Mucosal immunization with DNA vaccines

DNA vaccines represent a new approach to the control of infectious disease. Both cellular and humoral immune responses are induced without the attendant concerns associated with live, attenuated vaccines. The vast majority of DNA vaccines are delivered by parental routes, which rarely elicit immune responses at the mucosal epithelia, the primary sites of pathogen transmission. In view of the importance of mucosal and regional lymph node immunity in the control of pathogens transmitted across the mucosal epithelia, a number of groups, including our own, have developed immunization strategies that target plasmid DNA to mucosal inductive sites associated with the lymphoid tissues of the respiratory, gastrointestinal, and genital tracts. Here, we describe the procedures for the formulation and delivery of plasmid DNA to mucosal inductive tissues and address the theoretical basis to selection of particular mucosal locations for the induction of effective immune responses.     

Transepithelial transport and mucosal defence II: secretion of IgA

In this second article on mucosal defence and transepithelial transport, Jean-Pierre Kraehenbuhl and Marian Neutra discuss the part played by a special class of antibody, polymeric IgA, in the protection of mucosal surfaces lining the digestive, respiratory and genital tracts, and the implications for mucosal vaccines. Polymeric IgA crosslinks luminal antigens or pathogens, thus preventing their interaction with epithelial cells. Following stimulation by antigen in the organized mucosal lymphoid tissue, effector B lymphocytes enter the circulation and migrate to distant mucosal or glandular sites, where they differentiate into polymeric-IgA-producing plasma cells. These antibodies reach the environment by transport across the epithelial cells of mucosal and glandular tissues.     

Collection,Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.     

Respiratory Mucosal Immunization with Reovirus Serotype 1/L Stimulates Virus-Specific Humoral and Cellular Immune Responses, Including Double-Positive (CD4+/CD8+) T Cells

Respiratory virus infections are a serious health challenge. A number of models that examine the nature of the respiratory immune response to particular pathogens exist. However, many pathogens that stimulate specific immunity in the lung are frequently not effective immunogens at other mucosal sites. A pathogen that is an effective respiratory as well as gastrointestinal immunogen would allow studies of the interaction between the mucosal sites. Reovirus (respiratory enteric orphan virus) serotype 1 is known to be an effective gut mucosal immunogen and provides a potential model for the relationship between the respiratory and the gut mucosal immune systems. In this study, we demonstrate that intratracheal immunization with reovirus 1/Lang (1/L) in C3H mice resulted in high titers of virus in the respiratory tract-associated lymphoid tissue (RALT). High levels of reovirus-specific immunoglobulin A were determined in the RALT fragment cultures. The major responding components of the bronchus-associated lymphoid tissue were the CD8(+) T lymphocytes. Cells from draining lymph nodes also exhibited lysis of reovirus-infected target cells after an in vitro culture. The present study also describes the distribution of transiently present CD4(+)/CD8(+) double-positive (DP) T cells in the mediastinal and tracheobronchial lymph nodes of RALT. CD4(+)/CD8(+) DP lymphocytes were able to proliferate in response to stimulation with viral antigen in culture. Furthermore, these cells exhibited lysis of reovirus-infected target cells after in vitro culture. These results establish reovirus 1/L as a viable model for future investigation of the mucosal immune response in the RALT and its relationship to the common mucosal immune system.     

Connexins in respiratory and gastrointestinal mucosal immunity

The mucosal lining forms the physical and chemical barrier that protects against pathogens and hostile particles and harbors its own population of bacteria, fungi and archea, known as the microbiota. The immune system controls tolerance of this population of microorganisms that have proven to be beneficial for its host. Keeping its physical integrity and a correct balance with the microbiota, the mucosa preserves its homeostasis and its protective function and maintains host’s health. However, in some conditions, pathogens may succeed in breaching mucosal homeostasis and successfully infecting the host. In this review we will discuss the role the mucosa plays in the defense against bacterial pathogens by considering the gap junction protein connexins. We will detail their implication in mucosal homeostasis and upon infection with bacteria in the respiratory and the gastrointestinal tracts.     

Host‐microbiome interactions in acute and chronic respiratory infections

Respiratory infection is a leading cause of global morbidity and mortality. Understanding the factors that influence risk and outcome of these infections is essential to improving care. We increasingly understand that interactions between the microbial residents of our mucosal surfaces and host regulatory systems is fundamental to shaping local and systemic immunity. These mechanisms are most well defined in the gastrointestinal tract, however analogous systems also occur in the airways. Moreover, we now appreciate that the host–microbiota interactions at a given mucosal surface influence systemic host processes, in turn, affecting the course of infection at other anatomical sites. This review discusses the mechanisms by which the respiratory microbiome influences acute and chronic airway disease and examines the contribution of cross‐talk between the gastrointestinal and respiratory compartments to microbe–mucosa interactions.     

Distinct mechanisms for cross-protection of the upper versus lower respiratory tract through intestinal priming

A main feature of the common mucosal immune system is that lymphocytes primed in one mucosal inductive site may home to distant mucosal effector sites. However, the mechanisms responsible for such cross-protection remain elusive. To address these we have used a model of local mucosal infection of mice with reovirus. In immunocompetent mice local duodenal priming protected against subsequent respiratory challenge. In the upper respiratory tract this protection appeared to be mainly mediated by specific IgA- and IgG2a-producing B cells, whereas ex vivo active effector memory CTL were found in the lower respiratory tract. In accordance with these findings, clearance of reovirus from the lower respiratory tract, but not from the upper respiratory tract, of infected SCID mice upon transfer of gut-primed lymphocytes depended on the presence of T cells. Taken together this study reveals that intestinal priming leads to protection of both the upper and lower respiratory tracts, however through distinct mechanisms. We suggest that cross-protection in the common mucosal immune system is mediated by trafficking of B cells and effector memory CTL.     

Humoral immune responses to microbial infections in the genital tract

Human reproductive tracts represent components of the mucosal immune system with unique features. Although secretory IgA is present, IgG is more abundant, and typical mucosa-associated lymphoid tissue for generating common mucosal immune responses is absent. Antibody responses to genital infections or to locally applied vaccines are usually modest, but alternative strategies for eliciting genital tract antibodies are being developed.     

Local delivery of CpG oligodeoxynucleotides induces rapid changes in the genital mucosa and inhibits replication,but not entry,of herpes simplex virus type 2

Mucosal surfaces are the entry sites for the vast majority of infectious pathogens and provide the first line of defense against infection. In addition to the epithelial barrier, the innate immune system plays a key role in recognizing and rapidly responding to invading pathogens via innate receptors, such as Toll-like receptors (TLR). Bacterial CpG DNA, a potent activator of innate immunity, is recognized by TLR9. Here, we confirm that local mucosal, but not systemic, delivery of CpG oligodeoxynucleotides (ODN) to the genital tract protects mice from a subsequent lethal vaginal herpes simplex virus type 2 (HSV-2) challenge. Since these effects were so local in action, we examined the genital mucosa. Local delivery of CpG ODN induced rapid proliferation and thickening of the genital epithelium and caused significant recruitment of inflammatory cells to the submucosa. Local CpG ODN treatment also resulted in inhibition of HSV-2 replication but had no effect on HSV-2 entry into the genital mucosa. CpG ODN-induced protection against HSV-2 was not associated with early increases in gamma interferon (IFN-gamma) secretion in the genital tract, and CpG ODN-treated IFN-gamma(-/-) mice were protected from subsequent challenge with a lethal dose of HSV-2. Treatment of human HEK-293 cells transfected with murine TLR9 showed that the antiviral activity of CpG ODN was mediated through TLR9. These studies suggest that local induction of mucosal innate immunity can provide protection against sexually transmitted infections, such as HSV-2 or possibly human immunodeficiency virus, at the mucosal surfaces.     

Mucosal immunity and vaccination.

The gut mucosal immune system is a critical component of the body's defense against pathogenic organisms, especially those responsible for enteric infections associated with diarrhoeal disease. Attempts to vaccinate against infections of mucosal tissues have been less successful than vaccination against systemic infections, to a large extent reflecting a still incomplete knowledge about the most efficient means for inducing protective local immune responses at these sites. Secretory IgA (SIgA) is the predominating immunoglobulin along mucosal surfaces, and SIgA antibodies generated in gastrointestinal, respiratory or genito-urinary mucosal tissues can confer protection against infections affecting or originating in these sites. An efficacious intestinal SIgA immunity-inducing oral vaccine against cholera has been developed recently, and development of oral vaccines against other enteric infections such as those caused by enterotoxigenic Escherichia coli, Shigella and rotaviruses is in progress as well. Based on the concept of a common mucosal immune system through which activated lymphocytes from the gut can disseminate immunity to other mucosal and glandular tissues, there is currently also much interest in the possibility of developing oral vaccines against infections in the respiratory and urogenital tracts. However, the large and repeated antigen doses often required to achieve a protective immune response still makes this vaccination approach impractical for many purified antigens. There is, therefore, a great need to develop strategies for enhancing delivery of antigen to the mucosal immune system as well as to identify mucosa-active immunostimulating agents (adjuvants). These and other aspects of mucosal immunity in relation to immunization and vaccine development are discussed in this short review article.     

Mucosal immunity and vaccination

Abstract The gut mucosal immune system is a critical component of the body's defense against pathogenic organisms, especially those responsible for enteric infections associated with diarrhoeal disease. Attempts to vaccinate against infections of mucosal tissues have been less successful than vaccination against systematic infections, to a large extent reflecting a still incomplete knowledge about the most efficient means for inducing protective local immune responses at these sites. Secretory IgA (SIgA) is the predominating immunoglobulin along mucosal surfaces, and SIgA antibodies generated in gastrointestinal, respiratory or genito-urinary mucosal tissues can confer protection against infections affecting or originating in these sites. An efficacious intestinal SIgA immunity-inducing oral vaccine against cholera has been developed recently, and development of oral vaccines against other enteric infections such as those caused by enterotoxigenic Escherichia coli, Shigella and rotaviruses is in progress as well. Based on the concept of a common mucosal immune system through which activated lymphocytes from the gut can disseminate immunity to other mucosal and glandular tissues, there is currently also much interest in the possibility of developing oral vaccines against infections in the respiratory and urogenital tracts. However, the large and repeated antigen doses often required to achieve a protective immune response still makes this vaccination approach impractical for many purified antigens. There is, therefore, a great need to develop strategies for enhancing delivery of antigen to the mucosal immune system as well as to identify mucosa-active immunostimulating agents (adjuvants). These and other aspects of mucosal immunity in relation to immunization and vaccine development are discussed in this short review article.     

CD8+ T-cell-mediated cross-clade protection in the genital tract following intranasal immunization with inactivated human immunodeficiency virus antigen plus CpG oligodeoxynucleotides

Human immunodeficiency virus (HIV) is a mucosally transmitted infection that rapidly targets and depletes CD4+ T cells in mucosal tissues and establishes a major reservoir for viral persistence in gut-associated lymphoid tissues. Therefore, vaccines designed to prevent HIV infections must induce potent and durable mucosal immune responses, especially in the genital tract. Here we investigated whether intranasal (i.n.) immunization with inactivated gp120-depleted HIV-1 antigen (Ag) plus CpG oligodeoxynucleotide (ODN) as an adjuvant induced local immune responses in the genital tract and cross-clade protection against intravaginal (IVAG) challenge. Lymphocytes isolated from the iliac lymph nodes (ILNs) and genital tracts of female mice i.n. immunized with HIV-1 Ag plus CpG showed significant HIV-specific proliferation and produced significantly higher levels of gamma interferon (IFN-gamma) and beta-chemokines than mice immunized with HIV-1 Ag alone or mixed with non-CpG ODN. CD8+ lymphocytes were dramatically increased in the genital tracts of mice immunized with HIV-1 Ag plus CpG, and protection following IVAG challenge with recombinant vaccinia viruses (rVVs) expressing HIV-1 gag was shown to be CD8 dependent. Finally, cross-clade protection was observed between clades A, C, and G but not B following IVAG challenge with rVVs expressing HIV-1 gag from different clades. These studies provide evidence that mucosal (i.n.) immunization induced strong local T-cell-mediated immune responses in the genital tract and cross-clade protection against IVAG challenge.     

Chlamydia and apoptosis: life and death decisions of an intracellular pathogen

The chlamydiae are important obligate intracellular prokaryotic pathogens that, each year, are responsible for millions of human infections involving the eye, genital tract, respiratory tract, vasculature and joints. The chlamydiae grow in cytoplasmic vesicles in susceptible host cells, which include the mucosal epithelium, vascular endothelium, smooth muscle cells, circulating monocytes and recruited or tissue-specific macrophages. One important pathogenic strategy that chlamydiae have evolved to promote their survival is the modulation of programmed cell death pathways in infected host cells. The chlamydiae can elicit the induction of host cell death, or apoptosis, under some circumstances and actively inhibit apoptosis under others. This subtle pathogenic mechanism highlights the manner in which these highly successful pathogens take control of infected cells to promote their own survival - even under the most adverse circumstances.     

Combined oral/nasal immunization protects mice from Sendai virus infection

Based on the concept of a common mucosal immune system wherein mucosal associated lymphocytes traffic among the various mucous membranes, the murine gastrointestinal tract was immunized with Sendai virus antigens in order to elicit a virus-specific immune response in the respiratory tract. Multiple intragastric (oral) administration of live or killed Sendai virus induced IgA and IgG antiviral antibodies in both gastrointestinal secretions and serum. When cholera toxin as an adjuvant was included along with virus, gut IgA and IgG as well as serum IgA responses were enhanced. Antiviral antibodies induced in respiratory secretions by oral killed virus plus cholera toxin, however, were variable and protection from virus challenge was not demonstrated. Significantly higher levels of respiratory antiviral antibodies were induced if immunization with oral killed Sendai virus/cholera toxin was combined with intranasal administration of small amounts of killed virus. The combined immunization also resulted in protection of both the upper and lower respiratory tracts from virus infection. Protection of the upper respiratory tract was correlated with the presence of IgA antiviral antibodies in nasal washings. On the other hand, protection of the lower respiratory tract was correlated with IgG antiviral antibodies in bronchoalveolar lavage fluids. Immunization with intranasal killed virus alone conferred partial protection to the lower respiratory tract and no protection to the upper respiratory tract. Thus, oral immunization with killed virus antigen could prime for a protective immune response in the murine respiratory tract and this protective response included IgA antibodies.     

Cutting edge: dichotomy of homing receptor dependence by mucosal effector B cells: alpha(E) versus L-selectin

The common mucosal immune system may be compartmentalized because lymphocyte homing to the upper respiratory tract appears to be mediated by L-selectin interactions rather than alpha(4)beta(7) interactions, as is the case for gut-associated lymphoreticular tissue. To assess the role of L-selectin in effector B cell immunity, L-selectin-deficient mice were intranasally immunized with cholera toxin (CT), and mucosal immune responses were compared with C57BL/6 mice. The absence of L-selectin correlated with a reduction in CT-specific secretory-IgA responses in nasal passages and reproductive tract, but not intestinal lamina propria. Cell sorting experiments showed that an L-selectin-dependent subset was responsible for CT-specific responses in nasal passages and reproductive tract, whereas an alpha(E)beta(7)(+) B cell subset was responsible for L-selectin-independent intestinal immunity. This study provides evidence for compartmentalization of the common mucosal immune system into "intestinal" vs "nonintestinal" effector sites.     

M-cells: origin, morphology and role in mucosal immunity and microbial pathogenesis

M-cells are specialized cells found in the follicle-associated epithelium of intestinal Peyer's patches of gut-associated lymphoid tissue and in isolated lymphoid follicles, appendix and in mucosal-associated lymphoid tissue sites outside the gastrointestinal tract. In the gastrointestinal tract, M-cells play an important role in transport of antigen from the lumen of the small intestine to mucosal lymphoid tissues, where processing and initiation of immune responses occur. Thus, M-cells act as gateways to the mucosal immune system and this function has been exploited by many invading pathogens. Understanding the mechanism by which M-cells sample antigen will inform the design of oral vaccines with improved efficacy in priming mucosal and systemic immune responses. In this review, the origin and morphology of M-cells, and their role in mucosal immunity and pathogenesis of infections are discussed.     

Murine Models of Candida Gastrointestinal Colonization and Dissemination

Ninety-five percent of infectious agents enter through exposed mucosal surfaces, such as the respiratory and gastrointestinal (GI) tracts. The human GI tract is colonized with trillions of commensal microbes, including numerous Candida spp. Some commensal microbes in the GI tract can cause serious human infections under specific circumstances, typically involving changes in the gut environment and/or host immune conditions. Therefore, utilizing animal models of fungal GI colonization and dissemination can lead to significant insights into the complex pathophysiology of transformation from a commensal organism to a pathogen and host-pathogen interactions. This paper will review the methodologic approaches used for modeling GI colonization versus dissemination, the insights learned from these models, and finally, possible future directions using these animal modeling systems.     

提高黏膜免疫的方法及研究进展

黏膜是很多病原体入侵机体的重要入口,黏膜疫苗能诱导产生黏膜保护性免疫应答和系统性免疫应答,阻止病原微生物黏附、入侵和繁殖。但多数候选黏膜疫苗的安全性、稳定性、免疫效力及保护作用还无法达到理想的效果,佐剂或载体的使用改善了黏膜疫苗存在的不足,使黏膜疫苗有了广阔的发展前景。文章综述了提高黏膜免疫的方法及研究进展。     

The impact of aging on innate and adaptive immunity in the human female genital tract

Mucosal tissues in the human female reproductive tract (FRT) are primary sites for both gynecological cancers and infections by a spectrum of sexually transmitted pathogens, including human immunodeficiency virus (HIV), that compromise women''s health. While the regulation of innate and adaptive immune protection in the FRT by hormonal cyclic changes across the menstrual cycle and pregnancy are being intensely studied, little to nothing is known about the alterations in mucosal immune protection that occur throughout the FRT as women age following menopause. The immune system in the FRT has two key functions: defense against pathogens and reproduction. After menopause, natural reproductive function ends, and therefore, two overlapping processes contribute to alterations in immune protection in aging women: menopause and immunosenescence. The goal of this review is to summarize the multiple immune changes that occur in the FRT with aging, including the impact on the function of epithelial cells, immune cells, and stromal fibroblasts. These studies indicate that major aspects of innate and adaptive immunity in the FRT are compromised in a site‐specific manner in the FRT as women age. Further, at some FRT sites, immunological compensation occurs. Overall, alterations in mucosal immune protection contribute to the increased risk of sexually transmitted infections (STI), urogenital infections, and gynecological cancers. Further studies are essential to provide a foundation for the development of novel therapeutic interventions to restore immune protection and reverse conditions that threaten women''s lives as they age.