic cells and promotes antibody or cytotoxic T cell responses.

Proc Natl Acad Sci U S A

101

15440

15445

53. VossS

UlmerAJ

JungG

WiesmullerKH

BrockR

2007

The activity of lipopeptide TLR2 agonists critically depends on the presence of solubilizers.

Eur J Immunol

3489

3498

54. SumikawaY

AsadaH

HoshinoK

AzukizawaH

KatayamaI

2006

Induction of beta-defensin 3 in keratinocytes stimulated by bacterial lipopeptides through toll-like receptor 2.

Microbes Infect

1513

1521

55. SirskyjD

Diaz-MitomaF

GolshaniA

KumarA

AziziA

2010

Innovative bioinformatic approaches for developing peptide-based vaccines against hypervariable viruses.

Immunol Cell Biol

E-pub ahead of print. 11 May 2010

56. AziziA

AndersonDE

TorresJV

OgrelA

GhorbaniM

2008

Induction of broad cross-subtype-specific HIV-1 immune responses by a novel multivalent HIV-1 peptide vaccine in cynomolgus macaques.

J Immunol

180

2174

2186

57. LambertJS

KeeferM

MulliganMJ

SchwartzD

MesteckyJ

2001

A Phase I safety and immunogenicity trial of UBI microparticulate monovalent HIV-1 MN oral peptide immunogen with parenteral boost in HIV-1 seronegative human subjects.

Vaccine

3033

3042

58. TacketCO

2009

Plant-based oral vaccines: results of human trials.

Curr Top Microbiol Immunol

332

103

117

59. StreatfieldSJ

2005

Delivery of plant-derived vaccines.

Expert Opin Drug Deliv

719

728

60. StreatfieldSJ

2005

Plant-based vaccines for animal health.

Rev Sci Tech

189

199

61. HammondRW

NemchinovLG

2009

Plant production of veterinary vaccines and therapeutics.

Curr Top Microbiol Immunol

332

102

62. WalmsleyAM

ArntzenCJ

2000

Plants for delivery of edible vaccines.

Curr Opin Biotechnol

126

129

63. HaqTA

MasonHS

ClementsJD

ArntzenCJ

1995

Oral immunization with a recombinant bacterial antigen produced in transgenic plants.

Science

268

714

716

64. NochiT

TakagiH

YukiY

YangL

MasumuraT

2007

Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination.

Proc Natl Acad Sci U S A

104

10986

10991

65. NochiT

YukiY

KatakaiY

ShibataH

TokuharaD

2009

A rice-based oral cholera vaccine induces macaque-specific systemic neutralizing antibodies but does not influence pre-existing intestinal immunity.

J Immunol

183

6538

6544

66. AminM

JaafariMR

TafaghodiM

2009

Impact of chitosan coating of anionic liposomes on clearance rate, mucosal and systemic immune responses following nasal administration in rabbits.

Colloids Surf B Biointerfaces

225

229

67. BorgesO

TavaresJ

deSA

BorchardG

JungingerHE

Cordeiro-da-SilvaA

2007

Evaluation of the immune response following a short oral vaccination schedule with hepatitis B antigen encapsulated into alginate-coated chitosan nanoparticles.

Eur J Pharm Sci

278

290

68. KatzDE

DeLorimierAJ

WolfMK

HallER

CasselsFJ

2003

Oral immunization of adult volunteers with microencapsulated enterotoxigenic Escherichia coli (ETEC) CS6 antigen.

Vaccine

341

346

69. FreyA

GiannascaKT

WeltzinR

GiannascaPJ

ReggioH

1996

Role of the glycocalyx in regulating access of microparticles to apical plasma membranes of intestinal epithelial cells: implications for microbial attachment and oral vaccine targeting.

J Exp Med

184

1045

1059

70. MannJF

ShakirE

CarterKC

MullenAB

AlexanderJ

2009

Lipid vesicle size of an oral influenza vaccine delivery vehicle influences the Th1/Th2 bias in the immune response and protection against infection.

Vaccine

3643

3649

71. OwenRL

JonesAL

1974

Epithelial cell specialization within human Peyer's patches: an ultrastructural study of intestinal lymphoid follicles.

Gastroenterology

189

203

72. HathawayLJ

KraehenbuhlJP

2000

The role of M cells in mucosal immunity.

Cell Mol Life Sci

323

332

73. GebertA

GokeM

RothkotterHJ

DietrichCF

2000

[The mechanisms of antigen uptake in the small and large intestines: the roll of the M cells for the initiation of immune responses].

Z Gastroenterol

855

872

74. KuhnEM

KaupFJ

1996

Morphological characteristics of the ileal Peyer's patches in the rhesus macaque: a histological and ultrastructural study.

Anat Histol Embryol

75. ClarkMA

HirstBH

JepsonMA

1998

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

Infect Immun

1237

1243

76. GebertA

PabstR

1999

M cells at locations outside the gut.

Semin Immunol

165

170

77. GebertA

FassbenderS

WernerK

WeissferdtA

1999

The development of M cells in Peyer's patches is restricted to specialized dome-associated crypts.

Am J Pathol

154

1573

1582

78. HolmgrenJ

CzerkinskyC

2005

Mucosal immunity and vaccines.

Nat Med

S45

S53

79. YukiY

KiyonoH

2009

Mucosal vaccines: novel advances in technology and delivery.

Expert Rev Vaccines

1083

1097

80. BraydenDJ

JepsonMA

BairdAW

2005

Keynote review: intestinal Peyer's patch M cells and oral vaccine targeting.

Drug Discov Today

1145

1157

81. KuoleeR

ChenW

2008

M cell-targeted delivery of vaccines and therapeutics.

Expert Opin Drug Deliv

693

702

82. MeynellHM

ThomasNW

JamesPS

HollandJ

TaussigMJ

1999

Up-regulation of microsphere transport across the follicle-associated epithelium of Peyer's patch by exposure to Streptococcus pneumoniae R36a.

FASEB J

611

619

83. TyrerP

RuthFA

KydJ

HarveyM

SizerP

2002

Validation and quantitation of an in vitro M-cell model.

Biochem Biophys Res Commun

299

377

383

84. MannJF

FerroVA

MullenAB

TetleyL

MullenM

2004

Optimisation of a lipid based oral delivery system containing A/Panama influenza haemagglutinin.

Vaccine

2425

2429

85. ChouMY

HartvigsenK

HansenLF

FogelstrandL

ShawPX

2008

Oxidation-specific epitopes are important targets of innate immunity.

J Intern Med

263

479

488

86. ScibelliA

MatteoliG

RopertoS

AlimentiE

DipinetoL

2005

Flavoridin inhibits Yersinia enterocolitica uptake into fibronectin-adherent HeLa cells.

FEMS Microbiol Lett

247

87. SaltmanLH

LuY

ZahariasEM

IsbergRR

1996

A region of the Yersinia pseudotuberculosis invasin protein that contributes to high affinity binding to integrin receptors.

J Biol Chem

271

23438

23444

88. SinhaB

FrancoisPP

NusseO

FotiM

HartfordOM

1999

Fibronectin-binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin alpha5beta1.

Cell Microbiol

101

117

89. JangMH

KweonMN

IwataniK

YamamotoM

TeraharaK

2004

Intestinal villous M cells: an antigen entry site in the mucosal epithelium.

Proc Natl Acad Sci U S A

101

6110

6115

90. KozlowskiPA

WilliamsSB

LynchRM

FlaniganTP

PattersonRR

2002

Differential induction of mucosal and systemic antibody responses in women after nasal, rectal, or vaginal immunization: influence of the menstrual cycle.

J Immunol

169

566

574

91. GuptaPN

KhatriK

GoyalAK

MishraN

VyasSP

2007

M-cell targeted biodegradable PLGA nanoparticles for oral immunization against hepatitis B.

J Drug Target

701

713

92. LavelleEC

GrantG

PusztaiA

PfullerU

O'HaganDT

2000

Mucosal immunogenicity of plant lectins in mice.

Immunology

93. WangX

KochetkovaI

HaddadA

HoytT

HoneDM

2005

Transgene vaccination using Ulex europaeus agglutinin I (UEA-1) for targeted mucosal immunization against HIV-1 envelope.

Vaccine

3836

3842

94. ManochaM

PalPC

ChitralekhaKT

ThomasBE

TripathiV

2005

Enhanced mucosal and systemic immune response with intranasal immunization of mice with HIV peptides entrapped in PLG microparticles in combination with Ulex Europaeus-I lectin as M cell target.

Vaccine

5599

5617

95. ChionhYT

WeeJL

EveryAL

NgGZ

SuttonP

2009

M-cell targeting of whole killed bacteria induces protective immunity against gastrointestinal pathogens.

Infect Immun

2962

2970

96. GiannascaPJ

GiannascaKT

LeichtnerAM

NeutraMR

1999

Human intestinal M cells display the sialyl Lewis A antigen.

Infect Immun

946

953

97. KerneisS

BogdanovaA

KraehenbuhlJP

PringaultE

1997

Conversion by Peyer's patch lymphocytes of human enterocytes into M cells that transport bacteria.

Science

277

949

952

98. GramLK

RistGM

LennernasH

SteffansenB

2009

Impact of carriers in oral absorption: Permeation across Caco-2 cells for the organic anions estrone-3-sulfate and glipizide.

Eur J Pharm Sci

378

386

99. LimJS

NaHS

LeeHC

ChoyHE

ParkSC

2009

Caveolae-mediated entry of Salmonella typhimurium in a human M-cell model.

Biochem Biophys Res Commun

390

1322

1327

100. GullbergE

LeonardM

KarlssonJ

HopkinsAM

BraydenD

2000

Expression of specific markers and particle transport in a new human intestinal M-cell model.

Biochem Biophys Res Commun

279

808

813

101. HaseK

KawanoK

NochiT

PontesGS

FukudaS

2009

Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response.

Nature

462

226

230

102. TeraharaK

YoshidaM

IgarashiO

NochiT

PontesGS

2008

Comprehensive gene expression profiling of Peyer's patch M cells, villous M-like cells, and intestinal epithelial cells.

J Immunol

180

7840

7846

103. LangermannS

MollbyR

BurleinJE

PalaszynskiSR

AugusteCG

2000

Vaccination with FimH adhesin protects cynomolgus monkeys from colonization and infection by uropathogenic Escherichia coli.

J Infect Dis

181

774

778

104. PoggioTV

La TorreJL

ScodellerEA

2006

Intranasal immunization with a recombinant truncated FimH adhesin adjuvanted with CpG oligodeoxynucleotides protects mice against uropathogenic Escherichia coli challenge.

Can J Microbiol

1093

1102

105. BouckaertJ

BerglundJ

SchembriM

DeGE

CoolsL

2005

Receptor binding studies disclose a novel class of high-affinity inhibitors of the Escherichia coli FimH adhesin.

Mol Microbiol

441