Mucosal Inductive Sites Research Articles

Chitosan-polymer based nanovaccine as promising immersion vaccine against Aeromonas veronii challenge in red tilapia (Oreochromis sp.)

Red tilapia (Oreochromis sp.), one of the important freshwater fish species in fish farming in Thailand, has for long been suffering from a serious bacterial disease named epizootic ulcerative syndrome and hemorrhagic septicemia. The disease is mainly caused by Aeromonas veronii. Vaccine is proposed to be a major impact tool for sustainable control and prevention strategies. Vaccination by immersion has many benefits over injection. However, the conventional immersion method suffers from a low potency due to the inefficient uptake of antigens across mucosal tissue. Here, we developed a chitosan-polymer based nanovaccine together with an efficient delivery vehicle to enhance the immunogenicity of immersion vaccination, increasing bioavailability and inducing local immune responses during transit to mucosal inductive immune sites. The physiochemical properties of nanovaccine, which was modified on surface particle by using a mucoadhesive polymer, were assessed for size, zeta potential, and particle distribution. Our study demonstrated by SEM image and microscopic fluorescence image that nanovaccine greatly increased the binding and penetrating ability into gills when compared with formalin killed vaccine. The nano-sized particles were well dispersed in water and trapped in core nanoparticle as confirmed by TEM image. The efficacy of vaccine was performed by immersion challenge with virulent A.veronii after 30 days post vaccination in tilapia. The result revealed a high level of mortality in the control, empty-polymeric nanovaccine and formalin killed bacterin vaccine groups. A high relative percentage survival (RPS) of vaccinated fish was noted with chitosan-polymer based nanovaccine. Our studies indicated that this chitosan-polymer based nanovaccine derived from cell fragments and supernatant was the improved version of the conventional formalin killed vaccine. The chitosan polymer based particle could increase the efficacy of nanovaccine toward the target mucosal membrane and enhance protection against A. veronii infection in red tilapia.

Oral probiotic promotes indoleamine 2,3-dioxygenase- and TGF-β–Producing plasmacytoid dendritic cells to initiate protection against type 1 diabetes

Colonization factor antigen I (CFA/I) fimbria, an adhesin from enterotoxigenic Escherichia coli, confers protection in murine autoimmune models for type 1 diabetes (T1D), multiple sclerosis, and rheumatoid arthritis. Although CFA/I fimbriae's initial mode of action is in a bystander or in an antigen (Ag)-independent fashion, protection is ultimately dependent upon the induction and/or activation of auto-Ag-specific regulatory T cells (Tregs). However, little is known about how protection transitions from bystander suppression to Ag-specific Tregs. Since dendritic cells (DCs) play an integral role in fate decisions for T cells becoming inflammatory or tolerogenic, the described study tests the hypothesis that Lactococcus lactis expressing CFA/I (LL-CFA/I) stimulates DCs to establish a regulatory microenvironment. To this end, bone marrow-derived dendritic cells (BMDCs) were infected in vitro with LL-CFA/I. Results revealed increased production of IL-10, TGF-β, and indoleamine 2,3-deoxygenase (IDO). Although co-culture of LL-CFA/I infected BMDCs with naïve T cells did not promote Foxp3 expression, TNF-α and IFN-γ production was suppressed. NOD mice orally dosed with LL-CFA/I showed an increase in regulatory plasmacytoid DCs (pDCs) expressing IDO and TGF-β in pancreatic lymph nodes (PaLNs) and spleen three days post-treatment. However, Tregs did not appear in the mucosal inductive sites until much later. These findings show that LL-CFA/I influences specific DC populations to establish tolerance.

Cellular Basis for the Enhanced Efficacy of the Fms-Like Tyrosine Kinase 3 Ligand (FL) Adjuvanted VCG-Based Chlamydia abortus Vaccine.

Efficacious vaccines are needed to control genital chlamydial diseases in humans and the veterinary industry. We previously reported a C. abortus (Cab) vaccine comprising recombinant Vibrio cholerae ghosts (rVCG) expressing the conserved and immunogenic N-terminal region of the Cab polymorphic membrane protein D (rVCG-Pmp18.1) protein that protected mice against intravaginal challenge. In this study, we investigated the immunomodulatory effect of the hematopoietic progenitor activator cytokine, Fms-like tyrosine kinase 3-ligand (FL) when co-administered with the rVCG-Pmp18.1 vaccine as a strategy to enhance the protective efficacy and the potential mechanism of immunomodulation. Groups of female C57BL/6J mice were immunized and boosted twice intranasally (IN) with rVCG-PmpD18.1 with and without FL or purified rPmp18.1 or rVCG-gD2 (antigen control) or PBS (medium) per mouse. The results revealed that co-administration of the vaccine with FL enhanced antigen-specific cellular and humoral immune responses and protected against live Cab genital infection. Comparative analysis of immune cell phenotypes infiltrating mucosal and systemic immune inductive tissue sites following immunization revealed that co-administration of rVCG-Pmp18.1 with FL significantly enhanced the number of macrophages, dendritic and NK cells, γδ and NK T cells in the spleen (systemic) and iliac lymph nodes (ILN) draining the genital tract (mucosal) tissues compared to rVCG-Pmp18.1 alone. Furthermore, FL enhanced monocyte infiltration in the ILN, while CD19+ B cells and CD4+ T cells were enhanced in the spleen. These results indicate that the immunomodulatory effect of FL is associated with its ability to mobilize innate immune cells and subsequent activation of robust antigen-specific immune effectors in mucosal and systemic lymphoid tissues.

Bacterial community analysis on the different mucosal immune inductive sites of gastrointestinal tract in Bactrian camels.

The microbial communities colonize the mucosal immune inductive sites could be captured by hosts, which could initiate the mucosal immune responses. The aggregated lymphoid nodule area (ALNA) and the ileal Payer’s patches (PPs) in Bactrian camels are both the mucosal immune inductive sites of the gastrointestinal tract. Here, the bacteria community associated with the ALNA and ileal PPs were analyzed using of 16S rDNA-Illumina Miseq sequencing. The mutual dominant bacterial phyla at the two sites were the Bacteroidetes, Firmicutes, Verrucomicrobia and Proteobacteria, and the mutual dominant genus in both sits was Prevotella. The abundances of the Fibrobacter, Campylobacter and RFP12 were all higher in ALNA than in ileal PPs. While, the abundances of the 5-7N15, Clostridium, and Escherichia were all higher in ileal PPs than in ALNA. The results suggested that the host’s intestinal microenvironment is selective for the symbiotic bacteria colonizing the corresponding sites, on the contrary, the symbiotic bacteria could impact on the physiological functions of this local site. In ALNA and ileal PPs of Bactrian camel, the bacteria which colonized different immune inductive sites have the potential to stimulate different immune responses, which is the result of the mutual selection and adaptation between microbial communities and their host.

Oral Biologic Delivery: Advances Toward Oral Subunit, DNA, and mRNA Vaccines and the Potential for Mass Vaccination During Pandemics.

Oral vaccination enables pain-free and self-administrable vaccine delivery for rapid mass vaccination during pandemic outbreaks. Furthermore, it elicits systemic and mucosal immune responses. This protects against infection at mucosal surfaces, which may further enhance protection and minimize the spread of disease. The gastrointestinal (GI) tract presents a number of prospective mucosal inductive sites for vaccine targeting, including the oral cavity, stomach, and small intestine. However, currently available oral vaccines are effectively limited to live-attenuated and inactivated vaccines against enteric diseases. The GI tract poses a number of challenges,including degradative processes that digest biologics and mucosal barriers that limit their absorption. This review summarizes the approaches currently under development and future opportunities for oral vaccine delivery to established (intestinal) and relatively new (oral cavity, stomach) mucosal targets. Special consideration is given to recent advances in oral biologic delivery that offer promise as future platforms for the administration of oral vaccines.

The Mammary Gland as an Integral Component of the Common Mucosal Immune System.

The human mammary gland is an integral effector component of the common mucosal immune system. However, from physiological and immunological aspects, it displays several unique features not shared by other mucosal sites. The development, maturation, and activity of the mammary gland exhibits a strong hormonal dependence. Furthermore, in comparison to the intestinal and respiratory tracts, the mammary gland is not colonized by high numbers of bacteria of enormous diversity and does not contain mucosal inductive sites analogous to the intestinal Peyer's patches. Consequently, when exposed to antigens, local or generalized immune responses are low or not present. Comparative evaluations of various immunization routes effective in the induction of antibodies in human milk are limited. Systemic immunization induces IgG antibodies in plasma, but due to the low levels of total IgG in human milk, their protective effect remains unknown. Oral or intranasal immunization or infection induces secretory IgA in milk, as demonstrated in several studies. Other routes of mucosal immunization, such as sublingual or rectal exposure effective in the induction of antibodies in various external secretions, have not been explored in the mammary gland. Because secretory IgA in milk displays protective functions, alternative immunization routes and antigen delivery systems should be explored.

Convergent Evolution of Mucosal Immune Responses at the Buccal Cavity of Teleost Fish.

SummaryThe buccal mucosa (BM) is a critical first line of defense in terrestrial animals. To gain further insights into the evolutionary origins and primordial roles of BM in teleosts here we show that rainbow trout, a teleost fish, contains a diffuse mucosal associated lymphoid tissue (MALT) within its buccal cavity. Upon parasite infection, a fish immunoglobulin specialized in mucosal immunity (sIgT) was induced to a high degree, and parasite-specific sIgT responses were mainly detected in the buccal mucus. Moreover, we show that the trout buccal microbiota is prevalently coated with sIgT. Overall our findings revealed that the MALT is present in the BM of a non-tetrapod species. As fish IgT and mucus-producing cells are evolutionarily unrelated to mammalian IgA and salivary glands, respectively, our findings indicate that mucosal immune responses in the BM of teleost fish and tetrapods evolved through a process of convergent evolution.

Mucosal immunoglobulins protect the olfactory organ of teleost fish against parasitic infection.

The olfactory organ of vertebrates receives chemical cues present in the air or water and, at the same time, they are exposed to invading pathogens. Nasal-associated lymphoid tissue (NALT), which serves as a mucosal inductive site for humoral immune responses against antigen stimulation in mammals, is present also in teleosts. IgT in teleosts is responsible for similar functions to those carried out by IgA in mammals. Moreover, teleost NALT is known to contain B-cells and teleost nasal mucus contains immunoglobulins (Igs). Yet, whether nasal B cells and Igs respond to infection remains unknown. We hypothesized that water-borne parasites can invade the nasal cavity of fish and elicit local specific immune responses. To address this hypothesis, we developed a model of bath infection with the Ichthyophthirius multifiliis (Ich) parasite in rainbow trout, Oncorhynchus mykiss, an ancient bony fish, and investigated the nasal adaptive immune response against this parasite. Critically, we found that Ich parasites in water could reach the nasal cavity and successfully invade the nasal mucosa. Moreover, strong parasite-specific IgT responses were detected in the nasal mucus, and the accumulation of IgT+ B-cells was noted in the nasal epidermis after Ich infection. Strikingly, local IgT+ B-cell proliferation and parasite-specific IgT generation were found in the trout olfactory organ, providing new evidence that nasal-specific immune responses were induced locally by a parasitic challenge. Overall, our findings suggest that nasal mucosal adaptive immune responses are similar to those reported in other fish mucosal sites and that an antibody system with a dedicated mucosal Ig performs evolutionary conserved functions across vertebrate mucosal surfaces.

Some news from the unknown soldier, the Peyer’s patch macrophage

In mammals, macrophages (MF) are present in virtually all tissues where they serve many different functions linked primarily to the maintenance of homeostasis, innate defense against pathogens, tissue repair and metabolism. Although some of these functions appear common to all tissues, others are specific to the homing tissue. Thus, MF become adapted to perform particular functions in a given tissue. Accordingly, MF express common markers but also sets of tissue-specific markers linked to dedicated functions. One of the largest pool of MF in the body lines up the wall of the gut. Located in the small intestine, Peyer’s patches (PP) are primary antigen sampling and mucosal immune response inductive sites. Surprisingly, although markers of intestinal MF, such as F4/80, have been identified more than 30 years ago, MF of PP escaped any kind of phenotypic description and remained “unknown” for decades. In absence of MF identification, the characterization of the PP mononuclear phagocyte system (MPS) functions has been impaired. However, taking into account that PP are privileged sites of entry for pathogens, it is important to understand how the latter are handled by and/or escape the PP MPS, especially MF, which role in killing invaders is well known. This review focuses on recent advances on the PP MPS, which have allowed, through new criteria of PP phagocyte subset identification, the characterization of PP MF origin, diversity, specificity, location and functions.

C5a receptor targeting of partial non-structural protein 3 of dengue virus promotes antigen-specific IFN-γ-producing T-cell responses in a mucosal dengue vaccine model

Mucosal vaccination is an ideal strategy to induce protective immunity in both mucosal and parenteral areas. Successful induction of an antigen-specific immune response via mucosal administration essentially requires the effective delivery of antigen into a mucosal immune inductive site, which depends on antigen delivery into M cells. We previously reported that M cells specifically express C5aR, and antigen targeting to C5aR by using specific ligands, including Co1 peptide, promotes the antigen-specific immune response in both mucosal and systemic immune compartments. In this study, we found that application of the Co1 peptide to dengue virus antigen containing CD8 T cell epitopes effectively induced an antigen-specific IFN-γ-producing CD8+ T cell response after oral mucosal administration of antigen. Consequently, we suggest that Co1 peptide-mediated C5aR targeting of antigen into M cells can be used for the induction of an effective antigen-specific CD8+ T cell immune response in oral mucosal vaccine development.

Targeted Strategies for Mucosal Vaccination.

Mucosal immune responses are in the first line of defense against most infections and protective mucosal immunity can be achieved by mucosal vaccination. However, mucosal tolerance and physicochemical features of the mucosal environment pose challenging obstacles to the development of mucosal vaccines. Vaccine formulations must be designed to enhance stability at the mucosae and incorporate features that induce innate immunity at mucosal inductive sites. To face these challenges, a number of novel delivery systems for targeting of mucosal vaccines to specific mucosal locations have been developed. In addition, specific mucosal immune cell targeting can potentially be achieved with ligand-antigen bioconjugates, in particular, those directed to specific receptors expressed on Microfold (M) cells, mucosal epithelial cells, or mucosal antigen presenting cells (APCs). In this topical review, targeted strategies to enhance the effectiveness of mucosal vaccines are addressed, and obstacles to the design and progression of effective ligand-mediated mucosal vaccines are highlighted.

The Peyer's Patch Mononuclear Phagocyte System at Steady State and during Infection.

The gut represents a potential entry site for a wide range of pathogens including protozoa, bacteria, viruses, or fungi. Consequently, it is protected by one of the largest and most diversified population of immune cells of the body. Its surveillance requires the constant sampling of its encounters by dedicated sentinels composed of follicles and their associated epithelium located in specialized area. In the small intestine, Peyer’s patches (PPs) are the most important of these mucosal immune response inductive sites. Through several mechanisms including transcytosis by specialized epithelial cells called M-cells, access to the gut lumen is facilitated in PPs. Although antigen sampling is critical to the initiation of the mucosal immune response, pathogens have evolved strategies to take advantage of this permissive gateway to enter the host and disseminate. It is, therefore, critical to decipher the mechanisms that underlie both host defense and pathogen subversive strategies in order to develop new mucosal-based therapeutic approaches. Whereas penetration of pathogens through M cells has been well described, their fate once they have reached the subepithelial dome (SED) remains less well understood. Nevertheless, it is clear that the mononuclear phagocyte system (MPS) plays a critical role in handling these pathogens. MPS members, including both dendritic cells and macrophages, are indeed strongly enriched in the SED, interact with M cells, and are necessary for antigen presentation to immune effector cells. This review focuses on recent advances, which have allowed distinguishing the different PP mononuclear phagocyte subsets. It gives an overview of their diversity, specificity, location, and functions. Interaction of PP phagocytes with the microbiota and the follicle-associated epithelium as well as PP infection studies are described in the light of these new criteria of PP phagocyte identification. Finally, known alterations affecting the different phagocyte subsets during PP stimulation or infection are discussed.

Development of live attenuated bacterial vaccines targeting Escherichia coli heat-labile and heat-stable enterotoxins

Enterotoxigenic Escherichia coli (ETEC), defined by the production of heat labile (LT) and/or heat stable (ST) toxins, are major causes of diarrhea in animals, children in developing countries and to travelers. No broadly protective ETEC vaccine is available, largely because of the difficulty in inducing immunity to the small ST molecule. To take advantage of the demonstration (Liu et al., 2011; Zhang et al., 2013, 2010) that genetically produced fusions of mutant ST with LT subunits can induce effective immunity against both toxins, we engineered a live attenuated vaccine vector strain of E. coli (ZCR533), expressing the immunogenic LT-ST fusions. To present the LT-ST fusions to the mucosal immune system, we used restriction-free cloning to incorporate them into the passenger domain of the autotransporter protein (EspP) expressed on a medium copy number plasmid. This versatile system permits expression of incorporated antigens in either surface-bound or secreted forms by the ZCR533 vector, for delivery to the mucosal inductive sites. Incorporation of the fusions into EspP plasmids was confirmed by PCR and DNA sequencing. Protein expression was confirmed by Western blot of whole cell lysates and culture supernatents using polyclonal antisera to LT. Expression of the surface-targeted fusion on the surface of ZCR533 was confirmed by immuno-fluorescent staining. These studies show that antigenic LT-ST fusions can be surface-expressed or secreted, by our attenuated E. coli ZCR533 vaccine vector via the EspP autotransporter. These constructs could serve as broadly protective vaccine candidates to protect against both LT- and ST-producing ETEC.

Microbiota-derived butyrate negatively regulates RORγt+ ILC3 in Peyer’s patch of the terminal ileum

Abstract In the intestinal immune system, anatomical and physiological distinctions in the gut contribute to its regional specialization by microbiota and immunomodulatory agents from the diet. Given that the ileal Peyer’s patch (PP) belongs anatomically to the small intestine, while it is physiologically exposed to an environment similar to the large intestine with respect to microbes and microbial metabolites, its characteristics may differ from those in PPs in the jejunum. As the terminal ileal PP is a key mucosal organ where host defense and oral immune tolerance in the gut develop, identification of the specific factors regionally specialized in the tissue is essential to understand mucosal homeostasis. Among the various cell types present in PPs, group 3 innate lymphoid cells (ILC3s) are closely associated with the regulation of commensal bacteria through the suppression of commensal bacteria-specific CD4+ T cells, although the regulation of ILCs in ileal PPs is poorly defined. In this study, we found that butyrate plays a role as a regional specific factor involved in the repression of ILC3s in PPs of the terminal ileum. This butyrate-mediated negative regulation of ILC3s alleviates the tolerogenic mucosal microenvironment by suppressing regulatory T cells in PPs. Collectively, we conclude that the inhibition of ILC3s by microbiota-derived butyrate can confer the functional ability to induce antigen-specific immunity, and that this network contributes to homeostatic regulation of the mucosal immune inductive site.

Mucosal Immune Responses Associated with NKT Cell Activation and Dendritic Cell Expansion by Nasal administration of α-galactosylceramide in the Nasopharynx

Objective: Intranasal immunization is an effective method to induce mucosal immune responses in the upper respiratory tract.α-galactosylceramide (α-GalCer) is considered as one of the most potent candidates for a mucosal adjuvant. In the present study, mucosal immune responses in the nasopharynx associated with NKT cell activation by nasal administration of α-GalCer were examined for inducing protective immunity in the nasopharynx, with the ultimate goal of developing a mucosal vaccine for preventing upper respiratory infectious diseases. Methods: Mice were administered α-GalCer intranasally as a ligand for NKT cells, without any antigen, weekly total three times. One week after the final administration of α-GalCer, mice were killed, and nasal immune responses were examined. Dendritic cells (DCs) in nasal-associated lymphoid tissue (NALT), a mucosal inductive site, were examined by immunohistochemistry. DCs, NKT cells, and B cells in NALT and nasal passage (NP) were examined by flow cytometry. Cytokine-producing CD4+ T cells were also examined by flow cytometry. Quantification of immunoglobulin (Ig)-producing cells was examined by enzyme-linked immunospot (ELISPOT) assay. In addition, bacterial challenges with live Haemophilus influenzae (Hi) and Streptococcus pneumoniae (Sp) were performed, and bacterial clearance from nasopharynx was examined. Results: After nasal immunization of α -GalCer, DCs increased in NALT. Antibody-producing cells, mainly those that produce IgA, significantly increased in the NP, a mucosal effector site. Interleukin (IL)-17-producing Th 17 cells were also induced in the NP. Bacterial challenges with live Hi and Sp resulted in enhanced clearances of both bacterial species from the nasopharynx. It is interesting that bacterial clearance was impaired by IL-17 neutralization. Conclusion: Nasal vaccination is effective for the induction of protective immunity against upper respiratory infection. The results of the present study demonstrated that nasal administration of α-GalCer could activate NKT cells in the nasopharynx, followed by the maturation of DCs, B cells, and some cytokine-producing CD4+ T cells. These findings suggest that the activation of NKT cells by nasal administration of α-GalCer induced protective immunity in the nasopharynx, possibly involving the interaction with DCs and induction of Th17 cells.

Challenges in mucosal HIV vaccine development: lessons from non-human primate models.

An efficacious HIV vaccine is urgently needed to curb the AIDS pandemic. The modest protection elicited in the phase III clinical vaccine trial in Thailand provided hope that this goal might be achieved. However, new approaches are necessary for further advances. As HIV is transmitted primarily across mucosal surfaces, development of immunity at these sites is critical, but few clinical vaccine trials have targeted these sites or assessed vaccine-elicited mucosal immune responses. Pre-clinical studies in non-human primate models have facilitated progress in mucosal vaccine development by evaluating candidate vaccine approaches, developing methodologies for collecting and assessing mucosal samples, and providing clues to immune correlates of protective immunity for further investigation. In this review we have focused on non-human primate studies which have provided important information for future design of vaccine strategies, targeting of mucosal inductive sites, and assessment of mucosal immunity. Knowledge gained in these studies will inform mucosal vaccine design and evaluation in human clinical trials.

Expression and characterization of an M cell-specific ligand-fused dengue virus tetravalent epitope using Saccharomyces cerevisiae

A fusion construct (Tet-EDIII-Co1) consisting of an M cell-specific peptide ligand (Co1) at the C-terminus of a recombinant tetravalent gene encoding the amino acid sequences of dengue envelope domain III (Tet-EDIII) from four serotypes was expressed and tested for binding activity to the mucosal immune inductive site M cells for the development of an oral vaccine. The yeast episomal expression vector, pYEGPD-TER, which was designed to direct gene expression using the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, a functional signal peptide of the amylase 1A protein from rice, and the GAL7 terminator, was used to clone the Tet-EDIII-Co1 gene and resultant plasmids were then used to transform Saccharomyces cerevisiae. PCR and back-transformation into Escherichia coli confirmed the presence of the Tet-EDIII-Co1 gene-containing plasmid in transformants. Northern blot analysis of transformed S.cerevisiae identified the presence of the Tet-EDIII-Co1-specific transcript. Western blot analysis indicated that the produced Tet-EDIII-Co1 protein with the expected molecular weight was successfully secreted into the culture medium. Quantitative Western blot analysis and ELISA revealed that the recombinant Tet-EDIII-Co1 protein comprised approximately 0.1-0.2% of cell-free extracts (CFEs). In addition, 0.1-0.2mg of Tet-EDIII-Co1 protein per liter of culture filtrate was detected on day 1, and this quantity peaked on day 3 after cultivation. In vivo binding assays showed that the Tet-EDIII-Co1 protein was delivered specifically to M cells in Peyer's patches (PPs) while the Tet-EDIII protein lacking the Co1 ligand did not, which demonstrated the efficient targeting of this antigenic protein through the mucosal-specific ligand.

Neutrophils negatively regulate induction of mucosal IgA antibody responses to sublingual immunization (MUC4P.835)

Abstract Secretory IgA antibody plays an important role in the protection of mucosal surfaces against microbial pathogens. Induction of the IgA antibody has been known to be largely dependent upon the cytokine milieu. However, the importance of innate immune cell subsets for IgA responses has not been fully appreciated yet. We investigated the role of myeloid cell-subsets on IgA responses to sublingual immunization. In contrast with cholera toxin as an adjuvant which promotes IgA responses, sublingual application of the Bacillus anthracis edema toxin (EdTx) to C57BL/6 mice transiently increased the frequencies of neutrophils in sublingual tissue and cervical lymph nodes (CLNs), and promoted antigen-specific serum IgG, but not serum or mucosal IgA responses. However, EdTx as an adjuvant failed to increase frequencies of neutrophils in sublingual tissue and CLNs of mice lacking IKKβ in myeloid cells (IKKβΔMye), and these mice developed antigen-specific mucosal IgA responses. Interestingly, depletion of neutrophils in C57BL/6 mice promoted antigen-specific fecal and serum IgA responses, similar to those of IKKβΔMye mice, and this effect was associated with increased frequencies of surface IgA expressing plasma cells, α4β7+CCR9+ B cells in lamina propria and number of Peyer’s patch. Our results show that the presence of neutrophils at mucosal inductive sites negatively influences induction of IgA responses and underlying mechanisms are under investigations.

Characterization of Nasal Cavity‐Associated Lymphoid Tissue in Ducks

The nasal mucosa is involved in immune defense, as it is the first barrier for pathogens entering the body through the respiratory tract. The nasal cavity-associated lymphoid tissue (NALT), which is found in the mucosa of the nasal cavity, is considered to be the main mucosal immune inductive site in the upper respiratory tract. NALT has been found in humans and many mammals, which contributes to local and systemic immune responses after intranasal vaccination. However, there are very few data on NALT in avian species, especially waterfowl. For this study, histological sections of the nasal cavities of Cherry Valley ducks were used to examine the anatomical location and histological characteristics of NALT. The results showed that several lymphoid aggregates are present in the ventral wall of the nasal cavity near the choanal cleft, whereas several more lymphoid aggregates were located on both sides of the nasal septum. In addition, randomly distributed intraepithelial lymphocytes and isolated lymphoid follicles were observed in the regio respiratoria of the nasal cavity. There were also a few lymphoid aggregates located in the lamina propria of the regio vestibularis, which was covered with a stratified squamous epithelium. This study focused on the anatomic and histological characteristics of the nasal cavity of the duck and performed a systemic overview of NALT. This will be beneficial for further understanding of immune mechanisms after nasal vaccination and the development of effective nasal vaccines for waterfowls.

Novel vaccine regimen elicits strong airway immune responses and control of respiratory syncytial virus in nonhuman primates.

Induction of long-lasting immunity against viral respiratory tract infections remains an elusive goal. Using a nonhuman primate model of human respiratory syncytial virus (hRSV) infection, we compared mucosal and systemic immune responses induced by different DNA delivery approaches to a novel parenteral DNA prime-tonsillar adenoviral vector booster immunization regimen. Intramuscular (i.m.) electroporation (EP) of a DNA vaccine encoding the fusion protein of hRSV induced stronger systemic immune responses than intradermal EP, tattoo immunization, and conventional i.m. DNA injection. A single EP i.m., followed by two atraumatic tonsillar immunizations with the adenoviral vector, elicited strong systemic immune responses, an unique persistent CD4(+) and CD8(+) T cell response in the lower respiratory tract and protection from intranasal hRSV challenge. Thus, parenteral DNA priming followed by booster immunization targeted to a mucosal inductive site constitutes an effective vaccine regimen for eliciting protective immune responses at mucosal effector sites. The human respiratory syncytial virus (hRSV) is the most common cause of severe respiratory tract disease in infancy and leads to substantial morbidity and morality in the elderly. In this study, we compared the immunogenicity and efficacy of several gene-based immunization protocols in rhesus macaques. Thereby, we found that the combination of an initially parenterally delivered DNA vaccine with a subsequent atraumatic tonsillar adenoviral vector immunization results in a strong systemic immune response accompanied by an exceptional high T-cell response in the mucosa. Strikingly, these animals were protected against a RSV challenge infection controlling the viral replication indicated by a 1,000-fold-lower viral load in the lower respiratory tract. Since mucosal cellular responses of this strength had not been described in earlier RSV vaccine studies, this heterologous DNA prime-tonsillar boost vaccine strategy is very promising and should be pursued for further preclinical and clinical testing.