Mh

Aa

b

c

a

ARRAA

KITPDV

1

mweteaSm[crt

Cf

0d

Vaccine 29 (2011) 5304– 5312

Contents lists available at ScienceDirect

Vaccine

j ourna l ho me pag e: www.elsev ier .com/ locate /vacc ine

ucosal immunization with liposome-nucleic acid adjuvants generates effectiveumoral and cellular immunity

ngela Hendersona, Katie Propsta, Ross Kedlc, Steven Dowa,b,∗

Department of Microbiology, Immunology, and Pathology and the Rocky Mountain Regional Center of Excellence, Colorado State University, Ft. Collins, CO 80523, United StatesDept of Clinical Sciences, Colorado State University, Ft. Collins, CO 80523, United StatesIntegrated Dept of Immunology, National Jewish Health and the University of Colorado Denver, Denver, CO 80219, United States

r t i c l e i n f o

rticle history:eceived 14 January 2011eceived in revised form 15 April 2011ccepted 5 May 2011vailable online 19 May 2011

eywords:nnate immunity

cellulmonaryendritic cellaccine adjuvant

a b s t r a c t

Development of effective new mucosal vaccine adjuvants has become a priority with the increase inemerging viral and bacterial pathogens. We previously reported that cationic liposomes complexed withnon-coding plasmid DNA (CLDC) were effective parenteral vaccine adjuvants. However, little is knownregarding the ability of liposome-nucleic acid complexes to function as mucosal vaccine adjuvants, orthe nature of the mucosal immune responses elicited by mucosal liposome-nucleic acid adjuvants. Toaddress these questions, antibody and T cell responses were assessed in mice following intranasal immu-nization with CLDC-adjuvanted vaccines. The effects of CLDC adjuvant on antigen uptake, trafficking,and cytokine responses in the airways and draining lymph nodes were also assessed. We found thatmucosal immunization with CLDC-adjuvanted vaccines effectively generated potent mucosal IgA anti-body responses, as well as systemic IgG responses. Notably, mucosal immunization with CLDC adjuvantwas very effective in generating strong and sustained antigen-specific CD8+ T cell responses in the air-

ways of mice. Mucosal administration of CLDC vaccines also induced efficient uptake of antigen by DCswithin the mediastinal lymph nodes. Finally, a killed bacterial vaccine adjuvanted with CLDC inducedsignificant protection from lethal pulmonary challenge with Burkholderia pseudomallei. These findingssuggest that liposome-nucleic acid adjuvants represent a promising new class of mucosal adjuvants fornon-replicating vaccines, with notable efficiency at eliciting both humoral and cellular immune responsesfollowing intranasal administration.

. Introduction

Many pathogens attach to or invade mucosal surfaces anducosal immunity is often the key to controlling initial infectionsith such pathogens. Mucosal immune responses are typically gen-

rated most efficiently when vaccines are administered mucosally,hough the majority of vaccines today are administered par-nterally [1–4]. Indeed, only a few mucosal vaccines have beenpproved for human use, including poliovirus, influenza, rotavirus,almonella typhi, and Vibrio cholera vaccines [1,5]. Currently, mostucosal vaccines are prepared using live, attenuated organisms

6,7]. Though effective, such vaccines are costly to prepare, require

areful attention to storage conditions, and pose some potentialisk to immunosuppressed individuals. Therefore, there is con-inued interest in the development of effective, non-replicating

∗ Corresponding author at: Dept. of Microbiology, Immunology, and Pathology,olorado State University, Ft. Collins, CO 80523, United States. Tel.: +1 970 297 4014;

ax: +1 970 297 1275.E-mail address: sdow@colostate.edu (S. Dow).

264-410X/$ – see front matter © 2011 Published by Elsevier Ltd.oi:10.1016/j.vaccine.2011.05.009

© 2011 Published by Elsevier Ltd.

mucosal vaccines. However, most mucosal antigens are poorlyimmunogenic and require the use of potent mucosal vaccine adju-vants.

At present, several adjuvants have been used with non-replicating mucosal vaccines, including mutated cholera toxinand E. coli labile toxins, as well as synthetic TLR agonist, suchas CpG oligodeoxynucleotides (ODN) [4,5,8–11]. Cholera toxin(CT) adjuvants elicit strong humoral immunity following mucosaladministration, though the risk of systemic toxicity and especiallyneurotoxicity renders current CT adjuvants generally unsuitable foruse in human vaccines. A modified cholera toxin subunit B (CTB)adjuvant is relatively effective as a mucosal adjuvant and elimi-nates the risk of systemic toxicity. CpG ODN have been widely usedas parenteral vaccine adjuvants and as effective mucosal vaccineadjuvants [5,12–20]. Studies have shown that CpG ODN adjuvantspotently activate innate immune responses by stimulating innateimmune signaling via TLR9 [21–23]. While each of these adjuvants

has certain desirable properties, there are also some characteristicsabout CTB and CpG that raise efficacy and safety concerns [24–28].Therefore, there remains a need for more potent, more quicklyacting, and potentially safer mucosal adjuvants.

ccine

gmjlhtt(siiITmc

obaptCieCtclpavsirsinla

tiwrlcrnpaICu

2

2

aHcao

A. Henderson et al. / Va

Liposome-based mucosal adjuvants been thoroughly investi-ated, using a variety of different antigens [29–34]. The impact ofode of antigen association with the liposome (encapsulation, con-

ugation, and absorption) and the physiochemical properties of theiposome (size, charge, lipid composition) on immune responsesave also been studied [35]. At present, cationic liposomes are par-icularly advantageous as mucosal adjuvants due to their abilityo enhance the uptake of the vaccine by antigen presenting cellsAPC) and to induce APC activation [36–38]. Indeed, numeroustudies have shown that liposomes are essential to achieve efficientmmune responses [34,39,40]. Many liposome-based adjuvants cannduce mucosal production of IgA, and some also induce systemicgG production, but few have been shown to induce effective CD8+

cell responses. Therefore, there is still a need of broadly effectiveucosal vaccine adjuvants, capable of eliciting both humoral and

ellular immune responses.We previously reported that a vaccine adjuvant consisting

f cationic liposome–DNA complexes (CLDC) effectively elicitedalanced cellular and humoral immunity following parenteraldministration [41]. The effectiveness of the CLDC adjuvantedarenteral vaccines has been attributed to the combination ofhe liposome (carrier) and the plasmid DNA (immunostimulant).ombination vaccine adjuvants have recently become an area of

nterest due to the synergistic effect of combining antigen deliv-ry with potent stimulation of the innate immune system [42,43].ationic liposome–DNA complexes can be classified as a combina-ion adjuvant, as the need for physical association of all three of theomponents of the CLDC-based vaccines was demonstrated in ouraboratory [41]. In those experiments, mice immunized with Ovalus liposome alone or Ova plus plasmid DNA alone failed to gener-te significant immune responses [41]. The efficacy of CLDC-basedaccines for immunization against a variety of different antigens ineveral different species has also been reported, including studiesn guinea pigs, woodchucks, and non-human primates, and moreecently in normal human volunteers [44–49]. Moreover, recenttudies in our laboratory have also revealed that intranasal admin-stration of CLDC alone as an immune therapeutic generated rapid,on-specific, innate immune protection against inhalational chal-

enge with rapidly lethal bacterial pathogens including Burkholderiand Francisella [50,51].

Therefore, we wondered whether CLDC could also be used effec-ively as mucosal vaccine adjuvants. To address this question, wenvestigated the mucosal adjuvant properties of CLDC combined

ith soluble protein antigens, delivered by the intranasal (i.n.)oute. The ability of CLDC adjuvant to elicit humoral and cellu-ar immune responses was investigated, and experiments wereonducted to identify mucosal antigen presenting cells (APCs)esponsible for antigen uptake and trafficking to regional lymphodes. Finally, the ability of CLDC-adjuvanted vaccines to elicitrotective immunity against serious pathogens was assessed in

model of lethal pulmonary Burkholderia pseudomallei challenge.n the course of these studies, we identified properties shared byLDC adjuvants and other mucosal adjuvants, as well as propertiesnique to CLDC-based mucosal adjuvants.

. Materials and methods

.1. Mice

Specific pathogen-free 6–8-week-old female C57BL/6, BALB/c,nd ICR mice were purchased from the Jackson Laboratories (Bar

arbor, ME) or Harlan Laboratories (Indianapolis, IN). All proto-ols involving animal experiments described in this study werepproved by Institutional Animal Care and Use Committee at Col-rado State University.

29 (2011) 5304– 5312 5305

2.2. Reagents and biochemicals

Ovalbumin was purchased from Sigma–Aldrich (St Louis, MO)and was prepared as a 1 mg/ml solution in diH2O. Fluorescent AlexaFluor 647 ovalbumin was purchased from Invitrogen (San Diego,CA) and was resuspended in PBS at a concentration of 1 mg/mlprior to use. All cell preparations were resuspended in completeRPMI (Invitrogen, Carlsbad, CA) containing 10% FBS (Gemini Bio-Products, West Sacramento, CA), 2 mM l-glutamine (Invitrogen),1X non-essential amino acids (Invitrogen), 0.075% sodium bicar-bonate (Fisher Scientific, Pittsburgh, PA), 100 U/ml penicillin, and100 �g/ml streptomycin (Invitrogen).

2.3. Preparation of cationic liposomes–DNA complexes andvaccines

Liposomes were prepared by combining cationic liposomeDOTIM octadecenoyloxy(ethyl-2-heptadecenyl-3-hydroxyethyl)imidazolinium chloride and cholesterol in equimolar concen-trations as described previously [52]. Cationic liposome–DNAcomplexes (CLDC) were freshly prepared at room temperature andadministered within 30 min. Non-coding plasmid DNA (0.2 mg/ml,Juvaris Biotheraputics) was diluted in sterile Tris-buffered 5%dextrose water. The cationic liposomes were then added withgentle pipetting at a concentration of 100 �l of liposomes per 1 mlof solution, resulting in the spontaneous formation of CLDC. Toformulate the CLDC-adjuvanted vaccines, the protein antigen wasadded to the diluted plasmid DNA solution prior to the addition ofthe cationic liposomes.

2.4. Intranasal immunizations

Prior to immunization, mice were anesthetized by intraperi-toneal (i.p.) injection of ketamine (100 mg/kg) with xylazine(10 mg/kg). Each mouse was immunized with a total of 20 �l vac-cine, which was administered by an equal amount in each naresand allowing the mice to inhale the vaccine. For most experiments,mice were immunized with a total of 2 g ovalbumin (Ova). Micewere immunized once and boosted 10 days later. Serum was col-lected 5–7 days after the boost for analysis of cellular and humoralimmune responses. Saliva was collected following i.p. injection of10 �g pilocarpine (Sigma–Aldrich) in PBS.

2.5. Antibody response in serum, saliva, and BAL fluid

Antibody responses to Ova were assessed as described previ-ously [53,54]. Briefly, ELISA plates were coated with Ova, blockedto reduce non-specific binding, then incubated with serial dilutionsof serum from vaccinated and control mice. Antibody titers weredetermined using endpoint dilution assay and were expressed asthe log reciprocal of the highest dilution of a sample with an ODreading of 0.1 above background.

2.6. Cell collection

Bronchoalveolar lavage (BAL) cells were obtained by airwaylavage, as previously described [55]. Cells from the 3 to 4 washesper mouse were pooled, centrifuged at 1200 rpm for 5 min at 4 ◦C.The cells were further purified by NH4Cl lysis of the RBC. Lymphnode cells were prepared by mechanical disruption and screeningthrough a 70-�m nylon mesh screen (BD Biosciences), followed

by NH4Cl lysis. Lung cells were prepared by first mincing the tis-sues, then digesting in a solution of 5 mg/ml collagenase (type1A, Sigma–Aldrich) plus DNAase (50 U/ml) and soybean trypsininhibitor (10 mg/ml) for 20 min at 37 ◦C, as described previously

5 ccine

[gNpa

2

p(cEN((6mswFS

2

tCatli1Nuo

2

uhltpwaf

2

auAtw

2B

otfia

306 A. Henderson et al. / Va

55]. The cells were then mechanically disrupted through an 18-auge needle as previously described [56] and further purified byH4Cl lysis. Cells from each organ source were counted and resus-ended in complete medium on ice prior to immunostaining andnalysis.

.7. Antibodies and flow cytometric analysis

Directly conjugated antibodies used for these analyses wereurchased from eBioscience (San Diego, CA) or BD PharmingenSan Diego, CA). The following antibodies were used in variousombinations: anti-CD8b (APC, FITC; clone H35-17.2), anti-I-A/I-

(MHC class II, FITC; clone NIMR-4), anti-CD11c (PE-Cy7; clone418), anti-CD11b (Pacific Blue, biotin; clone M1/70), anti-Ly6G

PE; clone 1A8), anti-Ly6C (Biotin, FITC; clone AL-21), anti-PDCAPE; clone ebio927), anti-CD45R (B220, Pacific Blue; clone RA3-B2). Immunostaining was done as described previously [55]. Inost cases, cells were fixed in 1% paraformaldehyde for 20 min and

tored in FACS buffer at 4 ◦C for 1–2 days prior to analysis. Analysisas carried out with a Cyan ADP flow cytometer (Beckman Coulter,

ort Collins, CO). Data were analyzed using FlowJo software (Treetar, Ashland, OR).

.8. MHC-peptide tetramers

Soluble H-2Kb MHC class I tetramers containing the Ova8 pep-ide, SIINFEKL, were produced as described previously [57]. TheD8+ T cell response in mice vaccinated against ovalbumin wasssessed in C57BL/6 mice. Single cell suspensions (typically 5 × 105

o 1 × 106 cells suspended in 100 �l of complete media) from theung, peripheral bone marrow, and mediastinal lymph node werencubated with tetramer at 37 ◦C for 90 min. Splenocytes from OT-

mice (Ova8-specific TCR transgenic mice, provided by T. Potter,ational Jewish Medical and Research Center, Denver, CO) weresed as positive controls for tetramer staining. Staining and analysisf tetramer-labeled cells was done as described previously [41].

.9. Cytokine analysis

Cytokine production in lung and BAL samples was assessedsing a cytometric bead array (CBA; Becton Dickinson). Lungomogenates were prepped as described previously [58] and the

avage was performed using 1.5 ml of a PBS with EDTA (1 mM) solu-ion and processed as previously described [59]. The assay waserformed according to the manufacturer’s instructions. Analysisas carried out using a Cyan ADP flow cytometer and data were

nalyzed using FlowJo software. The limit of detection for this assayor each cytokine was reported by the manufacturer to be 5 pg/ml.

.10. Uptake of labeled ovalbumin in the draining lymph node

Uptake and trafficking of Ova by cells in the airwaysnd distribution to the draining lymph node was assessedsing Alexa647-labeled Ova (Invitrogen). Alexa647-Ova alone, orlexa647-Ova complexed to CLDC, were administered intranasally

o mice. Six hours after administration, the mediastinal lymphnodeas collected for immunostaining and analysis by flow cytometry.

.11. Vaccination with heat-killed bacteria for protection fromurkholderia pulmonary challenge

Heat killing of B. pseudomallei was performed as described previ-

usly [60]. Briefly, bacteria were washed and resuspended in PBS,hen heated to 80 ◦C for 1 h. Complete bacterial killing was con-rmed by agar plating on LB agar plates. To assess the ability of CLDCdjuvanted vaccines to elicit protection from a lethal infectious

29 (2011) 5304– 5312

challenge, BALB/c mice were vaccinated i.n. with CLDC adjuvantalone, 1 × 105 heat-killed B. pseudomallei organisms alone, or heat-killed bacteria mixed with 10 �l CLDC in a total volume of 20 �l.Mice were boosted in the same manner 10 days later, and thensubjected to lethal i.n. challenge with 7500 CFU live B. pseudomallei1026b (8 × LD50) 14 days after the boost, using a bacterial chal-lenge protocol described previously [50]. Mice were monitored fordisease symptoms twice daily and were euthanized according topre-determined humane endpoints.

2.12. Statistical analyses

Statistical analysis was performed using Prism 5.0 software(Graph Pad, La Jolla, CA). For comparisons between two groups,two-tailed non-parametric (Mann–Whitney) t-tests were per-formed. For comparison of more than two groups, a non-parametricANOVA (Kruskal–Wallis test) was done, followed by Dunn’s multi-ple means comparison test. Survival times were determined usingKaplan–Meier curves, followed by the log-rank test. The Bonfer-roni correction was applied for comparison of more than 2 survivalcurves. For all comparisons, differences were considered statisti-cally significant for p < 0.05.

3. Results

3.1. Mucosal immunization with CLDC adjuvant elicits systemicand local antibody responses

To assess the mucosal adjuvant properties of CLDC, we firstinvestigated the ability of vaccines delivered intranasally (i.n.)with the CLDC adjuvant to generate systemic humoral immuneresponses, using the model antigen ovalbumin (Ova). Mice weretypically immunized twice, 10 days apart. Mice immunized i.n.with a CLDC/Ova vaccine developed significant increases in totalserum Ova-specific IgG titers, compared to mice vaccinated withOva alone (Fig. 1A). CLDC adjuvanted vaccines also elicited signifi-cant increases in serum Ova-specific IgG1 titers (Fig. 1B and C).

The ability of CLDC-adjuvanted vaccines to induce local IgAresponses was assessed next. Intranasal immunization resulted ina significant increase in Ova-specific IgA titers in saliva of CLDC/Ovavaccinated mice, compared to mice vaccinated with Ova alone(Fig. 2A). CLDC/Ova also induced significant ova-specific IgA titersin the airways of mice, as assessed in the BAL fluid (Fig. 2B). Thus, itwas apparent that the mucosal administration of CLDC adjuvantedvaccines was capable of eliciting significant mucosal IgA responses,as well as significant systemic IgG responses.

3.2. Mucosal immunization with CLDC adjuvant inducesantigen-specific CD8 T cell responses

We reported previously that CLDC-adjuvanted vaccines admin-istered parenterally produced strong antigen specific T cellresponses, and were particularly effective in stimulating cross-priming and generation of antigen-specific CD8+ T cell responses[41]. Thus, it was of interest to determine whether the CLDCadjuvant could elicit similar responses following mucosal admin-istration. For these experiments, numbers of Ova-specific CD8+ Tcells in blood, BAL fluid, and lung tissues were enumerated using H-2Kb-ova8 tetramers and flow cytometry, as noted previously [41].Following i.n. immunizations, a significant increase in numbers ofCD8+ T cells was noted in all three sites evaluated (blood, lungparenchyma, and airways) (Fig. 3B). The expansion of Ova-specific

CD8+ T cells was particularly dramatic in the airways of vaccinatedmice, with 34.7% of all airway CD8+ T cells being Ova-specific. It wasclear therefore that CLDC-adjuvanted vaccines were quite effectivein generating CD8+ T cell responses in pulmonary mucosal tissues of

A. Henderson et al. / Vaccine 29 (2011) 5304– 5312 5307

Fig. 1. Mucosal immunization with CLDC adjuvant elicits systemic IgG. (A–C)C57BL/6 mice (5/group) were intranasally vaccinated twice with 2 �g ovalbuminprotein alone or in conjunction with a CLDC adjuvant as described in Section 2. Atthree weeks post-vaccination serum was collected. An ELISA for ova-specific anti-bodies was performed on serial dilutions using secondary antibodies to (A) totalIgG, (B) IgG1, and (C) IgG2a. The antibody titers are expressed as the reciprocal ofthe highest dilution of serum with an OD reading of 0.1 above background. Simi-lar results were seen in one additional experiment. Significant differences (*p < 0.05,**p < 0.01) were determined by non-parametric ANOVA followed by Dunn’s multiplemeans comparison.

Fig. 2. Mucosal immunization with CLDC adjuvant elicits local IgA responses. (A andB) C57BL/6 mice (10/group) were intranasally vaccinated twice with 2 �g ovalbuminprotein alone or in conjunction with a CLDC adjuvant as described in Section 2.At three weeks post-vaccination saliva and BAL fluid were collected. Results werepooled from two independent experiments. An IgA ELISA for ova-specific antibodieswas performed on serial dilutions. The antibody titers are expressed as the reciprocal

of the highest dilution of a sample with an OD reading of 0.1 above background.Significant differences (*p < 0.05, **p < 0.01, ***p < 0.001) were determined by non-parametric ANOVA followed by Dunn’s multiple means comparison.

vaccinated animals. The presence of antigen specific CD8+ T cells inthe airways could be very beneficial for inducing protection againstinhaled viral and bacterial pathogens.

3.3. Mucosal immunization with a CLDC adjuvanted vaccineinduces the production of IL-6 in the airways

Experiments were conducted next to assess the effects of CLDCadjuvant on local induction of innate immune responses in theairways and in lung tissues. In prior studies from our lab it was

reported that i.n. administration of CLDC stimulated pulmonaryproduction of pro-inflammatory cytokines, including IL-12, IFN-�,and MCP-1[51,58,61,62]. In the present study, we were interestedin examining CLDC induction of cytokines known to be involved

5308 A. Henderson et al. / Vaccine 29 (2011) 5304– 5312

Fig. 3. Mucosal immunization with CLDC-based vaccines results in the cross-priming of CD8+ T cells. C57BL/6 mice (5/group) were intranasally vaccinated twice with 2 �govalbumin protein alone or in conjunction with a CLDC adjuvant as described in Section 2. One week after the second immunization, CD8+ T cell responses were measuredusing H-2Kb/SIINFEKL tetramers as described in Section 2. (A) Representative FACS plot of SIINFEKL-specific CD8+ T cells elicited by vaccination with ova peptide in CLDCa cludin2 lear ce( ’s mu

i[cnnt

FtisTt2st

djuvant in the BAL fluid. (B) Total CD8+ T cells were gated for analysis (after exKb/SIINFEKL+ was plotted for the BAL fluid, lungs, and peripheral blood mononuc*p < 0.05, **p < 0.01) were determined by non-parametric ANOVA followed by Dunn

n IgA antibody class switching, including IL-6, IL-10, and TGF-�63–66]. While i.n. administration of CLDC did not induce signifi-

ant increases in IL-10 or TGF-� concentrations in the lungs (dataot shown); we found that administration of CLDC induced sig-ificant increases in IL-6 production in both the airways and lungissues (Fig. 4). For example, IL-6 concentrations in the airways of

ig. 4. Mucosal immunization with CLDC adjuvanted vaccines induces the produc-ion of IL-6 in the airways and lung tissues. C57BL/6 mice (5/group) were givenntranasal CLDC 24 h prior to collecting the BAL fluid and lung tissue. The lungupernatant was collected after tissue homogenization, as described in Section 2.he mouse inflammatory cytometric bead array was used to determine the concen-ration of IL-6 produced following stimulation with CLDC, as described in Section. Similar results were seen in one additional experiment, the asterisks denoteignificant differences (**p < 0.01) determined by non-parametric Mann–Whitney-test.

g MHC class II+ cells), and the percentage of the total CD8+ T cells that were H-lls. Similar results were seen in one additional experiment, significant differencesltiple means comparison.

mice treated with CLDC increased to 19.40 (pg/ml) ± 6.7, comparedto 4.21 (pg/ml) ± 0.01 in sham-treated control animals. Thus, theability of CLDC to elicit high levels of local IL-6 production mayaccount in part for the ability of the CLDC adjuvant to induce effi-cient IgA production.

3.4. Antigens complexed to CLDC are delivered efficiently to themediastinal lymph nodes

Given that the CLDC adjuvant could generate efficient humoraland cellular immune responses, it was important to try and under-stand how the adjuvant affected antigen presentation in the lungs.Therefore, experiments were conducted to directly assess the abil-ity of CLDC to enhance delivery of soluble antigens to draininglymph nodes. For these experiments Ova labeled with AlexaFluor647 was used to facilitate uptake and trafficking studies. Mice wereimmunized i.n. with Alexa647-Ova alone or Alexa647-Ova com-plexed to CLDC. Six hours later, antigen uptake in the mediastinallymph nodes (MLN) was assessed using flow cytometry. We foundthat administration of Alexa647-Ova complexed to CLDC resultedin significantly greater antigen delivery to the MLN, compared toadministration of Alexa647-Ova alone (Fig. 5A and B).

Next, the impact of CLDC on antigen uptake by pulmonary APCswas assessed. We found that the uptake of labeled Ova by CD11c+

DC in the MLN was significantly greater when the antigen was com-plexed to CLDC then when it was administered alone (Fig. 5C). Itshould also be noted that there was no significant difference inthe total number of CD11c+ DC in the MLN when mice adminis-

A. Henderson et al. / Vaccine 29 (2011) 5304– 5312 5309

Fig. 5. Antigens complexed to CLDC were delivered to the mediastinal lymph node following mucosal immunization and were taken up efficiently by dendritic cells.Mice (5/group) were given intranasal Alexa647 ovalbumin (5 �g) in association with CLDC 6 h prior to the collection of the draining mediastinal lymph node (MLN). (A)Representative FACS plots of Alexa647+ cells found in the MLN, the numbers represent the percent of Alexa647+ cells. (B) Quantification of Alexa647+ cells in the MLN.S ed bt cant dC

tpufatl

3ap

CiurwhmwwiC

3bbthcstFfp

Lastly, to place the potency of CLDC mucosal adjuvant prop-erties in context, CLDC-elicited vaccine responses were comparedto those generated by the conventional mucosal adjuvants choleratoxin B (CTB) and CpG oligonucleotides (CpG ODN) [9,10,12]. Mice

Fig. 6. Mucosal immunization with heat killed bacteria and CLDC adjuvant gen-erates effective protective immunity against lethal pulmonary challenge withBurkholderia pseudomallei. BALB/c mice (n = 4–5 mice per non-vaccinated controland CLDC groups, and 9 mice per HK Bp and HK Bp + CLDC groups) were primedintranasally with 1 × 105 CFU heat-killed B. pseudomallei 1026b suspended in D5Wbuffer or with heat-killed bacteria complexed to the CLDC adjuvant. Mice wereboosted in the same manner 10 days later. Mice in the CLDC alone group wereprimed and boosted with this adjuvant alone. All animals were then challengedintranasally with 7500 CFU live B. pseudomallei 1026b 14 days following the boost,and survival was monitored. Statistical differences in survival times were deter-

ignificant differences (*p < 0.05) were determined by non-parametric ANOVA followhe MLN. CD11c+ cells were analyzed for uptake of Alexa647 ovalbumin, and a signifiLDC group (**p = 0.007). Similar results were found in one additional experiment.

ered Alexa647-ova alone and mice administered Alexa647-Ovalus CLDC were compared (data not shown). The effect of CLDC onptake of Ova by other APCs in the lung was also investigated. Weound that administering Ova complexed to CLDC did not enhancentigen uptake by B cells or macrophages (data not shown). Thus,hese results show that CLDC are effective vaccine adjuvants in theungs because they enhance antigen uptake by pulmonary DC.

.5. Mucosal immunization with heat killed bacteria and CLDCdjuvant generates effective protective immunity against lethalulmonary challenge with B. pseudomallei

Experiments were conducted next to assess the potential forLDC-adjuvanted mucosal vaccines to generate robust, protective

mmunity against an inhaled pathogen. For these experiments, wesed a mouse model of lethal B. pseudomallei pneumonia, based onecent studies conducted by our laboratory [50,62]. BALB/c miceere vaccinated and boosted i.n. with CLDC adjuvant alone, 1 × 105

eat-killed B. pseudomallei organisms alone, or heat-killed bacteriaixed with 10 �l CLDC in a total volume of 20 �l. Control miceere not vaccinated. All mice were then subjected to i.n. challengeith 8 × LD50 (7.5 × 103) CFU B. pseudomallei 2 weeks after the last

mmunization and survival times were determined by the Animalare and Use Committee at Colorado State University.

All unvaccinated control mice reached end-point prior to day after challenge, and the CLDC alone mice succumbed to diseasey day 4. In contrast, 4 of the 9 mice vaccinated with heat-killedacteria alone survived for >40 days (Fig. 6). However, it is impor-ant to note that 100% of the surviving mice vaccinated only witheat-killed B. pseudomallei (without CLDC adjuvant) eventually suc-umbed to chronic disease by day 60 post-challenge (data nothown). In contrast, 100% of mice vaccinated with heat-killed bac-

eria plus CLDC survived bacterial challenge for >40 days (Fig. 6).ive of these 9 mice also survived past day 60 and were there-ore considered long-term survivors, though cultures were noterformed to confirm that in fact the organism had been erad-

y Dunn’s multiple means comparison. (C) Representative FACS plot of CD11c+ DC inifference was found between the Alexa647-ova alone group and the Alexa647-ova+

icated. These results indicate that mucosal vaccination using aCLDC-adjuvanted vaccine elicited significant protective local andsystemic immunity against a lethal challenge with a very virulentbacterial pathogen.

3.6. Potency of CLDC adjuvant equivalent or superior to that ofconventional mucosal vaccine adjuvants

mined by Kaplan–Meier curves followed by log-rank test. The Bonferroni correctedthreshold was applied and comparisons with p < 0.013 were considered significant(*p = 0.01 for mice vaccinated with heat-killed bacteria alone vs. those vaccinatedwith heat-killed bacteria complexed to CLDC). Data shown are representative of 2combined independent experiments.

5 ccine 29 (2011) 5304– 5312

wCuime(a(aiitsa

4

swaepih

ttco[apimd

appmIvasWfsa

rabaalaea

ctts

Fig. 7. Mucosal immunization with CLDC adjuvant elicits potent immune responsesequivalent to leading mucosal vaccine adjuvants. (A–C) C57BL/6 mice (5/group)were intranasally vaccinated twice with 2 �g ovalbumin protein alone or in con-junction with a CLDC adjuvant, a CTB adjuvant (5 �g), or a CpG adjuvant (10 �g),as described in Section 2. One week after the second immunization the BAL fluidwas collected. (A and B) ELISA for ova-specific antibodies was performed on serialdilutions of the BAL fluid using secondary antibodies to (A) IgA and (B) total IgG. Theantibody titers were expressed as the reciprocal of the highest dilution of serum withan OD reading of 0.1 above background. (C) Total CD8+ T cells were gated for analy-sis (after excluding MHC class II+ cells), and the percentage of the total CD8+ T cells

310 A. Henderson et al. / Va

ere therefore vaccinated i.n. with 2 �g Ova admixed with CLDC,TB (5 �g), or CpG (10 �g) [19,67–70]. Intranasal immunizationsing each of the three different adjuvants elicited significant

ncreases in ova-specific IgA titers in the BAL fluid of vaccinatedice (Fig. 7A). Of the three adjuvants only the CLDC adjuvant gen-

rated significant increases in Ova-specific IgG titers in the BAL fluidFig. 7B). However, it should also be noted that only the CpG ODNdjuvant elicited significant increases in ova-specific IgG2a titersdata not shown). Mucosal adjuvants were also compared for theirbility to generate CD8 T cell responses in the lungs. Intranasalmmunization with CLDC adjuvant appeared particularly effectiven generating antigen-specific CD8 T cell responses, especially inhe airways of vaccinated mice (Fig. 7C). Overall, these resultsuggested that at least for soluble protein antigens, CLDC baseddjuvants were as effective as current mucosal vaccine adjuvants.

. Discussion

After assessing both humoral and cellular immune responses tooluble antigens delivered intranasally using the CLDC adjuvant,e concluded that CLDC was indeed an effective mucosal vaccine

djuvant. CLDC adjuvanted vaccines were found to be particularlyffective at generating mucosal IgA responses, as well as intra-ulmonary T cell responses. The ability of the CLDC adjuvant to

ncrease the immunogenicity of a complex particulate antigen (i.e.,eat-killed bacteria) was also demonstrated.

A variety of immunological properties that have been attributedo cationic liposomes are likely to have contributed to the effec-iveness of CLDC as a mucosal vaccine adjuvant. For one, positivelyharged liposomes rapidly adhere to negatively charged surfacesf cells such as APCs and epithelial cells, increasing their uptake38,71]. In addition, cationic liposomes have been shown to directlyctivate APCs such as DC [38,72,73]. Finally, the size of the CLDCarticles used in this study (approximately 250 nm diameter) is

deal for uptake by DC, including pulmonary DC [72,74]. Thus, theucosal adjuvant properties of CLDC are likely dependent to a large

egree on cationic liposomes.The adjuvant activity of cationic liposome-nucleic acid-based

djuvants is also importantly influenced by the nucleic acid com-onent of the vaccine [75]. In the current study, the non-codinglasmid DNA used in the preparation of CLDC contains many CpGotifs and is known to activate innate immunity via TLR9 signaling.

ndeed, in our studies we found that cellular immune responses toaccination with CLDC adjuvanted vaccines were nearly completelybolished in MyD88−/− mice (data not shown), indicating that TLRignaling in the lungs was critical to the activity of CLDC adjuvants.

hile it is currently not known how well other TLR agonists mightunction as mucosal adjuvants when complexed to cationic lipo-omes, it is known that TLR-3 agonists, such as polyI:C, are effectivet stimulating immune responses with cationic liposomes [41].

The capacity of CLDC to modulate the airway cytokine envi-onment is another critical feature to consider when assessing thedjuvant activity. Intranasal administration of CLDC has previouslyeen shown to induce the production of IL-12, TNF-�, IFN-�, IFN-�nd IFN-� [51,58,61,62]. In the current study we found that CLDCdministration induced pulmonary expression of IL-6, a cytokineinked to the induction of IgA class-switching [66,76–80]. IL-6 haslso been shown to stimulate T cell proliferation [81–83], and tonhance generation of protective immunity following vaccinationgainst respiratory pathogens [84,85].

The unique ability of CLDC to elicit the cross-priming of CD8 T

ells to protein antigens has been explored previously in the con-ext of parenteral vaccines [41] as well in a therapeutic vaccine usedo suppress hyperresponsiveness in the airways [86]. In the presenttudy we found that mucosal vaccination using CLDC, as an adju-

that were H-2 Kb/SIINFEKL+ was plotted for BAL fluid. Similar results were seen inone additional experiment. Significant differences (*p < 0.05, **p < 0.01, ***p < 0.001)were determined by non-parametric ANOVA followed by Dunn’s multiple meanscomparison.

ccine

vricrlM

aapokaaowiCa

atoocmbfoa

bcweulc

A

Hpa

R

[

[

[

[

[

[

[

[

[

[

[[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

A. Henderson et al. / Va

ant was also capable of rapidly generating pulmonary CD8+ T cellesponses. While the mechanism of CLDC mediated cross-primings not fully understood, it is believed that the cationic liposomeomponent of CLDC results in the slight instability of the endosomeesulting in the leakage of endosomal contents into the cytoplasm,eading to the processing and presentation of peptide fragments via

HC class I [36].By using a labeled antigen (Ova), we were able to directly visu-

lize the interaction of the antigen with relevant APCs, as well asssess how CLDC affected that interaction. We found that com-lexing the antigen to CLDC resulted in the preferential targetingf antigen to resident pulmonary DC. We believe that DCs are theey cells responsible for increased antigen presentation followingntigen delivery with CLDC. We also noted the uptake of labeledntigen by B cells and macrophages in the MLN, but the additionf CLDC did not enhance antigen uptake by these cells. Therefore,e believe that the uptake of antigen by B cells and macrophages

n the MLN may have resulted from passive transport of soluble orLDC bound antigen directly through the lymphatics, without cellssociated transport.

The ability of the CLDC adjuvant to improve antigen processingnd presentation following mucosal administration is not confinedo the respiratory tract. For example, we have recently reported thatral administration of CLDC-adjuvanted vaccines is also capablef generating substantial protective immunity against pulmonaryhallenge with Yersinia pestis [87]. Thus, induction of efficientucosal immunity, particularly at pulmonary surfaces, appears to

e a general property of CLDC-based adjuvants. Moreover, we alsoound here that CLDC adjuvants performed well when compared tother conventional adjuvants in terms of potency of both humoralnd cellular immunity.

In summary, these findings suggest that liposome-nucleic acidased adjuvants are an important new category of mucosal vac-ine adjuvant that generates considerable activity when combinedith protein antigens. Properties that appear to contribute to the

ffectiveness of CLDC for mucosal administration include efficientptake by DC, rapid transit of antigen-CLDC complexes to regional

ymph nodes, and potent induction of cytokines involved in IgAlass-switching at mucosal surfaces.

cknowledgements

The authors wish to acknowledge the assistance of Dr. Scottafeman for preparation of liposomes. These studies were sup-orted in part by a grant from the NIH NIAID (U54 AI065357) and by

collaborative research agreement from Juvaris Biotherapeutics.

eferences

[1] Belyakov IM, Ahlers JD. What role does the route of immunization play inthe generation of protective immunity against mucosal pathogens? J Immunol2009;183(December (11)):6883–92.

[2] Harandi AM, Medaglini D, Shattock RJ. Vaccine adjuvants: a priority for vaccineresearch. Vaccine 2010;28(March (12)):2363–6.

[3] Lamm ME. Interaction of antigens and antibodies at mucosal surfaces. AnnuRev Microbiol 1997;51:311–40.

[4] Neutra MR, Kozlowski PA. Mucosal vaccines: the promise and the challenge.Nat Rev Immunol 2006;6(February (2)):148–58.

[5] Holmgren J, Czerkinsky C. Mucosal immunity and vaccines. Nat Med2005;11(April (4 Suppl.)):S45–53.

[6] Belshe RB, Edwards KM, Vesikari T, Black SV, Walker RE, Hultquist M, et al. Liveattenuated versus inactivated influenza vaccine in infants and young children.N Engl J Med 2007;356(February (7)):685–96.

[7] Viret JF, Favre D, Wegmuller B, Herzog C, Que JU, Cryz Jr SJ, et al. Mucosaland systemic immune responses in humans after primary and booster immu-

nizations with orally administered invasive and noninvasive live attenuatedbacteria. Infect Immun 1999;67(7):3680–5.

[8] Cox E, Verdonck F, Vanrompay D, Goddeeris B. Adjuvants modulating mucosalimmune responses or directing systemic responses towards the mucosa. VetRes 2006;37(May–June (3)):511–39.

[

[

29 (2011) 5304– 5312 5311

[9] Eriksson K, Holmgren J. Recent advances in mucosal vaccines and adjuvants.Curr Opin Immunol 2002;14(October (5)):666–72.

10] Moyle PM, McGeary RP, Blanchfield JT, Toth I. Mucosal immunisation: adju-vants and delivery systems. Curr Drug Deliv 2004;1(October (4)):385–96.

11] Oliveira ML, Areas AP, Ho PL. Intranasal vaccines for protection against res-piratory and systemic bacterial infections. Expert Rev Vaccines 2007;6(June(3)):419–29.

12] Freytag LC, Clements JD. Mucosal adjuvants. Vaccine 2005;23(March(15)):1804–13.

13] Harandi AM. The potential of immunostimulatory CpG DNA for inducingimmunity against genital herpes: opportunities and challenges. J Clin Virol2004;30(July (3)):207–10.

14] Hasegawa H, Ichinohe T, Tamura S, Kurata T. Development of a mucosal vaccinefor influenza viruses: preparation for a potential influenza pandemic. ExpertRev Vaccines 2007;6(April (2)):193–201.

15] Holmgren J, Adamsson J, Anjuere F, Clemens J, Czerkinsky C, Eriksson K, et al.Mucosal adjuvants and anti-infection and anti-immunopathology vaccinesbased on cholera toxin, cholera toxin B subunit and CpG DNA. Immunol Lett2005;97(March (2)):181–8.

16] Kensil CR, Mo AX, Truneh A. Current vaccine adjuvants: an overview of a diverseclass. Front Biosci 2004;September (9):2972–88.

17] Klinman DM, Klaschik S, Sato T, Tross D. CpG oligonucleotides as adjuvantsfor vaccines targeting infectious diseases. Adv Drug Deliv Rev 2009;61(March(3)):248–55.

18] McCluskie MJ, Weeratna RD, Clements JD, Davis HL. Mucosal immunizationof mice using CpG DNA and/or mutants of the heat-labile enterotoxin ofEscherichia coli as adjuvants. Vaccine 2001;19(June (27)):3759–68.

19] McCluskie MJ, Weeratna RD, Davis HL. The potential of oligodeoxynu-cleotides as mucosal and parenteral adjuvants. Vaccine 2001;19(March(17–19)):2657–60.

20] Stevceva L, Ferrari MG. Mucosal adjuvants. Curr Pharm Des 2005;11(6):801–11.21] Dittmer U, Olbrich AR. Treatment of infectious diseases with immunostim-

ulatory oligodeoxynucleotides containing CpG motifs. Curr Opin Microbiol2003;6(October (5)):472–7.

22] Jahrsdorfer B, Weiner GJ. CpG oligodeoxynucleotides for immune stimulationin cancer immunotherapy. Curr Opin Investig Drugs 2003;4(June (6)):686–90.

23] Krieg AM. Therapeutic potential of toll-like receptor 9 activation. Nat Rev DrugDiscov 2006;5(June (6)):471–84.

24] Fujihashi K, Koga T, van Ginkel FW, Hagiwara Y, McGhee JR. A dilemma formucosal vaccination: efficacy versus toxicity using enterotoxin-based adju-vants. Vaccine 2002;20(June (19–20)):2431–8.

25] Heikenwalder M, Polymenidou M, Junt T, Sigurdson C, Wagner H, Akira S,et al. Lymphoid follicle destruction and immunosuppression after repeated CpGoligodeoxynucleotide administration. Nat Med 2004;10(February (2)):187–92.

26] Klinman DM. Immunotherapeutic uses of CpG oligodeoxynucleotides. Nat RevImmunol 2004;4(April (4)):249–58.

27] van Ginkel FW, Jackson RJ, Yuki Y, McGhee JR. Cutting edge: the mucosal adju-vant cholera toxin redirects vaccine proteins into olfactory tissues. J Immunol2000;165(November (9)):4778–82.

28] van Ginkel FW, Nguyen HH, McGhee JR. Vaccines for mucosal immunity tocombat emerging infectious diseases. Emerg Infect Dis 2000;6(March–April(2)):123–32.

29] Acevedo R, Callico A, del Campo J, Gonzalez E, Cedre B, Gonzalez L, et al.Intranasal administration of proteoliposome-derived cochleates from Vibriocholerae O1 induce mucosal and systemic immune responses in mice. Methods2009;49(December (4)):309–15.

30] Benoit A, Huang Y, Proctor J, Rowden G, Anderson R. Effects of alveolarmacrophage depletion on liposomal vaccine protection against respiratory syn-cytial virus (RSV). Clin Exp Immunol 2006;145(July (1)):147–54.

31] Rosada RS, de la Torre LG, Frantz FG, Trombone AP, Zarate-Blades CR, FonsecaDM, et al. Protection against tuberculosis by a single intranasal administra-tion of DNA-hsp65 vaccine complexed with cationic liposomes. BMC Immunol2008;9:38.

32] Sakaue G, Hiroi T, Nakagawa Y, Someya K, Iwatani K, Sawa Y, et al. HIV mucosalvaccine: nasal immunization with gp160-encapsulated hemagglutinating virusof Japan-liposome induces antigen-specific CTLs and neutralizing antibodyresponses. J Immunol 2003;170(January (1)):495–502.

33] Tseng LP, Chiou CJ, Chen CC, Deng MC, Chung TW, Huang YY, et al.Effect of lipopolysaccharide on intranasal administration of liposomal New-castle disease virus vaccine to SPF chickens. Vet Immunol Immunopathol2009;131(October (3–4)):285–9.

34] Wang D, Christopher ME, Nagata LP, Zabielski MA, Li H, Wong JP, et al. Intranasalimmunization with liposome-encapsulated plasmid DNA encoding influenzavirus hemagglutinin elicits mucosal, cellular and humoral immune responses.J Clin Virol 2004;31(December (Suppl. 1)):S99–106.

35] Heurtault B, Frisch B, Pons F. Liposomes as delivery systems for nasal vaccina-tion: strategies and outcomes. Expert Opin Drug Deliv 2010;7(July (7)):829–44.

36] Chikh G, Schutze-Redelmeier MP. Liposomal delivery of CTL epitopes to den-dritic cells. Biosci Rep 2002;22(April (2)):339–53.

37] Copland MJ, Baird MA, Rades T, McKenzie JL, Becker B, Reck F, et al. Liposo-mal delivery of antigen to human dendritic cells. Vaccine 2003;21(February

(9–10)):883–90.

38] Torchilin VP. Recent advances with liposomes as pharmaceutical carriers. NatRev Drug Discov 2005;4(February (2)):145–60.

39] Baca-Estrada ME, Foldvari MM, Snider MM, Harding KK, Kournikakis BB,Babiuk LA, et al. Intranasal immunization with liposome-formulated Yersinia

5 ccine

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

312 A. Henderson et al. / Va

pestis vaccine enhances mucosal immune responses. Vaccine 2000;18(April(21)):2203–11.

40] Khatri K, Goyal AK, Gupta PN, Mishra N, Mehta A, Vyas SP. Surface modified lipo-somes for nasal delivery of DNA vaccine. Vaccine 2008;26(April (18)):2225–33.

41] Zaks K, Jordan M, Guth A, Sellins K, Kedl R, Izzo A, et al. Efficient immuniza-tion and cross-priming by vaccine adjuvants containing TLR3 or TLR9 agonistscomplexed to cationic liposomes. J Immunol 2006;176(June (12)):7335–45.

42] Guy B. The perfect mix: recent progress in adjuvant research. Nat Rev Microbiol2007;5(July (7)):505–17.

43] Mutwiri G, Gerdts V, van Drunen Littel-van den Hurk S, Auray G, Eng N, GarlapatiS, et al. Combination adjuvants: the next generation of adjuvants? Expert RevVaccines 2011;10(January (1)):95–107.

44] Bernstein DI, Cardin RD, Bravo FJ, Strasser JE, Farley N, Chalk C, et al. Potent adju-vant activity of cationic liposome–DNA complexes for genital herpes vaccines.Clin Vaccine Immunol 2009;16(May (5)):699–705.

45] Bernstein DI, Farley N, Bravo FJ, Earwood J, McNeal M, Fairman J, et al. Theadjuvant CLDC increases protection of a herpes simplex type 2 glycoprotein Dvaccine in guinea pigs. Vaccine 2009.

46] Chang S, Warner J, Liang L, Fairman J. A novel vaccine adjuvant for recombinantflu antigens. Biologicals 2009;37(June (3)):141–7.

47] Cote PJ, Butler SD, George AL, Fairman J, Gerin JL, Tennant BC, et al. Rapidimmunity to vaccination with woodchuck hepatitis virus surface antigen usingcationic liposome–DNA complexes as adjuvant. J Med Virol 2009;81(October(10)):1760–72.

48] Fairman J, Moore J, Lemieux M, Van Rompay K, Geng Y, Warner J, et al.Enhanced in vivo immunogenicity of SIV vaccine candidates with cationicliposome–DNA complexes in a rhesus macaque pilot study. Hum Vaccin2009;5(March (3)):141–50.

49] Lay M, Callejo B, Chang S, Hong DK, Lewis DB, Carroll TD, et al. Cationic lipid/DNAcomplexes (JVRS-100) combined with influenza vaccine (Fluzone) increasesantibody response, cellular immunity, and antigenically drifted protection.Vaccine 2009;27(June (29)):3811–20.

50] Goodyear A, Kellihan L, Bielefeldt-Ohmann H, Troyer R, Propst K, Dow S. Pro-tection from pneumonic infection with Burkholderia species by inhalationalimmunotherapy. Infect Immun 2009;77(April (4)):1579–88.

51] Troyer RM, Propst KL, Fairman J, Bosio CM, Dow SW. Mucosal immunotherapyfor protection from pneumonic infection with Francisella tularensis. Vaccine2009;27(July (33)):4424–33.

52] Templeton NS, Lasic DD, Frederik PM, Strey HH, Roberts DD, Pavlakis GN.Improved DNA: liposome complexes for increased systemic delivery and geneexpression. Nat Biotechnol 1997;15(July (7)):647–52.

53] Frey A, Di Canzio J, Zurakowski D. A statistically defined endpoint titer deter-mination method for immunoassays. J Immunol Methods 1998;221(December(1–2)):35–41.

54] Maloy KJ, Donachie AM, O’Hagan DT, Mowat AM. Induction of mucosal andsystemic immune responses by immunization with ovalbumin entrappedin poly(lactide-co-glycolide) microparticles. Immunology 1994;81(April(4)):661–7.

55] Bosio CM, Dow SW. Francisella tularensis induces aberrant activation of pul-monary dendritic cells. J Immunol 2005;175(November (10)):6792–801.

56] Fisher JH, Larson J, Cool C, Dow SW. Lymphocyte activation in the lungs of SP-Dnull mice. Am J Respir Cell Mol Biol 2002;27(July (1)):24–33.

57] Ahonen CL, Doxsee CL, McGurran SM, Riter TR, Wade WF, Barth RJ, et al. Com-bined TLR and CD40 triggering induces potent CD8+ T cell expansion withvariable dependence on type I IFN. J Exp Med 2004;199(March (6)):775–84.

58] Goodyear A, Jones A, Troyer R, Bielefeldt-Ohmann H, Dow S. Critical protec-tive role for MCP-1 in pneumonic Burkholderia mallei infection. J Immunol2011;184(February (3)):1445–54.

59] Lambrecht BN, Salomon B, Klatzmann D, Pauwels RA. Dendritic cells arerequired for the development of chronic eosinophilic airway inflammationin response to inhaled antigen in sensitized mice. J Immunol 1998;160(April(8)):4090–7.

60] Lauw FN, Simpson AJ, Prins JM, van Deventer SJ, Chaowagul W, White NJ, et al.The CXC chemokines gamma interferon (IFN-gamma)-inducible protein 10 andmonokine induced by IFN-gamma are released during severe melioidosis. InfectImmun 2000;68(July (7)):3888–93.

61] Freimark BD, Blezinger HP, Florack VJ, Nordstrom JL, Long SD, Deshpande DS,et al. Cationic lipids enhance cytokine and cell influx levels in the lung followingadministration of plasmid: cationic lipid complexes. J Immunol 1998;160(May(9)):4580–6.

62] Propst KL, Troyer RM, Kellihan LM, Schweizer HP, Dow SW. Immunotherapymarkedly increases the effectiveness of antimicrobial therapy for treatmentof Burkholderia pseudomallei infection. Antimicrob Agents Chemother (2010

February 22).

63] Cerutti A. The regulation of IgA class switching. Nat Rev Immunol 2008;8(June(6)):421–34.

64] Fagarasan S, Honjo T. Intestinal IgA synthesis: regulation of front-line bodydefences. Nat Rev Immunol 2003;3(January (1)):63–72.

[

29 (2011) 5304– 5312

65] Macpherson AJ, McCoy KD, Johansen FE, Brandtzaeg P. The immune geographyof IgA induction and function. Mucosal Immunol 2008;1(January (1)):11–22.

66] Okahashi N, Yamamoto M, Vancott JL, Chatfield SN, Roberts M, Bluethmann H,et al. Oral immunization of interleukin-4 (IL-4) knockout mice with a recombi-nant Salmonella strain or cholera toxin reveals that CD4+ Th2 cells producingIL-6 and IL-10 are associated with mucosal immunoglobulin A responses. InfectImmun 1996;64(May (5)):1516–25.

67] Fukuiwa T, Sekine S, Kobayashi R, Suzuki H, Kataoka K, Gilbert RS, et al. Acombination of Flt3 ligand cDNA and CpG ODN as nasal adjuvant elicits NALTdendritic cells for prolonged mucosal immunity. Vaccine 2008;26(September(37)):4849–59.

68] Kim HJ, Kim JK, Seo SB, Lee HJ, Kim HJ. Intranasal vaccination with peptides andcholera toxin subunit B as adjuvant to enhance mucosal and systemic immunityto respiratory syncytial virus. Arch Pharm Res 2007;30(March (3)):366–71.

69] Porgador A, Staats HF, Faiola B, Gilboa E, Palker TJ. Intranasal immunizationwith CTL epitope peptides from HIV-1 or ovalbumin and the mucosal adju-vant cholera toxin induces peptide-specific CTLs and protection against tumordevelopment in vivo. J Immunol 1997;158(January (2)):834–41.

70] Wakabayashi A, Nakagawa Y, Shimizu M, Moriya K, Nishiyama Y, TakahashiH. Suppression of an already established tumor growing through activatedmucosal CTLs induced by oral administration of tumor antigen with choleratoxin. J Immunol 2008;180(March (6)):4000–10.

71] Ahsan F, Rivas IP, Khan MA, Torres Suarez AI. Targeting to macrophages:role of physicochemical properties of particulate carriers – liposomes andmicrospheres – on the phagocytosis by macrophages. J Control Release2002;79(February (1–3)):29–40.

72] Foged C, Brodin B, Frokjaer S, Sundblad A. Particle size and surface chargeaffect particle uptake by human dendritic cells in an in vitro model. Int J Pharm2005;298(July (2)):315–22.

73] Nakanishi T, Kunisawa J, Hayashi A, Tsutsumi Y, Kubo K, Nakagawa S, et al.Positively charged liposome functions as an efficient immunoadjuvant ininducing immune responses to soluble proteins. Biochem Biophys Res Commun1997;240(November (3)):793–7.

74] Brewer JM, Pollock KG, Tetley L, Russell DG. Vesicle size influences the traf-ficking, processing, and presentation of antigens in lipid vesicles. J Immunol2004;173(November (10)):6143–50.

75] Dow S, Liposome-nucleic acid immunotherapeutics. Expert Opin Drug Deliv2008;5(January (1)):11–24.

76] Beagley KW, Eldridge JH, Lee F, Kiyono H, Everson MP, Koopman WJ, et al. Inter-leukins and IgA synthesis human and murine interleukin 6 induce high rate IgAsecretion in IgA-committed B cells. J Exp Med 1989;169(June (6)):2133–48.

77] DiCosmo BF, Geba GP, Picarella D, Elias JA, Rankin JA, Stripp BR, et al.Airway epithelial cell expression of interleukin-6 in transgenic mice uncou-pling of airway inflammation and bronchial hyperreactivity. J Clin Invest1994;94(November (5)):2028–35.

78] McGhee JR, Mestecky J, Elson CO, Kiyono H. Regulation of IgA synthesis andimmune response by T cells and interleukins. J Clin Immunol 1989;9(May(3)):175–99.

79] Ruedl C, Rieser C, Kofler N, Wick G, Wolf H. Humoral and cellular immuneresponses in the murine respiratory tract following oral immunization withcholera toxin or Escherichia coli heat-labile enterotoxin. Vaccine 1996;14(June(8)):792–8.

80] Sato A, Hashiguchi M, Toda E, Iwasaki A, Hachimura S, Kaminogawa S. CD11b+Peyer’s patch dendritic cells secrete IL-6 and induce IgA secretion from naiveB cells. J Immunol 2003;171(October (7)):3684–90.

81] DiCosmo B, Geba G, Picarella D, Elias JA, Rankin JA, Stripp B, et al. Expressionof interleukin-6 by airway epithelial cells effects on airway inflammation andhyperreactivity in transgenic mice. Chest 1995;107(March (3 Suppl.)):131S.

82] Jonuleit H, Kuhn U, Muller G, Steinbrink K, Paragnik L, Schmitt E, et al.Pro-inflammatory cytokines and prostaglandins induce maturation of potentimmunostimulatory dendritic cells under fetal calf serum-free conditions. EurJ Immunol 1997;27(December (12)):3135–42.

83] Rincon M, Anguita J, Nakamura T, Fikrig E, Flavell RA. Interleukin (IL)-6 directsthe differentiation of IL-4-producing CD4+ T cells. J Exp Med 1997;185(Febru-ary (3)):461–9.

84] Leal IS, Smedegard B, Andersen P, Appelberg R. Interleukin-6 and interleukin-12participate in induction of a type 1 protective T-cell response during vacci-nation with a tuberculosis subunit vaccine. Infect Immun 1999;67(November(11)):5747–54.

85] Picard C, Puel A, Bonnet M, Ku CL, Bustamante J, Yang K, et al. Pyogenic bac-terial infections in humans with IRAK-4 deficiency. Science 2003;299(March(5615)):2076–9.

86] Takeda K, Dow SW, Miyahara N, Kodama T, Koya T, Taube C, et al. Vaccine-

induced CD8+ T cell-dependent suppression of airway hyperresponsivenessand inflammation. J Immunol 2009;183(July (1)):181–90.

87] Jones A, Bosio C, Duffy A, Goodyear A, Schriefer M, Dow S. Protection againstpneumonic plague following oral immunization with a non-replicating vaccine.Vaccine 2010;28(August (36)):5924–9.