Title: Topical CpG adjuvantation of a protein-based vaccine induces protective immunity to 1 Listeria monocytogenes 2 3 Running Title: Topical CpG adjuvant 4 5 Authors: Wing Ki Cheng1, Kathleen Wee2, Tobias R. Kollmann2, and Jan P. Dutz1 6 7 1 Department of Dermatology and Skin Science, Faculty of Medicine 8 Child and Family Research Institute, The University of British Columbia, Vancouver, British 9 Columbia, Canada 10 11 2 Department of Pediatrics, Faculty of Medicine 12 Child and Family Research Institute, The University of British Columbia, Vancouver, British 13 Columbia, Canada 14 15 Address correspondence and reprint requests to Dr. Jan P. Dutz, Department of Dermatology and 16 Skin Science, Child and Family Research Institute, A4-122, 950 W 28th Avenue, Vancouver, 17 British Columbia, Canada, V5Z 4H4. Phone: +1 604-875-2245; Fax: +1 604-873-9919; Email: 18 [email protected] 19 20 21
ABSTRACT 22 23 Robust CD8+ T cell responses are essential for immune protection against intracellular 24 pathogens. Using parenteral administration of ovalbumin (OVA) protein as a model antigen, the 25 effect of the Toll-like receptor 9 (TLR9) agonist, CpG oligodeoxynucleotide (ODN) 1826, as an 26 adjuvant delivered either topically, subcutaneously, or intramuscularly on antigen-specific CD8+ 27 T cell responses in a mouse model was evaluated. Topical CpG adjuvant increased the frequency 28 of OVA-specific CD8+ T cells in the peripheral blood and in the spleen. The more effective 29 strategy to administer topical CpG adjuvant to enhance CD8+ T cell responses was single dose 30 administration at the time of antigen injection with a prime-boost regimen. Topical CpG adjuvant 31 conferred both rapid and long-lasting protection against systemic challenge with recombinant 32 Listeria monocytogenes (Lm) expressing the cytotoxic T lymphocyte (CTL) epitope of OVA257-33 264 (Lm-OVA) in a TLR9-dependent manner. Topical CpG adjuvant induced a higher proportion 34 of CD8+ effector-memory T cells than parenteral administration of the adjuvant. Although 35 traditional vaccination strategies involve co-formulation of antigen and adjuvant, split 36 administration using topical adjuvant is effective and has advantages of safety and flexibility. 37 Split administration of topical CpG ODN 1826 with parenteral protein antigen is superior to 38 other administration strategies in enhancing both acute and memory protective CD8+ T cell 39 immune responses to subcutaneous protein vaccines. This vaccination strategy induces rapid and 40 persistent protective immune responses against the intracellular organism Lm. 41 42 Word count: 227 43 44
INTRODUCTION 45 46 Infectious diseases are the leading cause of illness in humans. Immunization is the most cost-47 effective method to improve population health against infectious diseases. However, current 48 vaccines remain inefficient in part due to a poor ability to elicit cytotoxic T lymphocytes (CTLs), 49 delayed protection, and lack of long-lasting protective effect. An adjuvant is often added to 50 increase the immunogenicity of vaccine antigens. Adjuvants are routinely combined with 51 antigens for efficacy and ease of formulation (1). Split administration, where vaccine antigen and 52 adjuvant are delivered separately, has been less commonly studied (2) but has advantage of 53 flexibility and simplicity. Here, we explore split administration of antigen and adjuvant using the 54 skin as an administration site and a Toll-like receptor 9 (TLR9) agonist as the vaccine adjuvant. 55 56 CpG ODNs bind TLR9, an endosomal pattern recognition receptor that recognizes unmethylated 57 bacterial and viral ODNs with CpG motifs (3). CpG ODNs induce both potent CD8+ T cells and 58 Th1-biased CD4+ T cell immune responses in both mouse and human (4), (5). The enhanced 59 immune responses induced by CpG ODNs provide protective immunity in several infectious 60 disease models. For example, reduced bacterial burden was detected in mice immunized with 61 CpG ODN and OVA co-administered intramuscularly when challenged with Listeria 62 monocytogenes (Lm) expressing the CTL epitope of OVA257-264 (Lm-OVA) 10 days post-63 immunization (6). Also, intramuscular co-injection of recombinant Toxoplasma gondii protein 64 with CpG ODN as an adjuvant induces a Th1 biased humoral response demonstrated by 65 increased IgG2a to IgG1 antibody ratio and increased protection against oral T. gondii infection 66 in a mouse model (7). In a human clinical trial, intramuscular delivery of CpG 7909 induced 67
robust specific antibody response to a commercial hepatitis B vaccine (Engerix-B) (8). Although 68 studies have demonstrated the effectiveness of CpG ODNs as adjuvants, concerns remain about 69 the local and systemic side effects observed. In mice, CpG ODNs can induce tumor necrosis 70 factor alpha (TNF-α) release by macrophages resulting septic shock (9), (10). Strategies to limit 71 the systemic toxicity of CpG ODNs include conjugation of CpG ODN to antigen (11) and co-72 encapsulation of CpG ODN with antigen (12). These strategies are cumbersome and require 73 validation in humans. Split administration of CpG ODN as an adjuvant as a means of enhancing 74 vaccine efficacy and limiting toxicity has not been pursued. 75 76 The skin is the most accessible organ of our bodies and harbors many immune cells including 77 different subsets of dendritic cells (DCs) (13), mast cells (14), and resident lymphocytes (15), 78 (16), (17) that can be harnessed to induce immune responses. We, and others, have explored the 79 skin as a site of vaccination by co-administration of antigen and adjuvant. Topical peptide 80 vaccination with cholera toxin induces robust cellular immune responses in mice (18). However, 81 the toxicity of cholera toxin makes it difficult to handle. Topical CpG ODN co-administered with 82 topical antigen promotes CD8+ T cell production to the antigen (19). Topical compared to 83 subcutaneous administration of CpG ODN also promotes cross-presentation of injected soluble 84 OVA protein antigen with less systemic cytokine production and toxicity (20). When topical 85 CpG ODN administered epifocally to the antigen administration site, meaning that it is applied to 86 skin that has the same lymphatic drainage as the site to which antigen is given, the CpG adjuvant 87 augments CD8+ T cell responses against melanoma in a mouse model (21). The CpG has an 88 adjuvant effect only with epifocal administration and not when it is applied to a site contralateral 89 to the antigen. This demonstrates the ability of splitting antigen and vaccine antigen 90
administration and emphasizes the need for common lymph drainage of antigen and adjuvant for 91 maximum effect (21). The requirement of epifocal administration of adjuvant and antigen 92 suggests that topical CpG ODN instructs antigen-specific T cell generation in the skin draining 93 lymph nodes (SLNs). Here, we sought to examine a various strategies to administer CpG ODN 94 1826 onto the skin to enhance CD8+ T cell responses. We demonstrate that single topical CpG 95 ODN administration at the time of standard (parenteral) immunization is effective, preferentially 96 induces effector-memory T cells and may be used to induce protective immunity against the 97 intracellular pathogen Lm. 98 99
MATERIALS AND METHODS 100 101 Animals. C57BL/6 female mice 6-8 weeks of age were purchased from Charles River 102 Laboratories (Wilmington, MA) and housed in a specific pathogen-free animal care facility at the 103 Child & Family Research Institute (CFRI) (Vancouver, Canada). TLR9-deficient mice on 104 C57BL/6 background (TLR9 KO) were obtained from Oriental Bioservice (Tokyo, Japan), bred 105 and maintained in the animal care facility at CFRI. Animal experiments were conducted in 106 accordance with protocols approved by the Animal Care Committee of The University of British 107 Columbia and Canadian Council of Animal Care. 108 109 Deoxynucleotides and peptide. Synthetic HPLC-purified, single-stranded, phosphorothioated 110 CpG ODN 1826 (5'-TCCATGACGTTCCTGACGTT-3') was purchased from Sigma-Aldrich 111 Inc. (Saint Louis, MO). Control ODN without CpG motifs (5’-TCCAGGACTTCTCTCAGGTT-112 3’) was purchased from Integrated DNA Technologies, Inc. (San Diego, CA). Lyophilized CpG 113 ODN 1826 and control ODN were reconstituted to 5 mg/mL with PBS/DMSO (1:1 v/v). The 114 immunodominant Kb-restricted OVA8 peptide (OVA257-264, amino acid sequence SIINFEKL) 115 was synthesized by Kinexus (Vancouver, BC). 116 117 Immunization. C57BL/6 or TLR9 KO female mice 6-12 weeks of age were anesthetized by 118 intraperitoneal injection of 75 mg/kg ketamine (Ketalean, Bimeda-MTC Animal Health Inc., 119 Cambridge, ON) and 7.5 mg/kg xylazine (Rompun, Bayer Health Care Inc., Toronto, ON). Mice 120 were shaved on the dorsal back, tape-stripped fifteen times using cellophane tape (Staples, 121 Vancouver, BC), and the skin was wiped with acetone (Fisher Scientific, Edmonton, AB) using a 122
cotton swab. 100 μg of chicken ovalbumin protein grade V (OVA), from Sigma-Aldrich Inc. 123 (Saint Louis, MO) was either injected subcutaneously or intramuscularly. Next, 250 μg or 50 μg 124 of CpG ODN 1826, was administered topically, subcutaneously, or intramuscularly in an 125 epifocal manner (overlaying the antigen injection site). PBS/DMSO (1:1 v/v) or control ODN 126 was applied when no adjuvant was administered. The area was then covered with waterproof 127 tape to prevent oral ingestion of the topical adjuvant or control. When antigen and adjuvant (or 128 control) were administered by the same route, they were given together in a single injection. For 129 a prime-boost regimen, mice were immunized on days 0 and 7 while mice were immunized once 130 on day 0 for a one-time immunization regimen. Multiple applications of the adjuvant, CpG ODN 131 1826, was administered at the time of antigen administration and again at 1 and 2 days (for 3 132 consecutive days) post-immunization: Days 0, 1 and 2 for a one-time immunization regimen and 133 days 0, 1, 2, 7, 8 and 9 for a prime-boost regimen. 134 135 Bacterial strains, medium, and growth conditions. Wild-type Lm strain 10403s and mutant strain 136 Lm-OVA, which has been modified to express the CTL epitope of OVA257-264 (Lm-OVA) was 137 provided by Dr. H Shen (University of Pennsylvania, Philadelphia, PA). The construction of the 138 less virulent form of ΔactA-Lm-OVA with a targeted deletion in the virulence determinant ActA 139 has been described elsewhere (22). For immunization and infection experiments, frozen infection 140 aliquots of Lm strains were prepared as described previously (23). Briefly, Lm strains were 141 grown in brain-heart infusion (BHI) broth to mid logarithmic phase (OD600, 1.0) at 37°C, 142 washed twice with endotoxin-free, isotonic saline (0.9% NaCl), resuspended in 0.9% NaCl with 143 20% glycerol (v/v), and stored at -80°C until use. 144 145
Immunizing infection and animal challenge with Lm. Mice were infected with 5 x 105 colony 146 forming units (CFUs) of the Lm-OVA in 100 μl 0.9% NaCl systemically via the tail vein. Three 147 days after infection, mice were euthanized to collect the spleen and then homogenized 148 mechanically in 5 mL 0.9% NaCl containing 0.05% Triton-X100. To enumerate the number of 149 viable bacteria colonizing the spleen post-infection, serial dilutions of the mechanically lysed 150 cell suspensions were plated on BHI agar. For immunizing infection, mice were intraperitoneally 151 injected with 2 x 106 CFUs of ΔactA-Lm-OVA. 152 153 Flow cytometry antibodies and tetramer. Antibodies B220-PerCP (clone RA3-6B2), CD8-APC 154 (clone 53-6.7), and interferon (IFN)-γ-APC (clone XMG1.2) were purchased from BD 155 Biosciences Inc. (Mississauga, ON). Antibodies CD62L-FITC (clone MEL-14) and CD127-APC 156 (clone A7R34) were purchased from eBioscience Inc. (San Diego, CA) whereas CD8-A700 157 (clone 53.67) was purchased from the AbLab at The University of British Columbia (Vancouver, 158 BC). PE conjugated OVA class I MHC tetramer specific for the Kb-restricted OVA8 peptide 159 (OVA257-264, amino acid sequence SIINFEKL) was generated by conjugation of biotinylated 160 monomers to streptavidin-PE in Dr. R. Tan's laboratory (Child & Family Research Institute, 161 Vancouver, BC). The anti-Fc receptor monoclonal antibody (clone 2.4G2) was obtained from 162 American Type Culture Collection (Rockville, MD). OVA-specific CD8+ T cells were identified 163 as B220 negative and CD8 and Kb-OVA class I MHC tetramer double positive population. Flow 164 cytometry data was acquired using the BD LSRII Flow Cytometer from BD Biosciences Inc. 165 (Mississauga, ON) with the BD FACSDiva software (version 6.0). 166 167 Quantification of OVA-specific effector and memory CD8+ T cells. Spleen and blood were 168
collected from mice. Single cell suspensions were prepared and were subjected to osmotic lysis 169 to remove erythrocytes. Cells were then incubated with anti-Fc receptor monoclonal antibody to 170 block Fc-binding sites. The B220 negative population was analyzed to detect OVA-specific 171 CTLs ex vivo using Kb-OVA class I MHC tetramers with the B220 and CD8 cell surface marker. 172 OVA-specific memory CD8+ T cells were classified by the cell surface marker CD127 and 173 CD62L. Staining was performed at 4°C for 45 min. Flow cytometry data was analyzed by the 174 FlowJo flow cytometry analysis software for Macintosh (version 8.8.2, Tree Star, Inc., Oregon). 175 176 Intracellular cytokine staining. The ability of the OVA-specific CTL population to produce IFN-177 γ was determined by re-stimulating the cells with OVA8 peptide ex vivo at 37°C for 4 hours. 178 Cells were then incubated with anti-Fc receptor monoclonal antibody to block Fc-binding sites. 179 Surface marker staining was performed at 4°C for 30 min. Cells were fixed and permeabilized 180 with Fixation and Permeabilization buffer, respectively, from eBioscience Inc. (San Diego, CA). 181 Intracellular cytokine staining was performed subsequently at room temperature for 30 min. 182 183 Statistical analysis. All quantitative data were presented as mean ± standard error of the mean 184 (SEM). Statistical analyses were performed using Prism 4 for Macintosh (version 4.0b or 5.0e). 185 The D’Agostino and Pearson omnibus normality test was used to determine whether the data 186 followed Normal distribution. Data that followed Normal distribution was analyzed by unpaired 187 two-tailed Student t-test (two groups comparison) or one-way ANOVA test followed by Tukey 188 multiple comparison post-test (three or more groups comparison). Data that did not follow 189 Normal distribution was analyzed by Mann-Whitney test (two groups comparison) or by 190 Kruskal-Wallis test followed by Dunn multiple comparison post-test (three or more groups 191
RESULTS 194 195 Topical CpG ODN 1826 as adjuvant for subcutaneous protein vaccination enhances CD8+ 196 T cell response 197 198 Using CpG ODN 1826 as an adjuvant (CpG adjuvant) to protein-based antigen (OVA), we 199 sought to compare its effects on the generation of antigen-specific CD8+ T cells through different 200 routes of administration. Since a majority of current licensed protein or inactivated-pathogen 201 vaccines require booster doses, three routes of CpG adjuvant administration were tested with a 202 prime-boost immunization regimen (Figure 1A): topical or subcutaneous route with 203 subcutaneous OVA protein, and topical or intramuscular route with intramuscular OVA protein. 204 Frequencies of OVA-specific CTLs were determined 5 days post-immunization (after boost), a 205 time-point that corresponds to a detectable peak in CTL numbers post-immunization (24), (25). 206 Whether the enhanced production of OVA-specific CD8+ T cells with topical CpG adjuvant was 207 CpG motif-specific was first determined. Immunization with the control ODN (ODN 1982) 208 without CpG motifs did not enhance OVA-specific CTL generation (Figure 1B). With 209 subcutaneous OVA protein antigen, we then demonstrated that topical CpG adjuvant increased 210 the proportion of OVA-specific cells amongst CD8+ T cells. A dose response curve was 211 performed to determine the optimal subcutaneous and topical doses of CpG adjuvant. A 50 µg 212 subcutaneous dose of CpG adjuvant was optimal in inducing antigen-specific CD8+ T cells (data 213 not shown) while 250 µg was used as a comparative optimal topical CpG adjuvant dose. Topical 214 delivery of CpG adjuvant with subcutaneous OVA protein antigen induced 2.5-fold and 9-fold 215 higher proportions of these T cells in the spleen than subcutaneous delivery of 50 µg and 250 µg 216
of CpG adjuvant, respectively (Figure 1C). When OVA protein was injected intramuscularly, 217 addition of topical CpG adjuvant also increased the proportion of OVA-specific CD8+ T cells by 218 3-fold compared to intramuscular delivery of the adjuvant (Figure 1C). The same trend was 219 observed when the absolute numbers of OVA-specific CD8+ T cells in the spleen were 220 determined for each immunization group (Figures 1B & 1C). These results support the 221 speculation that the topical route is a better route to administer CpG adjuvant to enhance CTL 222 responses compared to parenteral routes. Data also confirmed that the adjuvant effect of CpG 223 ODN 1826 is CpG-motif-specific. 224 225 226 Toxicities caused by parenteral (intraperitoneal) CpG ODNs include follicle microarchitecture 227 disruption as well as splenomegaly due to erythroid and myeloid expansion in the red pulp of the 228 spleen (26), (27). Thus, the spleens of immunized and untreated (naive) mice were weighted as 229 an indirect method to evaluate potential toxicity of CpG adjuvant administered via the three 230 routes mentioned above. In contrast to subcutaneous injection of 50 µg and 250 µg of CpG 231 adjuvant that increased spleen weight by 3- and 7-fold respectively, no significant increase of 232 spleen weight was detected in mice treated with topical CpG adjuvant (Figure 2). Overall, topical 233 delivery of CpG ODN 1826 as adjuvant was more effective and less toxic than parenteral 234 administration when given at the same time as parenteral protein-based vaccine, by either the 235 subcutaneous or the intramuscular route. 236 237 A rapid (7 day) prime-boost immunization regimen can be used to enhance CTL responses 238 with topical CpG ODN 1826 239
240 Proper spacing between doses of a given vaccine series is essential for optimal immune 241 responses. A shorter interval between vaccine booster doses would be more practical. We thus 242 tested a 7-day (common in murine cancer vaccine protocols) and a 21-day (standard timing for 243 antibody production) prime-boost immunization schedule for the optimal induction of antigen-244 specific CD8+ T cells (Figure 3A). Percentages of antigen-specific cells amongst CD8+ T cells 245 were determined by tetramer staining 5 days post-immunization (after boost). In the spleen, a 3-246 fold higher proportion of OVA-specific CD8+ T cells was detected when CpG adjuvant was 247 administered topically compared to subcutaneously (Figure 3B) using the 7-day interval prime-248 boost immunization regimen. In addition, the prime-boost immunization regimen with a 7-day 249 interval between the initial and the booster dose induced higher level of OVA-specific CD8+ T 250 cells than with a 21-day interval (Figure 3B). Hence, the short interval (7 days apart) compared 251 to the long interval (21 days apart) prime-boost immunization regimen was more effective in 252 inducing antigen-specific CD8+ CTLs. 253 254 A single application of topical CpG ODN 1826 at the time of protein-based vaccine 255 administration is sufficient to enhance antigen-specific CTL response 256 257 TLR stimulation commonly results in short-term stimulation of innate immunity. Consistent with 258 this concept, repeated daily topical administration of TLR7 agonist has been shown to further 259 improve CD8+ T cell induction following immunization with protein antigen (28). Thus, whether 260 daily multiple doses of topical CpG ODN further enhance the adjuvant effect with subcutaneous 261 protein-based vaccines was investigated. Whether a regimen of single immunization induces 262
comparable frequencies of antigen-specific CD8+ T cells to a prime-boost regimen was also 263 investigated. OVA protein was injected subcutaneously with one-time application of topical CpG 264 adjuvant or three-time application on 3 consecutive days (Figure 4A). Using flow cytometry, the 265 frequencies of OVA-specific CD8+ T cells and IFN-γ-producing CTLs were determined. A 266 booster dose at 7 days increased the pool of OVA-specific and IFN-γ-producing CD8+ T cells as 267 detected in the spleen 5 days post-immunization compared to single immunization (Figure 4B). 268 Multiple doses of CpG adjuvant administered over 3 consecutive days did not further enhance 269 the generation of OVA specific or IFN-γ-producing CTLs over single dose (Figure 4B). The 270 frequency or IFN-γ-producing CD8+ T cells paralleled that of OVA-specific CTLs. Thus, a 271 single topical application of CpG adjuvant at the time of subcutaneous protein antigen 272 administration with a prime-boost regimen, 7 days apart, is effective for the induction of 273 functional antigen-specific CTLs. 274 275 Topical CpG ODN 1826 induces a higher proportion of antigen-specific CD8+ effector-276 memory T cells than the subcutaneous route 277 278 Effector T cells undergo contraction and differentiate into memory T cells. Memory T cells 279 provide sustained protection against invading pathogens in a faster and more effective manner 280 than primary effector T cells (29). The ability of topical and subcutaneous CpG adjuvant to 281 augment the generation of antigen-specific CD8+ memory T cells was thus determined. Mice 282 were immunized using the prime-boost immunization regimen with OVA protein administered 283 subcutaneously with CpG adjuvant administered either topically or subcutaneously. The level of 284 circulating OVA-specific CD8+ effector T cells (5 days after boost) and long-term memory T 285
cells (30 days after boost) in peripheral blood were determined post-immunization (Figure 5A). 286 Topical compared to subcutaneous administration of CpG adjuvant robustly increased the 287 frequency of OVA-specific CD8+ effector T cells detected in the peripheral blood by up to 6-fold 288 at 5 days post-immunization (after boost) (Figure 5B). At 30 days post-immunization (after 289 boost), no significant difference in the frequency of CD8+ memory T cells in the blood between 290 mice immunized with topical and subcutaneous adjuvant (Figure 5B). 291 292 Despite similar proportions of the memory T cells, does topical CpG adjuvant change the 293 phenotype of the memory T cells? There are two major subsets of memory T cells: central-294 memory and effector-memory T cells (30), (31). Central-memory T cells are CD127+CD62L+, 295 have a higher proliferative potential than naive cells and preferentially reside in lymphoid 296 organs. Effector-memory T cells are CD127+CD62L-, have a faster effector function and 297 preferentially reside in non-lymphoid (stromal) tissues. To further characterize the OVA-specific 298 memory CD8+ T cells induced by immunization with CpG adjuvant administered topically or 299 subcutaneously, the surface markers CD127 and CD62L were used to differentiate effector-300 memory from central-memory T cells (Figure 5C). Interestingly, the population of 301 CD127+CD62L- OVA-specific effector-memory T cells in peripheral blood was 1.5-fold higher 302 upon immunization in the presence of CpG adjuvant administered topically compared to 303 subcutaneously (Figure 5D). Also, a trend of lower proportion of CD127+ CD62L+ OVA-specific 304 central-memory T cells was detected with topical CpG adjuvant compared to subcutaneous 305 administration (Figure 5D). Thus, immunization in the presence of CpG adjuvant (either 306 subcutaneously or topically) induces long-lived antigen-specific memory CD8+ T cells. In 307 addition, topical compared to subcutaneous delivery of CpG adjuvant induces a higher 308
proportion of antigen-specific effector-memory CD8+ T cells. 309 310 Topical CpG ODN 1826 confers rapid TLR9-dependent protection against systemic Lm 311 infection 312 313 Topical CpG adjuvant induced rapid and lasting CD8+ T cell responses to subcutaneous antigen. 314 To determine whether these responses were adequate to protect against infection, we tested the 315 ability of this vaccination method to protect against Listeria monocytogenes (Lm), an 316 intracellular bacterium predominantly controlled by host CD8+ T cell responses (32), (33). The 317 prime-boost and the one-time immunization regimen were tested for their ability to protect 318 animals from systemic Lm challenge. Mice were immunized with OVA antigen subcutaneously 319 with or without topical CpG adjuvant and then challenged with Lm-OVA 5 or 7 days post-320 immunization (Figure 6A). A 5-log decrease of bacterial load was noted in the spleen following a 321 prime-boost immunization regimen with topical CpG adjuvant compared to immunization 322 without adjuvant (Figure 6B). Immunization with CpG adjuvant alone (i.e. without antigen) 323 reduced bacterial burden. However, this was not as effective as immunization with antigen and 324 adjuvant (Figure 6B). Thus repeated innate stimulation provides a modicum of rapid protection 325 that is improved upon with antigen vaccination. Unexpectedly, topical compared to subcutaneous 326 administration of CpG adjuvant did not further decrease bacterial load in the spleen after acute 327 systemic Lm-OVA challenge (Figure 6B). In addition, a 3-log decrease of bacterial burden in the 328 spleen was detected in mice immunized with topical CpG adjuvant compared to without adjuvant 329 using the one-time immunization regimen (Figure 6C). Thus, immunization using topical or 330 subcutaneous CpG adjuvant with subcutaneous protein antigen can provide protection against 331
Lm infections, which is not observed with immunization with antigen alone. Although a prime-332 boost immunization regimen provides optimal protection against Lm, a one-time immunization 333 strategy can confer protection as well. 334 335 The uptake of CpG ODNs by cells are CpG motif-independent and CpG ODNs have been 336 reported to induce TLR9-independent immune effects (34), (35). To confirm that the adjuvant 337 effect of CpG ODN 1826 on Lm bacterial burden was TLR9 dependent, we compared the 338 bacterial load 3 days post-infection by Lm-OVA between immunized wild type (WT) and TLR9-339 deficient (KO) mice (Figure 7A). Bacterial loads in WT and KO mice following Lm-OVA 340 infection were identical (Figure 7B, left panel) suggesting similar susceptibility to infection. 341 Unlike WT animals with a 4-log decrease in bacterial load in the spleen, bacterial load was not 342 reduced in the KO mice (Figure 7B, right panel), demonstrating TLR9 dependency of the topical 343 CpG adjuvant effect. 344 345 Topical CpG ODN 1826 improves long-term protection against systemic Lm infection 346 347 Topical CpG adjuvant induced persistent CTL responses to subcutaneously administered protein 348 vaccine. Hence, we assessed the ability of our proposed prime-boost immunization strategy with 349 protein antigen injected subcutaneously and topical CpG ODN 1826 as adjuvant to provide long-350 term protective immunity against intracellular bacterial infection (Figure 8A). Mice immunized 351 in the presence of CpG adjuvant administered topically but not subcutaneously showed a modest 352 but significant decrease in bacterial burden (1-log reduction) (Figure 8B). An attenuated ΔactA-353 Lm-OVA strain was used as a live vaccine to serve as a positive control (22). Thus, our proposed 354
prime-boost immunization strategy with soluble protein antigen and topical CpG adjuvant but 355 not subcutaneous adjuvant is able to confer long-term protection against systemic Lm infection. 356 In our experiments, topical adjuvant use at time of protein immunization approximated the 357 protective effect of a live vaccine. 358 359
DISCUSSION 360 361 Using the skin as a site of adjuvant administration, we demonstrate that a TLR9 agonist may be 362 applied onto the skin to enhance CTL responses to locally administered protein antigen and to 363 provide protective immunity to the intracellular pathogen Lm. The structural and immune 364 properties of the skin enable a split adjuvant and antigen vaccination strategy: Guebre-Xavier 365 first demonstrated that topical administration of an enterotoxin improved the amplitude of the 366 antibody response to intramuscular or subcutaneous influenza antigen in mice (36). This group 367 also showed that an enterotoxin-containing patch could induce a trend towards higher antibody 368 responses to influenza vaccination in the elderly (37), demonstrating relevance of this approach 369 in humans. Other groups showed that topical TLR7 agonists induce CTL responses to protein 370 antigen in mice (2), improve immunity to Leishmania in mice (38) and are able to induce 371 immune responses to tumor antigens in humans (39). 372 373 Separate administration of vaccine and adjuvant at immunization has several advantages over 374 standard co-administration. Such advantages include: Potentially more efficacious sites of 375 adjuvant administration, increased safety and the ability to tailor adjuvant use to specific 376 populations without a need for vaccine re-formulation. However, bacterial toxins, such as 377 enterotoxins, have persistent safety concerns. Further, even limited amounts of topical TLR7 378 agonist may induce systemic cytokine release and splenomegaly in mice (28) and may induce 379 systemic symptoms and side effects in humans (40). Synthetic ODNs with CpG motifs are larger 380 molecules that have less penetration after topical application than imiquimod and similar TLR7 381 agonists. We thus explored split skin administration of adjuvant and antigen using synthetic CpG 382
ODN 1826 as a vaccine adjuvant. 383 384 Synthetic ODNs with CpG motifs have been explored for immunotherapeutic uses, including the 385 reduction of allergic responses and the enhancement of cancer therapy and innate immune 386 responses (4). CpG ODNs have also been extensively studied for their ability to improve vaccine 387 efficacy. These studies have always used co-administration of antigen and adjuvant. Studies in 388 non-human primates indicate that CpG ODNs are as effective as TLR7 adjuvants in their ability 389 to induce long-lasting protective immunity but without the induction of associated skin 390 inflammation (1). Topical administration of CpG ODNs co-administered with parenteral antigen 391 enhances CTL responses to antigen (20) and induces CTL to tumors in a murine model (21). 392 The adjuvant must be administered within the same lymph node draining area as the antigen and 393 results in movement of Langerin+ (epidermal and dermal derived) dendritic cells (DCs) and 394 antigen-bearing DCs into the draining lymph nodes. This results in an effective adjuvant effect 395 allowing CTL priming without the cytokine toxicity and splenomegaly noted with parenteral 396 administration of CpG adjuvant (20): We have previously shown that subcutaneous 397 administration of CpG adjuvant results in rapid elevation of TNF-α, IL-12p70, IL-6 and 398 monocyte chemoattractant protein-1 (MCP-1), as well as IFN-γ release in the serum (20). All of 399 these did not occur with topical administration of CpG adjuvant supporting the contention that 400 topical administration induces less systemic inflammation. Here, we expanded on our previous 401 observations and explored the use of synthetic CpG ODN 1826 as topical adjuvant in the context 402 of intracellular infection. Our results indicate that topical CpG ODN administration is superior to 403 co-administration with antigen via the subcutaneous or intramuscular route in terms of CTL 404 induction, has less systemic effects and is better than parenteral CpG in terms of long-term 405
protection against systemic Lm infection. 406 407 Current protein vaccines require booster doses to achieve effective immunity. A protective CD8+ 408 T cell threshold is often not reached with a single immunization (41). Instead, prime-boost 409 regimens are required for long-term protection (42). Proper spacing between doses of a given 410 vaccine series is essential for optimal immune responses. In contrast, rapid induction of 411 immunity is desired as a response to pandemic infection or bioterrorist attack. We find that an 412 effective strategy of CpG ODN 1826 administration for antigen-specific CTL induction is to 413 administer a single dose of topical CpG ODN 1826 as an adjuvant with subcutaneous protein 414 antigen followed by a booster dose 7 days later. Unlike the TLR7 agonist R848, this strategy 415 does not result in splenomegaly and multiple applications are not required to increase efficacy 416 (28). In addition, a single immunization with topical CpG adjuvant also enhanced protective 417 immunity. High levels of inflammatory mediators, such as IFNγ that may be induced by CpG 418 adjuvant, inhibit durable memory T cell responses (43), (44). We show in this paper, and have 419 noted previously, that topical administration of CpG adjuvant, which does not induce 420 splenomegaly or systemic cytokine release (20) (and thus induces less systemic inflammation 421 than subcutaneous CpG), nevertheless induced memory CD8+ T cell responses. We propose that 422 the local adjuvant administration induces the activation of antigen presenting cells (20) without 423 the widespread inflammation and possibly direct cytokine stimulation of T cells that may occur 424 with parenteral administration. This may simulate much more technically demanding DC 425 vaccination, which has been shown to induce rapid memory CD8+ T cell responses (44). 426 427 Topical CpG adjuvant induces a rapid increase of antigen-specific CTL and a higher proportion 428
of effector-memory CD8+ T cells. We next asked whether these changes were sufficient to 429 protect against intracellular bacterial infection. CD8+ T cells are required for protection against 430 Lm infection (32), (33). Further, intraperitoneal CpG ODN injection alone offers non-antigen-431 specific short term protection against Lm infection possibly due to IL-12 and IFN-γ release (45). 432 We thus used Lm-OVA to assess whether antigen-specific CTLs induced by our immunization 433 strategy confers protection against systemic Lm infection. Topical administration of CpG 434 adjuvant at the time of OVA protein immunization induced rapid protection against 435 intravenously administered Lm-OVA indicated by the reduction of bacterial load when mice 436 were challenged shortly after immunization. This protection was similar in extent to that 437 provided by subcutaneous CpG adjuvant (despite higher CTL frequencies), possibly due to a 438 saturation in the efficacy of clearance of intravenously administered organisms by CTL. 439 Repeated administration of topical CpG without antigen also decreased bacterial load slightly, 440 suggesting that topical application can have a broad, antigen non-specific effect, as noted 441 following intraperitoneal administration (45). Phosphorothioate modification of CpG ODNs to 442 render them more resistant to nuclease degradation may result in phosphorothioate CpG but 443 TLR9-independent immune activation (46), (47), (48), (49). We confirmed that the protective 444 effect of topical ODN 1826 as adjuvant was TLR9-dependent using TLR9KO mice. 445 446 Memory T cells have higher affinity for antigen and decreased threshold for activation (50). 447 Memory CD8+ T cells are crucial in providing protective immunity against intracellular 448 pathogens such as Plasmodium (41) and human immunodeficiency virus (51). Topical CpG 449 adjuvant administration at the time of subcutaneous protein immunization induces antigen-450 specific memory T cells forming a stable pool of circulating CD8+ T cells for up to four months 451
(20). However, topical compared to subcutaneous administration of CpG adjuvant did not result 452 in a further increase in the frequency of blood circulating antigen-specific memory CD8+ T cells. 453 454 Two types of CD8+ memory T cells with differential proliferative and protective potential have 455 been identified based on surface expression of CD62L: CD62L+ central-memory T cells with 456 low proliferative potential and CD62L- effector-memory T cells with high proliferative potential 457 (52). Both central- and effector-memory T cells may be crucial to combat pathogens, acting at 458 different times following infection. Cui et al. showed that parenteral CpG-B administration at 459 time of vaccination increases CD127low KLRG1hi short-lived effector cells (SLEC) formation but 460 not CD127hi KLRG1low CD8+ memory T cells (MPEC) (53). Obar et al. showed that 461 intraperitoneal injection of CpG ODN 1826 at time of vaccination increases central-memory 462 CD62Lhi CD8+ T cells (54). We note that topical CpG initially induces a larger burst of blood-463 resident antigen-specific CD8+ T cells in an acute response when compared to parenteral CpG 464 but we did not detect a subsequent difference in the frequency of memory T cells within the 465 blood compartment one month later. Although we did not directly phenotype the memory T cells 466 into the CD127hi KLRG1low MPEC, we found that topical administration of CpG adjuvant, 467 unlike the intraperitoneal administration of CpG by Obar et al., resulted in a preferential 468 induction of a pool of CD127+ CD62L- effector-memory cells. Preferential tissue-distribution 469 and effector-cell population contraction within the blood compartment may explain why 470 protective immunity following subcutaneous or topical CpG adjuvant vaccination resulted in a 471 weaker protective response to delayed systemic Lm challenge (30 days post-immunization) 472 compared to acute challenge (5 days post-immunization). 473 474
Taken together, our work indicates that a rapid (7 day) prime-boost regimen with a single dose of 475 topical CpG adjuvant at the time of parenteral antigen administration is a good strategy to induce 476 protective CD8+ T cell response for protein-based vaccines. We extend these observations to an 477 in vivo mouse infection model using Lm-OVA and show rapid protection when mice are 478 immunized with subcutaneous antigen with topical CpG ODN 1826. Our work thus supports the 479 use of topical CpG adjuvant to increase the cell-mediated immune response to protein vaccines. 480 The rapid protection induced makes this strategy attractive for the protection against novel 481 intracellular pathogens, as in pandemic viral outbreaks. This strategy has the added advantage of 482 not requiring novel vaccine formulations for developed vaccine. Further, it may be applied 483 selectively to at risk populations such as the elderly or the very young. 484 485 CpG ODNs are excellent adjuvants in non-human primates when given subcutaneously (1). It is 486 important to note that TLR9 expression pattern differs between human and mice. Could topical 487 CpG ODN application have adjuvant effects in humans? TLR9 is constitutively expressed or can 488 be induced to express on murine myeloid DCs, plasmacytoid DCs, B cells, monocytes, and mast 489 cells (55), (56). In normal mouse skin, TLR9 mRNA expression is induced following intradermal 490 injection of CpG 1668 (57). In humans, TLR9 expression is more restricted than in mice to 491 plasmacytoid DCs and B cells but can also be induced in monocytes and keratinocytes (58), (59), 492 (60) suggesting that topical application may also have an additional adjuvant effect in humans. 493 Topical administration of adjuvant is a superior strategy to parenteral administration because 494 topical application of adjuvant limits distribution of the adjuvant (39). We speculate that topical 495 CpG adjuvant induces keratinocyte as well as DC activation, multiplying immunogenic stimuli 496 without promoting systemic inflammation. 497 498
ACKNOWLEDGEMENTS 499 WKC was supported by the CIHR Canada Graduate Scholarship Master’s Award and a CIHR 500 Skin Research Training Centre Training Scholarship. KW is supported by the NSERC industrial 501 postgraduate scholarship award. JPD is a Senior Scientist of the Michael Smith Foundation for 502 Research and the Child and Family Research Institute (CFRI). The work was supported by a 503 CIHR operating grant (FRN 89878). The authors declare no conflicts of interest. 504 505
REFERENCES 506 507 1. Wille-Reece U, Flynn BJ, Loré K, Koup RA, Miles AP, Saul A, Kedl RM, Mattapallil 508
JJ, Weiss WR, Roederer M, Seder RA. 2006. Toll-like receptor agonists influence the 509 magnitude and quality of memory T cell responses after prime-boost immunization in 510 nonhuman primates. J Exp Med 203:1249–1258. 511
512 2. Johnston D, Bystryn J-C. 2006. Topical imiquimod is a potent adjuvant to a weakly-513
immunogenic protein prototype vaccine. Vaccine 24:1958–1965. 514 515 3. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino 516
K, Wagner H, Takeda K, Akira S. 2000. A Toll-like receptor recognizes bacterial DNA. 517 Nature 408:740–745. 518
Immunol 4:249–259. 521 522 5. Rothenfusser S, Hornung V, Ayyoub M, Britsch S, Towarowski A, Krug A, Sarris A, 523
Lubenow N, Speiser D, Endres S, Hartmann G. 2004. CpG-A and CpG-B 524 oligonucleotides differentially enhance human peptide-specific primary and memory 525 CD8+ T-cell responses in vitro. Blood 103:2162–2169. 526
527 6. Liu Y, Luo X, Yang C, Yu S, Xu H. 2011. Three CpG oligodeoxynucleotide classes 528
differentially enhance antigen-specific humoral and cellular immune responses in mice. 529 Vaccine 29:5778–5784. 530
Frank FM, Goldman A, Corral RS, Martin V. 2011. Combination of CpG-533 oligodeoxynucleotides with recombinant ROP2 or GRA4 proteins induces protective 534 immunity against Toxoplasma gondii infection. Exp Parasitol 128:448–453. 535
536 8. Cooper CL, Davis HL, Angel JB, Morris ML, Elfer SM, Seguin I, Krieg AM, 537
Cameron DW. 2005. CPG 7909 adjuvant improves hepatitis B virus vaccine 538 seroprotection in antiretroviral-treated HIV-infected adults. AIDS 19:1473–1479. 539
540 9. Wagner H, Lipford GB, Häcker H. 2000. The role of immunostimulatory CpG-DNA in 541
efficacy of live vaccines. J Immunol 174:4373–4380. 550 551 12. Heit A, Schmitz F, Haas T, Busch DH, Wagner H. 2007. Antigen co-encapsulated with 552
adjuvants efficiently drive protective T cell immunity. Eur J Immunol 37:2063–2074. 553 554 13. Romani N, Thurnher M, Idoyaga J, Steinman RM, Flacher V. 2010. Targeting of 555
antigens to skin dendritic cells: possibilities to enhance vaccine efficacy. Immunol Cell 556 Biol 88:424–430. 557
558 14. Dawicki W, Marshall JS. 2007. New and emerging roles for mast cells in host defence. 559
Curr Opin Immunol 19:31–38. 560 561 15. Macleod AS, Havran WL. 2011. Functions of skin-resident γδ T cells. Cell. Mol. Life 562
Sci. 68:2399–2408. 563 564 16. Clark RA. 2010. Skin-resident T cells: the ups and downs of on site immunity. J Invest 565
Dermatol 130:362–370. 566 567 17. Nestle FO, Di Meglio P, Qin J-Z, Nickoloff BJ. 2009. Skin immune sentinels in health 568
and disease. Nat Rev Immunol 9:679–691. 569 570 18. Kahlon R, Hu Y, Orteu CH, Kifayet A, Trudeau JD, Tan R, Dutz JP. 2003. 571
Optimization of epicutaneous immunization for the induction of CTL. Vaccine 21:2890–572 2899. 573
602 27. Heikenwalder M, Polymenidou M, Junt T, Sigurdson C, Wagner H, Akira S, 603
Zinkernagel R, Aguzzi A. 2004. Lymphoid follicle destruction and immunosuppression 604 after repeated CpG oligodeoxynucleotide administration. Nat Med 10:187–192. 605
4:812–823. 623 624 33. Lara-Tejero M, Pamer EG. 2004. T cell responses to Listeria monocytogenes. Curr. 625
Opin. Microbiol. 7:45–50. 626 627 34. Saxena M, Busca A, Pandey S, Kryworuchko M, Kumar A. 2011. CpG protects human 628
monocytic cells against HIV-Vpr-induced apoptosis by cellular inhibitor of apoptosis-2 629 through the calcium-activated JNK pathway in a TLR9-independent manner. J Immunol 630 187:5865–5878. 631
632 35. Sanjuan MA, Rao N, Lai K-TA, Gu Y, Sun S, Fuchs A, Fung-Leung W-P, Colonna 633
M, Karlsson L. 2006. CpG-induced tyrosine phosphorylation occurs via a TLR9-634 independent mechanism and is required for cytokine secretion. J. Cell Biol. 172:1057–635 1068. 636
637 36. Guebre-Xabier M, Hammond SA, Ellingsworth LR, Glenn GM. 2004. 638
Immunostimulant patch enhances immune responses to influenza virus vaccine in aged 639 mice. J Virol 78:7610–7618. 640
37. Frech SA, Kenney RT, Spyr CA, Lazar H, Viret J-F, Herzog C, Glück R, Glenn GM. 642 2005. Improved immune responses to influenza vaccination in the elderly using an 643 immunostimulant patch. Vaccine 23:946–950. 644
645 38. Zhang W-W, Matlashewski G. 2008. Immunization with a Toll-like receptor 7 and/or 8 646
agonist vaccine adjuvant increases protective immunity against Leishmania major in 647 BALB/c mice. Infect Immun 76:3777–3783. 648
649 39. Adams S, O'Neill DW, Nonaka D, Hardin E, Chiriboga L, Siu K, Cruz CM, Angiulli 650
A, Angiulli F, Ritter E, Holman RM, Shapiro RL, Berman RS, Berner N, Shao Y, 651 Manches O, Pan L, Venhaus RR, Hoffman EW, Jungbluth A, Gnjatic S, Old L, 652 Pavlick AC, Bhardwaj N. 2008. Immunization of malignant melanoma patients with full-653 length NY-ESO-1 protein using TLR7 agonist imiquimod as vaccine adjuvant. J Immunol 654 181:776–784. 655
Lauer P, Reyes-Sandoval A, Hutchings CL, Moore AC, Gilbert SC, Hill AV, 662 Bartholomay LC, Harty JT. 2008. Memory CD8 T cell responses exceeding a large but 663 definable threshold provide long-term immunity to malaria. Proc Natl Acad Sci USA 664 105:14017–14022. 665
666 42. Jabbari A, Harty JT. 2006. Secondary memory CD8+ T cells are more protective but 667
slower to acquire a central-memory phenotype. J Exp Med 203:919–932. 668 43. Badovinac VP, Porter BB, Harty JT. 2004. CD8+ T cell contraction is controlled by 669
early inflammation. Nat Immunol 5:809–817. 670 671 44. Badovinac VP, Messingham KAN, Jabbari A, Haring JS, Harty JT. 2005. 672
Accelerated CD8+ T-cell memory and prime-boost response after dendritic-cell 673 vaccination. Nat Med 11:748–756. 674
675 45. Krieg AM, Love-Homan L, Yi AK, Harty JT. 1998. CpG DNA induces sustained IL-12 676
expression in vivo and resistance to Listeria monocytogenes challenge. J Immunol 677 161:2428–2434. 678
679 46. Vollmer J, Weeratna RD, Jurk M, Samulowitz U, McCluskie MJ, Payette P, Davis 680
HL, Schetter C, Krieg AM. 2004. Oligodeoxynucleotides lacking CpG dinucleotides 681 mediate Toll-like receptor 9 dependent T helper type 2 biased immune stimulation. 682 Immunology 113:212–223. 683
684 47. Roberts TL, Sweet MJ, Hume DA, Stacey KJ. 2005. Cutting edge: species-specific 685
TLR9-mediated recognition of CpG and non-CpG phosphorothioate-modified 686 oligonucleotides. J Immunol 174:605–608. 687
48. Sester DP, Brion K, Trieu A, Goodridge HS, Roberts TL, Dunn J, Hume DA, Stacey 688 KJ, Sweet MJ. 2006. CpG DNA activates survival in murine macrophages through TLR9 689 and the phosphatidylinositol 3-kinase-Akt pathway. J Immunol 177:4473–4480. 690
691 49. Luganini A, Caposio P, Landolfo S, Gribaudo G. 2008. Phosphorothioate-modified 692
oligodeoxynucleotides inhibit human cytomegalovirus replication by blocking virus entry. 693 Antimicrob. Agents Chemother. 52:1111–1120. 694
Woodland DL. 1999. Functionally heterogeneous CD8(+) T-cell memory is induced by 704 Sendai virus infection of mice. J Virol 73:7278–7286. 705
706 53. Cui W, Joshi NS, Jiang A, Kaech SM. 2009. Effects of Signal 3 during CD8 T cell 707
priming: Bystander production of IL-12 enhances effector T cell expansion but promotes 708 terminal differentiation. Vaccine 27:2177–2187. 709
710 54. Obar JJ, Jellison ER, Sheridan BS, Blair DA, Pham Q-M, Zickovich JM, Lefrançois 711
L. 2011. Pathogen-induced inflammatory environment controls effector and memory 712 CD8+ T cell differentiation. J Immunol 187:4967–4978. 713
714 55. Tran N, Koch A, Berkels R, Boehm O, Zacharowski PA, Baumgarten G, 715
Knuefermann P, Schott M, Kanczkowski W, Bornstein SR, Lightman SL, 716 Zacharowski K. 2007. Toll-like receptor 9 expression in murine and human adrenal 717 glands and possible implications during inflammation. J. Clin. Endocrinol. Metab. 718 92:2773–2783. 719
720 56. Krieg AM. 2007. Antiinfective Applications of Toll-like Receptor 9 Agonists. 721
Proceedings of the American Thoracic Society 4:289–294. 722 723 57. Liu L, Zhou X, Shi J, Xie X, Yuan Z. 2003. Toll-like receptor-9 induced by physical 724
trauma mediates release of cytokines following exposure to CpG motif in mouse skin. 725 Immunology 110:341–347. 726
727 58. Saikh KU, Kissner TL, Sultana A, Ruthel G, Ulrich RG. 2004. Human monocytes 728
infected with Yersinia pestis express cell surface TLR9 and differentiate into dendritic 729 cells. J Immunol 173:7426–7434. 730
731 59. Tripp CH, Ebner S, Ratzinger G, Romani N, Stoitzner P. 2010. Conditioning of the 732
injection site with CpG enhances the migration of adoptively transferred dendritic cells 733
FIGURE LEGENDS 740 741 Figure 1. Topical administration of CpG adjuvant is more effective in antigen-specific 742 effector CD8+ T cell generation. 100 μg OVA protein antigen was injected subcutaneously (sc) 743 or intramuscularly (im) in combination with CpG adjuvant administered topically (ec), sc, or im 744 as indicated. (A) Schematic diagram of prime-boost immunization regimen and T cell analysis 745 timeline. (B, C) Top panels show representative flow cytometric dot plots in log axis scale 746 identifying OVA-specific CD8+ T cells in the spleen. Cells were gated on B220 negative 747 population and OVA-specific CD8+ T cells were identified as CD8 and Kb-OVA class I tetramer 748 double positive population. Bottom panels show scatter plots of the percentage of OVA-specific 749 cells amongst CD8+ T cells and total OVA-specific cells detected in the spleen 5 days post-750 immunization (after boost). Animals labeled “No CpG” or “Ctl CpG” received antigen and had 751 skin treatment of tape-stripping and acetone (topical placebo treatment) without CpG or with 752 control deoxynucleotide (ODN 1982), respectively. Animals treated with “sc CpG” did not 753 receive any skin treatment. In parallel, naive mice that did not receive antigen or skin treatment 754 were analyzed. Data summarize at least two independent experiments (n = 6-9) and are 755 expressed in mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 756 757 Figure 2. Topical is less toxic than subcutaneous administration of CpG adjuvant. 100 μg 758 OVA protein antigen was injected subcutaneously (sc) with CpG adjuvant administered topically 759 (ec) or subcutaneously (sc) as indicated. (A) Schematic diagram of prime-boost immunization 760 regimen and T cell analysis timeline. (B) Toxicity analysis by spleen weight. A scatter plot of 761 spleen weights measured 5 days post-immunization (after boost). Data summarize at least two 762
independent experiments (n = 6-9) and are expressed in mean ± SEM. *** p < 0.001 763 764 Figure 3. Optimal intervals between doses of a vaccine series for the rapid generation of 765 antigen-specific CD8+ T cells. 100 μg OVA protein antigen was injected subcutaneously (sc) 766 with CpG adjuvant administered topically (ec, 250 μg) or subcutaneously (sc, 50 μg). (A) 767 Schematic diagrams of prime-boost immunization with interval of 7 days or 21 days and T cell 768 analysis timeline. (B) Left panels show representative flow cytometric dot plots in log axis scale 769 identifying OVA-specific cells CD8+ T cells in the spleen. Cells were gated as in Figure 1. Right 770 panel shows a scatter plot of the percentage of OVA-specific cells amongst CD8+ T cells 771 detected in the spleen. Data summarize at least two independent experiments (n= 4-10) and are 772 expressed in mean ± SEM. ** p < 0.01, ns = no significance 773 774 Figure 4. The better immunization strategy using topical CpG ODN as vaccine adjuvant to 775 elicit acute CD8+ T cell responses. (A) Schematic diagrams of single and prime-boost 776 immunization regimen and T cell analysis timelines. 100 μg OVA protein antigen was injected 777 subcutaneously (sc) with or without topical (ec) CpG adjuvant. Arrows indicate three 778 consecutive days of topical CpG adjuvant administration (250 μg per administration). (B) Top 779 panels show representative flow cytometric dot plots identifying OVA-specific CD8+ T cells and 780 IFN-γ-producing cells in the spleen after restimulation with OVA8 peptide ex vivo. Bottom panel 781 shows scatter plots of the percentage and the total number of OVA-specific CD8+ T cells and 782 IFN-γ-producing cells detected in the spleen on day 7 or day 12 post-initial immunization (after 783 prime) using a single or a prime-boost immunization regimen, respectively. Data summarize two 784
independent experiments (n = 8) and are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, ns = 785 no significance 786 787 Figure 5. Topical compared to subcutaneous administration of CpG adjuvant induced 788 higher proportion of antigen-specific CD8+ T effector memory cells. 100 μg OVA protein 789 antigen was injected subcutaneously (sc) with CpG adjuvant administered subcutaneously (sc, 50 790 μg) or topically (ec, 250 μg). (A) Schematic diagram of immunization regimen and T cell 791 analysis timeline. (B) Point graph of the percentage of OVA-specific cells amongst CD8+ T cells 792 detected in peripheral blood 5 days and 30 days post-immunization (after boost). Data 793 summarize two independent experiments (n = 3-6). (C) Representative flow cytometric density 794 plots in log axis scale of CD8+ T effector memory and central memory cells in the peripheral 795 blood 30 days post-immunization (after boost). (D) Left panel and right panel show a scatter plot 796 of the percentage of CD127+CD62L- effector memory cells and CD127+CD62L+ central memory 797 cells amongst OVA-specific CD8+ T cells detected in the peripheral blood 30 days post-798 immunization (after boost), respectively. Data represent two independent experiments (n = 6) 799 and are expressed in mean ± SEM. *** p < 0.001, ns = no significance 800 801 Figure 6. Acute protection against systemic Lm infection induced by immunization with 802 topical or subcutaneous CpG adjuvant. Naive mice, mice treated with topical (ec, 250 μg) 803 CpG adjuvant and mice immunized with 100 μg OVA protein antigen subcutaneously (sc, 50 μg) 804 with or without topical (ec, 250 μg) CpG adjuvant were challenged with 5 x 105 CFU Lm-OVA 805 intravenously. (A) Schematic diagram of immunization, infection and bacterial burden analysis 806 timelines. (B, C) Mice were immunized with a prime-boost or a single immunization regimen. 807
Mice were then challenged with Lm-OVA at time points indicated. Scatter dot plots in log axis 808 scale of bacterial counts in the spleen 3 days post-infection. Data summarize at least two 809 independent experiments (n = 2-10) and are expressed in mean ± SEM. * p < 0.05, ** p < 0.01, 810 *** p < 0.001, ns = no significance 811 812 Figure 7. Acute protection against systemic Lm infection induced by immunization with 813 topical or subcutaneous CpG adjuvant. Wild-type (WT) and TLR9-deficient (KO) mice were 814 unimmunized or immunized with 100 μg OVA protein subcutaneously (sc) with topical (ec, 250 815 μg) CpG adjuvant. All mice were challenged with 5 x 105 CFU Lm-OVA intravenously. (A) 816 Schematic diagram of immunization, infection and bacterial burden analysis timelines. (B) 817 Scatter dot plots in log axis scale of bacterial counts in the spleen 3 days post-infection. Data 818 summarize two independent experiments (n = 6-8) and are expressed as mean ± SEM. ** p < 819 0.01, ns = no significance 820 821 Figure 8. Long-term protection against systemic Lm infection induced by immunization 822 with topical or subcutaneous CpG adjuvant. Naive mice, mice treated with topical (ec, 250 823 μg) CpG adjuvant and mice immunized with 100 μg OVA protein antigen subcutaneously (sc) 824 without or with topical (ec, 250 μg) or subcutaneously (sc, 50 μg) CpG adjuvant were challenged 825 with 5 x 105 CFU Lm-OVA intravenously 30 days post-immunization (after boost). Mice 826 immunized with ΔactA-Lm-OVA intraperitoneally were analyzed in parallel as positive controls. 827 (A) Schematic diagram of immunization, infection, and bacterial burden analysis timeline. (B) 828 Scatter plots in log axis scale of bacterial counts in the spleen 3 days post-infection. Data 829 summarize at least two independent experiments (n = 5-19) and are expressed in mean ± SEM. 830
