A Listeria-based vaccine that secretes the sand fly salivary protein LJM11 confers long-term protection

IAI Accepts, published online ahead of print on 14 April 2014 Infect. Immun. doi:10.1128/iai.01633-14 Copyright 2014, American Society for Microbiology. All Rights Reserved. 1 2 A Listeria-based vaccine that secretes the sand fly salivary protein LJM11 confers long-term protection against vector-transmitted Leishmania major 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Delbert S. Abi Abdallah a, Alan Pavinski Bitar a, Fabiano Oliveira b, Claudio Meneses b, Justin J. Park a, Susana Mendez a,c *, Shaden Kamhawi b, Jesus G. Valenzuela b, and Hélène Marquis a# Department of Microbiology and Immunology, Cornell University, Ithaca, New York, USA a ; Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA b ; Baker Institute for Animal Health, Cornell University, Ithaca, New York, USA c Running title: Listeria-based vaccine against cutaneous leishmaniasis #Address correspondance to: Hélène Marquis, hm72@cornell.edu *Present address : National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Abstract Cutaneous leishmaniasis is a sand fly transmitted disease characterized by skin ulcers that carry significant scarring and social stigmatization. Over the past years, there has been cumulative evidence that immunity to specific sand fly salivary proteins confers a significant level of protection against leishmaniasis. In this study, we used an attenuated strain of Listeria monocytogenes as a vaccine expression system for LJM11, a sand fly salivary protein identified as a good vaccine candidate. We observed that mice were best protected against an intradermal needle challenge with Leishmania major and sand fly saliva when vaccinated intravenously. However, this protection was short-term. Importantly, groups of vaccinated mice were protected long-term when challenged with infected sand flies. Protection correlated with smaller lesion size, less scars, and better parasite control between 2 and 6 weeks post-challenge when compared to the control group of mice vaccinated with the parent L. monocytogenes strain not expressing LJM11. Moreover, protection correlated with high numbers of CD4 + IFN-γ + TNF-α +/- IL-10 - and low numbers of CD4 + IFN-γ +/- TNF-α - IL-10 + T cells at two weeks postchallenge. Overall, our data indicate that delivery of LJM11 by Listeria is a promising vaccination strategy against cutaneous leishmaniasis inducing long-term protection against ulcer formation following a natural challenge with infected sand flies. 2

36 INTRODUCTION 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 Leishmaniasis is a disease that is endemic in 98 countries of which most are developing countries (1). There are approximately 0.9 to 1.6 million new cases annually and 350 million people at risk of getting infected. The most common form of the disease is cutaneous leishmaniasis, which is characterized by skin ulcers. The lesions commonly self-cure but can leave significant scarring and lead to social stigmatization. The large majority of cases of cutaneous leishmaniasis occur in Afghanistan, Iran, Syria, Algeria, Brazil and Colombia (1). Leishmania spp. are protozoans that are transmitted by phlebotomine sand flies. During feeding, the fly injects saliva into its host. Some of the saliva proteins induce vasodilation and prevent coagulation to facilitate feeding, while others have immunomodulatory effects (2). There is strong evidence that the fly saliva enhances the ability of Leishmania to establish an infection by about a thousand fold (3, 4). Conversely, the development of a delayed-type hypersensitivity (DTH) response to salivary components from uninfected sand flies compromises the ability of Leishmania to establish an infection (5, 6). The immunoprotective value of pre-exposure to the saliva of uninfected sand flies is further emphasized by the observation that the influx of refugees, humanitarian aid workers, tourists, and military personnel in locations with previously low levels of infection leads to dramatic outbreaks of leishmaniasis (7-15). Following these observations, specific salivary proteins have been identified as potent protective immunogens against the establishment of an infection with Leishmania and are being considered as candidates for an anti-leishmania vaccine (16-19). Infected sand flies harbor the flagellated metacyclic promastigote form of Leishmania, which is transmitted into the dermis of a mammalian host during a blood meal. These parasites infect cells, predominantly professional phagocytes such as macrophages, in which they differentiate into nonflagellated amastigotes that divide and proliferate in parasitophorous vacuoles. Interestingly, the initial 3

59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 infection does not induce an inflammatory response thus enabling the parasite to expand in numbers (20). However, it has been well documented that resistance to Leishmania correlates with the development of a T H 1 type of immune response driven primarily by interferon gamma (IFN-γ)-producing CD4 + T cells (21). In this work, we used Listeria monocytogenes as a vector to vaccinate against a sand fly salivary protein. L. monocytogenes is an intracellular bacterial pathogen that has the ability of proliferating in the cytosol of infected cells and of spreading from cell to cell without exiting the intracellular milieu (22). Consequently, clearance of an infection is completely cell-mediated (23) and is dependent on the development of a T H 1 immune response that is dominated by IFN-γ-secreting T cells (24). Over the past decade, there has been a strong interest in using L. monocytogenes as a vaccine vector because of its many useful characteristics (25-27). The bacterium can easily be attenuated without affecting its immunogenicity and can secrete large amounts of heterologous proteins in infected cells, leading to the generation of a specific cell-mediated immune response against that heterologous antigen. Therefore, we generated an attenuated Listeria-based vector that expresses a gene coding for the salivary protein LJM11 from the sand fly Lutzomyia longipalpis to assess the efficacy of a Listeria-based vaccine against cutaneous leishmaniasis. We observed a significant decrease in disease burden in mice vaccinated intravenously with Listeria secreting LJM11 and challenged by needle injection with purified parasites and sand fly saliva, or with infected sand flies. Protection was associated with high numbers of CD4 + IFNγ + TNF-α ± IL-10 - and low numbers of CD4 + IFN-γ ± TNF-α - IL-10 + T cells at two weeks post-challenge. MATERIALS AND METHODS Vaccine strain. L. monocytogenes strain DP-L1942, which carries a deletion in the acta gene was used to alleviate the safety concerns regarding a Listeria-based vaccine (28). ActA is a surface protein that mediates actin polymerization, enabling L. monocytogenes to move in the cytosol of host cells and to 4

83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 spread from cell to cell without escaping the intracellular milieu (29). The acta deletion mutant is as efficient as the wild-type strain at escaping vacuoles formed upon initial entry into cells and at multiplying in the cytosol of infected cells. However, it is incapable of spreading from cell to cell without escaping from the intracellular milieu, resulting in a 3-log attenuation defect in a mouse model of infection (30). The acta deletion strain confers protective immunity at the same level as the wild-type strain. In an effort to improve vaccine safety and efficacy, the mdrm gene was deleted. This gene codes for a multiple drug resistance transporter that contributes to the activation of Type I interferon in infected cells (31). This innate immune response to a cytosolic infection by L. monocytogenes decreases the ability of the host to control the infection (32, 33). An internal in-frame deletion of the mdrm gene was introduced in strain DP-L1942 by allelic exchange as described (34), generating the control strain HEL- 1207. The vaccine strain (HEL-1325) was created by introducing in HEL-1207 a construct comprised of the sequences coding for the ActA signal sequence and N-terminal (100 aa) followed by a FLAG tag and the gene coding for LJM11 minus its signal sequence (Fig. 1A). Secretion of LJM11 by intracellular bacteria was verified by pulse-labeling infected J774 mouse macrophage-like cells as described (35). Fitness of the recombinant strains was verified by performing intracellular growth curves in J774 cells as previously described (36). Mice and sand flies. Six to eight week old C57BL/6 mice were used for this study. Lutzomyia longipalpis sand flies were reared at the Laboratory of Malaria and Vector Research (LMVR), NIAID, NIH. Four to seven day old adult flies were dissected to obtain salivary glands, which were stored in PBS at -80 C. Salivary gland homogenates (SGH) were prepared by sonication. Lysates were cleared by centrifugation. When used for animal infections, sand flies were pre-infected with L. major promastigotes as described (6). 5

106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 Parasites. Leishmania major WR 2885, a recent isolate originating from Iraq and typed at the Walter Reed Army Institute of Research, was used for this vaccine study (16). Frozen aliquots of L. major promastigotes were thawed, passaged in vitro once or twice and allowed to grow to stationary phase. Parasites were pelleted, washed once and re-suspended in DMEM. A discontinuous Ficoll gradient was prepared with 20% and 10% Ficoll type 400 in MilliQ water, and overlaid with the parasite suspension. After centrifugation at 2000 rpm for 10 min with slow deceleration, the metacyclic promastigotes were recovered above the 10-20% interface. The parasites were washed in DMEM and counts were determined using a hemacytometer. A final suspension was prepared in PBS. Crude Leishmania antigen (CLA). Parasites grown in flasks, as described above, were pelleted by centrifugation, washed three times in PBS and freeze-thawed multiple times to generate a crude lysate. Protein content was measured using a BCA assay and aliquots of CLA in PBS were stored at -80 C. Mouse experiments. Mice were anesthetized with isoflurane and vaccinated intradermally (ID) in the pinna of the ear (10 μl), intramuscularly (IM) in the hamstring (10 μl), subcutaneously at the base of the neck (100 μl), or intravenously in the retro-orbital sinus (100 μl). The vaccination dose was 5 x 10 6 CFU, three times at two-week intervals. Mice were challenged either 3 or 12 weeks after the last boost to assess short and long-term protection, respectively. Anesthetized mice were challenged intradermally in the pinna of the ear with 500 metacyclic promastigotes and SGH (equivalent to half a pair of salivary glands) in a total volume of 10 μl. Sand fly challenges were performed at the NIAID LMVR animal facility as described (6). Ear lesions were measured on a weekly basis starting at day 7 post-challenge. Needle challenges generate single ear lesions that were measured for thickness and diameter. These measurements were transformed into lesion area. Sand fly challenges generate multiple lesions that sometime coalesce. For this reason, we measured the thickness of the biggest ear lesion. 6

129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 Processing of infected ears and flow cytometry experiments. Ear pinna dermal sheets were collected, treated with ethanol, separated and digested in 500 μl of DMEM containing Liberase TL (250 μg/ml, Roche) at 37 C for 1.5 hours. Digested tissues were minced and homogenized. Ear cells were washed with complete tissue culture medium (CTCM: DMEM, 10% FBS, 1 mm Na Pyruvate, 0.1 mm nonessential amino acids, 2 mm L-Glutamine, 30 mm Hepes, and 50 μm -mercaptoethanol). The resulting suspension was filtered, spun down and resuspended in CTCM. Parasite loads in the challenged ears were determined by limiting dilution assays as described (37). Parasite titers were determined from the highest dilution at which growth was visible. Ear cells were mixed with CLA-pre-pulsed bone marrow-derived dendritic cells (DC) to stimulate cytokine production by immune cells, improving the sensitivity of FACS analysis. Briefly, DC were prepared as previously described (38) and incubated with or without CLA (25 μg/ml) for 6 to 7 hours before adding ear cells at a ratio of 1 DC per 5 ear cells. After an overnight incubation, cells were treated for 4 hours with Brefeldin A (10 μg/ml) and PMA (5 ng/ml), collected in FACS buffer (10% normal mouse serum, 0.5% BSA, 0.02%NaN 3 in PBS), and incubated with antibodies against surface and intracellular markers: CD4-APC-H7, CD8a-APC, TNF-PerCP.Cy5.5, IFN-PECy7, and IL10-PE. Cells were collected on a BD Canto flow cytometer and analyzed using FlowJo software. Isotype controls and representative plots of CD4+ T cells stained for IFNγ, TNFα, and IL-10 are shown in figure S1. Integrated median fluorescence intensities (imfi) were calculated for the cells by multiplying the MFI with the frequency of cells producing for the particular cytokine and the data are reported in table S1. Data and statistical analyses. Ratios were obtained by comparing values from each individual mouse from a vaccine group to the calculated mean from the corresponding control group. Statistical analyses were performed using a two-tailed, unpaired, t-test or a One-Way Anova test with Bonferroni post-test analysis. 7

152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 Ethics statement. All animal experimental procedures were reviewed and approved by Cornell University Institutional Animal Care and Use Committee, NIAID Animal Care and Use Committee, and US Army Medical Research and Material Command Animal Care and Use Review Office. All animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council. RESULTS L. monocytogenes vaccine vector confers the greatest protection against cutaneous leishmaniasis when administered intravenously. We generated an attenuated Listeria vaccine strain (HEL-1325) in which LJM11 was fused to the N-terminus of the Listeria ActA protein (Fig. 1A). This strain secreted large amounts of the fusion protein (55.4 kda) in infected cells (Fig. 1B), and had an intracellular growth rate that was similar to that of the parent strain (Fig. 1C). This attenuated Listeria vaccine strain persisted for several days in tissues of infected mice, inducing a specific T cell immune response against LJM11 (Fig. S2, 1D and S3). Initially, various routes of vaccine delivery were tested to identify parameters conferring the best level of protection in a mouse model of cutaneous leishmaniasis (3). Groups of mice were vaccinated intravenously, intradermally, subcutaneously, or intramuscularly, and challenged intradermally in the ear pinna with purified promastigotes and sand fly SGH. An age-matched unvaccinated group of mice was included in the challenge. Mice vaccinated intravenously, intradermally and intramuscularly had diminished lesion development when compared to unvaccinated mice (Fig. 2A). These differences were statistically significant at all time points for the intravenous group (p values between 0.0055 and <0.0001), between 4 and 11 weeks post-challenge for the intradermal group (p values between 0.0371 and 0.0007), and at 8 and 10 weeks post-challenge for the intramuscular group (p values of 0.0214 and 8

175 176 0.0156). Mice vaccinated by the subcutaneous route developed lesions that were similar in size to the unvaccinated group. 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 The data were also analyzed by calculating the area under the curve for each individual mouse as a measure of disease burden over the duration of the entire study (Fig. 2B). The group vaccinated intravenously suffered a disease burden that was about three folds less than the unvaccinated and subcutaneously vaccinated groups, and about two folds less than the intradermally and intramuscularly vaccinated groups. The overall protection was highly significant for the group vaccinated intravenously (p <0.001) when compared to the group vaccinated subcutaneously and to the control group. Protection was also significant for the group vaccinated intradermally (p <0.05) when compared to the control group. The experiment described above did not include a vaccine vector control for any of the four routes of vaccination tested. To test the possibility that protection conferred by intravenous vaccination was due to a non-specific immune response to the vaccine vector, we performed an experiment in which we vaccinated mice intravenously with the vaccine vector (HEL-1207) or with PBS. Mice were challenged as described three weeks post-vaccination with L. major and sand fly saliva, and lesion areas were recorded over a period of 11 weeks. The results show no difference between these two groups (Fig. 2C), suggesting that the L. monocytogenes vaccine vector does not confer protection against cutaneous leishmaniasis. These data taken together indicate that while most routes of vaccination confer a certain level of protection against challenge, the intravenous route of vaccination confers the greatest protection. 195 196 197 Vaccinated mice are protected short-term against a needle challenge. In this next set of experiments, mice were vaccinated intravenously with either L. monocytogenes expressing LJM11 (HEL-1325=Vaccine group) or the isogenic L. monocytogenes strain not expressing LJM11 (HEL-1207=Control group) to 9

198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 assess the specificity of the protective immune response. Short-term immune protection was assessed by challenging the two groups of mice intradermally three weeks after the last boost either with purified parasites mixed with sand fly SGH (needle challenge) or infected Lutzomyia longipalpis sand flies (sand fly challenge). Ear lesion sizes were recorded weekly. Because a needle challenge generates a single ear lesion, we measured lesion thickness and diameter, and transformed these measurements into lesion area. Contrarily, a sand fly challenge generates multiple lesions that sometime coalesce. For this reason, we measured the thickness of the biggest ear lesion. In addition to measuring lesion sizes, a subset of mice from each group was sacrificed at 2, 6, and 11 weeks post-challenge to determine parasite counts and characterize the immune response in the infected ears. Results from the short-term needle (STN) challenge groups indicated that mice in the vaccine group developed smaller lesions than those in the control group between 3 and 10 weeks post-challenge (p values between 0.0122 and <0.0001) (Fig. 3A), concurrently exhibiting a significantly smaller disease burden (p <0.0001) (Fig. 3B). Ear lesions were monitored carefully for the development of scars or ulcers and those that developed either over the duration of the experiment were scored. Five percent of mice in the vaccine group and 55% of mice in the control group developed scars or ulcers (p=0.010) (Fig. 3C). Ear parasite numbers were about the same in both groups of mice at 2 and 11 weeks post-challenge (Fig. 3D). However, between 2 and 6 weeks post-challenge, ear parasite numbers increased by three logs in the control group as opposed to one log in the vaccine group. The difference in ear parasite numbers between vaccine and control groups at 6 weeks post-challenge was statistically significant (p=0.0011). Overall, results from the STN challenge indicated that the vaccine is effective in decreasing overall disease burden, preventing the formation of scars, and decreasing parasite replication between 2 and 6 weeks post-challenge. 10

220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 Results from the short-term sand fly (STSF) challenge groups indicated that mice developed similar size lesions in vaccine and control groups (Fig. 3E) with no significant difference in disease burden (Fig. 3F). Scars developed in 65 and 40 percent of the mice in control and vaccine groups respectively (Fig. 3G), but this difference was not statistically significant. Ear parasite numbers were approximately 1.5 logs higher in the vaccine group compared to the control group at 2 weeks post-challenge (Fig. 3H). However, between 2 and 6 weeks post-challenge, ear parasite numbers increased by nearly three logs in the control group as opposed to one log in the vaccine group. By 11 weeks post-challenge, ear parasite numbers were similar in both groups of mice. Overall, results from the STSF challenge indicated that the vaccine did not confer protection, although the vaccine group controlled ear parasite numbers much more efficiently than the control group between 2 and 6 weeks post-challenge. Vaccinated mice are protected long-term against a sand fly challenge. Long-term protection assays were performed by challenging mice 12 weeks post-vaccination. Results from the long-term needle (LTN) challenge indicated that the vaccine group (HEL-1325) of mice had significantly smaller ear lesions than the control group (HEL-1207) at 6 and 7 weeks post-challenge (p values of 0.012 and 0.004, respectively) (Fig. 4A). However, the overall burden of disease was not significantly different between the two groups (Fig. 4B). Scars developed in 62 and 45 percent of the mice in control and vaccine groups respectively (Fig. 4C). Ear parasite numbers were similar between the two groups at all time points (Fig. 4D). Overall, results from the long-term needle challenge (LTN) indicated that the vaccine did not confer long-term protection in mice. Results from the long-term sand fly (LTSF) challenge indicated that the vaccine group (HEL-1325) developed significantly smaller ear lesions between 4 and 8 weeks post-challenge compared to the control group (HEL-1207) (p values between 0.027 and 0.0015) (Fig. 4E), concurrently exhibiting a significantly smaller disease burden (p=0.019) (Fig. 4F). Scars developed in 52 and 15 percent of the mice 11

243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 in control and vaccine groups respectively, and this difference was statistically significant (p=0.017) (Fig. 4G). Similarly to the STSF challenge, ear parasite numbers started 1.5 logs higher in the vaccine group compared to the control group (Fig. 4H). However, these numbers decreased by a log in the vaccine group, whereas they increased by 1.5 logs in the control group between 2 and 6 weeks post-challenge. Parasite numbers did not change in either groups between 6 and 11 weeks post-challenge. Overall, results from the LTSF challenge indicated that although parasite numbers were not significantly different between the vaccine and control groups by the end of the experiment, the vaccine is effective in decreasing overall disease burden, and preventing the formation of scars. A combination of high CD4 + IFN-γ + TNF-α ± IL-10 - and low CD4 + IFN-γ ± TNF-α - IL-10 + T cell populations at two weeks post-challenge is predictive of protection. Analysis of CD4 + and CD8 + specific T cells recruited at the site of challenge indicated that more than 97% on average of the specific immune response to Leishmania was mediated by CD4 + T cells (Fig. 5A). At two weeks post-challenge, there were significantly higher numbers of CD4 + IFN-γ + TNF-α ± IL-10 - T cells in every vaccine group compared to the respective control groups (Fig. 5B-E). The share CD4 + IFN-γ + TNF-α ± IL-10 - T cells occupied was 68% and 64% in the protected groups (STN and LTSF vaccine groups), and between 36% and 57% in the nonprotected groups (LTN and STSF vaccine groups and all control groups) (Fig. 5F-I). There were no significant differences between control and vaccine groups in the number of cells producing TNF-α for any of the experimental challenges, although the share CD4 + IFN-γ ± TNF-α + IL-10 - T cells occupied was lower, 42% and 38%, in the protected groups (STN and LTSF), and between 43% and 74% in the nonprotected groups (LTN and STSF vaccine groups and all control groups) (Fig. 5F-I). The data also indicates a significantly smaller number of CD4 + IFN-γ - TNF-α - IL-10 + T cells in the protected LTSF vaccine group (Fig. 5E) and a significantly larger number of CD4 + IFN-γ ± TNF-α - IL-10 + T cells in the non-protected STSF vaccine group compared to their respective control groups (Fig. 5C). Although the share IL-10 (with or 12

266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 without IFN-γ) occupied in the protected groups was not always lower than in the non-protected groups as a whole, the protected vaccine groups (STN and LTSF) had 3.5-4 fold less CD4 + IFN-γ ± TNF-α - IL-10 + T cells than their respective control groups, whereas this difference was 0.3 and 1.6 fold between the nonprotected vaccine groups (LTN and STSF) and their respective control groups (Fig. 5F-I). The balance between CD4 + IFN-γ + IL-10 - and CD4 + IFN-γ ± IL-10 + T cells has been shown to be an indicator of protection in the mouse model of cutaneous leishmaniasis (39-41). Therefore, to assess this balance we calculated the vaccine/control ratios for CD4 + T cells expressing these cytokines ± TNFα at 2 weeks post-challenge and for disease burden. The ratios eliminate the inherent cytokine staining variability between samples processed on different days and lesion size variability for challenges performed with different preparations of metacyclic promastigotes or infected sand flies. For example, control and vaccine groups from a STN challenge were challenged together and processed together, but on a different day than control and vaccine groups from a STSF challenge. The protected STN and LTSF challenge groups had nearly a six-fold difference in the ratios of CD4 + IFN-γ + TNF-α ± IL-10 - (ratios of 2.3) and CD4 + IFN-γ ± TNF-α - IL-10 + (ratios of 0.4) respectively, with low disease burden ratios of 0.2 and 0.4 respectively (Fig. 6). The unprotected LTN challenge group had ratios of 1.3 and 0.7 for CD4 + IFN-γ + TNFα ± IL-10 - and CD4 + IFN-γ ± TNF-α - IL-10 + respectively, with a disease burden ratio of 0.6. The other unprotected STSF challenge group had ratios of 1.9 and 3.5 for CD4 + IFN-γ + TNF-α ± IL-10 - and CD4 + IFN-γ ± TNF-α - IL-10 + respectively, with a disease burden ratio of 0.8. Overall, the results reveal a pattern in which a combination of high CD4 + IFN-γ + TNF-α ± IL-10 - and low CD4 + IFN-γ ± TNF-α - IL-10 + T cell numbers at 2 weeks post-challenge is predictive of lower disease burden over the course of the experiment. Remarkably, no significant differences or patterns were observed in the anti-leishmania immune response of protected and non-protected mice at 6 and 11 weeks post-challenge (data not shown). DISCUSSION 13

290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 In this study, we used L. monocytogenes as a vaccine delivery vector for LJM11, a sand fly salivary protein that has been shown to be a potent vaccine antigen (16, 42). Our results indicate that delivery of LJM11 by Listeria is a promising vaccination strategy against cutaneous leishmaniasis inducing long-term protection against the formation of ulcers and scars and resulting in an overall decrease in disease burden following a natural challenge with infected sand flies. In addition, these studies reinforce the value of LJM11 as a protective immunogen and validate the use of L. monocytogenes as a delivery vaccine vector. Given that the formulation for a vaccine can have great effects on its efficacy, we set out to determine whether Listeria could be used as an effective vaccine delivery model. Among the advantages of using this system for vaccination are that L. monocytogenes can be attenuated without affecting its ability to induce a strong cell-mediated T H 1 immune response (30), and the antigen of interest is secreted directly in the cytosol of host cells bypassing the need for DNA or protein purification. Our Listeria vaccine vector can express an abundant amount of LJM11 in infected cells and vaccinated mice developed a robust immune response to this protein. Cutaneous leishmaniasis is by definition an infection of the skin, an organ that is populated by specialized sets of dendritic cells that contribute to homing receptor imprinting on T cells (43-45). Accordingly, mice vaccinated intradermally were protected against disease, whereas those vaccinated intramuscularly or subcutaneously were not, presumably because intradermal delivery of the antigen lead to the imprinting of a larger number of T cells that readily migrated to the skin upon challenge. However, the best level of protection was observed in mice vaccinated intravenously. Upon intravenous injection, bacteria are delivered systemically to a large number of secondary lymphatic tissues within minutes of injection. Seemingly, this would lead to production of a larger quantity of antigen, exposition of the antigen to different subsets of dendritic cells, and a more robust induction of T cell proliferation at central and peripheral sites. However, an intravenous vaccine would not be practical, as it would 14

314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 require specialized technical personnel to administer, creating an increased level of complexity in areas where cutaneous leishmaniasis is endemic. A future challenge will be to find ways to improve protection conferred by an intradermal vaccine. An initial approach could be to combine the vaccine with an inhibitor of IL-10, as this cytokine activates regulatory T cells, compromising clearance of the infection (40). Two different types of challenges were used to determine the short and long-term protection effects of the vaccine: a needle challenge in which mice were injected with purified metacyclic promastigotes mixed with sand fly saliva, and a natural challenge in which mice were exposed to infected sand flies. The needle challenge is more practical, less time consuming, more economical, less traumatic for the mice, and more precise than a natural challenge. For these reasons, most experiments were performed using the needle challenge. Ultimately, when optimal vaccine parameters were determined, we aimed at testing vaccine efficacy against a natural challenge using infected sand flies. During a natural challenge, sand flies feed multiple times on the mouse ear, causing more inflammation than a needle challenge. In addition, the challenge dose cannot be controlled during a natural challenge, adding a variable that is absent from the needle challenge. Nevertheless, challenging mice with infected sand flies is imperative to the assessment of vaccine efficacy, as this type of challenge reproduces more closely what happens when an individual becomes infected. Conferring protection long after the last boost and preferably for the life of the host is of primary concern when developing a vaccine. Vaccinated mice were protected short-term against a needle challenge, but not long-term. On the contrary, vaccinated mice were protected long-term against a sand fly challenge, but not short-term. There are obvious differences in these two types of challenges as described above. Needle injections are administered quickly and at a single site, while sand flies bite repeatedly creating more trauma and inflammation at the site of infection. Furthermore, sand flies secrete a mucin-like gel that has been reported to exacerbate infection (46). It would be reasonable to 15

338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 speculate that the extra inflammatory reaction caused by sand fly bites combined with a strong recall response in the short-term challenge might have hastened the development of a regulatory T cell response, abrogating the protective immune response to infection (39). However, vaccination with the purified protein was shown to confer short and long-term protection against a sand fly challenge (16). Perhaps, the structure of LJM11 is modified when made as a chimeric protein by Listeria, potentially affecting the presentation of important amino acids. These results emphasize the need to use a challenge method that is as close as possible to a natural challenge for truly testing vaccine efficacy. At 11 weeks post-challenge, parasite numbers were equivalent in all the groups, despite the fact that the overall disease burden and the number of mice with scars were significantly lower in the protected groups (STN and LTSF). It was not uncommon to find mice with equal parasite numbers but very different pathology at 11 weeks post-challenge (Fig. S4). Nonetheless, parasite numbers were significantly lower at 6 weeks post-challenge in the protected vaccinated groups compared to their respective control groups, and did not significantly increase between 6 and 11 weeks. These results suggest that early control of parasite replication is most important to prevent lesion development and that parasite clearance is not essential to prevent the pathology. Vaccinated mice challenged with infected sand flies had higher parasite numbers than control mice at two weeks post-challenge. Control and vaccinated groups were challenged simultaneously and analysis of flies post-infection indicated no differences between the percent of fed flies between mice. Therefore, it appears that the recall response to LJM11 was favorable to initial parasite survival and/or growth following a sand fly challenge. The immune response in the hours and days following challenge ought to be characterized to address these differences. Crucial to the control of an infection with Leishmania is the development of a T H 1 immune response, characterized mainly by the production of IFN-γ by CD4 + T cells (16, 47). Although CD8 + T cells can also serve as a source of IFN-γ, their cytolytic effect exacerbates immunopathology and mediates the 16

362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 development of metastatic lesions (48). However, we focused our analysis on CD4 + T cells because they consistently accounted for more than 97% of the total numbers of T cells specific to L. major at 2 weeks post-infection. Induction of multifunctional CD4 + T cells producing IFN-γ with or without TNF-α, and few CD4+ T cells producing IL-10 with or without IFN-γ is also very important to disease control (40, 49). Accordingly, our results indicate that protected vaccinated groups generated high CD4 + IFN-γ + TNF-α ± IL- 10 - and low CD4 + IFN-γ ± TNF-α - IL-10 + T cell numbers. This immune profile at 2 weeks post-challenge was predictive of the outcome disease burden for these 11-week long experiments. We conclude that the Listeria-based vaccine secreting the sand fly salivary protein LJM11 during intracellular infection generates an immune response that contributes to curbing disease burden and reducing the number of scars and ulcers in mice challenged with infected sand flies three months postvaccination. Protection correlates with a high ratio of CD4 + IFN-γ + TNF-α ± IL-10 - to CD4 + IFN-γ ± TNF-α - IL- 10 + at 2 weeks post challenge and lower parasite numbers at 6 weeks post-challenge. ACKNOWLEDGEMENTS The study was funded by the Department of Defense Peer Reviewed Medical Research Program of the Office of the Congressionally Directed Medical Research Programs (W81XWH-10-2-0022), and in part by the Intramural Research Program of the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. We are thankful to Dr. Regis Gomes for advice during the duration of this project. REFERENCES 1. Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M. 2012. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7:e35671. 17

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506 FIGURE LEGENDS 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 FIG 1 Listeria vaccine vector. A: Schematic representation of the L. monocytogenes vaccine construct. The various sequence parts code for: acta P: acta promoter; ActA SS: ActA signal sequence; 100 amino acids of the ActA N-terminus; FLAG: Flag tag; and LJM11: Lutzomyia longipalpis salivary gland protein LJM11. B: Infected J774 cells were pulse labeled with 35 S-met in the presence of host protein synthesis inhibitors. Host cells were lysed and secreted bacterial proteins were resolved by SDS-PAGE and detected by autoradiography. Molecular mass markers in kda are indicated on the far left; the location of ActA/LJM11 chimera and a degradation product (*) are indicated on the far right; HEL-1207 (L. monocytogenes acta mdrm), and HEL-1325 (HEL-1207 acta/ljm11). C: Intracellular growth kinetics of L. monocytogenes in J774 cells. D: Immune response to LJM11 in splenocytes from vaccinated mice. Mice were vaccinated intravenously as described in Methods with the control vector (HEL-1207) or the vaccine vector (HEL-1325). Three weeks later, spleens were collected and splenocytes were incubated overnight with dendritic cells that had been pulsed with or without LJM11. Levels of secreted IFN-γ and IL-10 were measured by ELISA (limit of detection was 15 and 70 pg/ml for IFN-γ and IL-10, respectively). Data were analyzed using the One way Anova test with Bonferroni post-test analysis. ** p<0.01. n=3 per mouse group. FIG 2 L. monocytogenes vaccine vector confers the greatest protection against cutaneous leishmaniasis when administered intravenously. Lesion areas of mice vaccinated using different routes. Mice were primed and boosted twice at two-week intervals with the vaccine vector (HEL-1325) by the intradermal (ID, dark blue x), intramuscular (IM, magenta diamond), subcutaneous (SC, light blue circle) or intravenous (IV, red cross) route, while a group of age-matched mice was left unvaccinated (CTRL, black square). Three weeks after the last boost, mice were challenged with L. major plus half a pair of sand fly salivary glands. A: Ear lesion areas (diameter x thickness) were measured weekly and plotted. B: Disease burden of mice vaccinated and challenged. Area under the curve was measured for each individual 24

530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 mouse and plotted. Experiment was repeated twice with n=6 per mouse group. C: Mice were primed and boosted intravenously twice at two-week intervals with the vaccine vector control (HEL-1207) or PBS, and challenged three weeks later as described above. Ear lesion areas are reported. Data were analyzed using the One-way Anova statistical test with Bonferroni post-test analysis. *** p<0.001, * p<0.05. FIG 3 Short-term needle and sand fly challenges. Mice were vaccinated intravenously three times at two-week intervals, and challenged intradermally three weeks after the last boost. Control group (HEL- 1207); Vaccine group (HEL-1325). A-D: Mice were challenged by needle injection with L. major plus half a pair of salivary gland homogenate. E-H: Mice were challenged with infected sand flies. A and E: Lesion area measurements for 10 weeks post-challenge. B and F: Disease burden calculated by measuring the area under the curve for each individual mouse. C and G: Percent of mice that developed scars over the duration of 10 weeks. D and H: Parasite numbers per ear at 2, 6 and 11 weeks post challenge. Experiments were repeated twice and data shown are pooled from both experiments; n of 30 mice per group (10 processed at each time-point). Data were analyzed using the T-test comparing vaccine and control groups. *** p<0.001, **p<0.01, * p<0.05. The control group in 3A is the same as the vector control group in 2C. FIG 4 Long-term needle and sand fly challenges. Mice were vaccinated intravenously three times at twoweek intervals, and challenged intradermally three months after the last boost. Control group (HEL- 1207); Vaccine group (HEL-1325). A-D: Mice were challenged by needle injection with L. major plus half a pair of salivary gland homogenate. E-H: Mice were challenged with infected sand flies. A and E: Lesion area measurements for 10 weeks post-challenge. B and F: Disease burden calculated by measuring the area under the curve for each individual mouse. C and G: Percent of mice that developed scars over the duration of 10 weeks. D and H: Parasite numbers per ear at 2, 6 and 11 weeks post challenge. Experiments were repeated twice and data shown are pooled from both experiments; n of 30 mice per 25

554 555 group (10 processed at each time-point). Data were analyzed using the T-test comparing vaccine and control groups. *** p<0.001, **p<0.01, * p<0.05. 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 FIG 5 Recall response to vaccine at 2 weeks post-challenge. Cells collected from ears were incubated overnight with dendritic cells pre-pulsed with CLA and then treated for 4 hours with brefeldin A and PMA. Cells were stained for surface markers and intracellular cytokines, and analyzed by flow cytometry. Results were analyzed by gating for CD4 surface expression and intracellular cytokine expression (IFN-γ and TNF-α, IFN-γ, IL-10, or TNF-α). The total number of Leishmania-specific cytokine producing CD4 + T cells in both control (HEL-1207; black bars) and vaccine (HEL-1325; grey bars) groups of mice is shown for (A) short-term needle challenge (STN), (B) long-term needle challenge (LTN), (C) short-term sand fly challenge (STSF), and (D) long-term sand fly challenge (LTSF). E-H: Data were converted into pie charts showing the contribution of each cytokine combination to the total number of cytokine producing CD4 + T cells. Experiments were repeated twice and data shown are pooled from both experiments. N-10 (n=5 mice per group per experimental repeat). Data were analyzed using the T-test comparing vaccine and control groups. **p<0.01, * p<0.05. FIG 6 Cytokine production at 2 weeks post-challenge predicts disease burden. Total numbers of CD4 + IFN-γ + TNF-α ± IL-10 - and CD4 + IFN-γ ± TNF-α - IL-10 + T cells were calculated for control (HEL-1207) and vaccine (HEL-1325) groups of mice and plotted as Vaccine/Control ratios. Ratios for CD4 + IFN-γ + TNF-α ± IL-10 - are shown in blue (triangles), CD4 + IFN-γ ± TNF-α - IL-10 + ratios are shown in red (squares) and disease burden ratios are shown in grey (circles). Cytokine ratios were calculated from data at 2 weeks post-challenge while disease burden ratios are for the duration of the entire study. Cytokine ratios are plotted on the left y-axis while the disease burden ratios are plotted on the right y-axis. STN: short-term needle challenge, LTN: long-term needle challenge, STSF: short-term sand fly challenge, LTSF: long-term sand fly challenge. Experiments were repeated twice and data shown is pooled from both experiments. n=10 (n=5 mice per group per repeat). 26