Intranasal vaccines: forthcoming challenges

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Intranasal vaccines: forthcoming challenges Charalambos D. Partidos The mucosal epithelium of the upper respiratory tract constitutes an effective physical barrier to many pathogens. Its mucosal-associated lymphoid tissue is of particular importance for the protection and integrity of mucosal surfaces and the body’s interior. Understanding the factors that influence the induction and regulation of mucosal immune responses will facilitate the design of vaccines capable of eliciting the appropriate type of protective immune response.

Charalambos D. Partidos UPR 9021 CNRS Immunochimie des Peptides et des Virus Institut de Biologie Moleculaire et Cellulaire 15 rue René Descartes F-67084 Strasbourg Cedex France tel: 133 38 841 7028 fax: 133 38 861 0680 e-mail: h.partidos @ibmc.u-strasbg.fr

▼ Because the majority of human pathogens

gain entrance to the body via the mucosal surface, the importance of generating a ‘first line of defence’ by establishing pathogen-specific immunity at the site of entry has been well recognized. Over the past few decades, the intranasal route has received considerable attention for its potential for vaccine delivery. The administration of different vaccine formulations in rodent models has established methods for increasing their absorption via the nasal epithelium, protecting them from proteolytic degradation and inducing the desired type of immune response1. There are several advantages of using the nose as a route for immunization (Box 1) and an intense search to develop safe and immunogenic mucosal vaccines is underway worldwide. Nasal-associated lymphoid tissue and the induction of an active mucosal immune response Antigen uptake via the nose The nasal mucosa is the first site of contact with inhaled antigens, and therefore the role of nasalassociated lymphoid tissue (NALT), which is organized lymphoid tissue at the base of the nasal cavity2, is important to the defence of mucosal surfaces.This lymphoid tissue is considered to be the equivalent of the Waldeyer’s ring in humans3,

which consists of the adenoid or nasopharyngeal tonsils, the bilateral pharyngeal lymphoid bands, the bilateral tubal and faucial or palatine tonsils, and the bilateral lingual tonsils4. During the evolution of the mammalian mucosal immune system, NALT has adapted to fulfill several functions. For the protection of the nasal epithelium from colonization by invading pathogens and entry by inhaled antigens, barriers to macromolecular absorption, mechanisms of antigen sampling, production of secretory IgA (s-IgA) antibody responses, as well as means of distributing effector T- and B-cells to local and distant mucosal sites have been developed. In addition, mechanisms of tolerance have also evolved to prevent harmful allergic immune responses to inhaled antigens. The mucosal-associated lymphoid tissue (MALT) can be divided into two functionally distinct compartments or sites: (1) the inductive site, which is a defined lymphoid microcompartment, and (2) the effector site, which contains diffuse accumulations of large numbers of lymphoid cells that do not associate into apparently organized structures. The antigen is first encountered in the inductive sites and initial responses are induced. By contrast, in the effector sites, IgA plasma cells are found and the production of s-IgA antibodies result in local protection1. In the upper respiratory tract of rodents and humans, the major inductive sites are the NALT and the tonsils, respectively. These sites possess M cells, which are responsible for the sampling and transporting of antigens to the underlying lymphoid tissue5, germinal centers containing B- and T-cells, plasma cells and antigen presenting cells (APCs). These cells are involved in the regulation and induction of antigen-specific effector cells, which will ultimately mediate the protective humoral and cellular immune responses.

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Box 1. Reasons why the nose is an attractive route for immunization • • • • •

• •

Easily accessible Highly vascularized Presence of numerous microvilli covering the nasal epithelium generates a large absorption surface After intranasal immunization, both mucosal and systemic immune responses are induced Immune response can be induced at distant mucosal sites owing to the dissemination of effector immune cells in the common mucosal immune system Can be used for the immunization of large population groups Does not require needles and syringes, which are potential sources of infection

The balance between active immunity and tolerance greatly depends on the nature of the antigen and its interaction with the mucosal inductive sites, as well as on the dose, adjuvant, frequency of antigen administration and genetic background of the host2. Particulate antigens can be removed either by the mucociliary clearance system or can be sampled by specialized cells that are similar in appearance to microfold (M) cells, which overlay the NALT4 and are transported to the posterior cervical lymph nodes2. In the case of soluble antigens, they can penetrate the mucosal epithelium and reach the superficial cervical lymph nodes2, particularly when its permeability is increased by the cholera toxin or its B subunit6. High doses of antigen administered intranasally are likely to reach the intestinal tract7 or be drained directly by the posterior cervical lymph nodes instead of the superficial cervical lymph nodes2. The superficial cervical lymph nodes, which drain the nasal mucosa, appear to be instrumental in the induction of mucosal tolerance2. Posterior cervical lymph nodes are involved in the generation of s-IgA responses2. This suggests that the lymphocyte composition differs in different lymph nodes as a result of

Box 2. The significance of s-IgA in the defence of mucosal surfaces • • • •

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Most abundantly synthesized isotype in the body Resistant to endogenous protease activity, which makes it well suited to protecting the mucosa Provides protection against pathogens such as bacteria or viruses Neutralizes microbial toxins

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their ‘homing’ behavior, which underlies the observed divergence in the induction of either tolerance or immunity. Understanding the mechanisms that lead to mucosal immune responsiveness or tolerance will be crucial in formulating strategies to treat allergies or autoimmune diseases8,9 (induction of tolerance) or to develop effective mucosal vaccination strategies1 (induction of immunity). The induction of immune responses Following intranasal immunization, humoral and cellular immune responses can develop. After the antigen is sampled and passed through endocytic vesicles to the underlying lymphoid cells in the submucosa, antigen processing and presentation occurs. This results in the activation of T-cells, which in turn provide help to B-cells to develop into IgA plasma cells. Isolated NALT cells contain antigen-specific antibody-secreting cells that are predominantly of the IgA isotype10. In humans, there are two subclasses: IgA1 and IgA2. The nasal mucosa and lacrima glands contain mainly IgA1 plasma cells, whereas a greater proportion of IgA2 plasma cells are found in the lamina propria of the lower intestinal tract11. In mucosal defences12–15 (Box 2), s-IgA plays an important role. These functions appear to be facilitated by the high-affinity binding of s-IgA to mucus. Intranasal immunization induces s-IgA responses in a wider range of mucosal tissues than oral immunization. This is likely to be caused by the more promiscuous profile of homing receptors possessed by the circulating IgA-secreting cells induced after intranasal immunization16. Further, the type of vaccine antigen appears to affect the induction of memory s-IgA responses. Polysaccharide and protein antigens do induce s-IgA antibody responses, but in polysaccharide antigens these responses outbreak very quickly owing to the absence of memory B-cells11. Thus, for this type of antigen, continuous restimulation might be needed to ensure protection. For the induction of an effective mucosal immune response, the activation of T-cells is of paramount importance. They will provide: (1) help to B-cells to become IgA-producing cells, (2) release of cytokines and chemokines, which contribute to all aspects of mucosal immunity and (3) contribute to clearing viral infections. T-cells can be classified as CD41 and CD81 T-cells. The CD41 T-cells recognize peptides that are derived from the processing of foreign antigens by APCs (in the inductive sites these cells include the dendritic cells, macrophages and B-cells) in the context of class II major histocompatibility complex molecules (MHC). The activation of antigen-specific CD41 T-cells can lead to the secretion of a distinct array of cytokines depending on the tissue microenviroment17, the nature of the antigen and the cytokine milieu. There are two phenotypes of effector CD41 T-cells according to the cytokine profile released: (1) Th1, which results after infection with viruses or

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intracellular bacteria and produces IL-2, INFg and lymphotoxin, and (2) Th2, which is triggered by exogenous antigens and produces IL-4, IL-5, IL-6 and IL-10 (Ref. 18). Evidence provided from experimental work in mice suggests that the mucosal tissues favor the development of the Th2-type response11; in humans, however, it is not yet clear which type is preferable19. The CD81 T-cells recognize peptides derived from the processing of antigens in the context of class I MHC molecules, and they are the main effector cells responsible for clearing viral infections. They have been shown to be important during pulmonary influenza virus infection, promoting host recovery and virus clearance. The adoptive transfer of influenza-virusspecific CD81 T-cells20 or CD81 T-cell clones21 into lethally infected hosts results in a reduction in the pulmonary virus titre and the prevention of death. Similarly, effector CD81 T-cells protect mice from respiratory syncytial virus infection22. It is likely that CD81 T-cells also play a major role in the clearance of established respiratory virus infections in humans23. After the initial sensitization, antigen-specific B-lymphocytes and T-lymphocytes migrate from the NALT to regional lymph nodes and proceed via the lymph into the circulation, from which they ‘home’ distant mucosal effector sites, such as the lamina propria of the respiratory, gastrointestinal and reproductive tracts and glandular tissues (e.g. lacryma, salivary and mammary glands) where immune responses are being expressed1,24.This cell distribution from the inductive sites to the effector sites has been termed the common mucosal immune system24,25 and ensures that all mucosal surfaces are furnished with antigen-specific immunocompetent cells for protection. Anti-infection vaccines for intranasal immunization For the development of safer and better vaccines, much effort has been devoted to defining and producing protective antigens or epitopes from the appropriate pathogen. However, when these antigens were given intranasally they were shown to be weakly immunogenic, possibly because of their poor immunogenicity or the induction of immunological tolerance.To circumvent these problems, several strategies have been developed including delivery systems, adjuvants and targeting to the mucosal surfaces. Here we concentrate on several of the strategies that have shown promise for potential vaccine application against infectious diseases. Modulation of mucosal immune responses Bacterial enterotoxins Currently, there is a great need to develop new adjuvants that are safe for human use and that enhance immune responses to vaccine antigens26. In experimental animal models, the most potent mucosal adjuvants under investigation are the cholera

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toxin (CT) from Vibrio cholerae and the heat-labile enterotoxin (LT) from Escherichia coli. CT and LT are synthesized as multisubunit toxins composed of five B subunits (CTB and LTB, respectively) responsible for binding to cells via the GM1 ganglioside receptors.The B subunits are linked to an A subunit, which after binding and translocation into the cells, dissociates into A1 and A2 polypeptide chains. The A1 polypeptide is responsible for the ADP-ribosylation of the GTP-binding protein that activates adenyl cyclase and increases intracellular levels of adenosine 39, 59-monophosphate in epithelial cells27. As a consequence, water and chloride ions are released into the small intestine. Both CT and LT are strong mucosal immunogens and act as effective adjuvants to mucosally co-administered antigens by enhancing serum and secretory antibody responses28,29 or cytotoxic T-cell responses30,31. However, the enzymatic activity of these toxins in the gut epithelium (which results in a watery diarrhea) precludes them for human use. In addition, both toxins promote predominantly Th2-type responses that can exacerbate lung pathology rather than enhance protection. Thus, much effort has been devoted to dissociating their adjuvanticity from toxicity. The CTB and LTB subunits proved to be relatively poor adjuvants compared with the holotoxins32. An alternative approach was based on the genetic detoxification of these toxins, while retaining their ability to function as mucosal adjuvants. In LT the availability of the three-dimensional structure combined with the application of site-directed mutagenesis has resulted in the generation of various mutants with significantly reduced ADP-ribosylation activity and toxicity on Y-1 adrenal cells compared with the toxin33. One of these mutants bearing a single substitution of Ser-63 to Lys in the A subunit (LTK63) was shown to exert a strong adjuvant effect for serum IgG, s-IgA antibodies and cytotoxic T-cell responses after intranasal co-immunization with ovalbumin34 or measles synthetic peptides31,35. When LTK63 was compared with the LTB, it was found to be more immunogenic and a better adjuvant after intranasal immunization36. However, its adjuvanticity was less pronounced compared with LT31,35. Similarly, mutants of CT generated by a single amino acid substitution in the ADPribosyltransferase active center have been found to exert an adjuvant effect to intranasally co-immunized pneumococcal surface protein A. Serum and secretory antibody responses were enhanced and, more importantly, protection could be afforded against lethal challenge with capsular serotype-3 Streptococcus pneumoniae A66 (Ref. 37). The mechanism(s) by which these toxins exert their ad-juvant effect(s) are still not clear, and are likely to be complex. There appears to be strong evidence suggesting that ADPri-bosylating activity is important for adjuvanticity. Detoxified mutants of CT and LT with partial ADP-ribosylating activity were significantly more effective adjuvants than the 275

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enzymatically inactive mutants38. However, in the case of the CT mutant E112K, which lacks ADP-ribosyltransferase activity, the ad-juvanticity was a result of a direct effect on APCs and T-lymphocytes39.The fact that CT and LT use different receptors for binding suggests that they might target different cell populations, thus resulting in changes in their adjuvanticity. Recently, a fusion protein that combines the full enzymatic activity of the A1 subunit of CT with two Ig-binding domains of the staphylococal protein A (which allows targeting to B-cells) was shown to have mucosal adjuvanticity.This genetically engineered non-toxic construct enhances serum IgG and mucosal IgA antibody responses to antigens co-administered via the intranasal route40. Subsequent studies have demonstrated that its adjuvanticity is dependent on the ADP-ribosyltransferase and Ig-binding activities41. Cytokines The cytokine environment at the site of antigen delivery plays a critical role in the induction of immune responses. In the context of vaccine development, the activation of the appropriate phenotype of CD41 T-cells (Th1 or Th2) after intranasal immunization is an important priority. Cytokines such as IL-12, and IL-6 secreted by APCs, favor the development of Th1and Th2-type immune responses, respectively18. They could be combined with mucosal adjuvants and delivery systems to induce the desired type of immune responses42. For example, the intranasal co-immunization of IL-12 with tetanus toxoid (TT) was shown to enhance serum IgG and s-IgA anti-TT antibody responses43. Mucosal adjuvanticity was also demonstrated with IL-1, a cytokine through which CT appears to enhance APC activity44. Despite the potential of certain cytokines to modulate mucosal immune responses, it should be noted that cytokines (being predominantly pleotropic molecules) have complex effects that depend on the state of activation of different immunocompetent cells and the balance of other cytokines. CpG motifs Recently, bacterial DNA has been shown to induce B-cell proliferation, antibody secretion and Th1-type responses dominated by the IL-12 and INFg cytokines45,46. This was attributed to specific single-stranded oligonucleotide sequences containing unmethylated CpG dinucleotides (CpG motifs), which are far more common in bacterial DNA than in vertebrate DNA 47. A similar immunomodulating effect can be seen with synthetic oligodeoxynucleotides (ODN) containing CpG motifs46. Given these strong immunostimulatory properties, synthetic CpG ODNs have been extensively tested for therapeutic applications and for immunopotentiating activity to co-administered vaccine antigens48,49.When administered intranasally with recombinant HBsAg50 or formalin-inactivated influenza virus51, both antigen-specific serum IgG and s-IgA responses were en276

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hanced. In addition, when administered intranasally, CpG and CT were shown to act synergistically, inducing stronger immune responses than those observed with 10 times more of either adjuvant alone50. Because CpGs can be tolerated even at high concentrations without any toxic effects52, this enables the ease of production on a large scale at a minimum cost and can induce immune activation on human cells53. This makes them an attractive candidate for future clinical applications. Targeting antigens to the nasal mucosa Targeting antigens to the nasal epithelium, and particularly to the M cells, can be an advantageous approach to mucosal vaccine delivery. This is particularly important because M cells are specialized for sampling antigens and delivering them to the immunocompetent cells of the underlying NALT7. However, this will require a good knowledge of specific carbohydrate receptors on the surface of the M cells and their precise functional role in order to be used as targets to breach the barrier of the nasal epithelium. In recent studies by Giannasca et al.54, M-cell-selective lectins have been identified that when administered intranasally in hamsters, stimulated immune responses to the conjugated test antigen. An alternative approach for targeting antigens to the nasal epithelium is to use adhesive molecules that are used by bacteria for mucosal colonization. For example, the fibronectinbinding protein I, which is responsible for mediating the binding of Streptococcus pyogenes to epithelial cells, was shown to potentiate immune responses to conjugated ovalbumin (OVA) after intranasal immunization55. In particular, it enhanced the serum and the secretory antibody responses to OVA and generated OVA-specific cytotoxic T-cell responses. The role of the epithelial lining of the nasal cavity in sampling antigens is beginning to become clear, and hopefully this will facilitate the design of new strategies for more effective delivery of vaccines via the nose. Antigen delivery systems for intranasal immunization Typically, for the induction of an effective immune response after mucosal immunization, higher doses of antigen are required compared with systemic immunization. This could be caused by proteolytic degradation or poor absorption by the nasal epithelium, which results in poor bioavailability of the antigen. To overcome this problem, strategies using delivery systems have been developed. These delivery systems protect the antigen from degradation and present it to the immunocompetent cells. Replicating delivery systems The development of vaccines based on recombinant bacteria or viruses expressing protective proteins or epitopes from other

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pathogens can be advantageous for intranasal immunization. This stategy is based on the presentation of an antigen in the context of a live virus or bacteria infection, resulting in a much broader humoral and cellular immune response to the corresponding pathogen. For such a strategy to be successful it is important that the vector: (1) is a live attenuated organism with mucosal tropism, (2) adequately expresses the foreign antigen, and (3) does not revert to virulence. Because these vectored vaccines are live, they are likely to share the same antigen processing and presentation pathway as the pathogen, resulting in the desired type of immune response. They also induce long-lasting systemic and mucosal immunity, are easily administered, inexpensive and can be produced in large quantities: these are factors that are important for large-scale immunization programs in developing countries. Several bacterial species have been attenuated and tested as vector systems for mucosal vaccine delivery56. Among them, the Bacille Calmette-Guerin (BCG) of Mycobacterium bovis has received considerable attention as a vector system for intranasal immunization because it is currently one of the most widely used human vaccines in the world57. There are several features that makes this vector attractive for the mucosal delivery of antigens: (1) BCG binds specifically to M cells, (2) it can be administered at any time after birth even in the presence of maternal antibodies, (3) it has potent immunostimulatory properties, (4) it is heat stable and inexpensive to produce, and (5) it can induce, over the long-term, protective systemic IgG and a highly sustained secretory IgA response, which is disseminated throughout the mucosal immune system58. More recently, a highly attenuated Shigella flexneri vector was used to deliver intracellularly plasmid-DNA-encoding measles proteins after intranasal immunization59. A vigorous anti-measlesspecific humoral and cellular response was induced. The advantage of this vector system is that it can directly deliver the plasmid DNA into APCs60. Amongst viral vectors, the most widely tested virus is the vaccinia. Various genes encoding for protective antigens have been inserted into the genome of vaccinia and tested for immunogenicity and protective capacity61. Vaccinia recombinants have several attractive features for vaccine delivery: (1) there is previous experience with the smallpox vaccine, (2) there is a large capacity for inserted genes62, (3) as a live vector, vaccinia can stimulate both cellular and humoral immune responses63,64, (4) the induced immunity can be long lasting, and (5) it can be administered via the mucosa65. Adenovirus strains have also been extensively studied as vectors for vaccine delivery66, and more recently for gene therapy67. Their high cloning capacity, stability, ability to grow in high titres, ease of purification and tropism to mucosal epithelium makes them a potential candidate for vaccine

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delivery68. Furthermore, after intranasal immunization they have been shown to induce long-lasting memory cytotoxic T-cell responses in mucosal tissues69 and to enhance serum and s-IgA responses to the encoded antigen70. Despite the potential advantages of the live attenuated viral and bacterial vectors as vaccine delivery systems, their invasive capacity imposes several risks. The reversion to virulence is always a major concern for attenuated pathogens. Further, the effect of multiple use on vector efficacy has to be determined, although, in a recent report it has been demonstrated that previous immunity to the vector does not affect subsequent immune responses to the expressed antigen when the mucosal route of immunization is used71. The use of bacterial vectors, such as Salmonella or E. coli, to express glycoproteins can result in antigens with altered antigenic specificity caused by their inability to glycosylate. Nevertheless, attenuated recombinant live vectors have much promise for the mucosal delivery of antigens. By capitalizing on the advances in molecular biology, future efforts should focus not only on increasing the efficacy and safety of the live vectors, but also on achieving the expression of multiple epitopes from different pathogens in a single vector system. Non-replicating delivery systems An alternative approach to the use of attenuated viral or bacterial vectors for vaccine delivery is the use of non-replicating vectors. Therefore, one of the major concerns of reversion to virulence can be eliminated. For successful mucosal immunization, the non-replicating vector should: (1) protect the antigen from degradation, (2) allow its transport via the M cells to the underlying lymphoid tissue for presentation to the immunocompetent cells, and (3) promote the appropriate type of immune response required for protection. Amongst the various non-replicating vector systems, particles such as liposomes, immune-stimulating complexes (ISCOMs) and biodegradable microparticles have been extensively tested for vaccine delivery. Liposomes Liposomes are aqueous suspensions of spheroid vesicles, which are phospholipids organized in bilayer structures. They can be manufactured to vary in size or phospholipid composition, which are parameters that can have an important impact on the delivery of antigens within cells.The antigen can be encapsulated inside the aqueous compartment of the liposome or linked to the surface72. Their adjuvanticity can be further improved by formulating liposomes with cytokines, mucosal adjuvants such as CT, or immunomodulators72. Liposomes are safe to use in humans (e.g. they have been used for the parenteral administration of drugs), are non-immunogenic and 277

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preferentially target macrophages in vivo, which are professional APCs . The latter property might account for their immunopotentiating effect to various antigens73. The potential vaccine delivery capacity of liposomes has been demonstrated for protein and peptide antigens72 or DNA vaccines74 administered intranasally. In the case of DNA vaccines, cationic lipid complexes composed of a positively charged lipid with the plasmid DNA significantly enhanced both the humoral and cellular immune responses to the encoded antigen after intranasal delivery74. This was mainly attributed to the increase in expression of the encoded protein in mucosal tissues compared with the administration of naked plasmid DNA74. Clinical-scale, liposomemanufacturing processes and quality-control tests for purity, stability and safety have now been established, opening the door for licensing liposomes for vaccine delivery in humans. Immune-stimulating complexes Immune-stimulating complexes (ISCOMs) are negatively charged, cage-like structures. They are formed after mixing Quil A (a saponin extract from Quillaja saponaria Molina bark) with cholesterol and phospholipids, into which antigens with a hydrophobic moiety can be incorporated75. These formulations have been widely used in immunogenicity and protection studies after systemic and mucosal immunization76. One of the most striking properties of ISCOMs is their ability to induce strong cytotoxic T-cell responses76, which are typical of a Th1-type response. However, a major drawback of using ISCOMs as a vaccine delivery system in humans is their toxicity. It is known that saponins and Quil A elicit strong hemolytic and cytolytic activity even at low doses. As a result, current efforts have concentrated on isolating fractions of Quil A that retain adjuvanticity but are devoid of toxicity77. Biodegradable microparticles Particles based on polymers of the D,L or DL isomers of lactic and glycolic acids (PLG microparticles) have received considerable attention because of several interesting features that make them excellent candidates for vaccine delivery. (1) The polymers polylactide and polyglycolide undergo nonenzymatic hydrolysis producing lactic and glycolic acids that are eliminated in the body without causing any inflammation. PLG microparticles have proven to be safe for human use in drug delivery and absorbable suture material. (2) The degradation rate for polylactide is slower compared with polyglycolide. Thus, using mixtures of the two polymers at different ratios can be advantageous for vaccine development because the release rate of the entrapped antigen can be pre-determined with a direct effect on the immune response (single-dose vaccines). 278

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(3) The size of PLG microparticles can also influence the kinetics of the immune response. Small microspheres can be readily phagocytosed by APCs, thus giving a much faster onset of immune response compared with large microparticles78. (4) Immunogenicity studies using encapsulated viruses79, proteins78, peptides80,81 or DNA82 have confirmed the immunopotentiating properties of PLG microparticles; probably because of the direct delivery of antigen to the APCs78, both humoral and cellular (CD41 and CD81) responses can be induced. In the case of DNA vaccines it has been argued that the stability is greater when the plasmid DNA is bound on the surface of microparticles rather than when it is encapsulated.This is owing to the high shear that occurs during the encapsulation procedure. Recent data have demonstrated that cationic PLG microparticles can efficiently bind plasmid DNA via ionic interactions between the positively charged particle surfaces and the negatively charged phosphate backbone of the DNA83. The administration of plasmid DNA bound to the cationic PLG via the intramuscular route induced both humoral and cellular responses83. However, their potential for intranasal delivery has not yet been demonstrated. (5) PLG microparticles have been shown to be effective in delivering antigens via mucosal routes84. After the intranasal immunization of guinea pigs with TT adsorbed onto PLG microparticles, enhanced systemic and mucosal TT-specific antibody responses were observed as compared with the free toxoid 85. In another study, the intranasal administration of human parainfluenza type-3 virus encapsulated into PLG microparticles resulted in the induction of protective immune responses against a virus challenge86. Overall, there is a substantial body of experimental data that supports the view that microparticles are a promising vaccine delivery system for human use. However, there are several concerns with regard to the stability of the entrapped antigen during storage, the efficacy of microparticle uptake by the mucosal epithelium84 and the production of sterile preparations for human use. Conclusions The recent advances in the understanding of how the mucosal immune system works, combined with progress made in molecular biology and genetic engineering, have opened up new possibilities for the design of a novel generation of vaccines that can be administered via mucosal routes. Work in experimental animal models has shown that these new mucosal vaccines can indeed induce protective immune responses, and already several of them are being tested in human trials. Perhaps the major challenge for vaccine development lies in

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the delivery of antigens for the induction of optimum protective immune responses. It remains to be seen whether or not these new advances in vaccine development will eventually deliver new, safe, affordable and more effective vaccines for the control of infectious disease.

Differential homing commitments and cell surface differentiation markers. Eur. J. Immunol. 25, 322–327 17

influences over T-helper cell function. J. Exp. Med. 171, 979–996

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In the August issue of Drug Discovery Today…

Editorials – Money matters? by Raymond C. Rowe Isn’t combinatorial chemistry just chemistry? by Nick Hird Update – latest news and views Steering a course through the technology maze Tim Peakman and Yann Bonduelle

Designed chiral libraries for drug discovery Paul Beroza and Mark J. Suto

Inhibition of tumor angiogenesis by synthetic receptor tyrosine kinase inhibitors Li Sun and Gerald McMahon

Can peptide structures be mimicked? Nigel Beeley

The in silico world of virtual libraries Andrew R. Leach and Michael M. Hann

Monitor – new bioactive molecules, combinatorial chemistry, invited profile Products

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