Induction Of Mucosal Immunity Research Articles

I24 Innate and adaptive immunity in host-microbiota mutualism

The mammalian intestine harbours one of the most dense microbial communities on the planet. Experiments that compare germ-free and colonised mice show that the mucosal immune system is highly adapted to the presence of the commensal microbiota. To induce mucosal immune responses to commensal intestinal bacteria, small numbers of commensals are sampled by dendritic cells (DC) at the epithelial surface. These locally induce IgA B cells through T-dependent and T-independent pathways, but they do not penetrate beyond the mesenteric lymph nodes to reach systemic secondary lymphoid structures. Experiments where colonisation of the intestine has been uncoupled from mucosal immune induction with live commensals show that the resulting response is very long-lived unless another response is induced by stimulation with a different commensal microbe. Adaptive immunity in pathogen-free experimental mice is normally ignorant of intestinal commensals. Nevertheless, there is a threshold set by innate immunity, which can strengthen the epithelial barrier and allow clearance of small numbers of commensals that escape the permeability barrier or are released by ,commensal loaded’ DC. Defective innate clearance is compensated functionally by increased systemic adaptive responses to intestinal commensals, showing a continuum between innate and adaptive immunity.

An IKKβ-IL-17 axis fine-tunes Ab responses to sublingual immunization (120.7)

Abstract The nature of immune cells recruited at the sites of antigen exposure and the cytokine microenvironment play a key role at shaping immune responses. Using a model sublingual vaccine with Bacillus anthracis edema toxin as an adjuvant, we show that alteration of IKKβ signaling in myeloid cells (IKKβΔMye) enhanced the magnitude of Ab and T cell responses, compared to control mice. While this model vaccine failed to induce S-IgA Abs in control mice, significant S-IgA Ab responses were induced in mucosal secretions of IKKβΔMye mice. We also established that the sublingual vaccine recruited lower numbers of CD11b+F4/80+Gr1high and CD11b+F4/80-Gr1high, but higher number of CD11b+F4/80+Gr1- cells in the sublingual tissues of IKKβΔMye mice. In contrast, higher numbers of CD11b+F4/80+Gr1high and CD11b+F4/80-Gr1high, but lower number of CD11b+F4/80+Gr1- cells were recruited in the sublingual tissues of control and IL-17A KO mice. Lack of IL-17A signaling did not affect the magnitude of Ab responses to the sublingual vaccine. However, the profile of B cell epitope responses was affected in IL-17A KO mice. Furthermore, CD11b+ cells isolated from IKKβΔMye mice assisted B cell-differentiation and induced production of IgA Abs by IL-17Rhigh B lymphocytes in vitro. Our results support the existence of an IKKβ-IL-17 axis for regulation of mucosal immunity and identify IKKβ signaling as a potential target for improving induction of mucosal immunity by sublingual vaccines.

Novel vaccine development strategies for inducing mucosal immunity

To develop protective immune responses against mucosal pathogens, the delivery route and adjuvants for vaccination are important. The host, however, strives to maintain mucosal homeostasis by responding to mucosal antigens with tolerance, instead of immune activation. Thus, induction of mucosal immunity through vaccination is a rather difficult task, and potent mucosal adjuvants, vectors or other special delivery systems are often used, especially in the elderly. By taking advantage of the common mucosal immune system, the targeting of mucosal dendritic cells and microfold epithelial cells may facilitate the induction of effective mucosal immunity. Thus, novel routes of immunization and antigen delivery systems also show great potential for the development of effective and safe mucosal vaccines against various pathogens. The purpose of this review is to introduce several recent approaches to induce mucosal immunity to vaccines, with an emphasis on mucosal tissue targeting, new immunization routes and delivery systems. Defining the mechanisms of mucosal vaccines is as important as their efficacy and safety, and in this article, examples of recent approaches, which will likely accelerate progress in mucosal vaccine development, are discussed.

Mucosal Adjuvant Potential of Quillaja saponins and Cross-linked Dextran Microspheres, Co-administered with Liposomes Encapsulated with Tetanus Toxoid.

Intranasal vaccination is particularly a striking route for mucosal immunization, due to the ease of administration and the induction of both mucosal and humoral immunity. However, soluble antigens (Ag) are not sufficiently taken up after the nasal administration and need to be co-administered with adjuvants, penetration enhancers or encapsulated in particles. So, in this study, tetanus toxoid (TT) as a model Ag was entrapped in nonionic liposomes. The effect of the co-administration of Quillaja saponin (QS) as an adjuvant and cross-linked dextran microspheres (CDM) as penetration enhancer on immune responses was also studied. TT or TT + QS loaded liposomes were prepared by dehydration-rehydration method (DRV), followed by the extrusion through 400 nm filters. Some formulations were mixed with CDM. Liposomes were first characterized for their size range, mean diameter and morphology using particle size analyzer, optical and transmission electron microscopes. The volume mean diameter of liposomes was determined as 3836 ± 179 and 624 ± 114 nm before and after the extrusion, respectively. Structural efficiency of TT extracted from liposomes was confirmed by SDS-PAGE method. Encapsulation efficiencies of TT and QS were 44 ± 8.50% and 60 ± 6.02%, respectively. Rabbits were nasally immunized with various formulations and serum IgG titers and nasal lavage sIgA titers were determined by an ELISA method. TT + QS liposomes induced higher sIgA levels in comparison with TT liposomes (p < 0.05), but the difference in serum IgG levels was not significant. Results indicated that neutral liposomes administered nasally, have a good potential for induction of mucosal immunity and co-encapsulation of QS and TT in liposomes improved the systemic and mucosal immune responses.

Adjuvant effect of Korean mistletoe lectin on mucosal immunity induction following intranasal immunization with hemagglutinin antigen

To determine Korean mistletoe lectin (KML-C) functions as a mucosal adjuvant, we monitored its ability to modulate the immune response after intranasal immunization with hemagglutinin (HA) as an antigen. Mucosal and systemic immune responses with KML-C were compared to responses with cholera toxin B subunit (CTB) as an adjuvant. Intranasal co-administration of HA and KML-C in mice resulted in increased anti-HA antibody titers in both vaginal wash and serum. Isotyping analysis revealed that KML-C augmented HA-specific antibody titers of IgG1, IgG2a, and IgG2b isotypes. More than 5-fold of interleukin 2 (IL-2), 3-fold of interferon-γ (IFN-γ), and 4-fold of granulocyte macrophage colony-stimulating factor (GM-CSF) mRNA were detected using real time PCR in splenocytes of mice immunized with HA plus KML-C. Fluorescence activated cell sorting (FACS) analysis indicated that co-administration of KML-C increased the subpopulation of CD3/CD8 double positive T cells (16.4%) than control (10%). Taken together, these data demonstrate that KML-C has the ability to serve as a mucosal adjuvant.

Induction of Influenza-Specific Mucosal Immunity by an Attenuated Recombinant Sendai Virus

BackgroundMany pathogens initiate infection at the mucosal surfaces; therefore, induction of mucosal immune responses is a first level of defense against infection and is the most powerful means of protection. Although intramuscular injection is widely used for vaccination and is effective at inducing circulating antibodies, it is less effective at inducing mucosal antibodies.Methodology/Principal FindingsHere we report a novel recombinant, attenuated Sendai virus vector (GP42-H1) in which the hemagglutinin (HA) gene of influenza A virus was introduced into the Sendai virus genome as an additional gene. Infection of CV-1 cells by GP42-H1 resulted in cell surface expression of the HA protein. Intranasal immunization of mice with 1,000 plaque forming units (pfu) of GP42-H1 induced HA-specific IgG and IgA antibodies in the blood, brochoalveolar lavage fluid, fecal pellet extracts and saliva. The HA-specific antibody titer induced by GP42-H1 closely resembles the titer induced by sublethal infection by live influenza virus; however, in contrast to infection by influenza virus, immunization with GP42-H1 did not result in disease symptoms or the loss of body weight. In mice that were immunized with GP42-H1 and then challenged with 5LD50 (1250 pfu) of influenza virus, no significant weight loss was observed and other visual signs of morbidity were not detected.ConclusionsThese results demonstrate that the GP42-H1 Sendai virus recombinant is able to confer full protection from lethal infection by influenza virus, supporting the conclusion that it is a safe and effective mucosal vaccine vector.

The elucidation of the oral/respiratory axis in inducing protective immunity against Francisella tularensis (161.22)

Abstract Using a Francisella tularensis subspecies novicida live attenuated vaccine strain (U112ΔiglB), we have shown that oral vaccination of C57BL/6 mice induces significant protective immunity against pulmonary challenge with the highly human virulent F. tularensis strain SCHU S4. This protection is primarily mediated by antigen-specific CD4+ T cells and IFN-γ production and is dependent on mucosal IgA expression. We are examining the mechanism(s) by which the oral/respiratory axis induces optimal protective immunity against F. tularensis. Intestinal M-cells play an important role in oral vaccination by trafficking antigens from the lumen to the lamina propria and in presentation of antigens to underlying APCs for the subsequent induction of mucosal immunity. We have shown that mCherry-labeled U112ΔiglB co-localizes to intestinal mouse M-cells by 90 minutes following vaccination. Localization to M-cells was further verified with mouse ileal loops. Translocation of U112ΔiglB has also been demonstrated through M-cell-like-cells in an in vitro transwell co-culture system similar to M-cell trophic enteric bacteria. Current studies are examining the functional importance of intestinal M-cells in induction of mucosal immunity for the rational development of an effective oral vaccine against pulmonary tularemia. These studies will provide information for the general design of oral vaccines using live attenuated bacterial strains.

Inductive/Effector Mechanisms for Humoral Immunity at Mucosal Sites

Inductive and effector functions in the human mucosal immune system in relationship to protective immunity to HIV are poorly defined. A broader review of vaccine-induced immunity in mucosal systems was undertaken. A series of questions are posed. The limited answers to the questions indicate our profound lack of knowledge of some of the broader issues of the induction of mucosal immunity. The questions posed include the following: Is there a common mucosal immune system? Is IgA the critical immunoglobulin class in mucosal protection? Will systemic administration of antigen stimulate mucosal immunity? Is there true sterilizing mucosal immunity? What is the duration of mucosal immunity? The focus on mucosal immunity in HIV of this conference highlights recent remarkable advances in our understanding of the early events in HIV pathogenesis at the mucosal surface. This review identifies areas for further research.

The M Cell-Targeting Ligand Promotes Antigen Delivery and Induces Antigen-Specific Immune Responses in Mucosal Vaccination

Oral mucosal immunization can induce protective immunity in both systemic compartments and the mucosa. Successful mucosal immunization depends on Ag delivery to the mucosal immune induction site. The high transcytotic activity of M cells within the mucosa makes these cells attractive targets for mucosal Ag delivery, although it remains unclear whether delivery of Ag to M cells only can guarantee the induction of effective immune responses. In this study, we evaluated the ability of an M cell-targeting ligand with adjuvant activity to induce immunity against ligand-fused Ag. We selected M cell-targeting ligands through biopanning of a phage display library against differentiated in vitro M-like cells and produced the recombinant Ags fused to the selected ligands using the model Ag. One of the selected peptide ligands, Co1, promoted the binding of ligand-fused Ag to mouse Peyer's patch M cells and human M-like cells that had been defined by binding with the M cell-specific and anti-GP2 Abs. In addition, Co1 ligand enhanced the uptake of fused Ag by immunogenic tissue in an ex vivo loop assay and in vivo oral administration experiments. After oral administration, the ligand-fused Ag enhanced immune responses against the fused Ag compared with those of the control Ag without ligand. In addition, this use of the ligand supported a skewed Th2-type immune response against the fused Ag. Collectively, these results suggest that the ligand selected through biopanning against cultured M-like cells could be used as an adjuvant for targeted Ag delivery into the mucosal immune system to enhance immune induction.

경구백신의 효율적인 적용을 위한 면역 보조제 개발

Abstract Vaccine is one of the best known and most successful applications of immunological theory to human health and it protects human life through inducing the immune response in systemic compartment. However, when we consider the fact that mucosal epithelium is exposed to diverse foreign materials including viruses, bacteria, and food antigens and protects body from entry of unwanted materials using layer of tightly joined epithelial cells, establishing the immunological barrier on the lining of mucosal surfaces is believed to be an effective strategy to protect body from unwanted antigens. Unfortunately, however, oral mucosal site, which is considered as the best target to induce mucosal immune response due to application convenience, is prone to induce immune tolerance rather than immune stimulation. Since intestinal epithelium is tightly organized, a prerequisite for successful mucosal vaccination is delivery of antigen to mucosal immune induction site including a complex system of highly specialized cells such as M cells. Consequently, development of efficient mucosal adjuvant capable of introducing antigens to mucosal immune induction site and overcome oral tolerance is an important subject in oral vaccine development. In this review, various approaches on the development of oral mucosal adjuvants being suggested for effective oral mucosal immune induction.

Multiple tandem copies of conserved gp41 epitopes incorporated in gag virus-like particles elicit systemic and mucosal antibodies in an optimized heterologous vector delivery regimen

Induction of neutralizing antibodies to prevent HIV infection, especially at the mucosa, is a critical goal of future vaccines. In this study, we have designed chimeric HIV-gag virus-like particles (VLPs) that contain multiple copies of the two highly conserved gp41 membrane-proximal external region (MPER) epitopes, ELDKWA and NWFDIT, with the objective of generating high titers of MPER-specific antibodies. We have shown that the implementation of optimized vector design, delivery regimens and appropriate delivery methods is critical to significantly increase epitope-specific antibody titers. One goal of the methods that were tested and employed was to generate high levels of mucosal MPER-specific antibodies, as mucosal immune induction could play a key role in preventing HIV infection. We also tested a design strategy that incorporated multiple repeats of the MPER epitopes within gag, which significantly increased specific antibody titers, systemically and mucosally. This alternative design strategy and the implementation of optimized heterologous immunization regimens can serve to ‘immuno-focus’ and significantly increase epitope-specific titers.

Cell-secreted Gp96-Ig-peptide complexes induce lamina propria and intraepithelial CD8+ cytotoxic T lymphocytes in the intestinal mucosa

Induction of mucosal immunity is critical for protection from enteric pathogens. Heat shock protein gp96 is one of the primary peptide and protein chaperones located in the endoplasmic reticulum. We reported previously that a cell-secreted gp96-Ig fusion protein (gp96-Ig) mediated strong systemic, antigen-specific CD8-CTL expansion in vivo. We now evaluate the mucosal immune response to stimulation by secreted gp96 using allogeneic NIH-3T3 transfected with ovalbumin (OVA) and gp96-Ig. A single intraperitoneal NIH-3T3-OVA-gp96-Ig immunization caused significant homing of OVA-specific TCR transgenic CD8 cells (OT-I) to Peyer's patches, to the intraepithelial compartment and to the lamina propria. Intraperitoneal immunization with cells secreting gp96-Ig provided stronger mucosal immunity than the same dose instilled vaginally or rectally or injected subcutaneously or intradermally. Our results provide the first evidence that cell-based gp96-Ig-secreting vaccines may serve as a potent modality to induce mucosal immunity.

Reverse micelle-encapsulated recombinant baculovirus as an oral vaccine against H5N1 infection in mice

Induction of mucosal immunity through oral immunization is an effective way to control influenza infection. In this study, baculovirus displaying influenza hemagglutinin was encapsulated within a reverse micelle structure of phosphatidylcholine and delivered into the gastrointestinal tract of mice to study its efficacy as an oral vaccine against cross-clade H5N1 infection. Mice vaccinated with encapsulated baculovirus displaying HA (En-BacHA) showed significantly enhanced HA specific serum IgG and mucosal IgA antibodies, and higher hemagglutination inhibition (HI) titers, when compared to its non-encapsulated form (BacHA). Estimation of serum neutralizing antibodies also indicated that En-BacHA formulation was able to induce strong cross-clade neutralization against heterologous H5N1 strains (clade 1.0, clade 2.1, clade 4.0 and clade 8.0). Further, mice vaccinated with En-BacHA alone were able to confer 100% protection against 5 MLD50 of HPAI heterologous H5N1 strain (clade 1). Inclusion of recombinant cholera toxin B subunit as a mucosal adjuvant in the vaccine formulation did not show any significant effect in both systemic and mucosal immune responses. Oral delivery of encapsulated recombinant H5 HA expressed on baculovirus surface is an effective way to prime the immune system against H5N1 infection in mice and will have no biosafety concerns associated with their production or administration.

Recombinant porcine rotavirus VP4 and VP4-LTB expressed in Lactobacillus casei induced mucosal and systemic antibody responses in mice

BackgroundPorcine rotavirus infection is a significant cause of morbidity and mortality in the swine industry necessitating the development of effective vaccines for the prevention of infection. Immune responses associated with protection are primarily mucosal in nature and induction of mucosal immunity is important for preventing porcine rotavirus infection.ResultsLactobacillus casei expressing the major protective antigen VP4 of porcine rotavirus (pPG612.1-VP4) or VP4-LTB (heat-labile toxin B subunit from Echerichia coli) (pPG612.1-VP4-LTB) fusion protein was used to immunize mice orally. The expression of recombinant pPG612.1-VP4 and pPG612.1-VP4-LTB was confirmed by SDS-PAGE and Western blot analysis and surface-displayed expression on L. casei was verified by immunofluorescence. Mice orally immunized with recombinant protein-expressing L. casei produced high levels of serum immunoglobulin G (IgG) and mucosal IgA. The IgA titters from mice immunized with pPG612.1-VP4-LTB were higher than titters from pPG612.1-VP4-immunized mice. The induced antibodies demonstrated neutralizing effects on RV infection.ConclusionThese results demonstrated that VP4 administered in the context of an L. casei expression system is an effective method for stimulating mucosal immunity and that LTB served to further stimulate mucosal immunity suggesting that this strategy can be adapted for use in pigs.

Mucosal adjuvant activity of oligomannose-coated liposomes for nasal immunization

In the present study, we investigated the effectiveness of liposomes coated with a neoglycolipid consisting of mannotriose and dipalmitoylphosphatidylcholine (Man3-DPPE) as an adjuvant for induction of mucosal immunity. Immunization of BALB/c mice with ovalbumin (OVA)-encapsulated Man3-DPPE-coated liposomes (oligomannose-coated liposomes; OMLs) by a nasal route produced high levels of OVA-specific IgG and IgA antibodies in serum of immunized mice 1 week after the last nasal immunization, whereas no significant serum antibody responses were observed in mice that received OVA in uncoated liposomes or OVA alone. Seven weeks after the last nasal immunization, nasal challenge with an excess amount of OVA in mice that had received OVA/OMLs led to an anamnestic response to the antigen that resulted in 5- to 10-fold increases of antigen-specific serum IgG and IgA antibodies. Only mice immunized nasally with OML/OVA secreted antigen-specific secretory IgA in nasal washes and produced interferon-gamma secreting cells in nasopharyngeal-associated lymphoreticular tissue. Taken together, these results show that nasal administration of OMLs induces mucosal and systemic immunity that are specific for the entrapped antigen in the liposomes. Thus, liposomes coated with synthetic neoglycolipids might be useful as adjuvants for induction of mucosal immunity.

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

Liposomes have been identified as effective immunological adjuvants and have potential for the intranasal and oral delivery of protein antigen. Anionic MLV liposomes were prepared by dehydration–rehydration method. For coating, liposomes were incubated in chitosan solution. Efficiency of coating was confirmed by the evaluation of FITC-labelled chitosan-coated liposomes using a fluorescent microscope. Liposomes morphology and size were studied by optical microscope and size analyzer. Mucoadhesion potential of liposomes was evaluated in human nose by gamma-scintigraphy using 99mTc-labelled liposomes. Rabbits (4 animals per group) were nasally immunized in weeks 0, 2 and 4 by liposomes encapsulated with 40 Lf TT. Bleedings and lavage collections were taken place in weeks 3 and 6, and IgG and sIgA titers were measured by ELISA method. Liposomes had a mean diameter of 2.38 μm. Loading of TT was 58.7 ± 12.4%. The mucoadhesion (clearance rate from nose) of both coated and non-coated liposomes was similar ( P > 0.05). Among the immunized animals, the highest nasal lavage sIgA titers were seen in non-coated liposomes followed by coated ones. The serum IgG titers (2nd bleeding) in animals immunized by both kinds of liposome were similar ( P > 0.05), and were lower than the TT solution group ( P < 0.05). Immunization by i.m. injection of TT solution resulted in the lowest sIgA and highest IgG titers ( P < 0.05) compared with liposomal groups. The results were indicative of good potential of negatively charged liposomes in the induction of mucosal immunity. Coating of liposomes by chitosan, failed to increase both the residence time of liposomes in nasal cavity and systemic responses. Conversely, coated liposomes could not induce the mucosal responses as efficiently as non-coated liposomes. It seems that the coating of liposomes affected their interaction potential with nasal associated lymphoid tissue cells.

Evaluation of Recombinant Influenza Virus-Simian Immunodeficiency Virus Vaccines in Macaques

There is an urgent need for human immunodeficiency virus (HIV) vaccines that induce robust mucosal immunity. Influenza A viruses (both H1N1 and H3N2) were engineered to express simian immunodeficiency virus (SIV) CD8 T-cell epitopes and evaluated following administration to the respiratory tracts of 11 pigtail macaques. Influenza virus was readily detected from respiratory tract secretions, although the infections were asymptomatic. Animals seroconverted to influenza virus and generated CD8 and CD4 T-cell responses to influenza virus proteins. SIV-specific CD8 T-cell responses bearing the mucosal homing marker beta7 integrin were induced by vaccination of naïve animals. Further, SIV-specific CD8 T-cell responses could be boosted by recombinant influenza virus-SIV vaccination of animals with already-established SIV infection. Sequential vaccination with influenza virus-SIV recombinants of different subtypes (H1N1 followed by H3N2 or vice versa) produced only a limited boost in immunity, probably reflecting T-cell immunity to conserved internal proteins of influenza A virus. SIV challenge of macaques vaccinated with an influenza virus expressing a single SIV CD8 T cell resulted in a large anamnestic recall CD8 T-cell response, but immune escape rapidly ensued and there was no impact on chronic SIV viremia. Although our results suggest that influenza virus-HIV vaccines hold promise for the induction of mucosal immunity to HIV, broader antigen cover will be needed to limit cytotoxic T-lymphocyte escape.

Modern Mucosal Vaccines, Adjuvants and Microbicides

Preventing infection at the pathogen portal of entry through induction of mucosal immunity and the use of microbicides has always been an exciting prospect. Moreover, the promise of needle-free prophylaxis is attractive for many reasons. This meeting report highlights some of the critical issues that were discussed concerning recent advances in the field. New routes of vaccination and modalities of delivery are still being discovered, and important advances are occurring in the development of safe mucosal adjuvants. Protection of mucosal surfaces is likely to be particularly crucial to prevent infections with pathogens, such as HIV, that can be sequestered rapidly.

The contribution of type I interferon signaling to immunity induced by alphavirus replicon vaccines

The type I interferon (IFN) system is critical for protecting the mammalian host from numerous virus infections and plays a key role in shaping the antiviral adaptive immune response. In this report, the importance of type I IFN signaling was assessed in a mouse model of alphavirus-induced humoral immune induction. Venezuelan equine encephalitis virus replicon particles (VRP) expressing the hemagglutinin (HA) gene from influenza virus (HA-VRP) were used to vaccinate both wildtype (wt) and IFN α/β receptor knockout (RKO) mice. HA-VRP vaccination induced equivalent levels of flu-specific systemic IgG, mucosal IgG, and systemic IgA antibodies in both wt and IFN RKO mice. In contrast, HA-VRP vaccination of IFN RKO mice failed to induce significant levels of flu-specific mucosal IgA antibodies at multiple mucosal surfaces. In the VRP adjuvant system, co-delivery of null VRP with ovalbumin (OVA) protein significantly increased the levels of OVA-specific serum IgG, fecal IgG, and fecal IgA antibodies in both wt and RKO mice, suggesting that type I IFN signaling plays a less significant role in the VRP adjuvant effect. Taken together, these results suggest that (1) at least in regard to IFN signaling, the mechanisms which regulate alphavirus-induced immunity differ when VRP are utilized as expression vectors as opposed to adjuvants, and (2) type I IFN signaling is required for the induction of mucosal IgA antibodies directed against VRP-expressed antigen. These results shed new light on the regulatory networks which promote immune induction, and specifically mucosal immune induction, with alphavirus vaccine vectors.

Nonmucosal Alphavirus Vaccination Stimulates a Mucosal Inductive Environment in the Peripheral Draining Lymph Node

The strongest mucosal immune responses are induced following mucosal Ag delivery and processing in the mucosal lymphoid tissues, and much is known regarding the immunological parameters which regulate immune induction via this pathway. Recently, experimental systems have been identified in which mucosal immune responses are induced following nonmucosal Ag delivery. One such system, footpad delivery of Venezuelan equine encephalitis virus replicon particles (VRP), led to the local production of IgA Abs directed against both expressed and codelivered Ags at multiple mucosal surfaces in mice. In contrast to the mucosal delivery pathway, little is known regarding the lymphoid structures and immunological components that are responsible for mucosal immune induction following nonmucosal delivery. In this study, we have used footpad delivery of VRP to probe the constituents of this alternative pathway for mucosal immune induction. Following nonmucosal VRP delivery, J chain-containing, polymeric IgA Abs were detected in the peripheral draining lymph node (DLN), at a time before IgA detection at mucosal surfaces. Further analysis of the VRP DLN revealed up-regulated alpha4beta7 integrin expression on DLN B cells, expression of mucosal addressin cell adhesion molecule 1 on the DLN high endothelia venules, and production of IL-6 and CC chemokines, all characteristics of mucosal lymphoid tissues. Taken together, these results implicate the peripheral DLN as an integral component of an alternative pathway for mucosal immune induction. A further understanding of the critical immunological and viral components of this pathway may significantly improve both our knowledge of viral-induced immunity and the efficacy of viral-based vaccines.