Topical CpG Adjuvantation of a Protein-Based Vaccine InducesProtective Immunity to Listeria monocytogenes

Wing Ki Cheng,a Kathleen Wee,b Tobias R. Kollmann,b Jan P. Dutza

Department of Dermatology and Skin Science, Faculty of Medicine, Child and Family Research Institute, The University of British Columbia, Vancouver, British Columbia,Canadaa; Department of Pediatrics, Faculty of Medicine, Child and Family Research Institute, The University of British Columbia, Vancouver, British Columbia, Canadab

Robust CD8� T cell responses are essential for immune protection against intracellular pathogens. Using parenteral administra-tion of ovalbumin (OVA) protein as a model antigen, the effect of the Toll-like receptor 9 (TLR9) agonist, CpG oligodeoxynucle-otide (ODN) 1826, as an adjuvant delivered either topically, subcutaneously, or intramuscularly on antigen-specific CD8� T cellresponses in a mouse model was evaluated. Topical CpG adjuvant increased the frequency of OVA-specific CD8� T cells in theperipheral blood and in the spleen. The more effective strategy to administer topical CpG adjuvant to enhance CD8� T cell re-sponses was single-dose administration at the time of antigen injection with a prime-boost regimen. Topical CpG adjuvant con-ferred both rapid and long-lasting protection against systemic challenge with recombinant Listeria monocytogenes expressingthe cytotoxic T lymphocyte (CTL) epitope of OVA257–264 (strain Lm-OVA) in a TLR9-dependent manner. Topical CpG adjuvantinduced a higher proportion of CD8� effector memory T cells than parenteral administration of the adjuvant. Although tradi-tional vaccination strategies involve coformulation of antigen and adjuvant, split administration using topical adjuvant is effec-tive and has advantages of safety and flexibility. Split administration of topical CpG ODN 1826 with parenteral protein antigen issuperior to other administration strategies in enhancing both acute and memory protective CD8� T cell immune responses tosubcutaneous protein vaccines. This vaccination strategy induces rapid and persistent protective immune responses against theintracellular organism L. monocytogenes.

Infectious diseases are the leading cause of illness in humans.Immunization is the most cost-effective method to improve

population health against infectious diseases. However, currentvaccines remain inefficient in part due to a poor ability to elicitcytotoxic T lymphocytes (CTLs), delayed protection, and lack oflong-lasting protective effect. An adjuvant is often added to in-crease the immunogenicity of vaccine antigens. Adjuvants areroutinely combined with antigens for efficacy and ease of formu-lation (1). Split administration, whereby vaccine antigen and ad-juvant are delivered separately, has been less commonly studied(2) but has the advantage of flexibility and simplicity. Here, weexplore split administration of antigen and adjuvant using theskin as an administration site and a Toll-like receptor 9 (TLR9)agonist as the vaccine adjuvant.

CpG oligodeoxynucleotides (ODNs) bind TLR9, an endo-somal pattern recognition receptor that recognizes unmethylatedbacterial and viral ODNs with CpG motifs (3). CpG ODNs induceboth potent CD8� T cells and Th1-biased CD4� T cell immuneresponses in both mouse and humans (4, 5). The enhanced im-mune responses induced by CpG ODNs provide protective im-munity in several infectious disease models. For example, reducedbacterial burden was detected in mice immunized with CpG ODNand ovalbumin (OVA) coadministered intramuscularly whenchallenged with Listeria monocytogenes expressing the CTLepitope of OVA257–264 (designated Lm-OVA) 10 days postimmu-nization(6).Also, intramuscularcoinjectionofrecombinantToxo-plasma gondii protein with CpG ODN as an adjuvant induces aTh1-biased humoral response demonstrated by an increasedIgG2a to IgG1 antibody ratio and increased protection against oralT. gondii infection in a mouse model (7). In a human clinical trial,intramuscular delivery of CpG 7909 induced robust specific anti-body response to a commercial hepatitis B vaccine (Engerix-B)(8). Although studies have demonstrated the effectiveness of CpG

ODNs as adjuvants, concerns remain about the local and systemicside effects observed. In mice, CpG ODNs can induce tumor ne-crosis factor alpha (TNF-�) release by macrophages, resulting inseptic shock (9, 10). Strategies to limit the systemic toxicity of CpGODNs include conjugation of CpG ODN to antigen (11) and co-encapsulation of CpG ODN with antigen (12). These strategies arecumbersome and require validation in humans. Split administra-tion of CpG ODN as an adjuvant and as a means of enhancingvaccine efficacy while limiting toxicity has not been pursued.

The skin is the most accessible organ of our bodies and harborsmany immune cells including different subsets of dendritic cells(DCs) (13), mast cells (14), and resident lymphocytes (15–17) thatcan be harnessed to induce immune responses. We, and others,have explored the skin as a site of vaccination by coadministrationof antigen and adjuvant. Topical peptide vaccination with choleratoxin induces robust cellular immune responses in mice (18).However, the toxicity of cholera toxin makes it difficult to handle.Topical CpG ODN coadministered with topical antigen promotesCD8� T cell production to the antigen (19). Topical administra-tion of CpG ODN also promotes cross-presentation of injectedsoluble OVA protein antigen with less systemic cytokine produc-tion and toxicity than subcutaneous administration (20). Whentopical CpG ODN is administered epifocally to the antigen ad-

Received 18 November 2013 Returned for modification 18 December 2013Accepted 26 December 2013

Published ahead of print 3 January 2014

Editor: M. F. Pasetti

Address correspondence to Jan P. Dutz, dutz@interchange.ubc.ca.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/CVI.00734-13

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ministration site, meaning that it is applied to skin that has thesame lymphatic drainage as the site to which antigen is given, theCpG adjuvant augments CD8� T cell responses against melanomain a mouse model (21). CpG has an adjuvant effect only withepifocal administration and not when it is applied to a site con-tralateral to the antigen. This demonstrates the ability of splittingantigen and vaccine antigen administration and emphasizes theneed for common lymph drainage of antigen and adjuvant formaximum effect (21). The requirement of epifocal administrationof adjuvant and antigen suggests that topical CpG ODN instructsantigen-specific T cell generation in the skin draining lymphnodes (SLNs). Here, we sought to examine various strategies toadminister CpG ODN 1826 onto the skin to enhance CD8� T cellresponses. We demonstrate that single topical CpG ODN admin-istration at the time of standard (parenteral) immunization is ef-fective, preferentially induces effector memory T cells, and may beused to induce protective immunity against the intracellularpathogen L. monocytogenes.

MATERIALS AND METHODSAnimals. C57BL/6 female mice 6 to 8 weeks of age were purchased fromCharles River Laboratories (Wilmington, MA) and housed in a specific-pathogen-free animal care facility at the Child & Family Research Institute(CFRI) (Vancouver, Canada). TLR9-deficient mice on C57BL/6 back-ground (TLR9 knockout [KO]) were obtained from Oriental Bioservice(Tokyo, Japan) and bred and maintained in the animal care facility atCFRI. Animal experiments were conducted in accordance with protocolsapproved by the Animal Care Committee of The University of BritishColumbia and Canadian Council of Animal Care.

Deoxynucleotides and peptide. Synthetic high-pressure liquid chro-matography (HPLC)-purified, single-stranded, phosphorothioated CpGODN 1826 (5=-TCCATGACGTTCCTGACGTT-3=) was purchased fromSigma-Aldrich Inc. (St. Louis, MO). Control ODN without CpG motifs(5=-TCCAGGACTTCTCTCAGGTT-3=) was purchased from IntegratedDNA Technologies, Inc. (San Diego, CA). Lyophilized CpG ODN 1826and control ODN were reconstituted to 5 mg/ml with phosphate-bufferedsaline (PBS)– dimethyl sulfoxide (DMSO) (1:1, vol/vol). The immuno-dominant Kb-restricted OVA8 peptide (OVA257–264, amino acid sequenceSIINFEKL) was synthesized by Kinexus (Vancouver, BC).

Immunization. C57BL/6 or TLR9 KO female mice 6 to 12 weeks of agewere anesthetized by intraperitoneal injection of 75 mg ketamine/kg ofbody weight (Ketalean; Bimeda-MTC Animal Health Inc., Cambridge,ON) and 7.5 mg/kg xylazine (Rompun; Bayer Health Care Inc., Toronto,ON). Mice were shaved on the dorsal back, tape-stripped 15 times usingcellophane tape (Staples, Vancouver, BC), and the skin was wiped withacetone (Fisher Scientific, Edmonton, AB) using a cotton swab. One hun-dred micrograms of chicken ovalbumin protein grade V (OVA) fromSigma-Aldrich Inc. (St. Louis, MO) was injected either subcutaneously orintramuscularly. Next, 250 �g or 50 �g of CpG ODN 1826 was adminis-tered topically, subcutaneously, or intramuscularly in an epifocal manner(overlaying the antigen injection site). PBS-DMSO (1:1, vol/vol) or con-trol ODN was applied when no adjuvant was administered. The area wasthen covered with waterproof tape to prevent oral ingestion of the topicaladjuvant or control. When antigen and adjuvant (or control) were admin-istered by the same route, they were given together in a single injection.For a prime-boost regimen, mice were immunized on days 0 and 7, whilemice were immunized once on day 0 for a one-time immunization regi-men. Multiple applications of the adjuvant CpG ODN 1826 were admin-istered at the time of antigen administration and again at 1 and 2 days (for3 consecutive days) postimmunization: days 0, 1, and 2 for a one-timeimmunization regimen and days 0, 1, 2, 7, 8, and 9 for a prime-boostregimen.

Bacterial strains, medium, and growth conditions. Wild-type L.monocytogenes strain 10403s and strain Lm-OVA, which was modified

from the wild type to express the CTL epitope of OVA257–264, were pro-vided by H. Shen (University of Pennsylvania, Philadelphia). The con-struction of the less virulent form, strain �actA-Lm-OVA, with a targeteddeletion in the virulence determinant ActA has been described elsewhere(22). For immunization and infection experiments, frozen infection ali-quots of L. monocytogenes strains were prepared as described previously(23). Briefly, L. monocytogenes strains were grown in brain heart infusion(BHI) broth to mid-logarithmic phase (optical density at 600 nm[OD600], 1.0) at 37°C, washed twice with endotoxin-free, isotonic saline(0.9% NaCl), resuspended in 0.9% NaCl with 20% glycerol (vol/vol), andstored at �80°C until use.

Immunizing infection and animal challenge with L. monocytogenes.Mice were infected with 5 � 105 CFU of strain Lm-OVA in 100 �l 0.9%NaCl systemically via the tail vein. Three days after infection, mice wereeuthanized to collect the spleen, which was then homogenized mechani-cally in 5 ml 0.9% NaCl containing 0.05% Triton X-100. To enumerate theviable bacteria colonizing the spleen postinfection, serial dilutions of themechanically lysed cell suspensions were plated on BHI agar. For immu-nizing infection, mice were intraperitoneally injected with 2 � 106 CFU of�actA-Lm-OVA.

Flow cytometry antibodies and tetramer. Antibodies B220-PerCP(clone RA3-6B2), CD8-APC (clone 53-6.7), and gamma interferon (IFN-�)-APC (clone XMG1.2) were purchased from BD Biosciences Inc. (Mis-sissauga, ON, Canada). Antibodies CD62L-FITC (clone MEL-14) andCD127-APC (clone A7R34) were purchased from eBioscience Inc. (SanDiego, CA), whereas CD8-A700 (clone 53.67) was purchased from theAbLab at The University of British Columbia (Vancouver, BC, Canada).Phycoerythrin (PE)-conjugated OVA major histocompatibility complexclass I (MHC-I) tetramer specific for the Kb-restricted OVA8 peptide(OVA257–264, amino acid sequence SIINFEKL) was generated by conjuga-tion of biotinylated monomers to streptavidin-PE in R. Tan’s laboratory(Child & Family Research Institute, Vancouver, BC, Canada). The anti-Fcreceptor monoclonal antibody (clone 2.4G2) was obtained from Ameri-can Type Culture Collection (Rockville, MD). OVA-specific CD8� T cellswere identified as a B220-negative and CD8 and Kb-OVA MHC-Itetramer double-positive population. Flow cytometry data were acquiredusing the BD LSRII Flow Cytometer from BD Biosciences Inc. (Missis-sauga, ON) with the BD FACSDiva software (version 6.0).

Quantification of OVA-specific effector and memory CD8� T cells.Spleen and blood were collected from mice. Single-cell suspensions wereprepared and were subjected to osmotic lysis to remove erythrocytes. Cellswere then incubated with anti-Fc receptor monoclonal antibody to blockFc-binding sites. The B220-negative population was analyzed to detectOVA-specific CTLs ex vivo using Kb-OVA MHC-I tetramers with theB220 and CD8 cell surface marker. OVA-specific memory CD8� T cellswere classified by the cell surface markers CD127 and CD62L. Stainingwas performed at 4°C for 45 min. Flow cytometry data were analyzed bythe FlowJo flow cytometry analysis software for Macintosh (version 8.8.2;Tree Star, Inc., Ashland, OR).

Intracellular cytokine staining. The ability of the OVA-specific CTLpopulation to produce IFN-� was determined by restimulating the cellswith OVA8 peptide ex vivo at 37°C for 4 h. Cells were then incubated withanti-Fc receptor monoclonal antibody to block Fc-binding sites. Surfacemarker staining was performed at 4°C for 30 min. Cells were fixed andpermeabilized with fixation and permeabilization buffers, respectively,from eBioscience Inc. (San Diego, CA). Intracellular cytokine staining wasperformed subsequently at room temperature for 30 min.

Statistical analysis. All quantitative data are presented as means standard errors of the means (SEM). Statistical analyses were performedusing Prism 4 for Macintosh (version 4.0b or 5.0e). The D’Agostino andPearson omnibus normality test was used to determine whether the datafollowed a normal distribution. Data that followed a normal distributionwere analyzed by unpaired two-tailed Student’s t test (comparison be-tween two groups) or one-way analysis of variance (ANOVA) test fol-lowed by Tukey’s multiple-comparison posttest (three or more groups).

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Data that did not follow normal distribution were analyzed by the Mann-Whitney test (two groups) or by the Kruskal-Wallis test followed byDunn’s multiple-comparison posttest (three or more groups). P values of0.05 were considered to be statistically significant.

RESULTSTopical CpG ODN 1826 as adjuvant for subcutaneous proteinvaccination enhances CD8� T cell response. Using CpG ODN1826 as an adjuvant (CpG adjuvant) to protein-based antigen(OVA), we sought to compare its effects on the generation ofantigen-specific CD8� T cells through different routes of admin-istration. Since a majority of current licensed protein or inactivat-ed-pathogen vaccines require booster doses, three routes of CpGadjuvant administration were tested with a prime-boost immuni-zation regimen (Fig. 1A): topical or subcutaneous route with sub-cutaneous OVA protein, and topical or intramuscular route withintramuscular OVA protein. Frequencies of OVA-specific CTLswere determined 5 days postimmunization (after boost), a timepoint that corresponds to a detectable peak in CTL numberspostimmunization (24, 25). Whether the enhanced production of

OVA-specific CD8� T cells with topical CpG adjuvant was CpGmotif specific was first determined. Immunization with the con-trol ODN (ODN 1982) without CpG motifs did not enhanceOVA-specific CTL generation (Fig. 1B). With subcutaneous OVAprotein antigen, we then demonstrated that topical CpG adjuvantincreased the proportion of OVA-specific cells among CD8� Tcells. A dose-response curve was performed to determine the op-timal subcutaneous and topical doses of CpG adjuvant. A 50 �gsubcutaneous dose of CpG adjuvant was optimal in inducing an-tigen-specific CD8� T cells (data not shown), while 250 �g wasused as a comparative optimal topical CpG adjuvant dose. Topicaldelivery of CpG adjuvant with subcutaneous OVA protein antigeninduced 2.5-fold- and 9-fold-higher proportions of these T cells inthe spleen than subcutaneous delivery of 50 �g and 250 �g of CpGadjuvant, respectively (Fig. 1C). When OVA protein was injectedintramuscularly, addition of topical CpG adjuvant also increasedthe proportion of OVA-specific CD8� T cells 3-fold compared tointramuscular delivery of the adjuvant (Fig. 1C). The same trendwas observed when the absolute numbers of OVA-specific CD8�

FIG 1 Topical administration of CpG adjuvant is more effective in antigen-specific effector CD8� T cell generation. One hundred micrograms of OVA proteinantigen was injected subcutaneously (sc) or intramuscularly (im) in combination with CpG adjuvant administered topically (ec), sc, or im as indicated. (A)Schematic diagram of prime-boost immunization regimen and T cell analysis timeline. (B, C) Top panels show representative flow cytometric dot plots in log axisscale identifying OVA-specific CD8� T cells in the spleen. Cells were gated on a B220-negative population, and OVA-specific CD8� T cells were identified as CD8and Kb-OVA class I tetramer double-positive population. Bottom panels show scatter plots of the percentage of OVA-specific cells among CD8� T cells and totalOVA-specific cells detected in the spleen 5 days postimmunization (after boost). Animals labeled “No CpG” or “Ctl ODN” received antigen and had skintreatment of tape-stripping and acetone (topical placebo treatment) without CpG or with control deoxynucleotide (ODN 1982), respectively. Animals treatedwith “sc CpG” did not receive any skin treatment. In parallel, naive mice that did not receive antigen or skin treatment were analyzed. Data summarize at leasttwo independent experiments (n � 6 to 9) and are expressed in means SEM. *, P 0.05; **, P 0.01; ***, P 0.001.

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T cells in the spleen were determined for each immunizationgroup (Fig. 1B and C). These results support the speculation thatthe topical route is a better route to administer CpG adjuvant toenhance CTL responses than parenteral routes. Data also con-firmed that the adjuvant effect of CpG ODN 1826 is CpG motifspecific.

Toxicities caused by parenteral (intraperitoneal) CpG ODNsinclude follicle microarchitecture disruption as well as spleno-megaly due to erythroid and myeloid expansion in the red pulp ofthe spleen (26, 27). Thus, the spleens of immunized and untreated(naive) mice were weighed as an indirect method to evaluate po-tential toxicity of CpG adjuvant administered via the three routesmentioned above. In contrast to subcutaneous injection of 50 �gand 250 �g of CpG adjuvant, which increased spleen weight 3-and 7-fold, respectively, no significant increase of spleen weightwas detected in mice treated with topical CpG adjuvant (Fig. 2).Overall, topical delivery of CpG ODN 1826 as adjuvant was moreeffective and less toxic than parenteral administration when given

at the same time as parenteral protein-based vaccine, by either thesubcutaneous or the intramuscular route.

A rapid (7-day) prime-boost immunization regimen can beused to enhance CTL responses with topical CpG ODN 1826.Proper spacing between doses of a given vaccine series is essentialfor optimal immune responses. A shorter interval between vaccinebooster doses would be more practical. We thus tested a 7-day(common in murine cancer vaccine protocols) and a 21-day(standard timing for antibody production) prime-boost immuni-zation schedule for the optimal induction of antigen-specificCD8� T cells (Fig. 3A). Percentages of antigen-specific cellsamong CD8� T cells were determined by tetramer staining 5 dayspostimmunization (after boost). In the spleen, a 3-fold-higherproportion of OVA-specific CD8� T cells was detected when CpGadjuvant was administered topically than subcutaneously (Fig.3B) using the 7-day-interval prime-boost immunization regimen.In addition, the prime-boost immunization regimen with a 7-dayinterval between the initial and the booster dose induced a higherlevel of OVA-specific CD8� T cells than with a 21-day interval(Fig. 3B). Hence, the short-interval (7 days apart) prime-boostimmunization regimen was more effective than the long-interval(21 days apart) regimen in inducing antigen-specific CD8� CTLs.

A single application of topical CpG ODN 1826 at the time ofprotein-based vaccine administration is sufficient to enhanceantigen-specific CTL response. TLR stimulation commonly re-sults in short-term stimulation of innate immunity. Consistentwith this concept, repeated daily topical administration of TLR7agonist has been shown to further improve CD8� T cell inductionfollowing immunization with protein antigen (28). Thus, whetherdaily multiple doses of topical CpG ODN further enhance theadjuvant effect with subcutaneous protein-based vaccines was in-vestigated. Whether a regimen of single immunization inducesfrequencies of antigen-specific CD8� T cells comparable to thoseof a prime-boost regimen was also investigated. OVA protein wasinjected subcutaneously with a one-time application of topicalCpG adjuvant or a three-time application on 3 consecutive days(Fig. 4A). Using flow cytometry, the frequencies of OVA-specificCD8� T cells and IFN-�-producing CTLs were determined. A

FIG 2 Topical administration is less toxic than subcutaneous administrationof CpG adjuvant. One hundred micrograms of OVA protein antigen was in-jected subcutaneously (sc) with CpG adjuvant administered topically (ec) orsubcutaneously (sc) as indicated. (A) Schematic diagram of prime-boost im-munization regimen and T cell analysis timeline. (B) Toxicity analysis byspleen weight. Scatter plot of spleen weights measured 5 days postimmuniza-tion (after boost). Data summarize at least two independent experiments (n �6 to 9) and are expressed in means SEM. ***, P 0.001.

FIG 3 Optimal intervals between doses of a vaccine series for the rapid generation of antigen-specific CD8� T cells. One hundred micrograms of OVA proteinantigen was injected subcutaneously (sc) with CpG adjuvant administered topically (ec, 250 �g) or subcutaneously (sc, 50 �g). (A) Schematic diagrams ofprime-boost immunization with interval of 7 days or 21 days and T cell analysis timeline. (B) Left panels show representative flow cytometric dot plots in log axisscale identifying OVA-specific cells CD8� T cells in the spleen. Cells were gated as described for Fig. 1. Right panel shows a scatter plot of the percentage ofOVA-specific cells among CD8� T cells detected in the spleen. Data summarize at least two independent experiments (n � 4 to 10) and are expressed in means SEM. **, P 0.01; ns, no significance.

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booster dose at 7 days increased the pool of OVA-specific andIFN-�-producing CD8� T cells as detected in the spleen 5 dayspostimmunization compared to the single immunization (Fig.4B). Multiple doses of CpG adjuvant administered over 3 consec-utive days did not further enhance the generation of OVA-specificor IFN-�-producing CTLs over single dose (Fig. 4B). The fre-quency of IFN-�-producing CD8� T cells paralleled that of OVA-specific CTLs. Thus, a single topical application of CpG adjuvantat the time of subcutaneous protein antigen administration with aprime-boost regimen, 7 days apart, is effective for the induction offunctional antigen-specific CTLs.

Topical administration of CpG ODN 1826 induces a higherproportion of antigen-specific CD8� effector memory T cellsthan the subcutaneous route. Effector T cells undergo contrac-

tion and differentiate into memory T cells. Memory T cells pro-vide sustained protection against invading pathogens in a fasterand more effective manner than primary effector T cells (29). Theability of topical and subcutaneous CpG adjuvant to augment thegeneration of antigen-specific CD8� memory T cells was thusdetermined. Mice were immunized using the prime-boost immu-nization regimen with OVA protein administered subcutaneouslywith CpG adjuvant administered either topically or subcutane-ously. The levels of circulating OVA-specific CD8� effector T cells(5 days after boost) and long-term memory T cells (30 days afterboost) in peripheral blood were determined postimmunization(Fig. 5A). Topical administration of CpG adjuvant robustly in-creased the frequency of OVA-specific CD8� effector T cells de-tected in the peripheral blood up to 6-fold, compared to subcuta-

FIG 4 Better immunization strategy using topical CpG ODN as vaccine adjuvant to elicit acute CD8� T cell responses. (A) Schematic diagrams of single andprime-boost immunization regimens and T cell analysis timelines. One hundred micrograms of OVA protein antigen was injected subcutaneously (sc) with orwithout topical (ec) CpG adjuvant. Arrows indicate three consecutive days of topical CpG adjuvant administration (250 �g per administration). (B) Top panelsshow representative flow cytometric dot plots identifying OVA-specific CD8� T cells and IFN-�-producing cells in the spleen after restimulation with OVA8peptide ex vivo. Bottom panels show scatter plots of the percentage and the total number of OVA-specific CD8� T cells and IFN-�-producing cells detected inthe spleen on day 7 or day 12 post-initial immunization (after prime) using a single or a prime-boost immunization regimen, respectively. Data summarize twoindependent experiments (n � 8) and are expressed as means SEM. *, P 0.05; **, P 0.01; ns, no significance.

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neous administration, at 5 days postimmunization (after boost)(Fig. 5B). At 30 days postimmunization (after boost), no signifi-cant difference in the frequency of CD8� memory T cells in theblood of mice immunized with topical and subcutaneous adju-vant was observed (Fig. 5B).

Despite similar proportions of the memory T cells, does topicalCpG adjuvant change the phenotype of the memory T cells? Thereare two major subsets of memory T cells: central memory andeffector memory T cells (30), (31). Central memory T cells areCD127�CD62L�, have a higher proliferative potential than naivecells, and preferentially reside in lymphoid organs. Effector mem-ory T cells are CD127�CD62L�, have a faster effector function,and preferentially reside in nonlymphoid (stromal) tissues. Tofurther characterize the OVA-specific memory CD8� T cells in-duced by immunization with CpG adjuvant administered topi-cally or subcutaneously, the surface markers CD127 and CD62Lwere used to differentiate effector memory from central memoryT cells (Fig. 5C). Interestingly, the population of CD127�CD62L�

OVA-specific effector memory T cells in peripheral blood was1.5-fold higher upon immunization in the presence of CpG adju-vant administered topically than subcutaneously (Fig. 5D). Also, atrend of a lower proportion of CD127�CD62L� OVA-specificcentral memory T cells was detected with topical CpG adjuvantthan with subcutaneous administration (Fig. 5D). Thus, immuni-zation in the presence of CpG adjuvant (either subcutaneously ortopically) induces long-lived antigen-specific memory CD8� Tcells. In addition, topical delivery of CpG adjuvant induces ahigher proportion of antigen-specific effector memory CD8� Tcells than does subcutaneous delivery.

Topical CpG ODN 1826 confers rapid TLR9-dependent pro-tection against systemic L. monocytogenes infection. Topical

CpG adjuvant induced rapid and lasting CD8� T cell responses tosubcutaneous antigen. To determine whether these responseswere adequate to protect against infection, we tested the ability ofthis vaccination method to protect against Listeria monocytogenes,an intracellular bacterium predominantly controlled by hostCD8� T cell responses (32, 33). The prime-boost and the one-time immunization regimens were tested for their ability to pro-tect animals from systemic L. monocytogenes challenge. Mice wereimmunized with OVA antigen subcutaneously with or withouttopical CpG adjuvant and then challenged with mutant strain Lm-OVA 5 or 7 days postimmunization (Fig. 6A). A 5-log decrease ofbacterial load was noted in the spleen following a prime-boostimmunization regimen with topical CpG adjuvant compared toimmunization without adjuvant (Fig. 6B). Immunization withCpG adjuvant alone (i.e., without antigen) reduced bacterial bur-den. However, this was not as effective as immunization with an-tigen and adjuvant (Fig. 6B). Thus, repeated innate stimulationprovides a modicum of rapid protection that is improved uponwith antigen vaccination. Unexpectedly, topical administration ofCpG adjuvant did not further decrease bacterial load in the spleencompared to subcutaneous administration after acute systemicLm-OVA challenge (Fig. 6B). In addition, a 3-log decrease of bac-terial burden in the spleen was detected in mice immunized withtopical CpG adjuvant compared to those immunized without ad-juvant in the one-time immunization regimen (Fig. 6C). Thus,immunization using topical or subcutaneous CpG adjuvant withsubcutaneous protein antigen can provide protection against L.monocytogenes infections, which is not observed with immuniza-tion with antigen alone. Although a prime-boost immunizationregimen provides optimal protection against L. monocytogenes, aone-time immunization strategy can confer protection as well.

FIG 5 Topical administration of CpG adjuvant induced a higher proportion of antigen-specific CD8� T effector memory cells than did subcutaneousadministration. One hundred micrograms of OVA protein antigen was injected subcutaneously (sc) with CpG adjuvant administered subcutaneously (sc, 50 �g)or topically (ec, 250 �g). (A) Schematic diagram of immunization regimen and T cell analysis timeline. (B) Point graph of the percentage of OVA-specific cellsamong CD8� T cells detected in peripheral blood 5 days and 30 days postimmunization (after boost). Data summarize two independent experiments (n � 3 to6). (C) Representative flow cytometric density plots in log axis scale of CD8� T effector memory and central-memory cells in the peripheral blood 30 dayspostimmunization (after boost). (D) Left panel and right panel show a scatter plot of the percentage of CD127�CD62L� effector memory cells andCD127�CD62L� central-memory cells among OVA-specific CD8� T cells detected in the peripheral blood 30 days postimmunization (after boost), respectively.Data represent two independent experiments (n � 6) and are expressed in means SEM. ***, P 0.001; ns, no significance.

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The uptake of CpG ODNs by cells is CpG motif independent,and CpG ODNs have been reported to induce TLR9-independentimmune effects (34, 35). To confirm that the adjuvant effect ofCpG ODN 1826 on L. monocytogenes bacterial burden was TLR9dependent, we compared the bacterial load 3 days postinfection byLm-OVA between immunized wild-type (WT) and TLR9-defi-cient (KO) mice (Fig. 7A). Bacterial loads in WT and KO micefollowing Lm-OVA infection were identical (Fig. 7B, left panel),suggesting similar susceptibility to infection. Unlike WT animalswith a 4-log decrease in bacterial load in the spleen, bacterial loadwas not reduced in the KO mice (Fig. 7B, right panel), demon-strating TLR9 dependency of the topical CpG adjuvant effect.

Topical CpG ODN 1826 improves long-term protectionagainst systemic L. monocytogenes infection. Topical CpG adju-vant induced persistent CTL responses to subcutaneously admin-istered protein vaccine. Hence, we assessed the ability of our pro-posed prime-boost immunization strategy with protein antigeninjected subcutaneously and topical CpG ODN 1826 as adjuvantto provide long-term protective immunity against intracellularbacterial infection (Fig. 8A). Mice immunized in the presence ofCpG adjuvant administered topically but not subcutaneouslyshowed a modest but significant decrease in bacterial burden (1-log reduction) (Fig. 8B). An attenuated strain, �actA-Lm-OVA,

FIG 6 Acute protection against systemic L. monocytogenes infection induced by immunization with topical or subcutaneous CpG adjuvant. Naive mice, micetreated with topical (ec, 250 �g) CpG adjuvant, and mice immunized with 100 �g OVA protein antigen subcutaneously (sc, 50 �g) with or without topical (ec,250 �g) CpG adjuvant were challenged with 5 � 105 CFU Lm-OVA intravenously. (A) Schematic diagram of immunization, infection, and bacterial burdenanalysis timelines. (B, C) Mice were immunized with a prime-boost or a single-immunization regimen. Mice were then challenged with Lm-OVA at the timepoints indicated. Scatter dot plots in log axis scale of bacterial counts in the spleen 3 days postinfection. Data summarize at least two independent experiments(n � 2 to 10) and are expressed in means SEM. *, P 0.05; **, P 0.01; ***, P 0.001; ns, no significance.

FIG 7 Acute protection against systemic L. monocytogenes infection inducedby immunization with topical or subcutaneous CpG adjuvant. Wild-type(WT) and TLR9-deficient (KO) mice were unimmunized or immunized with100 �g OVA protein subcutaneously (sc) with topical (ec, 250 �g) CpG adju-vant. All mice were challenged with 5 � 105 CFU Lm-OVA intravenously. (A)Schematic diagram of immunization, infection, and bacterial burden analysistimelines. (B) Scatter dot plots in log axis scale of bacterial counts in the spleen3 days postinfection. Data summarize two independent experiments (n � 6 to8) and are expressed as means SEM. **, P 0.01; ns, no significance.

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was used as a live vaccine to serve as a positive control (22). Thus,our proposed prime-boost immunization strategy with solubleprotein antigen and topical CpG adjuvant but not subcutaneousadjuvant is able to confer long-term protection against systemic L.monocytogenes infection. In our experiments, topical adjuvant useat the time of protein immunization approximated the protectiveeffect of a live vaccine.

DISCUSSION

Using the skin as a site of adjuvant administration, we demon-strate that a TLR9 agonist may be applied onto the skin to enhanceCTL responses to locally administered protein antigen and to pro-vide protective immunity to the intracellular pathogen L. mono-cytogenes. The structural and immune properties of the skinenable a split adjuvant and antigen vaccination strategy: Guebre-Xabier et al. first demonstrated that topical administration of anenterotoxin improved the amplitude of the antibody response tointramuscular or subcutaneous influenza virus antigen in mice(36). This group also showed that an enterotoxin-containingpatch could induce a trend toward higher antibody responses toinfluenza vaccination in the elderly (37), demonstrating the rele-vance of this approach in humans. Other groups showed that top-ical TLR7 agonists induce CTL responses to protein antigen inmice (2), improve immunity to Leishmania in mice (38), and areable to induce immune responses to tumor antigens in humans(39).

Separate administration of vaccine and adjuvant at immuniza-tion has several advantages over standard coadministration. Suchadvantages include potentially more efficacious sites of adjuvantadministration, increased safety, and the ability to tailor adjuvantuse to specific populations without a need for vaccine reformula-tion. However, bacterial toxins, such as enterotoxins, have persis-tent safety concerns. Further, even limited amounts of topicalTLR7 agonist may induce systemic cytokine release and spleno-megaly in mice (28) and may induce systemic symptoms and sideeffects in humans (40). Synthetic ODNs with CpG motifs arelarger molecules that have less penetration after topical applica-tion than imiquimod and similar TLR7 agonists. We thus ex-plored split skin administration of adjuvant and antigen usingsynthetic CpG ODN 1826 as a vaccine adjuvant.

Synthetic ODNs with CpG motifs have been explored for im-

munotherapeutic uses, including the reduction of allergic re-sponses and the enhancement of cancer therapy and innate im-mune responses (4). CpG ODNs have also been extensivelystudied for their ability to improve vaccine efficacy. These studieshave always used coadministration of antigen and adjuvant. Stud-ies in nonhuman primates indicate that CpG ODNs are as effec-tive as TLR7 adjuvants in their ability to induce long-lasting pro-tective immunity but without the induction of associated skininflammation (1). Topical administration of CpG ODNs coad-ministered with parenteral antigen enhances CTL responses toantigen (20) and induces CTL response to tumors in a murinemodel (21). The adjuvant must be administered within the samelymph node draining area as the antigen and results in movementof Langerin� (epidermally and dermally derived) dendritic cells(DCs) and antigen-bearing DCs into the draining lymph nodes.This results in an effective adjuvant effect allowing CTL primingwithout the cytokine toxicity and splenomegaly noted with par-enteral administration of CpG adjuvant (20): we have previouslyshown that subcutaneous administration of CpG adjuvant resultsin rapid elevation of TNF-�, interleukin-12p70 (IL-12p70), IL-6,and monocyte chemoattractant protein-1 (MCP-1), as well asIFN-� release in the serum (20). None of these occurred withtopical administration of CpG adjuvant, supporting the conten-tion that topical administration induces less systemic inflamma-tion. Here, we expanded on our previous observations and ex-plored the use of synthetic CpG ODN 1826 as topical adjuvant inthe context of intracellular infection. Our results indicate that top-ical CpG ODN administration is superior to coadministrationwith antigen via the subcutaneous or intramuscular route in termsof CTL induction, has fewer systemic effects, and is better thanparenteral CpG in terms of long-term protection against systemicL. monocytogenes infection.

Current protein vaccines require booster doses to achieve ef-fective immunity. A protective CD8� T cell threshold is often notreached with a single immunization (41). Instead, prime-boostregimens are required for long-term protection (42). Proper spac-ing between doses of a given vaccine series is essential for optimalimmune responses. In contrast, rapid induction of immunity isdesired as a response to a pandemic infection or bioterrorist at-tack. We find that an effective strategy of CpG ODN 1826 admin-istration for antigen-specific CTL induction is to administer a

FIG 8 Long-term protection against systemic L. monocytogenes infection induced by immunization with topical or subcutaneous CpG adjuvant. Naive mice,mice treated with topical (ec, 250 �g) CpG adjuvant, and mice immunized with 100 �g OVA protein antigen subcutaneously (sc) without or with topical (ec, 250�g) or subcutaneous (sc, 50 �g) CpG adjuvant were challenged with 5 � 105 CFU Lm-OVA intravenously 30 days postimmunization (after boost). Miceimmunized with �actA-Lm-OVA intraperitoneally were analyzed in parallel as positive controls. (A) Schematic diagram of immunization, infection, andbacterial burden analysis timeline. (B) Scatter plots in log axis scale of bacterial counts in the spleen 3 days postinfection. Data summarize at least two independentexperiments (n � 5 to 19) and are expressed in means SEM. **, P 0.01; ns, no significance.

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single dose of topical CpG ODN 1826 as an adjuvant with subcu-taneous protein antigen followed by a booster dose 7 days later.Unlike what is observed with the TLR7 agonist R848, this strategydoes not result in splenomegaly and multiple applications are notrequired to increase efficacy (28). In addition, a single immuniza-tion with topical CpG adjuvant also enhanced protective immu-nity. High levels of inflammatory mediators, such as IFN-�, whichmay be induced by CpG adjuvant, inhibit durable memory T cellresponses (43, 44). We show in this paper, and have noted previ-ously, that topical administration of CpG adjuvant, which doesnot induce splenomegaly or systemic cytokine release (20) (andthus induces less systemic inflammation than subcutaneousCpG), nevertheless induced memory CD8� T cell responses. Wepropose that the local adjuvant administration induces the activa-tion of antigen-presenting cells (20) without the widespread in-flammation and possibly direct cytokine stimulation of T cells thatmay occur with parenteral administration. This may simulate themuch more technically demanding DC vaccination, which hasbeen shown to induce rapid memory CD8� T cell responses (44).

Topical CpG adjuvant induces a rapid increase of antigen-spe-cific CTL and a higher proportion of effector memory CD8� Tcells. We next asked whether these changes were sufficient to pro-tect against intracellular bacterial infection. CD8� T cells are re-quired for protection against L. monocytogenes infection (32, 33).Further, intraperitoneal CpG ODN injection alone offers non-antigen-specific short-term protection against L. monocytogenesinfection, possibly due to IL-12 and IFN-� release (45). We thusused Lm-OVA to assess whether antigen-specific CTLs induced byour immunization strategy confers protection against systemic L.monocytogenes infection. Topical administration of CpG adjuvantat the time of OVA protein immunization induced rapid protec-tion against intravenously administered Lm-OVA, as indicated bythe reduction of the bacterial load when mice were challengedshortly after immunization. This protection was similar in extentto that provided by subcutaneous CpG adjuvant (despite higherCTL frequencies), possibly due to a saturation in the efficacy ofclearance of intravenously administered organisms by CTL. Re-peated administration of topical CpG without antigen also de-creased the bacterial load slightly, suggesting that topical applica-tion can have a broad, antigen-nonspecific effect, as notedfollowing intraperitoneal administration (45). Phosphorothioatemodification of CpG ODNs to render them more resistant to nu-clease degradation may result in phosphorothioate CpG butTLR9-independent immune activation (46, 47, 48, 49). We con-firmed that the protective effect of topical ODN 1826 as adjuvantwas TLR9 dependent using TLR9 KO mice.

Memory T cells have higher affinity for antigen and a decreasedthreshold for activation (50). Memory CD8� T cells are crucial inproviding protective immunity against intracellular pathogenssuch as Plasmodium (41) and HIV (51). Topical CpG adjuvantadministration at the time of subcutaneous protein immunizationinduces antigen-specific memory T cells to form a stable pool ofcirculating CD8� T cells for up to 4 months (20). However, topicaladministration of CpG adjuvant did not result in a further in-crease in the frequency of blood-circulating antigen-specificmemory CD8� T cells compared to subcutaneous administration.

Two types of CD8� memory T cells with differential prolifer-ative and protective potential have been identified based on sur-face expression of CD62L: CD62L� central memory T cells, withlow proliferative potential, and CD62L� effector memory T cells,

with high proliferative potential (52). Both central and effectormemory T cells may be crucial to combat pathogens, acting atdifferent times following infection. Cui et al. showed that paren-teral CpG-B administration at the time of vaccination increasesCD127low KLRG1hi short-lived effector cells (SLEC) formationbut not CD127hi KLRG1low CD8� memory T cells (MPEC) (53).Obar et al. showed that intraperitoneal injection of CpG ODN1826 at the time of vaccination increases central memory CD62Lhi

CD8� T cells (54). We note that topical CpG initially induces alarger burst of blood-resident antigen-specific CD8� T cells in anacute response compared to parenteral CpG, but we did not detecta subsequent difference in the frequency of memory T cells withinthe blood compartment 1 month later. Although we did not di-rectly phenotype the memory T cells into the CD127hi KLRG1low

MPEC, we found that topical administration of CpG adjuvant,unlike the intraperitoneal administration of CpG by Obar et al.,resulted in a preferential induction of a pool of CD127�CD62L�

effector memory cells. Preferential tissue distribution and effectorcell population contraction within the blood compartment mayexplain why protective immunity following subcutaneous or top-ical CpG adjuvant vaccination resulted in a weaker protective re-sponse to delayed systemic L. monocytogenes challenge (30 dayspostimmunization) than to acute challenge (5 days postimmuni-zation).

Taken together, our work indicates that a rapid (7-day) prime-boost regimen with a single dose of topical CpG adjuvant at thetime of parenteral antigen administration is a good strategy toinduce protective CD8� T cell response for protein-based vac-cines. We extend these observations to an in vivo mouse infectionmodel using Lm-OVA and show rapid protection when mice areimmunized with subcutaneous antigen with topical CpG ODN1826. Our work thus supports the use of topical CpG adjuvant toincrease the cell-mediated immune response to protein vaccines.The rapid protection induced makes this strategy attractive forprotection against novel intracellular pathogens, as in pandemicviral outbreaks. This strategy has the added advantage of not re-quiring novel vaccine formulations for developed vaccine. Fur-ther, it may be applied selectively to at-risk populations such as theelderly or the very young.

CpG ODNs are excellent adjuvants in nonhuman primateswhen given subcutaneously (1). It is important to note that TLR9expression pattern differs between humans and mice. Could top-ical CpG ODN application have adjuvant effects in humans? TLR9is constitutively expressed or can be induced to express on murinemyeloid DCs, plasmacytoid DCs, B cells, monocytes, and mastcells (55, 56). In normal mouse skin, TLR9 mRNA expression isinduced following intradermal injection of CpG 1668 (57). In hu-mans, TLR9 expression is more restricted than in mice to plasma-cytoid DCs and B cells but can also be induced in monocytes andkeratinocytes (58, 59, 60), suggesting that topical application mayalso have an additional adjuvant effect in humans. Topical admin-istration of adjuvant is a superior strategy to parenteral adminis-tration because topical application of adjuvant limits the distribu-tion of the adjuvant (39). We speculate that topical CpG adjuvantinduces keratinocyte as well as DC activation, multiplying immu-nogenic stimuli without promoting systemic inflammation.

ACKNOWLEDGMENTS

W.K.C. was supported by the CIHR Canada Graduate Scholarship Mas-ter’s Award and a CIHR Skin Research Training Centre Training Schol-

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arship. K.W. is supported by the NSERC industrial postgraduate scholar-ship award. J.P.D. is a Senior Scientist of the Michael Smith Foundationfor Research and the Child and Family Research Institute (CFRI). Thework was supported by a CIHR operating grant (FRN 89878).

We declare that we have no conflicts of interest.

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