A novel bioadhesive intranasal delivery system for inactivated influenza vaccines

Journal of Controlled Release 70 (2001) 267–276www.elsevier.com/ locate / jconrel

A novel bioadhesive intranasal delivery system for inactivatedinfluenza vaccines

*Manmohan Singh, Maylene Briones, Derek T. O’HaganChiron Technologies, Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608, USA

Received 9 March 2000; accepted 8 September 2000

Abstract

The aim of the current studies was to evaluate a bioadhesive delivery system for intranasal administration of a flu vaccine,in combination with a mucosal adjuvant (LTK63). A commercially available influenza vaccine, containing hemagglutinin(HA) from influenza /A Johannesberg H1N1 1996, and LTK63 or LTR72 adjuvants, which are genetically detoxifiedderivatives of heat labile enterotoxin from Escherichia coli, were administered IN in a bioadhesive delivery system, whichcomprised esterified hyaluronic acid (HYAFF) microspheres, to mice, rabbits and micro-pigs at days 0 and 28. Forcomparison, additional groups of animals were immunized intranasally with the HA vaccine alone, with soluble HA1

LTK63, or IM with HA. In all three species, the groups of animals receiving IN immunization with the bioadhesivemicrosphere formulations, including LT mutants, showed significantly enhanced serum IgG responses (P,0.05) and higherhemagglutination inhibition (HI) titers in comparison to the other groups. In addition, the bioadhesive formulation alsoshowed a significantly enhanced nasal wash IgA response (P,0.05). Most encouragingly, in pigs, the bioadhesivemicrosphere vaccine delivery system induced serum immune responses following IN immunization, which were significantlymore potent than those induced by traditional IM immunization at the same vaccine dose (P,0.05). 2001 ElsevierScience B.V. All rights reserved.

Keywords: Bioadhesive; HYAFF microparticles; Flu antigen; Immunogenicity

1. Introduction proach for the development of new generationvaccines. Although a number of vaccines are com-

Since the majority of pathogens initially infect mercially available which control the spread oftheir hosts through mucosal surfaces, the induction influenza [1–5], these vaccines induce serum im-of mucosal immunity is likely to make an important munity, but do not induce mucosal immunity at thecontribution to protective immunity. In addition, site of infection in the nasal cavity. In addition,mucosal administration, which avoids the use of commercially available vaccines are ineffective forneedles, is becoming an increasingly attractive ap- the induction of cytotoxic T lymphocyte (CTL)

responses, which are responsible for killing virallyinfected cells. Therefore, the currently available

*Corresponding author. Tel.: 11-510-923-7662; fax: 11-510-vaccines are not considered to be optimal [6,7]. As a923-2586.consequence, work is currently underway to developE-mail address: derek o’[email protected] (D.T.

]O’Hagan). more effective influenza vaccines that induce mucos-

0168-3659/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0168-3659( 00 )00330-8

268 M. Singh et al. / Journal of Controlled Release 70 (2001) 267 –276

al IgA responses through local administration, and mucoadhesion can involve physical or chemicalalso induce more potent systemic responses [8–10]. interactions, including electrostatic or hydrophobicAn early study in mice showed that IN immunization bonding, van der Waal’s forces or hydrogen bonding.with a potent adjuvant induced superior cross-protec- Irrespective of the mechanisms involved, the maintive immunity than parenteral immunization, sup- advantages of bioadhesive delivery systems includeporting the use of the IN route for flu vaccine extended residence time at the site of action, localdevelopment [11]. The most potent mucosal ad- delivery to a selected site and enhanced interactionjuvants which are available for local immunization with the mucosal epithelium [20]. In a range ofare heat labile enterotoxin from Escherichia coli studies in recent years, several bioadhesive polymers(LT) and cholera toxin (CT) from Vibrio cholerae, have been described, including chitosans,and these molecules and their subunits have shown methacrylic acids, starch, gelatin, hyaluronic acidsome promise as intranasal adjuvants for flu [9–15]. and cellulose derivatives to enhance the absorptionHowever, since the native toxins CT and LT are the of co-administered protein drugs [22–25]. Hy-causative agents, respectively for cholera and travel- aluronic acid is a naturally occurring mucopolysac-er’s diarrhea, they are considered to be too toxic for charide consisting of residues of D-glucuronic aciduse in humans. Therefore, several groups have and N-acetyl-D-glucosamine. Through the esterifica-focused on the development of detoxified mutants of tion of the carboxyl groups of hyaluronic acid withLT and CT as mucosal adjuvants. Colleagues within alcohols, biodegradable polymers have been de-Chiron have focused on the development of LT veloped, called HYAFF [22]. The HYAFFE poly-mutants with reduced, or eliminated enzymatic ac- mers can be used to make microspheres using ativity, since it is the ADP-ribosylating enzymatic coacervation phase-separation process [26]. Theactivity of LT and CT which causes abnormal HYAFF microspheres have strong bioadhesive prop-intracellular accumulation of cAMP and excess fluid erties and have been used for delivery of calcitoninsecretion from intestinal cells. We have used site- and insulin following mucosal administrationdirected mutagenesis to replace single amino acids [23,24]. However, HYAFF microspheres have notwithin the enzymatic A subunit of LT and have previously been used for mucosal delivery of vac-developed mutants (LTK63 and LTR72) with re- cines.duced or eliminated enzymatic activity [16]. LTK63 In the current studies, we report the use of anis completely devoid of ADP-ribosyltransferase ac- influenza hemagglutinin (HA) vaccine administeredtivity and appears to be non-toxic both in vivo and in IN to mice, rabbits and micro-pigs along with LTvitro, while LTR72 has residual enzymatic activity mutants and a bioadhesive delivery system, compris-(,1% of the native LT) and has significantly ing HYAFF microspheres. The IN route has beenreduced, but detectable toxicity [16]. Both of these used previously to administer vaccines and adjuvantsmutants have been previously shown to be potent to animal models [8]. The responses to the bioadhe-mucosal adjuvants for antibody and CTL induction sive formulations were compared to HA combinedin a number of studies in mice [9,15–17]. with LT mutants by the IN route, and also HA

Since the early 1980s several groups have focused administered alone by the IN and IM routes. For IMon bioadhesion as a concept to improve local and administration, we used a commercially availablesystemic drug delivery [18,19]. In general, bioadhe- vaccine which like most other flu vaccines, issive delivery systems are designed to adhere to unadjuvanted.various tissue surfaces, mainly the mucosal epi-thelium. An alternative term, mucoadhesion is alsoused often to describe the interaction of a polymer 2. Materials and methodsdelivery system and a mucosal site. Mucoadhesionappears to require a highly expanded and hydrated 2.1. Materialspolymer network, which promotes an intimate mo-lecular contact between the delivery system and the A monovalent A/Johannesberg split vaccine prep-mucus layer [20–24]. The mechanisms of bio- or aration of purified influenza HA was provided by

M. Singh et al. / Journal of Controlled Release 70 (2001) 267 –276 269

Chiron Vaccines, Siena, Italy. Dosing of the vaccine LT mutants in PBS from the surface of the HYAFFwas based on HA content as assayed by single radial microspheres was also estimated by ELISA [9]immunodiffusion (SRID) as described previously[9]. The mucosal adjuvants, LTK63 and LTR72 were

2.5. Immunization protocolsfermented and purified at Chiron, Emeryville, USA.HYAFF microspheres were prepared by a coacerva-tion phase separation technique as previously de-

2.5.1. Micescribed [23,24,26] and were provided by Fidia

The first study was a preliminary evaluation inAdvanced Biopolymers, Padova, Italy. The bioadhe-

mice of the bioadhesive vaccine delivery systemsive properties of HYAFF microspheres has previ-

using HYAFF microspheres. The antigen and ad-ously been described both in rat [24] and sheep [23]

juvant doses used were 10 mg HA and 25 mgmodels.

LTK63. Four groups of Balb /C mice (10 per group)were immunized IN with either the HYAFF-HA-

2.2. MethodsLTK63 formulation, HYAFF-HA, HA1LTK63 solu-ble proteins or HA alone. The animals were boosted

The combined bioadhesive vaccine formulationsat day 28 with the same formulations and blood was

were prepared as follows; HA and LT mutants at thecollected at day 42.

doses described in the text were incubated withHYAFF microspheres in PBS. The suspension waskept at 48C for 6 h and then freeze dried overnight. 2.5.2. RabbitsPrior to administration to the animal models, the In the first study in rabbits, three groups of Newmicrosphere formulation was re-suspended in PBS, Zealand whites (five or six per group) were immun-to allow easy administration of the dose as a ized IN with either a HYAFF-HA-LTR72 formula-suspension. The HYAFF dose was 5 mg for mice (25 tion, HA1LTR72 soluble proteins or HA alone. Theml volume of administration) and 20 mg of micro- HA dose in all groups was 25 mg and the LTR72spheres per animal for rabbits (200 ml) and pigs (250 dose was 50 mg. The animals were boosted at day 28ml). The HA and LT mutant doses was changed and blood was collected at days 28 and 42. In aaccording to the animal species being tested and second smaller study, two groups of rabbits (five perdetails are included in the text. group), were immunized IN with a HYAFF-HA-

LTK63 combination, with doses of HA at 10 mg and2.3. Microparticle characterization LTK63 at 25 mg. For comparison, a second group of

rabbits were immunized IM with HA 25 mg. BloodThe HYAFF microspheres were sized on a Mal- samples were collected at days 14 and 42.

vern Mastersizer both before and after combinationwith the antigen and adjuvant. The HYAFF micro-spheres as provided by the manufacturer had a mean 2.5.3. Micro-pigssize of 8.4 mm, as previously described [25]. The In the pig study, three groups of four Yucatansize following hydration in vitro during association micro-pigs (8–10 kg) were used and were housed inwith the the antigen/adjuvant was 3262.3 mm. After pairs. In this study, the responses induced by INfreeze drying, the size of the microspheres in the immunization were compared to IM immunization.final formulation was determined to be 8.260.6 mm. The doses selected for pigs were 25 mg of HA for all

groups, and 100 mg of LTK63 for the two IN groups.2.4. Antigen and adjuvant integrity One group was immunized IN at weeks 0 and 4 with

the HYAFF-HA-LTK63 bioadhesive microsphereBoth the HA and the LT mutants were evaluated formulation. A second group was immunized IN

by an ELISA [9] to evaluate antigenic integrity with soluble HA1LTK63 at the same dose. Forfollowing formulation with the HYAFF micro- comparison, a third group of pigs were immunizedspheres. The rate of in vitro release of HA and the IM with 25 mg HA.


2.6. Immunoassays agglutination of goat red blood cells (RBC) in thepresence of HA antigen [28–30]. Fresh RBC were

Serum total anti-HA IgG were measured by diluted to 0.4% cell suspension using OD against540

ELISA as previously described [16]. Briefly, ELISA a cyanmethemoglobin reference standard. A/Johan-plates (Immulon-1, 96 well, U-bottom, obtained nesberg HA antigen stock was titered to 4 HA unitsfrom Dynatech Laboratories, Chantilly, VA) were defined as the highest concentration required tocoated with HA (10 mg/ml) overnight. After block- agglutinate a 0.2% RBC suspension. Serum samplesing (1% goat serum, 0.3% Tween 20 in phosphate- were serially diluted two-fold into an ELISA platebuffered saline), plates were coated with 1:3 serially then the HA antigen at a final concentration of 1 HAdiluted serum samples. After washing (blocking unit and 0.2% RBC was added. The HI titer was thenbuffer), the plates were coated with 1:4000 goat defined as the reciprocal dilution of the serumanti-pig IgG horseradish peroxidase conjugate required to completely inhibit agglutination. HI titers(Gibco, Grand Island, NY) and developed using are normally determined to reflect the potency of3,39,5,59-tetramethylbenzidine (TMB) substrate (Kir- influenza vaccines and these titers have been shownkegaard and Perry Laboratories, Gaithersburg, MD). to correlate closely with protective efficacy of vac-Absorbances were measured at A using a standard cines.490

ELISA reader. The titers represent reciprocal serumdilutions giving an A of 0.5 and were normalized490 2.7. Statisticsto a serum standard assayed in parallel.

Nasal wash samples from pigs were assayed for Analysis of variance was calculated by using theIgA using a bioluminescent immunosorbant assay StatView program for Macintosh computers. Differ-(BIA) as previously described [9,16]. Briefly, ELISA ences among groups of animals at significance levelsplates (MicroLite obtained from Dynatech) were first of 95% were calculated by analysis using Fisher’scoated with the HA antigen (5 mg/ml) overnight. protected least-significant-difference test.After blocking (5% goat serum, 25 mM Tris, 10 mMEGTA, 150 mM KCl, 2 mg/ml BSA, 0.3% Tween-20, pH 7.5), plates were coated with 1:3 serially

3. Resultsdiluted nasal wash samples in blocking buffer. Theplates were developed using 1:1000 diluted goatanti-pig IgA biotin conjugate (EY Labs, San Mateo, 3.1. Antigen and adjuvant integrity and releaseCA) pre-saturated with purified pig IgG (1 mg/ml, rates in vitroSigma Chemical Company, St Louis, MO) to reduceIgG cross-reactivity. Plates were then incubated with The HA antigen and the LT mutants both re-1:500 diluted streptavidin–jellyfish aequorin conju- mained largely intact after formulation and releasegate (SeaLite Sciences, Bogart, GA). Luminescence from HYAFF microspheres in vitro, with no signifi-was triggered with 10 mM calcium acetate and cant changes in antibody binding characteristics (datameasured using a luminometer (Dynatech ML3000). not shown). The rate of release of both HA and theQuantitation was based on relative light units (RLU) LT mutants indicated a fairly large burst release,representing total luminescence integrated over 3 s with 32% of LTK63 and 24% of HA released in day(arbitrary units). Titers represent log dilution values 1, followed by a more slow release phase, with 75%linearly extrapolated from the log RLU data to a of both HA and LTK63 released by day 10 in vitro.cut-off value at least two standard deviations above We do not consider that the in vitro release rate ofmean background. the formulations has any relevance for the likely rate

Serum samples for each animal were assayed for of release of antigen and adjuvant in vivo. However,hemagglutination inhibition (HI) titers by the Viral these studies were performed to determine if theand Rickettsial Disease Laboratory (Department of antigen and adjuvant were actually released from theHealth Services, Berkeley, CA) using a standard formulation and to allow an evaluation of integrity ofassay based on the ability of sera to inhibit the both following release.


3.2. In vivo immunogenicity

3.2.1. MiceThe group of mice receiving the bioadhesive

microsphere formulation (HYAFF-HA-LTK63),showed significantly enhanced serum IgG antibodyresponses in comparison to the other groups immun-ized IN with soluble antigen alone, with HA-HYAFF, or with HA1LTK63 (P,0.05) (Fig. 1).The HI titers were also highest in the group of miceimmunized with the bioadhesive microsphere formu-lation (Fig. 2). Mouse sera needed to be pooled toobtain sufficient serum to allow the HI titer to be

Fig. 2. Serum hemagglutination inhibition titers (HI) in groups ofobtained. Therefore, mean values are shown in Fig.mice (n510) immunized with either HA alone IN, HA1HYAFF

2. IN, HA1LTK63 IN or HA1LTK631HYAFF IN. The graphshows a single value obtained from pooled sera at day 42.

3.2.2. RabbitsThe group of rabbits immunized with the bioadhe- enhanced HI titers over the IM immunized group

sive HYAFF-HA-LTR72 formulation had significant- (Table 1).ly higher serum IgG titers than the groups immun-ized IN with soluble HA alone, or soluble HA1 3.2.3. Micro-pigsLTR72 at day 42 (P,0.05) (Fig. 3). In addition, in In the pig study, the group receiving the bioadhe-the second rabbit study, the group immunized IN sive formulation (HYAFF-HA-LTK63) had a sig-with HYAFF-HA-LTK63 showed a higher mean nificantly higher antibody response than the groupsserum IgG antibody responses than the group im- immunized IN with soluble HA1LTK63 and themunized IM, although there was no significant group immunized IM with HA at day 56 (P,0.05)difference (P.0.05) (Fig. 4). The IN immunized (Fig. 5). The nasal IgA titers were also significantlygroup in this study also showed a clear trend for

Fig. 3. Serum anti-HA serum IgG antibody titers in groups ofFig. 1. Anti-HA serum IgG antibody titers in groups of mice New Zealand white rabbits (n55 or 6) immunized with either HA(n510) immunized with either HA alone IN, HA1HYAFF IN, alone IN, HA1R72 IN or HA1R721HYAFF IN. The graphHA1LTK63 IN or HA1LTK631HYAFF IN. The graph repre- represents the geometric mean titers6S.E.M. for each group atsents the geometric mean titers6S.E.M. for each group at day 42. days 28 and 42. The HYAFF1LTR721HA formulation wasThe HYAFF1LTK631HA formulation was significantly better significantly better than soluble HA and HA1LTR72 (P,0.05) atthan all other groups (P,0.05). day 42.


Fig. 4. Anti-HA serum IgG antibody titers in two groups ofrabbits (n55) immunized with either HA alone IM, or HA1

LTK631HYAFF IN. The graph represents the geometric mean Fig. 5. Anti-HA serum IgG antibody titers in three groups oftiters6S.E.M. for each group at days 14 and 42. The groups were micro-pigs (n54 per group) immunized with either HA alone IM,not significantly different from each other at days 14 and 42 HA1LTK63 IN or HA1LTK631HYAFF IN. The graph repre-(P.0.05). sents the geometric mean titers6S.E.M. for each group at days 42

and 56. The HYAFF1LTK631HA formulation was significantlybetter than the HA (IM) and HA1LTK63 (IN) at day 56 (P,

higher in the group of animals immunized with 0.05).

bioadhesive microspheres (P,0.05) (Fig. 6). Inaddition, the HI titers also tended to be higher in the ministration of soluble HA with the potent adjuvantgroup of animals immunized IN with the bioadhesive LTK63. However, the limitations of small animalmicrosphere formulation (Table 2). models and their inability to accurately predict

responses in human subjects is well known andwidely acknowledged. This is particularly true in

4. Discussion relation to mucosal delivery, since mice have verysmall nasal cavities which can accommodate only

The initial observations in mice offered significant very low volumes of fluid (20–25 ml). Therefore,encouragement that the bioadhesive microsphere during drug or vaccine delivery studies, the mousedelivery system may offer some benefit over ad- nasal cavity is often completely filled following

Table 1Hemagglutination-inhibition (HI) titers and serum IgG titers at day 42 from individual animals in two groups of rabbits immunized witheither HA alone IM, or HA-LTK63-HYAFF IN

Formulation Route Serum IgG HemagglutinationELISA titers inhibition titers(Day 42) (Day 42)

HA alone 463112 1280IM 77087 160

68812 32028274 64099682 160

HA1LTK631HYAFF 85838 640IN n.d. n.d.

255575 1280263413 1280136493 640

n.d.5not done.


lation alone with HA, without the addition of LTmutants, was not evaluated further. The poor re-sponse to the bioadhesive formulation alone de-livered mucosally is consistent with observationsfrom an earlier study, in which only a marginalenhancement was seen with mucosal immunizationwith bioadhesive microspheres and mucosal deliverywas not comparable with IM immunization [31]. Incontrast, an alternative bioadhesive polymer,chitosan, which is a widely used pharmaceuticalexcipient [32], has shown encouraging results fol-lowing IN immunization in a small animal model[33]. Nevertheless, chitosan and related molecules

Fig. 6. Anti-HA nasal IgA antibody titers in three groups of have previously been described as potent adjuvantsmicro-pigs (n54) immunized with either HA alone IM, HA1 or immunomodulatory compounds following paren-LTK63 IN or HA1LTK631HYAFF IN. The graph represents the

teral immunization [34]. Therefore, the adjuvantgeometric mean titers6S.E.M. for each group at days 42 and 56.effect with IN chitosan may not come solely fromThe HYAFF1LTK631HA formulation was significantly betterthe bioadhesive properties of this polymer. It remainsthan the HA (IM) and HA1LTK63 (IN) at days 42 and 56

(P,0.05). to be seen if simple ‘bioadhesion’ is enough toimpart a potent adjuvant effect following mucosal

formulation administration, a situation that does not delivery, but our data would seem to indicate thataccurately reflect an approach that would prove inclusion of an adjuvant active molecule may also beacceptable in humans. Hence it was considered necessary to induce a potent response.necessary to further evaluate the bioadhesive formu- In the rabbit studies, the observations from thelations in more rigorous studies in larger animal mouse model were extended to a larger animal andmodels. In the mouse study, the bioadhesive micro- the second LT mutant, LTR72 was also evaluated. Inspheres alone did not provide an adjuvant effect and previous studies, LTR72 was shown to be a morethe presence of the LT mutant was necessary to effective mucosal adjuvant in mice than LTK63 for aachieve potent immune responses following intranas- model protein [15]. In addition, studies in mice withal administration. Therefore, the bioadhesive formu- HA have shown that IN HA1LTR72 was capable of

Table 2Hemagglutination-inhibition (HI) titers and serum IgG titers at day 42 from individual animals in three groups of micro-pigs immunizedwith either HA alone IM, HA1LTK63 IN or HA1LTK631HYAFF IN

Formulation Route Serum IgG HemagglutinationELISA titers inhibition titers(Day 42) (Day 42)

HA alone IM 137 802668 640273 160589 160

HA1LTK63 IN 97 401499 1280

86 320984 640

HA1LTK631HYAFF IN 1908 1280889 320

1764 640485 2560


inducing more potent serum immune responses than enhanced nasal wash IgA antibody response. InIM immunization with HA at similar dose [9]. addition, despite significant variability amongst in-Therefore, LTR72 was evaluated in the first rabbit dividual pigs, there was a clear trend for the bioadhe-study, which showed a clear demonstration of the sive formulation to also induce enhanced HI titersbenefit of delivering the HA-LTR72 combination in over IM immunization.a bioadhesive microsphere delivery system. This The observations from these studies are notable instudy also served to confirm the significant potential several ways. Firstly, they serve to illustrate theof LTR72 as an IN adjuvant for HA, both with and potency of the LT mutants as IN adjuvants for HA inwithout the HYAFF. Since the LTR72 bioadhesive three animal models. Studies already publishedformulation was very potent in rabbits, we also [9,14–17] and many unpublished observationsdecided to evaluate the LTK63 bioadhesive formula- (O’Hagan et al., in press) have shown that thesetion that had previously performed so well in the mutants are potent mucosal adjuvants for a widemouse study. In addition, we also decided to com- range of immunogens, to include recombinant pro-pare the IN bioadhesive formulation with the tradi- teins, protein polysaccharide conjugates, peptidestional approach to immunization, using HA alone by and DNA, when delivered by several differentthe IM route. In a small study, involving two groups routes. In a previous study, we compared a range ofof rabbits, the HYAFF-HA-LTK63 combination different antigen delivery systems and adjuvants,formulation was compared to IM immunization with including microparticles and Iscom’s, for IN deliverya similar dose of HA. Both the serum IgG and the of a recombinant protein and showed that LTK63serum HI titers induced by IN immunization in this was the most potent adjuvant for induction of serumstudy were comparable, or greater than those induced immunity [16]. In addition, the results described hereby IM immunization. These observations encouraged show that the potency of the HA1LT mutantus to continue with this formulation approach into a combination can be enhanced by formulation into alarger animal model, the pig. The omnivorous pig bioadhesive microsphere delivery system. IN im-has all the components of the ring of lymphoid munization with the bioadhesive microsphere formu-tissue, the Waldeyer’s ring, which are found in lation in pigs induced a significantly enhanced serumhumans and therefore represents a good animal immune response in comparison to traditional IMmodel to evaluate intranasal immunization ap- immunization. Since several flu vaccines are alreadyproaches [27]. commercially available, if they are eventually to be

For the pig study, we continued to use the LTK63 replaced by new vaccines administered by the INadjuvant, since it is completely non-toxic both in route, then the new vaccine must induce at least avitro and in vivo [16] and therefore, is the preferred similar level of serum immunity. This was achievedcandidate for subsequent human studies. In the pig in the current studies in both rabbits and pigs. Instudy, we undertook a rigorous evaluation of the addition, in the pig study, the IN approach alsocombination bioadhesive formulation and compared induced a significant IgA response in the nasalthe responses obtained IN, with those obtained after cavity, which might help to protect against initialIM immunization with the same dose of HA. Very infection. Although not evaluated in the currentencouragingly, IN immunization with soluble HA1 studies, IN immunization with LT mutants hasLTK63 induced comparable serum IgG antibody previously been shown to induce potent CTL re-responses to IM immunization with an equivalent sponses, which should also help with viral clearancedose of HA. This observation further confirmed the mechanisms ([17] and unpublished data). It was alsosignificant potential of LTK63 as a mucosal adjuvant notable that in the current studies, for easy adminis-and extended our earlier observations in mice [9,14– tration to all animal models, the microsphere formu-17] to a larger animal. Furthermore, the bioadhesive lations were used as suspensions in saline. TheHYAFF-HA-LTK63 formulation induced a signifi- bioadhesive properties would be expected to becantly enhanced serum IgG antibody response in enhanced if the formulations were administered ascomparison to IM immunization with HA. The dry powders, and this may have increased theirbioadhesive formulation also induced a significantly potency further.


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A novel bioadhesive intranasal delivery system for inactivated influenza vaccines