Recent advances in mucosal vaccine development

Journal of Controlled Release 67 (2000) 117–128 www.elsevier.com / locate / jconrel

Review

Recent advances in mucosal vaccine development Hongming Chen* AstraZeneca R& D Boston, 128 Sidney Street, Cambridge, MA 02139, USA Received 28 October 1999; accepted 27 December 1999

Abstract Proper stimulation of the mucosal immune system is critical for the effective protection of mucosal surfaces against colonization and invasion of infectious agents. This requires administration of vaccine antigens directly to various mucosal sites. Due to the low absorption efficiency of mucosally delivered vaccines, however, almost all of the currently marketed vaccines are administered parentally. In addition, sub-optimal immune responses are frequently induced by mucosal immunization and the use of mucosal adjuvants is commonly required. As a result, development of successful mucosal vaccines depends largely on the improvement of mucosal antigen delivery and on the discovery of new and effective mucosal adjuvants. In this review, recent advances in both areas are briefly discussed.  2000 Elsevier Science B.V. All rights reserved.

1. Mucosal immune system The subepithelial regions of mucosal surfaces such as the gastrointestinal, respiratory, and genital tracts contain an abundance of immunocompetent cells such as B and T lymphocytes [1]. These cells are organized into mucosal-associated lymphoid tissues (MALT), which are the main components of the mucosal immune system [2]. This immune system of the mucosal surfaces has different functions than the systemic immune system [3]. Numerous studies have indicated that induction of systemic immunity through parental immunization can effectively clear systemic infections, but it usually fails to protect the mucosal surfaces [4]. Induction of the mucosal immune system, particularly induction of mucosal *Correspondence address: Transform Pharmaceuticals, 1 Kendall Square, Building 700, Suite 712, Cambridge, MA 02139, USA. Tel.: 11-617-621-7905; fax: 11-617-679-0982. E-mail address: [email protected] (H. Chen)

immunity at the site of infection, on the other hand, provides the main protection against mucosal infections [4,5]. The mucosal immune system differs in several other ways from the systemic immune system. Mucosal immunization frequently results in the stimulation of both mucosal and systemic immune responses, while systemic immunization typically only induces systemic responses without activating the mucosal immune system. Induction of mucosal responses leads to production of secretory IgA (sIgA) antibodies, which are not usually produced by systemic immunization [3]. The production of sIgA on the mucosal surfaces is a result of the local exposure of antigens to the mucosal-associated lymphoid tissues, especially those in the upper respiratory tract (nasal lymphoid tissue, or NALT), and the gastrointestinal tract (gutassociated lymphoid tissue, or GALT) [6]. Anatomically, the GALT consists of the Peyer’s patches, the appendix, and solitary lymph nodes in the

0168-3659 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 00 )00199-1

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gastrointestinal tract, and the NALT contains the palatine and the pharyngeal tonsils [6]. The epithelial surfaces of both the NALT and the GALT contain specialized antigen-sampling cells known as the M cells. These cells can transport antigens from the mucosal surfaces into the underlying lymphoid tissues [7]. After entering into the MALT, the antigens are rapidly internalized and processed by antigen presenting cells such as subepithelial dendritic cells and macrophages, and presented to B cells and T cells located in the MALT [7]. Upon sensitization by the antigens, B cells proliferate and switch to IgAcommitted cells. These B cells eventually leave the MALT and migrate through the systemic circulation to various mucosal sites, including the initial induction site for terminal differentiation to sIgA-producing plasma cells [7]. This process of sIgA production is illustrated in Fig. 1. Evidence from many studies has confirmed that stimulation of the mucosal immune system at one

mucosal site can lead to sIgA production in the local as well as distal mucosal surfaces [2]. For example, antigen stimulation of the Peyer’s patches in the gastrointestinal tract produced sIgA-producing B cells in the intestine, but also in the bronchi as well as the genito–urinary tract [3]. This inter-connected mucosal system of sIgA induction and production has been given the name common mucosal immune system, or CMIS [3]. Several recent studies also suggested the existence of sub-compartmentalization within the CMIS. That is, IgA-committed B cells originated from a mucosal inductive site may preferentially migrate to certain effector sites rather than to all mucosal surfaces non-selectively [2,8]. It appears that the upper aerodigestive tract is primarily supplied by IgA-committed B cells from the NALT, while the genito–urinary tract preferentially receives IgA-committed B cells from the lower digestive tract. The gastrointestinal tract is mainly supplied by IgA-committed B cells from the GALT [2,8]. This sub-compartmentalization within the CMIS has a significant impact on the choice for route of immunization. For instance, oral immunization may not be the optimal choice for inducing sIgA in the genital tract, because it is less effective than rectal immunization. On the other hand, oral immunization would be superior to rectal immunization for inducing gastrointestinal immunity.

2. Mucosal immunization

Fig. 1. Induction of sIgA following mucosal exposure to antigens. Figure adapted from Nugent et al., 1998 [3] with modifications.

Mucosal surfaces, such as the gastrointestinal, respiratory and genital tracts, are the principal sites of entry and colonization for many pathogens. In order to effectively protect these surfaces, the mucosal immune system needs to be properly activated through mucosal immunization. Mucosal immunization also offers many advantages compared to parental immunizations. It can enhance vaccine efficacy by inducing mucosal and systemic immunity simultaneously. It can increase vaccine safety and minimize vaccine adverse effects by avoiding direct contact between potentially toxic vaccine components and the systemic circulation. Mucosal immunization also reduces the need for trained personnel required for vaccine administration. Finally, mucosal

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immunization allows for easy administration of multiple vaccines [6]. Effective stimulation of mucosal immune system requires the fulfillment of the following two requirements. First, vaccine antigens need to be delivered efficiently to mucosal inductive lymphoid tissues. Second, immune responses induced within the lymphoid tissues need to be enhanced through coadministration of appropriate mucosal adjuvants. Recent advances in these two areas are briefly reviewed in the following sections.

2.1. Mucosal antigen delivery Various physiological barriers on the mucosal surfaces prevent efficient absorption of mucosally delivered vaccines into the CMIS lymphoid tissues. These barriers include enzymatic degradation of antigens, mechanical clearance of antigens from the mucosal surfaces, and low uptake efficiency of antigens by the antigen-sampling cells. As a result, a large antigen dose with multiple administration is usually required for induction of immune responses on the mucosal surfaces. New strategies that are successful in overcoming these physiological barriers will significantly improve vaccine mucosal delivery efficiency.

2.1.1. Live recombinant vectors Recombinant viral or bacterial vectors are live organisms genetically engineered to express foreign antigens. These organisms are attenuated through mutation to render them avirulent or greatly reduced in virulence, but are still capable of populating and invading the mucosal surfaces. Therefore, a single mucosal inoculation of these vectors at a moderate dose can replicate to a very large immunogenic dose in vivo. This leads to long-lasting strong immune responses [8]. In addition, different antigens can be potentially delivered with a single vector and thus achieving protection against several diseases with a single immunization [8]. Recombinant viral vectors that have been explored for mucosal vaccine delivery include vaccina, adenovirus, poxvirus, and herpes simplex virus (HSV), etc. [9]. Among these, adenovirus vectors have shown the most promise for inducing mucosal immune

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responses to the foreign antigens encoded. Intranasal immunization of mice with recombinant adenovirus capable of expressing HSV glycoprotein B induced antigen-specific antibody responses both mucosally and systemically. Long term cytotoxic T cell response was detected in respiratory and genital tissues [10]. Recombinant adenoviruses expressing a simian immunodeficiency virus (SIV) envelope protein administered intranasally or orally to rhesus macaques induced both mucosal and systemic responses. In addition, animals challenged vaginally with infectious SIV showed a decreased viral burden compared to unimmunized animals [11]. Papp et al. investigated immune responses in mice immunized with human adenovirus type 5 vectors expressing glycoprotein D of bovine herpesvirus type 1, and found that intranasal immunization stimulated high levels of glycoprotein D specific sIgA in the lung and nasal washes [12]. Similar to viruses, bacterial pathogens such as Salmonella have also been explored as potential mucosal vaccine carriers to deliver foreign antigens. Several preclinical studies indicated that attenuated Salmonella successfully induced both mucosal and systemic immune responses. For example, Nayak et al. showed that oral immunization of mice with a live recombinant Salmonella vaccine strain expressing pneumococcal surface protein A resulted in colonization of the Peyer’s patches, spleens, and livers, and stimulation of systemic and mucosal antibody responses. Immunized mice were resistant to a subsequent challenge of S. pneumoniae [13]. Oral immunization of outbred New Zealand white rabbits with the same recombinant Salmonella strain also induced significant antigen-specific antibodies in serum and in vaginal secretions [13]. Recombinant attenuated Salmonella expressing SIV capsid protein induced antigen-specific lymphoproliferative responses in rhesus macaques following intragastric immunization [14]. Despite the large number of preclinical successes of these live vectors, none has been able to induce reproducible immune responses in human clinical trials so far [15]. Significant improvements in vector design, antigen selection and expression, as well as antigen stability and localization need to be made before live vectors can be commercialized as vaccine delivery vehicles [15].

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2.1.2. Biodegradable polymeric particles Micro- and nano-particles made from biodegradable polymers have been shown to effectively encapsulate some vaccine antigens and protect them from mucosal degradation. The main advantage of these polymers is their biodegradability. Release of encapsulated antigens is controlled by degradation of the polymers. Polymer degradation rate can be tailored to release antigens over an extended period of time and thus reduces the frequency of vaccination required to establish a long term immunity. Many different types of biodegradable polymers have been studied for vaccine delivery. These have been reviewed in detail by Michalek et al. [16]. Among them, poly( D,L-lactide-co-glycolide) (PLG) polymer is studied most extensively due to its long and safe history of human use as resorbable sutures. The PLG polymer is biodegradable through hydrolysis to give endogenous metabolites lactic and glycolic acids. The release rate of an encapsulated antigen from PLG particles is controlled by particle degradation rate, which is in turn determined by the polymer composition and its molecular weight [17]. Studies in various animals have demonstrated the potential of PLG particles as mucosal vaccine carriers. Some of the recent studies are summarized in Table 1. For example, fimbriae from B. pertusiss

encapsulated in PLG particles given orally to mice induced serum and mucosal antibody responses. Immunized animals also showed protection against live bacterial challenges [18]. Baras et al. showed in their recent work that a single nasal or oral immunization of glutathione S-transferase of S. mansoni in PLG microparticles induced a long-lasting antigenspecific antibody response in mice, with a peak at 9–10 weeks following immunization [19]. This study demonstrated the feasibility of a longterm in vivo release of antigens from PLG particles. Water-soluble biodegradable polymers have also been explored as potential vaccine delivery vehicles. The biggest advantage of water-soluble polymers is the elimination of organic solvents from the antigen encapsulation process. Organic solvents can adversely affect antigen stability during encapsulation. One such water-soluble biodegradable polymer, chitosan, has gained much attention in recent years. Chitosan is a polysaccharide composed of glucosamine and N-acetylglucosamine copolymer. It is derived from chitin in crustacean shells by partial deacetylation [26]. Chitosan has mucoadhesive property and is a mucosal absorption enhancer [26]. Antigens such as B. pertussis filamentous haemagglutinin and recombinant pertussis toxin have been mixed with chitosan and administered to mice intranasally. Serum IgG

Table 1 Recent studies on PLG particles for mucosal antigen delivery Antigen studied

Animals used

Route of immunization

Immune responses

Reference

H. pylori lysate

Mice

Oral

Serum IgG sIgA in gut wash

[20,21]

S. mansoni glutathione S-trasferase

Mice

Oral and nasal

Serum neutralizing antibody

[19]

HSV glycoprotein D2

Mice

Nasal

Serum IgG sIgA in nasal wash, saliva, and vaginal wash

[22]

Rotavirus VP6 DNA vaccine

Mice

Oral

Specific serum antibodies Intestinal sIgA

[23]

Phosphorylcholine

Mice

Oral

Intestinal sIgA

[24]

Ricin toxoid

Mice

Nasal

Serum IgG, lung sIgA Protection against challenge

[25]

B. pertussis fimbriae

Mice

Oral

Serum IgG sIgA in saliva and stools IgG in vaginal washes Protection against challenge

[18]

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and secretory IgA responses in lung lavage and nasal washes were significantly improved compared to when chitosan was absent [27]. Chitosan nanospheres have also been used in mice to orally deliver plasmid DNA encoding peanut allergen for inducing immunoprotection against peanut allergy [28]. Other examples of water-soluble polymers are starch, dextran, and alginate [29–31]. Recently, novel starch microparticles grafted with biocompatible silicone polymers have been shown to effectively deliver human serum albumin or bovine serum albumin orally or intranasally. Both systemic and mucosal responses were induced [32,33].

2.1.3. Lipid particles Lipid particles have also been explored as protective carriers for mucosal vaccines. The most common form of lipid particles is a liposome. A liposome is a spherical vesicle made of concentric lipid bilayers encasing an aqueous core [34]. It can carry lipid-soluble drugs in its lipid bilayers and at the same time water-soluble drugs in its aqueous cores [34]. Liposomes have been used for mucosal vaccine delivery with some success [35–39]. In most cases, it has been shown that co-administration of antigens and adjuvants in liposomes can significantly improve immune responses [35,37–39]. For example, interleukin-12 enhanced serum antibody responses when it was co-administered intranasally with liposomes containing recombinant glycoproteins from bovine herpesvirus type 1 [35]. Other adjuvants such as monophosphoryl lipid A, cholera toxin B subunit, and granulocyte–macrophage colony-stimulating factor have also been shown to improve immune responses induced by liposomal antigens [37–39]. Stability of liposomes has traditionally limited their usefulness as vaccine carriers, particularly for oral delivery, where the liposomes themselves are susceptible to dissolution by intestinal detergents, and to degradation by intestinal phospholipases [34,40,41]. Techniques used to stabilize liposomes include varying lipid compositions in the liposomes [42] and polymerizing liposome membranes [40,43]. Polymerized liposomes contain phosphatidylcholines with conjugated diene groups in both 1- and 2-acyl chains [44]. These conjugated diene groups can be polymerized upon exposure to initiators to create cross-linked networks in the bilayer membranes

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[40,43,45]. Stability of the polymerized liposomes was successfully demonstrated in vitro as well as in vivo [43]. Potential of polymerized liposomes as mucosal vaccine carriers has been demonstrated with tetanus toxoid (TT) [46]. ISCOMs, or immune stimulating complexes, are three-dimensional cage-like structures of 30–70 nm in diameter and are formed by mixing lipids, cholesterol, and Quil A [47,48]. Quil A is a mixture of Quillaja saponins extracted from the bark of the tree Quillaja sapanaria Molina, and is a potent immunoadjuvant. Hydrophobic antigens can be incorporated into the ISCOMs spontaneously [47]. Incorporation of hydrophilic antigens, on the other hand, may require modification of the antigens before they can be inserted into ISCOMs [47]. Ever since the discovery of ISCOMs more than a decade ago [49], ISCOMs have been shown by many research groups to effectively induce antibody and cell-mediated immunity during parental as well as mucosal immunizations [50–56]. ISCOMs containing envelope proteins of respiratory syncytial virus (RSV) induced potent serum IgG responses after two intranasal administrations to BALB / c mice. Strong sIgA responses locally in the lungs and the upper respiratory tract, and remotely in the genital and the intestinal tracts were also induced [50]. Intranasal immunization of influenza A virus nucleoprotein in ISCOMs induced strong antibody responses and cellmediated immunity. Immunized mice were fully protected against an otherwise lethal challenge infection of an unrelated influenza virus subtype [54]. The main advantage of ISCOMs stems from the fact that ISCOM structure combines the adjuvanticity of the Quillaja saponin and the immunogenicity of the incorporated antigen in the same entity. Potential application of ISCOMs in humans are currently under clinical evaluation [57].

2.1.4. Edible vaccines Another creative strategy to delivery mucosal antigen has been made possible through incorporation of genes encoding antigens into plant species. The concept of using plants for production and delivery of mucosal antigens was first introduced by Arntzn et al. in 1992, where tobacco plants were genetically transformed with the gene encoding hepatitis B surface antigen [58]. The antigen pro-

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duced in the transgenic tobacco plants was shown to be similar to those used in the commercial vaccines derived from recombinant yeast. Since then, antigens such as rabies virus glycoprotein [59], Norwalk virus capsid protein [60], VP60 of rabbit hemorrhagic disease virus [61], and heat-labile enterotoxin B subunit of E. coli (LT-B) [62] and V. cholerae (CT-B) [63] have all been produced in transgenic plants such as tomatoes and potatoes. Immunogenicity of these plant-derived antigens has been demonstrated by feeding animals with the transgenic plants and inducing both systemic and mucosal responses [61–63]. In some cases, protection against subsequent challenges was observed [61,63]. In a recent human clinical trial, transgenic potatoes expressing LT-B were consumed by volunteers and induced specific anti-LT-B mucosal and systemic immune responses [64]. Despite these highly promising results, expression levels of foreign antigens in plant tissues remain quite low. Typically, a large amount of plant tissues needs to be consumed to receive an adequate dose of the antigens [65]. Improving antigen expression level without compromising the health of the transgenic plants, and co-expressing appropriate adjuvants with the antigens may be the keys to the eventual success of an edible vaccine.

2.1.5. Plasmid DNA Since the discovery early this decade that intramuscular injection of plasmid DNAs encoding an antigen can induce antibody and cell-mediated immune responses, the concept of using DNAs as vaccines has been extensively explored. When delivered intracellularly, plasmid DNA vaccines can induce a transient expression of the encoded antigen inside the host cell. This allows access for the encoded antigen to the cellular MHC class I antigen presenting pathway, which is responsible for inducing cell-mediated immunity. Therefore, in addition to antibody responses which are induced by the MHC class II antigen presenting pathway, DNA vaccines are capable of inducing cellular immune responses, which are especially effective against viral and intracellular bacterial pathogens. Indeed, preliminary experiments in mice and chickens suggested that DNA encoding influenza HA protein produced antibody as well as cellular immune responses mucosally

and systemically. Immunized mice showed protection against a subsequent intranasal challenge of influenza virus [66]. Oral administration of a DNA vaccine encoding rotavirus VP6 encapsulated in PLG microparticles also induced systemic and mucosal immunity in mice [67]. In a recent study by Sasaki et al. in mice, immune responses to intranasally and intramuscularly delivered DNA vaccines against human immunodeficiency virus (HIV) type 1 was compared with or without QS-21 as an added adjuvant. Both routes produced similar levels of cell-mediated immunity, but intranasal immunization induced higher intestinal sIgA response than intramuscular immunization [68]. QS21 enhanced the response levels both intranasally as well as intramuscularly [68]. Several problems remain for the future development of DNA vaccination. Significant safety concerns remain over the potential for DNA genomic integration [69]. Moreover, efficacy of DNA vaccines is yet to be proven in large animal models [69].

2.2. Mucosal adjuvants Mucosally delivered antigens are frequently not immunogenic. Adjuvants are therefore required to be co-delivered with the antigens in order to enhance the immune responses. Many substances have been shown to act as vaccine adjuvants [3], the majority of which have only been used in parental immunizations. A few of these agents have also demonstrated adjuvanticity by the mucosal routes [3]. These are summarized in Table 2.

2.2.1. Bacterial toxins Two bacterial toxins with proven mucosal adjuvanticity are cholera toxin (CT) produced by V. cholerae and heat-labile enterotoxin (LT) produced by E. coli. The two molecules have many features in common. Both have multisubunits with A and B components. Mucosal toxicity of both toxins originates from the A component which can be dissociated into two pieces A1 and A2. A1 catalyzes the ADP-ribosylation of the stimulatory GTP-binding proteins on the basolateral surfaces of the epithelial cells, resulting in an increase in intracellular levels of cAMP. The increased cAMP level then leads to

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Table 2 Substances with demonstrated mucosal adjuvanticity a Adjuvant

Animals tested

Route of immunization

Antigens tested

Bacterial toxins

Mice Mice Mice

Oral or intranasal Intranasal Intranasal

Mice

Oral

Human papillomavirus [70] H. influenzae membrane protein [71] Synthetic peptides of human and bovine RSV [72] Recombinant H. pylori urease [73]

IL-12

Mice

Intranasal

Influenza vaccine [91] Tetanus toxoid [92]

Muramyl dipeptide (MDP)

Mice

Oral, intrarectal, or intranasal Intraintestinal or intra-Peyer’s patch

Sendai virus and rotavirus [93]

Rats

N. gonorrhoeae porin protein [94]

CpG oligodinucleotides

Mice

Intranasal

Hepatitis B surface antigen [90]

Avridine

Mice Rats

Oral Intraintestinal

Killed influenza vaccine [95] Cholera toxin or procholeragenoid [96]

Monophsphoryl lipid A

Mice Rats

Intrajejunal Intraintestinal

Ovalbumin [97] Cholera toxin [98]

Alum

Mice

Intranasal

Tetanus toxoid [99]

MF59

Mice

Intranasal

Subunit influenza vaccine [100]

a

Adapted from Nugent et al. (1998) [3] with modifications.

secretion of water and electrolytes into mucosal lumen. Studies with many antigens such as virus-like particles from human papillomavirus type 16 [70], outer membrane protein P6 of nontypeable H. influenzae [71], synthetic peptides of the G-glycoprotein from human and bovine RSV [72], and recombinant H. pylori urease [73] have demonstrated that both CT and LT are capable of enhancing mucosal immune responses when delivered with the antigens. Nevertheless, the fact that both toxins induce luminal secretion limits their practical use as mucosal adjuvants. In fact, as little as 5 mg of purified CT administered orally can result in severe diarrhea in humans [74]. Several different strategies have therefore been explored in search for a safe alternative. One popular approach is to use site-specific mutagenesis to specifically change one amino acid in the protein sequences to render them less toxic while maintaining their adjuvanticities. Specific examples of CT and LT mutants include CTF61 [75], CTK7, CTK112 and CTE118 [76], LTR72 [77], LTK63 [78], and LTG192 [79,80]. A different approach was

taken by Lycke et al., where a fusion protein CTA1DD was constructed from the A1 subunit of CT and a dimer of an Ig-binding domain of S. aureus protein A [81]. This fusion protein targets B cell surface receptors instead of the GM1 ganglioside receptors on all nucleated cells. This modification increased the molecule’s specificity and decreased its toxicity. It was shown that the adjuvant ability of CTA1-DD to antigens such as keyhole limpet hemocyanin was comparable to that of intact CT [81].

2.2.2. CpG oligodinucleotides CpG oligodinucleotides have shown some promise as vaccine adjuvants in recent years. These are synthetic oligodinucleotides containing a CpG motif, i.e., a central unmethylated CpG dinucleotide preferentially flanked by two 59 purines and two 39 pyrimidines [82]. These oligodinucleotides have been shown to induce T-cell independent B cell proliferation, and to activate monocytes, macrophages, and dendritic cells [83,84]. CpG oligodinucleotides are strong adjuvants when delivered systemically with a variety of antigens [85–89]. Mucos-

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al adjuvanticity of CpG oligodinucleotides was demonstrated in a recent study where hepatitis B surface antigen was co-delivered intranasally to mice with CpG oligodeoxynucleotides or CT. The results indicated that CpG was superior to CT for the induction of humoral and cell-mediated systemic immunity, and for the induction of mucosal sIgA responses in the lung and in feces. In addition, synergy was observed between CpG and CT. When administered together, CT and CpG induced stronger responses than ten times more of either CT or CpG administered alone [90].

2.2.3. Other adjuvants with mucosal activities Several other adjuvants have shown mucosal activities. These include interleukin (IL)-12, muramyl dipeptide (MDP), avridine, monophosphoryl lipid A (MPL), aluminum salts, and MF59. The use of IL-12 as a mucosal vaccine adjuvant was examined by Arulanandam et al. [91], where mice were immunized intranasally with an influenza vaccine plus IL-12. The treatment resulted in elevated antibody levels in serum and in bronchoalveolar lavage fluids compared to animals receiving vaccine alone. Mice immunized with vaccine plus and IL-12 also exhibited decreased weight loss and improved survival rate after a lethal challenge of infectious influenza viruses [91]. Boyaka et al. also demonstrated the mucosal adjuvanticity of IL-12 by intranasally administering TT and IL-12 to mice. Enhanced systemic and mucosal responses were observed [92]. Mucosal adjuvanticity of MDP and its derivatives was demonstrated with antigens such as Sendai virus and rotavirus [93], and N. gonorrhoeae porin proteins [94]. For example, N. gonorrhoeae major outer membrane porin proteins were purified and immunized with MDP to rats intraintestinally and subsequently induced mucosal sIgA and serum IgG responses [94]. Mucosal adjuvant effect of MDPLys(L18), a derivative of MDP, was demonstrated by its ability to enhance host resistance against mucosal challenges of Sendai virus and rotavirus in mice after its co-administration with either of the viruses mucosally [93]. Avridine, a synthetic lipoidal amine [N,N-dioctadecyl-N9,N9 -(2-hydroxymethyl) propanediamine], has shown mucosal adjuvanticity in several studies

when it was incorporated in liposomes [95,96]. For instance, liposomes containing avridine co-administered orally with killed influenza viruses in mice induced sIgA production in the respiratory tract [95]. Co-administration of avridine-containing liposomes intraintestinally to rats also enhanced mucosal immune responses induced by cholera toxin or procholeragenoid [96]. MPL is a derivative of bacterial lipopolysaccharide (LPS), which is a component of the gramnegative bacterial outer membranes. LPS has strong adjuvant effects on the immune system, including stimulation of macrophages, B cells, and antigen presenting cells [3]. However, it is too toxic for human use. MPL maintains many of the adjuvant activity of LPS but is significantly less toxic [3]. Several studies have tested MPL mucosally and reported enhanced immune responses when antigens were delivered with MPL [97,98]. Aluminum salts have been used in human and veterinary vaccines since early this century, and they remain as the only class of adjuvants approved for human application in the US. They have been used exclusively for parental immunizations until 1998, when Isaka et al. showed that aluminum-adsorbed TT induced stronger responses in mice after intranasal administration compared to aluminum-non-adsorbed TT. It was the first study to demonstrate mucosal adjuvanticity from an aluminum compound [99]. MF59 is a microfluidized oil-in-water emulsion developed by Chiron [100]. Its parental adjuvanticity was successfully proven when the first product, an influenza vaccine containing MF59 as an adjuvant, was approved for market in Italy in 1997 [69]. Recently, the mucosal adjuvanticity of MF59 was demonstrated in mice with intranasally administered subunit influenza vaccines [100]. Despite the progress in adjuvant research, nothing has been shown to have mucosal adjunticity in humans yet. Finding such as adjuvant will be the key to the success of mucosal vaccine development.

3. Summary Recent discoveries in both mucosal vaccine delivery and mucosal adjuvant research have significantly

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improved the effectiveness of mucosal immunization in animal models. New delivery strategies such as the utilization of live recombinant vectors, DNA plasmids, and transgenic plants to deliver antigens present promises to improve the efficiency of mucosal antigen delivery. New classes of mucosal adjuvants such as modified CT or LT and oligodinucleotides have demonstrated potent mucosal adjuvanticity without detectable toxicity in animals. Continued research to understand the inter-connection and sub-compartmentalization of the common mucosal system will certainly guide the rational selection for routes of mucosal administration. An efficient delivery vehicle, combined with an effective adjuvant given through an optimal route of administration, will ultimately allow for the development of a successful mucosal vaccine in humans.

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