REVIEWS Mucosal vaccines: the promise and the challenge Marian R. Neutra*‡ and Pamela A. Kozlowski* Abstract | Most infectious agents enter the body at mucosal surfaces and therefore mucosal immune responses function as a first line of defence. Protective mucosal immune responses are most effectively induced by mucosal immunization through oral, nasal, rectal or vaginal routes, but the vast majority of vaccines in use today are administered by injection. As discussed in this Review, current research is providing new insights into the function of mucosal tissues and the interplay of innate and adaptive immune responses that results in immune protection at mucosal surfaces. These advances promise to accelerate the development and testing of new mucosal vaccines against many human diseases including HIV/AIDS. Mucosal surfaces are enormous surface areas that are of mucosal T cells is labour intensive and technically vulnerable to infection by pathogenic microorganisms. challenging. As a result, only a few mucosal vaccines The adaptive immune system is designed to distinguish have been approved for human use in the United States antigens, pathogens and vaccines that enter the body or elsewhere. These include oral vaccines against polio- through mucosal surfaces from those that are introduced virus3, Salmonella typhi1, V. cholerae 1 and rotavirus4, directly into tissues or the bloodstream by injection and a nasal vaccine against influenza virus5. However, or injury. It is becoming increasingly clear that local research and testing of mucosal vaccines is currently mucosal immune responses are important for protection accelerating, stimulated by new information on the muco- against disease: for example, mucosal antibodies against sal immune system and by the threat of the mucosally Vibrio cholerae bacteria and cholera toxin are associated transmitted virus, HIV6,7. with resistance to cholera1. In this Review, we provide an overview of the events Mucosal immune responses are most efficiently within mucosal tissues that lead to protective mucosal induced by the administration of vaccines onto muco- immune responses, and we consider key biological and sal surfaces, whereas injected vaccines are generally technical aspects of mucosal vaccine design. We then poor inducers of mucosal immunity and are therefore summarize current progress in the development of less effective against infection at mucosal surfaces1,2. mucosal vaccines against HIV. Nevertheless, clinical vaccine research has been based largely on injection of antigens, and most vaccines Mechanisms of mucosal protection in use today are administered intramuscularly or Innate defences at mucosal surfaces. Mucosal surfaces sub cutaneously. This is understandable because an of the respiratory, gastrointestinal and urogenital tracts *GI Cell Biology Research injection delivers a known quantity of antigen into are separated from the outside world by delicate epithe- Laboratory, Children’s the body and results in the generation of specific anti- lial barriers. In the gastrointestinal tract, for example, a Hospital and Department bodies and lymphoid cells that are readily measured single layer of epithelial cells joined by tight junctions of Pediatrics, Harvard in blood samples. By contrast, our understanding faces a complex luminal environment that is rich in Medical School, Boston, of mucosal immunity and development of mucosal microorganisms. Epithelia and their associated glands Massachusetts 02115, USA. ‡Children’s Hospital, Enders vaccines has lagged behind, in part because admin- (such as the salivary glands) produce nonspecific 720, 300 Longwood Avenue, istration of mucosal vaccines and measurement of or innate defences including mucins and antimicrobial Boston, Massachusetts mucosal immune responses are more complicated. proteins8. Nevertheless, foreign antigens and micro- 02115, USA. The dose of mucosal vaccine that actually enters the organisms frequently breach the epithelial barrier and Correspondence to M.R.N. e-mail: marian.neutra@ body cannot be accurately measured because anti- mucosal tissues are sites of intense immunological childrens.harvard.edu bodies in mucosal secretions are difficult to capture activity. In the intestinal mucosa, dispersed lymphoid doi:10.1038/nri1777 and quantitate, and recovery and functional testing and antigen-presenting cells are particularly abundant; 148 | FEBRUARY 2006 | VOLUME 6 www.nature.com/reviews/immunol © 2006 Nature Publishing Group REVIEWS Antibody-dependent it has been estimated that there are more antibody- sterile environment, such as muscle, might be ‘ignored’ cell-mediated cytoxicity producing cells in the intestinal mucosa than in the when given mucosally, where the tissue is constantly (ADCC). A mechanism by spleen and lymph nodes combined9,10. exposed to microorganisms. which natural killer cells are Epithelial cells are active participants in muco- targeted to antibody-coated sal defence. They function as sensors that detect Adaptive immune protection at mucosal surfaces. cells, resulting in the lysis of the antibody-coated cells. dangerous microbial components through pattern- Diverse strategies are used by mucosal pathogens to recognition receptors such as Toll-like receptors (TLRs). infect humans. Some pathogens such as V. cholerae and They respond by sending cytokine and chemokine sig- enteropathogenic Escherichia coli cause disease by colo- nals to underlying mucosal cells, such as dendritic cells nizing epithelial surfaces. Pathogens such as rotavirus (DCs) and macrophages, to trigger innate, nonspecific and influenza virus infect the epithelium, whereas oth- defences and promote adaptive immune responses8,11,12. ers such as Shigella flexneri and S. typhimurium establish In the intestine, where bacteria are abundant, epithelial local infection in the lamina propria. Other pathogens, cells, together with intraepithelial lymphocytes and including HIV and S. typhi, use the intestinal mucosa underlying phagocytic cells, can modulate and dampen as a staging area for systemic spread of infection. these signals to prevent undesirable responses to non- Protection against such diverse threats involves multiple threatening nutrients and the normal intestinal flora immune effector strategies that operate on both sides of that could lead to mucosal inflammation13–15. Therefore, the epithelial barrier (FIG. 1). mucosal tissues are in a constant state of alert, but they An important characteristic of the mucosal adaptive are adapted to the presence of foreign microorganisms immune response is the local production and secretion and their products. As a result, vaccines that would of dimeric or multimeric immunoglobulin A (IgA) anti- produce vigorous immune responses if injected into a bodies that, unlike other antibody isotypes, are resistant to degradation in the protease-rich external environ- ments of mucosal surfaces. In humans, more IgA is Entrapment produced than all the other immunoglobulin isotypes 10 Lumen and clearance combined , and high concentrations of IgA antibodies (over 1 mg per ml) are present in the secretions that are 16,17 Block adherence sIgA associated with mucosal surfaces in normal humans . and invasion Epithelial cell Mucus The protease resistance of secretory IgA (sIgA) is a result of its dimerization and high degree of glycosylation during its synthesis in mucosal plasma cells18, and its association with a glycosylated fragment (the secre- Antigen DC tory component) derived from the epithelial polymeric capture Dimeric IgA immunoglobulin receptor (pIgR) that mediates transport by DCs of dimeric IgA across epithelial cells to the lumen19. B cell 2 Opsonization sIgA has multiple roles in mucosal defence . It promotes the entrapment of antigens or micro- Neutralization organisms in the mucus, preventing direct contact of pathogens with the mucosal surface, a mechanism that IgG ADCC is known as ‘immune exclusion’. Alternatively, sIgA of Egress the appropriate specificity might block or sterically Macrophage of cells NK cell hinder the microbial surface molecules that mediate 20 HEV epithelial attachment , or it might intercept incoming Egress of pathogens within epithelial-cell vesicular compart- antibody 2,19 CTL ments during pIgR-mediated transport . Interstitial fluids of mucosal tissues that underlie the epithelial barrier contain dimeric IgA that is synthesized by Lymph Fenestrated Blood local IgA-secreting plasma cells and this might prevent Cell-mediated capillary killing mucosal-cell infection, by mediating the transport of pathogens that have breached the epithelial barrier Figure 1 | Mechanisms of immune protection at mucosal surfaces. Multiple back into the lumen through pIgR21 or by mediating immune effector mechanisms contribute to protection at mucosal surfaces. antibody-dependent cell-mediated cytotoxicity (ADCC) Antigen-specific effector B and T cells in the bloodstream recognize mucosal that leads to the destruction of local infected cells10,22. high endothelial venules (HEVs) and enter the mucosa. Mucosal B cells terminally Local IgG synthesis also can occur in the mucosal differentiate to become mucosal plasma cells, most of which produce dimeric IgA tissues following the administration of antigen or that is exported into secretions as secretory IgA (sIgA) to intercept antigens and vaccine to mucosal surfaces17,18,23,24. Large numbers of pathogens, and to prevent mucosal invasion. Neutralizing IgG is also present within mucosal tissues; mucosal IgG might be derived from local