Microparticles for intranasal immunization

Advanced Drug Delivery Reviews 51 (2001) 127–141 www.elsevier.com / locate / drugdeliv

Microparticles for intranasal immunization Michael Vajdy*, Derek T. O’Hagan Chiron Vaccines, 4560 Horton Street, Emeryville, CA 94608, USA

Abstract Of the several routes available for mucosal immunization, the nasal route is particularly attractive because of ease of administration and the induction of potent immune responses, particularly in the respiratory and genitourinary tracts. However, adjuvants and delivery systems are required to enhance immune responses following nasal immunization. This review focuses on the use of microparticles as adjuvants and delivery systems for protein and DNA vaccines for nasal immunization. In particular we discuss our own work on poly(lactide co-glycolide) (PLG) microparticles with entrapped protein or adsorbed DNA as a vaccine delivery system. The possible mechanisms involved in the enhancement of immune responses through the use of DNA adsorbed onto PLG microparticles are also discussed.  2001 Elsevier Science B.V. All rights reserved. Keywords: Mucosal delivery; Nasal immunization; Adjuvants; Microparticles; DNA vaccines

Contents 1. Mucosal delivery of vaccines ................................................................................................................................................... 2. The advantages of IN immunization ......................................................................................................................................... 3. Poly(lactide co-glycolide) (PLG) microparticles for vaccine delivery .......................................................................................... 4. Immunity following IN immunization with protein antigens ....................................................................................................... 5. Alternative microencapsulation approaches for IN immunization ................................................................................................ 6. IN immunization with mutants of heat labile enterotoxin (LT) from Escherichia coli ....................................................... 7. Induction of cytotoxic T lymphocyte (CTL) responses through IN immunization ......................................................................... 8. IN immunization with DNA adsorbed onto PLG microparticles .................................................................................................. 9. Local presence of antigen following IN immunization with proteins or DNA............................................................................... 10. Concluding remarks............................................................................................................................................................... References ..................................................................................................................................................................................

1. Mucosal delivery of vaccines The origin of mucosal vaccination is several thousands years old and originates from the time when Chinese medicine men allowed children to *Corresponding author. E-mail address: michael [email protected] (M. Vajdy). ]

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inhale powders made from dried crusts of pox scars [1]. Even so, vaccination through the mucosal routes remains a challenging concept and has not yet been successfully commercialized for a wide range of products. Almost all of the currently available vaccines are injected systemically, with a few exceptions, e.g., live attenuated polio and Salmonella vaccines, which are administered orally and an

0169-409X / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 01 )00167-3

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adjuvanted flu vaccine, administered intranasally (IN). Although systemic immunization has been an outstanding success, significantly improving public health and resulting in the eradication of an important infectious disease, small pox, there have also been significant problems. The most notable of these, which has been a problem particularly in developing countries, is the transmission of infection due to the re-use of needles [40]. If mucosal vaccinations were to replace systemic immunization, the problem of infection due to the re-use of infected needles would be eliminated. Moreover, systemic administration of vaccines generally fails to induce mucosal immunity. Hence, perhaps, the most important advantage of mucosal immunization, is the ability to induce both local and systemic immunity. In addition, mucosal immunization is the most effective approach for the induction of local long-term immunological memory at sites of entry for most pathogens. Furthermore, many studies have highlighted the presence of a common mucosal immune system, which results in vaccine administration at one site inducing immunity at several distant mucosal sites [34]. Hence, mucosal immunization offers significant advantages in terms of safety and efficacy, in comparison to traditional systemic delivery of vaccines.

2. The advantages of IN immunization There are several mucosal routes available for local immunization including oral, nasal, pulmonary, vaginal and rectal. Of these, the nasal route is attractive for several reasons. The nose, like the mouth is a practical site for easy self-administration of vaccines, using commercially available delivery devices. Delivery of vaccines to the lower lung is much more difficult, requiring sophisticated technologies, and causes significant concerns in relation to potential toxicity. Although the oral route is much preferred, in comparison to oral immunization, IN immunization generally requires much lower doses of antigen, which has important implications for many recombinant antigens, which are often costly. Lower doses are possible by the IN route mainly because IN immunization does not expose antigens to low pH and a broad range of secreted degradative enzymes. A particularly attractive feature of IN

immunization, in comparison to oral immunization, is that IN immunization has been shown to induce potent responses both in the respiratory and the genital tracts, as a consequence of the common mucosal immune system [35]. In contrast to immunization of the genital tract, IN immunization is more convenient and acceptable, and has been shown to induce much more potent local and systemic responses [22,28,58,68]. Finally, compared to rectal immunization the nasal route is more readily accessible and culturally more acceptable.

3. Poly(lactide co-glycolide) (PLG) microparticles for vaccine delivery In recent years, the principal polymers used for the preparation of microencapsulated vaccines have been the aliphatic polyesters, the poly(lactide coglycolides) (PLGs). PLGs are the primary candidates for the development of microencapsulated vaccines because they are biodegradable and biocompatible, and have been used in humans for many years as suture material and as controlled release drug delivery systems [69]. PLG polymers have also been extensively evaluated for the development of controlled release single dose vaccines and this area has recently been reviewed [46]. One of the limitations of PLGs in relation to vaccine development is that these polymers are soluble in only a limited range of organic solvents, and are insoluble in water. The most commonly used solvent for PLG polymers is dichloromethane (DCM), although ethyl acetate and others may also be used. The methods most commonly used for the preparation of microencapsulated vaccines involves the emulsification of aqueous solutions of antigens into organic solvents containing the polymer, and extraction or evaporation of the solvent to form microparticles. The preparation methods commonly employed for the encapsulation of vaccines into PLG polymers have previously been described in detail [41]. A significant problem with PLG microencapsulation is the possibility of antigen denaturation as a consequence of exposure to organic solvents. In addition, during microencapsulation, vaccine antigens may also be exposed to high shear, aqueous–organic interfaces, cavitation and localized elevated temperatures. Nevertheless, despite these

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significant problems, a number of proteins have been successfully entrapped in PLG microparticles with full maintenance of structural and immunologic integrity [43]. Moreover, some proteins have also induced neutralizing antibodies and protective immunity following microencapsulation in PLG microparticles and mucosal delivery (see below). Hence, despite the problems and drawbacks with this technology, the use of polymeric microparticles offers significant potential for the development of mucosally administered vaccines. The main advantages of microparticles are that they can be designed to protect entrapped vaccines against degradation and to target vaccines for uptake into the mucosal associated lymphoid tissue (MALT). Moreover, microparticles can be prepared from a range of different polymers, including bioadhesive polymers, which can be designed to retain microparticles in the nasal cavity.

4. Immunity following IN immunization with protein antigens It is clear that the term ‘IN’ immunization has often been used loosely and inaccurately. For example, the term IN immunization has often been used to describe IN administration of vaccines to anesthetized mice. However, it is clear that anesthetizing mice prior to IN administration results in the delivery of the bulk of the vaccine into the lung. This results in relatively easy access of the vaccine to the systemic lymphoid tissue and the induction of potent systemic immunity. This cannot accurately reflect the likely immune responses that would be induced in humans following IN immunization, unless the vaccine is delivered to the lungs using a sophisticated and highly efficient device. Therefore, a more accurate term to describe IN immunization in anesthetized mice, particularly with larger volumes ( . 20 ml), is intratracheal (IT) immunization. In some studies, the term IT immunization has been used, but this normally involves a process in which the trachea is surgically opened and the vaccine is directly introduced, under anesthesia [30]. Following IN immunization of anesthetized mice, Eyles et al. found that if 10 ml of a vaccine formulation was applied through

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the nose most of it remained in the nasal passages. However, if 50 ml was administered, almost half was found in the lung. Interestingly, regardless of the volume used one third of the vaccine was recovered from the gastrointestinal tract, indicating that at least some of the vaccine was swallowed. Moreover, only the 50 ml volume resulted in vaccine-specific IgG and IgA antibodies in lung lavage, although no difference in serum IgG antibodies could be discerned whether soluble or encapsulated vaccines were used [19]. We believe that the term IN immunization in mice should be restricted to vaccine administration in small volumes ( , 20 ml), preferably in the complete absence of anesthesia. IN immunization in mice with microencapsulated antigens has been shown to induce protective immunity against pathogen challenge. For example, several antigens from Bordetella pertussis were entrapped in microparticles and following IN immunization, induced protective immunity against aerosol challenge with the bacteria [8,54]. Optimal protection was achieved when more than one antigen was administered simultaneously [54]. IN immunization in mice with ricin toxoid in microparticles also induced long lasting protection against aerosol challenge with the toxin [70]. Furthermore, IN immunization with protein-linked phosphorylcholine protected mice against a lethal challenge with Streptococcus pneumoniae [61]. The nasal associated lymphoid tissue (NALT), which has been well defined in mice [29], has been assumed to be the site of uptake of microparticles from the nasal cavity. However, in humans, the organized lymphoid tissue of the upper respiratory tract is represented by the tonsils and the Waldeyer’s rings of the trachea [7]. Although it has been claimed that the mouse is a good model to predict the likely outcome of IN immunization in humans [29], this remains to be proven. Oral or IT immunization with microparticles containing entrapped simian immunodeficiency virus (SIV) induced protective immunity in systemically primed macaques against repeated intravaginal challenge with SIV [31]. In contrast, systemic or oral immunization alone did not provide protection, and a parenteral prime was needed prior to mucosal immunization. In the limited numbers of animals evaluated, oral immunization (3 / 3 protected from

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initial challenge) appeared to be more effective than IT immunization (2 / 3). However, in another study, IT immunization following systemic priming induced optimal protection (4 / 4) in rhesus monkeys against aerosol challenge with staphylococcal enterotoxin B [62]. Again, a systemic prime was needed to induce optimal protection, since IT immunization alone (2 / 4) and oral immunization alone (1 / 4) only protected limited numbers of monkeys [62]. The vagina is considered to be a component of the common mucosal immune system and oral immunization in mice with microparticles has been shown to induce a vaginal antibody response [9]. In addition, IN immunization with microparticles also induced antibodies in the lower genital tract of mice [63]. Although there is no evidence to indicate the presence of aggregated lymphoid follicles or M cells in the vaginal mucosa [47], intravaginal (IVAG) immunization in humans induced local antibody responses [28]. However, IVAG immunization protocols in small animal models have not normally met with great success, despite the use of novel delivery systems and adjuvants [44,45,59]. Moreover, the local immune response in the vagina is subject to significant hormonal regulation, with major changes in local antibodies at different stages of the menstrual cycle [67]. A recent study in mice showed that the IN route of immunization was more effective than the IVAG route for the induction of immune responses in the vagina [14]. Consequently, the vaginal route of immunization appears unlikely to be successfully exploited for the development of novel vaccines. A more successful strategy for the induction of immunity in the lower genital tract is likely to involve oral, IN or rectal immunization [14,25,63]. A recent study has confirmed that the IN route of immunization may be exploited for the induction of genital tract antibody responses in female humans [6].

5. Alternative microencapsulation approaches for IN immunization Strong evidence for dissemination of antigen-specific antibody-secreting cells from NALT to the

cervical lymph nodes and spleen following IN immunizations has been provided by Heritage et al. [26]. These local and systemic humoral responses were generated by entrapment of human serum albumin (HSA) in polymer-grafted microparticles [3(triethoxysilyl)propyl-terminated polydimethylsiloxane (TS-PDMS)] with a size range of 1–100 mm. McDermott et al. reported that polymer-grafted starch microparticles have been used as an alternative to PLG particles and were shown to effectively deliver antigens following oral or IN immunizations and elicit local and systemic humoral responses [33]. However, in comparison to PLGs, these microparticles are poorly defined and their biocompatibility has not been tested in humans. In another study, a single IN or oral immunization with a Schistosoma mansoni antigen entrapped in PLG or polycaprolactone (PCL) microparticles resulted in sustained serum IgG responses [5]. However, this vaccine strategy failed to induce IgA responses in serum or BAL fluids following oral immunization, although IN immunization resulted in both serum and BAL fluid IgA responses. Interestingly, only PLG-entrapped, and not PCL-entrapped vaccine resulted in strong neutralizing antibody responses, following either IN or oral immunization. Moreover, the humoral responses were detectable earlier following PLG vs. PCL immunizations, presumably due to the physicochemical differences between the two polymers and different rates of antigen release. Following IN immunization of anesthetized mice with hemagglutinin (HA) from influenza virus entrapped in one of four microparticle resins (sodium polystyrene sulfonate, calcium polystyrene sulfonate, polystyrene benzyltrimetylammonium chloride, or polystyrene divinylbenzene) sized to 20–45 mm enhanced anti-HA serum hemagglutinin-inhibiting antibodies and nasal wash IgA antibodies were induced [27]. Importantly, this study showed that this immunization strategy reduced viral burden in the lungs following IN administration of virus to the lungs of anesthetized mice. Interestingly, these resins induced enhanced serum IFN-g levels following IT immunizations, while the levels of IL-4, IL-2 and IL-6 remained unchanged, suggesting a TH1-type response [27].

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6. IN immunization with mutants of heat labile enterotoxin (LT) from Escherichia coli As discussed in detail elsewhere in the current issue of Advanced Drug Delivery Reviews (Mucosal adjuvants for nasal vaccination by Rino Rappuoli) genetically detoxified mutants of heat labile enterotoxin (LT) have been shown to be potent adjuvants for inducing mucosal and systemic immune responses. LT is toxic in its native state and induces accumulation of intestinal fluid and diarrhea in human [51]. In order to retain the adjuvanticity of these molecules but reduce their toxicity, several mutants have been generated by site directed mutagenesis. Of these, two mutants of the enzymatic A subunit, LTK63 and LTR72, maintain a high degree of adjuvanticity [48]. LTK63 is the result of a substitution of serine 63 with a lysine in the A subunit, which renders it enzymatically inactive and nontoxic [11,12,50,51]. LTR72 is derived from a substitution of alanine 72 with an arginine in the A

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subunit and contains about 0.6% of the enzymatic activity of wild-type LT. LTR72 is shown to be 100 000-fold less toxic than wild-type LT in Y1 cells in vitro and 25–100 times less toxic than wild-type LT in the rabbit ileal loop assay [24]. We recently compared the potency of PLG microparticles with various alternative adjuvants and delivery systems for the induction of antibody responses against gD2 from herpes simplex virus following IN immunization [63]. In these studies, gD2 was administered in the water in oil emulsion, MF59, entrapped in PLG microparticles, associated with immunostimulatory complexes (ISCOMS) or mixed with LTK63. We demonstrated that gD2 entrapped in PLG induced the highest specific IgA titers in mucosal secretions (Fig. 1). In addition, gD2 entrapped in PLG also induced strong serum IgG titers, but LTK63 was more potent (Fig. 2). In fact, LTK63 and gD2 by the IN route, induced comparable serum antibody responses to IM immunization with MF59, a potent emulsion based adjuvant [63].

Fig. 1. Specific IgA responses in mucosal secretions 2 weeks following IN immunizations with 10 mg HSV gD2 in combination with MF59 emulsion, PLG microparticles, ISCOMs or LTK63 adjuvants.

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Fig. 2. Serum antibody responses following IN immunizations with HSV gD2 in combination with MF59 emulsion, PLG microparticles, ISCOMs or LTK63 adjuvants. The results are presented as gD2-specific serum ELISA units and neutralization titers.

These data show that IN immunization with protein antigens entrapped in PLG microparticles is one of the most effective means of enhancing specific antibody responses in various mucosal secretions, while also inducing strong systemic antibody responses. In addition, we also demonstrated that LTK63 was a potent mucosal adjuvant for induction of immune responses to recombinant antigens. In a subsequent study, we showed that IN immunization with recombinant gD2 with LTK63 induced humoral responses in serum as well as in mucosal secretions and reduced disease severity and mortality in guinea pigs, following viral challenge [42]. In recent studies we showed that IN immunization HA and LTR72 induced potent IgA antibody titers in nasal and vaginal secretions, whereas IM immunization with HA did not. Moreover, IN immunization with HA and LTR72 also induced higher serum IgG and hemagglutinin inhibition titers than IM immunization [4]. In a separate study, we compared the adjuvant effect of LTK63 and MF59 for IN immunization with HA and found that LTK63 induced

higher serum IgG, nasal wash IgA and hemagglutination inhibition titers (unpublished data). Thus, the LT mutants appear to be strong mucosal adjuvants for induction of humoral responses against protein antigens given IN. Since LT mutants are very potent antigens following mucosal immunization, there is a concern that immunity to LT might affect the potency of these molecules when used as adjuvants. Therefore, we recently evaluated the potency of LTK63 in mice and pigs with pre-existing immunity to the adjuvant [64]. We found that pre-existing immunity to LTK63 did not affect it’s potency as an adjuvant, when used for IN immunization with a second vaccine, soon after the first. In addition, these studies also showed the potency of LT mutants for a protein polysaccharide conjugate vaccine (Neisseria meningitidis group C CRM conjugate), and extended their use into a larger animal model, the pig. In a recent study, we showed that an influenza vaccine given IN together with LTK63 in a novel bioadhesive microsphere delivery system, induced

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Fig. 3. Induction of cytotoxic T lymphocyte responses following IN immunizations with HIV-1 p55 gag protein co-administered with LTK63. Mice were given IN immunizations of unadjuvanted protein (^) as a negative control or an intraperitoneal injection with vv-gag-pol (h) as a positive control. IN immunizations of p55 gag protein at range of doses of LTK63 induced potent CTL responses (m, j, d).

enhanced serum IgG as well as nasal IgA responses in mice and pigs [56] (Figs. 3 and 4). Thus, combination of LT mutant adjuvants with a microparticle system enhanced local and systemic humoral responses. Collectively, these data show that LT mutants are effective mucosal adjuvants in small and larger animal models and can be used in combination with microparticle formulations to enhance immune responses.

7. Induction of cytotoxic T lymphocyte (CTL) responses through IN immunization Although most studies on the induction of immune responses through IN immunization have involved induction of humoral immunity, IN immunization

can also result in induction of strong cell mediated immunity. Mora et al. [37] reported that IN immunization of anesthetized mice with a lipidated HIV-1 gp120 peptide entrapped in PLG particles induced gp120-specific CTL and antibody responses. Moreover, IN immunization with HIV-1 gp120 entrapped in microparticles induced systemic CTL responses and mucosal antibodies in mice [36]. Using a range of mucosal adjuvants including LTK63 and LTR72, Simmons et al. showed that IN immunization with ovalbumin resulted in the induction of systemic cell mediated immune responses [55]. Although this study did not demonstrate any local CTL responses following IN immunization with the LT mutants, it did demonstrate that wild type LT exerts its adjuvant activity for induction of CTL responses independent of IFN-g or IL-12. This

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Fig. 4. Anti-HA IgA titers in nasal secretions in three groups of pigs immunized with either HA alone IM, HA 1 LTK63 IN or HA 1 LTK63 1 HYAFF (bioadhesive microspheres) IN. The graphs represent the geometric mean titer6standard error of mean for each group.

study also showed that the ligand binding moiety of LT, LTB failed to effectively induce a CTL response. This observation was consistent with an earlier report, with a measles virus epitope combined with CTB [49]. However, in contrast, the same peptide entrapped in PLG microparticles did induce CTL responses. These studies showed that IN immunization with LT mutants can be an effective means for the induction of cell mediated immunity. However, some antigens may require optimization of the delivery systems as well as inclusion of adjuvants.

We recently described the ability of LT mutants to induce CTL responses against HIV-1 p55 gag, following IN, oral or IM immunization [38]. Interestingly, we found evidence that LTK63 and LTR72 had diverse effects when used as mucosal adjuvants for oral vs. IN immunizations. We found that LTK63 induced stronger CTL responses following IN immunization with p55 compared with LTR72. Conversely, LTR72 induced stronger CTL responses against p55 when given orally, and it also induced local CTL responses (Fig. 5). Thus, it appears that some ADP-ribosyl-transferase activity of the LT

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Fig. 5. Anti-hemagglutinin (HA) serum IgG titers in three groups of pigs immunized with either HA alone IM, HA 1 LTK63 IN or HA 1 LTK63 1 HYAFF (bioadhesive microsphers) IN. The graphs represent the geometric mean titer 1 standard error of mean for each group.

mutant may be required for oral, but not for IN immunizations, if induction of CTL responses is the objective.

8. IN immunization with DNA adsorbed onto PLG microparticles Although traditional vaccines have comprised proteins, live attenuated viruses, or killed bacteria, much attention has recently been focused on DNA vaccines. Immunization with DNA has several advantages over immunization with proteins, including

the induction of potent CTL responses in human and non-human primates [15,16]. The ruggedness and simplicity of DNA offers the potential for improved vaccine stability and reduced costs for vaccine production. Moreover, compared to attenuated viruses as delivery vehicles for HIV genes, plasmid DNA offers a safe alternative. Clinical trials involving IM immunization with DNA vaccines have already been performed in humans and appears to be safe and well-tolerated at the doses tested [53,66]. However, although DNA vaccines have proven potent in small animal models, the potency in larger primates, including humans, has been more disap-

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pointing. Consequently, there is a clear need to improve the potency of DNA vaccines for human immunization. We recently described the development of novel positively charged PLG microparticles with adsorbed DNA, which were used to induce enhanced immune responses following intramuscular (IM) immunization [57]. Although PLG microparticles appear to have significant potential for delivery of DNA, the formulations previously described have had significant limitations. For example, recent work has confirmed that DNA is significantly damaged following microencapsulation in PLG microparticles, due to exposure to high shear during particle preparation [3,43,60]. It has been reported that microencapsulation of DNA results in a significant reduction in the Table 1 Induction of local and systemic cell-mediated immune responses following IN immunization with DNA encoding HIV-1 gag adsorbed onto cationic PLG microparticles compared to naked DNA, as measured by an IFN-g ELISPOT assay Vaccine formulation

Cervical lymph nodes

Spleen

gag-DNA PLG-CTAB gag-DNA naked

247631 0

480674 157660

The data show the number of gag-specific IFN-g secreting cells per 10 million mononuclear cells and S.D. of a minimum of three wells from pools of five mice per group.

amount of supercoiled DNA, with only 10–20% of the encapsulated material retaining super-coiled structure [43]. Moreover, encapsulation efficiency of DNA in microparticles is low, with only 20–50% encapsulation efficiency reported [43]. To overcome these and other significant problems, we developed the novel approach of adsorbing DNA onto the surface of microparticles. Adsorption of DNA to cationic microparticles results in maintenance of super-coiled DNA and allows high efficiency of DNA association with the microparticles [57]. These important advantages of the adsorption approach, including enhanced DNA stability and high loading efficiency, make this process much more amenable to commercial development. We explored the potential of cationic PLG microparticles to induce local and systemic cell-mediated immunity following IN immunization with DNA encoding HIV-1 gag. In addition, we measured local and systemic gag-specific antibody responses. We found that cationic PLG microparticles with adsorbed DNA induced enhanced local (Table 1) and systemic (Fig. 6) cell-mediated, as well as humoral (Fig. 7), immunity against HIV-1 gag. Thus, IN immunizations with DNA adsorbed onto cationic PLG appeared to be a novel approach for induction of enhanced local and systemic cell mediated as well as humoral immune responses.

Fig. 6. Induction of systemic CTL responses in splenocytes following IN immunizations with DNA encoding HIV-1 gag adsorbed onto cationic PLG microparticles, prepared using either CTAB or DDA, compared to naked plasmid DNA as measured by a 51 Cr-release assay.

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Fig. 7. Induction of systemic humoral immune responses following IN immunizations with DNA encoding HIV-1 gag adsorbed onto cationic PLG microparticles, prepared using either CTAB or DDA, compared to naked plasmid DNA as measured by ELISA.

9. Local presence of antigen following IN immunization with proteins or DNA Antigens administered to the nasal cavity are believed to be taken up by M cells overlying the follicle associated epithelium of the nasal associated lymphoid tissue NALT [23,65]. It is well established that M cells are highly efficient in the uptake of particulate antigens and microparticles and delivery to underlying antigen-presenting cells in the local lymphoid structure [39,65]. Following IT delivery, it is likely that microparticles are engulfed by macrophages or dendritic cells and transported to bronchus-associated lymphoid tissue and then to local draining lymph nodes. Alternatively, microparticles may be cleared from the lung through the mucociliary elevator and swallowed. Nevertheless, delivery to the lungs is technically difficult and may be associated with potential toxicity issues, including hypersensitivity problems. Thus, in terms of vaccination strategies, the IN route appears more practical and feasible compared to delivery of vaccines to the lungs. In a study designed to determine the inductive sites for ocular IgA responses, IN immunization of PLG-entrapped haptenated proteins led to uptake and distribution of microparticles in NALT and CLN within minutes [52]. In this study, no evidence of

particle uptake into PP was found following IN immunization. Conversely, following oral immunization, no antigen uptake was evident in NALT or cervical lymphnode (CLN), although Peyer’s Patches (PP) was shown to have taken up the PLG-entrapped antigen. IN immunization led to induction of antigen-specific serum IgG as well as the presence of IgA- and IgG-secreting cells in CLN. Importantly, this study showed that IN immunization was superior to ocular immunization for the induction of antigenspecific IgA responses in tears [52]. Although some insight into vaccine uptake following IN immunization with protein antigens has been obtained, little information is available regarding the mechanisms of DNA uptake and expression following mucosal delivery. It is important to note that, although some studies have suggested an important role for dendritic cells (DCs) for uptake and expression of encoded protein following IM DNA injection [10,17] there are distinct differences in the anatomy, structure, and cellular constituents of nasal and upper-respiratory mucosa compared to muscle. To investigate a possible mechanism for the enhanced immune responses induced following IN immunizations with PLG-DNA, we localized and phenotypically identified the cells that expressed gag protein in local and systemic lymphoid tissues. Following a single IN immunization with PLG-DNA

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expressing HIV-1 gag, we localized and identified the cells that expressed the encoded gene by immuno-fluorescent staining (unpublished data). In the immunostaining studies of CLN and spleen, the majority of gag-expressing cells were CD11b 1 , suggesting that this population is responsible for uptake and expression of DNA following IN immunization with PLG / DNA. Although CD11b is expressed by many cell populations, it is primarily considered a marker for tissue macrophages (Macs) and DCs, which are both professional antigen-presenting cells (APCs) [29,36,70]. However, compared to Macs, DCs are more potent APCs [2,31]. Our data suggest that following IN immunization with DNA adsorbed onto PLG microparticles, monocyte lineage cells, Macs and / or DCs, are involved in the uptake and expression of gag-DNA, since we detected both CD11b 1 and CD11c 1 gag-expressing cells. Whether these cells also actively present gag peptides to ¨ T cells in vivo is an important neighboring naıve question which needs further investigation. Our previous in vitro data showed that bone marrowderived DC can take up PLG / DNA encoding HIV-1 gag and present it to a gag-specific T cell hybridoma [13]. The prolonged expression of DNA following IN immunizations with PLG-DNA may be in part due to protection of DNA from damage by tissue DNAse, which has previously been reported in vitro [57]. In addition, the presence of the cationic surfactant, CTAB, on the surface of PLG microparticles may contribute to disruption of endosomes and subsequent release of DNA into the cytoplasm, to enhance the response. Elucidation of these and other possible factors that contribute to the enhanced responses observed following IN delivery of PLGDNA is under investigation.

10. Concluding remarks IN immunization with protein or DNA encapsulated in or adsorbed onto PLG microparticles offers an attractive approach for enhancement of local and systemic cell mediated and humoral immune responses. The IN route of immunization has several advantages over other routes of mucosal immunization, including the potential for the induction of

enhanced immunity in the genitourinary, respiratory and gastrointestinal tracts. Polymeric delivery systems can be designed to enhance the efficacy of mucosally administered vaccines in a number of ways; they can protect antigens from degradation, concentrate them in one area of the mucosal tissue for better uptake, extend their residence time in the body, or target them to specific sites of antigen uptake (e.g., NALT). Significant progress has been made recently with PLGs, allowing the effective stabilization of proteins and DNA in or on microparticles during the preparation process. Thus, IN delivery of entrapped or adsorbed antigens may prove to be a practical and feasible approach for vaccination. Developments in the aerosolization of PLG microparticles also offers the potential of delivery of microencapsulated vaccines to the lungs [18,32]. The potential of targeting ligands to enhance particle uptake in larger animal models, including humans, is currently unknown, although in rodents, the extent of uptake of microparticles can be enhanced using targeting ligands [20,21]. Future research in this area, and in the use of bioadhesives to retain antigens in the nasal cavity may prove beneficial.

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